Gold-mediated selective cysteine modification of peptides using allenes

Article abstract of DOI:10.1039/c2cc38214h

A new approach for selective modification of cysteine-containing peptides through gold-mediated oxidative allene-thiol coupling reaction in aqueous medium is developed.

Full text of DOI:10.1039/c2cc38214h

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Gold-mediated selective cysteine modification of
peptides using allenes†
Anna On-Yee Chan,a Johnson Lui-Lui Tsai,a Vanessa Kar-Yan Lo,a Gai-Li Li,b
Man-Kin Wong*b and Chi-Ming Che*a
Cite this: Chem. Commun., 2013,
49, 1428
Received 14th November 2012,
Accepted 24th December 2012
DOI: 10.1039/c2cc38214h
www.rsc.org/chemcomm
A new approach for selective modification of cysteine-containing with nucleophiles to give a diversity of synthetically useful 1,2/
peptides through gold-mediated oxidative allene–thiol coupling 1,3-adducts (Scheme 1).7,8 In this work, a diversity of allenes were
reaction in aqueous medium is developed.
successfully activated by gold compounds to couple with the thiol
group of cysteine-containing peptides to afford novel hydroxy vinyl
Selective biomolecule modification is an important chemical biol- thioethers instead of the commonly observed 1,2/1,3-adducts
ogy tool for manipulating the properties of biomolecules and (Scheme 1).9 To our knowledge, this is the first gold-mediated reaction
investigating their functions in complex biological systems.1,2 Con- for cysteine modification of peptides using allenes.
ventional chemical approaches for bioconjugation of peptides and
At the outset, allene 1a (molecular mass of 192 Da, 1 mM) was
proteins involve covalent bond formation reactions between the treated with peptide STSSSCNLSK (2) (100 mM) in the presence of
nucleophilic amino (–NH2) and thiol (–SH) groups of amino acids AuCl–AgOTf (1 mM/1 mM) in 100 mL of CH3CN–H2O (3 : 7) solution
with electrophilic reagents.1,2 Yet, cross-reactivity with other amino at room temperature for 1 h. As confirmed by LC-MS/MS analysis of
acid residues is observed, especially in high pH medium. Transi- the crude reaction mixture, the cysteine residue of peptide 2 was
tion metal catalysis renders numerous novel organic transforma- modified to give peptide 2a in 73% conversion together with a trace
tion reactions possible and has been widely used in organic amount of dimerized peptide 2a0 (Fig. 1). The molecular mass
synthesis. Recently, promising strategies have been developed to increased by 208 Da revealing the incorporation of one allene 1a
apply transition metal catalysis for bioconjugation.3 The excellent and one oxygen atom on peptide 2. No cysteine modification was
reactivity and selectivity of transition metal catalysis significantly observed in the absence of AuCl and/or AgOTf, and no 1,2/
expand the scope of the chemical reaction tool boxes for bio- 1,3-adducts of peptide 2 with allene 1a (a mass increase by
molecule modification. In the past several years, we have developed 192 Da) were found. The identity of the residue generated from
efficient transition metal-catalyzed reactions for modification of the reaction was confirmed to be Au(0) and AgCl by XRD analysis.
proteins and peptides.4
Optimization of the reaction conditions for cysteine modifica-
Gold catalysis is presently an area of considerable interest in tion of peptide 2 using the gold-mediated allene coupling reaction
organic synthesis, owing to its excellent reactivity and selectivity, was conducted by varying the equivalents of AuCl–AgOTf, reaction
compatibility with aqueous reaction medium, and mild reaction temperature, and the gold compounds. Our findings suggested that
conditions.5,6 In particular, gold compounds are effective in 10 equivalents of AuCl–AgOTf gave the highest conversion to allene-
activating the p-system of allenes for subsequent addition reactions modified peptide 2a with the least amount of dimerized peptide
2a0. Increasing the reaction temperature to 37 or 55 1C gave no
a State Key Laboratory of Synthetic Chemistry, Department of Chemistry and Open
Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug
Discovery and Synthesis, The University of Hong Kong, Pokfulam Road,
Hong Kong, China. E-mail: cmche@hku.hk; Fax: +852-2857-1586
b Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hung Hom, Hong Kong, China.
significant increase in the conversion of 2, while decreasing the
reaction temperature to 4 1C led to a slight reduction of conversion
of 2 to 65%. Room temperature was thus chosen for subsequent
studies. Replacing AuCl with Au(PPh3)Cl, Au(TPP)Cl [H2TPP = meso-
tetraphenylporphyrin] or KAuCl4 in the modification of peptide
E-mail: bcmkwong@polyu.edu.hk; Fax: +852-2364-9932
† Electronic supplementary information (ESI) available: Experimental procedures
for preparation of allenes and modification of peptides, NMR spectra of allenes,
results of optimization of reaction conditions for cysteine modification of peptide
2, mass reconstruction spectrum of LC-MS analysis of modification of peptide 2,
MS/MS spectra of modified peptides, LC-MS spectra of DTT treated RNaseA and
modified RNaseA polypeptide, MS/MS spectrum of the trypsin digested modified
RNaseA polypeptide. See DOI: 10.1039/c2cc38214h
Scheme 1 Addition reactions of nucleophiles to allenes.
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Table 1 Cysteine modification of peptides using allene 1g in the presence of
AuCl–AgOTf a
Entry
Peptide sequence
Conversionb (%)
1
2
3
4
5
STSSSCNLSK (2)
AYEMWCFHQR (3)
GSH (4)
CSKFR (5)
KSTFC (6)
90
94
90
97
82
a
Peptide (0.01 mmol), allene 1g (0.1 mmol), AuCl (0.1 mmol) and AgOTf
b
(0.1 mmol) in CH3CN–H2O (2 : 1) solution (100 mL), 1 h, r.t. Determined
by total ion count (TIC) of LC-MS analysis and only a trace amount of
dimerized peptide was found.
Fig. 1 (a) Cysteine modification of peptide 2 using allene 1a in the presence of
AuCl–AgOTf. (b) Structure of allene-modified peptide 2a.
50 mL) at 70 1C for 10 min to obtain 0.5 mM of reduced RNaseA.
As confirmed by LC-MS analysis, 8 free cysteine residues were
generated after the DTT treatment of RNaseA. The DTT-treated
RNaseA was allowed to react with allene 1a (10 equivalents) in
the presence of AuCl–AgOTf (10 equivalents each) for 1 h at
room temperature (Scheme 2). LC-MS analysis showed that the
conversion to the singly modified DTT-treated RNaseA was 24%
whereas the conversion to di-modified RNaseA polypeptide was
9%. LC-MS/MS analysis of the trypsin-digested reaction
mixtures of the modified RNaseA polypeptides revealed that
the modification using allene 1a selectively occurred at Cys95.
To provide support for the structure of the allene–cysteine
adducts, we have performed the coupling reactions of thiophenol
(7) and benzyl thiol (8) with allene 1a (Scheme 3). Z-Hydroxy vinyl
thioether 7a10 was obtained in 82% yield using thiophenol (7) with a
trace amount of disulfide observed. The yield of the corresponding
Z-hydroxy vinyl thioether 8a obtained using benzyl thiol (8) was
22%. The benzyl thiol dimer was obtained in 59% yield.
2 using allene 1a resulted in a significant decrease in the
conversion of peptide 2 (from 73% to 9%, 8%, and 10%, respec-
tively). Thus, AuCl–AgOTf was used in the following studies. The
use of CH3CN in the reaction mixture could be reduced to 20%
without altering the conversion of peptide 2 (for details, please refer
to the ESI†).
Under the optimized reaction conditions, gold-mediated
selective cysteine modification of peptide 2 using allenes 1b–h
has been achieved. As confirmed by LC-MS/MS analysis, the
conversions of the corresponding allene-modified peptides were
found to be 60–90% (Fig. 2). Dimerized peptide 2a0 was obtained in
a trace amount, and no 1,2/1,3-adducts were observed. The present
gold-mediated bioconjugation reaction is compatible with aldehyde-,
alcohol-, and coumarin-containing allenes (1d, 1g, and 1h).
Apart from peptide 2, cysteine modification of peptides
AYEMWCFHQR (3), glutathione (GSH; 4), CSKFR (5) and KSTFC
(6) by the gold-mediated allene coupling reaction was examined
using allene 1g. The conversions to the corresponding allene-
modified peptides were found to be 94, 90, 97, and 82%,
respectively (Table 1, entries 2–5). As confirmed by LC-MS/MS
analysis, the excellent selectivity of the present gold-mediated
bioconjugation reaction is exemplified by the exclusive cysteine
modification of peptides 2–6. Moreover, no modification was
observed for a non-cysteine-containing peptide YTSSSKNVVR as
confirmed by LC-MS analysis, providing further support for the
excellent chemoselectivity of the present method.
The regiochemistry of the allene–cysteine adducts was
studied using the asymmetrically substituted allene 1g and
thiophenol (7) as the model reaction. Isomers 7b and 7c were
obtained in a ratio of 3 : 1 and their structures were confirmed
1
by H NMR analysis (Scheme 4).
In the present work, allenes (1a–h) were activated by AuCl–AgOTf
for oxidative coupling reaction with the cysteine thiol group. The
allene-modified peptides contain a hydroxy vinyl thioether moiety
instead of the literature reported 1,2/1,3-adducts. A proposed
reaction mechanism for this oxidative allene–thiol coupling is
depicted in Scheme 5. Thiol–ene radical intermediate is generated
from the reaction of AuCl–AgOTf, allene, and thiol. Through the
capture of molecular oxygen, hydrogen atom transfer, and dispro-
portionation, the thiol–ene radical intermediate is converted into a
hydroxy vinyl thioether.
Next we proceeded to study gold-mediated cysteine modifi-
cation of a more complex peptide generated from dithiothreitol
(DTT) treated RNaseA. RNaseA solution in H2O (1 mmol mLꢀ1
50 mL) was mixed with DTT aqueous solution (400 mmol mLꢀ1
,
,
This reaction mechanism is proposed on the basis of literature
reports11 and mechanistic studies. The coupling reaction conducted
Fig. 2 Structures of allenes 1b–h and the corresponding conversions of peptide 2
obtained in the modification using allenes 1b–h in the presence of AuCl–AgOTf.
Scheme 2 Cysteine modification of DTT-treated RNaseA.
Chem. Commun., 2013, 49, 1428--1430 1429
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We are thankful for the financial support from The University
of Hong Kong (University Development Fund), Hong Kong
Research Grant Council (PolyU 5027/09P) and RGC Collaborative
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1430 Chem. Commun., 2013, 49, 1428--1430
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