Fmoc-based synthesis of peptide thioesters for native chemical ligation employing a tert -butyl thiol linker

Article abstract of DOI:10.1021/ol1029723

tert-Butyl thioesters display an astonishing stability toward secondary amines in basic milieu, in contrast to other alkyl and aryl thioesters. Exploiting this enhanced stability, peptide thioesters were synthesized in a direct manner, applying a tert-butyl thiol linker for Fmoc-based solid-phase peptide synthesis.

Full text of DOI:10.1021/ol1029723

ORGANIC
LETTERS
2011
Vol. 13, No. 7
1606–1609
Fmoc-Based Synthesis of Peptide
Thioesters for Native Chemical Ligation
Employing a tert-Butyl Thiol Linker
,†,‡
Richard Raz‡,§ and Jorg Rademann*
€
Medicinal Chemistry, Department of Pharmacy, Leipzig University, Bru€derstrasse 34,
04103 Leipzig, Germany, Leibniz Institute of Molecular Pharmacology (FMP), Robert-
€
Rossle-Strasse 10, 13125, Germany, and Institute of Chemistry and Biochemistry, Free
University Berlin, Takustrasse 3, 14195 Berlin, Germany
rademann@uni-leipzig.de
Received January 4, 2011
ABSTRACT
tert-Butyl thioesters display an astonishing stability toward secondary amines in basic milieu, in contrast to other alkyl and aryl thioesters.
Exploiting this enhanced stability, peptide thioesters were synthesized in a direct manner, applying a tert-butyl thiol linker for Fmoc-based solid-
phase peptide synthesis.
Peptide thioesters are key molecules in the synthesis of
peptides and proteins, for example, as starting materials
for native chemical ligation (NCL).1 NCL is the trans-
thioesterification of a C-terminal thioester peptide with an
N-terminal cystein peptide, followed by an S f N acyl
shift2 to furnish a native amide linkage. While peptide
thioesters can be prepared by Boc chemistry,3 their synth-
esis with standard Fmoc-based methods is hampered by
the reactivity of thioesters against piperidine resulting in
rapid thioester cleavage during Fmoc deprotection. Direct
syntheses of peptide thioesters using Fmoc amino acids
have been attempted with limited success, e.g., by applying
milder deblocking techniques which suffer from low yields
and/or incomplete Fmoc deprotection.4 Instead “safety-
catch linkers” were used for the Fmoc-based synthesis of
peptide thioesters. These linkers are activated, e.g., by
alkylation of acyl sulfonamides (Kenner’s linker),5 oxida-
tion of acyl hydrazides,6 or intramolecular diacylation.7,8
(4) (a) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39,
8669–8672. (b) Kawakami, T.; Hasegawa, K.; Aimoto, S. Bull. Chem.
Soc. Jpn. 1993, 66, 2700–2706. (c) Futaki, S; Sogawa, K.; Maruyama, J.;
Asahara, T.; Niwa, M. Tetrahedron Lett. 1997, 38, 6237–6240. (d)
Clippingdale, A. B.; Barrow, C. J.; Wade, J. D. J. Pept. Sci. 2000, 6,
225–234. (e) Bu, X.; Xie, G.; Law, C. W.; Guo, Z. Tetrahedron Lett.
2002, 43, 2419–2422.
(5) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. Chem.
Commun. 1971, 636–637.
(6) Millington, C. R.; Quarrell, R.; Lowe, G. Tetrahedron Lett. 1998,
39, 7201–7204.
(7) Tofteng, A. P.; Sørensen, K. K.; Conde-Frieboes, K. W.; Hoeg-
Jensen, T.; Jensen, K. J. Angew. Chem., Int. Ed. 2009, 48, 7411–7414.
(8) Blanco-Canosa, J. B.; Dawson, P. E. Angew. Chem., Int. Ed. 2008,
120, 6957–6961.
‡ Leibniz Institute of Molecular Pharmacology (FMP).
† Leipzig University.
§ Free University Berlin.
(1) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science
1994, 266, 776–779.
(2) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H.
Justus Liebigs Ann. Chem. 1953, 583, 129–149.
(3) (a) Aimoto, S. Biopolymers 1999, 51, 247–265. (b) Bang, D.;
Pentelute, B. L.; Gates, Z. P.; Kent, S. B. H. Org. Lett. 2006, 8, 1049–
1052.
(9) (a) Keck, G. E.; Welch, D. S.; Poudel, Y. B. Tetrahedron Lett.
2006, 47, 8267–8270. (b) Fedor, L. R. J. Am. Chem. Soc. 1969, 91, 913–
917. (c) Mohrig, J. R.; Carlson, H. K.; Coughlin, J. M.; Hofmeister,
G. E.; McMartin, L. A.; Rowley, E. G.; Trimmer, E. E.; Wild, A. J.;
Schultz, S. C. J. Org. Chem. 2007, 72, 793–798.
r
10.1021/ol1029723
Published on Web 02/28/2011
2011 American Chemical Society

Herein, we describe the first Fmoc-based synthesis of
peptide thioesters using a tert-butyl thioester as a linker.
tert-Butyl thioesters are of exceptional stability against
nucleophilic bases and strong acids,9 which distinguishes
them from other alkyl thioesters.10 At the same time, tert-
butyl thioesters remain smoothly cleavable by thiolates.11
displayed a half-life of 6.5 days, strongly supporting the
feasibility of the envisaged linker system for the direct
synthesis of peptide thioesters.
In order to obtain a thioester linkage of sufficient
stability, a spacer was introduced and acetyl-protected
4-mercapto 4-methylpentanol 5 was used as the MMP-
linker precursor. Commercially available 4-methylpent-3-
en-1-ol 3 was O-Fmoc-protected, and indium trichloride12
catalyzed the regioselective introduction of thioacetic acid
yielding the tertiary thioacetate 4 (Scheme 1).
Scheme 1
Deprotection of the Fmoc group afforded the linker
precursor 5. O-Tritylation of 5 followed by deacetylation
yielded mercaptan 6 which was converted to the model
thioester 7a by S-acylation employing Boc-phenylalanine,
N,N0-dicyclohexylcarbodiimide (DCC), and N-dimethyla-
minopyridine (DMAP).
Thioester 7a was used as a model compound to investi-
gate the stability of the linker on trityl resin and possessed a
t1/2 of more than 9 days in the presence of 20% piperidine
in DMF. For comparison, the corresponding thioester of
Boc-glycine 7b was prepared and tested. The stability of 7b
toward piperidine was significantly reduced (t1/2 = 7.5 h),
reflecting the high reactivity of glycine thioesters in NCL.13
All other amino acid thioesters tested (His, Trp, Gln, Val)
showed stability similar to that of the Phe-derived thio-
ester. In conclusion, tert-butyl thioesters should be useful
as linkers for all C-terminal amino acids with the highest
reactivity for glycine.
The MMP linker 5 was attached to 2-chlorotrityl chlo-
ride resin in pyridine providing resin 8 in a high yield as
determined by elemental analysis of sulfur from a resin
sample (Scheme 2). The acetyl protecting group was
removed with hydrazine acetate14 furnishing resin 9, and
the first amino acid was coupled yielding thioester product
10. Acylation of the thiolinker resin with phenylalanine
was investigated with different condensation reagents and
Figure 1. Stability of various thioesters of Phe during treatment
with 20% piperidine in DMF treatment. tert-Butyl thioester 1a
yielded the piperidine amide 2a and the DMF-derived N-dimethyl
amide. Half-lives were determined by LC-MS at 220 nm.
In order to validate the strategy, various thioesters were
investigated under Fmoc-cleavage conditions (20% piper-
idine in DMF) by HPLC-MS, and the t1/2 of tert-butyl
was > isopropyl > trityl > ethyl > benzyl (Figure 1). The
results indicate that thioesters are stabilized by steric
hindrance as well by electron donation. For example, the
sterically most demanding triphenyl methyl (trityl) thio-
ester was cleaved faster than the smaller but more electron-
rich isopropyl and the more bulky benzyl thioester faster
then ethyl. The tert-butyl thioester of Boc-phenylalanine
~
(12) Weıwer, M.; Dunach, E. Tetrahedron. Lett. 2006, 47, 287–289.
(13) At the C-terminus of a thioester peptide, histidine, cysteine, and
glycine are most reactive in NCL: Hackeng, T. M.; Griffin, J. H.;
Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10068–10073.
(14) Roush, W. R.; Lin, X.-F. J. Am. Chem. Soc. 1995, 117, 2236–
2250.
(10) Morse, B. K.; Tarbell, D. S. J. Am. Chem. Soc. 1952, 74, 416–
419.
(11) Wallace, O. B.; Springer, D. M. Tetrahedron Lett. 1998, 39,
2693–2694.
Org. Lett., Vol. 13, No. 7, 2011
1607

RXbbXaa 11a-d were synthesized and cleaved off the resin
using various conditions. Application of sodium etha-
nethiolate with the cryptand 15-crown-5 in THF followed
by treatment with TFA led almost exclusively to the
hydrolyzed peptide acid. Applying sodium thiophenolate,
15-crown-5 with an excess of ethanethiol18 in THF liber-
ated the thioesters from the resin cleanly, and subsequent
treatment with TFA delivered thioesters 15a-c without
traces of hydrolyzed peptides, however, in only modest
yields (14-26%). Nucleophilic cleavage with thiols suc-
ceeded best with 3-mercaptopropionic acid methyl ester in
the presence of sodium thiophenolate as base and the
cryptand 15-crown-5. The protected peptidyl thioesters
were treated with TFA and precipitated in methyl tert-
butyl ether (MTBE) to furnish peptide thioesters 13a-d in
high purity (>90%) and excellent yields (65-90%)
(Figure 2). Potential formation of diketopiperazine
(DKP) products during Fmoc-deprotection of the second
amino acidwas examinedcarefully byanalyzing thefiltrate
of the deblocking step. For the C-terminal dipeptides GQ
and PV, no DKP formation was detected. Traces of DKP
were found only during the washing step after deblocking
the second coupled amino acid in the preparation of the
pentapeptide Ac-SYRGF 13a which was still obtained in
69% yield. Coupling of a dipeptide to the singly acylated
resin 10 using HATU/HOAt as condensation reagents
improved the yield of thioester peptides 13a,b.
Scheme 2
with acyl fluoride. Reactions were monitored by attenu-
ated total reflection infrared spectroscopy (ATR-IR), and
amino acid loadings were quantified by spectrophoto-
metric Fmoc determination following cleavage from a
resin sample.
Exclusion of air from the resin was crucial for the success
of the first acylation step. Treatment of air-flushed resin
with Ellman’s reagent15 indicated a significant decrease of
free thiol groups on the resin presumably through oxida-
tive cross-linking.17 Oxidation of resin 9 was found to be
reversible; when treated with sodium bis(2-methoxy-
ethoxy)aluminum hydride (Red-Al) and washed, the Ell-
man test indicated reduction of the disulfide bridges.
Likewise, the removal of water by azeotropic distillation
from the amino acids increased coupling yields of amino
acids. Yieldsbetween 70and 80%and finalloadings higher
than 0.7 mmol/g) were reached for double couplings of dry
amino acids16 to resin 9 with EDC/DMAP under an inert
atmosphere. Even better results were obtainedfrom Fmoc-
amino acid fluorides17 delivering yields of up to 90%. Free
thiols were capped after the second coupling with Ac2O
and DIPEA. Deprotection of the Fmoc group and coup-
ling of the second amino acid gave quantitative yields.
For a detailed validation of the linker concept and the
sequence-dependent comparison of reactions, a number of
N-acetylated pentapeptides of generic structure Ac-SY-
Figure 2. HPLC chromatograms of crude thioesters measured
at 220 nm: (a) nucleophilic displacement of 12a Ac-SYRGF-
S(CH2)2COOMe, (b) total deprotection to 13a, (c and d) crude
MMP thioesters of 14b and 14d obtained in good yields and purities.
Acidic cleavage and deprotection of the resin-bound
thioester peptides 11a-d using TFA and precipitation
with MTBE furnished clean products 14a-d in good yields
and purities >90% (Table 1). Cleavage of the MMP linker
was not observed in any of these products, neither were
truncated products or fail sequences detected. High-
resolution NMR spectra of 14a were fully assigned by
the use of TOCSY, HMQC, and HMBC spectra (see the
Supporting Information) and indicated partial racemiza-
tion of the C-terminal residue and conservation of the
thioester carbonyl after TFA cleavage, thus excluding an
(15) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70–71.
(16) Weik, S.; Rademann, J. Angew. Chem., Int. Ed. 2003, 42, 2491–
2494.
(17) Carpino, L. A.; Sadat-Aalaee, D.; Chao, H. G.; DeSelms, R. H.
J. Am. Chem. Soc. 1990, 112, 9651–9652.
(18) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc.
1999, 121, 11369–11374.
1608
Org. Lett., Vol. 13, No. 7, 2011

M phosphate buffer (pH 7.4) and delivered the ligation
product Ac-SYRGFCGRFLAVR-NH2 17 smoothly.
Alternatively, the MMP thioester 14a was activated in situ
with sodium thiophenolate and 15-crown-519 also furnish-
ing product 17upon addition of cysteinepeptide16(see the
Supporting Information). Persistence of the MMP linker
during synthesis of a longer peptide was finally tested for a
derivative of the 16mer penetratin-1, a cell-penetrating
peptide from the third helix of the homeodomain of the
antennapedia protein.20 The product Ac-RQIKIWF-
NRRMKWKKF-MMP 18 (Figure 3) was obtained in
67% yield after HPLC purification.
In summary, the MMP-linker can be obtained quickly
from well-accessible starting materials and can be applied
for the preparation of protected and unprotected peptide
thioesters displaying a broad range of sequences. The
products can be directly used in native chemical ligations
and possibly in other reactions of peptidyl thioesters.
Currently, the use of the linker for further C-terminal
modifications of peptides is under investigation.
Table 1. Yields of Selected Crude Peptide Thioesters
entry
peptide thioester
yielda (%)
1
2
Ac-SYRGF-S(CH2)2COOMe (13a)
Ac-SYRGW-S(CH2)2COOMe (13b)
Ac-SYRGQ-S(CH2)2COOMe (13c)
Ac-SYRPV-S(CH2)2COOMe (13d)
Ac-SYRGF-MMP (14a)
69/89b
78b
86
3
4
67
5
54/83b
90b
53
6
Ac-SYRGW-MMP (14b)
7
Ac-SYRGQ-MMP (14c)
8
Ac-SYRPV-MMP (14d)
88
9
Ac-SYRGF-SEt (15a)
14c
20
10
11
Ac-SYRPV-SEt (15b)
Ac-SYRGQ-SEt (15c)
26
a Yields were calculated relative to the loading of the first amino acid.
Purities of products were determined via HPLC at 220 nm with UV/vis
spectroscopy to be >90%. b This peptide yield was attained with the
coupling of a dipeptide to the initial amino acid. c After HPLC
purification.
Acknowledgment. We gratefully acknowledge the DFG
(RA895/2, FOR 806 and SFB 765) and the Fonds der
Chemischen Industrie for continuous support. We thank
ꢀ
Andre Horatscheck, Dr. Peter Schmieder, and Brigitte
Schlegel (Leibniz Institute of Molecular Pharmacology)
for technical support and Prof. Valentin Wittmann
€
(Universitat Konstanz) for helpful discussions.
Supporting Information Available. Experimental pro-
cedures, stability tests, NMR analyses for key com-
pounds, and LC-MS data for important reactions.This
material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 3. HPLC with accompanying mass spectrum of the crude
16mer penetratin derivative 18. A small portion of the peptide,
a, was oxidized at the methionine residue to give the sulfoxide
(þ16 m/z).
(19) Essentially, the peptide-phenyl thioester which has been shown
to accelerate the NCL reaction is created in situ. Dawson, P. E.;
Churchill, M. J.; Ghadiri, M. R.; Kent, S. B. H. J. Am. Chem. Soc.
1997, 119, 4325–4329.
(20) (a) Damante, G.; Pellizari, L.; Esposito, G.; Fogolari, F.;
Viglino, P.; Fabbro, D.; Tell, G.; Formisano, S.; Di Lauro, R. EMBO
J. 1996, 15, 4992–5000. (b) Derosssi, D.; Calvet, S.; Trembleau, A.;
Brunissen, A.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1996, 271,
18188–18193.
S f O transesterification of the peptide C-terminus. The
deprotected peptide thioester 13a was reacted with the
N-terminal cysteine peptide CGRFLAVR-NH2 16 in 0.1
Org. Lett., Vol. 13, No. 7, 2011
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