Peptidyl N,N-bis(2-mercaptoethyl)-amides as thioester precursors for native chemical ligation

Article abstract of DOI:10.1021/ol102735k

With twoΒ-mercaptoethyl groups on the N, a tertiary amide of structure 1 is always poised for intramolecular thioesterification however it flips about the C-N bond. It is shown that a peptide with such a C-terminal N,N-bis(2-mercaptoethyl)-amide (BMEA) can be used directly for native chemical ligation (NCL). These BMEA peptides are easily prepared with standard Fmoc-solid phase peptide synthesis protocols, thus giving a very convenient access to the thioester components for NCL.

Full text of DOI:10.1021/ol102735k

ORGANIC
LETTERS
2011
Vol. 13, No. 3
386-389
Peptidyl N,N-Bis(2-mercaptoethyl)-
amides as Thioester Precursors for Native
Chemical Ligation†
Wen Hou, Xiaohong Zhang, Fupeng Li, and Chuan-Fa Liu*
DiVision of Chemical Biology and Biotechnology, School of Biological Sciences,
Nanyang Technological UniVersity, 60 Nanyang DriVe, Singapore 637551
cfliu@ntu.edu.sg
Received November 3, 2010
ABSTRACT
With two ꢀ-mercaptoethyl groups on the N, a tertiary amide of structure 1 is always poised for intramolecular thioesterification however it flips
about the C-N bond. It is shown that a peptide with such a C-terminal N,N-bis(2-mercaptoethyl)-amide (BMEA) can be used directly for native
chemical ligation (NCL). These BMEA peptides are easily prepared with standard Fmoc-solid phase peptide synthesis protocols, thus giving
a very convenient access to the thioester components for NCL.
Peptide CR-thioesters are key building blocks for a number
of protein synthesis strategies,1 including notably native
chemical ligation.2 This has stimulated considerable interest
in developing new methods to prepare these important
compounds in recent years.3 Traditionally, thioester peptides
are prepared with the Boc-solid phase synthesis method
whereby the peptide chain is assembled directly on a thioester
linker.1a,4 Although this method is very effective, the need
for a highly hazardous strong acid such as HF at the final
cleavage step represents a deterring element to many research
laboratories. Direct Fmoc-solid phase synthesis of peptide
thioesters on a thioester linker is also possible using a
modified Fmoc-deprotection protocol; however its use is
restricted to relatively small peptides and racemization of
the C-terminal amino acid residue is a non-negligible
problem.5 Considerable efforts have been devoted to devel-
oping alternative strategies to obtain thioester peptides
indirectly from nonthioester precursors, the synthesis of
which is compatible with standard Fmoc chemistry.6-8 For
† This work was first presented at the 11th Chinese International Peptide
Symposium (or CPS-2010) held on July 5-8, 2010 in Lanzhou, China.
(1) For reviews, see: (a) Aimoto, S. Biopolymers 1999, 51, 247. (b)
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10.1021/ol102735k  2011 American Chemical Society
Published on Web 12/22/2010

example, certain activated amide systems based on Kenner’s
safety-catch sulfonamides,6a-c acylureas,6d and pyroglutamyl
imides6e have been developed. Other systems utilize NfS
acyl transfer to produce thioesters,9,10 which in a way are
mechanistically reminiscent of protein splicing.11 Most of
these systems require acidic conditions to catalyze amide-
to-thioester conversion which is followed by transthioesteri-
fication with a free thiol compound to generate a thioester.
Aimoto’s group reported the use of an autoactivating
C-terminal Cys-Pro ester (CPE) to mediate amide-to-thioester
conversion at neutral or slightly basic pH, which is driven
by diketopiperazine formation to trap the transiently exposed
R-amine of Cys through intramolecular aminolysis of the
prolyl ester.10a Despite an inconvenience in loading the first
amino acid to the CPE linker and the need for a relatively
reactive glycolic ester at the C-terminus, this method is
appealing for its clever design.
catalyst for thioesterification.11 NR-Cys alkylation (e.g.,
methylation and ethylation) would increase the ratio of the
productive cis form to the nonproductive trans form in the
resultant tertiary amide bond, but the conversion of the trans
isomer to the cis isomer would still need to overcome a
significant energy barrier no less than in the case of a normal
secondary amide peptide bond.12 For example, even an Xaa-
Pro tertiary amide has a high rotational barrier (∆G‡ )
18-21.5 kcal/mol) and a low cis/trans ratio of ∼0.05/1.0 in
unstructured polypeptide chains.12 For this reason, the
N-alkyl Cys systems, which require acidic conditions for
thioesterification, are usually very inefficient and give very
low yields of thioester products.9d,3b
These considerations have led us to propose a new NfS acyl
transfer system to generate thioesters for NCL, as shown in
Scheme 2. This system is based on the use of a peptide
In our efforts to develop new and convenient methods for
peptide thioester synthesis, we have paid particular attention
to the NfS acyl transfer reaction. Mechanistically, in order
for the NfS acyl transfer to take place, the planar amide
bond must be in the conformation where the thiol-bearing
N-substituent is anti to the carbonyl oxygen, as shown in
Scheme 1 for an Xaa-Cys peptide bond. This requires an
Scheme 2. N-to-S Acyl Transfer Using an
N,N-Bis(2-mercaptoethyl)-Substituted Tertiary Amide To
Generate a Thioester Peptide for NCL
Scheme 1
.
Mechanism of NfS Acyl Transfer Involving a
C-terminal tertiary amide 1, namely N,N-bis(2-mercaptoethyl)-
amide or BMEA. With this system, the NfS acyl transfer
reaction is twice as likely. It also obviates the need for trans-cis
amide isomerization prior to the NfS acyl transfer reaction
step because, with two ꢀ-mercaptoethyl (HS-Et) N-substitutions,
the BMEA amide will always have one HS group correctly
positioned for the intramolecular thiolysis reaction (Scheme 2),
and the relatively high basicity of the secondary amine in the
thioesterification product 2 would also make its trapping easier
via protonation, which might help drive the formation of 2 at
an NCL-operable pH. Coupling thioesterification with native
chemical ligation would then ultimately lead the overall reaction
in its forward direction (Scheme 2).
Peptidyl-Cys Amide Bond
energetically unfavorable cis isomer of the secondary amide,
a conformation it almost never adopts. And trans-cis
isomerization of a secondary amide peptide bond requires a
significant amount of activation energy (∆H‡ ∼20 kcal/
mol).12 Furthermore, to drive the reaction equilibrium toward
thioester formation, there must be a trapping mechanism,
e.g., protonation, for the newly exposed amine. In protein
splicing, trans-cis amide isomerization is catalyzed presum-
ably by the intein which also serves as a general acid-base
For the synthesis of a C-terminal BMEA peptide 1 shown
in Scheme 2, we designed a bis(2-mercaptoethyl)amine-
derived trityl resin 5 which was prepared in straightforward
reaction steps (Scheme 3). Thus, (2-aminoethyl)sulfanyl-trityl
(9) (a) Kawakami, T.; Sumida, M.; Nakamura, K.; Vorherr, T.; Aimoto,
S. Tetrahedron Lett. 2005, 46, 8805. (b) Ollivier, N.; Behr, J.-B.; El-Mahdi,
O.; Blanpain, A.; Melnyk, O. Org. Lett. 2005, 7, 2647. (c) Nagaike, F.;
Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki, A.; Nakahara, Y.; Nakahara,
Y. Org. Lett. 2006, 8, 4465. (d) Hojo, H.; Onuma, Y.; Akimoto, Y.;
Nakahara, Y.; Nakahara, Y. Tetrahedron Lett. 2007, 48, 25. (e) Ohta, Y.;
Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett. 2006,
8, 467. (f) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett. 2009,
Scheme 3. Synthesis of C-Terminal BMEA Peptides
11, 823
.
(10) (a) Kawakami, T.; Aimoto, S. Tetrahedron Lett. 2007, 48, 1903.
(b) Kang, J.; Reynolds, N. L.; Tyrrell, C.; Dorin, J. R.; Macmillan, D. Org.
Biomol. Chem. 2009, 7, 4918
.
(11) (a) Chong, S.; Mersha, F. B.; Comb, D. G.; Scott, M. E.; Landry,
D.; Vence, L. M.; Perler, F. B.; Benner, J.; Kucera, R. B.; Hirvonen, C. A.;
Pelletier, J. J.; Paulus, H.; Xu, M.-Q. Gene 1997, 192, 271. (b) Evans, T. C.;
Xu, M. Q. Chem. ReV. 2002, 102, 4869. (c) Muir, T. W. Annu. ReV.
Biochem. 2003, 72, 249.
(12) (a) Kessler, H. Angew. Chem., Int. Ed. 1970, 9, 219–235. (b)
Schiene-Fischer, C.; Fischer, G. J. Am. Chem. Soc. 2001, 123, 6227–6231.
Org. Lett., Vol. 13, No. 3, 2011
387

resin 3 was reacted with o-nitrobenzenesulfonyl chloride to
afford the sulfonamide resin 4. Alkylation of the sulfonamide
with Trt-SCH2CH2OH by Mitsunobu reaction and subsequent
thiolytic removal of the sulfonyl group yielded the desired
dialkylamine resin 5, ready for use in Fmoc SPPS.
with a yield of ∼75% after 24 h of reaction (Supporting
Information). pH 6 also gave a relative clean reaction with
just a slight increase in side products. The rate of ligation
was about the same at pH 4, 5, or 6 (Supporting Information).
We also found MESNa (HSCH2CH2SO3Na) to be a better
thiol additive for the ligation reaction presumably owning
to its excellent solubility in aqueous buffer. As we can see
from Figure 2A, when the reaction was conducted at 37 °C
Loading the first Fmoc-amino acid onto resin 5 was
achieved using DIC/HOAt, a coupling protocol known to
be effective for a sterically hindered secondary amine with
minimum risks of racemization.13 Subsequent assembly of
the peptide chain was carried out using standard Fmoc SPPS
procedures. Because of its inactivated nature, the tertiary
amide linkage is completely stable during Fmoc SPPS.
To test the BMEA-mediated ligation method, we first
prepared a small BMEA peptide with a Gly as the C-terminal
residue, H-LKSFG-BMEA. Interestingly, HPLC analysis of
the peptide showed the coexistance of the amide form 1 and
thioester form 2 (Scheme 2) as two peaks at a ratio of about
1:5 which gave the same molecular ion (Figure 1). The
Figure 2. Ligation between H-LKSFG-BMEA and H-CLKFA-
NH2 in the presence of MESNa (2% w/v) and 50 mM TCEP and
at pH 5. HPLC gradient: 0-50% buffer B in 50 min. (A) Reaction
at 37 °C. (B) Reaction under microwave irradiation. Peak a,
H-CLKFA-NH2; peak b, H-LKSFG-BMEA; peak c, ligation
product; peak d, H-LKSFG-MES thioester.
Figure 1. HPLC (A) and MS (B) analyses of LKSFG-BMEA. Peak
1, amide form; peak 2, thioester form. HPLC gradient: 0 - 45%
and pH 5, the yield was g80% after 8 h in the presence of
2% (w/v) MESNa. Interestingly, low-power microwave
irradiation could significantly accelerate the reaction, which
gave >90% yield after just 4 h (Figure 2B). NfS acyl transfer
appeared to be the rate-limiting step, as only a very small
amount of the MES thioester, but not the BMEA thioester 2
(Scheme 2), was seen in the HPLC (Figure 2), suggesting
that, once formed, the BMEA thioester was quickly reacted
with MESNa and/or the cysteinyl peptide H-CLKFA-NH2.
buffer B in 45 min.
smaller peak (Figure 1A, peak 2) appeared to be the thioester
form which, with a free amine in the BMEA portion, was
expected to be more hydrophilic and would elute out earlier
in HPLC, and the larger peak (peak 1) was the amide form.
This result was very encouraging as it clearly showed that
NfS acyl transfer with this BMEA peptide could readily
take place under these relatively acidic conditions (pH of
HPLC buffer: ∼2). In fact, when this peptide was treated
with 20% mercaptopropionic acid (MPA) in H2O, the MPA
thioester was obtained in ∼85% yield after 8 h at RT
(Supporting Information). In previous studies with the
N-alkyl-cysteine method, thiol exchange with MPA yielded
∼33% of the thioester product after 2-3 days of reaction.9d
Our results show that the presence of two HS-Et groups on
the amide nitrogen significantly increased the thioesterifying
capability of BMEA as compared with N-alkyl cysteine systems.
We next wanted to test whether the BMEA peptide could
be used directly for NCL, which would be ideal and is also
the focus of our present study. We first performed ligation
of H-LKSFG-BMEA (5 mM) with H-CLKFA-NH2 (15 mM)
at 37 °C using benzylmercaptan (1%) as the thiol additive.
A very clean ligation reaction was observed at pH 4 or 5
The above data clearly show that a peptide with a
C-terminal BMEA can be used as a thioester equivalent for
ligation with a cysteinyl peptide through a transiently formed
thioester. To investigate the scope of this BMEA-mediated
reaction system, we synthesized six other small BMEA
peptides with six representative C-terminal amino acid
residues: Ala, Phe, Ser, Leu, Asn, and Val, respectively
(Table 1). These peptides were then used in the ligation
reaction with H-CLKFA-NH2 at pH 5 and under microwave
irradiation. The results were summarized in Table 1. One
can see that excellent yields were obtained after 6 h of
reaction for almost all of these BMEA peptides with
H-LKSFV-BMEA being the only exception. The presence
of the Val ꢀ-branched side chain seemed to hinder the ability
for this BMEA peptide to undergo NfS acyl transfer. In
spite of this, the results show that BMEA peptides can tolerate
most amino acids at the C-terminal position for ligation with a
cysteinyl-peptide. Very clean ligation reactions were observed
for all of these BMEA peptides (Supporting Information). This
(13) Angell, Y. M.; Thomas, T. L.; Flentke, G. R.; Rich, D. H. J. Am.
Chem. Soc. 1995, 117, 7279.
388
Org. Lett., Vol. 13, No. 3, 2011

Table 1. Conversion (%) of H-LKSFXaa-BMEA Peptides in
a
Forming Ligation Products with H-CLKFA-NH2
Xaa/time (h)
Gly
Ala
Ser
Phe
Leu
Asn
Val
2
65
90
100
56
82
100
58
77
90
93
99
50
78
92
96
52
75
86
95
98
57
78
92
99
4
6
8
10
∼15
a Reaction conditions: H-LKSFXaa-BMEA (5 mM) and H-CLKFA-
NH2 (15 mM) in 100 mM acetate buffer (pH 5) containing 2% (w/v) MESNa
and 50 mM TCEP under microwave irradiation. Yields were based on HPLC
analysis and on the consumption of the BMEA peptides. See Supporting
Information for experimental details.
therefore demonstrates the broad application scope of the tertiary
amide BMEA system in native chemical ligation.
Having confirmed that BMEA peptides can be directly used
for NCL in the model studies, we then applied this method to
protein synthesis. The semisyntheses of a histone H3 protein
as well as an analog of it were successfully carried out. The
overall synthetic strategy involved the ligation between an H3
N-terminal BMEA peptide which was synthesized by Fmoc-
SPPS on the BMEA linker-derived resin and the H3 C-terminal
globular domain which was prepared by recombinant DNA
technology. So, a 13-residue BMEA peptide, H3(1-13)-BMEA
(∼6 mM), was ligated with the recombinant H3 globular
domain, H3(14-135)/K14C (∼2 mM), in a denaturing buffer
(6 M Gdn-HCl, pH 5) containing 2% (w/v) MESNa and 50
mM TCEP and under microwave irradiation. The HPLC
analysis and the mass spectral data of the ligation reaction were
shown in Figure 3 (top half). One can see that a significant
amount of ligation product H3/K14C was formed after 1 h and
that the yield reached about 55-65% after 16 h as estimated
from HPLC analysis. Similarly, an H3 analog containing a
methylated lysine at position 4, H3/K4me, was also pre-
pared through ligation between H3(1-13)/K4me-BMEA and
H3(14-135)/K14C (Figure 3, bottom half). The ligation
reaction was conducted at 37 °C (no microwave) under
otherwise identical conditions. As one can see from HPLC
analysis, about 70% of the ligation product was formed after 2
days of reaction (Figure 3A′). MALDI-TOF MS analysis clearly
confirmed the identity of the two purified ligation products
(Figure 3B and B′). These results indicated that the BMEA
method can also be applied to the synthesis of large polypeptide
molecules such as the histone proteins. These synthetic histones
Figure 3. (Top half) Synthesis of histone H3/K14C through ligation
between H3(1-13)-BMEA and H3(14-135)/K14C under micro-
wave irradiation. (A) HPLC monitoring of the ligation reaction;
(B) MALDI MS of the isolated ligation product H3/K14C (MW
calcd 15227.7). (Bottom half) Synthesis of a histone H3 analog
H3/K4me-K14C through ligation between H3(1-13)/K4me-BMEA
and H3(14-135)/K14C at 37 °C. (A′) HPLC monitoring of the
ligation reaction at 2 days; (B′) MALDI MS of the isolated ligation
product (MW calcd 15241.7). Peak I, unreacted H3(14-135)/
K14C; peak II or II′, ligation product. The BMEA peptide eluted
out much earlier before the HPLC range shown here.
intramolecular thiolysis (or N-to-S acyl transfer) to form a
thioester under very mild conditions. Although formed
transiently, the thioester has a lifetime that is long enough
to allow its capture for direct reaction with a cysteinyl peptide
at weakly acidic (or neutral15) pH. Notably, we show that
low-power microwave irradiation can significantly accelerate
BMEA-mediated ligation. The reaction system is applicable
to a wide range of C-terminal amino acid residues, pointing
therefore to its broad utility. The successful synthesis of
histone H3 proteins in our work further demonstrates the
practical value of this BMEA methodology in synthetic
protein chemistry. Compared to most other existing methods,
this system does not need a preactivation step or an intrinsic
driving device for the thioester formation reaction, making
it one of the simplest and most straightforward Fmoc-based
systems for the synthesis of thioester components for NCL.
are valuable reagents for studying histone epigenetics.14
A
preliminary bioassay showed that the synthetic H3 proteins were
fully functional to form histone octamers with the other three
core histone proteins (Supporting Information).
It is interesting to note that a very recent paper by Melnyk
et al. described the same system for peptide ligation.15 So
our two teams have independently shown that a seemingly
inert tertiary amide of the BMEA nature can undergo facile
Acknowledgment. We thank A*Star of Singapore (BMRC
08/1/22/19/588) and Nanyang Technological University for
financial support.
Supporting Information Available: Experimental pro-
cedures and compound characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
(14) Chatterjee, C.; Muir, T. W. J. Biol. Chem. 2010, 285, 11045.
(15) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Org.
Lett. 2010, 12, 5238.
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