Synthesis of a Cargo-Linked Peroxynitrite Cleavable Monomer
Shivani Avasarala, Andrea S. Carlini, and Nathan Gianneschi
The Department of Chemistry & Biochemistry, University of California San Diego, 9500 Gilman drive, La Jolla, California
Synthesis of Drug
ABSTRACT
d
a
Reactive oxygen species (ROS) responsive probes are of growing interest in the field of
biomedicine. Because of its extreme reactivity and selectivity over other endogenous ROS,
peroxynitrite is a promising target for the purpose of detecting and targeting the site of
diseased tissue, such as a myocardial infarct. In this study, we worked towards the synthesis
of a peroxynitrite-responsive cleavable monomer, which upon activation is expected to
release a bound cargo; in our study, this cargo was a therapeutic small molecule drug or a
fluorescent tag. The probe consists of a polymerizable moiety, a linker molecule, a
peroxynitrite substrate, and a cargo which possesses a 2˚ amine. In the drug-linked
monomer, it was determined that the 1,4-dihydropyridine amine was likely not nucleophilic
enough for a Buchwald-Hartwig (C-N) amination. In switching gears to a monomer linked to a
more nucleophilic fluorophore, 5(6)-carboxyrhodamine, initial protection and coupling steps
were completed. Synthesis of the new monomer is ongoing. Given successful
responsiveness to peroxynitrite, this probe can be polymerized and assembled into
nanoparticles used for the effective detection of peroxynitrite production in diseased tissue,
and may be altered to carry different cargos, such as drugs, labels, and targeting moieties.
Cargo: drug molecule,
fluorophore, contrast agent,
targeting peptide
410.31
a
100
[M+H+]1+
b
c
O
b
O
n
d
O
m
411.32
k
%
l
e
i
j
h
N
H
f
g
[M+Na+]1+
432.25
Protection of Fmoc-(4-iodo)-Phe-OH
0
300
400
100
500
Figure: Mass spectrometry (Left) and H’NMR in chloroform-d (Right) of drug molecule
Screen of Buchwald-Hartwig Reaction
•
No leakage during storage
of molecules
•
Tuned responsiveness and
activated release
H
N
O
OH
[M+Na+]+
O
O
O
593.19
%
I
I
594.23
Reagent
Catalyst
Identity
Pd(dba)2 (R)-BINAP XantPhos Xphos RuPhos I-Phe
eq
# aliquots
A
B
Ligand
0.05
4
C
0.1
1
D
0.1
1
Aryl-X Amine
0.2
1
0.2
1
Drug
1
4
Base
Product
0
580
Cs2CO3 Drug-Phe
1.2
4
1.4
4
m/z
620
600
1
4
E
Reaction A
408 m/z
Amine
85 °C
95 °C
339 m/z
637 m/z
Figure: Tert-Butyl Protection (Top Left), Mass spectrometry of product (Top Right), C’NMR of
product (Bottom)
Figure: Screen of Buchwald-Hartwig Reactions (Top), color change in reaction viles indicates the activation of the
catalyst by the ligand (Left), TLC and LCMS reveal consumption of Aryl-X but excess Amine drug remaining and
no product material. (Right)
Synthesis of Rhodamine
Modified Reaction Scheme
Figure: On-going reaction for the
synthesis of rhodamine-piperazine,
(Left), TLC Analysis under 365/254 nm
UV light of the starting reagents and
crude reaction solution (Middle and
Right)
Figure: Transition from using the amine drug
molecule to using a piperazine-taged
rhodamine, a flourescent dye, for its higher
nucleophilicity.
Advantages of Covalently
Bonded Cargos
High density packing
O
O
The Gianneschi Lab specializes in synthesizing responsive polymers and drug-loaded
particles. Covalently bound cargos offer several advantages over encapsulated cargos in
nanoparticle formulations.
•
H
N
O
Aryl-X
x
592.23
m/z
Figure: General structure of peroxynitrite
responsive cleavable monomer.
Background of ROS Responsive Materials
c
Conclusions and Future Directions
Conclusions:
L Pd
• Synthesized amine drug molecule with optimal purity and
recrystallization
Ar-NRR'
• Completed one screen of Buchwald-Hartwig reaction in a
LPd
vacuum-sealed glove box environment with the catalyst
Reductive
Elimination
Pd(dba)2 and four ligands: Xphos, XantPhos, RuPhos,
(R)BINAP
• Transitioned from using weak nucleophilic drug molecule to
synthesizing a highly nucleophilic secondary amine,
Deprotonation
rhodamine
Future:
Base-HX
• Complete full screen of Buchwald-Hartwig reaction,
incorporating several different reagents
Base
• Test responsivity of final probe in the presence of
O
peroxynitrite
O O
OH
2
Figure: Model demonstrates the efficiency of polymer with covalently bonded cargo
(Right) as opposed to trapped free-floating cargo (Left)
Drug-Loaded Peroxynitrite Responsive Monomer
Protection of Piperazine
Pd
O
Aryl-X
Base
O
O
CH2Cl2
85.1% yield
O
%
HN
Pd(dba)2
Ligand
Toluene/DMF
[M+H+]+
Figure: Amine drug molecule (left) and peroxynitrite-responsive cleavable monomer
bound to drug molecule (right).
b
a
Pd
X
L
Ar
L
210.16
200
220
L
CF3
d
X
Pd
L
Amine Binding
R
R'
Pd
m/z
180
c
Ar
Ar
HNRR'
N
0
e
Oxidative
Addition
R'
H
187.13
O
N
H
O
N
HN
209.15 [M+Na+]+
199.14
100
O
NH
Ar-X
R
N
O
L
Figure: Mono-boc piperazine
protection (Top Left), Mass
spectrometry of product (Top
Right),
C’NMR of product (Left),
H’NMR of product (Bottom)
O
ONOO-
CF3
O
O
O
Ar
X
CF3
Cargo: drug or fluorophore
References
1.
2.
3.
Yang, D.; Sun, Z.-N.; Peng, T.; Wang, H.-L.; Shen, J.-G.; Chen, Y.; Tam, P. K.-H., Synthetic Fluorescent Probes for Imaging of Peroxynitrite and Hypochlorous Acid
in Living Cells. In Live Cell Imaging: Methods and Protocols, Papkovsky, B. D., Ed. Humana Press: Totowa, NJ, 2010; pp 93-103.
1.
Peng, T.; Yang, D., HKGreen-3: A Rhodol-Based Fluorescent Probe for Peroxynitrite. Organic Letters 2010, 12 (21), 4932-4935.
1.
Surry, D. S.; Buchwald, S. L., Dialkylbiaryl phosphines in Pd-catalyzed amination: a user's guide. Chemical Science 2011, 2 (1), 27-50.