Selective lysine modification of native peptides: Via aza-Michael addition

Article abstract of DOI:10.1039/c7ob01866e

A series of vinylsulfonamides were synthesized and screened for site-selective modification of the ?-amino group of lysine-bearing free α-amine residues. N-Methyl-N-phenylethenesulfonamide has emerged as an applicable reagent and has been developed for efficient and highly selective modification of the lysine residue of native peptides in the presence of a free N-terminus via aza-Michael addition. We demonstrated that functional N-phenylvinylsulfonamide derivatives with a fluorescent moiety or drug could also be conjugated to the lysine residue of octreotide and insulin with high specificity, without modifying the N-terminus. Our method provides a promising strategy for site-selective lysine functionalization in native peptides with a free N-terminus.

Full text of DOI:10.1039/c7ob01866e

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Selective lysine modification of native peptides via aza-Michael
addition
Received 00th January 20xx,
Accepted 00th January 20xx
Hongli Chen,* Rong Huang, Zhihong Li, Wei Zhu, Jiakang Chen, Yuexiong Zhan, Biao Jiang*
A series of vinylsulfonamides were synthesized and screened for site-selective modification of the ε-amino group of lysine-
bearing free α-amine residues. N-methyl-N-phenylethenesulfonamide has emerged as an applicable reagent and has been
developed for efficient and highly selective modification of the lysine residue of native peptides in the presence of a free
N-terminus via aza-Michael addition. We demonstrated that functional N-phenylvinylsulfonamide derivatives with a
fluorescent moiety or drug could also be conjugated to the lysine residue of octreotide and insulin with high specificity,
without modifying the N-terminus. Our method provides a promising strategy for site-selective lysine functionalization in
DOI: 10.1039/x0xx00000x
www.rsc.org/
native
peptides
with
a
free
N-terminus.
and generation of byproducts are major problems
encountered when attempting to selectively label an amine.
The Michael-type addition is also an attractive strategy for
protein conjugation. Michael acceptors, such as maleimides,
vinyl sulfones and acrylamide, have been frequently utilized in
the modification of proteins. Vinyl sulfonamides, with a similar
structure to vinyl sulfones, are easier to prepare and have also
received some attention in applications for bioconjugation.23-26
Although the lysine ε-amino group requires a higher pH to
deprotonate, it has higher intrinsic nucleophilicity. Therefore,
selective lysine modification can be achieved by carefully
adjusting the nucleophilicity and basicity. Herein, we report a
class of vinylsulfonamides which site-specific modify the lysine
residue in native peptides rather than N-terminus via aza-
Michael addition.
Introduction
The modification of native peptides at a single amino acid or
specific site with synthetic moieties is significant
challenge 1-3
Among the approximately 20 primary amino
a
.
acids that compose proteins, only a subset can serve as
appropriate targets for bioconjugation. Many strategies have
been developed to target nucleophilic natural amino acid
residues, of which cysteine and lysine residues are the most
popular labeling sites. Cysteine is capable of being involved in
a chemoselective reaction due to its superior nucleophilic
properties.4-7 However, the low abundance of free cysteine
residues due to its tendency to form disulfide bonds limits its
application. Lysine residues are abundant in native proteins,
and methods to modify primary amines can provide versatile
techniques to label lysine residues.8,
However, lysine
9
conjugation methods often modify the N-terminus.10, 11 Site-
specific modification of the N-terminus is easier to achieve
because the ε-amino group of lysine is more difficult to
deprotonate due to its higher pKa (~10) compared to the N-
terminal α-amino group (pKa ~8). A number of strategies for
selective N-terminal modification of native peptides or
Results and discussion
We first synthesized a series of vinylsulfonamides (Fig. 1,
compounds
the easiest to use to produce divinylsulfonamides (Fig. 1,
10), which would be developed as efficient
1-7). We found that activated phenylamines were
proteins have been developed.12-18 In contrast, site-specific compounds
8-
functionalization of the ε-amino group of lysine is rarely
studied.19, 20Typically, the most common reagents for amine
modification, N-hydroxysuccinimides (NHS) esters, also react
with serine, tyrosine and histidine, affording heterogeneous
reagents for peptide stapling in our coming research. Then, we
began to screen the abilities of different vinylsulfonamides to
promote selective ε-amino group modification. Our initial
study found that triethylenediamine (DABCO) buffer was a
good media to take place the aza-Michael addition. Therefore
we employed an L-lysine methyl ester (11) to react with
bioconjugates.21
Although
stepwise
addition
of
substoichiometric amounts of an NHS ester can achieve a
single lysine-selective modification, tediousness and low
efficiency are problems22. Indeed, the balance of low reactivity
vinylsulfonamides (
PH=7. Based on RP-HPLC analysis, compounds
with Lys-OMe (ESI, Table S1, entry 1-2). Compounds
showed poor selectivity (ESI, Table S1, entry 3-4). Compounds
displayed good selectivity, however, the conversion was
poor (<20%) (ESI, Table S1, entry 5-6). N-methyl-N-
phenylethenesulfonamide (compound emerged as
1-7
) in DABCO buffer and CH3CN (1:1) at
1-2
did not react
3-4
5-6
7)
a
potential reagent with good selectivity and moderate
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conversion (~30%) (ESI, Table S1, entry 7). We further Compound 14 could be further labeled with fluorescein
screened a series of alkaline buffers at pH 7-9 for the reaction isothiocyanate (FITC) at the N-terminus to produce compound
of compound
Unfortunately, the selectivity or conversion became bad under compound 15 confirmed its structure and attachment site
7
and Lys-OMe (11) (ESI, Table S1, entry 8-17). 15 with high yield (87%). The MS and MS2 experiment for
all of those reaction conditions.
O
based on an m/z of 1606.5349 [M+H]+ and 803.7729 [M+2H]2+
and the presence of the fragment FITC-NH-D-Phe with m/z
537.11 (Fig. 2F, 2G, 2H).
H
H
O
O
O
N
N
S
S
N
O
S
S
N
N
HN
S
O
O
O
O
O
Cl
O
5
4
3
1
2
O
N
A
S
DPhe NH2
Phe Cys
O
DPhe NH2
Phe Cys
NO2
O
O
DTrp
Lys
DTrp
Lys
7
O
N
H
O
O
S
CH3CN:H2O=1:1
Et3N, rt, 4h, 89%
N
N
N
NH2
O
S
S
H
Cys Thr ol
Thr
Cys Thr ol
Thr
O
O
O
S
O
S
O
O
O
O
S
N
N
N
S
S
S
Octreotide , 13
14
O O
O O
8
O O
9
6
7
10
O
S
S
C
N
Fig. 1 The structure of vinylsulfonamides 1-10
C
NH FITC
O
DPhe NH
Phe Cys
DTrp
Lys
O
HO
O
FITC
OH
N
Compound
the presence of trimethylamine (Et3N 6 eq.) in CH3CN.
Compound was consumed, and only one high yield product
12, purified yield: 87%) was obtained, which was mono-
7 was allowed to react with Lys-OMe (11, 3 eq.) in
S
N
O
H
Cys Thr ol
Thr
PBS PH=9, rt, 2h, 87%
15
7
(
labeled based on the HRESIMS of the [M+H]+ and [M+Na]+ (ion
peaks at 358.1917 and 380.1615, respectively). The attached
position was the ε-amino group rather than the α-amino group
based on HSQC and HMBC analysis; key correlations were
observed between H-8 and C-10, C-13, C-14 (Scheme 1).
O
13
O
H
12
O
S
O
9
7
N
N
6
H2N
Et3N,CH3CN
rt, 8 h, 87%
S
O
1
NH2
O
N
10
8
2
14
O
O
NH2
12
7
11
Scheme 1 Selective modification of the ε-amino group
Having observed the high yield and good selectivity of the N-
methyl-N-phenylethenesulfonamide (compound )-based
7
conjugate, we next used this method to label peptides.
Octreotide (13), a synthetic octapeptide with the sequence
H2N-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge
Cys2-Cys7) that is one of the most extensively studied
somatostatin analogues, was selected to evaluate the lysine
site-specific modification. Octreotides have been widely
investigated for conjugation with toxic drugs for targeted
delivery into SSTR-selective cancer cells. The studies have
revealed that a mixture of products (modification at the N-
terminus, the lysine residue, or both) was produced when
octreotide was allowed to react with NHS esters.27-30
Octreotide was treated with compound
7 (3 eq.) in the
presence of Et3N (6 eq.) in a CH3CN and H2O (1:1) solution at
room temperature. The reaction proceeded smoothly, and
octreotide was almost completely converted to a single
product (14) after 4 h (Fig. 2A), as analyzed by RP-HPLC (Fig.
2B). The HRESIMS spectrum showed that product 14 was
mono-labeled, with signals at 1216.4995 [M+H]+ and 608.7549
[M+2H]2+ (Fig. 2C). After reduction of product 14 with TCEP,
peptide fragments were obtained and identified by MS2 (Fig.
2D). The peptide fragment Lys-Thr-Cys-Thr-ol with m/z 635.28
[M+H]+ carried the Michael acceptor moiety, because it
corresponds to the calculated M.W. of 634.28 g mol-1. The
other peptide fragment H2N-D-Phe-Cys-Phe-D-Trp with m/z
584.23 [M+H]+ was not modified (Fig. 2E). Therefore, the lysine
rather than the N-terminus was selectively functionalized.
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Fig. 3 Selective lysine modification of lanreotide. (A) The
reaction scheme. (B) The HRESIMS spectrum of product 18. (C)
The HRESIMS spectrum of reduced product of compound 18.
(D) The MS2 spectrum of reduced product of compound 18. (E)
Peptide fragments.
Fig. 2 Stepwise modification of the lysine residue and N-
terminus of octreotide. (A) The reaction scheme. (B) RP-HPLC
of the reaction. (C) The HRESIMS spectrum of product 14. (D)
The MS2 spectrum of product 14. (E) Peptide fragments of
compound 14. (F) The HRESIMS spectrum of product 15. (G)
The MS2 spectrum of product 15. (H) Peptide fragments of
Insulin (17), a peptide hormone consisting of two polypeptide
chains – the A chain (glycine-A1) and B chain (phenylalanine-
B1) – has a lysine in the B chain (lysine-B29). The B29 position
is generally tolerant with respect to insulin receptor affinity,
and B29-modified analogues such as detemir and degludec
(FDA approved drugs) have demonstrated excellent safety
profiles.31 B29 was found to react at a much slower rate than
the N-termini of A1 and B1 when insulin was treated with FITC
at pH 9.1, producing a mono-A1, mono-B1, di-A1B1, and tri-
substituted A1B1B29-labeled mixture.32 Studies have revealed
that NHS ester-based reagents are preferable to selectively
modify B29,33, 34 however, there is also report on the difficulty
to selectively attach a single group to available amine sites.35
Here, the ability of vinylsulfonamide-based aza-Michael
addition to site-specifically label insulin B29 was investigated.
To strengthen the applicability of this reaction, more suitable
reaction conditions were explored. First, insulin was treated
compound 15
.
To demonstrate the general applicability of our method to
selectively modify native peptides at lysine residues, we also
investigated the reaction of compound
7
with lanreotide (16
)
and insulin (17). Lanreotide, similar to octreotide, is
a
somatostatin analog with the sequence H-D-2Nal-Cys(2)-Tyr-D-
Trp-Lys-Val-Cys(7)-Thr-NH2. It was modified with compound
7
in a similar fashion to obtain the lysine-modified product 18
with high yield (84%) (Fig. 3A). The HRESIMS spectrum showed
that product 18 was mono-labeled, with signals at 647.2653
[M+2H]2+ (Fig. 3B). After reduction of product 18 with TCEP
(Fig. 3C), peptide fragments were obtained and identified by
MS2 (Fig. 3D and 3E). The result showed that the lysine residue
was modified.
with compound
7 in a buffer series containing various bases at
approximately pH 7 and room temperature. The buffer
adjusted with DBU was optimal at promoting site-specific B29
modification of insulin. Although the majority of the
conversion yielded a single product, the conversion of insulin
was poor (<10%). The effect of pH was examined based on the
DBU buffer. By screening buffers from pH 7 to pH 12, we found
that the conversion of insulin was low when the pH was below
8, and the reaction became complicated when the pH was
more than 10. Moderate conversion (52%) and excellent
selectivity (almost a single product 19 in 49% yield and 90%
yield based on recovered insulin) were observed in pH 9 buffer
adjusted with DBU (Fig. 4A). The isolated product 19 exhibited
signals with multiple charges at 1001.6223 [M+6H]6+,
1201.7446 [M+5H]5+ and 1502.1787 [M+4H]4+, demonstrating
that it was a mono-labeled product (Fig. 4B). After reduction of
the disulphide bonds and in-solution digestion of compound
19 by chymotrypsin, peptide fragments were obtained and
analyzed by MS2 (Fig. 4C) The modification site was
determined as B29 (lysine) based on the fragment (GFFYTPKT+
A
O
Tyr Cys D2Nal
N
NH2
Tyr Cys D2Nal
NH2
S
DTrp
Lys
DTrp
Lys
O
O
N
7
S
O
O
NH2
N
H
O
CH3CN:H2O=1:1
Et3N, 6h, rt, 84%
Cys Thr CNH2
Val
Cys Thr CNH2
Val
18
Lanreotide (16)
D
compound
7)
with m/z 579.2698 [M+2H]2+, which
corresponded well to the calculated M.W. of 1156.5263.
A
A1
O
N
GIVEQCCASVCSLYQLENYCN
H2N
S
GIVEQCCASVCSLYQLENYCN
H
2N
O
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
H2N
7
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
H2
N
O
DBU buffer : CH3CN=2:1
PH=9, rt, 12h, 49%
NH
NH2
B29
S
B1
N
O
E
Insulin (17)
19
B
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spectrum, matching its calculated M.W. of 1416.5341 g mol-1
(Fig. 5B). After in-gel digestion by chymotrypsin, the MS2
spectrum of compound 30 was determined. The degradation
product (m/z 1109.4239) was detected under MS2 conditions
due to the instability of the attached probe 20. Compound 20
was degraded to a residue with m/z 308.1147 when it was
directly used for the MS2 experiment under the same
conditions. The modified peptide fragment Lys-Thr-Cys-Thr-ol
with m/z 527.1955 included a fragment from probe 20 (m/z
399.04-308.11=90.93) and the unmodified peptide fragment
H2N-D-Phe-Cys-Phe-D-Trp with m/z 584.2334, indicating
labeling at lysine (Fig. 5C). For compound 31, the mono-lysine-
modified feature was also determined by HRESIMS with signals
at m/z 801.3308 [M+2H]2+ and 1602.6505 [M+H]+ (calculated
value: 1600.6441) and MS2 with an unmodified peptide
fragment H2N-D-Phe-Cys-Phe-D-Trp (m/z 584.2324) (Fig. 5D).
A
C
Fig. 4 Selective modification of insulin B29 lysine. (A) The reaction
scheme. (B) The HRESIMS spectrum of product 19. (C) The MS2
spectrum of product 19.
DPhe NH2
Phe Cys
DPhe NH2
Phe Cys
Compound 20
DTrp
Lys
DTrp
O
O
CH3CN:H2O=1:1,
Et3N, rt,
overnight, 83%
N
S
O
N
H
Lys
NH2
N
Cys Thr ol
Thr
Cys Thr ol
Thr
H
O
O
30
Octreotide (13)
An effective bioconjugation method has to have the ability to
attach functional groups of interest, such as fluorescent
probes and drugs. Therefore, we further explored
vinylsulfonamides as versatile building blocks for site-specific
preparation of more interesting bioconjugates. Coumarin- and
MeO
MeO
OMe
DPhe NH2
Phe Cys
NH
N
O
Compound 21
DPhe NH2
Phe Cys
DTrp
Lys
O
S
O
OMe
O
DTrp
Lys
CH3CN:H2O=1:1,
Et3N, rt,
overnight, 80%
NH2
HN
Cys Thr ol
Thr
Cys Thr ol
Thr
31
Octreotide (13)
combretastatin A-4- (CA4,
a
potent cytotoxic agent)
functionalized vinylsulfonamides (20 and 21) were synthesized.
The synthesis of product 20 was carried out by condensation
B
of N-(2-aminoethyl)benzeneamine
(22) and coumarin-3-
carboxylic acid (23) to form the amide (24), followed by
treatment with 2-chloroethanesulfonyl chloride (25) to afford
the desired product 20. Compound 21 was prepared in a
similar manner: conjugation of CA4 derivative 28, easily
obtained from CA4 (26) according to the reported procedure36
with compound 22 yielded compound 29, which was
subsequently allowed to react with compound 25 to obtain the
target product 21 (Scheme 2).
O
S
Cl
O
O
O
S
N
O
H2
N
O
O
O
N
Cl
NH
O
H
22
25
N
H
N
OH
H
Et3N, DCM, 0℃-
EDCI, HOBt, Et3N,
DCM, rt, 4h, 92%
O
O
O
O
C
reflux, 6h, 78%
23
24
20
MeO
MeO
MeO
MeO
MeO
MeO
O
LiOH,CH3OH,
H2O, rt, 3h
Br
O
OMe
26
OMe
27
O
OMe
OH
quantitative
yield
K2CO3, acetone,
OH
reflux, 8h, 85%
O
O
OMe
OMe
O
OMe
O
28
O
MeO
MeO
MeO
Cl
S
O
H2N
N
Cl
25
22
H
MeO
OMe
Et3N, DCM, 0℃
EDCI, HOBt, Et3N,
DCM, rt, 4h, 86%
OMe
NH
N
NH
N
H
O
O
-reflux, 83%
O
S
O
OMe
O
OMe
O
21
29
D
Scheme 2 Synthesis of functional vinylsulfonamides 20 and 21
Based on the successful incorporation of the probes to
vinylsulfonamides, their selective conjugation ability for the
peptides octreotide and insulin was investigated. As previously
described, octreotide was allowed to react with compounds 20
and 21 in the presence of Et3N, leading to the mono-labeled
derivatives 30 and 31 with high yield and successful selectivity
(Fig. 5A). For compound 30, the mass peak of m/z 709.2782
[M+2H]2+ and 1417.5403 [M+H]+ was found in the HRESIMS
Fig.
5 Selective lysine modification of octreotide with
functional vinylsulfonamides 20 and 21. (A) The reaction
scheme. (B) The HRESIMS spectrum of product 30. (C) The MS2
4 | J. Name., 2012, 00, 1-3
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spectrum of product 30. (D) The HRESIMS and MS2 spectrum
ARTICLE
Fig. 6 Selective lysine modification of insulin with functional
vinylsulfonamides 20 and 21. (A) The reaction scheme. (B) The
HRESIMS and MS2 spectrum of product 32. (C) The HRESIMS
and MS2 spectrum of product 33. (D) RP-HPLC of the reaction
showing that compound 21 was conjugated to insulin.
of product 31
.
In the case of insulin, to conjugate with compound 20 or 21
,
specific mono-modification of the B29 lysine was performed
using the above optimized method with DBU buffer at pH=9
(Fig. 6A). Products 32 and 33 were also confirmed by MS and
MS2 (Fig. 6B, 6C). Compound 21 was an excellent coupling
reagent for insulin that resulted in successful conversion
(>95%), yield (92%) and selectivity (almost a single product)
(Fig. 6D).
Somatostatin (34) which has a free N-terminus and two lysine
residues with the sequence H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-
Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (Disulfide Bridge Cys3-Cys14)
was employed to investigate whether there was a preference
for a single lysine residue. Somatostatin was reacted with
A
compound
7 in DBU buffer (PH=9) (Fig. 7A). The resulting
A1
GIVEQCCASVCSLYQLENYCN
H2N
HRESIMS spectrum revealed that a mono-labeled product 35
(9%) was obtained, with signals at 612.2921 [M+3H]3+ and
917.9351 [M+2H]2+ (Fig. 7B). Similarly, after reduction of
product 35 with TCEP (Fig. 3C), peptide fragments were
obtained and identified by MS2 (Fig. 7D and 7E).The single K9
residue of somatostatin was modified. However, the
conversion was low and the reaction system became
complicated with the prolonged reaction time. When
20
Compound
GIVEQCCASVCSLYQLENYCN
H2
H2
B1
N
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
H2
N
DBU buffer : CH3CN
=2:1, PH=9, rt,
overnight, 45%
O
O
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
N
O
H
NH
N
S
NH2
B29
N
O
O
Insulin (17)
32
GIVEQCCASVCSLYQLENYCN
H
2N
A1
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
H2
N
21
Compound
NH
GIVEQCCASVCSLYQLENYCN
H2
H2
N
DBU buffer : CH3CN
=2:1, PH=9, rt,
overnight, 92%
O
MeO
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
N
S
OMe
MeO
N
O
NH2
B29
B1
OMe
O
somatostatin (34) was reacted with compound
7 (3 eq.) in the
33
NH
Insulin (17)
presence of Et3N (6 eq.) in a CH3CN and H2O (1:1), a main di-
O
B
labelled product (36) was yielded (63%) with signals at
678.3126 [M+3H]3+ and 1016.9655 [M+2H]2+ (Fig. 7F). After
reduction of product 36 with TCEP (Fig. 7G), the resulting
HRESIMS spectrum showed that A1 and K9 were modified (Fig.
7H-I).
A
O
N
A G C K N F F W K T F T S C
S
O
7
A G C K N F F W K T F T S C
SO2
N
DBU buffer : CH3CN
=2:1, PH=7.4, rt, 2h,
9%
Somatostatin, 34
35
C
O
N
A G C K N F F W K T F T S C
S
O
7
A G C K N F F W K T F T S C
O2S
N
SO2
N
CH3CN:H2O=1:1
Et3N, 6h, rt, 63%
Somatostatin, 34
36
B
C
D
D
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Conclusions
Selective and effective modification of a lysine residue in the
presence of an N-terminus is anticipated to be difficult due to
the lower pKa of the N-terminal position. Here, a useful and
efficient method for utilizing vinylsulfonamide-based aza-
Michael addition to selectively modify lysine instead of the N-
terminus on peptides was developed. Furthermore, we
demonstrated that functional vinylsulfonamide derivatives
could also be produced via reaction with a fluorescent moiety
or drug and achieve the selective bioconjugation. The CA4-
conjugated probe showed excellent reactivity and selectivity to
modify the B29 lysine of insulin. This is a promising method for
native peptide modification where the N-terminus is crucial for
peptide bioactivity. However, peptides that contain multiple
lysine residues will result in a complicated situation. And our
method is more benefit for peptides that lack accessible free
Cys residue which has superior reactivity. Efforts to develop
more useful vinylsulfonamide reagents and application of this
method to modify peptides or proteins are currently
underway.
E
F
G
H
Acknowledgements
This work was supported by ShanghaiTech university.
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