Acid-labile cys-protecting groups for the Fmoc/tBu strategy: Filling the gap

Article abstract of DOI:10.1021/ol302550p

To address the existing gap in the current set of acid-labile Cys-protecting groups for the Fmoc/tBu strategy, diverse Fmoc-Cys(PG)-OH derivatives were prepared and incorporated into a model tripeptide to study their stability against TFA. S-Dpm proved to be compatible with the commonly used S-Trt group and was applied for the regioselecive construction of disulfide bonds.

Full text of DOI:10.1021/ol302550p

ORGANIC
LETTERS
2012
Vol. 14, No. 21
5472–5475
Acid-Labile Cys-Protecting Groups for the
Fmoc/tBu Strategy: Filling the Gap
†,‡
Miriam Gongora-Benıtez,†,‡ Lorena Mendive-Tapia,†,‡ Ivan Ramos-Tomillero,
ꢀ
ꢀ
´
Arjen C. Breman,† Judit Tulla-Puche,*,†,‡ and Fernando Albericio*,†,§,
Institute for Research in Biomedicine, 08028-Barcelona, Spain, CIBER-BBN,
08028-Barcelona, Spain, Department of Organic Chemistry, University of Barcelona,
08028-Barcelona, Spain, and School of Chemistry, University of KwaZulu Natal,
4001-Durban, South Africa
albericio@irbbarcelona.org; judit.tulla@irbbarcelona.org
Received September 16, 2012
ABSTRACT
To address the existing gap in the current set of acid-labile Cys-protecting groups for the Fmoc/tBu strategy, diverse Fmoc-Cys(PG)-OH
derivatives were prepared and incorporated into a model tripeptide to study their stability against TFA. S-Dpm proved to be compatible with the
commonly used S-Trt group and was applied for the regioselecive construction of disulfide bonds.
Since the early days of peptide chemistry, the effective
synthesis of natural or non-natural isomers, analogues, or
de novo designed peptides with complex disulfide bridge
patternshasbeena demanding task. Theoxidativefolding1
of fully deprotected linear peptides is a desirable and
commonly applied approach for the synthesis of complex
Cys-rich peptides. However, achievement of the desired
disulfide bond connectivity through this approach is not
always affordable. To overcome these challenging syn-
theses, a myriad of protecting groups for the β-thiol group of
Cys, along with efficient regioselective protection schemes,
have been developed.2
In recent years, several acid-labile Cys-protecting
groups have been developed for the Fmoc/tBu strategy
(Figure 1).3 Most of these are highly sensitive to acid, the
S-Trt group being one of the most commonly used in the
Fmoc/tBu approach. In contrast, the S-Mob group re-
quires a high TFA concentration and harsh conditions
(high temperature and long reaction times) to be fully
removed. In this regard, the current gap between S-Trt
and S-Mob groups captured our attention and prompted
us to browse through acid-labile protecting groups to find
Cys-protecting groups that, ideally, could be quantita-
tively removable under mild acidic conditions and, simul-
taneously, show compatibility with S-Trt for their further
application in synthetic strategies for the preparation
of Cys-rich peptides. Thus, three distinct scaffolds, namely
diphenylmethyl, biphenylmethyl, and benzyl groups, were
selected and finely tuned for this purpose. Twelve Fmoc-
Cys(PG)-OH (1aꢀl) were prepared and incorporated into
the model tripeptide Fmoc-Ala-Cys(PG)-Leu-NH2 (2aꢀl),
† Institute for Research in Biomedicine.
‡ CIBER-BBN.
§ University of Barcelona.
University of KwaZulu Natal.
(1) (a) Moroder, L.; Besse, D.; Musiol, H. J.; Rudolph-Bohner, S.;
€
Siedler, F. Biopolymers (Pept. Sci.) 1996, 40, 207. (b) Narayan, M.;
Welker, E.; Wedemeyer, W. J.; Scheraga, H. A. Acc. Chem. Res. 2000,
33, 805. (c) Anfinsen, C. B. Biochem. J. 1972, 128, 737.
(2) (a) Barany, G.; Merrifield, R. B. Solid-phase peptide synthesis. In
The Peptides; Gross, E., Meienhofer, J., Eds.; Academic Press: New York,
ꢀ
1979; Vol. 2, pp 1ꢀ84. (b) Andreu, D.; Albericio, F.; Sole, N. A.;
ꢀ
Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular
Biology: Peptide Synthesis Protocols; Pennington, M. W., Dunn, B. M.,
Eds.; Humana Press Inc.: Totowa, NJ, 1994; Vol. 35, pp 91ꢀ169.
(3) (a) Isidro-Llobet, A.; Alvarez, M.; Albericio, F. Chem. Rev. 2009,
ꢁ
109, 2455–2504. (b) Boulegue, C.; Musiol, H. J.; Prasad, V.; Moroder, L.
Chem. Today 2006, 24, 24.
r
10.1021/ol302550p
Published on Web 10/17/2012
2012 American Chemical Society

Figure 1. Acid-labile Cys-protecting groups for Fmoc/tBu chemistry.
and their lability against TFA was studied and compared
with the S-Mob group (2m) (Scheme 1).
Table 1. TFA-Lability Study of the Tripeptides 2
Scheme 1. Preparation of Fmoc-Cys(PG)-OH Derivatives and
Their Successive Incorporation into a Standard Tripeptide
After elongation on a Sieber amide resin, the standard
tripeptides were cleaved from the resin and TFA-lability
studies were carried out in solution with a range of reac-
tion times and temperatures, in the presence of 2.5% TIS
and 2.5% H2O as scavengers. The tripeptides were then
analyzed by RP-HPLC to determine the percentage of
deprotected Cys (Table 1).
As expected, the S-Mob protecting group was stable
against diluted TFA treatments and required a high con-
centration of TFA, a longer reaction time, and an increase
of temperature up to 40 °C to be totally removed (2m).
Pleasantly, the diphenylmethyl (Dpm) group 1a, along
with two decorated benzyl moieties;1h with a p-methoxy
and an o-methyl group and 1i with two o-methoxy groups;
exhibited the desired lability against TFA (2a, 2hꢀi), while
the others were not labile to TFA treatments (2dꢀf, 2j) or
were highly sensitive to TFA (2bꢀc, 2g, 2kꢀl). Further-
more, the three protecting groups 1a, 1h, and 1i were stable
under S-Trt cleavage conditions (10% TFA, 2a, 2hꢀi). This
outcome demonstrates the compatibility of these groups
with S-Trt for regioselective disulfide construction.
Among the three promising protecting groups for Cys,
the easily synthetically accessible S-Dpm was chosen as
an alternative to the S-Mob group for advanced studies.4
(4) S-Mob and S-Dpm groups are not compatible. Under the cleav-
age conditions of S-Dpm, 22% S-Mob removal was observed.
Org. Lett., Vol. 14, No. 21, 2012
5473

Although S-Dpm was described as a Cys-protecting group
by Photaki et al. back in 1970,5 use of this group in Fmoc/
tBu chemistry has not been tackled until now.
Table 2. Lability Study of the Acid-Labile S-Mmt, S-Trt, and
S-Dpm Groups on Solid Phasea
TFA
(%)
reaction
time
3a
3b
3c
3d
scavenger
(%)
(%)
(%)
(%)
Scheme 2. Compatibility Study of Protecting Groups
1
ꢀ
3 ꢁ 5 min
3 ꢁ 5 min
3 ꢁ 5 min
1 h
45
25
ꢀ
45
50
ꢀ
10
25
100
30
ꢀ
ꢀ
2
ꢀ
ꢀ
10
60
90
2.5% TIS
2.5% TIS
2.5% TIS
ꢀ
ꢀ
ꢀ
70
100
1 h
ꢀ
ꢀ
a All experiments were carried out with 15 mg of peptidyl-resin.
fragment 225ꢀ232/2250ꢀ2320 of the human immunoglobulin
G1 (IgG1)1a,8 combining S-Mmt and S-Dpm, along with the
preparation of the R-conotoxin ImI9 combining the S-Trt
and S-Dpm groups, were carried out.
The hingefragmentof IgG1was accomplished following
two strategies in parallel (Scheme 3). In the first approach
(Rink-amide resin), the S-Mmt group was selectively re-
moved by diluted TFA treatments and the first disulfide
bond was achieved by piperidine/DMF (1:4) on solid phase.
The anchored S-Dpm-protected dimer 4 was then treated
with TFA/TIS/H2O (95:2.5:2.5) for 1 h at 25 °C, and the
resultant fully deprotected intermediate 7 was redissolved
in DMSO/phosphate buffer (1:4) at pH 9 to render the
final cyclic parallel dimer 8 (see SI).
Before further studies, the absence of racemization dur-
ing Cys(Dpm) incorporation was proven (see Supporting
Information (SI)). Next, the compatibility of S-Dpm with
S-Trt and the highly sensitive acid-labile S-Mmt groups
were thoroughly examined through a single experiment.
Thus, a hexapeptide, which contained three Cys residues, was
elongated onto a DKPhandle linker, which allowed total free
acid cleavage6 (Scheme 2, hexapeptide 3). After TFA treat-
ment, the free thiol groups were methylated, and the Alloc
group was then removed from the R-Nof the Lys residue, and
the peptidyl-resin was treated with piperidine/THF to render
the C-terminal DKPhandle-protected hexapeptides (3aꢀd). At
10% TFA in the presence of 2.5% TIS as a scavenger, the S-
Trt and S-Mmt groups were fully removed on solid phase. In
contrast, S-Dpm was stable under these acidic conditions,
requiring up to 90% TFA and 2.5% TIS for its entire removal
(Table 2). It is worth mentioning that in any case the selective
removal of S-Mmt vs S-Trt was not achieved, thereby
showing the incompatibility of these protecting groups.7
After confirming the compatibility of S-Dpm with the
S-Trt and S-Mmt groups, we were encouraged to apply
the S-Dpm/S-Trt and S-Dpm/S-Mmt combinations in the
protection scheme for the regioselective construction of
intra- and intermolecular disulfide bridges. Thus, the
regioselective syntheses of a double-chain bis-cystinyl
Scheme 3. Regioselective Syntheses of Hinge Fragment of IgG1
Combining S-Mmt and S-Dpm Protecting Groupsa
a (i) TFA/TIS/CH2Cl2 (5:2.5:92.5) (5 ꢁ 1 min); (ii) piperidine/DMF
(1:4); (iii) H2O/ACN (1:9), 20% DMSO at pH 9; (iv) TFA/TIS/H2O
(95:2.5:2.5) for 1 h at 25 °C; and (v) DMSO/phosphate buffer (1:4) at pH
9 and 25 °C.
In the second approach (Sieber-amide resin), the linear
partial S-Dpm-protectedpeptide5wasobtained by diluted
TFA treatments. Subsequently, the first disulfide bondwas
accomplished in solution to render the protected dimer
intermediate 6. The second disulfide bridge was achieved
as described before to render the bis-cystinyl parallel dimer
8 (Figure 2).
(5) Photaki, I.; Taylor-Papadimitrious, J.; Sakarellos, C.; Mazarakis,
P.; Zervas, L. J. Chem. Soc. (C) 1970, 2683.
ꢀ
(6) Gongora-Benıtez, M.; Cristau, M.; Giraud, M.; Tulla-Puche, J.;
´
Albericio, F. Chem. Commun. 2012, 48, 2313.
(7) Although it has been reported in the literature that S-Mmt can be
selectively removed in the presence of S-Trt, actually it has no practical
use because the safety window is so narrow that conditions should be
carefully optimized and these are, therefore, not of general application.
(9) (a) McIntosh, J. M; Yoshikami, D.; Mahe, E.; Nielsen, D. B.;
Rivier, J. E.; Gray, W. R.; Olivera, B. M. J. Biol. Chem. 1994, 269, 16733.
(b) Nielsen, J. S.; Buczed, P.; Bulaj, G. J. Pept. Sci. 2004, 10, 249.
€
€
(8) Wunsch, E.; Moroder, L.; Gohring-Romani, S.; Musiol, H.-J.;
€
Gohring, W.; Bovermann, G. Int. J. Peptide Protein Res. 1988, 32, 268.
5474
Org. Lett., Vol. 14, No. 21, 2012

Figure 3. RP-HPLC analysis of the synthesized R-conotoxin ImI
11 combining S-Dpm and S-Trt protecting groups.
Figure 2. RP-HPLC analysis of the synthesized bis-cystinyl
parallel dimer 8 from the Sieber-amide approach.
point, various conditions were attempted to obtain the
final bicyclic peptide. When the total deprotection and
oxidation steps were performed consecutively, a mixture of
two isomers was identified by RP-HPLC and RP-HPLC-
ESMS analysis, while a single peak corresponding to the
expecting isomer 11 was observed when the two steps were
performed following a one-pot strategy (Figure 3).
In summary, the S-Dpm protecting group is an alternative
to the S-Mob group. S-Dpm can be fully deblocked under
the standard conditions used for cleavage and total deprotec-
tion steps in Fmoc chemistry, and it is fully compatible with
two commonly used acid-labile protecting groups such as S-
Trt and S-Mmt. Here we successfully applied S-Dpm for the
regioselective synthesis of peptides containing intra- and
intermolecular disulfide bonds. These results could be extra-
polated to the other two protecting groups 1h and 1i.
Although oxidative folding conditions can be carefully
refined to provide a major isomer, the regioselective synthe-
sis of the two-disulfide-containing R-conotoxin family10
members allows the correct construction of disulfide bond
pattern present in their biologically active isomers to be
ensured.9,11 Thus, the R-conotoxin ImI, a 12-mer peptide,
which contains two disulfide bridges (2Cys-8Cys and
3Cys-12Cys), was prepared by combining two S-Dpm
and two S-Trt for the protection of the Cys residues
(Scheme 4). After completion of the peptide elongation
Scheme 4. Regioselective Synthesis of R-Conotoxin ImI
Combining S-Trt and S-Dpm Protecting Groups
Acknowledgment. The work has been partially financed
by CICYT (CTQ2009-07758), the Generalitat de Catalu-
nya (2009SGR 1024), the Institute for Research in Biome-
dicine Barcelona (IRB Barcelona), and the Barcelona
Science Park. We thank Dr. Thomas Bruckdorfer from
IRIS Biotech GmbH for fruitful discussions.
Supporting Information Available. Experimental pro-
cedures, compound characterization, RP-HPLC analyses,
and spectral data of the Fmoc-Cys(PG)-OH derivatives.
This material is available free of charge via the Internet
at http://pubs.acs.org.
on a Sieber-amide resin, the partial S-Dpm-protected
intermediate 9 was cleaved from the resin by diluted
TFA treatments and the construction of the first disulfide
bond was achieved in H2O/ACN (3:7) at pH 8 for 16 h at
25°C, asdeterminedbyRP-HPLC analysis(seeSI). Atthis
(11) (a) Walewska, A.; Bulaj, G. Mechanisms of the Oxidative
Folding of Small, Disulfide-Rich Peptides. In Oxidative Folding of
Proteins and Peptides; Moroder, L., Ed.; Royal Chemical Society:
Cambridge, U.K., 2008. (b) Bulaj, G.; Olivera, B. M. Antioxid. Redox
Signal. 2008, 10, 141.
(10) (a) Myers, R. A.; Zafaralla, G. C.; Gray, W. R.; Abbott, J.; Cruz,
L. J.; Olivera, B. M. Biochemistry 1991, 30, 9370. (b) Arias, H. R.;
Blanton, M. P. Int. J. Biochem. Cell Biol. 2000, 32, 1017. (c) Janes, R. W.
Curr. Opin. Pharmacol. 2005, 5, 280.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 21, 2012
5475