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Graduate Theses and Dissertations
Graduate School
January 2012
Design, Synthesis and Evaluation of Novel
Diazirine Photolabels with Improved Ambient
Light Stability and Fluorous-Based Enrichment
Capacity
Arun Babu Kumar
University of South Florida, [email protected]
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Scholar Commons Citation
Kumar, Arun Babu, "Design, Synthesis and Evaluation of Novel Diazirine Photolabels with Improved Ambient Light Stability and
Fluorous-Based Enrichment Capacity" (2012). Graduate Theses and Dissertations.
http://scholarcommons.usf.edu/etd/4112
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Design, Synthesis and Evaluation of Novel Diazirine Photolabels with Improved Ambient
Light Stability and Fluorous-Based Enrichment Capacity
by
Arun Babu Kumar
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida
Major Professor: Dr. Roman Manetsch
Dr. Mark McLaughlin
Dr. Jon Antilla
Dr. Abdul Malik
Date of Approval
July 09, 2012
Keywords: Photoaffinity labeling, 3-Trifluoromethyl-3-phenyl diazirine, Proteomics,
BACE-1, β-secretase, Alzheimer’s disease, Small molecule
Copyright © 2012, Arun Babu Kumar
DEDICATION
I would like to dedicate this work to my entire family. The emotional and moral support
provided by my parents, wife and grandparents was of vital importance in making this
dissertation to fruition. The amusement and laughter provided by my daughter was the
much needed relaxation during the lean periods of research.
ACKNOWLEDGMENTS
I would like to thank my mentor Dr. Roman Manetsch for the great deal of guidance,
support and confidence that he has given me during the course of this work. I am
immensely grateful to his patience and open-mindedness when listening to my ideas,
how much ever immature they may be. The friendly and approachable interactions with
him had a great influence on me in nurturing the confidence to think like a researcher. It
is a great pleasure and honor to have him as my advisor.
I thank my committee members Dr. Mark McLaughlin, Dr. Jon Antilla, Dr. Abdul
Malik and Dr. Jeremiah Tipton for their valuable suggestions and guidance during the
course of my studies.
I would also like to thank my wife, Divya for the constant emotional and moral
support that she has provided me. I could always rely on her to comfort me, even during
the toughest periods. She had a colossal part in making this work to completion.
It is my pleasure to thank the undergraduates, Jordan Anderson and Anthony
Melendez, who worked with me and helped a great deal in finishing some of the
projects. I also like to thank the Manetsch lab members; Dr. Xiangdong Hu, Dr. Niranjan
Kumar Namelikonda, Dr. David Flanigan, Dr. Yijun Yiang, Dr. Richard Cross, Dr. Shikha
Mahajan, Lisa Malmgren, Sameer Kulkarni, Katya Nacheva, Kurt Van Horn, Andrii
Monastyrskyi, Jordany Maignan, Qiao Qiao, Iredia Iyamu and Cynthia Lichorowic for
their support and the valuable guidance that they shared with me during the course of
my work here.
͓OF͓CONTENTS
TABLE͓
List of Tables ................................................................................................................ iii
List of Figures ................................................................................................................ iv
List of Schemes ............................................................................................................... v
Abstract
................................................................................................................ vi
Chapter – 1:
1.1
1.2
1.3
Introduction .............................................................................................. 1
Photoaffinity labeling and diazirine as photolabel ..................................... 1
Alzheimer’s disease and BACE-1 ............................................................. 6
Research Aims ......................................................................................... 8
1.3.1 Development of ambient light stable 3-trifluoromethyl3-aryldiazirine photolabel .............................................................. 8
1.3.2 Enhancement of photoaffinity labeling via fluorous
enrichment .................................................................................... 9
1.3.3 1,3-disubstituted 2-propanols as small molecule
inhibitors of BACE-1...................................................................... 9
References ............................................................................................. 10
1.4
Chapter – 2:
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
Bringing 3-trifluoromethyl-3-aryldiazirine photolabeling out of
the “dark” age ......................................................................................... 17
Design of ambient light stable diazirine photolabel ................................. 18
Synthesis of modified 3-trifluoromethyl-3-aryl diazirine
photolabels 1 and 2 ................................................................................ 19
Photoactivation studies to compare photolabels 1 and 2
against original photolabel 3 ................................................................... 20
Ambient light stability of modified photolabels 1 and 2 ............................ 22
Enhancement of aqueous solubility ........................................................ 24
Design and synthesis of photoaffinity labels based on modified
trifluoromethylaryl diazirine photolabels 1 and 2 ..................................... 25
Photolabelling of Concanavalin A (Con A) with photoaffinity
label 14, 15 and 16 ................................................................................. 27
Experimental .......................................................................................... 29
References ............................................................................................. 46
Chapter – 3:
Design, synthesis and photoactivation studies of fluorous
photolabels ............................................................................................. 49
Chapter – 4:
4.1
Photolabeling with fluorous photoaffinity tag ........................................... 50
Photolabeling Con A with photolabel 1 ................................................... 51
i
4.2
4.3
4.4
4.5
Chapter – 5:
5.1
5.2
5.3
Approach-1: In-solution photoaffinity labeling of Con A........................... 52
Approach-2: Photoaffinity labeling of Con A on fluorous
derived slide ........................................................................................... 53
Experimental .......................................................................................... 55
4.4.1 General Procedure ..................................................................... 56
4.4.2 Approach-1: In-solution photoaffinity labeling with 1.................... 56
4.4.3 Photoaffinity labeling of Con A on fluorous derived glass slide .... 57
References ............................................................................................. 58
Synthesis and structure–activity relationship studies of 1,3disubstituted 2-propanols as BACE-1 inhibitors ...................................... 60
Previous findings on BACE-1 inhibitors: advancements and
hurdles ................................................................................................... 60
Results and discussion ........................................................................... 63
References ............................................................................................. 64
Appendices ............................................................................................................... 68
Appendix – 1 ...................................................................................................... 69
Appendix – 2 ...................................................................................................... 79
Appendix – 3 ...................................................................................................... 86
ii
LIST OF TABLES
Table 2.1.
Comparison of ambient light stability of modified
trifluoromethylaryl diazirines versus the conventional
trifluoromethylphenyl diazirine ................................................................ 23
Table 2.2.
Comparison of aqueous solubility of modified aromatic
diazirines versus the conventional aromatic diazirine ............................. 24
iii
LIST OF FIGURES
Figure 1.1.
Pictorial representation of photoaffinity labeling ........................................ 2
Figure 1.2.
Previously reported small molecules with potent BACE-1
inhibition ................................................................................................... 8
Figure 2.1.
Design of ambient light stable 3-trifluoromethyl-3-aryl diazirine .............. 19
Figure 2.2.
Photoactivation path of diazirine photolabels .......................................... 21
Figure 2.3.
Photoactivation product distribution at different time intervals
of UV exposure....................................................................................... 21
Figure 2.4.
Amount of diazirine in percentage as estimated by 19F NMR,
intact upon exposure to ambient light at different time intervals .............. 23
Figure 2.5.
Coomassie stain and western blot analysis of control and
photolabeled Con A ................................................................................ 28
Figure 2.6.
Biotin antibody Western blot of photolabeling with photoaffinity
labels 15 and 16, P2 and P3 respectively, in the presence of
mannose ................................................................................................ 29
Figure 4.1.
Fluorous photoaffinity label 1 .................................................................. 51
Figure 4.2.
Representation of the concept behind photolabeling of Con A
with photolabel 1 .................................................................................... 52
Figure 4.3.
Enrichment of Con A on fluorous glass slide using fluorous
tagged mannose..................................................................................... 54
Figure 4.4.
Design of photoaffinity labeling experiment using fluorous
derivatized glass slide ............................................................................ 54
Figure 5.1.
Transition state isostere inhibitors of BACE-1 ......................................... 62
Figure 5.2.
Small molecule inhibitors for BACE-1 ..................................................... 63
iv
LIST OF SCHEMES
Scheme 1.1. Photoactivation and labeling pattern of diazirine, aryl azide and
benzophenone photolabels ...................................................................... 3
Scheme 1.2. Formation of carbene and linear diazo intermediate during
photo activation of diazirine ...................................................................... 4
Scheme 2.1. Synthesis of modified photolabels 1 and 2.............................................. 20
Scheme 2.2. Synthesis of intermediates 20, 23, 24, 25 and 26 ................................... 26
Scheme 2.3. Synthesis of photoaffinity label 14, 15 and 16 ......................................... 27
v
ABSTRACT
Photoaffinity labeling is a quintessential technique in studying and analyzing the
interaction between a ligand and receptor. Diazirines are one of the important photolabile moieties used in photoaffinity labeling due to their superior photo labeling
characteristics. Herein, we report the investigations we conducted with diazirine
photolabels on (a) photochemical aspects leading to enhancement of their ambient light
stability and (b) equipping them with fluorous tags to enable fluorous enrichment of
labeled proteins. Furthermore, we report a pilot study to develop BACE-1 inhibitors,
which have potential to be developed into photoaffinity probes.
3-Trifluoromethyl-3-phenyldiazirine offers good selectivity and protection against
pseudolabeling but due to its photo lability, it undergoes decomposition even under
ambient light. Thus the laboratory handling, including synthesis, of 3-trifluoromethyl-3phenyldiazirine is cumbersome and restricted under constant darkness. Herein, we have
designed, synthesized and evaluated two photolabels with enhanced stability to ambient
light conditions in addition to the good selectivity and protection against pseudolabeling
as offered by 3-trifluoromethyl-3-phenyldiazirine. It was also found that the aqueous
solubility, a vital physical property for a photolabel, was also improved in the modified
ambient light stable photolabels.
Fluorous tags have found wide use in synthetic applications; herein we explore
the possibility of its application in photoaffinity studies. We designed, synthesized and
conducted photoactivation studies on two fluorous diazirine photolabels. The
photoactivation studies unraveled an unanticipated photoreaction when the fluorous tag
vi
was directly connected to the diazirine ring, yielding a fluorous alkene. The more
practical photolabel of the two was chosen as the target specific photoaffinity labeling
moiety for fluorous proteomics. Upon conducting photolabeling experiments under
various conditions, we found that the strong hydrophobic character of the fluorous tag
renders the photoaffinity label insoluble in aqueous solutions and significantly alters the
binding mode and affinity of the photoaffinity label to its target receptor.
A library of 1,3-disubstituted 2-propanols was combinatorially prepared and
tested as small molecule inhibitors of β-secretase (BACE-1). The initial screening of the
1,3-disubstituted 2-propanol library revealed a few low micromolar inhibitors for BACE-1.
The compound that showed the best activity was chosen for further SAR studies, which
resulted in a potent BACE-1 inhibitor with nanomolar inhibition. Investigation on the
selectivity of these compounds for BACE-1 inhibition over cathepsin D revealed that
these compound series possess very high selectivity. Furthermore, the physicochemical
properties study showed that these compounds possessed the calculated parameters
advantageous to cross the blood–brain barrier (BBB).
vii
CHAPTER – 1
Introduction
1.1
Photoaffinity labeling and diazirine as photolabel
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Research Aims
1.3.1
Development of ambient light stable 3-trifluoromethyl-3-aryldiazirine
photolabel
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Enhancement of photoaffinity labeling via fluorous enrichment
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1,3-Disubstituted 2-propanols as small molecule inhibitors of BACE-1
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CHAPTER – 2
Bringing 3-trifluoromethyl-3-aryldiazirine photolabeling out of the “dark” age
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Design of an ambient light stable diazirine photolabel
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Photoactivation studies to compare photolabels 1 and 2 against original
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45
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References
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GHYHORSPHQWTrends in Biotechnology :HEHU 3 - $ %HFN6LFNLQJHU $ * &RPSDULVRQ RI WKH SKRWRFKHPLFDO
EHKDYLRU RI IRXU GLIIHUHQW SKRWRDFWLYDWDEOH SUREHV Journal of Peptide Research 'DV - $OLSKDWLF 'LD]LULQHV DV 3KRWRDIILQLW\ 3UREHV IRU 3URWHLQV 5HFHQW
'HYHORSPHQWVChemical Reviews +DVKLPRWR 0 +DWDQDND < 5HFHQW SURJUHVV LQ GLD]LULQHEDVHG SKRWRDIILQLW\
ODEHOLQJEuropean Journal of Organic Chemistry /LX%/.DQJ'6$%LQLWLRVWXG\RIVWDELOLW\DQGVWUDLQRIF\FORSURSDQHDQG
LWVGHULYDWLYHVJournal of Chemical Information and Computer Sciences +H < * -XQN & 3 &DZOH\ - - /HPDO ' 0 $ UHPDUNDEOH SURSHOODQHJournal of the American Chemical Society 'DV-$GGRQD*+6DQGEHUJ:6+XVDLQ666WHKOH70LOOHU.:
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SURWHLQNLQDVH&GHOWDJournal of Biological Chemistry (UQL % .KRUDQD + * )DWW\DFLGV FRQWDLQLQJ SKRWRDFWLYDEOH FDUEHQH
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CHAPTER – 3
Design, synthesis and photoactivation studies of fluorous photolabels
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49
CHAPTER – 4
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References
*ODG\V] - $ ,QWURGXFWLRQ 5HFRYHUDEOH FDWDO\VWV DQG UHDJHQWV 3HUVSHFWLYH
DQG SURVSHFWLYH Chemical Reviews 2002, 102 <RVKLGD - ,WDPL .
7DJVWUDWHJ\IRUVHSDUDWLRQDQGUHFRYHU\Chemical Reviews 2002,102
=KDQJ : )OXRURXV /LQNHU)DFLOLWDWHG &KHPLFDO 6\QWKHVLV Chemical Reviews 2009,
109
58
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IOXRURXVUHYHUVHSKDVHVLOLFD JHOJournal of the American Chemical Society 1999, 121
&XUUDQ ' /HH = < )OXRURXV WHFKQLTXHV IRU WKH V\QWKHVLV DQG
VHSDUDWLRQRIRUJDQLFPROHFXOHVGreen Chemistry 2001,3**
%ULWWDLQ60)LFDUUR6%%URFN$3HWHUV(&(QULFKPHQWDQGDQDO\VLVRI
SHSWLGH VXEVHWV XVLQJ IOXRURXV DIILQLW\ WDJV DQG PDVV VSHFWURPHWU\ Nature
Biotechnology 2005,23
.R . 6 -DLSXUL ) $ 3RKO 1 / )OXRURXVEDVHG FDUERK\GUDWH PLFURDUUD\V
Journal of the American Chemical Society 2005,127
0RQWDQDUL 9 .XPDU . -XVW DGG ZDWHU $ QHZ IOXRURXV FDSSLQJ UHDJHQW IRU
IDFLOH SXULILFDWLRQ RI SHSWLGHV V\QWKHVL]HG RQ WKH VROLG SKDVH Journal of the American
Chemical Society 2004,126
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FDSWXUHPDVVGHWHFWLRQXVLQJIOXRURXVDIILQLW\WDJVJournal of Proteome Research 2007,
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VDFFKDULGHELRVHQVRUVChemistry-a European Journal 2003,9
59
CHAPTER – 5
Synthesis and structure–activity relationship studies of 1,3-disubstituted
2-propanols as BACE-1 inhibitors
5.1
Previous findings on BACE-1 inhibitors: advancements and hurdles
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