A general sequence independent solid phase method for the site specific synthesis of multiple sulfated-tyrosine containing peptides

Article abstract of DOI:10.1039/b823425f

In this communication, a new site specific synthesis of highly functionalized and multiple sulfated peptides using convential Fmoc-tBu solid phase peptide synthesis is described. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b823425f

Chemical Communications
www.rsc.org/chemcomm
Number 21 | 7 June 2009 | Pages 2953–3124
ISSN 1359-7345
COMMUNICATION
Rob M. J. Liskamp et al.
A general sequence independent solid Michael P. Doyle, Huai Sun et al.
phase method for the site specific Conformational isomers of
synthesis of multiple sulfated-tyrosine extraordinary ability: carboxamidate-
containing peptides bridged dimetalloorganic compounds
COMMUNICATION

COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A general sequence independent solid phase method for the site specific
synthesis of multiple sulfated-tyrosine containing peptidesw
a
a
Anton Bunschoten,z John A. W. Kruijtzer,z Johannes H. Ippel,a Carla J. C. de Haas,b
Jos A. G. van Strijp,b Johan Kemminka and Rob M. J. Liskamp*a
Received (in Cambridge, UK) 5th January 2009, Accepted 11th February 2009
First published as an Advance Article on the web 9th March 2009
DOI: 10.1039/b823425f
In this communication, a new site specific synthesis of highly
functionalized and multiple sulfated peptides using convential
Fmoc-tBu solid phase peptide synthesis is described.
cleaved from the resin by acidolysis and protection groups are
removed with the exception of the sulfate protection group
thereby preventing undesired acid induced removal of the
sulfate group(s) during this step.
Finally, the sulfate protecting groups were removed in a
slightly acidic (pH 6.4) reductive step, leaving the sulfate
groups untouched.
Although much less known than protein phosphorylation, the
significance of protein sulfation is rapidly gaining momentum.1
The importance of sulfation of for example GPCR proteins
as a key modulator of extracellular protein–protein interactions
requires the availability of methods for the preparation of
crucial sulfated peptide parts. However, so far no reliable
general sequence independent method is available for the site
specific incorporation of especially multiple sulfated tyrosines.2
Clearly, the absence of these methods reflects the relative
instability of sulfated tyrosine residues as compared to
phosphorylated tyrosine residues in peptides and proteins.3
In order to circumvent studies of ligands with the entire
and complex GPCR-C5a receptor, we needed the sulfated
N-terminal peptides as representative mimics of part of this
receptor, which contains two sulfated tyrosine residues.
However, upon evaluation of the relatively scarce literature
on the synthesis of multiple sulfated peptides,4 we concluded
that (i) a solid phase synthesis according to the Boc-strategy is
unfit in view of the acid lability of sulfated tyrosine residues,
(ii) evidently, global sulfation using e.g. DMF–SO3 does not
lead to sulfation of specific tyrosine residues when more of
these are present, (iii) thus far TFA stable sulfated tyrosine
building blocks for use in solid phase peptide synthesis (SPPS)
are unavailable and (iv) no general and convenient sequence
independent SPPS strategy is available for the synthesis of
highly functionalized and multiple sulfated peptides using
conventional Fmoc-tBu SPPS.5
Since sulfation is not limited to proteins, but also occurs on
bioactive peptides such as neuropeptides, for evaluation of
this protocol, we first attempted to synthesize sulfated-
Leu-enkephaline 7 (Scheme 1).6
In this protocol the tyrosine phenolic OH in 2 was protected
with a 2-Cl-Trt-group, which, after liberation with a low
percentage of TFA, was sulfated with 2,2,2-trichloroethyl
chlorosulfate leading to 5.7 After treatment with 95% TFA,
Leu-enkephaline 6 containing the protected sulfate moiety was
cleaved from the resin. These harsh acidic conditions do
not affect the 2,2,2-trichloroethyl sulfate ester,8 which was
successfully removed by a reductive beta-elimination reaction
using zinc-dust.7,9 The sulfated Leu-enkephaline 7 was obtained
pure, in an excellent yield of 63% (after HPLC purification).
Encouraged by these results, we embarked on our actual
target,10 that is, synthesis of the three possible sulfated
variants of the N-terminal peptide part of the C5a receptor
(C5aR7-28), which contains two tyrosine residues.11
Thus, the above protocol was used for this significantly
more complex peptide, containing more than 70% (16 out
of 22) of amino acid residues with a functional side chain,
requiring protection. Standard protection groups were used
for the side chains, which were all compatible with the strategy
Therefore, we describe in this communication a general
sequence independent solid phase method by which a peptide
is synthesized according to the Fmoc-tBu-strategy, followed
by selective deprotection of the tyrosine residues to be
sulfated and introduction of a protected sulfate group. Upon
completion of the synthesis of the sulfated peptide, it is
a Medicinal Chemistry & Chemical Biology, Utrecht Institute for
Pharmaceutical Sciences, Utrecht University, P. O. Box 80082,
Utrecht, 3508 TB, The Netherlands. E-mail: R.M.J.Liskamp@uu.nl;
Web: http://www.pharm.uu.nl/medchem
Fax: +31 30 2536655; Tel: +31 30 2537396
b Medical Microbiology, University Medical Center Utrecht,
Heidelberglaan 100, Utrecht, 3584 CX, The Netherlands
w Electronic supplementary information (ESI) available: Synthesis of
reagents and peptides; HPLC, mass and NMR data; ITC experiments.
See DOI: 10.1039/b823425f
z These authors contributed equally to this work.
Scheme 1 Solid phase synthesis of sulfated-Leu-enkephalin 7.
Chem. Commun., 2009, 2999–3001 | 2999
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This journal is The Royal Society of Chemistry 2009

exemplified by the synthesis of sulfated Leu-enkephaline 7
(Scheme 1), with the exception of the Trt-group. Unexpectedly,
the Trt-group was removed in part from histidine residue 13
during the selective deprotection of the tyrosine residue(s) to be
sulfated.12 After treatment with the sulfation agent, followed by
deprotection of all other side chains, concomitant cleavage from
the resin and reductive cleavage of the sulfate ester, this led to
undesired sulfation (30–50%) of the histidine residue.
The only way to prevent this side product was to use a
histidine residue with another protective group, which should
Scheme 2 Solid phase synthesis of a disulfated C5aR7-28 mimic 13.
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This journal is The Royal Society of Chemistry 2009
3000 | Chem. Commun., 2009, 2999–3001

Table 1 Binding of C5aR peptides to CHIPS
possible as was shown by the preparation of sulfated peptides 14
and 15 having sulfate groups at different positions. The
unexpected hurdle of premature cleavage of the commonly used
histidine protective group was taken by changing to another
convenient protection of the histidine side chain.
Peptide
Position of sulfation
Kd/nM
16: C5aR7-28
—
11
14
11 + 14
3200 ꢁ 100
460 ꢁ 30
37 ꢁ 5.5
+
14: C5aR7-28(SO3ꢂNH4+
15: C5aR7-28(SO3ꢂNH4+
13: C5aR7-28(SO3ꢂNH4
)
)
)
In our opinion this method will contribute to open up
research to study the importance of the sulfation post-
translational modification, which in terms of the functional
group appears to be related to phosphorylation, so that one
might denote it as an isosteric version of the phosphorylation
post-translational modification, albeit so far much less known
and investigated. Finally, we are confident that the presented
method is also amenable for the site specific sequence
independent synthesis of sulfated serine and threonine
containing peptides, which will be reported in due course.
This research was financed by the Technology Foundation
STW (UKG.06609). We thank Annemarie Dechesne and Kees
Versluis for recording the mass spectra and Dr E. J. Breukink
for assistance with the ITC measurements.
8.4 ꢁ 1.1
2
not be acid labile in the stage that the tyrosine residues were
selectively deprotected by acid, but also not be acid labile once the
sulfate moiety has been introduced and deprotected, since
acidolysis in the latter step will also lead to desulfation.
Fortuitously, the base-labile dimethoxy benzoyl (Dmbz) group
described by Zaramella et al. turned out to be a suitable
replacement for the Trt-group.13 This group can be removed by
7 M ammonia in methanol and it was found that these conditions
do not affect the sulfate group on a tyrosine residue (see ESIw).
The final stage was now reached for the successful
synthesis of three highly functionalized sulfated peptides
(Scheme 2 and Table 1). Instead of Fmoc-His(Trt)-OH, now
Fmoc-His(Dmbz)-OH was used. After solid phase peptide
synthesis for the preparation of 9, selective deprotection of
the tyrosine residues leading to 10, sulfation affording 11,
deprotection—with the exception of the sulfated tyrosine
residues and the histidine residue—and cleavage from the resin
to give 12 and removal first of the sulfate 2,2,2-trichloroethyl
ester and then of the Dmbz group led to the successful
completion of the sulfated peptide 13. When the same protocol
was used and also a tyrosine residue with the standard tBu
protecting group was incorporated, site specific sulfation was
achieved by selective removal of the 2Cl-Trt-group in the
presence of this tBu-group. In this way site specific synthesis
of sulfated tyrosine peptides 14 and 15 was realized in addition
to the multiple sulfated tyrosine peptide 13 (vide supra).
Notes and references
1 (a) C. Seibert and T. P. Sakmar, Protein Sci., 2008, 90, 459–477;
(b) A. S. Woods, H. J. Wang and S. N. Jackson, J. Proteome Res.,
2007, 6, 1176–1182; (c) K. L. Moore, J. Biol. Chem., 2003, 278,
24243–24246; (d) J. W. Kehoe and C. R. Bertozzi, Chem. Biol.,
2000, 7, R57–R61; (e) W. B. Huttner, Nature, 1982, 299, 273–276.
2 In addition to Tyr phosphorylation (1.8%), as much as 1% of all Tyr
residues of the total protein in an organism can be sulfated making
these the two most frequently occurring forms of post-translational
modification of this amino acid residue. In contrast to Ser (86%) and
Thr (12%) phosphorylation, sulfation of Ser and Thr is quite rare.
3 Compared to phosphorylated Tyr residues, the O–sulfate linkage is
highly acid labile which creates problems during conventional
Fmoc-tBu based SPPS, see: D. Balsved, J. R. Bundgaard and
J. W. Sen, Anal. Biochem., 2007, 363, 70–76.
4 (a) T. Young and L. L. Kiessling, Angew. Chem., 2002, 114,
3599–3451 (Angew. Chem., Int. Ed., 2002, 41, 3449–3451);
(b) H.-J. Musiol, A. Escherich and L. Moroder, in Houben-Weyl,
Methods of Organic Chemistry, Synthesis of Peptides and Peptido-
mimetics, ed. M. Goodman, A. Felix, L. Moroder and C. Toniolo,
Georg Thieme Verlag, Stuttgart and New York, 2003, ch. 6.6,
vol. E22b, pp. 425–453; (c) M. Ueki, S. Watanabe, R. Yamanaka,
M. Ohta and O. Okunaka, Pept. Sci., 1999, 117–120;
(d) K. Kitagawa, C. Aida, H. Fujiwara, T. Yagami, S. Futaki,
M. Kogire, J. Ida and K. Inoue, J. Org. Chem., 2001, 66, 1–10.
5 The less general Fmoc/benzyl SPPS strategy, using an azidomethyl
phenol protected Fmoc-Tyr-OH building block,4a for the synthesis
of sulfated PSGL-1 octapeptides may be problematic in asparagine
and glutamine containing peptides leading e.g. to aspartimide
formation and subsequent rearrangement.
6 C. Unsworth and J. Hughes, Nature, 1982, 295, 519–522.
7 Y. Liu, I. F. Lien, S. Ruttgaizer, P. Dove and S. D. Taylor, Org.
Lett., 2004, 6, 209–212.
8 The 2,2,2-trichloroethyl sulfate ester containing peptides are very
stable during HPLC analysis and purification with the standard
water–acetonitrile–TFA buffers.
9 S. Ram and R. E. Ehrenkaufer, Synthesis, 1988, 91–95.
10 J. H. Ippel, C. J. C. de Haas, A. Bunschoten, J. A. G. van Strijp,
J. A. W. Kruijtzer, R. M. J. Liskamp and J. Kemmink, J. Biol.
Chem., 2009, DOI: 10.1074/jbc.M808179200.
11 M. Farzan, C. E. Schnitzler, N. Vasilieva, D. Leung, J. Kuhn,
C. Gerard, N. P. Gerard and H. Choe, J. Exp. Med., 2001, 193,
1059–1065.
The resulting sulfated peptides were extensively character-
ized by both NMR spectroscopy and mass spectrometry. In
addition, in order to show that these sulfated peptides were
perfect mimics of the N-terminal part of the C5a receptor, we
carried out isothermal calorimetry (ITC) studies to determine
their binding ability to the immune evasive protein CHIPS,
which binds very strongly to the complete, sulfated, C5a
receptor.10 It was found that the disulfated C5a receptor
mimic 13 bound CHIPS with an affinity that is comparable
to that of the C5a receptor itself (Table 1). Upon evaluation of
the two mono sulfated C5a receptor mimics 14 and 15 as well
as the non-sulfated C5a N-terminal receptor mimic 16, it was
concluded that sulfation is essential for tight binding of a
ligand, since the non-sulfated receptor mimic bound CHIPS
much less strongly. This underlines the importance of sulfation
as a post-translational modification similar to the significance
of the phosphorylation post-translational modification.
Furthermore, selective sulfation is a means of tuning the
binding, since there was a clear difference in binding affinity
of mono sulfated peptide 14 and mono sulfated peptide 15.
To summarize, we present here the first general method for
the synthesis of sulfated peptides. This method allows the
introduction of multiple sulfate groups in highly functional
complex peptides as was illustrated by the synthesis of disulfated
C5a receptor mimic 13. In addition, site specific sulfation was
12 In contrast to His(Trt), Cys(Trt) is expected to be stable under the
used 2-Cl Trt deprotection conditions: K. Barlos, D. Gatos,
O. Hatzi, N. Koch and S. Koutsogianni, Int. J. Pept. Protein
Res., 1996, 47, 148–153.
13 S. Zaramella, R. Stromberg and E. Yeheskiely, Eur. J. Org. Chem.,
2003, 2454–2461.
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Chem. Commun., 2009, 2999–3001 | 3001