Native chemical ligation at phenylalanine

Article abstract of DOI:10.1021/ja072804l

Synthesis of erythro-N-Boc-β-mercapto-l-phenylalanine enables native chemical ligation at phenylalanine. In the form of the S-ethyldisulfanyl derivative, the N-Boc amino acid is used to cap a tetrapeptide generated by Fmoc-SPPS. The native chemical ligati

Full text of DOI:10.1021/ja072804l

Published on Web 07/21/2007
Native Chemical Ligation at Phenylalanine
David Crich*,† and Abhisek Banerjee
Department of Chemistry, UniVersity of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607-7061
Received April 22, 2007; E-mail: dcrich@chem.wayne.edu
Scheme 2. Precursor Synthesis
Native chemical ligation (NCL), a technique introduced by Kent
and co-workers for the synthesis of peptides and proteins, relies
on the combination of a N-terminal cysteine residue with a
C-terminal thioester to form a peptide bond.1 The method has two
steps: a trans-thioesterification, followed by a rapid intramolecular
S- to N-shift (Scheme 1). The power of this technique, or its
biochemical equivalent, expressed protein ligation,2 and the modi-
fication dubbed kinetically controlled ligation,3 has enabled the
synthesis of many moderate sized proteins4 and glycoproteins,5
culminating in the assembly of a 203 amino acid (AA) HIV protease
covalent dimer.6
Scheme 1. General NCL Mechanism
Scheme 3. Model Ligations
Extension of NCL beyond cysteine may be accomplished most
directly by post-ligation desulfurization of the pivotal cysteine
residue, affording ligation at alanine.7,8 Alternatively, a variety of
auxiliary thiols have been introduced for attachment to the
N-terminal AA and traceless cleavage after ligation (e.g., 1),9 but
none has complete generality.10 The phosphine 2 bridges the gap
with the Staudinger ligation but requires the synthesis of R-azido
acids for use in peptide and protein synthesis.11 Like Dawson,7 we
reasoned that NCL could be extended to further AAs if (i) a
convenient synthesis of the â-mercapto derivatives was available,
(ii) the trans-thioesterification step is not seriously affected by
additional steric hindrance, and (iii) the final desulfurization can
be conducted selectively. We report that the â-mercaptophenyla-
lanine derivative 3 meets these conditions, as demonstrated through
the synthesis of two complex decapeptides.
protected by reaction with S-ethyl ethanethiosulfinate,15 giving the
disulfide 6. Treatment with trifluoroacetic acid then afforded amino
ester 7, whereas saponification gave the acid 8 (Scheme 2).
The N-Cbz glycine and N-Boc-L-methionine thioesters 9 and 10
were obtained under standard carbodiimide conditions and were
ligated with the â-mercaptophenylalanine derivative 7 in the
presence of sodium 2-mercaptoethanesulfonate (MESNa) in a
mixture of acetonitrile and Tris buffer at pH 7.5-8.0. In this
manner, the glycine-based dipeptide 11 and the methionine analogue
12 were obtained in 75 and 50% yields, respectively, reflecting
the slower ligation at the more hindered thioester (Scheme 3). Blank
experiments with L-phenylalanine methyl ester in place of 7
established the critical role of the thiol in these couplings.
Desulfurization of 11 with nickel boride,16 obtained in situ by
sodium borohydride reduction of nickel chloride, finally gave
N-Cbz-Gly-L-Phe-OMe 13 in 80% yield. The cognate reduction of
12 afforded N-Boc-Met-L-Phe-OMe 14 in 70% isolated yield,
thereby demonstrating the preferential cleavage of the benzylic C-S
bond in the presence of methionine (Scheme 3). A comparable
competition experiment (Supporting Information) established selec-
We focused on phenylalanine as direct, selective functionalization
at the â-position was known and because we anticipated facile
reductive cleavage of the benzylic C-S bond would ensure
compatibility with other sulfur containing AAs.12 Adapting Easton’s
bromination protocol,13 we devised a convenient synthesis of the
threo-â-hydroxy-L-phenylalanine derivative 4 from L-phenylala-
nine14 and converted it to the erythro-thiol 5 by standard methods
(Scheme 2). To avoid oxidative dimerization, the thiol group was
† Current address: Department of Chemistry, Wayne State University, 5101 Cass
Avenue, Detroit, MI 48202.
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J. AM. CHEM. SOC. 2007, 129, 10064-10065
10.1021/ja072804l CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. We thank the University of Illinois at
Chicago for the Moriarty Fellowship (A.B.), the Protein Research
Laboratory at UIC for SPPS, and Professor Yasuhiro Kajihara,
Yokohama City University, for insightful discussions.
Scheme 4. Peptide Thioester Synthesis
Note Added after ASAP Publication. After this work was
published ASAP on July 21, 2007, we became aware that the
concept of native chemical ligation at phenylalanine had been
described previously: (a) Tchertchian, S.; Opligger, F.; Paolini, M.;
Manganiello, S.; Raimondi, S.; Depresle, B; Dafflon, N.; Gaertner,
H.; Botti, P. In Understanding Biology Using Peptides, Proceedings
of the 19th American Peptide Symposium, San Diego, June 18-
23, 2005; Blondelle, S. E., Ed.; Springer: New York, 2006; p 61.
(b) Botti, P.; Tchertchian, S. WO/2006/133962. We apologize for
this inadvertent oversight. This note was added on July 26, 2007.
Supporting Information Available: Full experimental details and
characterization data for all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
Scheme 5. Decapeptide Synthesis
References
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ethyl cysteine).17
With proof of principle established, we turned to the synthesis
of more challenging peptides. To probe the limits of the system,
we selected two LYRMXXRANK sequences,18 with their densely
packed array of the more reactive AA side chains, which we
modeled on Dawson’s NCL probes.19 The N-Boc acid 8 was
employed as the final AA in the Fmoc-based SPPS of a pentapeptide
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afforded the â-(SSEt)-FRANK peptide 15, thereby demonstrating
compatibility with the standard Fmoc-SPPS conditions.
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acid were converted to their S-benzyl thioesters under the non-
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of the target peptides LYRMGFRANK and LYRAMFRANK in
good overall yield (Scheme 5).
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be readily extended to NCL at histidine, tyrosine, and tryptophan.
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