Direct on-resin synthesis of peptide-αthiophenylesters for use in native chemical ligation

Article abstract of DOI:10.1021/ol052811j

A peptide-^αthiophenylester is a key reactant in native chemical ligation. Preformation of the peptide-^αthiophenylester could be useful for enhancing the ligation reaction. We report the direct on-resin preparation of preformed peptid

Full text of DOI:10.1021/ol052811j

ORGANIC
LETTERS
2006
Vol. 8, No. 6
1049-1052
Direct On-Resin Synthesis of
Peptide- Thiophenylesters for Use in
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Native Chemical Ligation
Duhee Bang,†,‡ Brad L. Pentelute,†,‡ Zachary P. Gates,†,‡ and
Stephen B. Kent*,†,‡,§
Institute for Biophysical Dynamics, Department of Chemistry, and
Department of Biochemistry and Molecular Biology, The UniVersity of Chicago,
Chicago, Illinois 60637
skent@uchicago.edu
Received November 21, 2005
ABSTRACT
r
r
A peptide- thiophenylester is a key reactant in native chemical ligation. Preformation of the peptide- thiophenylester could be useful for
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enhancing the ligation reaction. We report the direct on-resin preparation of preformed peptide- thiophenylesters using a simple and efficient
method. The peptide- thiophenylester reacted extremely rapidly with a Cys-peptide when compared to the peptide- thioalkylester.
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r
In native chemical ligation,1 an unprotected peptide-Rthioester
is reacted with a second peptide containing an N-terminal
cysteine residue to give a near quantitative yield of a single
product linked by an amide bond (Scheme 1A). Native
chemical ligation has led to the practical chemical syntheses
of a wide variety of different proteins.2 Until now, peptide-
thioesters were routinely synthesized in the form of peptide-
SCH2CH2CO-Leu (i.e. alkyl) thioester,3 and exogenous
thiophenol was added to the native chemical ligation reaction
mixture to generate a more reactive peptide-Rthiophenylester
by transthioesterfication (Scheme 1A).4
Recently, we have successfully used preformed peptide-
Rthiophenylesters for convergent chemical protein syntheses
by kinetically controlled ligation.5 We correctly anticipated
that the preformed peptide-Rthiophenylester could be used
for rapid ligation by eliminating the thiophenol exchange
† Institute for Biophysical Dynamics.
‡ Department of Chemistry.
§ Department of Biochemistry and Molecular Biology.
(1) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science
1994, 266, 776-779.
(3) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 10068-10073.
(4) Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent, S. B. H. J.
Am. Chem. Soc. 1997, 119, 4325-4329.
(2) Dawson, P. E.; Kent, S. B. Annu. ReV. Biochem. 2000, 69, 923-
960.
10.1021/ol052811j CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/25/2006

Scheme 1. (A) Native Chemical Ligation with
Peptide-RThioAlkylEster through Thiol Exchange during the
Ligation Reaction and (B) Native Chemical Ligation with a
Preformed Peptide-Rthiophenylester
Scheme 2. Synthetic Strategy for the On-Resin Preparation of
a Peptide-RThiophenylester
step during the native chemical ligation reaction (Scheme
1B). The peptide-Rthiophenylesters were generated by ex-
change of a peptide-thioalkylester with a large excess of
thiophenol in aqueous buffer, and then purified by reverse-
phase HPLC. However, this method of generating the
peptide-Rthiophenylester was slow and sometimes incom-
plete. Consequently, we felt this exchange method would
limit the potential use of the peptide-Rthiophenylester for
chemical protein synthesis. Here we report a method to
directly prepare peptide-Rthiophenylesters.
A simple and efficient chemistry for the generation of a
preformed peptide-Rthiophenylester was developed. A resin
linker was designed for the synthesis of peptide-Rthio-
phenylesters with a wide range of C-terminal amino acids
(Scheme 2). For the synthesis of peptide-Rthiophenylesters,
we adapted Dawson’s peptide-Rthioalkylester synthesis method
using a Boc chemistry-solid-phase peptide synthesis (SPPS)
protocol.3 The S-tritylmercaptophenylacetic acid was pre-
pared by treating 4-mercaptophenylacetic acid with trityl
chloride. (see the Supporting Information). Starting with a
p-methylbenzhydrylamine (MBHA) resin, glycine was coupled
followed by S-tritylmercaptophenylacetic acid. After removal
of the trityl protecting group, the resulting mercaptophenyl-
acetyl glycine-resin was used for polypeptide chain assembly
by the use of Boc chemistry in situ neutralization SPPS
protocols6 (Scheme 2).
During model peptide syntheses, we found that two major
byproducts were formed. First, we had 10-20% of byproduct
from slow first amino acid coupling to the mercaptophen-
ylacetyl-glycine-resin, even with the use of 1 h coupling in
the in situ neutralization protocol. Second, we had 20-30%
byproduct from the formation of diketopiperazine that
resulted in a two amino acid deletion at the C-terminus.7
We effectively prevented these side reactions by the use of
a modified in situ neutralization protocol (see the Supporting
Information for the synthesis of peptide-Rthiophenylester).
Model ligation of a peptide-Rthiophenylester and a Cys-
peptide was performed under standard native chemical
ligation conditions (aqueous buffer, 2 mM peptide concentra-
tion, and 1% thiophenol), and the model ligation was
compared with the ligation of a standard peptide-Rthioalkyl-
ester under identical conditions. For comparison, we prepared
Phe-Leu-Leu-Rthiophenylester and Phe-Leu-Leu-Rthioalk-
ylester, and we used Cys-Phe-Arg-Ala-Asn-Gly as a Cys-
peptide.
(5) We have shown that, in the absence of added thiophenol, reaction of
a peptide1-thiophenylester with a Cys-peptide2-thioalkylester gives a single
product, the peptide1-Cys-peptide2-thioalkylester (Bang, D.; Pentelute, B.;
Kent, S. B., unpublished data). This kinetically controlled ligation principle
has so far been used in the convergent chemical synthesis of proteins
containng 46, 70, and 99 residues..
(6) Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B.
Int. J. Peptide Protein Res. 1992, 40, 180-193.
(7) Gisin, B. F.; Merrifield, R. B. J. Am. Chem. Soc. 1972, 94, 3102-
3106.
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Org. Lett., Vol. 8, No. 6, 2006

Figure 2. Crude peptide (Thz-Ala-Leu-Gln-Ile-Val-Ala-Arg-Leu-
Lys-Asn-Asn-Asn-Arg-Gln-Val-mercaptophenylacetic acid-Gly) was
analyzed by HPLC. The UV profile at 214 nm is shown. The
chromatographic separations were performed as describe in Figure
1. The observed mass of the highest peak (t ) 9.7 min) was 2059.4
( 0.2 Da; the calculated mass of the peptide molecular structure
with use of average isotopes was 2059.3 Da.
The model ligation reactions between 2.2 mM concentra-
tions of peptide-Rthioesters and 2 mM of Cys-peptide were
carried out in pH 6.8 aqueous buffer (containing 200 mM
sodium phosphate, 6 M guanidine hydrochloride, and 20 mM
Tris(2-carboxyethyl)phosphine hydrochloride) in the presence
of 1% thiophenol for 9 h (Figure 1). We checked the pH
after 9 h and found that the pH dropped to 6.3 for both
reaction mixtures. Model ligation rates were monitored by
HPLC analysis. The analysis showed that the preformed
peptide-thiophenylester reacted significantly faster (>10×
initial rate under these conditions) than peptide-Rthioalk-
ylester with a Cys-peptide.
The generality8 of direct on-resin syntheis of peptide-
thiophenylesters was tested by using another peptide se-
quence (Thz-Ala-Leu-Gln-Ile-Val-Ala-Arg-Leu-Lys-Asn-
Figure 1. Model native chemical ligation of peptide-Rthiophen-
ylester (Phe-Leu-Leu-mercaptophenylacetic acid-Gly, 9) or peptide-
Rthioalkylester (Phe-Leu-Leu-mercaptopropionic acid-Gly, 2) and
CFRANG. (A) Observed FLL-CFRANG model-peptide formation
monitored by the use of HPLC-MS analysis. At each time point
during the ligation reactions (1/3, 1, 3, 9 h), an aliquot (20 µL)
from the ligation reaction was quenched by the addition of 5%
aqueous trifluoroacetic acid (8 µL). The aliquot was characterized
by analytical HPLC. Analysis of the ligated species was done by
integrating the areas from analytical HPLC profiles at each time
point {area of product peptide/(area of product peptide + area of
Cys-peptide)}. (B) Ligation reaction (1 h) of Phe-Leu-Leu-
thiophenylester and Cys-Phe-Arg-Ala-Asn-Gly was monitored by
analytical HPLC. (C) Ligation reaction (1 h) of Phe-Leu-Leu-
thioalkylester and Cys-Phe-Arg-Ala-Asn-Gly was monitored by
analytical HPLC. The UV profile at 214 nm is shown. The
chromatographic separations were performed on a narrow-bore
analytical HPLC C4 column, using a linear gradient (1-61%) of
buffer B in buffer A over 15 min (buffer A ) 0.1% trifluoroacetic
acid (TFA) in water; buffer B ) 0.08% TFA in acetonitrile) with
a flow rate of 0.5 mL/min. # ) residual TFA peak from acid
quenching; & ) thiophenol peak; % ) Phe-Leu-Leu-mercapto-
phenylacetic acid-Gly; * ) peptide-thiophenylester from thiol
exchange with thiophenol.
Figure 3. Native chemical ligation of peptide-Val-Rthiophenylester
and Cys-peptide: (A) at reaction time ) 0 h, Thz-Ala-Leu-Gln-
Ile-Val-Ala-Arg-Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-mercaptophe-
nylacetic acid-Gly (t ) 10.5 min; obsd 2059.2 ( 0.2 Da, calcd
2059.3 Da) and Cys-Ile-Asp-Pro-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-
Leu-Glu-Lys-Ala-Leu-Asn (t ) 12.8 min; obsd 2004.4 ( 0.2 Da,
calcd 2004.6 Da); (B) at reaction time ) 17 h, ligation was
essentially done and the product (Thz-Ala-Leu-Gln-Ile-Val-Ala-
Arg-Leu-Lys-Asn-Asn-Asn-Arg-Gln-Val-Cys-Ile-Asp-Pro-Lys-Leu-
Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn) is eluted at
13.5 min (obsd 4038.8 ( 0.3 Da, calcd 4038.6 Da) The UV profile
at 214 nm is shown. The chromatographic separations were
performed as described in Figure 1.
Org. Lett., Vol. 8, No. 6, 2006
1051

successfully obtained after HF cleavage, and analyzed by
analytical HPLC (Figure 2).
We ligated the peptide-Val-Rthiophenylester with a Cys-
peptide (Figure 3). The ligation was essentially done in 17
h. In contrast, peptide-Val-Rthioalkylester is known to ligate
very slowly (∼60% completion after 48 h) with a Cys-
peptide (as shown by Hackeng et al.).3
Occurrence of racemization in the synthesis and use of a
peptide-Rthiophenylester in native chemical ligation with a
Cys-peptide was evaluated as follows. A model pentapeptide
Ala-Leu-Phe-Ala-D-Phe-Rthiophenylester was prepared by the
modified in situ neutralization SPPS protocols. This peptide
was reacted with Cys-Gly-Pro-Ala-Ser, and the ligation
reaction was analyzed by reverse-phase HPLC under analyti-
cal conditions that separated the diastereomeric reaction
products that contained either -L-Phe5- or -D-Phe5- (see
Supporting Information). The results are shown in Figure 4.
Formation of the diastereomeric -L-Phe5- reaction byprod-
uct was barely detectable (<2%), in agreement with the low
racemization levels reported for the use of peptide-
Rthiophenylesters in native chemical ligation.3
In conclusion, we have developed a novel method for the
direct on-resin preparation of preformed peptide-Rthio-
phenylesters. The resulting peptide-Rthiophenylesters reacted
faster when compared to the widely used peptide-Rthio-
alkylester under native chemical ligation conditions. This
stragtegy may be useful in kinetically controlled convergent
ligation5 and for the reaction of slowly ligating peptide-Xxx-
thioesters (Xxx ) Ile, Val, Thr) with Cys-peptide.9
Figure 4. Evaluation of racemization in the synthesis and use of
peptide-Rthiophenylesters. (A) Analytical HPLC separation of the
diastereomeric peptides Ala-Leu-Phe-Ala-L-Phe-Cys-Gly-Pro-Ala-
Ser (t ) 13.2 min) and Ala-Leu-Phe-Ala-D-Phe-Cys-Gly-Pro-Ala-
Ser (t ) 14.5 min). (B) Analysis after 30 min of the native chemical
ligation reaction of Ala-Leu-Phe-Ala-D-Phe-Rthiophenylester with
Cys-Gly-Pro-Ala-Ser at pH 6.8, 2 mM for each peptide. The arrow
indicates the low level of L-Phe5 byproduct formed during the
reaction. Analytical HPLC conditions are described in the
Supporting Information.
Acknowledgment. We thank Vladimir Torbeev for his
reading and commenting on the manuscript. This research
was supported by the Department of Energy Genomes to
Life Genomics Program (Grant DE-FG02-04ER63786) and
by the National Science Foundation Materials Research
Science and Engineering Centers Program at the University
of Chicago (Grant DMR-0213745). Z.G. was supported by
the University of Chicago NIH Training Program in Physical
and Chemical Biology.
Asn-Asn-Arg-Gln-Val-mercaptophenylacetic acid-Gly) from
SDF1-R (Thz ) 1,3-thiazolidine-4-carboxylic acid). This
sequence was chosen to demonstrate the ease of preparation
of a peptide-Val-Rthiophenylester, because â-branching
amino aicds (Val, Thr, and Ile) as a C-terminal residue are
exceptionally difficult to exchange by transthioesterification
in aqueous solution. A crude peptide-Rthiophenylester was
Supporting Information Available: Experimental pro-
cedures for synthesis and characterization of S-trityl mer-
captophenyl acetic acid, for peptide synthesis, and for
evaluation of racemization. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL052811J
(8) We have used the modified in situ neutralization protocol to prepare
peptide-Rthiophenylesters ranging from 3 to 31 residues. We observed
unidentified byproduct peaks apart from our desired peaks. The peptides
were easily purified and used in chemical protein synthesis. These data
will be published elsewhere.
(9) Previously, because of their extremely slow reaction,3 beta branched
amino acid ligation sites were largely avoided or mutated to other amino
acid residues for the design of chemical protein synthesis.
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Org. Lett., Vol. 8, No. 6, 2006