Direct conversion of resin-bound peptides to C-terminal esters

Article abstract of DOI:10.1021/ol100471k

A mild and effective method was developed to convert peptides immobilized on the 2-chlorotrityl and Wang resins directly to C-terminal esters. After conventional Fmoc peptide synthesis, treatment with anhydrous HCl (0.2-3 M) in a variety of alcohols was shown to produce the corresponding peptide esters in good yield and purity. Under the mildest conditions investigated, acid-sensitive protection groups such as N-Boc, trityl, tert-butyl ether, tert-butyl ester, and Pbf remain intact.

Full text of DOI:10.1021/ol100471k

ORGANIC
LETTERS
2010
Vol. 12, No. 8
1852-1855
Direct Conversion of Resin-Bound
Peptides to C-Terminal Esters
Rushia A. Turner, Robert J. Weber, and R. Scott Lokey*
Department of Chemistry and Biochemistry, UniVersity of California at Santa Cruz,
1156 High Street, Santa Cruz, California 95064
lokey@chemistry.ucsc.edu
Received February 25, 2010
ABSTRACT
A mild and effective method was developed to convert peptides immobilized on the 2-chlorotrityl and Wang resins directly to C-terminal
esters. After conventional Fmoc peptide synthesis, treatment with anhydrous HCl (0.2-3 M) in a variety of alcohols was shown to produce
the corresponding peptide esters in good yield and purity. Under the mildest conditions investigated, acid-sensitive protection groups such
as N-Boc, trityl, tert-butyl ether, tert-butyl ester, and Pbf remain intact.
The development of peptides as therapeutic agents is on the
rise.1 Despite their suboptimal pharmacokinetic properties,
peptides demonstrate unparalleled potency and specificity
against a wide array of biological targets.2-6 The increasing
importance of peptides in drug discovery has produced a need
for high-throughput techniques that introduce the chemical
modifications known to improve the pharmacological proper-
ties of small molecule drugs.
One approach to improving membrane permeability of
drugs containing pharmacologically important carboxylic
acids is to mask the hydrophilic moiety as a alkyl ester.7-9
Upon membrane permeation, the ester prodrug is hydrolyzed
by intracellular esterases revealing the active carboxylic
acid.10-12 Applying this approach to the synthetic plan for
a peptide library may serve to improve the bioassay hit ratio.
However, a survey of the literature produced no viable
options for methods with which to convert resin bound
peptides directly into C-terminal methyl esters.
The few reported transesterifying cleavage methods gener-
ally require long reaction times and/or harsh conditions. For
example, one of the earliest methods used methanol or
ethanol under strongly basic conditions and required reaction
times of 2-24 h.13-17 Alternately, potassium cyanide was
used to promote transesterification with benzyl alcohol from
(8) Zablocki, J. A.; Tjoeng, F. S.; Bovy, P. R.; Miyano, M.; Garland,
R. B.; Williams, K.; Schretzman, L.; Zupec, M. E.; Rico, J. G.; Lindmark,
R. J.; Toth, M. V.; Mcmackins, D. E.; Adams, S. P.; Panzerknodle, S. G.;
Nicholson, N. S.; Taite, B. B.; Salyers, A. K.; King, L. W.; Campion, J. G.;
(1) McCoy, M. Chem. Eng. News 2005, 83, 19.
(2) Dancer, R. J.; Jones, A.; Fairlie, D. P. Aust. J. Chem. 1995, 48, 1835–
Feigen, L. P. Bioorg. Med. Chem. 1995, 3, 539–551
(9) Doh, H. J.; Cho, W. J.; Yong, C. S.; Choi, H. G.; Kim, J. S.; Lee,
C. H.; Kim, D. D. J. Pharm. Sci. 2003, 92, 1008–1017
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(3) Maisel, A. S. CardioVasc. Toxicol. 2003, 3, 37–42
(4) Bowersox, S. S.; Luther, R. Toxicon 1998, 36, 1651–1658
(5) Ahrendt, K. A.; Olsen, J. A.; Wakao, M.; Trias, J.; Ellman, J. A.
Bioorg. Med. Chem. Lett. 2003, 13, 1683–1686
(6) Schneider, S. E.; Bray, B. L.; Mader, C. J.; Friedrich, P. E.; Anderson,
M. W.; Taylor, T. S.; Boshernitzan, N.; Niemi, T. E.; Fulcher, B. C.; Whight,
S. R.; White, J. M.; Greene, R. J.; Stoltenberg, L. E.; Lichty, M. J. Pept.
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J. Am. Chem. Soc. 2002, 124, 12666–12667
(12) Clement, B.; Lopian, K. Drug Met. Disp. 2003, 31, 645–651
(13) Seebach, D.; Thaler, A.; Blaser, D.; Ko, S. Y. HelV. Chim. Acta
1991, 74, 1102–1118
(14) Ueki, M. I., S.; Handa, T. Bull. Chem. Soc. Jpn. 1981, 54, 3865–
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2003, 4, 461–485
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10.1021/ol100471k  2010 American Chemical Society
Published on Web 03/23/2010

Scheme 1. Fmoc SPPS of the N-Acyl-Leu-Leu Dipeptide Used
in the Optimization of Cleavage/ Esterification Procedure
Figure 1. Peptides used to investigate the stability of acid-sensitive
protecting groups during cleavage/esterification.
added. The cleavage and concomitant conversion to methyl
ester was complete within 2-5 h and gave yields of 95% or
greater (Table 1). The efficacy of the method with other
alcohols was investigated and showed good to excellent
results with both primary and secondary alcohols, such as
ethanol, 2-propanol, n-butanol, cyclopentanol, and benzyl
alcohol. In general, conversion to ester was greater than 95%,
with yields ranging from 73-99%. The workup of these
peptide esters entailed evaporation of the cleavage solution,
followed by trituration of the peptide esters with hexanes
and water.
the Merrifield resin at elevated temperatures.18 More recent
efforts to facilitate C-terminal modifications, in general, have
involved the use of specialized linkers and SPPS in the N to
C direction.19-21 To date, however, there have been no
reports of a transesterifying cleavage protocol that yields pure
and selectively protected peptides under mild conditions.
Methyl esters have been formed in solution by treating a
carboxylic acid with anhydrous methanolic HCl prepared
through the reaction between methanol and acetyl chlo-
ride.22,23 The procedure is usually high yielding, and
purification of ester products can be as simple as evaporating
the methanolic HCl solution. These attributes led us to
investigate this reagent in the one-pot cleavage and esteri-
fication of dipeptide model compounds 1 and 2 from the
2-chlorotrityl and Wang linkers, respectively (Scheme 1).
A methanolic HCl solution was prepared by the slow
addition of acetyl chloride to cold methanol. This solution
was stirred for 2 h before being added to aliquots of 1 and
2 that were pre-swelled with minimal methylene chloride.
Generally, 20-100 mg of resin was swelled in 0.5-1 mL
of methylene chloride, and 3-4 mL of methanolic HCl was
Typically, peptides are cleaved from 2-chlorotrityl and
Wang-type linkers by treatment with TFA, frequently result-
ing in the removal of some or all acid-sensitive side chain
protection groups. If a postcleavage side chain modification
is required, such as the installation of a fluorophore or a
saccharide, C-terminal esterification prior to side chain
deprotection may be necessary. Transesterification reactions
using peptides 3-5 synthesized on the 2-chlorotrityl linker
(Figure 1) were performed with HCl concentrations ranging
from 0.2-3 M, in order to determine the lowest concentration
of HCl that would afford both complete C-terminal esteri-
fication and preservation of side-chain protection groups such
as N-Boc, tert-butyl ester, tert-butyl ether, trityl, and pbf.
Using a 0.2 M methanolic HCl solution, peptides 3-5
showed good to excellent conversion to C-terminal methyl
ester within 3 h. The reaction resulted in good yields of
73-98% with only minor loss of the tyrosine tert-butyl ether
and the histidine trityl group (Table 2, entries 4-6). The
same peptides synthesized on the Wang resin transesterified
very slowly with a 0.2 M HCl solution, resulting in low
yields of protected peptides with a 2 h reaction time. A longer
reaction time improved the yield of the peptide ester but also
resulted in the loss of side-chain protection groups.
(15) Reddy, G. L.; Bikshapathy, E.; Nagaraj, R. Tetrahedron Lett. 1985,
26, 4257–4260.
(16) Pereira, W.; Close, V. A.; Jellum, E.; Patton, W.; Halpern, B. Aust.
J. Chem. 1969, 22, 1337–1340.
(17) Halpern, B.; Chew, L.; Close, V.; Patton, W. Tetrahedron Lett.
1968, 5163–5164.
(18) Moore, G. J.; Kwok, Y. C Can. J. Biochem 1980, 58, 641–643.
(19) Peters, C.; Waldmann, H. J. Org. Chem. 2003, 68, 6053–6055
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(20) Alsina, J.; Yokum, T. S.; Albericio, F.; Barany, G. J. Org. Chem.
1999, 64, 8761–8769
.
(21) Thieriet, N.; Guibe, F.; Albericio, F. Org. Lett. 2000, 2, 1815–
Higher concentrations of HCl (3 M) enabled removal of
the particularly acid-sensitive side chain protection groups
such as boc, tert-butyl ether, and the trityl groups from
cysteine and asparagine. The histidine trityl and arginine Pbf
1817
.
(22) Nudelman, A.; Bechor, Y.; Falb, E.; Fischer, B.; Wexler, B. A.;
Nudelman, A. Synth. Commun. 1998, 28, 471–474
.
(23) Ross, J. A.; Ross, B. P.; Rubinsztein-Dunlop, H.; McGeary, R. P.
Synth. Commun. 2006, 36, 1745–1750
Org. Lett., Vol. 12, No. 8, 2010
.
1853

Table 1. Results from Investigation into Scope of Cleavage/Esterification Procedure
peptide + linker
linker
R
HCl (M)
time (h)
% convna
% yieldb
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
Wang
Wang
Wang
Wang
Wang
methyl
ethyl
isopropyl
n-butyl
cyclopentyl
benzyl
methyl
ethyl
isopropyl
n-butyl
cyclopentyl
benzyl
3
3
3
3
3
3
3
3
3
3
3
3
2
2
3
2
3
2
5
5
5
5
5
5
98
99
99
98
99
99
99
97
95
98
98%
99%
97
99
80
99
96
99
98
91
78
94
73%
89%
Wang
a Percent conversion was determined by integration of peaks in the UV trace (λ ) 198 nm). b Yields of peptides purified by trituration with hexanes and
water.
Table 2. Investigation into the Stability of Acid-Sensitive Side-Chain Protection Groups in the Cleavage/ Esterification Reaction with
Methanol·HCl and t-Butyl Alcohol·HCl (3 h)
entry
peptide
sequence
linker
ROH·HCl (M)
% PG lossa
% convnb
% yieldc
1
2
3
4
5
3
4
5
3
4
5
3
4
5
3
4
5
Ac-C(Trt)-Y(t-Bu)-K(Boc)-L
Ac-R(Pbf)-H(Trt)-E(t-Bu)-L
Ac-W(Boc)-S(t-Bu)-N(Trt)-L
Ac-C(Trt)-Y(t-Bu)-K(Boc)-L
Ac-R(Pbf)-H(Trt)-E(tBu)-L
Ac-W(Boc)-S(t-Bu)-N(Trt)-L
Ac-C(Trt)-Y(t-Bu)-K(Boc)-L
Ac-R(Pbf)-H(Trt)-E(t-Bu)-L
Ac-W(Boc)-S(t-Bu)-N(Trt)-L
Ac-C(Trt)-Y(t-Bu)-K(Boc)-L
Ac-R(Pbf)-H(Trt)-E(t-Bu)-L
Ac-W(Boc)-S(t-Bu)-N(Trt)-L
Wang
Wang
Wang
methanol (0.2 M)
methanol (0.2 M)
methanol (0.2 M)
methanol (0.2 M)
methanol (0.2 M)
methanol (0.2 M)
methanol (0.2 M)
methanol (0.2 M)
10d
0
>99
>99
>99
82
77
98
62
50
77
6
8
5
0
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
2-chlorotrityl
8d
4e
0
74
73
98
53
31
74
74
92
80
6
7f
8f
9f
10
11
12
14d
35e
2g
0
methanol (0.2 M)
tert-butyl alcohol (0.2 M)
tert-butyl alcohol (0.2 M)
tert-butyl alcohol (0.2 M)
n/a
n/a
n/a
0
0
a Determined by integration of peaks in UV traces (λ ) 198 nm). b Percent conversion to ester was determined by integration of peaks in the UV trace
(λ ) 198 nm). c Yields of fully protected methyl esters were determined by integration of peaks in the UV trace (λ ) 198 nm) and comparison of product
ratios to the mass of peptide obtained. d Loss of t-Bu ether from tyrosine. e Loss of trityl group from histidine. f Two-step cleavage and esterification procedure
(see the Supporting Information for experimental details). g Loss of boc from tryptophan.
groups were considerably more robust and required longer
reaction times of 6-12 h for removal. High concentrations
of HCl promoted the transesterification of a tert-butyl ester,
though this problem can be circumvented, in principle, by
deprotecting side chain esters after the cleavage and C-
terminal esterification reaction is performed at a lower
concentration of HCl.
We found that when peptides 3-5 were cleaved from the
2-chlorotrityl resin with AcCl in tert-butyl alcohol (0.2 M),
peptides were obtained as the carboxylic acid in good yield
with all protecting groups intact (Table 2). The efficient
cleavage and absence of esterification under these conditions
may be due to the formation of acetic acid in tert-butyl
alcohol by way of elimination. Similarly mild conditions are
known that effect cleavage from the 2-chlorotrityl resin with
preservation of protecting groups (e.g., 1% TFA/DCM).
There are some noteworthy advantages to the one-pot
transesterification procedure described above. Synthesis of
peptide libraries requires reactions that are high yielding and
promote diversity among library members. The procedure
described here is effective with both primary and secondary
alcohols and is sufficiently mild as to enable the synthesis
of a variety of alkyl esters in a 96- or 384 well-plate format
utilizing robotic liquid handling. We also found that the one-
pot procedure was superior to a two-pot procedure with
respect to both efficiency and the ability to obtain consistently
high yields of protected peptide esters. A two-pot procedure
entailing cleavage of the peptide from the 2-chlorotrityl resin
1854
Org. Lett., Vol. 12, No. 8, 2010

with 1% TFA and subsequent esterification with 0.2 M
HCl·methanol (Table 2, Supporting Information) was a
considerably longer process and made the management of
side-chain protection groups a challenge. In addition, lower
concentrations of HCl resulted in incomplete conversion to
peptide ester, requiring longer reaction times that led to loss
of side-chain protection groups.
We investigated C-terminal racemization in the methanolic
HCl cleavage/esterification method by reacting both C-
terminal epimers of the AcN-Leu-Leu dipeptide 1 with 3 M
methanolic HCl. Optimized LCMS conditions enabled
separation of a mixture of the two epimers on a C18 column.
We followed the reactions of the individual stereoisomers
for 6 h and saw no evidence of C-terminal racemization (see
Figure 28 in the Supporting Information).
techniques reported previously. It employs commonly used
SPPS resins, involves shorter reaction times and milder
reaction conditions, and produces higher yields. This method
is adaptable and enables the synthesis of peptide esters from
a number of primary and secondary alcohols. Furthermore,
the concentration of HCl in the cleavage solution can be
optimized to selectively preserve or remove a number of acid-
sensitive side-chain protection groups. This method offers a
promising tool for the high-throughput synthesis of C-
terminal peptide ester libraries.
Supporting Information Available: Detailed synthetic
1
procedures; method for calculating product ratios; H and
13C NMR spectra and LCMS traces for compounds 1a-f
and 2a-f. This material is available free of charge via the
Internet at http://pubs.acs.org.
The transesterifying SPPS cleavage method described
above represents a significant improvement over other similar
OL100471K
Org. Lett., Vol. 12, No. 8, 2010
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