Fmoc-based synthesis of peptide thioacids for azide ligations via 2-cyanoethyl thioesters

Article abstract of DOI:10.1021/ol302247h

Rapid and efficient preparation of peptide thioacids from 2-cyanoethyl peptide thioesters has been accomplished. S-2-Cyanoethyl peptide thioesters were obtained cleanly without the need for purification from resin-bound tert-butyl peptide thioesters using

Full text of DOI:10.1021/ol302247h

ORGANIC
LETTERS
2012
Vol. 14, No. 19
5038–5041
Fmoc-Based Synthesis of Peptide
Thioacids for Azide Ligations via
2‑Cyanoethyl Thioesters
,†,‡
Richard Raz‡,§ and Jorg Rademann*
€
Medicinal Chemistry, Department of Pharmacy, Leipzig University, Bru€derstr.
34, 01403 Leipzig, Germany, Leibniz Institute of Molecular Pharmacology (FMP),
€
Robert-Rossle-Str. 10, 13125 Berlin, Germany, and Institute of Chemistry and
Biochemistry, Free University Berlin, Takustr. 3, 14195 Berlin, Germany
rademann@uni-leipzig.de
Received August 13, 2012
ABSTRACT
Rapid and efficient preparation of peptide thioacids from 2-cyanoethyl peptide thioesters has been accomplished. S-2-Cyanoethyl peptide
thioesters were obtained cleanly without the need for purification from resin-bound tert-butyl peptide thioesters using 3-mercaptopropionitrile as
a nucleophile. Elimination of the 2-cyanoethyl group proceeded rapidly (t1/2 < 8 min) under mild conditions and furnished peptide thioacids up to
the size of a 16-mer. Peptide thioacids could be isolated or formed in situ and reacted smoothly with electron-deficient azides yielding an amide as
the ligation product.
Peptide thioacids have recently received considerable
attention as reactive intermediates and building blocks.
For example, they have been applied successfully in the
amidation ligation reaction between thioacids and azides.1
Due to the speed and selectivity of the reaction,2 the liga-
tion reaction of thioacids and electron-deficient azides has
been exploited for protein modification.3 Furthermore, the
thiocarboxylic acid group is orthogonal to many other
chemical functionalities and this has been exploited for
ligations in more complicated peptide systems.4 The syn-
thesis of peptide thioacids has previously been described
using solid phase Boc-methodology,5 requiring HF for
cleavage from the solid support. Neutral or slightly basic
conditions provide a much milder approach to be applied
in order to release thioacids from their precursors. Crich
et al. introduced a base-labile, β-eliminable 9-fluorenyl-
methyl (Fm) linker, which enabled the synthesis of pep-
tide thioacids by Boc chemistry without the need for HF
cleavage.6 In contrast, Liu et al. have prepared peptide
thioacids by hydrothiolysis of peptide thioesters,7 which
‡ Leibniz Institute of Molecular Pharmacology.
§ Free University Berlin.
† Leipzig University.
(1) (a) Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J.
J. Am. Chem. Soc. 2003, 125, 7754–7755. (b) Rosen, T.; Lico, I. M.; Chu,
D. T. W. J. Org. Chem. 1988, 53, 1580–1582. (c) Hosein Hakimelahi, G.;
Just, G. Tetrahedron Lett. 1980, 21, 2119–2122.
(2) (a) Merkx, R.; Brouwer, A. J.; Rijkers, D. T. S.; Liskamp, R. B.
M. J. Org. Lett. 2005, 7, 1125–1128.
(4) (a) Tam, J. P.; Lu, Y.-A.; Liu, C.-F.; Shao, J. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 12485–12489.
(5) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B.
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Science 1994, 266, 776–779. (b) Schnolzer, M.; Kent, S. B. H. Science
1992, 256, 221–225. (c) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. U.S.A.
1981, 78, 4055–4058.
(6) Crich, D.; Sana, K. J. Org. Chem. 2009, 74, 7383–7388.
(3) (a) Zhang, X.; Li, F.; Liu, C. F. Chem. Commun. 2011, 47, 1746–
1748. (b) Zhang, X.; Li, F.; Lu, X. W.; Liu, C. F. Bioconjugate Chem.
2009, 20, 197–200.
r
10.1021/ol302247h
Published on Web 09/19/2012
2012 American Chemical Society

were prepared using an enzymatic approach.8 They found
that efficient release of peptide thioacids was possible at
an elevated temperature (42 °C, 2À4 h), whereas at rt
reactions remained incomplete after 7 h.
Scheme 2. Model Reaction with S-tert-Butyl Thioester 1
Recently, we have reported the application of a tert-
butyl thiol linker, 4-mercapto 4-methylpentanol (MMP),
for the Fmoc based synthesis of peptide thioesters.9 The
linker was used to construct peptide thioesters which were
simultaneously stable against standard Fmoc cleavage
conditions (piperidine in DMF (20% v/v)) while being
cleavable under mild conditions using thiolates.
Scheme 1. General Principle for Peptide Thioacid Synthesis
piperidine, the thioester was rapidly converted to the
piperidine amide and not the thioacid. In the presence of
DIPEA or 2,6-lutidine, the 2-cyanoethyl group was not
removed after 24 h and extended reaction times led to
hydrolysis of the thioester to the carboxylic acid.
We envisioned that application of this linker could be
extended to the synthesis of peptide thioacids by displacing
the peptide as a masked thioacid using an appropriate thio-
late for nucleophilic cleavage. Although base- and acid-labile
protecting groups for thioacids have been previously de-
scribed,10 we reasoned that a thiolate specifically activated
for the release of the thiocarboxylate functionality by
β-elimination should be especially favorable (Scheme 1).
Therefore, 2-cyanoethyl was selected for thioacid pro-
tection and investigated for the release of the desired
thioacid product.
The 2-cyanoethyl protecting group has been previously
used for the protection of carboxylic acids11 but, to our
knowledge, has not been used for the protection of thio-
acids. To test this idea, N-Boc-Phe-S-tert-butyl thioester 1
was prepared and used as a model compound (Scheme 2).
3-Mercaptopropionitrile 2 was generated by reduction of
its commercially available disulfide and was used to treat
thioester 1 in the presence of sodium thiophenolate under
conditions previously described.8 Thioester 3 was fur-
nished quantitatively via thiolytic cleavage of 1.
Initially, deprotection of 2-cyanoethyl thioester in 3 was
attempted with various amine bases. When treated with
Figure 1. Deprotection of the cyano ethyl protecting group per-
formed at room temperature was most efficient with (NH4)2S
(b) giving 96% conversion after 30 min. Conversion to the
thioacid was also effected with differing concentrations of DBU,
6.5% (2) and 13% (9) in DMF (v/v).
With the stronger, non-nucleophilic base DBU (6.5%, v/v)
deprotection of the cyanoethyl group deprotection occurred
in DMF with a reaction half-time of ∼1 h (Figure 1). In-
creasing the DBU concentration to 13% in DMF (v/v)
reduced the reaction half-time to 24 min. Conversion of
the model thioester 3 to the thioacid 4 occurred immedi-
ately with potassium tert-butoxide with the reaction finish-
ing in <1 h (see Supporting Information (SI) Table S1,
entry 4). As even shorter reaction times were desirable, we
investigated alternative conditions to accelerate the rate of
the reaction. Considering that the elimination of the
2-cyanoethyl group might be slowed down by the revers-
ibility of the reaction (Scheme 1), it should be possible to
accelerate the reaction by the addition of a sulfhydryl
nucleophile which can scavenge the released acrylonitrile.
Thus, deprotection of 2-cyanoethyl protecting group was
(7) Tan, X. H.; Zhang, X.; Yang, R.; Liu, C. F. ChemBioChem 2008,
9, 1052–1056.
(8) Tan, X. H.; Wirjo, A.; Liu, C. F. ChemBioChem 2007, 8, 1512–1515.
(9) Raz, R.; Rademann, J. Org. Lett. 2011, 13, 1606–1609.
(10) Crich, D.; Sharma, I. Angew. Chem., Int. Ed. 2009, 48, 7591–7594.
(11) (a) Kita, Y.; Maeda, H.; Takahashi, F.; Fukui, S.; Ogawa, T.;
Hatayama, K. Chem. Pharm. Bull. 1994, 42, 147–150. (b) Ogawa, T.;
Hatayama, K.; Maeda, H.; Kita, Y. Chem. Pharm. Bull. 1994, 42, 1579–
1589. (c) Misra, P. K.; Hashmi, S. A. N.; Haq, W.; Katti, S. B.
Tetrahedron Lett. 1989, 30, 3569–3572.
Org. Lett., Vol. 14, No. 19, 2012
5039

investigated employing ammonium sulfide as the base
(Figure 1). Under these conditions, the half-life of the
deprotection was <8 min although the base concentra-
tion was significantly reduced (1.5%) and the reaction
proceeded in water at pH 9. After 30 min 96% of the
thioester was already deprotected to the thioacid 4, which
was stable under HPLC conditions (see SI). Treating the
protected amino thioacid 4 with tosyl azide and 2,6-
lutidine1a afforded the sulfonamide product 5, which was
verified by LC-MS analysis. Thus, S-2-cyanoethyl was
proven to be suitable as a protecting group for thioacids.
Next, the efficacy of 2-cyanoethyl as a protecting group
for the synthesis and isolation of peptide thioacids was
investigated. The peptide sequence Ac-SYRGF was pre-
pared applying the MMP linker method furnishing the
resin-bound peptide thioester (Figure 2), which was dis-
placed from the resin with 3-mercaptopropionitrile as
2-cyano ethyl thioester 6a. Cleavage was accomplished
again in the presence of sodium thiophenolate and 15-crown-
5 and yielded the pure thioester, which could be employed for
thioacid release without further purification. Direct cleavage
attempts of the peptide from the resin with sulfhydryl
equivalents such as Na2S or NaHS in a THF/H2O mixture
in hopes of obtaining peptide thioacids led only to hydro-
lysis of the thioester.
occur via β-elimination of the protecting group. The acrylo-
nitrile released by the E mechanism can be expected to react
rapidly with a hydrosulfide anion to refurnish 1 equiv of
3-mercaptopropionitrile.
Figure 2. Synthesis of peptide thioacids using the MMP linker.
Next, the concept was extended toward the synthesis of a
selection of peptide thioesters (6aÀ6d) of the generic struc-
ture Ac-SYRXaaXbb containing different amino acids at the
C-terminus (Table 1). All these peptide thioesters could be
deprotected and isolated by preparative HPLC as peptide
thioacids (7aÀ7d) in good yields. Only under prolonged
treatment (>2 h) with diluted acid did slow decomposition to
peptide acids set in. NMR analysis indicated racemization of
the carboxy-terminal amino acid, which is due to the nature
of the thioester-linker we previously reported.9 In order to
test the efficiency of the synthesis protocol for an elongated
peptide thioacid, the 16-mer penetratin-1, a cell-penetrating
peptide from the third helix of the homeodomain of the an-
tennapedia protein,13 was prepared as MMP-linked thioester
8. Using the same methodology the penetratin 2-cyanoethyl
thioester 9a was afforded (Figure 3). After cleavage of
the protecting group with ammonium sulfide, the 16-mer
thioacid Ac-RQIKIWFNRRMKWKKF-COSH 9b was
obtained in a yield of 51% based on initial coupling of the
amino acid and was purified by preparative RP HPLC.
The application of either potassium tert-butoxide or
DBU as a base to the pentapeptide 6a gave impure
products, and no peptide thioacids could be isolated. Since
it is well described that both potassium tert-butoxide and
DBU are effective bases for β-eliminations,12 these failures
were unexpected. Possibly the deprotection reactions were
too slow under these conditions and the hydrolysis of
thioesters with residual water becomes dominant.
Therefore, again ammonium sulfide was investigated as
the base for the release of peptide thioacid 7a from the
2-CE thioester 6a. The sulfhydryl equivalentwas applied in
a phosphate buffer (pH 9) and furnished the clean thioacid
with barely any hydrolysis of the thioester (Figure 2).
Cleavage of the cyanoethyl group proceeded very rapidly
with a reaction half-time of <8 min, finishing in <30 min
at rt without the need for elevated temperatures (see SI).
For comparison, under the very same reaction conditions
Liu et al. had found that peptide thioesters of 3-mercapto-
propanoic acid were only partially cleaved after 7 h and
required elevated temperatures for completion (42 °C,
2 h).8 The observed drastic difference in the reactivities of
2-cyanoethyl and 2-carboxyethyl thioesters should be ad-
dressed. 13C NMR spectroscopy of the two thioesters
indicated virtually identical deshielding of the thioester
carbonyl carbon (200.96 ppm for the 2-carboxyethyl thio-
ester (10) vs 200.7 ppm for the 2-cyanoethyl thioester 3;
see the SI) and suggested comparable electrophilicity.
Thus, the reactivity difference of the two substrates might
be explained by different reaction mechanisms. While the
2-carboxyethyl group can be cleaved exclusively via an SN
mechanism, conversion of the 2-cyanoethyl group can
Table 1. Yields of Peptide CE-Thioesters and Thioacids with
Different Amino Acids at the C-Terminal Position Including the
16-mer Thioester and Thioacid Derivative of Penetratin
CE-thioester
yield (%)a
thioacid
yield (%)b
entry
peptide
1
2
3
4
5
Ac-SYRGF (6a,7a)
Ac-SYRPV (6b,7b)
Ac-SYRGQ (6c,7c)
Ac-SYRGS (6d,7d)
Ac-RQIKIWFNRR
MKWKKF (9a,9b)
95
83
75
61
97
79
62
73
83
51
a Yields relative to the loading of the first amino acid. Purities of
products were determined via HPLC at 220 nm with UV/vis spectros-
copy to be >90%. b Yields were determined after HPLC purification.
(12) (a) Krebs, A.; Swienty-Busch, J. Comp. Org. Synth. 1991, 6, 949–
974. (b) Bartsch, R. A.; Zavada, J. Chem. Rev. 1980, 80, 453–494.
5040
Org. Lett., Vol. 14, No. 19, 2012

Scheme 3
Figure 3. Synthesis of a 16-mer penetratin-1 peptide thioacid
from the precursor thioester peptide.
Table 2. Ligation of Ac-SYRGF-SH with Selected Azides
entry
1
substrate
product
yield (%)a
84
N3-SO2C6H4-
LYRAG-NH2
N3-SO2C6H4-
pCH3
Ac-SYRGF-NHSO2C6H4-
LYRAG-NH2 (12)
Finally, the viability of the pentapeptide thioacid 7a
synthesized via 2-cyanoethyl thioester was investigated in
thioacidÀazide ligation reactions (Table 2). For this purpose,
two alternative reaction paths were pursued (Scheme 3).
First, the isolated peptide thioacid 7a was ligated with a
model sulfonyl azide peptide N3-SO2C6H4-LYRAG-NH2
11 in an aqueous buffer system using 2,6-lutidine as a basic
catalyst as previously described.1a The reaction was com-
plete within 15 min and yielded the corresponding sul-
fonamide peptide Ac-SYRGF-[NHSO2C6H4COONH]-
LYRAG-NH2 12 in 84% yield after HPLC purification.
Alternatively, the direct conversion of the preceding
2-CE thioester 6a into the ligation product 13 in a two-
step one-pot reaction was explored. For the purpose 6a
was treated with DBU to effect elimination to the thioacid
7a in situ. Addition of tosyl azide to the reaction mixture
followed by 2,6-lutidine as the catalyst directly furnished
Ac-SYRGF-NHSO2C6H4CH3 13 as the ligation product.
This concept of in situ generation of thioacids for one-pot
ligation reactions has been recently applied using the Fm
protecting group for the synthesis of sulfonamides with
peptidic as well as nonpeptidic structures.14
2
Ac-SYRGF-NHSO2C6H4-
pCH3 (13)
55
a The product yield was determined by dry weight after HPLC
purification of the ligated peptide.
Insummary, wehave shownthatthe 2-cyanoethylgroup
is ideally suited for the protection of thiocarboxylic acids
and was applied in the preparation of thioacids and pep-
tide thioacids from their parent thioesters. Furthermore,
2-mercaptopropionitrile serves as an excellent source for
the introduction of the thiol functionality, in this case for
a masked peptide thioacid by nucleophilic displacement
from the MMP linker.
Acknowledgment. We gratefully acknowledge the DFG
(RA895/11, FOR806 and SFB 765) for continuous support.
Supporting Information Available. Experimental pro-
cedures, NMR analyses for key compounds, and LC-MS
data for important reactions. This material is available
free of charge via the Internet at http://pubs.acs.org.
(13) (a) Damante, G.; Pellizari, L.; Esposito, G.; Focolari, F.;
Viglino, P.; Fabbro, D.; Tell, G.; Formisano, S.; Di Lauro, R. EMBO
J. 1996, 15, 4992–5000. (b) Derossi, D.; Calvet, S.; Trembleau, A.;
Brunissen, A.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1996, 271,
18188–18193.
(14) Namelikonda, N. K.; Manetsch, R. Chem. Commun. 2012, 48,
1526–1528.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 19, 2012
5041