A universal strategy for preparing protected C-terminal peptides on the solid phase through an intramolecular click chemistry-based handle

Article abstract of DOI:10.1039/c2cc17222d

A new universal strategy exploits DKP formation in a dipeptide moiety whose C-terminal residue is blocked by a leaving group. It enables both synthesis of C-terminal protected peptides that are useful for convergent synthesis of large peptides and use of a C-terminal permanent protecting group that can be cleaved by catalytic hydrogenation to release the peptide. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2cc17222d

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COMMUNICATION
A universal strategy for preparing protected C-terminal peptides on the
solid phase through an intramolecular click chemistry-based handlew
a
b
b
ac
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Miriam Gongora-Benıtez, Michele Cristau, Matthieu Giraud,* Judit Tulla-Puche* and
Fernando Albericio*acd
Received 20th November 2011, Accepted 4th January 2012
DOI: 10.1039/c2cc17222d
A new universal strategy exploits DKP formation in a dipeptide
moiety whose C-terminal residue is blocked by a leaving group.
It enables both synthesis of C-terminal protected peptides that
are useful for convergent synthesis of large peptides and use of a
C-terminal permanent protecting group that can be cleaved by
catalytic hydrogenation to release the peptide.
The past decade has witnessed the so-called peptide revolution, in
which peptides have been promoted from mere biochemical
tools to real alternatives to small-molecule drugs.1 Furthermore,
peptides are becoming cornerstones of emerging fields such as
drug delivery, nanotechnology and materials.2 This is partly due
to the explosion of solid-phase peptide synthesis (SPPS), first
developed by Nobel Laureate Bruce Merrifield and then fine
tuned by numerous research groups.3 This method is now used
to prepare all peptides used in research as well as commercial
Scheme 1 Classical convergent strategy for peptide synthesis.
peptides comprising more than a few amino acids.
All synthetic strategies are based on the appropriate combination
of protecting groups together with an efficient method for
activating the carboxyl group prior to peptide coupling. While
extensive studies have yielded myriad protecting groups for
both the a-amino and the side-chain functions, which allow
synthesis of cyclic and/or complex peptides, far less effort has
been dedicated to the C-terminal function, which is blocked
with the insoluble polymer resin. Removal of the C-terminal
protecting group and concomitant liberation of the peptide
from the resin afford a free C-terminal functional group,
usually an acid or amide. However, a semi-permanent protecting
group for the C-terminal function would be desirable, as it
would enable cleavage of the peptide from the insoluble polymer
resin to render the C-terminal function protected for further
manipulation in solution.
Chemical synthesis of proteins and industrial preparation of
peptides longer than 20–30 residues are performed by a
convergent strategy in which different protected fragments
are first constructed on the solid phase and subsequently
coupled together in solution (Scheme 1).4
Protected peptides are currently prepared on a 2-Cl–
Trityl–Cl (2-CTC) resin using an Fmoc–tBu strategy. This
resin enables liberation of the protected peptide using 1–2% TFA
solution.5 A drawback of this methodology is the preparation of
C-terminal fragments whose C-terminal function requires to be
blocked. In the case of C-terminal acid peptides, this requires
reprotection of the C-terminal carboxyl group in solution, with a
consequent risk of racemization and low yields. For C-terminal
amide peptides, in addition to amidation or incorporation of the
amide form of the last residue, superlabile amide peptide resins
such as the Sieber-resin can be used.6 However, liberation of
protected peptides from these resins requires 2–5% TFA
solution, which is not totally compatible with all side-chain
protecting groups (e.g. the Trt of His).
a Institute for Research in Biomedicine, Barcelona Science Park
(PCB), Baldiri Reixac 10, 08028-Barcelona, Spain.
E-mail: albericio@irbbarcelona.org, judit.tulla@irbbarcelona.org
b Lonza Ltd, CH-3930 Visp, Switzerland.
Herein is described a new universal strategy for preparing
C-terminal protected peptides on solid phase. The strategy was
exemplified in two applications: (i) synthesis of C-terminal pro-
tected peptides useful for assembly of large peptides (convergent
approach); and (ii) use of a permanent C-terminal protecting group
that can be liberated by catalytic hydrogenation.
E-mail: matthieu.giraud@lonza.com
c CIBER-BBN, Networking Centre on Bioengineering, Biomaterials
and Nanomedicine, PCB, Baldiri Reixac 10, 08028-Barcelona, Spain
d Department of Organic Chemistry, University of Barcelona,
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Martı i Franques 1-11, 08028-Barcelona, Spain
w Electronic supplementary information (ESI) available: Experimental
details and characterization data. See DOI: 10.1039/c2cc17222d
c
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Chem. Commun., 2012, 48, 2313–2315 2313

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treatment with 0.1–0.3% TFA; if Alloc is used, then liberation
of the protected peptide is carried out with Pd(0); and if pNZ is
used, then cleavage is performed with Sn2+ 12
At this stage,
.
DKP formation does not take place. Moreover, during
successive treatments with 5% DIEA in DCM, which enables
washing/neutralization of the resin before liberation of the
protected peptide, DKP formation is negligible. This feature
is extremely important when Pd(0) is used, because extra washing
facilitates removal of any Pd salts, which can contaminate the
final product.
The absence of DKP formation during treatment of the
resin with DIEA is intriguing because, as it is well-known, in a
Boc/Bzl strategy any C-terminal sequence Aa–Pro can easily
render quantitative DKP formation after removal of the Boc
group of the Aa residue and subsequent neutralization with
5% DIEA in DCM.13 According to Goolcharran and Borchardt,14
the differences observed in the rate of DKP formation can be
explained by various factors: differences in the pKa values of the
terminal a-amino groups of the analogs, the steric bulk, the ability
of the Aa–Pro peptide bond to undergo cis–trans isomerization,
and/or the conformational stability of the resulting DKPs. In
our case, the presence of the target peptide and the linker as
the side-chain substituent of one of the DKP components can
affect said parameters—namely, by adding bulkiness to the
system and decreasing the polarity of the medium, thereby
resulting in the need for a stronger base; hampering cis–trans
isomerization of the Aa–Pro bond; and/or compromising the
conformational stability of the resulting DKP.
Scheme 2 Schematic representation of the universal DKP click
handle strategy.
Click reactions were defined by K.B. Sharpless as ‘‘those
inspired in nature, that give very high yields, generate inoffensive
side-products, are stereospecific, exhibit a large thermodynamic
driving force, have simple reaction conditions. . .’’7 In peptide
chemistry, the reaction that fits this definition is diketopiperazine
(DKP) formation, which often jeopardizes preparation of simple
C-terminal acid peptides as well as complex ones that contain
N-alkyl amino acids.8 After pioneering works of Geysen et al.9
and Atrash and Bradley,10 we have further elaborated the DKP
cleavage concept by incorporating a selectively cleavable linker
group in the DKP click handle (Scheme 2).
As a model to test our strategy, the fragment H-(17–36)-NH2 of
the drug Fuzeons (T-20) was chosen. The fragment is synthesized
in solution, by coupling of fragments Fmoc-(17–25)-OH and
H-(26–36)-NH2. The former is prepared using a 2-CTC resin,
and the latter, by coupling of H–Phe–NH2 to pre-formed
Fmoc-(26–35)-OH (also obtained using 2-CTC resin) in
solution.15 Scheme 3 shows the one-pot SPPS of C-terminal
protected fragment H-(27-36)-DKPhandle, using the new DKP
click handle strategy. The dipeptide Lys-D-Pro, the Fmoc-
Rink-amide linker, as well as the 10 Fmoc-amino acids of the
The present strategy exploits DKP formation in dipeptide
moieties whose C-terminus is blocked by a leaving group. As
the DKP itself becomes part of the permanent protecting
group of the C-terminal carboxyl function, the dipeptide
should contain a function to facilitate its connection to the
C-terminal fragment, such as the amino side-chain of Lys.z
Considering that DKP formation is favored by the presence of
an N-alkyl amino acid, which favors the cis-conformation, and
an L/D combination of amino acids, which stabilizes the
6-member ring DKP,11 a D-Pro as the other component was
chosen.y This D-Pro is attached directly to a hydroxyl resin
(e.g. hydroxy Merrifield resin). Finally, the connection between the
C-terminal fragment and the dipeptide moiety would be achieved
through a bifunctional linker (e.g. Rink-amide, Wang-type or
benzyl-type handles), which acts as a permanent protecting group,
and liberates the peptide with the expected functionality (acid or
amide) during the final global deprotection.
The cornerstone of this process is the protecting group of
the a-amino of the penultimate amino acid ( —in Scheme 2),
which dictates the conditions for releasing the protected peptide
from the resin. Thus, if Trt is used, cleavage is accomplished by
Scheme 3 Synthesis of C-terminal protected T-20 fragment
H-(27–36)-DKPhandle using the DKP click handle strategy.
c
2314 Chem. Commun., 2012, 48, 2313–2315
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further manipulation of the molecule in solution after release.
We are presently extending this strategy to side-chain anchoring,
which can involve other functional groups (e.g. hydroxyl, thiol
and carboxylic acid).
This study was partially supported by CICYT (CTQ2009-
07758), the Generalitat de Catalunya (2009SGR 1024), the
Barcelona Science Park, the Institute for Research in Bio-
medicine, and Lonza Ltd.
Notes and references
z Lysine was chosen due to its lower cost, but other amino acids with
amino side-chains were also used (see ESIw).
y Although D-Pro is an optimal component of the dipeptide part of the
handle, it can be replaced with L-Pro and other N-alkyl amino acids
(results not shown), which give similar results. Furthermore, g-amino-
Pro, which contains the two main features needed for this strategy
(an N-alkyl amino acid and an amino side-chain), can be used.
z Pyrrolidine can be used instead of piperidine.
8 BUBU Enkephalin was used to demonstrate the feasibility of this
strategy; however, this peptide can be synthesized using the 2-CTC
resin.
Scheme 4 Synthesis of BUBU Enkephalin using the DKP click
handle strategy.
fragment, were all incorporated smoothly using conventional
SPPS protocols (see ESIw). After elimination of the Fmoc
group from the last amino acid, the Trt group was removed.
The peptidyl-resin was neutralized with 5% DIEA in DCM,
and then the protected peptide was liberated by treatment with
5% piperidine in THFz, which was removed under reduced
pressure. The protected peptide was then washed with pre-cooled
Et2O. The protected T-20 fragment H-(27–36)-DKPhandle was
ready to be coupled with the fragment Boc-(17–26)-OH, which
was prepared using the 2-CTC resin, saving the two steps of the
classical convergent approach (incorporation of H–Phe–NH2
and removal of Fmoc in solution). Coupling of the fragments
using DIPCDI/HOBt, followed by global deprotection with
TFA–DMB(1,3-dimethoxybenzene)–TIS (92.5 : 5 : 2.5), afforded
the desired product, unprotected H-(17–36)-NH2, in a good
purity (see ESIw).
1 J. Reichert, Development trends for peptide therapeutics. 2010
Report Summary. Peptide Therapeutic Foundation, San Diego
(CA); Frost & Sullivan: Advances in Peptide Therapeutics (Technical
Insights), Frost & Sullivan, New York (NY), 2010.
2 C. A. E. Hauser and S. Zhang, Nature, 2010, 468, 516;
S. R. MacEwan, D. J. Callahan and A. Chiltoki, Nanomedicine,
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3 R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149; G. Barany and
R. B. Merrifield, Solid-Phase Peptide Synthesis, in The Peptides,
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vol. 2; Synthesis of Peptides and Peptidomimetics, (Houben-Weyl
E22a: Methods of Organic Chemistry)’’, ed. M. Goodman,
A. M. Felix, L. Moroder and C. Toniolo, Georg Thieme Verlag,
Stuttgart and New York, 2002; C. Haase and O. Seitz, Angew.
Chem., Int. Ed., 2008, 47, 1553.
Incorporation of a benzyl-type protecting group as a selectively
cleavable linker enables peptide synthesis through a totally acid-
free strategy, which has been exemplified by the synthesis of
BUBU Enkephalin, a highly potent and selective d-opioid agonist
whose D-Ser2 and Thr6 hydroxyl groups are tert-butylated.8
In this totally acid-free strategy, the a-amino function ( —in
Scheme 2) of Lys was protected with the Alloc group
(Scheme 4). After removal of Fmoc, 4-hydroxymethylbenzoic
acid (HMBA) was incorporated and the peptide sequence was
elongated (see ESIw). After cleavage of the protected peptide
from the resin, the benzyl-type protecting group was removed
by H2–Pd/C.
4 J. Y. Lee and D. Bang, Biopolymers, 2010, 9, 441.
5 K. Barlos, D. Gatos and W. Schaefer, Angew. Chem., Int. Ed.,
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6 P. Sieber, Tetrahedron Lett., 1987, 28, 2107.
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8 P. M. Fischer, J. Pept. Sci., 2003, 9, 9; J. Chatterjee, C. Gilon,
A. Hoffman and H. Kessler, Acc. Chem. Res., 2008, 41, 1331;
N. Bayo-Puxan, J. Tulla-Puche and F. Albericio, Eur. J. Org.
Chem., 2009, 18, 2957.
9 N. J. Maeji, A. M. Bray and H. M. Geysen, J. Immunol. Methods,
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11 G. N. Ramachandran and A. K. Mitra, J. Mol. Biol., 1976, 107, 85;
C. Grathwohl and K. Wuthrich, Biopolymers, 1976, 15, 2043;
¨
In conclusion, a new concept for protection of the C-terminus of
peptides has been developed. It overcomes some of the drawbacks
associated with SPPS, such as preparation of the C-terminal
fragment in a convergent strategy. Furthermore, it introduces more
flexibility into SPPS, by enabling the use of benzyl-type protecting
groups, which can be removed by catalytic hydrogenation. Finally,
it may enable better control in the solid-phase strategy, by allowing
D. E. Stewart, A. Sarkar and J. E. Wampler, J. Mol. Biol., 1990,
214, 253.
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12 A. Isidro-Llobet, J. Guasch-Camell, M. Alvarez and F. Albericio,
Eur. J. Org. Chem., 2005, 3031.
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13 M. Gairı, P. Lloyd-Williams, F. Albericio and E. Giralt, Tetrahedron
Lett., 1990, 31, 7363.
14 C. Goolcharran and R. T. Borchardt, J. Pharm. Sci., 1998, 87, 283.
15 B. Bray, Nat. Rev. Drug Discovery, 2003, 2, 587.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 2313–2315 2315