Chemoselective ligation of peptide phenyl esters with N-Terminal cysteines

Full text of DOI:10.1002/cbic.201000165

DOI: 10.1002/cbic.201000165
Chemoselective Ligation of Peptide Phenyl Esters with N-Terminal
Cysteines
Ge-Min Fang, Hong-Kui Cui, Ji-Shen Zheng, and Lei Liu*[a]
Protein chemical synthesis enables a level of control on protein
composition beyond what is attainable with protein expres-
sion. It can provide otherwise inaccessible insights into pro-
tein’s structure and function.[1] The development of increasing-
ly efficient and general methods for the ligation of complex
peptide fragments remains a central objective. The method of
choice is a Cys-based native chemical ligation (NCL) developed
by Kent et al.[2] NCL involves a chemoselective ligation reaction
between a C-terminal thioester and an N-terminal Cys to yield
a native peptide bond. Although being an extremely powerful
method, the applicability of NCL might sometimes be limited
in two respects: 1) The ligation reaction is restricted to the Cys
residue. Thus, in some cases special techniques must be imple-
mented by using a removable thiol-containing auxiliary, for
example.[3] 2) The rate of NCL is strongly dependent on the C-
terminal amino acid. Ligations at sterically hindered C-terminal
sites, such as Val, Ile, and Pro, are very slow and the corre-
sponding yields are often low.[4]
approach. Note that in earlier studies Kemp et al. and some
other groups showed the use of phenyl ester derivatives in
peptide couplings.[7,4c] Such couplings are different from the
ligation described in this study because the present reaction
requires the assistance of thiol capture and imidazole promo-
tion, whereas the previous couplings were direct aminolysis re-
actions.
Our study began with the examination of a number of pep-
tide oxo-esters for the Cys ligation in model systems
(Scheme 1). It was found that most oxo-esters could not pro-
To help solve the second problem, Danishefsky and co-work-
ers recently developed a highly interesting oxo-ester peptide
ligation method.[5] Through the use of a fairly activated C-ter-
minal para-nitrophenyl ester, it is possible to achieve direct Cys
ligations. Importantly, peptide substrates incorporating bulky
C-terminal amino acids can be accommodated with high reac-
tion efficiency. The success of this novel concept of peptide li-
gation opens new opportunities for studies on protein chemi-
cal synthesis. Nonetheless, Danishefsky’s pioneering method
has two drawbacks that require further improvement: 1) unde-
sired hydrolysis of the activated peptide para-nitrophenyl ester
is sometimes a major competing reaction; 2) a direct solid-
phase synthesis of peptide para-nitrophenyl ester remains very
difficult.
Scheme 1. Ligation efficiency (HPLC yields) of different oxo-esters. Bz=ben-
zoyl; TCEP=tris(2-carboxyethyl)phosphine; GnHCl=guanidine hydrochlo-
ride; MeCN=acetonitrile.
Herein we report that by using simple and less-activated
phenyl esters of peptides, chemoselective ligation between
two unprotected peptide fragments can also proceed directly
and smoothly under the promotion of imidazole.[6] Significant-
ly, it is found that ligations at sterically hindered C-terminal
sites (e.g., Val, Ile, Pro) are fairly efficient under the new condi-
tions. Moreover, the previous problems associated with the
para-nitrophenyl esters (that is, fast hydrolysis and difficult
solid-phase synthesis) are successfully overcome by the new
vide any ligation product. Surprisingly, when the phenyl ester
(1a) was tested, we obtained a very high ligation yield (98%)
in only 3 h. A more electron deficient phenyl ester (1b) reacted
faster, whereas electron rich phenyl esters (1c, d) reacted
slightly more slowly. Thiophenol was used as a promoter in
the above ligations, but the ligation did not proceed well with
the phenyl esters of more sterically hindered amino acids (e.g.,
Ile or Val).
To search for possible improvements, imidazole was added
to the ligation media.[8] To our surprise, we found that when
the imidazole concentration was 2.5m, the thiophenol promot-
er was no longer needed (Table 1). More interestingly, both
sterically unhindered and hindered amino acids could be suc-
cessfully ligated with Cys under the new conditions. For steri-
cally unhindered amino acids the ligation was very fast (1–2 h)
and the yields were very high (>96%; entries 1–4). For the
sterically demanding amino acids (Val, Ile, Pro), the ligation was
slower (7–10 h) but the yields remained good (70–94%). In
[a] G.-M. Fang, H.-K. Cui, J.-S. Zheng, Prof. Dr. L. Liu
Department of Chemistry, Key Laboratory of Bioorganic
Phosphorus Chemistry and Chemical Biology
(Ministry of Education), Tsinghua University
Beijing 100084 (China)
Fax: (+86)10-62771149
E-mail: lliu@mail.tsinghua.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000165.
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tion containing both Cys and nBuNH2 [Eq. 1]. Again Bz-Gly-Cys
was the only observed product; this indicates that the side
chain of Lys does not interfere the phenyl ester ligation. Finally,
the potential for racemization of the C-terminal amino acid in
the ligation was examined by analyzing the reaction between
Bz-Gly-Ala-OPh and Cys. The HPLC data (Supporting Informa-
tion) showed that less than 1% was racemized.
Table 1. Ligation (HPLC) yields of different amino acid esters.
Peptide-
t [h]
Yield [%]
99
1
2
3
4
5
6
7
Bz-Gly- (2a)
Bz-Ala- (2b)
Bz-Met- (2c)
Bz-Phe- (2d)
Bz-Val- (2e)
1
1
1
2
7
Kinetic measurements showed that in cases in which the
concentration of BzGly-OPh was 2 mm, the ligation rate with
4 mm Cys was similar to that with 16 mm Cys (Figure 1A).
However, when the imidazole concentration increased from 0.5
to 2.5m (in the presence of 2 mm BzGly-OPh and 4 mm Cys),
the ligation rate increased by about 3.4 times (Figure 1B).
Moreover, we found that the ligation rate in the presence of
4 mm Cys was almost identical to the rate of imidazole-pro-
moted hydrolysis of the phenyl ester in the absence of Cys
(Figure 1C). These data indicated that the phenyl ester reacts
first (in a rate-limiting step, see Scheme 3) with imidazole to
form a highly reactive intermediate (presumably acyl imida-
zole). Then the intermediate reacts rapidly with Cys through
thiol capture[9] or it is destroyed by H2O when Cys is absent.
To apply the phenyl ester ligation to protein chemical syn-
thesis, we developed a solid-phase method to prepare peptide
phenyl esters (Scheme 4).[10] The 4-methylbenzhydrylamine
(MBHA) resin was used to attach 4-hydroxyphenyl-
acetic acid. Subsequently the peptide phenyl ester
could be readily synthesized through standard Boc
chemistry. Thus, compared to peptide para-nitro-
phenyl esters, an important advantage of peptide
phenyl ester is that it can be prepared through solid-
phase synthesis. Note that we have tried to use
Fmoc chemistry to prepare peptide phenyl esters,
but failed.
97
96
96
94
89
70
Bz-Gly-Ile- (2 f)
Bz-Gly-Pro- (2g)
10
10
comparison with these data, the ligation of peptide para-nitro-
phenyl esters generally took 2–15 h with a yield of 50–80%,[5]
whereas the ligation between the thioester of Ile and Cys was
reported to take 72 h to generate the product in 58% yield.[5]
Interestingly, it was found that the traditional thioester-
mediated NCL could not be accelerated under the same imida-
zole conditions. To evaluate the selectivity of the phenyl ester
ligation for the Cys functionality, a competition experiment
was conducted in which Bz-Gly-OPh was added to a solution
containing both Cys and Ala (Scheme 2). Dipeptide Bz-Gly-Cys
To test whether the new ligation reaction might
Scheme 2. Reaction scheme of a competition experiment in which Bz-Gly-OPh was
added to a solution containing both Cys and Ala. Dipeptide Bz-Gly-Cys was the only
observed product.
constitute a practical way to chemoselectively con-
dense unprotected peptide segments, model protein
synthesis was conducted for hadrurin,[11] a basic anti-
microbial peptide. Native hadrurin does not contain
any Cys (Scheme 5), but to facilitate the synthesis Ala10 was
mutated to Cys (which can be readily converted back to Ala
after the ligation[3h, 12]). This created a ligation site of Ile-Cys. As
shown in Figure 2, the peptide phenyl ester (3) was ligated
was the only observed product (Supporting Information); this
strongly suggested that prior thiol capture of Cys predomi-
nates in the ligation. Furthermore, a competition experiment
was also carried out in which Bz-Gly-OPh was added to a solu-
Figure 1. Kinetic data of phenyl ester ligation.
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smoothly with peptide 4 to pro-
duce 5 (which was stable to
NaOH treatment) in 7 h with a
HPLC yield of 86%.
In the second example
gastrin[Ala8Cys] was synthesized.
Gastrin is a peptide hormone
functioning as a physiological
regulator of gastric acid secre-
tion.[13] The ligation site was
Scheme 3. Proposed mechanism of the phenyl ester ligation.
chosen to be Pro-Cys, an even
more challenging position that
has hardly ever been used in the traditional NCL. As shown in
Scheme 6 and Figure 3, in cases in which the phenyl ester 6
was used, the Pro-Cys ligation was accomplished in 7 h with a
HPLC yield of 63%. Both of the examples showed that the
phenyl ester ligation is especially useful for the ligations at dif-
ficult sites.
Scheme 4. Synthesis of peptide phenyl ester on the solid phase.
Scheme 6. Model synthesis of gastrin.
To examine whether an internal Cys residue might interfere
with the ligation,[14] we studied the reaction between Bz-Phe-
OPh and
a
model peptide Cys-Gly-His-Cys-Tyr-Gly-Ala
(Scheme 7). Only one product (which was stable to NaOH
treatment) was obtained with a high yield (90%) and an ex-
pected mass (Supporting Information). Furthermore, we syn-
thesized a peptide phenyl ester that contained an internal un-
protected Cys (Scheme 8). The ligation of this peptide phenyl
ester with Cys also provided one
Scheme 5. Model synthesis of hadrurin.
major product (which was stable
to NaOH treatment) with a high
yield (89%) and an expected
mass (Supporting Information).
Therefore, an internal unprotect-
ed Cys can be well tolerated in
the ligation of peptide phenyl
esters.
In summary, it was found that
simple phenyl esters of peptides
could undergo native chemical
ligation smoothly under the pro-
motion of imidazole. This new
ligation proceeded rapidly at
both sterically unhindered and
hindered C-terminal sites (e.g.,
Figure 2. Analytic HPLC data for the ligation between 4 and 3.
Val, Ile, Pro) to form the desired
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in an aqueous solution of Gn·HCl
(6m, 0.15 mL, containing 5 mL TFA).
N-terminal Cys peptide 4 (1.7 mg,
0.482 mmol) was dissolved in the li-
gation buffer (0.113 mL, 5.0m imi-
dazole, 4.0m Gn·HCl, 80 mm TCEP,
pH 7.14, pH was adjusted by using
neat TFA). The reaction mixture
containing both the solutions of 3
and 4 (0.1 mL each, pH 7.10) was
stirred at 36–388C under argon.
The reaction was monitored by
HPLC. The ligation product (5) was
confirmed by MALDI-TOF with a
HPLC yield of 86%.
Acknowledgements
This work was supported by NSFC
Figure 3. Analytic HPLC data for the ligation between 6 and 7.
(No.
20802040
and
No.
20932006), and the Specialized
Research Fund for the Doctoral Program of Higher Education of
China (Grant No. 200800030074).
Keywords: native chemical ligation · peptide phenyl ester ·
peptides · solid-phase synthesis
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Scheme 8. Synthesis of a peptide phenyl ester containing an internal unpro-
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Chemoselective ligation for the synthesis of hadrurin[Ala10Cys]
[Eq. (2)]: Peptide phenyl ester 3 (0.6 mg, 0.52 mmol) was dissolved
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Received: March 12, 2010
Published online on April 16, 2010
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