Traceless ligation of cysteine peptides using selective deselenization

Article abstract of DOI:10.1002/anie.201001900

Selective deselenization: Native chemical ligation at selenocysteine is followed by selective deselenization of the selenocysteine to alanine, in the presence of cysteine residues without protecting groups (see scheme). This selectivity is achieved by usi

Full text of DOI:10.1002/anie.201001900

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
keinan@scripps.edu
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
under http://dx.doi.org/10.1002/anie.201001900.
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Communications
slower than anticipated (17 h). We reasoned that the presence
of the aromatic thiol, MPAA (1 equiv), could interfere with
the deselenization reaction[27,28] by acting as a radical scav-
enger, thus competing with the deselenization of the Sec
residue.[27–29] To remove the aromatic thiol, compound 3a was
reduced with 50-fold excess DTT (dithiothreitol) to produce
the free selenol, which subsequently reoxidized to give the
dimeric diselenide peptide, 3b. This reaction sequence was
accomplished in one pot due to the high reactivity of the
selenol and low redox potential of diselenides.[19] Deseleniza-
tion of the diselenide dimer 3b with excess TCEP proceeded
rapidly, generating the Ala peptide 4 in less than 4 h, a time
comparable to previously reported deselenization reactions.
TCEP is a common reagent in protein chemistry that is
selective for disulfide reduction.[21–24] The chemoselectivity of
the deselenization conditions was further evaluated with
peptide 5a, H-Ala-Sec-Gly-Ser-Cys-Lys-Trp-Thr-Met-Ala-
NH2 which contains the potentially sensitive amino acids
Cys, Met and Trp. Peptide 5a oxidized under ambient
conditions to generate the cyclic selenylsulfide peptide 5b
(Scheme 2). Treatment of 5b with only TCEP (40-fold excess)
Figure 1. HPLC chromatogram for the deselenization reaction mixture
of peptide 5b, which was carried out with 9.4 equivalents DTT for 1 h,
and 2.2 equivalents TCEP for 2 h to give major product, 6, with traces
=
of the deselenized–desulfurized product, 7. Met( O) corresponds to
deselenized product with oxidized Met present in the starting material.
We propose that this deselenization of Sec to Ala occurs
through a radical-mediated mechanism (Scheme 3), similar to
the desulfurization of thiols by trialkyl phosphines (and
phosphites) under elevated temperatures or UV light pro-
posed first by Walling et al[27] and recently by Wan and
Danishefsky (in the presence of a radical initiator such as VA-
044).[11,32]
Scheme 3. Proposed deselenization mechanism of Sec-containing pep-
tides with TCEP in aqueous solution.
Scheme 2. Selective deselenization of peptide 5b. The deselenized
peptide 6 was the major product with only traces of side product, 7.
PB=NaH2PO4.
In principle, loss of selenium could occur through an
elimination mechanism to generate dehydroalanine.[33] To
rule out this possibility, we carried out two experiments. First,
the diselenide 1 was treated with either hydrogen peroxide[34]
or TCEP (Scheme 4). Under hydrogen peroxide the selenium
atom was converted to the corresponding selenoxide with
subsequent b-syn elimination to produce the modified
peptide 8 where the Sec has been converted to dehydroala-
nine (Scheme 4, bottom).[34] Peptide 8 was rapidly hydrolyzed
under the reaction conditions to the corresponding pyruvoyl
peptide 9a, which was observed together with its hydrate, 9b
(Figure S6B). In contrast, when diselenide dimer 1 was
reacted with TCEP under the deselenization conditions, the
corresponding Ala peptide, 10, was obtained without any
traces of either 9a or 9b (Scheme 4 top, Figure S6C).[33]
Second, we carried out the deselenization reaction of peptide
at pH 5.5 resulted in rapid deselenization of the Sec to yield
peptide 6. Surprisingly, analysis of the minor side product
identified peptide 7, H-Ala-Ala-Gly-Ser-Ala-Lys-Trp-Thr-
Met-Ala-NH2, consistent with a slow additional desulfuriza-
tion of the Cys residue.[30,31] However, when the reaction was
run (200 mm NaH2PO4, pH 5.1) with 9.4 equivalents DTT to
reduce the selenylsulfide, addition of just 2.2 equivalents
TCEP led exclusively to deselenization. Monoreduced dese-
lenized product 6, H-Ala-Ala-Gly-Ser-Cys-Lys-Trp-Thr-Met-
Ala-NH2, was obtained cleanly in 2 h with only traces of the
deselenized–desulfurized peptide
Figure 1 and Figure S4).
7 detected (Scheme 2,
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Scheme 5. Deselenization of peptide 5b in phosphate buffered D2O by
TCEP forms two products; the deselenized monodeuterated product,
[D]6 as the major product and the doubly deuterated deselenized–
desulfurized minor product, [D2]7.
together with H-Phe-Lys-(l)Ala-Ser-Asp-NH2 (FK(l)ASD.
amide, 12) and H-Phe-Lys-(d)Ala-Ser-Asp-NH2 (FK(d)
ASD.amide, 13, which by HPLC elutes at 8.8 min, 1 min earlier
than the l-enantiomer). Deselenization of 11 with TCEP was
complete within 2 h, producing only the l-enantiomer product
FK(l)ASD, which co-eluted with the synthetic peptide, and
has an identical mass. (Scheme 6, Figure S7 and S8).
Scheme 4. Reaction of dimer peptide 1 with H2O2 produced the
dehydroalanine analog, 8, which was rapidly hydrolyzed under the
reaction conditions to the corresponding pyruvoyl peptide 9a (in
equilibrium with its hydrate 9b). In contrast, reaction of peptide 1 with
TCEP gave only the deselenized product, 10, with an N-terminal Ala.
5b (Scheme 2) under the regular conditions using D2O
instead of H2O. The reaction mixture was worked up and
analyzed in H2O to reverse any deuterium incorporation at
acidic sites. The resulting deselenization product was found to
be consistent with [D]6, having a mass of M + 1 in comparison
to the product from H2O, 6 (Scheme 5, Figure S5). Further-
more, the chromatographically distinct minor side-product
(additional desulfurization) was found to be consistent with
[D2]7 having a mass of M + 2 in comparison with the above
described 7. Any mechanism involving dehydroalanine for-
mation would lead to an additional deuterium being incorpo-
rated at the a-carbon (Scheme 5, Figure S5).[31]
Scheme 6. The diselenide dimer of FKUSD, 11, was deselenized with
10 equivalents TCEP in 200 mm phosphate buffer, pH 5.1 to give only
FK(l)ASD product, 12, no traces of the of 13 were observed.
Epimerization is always a concern in the development of
new methods. While the desulfurization of Cys residues by
Raney Ni has been experimentally shown to be racemization-
free,[7,35] the radical-mediated mechanism could result in
epimerization.[11] The proposed deselenization mechanism
proceeds through a radical on the b-carbon, which is expected
to be the major intermediate.[11,32] However, radical migration
from b- to the a-carbon could lead to undesired epimerization
at the Sec residue. Furthermore, it has been experimentally
confirmed that thiyl radicals can abstract hydrogen from a-
carbons in peptides and proteins (as well as acidic protons in
lipids and DNA).[36,37] To test for epimerization, the penta-
peptide H-Phe-Lys-Sec-Ser-Asp-NH2 (FKUSD.amide, iso-
lated in the form of a diselenide dimer, 11) was synthesized
Taken together, these results support the formation of a
radical intermediate at the b-carbon of Sec, without migration
to the a-carbon, indicating a chemoselective process with
complete retention of stereoselectivity.
Finally, to evaluate selective deselenization of a complex
polypeptide following ligation, we prepared the 38-mer
peptide Grx3(1–38)(Cys11Sec–Cys14Sec–Ala38Cys) contain-
ing two Sec and one Cys residues by NCL (Scheme S1),
together with the all-Cys control, Grx3(1–38)(Ala38Cys).
Following removal of thiophenol by HPLC, deselenization of
Grx3(1–38)(Cys11Sec–Cys14Sec–Ala38Cys) by TCEP gave a
doubly
deselenized
product
Grx3(1–38)(Cys11Ala–
Cys14Ala–Ala38Cys) as the major product with a minor
Cys reduction side-product (Figure S9 to S12).[31] In contrast
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In conclusion, we have developed a straightforward
method for the selective reduction of selenocysteine to
alanine in the presence of unprotected cysteine. The desele-
nization reaction by TCEP is highly chemo- and enantiose-
lective. In effect, this strategy will allow ligation at both Ala
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Keywords: deselenization · desulfurization ·
native chemical ligation · peptides · protein modification
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