Early-Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2-Cyanobenzothiazole/1,2-Aminothiol Bioorthogonal Click Reaction

Article abstract of DOI:10.1002/open.201700191

Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2-cyanobenzothiazole (CBT) and a 1,2-aminothiol moiety. This methodology allows for the facile and site-specific modificat

Full text of DOI:10.1002/open.201700191

DOI: 10.1002/open.201700191
Early-Stage Incorporation Strategy for Regioselective
Labeling of Peptides using the 2-Cyanobenzothiazole/
1,2-Aminothiol Bioorthogonal Click Reaction
Kuo-Ting Chen, Christian Ieritano, and Yann Seimbille*[a]
Herein, we describe a synthetic strategy for the regioselective
labeling of peptides by using a bioorthogonal click reaction
between 2-cyanobenzothiazole (CBT) and a 1,2-aminothiol
moiety. This methodology allows for the facile and site-specific
modification of peptides with various imaging agents, includ-
ing fluorophores and radioisotope-containing prosthetic
groups. We investigated the feasibility of an early-stage incor-
poration of dipeptide 1 into targeting vectors, such as
c[RGDyK(C)] and HER2pep, during solid-phase peptide syn-
thesis. Then, the utility of the click reaction to label bioactive
peptides with a CBT-modified imaging agent (FITC–CBT, 9) was
assessed. The ligation reaction was found to be highly selec-
tive and efficient under various conditions. The fluorescently
labeled peptides 2 and 3 were obtained in respective yields of
88 and 82% under optimized conditions.
Low-molecular-weight peptides have received increasing inter-
est as molecular imaging probes and therapeutics for
cancer.[1,2] Their short blood half-life, low toxicity of degrada-
tive products, and high specificity for biomolecular targets
make them attractive candidates for use as diagnostic
agents.[3,4] Several short peptides, such as octreotide and
Figure 1. Synthetic strategies for peptide-based imaging probes labeling.
A) Direct labeling; B) late-stage incorporation; C) early-stage incorporation.
BBN(7–14) analogs, have been previously identified as efficient
ligands to image overexpressed receptors on tumor cell surfa-
ces.[5–7] Incorporation of a radionuclide or a fluorogenic dye
into peptides provides the functionality required for cancer di-
agnosis using real-time, non-invasive imaging technologies,
such as positron emission tomography (PET), single-photon
emission computed tomography (SPECT), and fluorescence
imaging.[8–10]
of a direct-labeling approach mainly based on the side-chain
reactivity of lysine (Lys) or cysteine (Cys) residues, where the
imaging agent is coupled to the targeting vector through the
formation of either a peptide bond, thioether, or thiourea link-
age. Direct labeling is often chosen because it allows us to
make use of commercially available peptides and imaging
agents. However, these conventional conjugations often lack
regioselectivity. The functionalized imaging agent (e.g. succini-
midyl ester, maleimide, isothiocyanate) can react with multiple
residues and generate a mixture of labeled products with vary-
ing yields. Labeling on the bioactive portion of the targeting
vector may compromise its bioactivity and, therefore, tedious
purification may be required to isolate the desired product.[11]
Moreover, a distorted stoichiometry, that is, nanomolar quanti-
ties of the imaging agent versus micromolar amounts of the
targeting peptide, is applied to favor the kinetics of the bio-
conjugation.[12] Furthermore, the labeling efficiency can be af-
fected by the distribution of the functional groups of the
lysine and cysteine residues in the peptide sequence.[13] In
some cases, these intrinsic residues can become buried within
the secondary and tertiary structural conformation, resulting in
For a labeled peptide to be effective, it must maintain affini-
ty and specificity for its target. The most common strategy to
incorporate an imaging agent into a peptide-based targeting
vector is a post-synthetic modification (Figure 1A). It consists
[a] Dr. K.-T. Chen, C. Ieritano, Dr. Y. Seimbille
Life Sciences Division, TRIUMF
Vancouver, British Columbia, V6T 2A3 (Canada)
E-mail: yseimbille@triumf.ca
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under https://doi.org/10.1002/
open.201700191.
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This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
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inefficient couplings.[14] Thus, for the successful development
of molecular imaging probes, strategies avoiding non-specific
labeling must be emphasized.
We describe, herein, the preparation of peptide-based imag-
ing probes with an early-stage incorporation strategy through
a rapid CBT/1,2-aminothiol click reaction. The scope of this ap-
proach was demonstrated through the labeling of two target-
ing vectors, a cyclic and a linear peptide, with well-established
biological activity. Joshi et al. recently identified a peptide se-
quence that specifically binds to an extracellular domain of the
human epidermal growth factor 2 receptor (HER2).[6] HER2 is a
member of the ErbB tyrosine kinase family, which is significant-
ly overexpressed in many tumors. A linker containing a termi-
nal lysine residue was introduced onto the HER2 binding se-
quence for on-resin derivatization of the peptide. A fluorescent
probe was installed on the lysine after selective removal of the
Ne protecting group. Although this approach enabled selective
labeling of the lysine residue, it is not adapted to radioactive
labeling. Indeed, deprotection and cleavage of the peptide
from the solid support is done post-labeling, the labeling effi-
ciency is quite low, and the reaction time is too long. We in-
stead envisioned that Fmoc—Lys–OH could be functionalized
with Boc–Cys(Trt)–OSuc to generate Fmoc–Lys(Boc–Cys(Trt))–
OH (1) for use directly in SPPS without the need for foreign re-
agents or expensive protecting groups in our synthesis of
HER2pep (S4). Analogously, we applied this strategy to the
synthesis of c[RGDyK(C)] (S3), where the RGD motif is a known
antagonist of the avb3 integrin, a transmembrane protein that
is also overexpressed in malignant tissue. As an initial proof of
concept, we evaluated the kinetics of the CBT/Cys ligation on
these two bioactive peptides with a fluorophore functionalized
with CBT. We propose that this bioconjugation strategy not
only provides maximal versatility and modularity in developing
peptide-based imaging probes, but also introduces a new
strategy for pre-targeting imaging and therapy.
An alternative strategy to the direct labeling is to functional-
ize inherent residues with a clickable handle for late-stage la-
beling (Figure 1B). The functionality is used to incorporate an
imaging agent through a rapid and chemoselective click reac-
tion. This strategy has been successfully used in the labeling of
peptides with sensitive fluorophores or short-lived radio-
nuclides.[15–18] However, similar to the direct-labeling approach,
incorporation of the clickable handle is not selective and re-
quires an isolation step prior to the conjugation of the imaging
agent. To overcome this critical limitation, an early-stage incor-
poration strategy (Figure 1C) has been advanced to enable re-
gioselective labeling of peptide-based molecular imaging
probes. With this method, a molecule functionalized with a
clickable handle is introduced into the peptide sequence
during the synthesis, followed by the chemoselective ligation
of an imaging agent containing the complementary click func-
tionality. This strategy not only avoids the formation of multi-
labeled products, but also allows for a flexible design where
the click handle is incorporated on a non-bioactive region of
the peptide, thus preserving its native function.
Synthetic approaches to regioselectively label peptides often
implement a variety of click reactions, including the Huisgen
cycloaddition (azide/alkyne), Staudinger ligation between an
azide and phosphite to yield an iminophosphorane, and the in-
verse electron demand Diels–Alder reaction (tetrazine/trans-cy-
clooctene).[19–21] While these synthetic routes are viable labeling
methods, they require the insertion of
a non-biogenic
functionality in the peptide motif for the click reaction. Such
functional groups are not always compatible with reagents
used in peptide synthesis (i.e. piperidine and trifluoroacetic
acid). Moreover, the click reaction may involve a toxic metal
catalyst, considered too slow for short-lived radionuclides or
the click reagents might be quite unstable. Consequently,
these reactions may not be the most suitable for the early-
stage strategy.
Our strategy for the development of the peptide-based tar-
geting probes 2 and 3 is illustrated in Figure 2. Preparation of
the linear peptide chains was performed by standard Fmoc
SPPS.[28] Dipeptide 1 was designed as a Fmoc-compatible sub-
strate. The acid-labile trityl and tert-butyloxycarbonyl protect-
ing groups on 1 are easily removed in the final cleavage/de-
protection step and release the 1,2-aminothiol click functionali-
ty for subsequent coupling to a CBT-bearing imaging agent.
Synthesis of dipeptide 1 is outlined in Scheme 1. Activation of
the carboxylic acid group of commercially available Boc–
Cys(Trt)–OH (4) was accomplished through coupling with N-hy-
droxysuccinimide in the presence of dicyclohexylcarbodiimide
(DCC) in ethyl acetate (EtOAc) to yield 5. The resulting ester 5
was conjugated with Fmoc–Lys–OH in mild basic conditions to
produce the CBT-conjugating building block 1 in a satisfying
yield of 75% over the two steps.
Over the last few years, we studied the naturally occurring
2-cyanobenzothiazole (CBT)/1,2-aminothiol click reaction for
the labeling of PET imaging probes.[22,23] The bioorthogonality
as well as the high selectivity, metabolic stability, and rapid for-
mation of the resulting luciferin conjugate (approximately
three orders of magnitude faster than the Staudinger ligation)
makes this synthetic approach an attractive option for radio-
chemistry and for general bioconjugations.[24–27] Indeed, consid-
ering the fragile nature of biomolecules and the relatively
short half-life of some positron-emitting radionuclides (18F, T1/2
:
109.8 min; 68Ga, T1/2: 67.6 min), the radiolabeling procedures
should be rapid and simple. As such, we envisioned to take ad-
vantage of the facile ligation between CBT and an N-terminal
cysteine (Cys) residue, which offers a nearly ideal labeling strat-
egy based on early-stage incorporation for peptides. Insertion
of the biogenic cysteine amino acid in a non-bioactive portion
of the targeting vector warrants the compatibility with stan-
dard solid-phase peptide synthesis (SPPS) reaction conditions
and the efficient coupling of the imaging agent.
Synthesis of the bioactive peptides c[RGDyK(C)] (S3) and
HER2pep (S4) containing dipeptide 1 were performed on an
automated synthesizer by following a standard Fmoc-based
SPPS strategy (structures and experimental details provided in
the Supporting Information).[29,30] In the synthesis of the linear
portion of S3, Fmoc–Gly–OH was selected as the initial residue
for loading onto the 2-chlorotrityl chloride solid support (S1)
to prevent unwanted C-terminal epimerization during the final
cyclization step (Scheme S1).[31] The following conjugations
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Scheme 1. Synthesis of Fmoc–Lys(Boc–Cys(Trt))–OH (1). Reagents and condi-
tions: a) N-hydroxysuccinimide, DCC, EtOAc, rt, 24 h; b) Fmoc–Lys–OH, tri-
ethylamine, ACN/DMF, 408C, 16 h, 75% over two steps.
protecting groups intact. Cyclization was carried out by treat-
ment of S2 with diphenylphosphoryl azide (DPPA) under basic
and diluted conditions. Subsequent global deprotection by
treatment of the cyclic peptide with a solution of TFA/H2O/
TIPS (v/v/v=95:2.5:2.5) yielded the avb3 antagonist S3 in 7%
yield over the last two steps. S4 was prepared by a similar
Fmoc-based SPPS strategy by using a Rink amide MBHA resin.
Preparation of S4 was initiated by loading 1 onto the solid
support, followed by a successive coupling/deprotection se-
quence with the corresponding Fmoc-protected amino acids.
After cleavage, HER2pep (S4) was isolated in a yield of 68%
based on initial resin loading.
We next turned our attention to the preparation of the CBT-
linked fluorophore 9, outlined in Scheme 2. Our synthesis was
initiated by the O-alkylation of 6-hydroxy-2-cyanobenzothia-
zole (6) with N-Boc-2-bromoethyl-amine under basic conditions
to afford the N-Boc-protected CBT 7 in 83% yield. Initial at-
tempts to remove the N-Boc protecting group with TFA in di-
chloromethane (DCM) at room temperature were unsuccessful.
We presumed that alkylation of the cyano moiety was occur-
ring because of the presence of the tert-butyl cation, followed
by a transformation into N-tert-butylamide side product via hy-
drolysis. To overcome the degradation of the cyano group
Figure 2. A) General strategy for the development of peptide-based imaging
probes through SPPS and CBT/Cys click reaction. AAn: amino acid, pAAs:
protected amino acids. B) Chemical structures of 2 and 3.
with Fmoc–Arg(Pbf)–OH, 1, Fmoc–d-Tyr(tBu)–OH and Fmoc–
Asp(OtBu)–OH were performed by using HBTU/N,N-diisopropy-
lethylamine (DIPEA) as coupling reagents. Cleavage of the
Fmoc protecting group was accomplished by treatment with
20% piperidine in DMF. The protected linear pentapeptide was
selectively cleaved from the resin by using an optimized solu-
tion of acetic acid/trifluoroethanol/dichloromethane (v/v/v=
1:1:10). S2 was obtained in a yield of 89% with all side-chain
Scheme 2. Preparation of FITC–CBT (9). Reagents and conditions: a) N-Boc-2-
bromoethyl-amine, NaI, K2CO3, acetone, reflux, 24 h, 83%; b) TFA, thioanisole,
DCM, 08C to rt, 4 h, 92%; c) Fluorescein isothiocyanate, DMF, rt, 4 h, 68%.
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during deprotection, thioanisole was utilized in the reaction as
a cation scavenger. After optimization of the reaction condi-
tions, the amino derivative 8 was obtained in 92% yield with
no indication of degradative product. 8 was then conjugated
to fluorescein isothiocyanate (FITC) to produce the FITC–CBT
analogue 9 in 68% yield.
shown in Figure 3, after 1 h at room temperature, the starting
material 9 was completely consumed. Formation of FITC–
HER2pep (2) was observed by reverse-phase HPLC (RP-HPLC)
and confirmed by electrospray ionization mass spectrometry
With the fluorescent probe 9 in hand, we studied the reac-
tivity of CBT derivatives 6 and 9 against 1,2-aminothiol to form
luciferin conjugates. We first determined the second-order rate
constant of the reaction between 6 and l-cysteine by using a
high-performance liquid chromatography (HPLC)-based
assay.[24] The assay was performed at pH 6.0, 7.4, and 9.0 in
phosphate-buffered saline (PBS) and gave rate constants of
0.004, 4.5, and 18.6mÀ1 sÀ1, respectively. The value at pH 7.4 is
consistent with what was previously reported by Godinat et al.
(4.5 vs. 3.2mÀ1 À1; Figure S1).[26] The results indicate that the
s
chemoselective ligation is pH dependent, as reaction rates at
neutral and basic pH conditions are 1125 and 4650 times
higher than the rate of conjugation under slightly acidic condi-
tions. From the mechanistic point of view, the thiol group is in-
itially involved in the attack of the CBT cyano group for the for-
mation of the luciferin ring. Our observation implied that the
increasing nucleophilicity of the thiol group could accelerate
the first step of the cyclization.[32]
Figure 3. HPLC monitoring of the click reaction between FITC–CBT (9) and
HER2pep. UVA: UV absorption at 254 nm. A.U.: absorption units.
We then analyzed the second-order kinetics of the reaction
between 9 and l-cysteine, which exhibited a rate constant of
2.1mÀ1 sÀ1 at pH 7.4 in PBS (Figure S2). The data indicate that 6
and 9 have similar reactivity towards free cysteine. It suggests
that modification at the 6 position has no detrimental effect
on the reactivity of CBT. Therefore, it is possible to exploit this
position to develop a wide range of highly reactive CBT-based
reagents for the bioorthogonal ligation with 1,2-aminothiol
containing molecules for biochemical and pharmacological
studies.
(ESI-MS). The reaction mixture was then purified by RP-HPLC to
give 2 in 71% yield. Purity of the bioconjugate was estimated
to be higher than 95% by HPLC (Figure S4). Next, we evaluat-
ed the click reaction at pH 9.0 in PBS. As previously observed
with l-cysteine, the reaction of 9 with HER2pep was faster at
pH 9.0 than pH 7.4. Total consumption of 9 was observed after
30 min of reaction, resulting in a similar yield of 2 (78%) after
purification by RP-HPLC (Table 1, entry 2). To mimic in vivo con-
ditions, the reaction was performed in the presence of 10%
human serum in PBS. Despite the complex medium, the bio-
conjugation reaction was extremely efficient. 2 was isolated
with a yield of 73% after 1 h incubation (Table 1, entry 3). Simi-
lar yields were obtained for the CBT/Cys click reaction both in
the presence or absence of human serum, thereby illustrating
the high degree of selectivity of CBT-based reagents towards
the N-terminal cysteine residue. Importantly, this highlights the
potential of this bioorthogonal reaction for in vivo applications,
such as a pre-targeting methodology. The reaction yields
(88%) were increased by using DMF as the solvent and DIPEA
as the base (Table 1, entry 4). This improvement is likely the
result of a better solubility of 2 in DMF than in aqueous solu-
tion. FITC–c(RGDyK) (3) was synthesized under the optimized
conditions (Table 1, entry 5) in a yield of 82% after HPLC purifi-
cation.
To illustrate the applicability of our methodology, we investi-
gated the bioconjugation reaction between 9 and our synthet-
ic peptides. 9 was found to be stable between pH 7.0 and 9.0
in PBS or DMF-containing DIPEA, but completely decomposed
at a higher pH in PBS after 1 h incubation at room temperature
(Figure S3). Thus, the coupling reactions were evaluated in dif-
ferent neutral to slightly basic conditions. A mixture of 9
(1.0 equiv) and HER2pep (1.0 equiv) at pH 7.4 in PBS and in
the presence of tris(2-carboxyethyl)phosphine hydrochloride
(TCEP·HCl; 2.0 equiv) was monitored (Table 1, entry 1). As
Table 1. Evaluation of the click reaction between 9 and peptides under
varying conditions
We contend that this bioconjugation strategy not only pro-
vides maximal versatility and modularity in developing pep-
tide-based theranostic agents, but also introduces a new strat-
egy for the generation of imaging or therapeutic conjugates
for pre-targeting applications in biological systems. The direct
application of 1 into routine Fmoc-based SPPS synthesis makes
it an ideal candidate for the construction of bioactive peptides
that, after resin cleavage and deprotection, will yield a reactive
target molecule that does not require further derivatization
Entry Conditions
Product Yield
[%][a]
1
2
3
4
5
9, HER2pep, TCEP, PBS, pH 7.4, 1 h
2
2
2
2
3
71
78
73
88
82
9, HER2pep, TCEP, PBS, pH 9.0, 0.5 h
9, HER2pep, 10% human serum, PBS, pH 7.4, 1 h
9, HER2pep, DIPEA, TCEP, DMF, 1 h
9, c[RGDyK(C)], DIPEA, TCEP, DMF, 1 h
[a] Isolated yield.
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temperature for 24 h. The reaction mixture was filtered off and the
solvent layers were concentrated and purified by flash column
chromatography (EtOAc/hexanes=1:4, silica gel) to give 5 as a
white solid (6.1 g, 10.8 mmol, quantitative yield). The crude prod-
uct 5 was directly used for the next step without further purifica-
tion. Compound 5 (6.1 g, 10.8 mmol) and triethylamine (2.0 mL,
14.3 mmol) were added to a suspension of Fmoc–Lys–OH (4.80 g,
13.0 mmol) in ACN (50 mL) and DMF (50 mL). The reaction mixture
was stirred at 408C. After stirring for 16 h, the reaction mixture was
diluted with EtOAc (200 mL) and washed with 1.0N HCl(aq) (2ꢂ
300 mL) and H2O (2ꢂ300 mL). The organic layers were dried over
MgSO4, concentrated, and purified by column chromatography
(EtOAc/hexanes=2:1, silica gel) to give 1 as a white solid (6.5 g,
prior to conjugation with an imaging agent. Moreover, the in-
corporation of the fluorophore at the 6 position of CBT did not
hamper its reactivity towards 1,2-aminothiol. It confirms the
possibility to functionalize CBT with other imaging agents, in-
cluding radio-metal chelators for PET or SPECT imaging. To our
delight, the bioconjugation proceeded extraordinarily well
under biological conditions. Even in the presence of human
serum, the specificity and efficiency of the CBT/1,2-aminothiol
ligation was unaltered. Potential of this bioorthogonal cycload-
dition for in vivo applications has been highlighted and efforts
are underway to optimize the kinetics of the ligation and to
apply this methodology to the preparation of theranostics by
conjugation of a chelator, enabling the coordination of an
imaging and a therapeutic radio metal.
1
7.9 mmol, 75% over 2 steps). H NMR (400 MHz, CDCl3) d 7.75 (d,
2H, J=7.5 Hz, ArH), 7.62 (d, 2H, J=7.0 Hz, ArH), 7.19–7.43 (m, 19H,
ArH), 6.46 (br, 1H, NH), 5.82 (br, 1H, NH), 5.36 (br, 1H, NH), 4.35–
4.42 (m, 3H), 4.21 (t, 1H, J=6.9 Hz), 3.89 (br, 1H), 3.19 (m, 2H),
2.49–2.72 (m, 2H), 1.82–1.94 (m, 1H), 1.72–1.79 (m, 1H), 1.36–1.55
(m, 2H), 1.42 (s, 9H, RC(CH3)3). LC-MS (ESI) m/z: [MÀH]À 812.5.
Experimental Section
General Information
tert-Butyl (2-((2-Cyanobenzo[d]thiazol-6-yl)oxy)ethyl)carba-
mate (7)
All chemicals were obtained from commercial suppliers and used
without further purification. All solvents were anhydrous grade
unless indicated otherwise. Hexanes are mixture of isomers. All
non-aqueous reactions were performed in oven-dried glassware
under a slight positive pressure of argon unless otherwise noted.
Reactions were magnetically stirred and monitored by thin-layer
chromatography (TLC) on Merck aluminum-backed pre-coated
plates (Silica gel 60 F254), and visualized with ultraviolet light or
by staining with 10% phosphomolybdic acid in neat ethanol. Flash
chromatography was performed on silica gel of 40–63 mm particle
size. Concentration refers to rotary evaporation. RP-HPLC was car-
ried out on an Agilent 1200 series system equipped with a diode
array detector. Yields are reported for spectroscopically pure com-
pounds. NMR spectra were recorded in D2O, CDCl3, or CD3OD in di-
luted solutions on a Bruker AVANCE 400 at ambient temperature.
Chemical shifts are given as d values in ppm and coupling con-
stants J are given in Hz. The splitting patterns are reported as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (dou-
blet of doublets), and br (broad signal). Low-resolution ESI mass
spectra were recorded on a Bruker HCT spectrometer. Fmoc-based
SPPS was conducted on an Aapptec Focus Xi automated peptide
synthesizer.
The mixture of 6-hydroxy-2-cyanobenzothiazole (6, 668 mg,
3.76 mmol), NaI (168 mg, 1.13 mmol), and K2CO3 (1.29 g,
9.41 mmol) in anhydrous acetone (20 mL) was stirred at room tem-
perature and treated with N-Boc-2-bromoethyl-amine (1.26 g,
5.64 mmol). The reaction mixture was heated under reflux for 24 h.
After reaction was completed (monitored by TLC), the reaction
mixture was concentrated, extracted with EtOAc (100 mL) and
washed with H2O (2ꢂ200 mL). The organic layer was dried over
MgSO4 and concentrated. Purification by column chromatography
(100% DCM to EtOAc/DCM=1:1, silica gel) afforded compound 7
1
as a white solid (1.0 g, 3.13 mmol, 83%). H NMR (400 MHz, CDCl3)
d 8.07 (d, 1H, metaJ=8.8 Hz, ArH), 7.33 (d, 1H, paraJ=2.4 Hz, ArH),
7.39 (dd, 1H, para,metaJ=2.4, 8.8 Hz, ArH), 4.96 (br, 1H, NH), 4.10 (t,
3
3
2H, J=5.2 Hz, OCH2R), 3.58 (q, 2H, J=5.2 Hz, RCH2N), 1.43 (s, 9H,
RC(CH3)3). LC-MS (ESI) m/z: [M+Na]+ 342.1.
6-(2-Aminoethoxy)benzo[d]thiazole-2-carbonitrile (8)
7 (791 mg, 2.48 mmol) and thioanisole (2.5 mL) in DCM (5 mL) was
stirred at 08C for 3 min. Trifluoroacetic acid (2.5 mL) in DCM (5 mL)
was slowly added into the solution. The reaction mixture was
warmed to room temperature and stirred for 4 h. The reaction mix-
ture was concentrated and purified by column chromatography
(EtOAc/hexanes=1:4 to 100% EtOAc to EtOAc/MeOH=4:1, silica
gel) to afford 8 as a yellow solid (500 mg, 2.28 mmol, 92%).
1H NMR (400 MHz, CD3OD) d 8.11 (d, 1H, metaJ=9.2 Hz, ArH), 7.74
(d, 1H, paraJ=2.4 Hz, ArH), 7.40 (dd, 1H, para,metaJ=2.4, 9.2 Hz, ArH),
HPLC Conditions
The analyses of FITC-labeled CBT or peptides were performed by
HPLC on an analytical RP-C18 column (Aquaꢁ, Phenomenex, 5 mm,
4.6ꢂ250 mm) with a gradient elution of acetonitrile (ACN; 10% to
90% in H2O, containing 0.1% TFA) at a flow rate of 1 mLminÀ1
over 30 min. The products were monitored at 254 nm by a UV de-
tector. The purification of the FITC-labeled peptides was performed
on a semi-preparative RP-C18 column (Lunaꢁ, Phenomenex, 5 mm,
10.0ꢂ250 mm) at a flow rate of 3 mLminÀ1 over 45 min. The prod-
ucts were monitored at 254 nm by a UV detector.
3
3
4.36 (t, 2H, J=7.8 Hz, OCH2R), 3.44 (t, 2H, J=7.8 Hz, RCH2N). LC-
MS (ESI) m/z: [M+H]+ 220.2.
1-(2-((2-Cyanobenzo[d]thiazol-6-yl)oxy)ethyl)-3-(3’,6’-dihy-
droxy-3-oxo-3H-spiro[isobenzofuran-1,9’-xanthen]-5-yl)thio-
urea (9)
Chemical Synthesis
To a solution of 8 (60 mg, 0.27 mmol) in DMF (4 mL) was added flu-
orescein isothiocyanate (100 mg, 0.25 mmol) at room temperature.
The reaction mixture was stirred in the dark. After stirring for 4 h,
the crude product was concentrated, re-dissolved in MeOH and
then co-evaporated with silica gel for coating. The crude com-
Fmoc–Lys(Boc–Cys(Trt))–OH (1)
N-Hydroxysuccinimide (1.35 g, 11.8 mmol) and DCC (2.67 g,
12.9 mmol) were added to a solution of Boc–Cys(Trt)–OH (4) (5.0 g,
10.7 mmol) in EtOAc (100 mL). The mixture was stirred at room
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pound on silica gel was purified by column chromatography
(EtOAc/DCM=1:4 to 100% EtOAc to EtOAc/MeOH=1:9 to 1:4) to
give 9 as an orange solid (112 mg, 0.18 mmol, 68%). 1H NMR
(400 MHz, CD3OD) d 8.13 (d, 1H, J=2.0 Hz, ArH), 8.07 (d, 1H, J=
9.2 Hz, ArH), 7.71–7.75 (m, 2H, ArH), 7.39 (dd, 1H, J=2.4, 9.2 Hz,
ArH), 7.14 (d, 1H, J=8.4 Hz, ArH), 6.63–6.67 (m, 4H, ArH), 6.53 (dd,
2H, J=2.4, 8.4 Hz, ArH), 4.38 (t, 2H, J=5.6 Hz, OCH2R), 4.10 (t, 2H,
3J=5.6 Hz, RCH2N). LC-MS (ESI) m/z: [MÀH]À 606.9.
3
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FITC–HER2pep (2)
The HER2 peptide (S4) (3.3 mg, 2.5 mmol), DIPEA (9.5 mL, 25 mmol)
and TCEP·HCl (1.4 mg, 5.0 mmol) were dissolved in DMF (2.0 mL)
and incubated for 30 min at room temperature.
2.5 mmol) was then added into the above solution, and stirred at
room temperature for 1 h. The reaction was monitored and puri-
fied by semi-preparative RP-C18 HPLC (retention time, tR =
16.1 min) to give compound 2 as a yellow solid (4.2 mg, 2.2 mmol,
88%). LC-MS (ESI) m/z: [M+2H]2+ 963.5 and [M+3H]3+ 642.6.
9 (1.6 mg,
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Biomol. Chem. 2016, 14, 4809.
FITC–c(RGDyK) (3)
The synthesis of 3 was carried out from S3 (1 mg, 1.4 mmol), as de-
scribed for the preparation of 2 (yield of 3: 1.5 mg, 1.1 mmol, 82%).
LC-MS (ESI) m/z: [M+H]+ 1314.6.
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Acknowledgements
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2011, 133, 11418.
We gratefully acknowledge the Leenaards Foundation (grant #
3699) and NSERC for financial support. We thank the NMR and
MS facilities in the Chemistry department at the University of Brit-
ish Columbia for providing support and resources. We would like
to thank the Life Sciences team at TRIUMF for technical
assistance.
Conflict of Interest
The authors declare no conflict of interest.
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Keywords: biorthogonal chemistry
·
click reactions
·
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fluorescent probes
peptide synthesis
·
peptide modifications
·
solid-phase
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3565.
Received: November 29, 2017
[2] X. Sun, Y. Li, T. Liu, Z. Li, X. Zhang, X. Chen, Adv Drug Deliv Rev 2017,
110, 38.
Version of record online && &&, 2018
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ÝÝ These are not the final page numbers!

COMMUNICATIONS
Pep talk: A strategy for directly labeling
peptides is described by using a 2-cya-
nobenzothiazole (CBT)/1,2-aminothiol
click reaction. Two peptides are modi-
fied with a clickable amino acid during
the synthesis by using Fmoc solid-phase
peptide synthesis. The following conju-
gation with a CBT-bearing fluorophore
is demonstrated to be rapid and che-
moselective under various conditions.
This synthetic strategy is anticipated to
be a useful tool in the development of
new peptide-based imaging probes for
biomedical applications.
K.-T. Chen, C. Ieritano, Y. Seimbille*
&& – &&
Early-Stage Incorporation Strategy for
Regioselective Labeling of Peptides
using the 2-Cyanobenzothiazole/
1,2-Aminothiol Bioorthogonal Click
Reaction
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&
These are not the final page numbers! ÞÞ