Angewandte
Communications
Chemie
Peptide Synthesis
Synthesis of Sulfotyrosine-Containing Peptides by Incorporating
Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy
Wentao Chen+, Jiajia Dong+, Suhua Li, Yu Liu, Yujia Wang, Leonard Yoon, Peng Wu,
K. Barry Sharpless,* and Jeffery W. Kelly*
Abstract: Tyrosine O-sulfation is a common protein post-
translational modification that regulates many biological
processes, including leukocyte adhesion and chemotaxis.
Many peptides with therapeutic potential contain one or
more sulfotyrosine residues. We report a one-step synthesis
for Fmoc-fluorosulfated tyrosine. An efficient Fmoc-based
solid-phase peptide synthetic strategy is then introduced for
incorporating the fluorosulfated tyrosine residue into peptides
of interest. Standard simultaneous peptide-resin cleavage and
removal of the acid-labile side-chain protecting groups affords
the crude peptides containing fluorosulfated tyrosine. Basic
ethylene glycol, serving both as solvent and reactant, trans-
forms the fluorosulfated tyrosine peptides into sulfotyrosine
peptides in high yield.
by a four-step synthesis in 66% overall yield and is currently
commercially available. Fmoc-based SPPS is used to incor-
porate the Np-protected sY diester building block into the
peptide of interest. After cleavage of the peptide from the 2-
chlorotrityl resin and removal of the standard side-chain
protecting groups, the Np group is removed in 1–2m
ammonium acetate at 378C over 6–12 h.[2] Another option
employs a five-step strategy to synthesize a dichlorovinyl
sulfate ester protected sY residue that is incorporated by
SPPS into the desired peptide employing an Fmoc-based
strategy.[5] Resin cleavage and cleavage of the side-chain
protecting groups are performed under typical conditions
(TFA:TIPS:H2O = 95:2.5:2.5 volume ratio; or TFA:phenol:
H2O:thioanisole:EDT= 82.5:5:5:5:2.5; where TFA = tri-
fluoroacetic acid, TIPS = triisopropylsilane, and EDT= 1,2-
ethanedithiol) and then the dichlorovinyl sulfate ester
protecting group(s) is removed in solution by hydrogenolysis
using 10% Pd/C (30 wt.%), H2, and ammonium formate
(9 equiv) in methanol at 258C for 1 h, minimizing desulfa-
tion.[5] The hydrogenolysis step precludes the incorporation of
cysteine (Cys) residues into the peptides. An even more
recent strategy involves the Fmoc-based solid-phase synthesis
of peptides containing Tyr residues with distinct phenol
protecting groups.[6] These protecting groups are selectively
removed while the peptide is still attached to the resin and the
phenol is subjected to tyrosine O-sulfation employing sulfuryl
imidazolium salt treatment (8 equiv/phenol functional
group). Acidic cleavage of the peptide from the resin and
removal of the standard side-chain protecting groups are
followed by removal of the 2,2,2-trichloroethyl protecting
group by means of catalytic hydrogenation using Pd(OH)2 on
carbon to afford the sY-containing peptide of interest.[6a]
Herein, we report a short and efficient route to sY-
containing peptides, wherein Fmoc-protected fluorosulfated
tyrosine (Y(OSO2F)) is incorporated into the peptide of
interest through an Fmoc-based solid-phase synthetic strat-
egy, either manually or by use of a peptide synthesizer. Like
other sulfur(VI) fluorides, aromatic fluorosulfates are redox
stable and hence do not serve as halogenation agents.[7] They
are also very stable toward hydrolysis under neutral and
acidic conditions, and moreover survive in basic milieu (for
example, in phosphate buffer at pH 10).[7] However, the
ArOSO2-F linkage becomes reactive in the presence of an
appropriate nucleophile only if the reaction conditions meet
the stringent requirements for the departure of the “FÀ” from
its covalent link to the SVI center.[7,8] In the case at hand, the
contrast between high stability alongside the activatable
sulfur(VI) fluoride exchange (SuFEx) reactivity pushes this
latest click reaction to the very top; only the copper-catalyzed
T
yrosine O-sulfation is a common enzymatic post-transla-
tional modification that occurs while the secreted and trans-
membrane proteome traffics through the Golgi compartment
of the cell.[1] Phosphorylation and sulfation of tyrosine (Tyr)
similarly modulate protein–protein interactions and affect
conformational changes within a protein.[2]
Currently, one of several approaches can be used for the
solid-phase peptide synthesis (SPPS) of protein fragments
and polypeptides comprising sulfotyrosine (sY) residues. In
À
the oldest approach, Fmoc-based (Tyr-OSO3 )+Na (Fmoc =
(9H-fluoren-9-ylmethoxy)-carbonyl) is simply coupled into
the growing peptide;[3] however, subsequent couplings can be
challenging and direct incorporation of more than one
sY residue often compromises resin swelling, impeding fur-
ther amino acid coupling steps.[3c,d] Moreover, acidic depro-
tection conditions often lead to desulfation. In an alternative
method, the neopentyl (Np) group is used to protect the
sY residue as a neutral sulfate diester, that is, as Tyr-O-SO2-
ONp.[2,4] The Fmoc-Tyr(OSO3Np)-OH amino acid is obtained
[*] W. Chen,[+] Dr. J. Dong,[+] Dr. S. Li, Dr. Y. Liu, Dr. Y. Wang, L. Yoon,
Dr. P. Wu, Dr. K. B. Sharpless, Dr. J. W. Kelly
Department of Chemistry
The Scripps Research Institute, La Jolla, CA 92037 (USA)
E-mail: sharples@scripps.edu
W. Chen,[+] Dr. J. W. Kelly
Department of Molecular and Experimental Medicine
The Scripps Research Institute, La Jolla, CA 92037 (USA)
Dr. Y. Liu, Dr. K. B. Sharpless, Dr. J. W. Kelly
The Skaggs Institute for Chemical Biology
The Scripps Research Institute, La Jolla, CA 92037 (USA)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2016, 55, 1835 –1838
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1835