Access to cyclic or branched peptides using bis(2-sulfanylethyl)amido side-chain derivatives of asp and glu

Article abstract of DOI:10.1021/ol300528r

Bis(2-sulfanylethyl)amido (SEA) side-chain derivatives of aspartic and glutamic acids enable the synthesis of tail-to-side chain cyclic or branched peptides using standard Fmoc-SPPS followed by SEA native peptide ligation.

Full text of DOI:10.1021/ol300528r

ORGANIC
LETTERS
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Vol. XX, No. XX
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Access to Cyclic or Branched Peptides
Using Bis(2-sulfanylethyl)amido Side-Chain
Derivatives of Asp and Glu
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Emmanuelle Boll, Julien Dheur, Herve Drobecq, and Oleg Melnyk*
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CNRS UMR 8161, Universite Lille Nord de France, Institut Pasteur de Lille, 59021,
Lille, France
oleg.melnyk@ibl.fr
Received March 1, 2012
ABSTRACT
Bis(2-sulfanylethyl)amido (SEA) side-chain derivatives of aspartic and glutamic acids enable the synthesis of tail-to-side chain cyclic or branched
peptides using standard Fmoc-SPPS followed by SEA native peptide ligation.
The importance of peptide cyclization for studying pep-
tide conformation, creating new structures, or for develop-
ing peptide therapeutics is well established.1 In particular,
side-chain lactam bridges linking two amino acid residues
that are several residues apart in the linear sequence or head-
to-tail backbone peptide cyclization enable rigidification of
the structure and improvement of in vivo stability.
Native chemical ligation (NCL),2 that is the reaction of a
C-terminal peptide thioester with an N-terminal cysteine
peptide, is now an established method for producing
backbone-cyclized peptides3 or proteins.4 Often, the rate
of cyclization is enhanced by proximity effects induced by
the folded state of the peptide or protein.5 The application
of NCL to the synthesis of side-chain cyclized peptides is
less frequent. Head-to-side-chain cyclization by ligating a
C-terminal thioester with a Cys residue located on a lysine
side chain was recently used by Kent’s group for creating a
novel protein scaffold.6 The alternative tail-to-side-chain
cyclization mode is rare, probably due to the difficulty of
installing a thioester group on amino acid side chains such
as aspartic or glutamic acids. Interestingly, this mode of
cyclization is found in the lasso peptides.7 Note that access
to peptides featuring a thioester group on the side chain of
Asp or Glu derivatives would also facilitate the synthesis of
branched peptides using NCL chemistry.8
(1) For a recent review on peptide macrocyclization, see: White, C. J.;
Yudin, A. K. Nat. Chem. 2011, 3, 509–24.
(2) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B.
Science 1994, 266, 776–9. (b) Kent, S. B. Chem. Soc. Rev. 2009, 38,
338–51.
Obviously, the design of masked side-chain thioester
derivatives of Asp and Glu compatible with Fmoc-SPPS is
an important goal which should facilitate access to various
(3) (a) Shao, Y.; Lu, W.; Kent, S. B. H. Tetrahedron Lett. 1998, 39,
3911–3914. (b) Camarero, J. A.; Muir, T. W. Chem. Commun. 1997,
1369–1370. For recent applications, see: (c) Clark, R. J.; Craik, D. J.
Biopolymers 2010, 94, 414–22. (d) Clark, R. J.; Craik, D. J. Methods
Enzymol. 2012, 503, 57–74. For a recent application using peptide
hydrazides as thioester precursors, see: (e) Zheng, J.-S.; Tang, S.; Guo,
Y.; Chang, H.-N.; Liu, L. ChemBioChem 2012, 13, 542–546.
(4) (a) Camarero, J. A.; Muir, T. W. J. Am. Chem. Soc. 1999, 121,
5597–5598. (b) Iwai, H.; Pluckthun, A. FEBS Lett. 1999, 459, 166–72. (c)
Camarero, J. A.; Fushman, D.; Sato, S.; Giriat, I.; Cowburn, D.;
Raleigh, D. P.; Muir, T. W. J. Mol. Biol. 2001, 308, 1045–62.
(5) Camarero, J. A.; Pavel, J.; Muir, T. W. Angew. Chem., Int. Ed.
1998, 37, 347–349.
(6) Mandal, K.; Pentelute, B. L.; Bang, D.; Gates, Z. P.; Torbeev,
V. Y.; Kent, S. B. H. Angew. Chem., Int. Ed. 2012, 51, 1283–1488.
(7) Wilson, K.-A.; Kalkum, M.; Ottesen, J.; Yuzenkova, J.; Chait,
B. T.; Landick, R.; Muir, T.; Severinov, K.; Darst, S. A. J. Am. Chem.
Soc. 2003, 125, 12475–12483.
(8) One report describes the synthesis of a branched peptide by NCL
of a Glu(SBz) containing peptide with a Cys peptide. See: Dolphin, G. T.
J. Am. Chem. Soc. 2006, 128, 7287–7290.
r
10.1021/ol300528r
XXXX American Chemical Society

cyclic or branched peptide scaffolds. We describe herein a
potential solution to this important problem.
bis[[2-[triphenylmethyl]sulfanyl]ethyl]]amine9 to the side
chain of Boc-Asp-OtBu or Boc-Glu-OtBu using bromo-
tripyrrolidinophosphonium hexafluorophosphate (PyBrop)/
N,N-diisopropylethylamine (DIEA) activation (Scheme 2).
Iodine oxidation yielded the corresponding SEAoff deri-
vatives 11a,b. Finally, the removal of the tert-butyloxy-
carbonyl (Boc) and tert-butyl (tBu) ester groups in tri-
The reaction of a bis(2-sulfanylethyl)amido (SEAon)
group with an N-terminal cysteine residue in water and
at neutral pH results in the formation of a native peptide
bond (Scheme 1).9,10 This reaction, called SEA ligation, is
triggered by an intramolecular N,S-acyl shift11 which leads
to the in situ formation of a transient thioester.12 The
transient thioester reacts subsequently with the N-terminal
Cys peptide as in NCL to give an X-Cys junction. Oxida-
tion of SEAon results in a cyclic disulfide called SEAoff
having a 1,2,5-dithiazepan-5-carbonyl structure. SEAoff is
a self-protected form of SEAon. SEAoff and SEAon can be
easily interconverted by reduction/oxidation.13
Scheme 2. Synthesis of Fmoc-Asp(SEAoff)-OH 1a and
Fmoc-Glu(SEAoff)-OH 1b
We anticipate that the SEAoff group should survive the
repetitive piperidine treatments used for removing the
Fmoc group during SPPS, given the known stability of
dialkyldisulfides in the presence of nitrogen nucleophiles.14
Consequently, we examined the utility of side-chain SEAoff
derivatives of Asp and Glu for accessing branched or tail-
to-side-chain cyclic peptides using Fmoc-SPPS and inter-
molecular or intramolecular SEA native peptide ligation
(Scheme 1).
fluoroacetic acid (TFA) followed by reaction with
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu)
furnished the target Fmoc-Asp(SEAoff)-OH and Fmoc-
Glu(SEAoff)-OH derivatives 1a,b.
Scheme 1. Side-Chain SEAoff Derivatives of Asp and Glu
Enable the Synthesis of Branched or Tail-to-Side Chain Cyclic
Peptides
With amino acids 1a,b in hand, we undertook in a first
approach the synthesis of model Asp(SEAoff) peptides 12a
and 13a and the synthesis of model Glu(SEAoff) peptide
12b (Scheme 3). These peptides were prepared under
standard Fmoc-strategy SPPS conditions and TFA cleav-
age steps. HPLC isolation yielded peptides 12a (42%),
12b (32%), and 13a (56%) in excellent purity.
Scheme 3. Synthesis of Branched Peptides 19a,b and 20a
The synthesis of Fmoc-Asp(SEAoff)-OH 1a and Fmoc-
Glu(SEAoff)-OH 1b was carried out by first coupling
(9) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Org.
Lett. 2010, 12, 5238–41.
(10) SEA ligation can proceed also at mildly acidic pH; see: Hou, W.;
Zhang, X.; Li, F.; Liu, C. F. Org. Lett. 2011, 13, 386–389.
(11) Ollivier, N.; Behr, J. B.; El-Mahdi, O.; Blanpain, A.; Melnyk, O.
Org. Lett. 2005, 7, 2647–50.
(12) (a) Dheur, J.; Ollivier, N.; Vallin, A.; Melnyk, O. J. Org. Chem.
2011, 76, 3194–3202. (b) Dheur, J.; Ollivier, N.; Melnyk, O. Org. Lett.
2011, 13, 1560–3.
(13) Ollivier, N.; Vicogne, J.; Vallin, A.; Drobecq, H.; Desmet, R.;
El Mahdi, O.; Leclercq, B.; Goormachtigh, G.; Fafeur, V.; Melnyk, O.
Angew. Chem., Int. Ed. 2012, 51, 209–213.
We next examined the utility of peptides 12a,b and 13a
for the synthesis of branched peptides using SEA native
peptide ligation in the presence of 4-(mercaptophenyl)-
acetic acid (MPAA)15 as shown in Scheme 3. For this,
Glu(SEAoff) peptide 12b was reduced in situ at pH 7.3 by
(15) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006, 128, 6640–6.
(16) (a) Ligations of 12a,b with 18 at pH 7.3 proceeded at a sig-
nificantly lower rate than ligation between C-terminal Ala-SEAoff
peptide H-ILKEPVHGA-SEAoff and 18 (see ref 9), for which t1/2
was ∼2 h.
(14) Parker, A. J.; Kharasch, N. Chem. Rev. 1959, 59, 583–628.
B
Org. Lett., Vol. XX, No. XX, XXXX

tris(2-carboxyethyl)phosphine (TCEP) into Glu(SEAon)
amide and thioester peptides 14b and 16b in the presence
of Cys peptide 18. HPLC analysis of the reaction mixture
showed that the ligation proceeded successfully without
significant side-reactions (Figure 1). The half-time (t1/2) of
the reaction, that is, the time required to reach 50% con-
version, was ∼19 h (93% conversion after 100 h, Figure 2).
The ligation product 19b was isolated in 39% yield after
HPLC purification. The reaction with Asp(SEAoff) pep-
tide 12a proceeded similarly, albeit with a significantly and
unexpected lower rate (t1/2 ∼48h) thanfor the Glu(SEAoff)
counterpart 12b.16a
Ligation of Asp(SEAoff) peptide 13a, featuring an Ala
residueinposition 2, withCyspeptide18atpH 7.3 wasalso
examined to probe the sensitivity of the reaction to aspar-
timide formation. Interestingly, Asp(SEAoff)-Ala peptide
13a reacted as efficiently as Asp(SEAoff)-Ile analogue 12a
(t1/2 ∼52h, Figure2), with onlyfew percentsofaspartimide
formation (see Supporting Information). Lowering the pH
to 5.5 led to a significant decrease of the time (t1/2 ∼12 h,
Figure 2) and permitted the isolation of the branched
product 20a in 84% yield after HPLC purification.
Figure 2. Time course of intermolecular (12a,b þ 18 or 13a þ 18)
or intramolecular (21a,b) ligations (see Scheme 3 or Figure 4 for
experimental conditions). Yields were determined by HPLC
(215 nm).
equilibration between 14 and 16 was determined similarly
at pH 1 (Figure 3B). Figure 3A shows that the proportion
of amide and thioester forms 14 and 16 was very similar for
Asp and Glu peptides, whereas Glu peptide amide 14b
equilibrated faster than Asp analog 14a. These data sug-
gest that the N,S-acyl shift is the rate limiting step for the
ligation of Asp or Glu(SEAoff) derivatives with Cys
peptides.17 In line with these results, previous studies have
Figure 1. HPLC analysis of the ligation of Glu(SEAoff) peptide
12b with Cys-peptide 18 (see Scheme 3 for experimental details).
Dithiol peptides 14 (Scheme 3), obtained by in situ
reduction of SEAoff derivatives 12, equilibrate with thio-
ester peptides 16. The lower reactivity of Asp(SEAoff
)
peptide 12a might be due to a destabilization of the
thioester form 16a or to a reduction of the N,S-acyl shift
kinetic rate compared to Glu(SEAoff). To clarify this point
we determined the fraction of dithiol amide 14 and thio-
ester 16 at equilibrium as a function of pH and the time
course for the equilibration. For this, peptides 12 were
reduced with TCEP atpH 4ꢀ5, thatisat a pH which favors
the amide form 14.12a For studying the equilibrium be-
tween 14 and 16, the pH was adjusted to the appropriate
value between pH 1 and 6, and the mixture was left for 24 h
at 37 °C. The yields of amide and thioester forms were
determined by HPLC (Figure 3A). The time course for the
Figure 3. Study of the position of equilibrium (A) and rate of
equilibration (B) for peptides 14/16.
Org. Lett., Vol. XX, No. XX, XXXX
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Scheme 4. Tail to Side-Chain Cyclization Using SEA Ligation
Figure 4. HPLC analysis of the cylization of Glu(SEAoff) pep-
tide 21b (21b 1 mM, 200 mM MPAA, 200 mM TCEP, 37 °C, pH
7.3). UV detection at 215 nm.
shown that the rate of transthioesterification of C-terminal
SEAoff peptides by 3-mercaptopropionic acid was corre-
lated with the rate of the N,S-acyl shift.12a
In conclusion, bis(2-sulfanylethyl)amido side-chain de-
rivatives of Fmoc-Asp-OH and Fmoc-Glu-OH are com-
patible with standard Fmoc-SPPS. The side-chain SEAoff
group enables the synthesis of branched or tail-to-side
chain cyclic peptides by intermolecular or intramolecular
SEA native peptide ligation with an N-terminal Cys
residue. Aspartimide formation was not significant when
Asp(SEAoff) residuewas followed byAla residue. TheN,S-
acyl shift of the bis(2-sulfanylethyl)amido group and SEA
ligations proceeded faster for Glu than for Asp derivatives.
Overall, the data presented here show that Fmoc-Asp-
(SEAoff)-OH and Fmoc-Glu(SEAoff)-OH are useful ami-
no acid derivatives for the synthesis of complex peptide
scaffolds.
We next examined the cyclization of peptides 21a,b,
featuring an N-terminal Cys(StBu) residue and an Asp
or Glu(SEAoff) group at the C-terminus (Scheme 4). In situ
reduction of both acyclic and cyclic disulfides by TCEP
triggered the SEA intramolecular ligation. Gratifyingly,
both reactions showed the formation of the cyclic peptides
26a,b. As for the intermolecular side chain ligations,
cyclization of Glu(SEAoff) peptide 21b proceeded faster
than cyclization of Asp analogue 21a (Figure 2). The
HPLC analysis of the reaction mixture for Glu(SEAoff)
peptide 21b shows the clean conversion of reduced peptide
23b into cyclic peptide 26b and the absence of significant
side reactions such as oligomerization (Figure 4). Cycliza-
tion of Asp derivative 21a proceeded similarly but was
complicated by the partial deamidation of the primary
amide group within cyclic peptide 26a. Consequently, the
isolated yield for 26a was lower. Besides careful mass
spectrometry and NMR analysis, the structure of cyclic
peptides 26a,b was confirmed by MALDI-TOF post
source decay fragmentation analysis after alkylation of
Cys thiol with iodoacetamide and trypsin digestion (see the
Supporting Information).
Acknowledgment. We acknowledge financial support
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^
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from Canceropole Nord-Ouest, Region Nord Pas de Calais,
ANR grant “click-unclick”, and the European Community.
We thank Jean-Michel Wieruszeski (CNRS) for helpful
discussions.
Supporting Information Available. Experimental pro-
cedures and characterization data for all compounds.
This material is available free of charge via the Internet
at http://pubs.acs.org.
(17) The small amounts of thioester 16b observed in Figure 1 are
formed during the acidic workup and extraction allowing removal of
MPAA.
The authors declare no competing financial interest.
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Org. Lett., Vol. XX, No. XX, XXXX