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COMMUNICATION
complex peptide. Upon exposure to our standard metal-free de-
sulfurization conditions, the γ-thioprolines containing peptides
11, 13, 19, and 21 were readily converted to the target peptide
products in less than 10 min.7a,10,12,24
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Since both native chemical ligation and direct aminolysis are
possible options for the synthesis of large peptides and small
proteins, we also directly compared these two methods for
synthesizing desulfurized 11.25,26 The direct aminolysis reaction
was conducted in DMSO using modified BlakeÀAimoto condi-
tions (Supporting Information, Figure S4).27 At room tempera-
ture, it proceeded well to give the coupling product in a slightly
higher yield. However, because of the difficulties related to the
preparation and purification of partially protected peptide frag-
ments28 and the risk of epimerization of the residue on the
C-terminal side of the condensation site,27 in many cases, proline
ligation would be a better choice for coupling two synthetic
fragments at the proline site.
In summary, an efficient and broadly useful two-stage proline
ligation protocol has been developed. The likely basis for the
different ability of the peptide esters to undergo proline ligation
has been rationalized. The proline ligation approach provides a
significant advance in the field of protein chemical synthesis and
will likely find broad application in the study of proline-rich
proteins and glycoproteins. It is of note that proline-rich proteins
frequently participate in diverse signaling pathways and serve
many crucial biological functions.29,30 The unique conforma-
tional properties of proline inserts provide the molecular basis for
highly discriminatory recognition during the formation of multi-
protein signaling complexes. Understanding how these interac-
tions contribute to the stability of the complex should prove
valuable in the rational design of peptide mimics that could
potentially be used to disrupt the interfaces between proline-rich
proteins and their binding partners.31 The methodology devel-
oped herein is expected to facilitate the preparation of proline-
rich polypeptide probe structures, thus enhancing opportunities
for detailed analysis of proteinÀprotein interactions.
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(22) Thioester intermediate 5 could be observed by UPLC analysis
and was differentiated from the N-linked product 6a by the addition of
MESNa, where the disappearance of 5 would be observed.
(23) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
characterization data. This material is available free of charge via
’ AUTHOR INFORMATION
Corresponding Author
(31) Kuriyan, J.; Cowburn, D. Annu. Rev. Biophys. Biomol. Struct.
1997, 26, 259.
’ ACKNOWLEDGMENT
Support for this research was provided by the National Institute
of Health (CA28824 to S.J.D.). We thank Rebecca Lambert for
valuable discussions. We also thank Dr. George Sukenick, Hui
Fang, and Sylvi Rusli of SKI’s NMR core facility for mass spectral
and NMR assistance and Laura Wilson and Jason Chan for
assistance with the preparation of the manuscript.
’ REFERENCES
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