N-terminal α-amino group modification of peptides by an oxime formation-exchange reaction sequence

Article abstract of DOI:10.1039/c3cc42261e

A site-specific and efficient method for N-terminal modification of peptides using oxone for selective oxidation of N-terminal α-amino groups of peptides to oximes followed by transoximation with O-substituted hydroxylamines has been developed.

Full text of DOI:10.1039/c3cc42261e

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N-terminal a-amino group modification of peptides by
an oxime formation–exchange reaction sequence†
Cite this: Chem. Commun., 2013,
49, 6888
Karen Ka-Yan Kung, Kong-Fan Wong, King-Chi Leung and Man-Kin Wong*
Received 28th March 2013,
Accepted 11th June 2013
DOI: 10.1039/c3cc42261e
www.rsc.org/chemcomm
A site-specific and efficient method for N-terminal modification of
Oxidation of peptides and proteins is a fundamental biological
peptides using oxone for selective oxidation of N-terminal a-amino process which involves the attack of reactive oxygen species (ROS) on
groups of peptides to oximes followed by transoximation with amino acid residues.1 Considerable effort has been spent on the
O-substituted hydroxylamines has been developed.
studies of oxidative damage. However, the use of oxidizing agents for
peptide modification still remains limited owing to the formation of
Site-specific modification of peptides and proteins via covalent attach- complex oxidized products via uncontrolled oxidation.
ment of synthetic molecules allows the formation of bioconjugates A major challenge is to find controllable oxidizing reagents for
under mild reaction conditions, which are useful for studies in chemoselective modification of all the N-terminal amino acids. Along
chemical biology.1,2 Current approaches include labeling of target with our ongoing effort on the development of bioconjugation
amino acid residues such as Lys (K) e-amino and Cys (C) thiol groups, reactions,14 we developed chemoselective methods for modification
but these reactions generally lead to multiple modifications and require of N-terminal a-amino groups of peptides via oxidative amide for-
genetic manipulation by site-directed mutagenesis, respectively.1,2 mation using the ‘‘[Mn(2,6-Cl2TPP)Cl]–alkyne–H2O2’’ protocol14a
Recent advances have been achieved by native chemical ligation,3 [H2(2,6-Cl2TPP) = meso-tetrakis(2,6-dichlorophenyl)porphyrin] and an
Staudinger ligation,4 1,3-dipolar cycloaddition,5 olefin metathesis6 and isolated alkyne-functionalized ketene.14f Through screening of the
imine (oxime and hydrazone) ligation,7 which require direct modifica- commonly used bench-top oxidizing reagents, we discovered an
tion or incorporation of unnatural functional groups into one or several efficient method for N-terminal a-amino group modification of
particular amino acid residues. There is continuous interest in the peptides using oxone15 (2KHSO5ꢀKHSO4ꢀK2SO4) as a convenient and
development of new methods for selective bioconjugation.
An appealing approach is N-terminal a-amino group ligation reaction16 with O-substituted hydroxylamines (Scheme 1).
for site-specific modification of peptides and proteins. As they We are pleased to find that N-terminal modification of peptide 1
chemoselective reagent, followed by subsequent oxime exchange
generally have only one N-terminal a-amino group, it usually has YTSSSKNVVR (100 mM) with oxone (200 mM, 2 equiv.) and NaHCO3
no significant impact on their biological activities. Thus, solution (1.5 mM, pH 8.3) at 25 1C for 1 h gave oxime-modified
N-terminal modification has potential applications in bioconjuga- peptide 2 in 99% substrate conversion (Fig. 1 and Table 1, entry 6).
tion,8 positional proteomics studies9 and immobilization.10 Tra- As confirmed by ESI-MS/MS analysis of the crude reaction mixture,
ditionally, the N-terminus of Ser (S) or Thr (T) can be treated with the N-terminal a-amino group of Tyr (Y) was modified while the
NaIO4 to form an aldehyde group for further functionalization.1,11 backbone amino acid residues such as Thr (T), Ser (S), Lys (K), Asn
Recently, selective oxidation of the N-terminal a-amino group to (N) and Arg (R) remained intact (Fig. S1, ESI†).
oximes and hydroxylamines has been utilized in the pyridoxal
Optimization of reaction conditions for oxidation of the
50-phosphate (PLP)-mediated biomimetic transamination–oxime N-terminus of peptide 1 was conducted using different oxidants
formation sequence12 and a-ketoacid–hydroxylamine amide-form- in aqueous solvents (Table 1). Our findings suggested that oxone
ing reaction,13 respectively. Note that oximes and hydroxylamines (200 mM, 2 equiv.) gave oxime-modified peptide 2 with excellent
are bioorthogonal to most amino acid residues. However, there is conversions in PBS (25 mM, pH 8.0) and NaHCO3 (1.5 mM, pH 8.3)
a lack of general methods for modification of all the N-terminal solutions, respectively (entries 4 and 6). Note that 70% and 40%
amino acids.
State Key Laboratory of Chirosciences and Department of Applied Biology and
Chemical Technology, The Hong Kong Polytechnic University, Hung Hom,
Hong Kong, China. E-mail: mankin.wong@polyu.edu.hk; Fax: +852-2364-9932
Scheme 1 N-terminal a-amino group modification by an oxime formation–
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc42261e
exchange reaction sequence.
c
6888 Chem. Commun., 2013, 49, 6888--6890
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Fig. 1 N-terminal a-amino group modification of peptide 1 using oxone in
NaHCO3 solution (1.5 mM, pH 8.3).
Fig. 2 Time course of N-terminal a-amino group modification of peptide 1 (100 mM)
with various concentrations of oxone in NaHCO3 solution (1.5 mM, pH 8.3) at 25 1C.
Table 1 Screening conditions for N-terminal modification of peptide 1 by oxime
formationa
N-terminal modified
Entry
Oxidant
Aqueous solvent
peptideb (%)
1
2
3
4
5
6
7
8
Oxone
Oxone
Oxone
Oxone
Oxone
Oxone
m-CPBA
m-CPBAc
PBS (pH 5.1)
PBS (pH 6.0)
PBS (pH 7.1)
PBS (pH 8.0)
0
0
70
96
40
99
29
85
Fig. 3 A model study using L-tyrosine methyl ester 3.
PBS (pH 9.3)
NaHCO3 (pH 8.3)
NaHCO3 (pH 8.3)
NaHCO3 (pH 8.3)
amino acids). These peptides were chosen with nucleophilic Ser
(S) and Lys (K) for studying chemoselectivity of this reaction. As
depicted in Table 2, N-terminal modifications were found in all
a
Reactions were carried out with peptide 1 (100 mM) and oxidant
(200 mM) in PBS (25 mM, pH 5.1–9.3) or NaHCO3 solution (1.5 mM,
b
pH 8.3) at 25 1C for 1 h. Conversions were determined by LC-MS for all
PBS solutions and by ESI-MS for NaHCO3 solution. m-CPBA (2 mM).
c
Table 2 N-terminal a-amino group modification of peptides (XSKFR) carrying
each of the 20 amino acids at the N-terminus by oxime formationa
conversions were obtained in PBS solutions at pH 7.1 and pH 9.3
(entries 3 and 5), while no conversion was observed under acidic
conditions (pH 5.1 and 6.0, entries 1 and 2). m-Chloroperoxyben-
zoic acid (m-CPBA, 200 mM, 2 equiv.) oxidized the N-terminus of
peptide 1 with 29% conversion in NaHCO3 solution (1.5 mM, pH
8.3), while 85% conversion was achieved by increasing the concen-
tration of m-CPBA to 2 mM (entries 7 and 8). No oxime-modified
peptide was found when H2O2, NaIO4, PhI(OAc)2, 2-bromopyridine
N-oxide, 2,6-dichloropyridine N-oxide or quinoline N-oxide was
used as the oxidant (200 mM, 2 equiv.) in NaHCO3 solution
(1.5 mM, pH 8.3). The pKa value of the Lys (K) e-amino group
(B10) is higher than that of the N-terminal a-amino group (B8).1
In this connection, the Lys (K) e-amino group is largely protonated
(B99%) under the reaction conditions (pH 8.3) and thus resistant
to oxidation. In contrast, around 50% of the N-terminal a-amino
group remains in its free amine form which is susceptible towards
oxidation, contributing to the N-terminal selectivity in oxidation.
Time course experiments for N-terminal a-amino group modifi-
cation of peptide 1 by varying oxone concentrations (1–10 equiv.)
and reaction temperatures (4 and 25 1C) were performed. Over 99%
conversions were found in 1 h at 25 1C when oxone (2–10 equiv.) was
used (Fig. 2). Overall conversions of 20–55% with oxone (1–5 equiv.)
could be achieved in 1 h when the time course experiments were
performed at 4 1C (Fig. S3, ESI†).
N-terminal a-amino
group modified
XSKFRb (%)
Both N-terminal a-amino
group and side chain
modified XSKFRb (%)
Entry
Peptide
1
2
3
4
5
6
7
8
QSKFR
RSKFR
YSKFR
HSKFR
ESKFR
FSKFR
NSKFR
ASKFR
ISKFR
LSKFR
VSKFR
GSKFR
KSKFR
DSKFR
SSKFR
TSKFR
PSKFR
WSKFR
WSKFR
WSKFR
MSKFR
CSKFR
CSKFR
99
91
87
87
86
85
85
80
80
80
80
67
52
45
40
21
99c
40
16
38
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
38d
6d
9
10
11
12
13
14
15
16
17
18
19e
20f
21
22
23i
41d
55g
0h
99j
a
Reactions were carried out with XSKFR (100 mM), oxone (200 mM) in NaHCO3
solution (1.5 mM, pH 8.3) at 25 1C for 1 h. b Conversions were determined by
ESI-MS. c The pyrrolidine ring of Pro (P) was oxidized to nitrone. d Both the
N-terminal a-amino group and indole of Trp (W) were oxidized to give a
1,3-dihydro-indol-2-one-oxime derivative. e m-CPBA (200 mM) was used instead
of oxone. f m-CPBA (500 mM) was used instead of oxone. g Both N-terminal
a-amino and thioether groups of Met (M) were oxidized to give a sulfone–
oxime derivative. h The thiol group of Cys (C) was oxidized to sulfonic acid.
i Oxone (500 mM) was used. j N-terminal a-amino and thiol groups of Cys (C)
To provide support for the structure of the oxime-modified
peptides, a model reaction with L-tyrosine methyl ester 3 was
performed, giving the corresponding oxime-modified product 4
in 91% conversion and 67% isolated yield (Fig. 3).
We then extended the scope of the N-terminal modification
to a series of unprotected peptides XSKFR14f (X = 20 natural were oxidized to oxime and sulfonic acid, respectively.
c
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Research Grants for Newly Recruited Junior Academic Staff and
SEG PolyU01).
Notes and references
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Fig. 4 Oxime exchange of oxime-modified peptide 2 by functionalized hydroxy-
lamines 5a–b.
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XSKFR peptides in a range of conversions (21–99%, entries 1–21 and
23), as confirmed by ESI-MS/MS analysis (see ESI†). The N-terminal
proline moiety of PSKFR was converted to nitrone (entry 17).17 Both
N-terminal and side-chain modifications were observed in peptides
with N-terminal Trp (W) and Met (M) (entries 18–21). Using 200 mM
oxone, only the thiol group in CSKFR was oxidized to give sulfonic
acid (entry 22). Both N-terminal and side-chain modifications of
CSKFR were found by increasing the oxone concentration to 500 mM
(entry 23). With the exception of the transamination methods
developed by Francis et al.,12 the common methods for modification
of N-terminal residues of peptides and proteins are limited to a few
N-terminal amino acids, for example, modification of N-terminal Ser
(S) or Thr (T) by NaIO4 and N-terminal Cys (C) by native chemical
ligation, respectively.2,3 Note that the present modification of the
N-terminal a-amino group of peptides by oxone provides a general,
chemoselective and convenient way to introduce oxime into the
N-terminal of virtually all the 20 natural amino acids via a simple
oxidation reaction under mild reaction conditions.
To achieve sequential peptide functionalization, an oxime
exchange16 between oxime-modified peptide 2 and O-substituted
hydroxylamines was investigated by varying promoters and reaction
temperatures (Table S1, ESI†). We found that the transoximation
of oxime-modified peptide 2 with O-benzylhydroxylamine (2 mM,
20 equiv.) could be achieved smoothly using trifluoroacetic acid16
(0.65 equiv.) at 50 1C for 20 h to give the O-benzyloxime-modified
peptide in 98% conversion. Besides, the transoximation also works
well with aniline and p-anisidine as promoters under neutral reaction
conditions,7 leading to O-benzyloxime-modified peptides in 96%
and 93% conversions, respectively. Furthermore, incorporation of
functionalized hydroxylamines such as dansyl (5a) and PEG (5b, av.
MV = 500) groups into oxime-modified peptide 2 was demonstrated to
give functionalized peptides 6a–b in 99% conversion (Fig. 4, Fig. S29
and S30, ESI†). Attachment of fluorophores onto peptides facilitates
molecular imaging for biological studies while PEGylation of peptides
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This work is supported by Hong Kong Research Grants
Council (PolyU 5031/11P), The Hong Kong Polytechnic University
(PolyU Departmental General Research Funds and Competitive
`
´
´
17 C. Gella, E. Ferrer, R. Alibes, F. Busque, P. de March, M. Figueredo
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c
6890 Chem. Commun., 2013, 49, 6888--6890
This journal is The Royal Society of Chemistry 2013