A general approach for the non-stop solid phase synthesis of TAC-scaffolded loops towards protein mimics containing discontinuous epitopes

Article abstract of DOI:10.1039/b817357e

In this Communication, the access to three different peptide loops attached to a small triazacyclophane (TAC) scaffold molecule for the mimicry of discontinuous epitopes present in, for example, antibodies is described for the first time. The Royal Societ

Full text of DOI:10.1039/b817357e

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COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A general approach for the non-stop solid phase synthesis of
TAC-scaffolded loops towards protein mimics containing
discontinuous epitopesw
Cristina Chamorro,a John A. W. Kruijtzer,a Marina Farsaraki,a Jan Balzarinib
and Rob M. J. Liskamp*a
Received (in Cambridge, UK) 3rd October 2008, Accepted 11th November 2008
First published as an Advance Article on the web 17th December 2008
DOI: 10.1039/b817357e
In this Communication, the access to three different peptide
loops attached to a small triazacyclophane (TAC) scaffold
molecule for the mimicry of discontinuous epitopes present in,
for example, antibodies is described for the first time.
One of the greatest challenges in the construction of protein
mimics is mimicry of the discontinuous epitope nature of
Fig. 1 (Sequential) continuous and discontinuous epitopes.
proteins. Discontinuous epitopes are peptide segments that
are located relatively closely in a protein’s spatial structure,
but can be located very distantly in its amino acid sequence
(Fig. 1). This is in contrast to continuous epitopes, which
consist of a single contiguous strand of amino acids.
Discontinuous epitopes are abundantly present in proteins
and are especially prominent at protein–protein interaction
sites. The most outstanding examples are probably the
discontinuous epitopes comprising the complementarity
determining region (CDR) loops of antibodies (see, for
example, Fig. 2). However, many other protein–protein inter-
actions include discontinuous epitopes, for example, those
present in the crucial HIV-gp120–CD4 interactions (Fig. 2),
in which we are interested.1
Fig. 2 The CDR loops involved in the interaction of Mab (1G7J) and
a lysozyme (left), and the gp120–CD4 interaction (middle and right;
overview and detail, respectively).1
These examples might lead to the fair assumption that when
an adequate mimicry of discontinuous epitopes can be
realized, efficient synthetic vaccines of, for example, gp120,
or even mimics of antibodies (‘‘synthetic antibodies’’), come
within reach.
Crucial for an adequate mimicry of discontinuous epitopes
in a protein is the availability of a molecular scaffold that is
capable of replacing most of the protein, and providing a
proper arrangement, orientation and perhaps even pre-
organization of the peptide segments that comprise the
discontinuous epitopes (Fig. 3). Another important character-
istic that is required for adequate mimicry is that the peptide
segments on the molecular scaffold should be a part of cyclic
peptides or peptide loops, since the discontinuous epitopes, for
example those in the CDRs, are present as loops. In some
cases, linear peptides might be satisfactory, and we have
Fig. 3 The replacement of most of a protein by a triazacyclophane
(TAC) scaffold and the mimicry of its discontinuous epitopes.
shown that this is indeed possible.2,3 However, for the best
possible loop mimicry, one would expect a cyclic peptide to be
superior.
A loop structure would also provide more
pre-organization than a linear peptide, which might also be
beneficial for its interactions.
From a chemical point of view, the molecular scaffold
should be robust, conveniently synthesized and preferably
accessible on a multigram scale. Furthermore, it is essential
that the different molecular fragments, i.e. the peptides, can be
introduced in an equally facile manner. Last but not least, a
handle for solid phase attachment or for the attachment of
auxiliary groups (e.g. a label) should be present.
a Medicinal Chemistry & Chemical Biology, Utrecht Institute of
Pharmaceutical Sciences, Faculty of Science, Utrecht University,
P.O. Box 80082, NL-3508 TB Utrecht, The Netherlands.
E-mail: r.m.j.liskamp@uu.nl; Fax: +31 30-2536655;
Tel: +31 30-2537396; www.pharm.uu.nl/medchem
b Rega Institute for Medical Research, K.U. Leuven,
Minderbroederstraat 10, B 3000 Leuven, Belgium
w Electronic supplementary information (ESI) available: Abbrevia-
tions and experimental details. See DOI: 10.1039/b817357e
Several scaffolds for peptides have been described in the
literature, of which the cyclic peptide, template assisted
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synthetic protein (TASP), is probably the most outstanding.4
This scaffold was introduced by Mutter and Vuilleumier in
their seminal work almost twenty years ago.5 Although there
have been many successful applications of TASP,4 we have
developed—in view of the above mentioned requirements—
the TAC scaffold, which can be conveniently synthesized on a
large scale.6 So far, this scaffold has been used for the
construction of synthetic receptor molecules,7 in an effective
mimic of the papain inhibitory protein,2 in an approach
towards a synthetic vaccine of pertussis3 and towards a mimic
of the copper-binding site of a protein.8
Crucial to the success of an approach to sequentially
introduce three peptide loops onto the TAC scaffold in a
continuous solid phase synthesis procedure is the availability
of an additional orthogonal protecting group, which can be
cleaved in the presence of the TAC protecting groups when-
ever cyclization to the individual peptide loop has to take
place. We found that the PTMSE group introduced by
Wagner and Kunz was highly suitable for this purpose, and
completely orthogonal to the o-NBS, Aloc and amino acid side
chain protecting groups.10 Therefore, it could be successfully
applied to the synthesis of cyclic peptides, independent of their
length and sequence.
In this Communication, we wish to describe a general
approach for the introduction of cyclic peptides onto the
TAC scaffold in a continuous solid phase synthesis. This
method provides access to protein mimics that contain up to
three different discontinuous epitopes, as are present in, for
example, antibodies (Fig. 3).
Thus, as a hinge for cyclization, a glutamic acid residue
containing the PTMSE group was used, i.e. Fmoc–
Glu(OPTMSE)–OH, which could be prepared on a multigram
scale from commercially available Cbz–Glu–OBzl.
This set the stage for the non-stop solid phase synthesis of
TAC-scaffolded loops derived from gp120 towards a protein
mimic containing discontinuous epitopes (Scheme 1). After
loading Tentagel S-RAM resin with the Fmoc–o-NBS–
Aloc-protected TAC scaffold, 1,6 construction of the first loop
using automatic peptide synthesis was begun by first introdu-
cing two glycine residues as a spacer.11 This was followed by
synthesis of the peptide arm sequence corresponding to resi-
dues 364–372 in loop 1 of gp120, leading to 3. After removal of
the PTMSE group with TBAF, an on-resin tail-to-side chain
cyclization to 4 was achieved using BOP.12 Next, o-NBS
removal by b-mercaptoethanol and DBU to give 5 was
followed by automatic peptide synthesis of the second peptide
As a promising target, we selected a mimic of the discontin-
uous epitope formed by the three loops of gp120 interacting
with the CD4 receptor.1 An effective mimic of this ensemble
might provide access towards a synthetic vaccine candidate or
even lead to a (protein) mimic of gp120, which might be
capable of interfering with the binding of HIV-gp120 to the
cellular CD4 receptor. Thus, based on the X-ray structure of
the gp120–CD4 complex and using data from relatively
straightforward molecular modelling, we selected the amino
acid loops 364SSGGDPEIV372
,
424INMWQKVG431 and
454LTRDGGN460 of gp120 for incorporation onto the TAC
scaffold.1,9
Scheme 1 The non-stop solid phase synthesis of TAC-scaffolded loops towards protein mimics containing discontinuous epitopes
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Fig. 4 The characterization of protein mimic 9 by MALDI-TOF (left), ES-MS (middle) and analytical HPLC (top right: TFA, acetonitrile, water;
bottom right: NH4OAc, acetonitrile, water). Mono-isotopic mass [M + H]+ calculated for C149H228N45O49S: 3463.65; found: 3463.58.
arm, comprising residues 424–431 in loop 2 of gp120. Upon
completion, the PTMSE group was removed, and cyclization
was effected with BOP to form the second loop mimic in 6.12
Introduction of the third loop took place after palladium-
assisted cleavage of the Aloc group, leading to 7, and
successful introduction of the third peptide arm, comprising
residues 454–460, was realized. The final cyclization was
performed after deprotection of the glutamic acid hinge, and
the resulting resin-bound gp120 discontinuous epitope mimic,
8, was ready for cleavage and simultaneous deprotection.
Thus, after acidolysis, the free gp120 mimic, 9, was obtained
as its trifluoroacetate salt in high purity (499%) after pur-
ification by preparative HPLC (Fig. 4).13
Financial support by the council for Chemical Sciences of
the Netherlands Organisation for Scientific Research
(CW-NWO) and by the GOA (Krediet nr. 05/19) of the
KULeuven. We thank Annemarie Dechesne for recording
the mass spectra and Anton Bunschoten for assistance with
the molecular modelling. The technical assistance of Mrs Leen
Ingels is greatly appreciated.
Notes and references
1 P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski and
W. A. Hendrickson, Nature, 1998, 393, 648.
2 D.-J. van Zoelen, M. R. Egmond, R. J. Pieters and R. M. J.
Liskamp, ChemBioChem, 2007, 8, 1950.
The identity of 9 was confirmed by MALDI-TOF and
ES-MS. The overall yield of the discontinuous epitope mimic
by this non-stop solid phase approach was 0.17% (ca. 1.5 mg),
which corresponds to an average yield of 97% per step
(see the ESIw).
The synthetic approach described in this paper allowed
reliable and convenient access for the first time to three
different peptide loops attached to a small TAC scaffold
molecule, leading to a protein mimic containing discontinuous
epitopes.
3 M. Hijnen, D.-J. van Zoelen, C. Chamorro, F. R. Mooi, P. van
Gageldonk, G. Berbers and R. M. J. Liskamp, Vaccine, 2007, 25,
6807.
4 For a recent review, see: Y. Singh, G. T. Dolphin, J. Razkin and
P. Dumy, ChemBioChem, 2006, 7, 1298.
5 M. Mutter and S. Vuilleumier, Angew. Chem., Int. Ed. Engl., 1989,
28, 535.
6 T. Opatz and R. M. J. Liskamp, Org. Lett., 2001, 3, 3499.
7 T. Opatz and R. M. J. Liskamp, J. Comb. Chem., 2002, 4, 275.
8 H. B. Albada, F. Soulimani, B. M. Weckhuysen and R. M. J.
Liskamp, Chem. Commun., 2007, 4895.
9 Recently, Eichler et al. attached similar, albeit linear, peptides of
gp120 for the design of immunogens: R. Franke, T. Hirsch,
H. Overwin and J. Eichler, Angew. Chem., Int. Ed., 2007, 46, 1253.
10 (a) M. Wagner and H. Kunz, Synlett, 2000, 400; (b) M. Wagner
and H. Kunz, Z. Naturforsch., B: Chem. Sci., 2002, 57b, 928.
11 A spacer consisting of two glycine residues was introduced,
because originally we experienced problems in attaching cyclic
peptides to the TAC scaffold, which we ascribed to steric
hindrance.
12 The cleavage and mass analysis of an aliquot of resin showed that
the synthesis had successfully reached this stage.
13 Ammonium acetate buffers were used for purification in view of
the presence of an Asp–Pro sequence in the first loop, which is
prone to cleavage under acidic conditions.
A key issue in this non-stop solid phase peptide synthesis
was the splendid synthesis of cyclic peptides, which could be
very efficiently achieved using the orthogonal PTMSE
group.10 The PTMSE group could be cleaved under almost
neutral conditions by treatment with TBAF in DCM, much
more rapidly than the well known TMSE group,14a thereby
leading to fewer sequence-dependent side reactions, such as
aspartimide formation and rearrangements of aspartyl/
glutamyl peptides.14
Although this protein mimic was not capable of preventing
HIV-1 (IIIB) and HIV-2 (ROD) infection in CEM cell cul-
tures,15 we plan to investigate its properties as a synthetic
vaccine, which is certainly one of the most attractive future
prospects of this and related protein mimics.
14 (a) P. Sieber, Helv. Chim. Acta, 1977, 60, 2711; (b) H.-G. Chao,
M. S. Bernatowicz, P. D. Reiss, C. E. Klimas and G. R. Matsueda,
J. Am. Chem. Soc., 1994, 116, 1746; (c) S. A. Kates and
F. Albericio, Lett. Pept. Sci., 1994, 1, 213; (d) J. D. Wade,
M. N. Mathieu, M. Macris and G. W. Tregear, Lett. Pept. Sci.,
2000, 7, 107.
15 J. Balzarini, L. Naesens, J. Slachmuijlders, H. Niphuis,
I. Rosenberg, A. Holy, H. Schellekens and E. De Clercq, AIDS,
1991, 5, 21.
In our opinion, with this approach and approaches under
development, we can successfully address the chemical chal-
lenges that lie ahead for the construction of protein mimics.
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Chem. Commun., 2009, 821–823 | 823