Synthesis of disulfated peptides corresponding to the N-terminus of chemokines receptors CXCR6 (CXCR61-20) and DARC (DARC8-42) using a sulfate-protecting group strategy

Article abstract of DOI:10.1002/psc.1220

Tyrosine sulfation is a post translational modification that occurs on integral membrane and secreted proteins, and is required for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmoc-based solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group (FmocTyr(SO₃DCV)OH, 1). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here we demonstrate that sTyr peptides containing Met or Trp residues can be prepared using our sulfate-protecting group strategy by preparing peptides corresponding to residues 1-20 of chemokine receptor CXCR6 and 8-42 of chemokine receptor DARC. Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp, by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for removing the DCV group and superiour to our original conditions using H₂, ammonium formate, Pd/C. The presence of Met was found not to interferewith the removal of the DCV group. The use of pseudoproline dipeptides and N-backbone protectionwith the 2,4-dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation during the synthesis of thesepeptides.Wealso report an alternative and more cost effective synthesis of amino acid 1. Copyright

Full text of DOI:10.1002/psc.1220

Research Article
Received: 26 November 2009
Revised: 19 January 2010
Accepted: 27 January 2010
Published online in Wiley Interscience: 1 March 2010
(www.interscience.com) DOI 10.1002/psc.1220
Synthesis of disulfated peptides
corresponding to the N-terminus
of chemokines receptors CXCR6
(CXCR61–20) and DARC (DARC8–42)
using a sulfate-protecting group
strategy
Ahmed M. Ali and Scott D. Taylor∗
Tyrosine sulfation is a post translational modification that occurs on integral membrane and secreted proteins, and is required
for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple
sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmoc-
based solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group
(FmocTyr(SO3DCV)OH, 1). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here
we demonstrate that sTyr peptides containing Met or Trp residues can be prepared using our sulfate-protecting group strategy
by preparing peptides corresponding to residues 1–20 of chemokine receptor CXCR6 and 8–42 of chemokine receptor DARC.
Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp,
by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for
removing the DCV group and superiour to our original conditions using H2, ammonium formate, Pd/C. The presence of Met was
found not to interfere with the removal of the DCV group. The use of pseudoproline dipeptides and N-backbone protection with
the 2,4-dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation
duringthesynthesisofthesepeptides. Wealsoreportanalternativeandmorecosteffectivesynthesisofaminoacid1. Copyright
c
ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: peptide synthesis; sulfotyrosine; protecting groups; chemokine receptors
Introduction
of the G-protein coupled receptor C5aR to the immune invasive
protein CHIPS [7], the interaction of the HIV-1 gp120 envelope
glycoprotein/CD4 complex with chemokine receptor CCR5 [8]
and have even been used to inhibit HIV infection in vitro [9].
Until very recently, the synthesis of sTyr peptides, especially
those containing multiple sTyr residues, were among the most
challenging peptides to prepare. Most early attempts to prepare
Tyrosine sulfation is a post translational modification that occurs
on integral membrane and secreted proteins. It is believed to
be widespread in nature and it has been estimated that as much
as 1% of all tyrosine residues in eukaryotes are sulfated [1].
The importance of this PTM is widely appreciated since it has
recently been demonstrated that tyrosine sulfation is required for
some important biological processes such as viral and parasitic
infection, hormone regulation, leukocyte trafficking and adhesion,
blood coagulation and the immune response [2]. A variety of
tools and techniques, such as bioinformatics studies, labeling
studies using radioactive 35S-enriched sulfate, sulfation inhibitors,
anti-tyrosine antibodies and mass spectrometry are used to
determine the presence, location and function of sTyr residues
in proteins [2]. sTyr-containing peptides have also proven to be
useful tools for studying tyrosine sulfation in proteins because
they allow one to assess the function of each individual sTyr
residue. Tyrosine-sulfated peptides have been used to assess the
affect of tyrosine sulfation on a variety of biological processes such
as chemokine binding to chemokine receptors [3–5], the binding
of P-selectin glycoprotein ligand 1 to P-selectin [6], the binding
∗
Correspondence to: Scott D. Taylor, Dept. of Chemistry, University of Waterloo,
200 University Ave. West. Waterloo, Ontario, Canada N2L3G1.
E-mail: s5taylor@sciborg.uwaterloo.ca
Dept. of Chemistry, University of Waterloo, 200 University Ave. West. Waterloo,
Ontario, Canada N2L3G1
Abbreviations used: 2-MP, 2-methylpiperidine; CHIPS, chemotaxis inhibitory
protein of Staphylococcus aureus; DARC, Duffy antigen and receptor for
chemokines; DCV, dichlorovinyl; EDT, ethanedithiol; HCTU, 2-(6-Chloro-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate; HRMS,
high resolution mass spectrometry; PvDBP, Plasmodium vivax Duffy binding
protein; PTM, post-translational modification; LRMS, low resolution mass
spectrometry; sTyr, sulfotyrosine; TIS, triisopropylsilane; QTOF, quadrupole
time-of-flight.
c
J. Pept. Sci. 2010; 16: 190–199
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

SYNTHESIS OF DISULFATED PEPTIDES FROM CXCR6 AND DARC
Cl
Cl
O
O S O
O
OSO3DCV
SPPS using
2-MP for Fmoc
removal
DCV group
+
HOOC (AA)n
PG
Rink amide resin
H
N
OH
H2N-(AA)n-COHN
PG
(AA)n
PG
FmocHN
O
O
1
1. TFA/TIS
2. Pd/C, H2,
HCO2-+NH4
-
OSO3
H
N
H2N-(AA)n-COHN
(AA)n CONH2
O
Scheme 1. Fmoc SPPS of sTyr peptides using FmocTyr(SO3DCV)OH as building block.
these peptides used a global sulfation strategy in which the Tyr
residues are sulfated, usually with a SO3 complex, after the peptide
has been constructed (For an excellent review on the synthesis
of sulfated peptides see: [10]). Among the problems associated
with this approach are selectivity and specificity: it is not possible
to introduce sTyr residues at specific locations when more than
one Tyr residue is present and the side chains of some other
amino acids, such as serine, are also sulfated. Special protecting
group strategies have been employed to deal with these issues;
however, these strategies tend to be fairly labor intensive and the
yield of sTyr peptide is, in general, quite poor [10,11]. Moreover,
if the sulfation is performed when the peptide is on the resin
then some desulfation occurs upon acid-promoted cleavage of
the final product from the support and side chain deprotection.
Perhaps the most common approach to sTyr peptides is one in
which the sTyr residue is incorporated as a free sulfate monoester
using Fmoc(SO3Na)OH [10,12]. However, couplings following the
incorporation of the sY residue can be sluggish and the synthesis
ofmultiplysulfatedpeptidesaredifficultduetopoorresinswelling
and long coupling times are often required [11]. Moreover, as
with the global sulfation strategy, acid-promoted cleavage of
the peptide from the resin and side chain deprotection results in
desulfation. To minimize desulfation, cleavage is performed using
90% aq. TFA at 0–4 ◦C. However, the reaction time for cleavage
and deprotection need to be optimized for each peptide and
even with these precautions some desulfation inevitably occurs
[12]. Additional problems with this procedure include incomplete
side chain deprotection and insufficient cleavage from the resin.
We recently introduced a new and highly effective approach to
Fmoc-based solid phase synthesis of sTyr peptides that eliminates
the problems associated with the methodologies mentioned
earlier [13] (While this work was in progress two other reports
appeared describing new approaches to Fmoc-based solid phase
synthesis of sTyr peptides. One of these (see Ref. 5) uses a
neopentyl group to protect the sulfate group during Fmoc solid
phase peptide synthesis. The neopentyl group is removed by
mild hydrolysis at the end of the synthesis. The other approach
(see Ref. 7) uses a global sulfation strategy in which the tyrosine
side chains that are to bear a sulfate group are initially protected
with a 2-chlorotrityl group (ClTrt). The ClTrt group is removed on
resin without affecting the less acid labile protecting groups used
for the protection of the side chains of the other residues. The
sulfate group is then introduced as a TCE-protected sulfodiester
using TCEOSO2Cl (6) and then the peptide is cleaved from the
resin and all side chain protecting groups are removed, with the
exception of the TCE group, using TFA/TIS/H2O. The resulting
peptide is purified and then the TCE groups are removed using
Zn/ammonium formate and the peptide purified again). In this
approach the sTyr residue is introduced during the synthesis
as a protected sulfodiester using FmocTyr(OSO3DCV)OH (1,
Scheme 1). The DCV group is used to protect the sulfate group and
2-MP is used to affect Fmoc removal. The peptide is cleaved from
the resin and all side chain protective groups are removed, with
theexceptionoftheDCVprotectinggroup, usingTFA/TIS. TheDCV
group is removed at the end of the synthesis by hydrogenolysis
using Pd/C, H2 and ammonium formate. Using this approach we
prepared some multisulfated peptides, such as a tetrasulfated
20-mer derived for the N-terminal region of the chemokine
receptor D6 and a disulfated 20-mer derived from the N-terminal
of the C5a-anaphylatoxin chemotactic receptor in good yield and
purity [13].
Among the sTyr peptides that we wished to construct are
those derived from the N-terminal regions of the DARC and the
chemokine receptor CXCR6. DARC can serve as an erythrocyte
receptor for the malaria parasite Plasmodium vivax [14]. P. vivax
expresses an erythrocyte-binding antigen called PvDBP [14]. The
first interaction between DARC and PvDBP is a crucial step in
erythrocyte invasion by the parasite and failure to complete such
interaction retards the invasion. Studies using Tyr to Phe mutants
and labeling studies strongly indicate that DARC is sulfated at
Tyr30 and Tyr41 and Tyr41 in particular plays a crucial role in the
DBP-DARC interaction and P. vivax infection of erythrocytes [15].
Chaudhuri et al., showed that a non-sulfated 35-mer N-terminal
peptide inhibited DBP binding to Duffy positive red blood cells
in vitro [16]. We are interested in examining the ability of sTyr
peptides to inhibit DBP-DARC interactions. Chemokine receptor
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J. Pept. Sci. 2010; 16: 190–199 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ALI AND TAYLOR
CXCR6 mediates the chemotaxis and adhesion of leukocytes to
soluble and membrane-anchored forms of its only known ligand
CXCL16, and is an HIV-1 co-receptor. Although bioinformatics
analysespredictthatoneTyrresidue(Tyr6)andpossiblytwo(Tyr10)
aresulfated[17],mutationoftheseresiduestoPheandinhibitionof
sulfation using sodium chlorate had no effect on receptor function
[18]. Sulfated peptides corresponding to the N-terminal region
of CXCR6 may provide additional insight as to whether or not
sulfation of these two Tyr residues contribute to receptor function.
Our specific synthetic targets were the disulfated peptides
AcMAEHDsY6HEDsY10GFSSFNDSSQNH2 (2) which corresponds
to residues 1–20 of the N-terminal region of CXCR6 and,
AcAELSPSTENSSQLDFEDVWNSSsY30GVNDSFPDGDsY41DNH2 (3)
which corresponding to residues 8–42 of the N-terminus region
of DARC. However, unlike previous sTyr peptides that we have
prepared [13] peptides 2 and 3 contain methionine (at position
1 in peptide 2) and Trp (at position 26 in peptide 3). This
raised concerns as to whether our approach to sTyr peptides
could be used to prepare these two peptides since the DCV
group is removed using hydrogenolysis. Although poisoning of
Pd catalysts by sulfur containing compounds is well known we
anticipated that this would not be an issue using our approach
since greater than stoichiometric (molar) amounts of Pd catalyst
compared to Met residue(s) can be used. However, reduction
of the indole ring in Trp residues has been shown to occur to
give 2,3-dihydrotryptophan or even octahydrotryptophan when
Trp-bearing peptides are subjected to a variety of hydrogenolysis
conditions [19–21]. In this report we describe the synthesis of
peptides 2 and 3 using our sulfate-protecting group strategy.
Reduction of the Trp residue was avoided by removing the DCV
group under basic hydrogenolysis conditions and Met did not
interfere with DCV removal. The use of pseudoproline dipeptides
to prevent aggregation during peptide synthesis and N-backbone
protection with the 2,4-dimethoxybenzyl (Dmb) group to prevent
aspartimide formation proved to be effective tactics for obtaining
these peptides in good yield. We also report an alternative and
more cost effective synthesis of the key amino acid 1.
Vydac218TP1022 C18column(10 µm, 22 mm ×250 mm)using an
8.0 ml/min flow rate. Flash chromatography was performed using
silica gel 60 Å (234–400 mesh) obtained from Silicycle (Laval, Que-
bec, Canada). Chemical shifts (δ) for 1H NMR spectra run in CDCl3
arereportedinppmrelativetotheinternalstandardtetramethylsi-
lane. For 13C NMR spectra run in CDCl3 chemical shifts are reported
in ppm relative to the CDCl3 (δ 77.0 for central peak). Electron
impact mass spectra were acquired with a JEOL HX110 double
focusing mass spectrometer. Positive and negative ion electro-
spray (ESI) experiments were performed with a Waters/Micromass
QTOF Ultima Global mass spectrometer. 1 : 1 CH3CN/H2O + 0.2%
formic acid is used as a solvent for positive ion spectra and 1 : 1
CH3CN/H2O + 0.5% ammonium hydroxide was used as solvent for
negative ion spectra.
tert-Butyl Nα-[tert-butoxylcarbonyl]-L-Tyrosine Dichlorovinyl
Sulfate, 8
To a solution of 5 (6.0 g, 17 mmol, 1.0 Eq) in dry THF (24 ml) at 0 ◦C
was added reagent 6 [24] (15.7 g, 53 mmol, 3.0 Eq) followed by a
solution of DMAP (2.1 g, 17 mmol, 1 Eq) and Et3N (4.9 ml, 34 mmol,
2.0 Eq) in dry THF (48 ml). The reaction was allowed to warm to
room temperature then stirred overnight and filtered. The filtrate
was diluted with EtOAc (200 ml) and the resulting solution was
washed with phosphate buffer (pH = 7.2, 2 × 100 ml) and brine
(2 × 100 ml) then dried (MgSO4), filtered concentrated by rotary
evaporation. The residue was dissolved in THF (72 ml) and 1 Eq of
DBU (2.5 ml, 17 mmol) was added at 1-h intervals over 4 h for a
total of 5 Eq DBU. After 6 h the reaction mixture was filtered and
the filtrate was diluted with EtOAc (2 × 100 ml). This solution was
washed with phosphate buffer (pH = 7.2, 2 × 100 ml), and brine
(2×100 ml)thendried(MgSO4),filteredandconcentratedbyrotary
evaporation. The residue was purified by flash chromatography
using ethylacetate: n-hexane (15 : 85) to yield 8.0 g of 8 as pale
1
yellow glassy semisolid (yield 88%). H NMR (300 MHz, CDCl3): δ
7.25–7.18 (m, 4H, HTyr), 7.12 (s, 1H, HDCV), 5.02 (d, J = 7.3 Hz, 1H,
NHTyr), 4.43–4.40 (m, 1H, CHTyr), 3.06–3.04 (m, 2H, CH2−Tyr), 1.39
(s, 9H, Htert−but), 1.36 (s, 9H, Htert−but); 13C NMR (75 MHz, CDCl3):
δ 170.5, 154.9, 148.8, 137.1, 133.6, 131.2, 120.8, 117.20, 82.2, 79.7,
54.7, 37.9, 28.2, 27.8; HRMS (ESI+): calculated for C20H28Cl2NO8S
(M+H)+ 512.0913, found 512.0917.
Materials and Methods
Rink amide resin, amino acids and coupling reagents used for
peptide synthesis were purchased from Novabiochem Corp. (San
Diego, CA, USA) and/or Advanced Chem Tech, Inc (Louisville,
KY, USA). L-amino acids were used for all peptide syntheses un-
less stated otherwise. Reagents used for the synthesis of amino
acid 1 were obtained from Aldrich Chemical Company (Oakville,
ON, Canada). PseudoprolinedipeptideFmocSer(tBu)ꢀMe,MeProOH
(12) and FmocAsp(OtBu)Gly(DMB)OH (14) were prepared accord-
ing to literature procedures [22,23]. Tetrahydrofuran (THF) was
distilled from sodium metal in the presence of benzophenone
under argon. 2-MP was obtained from Waterstone Technology
(Carmel, IN, USA) and was used without further purification. All
automated SPPS was performed using the Rink amide resin and
were performed on a Quartet peptide synthesizer from Protein
Technologies (Tucson, AZ, USA) on a 25 µM scale. Analytical and
semi-preparative RP-HPLC was achieved using Waters 600 con-
troller equipped with a Waters 2487 detector with the detector
set to 220 nm. Analytical HPLC was performed with a Vydac
218TP54 C18 column (5 µm, 4.6 mm × 250 mm) and/or Hig-
gins PROTO 200 C18 column (5 µm, 4.6 mm × 250 mm) using
a 1.0 ml/min flow rate. Semi-preparative HPLC was conducted on
Nα-[(Fluoren-9-yl)methoxylcarbonyl]-L-Tyrosine Dchlorovinyl
Sulfate, 1
Compound 8 (6.0 g, 11 mmol, 1.0 Eq) was dissolved in TFA (23 ml)
and the mixture was stirred at room temperature for 2 h then
concentrated by rotary evaporation. This process was repeated
using the same quantity of TFA. After rotary evaporation a third
portion of TFA (23 ml) was added and the mixture was stirred
overnight. The mixture was concentrated by rotary evaporation
and the residue was suspended in CHCl3 (50 ml) and concentrated
by rotary evaporation and this process was repeated several times
untilawhitesolidformedwhichwasdriedunderhighvacuum. The
residue was dissolved in an aqueous solution of sodium carbonate
(42 ml, 3.7 g, 34 mmol, 3.0 Eq) and the mixture was cooled using
an ice bath. A solution of Fmoc–OSu (5.9 g, 17 mmol, 1.5 Eq) in
dioxane (42 ml) was added and the reaction was allowed to warm
to roomtemperature then stirred overnight. The reaction mixture
was acidified using 1 M HCl (to pH = 2), extracted with EtOAc
(3 × 100 ml), dried (MgSO4), filtered and concentrated by rotary
evaporation. The residue was subjected to flash chromatography
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SYNTHESIS OF DISULFATED PEPTIDES FROM CXCR6 AND DARC
(100% CH2Cl2 to 5% MeOH in CH2Cl2) which gave pure 1 as a white
foam5.7 g(85%). 1HNMRand13CNMRdatawereidenticaltothose
previously reported for this compound [13]. The enantiopurity
of 1 was determined by constructing diastereomeric dipeptides
AcY(SO3DCV)A(DL)NH2, AcY(SO3DCV)A(L)NH2, and analyzing them
by HPLC and 1H-NMR as previously described [13]. See the
SupportingInformationfortheHPLCchromatogramsand1H-NMR
spectra of the diastereomeric peptides.
removed by dissolving them (10 mg) in H2O (1 ml) containing Et3N
(11 Eq for peptide 2 and 17 Eq for peptide 3 (1 Eq of Et3N per acidic
amino acid plus 2 Eq per DCV group) and the resulting solution
was diluted with HPLC grade methanol (1 ml). Pd(OH)2 (20% w/w,
5 mg) was added and the mixture was stirred at roomtemparature
under hydrogen gas (balloon pressure) overnight. The mixture
was transferred to a microcentrifuge tube and centrifuged. The
solution was decanted and the residue resuspended in 0.5 ml of
methanol and centrifuged and decanted again. The combined
supernatants were purified using semi-preparative RP-HPLC.
Peptide 2 was eluted from the semi-preparative HPLC column
using CH3CN/20 mM ammonium acetate as eluent and a linear
gradient of 10–15% CH3CN over 30 min. This gave 3.8 mg (41%
based on resin loading) of peptide 2 as a flocculent white
powder after repeated lyophilizations until a constant weight was
obtained. This material was determined to be approximately 97%
purebyanalyticalRP-HPLC(lineargradientof10 : 90,CH3CN:20 mM
ammonium acetate to 15 : 85 CH3CN : 20 mM ammonium acetate
over 30 min, tR = 25.92 min (see Figure 3). LRMS (ESI−): calculated
for C103H134N27O45S3 (M−H)−1 2564.8195, found 2564.5745.
Peptide 3 was eluted from the semi-preparative HPLC column
using CH3CN/20 mM ammonium acetate as eluent and a linear
gradient from 15% to 20% CH3CN over 60 min, (tR = 15.8 min).
This gave 2.9 mg (32% yield based on resin loading) of peptide 3
as a flocculent white powder after repeated lyophilizations until a
constant weight was obtained. This material was determined to be
approximately 98% pure by analytical RP-HPLC (linear gradient of
15 : 85 CH3CN : 20 mM ammonium acetate to 20 : 80 CH3CN : 20 mM
ammonium acetate over 60 min (tR = 22.46 min) (see Figure 6).
LRMS (ESI−): calculated for C169H234N41O74S2 (M−H)−1 4085.5255,
found 4085.2175.
General Procedure for the Solid Phase Synthesis of Peptides 2
and 3
Automated SPPS was used. Fmoc amino acids used for peptide as-
sembly were protected at their side chains with a tert-butyl group
for Asp, Gly, Thr, Ser, a trityl group for Asn, Gln and His, and a Boc
group for Lys. For peptide 2, Ser13 and Ser 14 were incorporated
using pseudoproline dipeptide FmocSer(tBu)ꢀMe,MeProOH (12).
For peptide 3, Asp38 and Glu39 were incorporated as dipeptide
FmocAsp(OtBu)Gly(DMB)OH (14) and residues Ser28 and Ser29
and Ser17 and Ser18 were incorporated as pseudoproline
dipeptide 12. The Rink amide resin was swollen in DMF for 30 min
prior to the attachment of the first amino acid. All couplings were
performed using 5-Eq amino acid in DMF and using Cl–HOBt (5
Eq)/HCTU (5 Eq)/DIPEA (5 Eq) unless stated otherwise. The DIPEA
was added as a separate solution to the reaction mixture. Double
couplings (2 × 20 min) were used throughout unless stated other-
wise. For the synthesis of peptide 2, Glu8, Asp9 and pseudoproline
dipeptide12wereincorporatedusing4-Eqaminoacidordipeptide
andHOAt(4Eq)/HATU(4 Eq)/DIPEA(4Eq)ascouplingagents(dou-
blecouplings, 2×45 min). Forthesynthesisofpeptide3, dipeptide
14 and pseudoproline dipeptide 12 were incorporated using 4 Eq
amino acid or dipeptide and HOAt (4 Eq)/HATU (4 Eq)/DIPEA (4 Eq)
as coupling agents (double couplings, 2 × 45 min). To conserve
on expensive dipeptides 12 and 14, these two dipeptides were
added manually to the reaction mixture rather than automatically
using the machine. A capping step using a 2 : 1 : 3 solution of
pyridine : acetic anhydride : DMF was performed (1 × 10 min) after
the incorporation of each amino acid. Fmoc deprotections were
performed using 2-MP (3×10 min). After each coupling and Fmoc
deprotection the resin was washed with DMF (6 ×30 s). After each
capping theresinwaswashedwithdichloromethane(6×30 s)and
DMF (6 × 30 s). After coupling and Fmoc deprotection of the last
amino acid, the peptide was acylated by subjecting it to a 2 : 1 : 3
solution of pyridine : acetic anhydride : DMF for 60 min. The resin
was washed with DMF (3 ×10 min), dichloromethane (3 ×10 min)
and then dried. The peptides were cleaved from the resin by sub-
jecting the resin to TFA : TIS : H2O : EDT (92.5 : 2.5 : 2.5 : 2.5, 2.5 ml)
for 2.5 h then filtered. The resin was subjected to the cleavage
cocktail again (2.5 ml) for 5 min and filtered again. The combined
filtrates were concentrated to half volume under reduced
pressure. The material was transferred to a 50 ml centrifuge tube
and diethyl ether was added which resulted in the precipitation
of the peptides. The mixture was cooled in a dry ace-acetone bath
for 30 min then centrifuged (5000 rpm, −4 ◦C). The supernatant
was decanted and acetonitrile was added to the resulting pellet
until the pellet dissolved. The solution was transferred to a
round bottom flask and concentrated to dryness. The residue
was dissolved or suspended in water and lyophilized. The crude
peptides 11 and 13 were analyzed by analytical RP-HPLC eluting
with a linear gradient of 5 : 95 CH3CN : H2O (0.1% TFA) to 95 : 5
CH3CN : H2O (0.1% TFA) over 60 min (λ = 220 nm) (See Figures 1B
and 4C). The DCV groups in crude peptides 11 and 13 were
Results and Discussion
Key to our approach to sTyr peptides is the use of amino acid
1. Our original approach to 1 involved reacting FmocTyrOtBu
with reagent 4 (Scheme 2) to introduce the DCV-protected sulfate
group [13]. Although this works very well, it necessitates the prior
preparation of reagent4. Reagent4 is readily prepared in excellent
yield but requires the use of trimethyloxonium tetrafluoroborate a
relatively expensive methylating agent [13]. We wished to develop
a more direct route to compound 1 that did not involve the use of
reagent 4. Toward this end amino acid 5 was reacted with readily
available trichloroethylsulfurylchloride (6) [24] (Scheme 3) in the
presence of DMAP/Et3N to give amino acid 7. After an aqueous
workup, crude 7 was reacted with DBU which gave DCV-protected
amino acid 8 in an 88% yield. Removal of the Boc and t-butyl
groups in 8 using TFA followed by reaction with Fmoc–OSu gave
compound 1 in an 85% yield. The enantiopurity of compound 1
prepared in this manner was found to be >98% as determined
by the synthesis of diastereomeric peptides followed by HPLC
analysis (see the Supporting Information).
Before embarking on a synthesis of peptides 1 and 2 it was
necessary to develop conditions for removal of the DCV group
that would not result in the reduction of the indole ring in Trp
residues. We initially examined conditions that have been used
to remove trichloroethyl-based protecting groups from amines
and phosphates. Treatment of hexapeptide Ac-Asp-Ala-Asp-Glu-
Tyr(SO3DCV)-L-NH2 (9) [13] with zinc/pyridine/acetylacetone [25],
tributylphospine [26], or tetra-n-butylammonium fluoride [27], all
failed to give Ac-Asp-Ala-Asp-Glu-Tyr(SO3−)-L-NH2 (10). Zn(Cu) in
c
J. Pept. Sci. 2010; 16: 190–199 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ALI AND TAYLOR
Figure 1. Analytical RP-HPLC chromatograms of the crude products obtained after cleavage from the resin and side chain deprotection from attempts
to prepare peptide 2. Chromatogram (A) using HBTU/HOBt as coupling reagents (1 × 1.5 h coupling time). Chromatogram (B) Glu8 and Asp9 were
incorporated using HATU/HOAt and double couplings (2 × 45 min). The remainder of the amino acids where incorporated using HCTU/Cl–HOBt and
double couplings (2 × 45 min). Chromatogram (C) Glu8, Asp9, and pseudoproline dipeptide 12 were incorporated using HATU/HOAt and double
couplings (2 × 45 min). The remainder of the amino acids where incorporated using HCTU/Cl–HOBt and double couplings (2 × 45 min). See Materials
and methods for details.
DMF [28] (entry 3) successfully removed the DCV group; however,
the reaction turned deep brown upon completion with the
formation of heavy precipitate which we anticipated would be
problematic when applied to peptides. Attempts to remove the
DCV group by hydrolysis in buffers ranging from pH 5 to 9 at 40 ◦C
were also unsuccessful. Subsequent to these studies we became
aware of a report by Medzihradszky-Schweiger who reported
that hydrogenolysis of carbobenzyloxy (Cbz) protecting groups in
Trp-bearing peptides using H2 and 10% P/C (MeOH as solvent)
did not result in the reduction of the indole ring when the
hydrogenolysis was performed in the presence of bases such as
Et3N [29] (It is also worthy of note that Medzihradszky-Schweiger
reported that poisoning of the Pd catalyst by sulfur-containing
aminoacidsdidnotoccurwhenthehydrogenolysiswasperformed
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SYNTHESIS OF DISULFATED PEPTIDES FROM CXCR6 AND DARC
O
amino acid, HBTU/HOBt as coupling reagents (1 × 1.5 h cou-
pling time) and using 2-MP for Fmoc removal (3 × 10 min)
followed by a capping step. After the assembly of the peptide
chain the peptide cleavage from the resin was achieved using
TFA : TIPS : H2O : EDT (92.5 : 2.5 : 2.5 : 2.5). However, the HPLC chro-
matogram of the crude material obtained after cleavage from the
support showed many peaks (Figure 1A). The mass spectrum of
the crude mixture indicated that the desired DCV-protected pep-
tide AcMAEHDY(SO3DCV)HEAY(SO3DCV)GFSSFNDSSQNH2 (11)
was present and this corresponded to the largest peak in
the HPLC chromatogram with a retention time of 30.7 min.
Two other significant peaks in the mass spectrum were identi-
fied as having resulted from two truncated acetylated peptides
corresponding to the residues 9–20 and 10–20. An attempt
to improve the synthesis of 11 was made by using the ap-
parently more potent coupling agent HCTU and performing
double couplings (2 × 45 min). However, this did not result
in an improvement as the HPLC chromatogram of the crude
peptide was very similar to our first attempt. Since two trun-
cated peptides corresponding to residues 9–20 and 10–20
were obtained in our previous syntheses, in our third attempt
we incorporated Glu8 and Asp9 using HATU/HOAt which has
been reported to be a highly efficient coupling agent [30]
The remainder of the amino acids where incorporated using
HCTU/Cl–HOBt. Although the HPLC trace of the crude mixture
showed some improvement many other peaks were still present
(Figure 1B).
One problem commonly encountered in SPPS is poor solvation
of the growing peptide chain which stems from aggregation of
hydrophobicresiduesorprotectinggroupsand/ortheformationof
secondary structures such as β-sheet formation. Such aggregation
leavesalimitednumberoffreeaminogroupsavailableforcoupling
resulting in poor coupling yields. One approach to prevent this
phenomenon is to use the so-called ‘‘pseudoprolines’’ in which
the hydroxyl group of a serine or threonine residue is reversibly
bound through an alkyl bridge to the nitrogen atom of the amide
backbone (Figure 2) [31–33]. Pseudoproline residues induce a
kink in the peptide backbone which disrupts intermolecular
or intramolecular aggregations commonly experienced during
peptide synthesis. Consequently, the use of pseudoproline often
results in an improvement in yield. The pseudoprolines are
converted back to serine or threonine during the acid conditions
used for cleaving the peptide from the support and removing
side chain protecting groups. To determine if this was an issue
CH
O
3
+
Cl
Cl
1.
O
S
FmocNH
OtBu
FmocNH
CH
N
N
O
3
-
OH
BF
O
(4)
4
2.TFA
OH
OSO3DCV
1
Scheme 2. Synthesis of FmocTyr(SO3DCV)OH (1) using reagent 4.
in the presence of bases such as Et3N). This prompted us to
examine whether the DCV group could be removed under basic
hydrogenolysis conditions. Peptide 9 was suspended in water and
5 Eq of triethylamine was added (1 Eq per acidic group in the
peptide plus one extra Eq) followed by the addition of 30–50 wt%
of 10% Pd/C or Pd(OH)2. Hydrogenolysis of the mixture using H2
(balloon pressure) for 16 h (overnight) gave the desired peptide 10
inessentiallyquantitativeyields(Toconfirmthatnohydrogenation
of the indole ring in Trp would occur when subjected to our basic
hydrogenolysisconditions,weprepareddipeptideAc-Trp-Ala-NH2
and subjected it to 50% wt% of Pd(OH)2, H2 (balloon pressure),
2 Eq Et3N, in H2O at roomtemperature for 16 h. No reduction
of the indole ring occurred as determined by 1H-NMR. See the
Supporting Information for details).
In our previous report on the synthesis of sTyr peptides,
we used 95% TFA/5% TIS to cleave the peptides from the
support and remove other side chain protecting groups [13].
However, for Met-bearing peptides it is usually essential that
scavengers such as thiols or disulfides like EDT or thioanisole
are present because these reagents help prevent the oxida-
tion of Met residues. To determine if the DCV group is stable
to cleavage cocktails containing EDT or thioanisole we sub-
jected Ac-Asp-Ala-Asp-Glu-Tyr(SO3DCV)-L-NH2 (9) to commonly
used cleavage cocktails containing these scavengers such as
TFA : TIS : EDT (95 : 2.5 : 2.5), TFA : TIS : EDT : H2O (92.5 : 2.5 : 2.5 : 2.5),
TFA : TIS : thioanisole (95 : 2.5 : 2.5) and TFA : TIS : Thioanisole : H2O
(92.5 : 2.5 : 2.5 : 2.5) for 2 h at roomtemperature. No peptide result-
ing from loss of the DCV group was detected (using analytical
HPLC) indicating that the DCV group is stable to these cleavage
cocktails.
Having established conditions for removal of the DCV group
that would be compatible with Trp residues and stable to sulfur-
containing scavenging cocktails we embarked on the synthesis
of peptides 2 and 3. Our first attempt to synthesize pep-
tide 2 employed our usual conditions [13] using automated
SPSS with the Rink amide resin (0.71 mmol/g), 5 Eq Fmoc
ClSO3CH2CCl3
O
O
O
(6)
BocHN
BocHN
OtBu
OtBu
BocHN
DBU
OtBu
DMAP/Et3N, THF
OH
OSO3TCE
OSO3DCV
8 (88%)
1. TFA/H2O
5
7
2. Fmoc-OSu
O
FmocHN
OH
OSO3DCV
1 (85%)
Scheme 3. Synthesis of FmocTyr(SO3DCV)OH (1) from amino acid 5.
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J. Pept. Sci. 2010; 16: 190–199 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ALI AND TAYLOR
cleavage from the support). Employing this strategy and after
the cleavage from the support, the crude mixture exhibited two
major peaks at tR = 23.7 and 26.3 min along with a variety of
minor peaks in the HPLC chromatogram (Figure 4A). Upon MS
H
R
O
HN
NH
R1
N
O
O
analysis of the two major peaks we found that the peak at tR
=
23.7 min corresponded to the desired DCV-protected precursor
to peptide 3, AcAELSPSTENS17S18QLDFEDVWNS28S29Y(SO3DCV)
GVNDSFPDGDY(SO3DCV)D-NH2 (13), while the peak at tR = 26.3
differed from 13 by just 18 mass units suggesting loss of a water
molecule. We reasoned that the peak at tR = 26.3 was due
to aspartimide formation at Asp38 and Gly39 because Asp–Gly
sequences are known to be very susceptible to this side reaction
during Fmoc SPPS (Scheme 4) [34]. When this reaction does occur
during Fmoc-based SPPS peptide synthesis some or all of the
resulting aspartimide often reacts with piperdine (if piperidine is
used for Fmoc removal) to give tertiary amides. The aspartimide
can also be hydrolyzed to the α-Asp and β-Asp peptides. If
aspartimide formation is indeed occurring during the synthesis
of peptide 13 then reaction of the aspartimide with 2-MP is not
occurring because the mass spectrum of the mixture did not
indicate that any products resulting from this reactions were
formed. This suggests that 2-MP is too sterically hindered to attack
the imide.
Figure 2. Incorporating pseudoproline (ꢀMe,Me Pro shown) into a peptide
induces a kink in the peptide backbone. R H (if derived from serine) or
R
CH3 (if derived from threonine).
with the synthesis of peptide 2 we decided to examine this tactic
as a means of increasing the quality of crude peptide 11. There
are four possible sites within peptide 2 where pseudoproline
can be incorporated: Ser13, Ser14, Ser18, and Ser19. Because
the latter two are near the C-terminus, we anticipated that
there would be little benefit to replace either of them with
pseudoproline. Pseudoprolines are incorporated into peptides as
dipeptides: Fmoc-AA-ꢀPro-OH. FmocSer(tBu)ꢀMe,MeProOH (12)
is a known compound and is commercially available. Therefore,
we elected to replace Ser14 with ꢀMe,MePro. A considerable
improvement in the quality of crude 11 was obtained when
we used dipeptide 12 and the conditions described above during
our third preparation of 2 as indicated by the HPLC trace of the
crude product which showed a single major peak corresponding
to our target compound (Figure 1C) as well as a few other
minor peaks. The DCV groups in crude 11 were removed by
subjecting it to 50% w/w Pd(OH)2 under H2 atmosphere (balloon
pressure) in water/methanol (1 : 1) containing 11 Eq Et3N and
stirring it overnight. After semi-preparative RP-HPLC purification
peptide 2 was obtained in a very respectable 41% overall yield
in approximately 97% purity as determined by analytical RP-HPLC
(Figure 3).
Because peptide 3 contains two Ser–Ser sequences at positions
17 and 18 and 28 and 29 our initial strategy for the synthesis of
peptide 3 employed HATU/HOAt to incorporate pseudoproline
dipeptide 12 at positions 17 and 18 and 28 and 29 while
incorporating all other amino acids using HCTU/Cl–HOBt. Double
coupling(2×20 min)andcappingstepswerealsoemployed.Fmoc
deprotection and cleavage from the support were done using
our usual protocols (using 2-MP for Fmoc removal (3 × 10 min)
and using TFA : TIPS : H2O : EDT (92.5 : 2.5 : 2.5 : 2.5) for 2 h for
Although there are a variety of ways to avoid aspartimide for-
mation perhaps the most effective way is N-backbone protection
with the Hmb group or the Dmb group [34,35]. Consequently, we
decided to incorporate residues Asp38 and Gly39 as the protected
dipeptide FmocAsp(OtBu)Gly(DMB)OH (14, Figure 5). Peptide syn-
thesis was performed using the same conditions as our previous
attempt except dipeptide 14 was substituted for Asp38 and Gly39.
The HPLC trace of the crude product after cleavage from the resin
indicated little or no aspartimide formation as indicated by the
absenceofasignificantpeakattR = 23.7 min(Figure 4B). Removal
of the DCV groups was achieved by subjecting crude 13 to 50%
w/w of Pd(OH)2 in presence of Et3N (17 Eq), and H2 (balloon pres-
sure) in water : methanol (1 : 1) for 24 h. No peaks corresponding
to peptide 3 containing a reduced Trp residue were evident in
the mass spectrum of the crude material. After semi-preparative
RP-HPLC purification peptide 3 was obtained in a 32% overall yield
in approximately 98% purity as determined by analytical RP-HPLC
(Figure 6).
Figure 3. Analytical RP-HPLC chromatogram of peptide 2 after RP-HPLC purification. See Materials and methods for details.
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SYNTHESIS OF DISULFATED PEPTIDES FROM CXCR6 AND DARC
Figure 4. Analytical RP-HPLC chromatograms of the crude products obtained after cleavage from the resin and side chain deprotection from attempts to
prepare peptide 3. Chromatogram (A) pseudoproline dipeptide 12 was incorporated using HATU/HOAt and double couplings (2×45 min). The remainder
of the amino acids where incorporated using HCTU/Cl–HOBt and double couplings (2 × 20 min). Chromatogram (B) Dipeptide 14 and pseudoproline
dipeptide 12 were incorporated using HATU/HOAt and double couplings (2 × 45 min). The remainder of the amino acids were incorporated using
HCTU/Cl–HOBt and double couplings (2 × 45 min). See Materials and methods for details.
O
O
H
N
O
Ot-Bu
H
N
O
O
O
N
H
N
COOH
O
N
N
H
O
O
N
H
N
H
O
O
O-tBu
aspartimide
O
14
Scheme 4. Aspartimide formation at an Asp-Gly sequence during peptide
synthesis.
Figure 5. Structure of FmocAsp(OtBu)Gly(DMB)OH (14).
c
J. Pept. Sci. 2010; 16: 190–199 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ALI AND TAYLOR
Figure 6. Analytical RP-HPLC chromatogram of peptide 3 after RP-HPLC purification. See Materials and methods for details.
Conclusions
were identical suggesting that deprotection times of 3 min +
11 min can be used with 2-MP (Ali, Taylor, unpublished results).
Interestingly, when piperidine was used no peptides resulting
from attack of piperidine on the sulfur atom of the DCV-protected
sTyr residue were detected. In any case, we suggest here that 2-MP
is a very practical and cost effective alternative to piperidine for
SPPS.
We have shown that sTyr peptides containing Met or Trp
residues can be readily prepared using our sulfate-protecting
group strategy. This was demonstrated by preparing a peptide
corresponding to residues 1–20 of CXCR6 (containing Met at
position 1) and a 35-mer peptide corresponding to residues 8–42
in DARC at (containing a Trp residue at position 26). Removing the
DCV groups at the end of the syntheses was readily achieved by
hydrogenolysis without any reduction of Trp by performing the
hydrogenolysis in the presence of triethylamine. Indeed, we have
found these conditions to be particularly efficient for removing
the DCV group and superiour to our original conditions using
H2, ammonium formate, Pd/C. The presence of Met was found
not to interfere with the removal of the DCV group. The use of
pseudoproline dipeptides and N-backbone protection with the
Dmb group were found to be effective tactics for preventing
aggregation and aspartimide formation during the synthesis of
these peptides. We also found that the key amino acid 1 can be
readily prepared in excellent yield from amino acid 5 and without
the use reagent 4.
Acknowledgements
This research was supported by a Discovery Grant from the Natural
Sciences and Engineering Research Council (NSERC) of Canada to
SDT. We thank the Egyptian Ministry of Higher Education for a
scholarship to AMA.
Supporting information
Supporting information may be found in the online version of this
article.
References
Key to the success of our approach to sTyr peptides is the
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piperidine is a controlled substance and so can be a nuisance
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not subjected to the restrictions and regulations that piperidine is
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have recently performed studies on the synthesis of a 12-residue
peptide (AcISDRDY(SO3DCV)MGWMDF-NH2) derived from CCK
using either 2-MP or piperidine and Fmoc deprotection times of
3 min + 11 min. The HPLC chromatograms of the crude peptides
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