Oxo-ester mediated native chemical ligation: Concept and applications

Article abstract of DOI:10.1021/ja804993y

A direct oxo-ester peptide ligation method has been developed. Through the use of an activated C-terminal para nitrophenyl ester (1), it is possible to achieve direct cysteine ligations (1 + 2 → 4). Peptide substrates incorporating bulky C-terminal amino acids (1) can be accommodated with high reaction efficiency. Copyright

Full text of DOI:10.1021/ja804993y

Published on Web 10/15/2008
Oxo-ester Mediated Native Chemical Ligation: Concept and Applications
Qian Wan,† Jin Chen,† Yu Yuan,† and Samuel J. Danishefsky*,†,‡
Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York AVenue,
New York, New York 10065, and Department of Chemistry, Columbia UniVersity, HaVemeyer Hall, 3000 Broadway,
New York, New York 10027
Received June 30, 2008; E-mail: s-danishefsky@ski.mskcc.org
Scheme 1
The landmark disclosure, in 1901, of the synthesis of the
dipeptide glycylglycine through hydrolysis of glycine is con-
sidered to mark the beginning of the field of peptide synthetic
chemistry.1 The past 100 years have seen major advances in
the development of enabling peptide assembly techniques.
Complex proteins are now viewed to be viable targets for total
synthesis. Perhaps the most innovative and central of the
advances, from a technical standpoint, was the introduction of
solid phase peptide synthesis (SPPS) by Merrifield, in 1963.2
Currently, automated SPPS is commonly employed for the
assembly of large, homogeneous peptide fragments. Despite the
significant gains made in SPPS methodology, practical issues
remain, as the technique is generally limited to peptide chains
incorporating fewer than ca. 50 amino acids. In 1994, another
major breakthrough in the field was made with the disclosure
by Kent and co-workers of a general technique, termed native
chemical ligation (NCL), which enables the coupling of large
peptidic fragments.3 This method, outlined in Scheme 1a, is
based on the principles employed in a 1953 disclosure of Wieland
and co-workers.4 As shown, the C-terminus of one fragment
(Peptide 1) is equipped with a thioester. The coupling partner
(Peptide 2) possesses an N-terminal cysteine residue. The rate
limiting phase of the sequence is widely accepted to be the
intermolecular transthioesterification (step 1), which generates
a unimolecular intermediate that is well-positioned to undergo
intramolecular SfN acyl transfer, generating the coupled peptide
fragment.5
It was in this context that our laboratory developed, in 2004, a
novel and broadly useful NCL variant, which allows for the merger
of glycopeptide fragments through the use of a relatively inert
C-terminal ortho-thiophenolic ester (Scheme 1b).7 Upon cleavage
of the S-S bond, the thiophenolic ester apparently undergoes
intramolecular OfS transfer, as shown. The intermediate thioester
is then trapped intermolecularly by an N-terminal cysteine residue
of the peptide or glycopeptide coupling partner. Finally, SfN acyl
transfer provides the final coupled peptide.
Our laboratory has been interested in developing novel and
reliable methods for the assembly of complex glycopeptides and
glycoproteins bearing multiple carbohydrate domains. Part of
this focus stems from our intensive program directed toward
the de novo total synthesis of the glycoprotein, erythropoietin
alpha (EPO) as a homogeneous entity (Figure 1). EPO, a 166-
residue glycoprotein bearing four different carbohydrate domains
(three of which are N-linked, and one of which is O-linked), is
a medicinally important agent that is commonly used in the
treatment of anemia.6 It is well understood that the carbohydrate
domains play a crucial role in conferring efficacy and stability
to the glycoprotein. However, rigorous comparisons of the
efficacy of various EPO glycoforms have been largely hindered
by difficulties associated with isolating homogeneous EPO from
natural sources. A de novo synthesis program could well provide
access to valuable homogeneous material for comparative
investigations. Our EPO total synthesis program has served to
stimulate ongoing efforts to develop enabling advances in peptide
elongation and ligation techniques.
Although cysteine-based NCL is a powerful method, it too suffers
from a number of limitations. First, cysteine residues are found
† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
Figure 1. Structure of erythropoietin alpha (EPO).
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15814 J. AM. CHEM. SOC. 2008, 130, 15814–15816
10.1021/ja804993y CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
a
Scheme 2
Scheme 3.
aReagents and Conditions: buffer at pH ) 6.3-6.5 (6.0 M Gn ·HCl, 188.8
mM Na2HPO4, 18.8 mM TCEP ·HCl), PhSH, TCEP, 30 °C.
Scheme 4
a
Table 1
entry
C-terminal AA
time (h)
yield
1
2
3
4
5
Thr (2)
Val (3)
Ile (4)
D-allo-Ile (5)
Pro (6)
2
6
7
6
15
79% (7)
69% (8)
70% (9)
60% (10)
50% (11)
and ꢀ-Ala. Dipeptide Boc-Phe-Cys-OH was the only observed
product, thus strongly suggesting that prior thiol capture of Cys
predominates under these conditions.
In our initial studies, we investigated NCL at less hindered
C-termini. However, though ligated products could be isolated,
undesired hydrolysis of the acyl donor was found to be a major
competing factor, even at lower pH ranges (data not shown).
We speculated that ester hydrolysis would be less problematic
in peptides with more hindered C-terminal amino acids. To
maximally suppress hydrolysis, p-NO2-phenol was added to
guanidine buffer. Ligation studies were conducted in a buffered
solution at pH 6.3-6.5, at 25-30 °C. As outlined in Table 1,
under these conditions, oxo-ester mediated ligation proceeded
smoothly with a variety of hindered C-terminal amino acid
residues, including Thr, Val, Ile, D-allo-Ile, and Pro (entries
1-5). In each case, the reaction times were relatively short and
the yields were generally quite good.
For comparative purposes, the efficiency of a traditional thioester-
mediated NCL was also examined (Scheme 3). While the analogous
nitrophenyl ester ligation proceeded in 7 h to afford 9 in 70% yield
(entry 3), the thioester ligation took 72 h to generate 9 in only
58% yield. It is known that such prolonged reaction times can lead
to the formation of undesired side products.
“Racemization” of the C-terminal amino acid residue is known
to be an issue in peptide coupling.17 Presumably, racemization
proceeds through the pathway outlined in Scheme 4. An analysis
of the extent of racemization in the peptides presented in Table 1
revealed less than 5-6% racemization at the C-terminal Leu, Ile,
and Val sites.18
Because of the high reactivity of the p-nitrophenolic ester, it is
conceivable that resident free lysine residues could be prone to
undergo competitive direct aminolysis with the ester to provide
undesired cyclic peptide adducts. To evaluate the selectivity of the
ligation protocol described above for the R-amino group over the
lysine ε-amine, we synthesized peptide 13, which incorporates an
unprotected Lys residue on the fragment bearing the C-terminal
nitrophenyl ester (Scheme 5). In the event, we were pleased to find
that ligation with 1 readily afforded coupled product, 14, in 61%
yield. Only a small amount of the undesired cyclic peptide was
observed under our conditions.
a Reagents and conditions: (a) buffer at pH ) 6.3-6.5 (6.0 M
Gn·HCl, 188.8 mM Na2HPO4, 7.2 mM p-NO2PhOH), TCEP, room
temp.
relatively infrequently in naturally occurring peptides and proteins.
There have been attempts to circumvent this issue through various
approaches, including the development of homocysteine8 and
seleno-cysteine9 ligation protocols, through postligation desulfur-
ization techniques,10 and through the use of cysteine-free auxiliaries
which may be cleaved following ligation.11
A second type of issue arises from the limitations imposed by
the C-terminal amino acid residue of the thioester coupling
fragment. Dawson and co-workers have demonstrated that the rate
of ligation is strongly dependent on the identity of the C-terminal
amino acid.12 Ligations at sterically hindered, ꢀ-branched C-
terminal sites, such as Pro, Val, Ile, and Thr, are prohibitively slow.
Even in the wake of NCL, the development of efficient and reliable
methods for achieving hindered peptide ligations remains an
immense practical challenge.
Since the rate-limiting SfS acyl transfer (Scheme 1a, step
1) appears to be highly dependent on the steric bulk of the
C-terminal amino acid, we pursued the possibility of developing
a modified ligation method that would proceed directly from an
oxo-ester C-terminal peptide, without the intermediacy of the
problematic thioester (see Scheme 1c).13 In this regard, we took
note of a 1955 disclosure by Bodanszky and co-workers on the
synthesis of small peptides through aminolysis of para nitro-
phenyl esters.14 Such esters are sufficiently activated to facilitate
direct peptide coupling.15,16 However, a number of issues had
limited the general applicability of the method, including (1)
the relative instability of the C-terminal nitrophenyl ester, (2)
the potential for racemization of the C-terminal amino acid
residue, and (3) the lack of strong selectivity for the R-amino
group versus the ε-amino group of Lys. These issues would
clearly need to be overcome in the development of a broadly
useful direct oxo-ester ligation method.
Activated esters, such as para nitrophenyl ester, are known to
be strong acyl donors. We first evaluated the selectivity of the
activated ester for the cysteine functionality through a simple
competition experiment. Thus, as outlined in Scheme 2, Boc-Phe-
p-NO2Ph was added to a solution containing equal parts Cys, Ala,
Finally, in an effort to probe the limits of our system in a
discerning way, we sought to investigate couplings of C-terminal
nitrophenyl ester peptides with the very hindered unnatural amino
acid penicillamine (Pen, 15).19 This commercially available amino
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C O M M U N I C A T I O N S
Scheme 5a
facility for mass spectral and NMR spectroscopic analysis (SKI
core grant no. CA02848). We thank Ms. Rebecca Wilson for
editorial counsel.
Note Added after ASAP Publication. After ASAP publication on
October 15, 2008, Scheme 1, Table 1, and the abstract graphic have
been corrected. The revised version was reposted on October 28, 2008.
a Reagents and conditions: buffer at pH ) 6.3-6.5 (6.0 M Gn ·HCl,
188.8 mM Na2HPO4, 7.2 mM p-NO2PhOH), TCEP, room temp, 1 h, 61%
isolated yield.
Supporting Information Available: Experimental procedures and
compound characterization data. This material is available free of charge
via the Internet at http://pubs.acs.org.
Scheme 6a
References
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a Key: (a) buffer at pH ) 6.3-6.5 (6.0 M Gn ·HCl, 188.8 mM Na2HPO4,
7.2 mM p-NO2PhOH), TCEP, 30 °C, 2-3 h; (b) buffer at pH ) 6.3-6.5
(6.0 M Gn ·HCl, 188.8 mM Na2HPO4, 18.8 mM TCEP ·HCl), TCEP, VA-
044, 37 °C, 2-3 h.
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acid, which may serve as a surrogate for Val, can provide a viable
alternative to Cys in cases where ligation at a Cys site is not
practical. In the event, we were pleased to observe that both peptides
16 and 2 readily underwent ligation with Pen (15) to afford the
corresponding peptides in acceptable yields (Scheme 6). Exposure
to thiol reduction conditions then served to efficiently transform
Pen to Val. A comparative coupling reaction was performed on
the corresponding thioester (Fmoc-RTGDSAG-Thr-SPh). After
24 h, only 4% of compound 19 was generated, based on LC-MS
integration.
In conclusion, we have described a major simplification in the
field of native chemical ligation. Hitherto, the success of this method
had been assumed to be dependent on the formation of a thioester
acyl donor. Through proper balancing of acyl donor potential with
substrate stability, we have demonstrated the feasibility of NCL
with oxo- rather than thioesters. Further applications of this method,
as well as this extended logic, to targets of major biological interest
will be described in due course.
(12) (a) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 10068–10073 Kemp also observed similar results. (b)
Kemp, D. S.; Galakatos, N. G.; Dranginis, S.; Asthon, C.; Fotouhi, N.;
Curran, T. P. J. Org. Chem. 1986, 51, 3320–3324.
(13) Oxo-esters were prepared according to the general procedures described
in references7a, , and 10e. See Supporting Information for full synthetic
details.
(14) Bodanszky, M. Nature 1955, 175, 685.
(15) Gagnon, P.; Huang, X.; Therrien, E.; Keillor, J. W. Tetrahedron Lett. 2002,
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(17) For discussion of C-terminal amino acid racemization in thioester-mediated
NCL, please see references 12a and 19a.
(18) Goodman, M.; McGahren, W. J. Tetrahedron 1967, 23, 2031–2050.
(19) Following submission of this communication Seitz and co-workers reported
a ligation at valine, using penicillamine as the valine precursor. (a) Haase,
C.; Rohde, H.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 6807-6810. On
the basis of the results of using penicillamine (15) as a valine surrogate,
described in this communication, our group performed further research
directed to a more reactive thiol-based acyl acceptor culminating in a valine
equivalence, which is described in the following reference. (b) Chen, J.;
Wan, Q.; Yuan, Y.; Zhu, J. L.; Danishefsky, S. J. Angew. Chem. Int. Ed.
2008, Early View, DOI: 10.1002/anie.200803523.
Acknowledgement. Support for this research was provided by
the National Institutes of Health (CA28824). A postdoctoral
fellowship is gratefully acknowledged by Q.W. (Mr. William H.
Goodwin and Mrs. Alice Goodwin and the Commonwealth
Foundation for Cancer Research, and the Experimental Therapeutics
Center, SKI). We thank Dr. George Sukenick, Ms. Sylvi Rusli,
and Ms. Hui Fang of the Sloan-Kettering Institute’s NMR core
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