Peptide αthioester formation using standard Fmoc-chemistry

Article abstract of DOI:10.1016/S0040-4039(03)00582-3

A highly efficient and simple Fmoc-based preparation of peptide ^αthioesters is presented. After Fmoc/t-butyl solid-phase synthesis on 2-chlorotrityl resin the C-terminal carboxylic group of the protected peptide is directly converted to the cor

Full text of DOI:10.1016/S0040-4039(03)00582-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 3551–3554
a
Peptide thioester formation using standard Fmoc-chemistry
Regula von Eggelkraut-Gottanka,a Annerose Klose,b Annette G. Beck-Sickingera,* and
Michael Beyermannb,*
aInstitute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany
bInstitute of Molecular Pharmacology, Peptide Chemistry Group, 13125 Berlin, Germany
Received 11 December 2002; revised 28 February 2003; accepted 1 March 2003
Abstract—A highly efficient and simple Fmoc-based preparation of peptide athioesters is presented. After Fmoc/t-butyl
solid-phase synthesis on 2-chlorotrityl resin the C-terminal carboxylic group of the protected peptide is directly converted to the
corresponding thioester. The method leads to very high yields, shows a low level of epimerization and can be easily applied also
a
for the preparation of long peptide thioesters as demonstrated for the 41 amino acid N-terminal fragment of pro-neuropeptide
Y (proNPY 1–40). © 2003 Elsevier Science Ltd. All rights reserved.
C-terminal peptide thioesters can be used in many
approaches1 as starting compounds for the chemical
synthesis of proteins particularly since the development
of the native chemical ligation method (NCL, Fig. 1).2,3
More than 300 biologically active proteins from numer-
ous different families have been successfully synthesized
by NCL.4 Expanding the applicability of the chemical
ligation via thioesters and N-terminally cysteine-bearing
peptides requires effective preparation methods for its
key intermediates: peptide thioesters. Whereas the
intein technology5,6 enables the molecular biological
access to protein thioesters, peptide thioesters (550
amino acids) can be better obtained by chemical syn-
thesis. This further provides the advantages of easily
achievable substitutions at any position of the peptide
sequence (e.g. by non canonical amino acids) or site-
specific modifications of the peptide molecule (e.g. by
fluorescent labels or biophysical probes). However, syn-
thetic difficulties still restrict the chemical preparation
of peptide athioesters. Up to now most peptide
thioesters are prepared by the so called Boc-strategy of
solid-phase synthesis (Boc=t-butyloxycarbonyl). Dis-
advantages include strong acid treatment and haz-
ardous operation conditions, which lead frequently to
undesired side-reactions.7–9 In contrast, Fmoc-strategy
thioester linkages to strong nucleophiles such as pipe-
ridine that is used for the removal of Fmoc groups.
Several strategies for solving this problem have been
reported. Specific cocktails for the removal of Fmoc
groups keeping the thioester intact have been
applied.10–12,8 Within the backbone amide linker (BAL)
strategy, an amino acid thioester is coupled to a fully
protected, C-terminally free peptide anchored to a solid
support through its backbone nitrogen. The final cleav-
age from the resin releases the peptide thioester.13 More
general seems to be a modification of Kenner’s sulfon-
amide ‘safety catch’ linker. After synthesis the sulfon-
amide linker is activated by alkylation followed by
cleavage of the peptide from the resin with a thiol
nucleophile.14 Without the need of a special linker a
method using alkylaluminum thiolate for the cleavage
of peptides from 4-hydroxymethylbenzoic acid
(HMBA)
and
4-hydroxymethyl-phenylacetamido-
methyl polystyrene (Pam) resins yields peptide
thioesters.15,16 All these methods either require special
agents/linkers or are reported to cause undesirable side-
reactions. A fast, efficient and simple method for the
preparation of peptide thioesters by Fmoc-based solid-
phase chemistry remained to be established.
(Fmoc=9-fluorenylmethyloxycarbonyl)
milder conditions and can be automated in parallel
synthesis, yet it is limited by the susceptibility of
implicates
Such an attractive route results from the Fmoc-based
synthesis of protected peptide acids on the commer-
cially available Cl-trityl-resin followed by a direct con-
version of the C-terminal carboxylic group to the
corresponding thioesters. The peptide thioester is suffi-
ciently stable towards acidic conditions, which allows
the deprotection in TFA and even the following chro-
matographic purification in acetonitrile/water systems.
* Corresponding authors. Tel.: +49 30 94793272, fax: +49 30
94793159 (M.B.); Tel.: +49 341 9736901; fax: +49 341 9736909
(A.G.B.-S.); e-mail: beyermann@fmp-berlin.de; beck-sickinger@
uni-leipzig.de
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00582-3

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R. 6on Eggelkraut-Gottanka et al. / Tetrahedron Letters 44 (2003) 3551–3554
MALDI-MS. As indicated in Table 1, activation by
phosphonium salts and carbodiimide, with or without
N-hydroxybenzotriazol (HOBt), resulted in a fast
thioester formation (more than 50% after 5 min)
whereas the highest rate was achieved by PyBOP-acti-
vation. After 16 h (over night) all reactions have been
run to completion with PyBOP or DIPCDI/HOBt,
respectively. In contrast, TBTU could hardly activate
Z-Gly-Ala-OH, a fact which can be attributed to a fast
reaction between TBTU and the thiol prior to the
activation of Z-Gly-Ala-OH (data not shown).
To optimize the reaction, different thiols were investi-
gated. Significant differences in the thioester formation
rate were observed in the rank order p-acetamidothio-
phenol>thiophenol>3-mercapto-propionic acid ethyl
ester (Table 2). Moreover, p-acetamidothiophenol is a
solid, shows convenient handling properties and has a
low toxicity profile which makes it to our favored
choice.
An effective thioester method comprises a low degree of
stereomutation during its formation and the ligation
step beside a high formation rate. We, therefore, carried
out epimerization studies for Z-Gly-Ala-thioester
obtained by activation with PyBOP or DIPCDI/HOBt,
respectively, in combination with p-acetamidothiophe-
nol. The resulting Z-Gly-Ala-S-acetamidophenyl esters
(0.005 mmol) were ligated to H-Cys-OEt×HCl (0.005
Figure 1. (A–D) aThioester formation of proNPY 1–40: (A)
Automated SPPS on Cl-Trt resin using Fmoc/tBu strategy;
(B) fully protected cleavage from resin; (C) reaction with
thiol; (D) deprotection of the peptide thioester; (E) NCL
yields the full-length, carboxyfluorescein (CF) labeled
proNPY analogue ([C41, K68(CF)]proNPY 1–69).
mmol) in
1
mL of 0.1
M
tris(hydroxy-
methyl)aminomethane (TRIS) buffer (pH 7.7) contain-
ing 3 M urea and i-mercaptoethanol (2 vol%). After
reaction for 30 min an aliquot of the reaction mixture
was applied to analytical RP-HPLC. (tR(LL): 19.7 min,
tR(DL): 20.3 min). According to the HPLC data, we
observed 53.5% stereomutation for PyBOP-activation
and 51% for DIPCDI/HOBt-activation. The Z-Gly-
Ala-OH used for the studies was optically pure
(>99.9%) as checked by a control experiment, coupling
Z-Gly-Ala-OH with DIPCDI/HOBt to H-Nle-OMe
(tR(LL): 23.00 min, tR(DL): 23.3 min).
So far, application of this approach was complicated by
the use of either a high excess of the activating agent
(diisopropylcarbodiimide (DIPCDI, 20 equiv.)),17
which requires an extra purification step before the
deprotection, or the level of epimerization.18 We have
investigated conditions for the athioester formation
using a very low or no excess of activating agent.
Different activating agents (benzotriazol-1-yl-N-oxy-
Table 1. Formation of Z-Gly-Ala-acetamidophenyl
thioester using different activation methods
tris-(pyrrolidino)phosphonium
(PyBOP), benzo-triazol-1-yl-N-oxy-tris-(dimethyl-
amino)phosphonium hexafluorophosphate (BOP),
DIPCDI, and N-[(1H-benzotriazol-1-yl)-(dimethyl-
amino)methylene]-N-methyl-methan-aminium tetra-
hexafluorophosphate
Activating agent Z-Gly-Ala-thioester,
after 5 min (%)
Z-Gly-Ala-thioester,
after 3 days (%)
PyBOP
BOP
TBTU
DIPCDI/HOBt 56
DIPCDI 60
72
57
3.5
96
90
13
99
88
fluoroborate N-oxide (TBTU)) and thiols (thiophenol,
p-acetamidothiophenol, 3-mercapto-propionic acid
ethyl ester) have been tested with respect to the rate of
thioester formation and epimerization.
Initial studies were performed with Z-Gly-Ala-OH as a
model substance. For analyzing different activating
agents, the corresponding agent (0.01 mmol) was added
to Z-Gly-Ala-OH (0.01 mmol) and p-acetamidothio-
phenol (0.01 mmol) dissolved in 0.5 ml of
dichloromethane (DCM). In the case of TBTU, BOP or
PyBOP reactions were started by the addition of N,N-
diisopropylethylamine (DIEA) (0.01 mmol). Progress of
the reaction was monitored by analytical RP-HPLC.
The product peaks were collected and identified with
Table 2. Formation of Z-Gly-Ala-thioester using PyBOP
and different thiols
Thiol
Yield of Z-Gly-Ala-thioester after 5
min (%)
p-Acetamidothiophenol
Thiophenol
3-Mercaptopropionic acid
ethyl ester
72
50
41

R. 6on Eggelkraut-Gottanka et al. / Tetrahedron Letters 44 (2003) 3551–3554
3553
In order to apply the method to longer peptides, we
have chosen the 21 amino acid (aa) segment of uro-
cortin, acetyl-DDPPLSIDLTFHLLRTLLEIA-OH,
and the 41 aa N-terminal fragment of pro-neuropeptide
Y (proNPY 1–40),YPSKPDNPGEDAPAEDLARYY-
SALRHYINLITRQRYGKRS-OH (Fig. 1).
Both peptide fragments were synthesized by Fmoc/t-
Butyl solid-phase strategy with an automated peptide
synthesizer on 2-chlorotrityl (Cl-Trt) resin which was
preloaded with the first C-terminal amino acid. The
protected peptides were cleaved from the resin with
acetic acid/trifluorethanol/DCM (1:1:8 v/v/v) for 1–2
hours at room temperature. To remove acetic acid, the
cleavage solution was in case of the 21-mer peptide
diluted with n-hexane, evaporated, redissolved in diox-
ane and lyophilized. For proNPY 1–40, extraction of
acetic acid with half-saturated NaHCO3-solution
proved to be superior to n-hexane; the DCM-phase was
evaporated and the residue weighed for further reaction
with thiol. A sample of each crude product was depro-
tected (21-mer peptide: TFA/phenol/triisopropylsilane/
H2O, 87.5/5/2.5/5; proNPY 1–40: TFA/thioanisole/
p-cresol, 90/5/5, 3 h, rt) and analyzed by RP-HPLC
and mass spectrometry revealing a content for the
Figure 2. RP-HPLC (lower panel; gradient from 10 to 70%
ACN in water over 30 min at a flow rate of 0.6 ml/min) and
MALDI-MS (upper panel) profiles of the crude proNPY 1–40
acetamidophenyl thioester, *corresponds to the hydrolyzed
thioester due to MALDI measurements.
urocortin segment thioester (0.5 mg, 0.18 mmol) dis-
solved in 2 mL NMP was mixed with 8 ml of a 0.2 M
solution of H-Cys-OEt×HCl in 0.1 M NaHCO3 buffer
(pH 8.5) containing 3 M urea. The progress of the
reaction was monitored by RP-HPLC. The reaction
was nearly completed (>90%) after 1 h. ESI-MS con-
firmed the correct mass of the ligation product ([M+
H]calc.: 2565.4, [M+H]found.: 2565.1). This model reaction
shows the applicability of the acetyl-urocortin segment
thioester for couplings to peptide libraries in connection
with structure–activity relationship studies of corti-
cotrophin-releasing-factor (CRF) receptor ligands.19
21-mer peptide of 54% (ESI-MS: Mcalc.: 2434.8; Mfound
:
2435.0) and ]70% for proNPY 1–40 (MALDI-MS:
[M+H]calc.: 4684.2, [M+H]found.: 4684.2).
The thioester of the 21-mer peptide was formed by
treating the crude protected peptide (380 mg, 0.11
mmol) with DIPCDI (1.5 equiv.) and p-acetamidothio-
phenol (15 equiv.) in DCM (20 mL) over night. The
solvent was evaporated and the residue deprotected as
described above. After precipitation with ice-cold
diethyl ether the deprotected peptide thioester was
purified by preparative RP-HPLC in a H2O–acetonitrile
(0.1% TFA) system. The purified peptide thioester ([M+
H]calc.: 2582.4, [M+H]found.: 2583.4) was obtained in a
considerable yield of 47% (76 mg) based on the peptide
content of the crude product.
Ligation of the thioester fragment of proNPY 1–40 and
the corresponding N-terminal cysteine fragment,
labeled with carboxyfluorescein (CF), [C41, K68(CF)]
proNPY
41–69,
was
performed
in
g-mor-
The optimum for the thioester formation of proNPY
1–40 was obtained by adding PyBOP (3 equiv.), p-acet-
amidothiophenol (3 equiv.) and DIEA (3 equiv.) to the
crude protected peptide (13 mg, 0.0016 mmol) dissolved
in DCM (20 mL). After 1.5–3 h reaction at rt the
solvent was evaporated and the residue deprotected as
described above. The peptide thioester was character-
ized by analytical RP-HPLC (Fig. 2) and MALDI-MS
([M+H]calc.:4833.4, [M+H]found: 4834.2). The thioester
was formed with a yield of >95%. With respect to the
peptide content in the crude product, a final yield of
70% (5.4 mg) of the thioester product was achieved.
The optical purity of the peptide thioesters was ana-
lyzed after hydrolysis in 6 N D2O/DCl by GC–MS. In
pholinopropanesulfonate (MOPS) buffer (0.1 M), pH
7.5, containing 6 M guanidine×HCl. After dissolving of
both reactants (1 mM) thiophenol (2%) and benzyl
mercaptan (2%) were added. The ligation mixture was
stirred at room temperature for 8 h and monitored by
analytical RP-HPLC. The ligation product was purified
by semi-preparative RP-HPLC and analyzed by
MALDI-MS ([M+H]calc.: 8425.4, [M+H]found: 8425.4).
In conclusion, the preparation method presented here
provides a highly efficient, fast and low-cost way for the
generation of peptide thioesters by Fmoc-based solid
phase strategy. The method can be applied also to the
thioester synthesis of long peptides as demonstrated by
the synthesis of the 41 aa proNPY 1–40. By using a
very low excess of activating reagents previous limita-
tions of the method could be overcome. We could show
for the first time that particularly phosphonium salt
based reagents in the presence of thiol are very suitable
for an excellent thioester formation in high yields
case of the acetyl-urocortin(1–21) thioester a
D-alanine
content of 0.2% was determined and for the C-terminal
amino acid of the proNPY 1–40 a content of 1.39% was
found (D-serine).
Native chemical ligation reactions demonstrated the
suitability of the generated peptide thioesters. The 21 aa

3554
R. 6on Eggelkraut-Gottanka et al. / Tetrahedron Letters 44 (2003) 3551–3554
(>95%) while keeping epimerization on a very low level
(<1.4%). Because of its simplicity and the usage of only
commercially available resin and reagents, the method
shows a wide applicability for the synthesis of the
thioester intermediate of NCL.
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Acknowledgements
The authors thank Regina Reppich for recording the
MALDI mass spectra. The financial contribution of
DFG (SFB 610/A1) and the Fond der Chemischen
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