Divergent and site-selective solid-phase synthesis of sulfopeptides

Article abstract of DOI:10.1002/asia.201100232

On solid ground: A solid-phase strategy for the efficient synthesis of sulfopeptides is described. Selective deprotection of orthogonally-protected tyrosine residues and solid-phase sulfation provided divergent access to differentially sulfated peptides in high yields. Copyright

Full text of DOI:10.1002/asia.201100232

COMMUNICATION
DOI: 10.1002/asia.201100232
Divergent and Site-Selective Solid-Phase Synthesis of Sulfopeptides
[
a]
[a]
[b]
[a]
Deni Taleski, Stephen J. Butler, Martin J. Stone, and Richard J. Payne*
[
7b,8]
Tyrosine (Tyr) sulfation represents one of the most
common post-translational modifications of peptides and
proteins. It has been estimated that up to 1% of the eukar-
yote proteome possesses sulfated Tyr (sTyr) residues, a
modification known to play a key role in human physiology
and pathology. Tyr sulfation, mediated by trans-Golgi lo-
calized tyrosylprotein sulfotransferase (TPST) enzymes, is
common on secreted and integral membrane proteins (e.g.
G-protein coupled receptors). These proteins are crucial to
a range of biological processes, including blood coagula-
ne
complexes, or the incorporation of pre-formed sTyr
[7a,9]
residues into peptides as the corresponding sodium
or
[10]
tetraalkylammonium salts. The problem with these strat-
egies is that the resulting sulfate monoester is labile under
the acidic conditions used for amino acid side-chain depro-
tection, often resulting in diminished yields of the desired
sulfopeptide. This drawback has led to the development of
new synthetic methods aimed at the sequence-independent,
site-specific incorporation of stabilized sTyr residues into
peptides. These approaches have utilized orthogonal pro-
tection of sulfate groups as diesters during Fmoc-based
solid-phase peptide synthesis.
employed include neopentyl,
and 2,2-dichlorovinyl (DCV) sulfate esters, each of which
are stable to the acidic conditions required for side-chain
deprotection and cleavage of the peptide from the resin,
and can be readily removed at the end of the synthesis. In
addition, methods have been reported for the synthesis of
preformed Fmoc-protected Tyr building blocks bearing
these protecting groups along with their successful incorpo-
[1]
[5]
[
2]
[3]
[4]
tion, cell–cell adhesion, viral entry into host cells, and
[5]
[11]
protein–protein interactions. In particular, Tyr sulfation is
necessary for the recognition of chemokine ligands by the
extracellular N-terminal domain of chemokine receptors, a
process integral to the mediation of inflammatory responses
Suitable protecting groups
2,2,2-trichloroethyl (TCE),
[12]
[13]
[6]
and the progression of infectious diseases.
To date, the exact structural and functional role(s) of sTyr
residues in protein–protein interactions remain largely un-
[1e]
known. This is due, in major part, to the difficulties asso-
ciated with accessing sulfopeptides and sulfoproteins in sig-
nificant quantities and purity for detailed biological study.
This problem is exacerbated by the inherent acid lability of
the sulfate ester linkage in sTyr residues, which in most
cases prevents direct synthesis by solid-phase peptide syn-
thesis (SPPS). Early methods for the preparation of sulfo-
peptides include global sulfation of peptides with sulfur tri-
[
11–14]
ration into sulfopeptides via Fmoc-strategy SPPS.
Lis-
kamp and co-workers have extended this concept by demon-
strating that Tyr residues (initially protected with an acid-
labile 2-chlorotrityl chloride group) could be globally sulfat-
ed with TCE-chlorosulfate whilst peptides are immobilized
on the solid-phase. The utility of this methodology was dem-
onstrated in the synthesis of a sulfated variant of the N-ter-
[7]
oxide–N,N-dimethylformamide or sulfur trioxide–pyridi-
[15]
minal domain of the C5a receptor. Despite these notable
advances, there remains the need for an efficient method for
the preparation of peptides bearing a variety of different
sulfation patterns. Ideally, the synthesis of such (sulfo)pepti-
des should be accessible from a single synthesis.
Because many sulfopeptides and sulfoproteins (including
the chemokine family of receptors) possess multiple sTyr
residues, we were interested in developing a general and ef-
ficient method to enable rapid access to peptide libraries
with well-defined sulfation patterns utilizing a divergent
Fmoc-strategy SPPS approach. To this end, we envisaged
the use of two orthogonally protected Tyr building blocks (1
and 2) in Fmoc-strategy SPPS, bearing side-chain tert-butyl-
dimethylsilyl (TBS) and allyl (All) ether groups, respectively
+
+
[
a] D. Taleski, Dr. S. J. Butler, Dr. R. J. Payne
School of Chemistry
The University of Sydney
New South Wales 2006 (Australia)
Fax : (+61)2-9351-3329
E-mail: richard.payne@sydney.edu.au
Homepage: http://www.chem.usyd.edu.au/~payne/
[
b] Prof. M. J. Stone
Department of Biochemistry and Molecular Biology
Monash University
Victoria 3800 (Australia)
+
[
] D.T. and S.J.B. contributed equally to this research.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100232.
1316
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Chem. Asian J. 2011, 6, 1316 – 1320

Scheme 1. Fmoc-protected Tyr building blocks 1 and 2 and TCE- and
DCV-imidazolium-based sulfating reagents 3 and 4. Fmoc=fluorenylme-
thyloxycarbonyl.
(
Scheme 1). Once installed at the desired positions in the
peptide sequence, these residues could be selectively and
differentially deprotected in the presence of each other (and
all side-chain protecting groups commonly employed in
Fmoc-strategy SPPS) thus allowing for site-selective manip-
ulation of Tyr residues within resin-bound peptides. We pro-
posed utilizing the imidazolium-based sulfating reagents 3
[14]
and 4, reported by Taylor and co-workers,
to access all
four variants of a given sulfopeptide (bearing two sulfation
sites) from a single solid-phase synthesis (for synthetic de-
tails, see the Supporting Information).
The first step in developing the proposed methodology in-
volved optimizing suitable conditions for the selective re-
moval of TBS and allyl ethers from orthogonally protected
peptides. To this end, we prepared resin-bound heptapeptide
5
, corresponding to the N-terminal region (residues 25–31)
of CC chemokine receptor 2 (CCR2), which encompasses
[6a,16]
both of the predicted sulfation sites (Tyr-26 and Tyr-28).
The peptide was assembled on a Rink amide resin using
Fmoc-strategy SPPS before the resin was split into eight
equimolar batches to allow for the solid-phase manipula-
tions at Tyr-26 and Tyr-28 (Scheme 2; for synthetic details,
see the Supporting Information).
Peptide 6, without sulfation, was the initial target which
allowed for optimization of the solid-phase deprotection
conditions. TBS-ether removal from Tyr-28 of 5 was smooth-
ly achieved using tetrabutylammonium fluoride (TBAF)
buffered with acetic acid (see Scheme 2 and the Supporting
Information). Optimal conditions for the deptrotection of
the allyl ether from Tyr-26 involved treatment of 5 with [Pd-
ACHTUNGTRENNUNG( PPh ) ], triethylsilane (TES), and, crucially, acetic acid (in
3 4
the absence of acetic acid, the reaction was sluggish and
failed to proceed to completion after extended treatment).
At this stage, acidolytic cleavage from the resin and con-
comitant side-chain deprotection afforded non-sulfated
Scheme 2. Divergent solid-phase synthesis of CCR2
sulfopeptides 10–12. Ac=acetyl, TIS=triisopropylsilane.
ACHTUNGTRENNUNG( 25–31) peptide 6 and
CCR2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(25–31) 6 in 90% yield after purification by HPLC.
Synthesis of CCR2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(25–31) with sulfation at Tyr-26 began by
Synthesis of the sTyr-28 form of CCR2 AHCTUNGTERNNUN(G 25–31) was achieved
removal of the allyl ether from 5 (employing the conditions
described above), followed by sulfation with 3 or 4 (8 equiv)
in the presence of triethylamine (8 equiv) for 24 hours. This
step was repeated to ensure complete sulfation of the free
phenol group. Acidolytic side-chain deprotection with con-
comitant TBS removal and cleavage from the resin afforded
TCE- and DCV-protected sulfate diesters 7a and 7b in
with TBAF-mediated removal of the silyl ether followed by
sulfation with 3 or 4. Deallylation of Tyr-26 followed by
side-chain deprotection and cleavage from the resin then af-
forded 8a and 8b in 48% and 53% yields respectively, fol-
lowing purification by HPLC. Finally, the solid-phase syn-
thesis of CCR2 ACHTUNGRNENUG( 25–31), sulfated at both Tyr-26 and Tyr-28,
was achieved by sequential removal of the silyl and allyl
70% and 64% yields, respectively, after HPLC purification.
ethers from 5 followed by global sulfation with 3 or 4 using
Chem. Asian J. 2011, 6, 1316 – 1320
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www.chemasianj.org
1317

R. J. Payne et al.
COMMUNICATION
the conditions described above.
After acidic cleavage and pu-
rification
by
reverse-phase
HPLC, protected sulfopeptides
9
a and 9b were furnished in
57% and 51% yields, respec-
tively. With 7a-9b in hand, all
that remained for the prepara-
tion of the desired sulfopepti-
des was the deprotection of
TCE- and DCV-sulfate esters.
This step was achieved by cata-
lytic
hydrogenolysis
with
Pd(OH) /H in the presence of
2
2
[13b]
triethylamine
three sulfated isoforms of
CCR2(25–31) 10–12 in excel-
lent yields (74–97%) following
purification by HPLC
Scheme 2).
Having established an effi-
to provide the
ACHTUNGTRENNUNG
(
cient route to the differentially
sulfated heptapeptides 10–12,
we now turned our attention to
the preparation of a more-com-
plex series of sulfopeptides,
namely the sulfated variants of
the N-terminal CCR2 fragment
CCR2 ACHTUNGTRENNUNG( 1–31). We envisaged the
convergent assembly of the de-
sired (sulfo)peptides from frag-
ments corresponding to CCR2-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(18–31) and a peptide thioester
corresponding to CCR2(1–17)
using recently developed cys-
AHCTUNGTRENNUNG
[17]
teine-free ligation chemistry.
To this end, resin bound pep-
tide 13 was first synthesized on
Rink amide resin using Fmoc-
strategy
SPPS
(Scheme 3).
Deallylation and acidic cleav-
age steps were then performed,
as described for 5, to afford
non-sulfated peptide 14. Simi-
larly, deprotection and solid-
phase sulfation of 13 with TCE-
protected
imidazolium-based
sulfating reagent 3, according to
the procedures developed for 5,
gave TCE-protected sulfopepti-
des 15–17 in 30–35% yields
(
based on the original resin
Scheme 3. Divergent solid-phase synthesis of CCR2 ACHTUNGRNENUG( 18–31) peptide 14 and sulfopeptides 18–20.
loading) after acidolytic cleav-
age and purification by HPLC.
At this stage, hydrogenolysis of the TCE protecting groups
from 15–17, followed by purification by HPLC, generated
the three differentially sulfated CCR2 AHCTUNGTERNNUN(G 18–31) peptides 18–
20 in 71–87% yields.
1318
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1316 – 1320

Solid-Phase Synthesis of Sulfopeptides
Scheme 4. Synthesis of CCR2
ACHTUNGERTNNUNG( 1–17) thioester 21, silver(I)-promoted ligation to CCR2 ACHTUNRTEGNNUNG( 18–31) peptide 14 and sulfopeptides 18–20 to afford CCR2 ACHTUNGTRENNUNG( 1–31)
peptide 22 and CCR2(1–31) sulfopeptides 23–25, respectively. DMSO=dimethyl sulfoxide.
ACHTUNGTRENNUNG
With the desired peptide 14 and sulfopeptides 18–20 now
in hand, we focused on the preparation of the CCR2(1–17)
peptide thioester 21 coupling partner for the proposed liga-
efficiently prepare a variety of sulfopeptides with defined
sulfation patterns. In turn, this should enable detailed struc-
tural and functional studies, so as to facilitate a better un-
derstanding of the role(s) of Tyr sulfation in biology, includ-
ing the importance of receptor sulfation in the mediation of
chemokine–receptor interactions.
AHCTUNGTRENNUNG
tion chemistry. The peptide sequence was first assembled on
2-chlorotrityl chloride resin by an Fmoc-strategy SPPS
before mild-acidic deprotection with hexafluoro-iso-propa-
nol (HFIP). Thioesterification and global side-chain depro-
tection using the method described by Kajihara and co-
[18]
workers provided the desired peptide thioester 21 in 13%
Acknowledgements
yield over 34 linear steps and purification by HPLC. Peptide
1
4 and sulfopeptides 18–20 were subsequently reacted with
This work was supported by the Australian Research Council (ARC Dis-
covery Project: 1094884).
peptide thioester 21 under silver(I)-promoted peptide-liga-
tion conditions (AgNO , HOOBt, iPr NEt, DMSO).
[17e]
The
3
2
reactions were monitored by LC-MS and were found to
have reached completion after 24 hours. At this point the N-
terminal Fmoc–carbamate moiety was deprotected using pi-
peridine in acetonitrile and the products purified by HPLC
Keywords: chemokine · peptides · receptors · solid-phase
synthesis · sulfation
to provide non-sulfated CCR2
CCR2(1–31) sulfopeptides 23–25 in 45–49% yields
Scheme 4). Gratifyingly, the silver(I)-promoted ligation re-
actions to afford the elaborated sulfopeptides did not hydro-
lyze the labile sulfate-ester linkage of the sTyr residues, and
therefore, this method serves as a general method for the
future preparation of longer sulfopeptides or sulfoproteins
for biological study.
In summary, we have developed a novel method for the
rapid and divergent synthesis of differentially sulfated pep-
tides from a single solid-phase synthesis. The efficiency of
the method was demonstrated in the high-yielding prepara-
tion of CCR2 peptides and sulfopeptides of various length.
In addition, we have demonstrated, for the first time, that
sulfopeptides can be elaborated using silver(I)-promoted li-
gation chemistry without affecting the labile sulfate ester
moieties. Using this new methodology, it is now possible to
ACHTUNGERTNNUNG( 1–31) 22 in 72% yield and
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Published online: April 20, 2011
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Chem. Asian J. 2011, 6, 1316 – 1320