A strategy for the synthesis of sulfated peptides

Article abstract of DOI:10.1002/1521-3773(20020916)41:18<3449::AID-ANIE3449>3.0.CO;2-U

Reversible blockage of the tyrosine phenol group leads to an efficient solid-phase strategy for the synthesis of sulfated peptides using an azidomethyl (Azm) protected tyrosine building block (1). The protected tyrosine derivative is compatible with stand

Full text of DOI:10.1002/1521-3773(20020916)41:18<3449::AID-ANIE3449>3.0.CO;2-U

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O
H
FmocN
A Strategy for the Synthesis of Sulfated
Peptides**
OH
Azm =
N3
Travis Young and Laura L. Kiessling*
OAzm
1
The sulfation of tyrosine residues is a posttranslational
modification that occurs in all eukaryotes.[1] This ubiquitous
modification has been identified on many secretory proteins
with diverse biological activities. Tyrosine O-sulfate (Tyr-
(SO3H)) residues often are important for facilitating specific
protein protein interactions.[2] However, there is little infor-
mation on the molecular basis for the importance of tyrosine
sulfation.[3] Characterization of the molecular interactions of
Tyr(SO3H) has been hampered by lack of access to the
necessary quantities of sulfated proteins and peptides. We
report a method for the efficient production of sulfated
peptides using a newprotecting-group strategy.
Scheme 1. Target tyrosine building block for the synthesis of sulfated
peptides.
tyrosine derivatives at the peptide-assembly stage would
allowthe synthesis of multiply sulfated peptides with various
sulfation patterns. Additionally, a solid-phase sulfation step
would avoid the complicated purification of partially pro-
tected, sulfated peptide intermediates and, in the case of
polysulfated peptides, mixtures with different sulfation pat-
terns. Excess reagents could also be used to drive the sulfation
reaction to completion. With this strategy, both sulfated and
unmodified peptides could be produced from the same
peptide synthesis. To implement this approach, we required
a protecting group for the tyrosine phenol that is stable to the
conditions used in Fmoc-SPPS, yet can be unmasked selec-
tively and quantitatively under mild conditions. Strongly
acidic or basic conditions for protecting-group removal must
therefore be avoided. We also wished to circumvent the
laborious process of cleavage and characterization of inter-
mediates to confirm the success of the deprotection reaction.
We therefore sought to design a protecting group that could
be removed under mild conditions and that possesses a
characteristic spectroscopic signal by which to monitor the
deprotection step.
We identified Loubinoux©s little-used azidomethyl (Azm)
phenol protecting group as an excellent candidate for
tyrosine protection.[10] The mild conditions required for
azide reduction were expected to release the phenol group
without cleavage of the Clt-ester bond or modification of
oxidation-sensitive amino acid residues such as cysteine and
methionine.[11] We therefore pursued the synthesis of Fmoc-
Tyr(OAzm)OH (Scheme 1).
The synthesis of target 1 proceeds from BocTyrOMe (2,
Boc ¼ tert-butyloxycarbonyl). Attempted alkylation of 2 with
dibromomethane or bromochloromethane affords only the
dimeric tyrosine methylene acetal, even when high concen-
trations of the electrophile are employed. Alkylation with an
alternate, bifunctional methylene equivalent, chloromethyl
methylsulfide, proceeds in good yield (Scheme 2). Initial
Although several strategies for the synthesis of sulfated
peptides have been reported,[4] a general, efficient approach is
still being sought. The primary obstacle in the synthesis of
sulfated peptides is the predilection of sulfotyrosine to
undergo desulfation under acidic conditions. Sulfotyrosine-
containing peptides generally decompose rapidly in the
presence of trifluoroacetic acid, the standard reagent used
for side-chain deprotection and cleavage from the solid
supports employed in Fmoc-based (Fmoc ¼ (9H-fluoren-9-
ylmethoxy)carbonyl) solid-phase peptide synthesis (Fmoc-
SPPS). The sulfate group counterion has been shown recently
to have a dramatic influence on the stability of sulfated
peptides toward acidic treatment.[5] Alternatively, acid-sensi-
tive substrates can be released from the 2-chlorotrityl- (2-Clt-)
derivatized resin without significant decomposition.[6] Indeed,
sulfated peptides may be liberated from this support without
significant accompanying desulfation.[7] Thus, the most attrac-
tive published method for sulfated peptide synthesis involves
stepwise incorporation of FmocTyr(SO3Na)OH into the
growing peptide.[8] Unfortunately, couplings using this salt
and subsequent reactions to elongate the resulting peptide can
be sluggish.[9] Moreover, our attempts to synthesize peptides
containing multiple sulfotyrosine residues were plagued by
poor resin swelling and the need for extended coupling times.
Given the lack of general methods for the synthesis of
sulfated peptides, we set out to develop a new approach. For
the greatest flexibility, we employed a postsynthetic sulfation
strategy. We envisioned that selective unmasking and sulfa-
tion of the desired tyrosine residues could be conducted on
the solid support. Incorporation of orthogonally protected
O
O
BocHN
BocHN
a-c
d-f
OMe
1
OMe
[*] L. L. Kiessling, T. Young
Departments of Chemistry and Biochemistry, University of Wiscon-
sin, Madison, Wisconsin 53706 (USA)
E-mail: kiessling@chem.wics.edu
O
X
OH
2
3: X = SMe
4: X = Cl
5: X = N3
[**] This research was supported by the NIH (GM49975). Use of the
ChemMAtCARS Sector 15 at the Advanced Photon Source is
supported by the NSF (CHE0087817), the DOE (contract no. W-31-
109-Eng-38), and by the Illinois Board of Education. We acknowledge
expert suggestions from Dr. D. P. Weicherding and the assistance of
Dr. R. V. Pawlick in the early stages of this work.
Scheme 2. Scheme for the synthesis of 1. Conditions: a) KOt-Bu, NaI,
CH3SCH2Cl, DMF, 82%; b) NCS, TMSCl, CH2Cl2; c) NaN3, DMF, H2O,
87% (over 2 steps); d) TMSOTf, CH2Cl2; e) FmocOSu, Et3N, THF, 84%
(over 2 steps); f) LiOH-H2O, THF-H2O, 0 8C, 88%. DMF ¼ N,N-dimethyl-
formamide, OSu ¼ N-hydroxysuccinimide.
Supporting information for this article is available on the WWW under
http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2002, 41, No. 18
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screening of precedented[12] methods for the selective activa-
tion of the O,S-acetal 3 failed to yield satisfactory results.[13]
For example, treatment of 3 with N-chlorosuccinimide (NCS)
in CH2Cl2 afforded only 48% yield of the desired compound,
4.
Our unsuccessful attempts to transform the O,S-acetal
prompted a search for more reactive electrophilic activators.
We reasoned that Lewis acid activation of NCS would
increase the consumption of starting material without com-
promising the stability of the product. Indeed, activation of
the O,S-acetal with NCS in the presence of tert-butyldime-
thylsilyl chloride (TMSCl) provides the chloride 4 in good
yield (Scheme 2).
The labile intermediate 4 can be purified by flash chroma-
tography on deactivated silica gel, but not without some
hydrolysis. A simple aqueous workup, however, provided 4
with little decomposition. The transformation of this material
to the azide 5 proceeds in high yield (87% for 2 steps).
The azidomethyl group is not impervious to acidic con-
ditions, a feature that complicates the synthesis of 1, as w e
were unable to remove the Boc carbamate selectively using
protic acids. We therefore employed TMSOTf to remove the
Boc group,[14] and protected the resulting amino group by
reaction with Fmoc-N-hydroxysuccinimide ester.[15] Selective
saponification of the methyl ester, the precursor to 1, provides
the target compound without loss of the Fmoc group[16] in
excellent yield. The structure of the protected tyrosine
derivative 1 was unequivocally established by X-ray crystallo-
graphic analysis.[17] The enantiopurity of this compound was
confirmed by the synthesis of diastereomeric dipeptides.[18]
With FmocTyr(Azm)OH (1) in hand, we set out to dem-
onstrate its use in sulfated peptide synthesis (Figure 1). Our
approach involves standard Fmoc-based solid-phase peptide
synthesis, followed by unmasking the desired phenols by
removal of the Azm group using homogeneous conditions for
azide reduction.[19] Complete removal of the Azm moiety is
confirmed by inspection of the IR absorption spectrum for the
strong, distinct band arising from the azide. The free phenolic
hydroxy groups are then sulfated by the action of DMF¥SO3 in
a pyridine/DMF mixture.[20] We opted for benzyl side-chain
protecting groups for other tyrosine residues: serine, threo-
nine, aspartic acid, and glutamic acid residues; this necessi-
tated a two-step cleavage/deprotection procedure. Cleavage
from the acid-labile 2-Clt resin under conditions compatible
with the labile sulfate esters is followed by routine hydro-
genolysis of the benzyl groups.
To test our newmethod, we synthesized a sequence derived
from PSGL-1, a sulfated glycoprotein. PSGL-1 is the relevant
ligand for P-selectin.[21] Significantly, a sulfated and glycosy-
lated epitope at the N-terminus of PSGL-1 is crucial for
binding.[22] We synthesized peptides related to this epitope.[23]
Access to such compounds could facilitate dissection of the
contributions caused by the distinct posttranslational mod-
ifications.[24] We synthesized a sequence; the corresponding
residues 5 12 of mature PSGL-1 that contains all three
putative sulfated tyrosine residues. To demonstrate the
flexibility of our strategy, we targeted the fully sulfated
octapeptide and a monosulfated octapeptide (Figure 2).
–
–
–
SO3 SO3
SO3
E
L
Ac
Y
5
Y
7
D
D
Y
D
F
F
6
7
10
–
OH
SO3
OH
E
L
Ac
Y
5
Y
7
Y
D
10
Figure 2. Sulfated octapeptide targets 6 and 7.
Synthesis of the precursor to the trisulfated sequence 6 by
automated continuous-flowFmoc-based chemistry proceeds
smoothly. Gratifyingly, amide-bond formation with our build-
ing block proceeds with a similar efficiency as with other
amino acids; no special reagents or conditions are required.
After the postsynthetic operations of Azm removal, sulfation,
resin cleavage, and benzyl-group hydrogenolysis, the desired
peptide 6 was isolated in 27% yield after HPLC purification.
Importantly, we observed no desulfated peptide in either the
MALDI-TOF mass spectrum (prior to HPLC) or during
HPLC purification. Synthesis of the monosulfated peptide 7
proceeds somewhat less efficiently, yielding several apparent
deletion peptide products as well as the desired product.
However, after HPLC purification, we isolated the target
peptide in 5% overall yield based on resin loading. Again, we
were unable to detect the corresponding desulfated peptide.
These achievements validate our strategy for the synthesis of
sulfated peptides.
Protein tyrosine sulfation has been pushed to the forefront
of research by its role in important physiological protein
protein recognition events. The ubiquity and emerging
significance of this posttranslational modification provides
an imperative for the development of efficient synthetic
approaches to sulfated peptides. We anticipate the strategy
outlined here will aid the study of tyrosine sulfation by
making sulfated peptide ligands and their analogues readily
available.
Stepwise Fmoc-SPPS
(AA)Y
OBn
Tyr
O
(AA)x
OBn
COO
2-Clt linker
N3
1. SnCl2/PhSH /Et3N
(Azm removal)
2. DMF•SO 3
(AA)Y
OBn
Tyr
(AA)x
OBn
COO
2-Clt linker
OSO3H
1. CH2Cl2/TFE/AcOH
2. H2, Pd(OH) 2/C
Sulfated Peptide
Figure 1. Overviewof sulfated peptide synthesis.
Received: March 31, 2002 [Z19013]
3450
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0044-8249/02/4118-3450 $ 20.00+.50/0
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COMMUNICATIONS
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Manashi Nath and C. N. R. Rao*
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[17] See the Supporting Information for the structure. Crystal data for
C26H24N4O5: Crystal size ¼ 0.09 î 0.02 î 0.02 mm3, orthorhombic,
P212121, a ¼ 5.0195(5), b ¼ 19.2028(12), c ¼ 24.566(2) ä, V¼
2367.9(3) ä3, Dcacl ¼ 1.325 mgmꢀ3, 2qmax ¼ 41.068, AgKa radiation (l ¼
0.5594 ä), w scans, T¼ 173(1) K, 26122 independent and 2803 unique
reflections. The data were corrected for Lorentz and polarization
effects, the empirical absorption correction was performed with
SADABS as described in R. H. Blessing, Acta Crystallogr. Sect. A
1995, 51, 33 38, m ¼ 0.058 mmꢀ1, Tmax/Tmin ¼ 0.9988/0.9948. The struc-
ture was solved and refined with the program package SHELXTL
(version 5.1) program library (G. Sheldrick, Bruker Analytical X-Ray
Systems, Madison, WI). All non-hydrogen atoms were refined with
anisotropic displacement coefficients. All hydrogen atoms were
included in the structure-factor calculation at idealized positions.
The final least-squares refinement of 317 parameters against 2803 da-
ta resulted in residual factors R (based on F2 for I ꢁ 2s) and wR (based
on F2 for all data) of 0.0667 and 0.2167, respectively. The final
difference Fourier map was featureless. CCDC-188441 (1) contains
the supplementary crystallographic data for this paper. These data can
be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrie-
ving.html (or from the Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB21EZ, UK; fax: (þ 44)1223-336-033; or
deposit@ccdc.cam.ac.uk).
Since the discovery of the carbon nanotubes, there has been
active interest in exploring whether other layered materials,
especially metal disulfides such as MoS2 and WS2, form
nanotubes and related structures. Tenne et al.[1,2] succeeded in
preparing nanotubes of MoS2 and WS2 by first heating the
metal oxide in a stream of forming gas (95% N2 þ 5% H2)
followed by reaction with H2S at elevated temperatures (700
10008C). Thermal decomposition of the ammonium thiome-
tallate (NH4)2MS4 (M ¼ Mo or W) in a H2 atmosphere has
been employed recently to obtain the disulfide nanotubes.[3]
Metal trisulfides are formed as intermediates in the formation
of the disulfide nanotubes in both of the above methods.
Accordingly, MoS2 and WS2 nanotubes could be prepared
directly from the decomposition of MoS3 and WS3 in a
hydrogen atmosphere.[3] NbS2 nanotubes have also been
prepared by the thermal decomposition of NbS3 in a hydrogen
atmosphere at 10008C.[4] These results suggested that it may
indeed be possible to prepare nanotubes of other layered
disulfides by the thermal decomposition of appropriate
trisulfide precursors. Since the disulfides of Group 4 metals,
such as Ti, Zr, and Hf, possess layered hexagonal struc-
tures,[5,6] we considered it feasible to prepare the nanotubes of
these materials starting from their trisulfides. Herein we
[*] Prof. Dr. C. N. R. Rao, M. Nath
CSIR Centre For Excellence In Chemistry and
Chemistry and Physics of Materials Unit
Jawaharlal Nehru Centre for Advanced Scientific Research
Jakkur P.O., Bangalore 560 064 (India)
E-mail: cnrrao@jncasr.ac.in
and
Solid State And Structural Chemistry Unit
Indian Institute of Science
Bangalore 560 012 (India)
Fax : (þ 91)80-846-2760
[**] The authors thank the DRDO (India) for research support and Mr.
Md. Motin Seikh and Dr. N. Chandrabhas for help with the Raman
measurements.
[18] For details, see the Supporting Information.
Angew. Chem. Int. Ed. 2002, 41, No. 18
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0044-8249/02/4118-3451 $ 20.00+.50/0
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