Epimerization-free C-terminal peptide activation

Article abstract of DOI:10.1002/chem.201303347

Smooth operation: C-terminal peptide activation with full stereointegrity was accomplished using a copper(II)-mediated coupling reaction of carboxylic acids with arylboroxines (see scheme, NCL = native chemical ligation, Boc = tert-butoxycarbonyl). This method allows epimerization-free activation and ligation of peptides with racemization-prone phenylglycine at the C terminus.

Full text of DOI:10.1002/chem.201303347

DOI: 10.1002/chem.201303347
Epimerization-Free C-Terminal Peptide Activation
Stanimir Popovic,[a] Hans Bierꢀugel,[a] Remko J. Detz,[b] Alexander M. Kluwer,[b]
Jelmer A. A. Koole,[a] Dieuwertje E. Streefkerk,[a] Henk Hiemstra,[a] and
Jan H. van Maarseveen*[a]
Peptides constitute a very important compound class in
drug research.[1] Thus, racemization-free peptide synthesis is
of key importance to acquire the required peptide chains in
diastereomerically pure form. In general, the way to synthe-
size peptides with full stereointegrity is by elongation at the
N terminus.[2] On the other hand, methods that allow ep
merization-free C-terminal peptide activation would greatly
ACHTUNGERTNiNUNG -
A
ACHTUNGTRENNUNG
enhance the available routes and would be a highly valuable
synthetic tool for obtaining the desired peptides.[3] Especial-
ly for the convergent solution-phase synthesis of peptides by
segment coupling a reliable and epimerization-free C-termi-
nal activation methodology is required.[4] Recently, Dani-
shefsky showed the utility of peptide 4-nitrophenyl esters in
peptide segment couplings.[5] These esters were prepared by
EDCl/HOBt-mediated coupling of the amino acid 4-nitro-
phenylesters to the C terminus of peptides.[5b] Due to other-
wise inevitable epimerization this methodology is restricted
by the requirement of a glycine residue at the C-terminal
penultimate position. Herein, we disclose our initial results
on the development of a racemization-free C-terminal pep-
tide activation through the copper(II)-mediated Chan–Lam-
type coupling between peptides and arylboroxines and sub-
sequent amine-coupling reactions (Scheme 1).[6]
Scheme 1. Copper(II)-mediated and classical peptide arylester synthesis
and subsequent elongation.
To evaluate and optimize the Chan–Lam-type esterifica-
tion of peptides, Boc-Trp-Phe-OH was selected as the sub-
strate. The first coupling attempt was performed using the
conditions reported by Cheng and co-workers, that is, stir-
ring of the carboxylic acid, phenylboronic acid (3 equiv),
CuACTHNUGTRENNG(U OTf)2 (0.4 equiv), and urea (1 equiv) at elevated temper-
Recently, the group of Cheng reported the Cu
(OTf)2-
ature (608C) under air using EtOAc as the solvent. Gratify-
ingly, after running the reaction for 12 h, Boc-Trp-Phe-OPh
could be isolated in a yield of 40%. The reaction proceeded
in a rather clean fashion, and besides the recovered peptide
phenol and diphenyl ether were isolated as the main side
products. It should also be noted that all three nitrogen
atoms bearing acidic protons, that is, the indole-NH, amide-
NH and Boc-NH, showed no reactivity under these mild
conditions. After screening several other bidentate N-cen-
tered ligands, ureas stood out as the ligand class of choice.
Replacing urea with 1,3-diethylurea gave a homogeneous re-
action mixture and the yield further improved to 50%. The
formation of phenol could be suppressed by starting from
phenylboroxine, further enhancing the yield to 69%. By de-
creasing the concentration of the carboxylic acid to 0.05m,
slightly increasing the temperature to 658C, and adding
Et3N as the base (1 equiv), yields of up to 82% were ob-
mediated reaction of benzoic acids with arylboronic acids to
provide facile access to arylesters.[7] The single example of
an aliphatic acid, that is, phenylacetic acid, that reacted effi-
ciently with phenylboronic acid to give the ester in near
quantitative yield prompted us to expand this chemistry to-
wards the peptide series. The suggested mechanistic course
of this reaction and the mild conditions make epimerization
very unlikely.[8] Therefore, the Chan–Lam approach may be
a suitable method for activation of peptides bearing a chiral
C-terminal amino acid.
[a] S. Popovic, H. Bierꢀugel, J. A. A. Koole, D. E. Streefkerk,
Prof. Dr. H. Hiemstra, Dr. J. H. van Maarseveen
Van ꢁt Hoff Institute for Molecular Sciences
University of Amsterdam, Science Park 904
1098XH Amsterdam (The Netherlands)
E-mail: j.h.vanmaarseveen@uva.nl
tained, albeit now requiring 1 equiv of CuACHTUNGTRENNUNG(OTf)2
Homepage: http://hims.uva.nl/soc
(Scheme 2). To check the stereointegrity of the copper(II)-
catalyzed peptide esterification, Boc-Trp-d-Phe-OPh was
prepared in 70% isolated yield, and after comparison of the
1H NMR spectra and chiral HPLC traces with Boc-Trp-Phe-
[b] Dr. R. J. Detz, Dr. A. M. Kluwer
InCatT, Science Park 904, 1098XH Amsterdam (The Netherlands)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201303347.
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Chem. Eur. J. 2013, 19, 16934 – 16937

COMMUNICATION
er to the sulfoxide was performed in quantitative yield using
mCPBA as the oxidizing agent. However, further oxidation
to the sulfone was not possible without affecting the trypto-
phan indole moiety. Fortunately, the synthesis of Boc-Phe-
Phe-4-(methylsulfonyl)phenyl ester proceeded without prob-
lems in 77% overall yield (entry 2), now using oxone as the
oxidizing agent, the remainder of which was easily removed
by non-basic aqueous extraction. To rule out the occurrence
of epimerization in the Chan–Lam/oxidation reaction se-
Scheme 2. Optimized copper(II)-mediated peptide arylester synthesis.
Reagents and conditions: (PhBO)3 (1 equiv), Cu(OTf)2 (1 equiv), Et3N
(1 equiv), 1,3-diethylurea (2 equiv), air, THF or EtOAc, 658C, 8 h.
AHCTUNGTRENNUNG
quence, the diastereomeric Boc-Phe-d-Phe-4-(methyl
ACHTUNGTRENNUNGsulACHTUNGTRENNUNGfo-
AHCTUNGTRENNUNG
al stereointegrity of this sequence was further demonstrated
by the synthesis of two epimeric dipeptides containing the
very racemization-prone phenylglycine at the peptide C ter-
minus. The optical purity of both Boc-Phe-Phg-4-(methylsul-
fonyl)phenyl ester and Boc-Phe-d-Phg-4-(methylsulfonyl)-
phenyl ester remained 99.9% (entries 4 and 5). Couplings at
sterically hindered positions offer another major challenge
in peptide synthesis. Recently, Danishefsky and co-workers
showed the efficiency of arylesters in sterically congested
cases, such as elongation of peptides bearing a C-terminal
valine.[5] According to the postulated mechanism of the
Chan–Lam reaction, the ester is obtained via s-bond forma-
tion between a carboxylic acid O atom and the aryl C atom
OPh no sign of epimerization was observed (de values of
99.8 and 97.7% for the ll- and ld-diastereoisomers, respec-
tively). This result is in stark contrast with the loss of stereo-
control observed after esterification of Boc-Trp-d-Phe-OH
with phenol using HATU—especially known for its low ra-
ACHTUNGTRENNUNGceACHTUNGTRENNUNGmization tendency—as the carboxyl activating agent, pro-
viding a 2:1 mixture of Boc-Trp-d-Phe-OPh and Boc-Trp-
Phe-OPh (data not shown).
Although unsubstituted arylesters have been used directly
in peptide ligation reactions, their mediocre reactivity re-
quires large amounts of activating additives lowering the
practicability.[9] Unfortunately, so far, all attempts to react
electron-poor arylboroxines that should deliver highly acti-
vated esters, such as the pentafluorophenyl or 4-nitrophenyl,
have given low yields. To overcome this, the 4-methylthio-
phenyl ester was considered, which can be transformed into
the 4-(methylsulfonyl)phenyl ester by oxidation. Previous
work has shown that 4-(methylsulfonyl)phenyl esters are
very suitable as optically stable activated esters in peptide
connected to the copperACTHNUGRTENUNG(III) species, which is one bond fur-
ther away from a potentially sterically hindered amino acid
a-carbon atom compared with classical ester formation
using peptide coupling reagents. Copper(II)-mediated reac-
tion of Boc-Phe-Val-OH with 4-(methylthio)phenyl borox-
ine using the optimized conditions gave the corresponding
ester in 68% yield (entry 6). Next, oxidation of the Chan–
Lam product with oxone gave Boc-Phe-Val-4-(methylsulfo-
elongation chemistry.[10] Cu
ACTHNUTRGENUN(G OTf)2-mediated coupling of
Boc-Trp-Phe-OH with 4-(methylthio)phenyl boroxine using
the optimized conditions gave the ester in the isolated yield
of 57% (Scheme 3, entry 1). Partial oxidation of the thioeth-
nyl)phenyl ester in quantitative yield. Also, Boc-TrpACHTUNGTRENNUNG(Boc)-
Val-4-(methylsulfonyl)phenyl ester was prepared in 70%
overall yield (entry 7). It is worth mentioning that due to
the presence of the indole-N-Boc protective group oxidation
to the sulfone proceeded uneventfully. Boc-Phe-Ala-4-
(methylsulfonyl)phenyl ester was prepared in 83% overall
yield (entry 8). Finally, also the tripeptide Boc-AspACHTUNGTRENNUNG(tBu)-
Phe-Phe-OH was esterified and oxidized in an overall yield
of 55% (entry 9).
Next, the peptide 4-(methylsulfonyl)phenyl esters were
evaluated in native chemical ligation (NCL) reactions.[11]
Treatment of Boc-Phe-Phe-4-(methylsulfonyl)phenyl ester
with excess cysteine only needed 1 h to reach full conversion
(Scheme 4a). This high rate is in accordance with results ob-
tained by Danishefsky starting from peptide 4-nitrophenyl
esters.[10] To overcome oxidative side reactions with the cys-
teine thiol moiety, after NCL capping by iodoacetamide was
performed allowing facile purification giving Boc-Phe-Phe-
Cys
ACHTUNGTRENNUNG
Phe-d-Phe-CysAHCTUNGTRENNUNG
Chiral HPLC and 1H NMR analysis revealed 98% de for
both diastereoisomers. Gratifyingly, also phenylglycine 4-
(methylsulfonyl)phenyl esters gave smooth NCL reactions
with cysteine and both Boc-Phe-Phg-Cys-OH and Boc-Phe-
d-Phg-Cys-OH could be isolated in yields of 68 and 66%,
Scheme 3. Peptide activated arylester synthesis.
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J. H. van Maarseveen et al.
Scheme 5. Direct peptide arylester aminolysis. A) H-AA-OtBu, THF, RT,
2–3 d; B) H-AA-OtBu (2.1 equiv, slow addition), THF, RT, 2–3 d;
C) HCl·H-AA-OtBu (1.1 equiv), DiPEA (2 equiv, slow addition), THF,
RT, 2–3 d; D) H-AA-OtBu (2.1 equiv), DMF, RT, 45 min (entry 5) or
18 h (slow addition, entry 11); E) tetrapeptide tert-butyl ester (1.1 equiv,
slow addition), THF, RT, 3–4 d.
Scheme 4. NCL reactions from peptide arylesters. Conditions and re-
agents: i) HCl·H-Cys-OMe, 0.2m Na2HPO4, 50 mm TCEP, pH 6.5, MeCN;
ii) 0.2m Na2HPO4, 50 mm TCEP, iodoacetamide, pH 7.5; iii) cysteine,
0.2m Na2HPO4, 50 mm TCEP, pH 6.5, MeCN (final pH 6.75); iv) 0.2m
Na2HPO4, 50 mm TCEP, 6m Gu·HCl, pH 7.5, MeCN (final pH 6.9).
respectively, in diasteromerically pure form (Scheme 4b).
Because peptide 4-(methylsulfonyl)phenyl esters only react
slowly with amines under neutral aqueous coupling condi-
tions (vide infra), they allow the presence of a free N termi-
nus for a short time. This facilitates their use as the N-termi-
nal fragment in NCL of fully unprotected peptides. To show
this, H-Asp-Phe-Phe-4-(methylsulfonyl)phenyl ester was li-
gated with H-Cys-Lys-OH to give, after purification by
preparative RP-HPLC, the pentapeptide H-Asp-Phe-Phe-
Cys-Lys-OH in 45% yield (Scheme 4c).
Because NCL with apolar peptides can be problematic
due to the poor solubility in aqueous buffered solutions, also
the direct aminolysis in organic solvents was investigated
(Scheme 5). Based on published epimerization experiments
of peptide esters, THF was selected as the solvent of
choice.[12] At first, Boc-Phe-Phe-4-(methylsulfonyl)phenyl
ester was treated with H-Gly-OtBu to give, after standing
for 1 day, Boc-Phe-Phe-Gly-OtBu in an isolated yield of 90
and 86% de (entry 1). To avoid epimerization, the basic H-
Gly-OtBu (2 equiv) had to be added by slow addition to
give the product in 91% yield and 99% de (entry 2). By
using the same slow addition conditions, Boc-Phe-d-Phe-
Gly-OtBu was isolated in a slightly lower yield of 85% with-
out any sign of epimerization (entry 3). Also, the HCl-salt
could be used as the starting material for which the amine
was liberated by slow addition of a base (DiPEA, 2 equiv;
entry 4). The choice of the solvent is crucial indeed for the
outcome of the reaction. By replacing THF with DMF, a dra-
matic aminolysis rate increase was observed giving full con-
version in 45 min only. However, the tripeptide Boc-Phe-
Phe-Gly-OtBu was isolated in the unacceptable 52% de
(entry 5). As could be expected, for peptide elongation
through a direct aminolysis reaction phenylglycine proved
to be sensitive to base-induced epimerization (entries 6 and
7). Both Boc-Phe-Phg-4-(methylsulfonyl)phenyl ester and
Boc-Phe-d-Phg-4-(methylsulfonyl)phenyl ester gave some
epimerization after treatment with H-Gly-OtBu to produce
the corresponding tripeptides in the yields of 86 and 75%,
respectively, and a de of 81% for both compounds. We were
glad to see that also the sterically congested H-Val-OtBu
could be used for the aminolysis reactions (entries 8–11).
Both slow addition of H-Val-OtBu to Boc-Phe-Phe-4-(meth-
ylsulfonyl)phenyl ester or liberation of the free amine of
HCl·H-Val-OtBu by slow addition of DiPEA gave the corre-
sponding tripeptides in high yields and optical purities. Also,
with H-Val-OtBu loss of de to 85% was observed when
using DMF as the solvent. Therefore, segment couplings of
two short peptides were achieved in THF giving the fully
protected hexapeptides Boc-Phe-Phe-Ala-Gly-Trp-Val-OtBu
and its epimer Boc-Phe-d-Phe-Ala-Gly-Trp-Val-OtBu in the
isolated yields of 85 and 80%, respectively, as pure stereo-
isomers (entries 12 and 13).
In conclusion, the copper(II)-mediated Chan–Lam reac-
tion is a versatile methodology for epimerization-free C ter-
minus peptide elongation and thus opens new horizons in
peptide synthesis. The peptide carboxylic acids were trans-
formed into peptide arylesters with complete stereointegrity.
This robust method allowed the synthesis of 4-(methylthio)-
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Chem. Eur. J. 2013, 19, 16934 – 16937

Epimerization-Free C-Terminal Peptide Activation
COMMUNICATION
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Keywords: aminolysis · aryl esterification · copper · peptide
activation · peptides
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Received: August 27, 2013
Published online: November 7, 2013
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