Angewandte
Chemie
DOI: 10.1002/anie.201001900
Peptide Ligation
Traceless Ligation of Cysteine Peptides Using Selective
Deselenization**
Norman Metanis, Ehud Keinan,* and Philip E. Dawson*
The synthesis of proteins with a fully native sequence is an
ongoing challenge in protein chemistry. Native chemical
ligation (NCL) approaches have proven to be generally
applicable where cysteine (Cys) residues are appropriately
positioned,[1,2] however, the synthesis of many proteins often
require ligation at non-Cys sites in the polypeptide
sequence.[3–7] Previously, we introduced a reductive strategy
for ligation at Ala sites[7] based on global desulfurization of
Cys[8] that has found widespread utility for the synthesis of
complex proteins by NCL.[9,10] Selective desulfurization can
[7]
be affected by both Rainey Ni and Pd/C/H2 and, more
recently, by the radical initiator VA-044 in combination with
the water soluble phosphine TCEP (tris(2-carboxyethyl)-
phosphine).[11] However, since these conditions result in
global desulfurization of all thiols in the protein, the
method requires protection and deprotection of all other
Cys residues in the native sequence.[6a,11,12] These additional
steps complicate the synthesis of larger polypeptides[13] and
limit the use of natural Cys residues for ligation.
Scheme 1. Native chemical ligation of N-terminal Sec-peptide 1 with C-
terminal thioester-peptide 2 gives the ligated product, 3a. Following
purification, 3a was deselenized to the alanyl peptide 4, using excess
TCEP. R=3-mercaptopropionyl-Leu. MPAA=4-mercaptophenylacetic
acid; DTT=dithiothreitol; TCEP=tris(2-carboxyethyl)phosphine.
Selenocysteine (Sec, U) has been shown to expand the
NCL method to Xaa-Sec site, allowing the synthesis of
selenoproteins.[14–16] Additionally, the resulting selenopepti-
des can be deselenized under similar conditions to that used
for Cys containing peptides to yield the corresponding Ala
peptide sequences.[11,17] We reasoned that the high propensity
of selenols to form radicals[18] could be harnessed for selective
reduction of selenols in the presence of thiols (Scheme 1),
thus avoiding the need for protection/deprotection steps. This
approach was inspired by our observation that synthetic
analogs of glutaredoxin 3 (Grx3) containing Sec were incom-
patible with reduction by the water soluble reducing agent
TCEP,[19] leading to the generation of significant levels of a
deselenized side products. By contrast, the wt-Grx3 was found
to be stable to TCEP. Indeed, the sensitivity of Sec in peptides
and proteins to reduction by TCEP has been previously noted
in the development of selenocysteine ligation methods and in
the context of selenoproteins.[15,16a,20] Importantly, TCEP[21] is
[*] Dr. N. Metanis, Prof. E. Keinan
Schulich Faculty of Chemistry
Technion—Israel Institute of Technology
Technion City, Haifa 32000 (Israel)
and
widely used to reduce disulfides to thiols in peptides and
[22–24]
À
Department of Molecular Biology
The Scripps Research Institute
10550 N. Torrey Pines Rd, La Jolla, CA 92037 (USA)
Fax: (+972)4-829-3913
E-mail: keinan@technion.ac.il
proteins without reduction of the C S bond.
Accordingly, we ligated the N-terminal Sec-peptide 1
(UGLEFRSI-amide, isolated in the form of a diselenide
dimer) to the thioester peptide
2 (Ac-LYRAG-SR)
(Scheme 1 and Figure S1 in the Supporting Information), to
produce 3a.[14–16] The ligation conditions of 6m GdmCl,
200 mm Na2HPO4, pH 7.5, saturated with 200 mm 4-mercap-
tophenylacetic acid (MPAA)[25] gave the best results. The
aromatic thiol acted as both a catalyst to activate the alkyl
thioester and as a mild reducing agent to generate a small
pool of free selenol to facilitate the ligation reaction. In
addition, under these conditions the product was spontane-
ously converted to selenylsulfide 3a, with MPAA, which was
beneficial for the HPLC purification.[26]
Prof. P. E. Dawson
Departments of Cell Biology and Chemistry
The Scripps Research Institute
10550 N. Torrey Pines Rd, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-7319
E-mail: dawson@scripps.edu
[**] This study was supported by the Israel–US Binational Science
Foundation, the German–Israeli Project Cooperation (DIP) (E.K.),
NIH GM059380 (PED), the Israeli Higher Education Planning and
Budgeting Committee and Israel Ministry of Science (N.M.), and
the Skaggs Institute for Chemical Biology. E.K. is the incumbent of
the Benno Gitter & Ilana Ben-Ami Chair of Biotechnology, Technion.
The purified selenylsulfide peptide 3a was treated with
50-fold excess TCEP at pH 5.5 to produce the deselenized
alanyl-peptide 4 (Scheme 1, Figure S2 and S3). Under these
conditions, the reaction proceeded to completion yet was
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2010, 49, 7049 –7053
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7049