Communications
DOI: 10.1002/anie.200805642
Peptide Synthesis
Efficient Solid-Phase Synthesis of Sulfotyrosine Peptides using a
Sulfate Protecting-Group Strategy**
Ahmed M. Ali and Scott D. Taylor*
Sulfation of tyrosine residues in certain proteins has been
shown to be crucial for the proper functioning of a variety of
crucial biological processes, such as viral entry into cells,
blood clotting, cell–cell adhesion, and progastrin processing.[1]
Although it appears that many proteins bear one or more
sulfotyrosine (sY) residues the precise biological roles of the
majority of these proteins have not been elucidated.[1] One of
the factors hindering the study of the role of sY in proteins
and peptides is that sY peptides are not readily available in
the quantities that are required for detailed investigations.
While a number of strategies have been developed for the
solid-phase synthesis of sY peptides their construction is by
no means straightforward and an efficient, general approach
has yet to be reported.[1,2] In the most widely employed
strategy sY residues are incorporated into peptides using
FmocTyr(SO3À+Na)OH (Fmoc = (9H-fluoren-9-ylmethoxy)-
carbonyl).[3a] However, couplings after the incorporation of
the sY residue can be sluggish and the synthesis of multiply
sulfated peptides are difficult due to poor resin swelling and
long coupling times are often required.[1,2,3b] Moreover,
cleavage of the peptide from the resin and side chain
deprotection is achieved under acidic conditions which can
result in desulfation. To minimize desulfation, cleavage is
performed using 90% aq. trifluoroacetic acid (TFA) at 0–48C
and the highly acid labile 2-chlorotrityl (2-ClTrt) resin is
preferably used. The reaction time for cleavage and depro-
tection need to be optimized for each peptide and even with
these precautions some desulfation inevitably occurs.[1,2,3a]
Additional problems with this procedure include incomplete
side-chain deprotection and insufficient cleavage from the
resin.[1,2] An alternative approach employing orthogonally
protected tyrosine residues has drawbacks, such as multiple
manipulations of the completed polymer-bound and free
peptide including treating the resin-bound peptide with
SnCl2/PhSH/Et3N and the use of only hydrogenolytically
labile protecting groups on the side chains of the other amino
acids.[3b] Given the problems associated with the above
methods we embarked upon a study to develop a general
and efficient approach to sY peptide synthesis.
One tactic that could potentially solve the problems
mentioned above is to incorporate the sY residue(s) at the
beginning of the synthesis as a protected sulfodiester(s). We
recently reported the use of the trichloroethyl (TCE) group as
a protecting group for sulfate esters.[4,5] This group is
introduced using easily prepared Cl3CCH2OSO2Cl (1)[4] or
its imidazolium derivative[5] and is readily removed under
very mild reducing conditions.[4,5] Although TCE-protected
sulfates are stable to a wide variety of conditions including
strong acid, they are not stable to an excess of the organic
bases that are commonly used to remove the Fmoc group
during solid-phase peptide synthesis (SPPS), such as piper-
idine, morpholine or 1,8-diazabicyclo[5.4.0.]undec-7-ene
(DBU) in DMF.[6] For example, 1H NMR spectroscopy
studies of compound 2 in 20% piperidine/[D7]DMF revealed
that it undergoes a relatively rapid elimination of HCl to give
dichlorovinyl (DCV) sulfate ester 3 followed by a slower
attack by piperidine on the sulfur atom of 3 and subsequent
formation of decomposition products (Scheme 1).
Scheme 1. Decomposition of ester 2 in piperidine/DMF.
Although the TCE group is unstable to the usual bases
that are used in Fmoc SPPS, we reasoned that if a base could
be found that would not attack the sulfur atom of a DCV-
protected sulfate ester yet be capable of rapid Fmoc removal
then the DCV group should be employable as a sulfate
protecting group during the SPPS of sY peptides. We further
reasoned that such a base would have to be more sterically
encumbered than piperidine yet have a basicity that was
similar to piperidine. Recently, Hachmann and Lebl reported
that Fmoc deprotection of FmocIle attached to chlorotrityl
resin using readily available 2-methylpiperidine (2-MP)[7]
occurred with a half-life that was only 1.5-times greater than
that of piperidine.[8] This prompted us to determine if DCV-
protected sulfate esters are stable to 2-MP and, if so, whether
2-MP could be used in place of piperidine for SPPS.
[*] A. M. Ali, Prof. Dr. S. D. Taylor
Department of Chemistry, University of Waterloo
200 University Ave. West, Waterloo, Ontario, N2L 3G1 (Canada)
Fax: (+1)519-746-0435
E-mail: s5taylor@sciborg.uwaterloo.ca
[**] This research was supported by a Discovery Grant from the Natural
Sciences and Engineering Research Council (NSERC) of Canada to
S.D.T. and by a scholarship from the Egyptian Government to
A.M.A.
Compound 3 was used as a model ester to determine the
stability of DCV-protected sulfates to base and acid. The
Supporting information for this article is available on the WWW
2024
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2009, 48, 2024 –2026