Insight into the SEA amide thioester equilibrium. Application to the synthesis of thioesters at neutral pH

Article abstract of DOI:10.1039/c6ob01079b

The bis(2-sulfanylethyl)amide (SEA) N,S-acyl shift thioester surrogate has found a variety of useful applications in the field of protein total synthesis. Here we present novel insights into the SEA amide/thioester equilibrium in water which is an essenti

Full text of DOI:10.1039/c6ob01079b

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Insight into the SEA amide thioester equilibrium. Application to
the synthesis of thioesters at neutral pH
Received 00th January 20xx,
Accepted 00th January 20xx
S. Pira, O. El Mahdi, L. Raibaut, H. Drobecq, J. Dheur, E. Boll, O. Melnyk*
The bis(2-sulfanylethyl)amide (SEA) N,S-acyl shift thioester surrogate has found a variety of useful applications in the field
of protein total synthesis. Here we present novel insights into the SEA amide/thioester equilibrium in water which is an
essential step in any reaction involving the thioester surrogate properties of the SEA group. We show also that the SEA
amide thioester equilibrium can be efficiently displaced at neutral pH for accessing peptide alkylthioesters, i.e. the key
components of the native chemical ligation (NCL) reaction.
DOI: 10.1039/x0xx00000x
www.rsc.org/
has been shown to proceed from pH 3 to 7.3,32 The SEA
thioester form 1b can also be trapped in situ by simple
alkylthiols such as 3-mercaptopropionic acid (MPA) to produce
Introduction
Modern protein total synthesis relies on the coupling of
unprotected peptide segments in water using chemoselective
amide bond forming reactions.1-6 Peptide thioester chemistry
plays a central role in the field since the discovery of the native
chemical ligation (NCL) by Dawson and coworkers.1,4 This
reaction is based on the chemoselective reaction of a C-
terminal peptide thioester with an N-terminal cysteinyl (Cys)
peptide in water at neutral pH. The reaction produces a native
peptide bond to cysteine and since its introduction in 1994 it
has been extended to the formation of nearly all types of
peptide junctions using desulfurization,7-9 deselenization10,11 or
thiol auxiliary approaches.12-14 During the last decade, several
N,S-acyl shift systems have been designed for acting as
thioester surrogates3,15-23 (for recent reviews see24,25). A few of
them have the capacity to act as thioester surrogates at
neutral pH, and thus to react spontaneously and
chemoselectively with Cys peptides in a manner analogous to
the NCL reaction.3,22,23,26 Although these N,S-acyl shift systems
are becoming more and more popular in the field of protein
total synthesis, their mechanism of action is far from being
fully understood.27
MPA peptide thioesters of type
4 which can be isolated
(Scheme 1).33 This transformation must be carried out at an
acidic pH of 4 to be of synthetic value.
This study provides novel insights into the SEA amide/thioester
equilibrium, which is the starting point of all the reactions
involving the SEA thioester surrogate. We characterized also
the SEA/MPA exchange reaction at different pH. Since the level
of SEA/MPA exchange was modest at neutral pH, we sought to
displace the equilibrium toward the MPA peptide thioester for
providing an access to these valuable peptide derivatives using
very mild conditions.
O
SH
N
SH
peptide 1
1a
SEA peptide amide
SH
O
N,S-
acyl shift
peptide 2
NH2
O
2
SH
peptide 2
O
MPAA (excess)
S
peptide 1
TCEP, pH 3-7
The bis(2-sulfanylethyl)amide (SEA3) group belongs to this
growing family of useful thioester surrogates which can
substitute classical peptide thioesters or be combined with
them for designing efficient protein synthetic strategies
(Scheme 1).5,28-31 SEA peptide amide 1a rearranges
spontaneously in water into the SEA thioester form 1b
Apparently, this rearrangement takes place in a wide range of
pH since SEA ligation with Cys peptides to produce peptide
peptide 1
N
NH
H
O
3
HS
CO2H
1b
SEA peptide thioester
MPA (excess)
HS
MPAA
TCEP
TCEP, pH 4
37°C
HO2C
CO2H
.
P
O
CO2H
S
2
3
peptide 1
MPA peptide thioester
CO2H
O
4
a. Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T - Mechanisms
of Tumorigenesis and Target Therapies, F-59000 Lille, France.
Université Sidi Mohamed Ben Abdellah, Morocco.
MPA
HS
OH
b
Scheme 1. Principle of SEA N,S-acyl shift system and of SEA native peptide
ligation.
†
Electronic Supplementary Informaꢀon (ESI) available: Procedures and
characterization for all new compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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with some bis(2-sulfanylethyl)amine added exogenously. To
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15
DOI: 10.1039/C6OB01079B
follow the rate of exchange by LC-MS we used the N-labelled
bis(2-sulfanylethyl)amine 10 (trifluoroacetate salt). Peptide
8
was used in this experiment since it gives only a singly charged
molecular ion by LC-MS, thereby facilitating the quantitation of
the exchange reaction. The data presented in Fig. 2 show that
Results and discussion
A. SEA amide/thioester equilibrium
The SEA amide/thioester equilibrium was studied using SEA
peptides 5a-d (Fig 1A) which were produced by Fmoc SPPS as
described elsewhere.3 The SEA cyclic disulfide was reduced
into the active dithiol form in the presence of TCEP at pH 6.5
and then diluted in the appropriate solution to fix the pH (HCl,
citrate phosphate buffer, citrate buffer or phosphate buffer
depending on the final pH, see Supporting Information). The
peptide solutions were equilibrated at 37 °C and then analyzed
by HPLC using UV detection at 215 nm to quantify the relative
amount of SEA amide and thioester forms. At pH ~2, peptide
5a equilibrated in about 2 h, peptides 5b,c in about 5 h while
peptide 5d required up to 30 h to reach the equilibrium. The
A)
2
Gly
Ala
Tyr
Val
1
0
logarithm of the ratio [SEA thioester
plotted as a function of the pH (Fig. 1B). Fig. 1B shows that
Log(SEA thioester /SEA amide ) is linearly correlated with
the pH for all the tested peptides. The lines corresponding to
the linear fits are nearly identical for peptides 5b-d featuring a
C-terminal Ala, Tyr or Val residue respectively. These lines
differ slightly from the one obtained for peptide 5a equipped
with a C-terminal Gly. No useful data could be obtained above
pH 5 by HPLC using UV detection due to the low proportion of
7/SEA amide 6] was
-1
B)
7
6
-2
-3
0
2
4
6
8
pH
Intensity (total
ion current, AU)
the SEA thioester form
7 in the mixture at equilibrium. A
higher signal-to-noise ratio could be achieved by analyzing the
mixtures by LC-MS and using the total ion current (TIC) for the
detection. Using this detection technique we could estimate
the value of Log(SEA thioester 7b/amide 6b) at pH 5.4 (-
2.1456) and pH 7.1 (-2.6566). These data are indicated by a red
C)
arrow in Fig. 1B. Fig. 1C shows the HPLC trace for the 6b/7b
mixture equilibrated at pH 7.1. The extrapolation of the linear
fit for Log(SEA thioester 7b/amide 6b) (continuous red line in
Fig. 1B) gives values of -2.06 at pH 5.4 and -3.23 at pH 7.1.
These calculated values are close to those determined
experimentally. Note that the experimental value for Log(SEA
thioester 7b/amide 6b) at pH 7.1 is probably overestimated
due to the partial rearrangement of the SEA amide peptide 6b
during the elution step, which is carried out at pH ~ 1.8). We
thus consider that at pH 7 a proportion of 0.1% for the SEA
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Time (min)
Figure 1. A) Study of the SEA thioester/SEA amide equilibrium using SEA peptides
5a-d. Peptides 1 mM, TCEP 50 mM, 37 °C under nitrogen atmosphere. B) Log(SEA
thioester /amide ) as a function of pH. Peak areas were determined by RP-
HPLC using UV detection at 215 nm. The data indicated by a red arrow were
determined by LC-MS using the total ionic current detection (peptide 5b). C) LC-
7
6
MS of the mixture 6b/7b equilibrated at pH 7.1, total ion current detection.
Waters XBridgeTM BEH300 C18 3.5 µm (4.6 × 150 mm). Eluent A: TFA 0.1% by
vol. in water; eluent B: TFA 0.1% by vol. in CH3CN/water 4:1 by vol. Gradient: 0-
100% B in 30 min; flow rate 1 mL/min.
thioester form
7 at equilibrium is a good estimate, meaning a
concentration of the order of a few µM if the SEA peptide
amide is solubilized at a mM concentration as is done usually.
SEA ligation proceeds at neutral pH, suggesting that thiol/SEA
thioester exchanges take place despite the very low
concentration of the SEA thioester form in these conditions.
SEA ligation is a transamidation process which is probably
driven by the nearly irreversible formation of the peptide bond
to cysteine.25 The experiment depicted in Scheme 2 was
designed to test if a SEA peptide can equilibrate at neutral pH
the level of exchange observed after 80 h at 37 °C was 90%,
that is the percent of exchange that was expected at
equilibrium based on the stoichiometry of the reaction (10
equiv of compound 10).
2 | J. Name., 2012, 00, 1-3
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B. SEA/thiol exchange
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Next we were wondering if the above propeDrtOieI:s10m.1ig0h39t/bCe6OexBp0l1o0i7t9eBd
for accessing peptide alkylthioesters at neutral pH. Accessing
peptide alkylthioesters using mild conditions might facilitate the
incorporation of sensitive groups into these useful building blocks.
As already mentioned before, this exchange reaction proceeds
efficiently at pH 4 and constitutes a useful entry toward
peptide alkylthioesters.30,33-35 Typical conditions for the
exchange, i.e. at pH 4.0 using 5% by vol (0.57 M) of 3-
mercaptopropionic acid (MPA), results usually in more than 95%
of exchange.33 However, we have observed that these
conditions can lead to the degradation of some functional
groups such as cysteine thiazolidine,36 which is a useful
protected form of cysteine for protein chemical synthesis.
Cysteine thiazolidine residue is highly stable at neutral pH but
not at acidic pH (pH 3-4), as already discussed by others.37
We re-examined the exchange of the SEA group by MPA using only
1% by vol of MPA (0.11 M) in the search for milder conditions (Fig.
3). MPAA was used as a catalyst of the thiol/thioester exchange
reaction.38,39 At pH 4, the level of exchange at equilibrium
reached ~80%. Increasing the pH from 4 to pH 7 resulted in a
gradual decrease of the level of exchange at equilibrium. At pH
7, the level of exchange was 20% only and thus too low for
being of synthetic value.
Scheme 2. Exchange of the SEA group of peptide
8
by 15N-labelled bis(2-
sulfanylethyl)amine 10 at neutral pH.
11
9
888.8
100
Intensity (AU)
148 h
889.9
889.9
890.0
890.9
887.9
887.9
891.9
0
888.9
889.0
100
42 h
890.9
890.9
0
A)
100
887.9
24 h
0
887.9
A)
100
0 h
888.9
889.9
890.9
0
100
883 884 885 886 887 888 889 890 891 892 893 894 895 896
m/z
pH 4.1
80
pH 5.0
pH 6.1
pH 7.1
1,0
0,8
0,6
0,4
0,2
0,0
60
B)
40
B)
20
0
0
20
40
Time (h)
60
80
0
20
40
60
80
100 120 140 160
Time (h)
Figure 3. The influence of the pH on the SEA/thiol exchange was studied using
SEA peptide 5b and 3-mercaptopropionic acid (MPA) as the exogeneous thiol.
Peptide 5b 4.6 mM, TCEP 30 mM, MPAA 62 mM, 1% MPA by vol (0.11 M), 6 M
Gdn.HCl, 37 °C under nitrogen atmosphere. The exchange was monitored by
HPLC (UV detection at 215 nm).
Figure 2. Exchange of the SEA group of peptide
sulfanylethyl)amine 10 at neutral pH. A) Monitoring by LC-MS (MS traces
showing the molecular ion for peptides and 11). B) The time course of the
8
by 15N-labelled bis(2-
9
exchange as monitored by LC-MS. Peptide
8 3 mM, TCEP 200 mM, compound 10
30 mM, 6 M Gdn.HCl 0.1 M sodium phosphate buffer pH 7, 37 °C under nitrogen
atmosphere.
The exchange of the SEA group by MPA favors the SEA amide form
at neutral pH probably due to the greater stability of tertiary
amides relative to MPA thioesters. One potential strategy for
displacing the equilibrium toward the MPA peptide thioester would
be to trap the bis(2-sulfanylethyl)amine which is released during the
exchange process. Previous work showed the usefulness of simple
alkylthioesters such as Gly-SCH2CH2CO2H (Gly-MPA 14, Fig. 4A) for
To conclude at this point, the SEA thioester form is present at
low (µM) concentration at neutral pH. Nevertheless, it can
participate in thiol/thioester exchanges and equilibrate with a
thiol added exogenously.
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scavenging the bis(2-sulfanylethyl)amine during the synthesis of C-
terminal peptide thioacids.39 We therefore examined the exchange
of the SEA group by MPA at pH 7 in the presence of Gly-MPA 14 (10
equiv, Fig. 4B). The use of Gly-MPA 14 as an additive resulted in a
significant increase of the level of exchange (80%, red dots, Fig. 4B)
compared to the experiment in the absence of Gly-MPA 14 (20% of
exchange, black dots, Fig. 4B). The level of exchange in the presence
of Gly-MPA 14 only (green dots, Fig. 4B) was 60%, showing the
importance of combining MPA and Gly-MPA 14 for obtaining high
levels of exchange. Increasing the concentration of MPA from 1 to
1.8% by vol led to no improvement that Gly-MPA 14 was present or
not. Similarly, increasing the amount of Gly-MPA 14 from 10 to 17
equivalents had no significant effect (data not shown). Therefore
we used 1% of MPA and 10 equivalents of Gly-MPA 14 for the
exchange at pH 7 for the rest of this study. A typical HPLC profile for
the exchange reaction is shown in Fig. 5 using peptide 5b. The
scavenging of the bis(2-sulfanylethyl)amine by Gly-MPA 14 results
in the formation of Gly-SEA compound 15, which elutes early by
HPLC and does not complicate the final HPLC purification step. A
small proportion of peptide thioester 12b hydrolysis into peptide
acid 13b was also observed. Nevertheless, the extent of hydrolysis
is low and does not impair the synthetic usefulness of the method.
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DOI: 10.1039/C6OB01079B
Intensity (light scattering, AU)
O
OH
ILKEPVHGA
13b
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00
Time (min)
Figure. 5. LC-MS characterization of the crude exchange mixture after 72 h.
Peptide 5b 4.6 mM, TCEP 30 mM, MPAA 62 mM, 1% MPA by vol (0.11 M), 10
equiv thioester 14, 6 M Gdn.HCl, pH 7 0.1 M sodium phosphate buffer, 37 °C
under nitrogen atmosphere.
The time course of the exchange reaction for different C-terminal
amino acid residues (Ala, Tyr, Leu, Arg) is shown in Fig. 6. The
exchange proceeds in 24-30 h and is slower for peptide 5c which
features a C-terminal Tyr residue. In any case, the target peptide
thioester 12 could be isolated in good yield (Table 1) with no
detectable racemization of the C-terminal residue.
A)
A)
100
80
100
80
60
X =
Ala
Tyr
Leu
40
B)
60
20
B)
Arg
40
0
20
0
10
20
30
40
50
60
1% MPA (0.11 M)
1.8% MPA (0.2 M)
10 equiv Gly-MPA 14
Time (h)
0
1% MPA (0.11 M), 10 equiv Gly-MPA 14
1.8% MPA (0.2 M), 10 equiv Gly-MPA 14
Figure 6. Time course of the SEA/thiol exchange reaction for different C-terminal
amino acids. See the legend of Fig. 5 for the experimental conditions.
0
20
40
60
80
100
Time (h)
Finally, the method was further validated by the synthesis of
the 24 amino acids peptide thioester 17, which features an
internal Cys residue (30%, Scheme 3). Note that the tert-butyl
sulfenyl protecting group present on the internal cysteine, that
is used temporarily to facilitate the synthesis of the SEA
peptide, is removed during the exchange reaction.
Figure 4. Displacement of the SEA/thiol exchange equilibrium using Gly-MPA
thioester 14. The reactions were monitored by HPLC (UV detection at 215 nm).
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Notes and references
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DOI: 10.1039/C6OB01079B
(1) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science
1994, 266, 776-779.
(2) Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew. Chem. Int. Ed.
2006, 45, 1248-1252.
(3) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O.
Org. Lett. 2010, 12, 5238-5241.
(4) Kent, S. B. Chem. Soc. Rev. 2009, 38, 338-351.
(5) Raibaut, L.; Ollivier, N.; Melnyk, O. Chem. Soc. Rev. 2012, 41,
7001-7015.
(6) Zhang, Y.; Xu, C.; Lam, H. Y.; Lee, C. L.; Li, X. Proc. Natl. Acad.
Sci. U. S. A. 2013, 110, 6657-6662.
Scheme 3. Synthesis of peptide thioester 17
.
(7) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526-
533.
(8) Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46,
9248-9252.
Table 1. Synthesis of peptide thioesters by SEA/thiol
exchange at neutral pH.a
(9) Rohde, H.; Seitz, O. Biopolymers 2010, 94, 551-559.
(10) Metanis, N.; Keinan, E.; Dawson, P. E. Angew. Chem. Int. Ed.
2010, 49, 7049-7053.
(11) Malins, L. R.; Mitchell, N. J.; Payne, R. J. J Pept Sci 2014, 20,
64-77.
entry
thioester
yield (%)b
D-AA
(%)c
0.96
0.74
0.22
0.50
nd
1
2
3
4
5
12b
12c
12e
12f
17
54
53
60
64
(12) Offer, J.; Boddy, C. N.; Dawson, P. E. J. Am. Chem. Soc. 2002
124, 4642-4646.
,
30
a
b
c
For the experimental conditions see the legend of Fig. 5. Isolated by HPLC.
The percentage of racemization for the C-terminal residue was determined by
chiral GC-MS after acid hydrolysis of the peptide thioesters.40
(13) Botti, P.; Carrasco, M. R.; Kent, S. B. H. Tetrahedron Lett.
2001, 42, 1831-1833.
(14) Malins, L. R.; Payne, R. J. Curr Opin Chem Biol 2015, 22, 70-
78.
(15) Ollivier, N.; Behr, J. B.; El-Mahdi, O.; Blanpain, A.; Melnyk, O.
Org. Lett. 2005, 7, 2647-2650.
(16) Nagaike, F.; Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.;
Nakahara, Y.; Nakahara, Y. Org. Lett. 2006, 8, 4465-4468.
(17) Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y.
Tetrahedron Lett. 2007, 48, 25-28.
(18) Kawakami, T.; Aimoto, S. Tetrahedron Lett. 2007, 48, 1903-
1905.
(19) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett.
2009, 11, 823-826.
(20) Hou, W.; Zhang, X.; Li, F.; Liu, C. F. Org. Lett. 2011, 13, 386-
389.
(21) Taichi, M.; Hemu, X.; Qiu, Y.; Tam, J. P. Org. Lett. 2013, 15,
2620-2623.
(22) Burlina, F.; Papageorgiou, G.; Morris, C.; White, P. D.; Offer,
J. Chem. Sci. 2014, 5, 766-770.
Conclusions
The study of the SEA amide/thioester equilibrium shows that
the proportion of the SEA thioester is only ~0.1% at pH 7.
Nevertheless, it can participate in thiol/thioester exchange
reactions and equilibrate with a thiol added exogenously such
as the bis(2-sulfanylethyl)amine or 3-mercaptopropionic acid
(MPA). The level of exchange with MPA at pH 7 is modest
(20%) but can be increased significantly up to 80% by
scavenging the bis(2-sulfanylethyl)amine which is released
during the exchange. This can be done by using Gly-MPA
thioester as an additive in combination with MPA. The method
was validated by the synthesis of several peptide thioesters
equipped with a C-terminal Ala, Tyr, Leu, Arg or Lys residue.
(23) Terrier, V. P.; Adihou, H.; Arnould, M.; Delmas, A. F.;
Aucagne, V. Chem. Sci. 2016, 7, 339-345.
(24) Zheng, J.-S.; Tang, S.; Huang, Y.-C.; Liu, L. Acc. Chem. Res.
2013, 46, 2475–2484.
(25) Melnyk, O.; Agouridas, V. Curr. Opin. Chem. Biol. 2014, 22,
137-145.
(26) Sato, K.; Shigenaga, A.; Tsuji, K.; Tsuda, S.; Sumikawa, Y.;
Sakamoto, K.; Otaka, A. ChemBioChem 2011, 12, 1840-1844.
(27) Raibaut, L.; Cargoet, M.; Ollivier, N.; Chang, Y. M.; Drobecq,
H.; Boll, E.; Desmet, R.; Monbaliu, J.-C. M.; Melnyk, O. Chem. Sci.
2016, 7, 2657-2665
Acknowledgements
We acknowledge the financial support from CNRS, Institut
Pasteur de Lille and Université de Lille. This work was partly
financed by ANR (grant ANR 11-BS07-022-01). We thank
François-Xavier Cantrelle for performing some NMR
experiments. Institut Pasteur de Lille is gratefully
acknowledged for giving us access to the NMR spectrometers.
(28) Boll, E.; Drobecq, H.; Ollivier, N.; Raibaut, L.; Desmet, R.;
Vicogne, J.; Melnyk, O. Chem. Sci. 2014, 5, 2017-2022.
This journal is © The Royal Society of Chemistry 20xx
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(29) Boll, E.; Ebran, J. P.; Drobecq, H.; El-Mahdi, O.; Raibaut, L.;
Ollivier, N.; Melnyk, O. Org. Lett. 2015, 17, 130-133.
(30) Boll, E.; Drobecq, H.; Ollivier, N.; Blanpain, A.; Raibaut, L.;
Desmet, R.; Vicogne, J.; Melnyk, O. Nat. Protocols 2015, 10, 269-
292.
View Article Online
DOI: 10.1039/C6OB01079B
(31) Ollivier, N.; Vicogne, J.; Vallin, A.; Drobecq, H.; Desmet, R.;
El-Mahdi, O.; Leclercq, B.; Goormachtigh, G.; Fafeur, V.; Melnyk,
O. Angew. Chem., Int. Ed. 2012, 51, 209-213.
(32) Desmet, R.; Pauzuolis, M.; Boll, E.; Drobecq, H.; Raibaut, L.;
Melnyk, O. Org. Lett. 2015, 17, 3354–3357.
(33) Dheur, J.; Ollivier, N.; Vallin, A.; Melnyk, O. J. Org. Chem.
2011, 76, 3194-3202.
(34) Ollivier, N.; Raibaut, L.; Blanpain, A.; Desmet, R.; Dheur, J.;
Mhidia, R.; Boll, E.; Drobecq, H.; Pira, S. L.; Melnyk, O. J. Pept.
Sci. 2014, 20, 92–97.
(35) Drobecq, H.; Boll, E.; Senechal, M.; Desmet, R.; Saliou, J. M.;
Lacapere, J. J.; Mougel, A.; Vicogne, J.; Melnyk, O. Bioconjugate
Chem. 2016, 10.1021/acs.bioconjchem.1026b00211.
(36) Bang, D.; Kent, S. B. Angew. Chem. Int. Ed. 2004, 43, 2534-
2538.
(37) Fang, G.-M.; Wang, J.-X.; Liu, L. Angew. Chem. Int. Ed. 2012
51, 10347-10350.
,
(38) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006, 128, 6640-
6646.
(39) Pira, S. L.; Boll, E.; Melnyk, O. Org. Lett. 2013, 15, 5346–
5349.
(40) Amelung, W.; Brodowski, S. Anal. Chem. 2002, 74, 3239-
3246.
6 | J. Name., 2012, 00, 1-3
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