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

ABSTRACT

Background of ROS Responsive Materials

Synthesis of Drug

Screen of Buchwald-Hartwig Reaction

Drug-Loaded Peroxynitrite Responsive Monomer

Synthesis of Rhodamine

References

Conclusions and Future Directions

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

Figure: General structure of peroxynitrite

responsive cleavable monomer.

NH

O

OO

Figure: Amine drug molecule (left) and peroxynitrite-responsive cleavable monomer

bound to drug molecule (right).

Advantages of Covalently

Bonded Cargos

• High density packing

• No leakage during storage

of molecules

• Tuned responsiveness and

activated release

Figure: Model demonstrates the efficiency of polymer with covalently bonded cargo

(Right) as opposed to trapped free-floating cargo (Left)

The Gianneschi Lab specializes in synthesizing responsive polymers and drug-loaded

particles. Covalently bound cargos offer several advantages over encapsulated cargos in

nanoparticle formulations.

Conclusions:

• Synthesized amine drug molecule with optimal purity and

recrystallization

• Completed one screen of Buchwald-Hartwig reaction in a

vacuum-sealed glove box environment with the catalyst

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,

rhodamine

Future:

• Complete full screen of Buchwald-Hartwig reaction,

incorporating several different reagents

• Test responsivity of final probe in the presence of

peroxynitrite

Modified Reaction Scheme

x

L2Pd

LPd

Ar-X

PdL

Ar

X

Pd

Ar

L

X

HNRR'N

RR'H

Pd

L

Ar

XBase

Base-HX

Ar-NRR'

N

R

R'

PdAr

L

L

Oxidative Addition

Amine BindingDeprotonation

Reductive Elimination

Reagent Catalyst Ligand Aryl-X Amine Base Product

Identity Pd(dba)2 (R)-BINAP XantPhos Xphos RuPhos I-Phe Drug Cs2CO3 Drug-Phe

eq 0.05 0.1 0.1 0.2 0.2 1 1.2 1.4 1

# aliquots 4 1 1 1 1 4 4 4 4

E

85 °C 95 °C

A B C D

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)

Aryl-X

Base

Pd(dba)2

Ligand

Toluene/DMFCF3

O

O

CF3

O

CF3

OH

O

O

OO

ONOO-

Cargo: drug or fluorophore

300 400 500

m/z0

100

%

410.31

411.32

432.25

[M+H+]1+

[M+Na+]1+

Protection of Piperazine

Figure: Transition from using the amine drug

molecule to using a piperazine-taged

rhodamine, a flourescent dye, for its higher

nucleophilicity.

NH

O

OO

l

k

j

i

m

h

g

f

e

d

c

b

a

n

Figure: Mass spectrometry (Left) and H’NMR in chloroform-d (Right) of drug molecule

NH

HNN

HN

O

O

O

O

O

Protection of Fmoc-(4-iodo)-Phe-OH

HN

O

OH

I

O

OHN

O

O

I

O

O

Figure: Tert-Butyl Protection (Top Left), Mass spectrometry of product (Top Right), C’NMR of

product (Bottom)

Figure: Mono-boc piperazine

protection (Top Left), Mass

spectrometry of product (Top

Right),

C’NMR of product (Left),

H’NMR of product (Bottom)

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)

Amine

408 m/z

339 m/z637 m/z

Reaction A

Aryl-X

209.15

180 200 220m/z0

100

%

199.14

187.13210.16

[M+H+]+

[M+Na+]+

a

b

c d

e

CH2Cl285.1% yield

a

b

c

d

[M+Na+]+

580 600 620m/z0

100

%

592.23

593.19

594.23

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