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Graduate Theses and Dissertations
Graduate School
January 2012
Development of a Bio-Molecular Fluorescent
Probe Used in Kinetic Target-Guided Synthesis for
the Identification of Inhibitors of Enzymatic and
Protein-Protein Interaction Targets
Katya Pavlova Nacheva
University of South Florida, [email protected]
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Nacheva, Katya Pavlova, "Development of a Bio-Molecular Fluorescent Probe Used in Kinetic Target-Guided Synthesis for the
Identification of Inhibitors of Enzymatic and Protein-Protein Interaction Targets" (2012). Graduate Theses and Dissertations.
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Development of a Bio-Molecular Fluorescent Probe Used in Kinetic Target-Guided Synthesis for
the Identification of Inhibitors of Enzymatic and Protein-Protein Interaction Targets
by
Katya P. Nacheva
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
Collage of Arts and Science
University of South Florida
Major Professor: Roman Mantesch, Ph.D.
Jon Antilla, Ph.D.
Wayne Guida, Ph.D.
Jianfeng Cai, Ph.D.
Date of Approval
November 14, 2012
Keywords: Fluorescence, Förster resonance energy transfer (FRET), Fragment-based lead
discovery, in situ click chemistry, Protein-protein interactions, Sulfo-click chemistry
Copyright © 2012, Katya P. Nacheva
Note to Reader
The entire work described in Chapter 2 has been published in the Organic and
Bimolecular Chemistry in 2012, Org. Biomol. Chem., 2012, 10 (38), 78407846, http://pubs.rsc.org/en/content/articlepdf/2012/ob/c2ob25981h. Reproduced by
permission of the Royal Society of Chemistry
Dedication
I would like to dedicate this dissertation to my parents, my sister, and my
daughter Sophia. Without their moral support, encouragement and love I would not have
been able to pursue this degree. My parents have always been a role model for me and
taught me to be persistent and to achieve my goals. I am very grateful to them for being
always next to me and for showing me how much faith they have in me. They both
worked very hard and made a lot of sacrifices to ensure that I and my sister had an
excellent education. I am proud to have such loving and supportive parents and I deeply
hope that I will make them proud.
It is always difficult to find the right words to thank my sister. She is an
extraordinary person whom I treasure very much and my life would have been different
without her support and friendship. She is my greatest motivator with her excellent
quality of work and great achievements. I am so grateful to have my little precious
daughter, Sophia, who brought so much joy to my life and gave meaning to everything I
do.
Acknowledgments
I would like to express my gratitude to my advisor, Dr. Roman Manetsch, for his
guidance and support through my research endeavors. His great expertise has had a huge
impact on me as a chemist and on my research. Dr. Manetsch has been a great mentor to
me and I appreciate everything he has done to direct and guide my work with valuable
suggestions and ideas. He has worked extremely hard to bring the lab to a high level of
research quality, and his persistence has been a driving force for all of us. His confidence
in me will always inspire me and carry me through my future work. Dr. Manetsch has
been always open for discussion, and the time and attention he devoted to me has been
really valuable. Personally, I highly appreciate his warm words and kindness in
challenging moments of my life. He encouraged me to keep going despite personal
difficulties.
I would like to recognize my committee members, Dr. Jon Antilla, Dr. Wayne
Guida, and Dr. Jianfeng Cai, for their support, attention and time. Their insightful
suggestions have been valuable during my graduate studies. I also thank Dr. Randy
Larsen for his great assistance and welcoming me in his lab to perform some of my
research studies. I appreciate the assistance of Dr. Jeremiah Tipton with the instrumental
measurements and helpful suggestions. I would also like to thank my group members in
the Manetsch lab from the past and present. The post-docs Dr. Xiangdong Hu, Dr.
Niranjan Kumar Namelikonda, Dr. David Flanigan, and Dr. Raghupathi
Neelarapu, who always found time for research discussions and provided me with great
ideas.
Also, I had a great experience working with the graduate students in the lab, and
am grateful for such a wonderful group of colleagues – so I would like to thank to Dr.
Arun Babu Kumar, Dr. R. Matthew Cross, Dr. Shikha Mahajan, Sameer Kulkarni,
Jordany Maignan, Andrii Monastyrskyi, Iredia Iyamu, Kurt Van Horn, Cynthia
Lichorowic, and Megan Barber. I am happy to thank Willima Maza for his great
assistance with the published manuscript, and for his guidance while I used some of the
instruments in Dr. Larsen’s lab. I would like to thank the undergraduate student Zac
Myers for his contributions to some of the research projects.
I also thank my family and friends for their support, reassurance and keeping me
on track with my study.
Table of Contents
List of Tables
v
List of Figures
vi
List of Schemes
xiv
Abstract
xv
Chapter 1: Introduction
1.1
1
Fluorescence and Design of Florescent Probes
1.1.1
1.1.2
1.1.3
1.2
1.3
1.4
1.5
1.6
Fluorescence Mechanism
Fluorophores and Related Applications
Major Classes of Fluorophores
1.1.3.1 Fluorophores with Emission up to 500 nm
1.1.3.1a Oxygen Containing Heterocyclic
Fluorophores
1.1.3.1b Nitrogen Based Fluorophores
1.1.3.1c Naphthalene Fluorophores
1.1.3.2 Fluorophores with Emission Beyond 500 nm
1.1.3.2a Fluorescein dyes
1.1.3.2b Cyanine dyes
Förster Resonance Energy Transfer (FRET) Systems
1.2.1 FRET Mechanism
1.2.2 FRET Applications in Biological Systems
1.2.3 FRET Mechanism in the Screening of Libraries of Small
Molecules
Targeting the ‘Undruggable’ Targets – Protein-Protein
Interactions (PPIs)
Targeting the Protein-Protein Interactions of Bcl-2 Family
Various Methods Used in Drug Discovery
Fragment-Based Lead Discovery (FBLD)
1.6.1 Fragment-Based Lead Discovery Using Biophysical
Approaches
i
1
1
3
4
5
5
6
6
7
7
8
9
9
12
16
18
19
22
24
24
1.6.2
1.7
1.8
Chapter 2:
2.1
2.2
2.3
2.4
2.5
2.6
Target Guided Synthesis (TGS)
1.6.2.1 Dynamic Combinatorial chemistry (DCC)
1.6.2.2 Kinetic TGS
1.6.3 Click chemistry and in situ click chemistry
1.6.3.1 In situ Click Chemistry using
Acetylcholinesterase as a template
1.6.3.2 In situ Click Chemistry using Carbonic
Anhydrase as a template
1.6.3.3 In situ Click Chemistry using HIV-1 Protease
(HIV-1-Pr) as a template
1.6.3.4 Sulfo-Click Amidation Reaction
Research Aims
1.7.1 Designing a Fluorescent Probe and Its Implementation in
FRET Systems
1.7.2 In Situ Generated Novel Carbonic Anhydrase Inhibitory
Compounds
1.7.3 Parallel Sulfo-Click Kinetic TGS Screening of Multiple
Protein-Protein Interactions
References
25
26
27
28
31
33
35
36
40
40
41
42
43
Fluorescent Properties and Resonance Energy Transfer of
3,4-bit(2,4-difluorophenyl)-maleimide
62
Overview
Photophysical Properties and Characterization
pH Sensitivity and Photobleaching Stability
Designing of FRET Systems with 3,4-diaryl-substituted
Maleimide Moiety as a Donor Partner
2.4.1 FRET System with Quencher Moiety as an Acceptor
2.4.2 FRET System with Bright Fluorophore Moiety
as an Acceptor
Conclusions
Experimental Section
2.6.1 General Information
2.6.2 Spectroscopic Analysis and Characterizations of
2.1, 2.1a, 2.1b and 2.1c
2.6.2.1 Quantum Yields and Extinction Coefficients in
Different Solvents
2.6.2.2 Solvatochromism
2.6.2.3 pH Dependence of the Emission
2.6.3 Spectroscopic Analysis and Characterization of the
FRET Peptide System with Dark Quencher as an Acceptor
2.6.3.1 Spectral Overlap
2.6.3.2 Absorption and Emission Spectra of 2.9
2.6.4 Fluorimetric Titration Experiment
2.6.5 Conditions for the incubations of the FRET peptide with
62
63
67
ii
69
69
73
75
76
76
77
77
78
79
81
81
82
82
83
β-secretease and Lineweaver-Burk plot
2.6.5.1 Incubation conditions of the FRET peptide with
β-secretease
2.6.5.2 Lineweaver-Burk Plot of the Enzymatic Reaction
2.6.6. Spectroscopic Analysis and Characterization of the
FRET System with Bright Acceptor
2.6.6.1 Absorption of the Acceptor (TPP)
2.6.6.2 Emission Spectra of the Acceptor (TPP)
2.6.6.3 FRET Titration Experiment between
the Donor (2.1a) and the Acceptor (TPP)
2.6.6.4 Representation of the Resonance Energy Ration
Change (RRC)
2.7
Chapter 3:
3.1
3.2
3.3.
3.4
3.5
Chapter 4:
4.1
83
84
85
85
85
86
87
2.6.7
Calculation of the Förster-Radius (R 0 )
88
2.6.8
Synthetic Procedures
88
References
96
In situ Generated Novel Carbonic Anhydrase
Inhibitory Compounds
Overview
Result and Discussion
3.2.1 Design of Non-Sulfonamide Containing Inhibitors
3.2.2 Kinetic TGS
3.2.3 Validation of Kinetic TGS Hits
3.2.4 Determination of Selectivity of the CA-Templated Reaction
3.2.5 Binding Measurements
Conclusion
Experimental Section
3.4.1 General Information
3.4.2 General Procedure for In situ Click Chemistry Experiments
3.4.3 Regioisomer determination for bCAII-derived in situ hits
3.4.4 In situ Click Chemistry Control experiments with Apo-CA
3.4.5 Synthetic Procedures
References
Parallel Sulfo-Click Kinetic TGS Screening of Multiple
Protein-Protein Interactions
Overview
4.2
Results and Discussion
4.2.1 Library of Thio Acids and Sulfonylazides Used in
A Multi-Component Kinetic TGS Screening
4.2.2 Optimization of Multi-Fragment Sulfo-Click Kinetic TGS
Screening with Triple Quadrupole Mass Spectrometry
Detection
4.2.3 Expansion of the Thio Acid Library and
iii
98
98
103
103
106
108
109
113
113
115
115
115
117
121
124
139
145
145
153
153
156
168
Multi-Component Kinetic TGSx against Mcl-1 and Bcl-X L
Conclusion
Experimental section
4.4.1 General information
4.4.2 General Protocol for Multi-Fragment
“Sulfo-Click” Kinetic TGS Experiments with Mcl-1 and Bcl-X L
4.4.3 General protocol for control incubations of mutant
R139A
Bcl-X L
4.4.4 General Procedure to Synthesize Fluorenylmethyl Thioesters
4.4.5 General Procedure to Synthesize Acylsulfomamides
4.4.6 Synthetic Procedures
176
176
176
177
4.5.
References
186
5.
Appendices
193
5.1 Appendix 1
194
4.3
4.4
iv
173
174
174
175
List of Tables
Table 1.1
Typical values of RF for well-known donor/acceptor pairs
Table 2.1.
Solvent dependent absorption and emission maxima of
compounds 2.1, 2.1a – 2.1c
Table 2.2.
10
66
Extinction coefficients of 2.1, 2.1a, 2.1b, and 2.1c
(λmax = 340 nm)
77
Table 2.3.
Quantum yield of 2.1, 2.1a, 2.1b, and 2.1c
77
Table 3.1
Solvent gradient used for TQMS methods for
AK1AZ2 and AK4AZ2
Table 3.2
116
Solvent gradient used for TQMS methods for
AK1AZ3 and AK4AZ3
116
Table 4.1.
Collision energy (eV) at which the corresponding acylium ion is
159
Table 4.2.
Fragment distribution in the initial studies of multi-fragment
kinetic TGS screening
Table 4.3.
161
Potential hit combinations identified through multi-fragment
v
Table 4.4.
kinetic TGS approach (SZ 1-38 and TA 1-45)
172
Elution gradient employed for TQMS-MRM analysis
174
v
List of Figures
Figure 1.1.
Jablonski Diagram
2
Figure 1.2.
Schematic representation of FRET mechanism which occurs
between A. a donor (D) and an acceptor (A) chromophorese and
B. a donor fluorophore andan acceptor quencher (Q)
11
Figure 1.3.
Detection of conformational changes induced by small molecules
12
Figure 1.4.
FRET system with CFP-YFP pair designed to measuring intracellular 14
calcium level
Figure 1.5.
FRET system utilized as a sensor to detect phosgene (or triphosgene)
15
Figure 1.6.
Examples of Bcl-2/Bcl-X L protein interaction modulators
21
Figure 1.7.
Example of Dynamic Combinatorial Chemistry (DCC) used to
generate inhibitors of bovine carbonic anhydrase II
27
Figure 1.8.
Kinetic Target-Guided Syntheses
27
Figure 1.9.
Examples of kinetic TGS reactions used against various biological
targets
28
Figure 1.10. Synthesis of syn- and anti-triazoles through stereoselective methods
or in situ click chemistry
30
Figure 1.11. Hit triazole combinations for AChE, identified through the in situ
vi
click chemistry approach
Figure 1.12.
34
Application of in situ click chemistry for the synthesis of novel
carbonic anhydras inhibitors
34
Figure 1.13. In situ click chemistry used to identify HIV-1 protease inhibitors
via a multi-component screening of fragments
Figure 1.14. Amdiation reaction between sulfonyl azides and thio acids
36
37
Figure 1.15. Kinetic TGS of Bcl-XL-templated amidation reaction between
sulfonyl azide and thio acid
38
Figure 2.1.
Normalized absorption and emission of 2.1 in dichloromethane
64
Figure 2.2.
X-ray crystal structure of 1 in dichloromethane
65
Figure 2.3.
Normalized emission spectra of 1 (DCM, 1 x 10-5 M) after
rradiation with Xe source for 6 – 18 hours, λ exc = 340 nm
Figure 2.4.
67
Fluorescence pH dependence of 1 under A. acidic conditions,
TFA (CH 3 CN, 5 x 10-6 M) and B. basic conditions, 2,6- lutidine
(CH 3 CN, 5 x 10-6 M), λ exc = 340 nm
Figure 2.5.
68
Emission spectra of A. The tethered donor/acceptor FRET peptides
and B. FRET peptide after enzymatic cleavage
71
Figure 2.6.
FRET peptide cleaved by β-secretase.
72
Figure 2.7.
Synthesis of the FRET paired system
73
Figure 2.8.
Diffusional FRET between the non-covalently bound
donor/acceptor pair
Figure 2.9.
74
Emission spectra of 2.1, 2.1a, 2.1b, and 2.1c recorded in various
solvents (λ ex = 340 nm)
78
vii
Figure 2.10. Emission spectra of 2.1 (λ ex = 340 nm) in CH 3 CN recorded through
titration of acid
80
Figure 2.11. Emission spectra of 2.1 (10 μM in CH 3 CN, λ ex = 340 nm) titrated
with Et 3 N (0 eq -2060 eq)
80
Figure 2.12. Emission spectra of 1b (5 μM in CH 3 CN, λ ex = 340 nm)) obtained
from the titration of 2,6-lutidine (0 eq -5600 eq)
81
Figure 2.13. Spectral overlap of the emission profile of 1c (donor, blue line) and
the absorption profile of the dark quencher (acceptor, black line)
81
Figure 2.14. Absorption spectrum of the covalently linked donor (2.1c) and the
acceptor (Dabcyl)
82
Figure 2.15. Normalized emission spectra the covalently linked donor (2.1c) and
the acceptor (Dabcyl)
82
Figure 2.16. Emission spectra recorded in a titration experiment in which
the quencher molecule, Dabcyl, (0 eq – 2.5 eq in DCM) was
Figure 2.17.
titrated to the acceptor 1c (10 μM in DCM, λ ex = 340 nm)
83
Lineweaver-Burk plot of the enzymatic reaction
84
Figure 2.18. Absorption of the acceptor (TPP)
85
Figure 2.19. Emission spectra of the acceptor (TPP) alone in toluene A
excited at λ exc = 270 nm and B. excited at λ exc = 420 nm
viii
85
Figure 2.20. Emission spectra obtained by titrating 2.1a (10 – 150 μM)
(25 μM) in toluene (λ exc = 270 nm), demonstrating the
FRET diffusion mechanism between the donor/acceptor
86
Figure 2.21. Emission spectra of A. FRET pairs 2.1a and TPP free in solution
(toluene) and B. FRET pairs 2.1a and TPP covalently attached
at the same concentration
86
Figure 2.22. Synthetic approach to generate the FRET peptide with attached
89
donor and acceptor moiety
Figure 3. 1.
Application of in situ click chemistry for the synthesis of novel
sulfonamide-based bCA II inhibitors
Figure 3.2.
Hydrolysis of the he coumarin natural product 6-(1S-hydroxy-3methylbutyl)-7-methoxy-2H-chromen-2-one
Figure 3.3.
109
Synthesis of anti-AK1AZ2, anti-AK1AZ3, anti-AK4AZ2,
anti-AK4AZ3
Figure 3.6.
107
General synthetic route used to generate the anti-, and syn-triazole
isomers
Figure 3.5.
104
Library of alkynes AK and azides AZ fragments utilized in
a kinetic TGS screening of bCAII
Figure 3.4.
102
110
Synthesis of syn-AK1AZ2, syn-AK1AZ3, syn-AK4AZ2,
ix
syn-AK4AZ3
Figure 3.7.
111
In situ hit identification by LC/MS-SIM, exemplified by
the AK1/AZ3 pair
Figure 3.8.
112
In situ click chemistry hit identificationo of AK4AZ3 triazole
combination.
Figure 3.9.
117
In situ click chemistry hit identificationo of AK4AZ2 triazole
combination.
Figure 3.10
118
In situ click chemistry hit identificationo of AK1AZ2 triazole
combination.
Figure 3.11
119
In situ click chmtisty control experiment of AK4 and AZ2
with hCAII and Apo CA
120
Figure 3.12. In situ click chmtisty control experiment of AK1 and AZ2
with hCAII and Apo CA
120
Figure 3.13. In situ click chmtisty control experiment. of AK1 and AZ3
with hCAII and Apo CA
121
Figure 3.14. In situ click chmtisty control experiment of AK4 and AZ3
with hCAII and Apo CA
Figure 4.1.
122
Protein-protein interaction modulators targeting: A) multiple
members of the Bcl-2 family and B) Mcl-1 selectively
Figure 4.2.
146
Kinetic TGS approach using a sulfo-click reaction to identify
inhibitors of Bcl-X L
148
x
Figure 4.3.
Schematic representation of mass spectrometry analysis with
Different single and triple quadrupole scanning modes
151
Figure 4.4 A. Library of thio acids TA1-TA10 used for the initial
proof-of-concept study of multi-fragemen screening
via sulfo-click kinetinc TGS
153
Figure 4.4 B. Library of sulfonyl azides SZ1-SZ38
Figure 4.5.
Synthetic approaches to generate sulfonyl azides
Figure 4.6.
Schematic representation of Multiple Reaction Monitoring
154
(MRM) mode of analysis performed with a triple quadrupole
mass spectrometer
Figure 4.7.
157
Collision-activated defragmentation curve of acylsulfonamide
SZ8TA4
Figure 4.8.
158
Library of nine sulfonyl azides (SZ1-SZ9) and nine thio acids
(TA1-TA9) used in a proof-of-concept study of multi-fragment
kinetic TGS screening
160
Figure 4.9.A. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5 using
multi-fragment kinetic TGSscreening with a TQMS.
xi
Incubation of five thio acids and nine sulfonyl azides
together in one well (45 possible acylsulfonamides) with buffer
163
Figure 4.9.B. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5 using
multi-fragment kinetic TGSscreening with a TQMS. Incubation
of five thio acids and nine sulfonyl azides together in one well (45
possible acylsulfonamides) with Bcl-XL.
164
Figure 4.10.A Identification of the four previously identified kinetic TGShit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5
using multi-fragment kinetic Incubation of 19 fragments
together inone well (81possible acylsulfonamides) with buffer
165
Figure 4.10.B. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5
using multi-fragment kinetic TGS screening with a TQMS.
Incubation of 19 together fragments inone well (81possible
acylsulfonamides) with Bcl-X L
166
Figure 4.11. Library of forty five thio acids (TA1 – TA45) used for
multi-component kinetic TGS against Bcl-X L and Mcl-1
xii
169
Figure 4.12
Multi-fragment kinetic TGS screening of 38 sulfonyl azides
(38 SZ) and45 thio acids (45 TA) giving rise to 1710
possible fragment combinations
xiii
170
List of Schemes
Scheme 2.1. Synthesis of 3,4-bis(2,4-difluorophenyl)-maleimide 2.1
and the analogs 2.1a, 1b, and 2.1c
xiv
65
Abstract
Fluorescent molecules used as detection probes and sensors provide vital
information about the chemical events in living cells. Despite the large variety of
available fluorescent dyes, new improved fluorogenic systems are of continued interest.
The Diaryl-substituted Maleimides (DMs) exhibit excellent photophysical properties but
have remained unexplored in bioscience applications. Herein we present the
identification and full spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)maleimide and its first reported use as a donor component in Förster resonance energy
transfer (FRET) systems. The FRET technique is often used to visualize proteins and to
investigate protein-protein interactions in vitro as well as in vivo. The analysis of the
photophysical properties of 3,4-bis(2,4-difluorophenyl)-maleimide revealed a large
Stokes shift (Δ) of 140 nm in MeOH, a very good fluorescence quantum yield in DCM
(Φ fl 0.61), and a high extinction coefficient ε (340) 48,400 M-1cm-1, thus ranking this
molecule as superior over other reported moieties from this class. In addition, 3,4-bis(2,4difluorophenyl)-maleimide was utilized as a donor component in two FRET systems
wherein different molecules were chosen as suitable acceptor components - a fluorescent
quencher (DABCYL) and another compatible fluorophore, tetraphenylporphyrin (TPP). It
has been demonstrated that by designing a FRET peptide which contains the DM donor
moiety and the acceptor (quencher) motif, a depopulation of the donor excited state
occurred via intermolecular FRET mechanism, provided that the pairs were in close
xiv
proximity. The Förster-Radius (R 0 ) calculated for this FRET system was 36 Å
and a Förster-Radius (R 0 ) of 26 Å was determined for the second FRET system which
contained TPP as an acceptor. The excellent photophysical properties of this fluorophore
reveal a great potential for further bioscience applications.
The
3,4-bis(2,4-difluorophenyl)-maleimide
fluorescent
moiety
was
also
implemented in an alternative application targeting the enzyme carbonic anhydrase (CAs)
are metalloenzymes that regulate essential physiologic and physio-pathological processes
in different tissues and cells, and modulation of their activities is an efficient path to
treating a wide range of human diseases. Developing more selective CA fluorescent
probes as imaging tools is of significant importance for the diagnosis and treatment of
cancer related disorders. The kinetic TGS approach is an efficient and reliable lead
discovery strategy in which the biological target of interest is directly involved in the
selection and assembly of the fragments together to generate its own inhibitors. Herein,
we investigated whether the in situ click chemistry approach can be implemented in the
design of novel CA inhibitors from a library of non-sulfonamide containing scaffolds,
which has not been reported in the literature. In addition, we exploit the incorporation of
the (recently reported by us) fluorescent moiety 3,4-bis(2,4-difluorophenyl)–maleimide)
as a potential biomarker with affinity to CA, as well as two coumaine derivatives
representing a newly discovered class of inhibitors. The screening of a set of library with
eight structurally diverse azides AZ1–AZ8 and fifteen functionalized alkynes AK1–
AK12 led to the identification of 8 hit combinations among which the most prominent
ones were those containing the coumarine and fluorescent maleimide scaffolds. The synand anti-tirazole hit combinations, AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were
xv
synthesized, and in a regioisomer-assignment co-injection test it was determined that the
enzyme favored the formation of the anti-triazoles for all identified combinations. The
mechanism of inhibition of these triazoles was validated by incubating the alkyne/azide
scaffolds in the presence of Apo-CA (non-Zn containing) enzyme. It was demonstrated
that the Zn-bound water/hydroxide was needed in order to hydrolyze the coumarins
which generated the actual inhibitor, the corresponding hydroxycinnamic acid. The time
dependent nature of the inhibition activity typical for all coumarine-based inhibitors was
also observed for the triazole compounds whose inhibition constants (Ki) were
determined in two independent experiments with pre-incubation times of 3 and 25
minutes, respectively. It was observed that the lower Ki values were determined, the
longer the pre-incubations lasted. Thus, a novel type of coumarin-containing triazoles
were presented as in situ generated hits which have the potential to be used as fluorescent
bio-markers or other drug discovery applications.
The proteins from the Bcl-2 family proteins play a central role in the regualtion of
normal cellular homeostasis and have been validated as a target for the development of
anticancer agents. Herein, in a proof-of-concept study based on a previous kinetic TGS
study targeting Bcl-X L , it was demonstrated that a multi-fragment kinetic TGS approach
coupled with TQMS technology was successfully implemented in the identification of
known protein-protein modulators. Optimized screening conditions utilizing a triple
quadruple mass spectrometer in the Multiple Reaction Monitoring (MRM) mode was
demonstrated to be very efficient in kinetic TGS hit identification increasing both the
throughput and sensitivity of this approach. The multi-fragment incubation approach was
studied in detail and it was concluded that 200 fragment combinations in one well is an
xvi
optimal
and
practical
number
permitting
good
acylsulfonamide
detectability.
Subsequently, a structurally diverse liberty of forty five thio acids and thirty eight
sulfonyl azides was screened in parallel against Mcl-1 and Bcl-X L, and several potential
hit combinations were identified. A control testing was carried out by substituting Bcl-X L
with a mutant
R139A
Bcl-X L, used to confirm that the potential kinetic TGS hit
combinations were actually forming at the protein’s hot spot and not elsewhere on the
protein surface. Although, the synthesis of all these kinetic TGS hit compounds is
currently ongoing, preliminary testing of several acylsulfonamides indicate that they
disrupt the Bcl-X L /Bim or Mcl-1/Bim interaction.
xvii
Chapter 1
Introduction
1.1
Fluorescence and Design of Florescent Probes
1.1.1
Fluorescence Mechanism
Fluorescence is a three-stage process that occurs in certain type of molecules
(fluorophores) that typically possess heterocyclic or polyaromatic structures.1 The
fluorescent process is illustrated by Jablonski’s simple electronic state diagram
(Figure1.1) and can be summarized in the following sequence of transformations: a)
photon absorption by a fluorophore (excitation); b) existence of an excited state (life
time); c) relaxation of the excited state by the emission of a photon.
Stage 1: Excitation
This process is initiated when a molecule in a singlet ground state (S0) absorbs a
photon with energy hυex, supplied by an external source (incandescent lamp or a laser)
and promotes an excited electronic singlet state (S2).
Stage 2. Excited-State Lifetime
The excited state exists for a very limited time (usually 1-10 nanoseconds) and
quickly relaxes to the first excited state (S1) from which fluorescence emission
originates.
Stage 3. Fluorescence Emission
1
It is important to note that not all excited molecules decay through a photon emission
(i. e., fluorescence) but some of the energy dissipates through a non-radiative mechanism.
Examples of non-radiative processes are: collisional quenching, bond rotation or
vibration, intersystem crossing, and fluorescence resonance energy transfer (FRET) to a
suitable acceptor molecule.2 The intersystem crossing occurs from a singlet excited state
(S1) to a triplet excited state (T1) with a subsequent relaxation through either a nonradiative decay or a photon emission (i.e., phosphorescence). Due to the fact that energy
dissipates at the excited stage, the energy of the emitted photon, hυem, is lower than the
absorbed energy and thus has a longer wavelength.
Figure 1.1. Jablonski Diagram2: (1) absorption of a photon to an excited state; (2)
internal conversion to excite state S1; (3) fluorescence; (4) intersystem crossing to T1; (5)
phosphorescence.
In fluorescence, the difference between the absorption maxima and the emission maxima
is known as the Stokes shift and it is a distinct property for each fluorophore. The Stokes
shift is essential for the sensitivity of various fluorescent techniques and often
2
fluorophores with small Stokes shift are susceptible to self-quenching. Another critical
property, which determines the fluorescence output of a given fluorophore, is the
efficiency with which it absorbs and emits photons, termed as the fluorescent quantum
yield (Φ). The absorption efficacy is usually quantified in terms of molar extinction
coefficient (ε) and it ranges from 5000 to 200,000 cm-1M-1 (phycobilin-proteins have
multiple fluorophores on each protein and have ε = 200,000 cm-1M-1)3. The product of the
molar extinction coefficient and the fluorescent quantum yield (ε * Φ) has been used as an
accurate comparison of the brightness of fluorescent molecules. Another important
characteristic of the fluoresence process is related to the fluorophore’s ability to be
repeatedly excited and detected, which is a cyclic and continues process, unless the
fluorophore is irreversibly destroyed in the excited state (“photobleached”). The
fluorophore’s susceptibility to photobleaching significantly limits its detectability and
overall application.
1.1.2
Fluorophores and Related Applications
Fluorescent chromophores find extensive applications as detection probes and
sensors4 in the profiling and visualization of protease activity,5 and protein kinetics.6
Fluorescence labeling has been recognized as an important tool to probe cellular
biochemistry due to its non-invasive and highly sensitive character.7
In recent years,
fluorescent detection techniques received special attention contributed to their flexibility
in implementation, selectivity, and simplicity.8 These detection techniques can also
provide real-time observation of chemical or physical changes of molecules of interest
and suggest a better understanding of the chemical events in living cells.9 Thus,
3
significant progress has been made in both fluorescence instrumentation as well as design
of improved and diverse fluorophores.
Generally in biology, there are three types of fluorescent chromophores: intrinsic,
coenzymatic, and extrinsic.1 The intrinsic and coenzymatic types are based on the
presence of specific amino acid residues with fluorescent properties and they have
provided important information about the structure and the functions of proteins.
However, the types of chromophores designed by nature are less versatile, and can not be
always introduced at a desired location in the protein. The extrinsic chromophores,
commonly referred as fluorescence probes (synthetic small molecules), often can be
covalently combined with the enzyme or the protein of interest.8b Thus, the respective
conjugates are able to exhibit fluorescence from a short to a long wavelength, depending
on the type of the chromophor. A key point in the process of fluorescent labeling with
small molecules is to ensure that the reactive fluorescent derivative selectively bind to a
particular functionality in the biological target of interest. Commonly utilized reactive
functionalities include the amine-reactive isothiocyanate derivatives (for example FITC),
the amine-reactive succinimiyl ester (for example NHS-fluorescein, NHS-rhodamine)
and sulfhydryl-reactive maleimides.10 Conveniently, the reaction of such functionalized
dyes with the targeted molecule results in the formation of a stable covalent bond
between the label protein and the fluorophore.
1.1.3
Major Classes of Fluorophores
The organic fluorophores are broadly divided according to the absorption and
emission wavelength values of their conjugates and thus rendered from near-ultraviolet to
4
500 nm and from 500 nm to near-infrared, 900 nm.11 One of the most prominent
representatives in the first group are: oxobenzopyrans, naphthofurans, oligothiophenes,
4,7-phenanthroline-5,6-diones, benzooxadiazoles, dansyl chloride, naphthalene 2,3dicarboxaldehyde, and 6-propionyl-2 (dimethylamino)naphthalene. The most important
type of chemophores that fall in the second category are: fluoresceins, rhodamines, 4,4difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY dyes), squaraines, and cyanines.
1.1.3.1
Fluorophores with Emission up to 500 nm
1.1.3.1a Oxygen Containing Heterocyclic Fluorophores
3-Oxo-3H-benzopyrans, commonly designated as coumarins, are one of the most
explored classes of fluorophores. Their modified fluorogenic acids are of particular
interest due to improved photophysical properties and especially the solubility.12 For
example, 2-amino-3-(6,7-dimethoxy-3-oxo-3H-benzopyran) propanoic acid (Dmca, 1.1)
is characterized with relatively good photostability, large extinction coefficient (10,900
M-1 cm-1), high emission quantum yield (ΦF 0.52), and good solubility in several
solvents.13 The aforementioned characteristics rank this molecule as an excellent tool for
labeling peptides and thus permit detection of the labeled protein at a picomolar scale,
sensitivity similar to that of radiolabeling.13
λabs = 345 nm
λem = 440 nm
-1 -1
ε = 10,900 M cm
Φ = 0.52
5
Importantly, the spectroscopic properties of coumarins facilitate selective determination
of its labeled proteins even in the presence of the fluorescent tryptophan residue. Also, it
has been suggested that a lysine residue labeled with oxobenzopyran derivatives is
suitable for solid-phase peptide synthesis (SPPS).14
1.1.3.1b Nitrogen Based Fluorophores
Two important classes of nitrogen based heterocyclic fluorophores are the 4,7phenanthroline-5,6-diones (phanquinones) and benzooxadiazoles. These fluorophores are
often utilized as fluorogenic labeling reagents in pre-column derivatizations of highperformance liquid chromatography (HPLC) used for the separation of amino acids.15 For
example the fluorophore, 4,7-phenanthroline-5,6-dione 1.2, is known for its ability to
selectively react with the primary amino function of L-amino acids to produce 1.3.16 The
fluorescent conjugate 1.3 with λabs = 400 nm, λem = 460 nm can be easily separated by
reverse phase HPLC.
Gatti et al. recently demonstrated that phanquinone 1.2 was
successfully used for the analysis of D,L-p-serine.16
N
N
O
NH2
CHR
COOH
N
68 oC / water
N
OH
N
O
R
Non-fluorescent
1.2
CO2H
Fluorescent
λabs1.3
= 400 nm
λem = 460 nm
6
1.1.3.1c Naphthalene Fluorophores
Naphthalene derivatives have been extensively investigated to label amino acids
and peptides. Dansyl chloride 1.4 was used to synthesize the fluorescent amino acid
dansylamine 1.5, which was easily incorporated into proteins, via the attachment of
specific amino acid sites.17 In addition, due to its small size, the amino acid 1.5 showed
potentials for in vivo studies to investigate protein structures and biomolecular
interactions.
1.1.3.2.
Fluorophores with Emission Beyond 500 nm
1.1.3.2a Fluorescein dyes
Among all polycyclic fluorophores, the xanthene dye fluorescein (λabs 490 nm and
λem 512 nm) and its derivatives still remain one of the most widely utilized fluorophores
in modern biochemical and medicinal research.10 Fluorescein exhibits relatively high
extinction coefficient, excellent fluorescent quantum yield and good solubility in water
but faces some disadvantages related to its photostability and pH sensitivity. The major
drawbacks of fluorescein and its macromolecular conjugates are summarized as:11 a) high
susceptibility to photobleaching; b) pH-dependent fluorescence; c) tendency to selfIt has been reported that the exact mechanism of the amidation reaction could follow
two different routes, controlled by the electronic nature of the azide used in the reaction
7
localization of low abundant receptors. Thus, the development of fluorescein derivatives
with improved photophysical characteristics is of significant importance for the field. The
most popular fluorescence derivatives used in conjugation with protein is fluorescein
isothiocyanate (FITC, 1.6) as an amine reactive bio-probe.11 Fluorescein-based
chromophores have been used for labeling of iron-free human serum transferrin, as
calcium18 or zinc indicators,19 as nitric oxide sensor based on copper (II) chelates,20 and
others.
1.1.3.2b
Cyanine dyes
Cyanine dyes are considered to be the main source of fluoreophores with longwavelength excitation band in the range of 600-900 nm with applications as labels, DNA
stains, membrane potential sensors and others.21 Manders and co-workers reported that a
fluorescent probe based on Cy5-dUTP has been successfully incorporated into enzymes
involved in the DNA biosynthesis to monitor nucleic acid movements in a cell.22 As a
result, a visualization of the DNA strands has been achieved, providing a real time
observation of the DNA during the cell cycle (the DNA assembly into chromosomes).22
8
1.2
Förster Resonance Energy Transfer (FRET) Systems
1.2.1 FRET Mechanism
Förster Resonance Energy Transfer (FRET) is a well-established technique that plays an
important role in drug discovery and bio-medicinal research. FRET is extensively used to
investigate protein-protein interactions, protein folding kinetics, enzyme activity, and
structural features of lipids and proteins.23 FRET is a non-radiative, distance-dependent
process occurring between compatible chromophores, a donor and an acceptor pair.24 In
this mechanism, energy is transferred from the excited singlet state of the donor molecule
to the acceptor, resulting in a fluorescence emission at wavelength typical for the
acceptor. While there are several factors that influence FRET, the primary criteria that a
FRET system must fulfill are the compatibility and the proximity between the donor and
the acceptor molecules. A compatible donor-acceptor pair is the one in which the
acceptor’s absorbance spectrum overlaps with the emission spectrum of the donor. The
degree to which the donor-acceptor profiles overlay is referred to as the spectral overlap
integral (J).25 If this requirement is not met, the energy emitted by the donor will not be
able to excite the acceptor partner and the FRET will not occur. Proximity is less
precisely defined and is deemed as the donor fluorophore being “close enough” to the
acceptor in order for the direct excitation of the acceptor fluorophore to take place
(typically 10-100 Å).24 Another important requirement is that the donor and the acceptor
transition dipole orientations are approximately parallel. It is accepted that the
mechanism of excitation energy transfer between a donor-acceptor pair follows the
Förster mechanism in which the singlet energy transfer rate, k(R), is:
9
k(R) = kF(1/(1+(R/RF)6),
(Eq. 1)
where R is the actual distance between a donor and an acceptor, RF is the Förster radius
and kF is the rate of transfer between donor and acceptor when the distance between them
is small; when R=RF, k(R)=1/2, thus the Förster radius is defined as the distance at
which the FRET efficiency between the donor-acceptor pairs is 50%. This equation
shows that the FRET process depends on the inverse sixth power of the distance between
the donor and the acceptor pair. Typical values of Förster radius for some well-known
FRET pairs are presented on Table 1.25
Table 1.1. Typical values of RF for well-known donor/acceptor pairs.
Donor
Acceptor
RF (Å)
Fluorescein
Tetramethylirodamine
55
EDANS
Dabcyl
33
Fluorescein
Fluorescein
44
BODIPY FL
BODIPY FL
57
Fluorescein
QSY 7 and QSY 9 dyes
61
In addition, the Förster distance (RF) is dependent on a few factors, including the
fluorescence quantum yield of the donor in the absence of an acceptor (fd), the refractive
10
index of the solution (n), the spectral overlap integral of the donor and the acceptor (J),
and the dipole angular orientation of each molecule (k), Eq. 2.
RF=9.78 x 103(n-4*fd*k2*J)1/6 Å
(Eq. 2)
Consequently, any changes in the distance between the donor-acceptor pair will result in
change in the FRET rate, thereby allowing this technique to be quantified.24-25 FRET is
often termed a ‘spectroscopic ruler’ and it provides valuable information about events
occurring in biological systems triggered by changes in the distance between the
appended chromophors. The FRET phenomena can be detected either by the appearance
of an increased emission signal from the acceptor and simultaneous decrease of the donor
emission, or by quenching of the donor emission in case when the acceptor is a
fluorescence-quenching molecule (Figure 1.2)
Figure 1.2. Schematic representation of FRET mechanism which occurs between: A. a
donor (D) and an acceptor (A) chromophorese and B. a donor fluorophore and an
acceptor quencher (Q)
11
Quenchers have the unique properties to absorb energy over a broad range of
wavelengths and dissipate the absorbed energy to heat.25 As a result of these properties,
quencher molecules have become suitable partners in FRET systems.
1.2.2
FRET Applications in Biological Systems
The most common applications of FRET systems include:23d a) detection of
protease activity, b) characterization of protein–protein interactions, c) small molecule
detection (Figure 1.3). In the later example, the donor-acceptor pair is appended to the Cor N-termini of a protein, enzyme, or DNA fragment which may undergo liganddependent conformational changes, triggered by surrounding biomolecular interactions
and processes.26 Such conformational shifts often result in a change of the relative
distance between the two fluorophores and a further change in the FRET signal and
efficiency is observed.
Figure 1.3. Detection of conformational changes induced by small molecules
12
A site-specific attachment of FRET probes is a very important task that requires a careful
selection of suitable chromophore-partners, as well as a good knowledge of specific
binding affinity to the target of interest. It has been demonstrated that genetically encoded
dyes (fluorescent proteins)27 as well as small synthetic fluorophores11 were successfully
implemented into in vitro or in vivo FRET applications.
Recent advances made by the implementation of the green fluorescent proteins
(GFP) in FRET-based imaging microscopy allowed a real-time monitoring of proteinprotein interactions in vivo. The green fluorescent proteins and derivatives (blue BFP;
cyan, CFP; and yellow, YFP), paired with each other or with small fluorophores can be
attached to various proteins of interest.28 It has been demonstrated that modulated GFP
that exhibits pH-sensitivity were attached to specific proteins and used to measure the pH
in some cellular compartments.8a, 28
Tsien et al. and other research groups developed several GFP-based FRET
systems to monitor biochemical interactions in living cells among which the FRET
construct to measure intracellular calcium is of significant importance.29 The flux of Ca2+
within the cell controls important biological processes related to the neuronal and
muscular functions and the in vivo visualization of these ions is of great importance.
Thus, CFP was attached to the N-terminus of calmodulin and YFP to the C- terminus of
M13, the caldmonium binding peptide.29a An increased concentration of Ca2+ caused the
binding of the M13 peptide to calmodulin which resulted in a change in the FRET signal
due to altered distance or orientation between the donor-acceptor pair. Thereby, high Ca2+
13
level lead to a FRET emission of YFP (acceptor) in the red spectrum range, whereas low
Ca2+ levels resulted in a low FRET and mostly blue emission from the donor (Figure 1.4).
Figure 1.4. FRET system with CFP-YFP pair designed to measuring intracellular
calcium level
The fluorescent protein-based conjugates face some limitations related primarily to their
size and small spectral range. Thus, the bulky fluorescent proteins (i.e. 27 kDA for GFP)
have a potential to interfere with the structure or the function of the host protein to which
they are appended.8a The broad excitation and emission spectra of FPs, and the possibility
of mutual dimerization may often result in low FRET accuracy.
Some of these limitations can be overcome by implementing small-moleculefluorophores as FRET pairs. Such type of fluorophores can be easily appended to C-, or
N-termini of proteins and exhibit higher sensitivity and photostability. Tsien and coworkers developed several membrane-permeant fluorophores that were successfully
implemented in FRET systems within the living cells.30 The blue-emitting coumarin
14
donor was coupled via a cleavable linkage to a green-emitting fluorescence derivative
and an efficient energy transfer was observed with primarily green emission produced by
the acceptor.30 The lactamase, present in the cell, was able to cleave the linker and
thereby disrupts the FRET mechanism by generating two separate fragments. The FREToff state induced by the enzymatic cleavage resulted only in blue emission from the donor
to be recorded. Through this technique the gene expression of a single cell can be
monitored and co-translated to other gene of interests.
In another application, a FRET technique was successfully utilized to design a
phosgene detector by using a chemical reaction between phosgene (or triphosgene) and a
complementary coumarine based donor/acceptor fluorophores (Figure 1.5).31
Figure 1.5. FRET system utilized as a sensor to detect phosgene (or triphosgene).
If phosgene is present in the system, the pre-arranged chemical reaction will bring the
donor/acceptor pairs within the appropriate Forster distance and emission typical for the
acceptor will be recorded. Thus, if the donor coumarin is excited at 345 nm (λex=345) and
emission typical for the acceptor at 468 (λem=468 nm) is recorded, an FRET-on state will
be indicating the presence of phosgene which will serve as a cross-linker agent.
15
Of particular interests are the far-red emitting (λem > 650 nm) cyanine dyes such
as Cy5, Cy5.5 and Cy7. The advantages of these fluorophores is that although there is a
small spectral overlap (due to a large separation of donor-acceptor emission spectra), a
good Forster radius is achieved due to their excellent absorbance and relatively good
quantum yield. For example, the Forster radius of the Cy3–Cy5 pair is >50 Å, with Cy3
emitting at 570 nm and Cy5 at 670 nm.28 These well-separate emission profiles allow for
accurate FRET measurements with no donor emission “contamination” and lower
possibility of direct excitation of the acceptor. These advantages rank the cyanine dyes
along with the rhodamine-based dyes as suitable for single-molecule FRET studies.32 For
example the Cy3-Cy5 pair was used to visualize the dimerization of a single EGF
receptor in a living cell.33 However, the limited commercial source of thiol-reactive
cyaninie chromorphores significantly diminished their popularity.34
New advances in the FRET detection are made by combining the instrumental
technology with new and optimized fluorophores. For example techniques such as
advanced laser and charged-coupled devices (CCD) allow measuring FRET on a pixel by
pixel basis delivering a general picture of molecular interactions such as seen between a
receptor and a ligand interaction.23f
1.2.3
FRET Mechanism in the Screening of Libraries of Small Molecules
In high-throughput screenings based on fluorescent probes, FRET systems are
used not only for investigating known protein-protein interactions in their native cellular
environment, but also for identifying new binding activities of proteins towards small
molecule ligands. 23d,35-36 In a proof-of-concept experiment, a FRET system with a CFP-
16
YFP sensor was utilized to screen 480 known compounds for pro-apoptosis-induced
activity in Jurkat cells through microtiter plates.23d, 37 Two novel apoptosis inhibitors of
caspases were identified using Hela cells in 96-well plates, demonstrating that the CFPYFP sensor provides a robust cell-based assay. 35-36a Due to the essential role of caspases
for the proper regulation and execution of apoptosis, significant efforts were made
towards the design of compounds that modulate the activation of the caspase cascade.
The FRET technology with a fluorescent protein has proven to be a suitable approach for
real time in situ monitoring of apoptosis, thus generating small-molecule-inducers of
apoptosis. Interestingly enough, the potency order of the hit compounds detected through
this CFP-YFP assay was confirmed with a conventional cell lysis-based assay. 35
A FRET-based assay was successfully implemented in the identification of
efficient protein-protein modulators for the c-Myc/Max interaction, which has been
validated as a potential target for breast and lung cancer treatment.38 An efficient FRET
signal between the two fluorophores fused to the dimerization domain of c-Myc and Max
indicated a close proximity and no disruption of the protein-protein interaction. When the
interaction between c-Myc/Max was modulated by binding of potential inhibitory
compounds, the average distance between the proteins resulted in an increase in the
actual distance between the c-Myc and Max and decrease in the FRET efficiency. It was
reported that this FRET-based assay was an efficient way to screen library with a large
number of compounds (< 1000) and to deliver small-molecular inhibitors for c-Myc/Max.
17
1.3
Targeting the ‘Undruggable’ Targets – Protein-Protein Interactions (PPIs)
Protein-protein interactions mediate a large number of biological processes and
thus are recognized as an attractive class of targets for developing human therapeutics.39
Protein-protein interactions (PPI) are essential for the regulation of a number of cellular
functions linked to signal transduction, gene transcription, initiating programed cell death
(apoptosis), and others.40, 41 The proper functioning of living cells is directly dependent
on the execution of these pathways, thus related abnormalities may lead to various
diseases such as autoimmune disorder, Alzheimers disease, and cancer.42 In the past two
decades it has been recognized that using small molecules that specifically modulate or
disturb a particular PPI has a tremendous potential in the biomedical field but it also
presents a challenging undertake.39,
43-44
The difficulties in the design of such small
molecules are closely related to the unfavorable topography of the protein interface – it is
relatively flat, large (1500-3000 Å), and lacks well-defined binding pockets.45,
46, 47
In
contrast, enzymatic targets often contain deep active site pockets and are easily inhibited
by small molecules which structurally resemble their natural substrate. However proteins
do not retain such complementary molecules or ligands which further impedes the leaddiscovery process.
The flexible and adaptive nature of the amino acids of the protein interface is another
burden to be overcome. Because of these challenges, it was initially considered that the
PPIs were an ‘undruggable’ target. However, a major break-through in this aspect was
made by Wells and co-workers who demonstrated that although there is a large
interaction surface between the proteins, only a small subregion of amino acids
contributed to most of the binding affinity.48, 49 These clusters of amino acids are defined
18
as ‘hot spots’ and are usually found at the center of the contact interface.48 Thus, the
dimensions of the hot spots are more compatible with the size of the small molecules,
hence providing a starting point in the design of drug-like leads, targeting specific PPIs.
Clackson and Wells proved that the hot spot is not just an artifact of the analysis by
examining the interaction between human growth hormone (hGH) and its domain
(hGHbp), where the amino acid side chains were systematically replaced by alanine.50 By
analyzing the changes in the binding free energy of each mutation it was concluded that
only a small number of all alanine residues buried at the interface are actually
contributing in the binding interaction. Thus, eight out of the thirty one alanine residues
were accounted for 85 % of the entire binding energy.50b Interestingly, in this study it was
found that the critically important residues were situated close to the center of the
interface but not in the peripheral region.
A number of small protein-protein interaction modulators (PPIM) have been
developed for variety of targets, majority of which are directly related to cancer
treatment. Examples of such targets are the following: HIV-1 protease dimerization,
p53/MDM2, Bcl-xL or Bcl-2/Bak-BH3 domains, IL-2/IL-2Rα, Mcl-1/Bim and others.51,
51b
1.4
Targeting the Protein-Protein Interactions of Bcl-2 Family
Members of the B-cell lymphoma 2 (Bcl-2) family are important regulators of
apoptotic cell death and consist of both anti-apoptotic (Bak, Bax, Bok and BH3-only
proteins) and pro-apoptotic members (Bcl-2, Bcl-XL, Mcl-1, and Bcl-w).39-41 The antiapoptotic proteins from the Bcl-2 family heterodimerizes with the pro-apoptotic
19
constituents and their ratio regulates the apoptotic signaling process in cells.52
Overexpressing of the anti-apoptotic Bcl-2 and Bcl-XL is observed in a number of human
tumors which is explained by the ability of Bcl-XL to prevent apoptosis by inhibiting the
pro-apoptotic Bax and Bak by binding to their BH3 domain.53 Positive results in the
modulating of PPIs were achieved by using monoclonal antibodies due to their high
affinity to the protein target and low toxicity.54 However, significant drawbacks of this
class of therapeutics are the low oral bioavailability, low cell/brain permeability, and the
high cost of manufacturing.51a Alternatively, fragment-based drug discovery strategies
using bio-molecular NMR, X-ray crystallography, or surface plasmon resonance (SPR),
lead to the identification of small molecules that mimic the BH3 domain of pro-apoptotic
Bcl-2 proteins and successfully disrupt its interaction with Bcl-XL.55,
56
NMR studies
suggested that the Bak-derived peptide forms an α-helix and binds in a hydrophobic
groove, formed by the α-helices of Bcl-XL.52
Hamilton et al. reported that small molecules were designed by mimicking a short
secondary structure of the domain BH3 by reproducing the interactions of the key amino
acids (Val74, Leu 78, and Ile 81) involved in the hot spot region of Bcl-XL/Bak.57, 58 The
most active α-helix mimic, compound 1.7, revealed a good structural complementarity
with Bcl-XL, high affinity (Ki = 114 nM ), and selectivity for Bcl-xL/Bak over related
p53/MDM2 interaction (Figure 1.6). Other example of reported protein-protein
interaction modulators (PPIMs) that mimic a BH3 domain are presented below.59
20
Figure 1.6. Examples of Bcl-2/Bcl-XL protein interaction modulators
Among these, worth mentioning are the compounds designed by Abbot Laboratories.
Over the last ten years, Fesik and Hajduk with Abbott laboratories were able to design
several highly potent small molecular inhibitors against Bcl-2 and Bcl-XL by using a
fragment-based lead discovery (FBLD) approach, in specific SAR-by-NMR, in
conjunction with synthesis and structure-guided lead design.55-56, 60
The most potent inhibitor generated through this approach is ABT-737 with Ki of
0.6 nM and good selectivity against Bcl-2, Bcl-XL and Bcl-W, in addition to its orally
active analog, ABT-263, which underwent clinical trials for cancer treatment.61 However,
the delivery of such potent compounds was a result of a cumbersome process involving
15
N HSQC screening of large number of compounds and a further optimization through
covalent linking of fragments that bind to adjacent binding sites. For example, the initial
21
NMR-based screening was performed over a library of 10,000 compounds to yield a
fluoro biaryl acid as a binding affinity ligand with Kd ~ 300 μM. This ligand was found
to be bound at the center of a hydrophobic groove of Bcl-XL resembling a key leucine
residue of the Bak peptide.61 In addition an NMR-binding study revealed the existence of
an adjacent subpocket, usually occupied by the side chain of Ile 85 of Bak.52 Thus, a
follow–up screening with another set of library led to the identification of a second-site
ligand, bound in close proximity to the first. The fragments were then optimized and
bridged through an acylsulfonamide linker, thus producing the high-affinity ligand. It is
important to mention that ABT-737 and ABT-263 exhibit lower binding affinity
(micromolar range) against other anti-apoptotic proteins such as Mcl-1, and Bcl-B.62
1.5
Various Methods Used in Drug Discovery
In the last two decades there has been a substantial interest toward development of
methods that improve the efficiency and reliability of the lead-discovery process.63-64A
surprisingly low number of new molecular entities met the approval of the US Food and
Drug Administration (FDA) in the last several years. For example, in 2006 in North
America the large investment of $55.2 billion in the biopharmaceutical research and
development (R&D) yielded only 22 molecules with approved therapeutic quality.64b In
the past twenty years high-throughput screening (HTS) and combinatorial chemistry have
been extensively explored in the drug discovery, hence ranked as main sources for most
of the current therapeutic candidates. However, these methods required the synthesis and
analysis of large chemical libraries which showed a very low rate of successfully
generated leads with drug-like properties. In addition, the process of handling and
22
screening these libraries acquires large amounts of proteins as well as labor-demanding
sample preparation. In contrast, the Fragment-Based Lead Discovery (FBLD) approaches
emerged as a simplified technique to explore larger chemical space by using fewer
compounds and a more rapid hit-to lead optimization phase.65 In addition, it has been
reported that lead compounds derived from the FBLD approaches exhibit higher ligand
efficiency (high values for the ratio of free energy of binding to the number of heavy
atoms) in comparison to molecules discovered thought HTP screening.65b
FBLD approaches can be broadly divided into two categories. The first category
comprises
of
biophysical
techniques
such
as:
NMR
spectroscopy,66
X-Ray
crystallography,67 mass spectrometry (MS),68 surface plasmon resonance (SRP)69 and
isothermal titration calorimetry.70 Thus, in these techniques first the binding interaction
of low-affinity fragments is structurally or biophysically validated and then they are
subsequently expanded or combined to generate more complex drug-like molecules. The
second category is driven by template-assisted strategies also known as Target-Guided
Synthesis (TGS). In TGS the biological target serves as a template for the assembly of its
own bidentate inhibitors from a collection of building blocks with complementary
reactive functionality. TGS can be divided into three major subcategories: (a) dynamic
combinatorial chemistry (DCC),71,72,73 (b) catalyst/reagent-accelerated TGS,74 and (c)
kinetic TGS.74b
23
1.6
Fragment-Based Lead Discovery (FBLD)
1.6.1
Fragment-Based Lead Discovery Using Biophysical Approaches
The starting point in this fragment-based approach are small fragments or low-
molecular-weight compounds (150 - 300 Da) with weak binding affinity to particular
biological target (milli- to micromolar).75,65 The large hydrophobic leads produced with
HTS methods often do not progress into drug molecules as they lack some advantages
that the small molecules have. Usually, small fragments or molecules fulfill important
requirements such as aqueous solubility, reduced structural complexity, high ligand
efficiency, and chemical diversity.64b 63 However, fragments with weak binding affinities
are difficult to detect for which the above listed biophysical approaches showed to be
suitable for this task. The most common approach, SAR-by-NMR, entails screening of
small molecules against the protein of interest and identifying binding motifs which are
subsequently linked to generated optimized lead molecules.55,
66
SAR-by-NMR was
developed in 1990s by Fesik and colleagues and since then it has gained momentum. In
this technique, a structure-activity relationship is determined by first screening a library
of fragments against the
15
N-labeled biological target with pre-assigned chemical shifts.
Subsequent analysis of the heteronuclear single quantum correlation (HSQC) spectra is
performed in order to determine the changes of the amide chemical shifts at the binding
site, triggered as a result of the fragment interactions with the labeled protein. Thus, this
SAR-by-NMR provides information as to where particular fragments bind at the protein
binding site. However, this method does not offer a best way to link or optimize the
identified low-affinity fragments.66,76 Other disadvantages of SAR-by-NMR are related to
the usage of significant amount of
15
N-labeled protein, as well as the presence of
24
fragments with good solubility even at high concentration. The process of assigning the
protein backbone residues before the screening experiment and the interpretation of the
HSQC spectra are time consuming and limiting. 66, 64b
1.6.2
Target Guided Synthesis (TGS)
As demonstrated above, some drawbacks in the biophysical FBLD approaches are
related to the requirement of specially designed methodologies, expensive equipment, as
well as a relatively low throughput. Also, none of the biophysical techniques provides the
crucial information about the process of connecting and optimizing the active
fragments.76, 77 Thus, these methods involve significant research effort and resources.
The template-assisted strategies (also known as TGS) offer an alternative approach
which overcomes the above mentioned pitfalls. TGS approach aims to accelerate the
process of discovery and identification of potential pharmaceutical candidates by
combining the screening and the synthesis of libraries of low-molecular-weight
compounds in one step.74b In this approach, the biological target is directly involved and
templates the assembly of its own bidentate ligand from a library of complementary
building blocks that able to react though a pre-arranged reaction. TGS is a very appealing
technique as it generates only the active compounds. The TGS can be broadly divided
into three major subcategories: (a) dynamic combinatorial chemistry (DCC), (b)
catalyst/reagent-accelerated TGS, and (c) kinetic TGS.
25
1.6.2.1
Dynamic Combinatorial Chemistry (DCC)
In dynamic combinatorial chemistry the building blocks are in constant
equilibrium, controlled through a pre-arranged reversible reaction between their reactive
functionalities. When the biological target is introduced, the equilibrium shifts towards
the compounds that show the highest affinity to the biological target and as a result the
concentration of that complex is amplified.68,
71b
Conceivably, the formation of the
compound that possess the best binding properties to the active site of the biological
target are favored above the other possibilities that co-exist in the solution. In order to
determine the best binding compounds, the equilibrium has to be “frozen” (by hydride
reduction or by lowering the pH), before being analyzed by HPLC or MS.78, 71a Common
reactions utilizing DCC include trans-esterification, olefin metathesis, boronic ester
formation, and disulfide exchange.79, 80, 81
Applying the concept of DCC, Huc and Lehn demonstrated that the
metaloenzyme bovine carbonic anhydrase II is able to synthesize its own inhibitors via
an imine generating reversible condensation reaction between various aldehyde and
amine building blocks.71a Concurrently with the concept of TGS, it was confirmed that
the enzyme bCA II favors the formation of these condensation compounds that were to
express the best binding affinity to the enzyme’s active site by preferentially amplifying
their concentration. Once the imines were formed, they were subsequently reduced with
NaBH3CN and the generated amines were analyzed by liquid chromatography with mass
spectroscopic detector (LC/MS) (Figure 1.7).71a
26
Figure 1.7. Example of Dynamic Combinatorial Chemistry (DCC) used to generate
inhibitors of bovine carbonic anhydrase II
1.6.2.2
Kinetic TGS
In this particular approach the biological target synthesizes its own inhibitors by
sampling several possible pairs of fragments until an irreversible reaction connects the
pair that best fits in the binding pocket, termed as click chemistry (Figure 1.8). This
ligand complex exhibits much higher binding affinities to the target than the individual
fragments and its detection can be simply accomplished by analyzing the reaction
mixture with analytical instruments, such as liquid chromatography with mass
spectroscopic detector (LC/MS).82, 83
Figure 1.8. Kinetic Target-Guided Syntheses
27
1.6.3
Click Chemistry and In Situ Click Chemistry
Examples of several reactions that were successfully implemented in kinetic TGS
include: a) a carbon-nitrogen bond formation, templated by glycinamide ribonucleotide
transformylase (GAR Tfase);84, 85 b) a carbon-sulfur bond formation via bCA II template
SN2-mediated reaction;86 c) a carbon-carbon bond formation template by the presence of
sirtuin;87 d) a sulfonamide formation template by Bcl-XL88, 89; e) a multi-component Ugi
reaction templated by thrombin90 (Figure 1.9).
Figure 1.9. Examples of kinetic TGS reactions used against various biological targets
Benkovic and Boger were among the first to apply the kinetic TGS approach
towards developing an adduct inhibitor of the enzyme glycinamide ribonucleotide
transformylase which templated the alkylation of one of its substrates with a folate28
derived electrophile (Figure 1.9A).84 An interesting observation was made by Chase and
Tubbs who reported that carnitine acetyltransferase was irreversibly inhibited by the
templated C-S bond formation between bromoacetyl-carnitine and coenzyme-A (Co-A).91
In a similar manner, kinetic TGS has been entailed to generate inhibitors of carbonic
anhydrase II by using the enzyme’s active site as a template of an SN2-mediated reaction
between 4-mercaptomethyl-benzenesulfonamide and different alkyl halides (Figure 1.9
B).86 Importantly, a competition study revealed that the enzyme favors the formation of
the bidentate compound, derived from the building blocks with the highest binding
affinity when two alkyl halides are incubated along with one thiol in the presence of
bCAII
For a reaction to be considered as a click reaction, these important criteria should
be met: a) high yield, b) wide in scope, c) simple product isolation, e) producing no or
minimal
amounts
of
byproducts,
f)
oxygen
and
water
compatibility,
g)
stereospecificity.82, 92 As of today, the best example of a click chemistry reaction is the
Huisgen 1,3-dipolar cyclodition reaction between azides and acetylenes resulting in
1,2,3-triazoles.92 This reliable triazole formation reaction is especially important for drug
discovery as its application is enhanced by the favorable physicochemical properties of
the triazoles. The relative stability of the azide and alkyne functionalities determines their
inertness to most biological and organic systems, as well as to molecular oxygen and
water. The sluggishness of the un-catalyzed trizole formation (activation barrier of ~ 25
kcal/mol) makes this transformation an excellent candidate for in situ click chemistry as
it reduces the possibility of false negatives (the connection of azide and alkyne blocks
without guidance from the target).93 Importantly, the triazole formation does not involve
29
any catalysts or external reagents which may disturb the binding sites.92 The triazole unit
in these moieties is readily engaged in hydrogen-bonding and dipole-dipole interactions
with the biological target, which enhances its pharmacophoric properties. Rated as
extremely slow at room temperature, the triazole formation was dramatically accelerated
(up to 107 times) by the recently discovered copper-(I) catalysis.94 Several synthetic
procedures are available in order to generate disubstituted triazoles. For example, copper(I) catalysts are specifically used to synthesize 1,4-disubstituted triazoles (antitriazoles),94 whereas the 1,5-disubstituted triazoles (syn-triazoles) can be obtained by
using magnesium acetylides or ruthenium catalysts95 (Figure 1.10).
Figure 1.10. Synthesis of syn- and anti-triazoles through stereoselective methods or in
situ click chemistry
Mock and co-workers discovered that the 1,3-dipolar cycloadition reaction
between azide and alkyne was dramatically accelerated (~105-folds) when they are
incorporated into the structure of cucurbituril, a nonadecacyclic cage built out of
substituted urea scaffolds.96 The ‘entropic trap’ induced by the hydrogen bonging within
the cucurbituril moiety served as a cycloadition template and largely increased the rate of
30
triazole formation due to the resulted close proximity between the alkyne and azide.
These results prompted Sharpless and co-workers to examine this approach in the concept
of kinetic TGS and since then several example of in situ click chemistry were
demonstrated. A significant members of the 1,2,3-triazole family have shown valid
biological properties such as anti-allergic, anti-bacterial, and anti-HIV activities.97 The in
situ click chemistry has been widely used to discover inhibitors for enzymes like
acetylcholinesterase (AChE),98,
99, 100
, HIV-1 protease,101 bovine carbonic anhydrase
(bCAII),102 and others.
1.6.3.1
In situ Click Chemistry Using Acetylcholinesterase (AChE) as a
Template
Acetylcholinesterase (AChE) was the first enzymatic target used in a proof-of-concept
experiment to generate inhibitory compounds via in situ click chemistry. The
acetylcholinesterase is an important enzyme that plays a key role in the central and
peripheral nervous system, catalyzing the hydrolysis of the neurotransmitter
acetylcholine.103 The AChE has an active site located at the bottom of 20 Å narrow gorge
and an adjacent binding site located the periphery of the gorge.103 By in situ click
chemistry, reactive site-specific-ligands were designed that effectively combined into
selective inhibitors, interacting with both binding sites of the enzyme.98 Thus, two known
AChE site-specific-inhibitors, tacrine and propidium compounds, were derivatized with
alkyne and azide functionalities and incubated as binary mixtures in presence of eel
AChE for 6 days at room temperature. Out of the ninety eight possible azide-alkyne
cycloaddition products (representing 49 syn- and 49 anti-isomers), only one hit
31
compound, syn-TZ2PA6, was identified in detectable amount. The syn-TZ2PA6 triazole
displayed an extremely high binding affinity to eel AChE with dissociation constant of 77
fM, whereas the anti-TZ2PA6 was not generated by the enzyme and exhibited lower
binding affinity.99 The protein-ligand interaction was further investigated through X-ray
crystallography. It was observed that the triazole unit was not just a passive linker but
was actively engaged in hydrogen bonding and
stacking interactions within the
active site cavity thus, significantly contributing to the overall binding. In a subsequent
study, the screening of tacrrine and propidium-based derivatives was performed by
employing an improved LC/MS-based analytical method, which led to the detection of
three new potent inhibitors (syn-TA2PZ6, syn-TZ2PA5, syn-TA2PZ5)98 (Figure 1.11)
Figure 1.11. Hit triazole combinations for AChE, identified through the in situ click
chemistry approach
These results clearly demonstrated the high efficiency and reliability of the in situ click
chemistry in the process of screening and delivering potential therapeutic agents. The
critical importance of the enzyme templation effect was again proved by the observation
that the trizoles with higher potency were preferentially formed by the enzyme.
32
1.6.3.2
In situ Click Chemistry Using Carbonic Anhydrase (CA) as a Template
Carbonic anhydrases (CAs) are Zn-containing metalloenzymes that catalyze the
reversible process of hydration of carbon dioxide and dehydration of bicarbonate.
Carbonic anhydrases play essential physiological role in pH control, bone respiration,
calcification, diffusion of CO2 and it is a validated target for treatment of various
diseases. The modulation of the CA activity through inhibition 104, 105 or activation106 has
shown a great potential for treatment of diseases related to elevated intraocular pressure,
obesity, memory loss, also as diuretics to treat certain edematous conditions.107,
108
At
least thirteen different classes of mammalian isozymes with various activities and tissue
specificity have been identified (CAI to CA XIII), but CAII is of particular
pharmaceutical interest.109
Recently, Mocharla and co-workers presented a study in which potent bidentate
inhibitors of bovine carbonic anhydrase II were designed by employing the in situ click
chemistry between one acetylenic benzene sulfonamide 1.8 and complementary azide
building blocks.102 Para-substituted aromatic and heteroaromatic sulfonamides are well
known for their ability to coordinate to the Zn2+ and represent a class of potent CA
inhibitors.104,
105
Thus, acetylene benzene sulfonamide 1.8 (Kd = 37 nM), used as an
active-site-anchoring motif, was incubated with a library of 44 azides in the presence of
bCA II and each binary mixture was analyzed by HPLC and mass spectrometry in
selected-ion mode (LCMS-SIM).102 A total of 12 hits were initially identified, 11 of
which were validated through control experiments held in the presence of a known CA
inhibitor (ethoxazolamide) or by replacing the bCA II with bovine serum albumin.102
33
Figure 1.12. Application of in situ click chemistry for the synthesis of novel carbonic
anhydrase inhibitors
It is important to mention that all in situ generated triazole compounds were about ~200
times more potent than the corresponding building blocks. In this study the authors
suggested that compounds identified through the in situ approach overall do not generate
‘false positives’, whereas ‘false negatives’ may be obtained. However, this observation
was not taken as a drawback of the method, because when the compounds missed in the
in situ screening were synthesized, they showed only week binding affinity.102
Interestingly, it was reported that the triazole unit in this case does not contribute to the
overall interaction with the enzyme and hence serve only as a fragment linker but not as a
pharmacophore.102
1.6.3.3
In situ Click Chemistry Using HIV-1 Protease (HIV-1-Pr) as a Template
The HIV-1 Protease (HIV-1-Pr) is an important target for the inhibition of viral
replications and it plays an essential role in the fight against AIDS.110 The rate with
which strains of HIV-1 become resistant to the currently available drugs explains the
34
need of rapid and efficient process to generates broad spectrum inhibitors effective
against new mutants, as well as the wild-type virus.111
Fokin and coworkers first demonstrated the feasibility of the in situ click
chemistry in the assembly of a known HIV-1-Pr inhibitor 1.11, which was previously
identified through an activity-based screening.101,
112
Alkyne 1.9 and azide 1.10 were
incubated in the presence of HIV-1 protease (SF-2) and the analysis with HPLC and mass
spectrometry in selected-ion mode (LCMS-SIM) resulted in the identification of the anittrizole 1.11 (Ki = 1.7 nM, IC50 = 6 nM) (Figure 1.12).101 The starting fragments in this
case exhibited significantly lower binding affinity in comparison to the building blocks
used in the studies with AChE and bCA II (Ki < 37 nM). Nevertheless, it was observed
that the enzyme could still synthesize its own inhibitors but a higher enzyme
concentration (15 μM) and a higher building block concentration (100 μM to 500 μM)
were necessary in order to saturate the enzyme’s active site and enhance the templation
process. Although, the higher fragment concentration resulted in an increased rate of the
background trazole formation, the enzyme templation effect was still dominating and
resulted in an enhanced 18:1 ratio in favor of the anti-isomer. In contrast, the background
reaction displayed ~ 1:1 ratio between the regio-isomers which certainly indicated that
the enzyme is able to selectively deliver its own inhibitors.101
35
Figure 1.13. In situ click chemistry used to identify HIV-1 protease inhibitors via a
multi-component screening of fragments
Subsequently in a multi-component study, azide 1.10 was incubated with five
different alkynes in the presence of HIV-1 protease.101 The comparison of the enzymatic
and background traces revealed that only triazole 1.11 was preferentially formed by the
enzyme and showed increased product amount in the enzyme reaction. Similar multifragment study has been demonstrated to discover inhibitory compounds against another
target, AChE.
1.6.3.4
Sulfo-Click Amidation Reaction
Williams and coworkers recently developed a reaction in which alkyl azides or
sulfonyl azides react with thio acids to produce the corresponding amides.113,
114
It has
been reported that the exact mechanism of the amidation reaction could follow two
different routes, controlled by the electronic nature of the azide used in the reaction
36
Figure 1.13.114 Thus, electron-poor azides first generate a linear intermediate which
subsequently leads to a cyclic thiatriazoline, whereas the electron-rich azides formed the
cyclic intermediate directly as a [3+2] process.114 This type of amidation reaction, termed
as a ‘sulfo click reaction' exhibits characteristics that rank it to be suitable for the kinetic
TGS approach. William and coworkers reported that the amidation reaction is high
yielding, requires mild conditions and is effective in both organic and aqueous media.114
Figure 1.14. Amdiation reaction between sulfonyl azides and thio acids
In a recent proof of concept study, it was demonstrated by the Manetsch laboratory that
this amidation reaction can be successfully implemented in a kinetic TGS approach to
produce modulators for protein-protein interactions.115 Thus, a set of three thio acids
(TA1-TA3) and six sulfonyl azides (SZ1-SZ6) were incubated at 37 °C for six hours in
the presence the target protein Bcl-XL (Figure 1.14).115 The library was designed in a
way that some building blocks structurally resembled the potent Bcl-XL inhibitor, ABT737 originally reported by Abbott Laboratories. Analysis of each binary mixture by liquid
chromatography combined with mass spectrometry detection in the selected ion mode
37
(LC/MS-SIM) revealed that out of the eighteen possible combinations only Nacylsulfonamide SZ4TA2 was identified as a TGS hit which was originally reported by
the Abbott Laboratories.115
IC = 106 nM
50
Reported : K = 19 nM
IC50 = 28.6 µM
IC = 28.8 µM
50
IC50 = 37.2 µM
i
Figure 1.15. Kinetic TGS of Bcl-XL-templated amidation reaction between sulfonyl
azide and thio acid
Two independent control experiments were performed to determine whether SZ4TA2
was assembled within the protein hydrophobic groove or randomly, elsewhere on the
protein surface. Thus, incubations were conducted either in the presence of wildtype proapoptotic BH3 peptide (WT BH3) or its mutant analog. The authenticity of the hit
combination was confirmed by demonstrating that only WT BH3 is able to out-compete
38
the reactive fragments and thus suppressed the Bcl-XL-templated effect, but the mutant
BH3 peptide did not bind to Bcl-XL and permit the assembly of SZ4TA2.115
In a subsequent study, the kinetic TGS based on sufo-click chemistry was further
validated and the screening of an expanded library of nine thio acids and nine sulfonyl
azides resulted in the identification of three new protein-protein modulators, selectively
assembled by the target protein, Bcl-XL.116 Thus, eighty one binary mixtures containing a
single thio acid (TA1-TA9) and a single sulfonyl azide (SZ1-SZ9) were incubated with
the target protein Bcl-XL for 6 hours at 38°C (Figure 1.15). The analysis of the LC/MSSIM traces led to the identification of the previously reported fragment combination
SZ4TA2 and three new hit combinations SZ7TA2, SZ9TA1 and SZ9TA5. 116 Control
experiments were carried out with two mutants of Bcl-XL in order to validate that the
aforementioned combinations actually modulate the Bcl-XL/BH3 interaction within the
protein hot spot. For example, an experiment in which the reactive fragments were
incubated with a known mutant of Bcl-XL, in which critically important residues were
substituted by alanines (Phe131 and Asp133) showed substantial reduction of the
templation effect. In addition, similarly to the proof of concept study, the control
incubations with wildtype and mutant pro-apoptotic Bim BH3 peptides were utilized in
ordered to confirm that the corresponding acylsulfonamides were assembled in the
protein hot spot.116 The identified hit combinations were subsequently synthesized and
the retention time of the corresponding LC/MS peak observed by authentic samples was
correlated to the one generated in the incubation reaction. The IC50 values of the three
PPIMs were determined by implementing a fluorescence polarization assay and the three
acylsulfonamides, SZ7TA2, SZ9TA1 and SZ9TA5 showed IC50s in the low micromolar
39
range116 (Figure 1.15). These results validated the kinetic TGS using the sulfo-click
reaction as a reliable platform to identify PPIMs.
1.7
Research Aims
1.7.1
Designing a Fluorescent Probe and Its Implementation in FRET Systems
Fluorescent chromophores find extensive application as detection probes and sensors,4
and play an important role in the profiling and visualization of biological activity such as
protease activity.5 Despite the large variety of available fluorescent dyes, improved
fluorogenic systems with a large Stokes shift, high quantum yield, and high photostability
are constantly sought. Although the Diaryl-substituted Maleimides (DMs) possess
promising photophysical properties such as high fluorescent quantum yields (fl > 0.10),
good extinction coefficients (ελ > 5,000 M-1cm-1), and large Stokes shifts ( > 90 nm),
they still remained unexplored as fluorescence probes in bioscience applications.
Fluorescent probes are often implemented in FRET-based techniques and provide
important information in drug discovery and bio-medicinal research. However, some
well-known fluorophores have limited applications as FRET partners due to their small
Stokes shifts and susceptibility to photo bleaching while incorporated in biological
conjugates. The ongoing efforts to generate small-molecule fluorescent probes with
enhanced spectroscopic characteristics prompted us to investigate the synthesis and
spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)-maleimide and its first
reported use as a donor component in FRET systems.
40
1.7.2
In Situ Generated Novel Carbonic Anhydrase Inhibitory Compounds
Carbonic anhyrases (CA) are essential for the regulation of numerous physiologic and
physio-pathological processes in different tissues and cells. The modulation of the CA
activities through inhibition or activation has been used as an efficient path to treat a wide
range of human diseases. Although sulfamates and sulfonamide have been in clinical use
for over 50 years, the systematic use of these therapeutics leads to various side effects
due to the lack of isozyme specificity of these inhibitors. Thus, the development of
structurally diverse non- sulfonamide-based inhibitors for CAs has potentials to introduce
improved therapeutic treatments. Despite the advances made in this direction, there has
not been a robust and uniform technique in place for discovering novel non-sulfonamidebased bioactive molecules. In contrast, kinetic TGS approach aims at accelerating the
process of drug discovery by combining the screening and synthesis of libraries of low
molecular weight compounds in one step. In this approach the biological target of interest
is directly involved in the selection and assembly of the fragments together to generate its
own inhibitors. However, all literature examples utilizing the kinetic TGS approach for
designing bovine carbonic anhydrase inhibitors required that a sulfonamide-containing
anchoring scaffold was present.
Our research efforts are focused on investigating whether the in situ click
chemistry approach between complementary reacting alkynes and azides can be
successfully applied to design novel CA inhibitors from a library of non-sulfonamide
containing scaffolds. In addition, we exploit the incorporation of the recently by us
reported fluorescent moiety ((2,4-difluorophenyl)–maleimide) as a potential biomarker
with affinity to CA.
41
1.7.3
Parallel Sulfo-Click Kinetic TGS Screening of Multiple Protein-Protein
Interactions
Protein-Protein interactions are essentical for the regualtion of normal cellular
homeostasis, and are recognized as an attractive class of targets for developing human
therapeutics. Despite the advances made in the discovery of small molecules which
modulate or disturb specific PPIs, the conventional drug discovery approaches fell short
of providing a straightforward methodology. Recent reports demonstrated that a kinetic
TGS approach based on the sulfo-click reaction between thio acids and sulfonyl azides
was successfully implemented in the identification of high-quality PPIMs targeting the
anti-apoptotic protein, Bcl-XL. In addition, recent studies suggested that Mcl-1 also
contributes to cancer cell proliferation, and that Bcl-2, Bcl-XL and Mcl-1 must be
simultaneously targeted for apoptosis induction in most types of cancers. In an ongoing
study of the Manetsch laboratory, several structurally diverse acylsulfonamides were
identified as selective Mcl-1 inhibitors by using a sulfo-click kinetic TGS approach
(unpublished data S. Kulkarni). Although it has been demonstrated that the kinetic TGS
has the potential to be performed with a large number of fragments incubated in a single
well, a literature report revealed that more than 7 fragments in one well failed to provide
definite results, thus suggesting the testing should be performed in an enhanced screening
throughput.
Inspired by the successfully implemented multi-component screening in the
identification of a known Bcl-XL inhibitor and the encouraging results from the recently
discovered PPIMs against Mcl-1, we aimed: (a) to optimize the throughput of the sulfoclick kinetinc TGS reaction by implementing a multi-fragement screening technique
42
performed with a triple quadrupole mass spectrometer, (b) to probe a larger chemical
space and to expand the existing library of 310 fragment combinations to 1710 fragmentcombinations, and (c) to seek for new PPIMs against Bcl-XL and Mcl-1 by implementing
the optimized experimental conditions to the newly developed library.
1.8
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human immunodeficiency virus. New York state journal of medicine 1987, 87 (5), 286-9.
104.
Supuran, C. T.; Scozzafava, A.; Casini, A., Carbonic anhydrase inhibitors.
Medicinal research reviews 2003, 23 (2), 146-89.
105.
(a) Innocenti, A.; Antel, J.; Wurl, M.; Vullo, D.; Firnges, M. A.; Scozzafava, A.;
Supuran, C. T., Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V and IX
with complex fluorides, chlorides and cyanides. Bioorganic & medicinal chemistry letters
2005, 15 (7), 1909-13; (b) Innocenti, A.; Antel, J.; Wurl, M.; Scozzafava, A.; Supuran, C.
T., Carbonic anhydrase inhibitors: inhibition of human cytosolic isozyme II and
mitochondrial isozyme V with a series of benzene sulfonamide derivatives. Bioorganic &
medicinal chemistry letters 2004, 14 (22), 5703-7.
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Temperini, C.; Scozzafava, A.; Supuran, C. T., Carbonic anhydrase activation and
the drug design. Current pharmaceutical design 2008, 14 (7), 708-15.
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107.
Pastorekova, S.; Vullo, D.; Casini, A.; Scozzafava, A.; Pastorek, J.; Nishimori, I.;
Supuran, C. T., Carbonic anhydrase inhibitors: Inhibition of the tumor-associated
isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides. Journal of
enzyme inhibition and medicinal chemistry 2005, 20 (3), 211-7.
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Vullo, D.; Innocenti, A.; Nishimori, I.; Pastorek, J.; Scozzafava, A.; Pastorekova,
S.; Supuran, C. T., Carbonic anhydrase inhibitors. Inhibition of the transmembrane
isozyme XII with sulfonamides-a new target for the design of antitumor and
antiglaucoma drugs? Bioorganic & medicinal chemistry letters 2005, 15 (4), 963-9.
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Breton, S., The cellular physiology of carbonic anhydrases. JOP : Journal of the
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Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J.; Heimbach, J. C.; Dixon, R.
A.; Scolnick, E. M.; Sigal, I. S., Active human immunodeficiency virus protease is
required for viral infectivity. Proc Natl Acad Sci U S A 1988, 85 (13), 4686-90.
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Condra, J. H.; Schleif, W. A.; Blahy, O. M.; Gabryelski, L. J.; Graham, D. J.;
Quintero, J. C.; Rhodes, A.; Robbins, H. L.; Roth, E.; Shivaprakash, M.; et al., In vivo
emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 1995, 374
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Sanchez-Pescador, R.; Power, M. D.; Barr, P. J.; Steimer, K. S.; Stempien, M. M.;
Brown-Shimer, S. L.; Gee, W. W.; Renard, A.; Randolph, A.; Levy, J. A.; et al.,
Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science
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thio acids with azides: a new mechanism and new synthetic applications. Journal of the
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Chem Biol 2011, 6 (7), 724-32.
61
Chapter 2: Fluorescent Properties and Resonance Energy Transfer of
3,4-bis(2,4-difluorophenyl)-maleimide
2.1
Overview
Fluorescent molecules find important applications as sensor elements,
biomolecular probes, and substrates in material and life sciences. Despite the large
variety of available fluorescent dyes, new fluorophoric systems possessing a large Stokes
shift, a high quantum yield, and a high photostability are of continued interest.1 Well
known fluorophores such as cyanines, tetramethyl rhodamines (TMR), and fluoresceins
are characterized by broad excitation and emission ranges and small Stokes shifts (less
than 30 nm in some cases), which limit their application in Förster resonance energy
transfer (FRET) systems due to partial absorption of the incident light by the FRET
acceptor partner.2a At the same time while Alexa 350 and Alexa 430 dye are
characterized with a high Stokes shift, greater than 70 nm, they exhibit lower extinction
coefficient.2b In addition, fluorescein conjugates appended to biological molecules are
quite often photolabile and their fluorescence is significantly quenched prior to
photodissociation. In contrast, the use of the Diaryl-substituted Maleimides (DMs) as
fluorescence probes in bioscience applications have not been extensively explored.
Despite their promising photophysical characteristics such as high fluorescent quantum
yields (fl > 0.10), good extinction coefficients (ελ > 5,000 M-1cm-1), and large Stokes
shifts ( > 90 nm), the DM molecules have been investigated primarily for material
62
science applications.3 Recent reports demonstrated the design and the application of
fluorescent metal sensors based on monoindole-substituted maleimides and the
development of an efficient organic light-emitting diode (OLED) based upon a
naphtylphenylamino substituted N-methyl-3,4-diphenyl-maleimide.4 The ongoing effort
to generate small-molecule fluorescent probes with high fluorescent quantum yield, a
large Stokes shift and high photostability, prompted us to investigate the photophysical
characteristics of a new fluorescent dye from the DM family, 3,4-bis(2,4-difluorophenyl)maleimide, and its functionalized derivatives. The simple and efficient synthetic route to
yield 2.1 and the convenient way to functionalize that moiety with appropriately installed
chemical handles, makes this fluorophore easily accessible. Herein we present the
identification
and
spectroscopic
characterization
of
3,4-bis(2,4-difluorophenyl)-
maleimide 2.1 and its first reported use as a donor component in FRET systems.
2.2.
Photophysical Properties and Characterization
The analysis of the photophysical properties of 2.1 revealed a high extinction
coefficient ε(340) 48,400 M-1cm-1, a large Stokes shift (Δ) of 140 nm in MeOH (λabs 341
nm; λem 481 nm), and a very good fluorescence quantum yield in DCM (fl 0.61) (Figure
1). It is important to note that when compared to the reported DM molecules (ε(λ) 2,180 to
14,000 M-1cm-1 and fl 0.02 to 0.52), 2.1 exhibits a higher extinction coefficient (ε(λ)
13,490 to 48,400 M-1cm-1) and excellent quantum yield in a variety of solvents (fl 0.08
to 0.61) in addition to the unusually large Stokes shift (Figure 2.1). These advantageous
spectral characteristics render 2.1 as a suitable donor component in a FRET system,
63
exemplified in the large Stokes shift which is essential for an efficient energy transfer
between the donor and acceptor with a minimal direct excitation of the donor. Therefore,
having a donor with an excitation maxima being sufficiently apart from the excitation
acceptor, results in a more accurate measurement of the energy transfer phenomena. An
efficient one step synthesis
F
2.0
F
O
Normalized Emission (a.u.)
Normalized Absorbance (a.u.)
2.0
1.5
NH
1.5
F
F
O
1
1.0
1.0
0.5
0.5
0.0
0.0
300
400
500
600
Wavelength (nm)
Figure 2.1 Normalized absorption and emission of 2.1 in dichloromethane
An efficient one step synthesis of 2.1 was achieved by allowing (2,4-difluorophenyl)-acetonitrile to react with iodine and sodium methoxide at -85 oC (Scheme 2.1).3
The exact structure of 2.1 was confirmed by 1H-,
13
C-,
19
F-NMR spectroscopy, and by
high resolution mass spectrometry. An X-ray diffraction structure was also obtained,
demonstrating that 2.1 has noncrystallographic C2-symmetry with the two difluorophenyl
rings not being coplanar to the maleimide ring plane (Figure 2.2).
64
Figure 2.2 X-ray crystal structure of 1 in dichloromethane
In an attempt to implement the fluorescent probe into a FRET system, we generated
derivatives 2.1a, 2.1b, and 2.1c containing chemical handles, enabling the linkage to a
FRET partner of interest. Conceivably, such types of derivatives were easily obtained by
substituting the DM fluorophore with various functionalities tethered to the nitrogen.
Scheme 2.1. Synthesis of 3,4-bis(2,4-difluorophenyl)-maleimide 2.1 and the analogs
2.1a, 2.1b, and 2.1c
65
Much like the parent compound 2.1, the analogs 2.1a, 2.1b, and 2.1c display high
fluorescence quantum yields, high molar extinctions (ε(λ) 5,990 to 23,000 M-1cm-1), and a
large Stokes shifts (Δ 115 to 143 nm depending on solvent). Bathochromic shifts were
observed in the emission spectra for 1 and its derivatives when moving from nonpolar to
polar solvents (Table 1).The largest bathochromic shift in the emission spectra, as well as
the largest values for the Stokes shift for all compounds was recorded in the presence of
the protic polar solvent methanol (Δ 135 nm for 2.1c to Δ 143 nm for 2.1b) while the
lowest values were observed for the more polar aprotic solvent CH3CN (Δ 122 nm for
2.1b to Δ 130 nm for 2.1).
Table 2.1. Solvent dependent absorption and emission maxima of compounds
2.1, 2.1a –2.1c
ɛ
hexanes
toluene
DCM
CH3OH
CH3CN
1.89
2.38
8.93
32.7
37.5
1
λabs/λfl a
(Stokes)
1a
λabs/λfl a
(Stokes)
1b
λabs/λfl a
(Stokes)
1c
λabs/λfl a
(Stokes)
 fl b
345/460 (115)
345/463 (118)
345/468 (123)
341/481 (140)
338/468 (130)
334/445 (111)
331/448 (117)
332/454 (122)
332/469 (137)
325/452 (127)
346/465 (119)
346/469 (123)
348/467 (119)
340/483 (143)
342/464 (122)
342/461 (119)
346/464 (118)
350/469 (119)
346/481 (135)
344/469 (125)
0.083
0.06
0.61
0.313
0.296
Absorption and emission spectra were recorded in 5 x 10-6 M solutions, λex 340 nm.
measured against peryline
a
b
Quantum yield of 1 was
Seliskar and McGlynn5a characterized the lowest lying singlet state of the unsubstituted
maleimide as a 11A1→11B2 transition of primarily n→π* character. However, it is well
known that transitions to n→π* states are strongly dependent upon the nature of the
solvent while transitions to π→π* states are not as strongly influenced.5b Thus, the fact
that the absorption maxima of 2.1, 2.1a-2.1c do not exhibit a significant solvent
66
dependence would suggest that the excitation is largley of π→π* origin while the lowest
energy emitting state is of n→π* character thus accounting for the significant solvent
dependence of the Stokes shift. The re-ordering of the excitation state may be a result of
the phenyl substitution on the parent maleimide ring and this is currently under
investigation.
2.3
pH Sensitivity and Photobleaching Stability
The pH sensitivity and stability towards photobleaching of 2.1 were explored as
such preconditions are important when selecting a fluorophore for general biological or
FRET applications.5a First, the photobleaching test was performed by monitoring the
intensity of the absorption and fluorescence spectra of 2.1 in DCM irradiated by a broad
spectrum Xe source (150 W) for an extended period of time (6-18 hours). This
experiment indicated that 2.1 has a very good photo-stability with a negligent loss of
fluorescence (~2 %) after 18 hours of illumination (Figure 2.3). Maleimides are known to
undergo intersystem crossing leading to formation of an excited triplet state.50 hours
6 hours
18hours
Normalized intensity
1000000
800000
600000
400000
200000
0
400
450
500
550
Wavelenght (nm)
Figure 2.3. Normalized emission spectra of 1 (DCM, 1 x 10-5 M) after irradiation with
Xe source for 6 – 18 hours, λexc = 340 nm
67
6
If the energy of the triplet of 2.1 is estimated to be close to that of the parent maleimide
structure, the quantum yield of triplet formation of 2.1 in CH3CN is ~0.37 and increases
with less polar solvents.5 The low photobleaching susceptibility of 2.1, despite the
substantial efficiency of triplet formation, may be an intrinsic property related to the
polyfluorination of the aromatic rings. It has been reported that substitution by heavy
atoms, such as fluorine in the case of 2.1 and it’s derivatives, significantly improves the
photobleaching stability of cyanine dyes substituted with sulfonyl groups despite the
sulfonyl group’s propensity to increase triplet yields.7
Due to the low aqueous solubility of 2.1, the pH dependence of the fluorescence
could not be accurately recorded in aqueous media. Therefore, experiments to determine
a potential pH dependence were carried out in organic solvents by analyzing the
photophysical properties of 2.1 under acidic (TFA) and basic (2,6-lutidine) environments.
A
0.8
Relative intensity
1.0
TFA (eq)
0
259
620
1420
0.6
0.4
0.2
0.0
400
450
500
B
2,6-lutidine (eq)
0
320
841
1684
2560
4205
0.8
Relative intensity
1.0
0.6
0.4
0.2
0.0
550
400
450
500
550
Wavelength (nm)
Wavelength (nm)
Figure 2.4. Fluorescence pH dependence of 1 under A. acidic conditions, TFA (CH3CN,
5 x 10-6 M) and B. basic conditions, 2,6- lutidine (CH3CN, 5 x 10-6 M), λexc = 340 nm
68
It was observed that the spectral profile of 2.1 was not influenced by the acidic media
(Figure 2.4A), while a steady decrease of the fluorescence intensity was observed when
2.1 was titrated with 2,6-lutidine (Figure 2.4B).
The quenching effect was even more pronounced when a strong base such as
triethylamine was titrated to 2.1. Rehm-Weller analysis using Eox(lutidine) of 1.89 V
(SCE) and Ered(maleimide)=-1.03 V (SCE) and 2.65 V for the energy of the singlet
excited state of 1 in DCM (λmax= 468nm) results in a positive potential ~0.3 V indicating
the mechanism of quenching is not electron trnsfer between 1 and 2,6 lutidine.8 The
decrease in the fluorescence intensity of 1 at higher pH was attributed to the
deprotonation of the bisamido group and the generation of a non-fluorescent monoanion.
In addition, the fact that the alkylated analog 1b displayed higher stability to basic media
confirmed the above mechanism of a base-induced fluorescence quenching. The
fluorescence reduction was found to be reversible upon treatment of the anion with acid.
This suggests that the n→π* transition of the excited singlet state to the ground state
primarily involves the lone pair on the nitrogen while also confirming the role of
hydrogen bonding in stablizing the excited state.
2.4
Designing of FRET Systems with 3,4-diaryl-substituted Maleimide Moiety as
a Donor Partner
2.4.1
FRET System with Quencher Moiety as an Acceptor
Considering the advantageous photophysical properties of DMs 2.1 and 2.1a-2.1c,
we evaluated 1c as a donor component in a FRET peptide substrate of β-secretase, a key
enzyme responsible for Alzheimer’s disease.9 The sequence of the peptide was derived
69
from the β-secretase cleavage site of the Swedish APP mutation (previously reported by
Anna Spec). The envisioned FRET peptide contains DM donor moiety and an acceptor
(quencher) motif allowing for depopulation of the donor excited state via an
intramolecular FRET mechanism. The peptide synthesis was performed by using
standard Fmoc chemistry on solid phase with subsequent coupling of the donor/ acceptor
pair. The fluorophore donor 2.1c was appended to the N-terminus, and the fluorescence
quenching energy acceptor, dimethylaminoazobenzene-2'-carboxylic acid (DABCYL)
was attached at the C-terminus.
The donor emission which displays a maxima centered at λem 455 nm suitably
overlaps with the absorption of the quencher centered at λabs 440 nm, fulfilling one of the
major requirements for an efficient intramolecular FRET system. The Förster-Radius (R0)
calculated for this FRET system is 36 Å. The proper quencher selection was confirmed in
a preliminary experiment in which the two molecules, 2.1b and the acceptor (Dabcyl)
were covalently linked through a coupling reaction (compound 2.9 in the Suplimentary
Materials) and the FRET-based quenching mechanism was observed due to the close
proximity between the FRET partners (λex 340 nm). The absorption and the emission
profile of 2.9 is presented in the Experimental Section and clearly indicates that
fluorescence quenching occures. These results were consistent with the spectral profile
obtained from the actual FRET peptide system (Figure 2.5A and 2.5B).
70
14000
A
14000
12000
12000
Resonance Energy Transfer
10000
8000
F
Linker
Intensity
Intensity
10000
B
Q
6000
+
F
Q
8000
6000
4000
4000
2000
2000
350
0
400
450
500
Wavelength (nm)
350
550
400
450
500
Wavelength (nm)
550
Figure 2.5. Emission spectra of A. The tethered donor/acceptor FRET peptides and B.
FRET peptide after enzymatic cleavage, (DMSO 1 x 10-5 M), λexc= 340 nm
The FRET substrate entails a “fluorescence dequenching” motiff in which the quenched
fluorophore participating in the intramolecular FRET becomes fluorescent once the
substrate/acceptor is cleaved by the enzyme, β-secretase. Incubation of the FRET peptide
with β-secretease (for 1h at room temperature) results in the cleavage of the Leu-Asp
bond and the formation of two fragments 2.4 and 2.5 that separate the fluorophore from
the quencher (Figure 2.6). Therefore, the fluorescence from the DM containing fragment
is recovered as the two pairs are no longer in the proximity required for the intramolcular
FRET to occur. The emission profile of the FRET system before and after the enzymatic
cleavage are presented on Figure 2.5A and 2.5B and clearly indicate a change in the
flourescnce signal attributed to the efficient energy transfer between the compatible
donor/acceptor partners.
71
HN Glu Val Asn Leu Asp Glu Phe
COOH
NH
O
O
2.10
N
O
O
O
NH
F
F
- secretase
cleavage
N N
F
F
N
HN Glu Val Asn Leu
O
+
Asp Glu Phe
NH
COOH
O
2.5
2.4
O
F
N
O
NH
O
F
N N
F
F
N
Figure 2.6. FRET peptide cleaved by β-secretase.
Therefore, the fluorescence from the DM containing fragment is recovered as the two
pairs are no longer in the proximity required for the intramolcular FRET to occur.
Diffusional quenching between the two FRET components in solution was found to be
negligible based upon titration experiments in which the quencher was gradually
introduced to a solution containing the donor. No significant quenching was observed
even at 1.5 equivalents of the quencher.
A kinetic study of the enzymatic cleavage of the peptide system revealed that the
intensity of the restored fluorescence (in RFU) is enhanced in a concentration-dependant
fashion with a Km 0.29 µM and a Vmax 28.9 µM/min. These results confirmed our
expectation that the DM derivatives can be sucessfully incorporated into FRET
experiments.
72
2.4.2
FRET System with a Bright Fluorophore Moiety as an Acceptor
Encouraged by these results, the application of 2.1 was expanded by the design of
a second FRET system using another chromophore as an acceptor - free-base
tetraphenylporphyrin (TPP).10 This molecule was chosen as a suitable acceptor due to the
TPPs high molar extinction coefficient 417) 470,300 M-1cm-1 (in THF), sufficient spectral
overlap with the emission band of the donor molecule centered at λabs 450 nm and to the
large Stokes shift of the donor. The low energy emission (λmax  640 nm) of the TPP
avoids any undesired overlap between the donor and the acceptor emissions and the
primary absorption band of TPP (Soret band ~416nm) is red shifted relative to the
absorption band of 2.1 which also minimizes the partial absorption of the excitation
energy by the porphyrin (primary inner-filter effect). The synthetic pathway to the final
FRET complex was done as a result of a spontaneous reaction between 2.1a and the preactivated succinimidyl ester of TPP 2.8 (Figure 2.7).
Figure 2.7. Synthesis of the FRET paired system.
73
When compound 2.7 was excited with the donor excitation wavelength (270 nm or 340
nm), siginificant flourescence enhancement typical for the acceptor emission was
observed (λmax  640 nm). This is particullry important since in a control experiment it
was confirmed that the acceptor unit alone does not emit under these conditions.* The
Förster-Radius (R0) calculated for this FRET system is 26 Å. The FRET efficiency was
estimated based upon the resonance energy ratio change (RRC) between the intensity of
the donor and the acceptor’s emissions for the covalently attached entity 2.7 and the free
components in solution kept at equal concentration. The experimental data revealed that
there was a 4.5-fold signal increase in the case of the covalently linked complex* which is
in a similar range as SFP/YFP (RRC=3), Cerulean/YFP (RRC=2), and BFP/GFP
(RRC=5) FRET pairs.11
TPP concentration
92 µM
80 µM
68 µM
55 µM
43 µM
30 µM
18 µM
Intensity
of 1a
10000
Intensity
Intensity
1400
5000
700
0
660
720
780
Wavelength (nm)
0
400
600
Wavelength (nm)
Figure 2.8. Diffusional FRET between the non-covalently bound donor/acceptor pair.
Emission spectra were obtained by gradual titration of the acceptor into a donor
containing sample (λexc = 340 nm, 2.5 x 10-5 M of 2.1a in toluene).
74
In addition, diffusional FRET was explored in a concentration dependant manner by two
parallel titration experiments in which one of the chromophores was kept at a constant
concentration, and the other was titrated, monitoring the emission signal after each
titration step. In both experiments, regardless of which component was kept at a constant
concentration, an increase in the fluorescence intensity of the acceptor emission and a
simultaneous decrease in the emission from the donor were observed due to diffusional
quenching/FRET (Figure 2.8).
2.5
Conclusions
In summary, we have presented new flourophore 3,4-bis(2,4-difluorophenyl)-maleimide
2.1 and its derivatives that possess favorable flourescence properties suitable for
biological and biomedical applications. In addition we demonstrated that 2.1 can easily
be decorated with functional handles for its conjugation to the biological target of
interest, without altering its fluorescent properties. Furthermore, for the first time we
demonstrated the implementation of the DM moiety as an acceptor unit in two FRET
systems.
75
2.6
2.6.1
Experimental Section
General Information
All of the reagents and solvents are commercially available and were used as purchased,
without further purification. Column chromatography was carried out using Merck
Kieselgel 60 H silicagel. 1H NMR,
13
C NMR were recorded on a Bruker 250 MHz and
Varian 400 MHz NMR spectrometer. All 1H NMR experiments were reported in δ units,
parts per million (ppm) downfield of TMS and were measured relative to the signals for
chloroform (7.26 ppm) and deuterated methanol (3.35, 4.78 ppm). All 13C NMR spectra
were reported in ppm relative to the signals for chloroform (77 ppm) and
dimethylsulfoxide (49.3 ppm) with 1H decoupled observation. HRMS were taken on an
Agilent G1969A LC/MSD TOF. Fluorescence measurements were conducted using a
Photon Counting Spectrophotometer ISS (PC1) and the absorption measurements were
recorded on Shimadzu UV-2401-PC. Absorption and fluorescence spectra were recorded
in 1 cm path length quartz cuvette. All solvents used for spectroscopy experiments were
spectrophotometric grade.
76
2.6.2
Spectroscopic Analysis and Characterizations of 2.1, 2.1a, 2.1b and 2.1c
2.6.2.1 Quantum Yields and Extinction Coefficients in Different Solvents
Table 2.2. Extinction coefficients of 2.1, 2.1a, 2.1b, and 2.1c (λmax = 340 nm)
Extinction
coefficient
2.1
2.1a
2.1b
2.1c
20,580
22,980
17,400
15,800
17,099
15,430
15, 400
15,320
48,400
28,320
24,150
23,110
20,536
17,620
16,950
16,720
13,490
18,560
11,200
12,530
ε(340) (M-1 cm-1)
hexanes
toluene
DCM
CH3OH
CH3CN
Table 2.3 Quantum yield of 2.1, 2.1a, 2.1b, and 2.1c measured in DCM against perylene
as a reference (λex = 340 nm, flperylene=0.72).14
Quantum yield
(fl)
2.1
2.1a
2.1b
2.1c
DCM
0.61
0.24
0.26
0.29
77
2.6.2.2
Solvatochromism
A
F
F
O
1.0
NH
F
F
O
NH 2
N
O
in CH3OH
F
F
0.8
Normalized intensity
Normalized intensity
0.8
in hexane
in toluene
in DCM
in CH3CN
F
1.0
in CH3OH
O
F
B
in hexane
in toluene
in DCM
in CH3CN
0.6
0.4
0.2
0.6
0.4
0.2
0.0
0.0
350
400
450
500
550
350
Wavelength (nm)
400
450
500
550
Wavelength (nm)
C
F
O
1.0
OH
N
D
0.8
F
F
in hexane
in toluene
in DCM
in CH3CN
F
O
COOH
N
in CH3OH
O
O
0.8
F
1.0
Normalized intensity
Normalized intensity
in hexane
in toluene
in DCM
in CH3OH
F
0.6
0.4
0.2
F
F
0.6
0.4
0.2
0.0
0.0
350
400
450
500
350
550
400
450
Wavelength (nm)
Wavelength (nm)
500
550
Figure 2.9 Emission spectra of 2.1, 2.1a, 2.1b, and 2.1c recorded in various solvents (λex
= 340 nm), A. emission spectra of 2.1, B. emission spectra of 2.1a, C. emission spectra of
2.1b, D. emission spectra of 2.1c
78
2.6.2.3
pH Dependence of the Emission
Procedure used for a titration of 2,6-lutidine to 5 uM solution of 1 in CH3CN
The pH dependence test was carried out in 1 cm path length quartz cuvette. A 10 mM
stock solution of the fluorophore 2.1 in CH3CN was prepared and 1 uL of 10 mM stock
solution was added to 2 mL CH3CN in a glass cuvette. The 5 uM final concentration of
2.1 in 2 mL CH3CN in the cuvette was kept consistent through the entire titration
experiment. A 8 M stock solution of the base, 2,6-lutidine was prepared by mixing 15 ml
of 2,6-lutidine (MW = 107.15g/mol; density (d) = 0.92 g/cm3) with 10 mL of CH3CN.
The base, 2,6-lutidine was gradually titrated to the solution of the fluorophore in CH3CN
in small increments: 320 eq = 1.6 mM 2,6-lutidine (0.5 uL of 8 M stock solution); 841 eq
= 4.2 mM 2,6-lutidine (1 uL of 8 M stock solution); 1684 eq = 8.4 mM 2,6-lutidine (2 uL
of 8 M stock solution); 2560 = 12.8 mM 2,6-lutidine (3 uL of 8 M stock solution); 4205
eq = 21 mM 2,6-lutidine (5 uL of 8 M stock solution).
2.6.2.3a Reversibility of the Base-Induced Fluorescence Quenching
Reversibility of the base-induced fluorescence quenching demonstrated through the
neutralization of the basified solution of 2.1 with acid (TFA).
79
TFA (eq.) / 2,6-lutidine (eq)
Relative intensity
1.0
0 / 0 (ini)
7280 / 4205
6240 / 4205
5200 / 4205
4160 / 4205
3240 / 4205
2080 / 4205
1040 / 4205
520 / 4205
0 / 4205
[acid] intensity
0.5
0.0
400
450
500
550
Wavelength (nm)
Figure 2.10. Emission spectra of 2.1 (λex = 340 nm) in CH3CN recorded through titration
of acid, TFA (0 eq – 7280 eq) to the basified solution of 2.1 (10 uM of 2.1 in CH3CN;
4205 eq of 2,6-lutidine). "Ini" marked curve refers to initial solution before addition of
acid or base.
Et3N (eq)
0 eq
340 eq
680 eq
1380 eq
2060 eq
1400000
1200000
Intensity
1000000
800000
600000
400000
200000
0
350
400
450
500
Wavelength (nm)
550
Figure 2.11. Emission spectra of 2.1 (10 μM in CH3CN, λex = 340 nm) titrated with Et3N
(0 eq -2060 eq)
80
F
Relative intensity
2,6-lutidine (eq.)
F
0
800
1600
2400
4000
5600
O
1.0
NH2
N
0.8
O
F
F
1b
0.6
0.4
0.2
0.0
350
400
450
500
550
600
Wavelength (nm)
Figure 2.12. Emission spectra of 2.1b (5 μM in CH3CN, λex = 340 nm)) obtained from
the titration of 2,6-lutidine (0 eq -5600 eq) demonstrating the smaller fluorescence
quenching effect due to the functionalization at the N-position
2.6.3
Spectroscopic Analysis and Characterization of the FRET Peptide System
with Dark Quencher as an Acceptor
2.6.3.1
Spectral Overlap
1.2
Acceptor Donor
1.0
1.0
0.8
0.6
0.5
0.4
0.2
Normalized Emission (a.u.)
Normalized Absorbance (a.u.)
1.4
0.0
0.0
350
400
450
500
Wavelength (nm)
550
600
Figure 2.13. Spectral overlap of the emission profile of 2.1c (donor, blue line) and the
absorption profile of the dark quencher (acceptor, black line), Dabcyl
81
2.6.3.2
Absorption and Emission Spectra of 2.9, the Covalently Attached Donor
(2.1b) and Quencher (Dabcyl)
Absorption
0.4
0.2
0.0
300
400
500
600
700
800
Wavelength (nm)
Figure 2.14. Absorption spectrum of the covalently linked donor (2.1c) and the acceptor
(Dabcyl) (λexc = 340 nm) (10 μM in DCM, used as a preliminary control to determine the
proper quencher selection
Normalized emission
2500
2000
1500
1000
500
400
440
480
520
Wavelength (nm)
560
Figure 2.15. Normalized emission spectra the covalently linked donor (1c) and the
acceptor (Dabcyl) (λexc = 340 nm, 10 μM in DCM), used as a preliminary control to
determine the proper quencher selection
82
2.6.4
Fluorimetric Titration Experiment Used to Rule out Diffusional
Quenching Between the Donor (2.1c) and the Dark Quencher (Dabcyl), Free in
Solution
Donor (eq) : Quencher (eq)
1:0
1:1
1 : 2.5
Intensity
1200000
800000
400000
0
400
450
500
550
Wavelength (nm)
600
Figure 2.16. Emission spectra recorded in a titration experiment in which the quencher
molecule, Dabcyl, (0 eq – 2.5 eq in DCM) was titrated to the acceptor 2.1c (10 μM in
DCM, λex = 340 nm)
2.6.5
Conditions for the Incubations of the FRET Peptide with β-Secretease
and Lineweaver-Burk Plot
2.6.5.1
Incubation Conditions of the FRET Peptide with β-Secretease
The sequence of this β-secretase FRET peptide is derived from the β-secretase
cleavage site of the “Swedish” APP mutation and has been used previously in the
AnaSpec Senso Lyte® 520 β-secretase Assay Kit. The incubation of the FRET substrate
with β-secretase leads to the cleavage of the Leu-Asp bond and regaining of the
fluorescence from the liberated fragment, containing the chromophore. All incubations
were performed in 384-well micro plate at room temperature, for 1 hour and each well
contained β-secretase, sodium acetate buffer (pH 4.5), and substrate in DMSO in total
volume of 30 µL. Once the FRET off state was initiated due to the enzymatic cleavage,
the fluorescent signal in relative fluorescent units (RFU) was continuously monitored
83
with spectrofluorimeter upon λexc / λem = 340 nm / 460 nm. The progression of the
enzymatic reaction was also monitored in a concentration dependent manner with the
following substrate concentrations: 2.93 μM, 2.7 μM, 2.4 μM, 2.13 μM, 1.86 μM, 1.6
μM, 1.33 μM, 1.06 μM, 0.66 μM, 0.33 μM, 0.017 μM. The experiment was performed in
three independent trials, carried out under the same incubation conditions which revealed
that the enzymatic reaction followed Michaelis–Menten kinetics
2.6.5.2
Lineweaver-Burk Plot of the Enzymatic Reaction
Linweaver-Burk plot15 was used to calculate two important characteristics of the rate of
the enzyme-mediated reaction, Vmax and Km (Vmax = 27 μM /min and Km = 0.24 μM).
Lineweaver ‐ Burk plot
0.15
y = 0.0085x + 0.037
R² = 0.9494
Km = 0.24 µM
Vmax = 27 µM/min
0.1
0.05
0
‐6
‐4
‐2
0
2
4
‐0.05
‐0.1
Figure 2.17. Lineweaver-Burk plot of the enzymatic reaction.
84
2.6.6
Spectroscopic Analysis and Characterization of the FRET System with
Bright Acceptor
Absorption of the Acceptor (TPP)
500000
400000
NH
-1
-1
cm )
2.6.6.1
Molar Extinction (M
N
N
HN
300000
200000
100000
0
350
400
450
500
550
600
650
Wavelength (nm)
Figure 2.18. Absorption of the acceptor (TPP).
2.6.6.2
Emission Spectra of the Acceptor (TPP) Alone in Solution Excited at 270
nm and at 420 nm
3500
Emission intensity
3000
Emission intensity
80000
A
2500
2000
1500
1000
500
B
60000
40000
20000
0
0
600
650
700
750
Wavelength (nm)
600
800
650
700
Wavelength (nm)
750
800
Figure 2.19. Emission spectra of the acceptor (TPP) alone in toluene A. excited at λexc =
270 nm and B. excited at λexc= 420 nm.
85
2.6.6.3
FRET Titration Experiment between the Donor (1a) and the Acceptor
(TPP)
[1b] (µM)
8000
7000
6000
2000
5000
Intensity
Intensity
10 µM
15 µM
25 µM
50 µM
75 µM
100 µM
125 µM
150 µM
[1b] Intensity
4000
Intensity
1000
3000
0
2000
630
700
770
Wavelength (nm)
1000
0
400
500
600
700
800
Wavelength (nm)
Figure 2.20. Emission spectra obtained by titrating 2.1a (10 – 150 μM) to TPP (25 μM)
in toluene (λexc = 270 nm), demonstrating the FRET diffusion mechanism between the
donor/acceptor. The intensity of the donor emission decreased and the intensity of the
acceptor emission increased; the acceptor concentration was constant.
2.6.6.4
Representation of the Resonance Energy Ratio Change (RRC).
FRET pairs free in solution (A)
FRET pairs covalently attached (B)
16000
14000
Intensity
12000
10000
8000
6000
4000
2000
0
400
500
600
700
Wavelength (nm)
800
Figure 2.21. Emission spectra of A. FRET pairs 2.1a and TPP free in solution (toluene)
and B. FRET pairs 2.1a and TPP covalently attached at the same concentration (5M in
toluene).
86
2.6.7
Calculation of the Förster-Radius (R0)
The Förster radius (R0) was calculated according to the following equation:16
 9000ln 10 K 2 Qd J 
3
2 4
R0  
  9.78  10 Qd K n J
5 4
 128 n N av

1

6

1
6
Å
where K2 is the orientation factor and is assumed to be 2/3 for randomLy oriented
molecules, Qd is the quantum yield of the donor molecule (2.1c), n is the index of
refraction, NA is the Avogadro’s number, and J is the spectral overlap integral between
donor and acceptor, represented as:
 F     d 
 F  d
4
J DA
D
A
D
where FD(λ) is the fluorescence spectrum of the donor, and εA is the extinction spectrum
of the acceptor.17
2.6.8 Synthetic Procedures
Synthesis of FRET peptide 2.10
The FRET peptide was generated by a solid-phase synthesis approach, followed by the
subsequent coupling of the donor (fluorophore) and the acceptor (quencher) molecules.
The fluorophore 2.1c was coupled to the N-terminus and the DABCYL component
(quencher) was linked to the amino group at the Lysine residue at the C-terminus. The
peptide was generated in 25 µmol by using peptide synthesizer (Symphony, Peptide
Technology Inc.). Fmoc-protective group was installed at the N-terminus with an Alloc
87
protective group at the lysine residue on the C-terminus. In the first step the peptide resin
was washed with DCM (3 x 5 mL x 30 s) and allowed to expand for 10 minutes in DCM
under Ar atmosphere. Subsequent Fmoc - removal was performed by reacting the peptide
resin with 20 % piperidine in DMF under Ar atmosphere for 20 min, followed by wash
with DMF (3 x 30 s) and DCM (3 x 30 s).12 Upon removal of Fmoc, while still in
solution under Ar atmosphere, the peptide resin was reacted with the fluorophore 2.1c
(40.7 mg, 4 eq), HCTU (41 mg, 4 eq), and DIPEA (17.4 μl, 4 eq). The reaction continued
for 120 min under Ar atmosphere and the system was stirred occasionally. After the
completion of the coupling reaction, the peptide resin was again washed with DMF (3 x
30 s) and then DCM (3 x 30 s). Subsequent Alloc removal was performed by allowing
the peptide resin to react with PhSiH3 (24 eq, 81µL) in DCM (1 mL) and solution of
Pd(PPh3)4 (3 mg, 0.10 eq) in DCM (3 mL) with Ar passing through and mechanical stir
for about 15 min.13 88
Figure 2.22. Synthetic approach to generate the FRET peptide with attached donor and
acceptor moieties.
89
The resin was washed twice with DCM (8 x 30 s) and Kaiser test was performed to
confirm the presence of free amine as a result of the successful removal of the Alloc
group. Subsequently, the pre-activated quencher, 4-(4-dimethylamino-phenylazo)benzoic acid 2,5-dioxo-2,5-dihydro-pyrrol-1-yl ester (35 mg, 4 eq) in DCM (3 mL) was
reacted for 120 min with the resin peptide. The peptide resin was washed with DCM (8 x
30 s) and that process was repeated once more. The peptide was cleaved from the resin
with 95 % THF, 2.5% H2O, 2.5% triisopropylsilane and subsequently washed with DCM
(5 x 30 s). The peptide was precipitated from cold diethyl ether and subsequently
segregated from the solvent by centrifugation. The isolated peptide was dissolved in
acetonitrile : water (30 : 70) and it was isolated as red powder after lyophilization. The
mass of the final peptide was confirmed by MALDI–TOF, calculated 1647.66, and found
1648.72
3,4-bis(2,4-difluorophenyl)-maleimide 2.1
A suspension of (2,4-difluoro-phenyl)-acetonitrile (153 mg, 1 mmol) and iodine (254 mg,
1 eq) in diethyl ether was reacted with freshly prepared sodium methoxide in methanol
solution (113 mg, 2.1 mmol in 10 mL dry methanol) at -85 oC. The reaction mixture was
brought to room temperature and after two hours it was treated with 3% HCl until
reaching pH 7. The reaction was completed after stirring at room temperature for 24
hours and the crude mixture was washed with water (20 mL), saturated solution of
sodium chloride (15 mL), and extracted with diethyl ether (30 mL  3). After extraction
with diethyl ether, the combined organic phases were dried over anhydrous sodium
sulfate and concentrated. The product 1 (177 mg, 55 %) was obtained by flash
chromatography (DCM : methanol = 10 : 2). Rf = 0.55 (DCM : methanol = 9 : 2). 1H
90
NMR (250 MHz, CDCl3) δ 8.44 (s, 1H), 7.50 (td, J = 8.3, 6.4 Hz, 2H), 6.97 (td, J = 8.0,
2.4 Hz, 2H), 6.85 – 6.77 (m, 2H).
13
C NMR (63 MHz, CDCl3) δ 169.49 (s), 166.37 -
158.64 (m), 134.39 (s), 132.48 (dd, J = 9.9, 4.3), 113.34 (ddd, J = 15.2, 4.0), 112.10 (dd,
J = 21.6, 3.6 Hz, 7H), 104.84 (t, J = 25.4 Hz). DEPT 90 13C NMR (63 MHz, CDCl3) δ
132.37 (dd, J = 10.0, 4.3 Hz), 111.99 (dd, J = 21.6, 3.7 Hz), 104.74 (t, J = 25.4 Hz).
HRMS (ESI) calcd. for C16H7F4NO2 [M+H]+: 322.0412, found: 322.0485.
1-(2-aminoethyl)-3,4-bis(2,4-difluorophenyl)-1H-pyrrole-2,5-dione 2.1a
The synthesis of (2-bromo-ethyl)-carbamic acid tert-butyl ester were performed
according to a reported procedure.1 The suspension of 3,4-bis-(2,4-difluorophenyl)maleimide 2.1 (321 mg, 1 mmol) in 10 mL dry DMF was cooled to 0o C and reacted with
(2-bromo-ethyl)-carbamic acid tert-butyl ester (336 mg, 1.5 eq) and potassium tertbutoxide (2 eq, 225 mg) under inert atmosphere. The reaction was completed after 36
hours and the crude mixture was washed with saturated solution of sodium bicarbonate
and extracted with ethyl acetate (3 x 20 mL). After extraction with ethyl acetate the
combined organic phases were dried over anhydrous sodium sulfate and concentrated.
The protected crude 2-[3,4-Bis-(2,4-difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]ethyl}-carbamic acid tert-butyl ester 2.2 was not purified but further reacted with 5%
TFA solution in DCM and stirred at room temperature for 3 hours in order to remove the
Boc group. The crude mixture was washed with water (20 mL) and extracted with DCM
(3 x 20 mL). The combined organic phases were dried over anhydrous sodium sulfate
and concentrated. The product (323 mg, 89 %) was obtained by chromatography (DCM :
methanol = 7 : 3). Rf = 0.55 (DCM : methanol = 8 : 2 ). 1H NMR (400 MHz, CD3OD) δ
7.57 (td, J = 8.4, 6.4 Hz, 2H), 7.09 – 7.03 (m, 2H), 7.03 – 6.95 (m, 2H), 3.99 – 3.94 (m,
91
2H), 3.27 – 3.23 (m, 2H), 1.26 (s, 2H). 13C NMR (63 MHz, CDCl3) δ 169.55 (s), 166.22 158.62 (m), 133.29 (s), 132.47 (dd, J = 9.9, 4.4 Hz), 113.67 (ddd, J = 15.1, 3.9, 2.2 Hz),
111.94 (dd, J = 21.6, 3.6 Hz), 104.70 (t, J = 25.6 Hz), 38.31 (s), 18.03 (s). HRMS (ESI)
calcd. for C18H12F4N2O2 [M+H]+: 365.0835, found: 365.0896
3,4-bis(2,4-difluorophenyl)-1-(3-hydroxypropyl)-1H-pyrrole-2,5-dione 2.1b
The suspension of 3,4-bis(2,4-difluorophenyl)-maleimide 1 (321 mg, 1 mmol) in dry 10
mL DMF was cooled to 0 oC and was reacted with potassium tert-butoxide (2 eq, 225
mg) and 3-bromopropan-1-ol (1.5 eq, 207 mg) under inert atmosphere. The reaction was
completed after 24 hours at room temperature and the crude mixture was washed with
saturated solution of sodium bicarbonate (15 mL) and extracted with DCM (3 x 30 mL).
After extraction with ethyl acetate the combined organic phases were dried over
anhydrous sodium sulfate and concentrated. The desired product 2.1b (322 mg, 85%) was
obtained by chromatography (DCM : methanol = 10 : 3). Rf = 0.45 (DCM : methanol = 8
: 2). 1H NMR (250 MHz, CDCl3) δ 7.49 (td, J = 8.3, 6.4 Hz, 2H), 6.96 (ddd, J = 8.0, 2.5,
1.3 Hz, 2H), 6.86 – 6.74 (m, 2H), 3.82 (t, J = 6.4 Hz, 2H), 3.66 (t, J = 5.7 Hz, 2H), 1.93 –
1.82 (m, 2H). 13C NMR (63 MHz, CDCl3 δ 169.93 (s), 166.22 - 158.57 (m), 133.39 (s),
132.41 (dd, J = 9.9, 4.3 Hz), 113.33 (ddd, J = 15.2, 4.0), 111.94 (dd, J = 21.6, 3.6 Hz),
104.69 (t, J = 25.4 Hz), 59.33 (s), 35.36 (s), 31.33 (s). HRMS (ESI) calcd. for
C19H13F4NO3, [M+H]+: 380.0832, found: 380.0812.
4-(3,4-bis-(2,4-difluorophenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)butanoate 2.3
The suspension of 3,4-bis(2,4-difluorophenyl)-maleimide 1 (321 mg, 1 mmol) in 10 mL
92
dry DMF was cooled to 0o C and was reacted with potassium tert-butoxide (2 eq, 225 mg)
and with 4-bromo-butyric acid (293 mg, 1.5 eq) under inert atmosphere. The reaction was
stirred for 24 hours at room temperature and the crude mixture was washed with saturated
solution of sodium chloride (15 mL) and extracted with ethyl acetate (3 x 30 mL). After
extraction with ethyl acetate the combined organic phases were dried over anhydrous
sodium sulfate and concentrated. The desired product 2.3 was (357 mg, 82%) was
obtained by chromatography (hexane : ethyl acetate = 6 : 4). Rf = 0.45 (hexane : ethyl
acetate = 7 : 3). 1H NMR (250 MHz, CDCl3) δ 7.43 (dd, J = 8.2, 6.4 Hz, 2H), 6.94 (td, J
= 8.0, 2.4 Hz, 2H), 6.78 – 6.65 (m, 2H), 4.14 – 3.95 (m, 2H), 3.65 (t, J = 6.7 Hz, 2H),
2.31 (t, J = 7.3 Hz, 2H), 2.04 – 1.86 (m, 2H), 1.15 (dd, J = 10.4, 3.8 Hz, 3H).13C NMR
(63 MHz, CDCl3) δ 172.63 (s), 169.47 (s), 162.40-158.51 (m), 133.29 (s), 132.48 (dd, J =
10.0, 4.2 Hz), 113.62 (ddd, J = 15.0, 3.8, 2.2 Hz), 111.91 (dd, J = 21.6, 3.6 Hz), 104.65
(t, J = 25.5 Hz), 60.59 (s), 38.08 (s), 31.64 (s), 23.87 (s), 14.21 (s). HRMS (ESI) calcd.
for C22H17F4NO4, [M+H]+: 436.1094, found: 436.1078.
4-[3,4-bis-(2,4-difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]-butyric
acid 2.1c
The suspension of 4-[3,4-bis-(2,4-difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]butyric acid ethyl ester 3 (435 mg, 1 mmol) in 15 mL HCl : AcOH = 1 : 1 was refluxed at
90 oC for 20 hours. The crude product was extracted with DCM (20 mL  3) and the
combined organic phases were dried over anhydrous sodium sulfate and concentrated.
The desired product 1c (326 mg, 80%) was obtained by chromatography (DCM :
methanol = 7 : 3). Rf = 0.52 (DCM : methanol = 8 : 2). 1H NMR (250 MHz, CDCl3) δ
7.43 (td, J = 8.3, 6.5 Hz, 2H), 6.97 (td, J = 8.0, 2.4 Hz, 2H), 6.80 – 6.69 (m, 2H), 3.68 (t,
93
J = 6.8 Hz, 2H), 2.39 (t, J = 7.3 Hz, 2H), 1.98 (dd, J = 14.2, 7.2 Hz, 2H). 13C NMR (63
MHz, CDCl3) δ 178.91 (s), 169.60 (s), 167.25 – 156.95 (m, 2H), 133.38 (s), 132.50 (dd, J
= 10.0, 4.1 Hz), 113.58 (ddd, J = 15.3, 3.9, 2.2 Hz), 112.03 (dd, J = 21.6, 3.5 Hz), 104.77
(t, J = 25.6 Hz), 38.01 (s), 31.40 (s), 23.49 (s). HRMS (ESI) calcd. for C20H13F3NO4,
[M+H]+: 408.0781, found: 408.0742.
N-{2-[3,4-bis-(2,4-difluoro-phenyl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]-ethyl}4-(10,15,20-triphenyl-porphyrin-5-yl)-benzamide 2.7
The
synthesis
of
4-(10,15,20-triphenyl-porphyrin-5-yl)-benzoic
acid
2,5-dioxo-
pyrrolidin-1-yl ester 2.8 was performed by following a reported procedure2 and the
precursor of 2.8,
4-(10,15,20-triphenyl-porphyrin-5-yl)-benzoic acid was obtained
through hydrolysis following a previously reported procedure3. The suspension of 4(10,15,20-triphenyl-porphyrin-5-yl)-benzoic acid 2,5-dioxo-pyrrolidin-1-yl ester 2.8 (755
mg, 1 mmol) in 10 mL dry DCM was reacted with 1-(2-amino-ethyl)-3,4-bis-(2,4difluoro-phenyl)-pyrrole-2,5-dione (2.1a) (364 mg, 1 eq). The reaction mixture was
stirred for 24 hours at room temperature and the product formation was monitored by
TLC and MALDI–TOF. The desired product 7 (904 mg, 90%) was obtained by
chromatography (DCM: methanol = 9 : 1). Rf = 0.50 (100% DCM). MALDI –TOF,
calculated for: 1006.309, found: 1006.310.
1
H NMR (250 MHz, CDCl3) δ 8.89 (d, J =
5.5 Hz, 5H), 8.81 (d, J = 4.8 Hz, 2H), 8.46 (d, J = 8.2 Hz, 2H), 8.33 (d, J = 8.1 Hz, 2H),
8.29 – 8.18 (m, 6H), 7.77 (td, J = 4.7, 2.3 Hz, 9H), 7.51 (td, J = 8.3, 6.5 Hz, 2H), 6.99 (td,
J = 8.2, 2.1 Hz, 2H), 6.82 (ddd, J = 11.0, 8.9, 2.4 Hz, 2H), 3.78 (t, 2H), 2.46 (t, J = 7.3
Hz, 2H), -2.73 (s, 1H). 13C NMR (63 MHz, CDCl3) δ 169.99 , 169.24 , 167.47 - 158.54
(m), 147.16 (s), 142.12 (s), 134.64 (s), 133.40 (s) 132.44 (dd, J = 11.0, 5.1 Hz), 129.62
94
(s), 129.55 (s) 128.05 (s), 128.87 (s) 126.81 (s), 120.70 (s), 120.51 (s), 118.68 (s), 113.55
(m), 112.01 (dd, J = 21.6, 3.6 Hz), 104.77 (t, J = 25.6 Hz ), 52.52 (s, ), 31.33 (s).
MALDI-TOF, calculated 1004.31, found 1005.33
4-(4-dimethylamino-phenylazo)-benzoic acid 3-[3,4-bis-(2,4-difluoro-phenyl)2,5-dioxo-2,5-dihydro-pyrrol-1-yl]-propyl ester 2.9
A suspension of 2.1b, 3,4-bis-(2,4-difluoro-phenyl)-1-prop-2-ynyl-pyrrole-2,5-dione,
(379 mg, 1 mmol) in dry 10 mL DCM was reacted with 1-ethyl-3-(3'dimethylaminopropyl) carbodiimide (287 mg,1.5 eq), 4-dimethylaminopyridine (61 mg,
0.5 eq) and 4-(4-dimethylamino-phenylazo)-benzoic acid (DABCYL) (269 mg, 1 eq).
The reaction mixture was stirred for 24 hours at room temperature and the product
formation was monitored by TLC and LC/MS. The cruder material was washed with
water and extracted with DCM (20 mL x 3). The combined organic phases were dried
over anhydrous sodium sulfate and concentrated.s The desired product 2.9 (560 mg,
80%) was obtained by chromatography (DCM : methanol = 8 : 2). Rf = 0.52 (DCM :
methanol = 8 : 2). 1H NMR (250 MHz, CDCl3) δ 8.13 (d, J = 8.6 Hz, 2H), 7.86 (dd, J =
18.7, 8.8 Hz, 4H), 7.44 (td, J = 8.2, 6.5 Hz, 2H), 6.75 (td, J = 8.0, 2.4 Hz, 2H) , 6.77 (ddd,
J = 9.2, 3.5, 1.8 Hz, 4H), 4.43 (t, J = 5.9 Hz, 2H), 3.90 (t, J = 6.7 Hz, 2H), 2.30 – 2.14 (m,
2H).
13
C NMR (63 MHz, CDCl3) δ 169.47 (s), 166.53 (s), 162.46 - 156.14 (m), 153.02
(s), 143.81 (s), 133.40 (s), 132.49 (dd, J = 10.0, 4.1 Hz), 130.62 (s), 130.06 (s), 125.63
(s), 122.14 (s), 113.77 (ddd, J = 15.1, 3.9, 2.2 Hz), 112.19 (dd, J = 21.6, 3.6 Hz), 104.74
(t, J = 25.6 Hz), 104.34 (s), 62.70 (s), 40.38 (s), 36.29 (s), 27.81. HRMS (ESI) calcd. for
C34H26F4N4O4, [M+H]+: 631.1890, found: 631.1855.
95
2.7
References:
1. (a) Xiao, Y., Liu, F.; Qian, X.; Cui, J. ,
Chem. Commun. 2005, 239-241. (b)
Rajasekharan, K. N.; Bruke M., J. Biol. Chem. 1989, 264. (c) Corrie J. E. T.;
Munasinghe V. R. N.; Rettig W.; J. Heterocyclic Chem., 2000, 37, 1447. d) Tanaka, K.;
Miura, T.; Umezawa, N.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. J. Am. Chem.
Soc. 2001, 123, 2530.
2. Sung, K.; Gunther, J.; Katzenellenbogen, J., Org. Lett. 2008, 10, 4931-4934; (b)
Nataliya Panchuk–Voloshina, Rosaria P. Haugland, Janell Bishop–Stewart, Mahesh K.
Bhalgat,. Paul J. Millard, Fei Mao, Wai-Yee Leung, and Richard P. The Journal of
Histochemistry & Cytochemistry, 1999, 47(9),1179–1188
3. Yeh, H.; Wu, W.; Chen, C. , Chem Comm 2003, 404-405.
4. (a) Wu, W; Yeh, H.; Chan, L.; and. Chen, C. , Adv. Mater 2002, 14, 1072. (b) Yeh,
H.; Chan, L.; Wu, W.; Chen, C., J. Mater, Chem. 2004, 14, 1293-1298.
5. (a) Seliskar, C.J.; McGlynn, S.P., J. Chem. Phys., 1972, 56, 1417-1422.
(b)
Haberfield, P.; Lux, M.S.; Rosen, D., J. Am. Chem. Soc.1977, 99, 6828-6831. (c) Wang,
B.C.; Liao, H.R.; Yeh, H.C.; Wu, W.C.; Chen, C.T., J. Lumin. 2005, 113, 321-328. (d)
Climent, T.; González-Luque, R.; Merchán, M., J. Phys. Chem. A. 2003, 107, 6995-7003.
6. von Sonntag, J; Knolle, W.; Naumov, S.; Mehnert, R., Chem. Eur. J. 2002, 8, 41994209.
7.(a) Koziar, J.C.; Cowan, D.O., Acc. Chem. Res., 1978, 11, 334-341. (b)Toutchkine, A.;
Nguyen, D.; Hahn, K., 6 Org. Lett. 2007, 9, 2775-2777.
8. (a) Knibbe, H.; Rehm, D.; Weller, A., 1967, Berichte der Bunsen-Gesellschaft Fur
Physikalische Chemie,1968, 72, 257-263. (b) Zeng, C.C; Zhang, N.T.; Lam, C.M.; Little,
96
R.D., Org. Lett., 2012, 14, 1314-1317. (c) Guy, J.; Caron, K., Dufresne, S.; Michnick,
S.W.; Skene, W.G.; Keillor, J.W., J. Amer. Chem. Soc., 2007, 129, 11969-11977.
9. (a) Wang, Y.; Du, G., Journal of Asian Nat. Prod. Research, 2009, 11, 604-612. (b).
Felice, F.; Ferreira, S., Cell Mol. Neurobiol. 2002, 22, 545. (c). Calderon, F; Bonnefont,
F., J. Neurosci Res. 1999, 56, 620.
10. (a) H. Du, R. A. Fuh, J. Li, A. Corkan, J. S. Lindsey,
Photochemistry and
Photobiology 1998, 68, 141-142. (b) G. H. Barnett, M. F. Hudson, and K. M. Smith, J.
Chem. Soc. Perkin Trans. 1975, I, , 1401-1403.
11. (a) Nguyen, A.W.; Daugherty, P.S., Nature Biotech. 2005, 23, 355-360. (b) Nguyen,
A.; You, X.; Jabaiah, A.; Daugherty, P., Reviews in Fluorescence 2008, 321-335
12. Carpino, L. A. and Han, G. Y.; J. Org. Chem. 1972, 37, 3404-3409.
13. Thieriet, N.; Alsina, J.; Giralt, E.; Guibé, F.; Albericio, F.; Tetrahedron Letters 1997,
38, 7275-7278.
14. Williams, A.; Winfield, S.; Miller, J.N.; Analyst, 1983, 108, 1067. (b). N.C. Ganguli;
K. Chakravery; J. Phys. Chem. 1979, 83, 2581
15. Lineweaver, H and Burk, D.; J. Am. Chem. Soc., 1934, 56, 658–666.
16. Stryer L; Ann. Rev. Biochem. 1978, 47, 819-846. (b). Selvin PR; Methods Enzymol.
1995, 246, 300-334.
17. Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 3 ed.; Springer: New York,
2006.
97
Chapter 3
In situ Generated Novel Carbonic Anhydrase Inhibitory Compounds
3.1
Overview
The metalloenzymes carbonic anhydrases (CAs) are directly involved in the
global carbon cycle and catalyze the simple but critical physiological reaction, the
interconversion between carbon dioxide and the bicarbonate anion:1-3
This essential reaction regulates numerous important physiologic and physio-pathological
processes in different tissues and cells, and the modulation of the CA activities through
inhibition or activation can be used as an efficient path to treat a wide range of human
diseases. The CAs play a central role in homeostasis, bone respiration, calcification,
tumorigenicity, and electrolyte secretion in variety of tissues and organs.1-3,
4-7
In
mammals, out of the 16 known isoforms, 13 are catalytically active (CAs I-VA, CA VB,
CAVI, CAVII, CA IX and CAs XII-XV), while the CA-related proteins (CARPs VIII,
IX, and XI) lack catalytic activity. Among these, the CA I and CA II isozymes were
widely explored due to their physiological abundance and tissue distribution, in addition
to the fact that CA II is ranked as one of the most effective catalysts known in nature
(Kcat/KM values >10
classical
type
or
8
M-1s-1, exhibiting near diffusional-limited kinetics).1,2,16 The
CA
inhibitors
(CAIs),
98 such
as
sulfonamides,
sulfamates,
hydroxysulfonamides, and hydroxamates, showed significant CA inhibitory activity and
found clinical use as diuretics and antiglaucoma agents with demonstrated potential for
anti-convulsant, anti-obesity, anti-cancer, and anti-infective applications.8-15 This class of
inhibitory compounds coordinates to the catalytically active Zn (II) ion in their
deprotonated form, and further engage in extensive hydrogen bonding and van der Waals
interactions with the surrounding amino acids located in the hydrophobic and hydrophilic
spheres of the active side.17-19 While the inhibition mechanism of these scaffolds is
thoroughly explored and well understood, there are some health related issues caused by
the currently marked sulfonamide drugs. To a large extent this is contributed to the lack
of isozyme specificity of the available inhibitors and the large number of isoforms in
human diffused in different tissues and organs. The systemic use of such sulfonamide
therapeutics leads to various side effects and allergic reaction in the patients. For
example, the use of the known antiglucoma agent, acetazolamide (Diamox) leads to
numerous side effects derived by inhibition of other CAs besides those present in the eye
ciliary processes (i.e., CA II, IV, and XII), causing fatigue, paresthesias, and kidney
stones.1,3,18 Also, side effects in obese epileptic patients treated with topiramate or
zonisamide were expressed by significant weight loss. Thus, the development of
structurally diverse non-sulfanilamide-based inhibitors for CAs reveals a considerable
potential for the discovery of novel therapeutic treatments. However, despite the
advances made in this direction, there has not been a uniform (or robust), efficient and
rapid technique in place which can be used to discover novel, non-sulfonamide-based
bioactive molecules. Most often, the discovery and identification of a novel inhibitory
class of compounds evolves as a result of labor-intensive procedures and screening
99 techniques. For example, the screening of 960 structurally diverse small molecules
against human carbonic anhydrase II (hCA II) yielded only thioxolone as a genuine CA
inhibitor in addition to two other representatives of non-sulfonamide inhibitors
(merbromin and tannic acid) which were ruled out due to the lack of determination of
their specificity.19 The ~ 2% “hit” rate efficiency reported through this screening assay
further emphasizes the need for improvements in the methods for screening and handling
large number of compounds. The kinetic target-guided synthesis (TGS) is an approach
that aims at accelerating the process of discovery and identification of potential
pharmaceutical candidates by combining the screening and synthesis of libraries of low
molecular weight compounds in one step. Through this approach the biological target of
interest is directly involved in the selection and assembly of its own bidentate inhibitors
by binding to the building blocks which exhibit the highest affinity to the enzyme’s active
site, and by introducing them in the correct alignment needed for their covalent
interaction. This methodology has been successfully applied and led to the identification
of highly potent inhibitors (some in fM range) in a variety of biological systems including
protein-protein interaction.20 Several applications of kinetic TGS were reported where
bovine carbonic anhydrase assembled its own potent inhibitory compounds via suitable
pre-arranged chemical reactions between a sulfonamide anchoring scaffold and a
complementary reacting partner. Interestingly enough, in all reported examples the
capturing sulfonamide scaffold was required, thus limited the scope of this approach by
generating only sulfonamide-based CA active compounds. One of the first examples
addressing the CA as a targeted biological system has been pioneered by Lehn at al. By
applying the concept of dynamic combinatorial chemistry (DCC), Lehn at al
100 demonstrated that bCA II synthesized its own inhibitors via an imine generating
reversible condensation reaction between constituents containing Zn (II) coordinating
groups and a number of aromatic moieties.21 Through this study it was concluded that the
enzyme bCA II favors the formation of these condensation compounds which were to
express the strongest binding affinity to the enzyme’s active site. More recently, Huc
described a similar approach in which the kinetic TGS was exploited to assemble
inhibitors of bCA II (770 nM – 59 nM) by using an alkylation reaction of a 4mercaptomethyl-benzenesulfonamide with various α-chloroketones.22 Competition
experiments revealed that the enzyme templated the reaction in a way that it facilitated
the formation of the alkylation products which exhibit the highest affinity for the target.
The most efficient variant of kinetic TGS, the in situ click chemistry approach,
relays on the 1,3-dipolar cycloaddition of azides and alkynes as the ligation reaction.22b22d
This cycloaddition approach has been rendered as the ideal click chemistry reaction
due to its slow reactivity profile which combines compatible functionalities in a stable
covalent bond generating no side products. This reaction is dramatically accelerated (up
to 107 times) by the recently discovered copper-(I) catalysis, and the obtained full
conversion and regio-selectivity makes the 1,2,3-triazole transformation very suitable for
many novel applications in the drug discovery field. Several members of the 1,2,3triazole family have shown valid biological properties such as anti-allergic, anti-bacterial,
and anti-HIV activities. Recently Kolb and Sharpless presented a successful application
of in situ click chemistry for the assembly of potent bidentate inhibitors for bCA II by
utilizing the cycloaddition reaction between acetylene benzensulfonamide and
complimentary azide building blocks (Figure 3.1).23
101 O
H 2N S
O
+
N3
R
bovine carbonic anydrase II
44 azide
building blocks
aq. buf fer pH 7.4
37oC, 40 hrs
O
H 2N S
O
N R
N
N
12 hit compounds
Figure 3. 1. Application of in situ click chemistry for the synthesis of novel sulfonamidebased bCA II inhibitors.
Importantly, in concurrence with the in situ click chemistry concept, the resulting triazole
compounds displayed much higher binding affinities to the target (Kd = 0.2 - 7.1 nM)
than the individual (starting) fragments (acetylene scaffold, Kd = 37 nM +/- 6).
Furthermore, it was demonstrated that the newly-formed triazole unit does not
significantly affect the binding activity which leads to the conclusion that the increased
affinity of the hit moieties to a large extent is related to the favorable binding interaction
with the more distant functionalities in the azide component. The incubation of the
anchoring scaffold, 4-ethynyl-benzenesulfonamide with a library of structurally diverse
azides in the presence of bCA II led to the identification of 12 hits out of 24 regent
combinations, 11 of which were validated and confirmed through control experiments.
Interestingly, one of the most potent hits among all was triazole 3.1 which effectively
combined the azide with anchoring sunlfonamides functionality, and a coumarin moiety
which recently was reported to be a very potent CA inhibitor as a member of a novel
inhibitory class.
102 We were interested to investigate the contribution of the two active scaffolds in the
inhibitory process and to probe whether the in situ click chemistry approach can be
successfully applied to design novel CA II inhibitors from a library of non-sulfonamide
containing scaffolds with coumarins as leading fragments.
3.2
Result and Discussion
3.2.1
Design of Non-Sulfonamide Containing Inhibitors
The above findings set the stage for performing the first in situ search for CA
inhibitory compounds from a library of non-sulfonamide containing fragments. The in
situ click chemistry experiments between alkynes (AK) and azides (AZ) complementary
scaffolds were conducted by utilizing the completely bioorthogonal [1,3]-dipolar
cycloadition reaction for the assembly of triazole compounds within the CA II active site.
A library of twenty (20) structurally diverse scaffolds was developed and among these
the coumarin based azides (AZ2, AZ3), and the fluorescent 3,4-bis (2,4-difluorophenyl)–
maleimide-alkynes (AK1, AK4), were chosen as the leading fragments.
Recently it has been shown that coumarins, thiocoumarins and phenols were the
most promising molecules in the design of novel non-sulfonamide-based inhibitors due to
the high affinity and specificity that they display to some CA isoforms. The natural
product 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one 3.2 was identified
as a bCA II inhibitor by employing electrospray ionization Fourier transform ion
103 cyclotron resonance mass spectrometry (ESI-FTICR-MS) coupled with online size
exclusion chromatography (SEC), a technique used to detect and identify intact targetligand complexes.24 In comparison to the bioassay-guided fractionation of natural product
extractions, the ESI-FTICR-MS is highly sensitive and less labor intensive but the
structural elucidation remains somewhat a challenge. The mechanism of inhibition of
these potent non-sulfonamide chemotypes has been deciphered in detail by Supuran and
Paulsen groups, and X-ray crystal structure of several coumarin • CA II complexes were
presented. It has been reported that the coumarin natural product 6-(1S-hydroxy-3methylbutyl)-7-methoxy-2H-chromen-2-one 3.2 exhibits a very unusual binding mode in
which its hyrdolized derivative, the 3.2 Z the cis-hydroxycinnamic acid acts as the actual
inhibitor binding at the entrance of the active site cavity (Figure 3.2).25
Figure 3.2. Hydrolysis of the he coumarin natural product 6-(1S-hydroxy-3methylbutyl)-7-methoxy-2H-chromen-2-one, assisted by the nucleophilic Zn-bound
water/hydroxide of the CA active site
Unlike the Zn-binding inhibitors, the coumarin class of inhibitors follows a non-zinc
mediated mechanism of inhibition in which the Zn-bound, nucleophilic water/hydroxide
hydrolyzes the lactone ring. Therefore, no actual interaction between the catalytically
essential metal ion and coumarin moiety was present. It has been demonstrated that the
104 inhibitory activity of the coumarin compounds is time dependent, in which a longer preincubation time of the enzyme with the inhibitor results in lower KI values, with 6 hours
deemed as an optimal required time. Therefore, these recent findings raised the question
about the detailed inhibitory process that moiety 1 may follow as it combines two
fragments with functionality which have known affinity to CA, the sulfonamide and the
coumarine, both able to inhibit the metalloenzyme through their own mechanism of
action. Therefore, these recent findings raised the question about the detailed inhibitory
process that compound 3.1 may follow, as it combines two fragments with a sulfonamide
and a coumarin, both able to inhibit the metalloenzyme through their own distinct
mechanism of action.
A similar type of binding interaction in which two reactive functionalities into the
moiety simultaneously exhibit affinity to the active site of the enzyme was recently
reported by introducing the X-ray crystal structure of a fluorescent antitumor CA
inhibitor 3.3 with excellent properties as an imaging tool.26 The X-ray crystal structure of
3.3 with the cytosolic isoform hCA II revealed a strong interaction between the bulky
tricyclic fluorescein scaffold with the α-helix formed by residues Asp 130–Val 135,
situated at the rim of the active site in addition to the interaction of the sulfonamide with
the Zn (II) ion within the enzyme active site (Kd = 0.64 nM hCA II).
105 The design and development of new potent CA fluorescent probes which possess
photophysical properties suitable for their application as imaging tools, opens the stage
for diagnosis and treatment of cancer related disorders due to the CA overexpression in
some tumor cells. Only recently we reported the synthesis and full spectroscopic
characterization of a new fluorescent moiety 3,4-bis(2,4-difluorophenyl)-maleimide with
excellent photophysical properties which rank it suitable as a biomarker and a FRET
partner: a) excellent quantum yield of 0.61 (in DCM); b) a large Stokes shift (140 nm in
DCM); c) high extinction coefficient of 48,400 M-1cm-1; and d) good photostability.27
Considering the aforementioned photophysical characteristics, we were prompted to
investigate the implementation of the maleimide molecule as a potential biomolecular
probes as an alternative to the fluorescein derivatives, which are susceptible to
fluorescence quenching when introduced to bio-conjugates. Furthermore, the (2,4difluorophenyl)–maleimide derivatives were also elected because of their structural
features which pre-determine the possibility of favorable hydrogen bonding interactions
with the maleimide core and Thr 199, adjacent to the Zn (II) ion in the enzyme active
site.
3.2.2 Kinetic TGS
A library of eight (8) structurally diverse azides among which two coumarin
based fragments, AZ2 and AZ3 were screened against fifteen (15) functionalized alkynes
with 3,4-bis (2,4-difluorophenyl)–maleimides, AK1 and AK4, chosen as lead fluorescent
probes, in the presence of bCA II in buffer (Figure 3.3).
106 X
Figure 3.3. Library of alkynes AK and azides AZ fragments utilized in a kinetic TGS
screening of bCAII.
All possible combinations of azides, AZ (400 M) and alkynes, AK (400 M)
were incubated with bCA II (33 M) in buffer (pH = 7.4) at 37o C for 40 hours to give
rise to one hundred and twenty (120) potential triazole products. A screening analysis of
each reaction mixture was performed by using LC/MS-SIM mode in order to determine
whether a given combination of building blocks has resulted in a product formation,
which was unambiguously identified by their molecular weight and retention time. In
addition to that, a control experiment was carried out by incubating each AK/AZ
combination under the same conditions but without the biological target bCA II
(background reaction). That was used to confirm that when the product formation was
induced by the enzyme, whereas incubation conditions without the assistance of BCA II,
there was an insignificant amount of triazole detected. Potential “hit” combinations were
107 identified by searching for significant differences in the peak area between the
chromatograms of the enzyme reaction and the background reaction. The analysis of the
LC/MS-SIM traces for all AZ/AK combinations revealed eight hit triazoles, AZ7AK4,
AZ1AK1, AZ4AK1, AZ2AK12, AK1AZ2, AK1AZ3, AK4AZ2, AK4AZ3, with
distinct product signals for molecular weight and retention time. Among these 8 hits, the
most prominent ones were selected based on the value of their amplification coefficients
(peak intensity of the product in the enzymatic reaction divided by the peak area of the
product in the background reaction) and only the triazoles that showed amplification
coefficient higher than 4.5, were further investigated, synthesized and tested for activity
against CA. Therefore, the following four triazoles met the above criteria and were the
hits of interest: AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3. Furthermore, in all
enzyme-generated triazole combinations, the anchoring azides (AZ2 and AZ3) and
alkynes (AK1 and AK4) were present either linked to other complementary scaffolds or
among themselves, with the latter case producing the most potent hit combinations. This
observation confirmed our initial hypothesis that the leading, non-sulfonamide anchoring
scaffolds are able to design CA inhibitors through the use of the reagents with the highest
affinity to the biological target.
3.2.3
Validation of Kinetic TGS Hits
Even though the inhibitory compounds were first detected with bovine CA II,
they also exhibited strong affinity to the human CA II (hCA II). When incubated under
the previously described conditions, a formation of the four triaozoles of interest,
AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3, was detected by using a triple quadrupole
108 mass spectroscopy. The mechanism of inhibiton of these coumarin-containing triazoles
was validated by substituted the hCA II with Apo CA II and performing the incubations
under the same conditions with the hit generating azide/alkyne fragments. The analysis of
the spectroscopic traces revealed that the triazole formation reaction was not catalyzed in
the presence of the Apo CA II, and the product formation resembled the one in the
background un-templated reaction. This observation supported the premises that the
catalytically active Zn (II) containing enzyme is needed for the assembly of its own
inhibitors.
3.2.4
Determination of Regio-Selectivity of the CA-Templated Reactions
All 1,5-disubstituted ‘syn-‘ and 1,4-disubstituted ‘anti-‘ hit triazoles AK1AZ2,
AK1AZ3, AK4AZ2, and AK4AZ3, were synthesized in order to determine the
regioisomer which is preferentially generated by the enzymatic reaction. The antitriazoles were prepared according to a recently reported azide-alkyne coupling protocol
under copper (I) catalysis in the presence of sodium ascorbate and t-BuOH, a protocol
used
to generate exclusively the anti-isomer.28 The syn-triazoles were obtained by
ruthenium catalyzed “fusion” of azides with alkynes in the presence of Cp*RuCl(PPh3)229
(Figure 3.4).
Figure 3.4. General synthetic route used to generate the anti-, and syn-triazole isomers
109 Therefore, the four anti-isomers of the hit triazole combinations, anti-AK1AZ2, antiAK1AZ3, anti-AK4AZ2, and anti-AK4AZ3, were obtained by reacting a suspension of
the corresponding alkyne/azide fragments in 2:1 mixture of t-BuOH : H2O with 30 mol%
sodium ascorbate and 10 mol% copper sulfate (CuSO4.5H2O) under inert atmosphere for
6-12 hours at room temperature (Figure 3.5). The product formation was monitored by
TLC and LC/MS. The four anti-triazoles were purified via flash chromatography,
isolated in very good yields and fully characterized by, 1H-NMR, 13C-NMR and HRMS.
Figure 3.5. Synthesis of anti-AK1AZ2, anti-AK1AZ3, anti-AK4AZ2, anti-AK4AZ3
The synthesis of the syn-triazoles was carried out under nitrogen atmosphere using a
standard Schlenk technique. A suspension of the corresponding azide and alkyne moieties
in dry THF was reacted with 2 mol% Cp*RuCl(PPh3)2 and stirred at 80 C for 6-10 hours
(Figure 3.6). The product formation was monitored by TLC and LC/MS. The four syntriazoles were purified via flash chromatography, isolated in very good yields and fully
characterized by, 1H-NMR, 13C-NMR and HRMS.
110 Figure 3.6. Synthesis of syn-AK1AZ2, syn-AK1AZ3, syn-AK4AZ2, and syn-AK4AZ3
A NOE experiment for the syn- and anti-AK1AZ3 was conducted in order to
verify the asserted structural differences at the trizole unit (or conjunction) between the
two isomers.
Regioisomer assignment for the in situ generated triazoles was accomplished by
comparing the HPLC traces from the in situ reactions with the authentic anti- trizoles and
also with a mixture of the authentic syn- and anti-triazoles. We were able to conclude that
in all AZ/AK hit combinations, the enzymatic reaction preferentially catalyzed the
formation of the anti-triazoes over the corresponding syn-isomer. An additional
confirmation of the regio-selectivity was achieved by LC/MS-SIM co-injection analysis
of a mixture containing the bCA II in situ reaction with the corresponding authentic antitriazole (Figure 3.7). The co-injection experiment for all four combinations confirmed the
same retention time between the anti-triazole, generated through the enzymatic reaction
and the retention time of the anti-triazole, synthesized via Cu(I) catalysis (see the
experimental section for the rest of the traces). In a similar manner, a second LC/MS-SIM
co-injection experiment was performed, in which a mixture containing the in situ reaction
along with a mixture of synthesized triazoles of a known syn/anti-ratio. The LC/MS
traces obtained from these co-injections clearly showed a change in syn/anti-ratio for the
111 template reaction indicating an enhancement of the peak corresponding to the anti-
20
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:6
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E
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1500
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500
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isomer.
Figure 3.7. In situ hit identification by LC/MS-SIM, exemplified by the AK1/AZ3 pair:
A. Building blocks AK1 and AZ3 in buffer
B. Building blocks AK1 and AZ3 in bCA II
C. Authentic sample anti-AK1AZ3
D. Co-injection between enzymatic reaction B and anti-AK1AZ3
E. Mixture of authentic syn-AK1AZ3 and anti-AK1AZ3
F. Co-injection between enzymatic reaction and the authentic syn-, anti-AK1AZ3
112 3.2.5
Binding Measurements
The time dependent nature of the inhibition activity typical for all coumarine-
based inhibitors was also observed for these triaozle compounds and that was assessed by
determining the inhibition constants (Ki) in two independent preliminary experiments
with pre-incubation time with the enzyme of 3 and 25 minutes, respectively. It was
observed that the longer pre-incubation time with the enzyme led to lower Ki values, for
example the inhibition constants for AK1AZ2 recorded with 3 min of pre-incubation
time was of 22 μM, whereas when the pre-incubation time was increased to 25 min a Ki
of 0.4 μM was observed. The activity determination of all trizoles by using stop-flow
assay and longer pre-incubation time with the enzyme (4-6 hours) is currently ongoing.
3.3. Conclusion
In this research study in situ click chemistry has been entailed in the discovery of
non-sulfonamide containing inhibitors for carbonic anhydrase II. Recently, coumarins
have been identified as a novel class of inhibitors with an unusual binding mode which
does not involve coordination to the Zn 2+ ion but instead the the coumarin is hydrolyzed
by the Zn-bound water to generate the actual inhibitory compound. A library of twenty
three structurally diverse alkyne/azide scaffolds was developed among which the
coumarin based azides (AZ2, AZ3), and the fluorescent 3,4-bis (2,4-difluorophenyl)–
maleimide-alkynes (AK1, AK4), were chosen as the leading fragments. Out of 120
possible triazoles 8 hit combinations were identified as generated preferentially by the
enzyme, among which the coumarin-containing triazoles exhibited higher in situ
amplification coefficient and they were further investigate. The syn- and anti-isomers of
AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were synthesized and it was determined
113 that the enzyme favors the formation of the anti-triazole over the syn-trizole for all of the
above hit combinations. The hit generating azide/alkyne fragments were also identified
by using hCA II as a target. The mechanism of inhibition of these triazoles was validated
by substituting the hCA II with Apo-CA (non-Zn containing enzyme) and demonstrating
that enzyme Zn-bound water/hydroxide is needed in order to hydrolyze the coumarins
and generates the actual inhibitors. The time dependent nature of the inhibition activity
typical for all coumarine-based inhibitors was also observed for the triaozle compounds
and that was assessed by determining the inhibition constants (Ki) in two independent
preliminary experiments with pre-incubation time of 3 and 25 minutes, respectively. It
was observed that the longer pre-incubation time with the enzyme led to lower Ki values;
however a stop-flow assay is currently in use in order to determine the inhibition activity
for more than 4 hour of pre-incubation time. Thus, a novel type of coumarin-containing
triazoles were presented as in situ generated hits which have the potential to be used as
fluorescent bio-markers or other drug discovery-related applications.
114 3.4
Experimental section
3.4.1 General information
All reagents and solvents were purchased from commercial sources and used without
further purification. Column chromatography was carried out using Merck Kieselgel 60
H silicagel. 1H NMR, 13C NMR were recorded on a Bruker 250 MHz and Varian 400
MHz NMR spectrometer. All 1H NMR experiments were reported in δ units, parts per
million (ppm) downfield of TMS and were measured relative to the signals for
chloroform (7.26 ppm) and deuterated methanol (3.35, 4.78 ppm). All 13C NMR spectra
were reported in ppm relative to the signals for chloroform (77 ppm) and
dimethylsulfoxide (49.3 ppm) with 1H decoupled observation. HRMS were taken on an
Agilent G1969A LC/MSD TOF. The LC/MS data weremeasured on an Agilent 1100
LC/MSD-VL with electrospray ionization.
3.4.2
General Procedure for In situ Click Chemistry Experiments
Stock solutions of azides (20 mM) and (20 mM) were prepared in DMSO. The alkyne (1
µL) was added to wells of 96-well microtiter plates, containing the enzyme (96 µl of a 1
mg/mL solution, 33 mM final bCAII concentration in phosphate buffer: 58 mM
Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl, 1 mM NaN3, pH 7.4), followed by the azide
reagent (1 µL). The reaction plate was incubated at 37°C for 40 hours. Compound
detection was accomplished by electrospray mass spectroscopy in positive selected ion
mode (LC/MS-SIM). The injection volume was 20 µL at a flow rate of 0.7 mL/min. The
chromatography was performed on a Microb C8 column (4.6 mm x 250 cm) for
115 AK1AZ3, AK4AZ3, and AK4AZ2, and Allumina Cyano (4.6 mm x 250 cm) for
AK1AZ2.
The following elution gradient was employed for AK1AZ2 and AK4AZ2:
Table 3.1 Solvent gradient used for TQMS methods for AK1AZ2 and AK4AZ2
Time
Flow rate
(min)
B %*
(mL min-1)
0
30
0.7
50
40
0.7
55
100
0.7
60
30
0.7
* eluent A: H2O; eluent B: CH3CN
The following elution gradient was employed for AK1AZ3 and AK4AZ3:
Table 3.2 Solvent gradient used for TQMS methods for AK1AZ3 and AK4AZ3
Time
Flow rate
(min)
B %*
(mL min-1)
0
50
0.7
50
54
0.7
55
100
0.7
60
50
0.7
* eluent A: H2O; eluent B: CH3CN
116 3.4.3
Regioisomer determination for bCAII-derived in situ hits
Figure 3.8. In situ click chemistry hit identificationo of AK4AZ3 triazole combination.
Regioisomer determination os syn- and anti-AK4AZ3
A. Building blocks AK4 and AZ3 in buffer
B. Building blocks AK4 and AZ3 in bCA II
C. Authentic sample anti-AK4AZ3
D. Co-injection between enzymatic reaction (B) and anti-AK4AZ3
E. Mixture of authentic syn-, anti-AK4AZ3
F. Co-injection between enzymatic reaction (B) and authentic syn-, anti-AK4AZ3
117 Figure 3.9. In situ click chemistry hit identificationo of AK4AZ2 triazole combination.
Regioisomer determination os syn- and anti- AK4AZ2
A. Building blocks AK4 and AZ2 in buffer
B. Building blocks AK4 and AZ2 in bCA II
C. Authentic sample anti-AK4AZ2
D. Co-injection between enzymatic reaction (B) and anti-AK4AZ2
E. Mixture of authentic syn-, anti-AK4AZ2
F. Co-injection between enzymatic reaction (B) and authentic syn-, anti-AK1AZ3
118 Figure 3.10. In situ click chemistry hit identificationo of AK1AZ2 triazole combination.
Regioisomer determination os syn- and anti-AK1AZ2
A. Building blocks AK1 and AZ2 in buffer
B. Building blocks AK1 and AZ2 in bCA II
C. Authentic sample anti-AK1AZ2
D. Co-injection between enzymatic reaction (B) and anti-AK1AZ2
E. Mixture of authentic syn-, anti-AK1AZ2
F. Co-injection between enzymatic reaction (B) and authentic syn-, anti-AK1AZ2
119 3.4.4
In situ Click Chemistry Control experiments with Apo-CA
Figure 3.11. In situ click chmtisty control experiment of AK4 and AZ2 with hCAII and
Apo CA
Figure 3.12. In situ click chmtisty control experiment of AK1 and AZ2 with hCAII and
120 Apo CA
Figure 3.13. In situ click chmtisty control experiment. of AK1 and AZ3 with hCAII and
Apo CA
121 Figure 3.14. In situ click chmtisty control experiment. of AK4 and AZ3 with hCAII and
Apo CA
122 3.4.5
Synthetic Procedures
Synthesis of 7-(2-hydroxyethoxy)-4-methyl-2H-chromen-2-one 3.5 and synthesis of 7-(3hydroxypropoxy)-4-methyl-2H-chromen-2-one 7 have been previously reported.30
Synthesis of 7-(2-azidoethoxy)-4-methyl-2H-chromen-2-one AZ2
To the solution of 7-(2-hydroxyethoxy)-4-methyl-2H-chromen-2-one 3.5 (0.5 mmol,
0.111g) in dry DMF (8 mL) under inert atmosphere was added triethylamine (3 eq, 1.5
mmol, 0.151 g) and the reaction mixture was cooled down to 0 oC Methanesulfonyl
choloride (1.5 eq, 0.758 mmol, 0.085 g) was introduced dropwise and the reaction
mixture was stirred at 0 oC for 30 min then brought to room temperature. The product
formation was monitored by LC/MS and TLC (ethyl acetate : hexanes 8 : 2- Rf = 0.55).
Compound 3.6 was not purified with flash chromatography due to its low stability and
123 tendency to decompose on silica gel. The crude mixture of 2-((4-methyl-2-oxo-2Hchromen-7-yl)oxy)ethyl methanesulfonate 3.6 was directly used for the next step and
reacted with solution of sodium azide in water (5 eq, 2.2 mmol, 0.158 g of sodium azide
dissolved in 10 mL of water) and heated at 40 oC for 15 hours. The crude reaction
mixture was washed with water (3 x 20 mL), extracted with DCM and the combined
organic phase was dried over Na2SO4. The crude product was purified by flash
chromatography (DCM : MeOH / 95 : 5) to give the desired azide AZ2 in 75% yield (Rf
= 0.55 in 90 : 10 = DCM : MeOH). 1H NMR (250 MHz, CDCl3) δ 7.42 (d, J = 8.8, 1H),
6.80 (dd, J = 8.8, 2.5, 1H), 6.70 (d, J = 2.5, 1H), 6.04 (d, J = 1.0, 1H), 4.18 – 4.05 (m,
2H), 3.63 – 3.47 (m, 2H), 2.33 (dd, J = 12.8, 2.7, 3H).
13
C NMR (63 MHz, CDCl3) δ
161.20, 161.16, 155.23, 152.56, 125.82, 114.16, 112.67, 112.37, 101.54, 77.67, 77.16,
76.65, 67.48, 50.01, 18.75, HRMS (ESI) calcd for C12H11N3O3, [M+H]+ : 246.0800,
found: 246.0780.
Synthesis of 7-(3-azidopropoxy)-4-methyl-2H-chromen-2-one AZ3
To the solution of 7-(2-hydroxyethoxy)-4-methyl-2H-chromen-2-one 7 (0.5 mmol, 0.117
g) in dry DMF (8 mL) under inert atmosphere was added triethylamine (3 eq, 1.5 mmol,
0.151 g) and the reaction mixture was cooled down to 0 oC. Methanesulfonyl choloride
(1.5 eq, 0.758 mmol, 0.085 g) was introduced dropwise and the reaction mixture was
124 stirred at 0 oC for 30 min then brought to room temperature. The product formation was
monitored by LC/MS and TLC (ethyl acetate : hexanes - 8 : 2- Rf = 0.50). Compound
3.8 was not purified with flash chromatography due to its low stability and tendency to
decompose on silica gel. The crude mixture of 2-((4-methyl-2-oxo-2H-chromen-7yl)oxy)ethyl methanesulfonate 3.8 was directly used for the next step and subsequently
reacted with solution of sodium azide in water (5 eq, 2.2 mmol, 0.158 g of sodium azide
dissolved in 10 mL of water) and heated at 40 oC for 15 hours. The crude reaction
mixture was washed with water (3 x 20 mL), extracted with DCM and the combined
organic phase was dried over anhydrous Na2SO4. . The crude product was purified by
flash chromatography (DCM : MeOH / 95 : 5) to give the desired azide AZ3 in 70%
yield (Rf = 0.55 in 90 : 10 = DCM : MeOH). 1H NMR (250 MHz, CDCl3) δ 7.40 (t, J =
13.0, 1H), 6.85 – 6.68 (m, 2H), 6.06 (d, J = 0.8, 1H), 4.05 (q, J = 5.8, 2H), 3.47 (t, J =
6.5, 2H), 2.30 (t, J = 9.0, 3H), 2.10 – 1.91 (m, 2H). 13C NMR (63 MHz, CDCl3) δ 161.76,
161.32, 155.33, 152.61, 125.72, 113.88, 112.53, 112.19, 101.60, 77.67, 77.16, 76.65,
65.18, 48.15, 28.66, 18.77. HRMS (ESI) calcd for C13H13N3O3, [M+H]+ : 260.0957,
found: 260.0857.
Synthesis of 3,4-bis(2,4-difluorophenyl)-1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione
AK1
125 A solution of (difluoro-phenyl)-pyrrole-2,5-dione 3.9 in dry DMF was reacted with
potassium tert-butoxide, and 3-bromoprop-1-yne under inert atmosphere. The reaction
mixture was heated to 75 oC for 4 hours and the product formation was monitored by
TLC and LC/MS. The crude reaction mixture was washed with brine (2 x 20 mL),
extracted with ethyl acetate (3 x 50 mL) and the combined organic phase was dried over
anhydrous Na2SO4.The crude product was purified by flash chromatography (Hexanes :
Ethyl acetate - 80 : 20) to give the desired azide AK1 in 78% yield (Rf = 0.55 in
Hexanes : Ethyl acetate = 85 : 25). 1H NMR (250 MHz, CDCl3) δ 7.43 (dd, J = 14.8, 8.3,
2H), 6.86 (td, J = 8.2, 2.0, 2H), 6.76 – 6.63 (m, 2H), 4.33 (d, J = 2.3, 2H), 2.18 (t, J = 2.3,
1H).
13
C NMR (63 MHz, CDCl3) δ 168.19, 166.43, 166.25, 162.79, 162.60, 162.40,
162.21, 158.72, 158.54, 133.67, 132.62, 132.55, 132.46, 132.39, 113.63, 113.57, 113.39,
113.35, 113.29, 112.24, 112.18, 111.89, 111.83, 105.16, 104.75, 104.35, 77.67, 77.16,
76.98, 76.65, 72.01, 27.80. HRMS (ESI) calcd for C19H9F4NO2 [M+H]+ : 360.0569,
found: 360.0642.
A solution of (difluoro-phenyl)-pyrrole-2,5-dione 3.9 (0.161 g, 0.5 mmol) in dry DMF
was reacted with potassium tert-butoxide (2 eq, 0.112 g), and 6-bromohex-1-yneunder
(2.5 eq, 0.201 g) under inert atmosphere. The reaction mixture was heated to 75 oC for 3
hours and the product formation was monitored by TLC and LC/MS. The crude reaction
126 mixture was washed with brine (2 x 20 mL), extracted with ethyl acetate (3 x 50 mL) and
the combined organic phase was dried over anhydrous Na2SO4.The crude product was
purified by flash chromatography (Hexanes : Ethyl acetate / 80 : 20) to give the desired
azide AK4 in 77% yield, (Rf = 0.45 in hexanes : EtOAc = 85 : 25). 1H NMR (250 MHz,
CDCl3) δ 7.60 – 7.39 (m, 2H), 7.08 – 6.88 (m, 2H), 6.80 (ddd, J = 10.4, 8.8, 2.5, 2H),
3.80 – 3.55 (m, 2H), 2.33 – 2.11 (m, 2H), 1.99 – 1.91 (m, 1H), 1.91 – 1.71 (m, 2H), 1.70
– 1.43 (m, 2H). 13C NMR (63 MHz, CDCl3) δ 169.55, 166.31, 166.12, 162.78, 162.61,
162.28, 162.09, 158.71, 158.54, 133.29, 132.59, 132.52, 132.43, 132.36, 113.78, 113.54,
112.14, 112.09, 111.80, 111.74, 105.11, 104.70, 104.30, 83.78, 77.67, 77.16, 76.65,
68.99, 53.53, 38.31, 27.67, 25.67, 18.03. HRMS (ESI) calcd for C22H15F4NO2 [M+H]+ :
402.1039, 402.1112
General procedure for the synthesis of 1,5-disubstituted (‘syn’) triazoles:31
The synthesis of the syn-triazoles was carried out under nitrogen atmosphere using a
standard Schlenk technique. A mixture of azide (1eq), alkyne (1eq), and 2 mol%
Cp*RuCl(PPh3)2 in dry THF was stirred at 60 C for 6 - 8 hour. The completion of the
reaction was monitored by TLC and LC/MS. The crude reaction mixture was quenched
with water and extracted with ethyl acetate. All syn-triazoles were purified by silica gel
chromatography. The syn-regioisomers were obtained by following this procedure and
were subsequently characterized by 1H NMR and 13C NMR, and HRM.
Synthesis of syn-AK1AZ2
127 This compound was obtained by following the general synthetic procedure for syntriazoles. Rf = 0.48 (dichloromethane : methanol = 4:1), isolated in 75 % yield. 1H NMR
(250 MHz, CDCl3) δ 7.75 (s, 1H), 7.40 (ddd, J = 8.5, 7.5, 4.8, 3H), 6.97 – 6.83 (m, 2H),
6.80 – 6.61 (m, 4H), 6.07 (d, J = 1.1, 1H), 5.12 – 4.91 (m, 4H), 4.43 (t, J = 4.8, 2H), 2.31
(d, J = 1.0, 3H). 13C NMR (63 MHz, CDCl3) δ 168.74, 162.91, 162.56, 162.39, 161.03,
160.67, 158.50, 155.20, 152.37, 133.83, 132.49, 132.43, 132.26, 132.22, 125.94, 114.58,
114.58, 113.28, 113.00, 112.77, 112.39, 112.01, 112.01, 105.38, 104.97, 104.57, 104.57,
102.06, 67.58, 67.58, 47.54, 30.15, 30.15, 18.77.
DEPT 135:
13
C NMR (63 MHz, CDCl3) δ 132.72, 132.30, 132.21, 132.15, 125.82,
112.65, 112.33, 112.27, 111.99, 111.89, 105.26, 104.86, 104.46, 101.94, 67.46, 47.43,
30.03, 18.65. HRMS (ESI) calcd for C31H20F4N4O5 [M+H]+ : 605.1370, found: 605.1443
Synthesis of syn-AK4AZ2:
128 This compound was obtained by following the general synthetic procedure for syntriazoles. Rf = 0.52 (dichloromethane : methanol = 4:1), isolated in 78 % yield. 1H NMR
(250 MHz, CDCl3) δ 7.58 – 7.35 (m, 1H), 6.94 (td, J = 8.0, 2.0, 1H), 6.86 – 6.64 (m, 1H),
6.10 (d, J = 0.9, 0H), 4.68 (t, J = 4.9, 1H), 4.47 (t, J = 5.0, 1H), 3.72 (t, J = 6.3, 1H), 2.83
(t, J = 7.0, 1H), 2.33 (s, 1H), 1.80 (s, 1H). 13C NMR (63 MHz, CDCl3) δ 169.48, 166.28,
166.10, 162.47, 162.25, 162.06, 160.94, 160.69, 158.46, 155.06, 152.35, 133.38, 132.46,
132.39, 132.30, 132.23, 125.82, 114.26, 113.52, 113.31, 112.44, 112.15, 112.09, 111.92,
111.80, 111.75, 105.09, 104.68, 104.28, 101.73, 77.60, 77.30, 77.09, 76.59, 67.18, 46.75,
37.92, 28.05, 25.38, 22.55, 18.61. DEPT135:
13
C NMR (63 MHz, CDCl3) δ 132.45,
132.39, 132.30, 132.23, 125.82, 112.44, 112.15, 112.09, 111.91, 111.81, 111.75, 105.09,
104.69, 104.28, 101.73, 77.30, 67.18, 46.74, 37.92, 28.05, 25.38, 22.55, 18.61. HRMS
(ESI) calcd for C34H26F4N4O5 [M+H]+ : 647.1839, found: 647.18393
Synthesis of syn-AK4AZ3:
This compound was obtained by following the general synthetic procedure for syntriazoles. Rf = 0.56 (dichloromethane : methanol = 4:1), isolated in 79 % yield. 1H NMR
(250 MHz, CDCl3) δ 7.47 (ddt, J = 12.3, 8.3, 4.1, 4H), 6.95 (tt, J = 14.8, 7.4, 2H), 6.87 –
129 6.71 (m, 4H), 6.16 – 6.06 (m, 1H), 4.47 (t, J = 6.8, 2H), 4.02 (t, J = 5.6, 2H), 3.72 – 3.57
(m, 2H), 2.69 (t, J = 7.1, 2H), 2.46 (dd, J = 12.2, 6.2, 2H), 2.39 (d, J = 3.7, 3H), 13C NMR
(63 MHz, CDCl3) δ 169.53, 166.18, 165.97, 162.41, 162.38, 162.20, 161.43, 161.21,
160.40, 160.32, 155.30, 152.55, 136.79, 133.50, 132.54, 132.48, 132.38, 132.31, 132.25,
125.87, 114.09, 113.60, 112.34, 112.14, 111.87, 105.22, 104.82, 104.41, 101.83, 77.67,
77.16, 76.65, 64.78, 44.11, 37.97, 29.53, 28.12, 25.48, 22.52, 18.75. HRMS (ESI) calcd
for C35H28F4N4O5 [M+H]+ : 661.1996, found: 661.2078
Synthesis of syn-AK1AZ3:
This compound was obtained by following the general synthetic procedure for syntriazoles. Rf = 0.45 (dichloromethane : methanol = 4:1), isolated in 80 % yield. 1H NMR
(250 MHz, CDCl3) δ 7.75 (s, 1H), 7.38 (td, J = 8.1, 6.0, 3H), 6.90 (td, J = 8.0, 1.9, 2H),
6.80 – 6.63 (m, 4H), 6.07 (d, J = 1.0, 1H), 4.82 (s, 2H), 4.70 (t, J = 6.7, 2H), 3.99 (t, J =
5.6, 2H), 2.50 – 2.35 (m, 2H), 2.31 (d, J = 0.9, 3H).
13
C NMR (63 MHz, CDCl3) δ
168.80, 168.50, 166.29, 162.43, 162.26, 161.28, 161.11, 158.58, 158.41, 155.15, 152.47,
135.05, 133.65, 133.33, 132.30, 132.16, 132.00, 131.94, 131.75, 131.67, 128.61, 128.42,
130 125.72, 113.95, 112.93, 112.19, 112.11, 111.88, 105.21, 104.80, 104.40, 101.71, 77.61,
77.10, 76.59, 64.82, 44.79, 29.86, 29.66, 18.64. HRMS (ESI) calcd for C32H22F4N4O5
[M+H]+ : 619.153, found: 619.1596
General procedure for the synthesis of 1,4-disubstituted (‘anti’) triazoles:32,33
A suspension of alkyne (1eq) and azide (1eq) in t-BuOH : H2O (2 : 1) was reacted with
30 mol% sodium ascorbate and 10 mol% copper sulfate (CuSO4.5H2O) under inert
atmosphere for 6-12 h at room temperature. The reaction progression was monitored by
TLC and LC/MS. The crude reaction mixture was washed with treated with solution of
Na2CO3 and extracted with dichloromethane. All anti-triazoles were purified by silica gel
chromatography characterized by 1H NMR and 13C NMR and HRM.
Synthesis of anti-AK1AZ2:
This compound was obtained by following the general synthetic procedure for antitriazoles.1,
2
Rf = 0.48 (dichloromethane : methanol = 4:1), isolated in 80 % yield. 1H
NMR (250 MHz, CDCl3) δ 7.84 (s, 1H), 7.47 (ddd, J = 10.8, 8.4, 5.4, 3H), 6.94 (ddd, J =
131 8.9, 7.9, 1.4, 2H), 6.83 – 6.63 (m, 4H), 6.13 (dd, J = 7.0, 0.8, 1H), 4.96 (s, 2H), 4.78 (t, J
= 4.9, 2H), 4.41 (t, J = 5.0, 2H), 2.33 (t, J = 8.8, 3H).
13
C NMR (63 MHz, CDCl3) δ
168.87, 166.38, 166.19, 162.76, 162.59, 162.34, 162.15, 161.02, 160.68, 158.70, 158.51,
155.16, 152.41, 142.64, 133.50, 132.60, 132.54, 132.45, 132.38, 125.87, 124.48, 114.44,
113.65, 113.62, 113.38, 112.61, 112.20, 112.15, 111.86, 111.81, 105.13, 104.73, 104.32,
101.89, 77.67, 77.16, 76.65, 66.72, 49.56, 33.74, 18.71. HRMS (ESI) calcd for
C31H20F4N4O5 [M+H]+ : 605.1370, found: 605.1435
Synthesis of anti-AK1AZ3:
This compound was obtained by following the general synthetic procedure for antitriazoles.1,
2
Rf = 0.46 (dichloromethane : methanol = 4:1), isolated in 85 % yield. 1H
NMR (250 MHz, CDCl3) δ 7.64 (d, J = 12.2, 1H), 7.48 (ddd, J = 8.3, 7.3, 4.3, 3H), 7.04 –
6.87 (m, 2H), 6.79 (dtd, J = 10.9, 4.3, 2.5, 4H), 4.95 (s, 2H), 4.57 (t, J = 6.9, 2H), 4.04 (t,
J = 5.7, 2H), 2.44 (dd, J = 12.4, 6.3, 2H), 2.37 (s, 3H).
13
C NMR (63 MHz, CDCl3) δ
168.89, 166.39, 166.20, 162.77, 162.57, 162.17, 162.09, 161.43, 161.20, 158.70, 158.51,
155.27, 152.54, 142.52, 133.47, 132.55, 132.45, 125.79, 123.59, 114.05, 113.65, 113.39,
112.35, 112.31, 112.22, 112.16, 111.87, 111.82, 105.14, 104.74, 104.33, 101.68, 77.67,
77.16, 76.65, 64.83, 47.17, 33.55, 30.18, 18.88. HRMS (ESI) calcd for C32H22F4N4O5
[M+H]+ : 619.153, found: 619.1596
132 Synthesis of anti-AK4AZ3:
This compound was obtained by following the general synthetic procedure for antitriazoles.1,
2
Rf = 0.55 (dichloromethane : methanol = 4:1), isolated in 78 % yield. 1H
NMR (250 MHz, CDCl3) δ 7.56 – 7.34 (m, 3H), 7.29 (d, J = 13.3, 1H), 6.99 – 6.82 (m,
2H), 6.82 – 6.62 (m, 4H), 6.06 (t, J = 4.3, 1H), 4.48 (t, J = 6.8, 2H), 4.09 – 3.87 (m, 2H),
3.58 (dd, J = 16.5, 10.1, 2H), 2.79 – 2.58 (m, 2H), 2.43 – 2.19 (m, 5H), 1.78 – 1.55 (m,
4H).
13
C NMR (63 MHz, CDCl3) δ 169.53, 166.29, 166.10, 162.82, 162.73, 162.25,
162.07, 161.49, 161.18, 160.83, 160.58, 155.25, 152.54, 147.65, 133.36, 132.56, 132.49,
132.41, 132.33, 125.79, 121.31, 114.01, 113.47, 112.25, 112.15, 112.09, 111.80, 111.74,
105.10, 104.70, 104.29, 101.72, 82.67, 77.67, 77.16, 76.65, 64.86, 46.82, 38.48, 29.81,
28.06, 26.72, 25.12, 18.72. HRMS (ESI) calcd for C35H28F4N4O5 [M+H]+ : 661.1996,
found: 661.2068
Synthesis of anti-AK4AZ2:
133 This compound was obtained by following the general synthetic procedure for antitriazoles.1,
2
Rf = 0.52 (dichloromethane : methanol = 4:1), isolated in 79 % yield. 1H
NMR (250 MHz, CDCl3) δ 7.54 – 7.38 (m, 4H), 6.94 (td, J = 8.3, 2.2, 2H), 6.87 – 6.70
(m, 4H), 6.17 – 6.08 (m, 1H), 4.75 (t, J = 5.0, 2H), 4.41 (t, J = 5.0, 2H), 3.65 (d, J = 6.2,
2H), 2.76 (d, J = 6.5, 2H), 2.43 – 2.27 (m, 3H), 1.74 (d, J = 2.9, 4H). 13C NMR (63 MHz,
CDCl3) δ 169.53, 166.28, 166.11, 162.73, 162.55, 162.25, 162.05, 161.02, 160.79,
158.66, 158.47, 155.15, 152.42, 147.79, 133.36, 132.56, 132.49, 132.40, 132.34, 125.89,
122.02, 114.38, 113.70, 113.46, 112.54, 112.16, 112.09, 111.80, 105.10, 104.69, 104.29,
101.87, 77.67, 77.16, 76.65, 66.92, 49.34, 38.48, 28.08, 26.71, 25.11, 18.69. DEPT 90
13
C NMR (63 MHz, CDCl3) δ 132.49, 132.42, 132.33, 132.26, 125.83, 121.95, 112.51,
112.09, 112.03, 111.74, 111.69, 105.04, 104.64, 104.24, 101.84. HRMS (ESI) calcd for
C34H26F4N4O5 [M+H]+ : 647.1839, found: 647.1909
The following building blocks were obtained from commercially available source: AK4,
AK11, AK12, AK12, AK14, AZ1, AZ4, AZ5, AZ6, AZ7,AZ8.
The following building blocks were reported on a patent submitted by the Mantesch lab
(WO 2009105746 A2 20090827):
Synthesis of AK2
(72%, Rf = 0.32 (Hx:EtOAc, 1:1)) 1H-NMR (400 MHz, CDCl3) : 7.20-7.05 (m, 5H),
3.11 (s, 2H), 2.82 (d, J = 10.8 Hz, 2H), 2.46 (d, J = 6.8 Hz, 2H), 1.96 (t, J =11.2 Hz, 2H),
134 1.60 (s, 3H), 1.58 (d, J = 12.0 Hz, 2H), 1.45-1.43 (m, 1H), 1.33-1.27 (m, 2H) ppm. 13CNMR (100 MHz, CDCl3) : 140.7, 129.3, 128.3, 125.9, 80.4, 74.9, 53.0, 47.9, 43.4, 37.7,
32.3, 3.7 ppm. HRMS (ESI+) for [M+H]+; calculated: 228.1752, found: 228.1741 (error
m/z =4.8 ppm)
Synthesis of AK3
(62%, Rf = 0.25 (Hx:EtOAc, 1:1)) 1H-NMR (400 MHz, CDCl3) : 3.32 (d, J = 2.0 Hz,
2H), 2.03 (s, 3H), 1.77 (d, J = 2.4 Hz, 3H), 1.65-1.55 (m, 12H) ppm.
13
C-NMR (100
MHz, CDCl3) : 78.4, 78.2, 50.7, 42.5, 39.9, 36.6, 30.3, 29.5, 3.6 ppm. HRMS (ESI+) for
[M+H]+; calculated: 204.2752, found: 204.1745(error m/z = 3.4 ppm)
Synthesis of AK5
(32%, Rf = 0.45 (DCM:MeOH, 4:1)) 1H-NMR (400 MHz, CDCl3) : 3.10 (d, J=2.4Hz,
2H), 2.90 (d, J=12 Hz, 2H), 2.46-2.43 (m, 4H), 2.22 (tt, J=12, 3.6Hz, 1H), 2.03(td, J=12,
1.6Hz, 2H), 1.75-1.71(m, 5H), 1.60-1.49(m, 6H), 1.38-1.35(m, 2H) ppm. 13C-NMR (100
MHz, CDCl3) : 80.6, 74.9, 62.8, 52.6, 50.2, 47.5, 27.6, 26.4, 24.9, 3.7 ppm. HRMS
(ESI+) for [M+H]+; calculated: 221.2017, found: 221.2018(error m/z = 0.4 ppm)
135 Synthesis of AK6
(72.2%, Rf = 0.45 (Hx:EtOAc, 1:1)) 1H-NMR (400 MHz, CDCl3) : 4.10-4.03 (m, 2H),
3.12 (d, J = 2.4 Hz, 2H), 2.82 (d, J = 11.2 Hz, 2H), 2.10 (t, J = 10 Hz, 2H), 1.86 (d, J =
10.8 Hz, 2H), 1.75 (s, 3H), 1.72 (d, J = 10.8 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H) ppm. 13CNMR (100 MHz, CDCl3) : 174.9, 80.5, 74.1, 60.1, 51.8, 47.5, 40.7, 28.1, 14.1, 3.4 ppm.
HRMS (ESI+) for [M+H]+; calculated: 210.1494, found: 210.1494 (error m/z = 0 ppm)
Synthesis of AK7
NH
N
N
+
MsO
CH3CN: H2O=9:1
N
K2CO3
(94%) 1H-NMR (400 MHz, CDCl3) : 7.25 (t, J = 7.2 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H),
6.84 (t, J = 7.2 Hz, 2H), 3.26 (s, 2H), 3.22 (s, 4H), 1.82 (s, 3H) ppm.
13
C-NMR (100
MHz, CDCl3) : 151.0, 128.8, 119.5, 115.8, 80.7, 73.7, 51.8, 48.8, 47.1, 3.3 ppm. HRMS
(ESI+) for [M+H]+; calculated: 215.1548, found: 215.1551 (error m/z = 1.4 ppm)
Synthesis of AK8
136 (65%, Rf = 0.45 (Hx:EtOAc, 4:1)) 1H-NMR (400 MHz, CDCl3) : 7.11-7.03 (m, 4H),
3.74 (s, 2H), 3.41 (d, J = 1.2 Hz, 2H), 2.93 (t, J = 5.6 Hz, 2H), 2.79 (t, J = 5.6 Hz, 2H),
1.84 (s, 3H) ppm.
13
C-NMR (100 MHz, CDCl3) : 134.4, 133.6, 128.3, 126.3, 125.8,
125.3, 80.6, 73.9, 54.3, 49.6, 47.0, 29.0, 3.3 ppm. HRMS (ESI+) for [M+H]+; calculated:
186.1282, found: 186.1285 (error m/z = 1.6 ppm)
Synthesis of AK9
(85%, Rf = 0.25 ((Hx:EtOAc, 5:1)) 1H-NMR (two isomers due to the amide function
group, 400 MHz, CDCl3) : 7.13-7.03 (m, 8H), 4.80 (s, 2H), 4.66 (s, 2H), 3.89 (t, J = 7.0
Hz, 2H), 3.75 (t, J = 7.0 Hz, 2H), 2.83 (t, J = 7.0 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H), 1.96
(s, 6H) ppm. 13C-NMR (two isomers due to the amide function group, 100 MHz, CDCl3)
:153.7, 153.4, 134.4, 133.8, 132.4, 132.2, 129.4, 128.8, 128.5, 126.8, 126.5, 126.4,
126.3, 125.9, 89.9, 89.1, 73.2, 73.0, 48.4, 44.4, 43.7, 39.3, 29.4, 28.2, 4.0 ppm. HRMS
(ESI+) for [M+H]+; calculated: 200.1075, found: 200.1072 (error m/z = 1.5 ppm)
Synthesis of AK10 (SA16)
(63%, Rf = 0.65 ((Hx:EtOAc, 3:1)). 1H-NMR (400 MHz, CDCl3) : 7.53-7.41 (m, 6H),
7.13-7.09 (m, 2H), 5.20 (s, 2H), 1.97 (s, 3H) ppm. 13C-NMR (100 MHz, CDCl3) :163.8,
137 161.3, 153.5, 140.7, 136.7, 134.0, 129.0, 128.7, 128.6, 127.2, 115.7, 115.5, 86.1, 72.2,
67.0, 3.7 ppm. HRMS (ESI+) for [M+H]+; calculated: 286.1238, found: 286.1246 (error
m/z = 2.8 ppm)
Synthesis of AK13 (SA5)
1
H-NMR (400 MHz, CDCl3) : 4.19-4.08 (m, 9H), 3.37 (s, 2H), 3.15 (s, 2H), 2.22 (s,
3H), 1.85 (s, 3H) ppm. 13C-NMR (100 MHz, CDCl3) : 83.2, 80.6, 74.1, 69.9, 68.4, 68.0,
55.2, 44.9, 41.4, 3.5 ppm. HRMS (ESI+) for [M+H]+; calculated: 282.0945, found:
282.0953 (error m/z = 2.8 ppm)
Synthesis of AK15
1
H-NMR (400 MHz, CDCl3) : 7.30-7.22 (m, 5H), 6.58(s, 1H), 6.09 (s, 1H), 4.73 (s, 1H),
3.83 (s, 3H), 3.54 (s, 3H), 3.34 (dd, J= 17.2, 2.4Hz, 1H), 3.26-3.15(m, 2H), 3.10-3.05 (m,
1H), 2.94 (td, J= 11.2, 3.2 Hz, 1H), 2.73 (d, J=12Hz, 1H), 2.24 (t, J=1.8 Hz, 1H) ppm.
13
C-NMR (100 MHz, CDCl3) : 147.3, 147.0, 143.1, 130.3, 129.5, 128.4, 127.5, 126.4,
111.5, 110.6, 78.4, 73.4, 65.7, 55.7, 48.9, 43.9, 29.0 ppm. HRMS (ESI+) for [M+H]+;
calculated: 308.1650, found: 308.1654 (error m/z = 1.3ppm)
138 3.5
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143 Chapter 4
Parallel Sulfo-Click Kinetic TGS Screening of Multiple Protein-Protein Interactions
4.1
Overview
The proteins from the Bcl-2 family consist of both anti-appoptotic (Bcl-2, Bcl-XL,
and Mcl-1) and proappoptotic members (Bax and Bak, and BH3-only proteins), and they
play a central role in the regualtion of normal cellular homeostasis.1 The anti-apoptotic
Bcl-2 family proteins, in particular Bcl-2, Bcl-XL and Mcl-1, were validated as promising
targets for the development of anticancer agents.1-2 Overexpressed in in some types of
cancer cells, they are considered to be responsible for tumor initiation, tumor progression,
and drug resistance.1, 2d Although there is a number of small molecules that have been
developed to disrupt the interactions between pro-apoptotic proteins and Bcl-2/Bcl-XL,
only few compounds are known to interact with Mcl-1.3 Interestingly enough, recent
studies demonstrated that Mcl-1 also contributes to cancer cell proliferation, and that Bcl2, Bcl-XL and Mcl-1 must be simultaneously targeted for apoptosis induction in most
types of cancers.4 The potent inhibitors designed by Abbot Laboratories, ABT-737 (Ki =
0.6 nM), and its orally active analog ABT-263, exhibit selectivity only to Bcl-2, Bcl-XL
and Bcl-w, but do not target Mcl-1 (Ki >1000 nM for ABT-737). Therefore, these
pharmacophores are not effective against cancer cells with up-regulated Mcl-1.5 It has
been demonstrated in a non-Hodgkin’s lymphoma model that resistance toward ABT-737
was acquired after three weeks of treatment, resulting in an up-regulation of Mcl-1.6 This
145
observation clearly indicated that a new treatment approach combining ABT-737 and a
Mcl-1-specific inhibitor may be needed to overcome the resistance and reactivate
apoptosis in cancer cells. Consequently, recent reports revealed that the efficiency of
ABT-263 and ABT-737 in cancer cells was restored by down-regulating Mcl-1 in the
cells.7 Cumulatively, these results evidenced the existing need to develop small
molecules that regulate the interaction between Mcl-1 and the BH3-only proteins, and to
selectively target tumors with elevated Mcl-1 in the cells. For example, among the BH3only proteins, Noxa binds to Mcl-1 but not to Bcl-2 and Bcl-XL, thus providing a way to
selectively target this PPI leading to apoptosis.4a In addition, the design of small
molecules, that simultaneously target the interaction of multiple members of the Bcl-2
family (Bcl-2, Bcl-XL and Mcl-1) and BH3-only proteins, is another appealing approach.
Figure 4.1. Protein-protein interaction modulators targeting: A) multiple members of the
Bcl-2 family and B) Mcl-1 selectively.
146
Examples of reported structurally diverse PPIMs targeting multiple members of the Bcl-2
family or Mcl-1 selectively, are presented in Figure 4.1.8
In an ongoing study of the Manetsch laboratory, several structurally diverse
acylsulfonamides were identified as selective Mcl-1 inhibitors by using the sulfo-click
kinetic TGS approach (unpublished data S. Kulkarni). A library of ten thio acids and
thirty one sulfonyl azides (310 possible fragment combinations) was incubated as binary
mixtures against Mcl-1, and the samples were analyzed using liquid chromatography in
combination with mass spectrometry equipped with electrospray ionization in the positive
selected ion mode (LC/MS-SIM). The detected hit combinations were subsequently
validated through control experiments, synthesized and tested for activity against Mcl-1.
The obtained data revealed that these sulfonamides displayed low micromolar IC50s and
approximately 3- to 7-fold less potency against Bcl-XL, in addition to a respectable ligand
efficiency (0.16-.018). It is noteworthy mentioning that in addition to the Abbot
laboratories there is only one literature reported by Pellecchia et al.,8c in which an
acylsulfonamide-derivative was discovered with nanomolar binding affinitiy against BclXL and Mcl-1. Utilizing SAR by ILOE (structure activity relationship by interligand
nuclear Overhauser effect), acylsulfonamide 4.4 was generated which binds to Bcl-xL
and Mcl-1 with IC50 values of 86 nM and 140 nM, respectively. It was demonstrated that
through SAR by ILOE, an efficient structural optimization of low-affinity fragments was
achieved which resulted in 175-fold improvement in potency to Bcl-xL and 181-fold
improvement in potency to Mcl-1 over an original compound. These encouraging results
set the stage for further investigation of the acylsulfonamides in drug discovery and
development.
147
In the last several years the development of potent protein-protein modulators
(PPIMs) relied primarily on conventional fragment-based lead discovery approaches.8 In
the Manetsch laboratory, it was demonstrated for the first time that the efficient and
straightforward approach of kinetic target-guided synthesis (TGS) can be successfully
extended for the identification of high-quality protein-protein interaction modulators.
Thus, in the two related studies, the kinetic TGS approach based on a sulfo-click reaction
between sulfonyl azides and thio acids was validated as a screening platform, targeting
Bcl-XL-protein interactions.9 A set of 9 thio acids and 9 sulfonyl azides was incubated as
binary mixtures at 37 °C for six hours in the presence and absence of Bcl-XL, and the
resulting asylsulfonamide was identified by liquid chromatography combined with mass
spectrometry detection in the selected ion mode (LC/MS-SIM).
Figure 4.2. Kinetic TGS approach using the sulfo-click reaction to identify inhibitors of
Bcl-XL
148
The comparison of the obtained traces led to the discovery of three new hit
combinations SZ7TA2, SZ9TA1, and SZ9TA5, in addition to SZ4TA2, which was
previously reported by Abbot Laboratories (Figure 4.2). It is worth mentioning that the
report showed the acylsulfonamides assembled by the protein were the more potent
combinations out of all other potential products from the library, which were not
identified as TGS hits. This was proven in an experiment wherein the hit compounds
SZ7TA2, SZ9TA1, and SZ9TA5, along with 33 randomly selected acylsulfonamides,
were synthesized and subsequently tested in order to assess their ability to modulate the
Bcl-XL/BH3 interactions. Strikingly, the 4 kinetic TGS hits were the most potent
compounds tested, disrupting the Bcl-XL/BH3 interaction by 60% or more, while the
randomly selected acylsulfonamides showed an average activity of 15%. The unique
properties of this efficient and selective TGS technique are derived from a simple
strategy such as to leave the entire last step of assembling and synthesis of PPIMs to be
performed by the protein. Unlike the conventional fragment-based approaches, the kinetic
TGS significantly lowers the time and resource investments as it eliminates the synthesis
of compounds which may not be active in subsequent confirmatory studies. Accumulated
results suggested that the PPIM kinetic TGS screening generates mainly the active
compounds and results in a low number of false positives.
A theoretical advantage of the kinetic TGS is the option to perform the proteintemplated incubation with a large number of fragments in a single well.9a In an in situ
click chemistry study, it was demonstrated that a multicomponent screening was feasible
with one thio acid and six sulfonyl azides incubated together along with the enzyme of
interest, AChE.10 The LC/MS-SIM analysis revealed that the enzyme was able to select
149
and assemble its best inhibitors, even in the complex mixtures of several azide and
acetylene building blocks, although a lower sensitivity was observed in comparison to the
corresponding binary reactions. In a similar manner, multicomponent HIV-1 protease
incubations comprised of one azide and five different alkynes have proven to be effective
in the delivery of HIV-1 inhibitory compounds.11
Despite the successful reports with enzymatic targets, a multi-fragment screening
is perceived to be more challenging when applied to protein-protein interaction targets.
Although, the recent report by Hu and co-workers presented evidence that incubation of
one thio acid and six sulfonyl azides permitted a successful detection of the known BclXL hit combination SZ4TA2 by using LC/MS-SIM screening approach, the attempt to
perform the experiment with all nine building blocks incubated at the same time (three
thio acids and six sulfonyl azides) failed to provide definite results.9a Nevertheless, the
obstacles due to the instrumental limitations could be overcome by a screening using a
more advanced mass spectrometry technology. For example, the triple quadrupole mass
spectrometry (TQMS) is a proven technique with numerous advantages offering superior
sensitivity over the single quadrupole instrumentation. TQMS offers a better signal-tonoise ratio (S/N), resulting in a significantly lower limit of detection and quantitation.
Moreover, the analysis with this highly sensitive technology allows for a simultaneous
detection of several targeted reactions by using a Multiple Reaction Monitoring (MRM)
mode as opposed to Selected Ion Mode (SIM). In comparison to the full-mass scan
(Figure 4.3A), the SIM mode (Figure 4.3B) provides higher sensitivity due to a longer
instrument dwells time on a narrower mass range with a pre-set m/z ratio of a precursor
ion. However, in the MRM experiment (Figure 4.3C), in addition to the pre-set precursor
150
ion, a unique fragment ion is delivered which can be easily monitored to provide
quantified analysis of more complex mixtures. Moreover, unlike the MRM mode, the
SIM mode can not be used to analyze reaction mixtures of compounds which have the
same m/z ratio. The MRM mode can easily distinguish between the same m/z ratio of
precursor ion as they would generate different fragment ions, distinct for each compound.
Figure 4.3. Schematic representation of mass spectrometry analysis with different single
and triple quadrupole scanning modes: A) a full scan with MS, B) Selected Ion Mode
(SIM), C) Multiple Reaction Monitoring (MRM).
151
The high reproducibility and better accuracy obtained even at low analyte concentrations
are other beneficial features of TQMS in the MRM scan mode.
Conceivably, employing the sulfo-click multi-fragment screening approach,
coupled with triple quadrupole mass spectrometry, is an opportunity to immensely
improve the throughput and to take the kinetic TGS to its next level of efficiency and
productivity. Such a multi-component screening strategy has the potential to drastically
increase the number of screened reactions, to reduce the time used for preparation of a
single sulfo-click reaction, and to also cut down the amount of the consumed proteins and
reagents. By using a triple-quadrupole mass spectrometer, the Manetsch laboratory
demonstrated that acylsulfonamide products can be reliably detected in incubation
samples containing 200 or more fragment combinations in one single well. The
simultaneous incubation of 200 fragment combinations (a) increases the screening time
efficiency by about ~150-times, (b) ensures a 15- to 20-fold improvement of
sample/reagents economy; c) cuts down the time required for the preparation of each
incubation reaction by about 200-times; d) ensures better hit detectability by utilizing the
enhanced multi-reaction-monitoring mode of sample detections.
Herein, we present our findings towards optimization of the screening process of
multiple PPIs via sulfo-click kinetinc TGS utilizing a triple-quadrupole mass
spectrometer. Furthermore, we report results obtained from the screening of an expanded
library of about 1,700 fragment combinations against the two targets BcL-XL, and Mcl-1,
and the identification of several acylsulfonamides as potential PPIMs.
152
4.2
Results and Discussion
Inspired by the successfully implemented multi-component screening (7
fragments in one well) in the identification of a known Bcl-XL inhibitor and the
encouraging results from the recently discovered PPIMs against Mcl-1, we aimed: (a) to
optimize the throughput of the sulfo-click kinetinc TGS reaction by implementing a
multi-fragement screening technique with a TQMS, (b) to probe a larger chemical space,
and to expand the existing library of 310 fragment combinations to 1710 fragmentcombinations, and (c) to seek for new PPIMs against Bcl-XL and Mcl-1 by implementing
the optimized experimental conditions to the newly developed library.
4.2.1
Library of Thio Acids and Sulfonylazides Used in a Multi-Componet Kinetic
TGS Screening
The library used in our initial experiments contains a structurally diverse set of
thirty eight sulfonyl azides (SZ1-SZ38) and ten thio acids (TA1-TA10) (Figure 4.4 A-B).
Figure 4.4 A. Library of thio acids TA1-TA10 used for the initial proof-of-concept study
of multi-fragment screening via sulfo-click kinetinc TGS
153
The ten thio acids (TA1-TA10) and thirty one of the sulfonyl azides (SZ1-SZ31) were
previously utilized in the kinetic TGS screening against Mcl-1 (unpublished results S.
Kulkarni), whereas additional seven sulfonyl azides (SZ32-SZ38) were synthesized to
further increase the number of fragment combinations for the initial multi-component
kinetic TGS proof-of-concept studies.
Ph
O O
S
N3
N
N
O O
S
N3
SZ1
Ph
S
O O
S
O O
S
N
H
SZ7
Ph
S
SZ4
SZ5
Ph
O O
S
S
O O
S
N3
N3
N
Ph
SZ8
O O
S
N3
O O
S
N3
O O
S
N3
N3
N
H
N
O
O O
S
O2N
O O
S
N3
O
N3
SZ6
O O
S
H2N
N
H SZ3
SZ2
O O Ph
S
S
N3
H
N
O
N
Ph
O O
S
N3 Ph
S
O O
S
N3
H
N
SZ9
O O
S
N3
NO2
O
N
SZ10
SZ11
O O
S
N3
O
Ph
O
Ph
F
S
O O
S
N3
Cl
O O
S
N3
O
Ph
O O
S
N3
N
S
SZ16
SZ17
O O
S
N3
N
N
O O
S
N3
O O
S
N3
N
O
SZ18
SZ19
O O
S
N3
N
Ph
SZ20
O
N
O O
S
N3
O
O O
S
N3
SZ35
S
O
N
Ph
SZ36
O O
S
N3
O
SZ32
O O
S
N3
NH
O
SZ28
O O
S
N3
SZ31
O O
S
N3
O O
S
N3
S
Ph
N
SZ27
N
SZ30
O
O
O O
S
N3
O
N
SZ29
N
SZ23
O O
S
N3
O
O
N
H
SZ34
Ph
O O
S
N3
N
H
Br
SZ22
O O
S
N3
Ph
SZ26
SZ25
SZ24
N
SZ21
O O Ph
S
N3 N
N
O
O
F3C
SZ15
O O
S
N3
SZ13
O O
S
N3
O O
S
N3
N
SZ14
O
SZ12
NO2
Cl
O
SZ33
O O
S
N3
S
SZ37
O O
S
N3
S
O O
SZ38
Figure 4.4 B. Library of sulfonyl azides SZ1-SZ38 used for the initial proof-of-concept
study of multi-fragmen screening via sulfo-click kinetic TGS
154
The sulfonylazides were obtained through one of the three general synthetic routes: (a)
reacting the corresponding sulfonamides with triflyl azide (TfN3)12,
13
, (b) alkylating
amines with 4-(bromomethyl)benzenesulfonyl azide8b, and (c) alkylating through an in
situ generated 2-chloroethanesulfonyl azide (Figure 4.5 A-C).14 The reported obstacles
associated to the challenges of isolation and low stability of the thio acids were easily
overcome by the use of the improved synthetic route using fluorenylmethyl protected thio
acids, which were rapidly interconverted into the corresponding thio acid prior to the
kinetic TGS (Figure 4.5 D).14
Figure 4.5. Synthetic approaches to generate sulfonyl azides: a) K2CO3, KI (cat.),
CH3CN:H2O (9:1), RT, 3 h; (b) K2CO3, CH3CN:H2O (9:1), RT, 12 h; c) aq. CuSO4, Et3N,
H2O, CH2Cl2/MeOH, RT; d) EDCI, DMAP, DCM, RT; e) 3.5% DBU, DMF, RT, 2min
155
The carboxylic acid containing fragments were first reacted with 9-fluorenyl
methanethiol to yield corresponding thioesters, which were then deprotected using 5%
piperidine in DMF (Figure 4.4 D). Conveniently, the thio acids generated in the
deprotection step were further utilized as methanolic solutions in the kinetic TGS
screening without further purification.14
4.2.2
Optimization of Multi-Fragment Sulfo-Click Kinetic TGS Sreening with
Triple Quadrupole Mass Sspectrometry Detection
For quantitative analysis, the improved sensitivity offered by the triple quadrupole mass
technology is further enhanced by utilizing the instrument in the multiple reaction
monitoring mode (MRM).15 The MRM mode significantly improves the quality of the
detected signal by serving as a ‘double filter’ which allows only a pre-set precursor ion
with a specified product ion to pass through the three quadrupoles ultimately reaching the
mass analyzer (Figure 4.6).16 A quadrupole mass analyzer is comprised of four parallel
rods in which fixed DC and alternating RF potentials are applied. The first filtering
process is conducted within the first quadrupole wherein only a precursor ion with a preset mass to charge ratio (m/z) has a stable trajectory reaching the second quadrupole
(Q2). In the second quadrupole, the precursor ion is collisionally fragmented into a
characteristic product ion, which passes the third quadrupole (Q3) and reaches the mass
analyzer, only if its m/z ratio corresponds to the specifically defined m/z settings of the
third quadrupole. All other product ions that do not resonate with the specific DC/RF
voltage combination will be filtered out in the third quadrupole. Thus, this advanced
engineering design coupled with an MRM screening mode reduces the noise level, and
increases the selectivity and sensitivity of the analysis.
156
Figure 4.6. Schematic representation of Multiple Reaction Monitoring (MRM) mode of
analysis performed with a triple quadrupole mass spectrometer
As described above, utilizing the MRM method requires knowledge of not only
the m/z ratio of the precursor ion of interest but also that of the corresponding product ion
generated at particular collision energy (eV). In order to correlate the collision energies to
fragmentation pathways, a direct infusion experiment was performed in which a
methanolic solution of an acylsulfonamide was injected into the collision cell, providing
information of the fragmentation patterns at corresponding collision energies (example of
SZ8TA4 is presented on Figure 4.7). From this particular experiment and the several
others performed with various acylsulfonamides (SZ1TA3, SZ2TA2, SZ7TA2,
SZ2TA4, SZ9TA7, SZ7TA7, SZ6TA7, SZ9TA1, and SZ8TA8), it was concluded that
among all tested acylsulfonamides there was a common similarity in the collisionactivated breaking of the acylsulfonamide linkage. It was observed that the acylium ion
was present in all investigated acylsulfonamides, and that this particular product ion was
often the most abundant species among all other fragments.
157
Figure 4.7. Collision-activated defragmentation curve of acylsulfonamide SZ8TA4.
Note, of all possible fragmentation pathways, the one leading to an acylium ion has the
highest relative ion abundance and requires the least collision energy (among the most
abundant ions). The relative abundance of each ion represents the number of times an ion
of that m/z ratio strikes the detector.
The intensity of each defragmentation curve corresponds to the abundance of a particular
product ion at that collision energy, relative to the amount of the rest of fragments
generated at the defragmentation process. The relative abundance of each ion is
determined by the number of times an ion of particular m/z ratio strikes the detector. The
average collision energy corresponding to the formation of the acylium ion for all the
tested acylsulfonamides was determined to be between 30 – 35 V (Table 4.1), which was
158
extrapolated to the rest of the entire acylsulfonamide library which were not tested
individually.
Table 4.1. Collision energy (eV) at which the corresponding acylium ion is generated
through the defragmentation process
Acylsulfonamide
Collision energy (eV) required to
generate the acylium ion
SZ1TA3
29
SZ2TA2
26
SZ7TA2
28
SZ2TA4
22
SZ8TA4
26
SZ9TA7
37
SZ7TA7
31
SZ6TA7
31
SZ9TA1
48
SZ8TA8
36
The fragmentation pathway leading to the acylium ion was advantageous to establish a
practical way to set up and analyze multi-component incubation samples. The instrument
sensitivity and the data analysis of multi-component kinetic TGS screening samples
containing a smaller number of thio acid fragments and a larger number of sulfonyl
azides are better and easier in comparison to an incubation sample containing a larger
159
number of thio acid fragments and a smaller number of sulfonyl azides. If several
acylsulfonamides generated from the same thio acid will be kept in the same kinetic TGS
screening sample, they all commonly generate the identical acylium ion as the product
ion. In contrast, an incubation sample containing acylsulfonamides structurally differing
in their carbonyl moieties, MRM will generate a multitude of acylium ions, which
decreases the overall sensitivity of the instrument and increases the complexity of the raw
data analysis.
The feasibility of kinetic TGS using a MRM-screening mode with a TQMS was
investigated for the screening of the previously reported library of nine sulfonyl azides
(SZ1-SZ9) and nine thio acids (TA1-TA9) containing four known Bcl-XL inhibitory
compounds (highlighted in a green color) (Figure 4.8).9b For optimization purposes, the
entire fragment library was arranged in various screening batches, which differ in their
total number of possible acylsulfonamides but contain at least one of the kinetic TGS hit
acylsulfonamides.
M+1
TA1
TA2
TA3
TA4
TA5
TA6
TA7
TA8
TA9
SZ1
436.16
547.27
533.16
641.27
496.18
481.15
486.18
520.14
426.14
SZ2
409.15
520.26
506.15
614.26
469.17
454.14
459.17
493.13
399.13
SZ3
319.07
430.17
416.07
524.18
379.09
364.05
369.08
403.05
309.05
SZ4
458.08
569.18
555.08
663.19
518.10
503.06
508.09
542.06
448.06
SZ5
276.06
387.17
373.06
481.17
336.08
321.05
326.08
360.04
266.04
SZ6
427.11
538.21
524.11
632.22
487.13
472.09
477.12
511.09
417.09
SZ7
527.07
638.17
624.07
732.18
587.09
572.05
577.08
611.05
517.05
SZ8
543.19
654.29
640.19
748.30
603.21
588.17
593.20
627.17
533.17
SZ9
622.12
733.22
719.12
827.23
682.14
667.10
672.13
706.10
612.10
Figure 4.8. Library of nine sulfonyl azides (SZ1-SZ9) and nine thio acids (TA1-TA9)
used in a proof-of-concept study of multi-fragment kinetic TGS screening. The numbers
on the table corresponds to the m/z of the parent ions. The compounds marked in green
represent previously identified acylsufonamide inhibitors against Bcl-XL9b
160
The Bcl-XL incubations conditions used for the screening of the binary mixtures were
kept unchanged at this multi-fragment approach (2 µM concentration of Bcl-XL, 20 µM
sulfonyl azide, 20 µM thio acids, and 37 °C for six hours).9b
Starting with 9 fragment combinations in one well, we quickly progressed to 45
fragment combinations per well without sacrificing the high quality of the LC/MSMS
traces and the detectability of the previously identified kinetic TGS hit combinations.
Each kinetic TGS hit combination was detected in the multiple-fragment screening and
was further validated by comparing its peak retention time with that of the corresponding
authentic sample. The pre-arranged blocks of multi-fragment combinations of thio acids
and sylfonyl azides are presented in Table 4.2.
Table 4.2. Fragment distribution in the initial studies of multi-fragment kinetic TGS
screening
Number of possible
Fragments combined
fragment combinations
TA x SZ
9
one TA x SZ1-SZ9
15
TA2-TA4 x SZ1-SZ9
18
TA1-TA2 x SZ1-SZ9
27
TA2-TA4 x SZ1-SZ9
36
TA2-TA5 x SZ1-SZ9
45
TA1-TA5 x SZ1-SZ9
81
TA1-TA9 x SZ1-SZ9
161
Encouraged by these results we incubated and analyzed the entire library of eighteen
fragments (eighty one possible sulfonylazides). Importantly, using the enhanced MRMscreening mode with a TQMS, simultaneous monitoring of all 81 potential combinations
was conducted in a single kinetic TGS incubation reaction. Strikingly, the comparison of
the TQMS-MRM data for samples in the presence and absence of Bcl-XL revealed that
the four previously identified kinetic TGS hit acylsulfonamides were detected even when
they were all present together in the same incubation mixture. Thus, the number of the
incubated samples to be analyzed by LC/MS-SIM was drastically reduced from 162 in a
binary set-up (81 incubation mixtures with Bcl-XL and 81 incubation mixtures with buffer
only) down to only 2 wells (1 incubation mixture with Bcl-XL and 1 incubation mixture
with buffer only). Furthermore, the time used for the screening and identification of the
active acylsulfonamides and the required amount of protein/reagents was all together
significantly reduced. The LC/MSMS traces of the four previously identified kinetic TGS
hit acylsulfonamides identified in a multi-component screening containing 45 fragment
combinations (Figure and 4.9A-B) or 81 fragment combinations (Figure 4.10A-B) are
presented below.
162
Figure 4.9.A. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5 using multi-fragment kinetic
TGS screening with a TQMS. Incubation of five thio acids and nine sulfonyl azides
together in one well (45 possible acylsulfonamides) with buffer
163
Figure 4.9.B. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5 using multi-fragment kinetic
TGS screening with a TQMS. Incubation of five thio acids and nine sulfonyl azides
together in one well (45 possible acylsulfonamides) with Bcl-XL. The values of the
amplification coefficients are marked on each LC/MSMS trace. The amplification
coefficients is calculated as the peak area of the product in the enzymatic reaction is
divided by the peak area of the corresponding product in the background (buffer) reaction
164
TA1,2,3,4,5,67,8,9_SZ1-9_Buff_10ulinj
9/5/2011 11:38:16 AM
RT: 0.00 - 15.01
Intensity
RT: 8.30
MA: 2043
SZ9TA1
Buffer
400
200
0.91
0
2.01
3.59 4.37
6.13 6.98
8.84 9.72
RT: 8.17
AA: 101627
Intensity
30000
20000
10000
12.45
13.62
SZ4TA2
Buffer
NL: 3.52E4
TIC F: + c ESI SRM ms2
569.000 [215.750-216.250]
MS ICIS
TA1,2,3,4,5,67,8,9_SZ19_Buff_10ulinj
SZ7TA2
Buffer
NL: 7.37E4
TIC F: + c ESI SRM ms2
638.000 [215.750-216.250]
MS ICIS
TA1,2,3,4,5,67,8,9_SZ19_Buff_10ulinj
SZ9TA5
Buffer
NL: 4.61E3
TIC F: + c ESI SRM ms2
682.000 [136.650-137.150]
MS ICIS
TA1,2,3,4,5,67,8,9_SZ19_Buff_10ulinj
RT: 7.93
AA: 252016
60000
Intensity
11.20
RT: 9.15
AA: 1376
0
40000
20000
RT: 9.81
AA: 4750
0
RT: 8.46
AA: 11109
4000
Intensity
NL: 5.60E2
TIC F: + c ESI SRM ms2
622.000 [108.550-109.050]
MS
TA1,2,3,4,5,67,8,9_SZ19_Buff_10ulinj
3000
2000
1000
0
0
2
4
6
8
Time (min)
10
12
14
Figure 4.10.A. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5 using multi-fragment kinetic
TGS screening with a TQMS. Incubation of 19 fragments together in one well (81
possible acylsulfonamides) with buffer
165
TA1,2,3,4,5,67,8,9_SZ1-9_BXL_10ulinj
9/5/2011 11:54:37 AM
RT: 0.00 - 15.01
NL: 6.22E3
TIC F: + c ESI SRM ms2
622.000 [108.550-109.050]
MS
TA1,2,3,4,5,67,8,9_SZ19_BXL_10ulinj
RT: 8.30
MA: 17936
Intensity
6000
SZ9TA1
x 8.7
4000
2000
Intensity
0
SZ4TA2
x 16.9
400000
200000
RT: 10.94
AA: 8118
0
Intensity
NL: 7.18E5
TIC F: + c ESI SRM ms2
638.000 [215.750-216.250]
MS ICIS
TA1,2,3,4,5,67,8,9_SZ19_BXL_10ulinj
RT: 7.93
AA: 2535448
600000
SZ7TA2
x 10
400000
200000
RT: 9.25
AA: 13292
RT: 8.46
AA: 110920
0
30000
Intensity
NL: 5.54E5
TIC F: + c ESI SRM ms2
569.000 [215.750-216.250]
MS ICIS
TA1,2,3,4,5,67,8,9_SZ19_BXL_10ulinj
RT: 8.17
AA: 1721082
NL: 3.79E4
TIC F: + c ESI SRM ms2
682.000 [136.650-137.150]
MS ICIS
TA1,2,3,4,5,67,8,9_SZ19_BXL_10ulinj
SZ9TA5
x 9.9
20000
10000
0
0
2
4
6
8
Time (min)
10
12
14
Figure 4.10.B. Identification of the four previously identified kinetic TGS hit
combinations SZ9TA1, SZ4TA2, SZ7TA2, and SZ9TA5 using multi-fragment kinetic
TGS screening with a TQMS. Incubation of 19 fragments together in one well
(81possible acylsulfonamides) with Bcl-XL. The values of the amplification coefficients
are marked on each LC/MSMS trace. The amplification coefficients is calculated as the
peak area of the product in the enzymatic reaction is divided by the peak area of the
corresponding product in the background (buffer) reaction
166
Next, to ensure a good reproducibility and an acceptable signal-to-noise ratio, the
screening of the library consisting of 38 SZs and 10 TAs (Figure 4.1A-B) was performed
in order to determine whether incubation mixtures containing more than 81 fragment
combinations in one well could be reliably tested. Thus, 38 SZs and 5 TAs (190 possible
acylsulfonamides) were incubated together in one well at 37 oC for 6 hours in in presence
and absence of Bcl-XL. The subsequent analysis of the obtained LC/MSMS data revealed
that an overall decrease in the peak intensities in the background reaction was observed
when the incubations were held only for 6 hours. This in turn lead to underestimations of
the extent of the acylsuflonamide formation in the background reactions and thus
overestimations of the templation coefficient leading to false positives. In order to gain a
better understanding of the progression of the uncatalyzed as well as the enzymatic
reaction, we designed a kinetics study where the same incubation mixture was analyzed
with TQMS at 4 hours, 8 hours, 12 hours, and 24 hours. The comparison of the peak
areas of the acylsulfonamide products for the monitored reactions in the incubation
mixture revealed a steady increase in the peak area until 10 – 12 hours of incubation and
an improved characterization of the background reaction. Based on this experimental
data, the incubation time for multi-component screening of 18 and more fragments in a
well was extended from 6 hours to 12 hours. By using these incubation conditions the
previously identified hit compounds SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5 were
detected in incubation of 38 SZs and 5 TAs in a well (190 possible acylsulfonamides)
with good signal-to-noise ratio. However when 310 fragment combinations were
incubated in a well (10 TAs and 31 SZs), a lower quality of the LC/MSMS traces was
observed with an increased noise level, possibly related to the reduced instrument dwell
167
time allotted for each of the simultaneously monitored transitions (310 reactions
monitored simulteniously). Although the previously identified hit compounds were
detectable even at 310 fragment combinations in a well, these conditions were considered
to be suboptimal due to the lower sensitivity and reproducibility. Also, testing of 310
templated reactions simultaneously approaches the full capacity of the instrument, which
can monitor maximally 340 different masses in the MRM mode. Thus it was concluded
that 190 fragment combinations in a well, comprised of five thio acids and thirty eight
sulfonyl azides, was a manageable number of simultaneously monitored reactions which
provides acceptable signal-to-noise ratios and good quality LCMSMS traces.
Subsequently, the entire library of 1700 possible acylsulfonamides was screened as 190
fragment combinations in a well, distributed as five thio acids (5 TAs) and thirty eight
sulfonyl azides (38 SZs) incubated together with the target protein.
4.2.3
Expansion of the Thio Acid Library and Multi-Component Kinetic TGS
against Mcl-1 and Bcl-XL
In order to probe larger chemical space through the optimized multi-component
kinetic TGS approach, we decided to expand the existing library of thio acids by
introducing several new and structurally more diverse reactive fragments. The first step
in the design and selection of the thio acid scaffolds was achieved by performing an
exhaustive literature search for reported examples of PPIMs against various protein
targets. Therefore, the thio acid fragment design was based on structural moieties
implemented in PPIM designs for a variety of targets such as p53/MDM2, IL-2/IL2Ralpha, and others.18-20 Structural motifs of previously reported PPIMs were
incorporated in our thio acid fragment library, given that a synthetic route showed it to be
168
plausible and less than 6 consecutive steps. Structurally, the new thio acids in the
fragment library were comprised of indole or bi-indole systems, an array of bi-phenyl
rings linked with alkyl chains, N- and O-heterocyclic systems, and others (Figure 4.11).
These molecules were derivatized with a carboxylic acid functionality which was
conveniently interconverted into the corresponding fluorenylmethyl thioesters. The
thioesters were isolated in relatively good yields and rapidly deprotected with 5%
piperidine prior to the kinetic TGS.
Figure 4.11. Library of forty five thio acids (TA1 – TA45) used for multi-component
kinetic TGS screening against Bcl-XL and Mcl-1
169
The entire library of fragments (38 sulfonyl azides and 45 thio acids yielding 1710
possible combinations) was incubated as multi-component mixtures against both Bcl-XL
and Mcl-1 (10 μM concentration of each, at 37 °C, for twelve hours). The multicomponent incubations of the 38 sulfonyl azides and 45 thio acids were performed by
combining 5 thio acids and 38 sulfonly azides in one well (20 μM final concentration of
each fragment; 190 possible fragment combinations). The entire library was divided into
9 individual screening blocks, each containing 5 thio acids and 38 sulfonly azides in a
well (Figure 4.12). In addition, these multi-component mixtures were incubated in a
phosphate buffer in the absence of Mcl-1 or Bcl-XL. Subsequently, the samples were
analyzed using liquid TQMS in the enhanced MRM detection mode, and comparison of
the obtained chromatograms in the presence and absence of Mcl-1 or Bcl-XL revealed
that about 150 potential hit combinations were identified all together for both targets.
Figure 4.12 Multi-fragment kinetic TGS screening of 38 sulfonyl azides (38 SZ) and 45
thio acids (45 TA) giving rise to 1710 possible fragment combinations. The entire library
has been divided in 9 incubation mixtures, each containing 5 thio acids and 38 sulfonayl
azides
170
In order to validate the screening and also sort out only the most promising and consistent
kinetic TGS hit combinations, two follow-up experiments were conducted wherein
analyses were performed with two different screening patterns with a reduced number of
fragments in a well. Thus, the entire library was analyzed into two trails. The first one
was performed in incubation samples containing 38 sulfonyl azides and two or three thio
acids (38 SZs x 2 TAs and 38 SZs x 2 TAs), and the second was conducted between one
single thio acid and all thirty eight sulfonyl azides (38 SZs x 1 TA) in a well. An
additional layer of a control testing was carried out by substituting Bcl-XL with a mutant
R139A
Bcl-XL which was used to confirm that the potential kinetic TGS hit combinations
were actually forming at the protein’s hot spot and not elsewhere on the protein surface.
The number of potential hits, selected at the end of the third set of experiments was close
to 100 acylsulfomanamide hits. These hit combinations were further re-evaluated in
binary incubation mixtures, as well as their retention times in the LC/MSMS compared to
the ones of the corresponding authentic samples. It was interesting to find out that while
some thio acids delivered higher number of kinetic TGS hits (TA23, TA17, TA19, and
others), there were several thio acids (TA27, TA28, TA37, TA38) which showed no
templation effect with Mcl-1 or Bcl-XL as a target. Currently, fifty acylsulfonamides
identified through this screening protocol are synthesized and tested for biological
activity. The distribution of the potential hit combination is presented in Figure 4.14.
171
Table 4.3. Potential hit combinations identified through multi-fragment kinetic TGS approach (SZ 1-38 and TA 1-45)
M+1
SZ1
SZ2
SZ3
SZ4
SZ5
SZ6
SZ7
SZ8
SZ9
SZ10
SZ11
SZ12
SZ13
SZ14
SZ15
SZ16
SZ17
SZ18
SZ19
SZ20
SZ21
SZ22
SZ23
SZ24
SZ25
SZ26
SZ27
SZ28
SZ29
SZ30
SZ31
SZ32
SZ33
SZ34
SZ35
SZ36
SZ37
SZ38
TA1
TA2
TA3
TA4
TA5
TA6
TA7
TA8
TA9
TA10
TA11
TA12
TA13
TA14
TA15
TA16
TA17
TA18
TA19
TA20
TA21
TA22 TA23
TA24
TA25
TA26
TA27
TA28
TA29
TA30
TA31
TA32
TA33
TA34
TA35
TA36
TA37
TA38
TA39
potential hits for Mcl‐1 and Bcl‐XL potential hits for Bcl‐XL (for Bcl‐XL, amplification coefficient is twice as high as the one for Mcl‐1) potential hits for Mcl‐1 (for Mcl1, amplification coefficient is twice as high as the one for Bcl‐XL) 172
TA40
TA41
TA42
TA43
TA44
TA45
4.3.
Conclusion
In a proof-of-concept study based on a previous kinetic TGS study targeting Bcl-XL, it
was demonstrated that a multi-fragment kinetic TGS approach coupled with TQMS
technology was successfully implemented in the identification of known protein-protein
modulators. Optimized screening conditions utilizing a triple quadruple mass
spectrometer in the Multiple Reaction Monitoring (MRM) mode was proven to be very
efficient in kinetic TGS hit identification increasing both the throughput and the
sensitivity of this approach. The multi-fragment incubation approach was studied in detail
and it was concluded that 200 fragment combinations in one well is an optimal and
practical number permitting good acylsulfonamide detectability. Overall, the presented
multi-component kinetic TGS technique has shown to have a great potential in
streamlining the kinetic screening method as well as accelerating the hit identification
process. In a subsequent study, a structurally diverse library of forty five thio acids and
thirty eight sulfonyl azides was screened in parallel against Mcl-1 and Bcl-XL, and
several potential hit combinations were identified. Although, the synthesis of all these
kinetic TGS hit compounds is currently ongoing, preliminary testing of several
acylsulfonamides indicate that they disrupt the Bcl-XL/Bim or Mcl-1/Bim interaction.
Importantly, this multi-fragment kinetic TGS screening is generally applicable and it has
the potential to be utilized for the screening of other protein-protein interaction targets
(MDM2/p53, IL-2–IL-2Rα, and other).
173
4.4
Experimental section
4.4.1 General information
All reagents and solvents were purchased from commercial sources and used without
further purification. Column chromatography was carried out using Merck Kieselgel 60
H silica gel. 1H NMR,
13
C NMR were recorded on a Bruker 250 MHz and Varian 400
MHz NMR spectrometer. All 1H NMR experiments were reported in δ units, parts per
million (ppm) downfield of TMS and were measured relative to the signals for
chloroform (7.26 ppm) and deuterated methanol (3.35, 4.78 ppm). All 13C NMR spectra
were reported in ppm relative to the signals for chloroform (77 ppm). The HRMS data
were measured on an Agilent 1100 Series MSD/TOF with electrospray ionization. The
LC/MS data were measured on Thermo Scientific TSQ 8000 Triple Quadrupole LC/MS
and Agilent Technology 6460 Triple Quad LC/MS, using an Agilent column Kinetex 2.6
µm PFP, 4.60 mm x 50 mm.
The elution gradient employed for TQMS-MRM analysis is shown below:
Table 4.4. Elution gradient employed for TQMS-MRM analysis
Time
% B*
Flow rate
0.00
10.0
0.7 mL min-1
2.00
10.0
0.7 mL min-1
15.00
95
0.7 mL min-1
17.00
10
0.7 mL min-1
* eluent A: H2O (0.05% TFA); eluent B: CH3CN (0.05% TFA)
174
4.4.2
General
Protocol
for
Multi-fragment
“Sulfo-Click”
Kinetic
TGS
Experiments with Mcl-1 and Bcl-XL
2 mM stock solutions of the required sulfonyl azide building blocks in methanol was
prepared in advance and 100 L of each stock solution was combined in a via (using all
sulfonyl azides needed for the multi-fragment incubation). The solvent was evaporated
and 100 L fresh MeOH was added in the vial in order to obtain multi-component
mixture of sulfonyl azides with 2 mM final concentration of each suflonyl azide. Each
thio acid used for the multi-fragment screening was prepared by individually deprotecting
the corresponding fluorenylmethyl thioesters (~ 500 g weighted in a 2 mL eppendorf)
with 5 % piperidine in DMF for 2-4 min (1 L solution used for 4.7 mol thioester) and
each reaction was diluted with methanol in order to generate 20 mM stock solution of the
corresponding thio acid. The thio acids required for the multi-component screening were
combined in an eppendorf vial and further diluted with methanol to 2 mM final solution
of each thioacid. In a 96-well plate 1 L of a the prepared multi-thio acids solution (2
mM stock solution) and 1 L of a the prepared multi-sulfonyl azides solution (2 mM
stock solution) were added to a solution of Mcl-1 (98 L of 10 M Mcl-1 solution in
buffer) or to solution of Bcl-XL (98 L of 10 M Bcl-XL solution in buffer). The
phosphate buffer used for the incubations (pH = 7.40) has the following composition: 58
mM Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl, 1 mM NaN3. In addition to that, a set of
incubation was carried out under the same conditions but in the presence of buffer only
(background reaction). The 96-well plate was incubated at 37 °C for twelve hours. The
incubation mixtures were then subjected to Liquid Chromatography combined with triple
quadrupole mass spectrometry in the the Multiple Reaction Monitoring mode (MRM),
175
Kinetex PFP (4.60 mm x 50 mm) proceeded by a Phenomenex C18 guard column. The
injection volume was 20 µL at a flow rate of 0.7 mL/min (see Table 4.4).
4.4.3
General protocol for control incubations of mutants R139ABcl-XL17
A control experiment with
R139A
Bcl-XL was carried out by following the described
incubation protocol (see subchapter 4.4.2) wherein wildtype Bcl-XL was substituted with
mutant
R139A
Bcl-XL. This control experiment was carried out in parallel with wildtype
Bcl-XL containing incubation mixtures. In addition to that, a set of incubation reactions
was carried out under the same conditions but in the presence of buffer only (background
reaction). The three incubation reactions were subjected to LC/MS Triple QuadrupoleMRM analysis.
4.4.4 General Procedure to Synthesize Fluorenylmethyl Thioesters
A solution of a corresponding carboxylic acid derivative in dry DCM was reacted with
EDCI (2 eq), DMAP (0.5 eq), and (9H-fluoren-9-yl)methanethiol (1eq). The completion
of the reaction was followed by TLC and LC/MS. The crude reaction mixture was
quenched with water end extracted twice with EtOAc. The combined organic layers were
dried over anhydrous sodium sulfate and concentrated. The thioester was then obtained
by flash chromatography.
4.4.5 General Procedure to Synthesize Acylsulfomamides18
All the reactions were performed at 50-80 mg scale in an eppendorf.
The 9-
fluorenylmethyl thioester was taken in a 1.5 mL eppendorf in dry DMF, (per 1 μmol of
176
thioester, 4.7 μL of DMF was added) 5 equivalents of solid Cs2CO3 was added and the
reaction was stirred for a 10 minutes at room
temperature. Then the sulfonylazide (1 eq w.r.t. thioester) was added to the reaction
mixture and stirred for 15 minutes. After the reaction was complete, (monitored by LCMS) water (200 μL) was added and the pH was adjusted to 7.0 using 1M HCl solution.
The reaction mixture was extracted with DCM until there was no product in aqueous
layer (checked by TLC or LC-MS).The product was purified by flash chromatography.
4.4.6 Synthetic Procedures
Syntesis of TE20
The acid derivative of 3,4-bis(2,4-difluorophenyl)-maleimide 4.6 was synthesized
according to a reported protocol.19 TE20 was obtained by following the general
procedure in section 4.4.4
(Rf = 0.48 in hexanes : EtOAc = 85 : 25), isolated in 62 % yield. 1H NMR (250 MHz,
CDCl3) δ 7.63 – 7.44 (m, 8H), 7.41 – 7.30 (m, 4H), 7.26 – 7.11 (m, 8H), 6.80 (ddd, J =
8.0 Hz, 2.5, 1.3, 4H), 6.63 (ddd, J = 11.1 Hz, 7.2, 2.5, 4H), 4.05 – 3.92 (m, 2H), 3.51 (dd,
J = 8.4 Hz, 5.1, 4H), 3.41 – 3.29 (m, 4H), 2.44 (t, J = 7.3 Hz, 4H), 1.92 – 1.79 (m, 4H).
13
C NMR (63 MHz, CDCl3) δ 197.88, 169.38, 166.08, 162.73, 162.23, 158.69, 145.44,
141.12, 133.19, 132.53, 132.44, 127.74, 127.15, 124.72, 119.93, 113.75, 113.45, 112.10,
111.70, 46.75, 41.36, 37.94, 32.26, 24.23.
177
Syntesis of TE28
TE28 was obtained by following the general procedure in section 4.4.4
Isolated in 64 % yield, (Rf = 0.55 in hexanes : EtOAc = 95 : 15). 1H NMR (400 MHz,
cdcl3) δ 8.68 (s, 1H), 7.74 (d, J = 7.5 Hz, 2H), 7.68 – 7.61 (m, 2H), 7.41 – 7.35 (m, 2H),
7.30 (qd, J = 7.4 Hz, 1.2, 2H), 6.96 – 6.85 (m, 3H), 6.80 – 6.74 (m, 1H), 4.99 (dd, J = 8.1
Hz, 3.8, 1H), 4.18 (t, J = 5.9 Hz, 1H), 3.57 (d, J = 6.0 Hz, 2H), 3.24 (dd, J = 16.1 Hz, 3.8,
1H), 3.12 (dd, J = 16.1 Hz, 8.1, 1H).
13
C NMR (101 MHz, cdcl3) δ 194.80, 166.23,
145.27, 142.78, 141.03, 127.73, 127.12, 126.03, 124.68, 124.65, 124.28, 122.91, 119.87,
117.26, 115.72, 77.31, 76.99, 76.68, 73.37, 46.56, 44.26, 32.52.
Syntesis of TE29
The synthesis the corresponding acid were performed by following a reported
procedure20 and the general procedure in section 4.4.4
178
Isolated in 64 % yield, (Rf = 0.49 in hexanes : EtOAc = 90 : 10). 1H NMR (250 MHz,
CDCl3) δ 7.69 (d, J = 7.4 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 7.44 – 7.33 (m, 4H), 7.29 –
7.17 (m, 5H), 7.05 (s, 1H), 4.17 (t, J = 5.5 Hz, 1H), 3.63 (d, J = 5.5 Hz, 2H). 13C NMR
(101 MHz, cdcl3) δ 145.38, 144.73, 141.23, 139.84, 130.12, 129.72, 129.46, 129.24,
128.95, 128.08, 127.93, 127.73, 127.17, 125.82, 125.60, 124.71, 124.56, 123.81, 120.23,
120.03, 119.93, 109.27, 108.24, 77.33, 77.01, 76.70, 46.66, 46.32, 32.36, 31.79.
Syntesis of TE30
The synthesis the corresponding acid were performed by following a reported
procedure21 and the general procedure in section 4.4.4
Isolated in 65 % yield, (Rf = 0.55 in hexanes : EtOAc = 90 : 10). 1H NMR (250 MHz,
CDCl3) δ 8.28 (d, J = 1.3 Hz, 1H), 7.89 (s, 1H), 7.76 – 7.59 (m, 5H), 7.47 (d, J = 8.0 Hz,
1H), 7.35 – 6.97 (m, 7H), 6.64 (d, J = 3.0 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 5.48 (s, 2H),
4.17 (t, J = 6.0 Hz, 1H), 3.58 (d, J = 6.1 Hz, 2H). 13C NMR (101 MHz, cdcl3) δ 191.33,
145.79, 141.01, 139.90, 138.79, 130.02, 129.92, 129.38, 128.28, 127.93, 127.61, 127.08,
179
124.81, 123.37, 122.10, 121.69, 119.85, 109.18, 104.74, 77.33, 77.01, 76.70, 46.98,
46.40, 32.62.
Syntesis of TE32
TE32 was obtained by following the general procedure in section 4.4.4
Isolated in 75 % yield, (Rf = 0.50 in hexanes : EtOAc = 85 : 15). 1H NMR (250 MHz,
CDCl3) δ 7.61 (d, J = 7.4 Hz, 2H), 7.48 (d, J = 7.3 Hz, 2H), 7.22 (tt, J = 11.2 Hz, 5.6,
4H), 6.92 – 6.74 (m, 3H), 4.50 – 4.33 (m, 2H), 4.12 – 3.92 (m, 3H), 3.58 – 3.34 (m, 2H),
2.85 – 2.56 (m, 2H).
13
C NMR (101 MHz, cdcl3) δ 196.24, 164.01, 147.02, 145.12,
143.85, 141.09, 129.52, 129.08, 127.96, 127.78, 127.13, 124.59, 123.86, 123.78, 120.05,
119.92, 118.22, 114.85, 77.41, 77.09, 76.78, 67.39, 46.48, 40.94, 37.57, 32.45.
Syntesis of TE34
TE34 was obtained by following the general procedure in section 4.4.4
O
O
H
N
S
S
TA34
180
Isolated in 70 % yield, (Rf = 0.55 in hexanes : EtOAc = 85 : 15).
1
H NMR (400 MHz, cdcl3) δ 8.88 (s, 1H), 7.81 – 7.71 (m, 2H), 7.66 – 7.60 (m, 2H), 7.46
– 7.19 (m, 6H), 7.13 (td, J = 7.6 Hz, 1.4, 1H), 7.07 – 6.95 (m, 1H), 6.83 (dt, J = 33.9 Hz,
18.9, 1H), 4.27 – 4.14 (m, 1H), 3.99 (dt, J = 8.6 Hz, 5.4, 1H), 3.55 (dt, J = 8.7 Hz, 6.2,
2H), 3.20 (dt, J = 16.0 Hz, 5.4, 1H), 2.82 – 2.68 (m, 1H). 13C NMR (101 MHz, cdcl3) δ
195.08, 166.59, 145.23, 141.08, 135.76, 128.21, 127.75, 127.49, 127.16, 124.70, 124.66,
124.08, 119.88, 118.79, 117.14, 77.33, 77.01, 76.69, 46.60, 42.65, 38.08, 32.55.
Syntesis of TE35
TE35 was obtained by following the general procedure in section 4.4.4
Isolated in 70 % yield, (Rf = 0.50 in hexanes : EtOAc = 85 : 15). 1H NMR (250 MHz,
CDCl3) δ 7.85 (dd, J = 7.7 Hz, 1.8, 4H), 7.57 (d, J = 7.1 Hz, 4H), 7.47 (d, J = 7.3 Hz,
4H), 7.36 – 7.10 (m, 15H), 4.09 – 3.96 (m, 2H), 3.50 – 3.34 (m, 4H), 3.08 – 2.98 (m, 4H),
2.96 – 2.85 (m, 4H).
13
C NMR (63 MHz, CDCl3) δ 196.41, 164.96, 164.83, 145.12,
141.04, 131.60, 128.97, 127.74, 127.09, 126.73, 124.57, 123.76, 119.87.
181
Syntesis of TE41
Isolated in 70 % yield, (Rf = 0.48 in hexanes : EtOAc = 85 : 10). The synthesis the
corresponding acid were performed by following a reported procedure22 and the general
procedure in section 4.4.4. 1H NMR (250 MHz, CDCl3) δ 8.12 (s, 1H), 7.71 (ddd, J =
34.0 Hz, 17.8, 4.5, 8H), 7.44 – 7.21 (m, 10H), 6.95 (s, 1H), 4.20 (t, J = 6.0 Hz, 1H), 3.62
(d, J = 6.1 Hz, 2H).
13
C NMR (101 MHz, cdcl3) δ 157.67, 145.68, 141.05, 134.67,
132.59, 129.73, 129.34, 129.14, 129.07, 128.89, 127.70, 127.14, 125.10, 124.80, 124.32,
123.90, 120.90, 120.29, 119.91, 111.19, 101.54, 46.89, 32.80.
All acylsulfonamides according to the protocol described in subchapter 4.4.518
Acylsulfonamide SZ17TA24:
1
H NMR (400 MHz, DMSO-d6)  ppm 1.19 (s, 2 H) 2.47 (d, J=1.95 Hz, 1 H) 2.52 - 2.64
(m, 2 H) 3.06 - 3.18 (m, 2 H) 3.57 (br. s., 5 H) 3.58 (s, 5 H) 3.62 - 3.74 (m, 12 H) 3.84 (s,
2 H) 6.68 (d, J=8.59 Hz, 1 H) 6.73 - 6.85 (m, 3 H) 6.88 - 6.99 (m, 3 H) 7.08 - 7.14 (m, 4
182
H) 7.16 - 7.23 (m, 3 H) 7.54 (m, J=8.59 Hz, 2 H) 7.75 (m, J=8.20 Hz, 2 H) 7.89 (d,
J=8.20 Hz, 2 H).
13
C NMR (101 MHz, DMSO-d6)  ppm 35.59 (s, 1 C) 39.33 (s, 1 C)
39.54 (s, 1 C) 39.75 (s, 1 C) 39.96 (s, 1 C) 40.17 (s, 1 C) 40.38 (s, 1 C) 40.58 (s, 1 C)
55.34 (s, 1 C) 56.21 (s, 1 C) 60.66 (s, 1 C) 60.86 (s, 1 C) 108.18 (s, 1 C) 112.86 (s, 1 C)
114.43 (s, 1 C) 121.15 (s, 1 C) 124.90 (s, 1 C) 125.69 (s, 1 C) 126.30 (s, 1 C) 127.91 (s, 1
C) 128.03 (s, 1 C) 128.91 (s, 1 C) 128.94 (s, 1 C) 129.00 (s, 1 C) 129.27 (s, 1 C) 129.68
(s, 1 C) 136.46 (s, 1 C) 142.29 (s, 1 C) 151.64 (s, 1 C) 152.70 (s, 1 C) 159.70 (s, 1 C)
Acylsulfonamide SZ35TA24:
1
H NMR (400 MHz, DMSO-d6)  ppm 3.59 (s, 3 H) 3.68 (s, 3 H) 3.72 (s, 3 H) 3.85 (s, 2
H) 6.69 (d, J=8.59 Hz, 1 H) 6.83 (d, J=8.59 Hz, 1 H) 7.03 - 7.11 (m, 4 H) 7.12 - 7.19 (m,
1 H) 7.22 (m, J=8.20 Hz, 2 H) 7.36 - 7.44 (m, 2 H) 7.70 - 7.75 (m, 2 H) 7.77 (d, J=8.59
Hz, 2 H) 7.82 (m, J=8.59 Hz, 2 H) 7.96 - 8.03 (m, 2 H). 13C NMR (101 MHz, DMSO-d6)
 ppm 35.60 (s, 1 C) 39.33 (s, 1 C) 39.54 (s, 1 C) 39.75 (s, 1 C) 39.96 (s, 1 C) 40.17 (s, 1
C) 40.38 (s, 1 C) 40.58 (s, 1 C) 56.22 (s, 1 C) 60.66 (s, 1 C) 60.88 (s, 1 C) 108.20 (s, 1 C)
119.12 (s, 1 C) 119.62 (s, 1 C) 124.39 (s, 1 C) 124.91 (s, 1 C) 126.30 (s, 1 C) 127.13 (s, 1
C) 128.69 (s, 1 C) 128.92 (s, 1 C) 128.96 (s, 1 C) 129.28 (s, 1 C) 130.60 (s, 1 C) 133.90
(s, 1 C) 142.30 (s, 1 C) 144.16 (s, 1 C) 147.13 (s, 1 C) 151.65 (s, 1 C) 152.70 (s, 1 C)
156.50 (s, 1 C) 157.87 (s, 1 C) 166.45 (s, 1 C).
183
Acylsulfonamide SZ8TA24:
13
C NMR (126 MHz, DMSO-d6)  ppm 35.62 (s, 1 C) 39.46 (s, 1 C) 39.64 (s, 1 C) 39.80
(s, 1 C) 39.97 (s, 1 C) 40.14 (s, 1 C) 40.23 (s, 1 C) 40.30 (s, 1 C) 40.46 (s, 1 C) 55.83 (s,
1 C) 55.87 (s, 1 C) 56.22 (s, 1 C) 56.78 - 59.42 (m, 1 C) 56.83 (s, 1 C) 57.81 (s, 1 C)
60.68 (s, 1 C) 60.89 (s, 1 C) 67.38 (s, 1 C) 108.19 (s, 1 C) 111.90 (s, 1 C) 112.37 (s, 1 C)
124.94 (s, 1 C) 126.28 (s, 1 C) 127.71 (s, 1 C) 128.13 (s, 1 C) 128.68 (s, 1 C) 128.97 (s, 1
C) 129.63 (s, 1 C) 142.31 (s, 1 C) 147.24 (s, 1 C) 147.79 (s, 1 C) 151.67 (s, 1 C) 152.73
(s, 1 C) 166.09 (s, 1 C)
Acylsulfonamide SZ4TA21:
1
H NMR (400 MHz, DMSO-d6)  ppm 3.22 - 3.28 (m, 3 H) 3.64 (q, J=6.38 Hz, 2 H) 3.72
(s, 3 H) 7.01 (td, J=7.42, 1.17 Hz, 1 H) 7.09 (d, J=7.81 Hz, 1 H) 7.11 - 7.16 (m, 1 H) 7.17
(s, 1 H) 7.18 - 7.22 (m, 1 H) 7.22 - 7.26 (m, 2 H) 7.27 (dd, J=7.62, 1.76 Hz, 1 H) 7.29 7.37 (m, 4 H) 7.52 - 7.57 (m, 2 H) 7.83 - 7.88 (m, 2 H) 7.90 (dd, J=9.18, 2.15 Hz, 1 H)
8.59 (d, J=2.34 Hz, 1 H) 8.76 (t, J=5.86 Hz, 1 H).
13
C NMR (101 MHz, DMSO-d6) 
ppm 14.45 - 191.12 (m, 100 C) 31.53 (s, 1 C) 39.36 (s, 1 C) 39.57 (s, 1 C) 39.78 (s, 1 C)
184
39.99 (s, 1 C) 40.20 (s, 1 C) 40.41 (s, 1 C) 40.61 (s, 1 C) 42.45 (s, 1 C) 56.03 (s, 1 C)
112.34 (s, 1 C) 121.35 (s, 1 C) 126.50 (s, 1 C) 128.35 (s, 1 C) 128.64 (s, 1 C) 128.87 (s, 1
C) 129.13 (s, 1 C) 129.49 (s, 1 C) 129.82 (s, 1 C) 130.30 (s, 1 C) 130.81 (s, 1 C) 134.46
(s, 1 C) 135.44 (s, 1 C) 142.73 - 153.10 (m, 1 C) 156.56 (s, 1 C)
Acylsulfonamide SZ16TA21:
1
H NMR (400 MHz, DMSO-d6)  ppm 3.70 - 3.74 (m, 3 H) 6.97 - 7.04 (m, 1 H) 7.09 (d,
J=7.81 Hz, 1 H) 7.28 (dd, J=7.62, 1.76 Hz, 1 H) 7.32 - 7.38 (m, 1 H) 7.53 - 7.58 (m, 2 H)
7.84 (d, J=8.20 Hz, 2 H) 7.86 - 7.91 (m, 2 H) 7.92 - 8.00 (m, 4 H) 8.07 - 8.12 (m, 2 H).
13
C NMR (101 MHz, DMSO-d6)  ppm 39.32 (s, 1 C) 39.53 (s, 1 C) 39.74 (s, 1 C) 39.95
(s, 1 C) 40.15 (s, 1 C) 40.37 (s, 1 C) 47.77 - 48.15 (m, 1 C) 55.97 (s, 1 C) 107.33 - 107.71
(m, 1 C) 108.47 - 109.23 (m, 1 C) 112.30 (s, 1 C) 121.31 (s, 1 C) 125.98 (s, 1 C) 126.36
(s, 1 C) 126.40 (s, 1 C) 128.24 (s, 1 C) 128.53 (s, 1 C) 128.66 (s, 1 C) 128.84 (s, 1 C)
129.14 (s, 1 C) 129.46 (s, 1 C) 129.79 (s, 1 C) 130.18 (s, 1 C) 130.40 (s, 1 C) 130.78 (s, 1
C) 139.92 (s, 1 C) 142.87 (s, 1 C) 143.47 (s, 1 C) 143.75 (s, 1 C) 156.53 (s, 1 C) 165.93
(s, 1 C) 192.31 - 192.69 (m, 1 C) 204.64 - 205.96 (m, 1 C) 227.40 - 227.97 (m, 1 C)
185
Acylsulfonamide SZ11TA25:
1
H NMR (400 MHz, DMSO-d6) ppm 1.29 (s, 10 H) 7.30 - 7.37 (m, 1 H) 7.48 (m,
J=8.59 Hz, 2 H) 7.61 (m, J=8.59 Hz, 2 H) 7.69 (d, J=8.20 Hz, 2 H) 7.80 - 7.95 (m, 3 H)
7.95 - 8.03 (m, 3 H) 8.03 - 8.09 (m, 2 H) 8.62 - 8.68 (m, 1 H).
13
C NMR (101 MHz,
DMSO-d6) ppm 7.18 - 182.44 (m, 95 C) 39.33 (s, 1 C) 39.54 (s, 1 C) 39.75 (s, 1 C)
39.96 (s, 1 C) 40.17 (s, 1 C) 40.38 (s, 1 C) 40.58 (s, 1 C) 120.96 (s, 1 C) 123.26 (s, 1 C)
126.20 (s, 1 C) 126.23 (s, 1 C) 126.37 (s, 1 C) 127.02 (s, 1 C) 128.11 (s, 1 C) 129.24 (s, 1
C) 136.90 (s, 1 C) 137.68 (s, 1 C) 140.79 (s, 1 C) 150.00 (s, 1 C) 150.88 (s, 1 C) 155.92
(s, 1 C)
Acylsulfonamide SZ1TA23:
1
H NMR (400 MHz, DMSO-d6) ppm 1.29 (s, 10 H) 7.30 - 7.37 (m, 1 H) 7.48 (m,
J=8.59 Hz, 2 H) 7.61 (m, J=8.59 Hz, 2 H) 7.69 (d, J=8.20 Hz, 2 H) 7.80 - 7.95 (m, 3 H)
7.95 - 8.03 (m, 3 H) 8.03 - 8.09 (m, 2 H) 8.62 - 8.68 (m, 1 H).
13
C NMR (101 MHz,
DMSO-d6) ppm 7.18 - 182.44 (m, 95 C) 39.33 (s, 1 C) 39.54 (s, 1 C) 39.75 (s, 1 C)
186
39.96 (s, 1 C) 40.17 (s, 1 C) 40.38 (s, 1 C) 40.58 (s, 1 C) 120.96 (s, 1 C) 123.26 (s, 1 C)
126.20 (s, 1 C) 126.23 (s, 1 C) 126.37 (s, 1 C) 127.02 (s, 1 C) 128.11 (s, 1 C) 129.24 (s, 1
C) 136.90 (s, 1 C) 137.68 (s, 1 C) 140.79 (s, 1 C) 150.00 (s, 1 C) 150.88 (s, 1 C) 155.92
(s, 1 C).
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192
5. Appendices
193
5.1 Appendix 1
194