Synthesis of peptide alkylthioesters using the intramolecular N, S-acyl shift properties of bis(2-sulfanylethyl)amido peptides

Article abstract of DOI:10.1021/jo200029e

The design of novel methods giving access to peptide alkylthioesters, the key building blocks for protein synthesis using Native Chemical Ligation, is an important area of research. Bis(2-sulfanylethyl)amido peptides (SEA peptides) 1 equilibrate in aqueou

Full text of DOI:10.1021/jo200029e

ARTICLE
pubs.acs.org/joc
Synthesis of Peptide Alkylthioesters Using the Intramolecular N,S-Acyl
Shift Properties of Bis(2-sulfanylethyl)amido Peptides
Julien Dheur, Nathalie Ollivier, Aurꢀelie Vallin, and Oleg Melnyk*
CNRS UMR 8161, Universitꢀe Lille Nord de France, Institut Pasteur de Lille, IFR 142, 1 rue du Pr Calmette, 59021 Lille, France
S Supporting Information
b
ABSTRACT: The design of novel methods giving access to peptide alkylthioesters, the key
building blocks for protein synthesis using Native Chemical Ligation, is an important area of
research. Bis(2-sulfanylethyl)amido peptides (SEA peptides) 1 equilibrate in aqueous solution
with S-2-(2-mercaptoethylamino)ethyl thioester peptides 2 through an N,S-acyl shift mechanism.
HPLC was used to study the rate of equilibration for different C-terminal amino acids and the
position of equilibrium as a function of pH. We show also that thioester form 2 can participate
efficiently in a thiolꢀthioester exchange reaction with 5% aqueous 3-mercaptopropionic acid. The
highest reaction rate was obtained at pH 4. These experimental conditions are significantly less
acidic than those reported in the past for related systems. The method was validated with the
synthesis of a 24-mer peptide thioester. Consequently, SEA peptides 1 constitute a powerful
platform for access to native chemical ligation methodologies.
’ INTRODUCTION
the instability of the thioester group in the presence of bases such
as piperidine used for Fmoc removal.
Ligation chemistries are powerful synthetic tools for the con-
vergent and controlled assembly of complex molecular scaffolds. In
particular, native peptide ligation methods allow the formation of
native peptide bonds between unprotected peptides and thus the
total or hemi synthesis of proteins.1ꢀ4 Native chemical ligation
(NCL)1,5 and related extended methodologies6ꢀ8 are certainly the
most frequently used techniques for protein total synthesis. NCL is
based on the reaction of peptide thioesters with cysteinyl peptides.
The reaction proceeds through a series of thiolꢀthioester exchanges
first with an exogenous aromatic thiol used in excess and then with
the side-chain thiols of cysteine residues. N-terminal cysteine leads
to a key thioester-linked intermediate which rearranges sponta-
neously by acyl migration from sulfur to nitrogen, resulting in the
formation of a native peptide bond.
Peptide alkylthioesters are frequently used for protein synthe-
sis, using NCL or extended methodologies. In this case, NCL is
carried out in the presence of an exogenous aromatic thiol such as
4-mercaptophenyl acetic acid (MPAA). MPAA converts the
alkylthioester into a more reactive aryl thioester by thiolꢀthioe-
ster exchange.9 MPAA maintains also cysteine thiols in a reduced
form and permits the reversal of nonproductive thioester inter-
mediates formation with internal cysteine residues.
The crucial importance of peptide alkylthioesters in NCL-
based synthetic strategies has stimulated the development of
various methods allowing their synthesis. The field has been
reviewed very recently.10 Historically, peptide alkylthioesters
were first synthesized by using Boc/benzyl solid phase peptide
synthesis (SPPS) methods.11 This past decade, various Fmoc-
SPPS methods have been reported that give also access to these
key peptide derivatives. Fmoc-SPPS is by far the most popular
technique for peptide synthesis.12 However, the synthesis of
peptide thioesters with Fmoc-SPPS is highly challenging due to
To circumvent this problem, additives such as hydroxybenzo-
triazole (HOBt) were used in combination with piperidine or
other bases to remove the Fmoc group on thioester-linked
peptidyl resin.13,14 However, the most frequently used strategy
is to introduce the thioester functionality after the peptide
elongation step. This can be done either on the solid phase,15ꢀ19
during the cleavage from the solid support,20ꢀ28 in solution or
in situ during NCL.22,29ꢀ39 Among the various methods de-
scribed, the intramolecular O,S-acyl shift31,34 and later on the N,
S-acyl shift-based strategies23,29,30,32,33,35ꢀ39 have gained increas-
ing importance in this field during the past few years. In
particular, N,S-acyl shift-based methods were used either for
peptide thioester synthesis17,23,29,30,38,39 or for designing novel
native ligation methods relying on the in situ generation of
peptide thioesters.32,33,35ꢀ37 In particular, peptides featuring
a C-terminal N-alkylcysteine rearrange in acidic media into
a transient peptide thioester that can be exchanged with an
external thiol.29,38,39 An application of this chemistry has been
reported recently by Brik et al.40 In this work, N-methylcysteine-
functionalized resin was prepared by methylation of a supported
o-nitrobenzenesulfonylcysteine residue in the presence of DBU
and methyl p-nitrobenzenesulfonate. Removal of the o-nitroben-
zenesulfonyl group in the presence of DBU and mercaptoethanol
was followed by the remaining assembly of the target peptide.
Conversion of the N-methylcysteine-terminated peptide into the
corresponding 3-mercaptopropionic acid (MPA)ꢀthioester was
carried out at pH 1 in 20% aqueous 3-mercaptopropionic acid
during 12 h.
Received: January 13, 2011
Published: March 21, 2011
r
2011 American Chemical Society
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Scheme 1. Conversion of SEA Peptides 1,2 into
MPAꢀThioesters 3
Scheme 2. Synthesis of SEA Peptides 4aꢀda
Recently also, microwave-assisted tandem N,S-acyl migration
and MPAꢀthioester exchange permitted the preparation of a
glycosylated peptide thioester.23 Microwave irradiation was also
found to induce an N,S-acyl shift within Cys peptides and the
preparation of peptide thioesters by MPAꢀthioester exchange.41
Even more recently, peptide anilides featuring a thiol group in the
β position relative to the anilide were rearranged in 4 M HCl/
DMF for 8 h into peptide thioesters.30
a The open form 1aꢀd was cyclized by air oxidation. Reduction of cyclic
form 4aꢀd into open form 1aꢀd was carried out at pH 5 in the presence
of 0.1 M TCEP.
form 2 increases by decreasing the pH of the solution. We
reasoned that conversion of peptide amide 1 into MPAꢀthioe-
ster 3 could proceed through transthioesterification of the
transient thioester form 2 by MPA.
Clearly, N,S-acyl shift strategies constitute a promising access
to peptide thioester building blocks. A significant improvement
would be to design chemical systems able to rearrange with use of
mild experimental conditions, rather than in strong acid media as
reported before. We have described very recently the synthesis
of bis(2-sulfanylethyl)amido peptides using Fmoc-SPPS.33,42
These peptide derivatives, called SEA peptides, ligate chemose-
lectively and regioselectively with cysteinyl or homocysteinyl
peptides at pH 7 to give a native peptide bond at the ligation site.
Later on, Liu et al. described a similar system.43 Interestingly, this
group reported the synthesis of a pentapeptide thioester by
transthioesterification of a bis(2-sulfanylethyl)amido peptide in
20% MPA at pH 2. It described also the direct use of SEA
peptides in NCL at pH 4ꢀ6, suggesting that conversion of SEA
peptides into alkylthioesters might be carried out with milder
experimental conditions. We independently discovered the ease
of transthioesterification of bis(2-sulfanylethyl)amido peptides
by MPA and wish to disclose our findings in this field. We first
describe hereinafter a study of the N,S-acyl shift equilibrium (rate
of equilibration, effect of pH) using model nonapeptides featur-
ing Gly, Ala, Tyr, or Val C-terminal residues. Next, we show that
SEA peptides are cleanly converted into the corresponding
MPAꢀthioesters in 5% aqueous MPA. The effect of pH on the
rate of MPAꢀthioester formation was examined. The highest
rate was obtained at pH 4. These experimental conditions are
significantly less acidic than those required for the N,S-acyl shift
chemical systems reported to date. Importantly, lowering the pH
to 3.2 increased the proportion of thioester form in equilibrium
with bis(2-sulfanylethyl)amido peptide but reduced the rate of
thioester formation. The method permitted the synthesis of a 24
amino acid long MPAꢀthioester derived from HGF K1 domain,
showing the interest of SEA peptides for the synthesis of large
peptide thioesters.
We first characterized the equilibrium between peptide amide
1 and thioester 2. Indeed, the rate of equilibration as well as the
fraction of thioester form 2 at equilibrium is expected to influence
the rate of MPAꢀthioester formation. We used for this purpose
model SEA peptides 4aꢀd (Scheme 2). Their synthesis was
already described elsewhere.33 In brief, we used a supported
bis(2-sulfanylethyl)amino reagent linked to a trityl polystyrene
resin through both sulfur atoms. This solid support is fully
compatible with standard automated Fmoc-SPPS. Deprotec-
tion and cleavage of the peptide from the resin furnished
peptide amides 1aꢀd. To avoid any interference of peptide
amide ꢀ-peptide thioester 2 equilibrium during RP-HPLC
purification, 1aꢀd were converted into the cyclic disulfides
4aꢀd by air oxidation in basic medium. The isolated yields were
in the range 23ꢀ47%.
The cyclic disulfides 4aꢀd were reduced into peptides 1aꢀd
in the presence of 0.1 M TCEP at pH 5. At this pH, the amide
form 1 is the unique species detected by RP-HPLC. Thioester
forms 2aꢀd were observed below pH 4.5 and eluted earlier by
RP-HPLC than the corresponding amide forms 1aꢀd, probably
due to the protonated thiol handle present within thioester form
2. LC-MS analysis of the 1b/2b mixture showed the same
molecular ion for the two peaks (see the Supporting Information),
in accord with the proposed structures. Additional evidence
for structure 2 is provided by the capacity of the β-amino thiol
moiety within thioesters 2aꢀd to form a thiazolidine by reacting
with glyoxylic acid at pH 1.44
To study the rate of equilibration between peptide amide form
1 and thioester form 2, the pH was rapidly adjusted to 1ꢀ2 after
the reduction step with TCEP. The time course of the equil-
ibration between forms 1aꢀd and 2aꢀd was monitored by
RP-HPLC using a linear waterꢀacetonitrile gradient at pH 2
(Figure 1). We have verified that equilibration between forms 1
and 2 during RP-HPLC analysis was not significant. Thus, the
fraction of forms 1 and 2 determined by using this analytical
’ RESULTS AND DISCUSSION
The conversion of SEA peptides into MPAꢀthioesters is
illustrated in Scheme 1. SEA peptide amide form 1 is in
equilibrium with thioester form 2. The proportion of thioester
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Figure 1. The rate of equilibration of peptide amide 1 into peptide thioester 2 was monitored by RP-HPLC (detection at 215 nm). The fraction of each
form 1 and 2 in the mixture is plotted as a function of time (h). Equilibration of (A) Gly analogue 1a, (B) Ala analogue 1b, (C) Tyr analogue 1c, and (D)
Val analogue 1d.
method (Figure 1) corresponds to the fraction of 1 and 2 in the
reaction mixture.
MPAꢀthioester 3b appeared to be optimal at pH 4.0. The time
required to reach 50% conversion (t1/2) was 120 min. Interest-
ingly, the rate of MPAꢀthioester 3b formation was only slightly
diminished at pH 5. Consequently, the conversion of amide
form 1 into thioester 3 takes place efficiently even if thioester
form 2 is barely detectable by RP-HPLC. The fraction of
thioester form 2b is significantly higher at pH 3.2 (∼20%) than
at pH 4.0 (8%) as shown in Figure 2. However, lowering the pH
from 4.0 to 3.2 decreased significantly the rate of thioester 3b
formation (t1/2 = 180 min). The effect of pH on the rate of
MPAꢀthioester formation suggests that the secondary amine
in the thiol handle within 2b might facilitate the attack of the
external thiol by an intramolecular general-base catalysis mecha-
nism. Thus a balance between the fraction of thioester form 2b in
the mixture and the fraction of free secondary amine might govern
the rate of MPAꢀthioester 3b formation. In the rest of this study,
conversion of peptides 4 into MPAꢀthioesters 3 was carried out
at pH 4.0.
Next, the optimal experimental conditions determined above
were used for the synthesis of MPAꢀthioesters 3aꢀd (Table 1).
Isolated yields were in the range 62ꢀ87%, showing the efficiency
of the method. The lowest yield (62%, Table 1, entry 4) as well as
the longest reaction time was obtained for Val C-terminal
residue. This result is in accord with the time required for Val
derivative 1d to equilibrate compared to Gly, Ala, and Tyr
derivatives 1aꢀc (Figure 1). Typical RP-HPLC traces for the
conversion of Gly analogue 1a into MPAꢀthioester 3a are
shown in Figure 4. MPAꢀthioesters 3bꢀd were also analyzed
Gly analogue 1a equilibrated rapidly in about 2 h (Figure 1A).
Ala and Tyr analogues 1b and 1c equilibrated in about 5 h
(Figure 1B and C), whereas Val analogue required up to 30 h to
reach equilibrium (Figure 1D). Clearly, the time required to
reach equilibrium is in the order Gly > Ala, Tyr > Val and is
correlated with the bulkiness of the amino acid residue.
Next, the fraction of peptide amide 1 and peptide thioester 2 at
equilibrium for different pH values was determined by RP-HPLC
(Figure 2). The relationship between the fraction of peptides 1,2
and pH is very similar for Ala 1b, Tyr 1c, and Val 1d analogues.
Typically, the proportion of peptides 1,2bꢀd is equal at pH ∼3,
whereas peptide thioesters 2bꢀd are almost the unique specie
detected at pH ∼1. Gly analogue behaves slightly differently as both
forms 1a and 2a are in equal proportion at pH ∼2, whereas peptide
amide 1a is still present in significant amounts at pH below 1.
Another interesting point to mention is that the fraction of peptide
thioester 2 at equilibrium at pH 4 is in the range 4ꢀ10% for the four
analogues studied. This result suggests that the conversion of
peptide 1 into MPAꢀthioester 3 may take place at pH 4.
To test this hypothesis, peptide 4b was reacted with MPA
(5% by volume) at pH 3.2, 4.0, or 5.0. The reaction was carried
out in the presence of 0.1 M TCEP to maintain the thiol groups
in reduced form. The time course of MPAꢀthioester 3b forma-
tion was followed by RP-HPLC (Figure 3).
This experiment revealed the rapid conversion of peptide 4b into
MPAꢀthioester 3b for the three pH values tested. Formation of
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Figure 2. The fraction of peptide amide 1 and peptide thioester 2 at equilibrium at different pH was determined by RP-HPLC (detection at 215 nm).
The fraction of each form 1 and 2 in the mixture is plotted as a function of pH. Data for (A) Gly analogue 1a, (B) Ala analogue 1b, (C) Tyr analogue 1c,
and (D) Val analogue 1d.
Figure 3. Rate of conversion of peptide 4b into MPAꢀthioester 3b for different pH values: pH 3.2 ([), pH 4.0 (9), or pH 5.0 (2). Peptide 4b (1 mM
final concentration) was solubilized in 0.2 M pH 7.0 phosphate buffer at 37 °C containing 0.1 M TCEP and 5% MPA by volume. The pH was adjusted
with NaOH 5 N and 1 N.
by chiral GC-MS analysis after acid hydrolysis (Table 1).45 No
racemization was observed for C-terminal Ala, Tyr, or Val
residues.
in Scheme 3. NCL of MPAꢀthioesters 3c,d with cysteinyl peptide 5
in the presence of MPAA9 furnished successfully the corresponding
native peptides 6c,d as expected.
Finally, to illustrate the utility of the method for the syn-
thesis of large peptide thioesters we undertook the synthesis of
Next, we have verified that MPAꢀthioesters synthesized in this
study were efficient partners in NCL. Typical examples are provided
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Table 1. Conversion of Peptides 4aꢀd into MPAꢀThioesters 3aꢀd
entry
MPAꢀthioester 3
C-terminal residue
isolated yield (%)a
reaction time (h)
D-enantiomerb (%)
1
2
3
4
a
b
c
Gly
Ala
Tyr
Val
70
71
87
62
16
16
16
94
0.37
0.6
d
0.1
a Peptides were purified by RP-HPLC. b Determined by chiral GC-MS analysis after acid hydrolysis.
Scheme 4. Synthesis of MPAꢀThioester 3e
Figure 4. RP-HPLC monitoring of the conversion of Gly analogue 1a
into MPAꢀthioester 3a (4a 1 mM, TCEP 0.1 M, MPA 5% by volume,
pH 4.0, 37 °C under inert atmosphere).
Scheme 3. NCL of Peptides 3c,d with Cysteinyl Peptide 5
and Synthesis of Peptides 6c,d
position was introduced as Cys(StBu) during SPPS to avoid
the formation of unwanted disulfide bonds during this oxida-
tion step. Next, peptide amide 4e was reacted with MPA/
TCEP, using the experimental conditions described above
(pH 4.0). Again, we observed the clean conversion of 4e into
MPAꢀthioester 3e. The RP-HPLC trace of the crude reaction
mixture is shown in Figure 5A. The isolated yield for peptide
3e after RP-HPLC purification was 41% overall. Finally, chiral
GC-MS analysis of peptide 3e after acid hydrolysis showed the
absence of racemization for C-terminal lysine residue.45
In conclusion, bis(2-sulfanylethyl)amido peptides (SEA
peptides) constitute a powerful platform for native chemical
ligation methodologies. By themselves, SEA peptides ligate
with Cys peptides to give the corresponding native peptides.
Alternately, SEA peptides can be converted efficiently into
MPAꢀthioesters that are commonly used in NCL or extended
methodologies. The important finding reported here is that
conversion of SEA peptides into MPAꢀthioesters takes
place efficiently at pH 4. These experimental conditions are
much milder than those reported before for related systems.
MPAꢀthioester 3e (Scheme 4). The sequence of peptide 3e
corresponds to the first 24 amino acid residues of K1 domain
of human hepatocyte growth factor.46 For this, Fmoc-L-Lys-
(Boc)-OH was coupled to bis(2-sulfanylethyl)amino trityl
polystyrene resin, using PyBrop/DIEA activation as described
above. Then, the peptide was assembled by using standard
Fmoc-SPPS. Deprotection and cleavage furnished amide form
1e, which was directly oxidized into the cyclic derivative 4e to
facilitate RP-HPLC purification. Oxidation was carried out
with iodine in aqueous acetic acid. Cys residue in the fourth
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Figure 5. RP-HPLC traces of peptide 3e: (A) crude reaction mixture after 18 h and (B) C18 RP-HPLC purified.
Importantly, SEA peptides are easily synthesized by using Fmoc-
SPPS. Consequently, the method described here should facilitate
the synthesis of these key building blocks and especially of acid-
sensitive MPAꢀthioesters, and extends the usefulness of N,S-
acyl shift methodologies.
Analytical HPLC was performed by using a Waters Alliance instrument
(column: Waters XBridge BEH300 C18 Å, 5 μm, 4.6 ꢁ 250 mm) and
solvents A (0.05% TFA in H2O) and B (80% acetonitrile, 20% H2O,
0.05% TFA) in a linear gradient (0ꢀ100% B in 30 or 60 min at 30 °C)
with a flow rate of 1 mL/min. Detection was achieved with a UVꢀvis
detector at wavelength λ = 215 nm.
The crude peptide was purified by semipreparative RP-HPLC on a
C18 nucleosil 120 Å, 5 μm column (230 nm, 6 mL/min, rt, buffer A
water containing 0.05% TFA, buffer B CH3CN/water 4/1 containing
0.05% TFA, linear gradient of 0ꢀ30% B in 40 min) to give 13.7 mg of
peptide 3a (70% yield).
’ EXPERIMENTAL SECTION
Synthesis of Peptide 3a. Peptide 4a was prepared as described
elsewhere.33 Reaction buffer was prepared by dissolving 500 mg of
tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl, 2 mmol)
3
MALDI-TOF matrix: R-cyano-4-hydroxycinnaminic acid. Calcd for
and 1 mL of 3-mercaptopropionic acid (MPA) (5% by volume, 11.5
mmol) in 20 mL of degassed sodium phosphate buffer (pH 7.0, 0.2 M).
The pH of the solution was then adjusted at 4.0 by adding 5 M aqueous
NaOH. Peptide 4a (20 mg, 14.2 μmol) was dissolved in the above
solution and placed under nitrogen atmosphere at 37 °C under magnetic
stirring. The reaction was monitored by RP-HPLC. Prior to the analysis,
the excess of MPA was removed from 50 μL aliquots by adding 50 μL of
10% aqueous TFA and then extracting with diethyl ether (3 ꢁ 1 mL).
C46H76N12O13S [M þ H]þ 1037.5, found 1037.6.
1H and 13C NMR for peptide 3a: 1H NMR (300 MHz, H2O/D2O: 9/1
by volume, 20 °C) δ 8.68 (d, J = 7.6 Hz, 1H), 8.61 (s, 1H), 8.59 ꢀ 8.45 (m,
4H), 8.34 (d, J = 6.9 Hz, 1H), 8.25 (d, J = 6.8 Hz, 1H), 7.32 (s, 1H), 4.44
(m, 2H), 4.31 (q, J = 7.0 Hz, 1H), 4.16 (d, J = 6.0 Hz, 2H), 4.01ꢀ3.85 (m,
3H), 3.78ꢀ3.64 (m, 3H), 3.35ꢀ3.18 (m, 2H), 3.13 (t, J = 6.8 Hz, 2H), 2.98
(s, 2H), 2.65 (t, J = 6.8 Hz, 2H), 2.47 (q, J = 6.5 Hz, 2H), 2.41ꢀ2.18 (m,
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2H), 2.14ꢀ1.57 (m, 18H), 1.52ꢀ1.39 (m, 4H), 1.28ꢀ1.13 (m, 2H),
0.98ꢀ0.83 (m, 16H); 13C NMR (75 MHz, H2O/D2O: 9/1 by volume,
20 °C) δ 203.2, 180.4, 179.8, 176.8, 176.6, 176.3, 176, 175.4, 174.7, 174.6,
174.1, 172, 136.4, 131.2, 120.2, 103.8, 60.41, 6.12, 55.3, 54.5, 53.6, 51.8, 45.5,
45.2, 44, 42.5, 42.1, 39.2, 36.9, 32.8, 32.2, 32.2, 29.1, 29.1, 28.7, 27.4, 27, 26.7,
26.5, 24.8, 24.6, 23.9, 21, 20.6, 16.8, 13.2.
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 3b. The synthesis of peptide 3b was
performed similarly on a 13.9 μmol scale. Purification was done by
RP-HPLC on a C18 nucleosil 120 Å, 5 μm column (230 nm, 6 mL/min,
rt, buffer A water containing 0.05% TFA, buffer B CH3CN/water 4/1 by
volume containing 0.05% TFA, linear gradient of 0ꢀ30% B in 40 min) to
give 13.8 mg of peptide 3b (71% yield).
Chiral GC-MS analysis: 0.1% of D-Val.
MALDI-TOF matrix: 2,5-dihydroxybenzoic acid. Calcd for C49H82-
N12O13S [M þ H]þ1079.6, found 1079.7.
1H and 13C NMR analysis of 3d (selected NMR data): 1H NMR (300
MHz, H2Oþ D2O) δ8.62 (t, J= 4.1 Hz, 2H), 8.57 (d, J= 6.7 Hz, 1H), 8.46
(m, 3H), 8.34 (d, J = 7.0 Hz, 1H), 8.24 (d, J = 7.2 Hz, 1H), 7.32 (s, 1H),
4.49ꢀ4.25 (m, 3H), 4.11ꢀ3.92 (m, 3H), 3.82 (m, 2H), 3.75ꢀ3.63 (m,
1H), 3.36ꢀ3.17 (m, 2H), 3.12 (t, J = 6.8 Hz, 2H), 2.98 (s, 2H), 2.65 (t, J =
6.8 Hz, 2H), 2.47 (q, J = 6.7 Hz, 2H), 2.34ꢀ2.18 (m, 2H), 2.15ꢀ1.54 (m,
14H), 1.53ꢀ1.31 (m, 3H), 1.28ꢀ1.10 (m, 1H), 1.02ꢀ0.81 (m, 23H); 13C
NMR (75 MHz, H2O þ D2O) δ 205.9, 180.4, 179.8, 176.7, 176.6, 176.2,
176, 174.7, 174.2, 174.1, 172, 136.4, 131.2, 120.2, 68, 62.9, 62.5, 60.4, 56.1,
55.2, 53.6, 42.5, 42.1, 39.2, 37, 33, 32.8, 32.2, 29.1, 27.4, 27, 26.7, 24.8, 24.6,
23.9, 21.2, 21.1, 20.7, 19.7, 16.8, 13.2.
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 5. Peptide elongation was performed on
Rink-PEG-PS resin (NovaSyn TGR, 0.25 mmol/g, 500 μmol scale) by
using standard Fmoc/tert-butyl chemistry on an automated peptide
synthesizer with HBTU/DIEA activation in DMF. A capping step was
performed after each coupling with Ac2O/DIEA. At the end of the
synthesis, the Fmoc protecting group of the last amino acid was removed
with 20% piperidine in DMF.
Final deprotection and cleavage from the solid support were per-
formed with TFA/water/1,2-ethanedithiol/TIS 94.5/2.5/2.5/1 by
volume for 2 h (50 mL). The crude peptide was precipitated in diethyl
ether/heptane 1/1 by volume (500 mL), solubilized in 10 mL of
deionized water, and lyophilized.
Chiral GC-MS analysis: 0.37% of D-Ala.
MALDI-TOF matrix: 2,5-dihydroxybenzoic acid. Calcd for C47H78-
N12O13S [M þ H]þ1051.6, found 1051.5; [M þ Na]þ 1073.5, found
1073.5.
1H and 13C NMR for peptide 3b (selected NMR data): 1H NMR (600
MHz, H2O þ D2O, 20 °C) δ 8.66 (d, J = 7.4 Hz, 1H), 8.61 (s, 1H), 8.58
(m, 2H), 8.51 (d, J = 6.1 Hz, 1H), 8.36 (d, J = 7.0 Hz, 1H), 8.27 (d, J = 7.1
Hz, 1H), 7.32 (s, 1H), 4.52 (m, 1H), 4.46ꢀ4.39 (m, 2H), 4.31 (q, J = 7.2
Hz, 1H), 4.01 (m, 1H), 3.97 (t, J = 5.9 Hz, 2H), 3.86ꢀ3.79 (m, 2H),
3.73ꢀ3.67 (m, 1H), 3.24 (ddd, J = 23.7, 15.5, 7.2 Hz, 2H), 3.11 (t, J = 6.8
Hz, 2H), 3.02ꢀ2.95 (m, 2H), 2.76ꢀ2.69 (m, 1H), 2.68 (t, J = 6.8 Hz, 2H),
2.56ꢀ2.43 (m, 2H), 2.41ꢀ2.22 (m, 2H), 2.13ꢀ1.54 (m, 18H), 1.52ꢀ1.31
(m, 8H), 1.21 (m, 1H), 0.98ꢀ0.85 (m, 23H); 13C NMR (151 MHz, H2O
þ D2O, 20 °C) δ 179.9, 179.2, 176.8, 176.6, 176.3, 176, 174.8, 174.7, 174.1,
173.9, 173.8, 172.1, 165.8, 165.6, 137, 136.9, 136, 135.9, 121.9, 121.4, 120.5,
120, 118.1, 116.1, 103.8, 63, 62.9, 58.6, 58.6, 58.6, 58.5, 56.2, 56.1, 56.1, 55.3,
55.2, 55.2, 53.5, 45.3, 45.1, 42.1, 39.5, 39.2, 39, 37.9, 36.9, 36.8, 36.5, 36.1,
35.3, 33.1, 33.1, 32.3 32.2, 32.2, 31.9, 29.9, 29.4, 29.1, 28.8, 27.9, 27.4, 27.2,
26.8, 26.7, 26.3, 26, 25.3, 24.8, 19.8, 19.3, 13.5, 12.9.
Purification was performed by RP-HPLC on a C18 Nucleosil 120 Å,
5 μm column (215 nm, 6 mL/min, rt, buffer A water containing 0.05%
TFA, buffer B CH3CN/water 4/1 by volume containing 0.05% TFA,
0ꢀ25% B in 30 min) to give 495 mg of peptide 5 (69%).
MALDI-TOF matrix: R-cyano-4-hydroxycinnaminic acid. Calcd for
C49H84N14O12S [M þ H]þ 1093.6, found 1093.4; [M þ Na]þ 1115.6,
found 1115.4.
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 3c. Synthesis was done on a 8.5 μmol scale.
Purification was done by RP-HPLC on a C18 nucleosil 120 Å, 5 μm
column (215 nm, 6 mL/min, rt, buffer A water containing 0.05% TFA,
buffer B CH3CN/water 4/1 by volume containing 0.05% TFA, linear
gradientof 0ꢀ30%Bin40min) togive10.9mgof peptide 3c(87%yield).
Chiral GC-MS analysis: 0.6% of D-Tyr.
Synthesis of Peptide 6c. 4-Mercaptophenylacetic acid (MPAA,
33.63 mg, 0.2 mmol) and tris(2-carboxyethyl)phosphine hydrochloride
(TCEP. Cl, 459 mg, 1.6 mmol) were solubilized in 0.2 M sodium
3
phosphate buffer pH 7.3 (10 mL). NaOH (6 M) was added to adjust the
pH to 7.2. Then, peptide 3c (5.3 mg, 3.6 μmol) and peptide 5 (7.7 mg,
5.4 μmol, 1.5 equiv) were dissolved in the pH 7.3 phosphate buffer
(1.8 mL). The two solutions were mixed and the reaction mixture was
agitated at 20 °C. Final reaction conditions: 1 mM for peptide 3c,
1.5 mM for peptide 5, 10 mM MPAA, 80 mM TCEP, 0.1 M phosphate,
pH 7.2. Purification was performed by RP-HPLC on a C18 Atlantis
column 120 Å, 5 μm (215 nm, 25 mL/min, rt, buffer A water containing
0.05% TFA, buffer B CH3CN/water 4/1 by volume containing 0.05%
TFA, 0ꢀ35% B in 40 min) and afforded 5.2 mg of peptide 6c (54%).
LC-MS analysis of 6c: C99H160N26O24S [M þ H]þ calcd 2131.6,
found 2132.01; [M þ 2H]2þ calcd 1066.3, found 1066.5.
MALDI-TOF matrix: 2,5-dihydroxybenzoic acid. Calcd for C53H82-
N12O14S [M þ H]þ1143.6, found 1143.4; [M þ Na]þ 1165.6, found
1165.4.
1H and 13C NMR analysis of 3c (selected NMR data): 1H NMR (300
MHz, H2O þ D2O) δ 8.59ꢀ8.5 (m, 3H), 8.47 (t, J = 7.1 Hz, 2H), 8.36 (m,
2H), 8.24 (d, J = 6.9 Hz, 1H), 7.26 (s, 1H), 7.13 (d, J = 8.5 Hz, 2H), 6.83 (d,
J= 8.6 Hz, 2H), 4.49ꢀ4.38 (m, 2H), 4.31 (q, J= 6.6 Hz, 1H), 3.98 (t, J= 7.2
Hz, 1H), 3.93ꢀ3.82 (m, 4H), 3.82ꢀ3.73 (m, 1H), 3.72 (s, 1H), 3.63 (d, J =
10.2 Hz, 1H), 3.28ꢀ3.05 (m, 7H), 2.94 (m, 4H), 2.63 (t, J = 6.7 Hz, 2H),
2.45 (t, J = 7.2 Hz, 2H), 2.32ꢀ1.10 (m, 28H), 1.,02ꢀ0.85 (m, 24H); 13C
NMR (75 MHz, H2O þ D2O) δ 205.5, 180.3, 179.7, 176.8, 176.6, 176.3,
176, 174.6, 174.1, 173.8, 172, 169.7, 157.4, 136.4, 133.4, 131.2, 130.6, 121,
120.1, 118.3, 117.1, 103.8, 63.8, 62.9, 62.7, 60.4, 56.1, 55.2, 55.1, 53.6, 50.7,
45, 42.5, 42.1, 39.2, 39, 36.8, 33.1, 32.7, 32.1, 29.1, 28.6, 27.4, 27, 26.8, 26.7,
24.8, 24.6, 23.9, 21, 20.7, 16.8, 13.2.
Synthesis of Peptide 6d. Peptide 6d was synthesized by using the
same procedure as described above on a 2.2 μmol scale of peptide 3d
(3.1 mg). Purification by RP-HPLC on a C18 Atlantis column 120 Å, 5
μm (215 nm, 25 mL/min, rt, buffer A water containing 0.05% TFA,
buffer B CH3CN/water 4/1 by volume containing 0.05% TFA, 15ꢀ35%
B in 40 min) afforded 1.9 mg of peptide 6d (33%).
LC-MS analysis of 6d: C95H160N26O23S [M þ H]þ m/z calcd
2067.5, found 2068.02; [M þ 2H]2þ m/z calcd 1034.3, found 1034.6.
Synthesis of 4e
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 3d. Synthesis was done on a 7.4 μmol scale.
Purification was done by RP-HPLC on a C18 nucleosil 120 Å, 5 μm
column (230 nm, 6 mL/min, rt, buffer A water containing 0.05%
TFA, buffer B CH3CN/water 4/1 by volume containing 0.05% TFA,
linear gradient of 0ꢀ35% B in 40 min) to give 6.5 mg of peptide 3d
(62% yield).
Preparation of bis(2-sulfanylethyl)amino trityl polystyr-
ene resin: Bis({2-[(triphenylmethyl)sulfanyl]ethyl})amine47 (933 mg,
1.5 mmol) was deprotected by treatment with TFA/TIS (97.5/2.5,
v/v, 150 mL) during 30 min. The solvent was evaporated under
reduced pressure and residual TFA was coevaporated with cyclohexane
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ARTICLE
(2 ꢁ 50 mL). The resulting white solid was dissolved in anhydrous DMF
(36 mL) and added immediately to trityl chloride polystyrene resin
cross-linked with 1% divinylbenzene (10.71 g, 1.4 mmol/g) under argon.
The suspension was shaken overnight. Then, the remaining trityl
chloride groups were capped with methanol (500 μL) in the presence
of 2,6-lutidine (1.4 mL) during 30 min. Finally, the resin was washed
with DMF (2 ꢁ 2 min), methanol (2 ꢁ 2 min), DMF (2 ꢁ 2 min), 5%
(v/v) DIEA in DMF (2 ꢁ 2 min), and DMF (2 ꢁ 2 min). The loading
was determined by coupling Fmoc-Gly-OH to a resin aliquot, using
standard PyBrop/DIEA activation in DMF, and then by measuring the
absorbance at 290 nm of the dibenzofulveneꢀpiperidine adduct released
by using 20% (v/v) piperidine in DMF.
volume containing 0.05% TFA, 0ꢀ10% B in 5 min, then 10ꢀ100% B in
150 min) afforded 9.7 mg of 3e (41%).
MALDI-TOF matrix: sinapinic acid. C115H203N35O33S2 [M þ H]þ
m/z calcd 2667.48, found 2668.3.
’ ASSOCIATED CONTENT
S
Supporting Information. RP-HPLC, CZE, and MS data
b
for all compounds, and H and 13C NMR spectra for peptides
1
3aꢀd. This material is available free of charge via the Internet at
http://pubs.acs.org.
Peptide elongation: Bis(2-sulfanylethyl)amino trityl polystyrene
resin (0.5 mmol, 0.175 mmol/g) was conditioned in CH2Cl2. Fmoc-
Lys(Boc)-OH (2.342 g, 5 mmol) was dissolved in CH2Cl2 (a few drops
of DMF were added to favor dissolution). PyBrop (2.331 g, 5 mmol) was
dissolved in the minimal volume of CH2Cl2 and added to the resin.
DIEA (2.613 mL, 15 mmol) was added to the resin in one portion. The
resin was shaken during 2 h, and then washed with CH2Cl2 (3 ꢁ 2 min).
The resin was treated with Ac2O/DIEA/CH2Cl2 10/5/85 by volume
(10 mL, 2 min then 10 mL, 20 min) to cap unreacted amino groups.
Finally, the resin was washed with CH2Cl2 (5 ꢁ 2 min).
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: oleg.melnyk@ibl.fr. Homepage: http://csb.ibl.fr.
’ ACKNOWLEDGMENT
We than the French Ministry of Research, grants from the
EEC, CNRS, Rꢀegion Nord Pas-de-Calais, and Cancꢀerop^ole
Nord-Ouest for financial support. This work was performed in
the Chemistry Systems Biology platform facility of the Institute
of Biology (Lille, France).
The peptide was assembled on a 0.25 mmol scale (0.175 mmol/g) by
using standard automated Fmoc-SPPS. The couplings were performed
with protected amino acids (0.2 M in DMF, 4 equiv), HBTU (0.5 M in
DMF, 3.6 equiv), and DIEA (2 M in DMF, 8 equiv).
Peptide deprotection and cleavage: Deprotection and clea-
vage were performed with TFA/TIS/DMS/thioanisole/H2O (90/
2.5/2.5/2.5/2.5 by volume, 25 mL) during 2 h 30 min. The peptide
was precipitated in cold Et2O/heptane 1/1 by volume, dissolved in
deionized water, and lyophilized to give 288 mg of crude peptide 1e
(32%).
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LC-MS C120H216N36O31S4 [M þ H]þ m/z calcd (mean) 2788.5,
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Peptide 1e was directly oxidized into 4e before RP-HPLC purifica-
tion. For this, crude 1e (95.7 mg) was dissolved in AcOH/eau 1/4 by
volume (47.9 mL). I2 (197 mM) in DMSO (270 μL, 50 μmol, 2 equiv)
was added to this solution. After 30 s of mixing, 64.8 mM dithiothrꢀeitol
(DTT, 823 μL, 50 μmol, 2 equiv) was added to decompose the excess of
I2. Then, the crude peptide was immediately purified by RP-HPLC.
(C18 nucleosil 120 Å, 5 μm, buffer A 100% water containing 0.05% TFA,
buffer B CH3CN/water 4/1 by volume containing 0.05% TFA, gradient:
0ꢀ20% B in 5 min, then 20ꢀ40% B in 40 min, flow rate 6 mL/min,
detection at 215 nm) to give 25.8 mg of peptide 4e (27%).
MALDI-TOF C120H214N36O31S4 [M
(monoisotopic) 2784.52, obsd 2784.6.
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H]þ m/z calcd
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sodium phosphate buffer pH 7.3 (10 mL). 3-Mercaptopropionic acid
(MPA, 0.5 mL) was added. NaOH (5 M, 340 μL) was then added to
adjust the pH to 3.98.
Peptide 4e (24.67 mg, 6.9 μmol) was dissolved in the above solution
(10.3 mL). The reaction was agitated at 37 °C under argon atmosphere.
The reaction was monitored by RP-HPLC on an C18 Xbridge BEH
column (215 nm, 1 mL/min, 30 °C, buffer A water containing 0.05%
TFA, buffer B CH3CN/water 4/1 by volume containing 0.05% TFA,
0ꢀ100% B in 30 min). For this, aliquots were acidified with 10%
aqueous TFA and extracted with Et2O to remove MPA in excess prior to
analysis.
After completion of the reaction, the mixture was diluted with water
(10 mL) and 10% aqueous TFA (5 mL) was added. After 3 extractions
with Et2O (3 ꢁ 15 mL) and argon bubbling during 15 min, purification by
RP-HPLC on a C18 Nucleosil column 120 Å, 5 μm (215 nm, 6 mL/min,
rt, buffer A water containing 0.05% TFA, buffer B CH3CN/water 4/1 by
3201
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