Modular synthesis of photocleavable peptides using click chemistry

Article abstract of DOI:10.1016/j.tetlet.2012.02.002

A modular synthesis of photocleavable peptides was developed. Peptide based kinase substrates were modified on solid support with a traceless linker derived from 1-(2-nitrophenyl)propargyl alcohol and coupled to azide functionalized cell-penetrating pepti

Full text of DOI:10.1016/j.tetlet.2012.02.002

Tetrahedron Letters 53 (2012) 1933–1935
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Modular synthesis of photocleavable peptides using click chemistry
⇑
Joshua A. Baccile, Mark A. Morrell, Ross M. Falotico, Brandon T. Milliken, Daniel L. Drew, Francis M. Rossi
Department of Chemistry, SUNY Cortland, Cortland, NY 13045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A modular synthesis of photocleavable peptides was developed. Peptide based kinase substrates were
modified on solid support with a traceless linker derived from 1-(2-nitrophenyl)propargyl alcohol and
coupled to azide functionalized cell-penetrating peptides using Cu(I) catalyzed click chemistry.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 January 2012
Revised 31 January 2012
Accepted 1 February 2012
Available online 9 February 2012
Keywords:
Click chemistry
Caged compounds
Cell-penetrating peptides
Photocleavable linkers
Introduction
disulfide linkers can be effective, it would be desirable to release
the bioactive molecule independently of the bioreductive capacity
Because of its ease of use and orthogonality to numerous func-
tional groups, the copper catalyzed azide alkyne cycloaddition
(CuAAC), the most common example of click chemistry, has be-
come a method of choice for linking complex molecules.1,2 In addi-
tion to its widespread use with molecules of biological interest,3,4
the reaction has also seen applications in areas such as polymer
and surface chemistry.5–7
of a particular sub-cellular compartment and with the additional
spatial and temporal control afforded by photo-induced cleavage.
In addition, because the efficacy of CPPs is known to vary consid-
erably between cell types, it is often necessary to prepare and
evaluate multiple CPP-bioactive peptide conjugates.29–31 Thus, a
modular method for attaching CPPs to bioactive peptides is
attractive.
In some instances it is desirable to couple complex molecules
through a linker that can be subsequently cleaved. Photocleavable
linkers, which can be cleaved with spatial and temporal control,
have been used to conjugate polyethylene glycols to proteins,8
transiently link biotin to biomolecules,9–11 create ‘caged’ fluores-
cent biomolecular probes,12,13 attach a leader sequence to pep-
tides,14 control the sub-cellular localization of peptides,15 attach
mass spectrometry tags to bioanalytical probes,16 and modify sur-
faces.17–20
We became interested in using a photocleavable click linker
to transiently conjugate cell-penetrating peptides (CPPs) to bioac-
tive peptides. CPPs can facilitate the transport of normally
membrane-impermeant molecules.21–23 Unfortunately, the CPP
approach has drawbacks. CPP conjugates frequently become local-
ized in sub-cellular compartments and in the cell membrane.24–26
In addition, the presence of a CPP can alter or attenuate the activ-
ity of the attached cargo.27,28 Typically these problems are ad-
dressed by conjugating the CPP to the molecule of interest with
a cleavable linker such as disulfide. Once in the cell, disulfide is
cleaved by the reductive environment of the cytoplasm. Although
Linker synthesis and model study
Click chemistry is ideal for the modular assembly of peptides.
Previous reports demonstrated the non-modular synthesis of
CPP-linked bioactive peptides and have shown the utility of CuAAC
for the attachment of CPPs to bioactive molecules.32–35
The extension of this chemistry to the preparation of photocleav-
able peptides requires a readily available photocleavable linker. The
ideal linker should be easily prepared, react readily with amines,
and upon photocleavage be traceless with respect to the bioactive
peptide. Recently several photocleavable click chemistry linkers
have been reported but their preparations require multi-step syn-
theses.8,10,12,14,36 We envisioned that 1-(2-nitrophenyl)propargyl
alcohol 1, which can be prepared in one step from commercially
available starting materials,37 could serve as a photocleavable
linker. As shown in Scheme 1, treatment of 2-nitrobenzaldehyde
with ethynylmagnesium bromide gave propargyl alcohol 1 in a
93% yield. Treatment of 1 with carbonyldiimidazole, afforded the
amine-reactive imidazolide 2 with an isolated yield of 95%.
Before applying our method to peptide synthesis, we decided to
demonstrate the feasibility of the approach with compounds that
could be spectroscopically characterized as shown in Scheme 2.
⇑
Corresponding author. Tel.: +1 607 753 5580; fax: +1 607 753 5928.
E-mail address: frank.rossi@cortland.edu (F.M. Rossi).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.002

1934
J. A. Baccile et al. / Tetrahedron Letters 53 (2012) 1933–1935
Synthesis of photocleavable peptides
We applied our method to the modular synthesis of CPP-linked
photocleavable bioactive peptides as shown in Scheme 3. The
sequences of the peptides used are shown in Table 1.
Scheme 1. Synthesis of photocleavable linker.
We chose to link the cell-penetrating peptides TAT and MyrK4
to peptide substrates for Abl kinase and CaM kinase II. Both TAT
and MyrK4 have been shown to transport the Abl kinase substrate
into cells,26 and TAT is known to be effective at transporting a CaM
kinase II substrate across cell membranes.32 Peptide substrates 5a
(Abl) and 5b (CaM kinase II) were synthesized on Rink amide resin
using Fmoc-solid phase peptide synthesis. The photocleavable
linker was attached to the N-terminus by reacting the peptide of
interest with imidazolide 2 and HOBt. Alternately, the linker-
labeled peptide was prepared by generating compound 2 in situ
from the reaction of 1 and carbonyldiimidazole, effectively giving
a linker that could be prepared in one step from commercially
available starting materials. Cleavage of the peptides from solid
support and deprotection of the amino acid side chains gave the
desired linker-modified peptides 6a and 6b as a mixture of diaste-
reomers, which in the case of the CaM kinase II substrate could be
partially resolved by HPLC.
Scheme 2. Model study.
Peptides 6a and 6b were each coupled to the azide labeled CPPs
7a (TAT) and 7b (MyrK4) using copper catalyzed click chemistry to
give photocleavable peptide conjugates 8a–d.
Scheme 3. Peptide coupling.
Reaction of 2 with benzyl amine gave carbamate 3. Coupling of 3
with 6-azidohexanoic acid under click conditions gave 4 in a 70%
yield. Interestingly, we saw no evidence of the by-products ob-
served by Bertrand and Gesson with the click reaction of the struc-
turally related dimethyl propargyl benzyl carbamate.38
Figure 1. Photolysis of photocleavable peptide 8d. Peptide was irradiated with UV
light for the indicated period of time and analyzed by HPLC (monitored at 210 nm).
Peaks at 16.68 and 16.78 min correspond to peptide 8d; peak at 12.69 min
corresponds to released peptide 9; top trace, authentic sample 9.
Table 1
Peptides prepared in Scheme 3
Entry Peptidea
Cell-penetrating peptidea (CPP) Yieldb (%) HPLC retention time (min)
Molecular formula
m/z calcd m/z obsd
6a
6b
7a
7b
8a
8b
8c
8d
9
EAIYAAPFAKKK (Abl)
KKALHRQETVDAL (CaMKII) N/A
N/A
N/A
EAIYAAPFAKKK (Abl)
EAIYAAPFAKKK (Abl)
KKALHRQETVDAL (CaMKII) YGRKKRRQRRR (TAT)
KKALHRQETVDAL (CaMKII) K( -myristoyl)KKK (MyrK4)
KKALHRQETVDAL N/A
N/A
28
16.14e
C74H108N17O19 [M+H]+
C75H120N23O23 [M+H]+
C70H128N35O15 [M+H]+
C44H87N12O6 [M+H]+
1538.801 1538.917
1710.893 1710.945
1699.033 1698.613
46, 37c
13
9.23, 9.30d,f 14.46, 14.52e,f
YGRKKRRQRRR (TAT)
K( -myristoyl)KKK (MyrK4)
YGRKKRRQRRR (TAT)
K( -myristoyl)KKK (MyrK4)
12.95e
19.11d
13.93e
12.54d
13.38e
e
16
29
38
44
39
33
879.687
879.690
1618.9
1209.2
1136.6
C144H236N52O34 [M+2H]2+ 1618.9
C118H195N29O25 [M+2H]2+ 1209.2
C145H250N59O37 [M+3H]3+ 1136.6
e
e
11.70, 11.84d,f16.68, 16.78e,f C119H207N35O29 [M+2H]2+ 1295.290 1295.289
12.69e
C65H114N22O19 [M+H]+
1507.87
1507.86
a
b
c
Peptides C-terminal amidated.
Based on the weight of HPLC purified, lyophilized powder.
In situ generation of 2.
d
HPLC gradient A: 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B); 95% A, 2 min; 95% A to 20% A, 20 min; flow rate = 2 mL/min; Jupiter Proteo column
, 150 Â 4.6 mm).
(4
l
e
HPLC gradient B: gradient C 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B); 100% A, 4 min; 100% A to 20% A, 20 min; flow rate = 1 ml/min; Jupiter
Proteo column (4
l
, 150 Â 4.6 mm).
f
Mixture of diastereomers.

J. A. Baccile et al. / Tetrahedron Letters 53 (2012) 1933–1935
1935
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nitroveratryl) with commercially available aldehyde precursors.
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Acknowledgments
The authors would like to thank Dr. Rick Geier and the Colgate
University Chemistry Department for the use of their MALDI-TOF
spectrometer, and Dr. Nancy Allbritton of the University of North
Carolina, Chapel Hill for the generous gift of peptides 5a and 5b.
This research was partially supported by a Grant from the Research
Corporation for Science Advancement to FMR.
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Supplementary data (experimental details for the preparation
of compounds 1–9, NMR spectra of compounds 1–4 and HPLC
chromatographs of compounds 6a–9) associated with this Letter
can be found, in the online version, at doi:10.1016/j.tetlet.
2012.02.002.
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