Triblock peptide and peptide thioester synthesis with reactivity- differentiated sulfonamides and peptidyl thioacids

Full text of DOI:10.1002/anie.200903050

Angewandte
Chemie
DOI: 10.1002/anie.200903050
Chemical Ligation
Triblock Peptide and Peptide Thioester Synthesis With
Reactivity-Differentiated Sulfonamides and Peptidyl Thioacids**
David Crich* and Indrajeet Sharma
Dedicated to Professor A. Paul Schaap
With the construction of peptides by solid-phase peptide
synthesis limited, for practical reasons, to chains of around
fifty residues,[1] the development of methods for the assembly
of peptide blocks into longer sequences is of importance.
Kentꢀs concept[2] of native chemical ligation was a major
advance in this area, and has been considerably extended and
optimized since its introduction in 1994.[1,3] A significant
number of these improvements have addressed the develop-
ment of methods for peptidyl thioester synthesis and the
limitations posed by the mechanistic requirement of using an
N-terminal 2-mercaptoethylamine, typically cysteine.[4] The
Scheme 1. Amide-forming reaction.
most important modification, however, was the introduction,
by Kent and co-workers,[5] of the thiazolidine group as a
means of protection for N-terminal cysteine moieties. This
approach is compatible with the native chemical ligation, and
permits the block assembly of three or more peptides into a
single entity. Our group has been engaged in the development
of an alternative method of block synthesis for peptides in
which a C-terminal peptidyl thioacid reacts with an electron-
deficient N-terminal sulfonamide to yield a native amide
bond.[5] The mechanism of this reaction, which is not limited
to the use of any particular amino acid, involves nucleophilic
aromatic substitution by the thiocarboxylate on the electron-
deficient sulfonamide to give a highly reactive thioester and,
after loss of sulfur dioxide, an amine leading ultimately to the
amide product (Scheme 1). A variant on the method employs
a free amine and an electron-deficient aryl halide, such as the
Sanger or Mukaiyama reagents, as the condensing agent in
place of the sulfonamide.[7,8]
Scheme 2. Triblock peptide synthesis.
To convert this method into one capable of enabling the
controlled coupling of three blocks into a single segment with
minimal protecting-group manipulation we required a set of
two sulfonamides with differential reactivity toward thiocar-
boxylates (Scheme 2).
A series of N-sulfonylphenylalanine derivatives was
therefore prepared (see Supporting Information) and
screened for reactivity toward thioacetic acid under a
standard set of conditions related to those used in our peptide
synthesis (Table 1).
Under the conditions employed, a single electron-with-
drawing group was found to be insufficient to induce reaction,
as was the presence of two trifluoromethyl groups in the 3-
and 5-positions. However, 2,4-disubstituted systems in which
a single nitro group was complemented by a second, but less
potent electron-withdrawing group functioned well (Table 1).
Nevertheless, all three such systems investigated (denoted as
ENS, CNS, and FNS) proved significantly less reactive than
the 2,4-dinitrobenzenesulfonamide (DNS) employed origi-
nally, and therefore met our reactivity criteria.
[*] Prof. Dr. D. Crich
Centre de Recherche de Gif, Institut de Chimie des Substances
Naturelles, Centre National de la Recherche Scientifique, Avenue de
la Terrasse, 91198 Gif-sur-Yvette (France)
Fax: (+33)1-6907-7752
E-mail: dcrich@icsn.cnrs-gif.fr
Prof. Dr. D. Crich, I. Sharma
Department of Chemistry, Wayne State University
5101 Cass Avenue, Detroit, MI 48202 (USA)
[**] We thank the NIH (GM62160) for partial support of this work and
Albert A. Bowers for preliminary experiments with heteroaryl
sulfonamide systems.
A further series of experiments with more elaborate
thioesters revealed the reactivity of both the CNS and ENS
sulfonamides to be adequate for coupling with primary
thioacids but not with electron-deficient or more hindered
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200903050.
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Table 1: Reactivity of thioacetic acid towards N-sulfonylphenylalanine
derivatives.[a]
This overall reactivity pattern was sufficient to enable a
first series of triply convergent reactions in which an amino
acid or peptide, protected at the N-terminus by an ENS or
CNS group and carrying a 2,4,6-trimethoxybenzyl[9] (Tmob)
thioester at the C-terminus, was first treated with triethylsi-
lane and trifluoroacetic acid to release the C-terminal
thioacid before exposure to a DNS-protected peptide and a
mild base (Table 2). This first coupling resulted in the
formation of a new peptide bearing the ENS or CNS group
at the N-terminus ready for a final coupling with a thioacid,
albeit necessarily a primary one (Table 2). This critical series
of experiments established the feasibility of generation of a
thioacid in the presence of a moderately electron-deficient
sulfonamide and the ability of that thioacid to undergo
subsequent and selective condensation with the more reactive
DNS class of sulfonamides. The reaction sequence was
applied successfully to the synthesis of simple di- and
tripeptides (Table 2) and to the synthesis of model octapep-
tides (Table 2, entries 5 and 6). Finally, the sequence was
shown to be amenable to the use of aqueous buffered media,
rather than DMF as solvent, with little loss of yield as is clear
from a comparison of entries 4 and 7 of Table 2.[10]
Entry
R1
R2
R3
R4
t [h]
Yield [%]
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
CF3
H
H
H
H
Ac
CN
H
H
H
NO2
NO2
CF3
NO2
H
H
H
H
CF3
H
H
H
H
12
12
12
12
12
6
–
–
–
–
CF3
NO2
H
COOMe
CN
–
85
87
88
94
6
4
NO2
NO2
0.07[b]
[a] All reactions employed 0.15m sulfonamide in DMF with thioacetic
acid (1.5 equiv), and Cs2CO3 (1.5 equiv) as base (See Supporting
Information). [b] 4 min.
Further investigation revealed the FNS group to be
somewhat more reactive than the CNS and ENS groups
toward the less reactive a-amino-derived thiocarboxylates as
illustrated by the examples in Scheme 5. A critical point in the
use of the FNS group in this manner, however, was the switch
from cesium carbonate to cesium hydrogencarbonate at the
level of the first coupling reaction.[11]
ones. Thus, while both the CNS and ENS class of sulfona-
mides were amenable to reaction with primary thioacids
(Schemes 3 and 4) they either failed to react or reacted only
very slowly with peptide-based thioacids such as Alloc-Gly-
Gly-SH.
Scheme 5. Tricomponent couplings employing the FNS group. TFA=
trifluoroacetic acid, DCM=dichloromethane.
Scheme 3. Three-component coupling.
The block synthesis strategy that we present here is
complementary to the methods developed by Kent
based on native chemical ligation. However, for
maximum flexibility in approaching future targets,
the ability to incorporate both approaches into a
single scheme is desirable. For this, the compatibility
of thioesters with our thiocarboxylate–sulfonamide
coupling approach is required. The triblock synthesis
set out in Scheme 6, in which a fluorenylmethyl
(Fm)[6a] thioester is carried through two coupling
steps, nicely illustrates that such is the case.
In addition to the “right to left” strategy for block
peptide synthesis set out above, with its necessary
reliance on the use of a series of sulfonamides of
decreasing reactivity, we have briefly investigated an
Scheme 4. Reaction with glutamic and aspartic acid side chain thioacids.
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Table 2: Triply convergent synthesis.
Entry Peptide 2-
STmob
DNS-Peptide 1
Peptide 2-Peptide 1
(yield [%])
RCO-SH
RCO-Peptide 2-Peptide 1
(yield [%])
1
2
ENS-Trp-STmob DNS-Phe-OMe
CNS-Phe-STmob DNS-Phe-OMe
ENS-Trp-Phe-OMe
CNS-Phe-Phe-OMe (84)
Ac-SH
Boc-Glu(SH)-
OtBu
Ac-Trp-Phe-OMe (60)
Boc-Glu(Phe-Phe-OMe)-OtBu (81)
3
CNS-Phe-STmob DNS-Phe-OMe
CNS-Phe-Phe-OMe (84)
Boc-Asp(SH)- Boc-Asp(Phe-Phe-OMe)-OBn (78)
OBn
4
ENS-Ala-Phe-
STmob
DNS-Phe-OMe
ENS-Ala-Phe-Phe-OMe (81)
Boc-Glu(SH)-
OtBu
Boc-Glu(Ala-Phe-Phe-OMe)-OtBu (83)
5
ENS-Ala-Phe-
STmob
DNS-Val-Met-Val-Pro-
Ala-OEt
ENS-Ala-Phe-Val-Met-Val-Pro-Ala- Boc-Glu(SH)-
Boc-Glu(Ala-Phe-Val-Met-Val-Pro-Ala-OEt)-
OtBu (71)
OEt (81)
OtBu
6
ENS-Ala-Val-
STmob
ENS-Ala-Phe-
STmob
DNS-Val-Met-Val-Pro-
Ala-OEt
DNS-Phe-OMe
ENS-Ala-Val-Val-Met-Val-Pro-Ala-
OEt (78)
ENS-Ala-Phe-Phe-OMe (74)
Boc-Glu(SH)-
OtBu
Boc-Glu(SH)-
OtBu
Boc-Glu(Ala-Val-Val-Met-Val-Pro-Ala-OEt)-
OtBu (65)
Boc-Glu(Ala-Phe-Phe-OMe)-OtBu (80)
7[a]
[a] Both coupling steps in this example were conducted in an aqueous buffer: 4:1 v/v NMP: 6m Gn·HCl, 1m HEPES, pHꢀ8, NMP=N-
Methylpyrrolidone; Gn=guanidine.
Overall, we present a combination of versatile new
methods for the block synthesis of peptides based on the
reaction of thioacids with electron-deficient sulfonamides.
The assembly of the various blocks may be conducted in a
“right to left” or “left to right” manner and may be arranged
in such a way as to provide a peptide thioester ready for
incorporation in a native chemical ligation sequence.
Received: June 6, 2009
Revised: August 11, 2009
Published online: September 8, 2009
Scheme 6. Compatibility of the thioesters with the thioacid–sulfona-
mide block synthesis.
Keywords: chemical ligation ·
nucleophilic aromatic substitution · peptide synthesis ·
thioacids · thioesters
.
alternative “left to right” strategy. This approach enables the
more reactive DNS sulfonamide to be employed in all
coupling steps but requires the compatibility of the Tmob
thioester function with the sulfonamide coupling reaction.
The triblock syntheses set out in Scheme 7 show such a
sequence and thereby establish the compatibility of the Tmob
thioester. Again with a view to potential interconnection with
a native chemical ligation strategy, one of the examples is
terminated by the incorporation of a further thioester.
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Scheme 7. “Left to right” strategy showing compatibility with thioesters.
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[10] Within the limits of our NMR and UPLC detection methods the
couplings reported in this manuscript proceed without racemi-
zation. In preliminary studies on the thioacid–sulfonamide
method, and on the related thioacid–amine–Sangers/
Mukaiyama approach, this was rigorously established through
the synthesis of authentic samples of epimeric products.[6a, 7]
[11] With cesium carbonate the N-FNS protected di- and higher
peptide thioacids were observed by mass spectrometry to
undergo cyclization, with retention of the FNS group, rather
than condensation when exposed to a DNS peptide and cesium
carbonate. This was attributed to the acidity of the FNS
sulfonamide NH group and so was circumvented by the use of
the milder base. The use of pyridine as base afforded a similar
result to cesium carbonate.
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