On-resin peptide macrocyclization using thiol-ene click chemistry

Article abstract of DOI:10.1039/c001375g

A versatile and rapid synthetic strategy has been developed for the on-resin cyclization of peptides using thiol-ene photochemistry. This unique method exploits the thiol group of natural cysteine amino acids and allows for various alkenes to be incorporated orthogonal to the peptide backbone.

Full text of DOI:10.1039/c001375g

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Chemical Communications
www.rsc.org/chemcomm
Volume 46 | Number 23 | 21 June 2010 | Pages 4001–4204
ISSN 1359-7345
COMMUNICATION
Kristi S. Anseth et al.
FEATURE ARTICLE
Yong-Gui Zhou et al.
On-resin peptide macrocyclization
using thiol–ene click chemistry
Bifunctional AgOAc-catalyzed
asymmetric reactions

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COMMUNICATION
www.rsc.org/chemcomm | ChemComm
On-resin peptide macrocyclization using thiol–ene click chemistryw
Alex A. Aimetti,a Richard K. Shoemaker,b Chien-Chi Linc and Kristi S. Anseth*ac
Received 20th January 2010, Accepted 23rd March 2010
First published as an Advance Article on the web 8th April 2010
DOI: 10.1039/c001375g
A versatile and rapid synthetic strategy has been developed for
the on-resin cyclization of peptides using thiol–ene photo-
chemistry. This unique method exploits the thiol group of
natural cysteine amino acids and allows for various alkenes to
be incorporated orthogonal to the peptide backbone.
Our strategy utilizes the natural amino acid, cysteine, as the
thiol source for the reaction. However, peptides can be
designed to contain cysteine residues that do not participate
in the cyclization reaction by exploiting highly selective
orthogonal Cys protecting groups. Various alkenes and their
effect on the cyclization were studied. Commercially available
Fmoc-Lys(Alloc)-OH was used as a building block to incorporate
an allyl ester within the peptide sequence. The allyloxycarbonyl
(Alloc) functional group is traditionally used as an orthogonal
protecting group for lysine amino acids during peptide synthesis.
However, recently the Lys(Alloc) monomer has been included
Peptides have gained interest in the fields of chemical biology
and drug discovery1 due to their ability to bind to highly
selective receptors of therapeutic interest. Additionally,
peptides can be synthetically produced and optimized allowing
for large-scale production. However, the drawbacks of
peptide therapeutics are their short half-lives in vivo, poor
bioavailability, and decreased cell permeability. Peptide
macrocyclization, including peptide stapling,2 has been shown
to address these limitations and result in a molecule with
increased potency. Cyclic peptides show increased stability
to proteolytic degradation3 relative to their linear precursors
and are able to bind to their intended target at decreased
effective concentrations, mainly attributed to the constrained
conformation lowering the entropic cost of binding.4
The synthesis of cyclic peptides has been traditionally
achieved, both on resin or in solution, by the formation of
disulfide,5 amide,6 ester,7 olefin,8 and C–C bonds.9 While these
strategies result in peptide cyclization, the process can be
extensive with reaction times ranging from hours to days.
Due to the potential clinical advantages of cyclic peptides,
there remains an unmet need to develop rapid and efficient
synthetic strategies for their formation. As a result, researchers
have become increasingly interested in using alternative
synthetic strategies based on ‘‘click’’ reactions, termed by
Sharpless, to achieve site-specific conjugation between two
functional groups in very high yields. For example, the classic
copper(I)-catalyzed azide–alkyne cycloaddition click reaction
has been recently exploited for the on-resin formation of
peptide macrocycles.10 Alternatively, the thiol–ene click reaction
is a radical mediated addition of a thiol to an alkene and has
been utilized for the formation of dendrimers,11 polymeric
networks,12,13 and disaccharides.14 This contribution demon-
strates the thiol–ene click reaction as an efficient and unique
method for rapid formation of cyclic peptides on-resin.
a Department of Chemical & Biological Engineering,
University of Colorado, Boulder, CO 80309, USA.
E-mail: Kristi.Anseth@colorado.edu; Fax: +1 303-492-4341;
Tel: +1 303-492-7471
Scheme 1 Synthetic route to on-resin peptide macrocyclization using
thiol–ene photochemistry. Conditions: 1. (i) 2% TFA/CH2Cl2. 2. (ii)
DMPA (1 equiv.), hn (365 nm, 20 mW cmꢀ2), 20–60 min; (iii) TFA/
TIPS/H2O. Alkenes were incorporated using Fmoc-Lys(Alloc)-OH
monomer (1, 3) or on-resin modification using 5-norbornene-
2-carboxylic acid (2, 4).
b Department of Chemistry & Biochemistry, University of Colorado,
Boulder, CO 80309, USA
c Howard Hughes Medical Institute, University of Colorado, Boulder,
CO 80309, USA
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c001375g
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within a peptide sequence to participate in a thiol–ene photo-
reaction to pattern within a hydrogel.15 We have exploited this
commercially available building block as a facile method
to incorporate an alkene within the peptide sequence.
Alternatively, a strained, bicyclic alkene (norbornene) was
incorporated orthogonally to the peptide backbone, as this
alkene exhibits higher reactivity.12 Arg-Gly-Asp (RGD), a
peptide ligand of the avb3 integrin,16 was synthesized as a
model peptide to demonstrate proof of concept. Scheme 1
illustrates the general procedure for cyclic peptide formation.
Linear Ac-C(Mmt)RGDSfK(alkene)-NH2 (1–2) was built on
the solid phase using Fmoc chemistry. The monomethoxytrityl
(Mmt) sulfhydryl protecting group was selectively removed
on-resin using 2% TFA/CH2Cl2. A type I photoinitiator,
2,2-dimethoxy-2-phenylacetophenone (DMPA), was added
to the peptidyl resin and exposed to 365 nm light. The reaction
was well mixed to minimize the impact of light attenuation. An
on-resin Ellman’s test (2.5 mM 5,50-dithiobis-(2-nitrobenzoic
acid) and diisopropylethylamine in methanol) was utilized to
qualitatively monitor the extent of thiol conversion.17 The
reaction with the allyl ester reached completion (based on the
Ellman’s test) at 1 h. However, the reaction with the strained
norbornene reached completion after just 20 min. On-resin
photocyclization reactions to achieve 4 and 3 were recovered
at 37% and 24% yield, respectively (calculated based on initial
resin substitution).
Cyclized products were characterized using various NMR
techniques. Fig. 1b illustrates unambiguous evidence of
cyclization in regard to 4. The HMBC spectrum shows proton
shifts at the #4 position (1H d 2.97–2.85 ppm, diastereotopic
protons influenced by the endo/exo conformation of the
norbornane ring) that correlate with the carbon assignment
at position #62 (13C d 47.87, 47.08 ppm) as shown using
HSQC data. Further, Fig. S14 (Supporting Informationw)
confirms the presence of NOEs between protons 4, 2, 51
(Fig. 1a) and protons associated with the norbornane ring.
As expected, cyclic peptides eluted earlier than their linear
counterpart using RP-HPLC (Fig. 1c). Spectral characterization
for 1–3 can be found in Supporting Information.w
To confirm the thiol–ene reaction did not exhibit deleterious
effects on the cyclic peptides’ activity, a competitive binding
ELISA was performed. Glycoprotein IIb-IIIa (GPIIb/IIIa) is
present on platelets and its binding to fibrinogen has been
associated with platelet aggregation. RGD has been shown to
inhibit the binding of GPIIb/IIIa to fibrinogen.18 Briefly,
RGD peptide derivatives and fibrinogen were added to a
Maxisorp 96-well plate coated with GPIIb/IIIa and incubated
for 3 h. A goat polyclonal to fibrinogen antibody labelled with
HRP was added (1 h) and later detected using ABTS substrate
and the absorbance measured at 405 nm. Fig. 2 shows the
inhibition of fibrinogen binding to GPIIb/IIIa in response to 3
Further, cyclic products were also obtained using a thermal
initiation scheme as well as a solution phase photoreaction
using unprotected linear peptides (1 and 2). Isolated yield
calculations for each synthetic route can be found in Supporting
Information Table S1.w Thermal reactions performed at 65 1C
using azobisisobutyronitrile (AIBN; t1/2, 65 1C B 10 h) required
longer reaction times (B48 h) and resulted in decreased
yields relative to the on-resin photoreaction. Additionally,
linear peptide precursors were purified and solubilized in
dilute concentrations (2 mM) with DMPA and exposed
to 365 nm light to form their cyclic products. Although the
yields for the cyclization reaction were comparable to the
on-resin photoreaction, the need for two purification steps
and additional work-up led to decreased overall yield of the
final product.
Fig. 2 Inhibition of fibrinogen binding to GPIIb/IIIa in the presence
of cyclic RGD derivatives formed via thiol–ene click chemistry.
Fig. 1 (a) Chemical structure of 4 containing numbered atoms. (b) NMR spectrum (HMBC (black)/HSQC (blue) overlay) confirming bond
correlations across the thioether bond. (c) RP-HPLC chromatogram highlighting varying elution times between cyclic (4) and linear (2) purified
peptides.
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and 4 exhibiting IC50 values of 0.20 ꢂ 0.09 and 0.36 ꢂ 0.09 mM,
respectively, which are comparable to reported literature
values.18 The linear peptide (1) was less potent in inhibiting
fibrinogen binding (IC50 = 1.41 ꢂ 0.28 mM).
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In summary, we report the use of the thiol–ene click reaction
for the on-resin macrocyclization of peptides. Peptides can be
formed using commercially available amino acids without any
post-synthetic modification (1, 3). Alternatively a strained
alkene (norbornene; 2, 4) can be utilized to achieve
enhanced reaction kinetics and cyclization within 20 min. This
Communication demonstrates the thiol–ene click photo-
reaction as a facile method for the rapid synthesis (B20 min)
of cyclic peptides with improved yields relative to other
on-resin reactions.
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The authors would like to thank Cole DeForest for initial
conceptual discussions and Kristen Feaver for assistance with
the binding ELISA. This work was funded by the NIH (Grant
RO1 DK076084), HHMI, and Graduate Assistance in Areas
of National Need fellowship (A.A.A.).
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Chem. Commun., 2010, 46, 4061–4063 | 4063