Electron-deficient alkynes as cleavable reagents for the modification of cysteine-containlng peptides in aqueous medium

Article abstract of DOI:10.1002/chem.200800669

An efficient method has been developed for the chemoselective cysteine modification of unprotected peptides and proteins in aqueous media through the formation of a vinyl sulfide linkage by using electron-deficient alkynes, including alkynoic amides, esters and alkynones. The terminal alkynone-modified peptides could be converted back into the unmodified peptides (81 % isolated yield) by adding thiols under mild conditions. The usefulness of this thiol-assisted cleavage of the vinyl sulfide linkage in peptides has been exemplified by the enrichment of a cysteine-containing peptide (71% recovery) from a mixture of cysteine-containing and non-cysteine-containing peptides.

Full text of DOI:10.1002/chem.200800669

FULL PAPER
DOI: 10.1002/chem.200800669
Electron-Deficient Alkynes as Cleavable Reagents for the Modification of
Cysteine-Containing Peptides in Aqueous Medium
Hoi-Yan Shiu, Tak-Chung Chan, Chi-Ming Ho, Yungen Liu, Man-Kin Wong,* and
Chi-Ming Che*[a]
Abstract: An efficient method has
been developed for the chemoselective
cysteine modification of unprotected
peptides and proteins in aqueous
media through the formation of a vinyl
sulfide linkage by using electron-defi-
cient alkynes, including alkynoic
amides, esters and alkynones. The ter-
minal
alkynone-modified
peptides
by adding thiols under mild conditions.
The usefulness of this thiol-assisted
cleavage of the vinyl sulfide linkage in
peptides has been exemplified by the
could be converted back into the un-
modified peptides (81% isolated yield)
enrichment of
a cysteine-containing
Keywords: alkynes · chemoselectiv-
ity · cleavage reactions · cysteine ·
peptides
peptide (71% recovery) from a mix-
ture of cysteine-containing and non-
cysteine-containing peptides.
Introduction
cation can be divided into two categories. Alkylating re-
agents form the first category, including haloacyl compounds
and maleimides, which modify cysteine units by nucleophilic
substitution and conjugate addition reactions, respectively,
as depicted by Equations (1) and (2) of Scheme 1. Both
Site-selective modification of proteins is of growing impor-
tance because it leads to novel bioconjugates that are useful
for biological studies of, for example, protein–protein inter-
actions and live cell imaging.[1] Despite the advancements in
chemical synthesis over the past few decades, there are only
a handful of synthetic methods that can be applied to pro-
tein modification. Most of the chemical reactions used in or-
ganic synthesis are not compatible with aqueous media or
the sensitive functional groups of proteins. Thus, there is a
continued interest in the development of new methods for
the selective modification of peptides and proteins under
mild conditions.[2] Selective cysteine modification is a partic-
ularly useful bioconjugation reaction owing to the high reac-
tivity of the cysteine sulfhydryl group and to the relatively
sparse occurrence (1.7%) of cysteine units in peptides and
proteins.[1] The reagents commonly used for cysteine modifi-
[a] H.-Y. Shiu,+ Dr. T.-C. Chan,+ Dr. C.-M. Ho, Dr. Y. Liu,
Dr. M.-K. Wong, Prof. Dr. C.-M. Che
Scheme 1. Methods for cysteine modification.
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)
Fax : (+852)-2857-1586
À
methods involve sulfur carbon bond formation. However,
cross-reactivity with other amino acids such as histidine, me-
thionine, lysine and tyrosine has been reported, especially in
media at high pH.[1] Thiol–disulfide exchange reagents are
another class of reagents used for cysteine modification and
E-mail: mkwong@hkusua.hku.hk
cmche@hku.hk
[+] Contributed equally to this work.
À
they act through the reversible formation of a sulfur sulfur
bond [Scheme 1, Eq. (3)]. They can lead to specific modifi-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800669.
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3839

cation of biomolecules, but the
inherent instability of the disul-
fide bond limits their applica-
tions in this endeavour. Kent
and co-workers pioneered the
selective coupling of N-terminal
cysteine residues with C-termi-
nal thioesters (native chemical
ligation) to achieve chemical
protein synthesis.[3] Recently,
Crich and co-workers reported
the use of allylic selenosulfides
for chemical ligation of cysteine
derivatives in protic media.[4]
Despite these advances, the de-
velopment of new reagents for
the cysteine modification of
peptides and proteins with che-
moselectivity remains a chal-
lenge.
Scheme 2. Chemoselective cysteine modification and cleavage of the vinyl sulfide linkage.
Conjugate addition of thiols
to electron-deficient alkynes
has been described in the litera-
ture, but most of the reported
reactions were conducted in or-
ganic solvents.[5] Tsou and co-
workers reported selective cys-
teine modifications by a series
of reactions conducted in aque-
ous medium. The alkynoic
amides could function as irre-
versible inhibitors of epidermal
Scheme 3. Modification of peptide 1 by alkyne 2a.
growth factor receptor kinase (EGFR) by forming a cova-
lent linkage with the sulfhydryl group of the cysteine of the
enzyme.[6] Arjona and co-workers reported a cleavage reac-
tion for the deprotection of an electron-withdrawing tosyl-
vinyl group from a thiol. The corresponding cleavage of the
vinyl sulfide linkage was achieved by a base-induced thiolate
exchange in organic solvents.[7]
Herein, we report the use of electron-deficient alkynes
such as alkynoic amides, esters and alkynones for chemose-
lective cysteine modification of unprotected peptides
through the formation of a vinyl sulfide linkage in an aque-
ous medium [Scheme 2, Eq. (1)]. Notably, with R1 =aryl/
alkyl and R2 =H, the vinyl sulfide linkage could be cleaved
to regenerate the unmodified peptides by treatment with
thiols under mild conditions [Scheme 2, Eq. (2)]. The useful-
ness of this cleavage reaction has been demonstrated by the
enrichment of a cysteine-containing peptide from a mixture
of cysteine-containing and non-cysteine-containing peptides.
with alkynoic amide 2a (0.1 mm) in 100 mL of H2O/CH3CN
(9:1) at room temperature was examined (Scheme 3).[8]
A
25% conversion of 1 to 2a-modified 1 was observed after
500 min by HPLC analysis. Selective attachment of 2a to
the cysteine sulfhydryl group of 1 was confirmed by LC–
MS/MS. Neither the N-terminal a-amino group nor the side-
chains of serine and lysine were modified. Note that conju-
gate addition of the nucleophilic functional groups of serine,
cysteine and lysine derivatives to alkynoic amides and esters
and alkynones in organic solvents have been reported.[9]
However, no selectivity of cysteine over serine or lysine de-
rivatives in the reported coupling reactions was mentioned.
The conjugate addition of thiols to electron-deficient al-
kynes is generally facilitated in solutions at alkaline pH, for
example, by the addition of imidazole as a base or by using
alkaline Tris-HCl buffer (pH 7.0–9.0). Presumably this is
due to the increase in concentration of thiolate ions in alka-
line solution. As depicted in Figure 1, the reaction rate of
the coupling reaction depicted in Scheme 3 increased with
pH. Up to 80% conversion of 1 could be obtained when the
reaction was conducted in a pH 8.0 buffer for 500 min.
The cysteine modification of peptide 1 also proceeded se-
lectively with other electron-deficient alkynes, including al-
kynoic ester 3a and alkynones 4a–d, to give 3a- and 4a–d-
modified 1, respectively (Scheme 4).
Results
Cysteine-selective modification of peptide STSSSCNLSK
(1) by electron-deficient alkynes: Initially, the coupling reac-
tion of an unprotected peptide STSSSCNLSK (1; 0.1 mm)
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Cleavage of Modified Cysteine-Containing Peptides
FULL PAPER
Figure 1. Time course of the modification of peptide 1 by alkyne 2a
~
&
under various conditions. : 2a (0.1mm), pH 6.3; : 2a (0.1mm), imida-
&
Figure 2. Time course of the modification of 1 with 3a and 4a–d. : 3a
!
zole (0.2 mm), pH 7.4; : 2a (0.1 mm), imidazole (1mm), pH 7.7; &: 2a
(0.1mm), pH 7.0 buffer; : 2a (0.1mm), pH 8.0 buffer;
~
(0.1mm), pH 8.0 buffer; : 4a (0.1mm), pH 8.0 buffer; &: 4b (0.1mm),
*
: 2a (0.1mm),
n
*
pH 8.0 buffer; : 4c (0.1mm), pH 8.0 buffer;
: 4d (0.1mm), pH 8.0
n
pH 9.0 buffer.
buffer.
itial reaction rate was observed
in the modification of 1 by 4a
or 4b. By using 4a, over 90%
conversion of 1 was obtained
within 5 min. Modifications by
both 4a and 4b could reach
100% conversion in 30 min.
The reaction rates decrease in
the following order: terminal
alkynone@internal alkynoneꢀ
alkynoic ester>alkynoic amide.
To examine the compatibility
of electron-deficient alkynes
with other nucleophilic amino
acids, we conducted several
coupling reactions with the un-
protected
peptide
AYEMWCFHQK, which con-
tains histidine, methionine,
lysine and tyrosine, with differ-
ent alkynes (Scheme 5).[10] The
reactions of AYEMWCFHQK with 2a, 3a, 4a–d and com-
monly used cysteine modification reagents (iodoacetamide
and N-methylmaleimide) (1 equiv) in H2O and pH 8.0
buffer were attempted and exclusive cysteine modification
was observed in all cases (Table 1). Note that only 1 equiv
of 4a or 4b was required to achieve complete conversion in
H2O (pH 6.3). The reactivities of 4a and 4b are comparable
to that of N-methylmaleimide and yet significantly higher
than that of iodoacetamide (<5% conversion). By using
pH 8.0 buffer, a significant increase in the reaction rate was
observed: 60% conversion for 2a (entry 1) and complete
conversion for all the other substrates (entries 2–8). When a
large excess (50 equiv) of electrophile was used in pH 8.0
Scheme 4. Structures of 2a-, 3a- and 4a–d-modified 1.
Figure 2 shows the reaction rate profile of the reactions
between peptide 1 (0.1 mm) and alkynes 3a and 4a–d
(0.1 mm). The reaction medium was Tris-HCl buffer
(pH 8.0) with 10% (v/v) of CH3CN as co-solvent. A high in-
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3841

M.-K. Wong, C.-M. Che et al.
and 9.0 buffers, 2a, 3a, 4c–d and iodoacetamide were able
to give exclusive cysteine modification. In addition to cys-
teine modification, cross-reactivity with the N-terminal ala-
nine residue was observed for 4a, 4b and N-methylmalei-
mide; however, the nucleophilic side-chains of histidine, me-
thionine, lysine and tyrosine remained intact.
To demonstrate the practicality of this bioconjugation re-
action, two one-pot peptide modifications were conducted;
one by reacting 10 mg of AYEMWCFHQK with 2a
(1 equiv) in pH 8.0 buffer and the other with 4a (1 equiv) in
H2O. Both reactions afforded the corresponding cysteine-
modified peptides in 72% (7.9 mg) and 70% (7.6 mg) isolat-
ed yields, respectively.
Dansyl-, biotinyl- and farnesyl-linked alkynes 2b,c and
3b,c (5 equiv) individually attached to the cysteine unit of
AYEMWCFHQK within 6 h in pH 8.0, which was confirmed
by MALDI-TOF and LC–MS/MS analysis. Dialkyne-amide
2d allowed coupling of two AYEMWCFHQK units through
the cysteine side-chain.
By using 2a in pH 8.0 buffer, cysteine modification of
other unprotected peptides, including CALNN (N-terminal
cysteine), LHQRRGAIKQAKVHHVKC (C-terminal cys-
teine) and SSCSSCNLSK (two cysteine units), was accom-
plished (Scheme 6).
Scheme 5. Structures of 2a-, 3a-, 4a–d-modified AYEMWCFHQK.
Table 1. Cysteine modification of AYEMWCFHQK.[a]
Entry
Electrophile
Conversion [%][b]
pH 8.0 pH 8.0 or 9.0
(1 equiv) (50 equiv)
H2O
(1 equiv)
G
N
ACHTUNGTRENNUNG
1
2
3
4
5
6
7
8
2a
3a
4a
4b
4c
4d
<5
30
60
100
100
100
100
100
100
100
100
100
100
100
<5
<5
<5
100
100[c]
100[c]
100
100
Modification of bovine serum albumin with fluorescent
dansyl-linked alkyne 2b: The ligation of the single, surface-
exposed cysteine residue of bovine serum albumin (BSA) to
fluorescent dansyl-linked alkyne 2b was confirmed by
MALDI-TOF MS analysis. The peaks at 66545 and
iodoacetamide
N-methylmaleimide
100
100[c]
[a] The reaction mixtures of AYEMWCFHQK and electrophiles in aque-
ous CH3CN solutions (10% CH3CN) were maintained at 258C for 6 h.
[b] The conversion [%] was determined by LC–MS/MS. [c] The amino
acid residues of alanine (A) and cysteine (C) were modified.
Scheme 6. Structures of 2a-modified CALNN, LHQRRGAIKQAKVHHKC and SSCSSCNLSK.
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Cleavage of Modified Cysteine-Containing Peptides
FULL PAPER
Table 2. Z/E ratios of 7, 8 and 9a–d obtained by the coupling of 6 with alkynes 2a, 3a and 4a–d.[a]
66912 Da for native BSA and
2b-modified BSA, respectively,
revealed the addition of one
molecule of 2b per BSA. In ad-
dition, trypsin digestion of both
the native BSA and the 2b-
modified
BSA was
con-
ducted. The shift in the mass
of the cysteine-containing pep-
Conditions
Z/E ratio of alkyne-modified 6[b]
7
8
9a
9b
9c
9d
tide
fragment
Gly45-Lys65
H2O/CH3CN
H2O/CH3CN+imidazole[d]
pH 7.0[e]
4:1[c]
8:1
16:1
18:1
22:1
1:2
6:1
5:1
8:1
5:1
3:1
5:1
5:1
5:1
5:1
2:1
3:1
3:1
2:1
1:1
4:1
3:1
2:1
2:1
3:1
1:2
1:1
1:1
2:1
2:1
(GLVLIAFSQYLQQCPF-
DEHVK) from m/z=2435.2 to
2780.3 after modification con-
firmed the incorporation of one
molecule of 2b (MW=345)
into the BSA peptide sequence.
Notably, no modification was
observed with lysozyme, which
contains no free cysteine resi-
pH 8.0[e]
pH 9.0[e]
[a] Reaction conditions: 6 (0.4 mmol), alkyne 2a, 3a, 4a–d (0.48 mmol) in H2O (9 mL) and CH3CN (1 mL),
t=16 h, room temperature unless otherwise stated. [b] Determined by 1H NMR analysis of the crude reaction
mixture. [c] Incomplete conversion after 16 h reaction. [d] Imidazole (0.8 mmol) was added. [e] H2O was re-
placed by 9 mL of pH 7.0, 8.0 or 9.0 Tris-HCl buffer, respectively.
À
due. It was found that 2b-modified BSA gave a fluorescent
signal under UV excitation in SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel electrophoresis) protein analysis.
However, treatment of 2b with lysozyme gave no fluores-
cent signal (Figure 3).
the carbon carbon double bond of the vinyl sulfide via in-
termediates such as allenol and ketene hemiacetal.[7b] In this
connection, the weaker the electron-withdrawing ability of
the activating group (alkynoic amides<alkynoic esters<al-
kynones), the less isomerisation of Z to E isomers would be
observed. This trend is consistent with our findings: the Z/E
ratios of the amide 7, ester 8 and ketones 9a–d are up to
22:1, 8:1 and 5:1, respectively.
Cleavage reaction of electron-deficient alkyne-modified
STSSSCNLSK by thiols: A unique feature of the present
method is that vinyl sulfide linkages are generated from the
coupling of cysteine residues with alkynes. We envisioned
that the unsaturated vinyl sulfide units may allow further
synthetic manipulation. We examined the reactions of 2a-,
3a- and 4a–d-modified 1 (Scheme 4) with thiols under dif-
ferent reaction conditions. As depicted in Figure 4, treat-
ment of 4a-modified 1 with excess PhSH (30 equiv) afforded
the intermediate adduct A, which was then converted into 1.
Similarly, the cleavage of 4a-modified 1 to regenerate 1
was achieved in the presence of 1 equiv of imidazole or in
alkaline buffer solutions (pH 7.0–9.0; Figure 5). The highest
conversion (over 80%) was achieved by using pH 8.0 buffer
and over 70% regeneration could be obtained in 25 min.
In addition, 4b-modified 1 could be converted into 1
under the same reaction conditions. In contrast, 2a-, 3a- and
4c,d-modified 1 and 2b-modified BSA were stable in the
presence of PhSH.
Of the nucleophiles examined, including HOCH2CH2SH,
l-cysteine, PhOH, EtOH, PhSeH, C6H13NH2 and two com-
monly used reducing agents, DTT (dithiothreitol) and
TCEP (tris(2-carboxyethyl)phosphine), only HOCH2CH2SH,
l-cysteine and DTT were able to regenerate the unmodified
peptide.
The reactivities of the three thiols, PhSH, l-cysteine and
HOCH2CH2SH, towards the cleavage of 4a-modified 1 were
examined and compared (Figure 6). The three thiols exhibit-
ed similar reactivity in the cleavage reaction (up to 45% re-
Figure 3. SDS-PAGE analysis of 2b-modified BSA.
Model studies of the stereochemistry of the vinyl sulfide
linkages formed in peptide ligation: As mentioned in the
previous section, vinyl sulfide linkage was formed in the
modification of peptides and proteins with electron-deficient
alkynes. To model the stereoselectivity of the vinyl sulfide
linkage formed in the peptide modification, coupling reac-
tions of the protected cysteine 6 with alkynes 2a, 3a and
4a–d were performed (Table 2). Z Stereoisomers were
found to be the major coupling products for amide 7 (Z/E
up to 22:1), ester 8 (Z/E up to 8:1) and ketones 9a–d (Z/E
up to 5:1). Our findings are consistent with previous work
by Crisp and Millan who found that the ratio of Z/E stereo-
isomers formed in the conjugate addition of protected
amino acids to electron-deficient alkynes was dependent on
the solvent system used.[9a] In general, Z isomers are the
predominant products formed from the trans addition of
thiols to electron-deficient alkynes. Nevertheless, the elec-
tron-withdrawing group could lead to the isomerisation of
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M.-K. Wong, C.-M. Che et al.
generation of 1 in 5 min and up
to 85% regeneration of 1 in
75 min). PhSH was found to
have a slightly higher reactivity
than
l-cysteine
and
HOCH2CH2SH.
Treatment of 10 mg of 4a-
modified 1 with 30 equiv of l-
cysteine afforded the unmodi-
fied peptide in 81% isolated
yield. The regeneration of the
starting peptide from 4a- and
4b-modified AYEMWCFHQK
was also accomplished by the
Figure 4. Cleavage of 4a-modified 1 by PhSH.
addition
of
PhSH,
HOCH2CH2SH or l-cysteine.
Peptide enrichment using
a
cleavable biotin-linked alkyne: In proteomics applications,
the enrichment of specific peptide targets from complex bio-
logical mixtures is important for reducing the complexity of
peptide samples and for enhancing specific subsets of low-
abundance proteins.[11] Cysteine is a widely used amino acid
residue for the attachment of specific affinity tags for
sample enrichment. This is generally achieved by covalent li-
gation of cysteine-containing peptides/proteins by biotin-
linked tags. The biotin-tagged samples can be enriched by
binding to immobilised streptavidin. However, harsh condi-
tions, such as incubation of the biotin–streptavidin complex
at 60–908C under denaturing conditions, are required to re-
lease the biotin-tagged samples.[12] On the other hand, on-
bead digestion may lead to contamination by streptavidin,
natively biotinylated proteins and/or non-specific resin-
bound proteins.
In this work we found that exclusive ligation of alkyne 5
to AYEMWCFHQK occurred in the presence of a mixture
of two non-cysteine-containing peptides, RPKPQQF and
ASHLQLAR (Scheme 7). After coupling of the peptide
mixture with a biotin azide by a copper(I)-catalysed Sharp-
less–Huisgen [3+2] cycloaddition reaction,[13] the biotin-
tagged AYEMWCFHQK was subsequently captured by
streptavidin beads. Treatment of the beads with an aqueous
solution of PhSH/DTT at pH 8 at 378C for 2 h regenerated
AYEMWCFHQK, as confirmed by LC–MS/MS analysis. A
71% recovery of the peptide AYEMWCFHQK was ach-
ieved.
Figure 5. Cleavage of 4a-modified 1 by PhSH under various conditions.
~
&
: PhSH (3mm), pH 6.3; : PhSH (3mm), imidazole (0.1mm), pH 7.2; &:
*
PhSH (3mm), pH 7.0 buffer; : PhSH (3mm), pH 8.0 buffer;
: PhSH
n
(3mm), pH 9.0 buffer.
Discussion
Electron-deficient alkynes as a new class of cleavable re-
agents for chemoselective cysteine modification: Conjugate
addition of thiols to electron-deficient alkynes under alka-
line conditions has been known for decades. However, the
reported reactions involved simple thiols and were conduct-
ed in organic solvents.[5] Apart from thiols, amines and alco-
hols can also react with alkynes. In this work electron-defi-
&
Figure 6. Cleavage of 4a-modified 1 by three different thiols at pH 8.0.
:
~
*
PhSH (3mm); : l-cysteine (3mm), : HOCH2CH2SH (3mm).
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Cleavage of Modified Cysteine-Containing Peptides
FULL PAPER
presence of thiols leading to ir-
reversible modification.
As depicted in Scheme 8, the
key step in the cleavage reac-
tion is the addition of the thiol
to the vinyl sulfide linkage to
give a dithioacetal, which sub-
sequently releases the peptide
by elimination. In general, the
addition of thiol to vinyl sulfide
(k2) has a lower reaction con-
stant than that for the coupling
of the cysteine residue with the
electron-deficient
alkyne
(k1).[14] In this work the rates of
cysteine modification by differ-
ent alkynes are in the following
order: terminal alkynone@in-
ternal
alkynoneꢀalkynoic
ester>alkynoic amide (Figure 2
and Scheme 5). Thus the forma-
tion of dithioacetal from the
second conjugate addition of
thiols to the less reactive alky-
noic amide-, alkynoic ester- and
Scheme 7. Peptide enrichment experiment.
internal
alkynone-modified
peptides would be less favoura-
ble, which accounts for their stability in the presence of
excess thiols.
As mentioned previously, the conjugate addition of thiols
to electron-deficient alkynes is generally assisted by the ad-
dition of imidazole as a base or by using alkaline Tris-HCl
buffer (pH 7.0–9.0). Similarly, the cleavage of alkynone-
modified peptides by thiols could also be facilitated in alka-
line solution as the cleavage reaction involves a second addi-
cient alkynes have been found to selectively modify unpro-
tected cysteine-containing peptides and proteins in aqueous
media with other amino acid groups such as histidine, me-
thionine, lysine and tyrosine remaining intact.
By using the present method, an unsaturated vinyl sulfide
linkage is generated from the coupling of a cysteine residue
with electron-deficient alkynes. We found that the unsaturat-
ed vinyl sulfide units could be cleaved by an addition/elimi-
nation mechanism by treatment
with thiols. In contrast, the cur-
rently used alkylation reagents
(haloacyl compounds and male-
imides) modify cysteine by the
formation of non-cleavable
À
sulfur carbon bonds.
By
a judicious choice of
alkyne substituents (R1 and R2),
we were able to tune the reac-
tivity of the alkynones towards
the thiol-assisted cleavage of
vinyl
sulfide
linkages
(Scheme 8). In this work treat-
ment of terminal alkynone-
modified peptides with thiols
led to the recovery of unmodi-
fied peptides under mild reac-
tion conditions. In contrast, al-
kynoic amide-, alkynoic ester-
and internal alkynone-modified
peptides were stable in the Scheme 8. Different reactivities in the thiol-assisted cleavage of vinyl sulfide linkages.
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M.-K. Wong, C.-M. Che et al.
tion of thiolate to the unsaturated vinyl sulfide linkage.
Both the cysteine modification and cleavage reaction could
be accelerated by an increase in thiolate ion concentration
in alkaline solution.
oxidation. The functionalised alkynes such as dansyl-, bio-
tinyl- and farnesyl-linked alkynes 2b,c and 3b,c were pre-
pared by simple organic reactions. By using click chemistry,
the 5-modified peptides could be further functionalised by
coupling with organic azides, allowing a convenient access
to a wide variety of structurally diverse bioconjugates.
Given their excellent chemoselectivity in cysteine modifica-
tion and ease of synthesis, we envision that electron-defi-
cient alkynes could be developed into a class of reliable and
practical reagents for cysteine modification.
In this work, alkynoic amides, including 2a–d, were dem-
onstrated to be efficient reagents with excellent chemoselec-
tivity for the modification of cysteine-containing peptides/
proteins. As depicted in Table 1, exclusive cysteine modifica-
tion could be achieved even with a large excess (50 equiv)
of alkynoic amides at pH 9.0. In contrast, cross-reactivity
with the N-terminal a-amino group of alanine was observed
for the commonly used N-methylmaleimide under the same
reaction conditions. As depicted in Table 2, high stereoselec-
tivity (Z/E up to 22:1) for vinyl sulfide formation could be
obtained in alkaline solution. This high stereoselectivity is
important for minimising the formation of heterogeneous
mixtures of small molecule–biomolecule conjugates. Our
studies indicated that alkynoic amides tolerated a wide
range of reaction conditions: 1 or 50 equiv of reagent used,
H2O, imidazole or pH 7.0–9.0 buffer, and room temperature
to 378C. The practicality of alkynoic amides in bioconju-
gation has been exemplified by the following one-pot pep-
The use of a cleavable biotin-linked alkyne in peptide en-
richment experiments: In this work the use of electron-defi-
cient alkynes allowed modification of cysteine-containing
peptides and proteins in a selective manner. We have there-
fore developed a cleavable biotin-linked alkyne for specific
enrichment of cysteine-containing peptide samples with high
recovery (71%) under mild conditions (Scheme 7). Cleavage
of the vinyl sulfide linkage is thiol-specific and yet the vinyl
sulfide linkage remains stable upon treatment with phos-
phine-based reducing reagents such as TCEP. This orthogo-
nal reactivity towards phosphine-based reducing reagents
allows sample preparation for proteomics applications using
phosphine-based reducing reagents. In particular, this would
allow subsequent modification of the alkyne-modified pep-
tides by copper(I)-catalysed Sharpless–Huisgen [3+2] cyclo-
addition in which TCEP is needed for in situ generation of
copper(I) ions. The enrichment procedure described in this
work is compatible with the conventional biotin–streptavidin
affinity technique.[12b] After cleavage of the vinyl sulfide
linkage, the free cysteine sulfhydryl group is recovered in
the enriched peptides. Thus, the use of these cleavable
biotin-linked alkynes could lead to the recovery of the
native peptides/proteins. This would simplify subsequent
mass spectrometry analysis of the enriched samples by elim-
inating undesirable effect(s) associated with tagging groups
such as reduced sensitivity and tag fragmentation.
An important factor governing the utility of these cleava-
ble reagents in peptide enrichment experiments is the kinet-
ics of reversibility of the biotinylated peptides. In this work
no scrambled product was found when biotinylated peptide
AYEMWCFHQK was treated with a mixture of peptides
containing free cysteine residues. Furthermore, the biotiny-
lated peptide (60 mm) as well as the 4a- and 5-modified pep-
tides (10 mm) in pH 8.0 Tris-HCl buffer/CH3CN (9:1) re-
mained stable after storage at 258C for 30 days. These ex-
periments revealed that cleavable electron-deficient alky-
nones should have potential in proteomics studies of com-
plex biological samples.
tide
modification:
the
reaction
of
10 mg
of
AYEMWCFHQK with 2a (1 equiv) in pH 8.0 buffer afford-
ed the corresponding cysteine-modified peptide in 72%
(7.9 mg) isolated yield. Finally, the alkynoic amide modified
peptides remained stable upon treatment with thiols and re-
ducing agents, including DTT and TCEP, under various con-
ditions.
Terminal alkynones, including 4a and 4b, exhibited the
highest reactivity in cysteine modification. As shown in
Figure 2, when 4a (1 equiv) in pH 8.0 buffer was used, over
90% conversion of 1 was obtained within 5 min and 100%
substrate conversion was achieved in 30 min. One-pot pep-
tide modification by the reaction of 10 mg of
AYEMWCFHQK with 4a (1 equiv) in H2O gave the cys-
teine-modified peptide in 70% (7.6 mg) isolated yield. The
high reactivity of terminal alkynones allows high-throughput
cysteine modification and obviates the need of using excess
labelling reagents. Notably, we found that terminal alky-
none-modified peptides could be converted back into un-
modified peptides by thiol-assisted cleavage under mild re-
action conditions. Note that treatment of 10 mg of 4a-modi-
fied 1 with 30 equiv of l-cysteine afforded the unmodified
peptide in 81% isolated yield. In this preparative-scale
cleavage reaction, odourless l-cysteine could be used in-
stead of the strongly odorous PhSH. Indeed,
HOCH2CH2SH, a commonly used reagent in biological ex-
periments, could also be employed.
Most of the alkynes used in our experiments are not com-
mercially available, but they can be prepared by relatively
simple procedures, as depicted in the Supporting Informa-
tion. Alkynoic amides 2 and alkynoic esters 3 could be pre-
pared in good yields by coupling propiolic acid or propynoyl
chloride with amines and alcohols, respectively. Alkynones 4
could be synthesised by nucleophilic addition of lithiated al-
kynes/Grignard reagents to aldehydes followed by alcohol
Biotin disulfide N-hydroxysuccinimide ester (Biotin-S-S-
NHS) is a commercially available biotin–disulfide linker
used for the recovery of protein–DNA complexes after
avidin affinity column purification.[15] The NHS moiety
allows ligation of the probe to the amino groups of the
target proteins, whereas the biotin group is used for specific
binding onto the streptavidin beads. Cleavage of the disul-
fide bond could be achieved by treatment with thiol-based
3846
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 3839 – 3850

Cleavage of Modified Cysteine-Containing Peptides
FULL PAPER
Procedure for the modification of peptide 1 by electron-deficient alkyne
2a: In a 1 mL vial equipped with a 500 mL insert, peptide 1 (40 mL,
1 mmolmLÀ1 in H2O), electron-deficient alkyne 2a (4 mL, 10 mmolmLÀ1 in
CH3CN), H2O (320 mL) and CH3CN (36 mL, final volume 400 mL, H2O/
CH3CN=9:1) were mixed at room temperature. The reaction mixture
was then monitored by reversed-phase HPLC. Every 30 min, a sample of
the reaction mixture (30 mL) was injected into the HPLC apparatus. The
peptides were eluted at a flow rate of 0.5 mLminÀ1 with the solvent gra-
dient shown in Figure S1 of the Supporting Information and the column
effluent was monitored at 210 nm. The peptide sequences were deter-
mined by LC–MS/MS.
reducing agents. However, the NHS group is amine-reactive
such that lysine and a-amino groups can react with the
linker leading to non-specific modification. There is no se-
lectivity in the cleavage of the disulfide linker by thiol- and
phosphine-based reducing agents. Proteins obtained after
cleavage of the disulfide linker are attached with an amine-
modified residue that precludes the recovery of native pro-
teins and causes complications in subsequent mass spectro-
metric analyses.
The above coupling reaction between 1 and 2a was repeated under vari-
ous conditions and was also performed with 1 and 3a as well as 4a–d at
pH 8.0 (see the Supporting Information).
Conclusion
Procedure for the coupling reaction of protected cysteine 6 with the elec-
tron-deficient alkyne 2a: Imidazole (55 mg, 0.8 mmol) was added to a
We have developed an efficient and versatile method for the
selective cysteine modification of unprotected peptides and
proteins in aqueous media using electron-deficient alkynes
through the formation of a vinyl sulfide linkage. Notably,
peptides modified by terminal alkynones could be converted
back into the unmodified peptides by treatment with thiols
under mild reaction conditions. The usefulness of this thiol-
assisted cleavage of the vinyl sulfide linkage has been dem-
onstrated by the selective enrichment of a cysteine-contain-
ing peptide from a mixture of peptides.
round-bottom flask containing
6 (100 mg, 0.4 mmol) and alkyne 2a
(70 mg, 0.48 mmol) in a solution of H2O (9 mL) and CH3CN (1 mL).
After stirring at room temperature for 16 h, the reaction mixture was ex-
tracted with CH2Cl2 (3ꢁ20 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The residue was purified by flash column chromatography (30–40%
EtOAc in n-hexane) to provide (Z)-/(E)-7 (142 mg, 90% isolated yield
based on 100% conversion) as a solid.
The above reaction conditions were used for the coupling of 6 with 3a
and 4a–d.
General procedure for cysteine modification of AYEMWCFHQK with
2a, 3a, 4a–d, iodoacetamide and N-methylmaleimide (1 or 50 equiv) in
H2O or pH 8.0 Tris-HCl buffer: AYEMWCFHQK (10 mL, 1 mmolmLÀ1
in H2O), electrophile (1 mL, 1 equiv from a stock solution: 10 mmolmLÀ1
in CH3CN), CH3CN (9 mL) and H2O or pH 8.0 Tris-HCl buffer (80 mL)
were mixed in a 1.0 mL Eppendorf tube. When 50 equiv of alkyne was
needed, 5 mL of the electrophile (from a stock solution: 100 mmolmLÀ1 in
CH3CN) was used. The reaction mixture was kept at 258C for 6 h. The
cysteine-modified peptides were characterised by LC–MS/MS.
Experimental Section
General: Chemicals purchased from commercial sources were used with-
out further purification. Double-distilled water (ddH2O) used as reaction
solvent and in peptide modifications was deionised by using a NANO-
pure purification system (Barnstead, USA). Flash column chromatogra-
phy was performed by using silica gel 60 (230–400 mesh ASTM) with
ethyl acetate/n-hexane or methanol/dichloromethane as eluent. HPLC
analysis was conducted by using a Waters 2690 Separations Module
equipped with a Waters 996 Photodiode Array Detector and a XTerra
General procedure for cysteine modification of N- and C-terminal cys-
teine-containing peptides (CALNN and LHQRRGAIKQAKVHHVKC)
with 2a: The peptide (10 mL, 1 mmolmLÀ1 in H2O), 2a (1 mL,
10 mmolmLÀ1 in CH3CN), CH3CN (9 mL) and pH 8.0 Tris-HCl buffer
(80 mL) were mixed in a 1.0 mL Eppendorf tube. The reaction mixture
was kept at 258C for 6 h. The cysteine-modified peptides were character-
ised by LC–MS/MS.
1
RP18 5 mm column. H and 13C NMR spectra were recorded on a Bruker
Procedure for cysteine modification of SSCSSCNLSK with 2a: The pep-
tide (10 mL, 1 mmolmLÀ1 in H2O), 2a (10 mL, 10 mmolmLÀ1 in CH3CN)
and pH 8.0 Tris-HCl buffer (80 mL) were mixed in a 1.0 mL Eppendorf
tube. The reaction mixture was kept at 258C for 6 h. The cysteine-modi-
fied peptide was characterised by LC–MS/MS.
DPX-300 or DPX-400 spectrometer. Chemical shifts (ppm) are refer-
enced to TMS. Mass spectra were measured by using a Finnigan MAT 95
or LCQ mass spectrometer.
Mass spectrometric analysis of peptides: All peptide samples for
MALDI-TOF MS analysis were co-crystallised by using a a-cyano-4-hy-
droxycinnamic acid (Aldrich) solution (10 mgmLÀ1 CH3CN/ddH2O (1:1)
with 0.1% TFA). MALDI-TOF mass spectra of peptides were recorded
in reflectron mode by using a Voyager-DE STR system (ABI, USA)
equipped with a N2 laser (337 nm). LC–MS/MS analyses were performed
by using a hybrid Q-TOF mass spectrometer (QSTAR-XL system, ABI,
USA) equipped with an ionspray source and an Agilent 1100 series cap-
LC pump. Chromatography of the reaction mixture was performed by
using an Agilent ZORBAX 300SB-C18 (0.3 mmꢁ150 mm) reversed-
phase column with a CH3CN/ddH2O gradient mobile phase containing
0.1% formic acid (flow rate: 5 mLminÀ1).
General procedure for cysteine modification of AYEMWCFHQK with
2b–d and 3b,c: The peptide AYEMWCFHQK (10 mL, 1 mmolmLÀ1 in
H2O), alkyne (5 mL, 10 mmolmLÀ1 in CH3CN), CH3CN (5 mL) and pH 8.0
Tris-HCl buffer (80 mL) were mixed in a 1.0 mL Eppendorf tube. The re-
action mixture was kept at 258C for 6 h. The cysteine-modified
AYEMWCFHQK was characterised by LC–MS/MS.
Procedures for the large-scale synthesis of 2a- and 4a-modified
AYEMWCFHQK: A solution of the peptide AYEMWCFHQK (10 mg)
and 2a (2.3 mg, 1.5 equiv) in pH 8.0 Tris-HCl buffer/CH3CN (100 mL,
9:1) was prepared and placed in two centrifugal tubes (size 50 mL). The
reaction mixtures were kept at 258C overnight. The aqueous phases were
freeze-dried and the residues purified by preparative reversed-phase
HPLC using a C18 column and CH3CN/H2O/TFA as the solvent system.
The 2a-modified AYEMWCFHQK was isolated in 72% yield (7.9 mg).
The sequence of 2a-modified AYEMWCFHQK was confirmed by
MALDI-TOF and LC–MS/MS analyses.
Solid-phase synthesis of peptides: Peptides were synthesised according to
the standard solid-phase Fmoc-peptide synthesis procedure using the
Wang resin as solid support. The peptides were purified by preparative
reversed-phase HPLC using a C18 column with CH3CN/H2O/TFA as the
solvent system. The amino acid sequences of the peptides were con-
firmed by tandem mass spectrometric (MS/MS) analysis.
For the synthesis of 4a-modified AYEMWCFHQK,
a solution of
The peptides STSSSCNLSK, AYEMWCFHQK, CALNN and
SSCSSCNLSK were synthesised, whereas the peptides LHQRRGAIK-
QAKVHHVKC, ASHLGLAR and RPKPQQF were obtained from
commercial sources.
AYEMWCFHQK (10 mg) and 4a (2 mg, 1.5 equiv) in H2O/CH3CN
(100 mL, 9:1) was prepared. Following the procedure described above,
4a-modified AYEMWCFHQK was isolated in 70% yield (7.6 mg).
Chem. Eur. J. 2009, 15, 3839 – 3850
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3847

M.-K. Wong, C.-M. Che et al.
General procedure for modification of bovine serum albumin (BSA) and
lysozyme with 2b: In the case of BSA, a solution of BSA (10 mL,
1 mmolmLÀ1 in H2O), 2b (10 mL, 10 mmolmLÀ1 in CH3CN) and pH 8.0
Tris-HCl buffer (80 mL) were mixed in a 1.0 mL Eppendorf tube. In the
case of lysozyme, a solution of lysozyme (10 mL, 1 mmolmLÀ1 in H2O), 2b
(10 mL, 10 mmolmLÀ1 in CH3CN) and pH 8.0 Tris-HCl buffer (80 mL)
were mixed in a 1.0 mL Eppendorf tube. The reaction mixture was kept
at 258C for 16 h. The cysteine-modified BSA was subjected to SDS-
PAGE analysis and trypsin digestion for MALDI-TOF analysis.
column and CH3CN/H2O/TFA as the solvent system. The 4a-modified 1
was isolated in 55% yield (12.4 mg). The 4a-modified 1 was confirmed
by MALDI-TOF MS analysis.
A solution of 4a-modified 1 (10 mg) and l-cysteine (32 mg, 30 equiv) in
pH 8.0 Tris-HCl buffer/CH3CN (100 mL, 9:1) was prepared and placed in
two centrifugal tubes (size 50 mL). The reaction mixtures were kept at
258C for 4 h. The aqueous phases were freeze-dried and purified by prep-
arative reversed-phase HPLC using a C18 column and CH3CN/H2O/TFA
as the solvent system. The unmodified 1 was isolated in 81% yield
(7.2 mg) and was confirmed by MALDI-TOF MS analysis.
Procedure for SDS-PAGE analysis: The 2b-modified protein mixture
(10 mL) was mixed with 2X loading buffer (90 mL) in a 0.5 mL Eppendorf
tube and then boiled for 5 min. Samples were analysed by SDS-PAGE by
loading a sample of the boiled solution (10 mL) in each lane of a 12.5%
SDS-PAGE gel and running in a SE 250 Mini-Vertical Unit (Amersham,
USA) at 100 V at room temperature until the front of the dye reached
the bottom of the gel. After SDS-PAGE separation, the 2b-modified pro-
tein mixture was visualised with an Alphamager 2200 Multilmage Light
Cabinet and finally stained with Coomassie blue.
Procedure for peptide enrichment using a cleavable biotin-linked alkyne:
Alkyne
5 a mixture of peptides,
(6 mL, 10 mmolmLÀ1 in THF),
RPKPQQF (60 mL, 1 mmolmLÀ1 in H2O), ASHLQLAR (60 mL,
1 mmolmLÀ1 in H2O) and AYEMWCFHQK (60 mL, 1 mmolmLÀ1 in
H2O), and THF (14 mL, final volume of 200 mL with H2O/THF=9:1)
were mixed in a 1.0 mL Eppendorf tube. The reaction mixture was kept
at 258C for 24 h. The sequence of 5-modified AYEMWCFHQK and the
other two unmodified peptides were characterised by LC–MS/MS analy-
sis.
Procedure for trypsin digestion: For the trypsin digestion of BSA, a ratio
of 1:50 (w/w) of trypsin to BSA was used. The 2b-modified BSA mixture
(10 mL) was mixed with an ammonium bicarbonate solution (100 mL,
The above 5-modified peptide mixture (20 mL), phosphate-buffered
saline (PBS; 58 mL, pH 8.10), biotin-N3 (1 mL, 10 mmolmLÀ1 in DMSO),
100 mm) in
a 0.5 mL Eppendorf tube. The trypsin solution (1 mL,
tris(2-carboxyethyl)phosphine
hydrochloride
(TCEP;
10 mL,
1 mgmLÀ1 in 1 mm HCl) was added to the Eppendorf tube. The reaction
mixture was incubated at 378C for 2 h and the trypsin-digested mixture
was then ready for MALDI-TOF analysis.
10 mmolmLÀ1 in H2O), tris(benzyltriazolylmethyl)amine (TBTA; 1 mL,
10 mmolmLÀ1 in DMSO/tert-butyl alcohol=1:4) and CuSO4·5H2O
(10 mL, 10 mmolmLÀ1 in H2O) were mixed in a 1.0 mL Eppendorf tube.
The reaction mixture was kept at 258C for 1 h. The sequence of the
biotin-tagged AYEMWCFHQK and the other two unmodified peptides
were characterised by LC–MS/MS and MALDI-TOF MS/MS analyses.
Procedure for cleavage of 4a-modified 1 with PhSH to regenerate un-
modified 1
Preparation of 4a-modified 1: Peptide 1 (40 mL, 1 mmolmLÀ1 in H2O), 4a
(4 mL, 10 mmolmLÀ1 in CH3CN), CH3CN (4 mL) and H2O (32 mL) were
mixed in a 1.0 mL Eppendorf tube. The reaction mixture was kept at
258C for 24 h. HPLC analysis confirmed that all of 1 had been consumed.
A new peak was found at 11.7 min with m/z=1143, which was assigned
to 4a-modified 1. The 4a-modified 1 was characterised by MALDI-TOF
MS analysis.
A solution of Dynabeads M-280 Streptavidin# (300 mL, 10 mgmLÀ1) was
washed by PBS several times before use to remove preservatives. The
washing procedure was facilitated by using the Dynac MPC-S Magnetic
Particle Concentrator. The washed Dynabeads were added to the biotin-
tagged AYEMWCFHQK reaction mixture and kept at 258C for 30 min.
The supernatant containing the unbound peptides (RPKPQQF and
ASHLQLAR) was removed and labelled as the mother liquor. Beads
that stuck to the wall of the Eppendorf tube were washed with H2O sev-
eral times.
To cleave the peptide from the beads, PhSH (2 mL, 100 mmolmLÀ1 in
CH3CN), DTT (5 mL, 100 mmolmLÀ1 in H2O) and pH 8.0 Tris-HCl/
CH3CN (30 mL, 9:1) were mixed and the beads added. The reaction mix-
ture was kept at 378C for 2 h. The resulting supernatant was character-
ised by LC–MS/MS. Recovery of AYEMWCFHQK was confirmed by
LC–MS/MS.
The above procedure was repeated for the preparation of 4b-modified 1
and for the modification of AYEMWCFHQK by 4a.
Cleavage reaction of 4a-modified 1 by PhSH: The 4a-modified 1 (80 mL),
PhSH (12 mL, 100 mmolmLÀ1 in CH3CN), CH3CN (28 mL) and H2O
(280 mL) were mixed in a 1.0 mL Eppendorf tube at room temperature.
The reaction mixture was then monitored by reversed-phase HPLC.
Every 24 min, a sample of the reaction mixture (30 mL) was injected in
the HPLC apparatus. The peptide was eluted at
a flow rate of
0.5 mLminÀ1 with the solvent gradient shown in Figure S1 of the Sup-
porting Information (the column effluent was monitored at 210 nm) and
characterised by MALDI-TOF MS analysis.
To determine the percentage recovery of AYEMWCFHQK from the
peptide enrichment experiment, a calibration curve of different concen-
trations of AYEMWCFHQK (125, 150, 180, 210, 240 and 300 mm) was es-
tablished by LC–MS/MS. The area under the total ion count was plotted
against the concentration of AYEMWCFHQK.
The above procedure was repeated for the cleavage of 4b-modified 1 and
4a-modified AYEMWCFHQK.
General procedure for the cleavage reactions of 4a-modified 1 by nucleo-
philes to regenerate unmodified 1 in pH 8.0 Tris-HCl buffer: The 4a-
modified 1 (20 mL), the water-soluble nucleophile stock solution (30 mL
of HOCH2CH2SH or l-cysteine), CH3CN (10 mL) and pH 8.0 Tris-HCl
buffer (40 mL) were mixed in a 1.0 mL Eppendorf tube. The reaction
mixture was kept at 258C for 8 h. The reaction mixture was analysed by
MALDI-TOF MS.
A 25 mL sample of the reaction mixture was injected into the HPLC ap-
paratus. The peptide was eluted at a flow rate of 0.5 mLminÀ1 with the
solvent gradient shown in Figure S1 of the Supporting Information and
the column effluent was monitored at 210 nm. The AYEMWCFHQK
fraction was collected, freeze-dried and resuspended in H2O (10 mL). The
suspension was characterised by LC–MS/MS. The area under the total
ion count was obtained and compared with the calibration curve to quan-
tify the percentage recovery of AYEMWCFHQK in the peptide enrich-
ment experiment. The percentage recovery of native AYEMWCFHQK
recovered after cleavage from the beads was found to be 71%. # Dyna-
beads M-280 Streptavidin was purchased from Invitrogen Dynal AS
(Oslo, Norway).
The above procedure was used for the cleavage of 4a-modified
AYEMWCFHQK by HOCH2CH2SH or l-cysteine.
The 4a-modified 1 (20 mL), the water-insoluble nucleophile stock solution
(3 mL of PhOH, ethanol, PhSeH or hexylamine), CH3CN (7 mL) and
pH 8.0 Tris-HCl buffer (70 mL) were mixed in a 1.0 mL Eppendorf tube.
The reaction mixture was kept at 258C for 8 h. The reaction mixture was
analysed by MALDI-TOF MS.
Characterisation data for 7, 8 and 9a–d: (Z)-7: Analytical TLC (silica gel
1
60, 50% EtOAc in n-hexane): Rf =0.40; H NMR (400 MHz, CDCl3): d=
Procedure for the large-scale synthesis of 4a-modified STSSSCNLSK
and recovery of unmodified STSSSCNLSK: A solution of peptide 1
(20 mg) and 4a (2.6 mg, 1 equiv) in H2O/CH3CN (200 mL, 9:1) was pre-
pared and placed in four centrifugal tubes (size 50 mL). The reaction
mixtures were kept at 258C overnight. The aqueous phases were freeze-
dried and purified by preparative reversed-phase HPLC using a C18
7.58–7.55 (m, 2H), 7.33–7.29 (m, 2H), 6.95 (d, J=9.9 Hz, 1H), 5.90 (d,
J=9.9 Hz, 1H), 5.41 (br d, J=7.2 Hz, 1H), 4.60–4.57 (m, 1H), 4.23 (q,
J=7.1 Hz, 2H), 3.27 (br d, J=4.3 Hz, 2H), 1.45 (s, 9H), 1.31 ppm (t, J=
7.1 Hz, 3H); 13C NMR (75.48 MHz, CDCl3): d=170.2, 164.2, 155.1, 147.0,
137.9, 128.9, 124.1, 119.5, 116.2, 80.3, 62.1, 53.9, 38.8, 28.3, 14.1 ppm;
3848
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 3839 – 3850

Cleavage of Modified Cysteine-Containing Peptides
FULL PAPER
EIMS: m/z: 394 [M]+; HRMS (EI): calcd for C19H26N2O5S: 394.1562;
found: 394.1550.
(E)-9c: Analytical TLC (silica gel 60, 40% EtOAc in n-hexane): Rf =
0.47; 1H NMR (500 MHz, CDCl3): d=7.97–7.95 (m, 2H), 7.54–7.50 (m,
1H), 7.46–7.43 (m, 2H), 6.77 (s, 1H), 5.32 (brd, J=7.4 Hz, 1H), 4.70–
4.67 (m, 1H), 4.27–4.21 (m, 2H), 3.40–3.26 (m, 2H), 2.90–2.82 (m, 2H),
1.66–1.56 (m, 2H), 1.48–1.41 (m, 2H), 1.43 (s, 9H), 1.29 (t, J=7.1 Hz,
3H), 0.94 ppm (t, J=7.3 Hz, 3H); 13C NMR (75.47 MHz, CDCl3): d=
187.3, 170.4, 164.6, 155.0, 139.7, 132.2, 128.5, 128.2, 113.7, 62.2, 52.4, 35.0,
33.9, 32.0, 28.3, 22.8, 14.2, 13.9 ppm; EIMS: m/z: 435 [M]+; HRMS (EI):
calcd for C23H33NO5S: 435.2079; found: 435.2078.
(E)-7: Analytical TLC (silica gel 60, 50% EtOAc in n-hexane): Rf =0.49;
1H NMR (400 MHz, CDCl3): d=7.59 (d, J=14.8 Hz, 1H), 7.55–7.53 (m,
2H), 7.37 (brs, 1H), 7.34–7.30 (m, 2H), 7.10 (t, J=7.4 Hz, 1H), 6.08 (d,
J=14.8 Hz, 1H), 5.40 (brd, J=7.0 Hz, 1H), 4.63–4.61 (m, 1H), 4.25 (q,
J=7.1 Hz, 2H), 3.32–3.24 (m, 2H), 1.46 (s, 9H), 1.31 ppm (t, J=7.1 Hz,
3H); 13C NMR (75.48 MHz, CDCl3): d=170.1, 142.6, 138.1, 129.0, 124.2,
119.8, 118.2, 80.6, 62.3, 52.9, 35.2, 28.3, 14.1 ppm; EIMS: m/z: 394 [M]+;
HRMS (EI): calcd for C19H26N2O5S: 394.1562; found: 394.1550.
(Z)-9d: Analytical TLC (silica gel 60, 40% EtOAc in n-hexane): Rf =
0.60; 1H NMR (500 MHz, CDCl3): d=7.29–7.26 (m, 2H), 7.18–7.16 (m,
3H), 6.23 (s, 1H), 5.34–5.31 (m, 1H), 4.57–4.54 (m, 1H), 4.23 (q, J=
7.1 Hz, 2H), 3.37–3.23 (m, 2H), 2.95–2.92 (m, 2H), 2.78–2.74 (m, 2H),
2.46–2.43 (m, 2H), 1.52–1.48 (m, 2H), 1.44 (s, 9H), 1.39–1.33 (m, 2H),
1.29 (t, J=7.1 Hz, 3H), 0.93 ppm (t, J=7.3 Hz, 3H); 13C NMR
(75.47 MHz, CDCl3): d=197.3, 170.2, 160.6, 141.5, 128.5, 128.4, 126.0,
120.5, 62.1, 53.3, 44.8, 36.2, 32.3, 31.9, 30.4, 29.7, 28.3, 22.2, 14.2,
13.8 ppm; EIMS: m/z: 463 [M]+; HRMS (EI): calcd for C25H37NOS:
463.2392; found: 463.2396.
(Z)-8: Analytical TLC (silica gel 60, 30% EtOAc in n-hexane): Rf =0.45;
1H NMR (400 MHz, CDCl3): d=7.04 (d, J=10.0 Hz, 1H), 5.85 (d, J=
10.0 Hz, 1H), 5.45 (brd, J=7.0 Hz, 1H), 4.60–4.56 (m, 1H), 4.23 (q, J=
7.1 Hz, 2H), 3.74 (s, 3H), 3.35–3.24 (m, 2H), 1.45 (s, 9H), 1.30 ppm (t,
J=7.1 Hz, 3H); 13C NMR (75.48 MHz, CDCl3): d=170.0, 166.8, 155.0,
149.9, 113.4, 80.2, 62.0, 53.9, 51.3, 38.4, 28.2, 14.1 ppm; EIMS: m/z: 333
[M]+; HRMS (EI): calcd for C14H23NO6S: 333.1246; found: 333.1233.
(E)-8: Analytical TLC (silica gel 60, 30% EtOAc in n-hexane): Rf =0.51;
1H NMR (400 MHz, CDCl3): d=7.59 (d, J=15.3 Hz, 1H), 5.86 (d, J=
15.3 Hz, 1H), 5.38 (brd, J=7.0 Hz, 1H), 4.65–4.58 (m, 1H), 4.23 (q, J=
7.1 Hz, 2H), 3.71 (s, 3H), 3.38–3.20 (m, 2H), 1.45 (s, 9H), 1.31 ppm (t,
J=7.1 Hz, 3H); 13C NMR (75.48 MHz, CDCl3): d=169.9, 146.4, 114.9,
80.5, 62.2, 53.2, 51.5, 35.3, 28.2, 14.1 ppm; EIMS: m/z 333 [M]+; HRMS
(EI): calcd for C14H23NO6S: 333.1246; found: 333.1233.
(E)-9d: Analytical TLC (silica gel 60, 40% EtOAc in n-hexane): Rf =
0.62; 1H NMR (500 MHz, CDCl3): d=7.29–7.26 (m, 2H), 7.21–7.18 (m,
3H), 6.01 (s, 1H), 5.26–5.22 (m, 1H), 4.57–4.54 (m, 1H), 4.22 (q, J=
7.1 Hz, 2H), 3.24–3.08 (m, 2H), 3.04–2.98 (m, 2H), 2.94–2.88 (m, 2H),
2.76–2.72 (m, 2H), 1.52–1.48 (m, 2H), 1.44 (s, 9H), 1.32–1.24 (m, 5H),
0.94–0.86 ppm (m, 3H); 13C NMR (75.47 MHz, CDCl3): d=195.6, 170.4,
162.9, 141.4, 128.4, 128.3, 126.0, 116.3, 61.9, 52.2, 46.0, 34.5, 33.8, 31.9,
30.4, 29.7, 27.4, 22.7, 14.2, 13.9 ppm; EIMS: m/z: 463 [M]+; HRMS (EI):
calcd for C25H37NOS: 463.2392; found: 463.2390.
(Z)-9a: Analytical TLC (silica gel 60, 30% EtOAc in n-hexane): Rf =
0.44; 1H NMR (400 MHz, CDCl3): d=7.97–7.94 (m, 2H), 7.56–7.52 (m,
1H), 7.48–7.44 (m, 2H), 7.33 (d, J=9.7 Hz, 1H), 7.08 (d, J=9.7 Hz, 1H),
5.45 (brd, J=7.2 Hz, 1H), 4.63–4.61 (m, 1H), 4.24 (q, J=7.2 Hz, 2H),
3.32 (brd, J=3.7 Hz, 2H), 1.46 (s, 9H), 1.30 ppm (t, J=7.2 Hz, 3H);
13C NMR (100.62 MHz, CDCl3): d=189.0, 170.1, 155.0, 152.3, 137.7,
132.5, 128.6, 128.0, 80.3, 62.1, 53.9, 39.5, 28.3, 14.1 ppm; EIMS: m/z: 379
[M]+; HRMS (EI): calcd for C19H25NO5S: 379.1453; found: 379.1437.
Acknowledgements
(E)-9a: Analytical TLC (silica gel 60, 30% EtOAc in n-hexane): Rf =
0.53; 1H NMR (400 MHz, CDCl3): d=7.96 (d, J=8.0 Hz, 2H), 7.83 (d,
J=14.9 Hz, 1H), 7.56 (t, J=7.4 Hz, 1H), 7.47 (t, J=7.4 Hz, 2H), 7.10 (d,
J=14.9 Hz, 1H), 5.41 (brd, J=7.0 Hz, 1H), 4.69–4.67 (m, 1H), 4.25 (q,
J=7.1 Hz, 2H), 3.46–3.35 (m, 2H), 1.45 (s, 9H), 1.31 ppm (t, J=7.1 Hz,
3H); 13C NMR (75.48 MHz, CDCl3): d=186.7, 170.0, 155.0, 147.7, 137.8,
128.6, 128.4, 119.3, 80.6, 62.3, 53.2, 35.6, 28.2, 14.1 ppm; EIMS: m/z: 379
[M]+; HRMS (EI): calcd for C19H25NO5S: 379.1453; found: 379.1437.
This work was supported by The University of Hong Kong (University
Development Fund) and the Area of Excellence Scheme (AoE/P-10-01)
established under the University Grants Committee (HKSAR) and the
Research Grants Council (HKSAR) (HKU 7052/07P). We thank Dr. W.-
H. Cheung for providing some of the peptide samples and Dr. C.-N. Lok
for providing advice and materials for the peptide enrichment experi-
ment. We thank the reviewers for their helpful comments and sugges-
tions.
(Z)-9b: Analytical TLC (silica gel 60, 40% EtOAc in n-hexane): Rf =
0.47; 1H NMR (300 MHz, CDCl3): d=7.27–7.23 (m, 2H), 7.17–7.15 (m,
3H), 6.99 (d, J=9.7 Hz, 1H), 6.25 (d, J=9.7 Hz, 1H), 5.40 (brd, J=
7.2 Hz, 1H), 4.55–4.50 (m, 1H), 4.19 (q, J=7.1 Hz, 2H), 3.22 (brs, 2H),
2.95–2.90 (m, 2H), 2.79–2.74 (m, 2H), 1.42 (s, 9H), 1.26 ppm (t, J=
7.1 Hz, 3H); 13C NMR (75.48 MHz, CDCl3): d=198.2, 170.1, 155.1, 149.4,
145.6, 141.2, 128.5, 128.4, 126.0, 123.5, 120.3, 80.3, 62.1, 53.9, 44.5, 39.2,
31.6, 28.1, 14.2 ppm; EIMS: m/z: 407 [M]+; HRMS (EI): calcd for
C21H29NO5S: 407.1766; found: 407.1767.
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(E)-9b: Analytical TLC (silica gel 60, 40% EtOAc in n-hexane): Rf =
1
0.50; H NMR (300 MHz, CDCl3): d=7.54 (d, J=15.3 Hz, 1H), 7.27–7.25
(m, 2H), 7.20–7.17 (m, 3H), 6.23 (d, J=15.3 Hz, 1H), 5.34 (brd, J=
7.0 Hz, 1H), 4.63–4.58 (m, 1H), 4.21 (q, J=7.2 Hz, 2H), 3.35–3.20 (m,
2H), 2.95–2.91 (m, 2H), 2.81–2.77 (m, 2H), 1.43 (s, 9H), 1.29 ppm (t, J=
7.2 Hz, 3H); 13C NMR (75.48 MHz, CDCl3): d=195.4, 170.0, 155.0, 145.6,
141.1, 128.5, 128.4, 126.1, 123.5, 80.6, 62.3, 53.2, 42.7, 35.5, 31.6, 28.3,
14.1 ppm; EIMS: m/z: 407 [M]+; HRMS (EI): calcd for C21H29NO5S:
407.1766; found: 407.1761.
(Z)-9c: Analytical TLC (silica gel 60, 40% EtOAc in n-hexane): Rf =
0.57; 1H NMR (500 MHz, CDCl3): d=7.94–7.92 (m, 2H), 7.53–7.50 (m,
1H), 7.46–7.43 (m, 2H), 7.03 (s, 1H), 5.40 (brd, J=7.6 Hz, 1H), 4.62–
4.58 (m, 1H), 4.24 (q, J=7.1 Hz, 2H), 3.44–3.30 (m, 2H), 2.63–2.60 (m,
2H), 1.65–1.56 (m, 2H), 1.48–1.40 (m, 2H), 1.44 (s, 9H), 1.28 (t, J=
7.1 Hz, 3H), 0.97 ppm (t, J=7.3 Hz, 3H); 13C NMR (75.47 MHz, CDCl3):
d=188.5, 170.2, 163.6, 155.0, 138.7, 132.1, 128.5, 128.0, 117.4, 80.3, 62.1,
53.3, 36.9, 32.5, 32.1, 28.3, 22.3, 14.1, 13.9 ppm; EIMS: m/z: 435 [M]+;
HRMS (EI): calcd for C23H33NO5S: 435.2079; found: 435.2088.
Chem. Eur. J. 2009, 15, 3839 – 3850
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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Received: April 8, 2008
Published online: February 19, 2009
3850
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Chem. Eur. J. 2009, 15, 3839 – 3850