Tyrosine-sulfate isosteres of CCR5 N-terminus as tools for studying HIV-1 entry

Article abstract of DOI:10.1016/j.bmc.2008.10.005

The HIV-1 co-receptor CCR5 possesses sulfo-tyrosine (TYS) residues at its N-terminus (Nt) that are required for binding HIV-1 gp120 and mediating viral entry. By using a 14-residue fragment of CCR5 Nt containing two TYS residues, we recently showed that CCR5 Nt binds gp120 through a conserved region specific for TYS moieties and suggested that this site may represent a target for inhibitors and probes of HIV-1 entry. As peptides containing sulfo-tyrosines are difficult to synthesize and handle due to limited stability of the sulfo-ester moiety, we have now incorporated TYS isosteres into CCR5 Nt analogs and assessed their binding to a complex of gp120-CD4 using saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). STD enhancements for CCR5 Nt peptides containing tyrosine sulfonate (TYSN) in complex with gp120-CD4 were very similar to those observed for sulfated CCR5 Nt peptides indicating comparable modes of binding. STD enhancements for phosphotyrosine-containing CCR5 Nt analogs were greatly diminished consistent with earlier findings showing sulfo-tyrosine to be essential for CCR5 Nt binding to gp120. Tyrosine sulfonate-containing CCR5 peptides exhibited reduced water solubility, limiting their use in assay and probe development. To improve solubility, we designed, synthesized, and incorporated in CCR5 Nt peptide analogs an orthogonally functionalized azido tris(ethylenoxy) l-alanine (l-ate-Ala) residue. Through NMR and SPR experiments, we show a 19-residue TYSN-containing peptide to be a functional, hydrolytically stable CCR5 Nt isostere that was in turn used to develop both SPR-based and ELISA assays to screen for inhibitors of CCR5 binding to gp120-CD4.

Full text of DOI:10.1016/j.bmc.2008.10.005

Bioorganic & Medicinal Chemistry 16 (2008) 10113–10120
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tyrosine-sulfate isosteres of CCR5 N-terminus as tools for studying HIV-1 entry
Son N. Lam a, Priyamvada Acharya b, Richard Wyatt b, Peter D. Kwong b, Carole A. Bewley a,
*
a Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
b Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The HIV-1 co-receptor CCR5 possesses sulfo-tyrosine (TYS) residues at its N-terminus (Nt) that are
required for binding HIV-1 gp120 and mediating viral entry. By using a 14-residue fragment of CCR5
Nt containing two TYS residues, we recently showed that CCR5 Nt binds gp120 through a conserved
region specific for TYS moieties and suggested that this site may represent a target for inhibitors and
probes of HIV-1 entry. As peptides containing sulfo-tyrosines are difficult to synthesize and handle
due to limited stability of the sulfo-ester moiety, we have now incorporated TYS isosteres into CCR5
Nt analogs and assessed their binding to a complex of gp120–CD4 using saturation transfer difference
(STD) NMR and surface plasmon resonance (SPR). STD enhancements for CCR5 Nt peptides containing
tyrosine sulfonate (TYSN) in complex with gp120–CD4 were very similar to those observed for sulfated
CCR5 Nt peptides indicating comparable modes of binding. STD enhancements for phosphotyrosine-con-
taining CCR5 Nt analogs were greatly diminished consistent with earlier findings showing sulfo-tyrosine
to be essential for CCR5 Nt binding to gp120. Tyrosine sulfonate-containing CCR5 peptides exhibited
reduced water solubility, limiting their use in assay and probe development. To improve solubility, we
designed, synthesized, and incorporated in CCR5 Nt peptide analogs an orthogonally functionalized azido
Received 12 August 2008
Revised 1 October 2008
Accepted 1 October 2008
Available online 5 October 2008
Keywords:
HIV-1 gp120
Phenylmethanesulfonic acid
Tyrosine sulfonate
Saturation transfer difference NMR
Surface plasmon resonance
Enzyme-linked immunosorbent assay
Small molecule inhibitor
tris(ethylenoxy)
L-alanine (L-ate-Ala) residue. Through NMR and SPR experiments, we show a 19-residue
TYSN-containing peptide to be a functional, hydrolytically stable CCR5 Nt isostere that was in turn used
to develop both SPR-based and ELISA assays to screen for inhibitors of CCR5 binding to gp120–CD4.
Published by Elsevier Ltd.
1. Introduction
Despite interesting and important biological roles, Tyr(SO4)-
containing peptides are not without liabilities. The ArOASOꢀ3 bond
HIV-1 infection is initiated through interactions between the
viral surface envelope (Env) glycoprotein gp120 and cellular recep-
tors CD4 and CCR5 or CXCR4.1,2 CCR5 and CXCR4, also referred to as
HIV-1 co-receptors, are 7-transmembrane spanning G-protein cou-
pled receptors that have the unusual feature of containing sulfo-
tyrosine (TYS) residues in their extracellular N-terminal domains.3
CCR5 N-terminus (Nt) contains four tyrosine residues at positions
3, 10, 14, and 15, and sulfation of at least residues 10 and 14 is
essential for mediating viral entry.4 Moreover, CCR5 Nt peptides
containing sulfo-tyrosine residues at positions 10 and 14 inhibit
HIV-1 membrane fusion.5,6 Recently we showed by NMR that a
CCR5 Nt peptide comprising residues 2–15 (1, Nt2–15), where
Tyr10 and Tyr14 are sulfated, binds CD4-activated HIV-1 gp120
(CD4–gp120) but not gp120 or CD4 alone; and that residues 9–
15 adopt an ordered, alpha helical structure upon binding.7 Molec-
ular docking of the minimized mean structure showed CCR5 Nt to
dock in a single orientation to a conserved region on gp120 specific
for sulfo-tyrosine. Those studies demonstrated that a CCR5 Nt pep-
tide fragment can function as a natural co-receptor mimic.
is prone to hydrolysis.8 Lack of robust protecting groups orthogo-
nal to the sulfate group combined with the need for acid catalyzed
cleavage of side chain protecting groups and peptide from resin
can make peptide synthesis difficult and yields low. Furthermore,
the need to prevent hydrolysis during chromatographic separa-
tions can make purification of crude peptides
a challenge.
Together, these factors place limitations on the use of Tyr(SO4)-
containing peptides as versatile biochemical probes and tools.
We thus sought to develop a functional, non-hydrolyzable CCR5
Nt peptide analog amenable to orthogonal modifications and
whose synthesis can be scaled up for assay development. To this
end, we used saturation transfer difference9 (STD) NMR and/or sur-
face plasmon resonance (SPR) techniques to investigate the effects
on gp120 binding of CCR5 Nt peptides where (i) sulfo-tyrosine (re-
ferred to hereafter as TYS) residues were replaced by tyrosine
phosphate [TyrðPO23ꢀ), referred to as TYP] and tyrosine sulfonate
[TyrðCH2SOꢀ), referred to as TYSN] isosteres (Fig. 1); (ii) their
3
length was increased to 17 or 19 residues; (iii) a third TYS or TYSN
isostere at residue Tyr3 was incorporated; and/or (iv) a C-terminal
biotinylated polyethylene glycol linker was introduced. On the ba-
sis of these results, we have synthesized a functional CCR5 Nt ana-
log (8) bearing chemically stable TYSN residues, and shown that
* Corresponding author. Tel.: +1 301 594 5187; fax: +1 301 402 4182.
E-mail address: caroleb@mail.nih.gov (C.A. Bewley).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.10.005

10114
S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
Nt(2-15)
R1
3
OH
OH
NH2
O
O
O
O
1 R1 = OH, R2 = R3 = OSO3
TYS10 / TYS10
-
O
O
H
N
H
N
2
OH
AcHN
HO
O
N
-
2-
O
H
N
2 R1 = OH, R2 = OSO3 , R3 = OPO3 TYS10 / TYP14
N
N
H
N
H
O
H
N
H
15
NH2
H
O
O
O
O
N
-
N
H
N
H
N
H
3 R1 = OH, R2 = OPO32-, R3 = OSO3
TYP10 / TYS14
TYSN10 / TYSN14
TYP10 / TYP14
N
H
OH
O
O
O
-
4 R1 = OH, R2 = R3 = CH2SO3
O
H2N
O
10
14
2-
5 R1 = OH, R2 = R3 = OPO3
R2
R3
Nt(2-18)
R1
OH
O
3
OH
O
O
O
O
O
H
N
H
N
2
OH
NH2
-
6 R1 = OH, R2 = R3 = OSO3
AcHN
HO
N
OH
18
O
TYS10 / TYS14
TYS3 / TYS10 / TYS14
TYSN3 / TYSN10 / TYSN14
O
H
N
N
N
H
N
H
O
H
N
O
H
H
H
H
O
-
7 R1 = R2 = R3 = OSO3
O
O
O
N
N
H
N
N
N
H
N
N
H
OH
N
NH2
O
O
H
O
8 R1 = R2 = R3 = CH2SO3
H
-
O
O
O
O
H2N
O
OH
10
14
HO
R2
R3
Figure 1. CCR5 Nt peptides and analogs.
when biotinylated through a PEG linker (12) the TYSN-containing
construct can be used to screen in both SPR-based and ELISA plat-
forms for molecules that specifically inhibit CCR5 Nt binding to
HIV-1 gp120. Additionally, STD NMR of peptides containing one
TYS and one TYP residue validate our model of CCR5 Nt binding
to gp120.
tively, exhibited the strongest enhancements within the entire
spectrum (normalized to 100%), while those for TYS10 were esti-
mated at ꢁ66% (spectral overlap occurs between TYS10 Hd and
TYS10 He), consistent with our docked model. Relative to these
aromatic protons, enhancements for all other residues, including
Tyr3, were negligible in comparison.
Having mapped by STD NMR the epitope used by CCR5-Nt to bind
gp120, we next assessed binding to gp120–CD4 of Nt2–15 peptides
bearing a single substitution of TYP in place of TYS at either position
10 or 14. As seen in the expansion of the aromatic region of the STD
NMR spectrum for analog 2 which contains phosphotyrosine at res-
2. Results and discussion
Recently we solved by NMR the solution structure of a func-
tional 14-residue CCR5 Nt peptide comprising residues 2–15 (1)
in complex with gp120–CD4.7 When bound to this complex, the
backbone of residues 9–15 adopt an alpha helical structure that
displays TYS10 and TYS14 from the same face of the helix
(Fig. 2). Molecular docking of CCR5 Nt to the crystal structure of
gp120–CD4 in complex with sulfated monoclonal antibody 412d
showed Nt to bind to the base of the third variable loop (V3) in a
manner similar to that of 412d. In the model, the side chain of
TYS14 makes numerous hydrogen bonds and extensive van der
Waal’s interactions with positively charged and hydrophobic resi-
dues forming this site (Fig. 2, orange surface), and Tyr15 packs clo-
sely to several neighboring hydrophobic residues (Fig. 2, green
surface). The side chain of TYS10 on the other hand is positioned
further from gp120, possibly making hydrogen bonds through its
sulfate group (Fig. 2, blue surface). This model was validated by
STD NMR, a technique that gives rise to a difference spectrum
whose proton signal intensities are proportional to their proximity
to receptor.9 As seen in the expansion of the aromatic region of a
1D 1H-STD NMR spectrum of 1 binding to gp120–CD4 (Fig. 3a), res-
idue 14, STD enhancementsfor TYP14Hd and Hedecrease from 100%
in1 to45%and54%in2, respectively(Fig. 3b, Table1). Enhancements
for Tyr3 and Tyr15 were unaffected by the presence of TYP14, while
enhancements for TYS10 increased slightly from 66% in 1 to 88% in 2.
In contrast, relative enhancements observed for tyrosine residues in
analog 3 (Fig. 3c) containingTYP at position 10 and TYS at position 14
were more similar to those of 1 (Fig. 3a) with strongest enhance-
ments observed for TYS14 and Tyr15 followed by TYP10. Although
overlap between signals for Tyr3 Hd and TYP10 He, and TYP10 He
and TYP10 Hd prevented accurate integration of the difference
spectrum for 3, a moderate decrease in intensity for signals corre-
sponding to TYP10 Hd and He relative to the same atoms in 1 is
apparent from comparison of Figure 3c with 3a. Incorporation of
two TYP residues at positions 10 and 14 (5) resulted in complete loss
of binding to gp120–CD4 (data not shown), in agreement with
previous studies.4 Together, these results indicate that the Tyr14
binding site on CD4-activated gp120 has a strong preference for
TYS while that of Tyr10 is less stringent and will accommodate
phosphotyrosine, albeit with diminished interactions.
onances corresponding to Hd and He of TYS14, and Tyr15, respec-
Figure 2. gp120–CCR5 interactions. (A) Model of CCR5 Nt (gray) docked to gp120 (beige)–CD4 (yellow). (B and C) Close-up of CCR5 Nt binding surface with gp120 rendered as
a surface, and CCR5 Nt backbone a cartoon. Side chains of TYS10, TYS14, and Tyr15 (labeled) are drawn as sticks. In (C), the structure is rotated 90° about the z axis relative to
(B). Contact surfaces unique to and within 5 Å of TYS10, TYS14 and TYR15 are colored light blue, orange, and green, respectively. Contact surfaces that overlap between TYS10
and TYS14 are colored dark blue, and surfaces that overlap between TYS14 and TYR15 are colored cyan.

S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
10115
Figure 3. Aromatic region of 1H STD NMR spectra of 14-residue CCR5 Nt2–15 (1) and analogs 2–4. Reference and difference spectra are shown in black and red, respectively. (a)
CCR5 Nt2–15 (1) containing TYS10 and TYS14; (b) analog 2 containing TYS10 and TYP14; and (c) analog 3 containing TYP10 and TYS14; (d) analog 4 containing TYSN10 and
TYSN14. Spectra were recorded at 298 K on samples containing 20 lM gp120–CD4 in the presence of 800 lM peptide at pH 6.8 with spectra normalized to 15e (ꢂ).
Table 1
A mode of binding of TYSN to gp120 that is similar to that of nat-
ural TYS and consistent with our NMR data is presented in Figure 4.
CCR5 TYS14 binds in a deep pocket on gp120 containing several po-
lar and one positively charged residue positioning the sulfate ester
to make a full complement of hydrogen bonds with the side chains
of Arg298, Asn302, and Thr303, and the backbone N atoms of
Asn302, Thr303, and Gly441 (Fig. 4B). In the case of TYSN, the
hydrogen bond between Asn302 and the ester oxygen would be
lost, and steric clash between the sulfonate methylene and sur-
rounding residues may be imposed (Fig. 4C). Any changes in the
binding pocket resulting from these differences would be expected
to reduce slightly the interactions with TYSN and gp120. Addition-
ally, the presence of the single positive charge, provided by Arg298,
may contribute to the selectivity of sulfate over phosphate.
STD enhancements for peptides 1–4 binding to gp120–CD4a
Peptide
10d
10e
14d
14
e
15d
15
e
Relative STDb
1
2
3
4
ꢁ70c
ꢁ70c
100
45
95
100
54
95
100
95
95
100
100
100
100
+++
+
+++
++
88
87
d
d
86
70
56
86
100
a
Aromatic proton resonances designated in bold. Enhancements for Tyr3 (not
shown) ranged from 5% to 35% for peptides 1–4.
b
STD enhancements of residue 14 relative to TYS in Nt2–15 (1).
Integration is approximate due to overlap between these signals.
Overlap prevented integration; qualitative comparisons of spectra shown in
c
d
Figure 3a and c indicate diminished enhancements relative to TYS10 in Figure 3b.
2.1. Sulfate versus sulfonate in the gp120 TYS binding pocket
At neutral pH, sulfo-tyrosine and phosphotyrosine bear formal
charges of ꢀ1 and ꢀ2, respectively. Although sulfate and phos-
phate groups differ slightly in size, because the Tys14 binding site
on gp120 carries a single positive charge, we reasoned that the dis-
crimination in binding sulfate versus phosphate might be attrib-
uted to this difference in charge.10 We next investigated the
singly charged non-hydrolyzable phenylmethanesulfonic acid moi-
2.2. Binding of CCR5 Nt(2–18) peptides
The predicted extracellular region of CCR5 N-terminus extends
at least to residue 20, with Cys20 forming a disulfide bridge with
Cys269 of the third extracellular loop and Tyr3 likely being sul-
fated.13 To examine the effects on binding gp120 longer CCR5 Nt
peptides bearing two or three sulfo-tyrosine residues, samples
comprising gp120–CD4 in complex with the 17-residue peptides
6 and 7, where sulfo-tyrosines are present at positions 10/14 and
3/10/14, respectively, were prepared and investigated by NMR
using conditions identical to those for 1–4. Relative to 14-mer 1,
1H NMR spectra for both constructs showed increased line broad-
ening in the presence of gp120–CD4, especially for TYS14 and
Tyr15 (Fig. 5a and b; Table 2) suggesting that gp120 binds these
constructs in slower exchange and with higher affinity than pep-
tides 1–4. 1H STD NMR revealed strong enhancements for TYS10,
ety Tyr ꢀ CH2 ꢀ SOꢀ (referred to as tyrosine sulfonate, or TYSN) as
3
a TYS isostere.11 Fmoc-protected tyrosine sulfonate was synthe-
sized12 and incorporated into CCR5 Nt analog 4, and its binding
to gp120–CD4 was assessed by NMR. The difference spectrum
(Fig. 3d) for 4 in the presence of gp120–CD4 clearly indicated bind-
ing to the complex. In particular, enhancements for the aromatic
protons of TYSN10 were comparable to those of TYS10 with values
ranging from 70% to 86%, while slightly reduced binding of TYSN to
the gp120 TYS14 binding site was apparent from an enhancement
of 86% for H
e
of TYSN14 relative to 100% for TYS14. On the other
of TYP versus
TYS14, and Tyr15 in Nt2–18 (6). Additionally, the
c-methyl of
Thr16 showed appreciable STD enhancements (data not shown),
hand, direct comparison of values for both Hd and H
e
TYSN reveals stronger enhancements for TYSN regardless of posi-
tion. Providing further support that tyrosine sulfonate can act as
a functional TYS isostere and bind in close proximity to gp120,
strong STD enhancements also were observed for the methylene
protons (–CH2–SO3) in both TYSN units.
while Tyr3 resonances did not. In triply sulfated CCR5 Nt peptide
7, line broadening of Hd and He resonances of TYS14 and Tyr15
was even greater than in peptide 6 (Fig. 5b), suggesting the pres-
ence of TYS3 further stabilized the ternary complex of CD4-HIV-1
gp120-CCR5 Nt peptide and effectively reduced the peptide’s off

10116
S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
Figure 4. Comparison of TYS and TYSN binding to gp120. (A) Close-up of TYS-gp120 binding pocket with CCR5 Nt displayed as a gray ribbon; (B) hydrogen bonding network
for TYS binding to gp120; and (C) schematic showing potential differences in interactions between TYS and TYSN binding to gp120, generated by simple atom replacement of
CH2 in TYSN for O in TYS. Dashed red line indicates a potential clash between methylene of TYSN and the side chain amide of Asn302. Residue 14 shown in purple and gp120
residues in gold.
Figure 5. Expansions of NMR spectra of 17-residue CCR5 Nt (2–18) and analogs. 1H NMR spectra of free and bound peptides are shown in black and blue, respectively, and 1
H
STD spectra of complexes are red. Number and position of TYS and TYSN residues are shown above respective spectra and in Figure 1 and in this figure.
Table 2
log 8 where TYSN residues were present at positions 3, 10, and 14.
Despite containing three sulfonate residues, analog 8 was only
sparingly soluble in water hindering extensive binding studies.
To overcome this solubility problem, we sought to design and syn-
thesize an alpha amino acid that would be compatible with the
conditions used during peptide synthesis, and incorporate both
an ethylene glycol side chain and an orthogonal functional group
amenable to late-stage chemical modifications (such as Hüisgen
cycloaddition14,15 or Staudinger ligation16,17 reactions). Azidation
of commercially available chlorohydrin 9 provided the desired
masked amino moiety.18 Bromination of the azido alcohol gave
quantitatively the azido bromide,19 that was used directly to O-
Increase in line width (Hz) between free and bound peptidesa
Peptide
10d
10
e
14d
0.8
14
e
15d
15
e
b
1
4
6
7
14
1.0
1.1
0.8
2.3
2.4
—
—
—
1.4
2.8
—
0.6
—
—
2.8
3.2
—
0.8
1.2
1.3
2.2
1.0
1.0
2.9
1.9
ꢁ3c
—
2.8
—
a
NMR solutions contained 40:1 peptide/gp120–CD4.
Overlapped signals not measured.
Conservative estimate due to slight overlap between 14d and side chain amide
b
c
with severe line broadening apparent in Fig. 5b, blue spectrum.
rate from complex. Overlap of several Hd and H
e
peaks of TYS3 and
alkylate Boc-
L
-serine20 to give N-protected azido tris(ethylenoxy)
-ate-Ala).21 This 3-step sequence of reactions
TYS10 prevented quantitative measure of STD enhancements for
these residues. However, measurement of line widths in 1H spectra
of free versus bound peptides showed that all resolved aromatic
signals undergo line broadening ranging from 0.8 to greater than
3.0 Hz in the presence of complex (Table 2) indicating the combi-
nation of increasing peptide length and sulfation of Tyr3, Tyr10,
and Tyr14 enhances binding to gp120.
L-alanine 10 (Boc L
thus allowed synthesis of multigram quantities of an unnatural
amino acid suitable for peptide synthesis using Boc chemistry
(see Scheme 1).
Alternatively, TFA deprotection of 10 followed by treatment
with FmocCl afforded 11 for use in Fmoc-based solid phase synthe-
sis. For the purpose of immobilization, it was desirable to introduce
the PEG amino acid anchor in a position equivalent to CCR5
transmembrane helix 1 (TM1). Thus, the synthesis of a second gen-
eration CCR5 TYSN analog 12 was initiated with 11 at the C-termi-
2.3. Synthesis of polyethylene glycol-containing alpha amino
acid
nus. Inclusion of one L-ate-Ala residue increased water solubility of
To determine whether a triply sulfonated peptide also would
show increased binding to complex, we synthesized CCR5 Nt ana-
triply sulfonated peptides by greater than 50-fold allowing for
preparations of stock solutions exceeding 30 mM compared to a

S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
10117
O
O
O
a. NaN3
b. PBr3
c. NaH
Boc-Ser
d. TFA
BocHN
FmocHN
HO
Cl
e. FmocCl
f. SPPS
OH
OH
O
2
N3
3
N3
3
10
11
O
9
O
O
O
O
H
N
H
N
DYQVSSPIYDINYYTSE
DYQVSSPIYDINYYTSE
N
H
NH2
N
H
NH2
O
O
O
h. [2+3] - Cu(II)
NaAscorbate,
propargyl biotin
H
N N
O
N
R
N
O
S
O
O
(CH2)3
HN
O
12 R=N3
14
NH
g. Pd/C, H2
13 R=NH2
Scheme 1. Synthesis of the alpha amino acid azido tris(ethylenoxy)
L-alanine (L-ate-Ala). (a) NaN3, DMF, quant (b) PBr3, THF, 85% (c) N-Boc L-Ser, NaH, DMF, 75% (d) TFA, TIS,
CH2Cl2 (e) FmocCl, DIPEA, H2O/dioxane, 75% 2 steps (f) SPPS (g) H2, Pd/C (h) Cu(II)SO4, Na ascorbate, propargyl biotin, H2O/MeOH.
maximum solubility of 0.4 mM observed for peptide 8. To further
demonstrate the utility of the -ate-Ala linker, [2 + 3] Hüisgen
due, demonstrating that addition of this linker as well as biotin
do not contribute to binding of 14 to gp120–CD4.
L
cyclization of propargyl biotin onto analog 12 readily provided
14 for immobilization or labeling. For the sequence of CCR5 Nt,
access to this modification proved especially useful as attempts
to biotinylate amine 13 by direct coupling to NHS-biotin resulted
in near quantitative intramolecular cyclization between Glu18
The enhanced binding observed by NMR for longer and triply
sulfated or sulfonated CCR5 Nt peptides was corroborated by
SPR. To assess binding of Nt peptides to gp120–CD4, CD4 was first
immobilized onto a carboxymethylated dextran surface. Prior to
each injection of Nt peptide, HIV-1 gp120 was introduced to the
CD4 surface to assure generation of fresh complex. As seen in Fig-
ure 6a, CCR5 Nt peptide 1 bound gp120–CD4 in a concentration
dependent manner and in fast exchange with sensorgrams char-
acteristic of fast association and fast dissociation. Steady-state
analysis of the binding curves of 1 using Michaelis–Menten kinet-
and the amine of L-ate-Ala.
2.4. Evaluation of triply sulfonated CCR5 Nt
Once in hand, CCR5 Nt tyrosine sulfonate analogs were evalu-
ated for binding to gp120–CD4 by NMR and SPR. Similar to results
for triply sulfated peptide 7, in the presence of gp120–CD4 line
ics provided an equilibrium dissociation constant of 17
2 lM.
Triply sulfonated CCR5 Nt analog 12 exhibited a vastly different
kinetic profile. As seen in Figure 5b, relative to 1 association
and dissociation of 12 to gp120–CD4 is slower, and a higher level
of late-stage binding is observed. Least squares best fitting of
these binding curves to a 1:1 Langmuir binding kinetic model
broadening of 14’s aromatic Hd and H
Once again, the strongest STD enhancements corresponded to Hd
and H of TYSN 14 and Tyr15, and in contrast to all other sulfated
peptides studied, the Hd and H resonances of TYS3 of 14 were
e signals occurred (Fig. 5c).
e
e
completely resolved showing an ꢁ50% enhancement relative to
Tyr15. In addition, STD enhancements of the aryl sulfonate meth-
ylenes were observed for all three sulfonate residues (data not
shown) providing further evidence that TYSN functions as a TYS
isostere in the context of CCR5 Nt peptides. Last, no enhancements
gave an equilibrium dissociation constant of 1.9 0.02 lM for sul-
fonated peptide 12. These SPR data were consistent with the vari-
ations in binding profiles seen in our NMR experiments where
17- and 19-residue triply sulfated and sulfonated Nt peptides
exhibited greater line broadening than the various 14-residue
peptides.
were observed for protons within the biotinylated L-ate-Ala resi-
Figure 6. Surface plasmon resonance experiments of CCR5 Nt peptides binding to gp120–CD4. Doubly referenced sensorgrams for (a) doubly sulfated Nt peptide 1 and (b)
triply sulfonated Nt peptide 12 binding to CD4-captured HIV-1 gp120 with peptide concentrations ranging from 50 to 3.1 M (twofold serial dilutions). Maximum response
(Rmax) plots for immobilized peptide 14 binding to (c) gp120–CD4 (blue, left), CD4 (red, center), and gp120 (orange, right); and (d) gp120–CD4 in the presence of varying
concentrations of mAb 412 d. Protein concentrations in 6 c ranged from 50 to 1.6 M (twofold serial dilutions), and solutions containing 1 M gp120–CD4 in the presence of
l
l
l
100–0.001 nM 412 d were used in 6 d. Response units (RUs) are shown on the y-axis with doubly referenced sensorgrams plotted on the same scale for panels (a) and (b). Rmax
points in panels (c) and (d) represent the maximum steady state response observed for each concentration of protein and/or antibody used.

10118
S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
logs such as 14 will enable the rapid discovery of small molecule
inhibitors of HIV-1 gp120 and CCR5 interactions.
3. Experimental
3.1. General
All reactions were performed under a positive flow of nitrogen
in flasks that were flame dried under vacuum. Aldrich SureSealÒ
anhydrous solvents (CH2Cl2 and DMF) and NMP were used without
further purification and stored under nitrogen. Glass-backed TLC
plates (EMD Silica Gel 60 with a 254 nm fluorescent indicator)
were cut into 2 ꢃ 5 cm portions and stored over desiccant. Devel-
oped TLC plates were visualized at 254 nm, stained with an I2–
SiO2 mixture, and/or treated with Hanessian’s stain or PMA and
charred. Column chromatography was conducted using a Biotage
Sp4 automated purification system and fractions collected by mon-
itoring at 254 nm. NMR experiments (1D and 2D) were conducted
on Varian Gemini 300 and Bruker AVANCE 500 and 600 MHz spec-
trometers using specified solvents at 298 K. FT-IR data were ac-
quired using a Perkin Elmer ATR-FTIR attachment. All Fmoc
amino acids, resins, and coupling reagents used for peptide synthe-
sis were purchased from Peptides International; and peptides 1–3
and 5 were purchased from American Peptide Company. Figures
were made using the programs Pymol23 and LigPlot.24
Figure 7. ELISA data showing inhibition of binding of gp120–CD4 to immobilized
14 by sulfated, sulfonated and phosphorylated Nt peptides.
2.5. Development of screening assays using sulfonate isostere
14
The discovery that the CD4-bound conformation of gp120 pos-
sesses a highly conserved sulfo-tyrosine binding pocket potentially
presents a new target for HIV-1 Entry inhibitors.7 For the purposes
of rapid screening and identification of compounds that might in-
hibit binding of CCR5 to the gp120 TYS binding site, we employed
our functional, chemically stable, biotinylated Nt analog 14 to de-
velop rapid screening assays using SPR and ELISA platforms. To test
the plausibility of screening by SPR, 14 was immobilized onto a
streptavidin-coated chip22 and analyzed for binding to soluble pro-
teins. As shown in the plot of maximum response units (Rmax) as a
function of varying protein concentrations (Fig. 6c), immobilized
14 bound neither CD4 (red) nor gp120 (orange) on their own, but
showed concentration dependent binding to the complex of
gp120–CD4 (blue). Both CCR5 Nt and sulfated antibody 412d bind
CD4-activated gp120 at the same site at the base of gp120’s third
variable loop. Consistent with this mode of binding, mAb 412d
was able to compete with binding of 14 to CD4-activated gp120
in a dose dependent manner (Fig. 6d). Peptide 14 was further em-
ployed to develop an enzyme-linked immunosorbent assay (ELISA)
using soluble gp120 and CD4 with colorimetric detection using
horseradish peroxidase. Dose dependent binding of gp120–CD4
to immobilized 14 was confirmed, and binding to this complex
was inhibited by mAb 412d (plot not shown). To validate the use
of this assay for screening of inhibitors, the effect of several sul-
fated, sulfonated and phosphorylated peptides was assessed. As
seen in Figure 7, doubly sulfated peptides 6 and 1 were the stron-
gest inhibitors of 14 binding to gp120 followed by triply sulfonated
peptide 12, and phosphorylated peptide 5 was non-inhibitory.
3.2. Reagents and conditions for NMR experiments
Peptides were dissolved in freshly prepared and sterile filtered
‘‘NMR Buffer” (20 mM sodium phosphate, 50 mM NaCl, pH 6.8),
and peptide stock solutions were adjusted to pH 6.8. Soluble CD4
(two-domain) was obtained from the AIDS Research and Reference
Reagent Program (Cat. No. 4615: Progenics Pharmaceuticals). YU2
gp120, with a Gly-Ala-Gly tripeptide replacing the V1/V2 region,
was produced by a Drosophila Schneider 2 line under control of an
inducible metallothionein promoter25 and purified by affinity chro-
matography as described previously.26 All proteins and protein
complexes were passed through a Superdex 200 16/60 gel filtration
column in NMR Buffer, and quantified by A280 measurements prior
to use. Typical NMR samples contained 800
lM peptide with or
without 20 M protein (270 L + 30
l
l
l
L D2O). 1H, 13C, and 15N
NMR assignments were made using data from 2D HOHAHA, ROESY,
1H-13C HSQC, 1H-13C HSQC-TOCSY, and 1H-1H ROESY experiments
recorded at 298 K using standard Bruker pulse programs with water
suppression. (Complete resonance assignments are provided in the
Supporting Information.) Saturation transfer difference (STD) NMR
experiments were recorded with the carrier set at ꢀ1 or 12 ppm for
on-resonance irradiation and 50 ppm for off-resonance irradiation.
Selective protein saturation (duration ranging from 1 to 5 s) was
accomplished using a train of 50 ms Gauss-shaped pulses, each sep-
arated by a 1 ms delay, at an experimentally determined optimal
2.6. Conclusion
In summary, we have analyzed by 1H NMR, STD NMR and SPR a
series of 14-, 17- and 19-residue CCR5 Nt peptides containing vari-
ations such as site specific introduction of Tyr(PO3) and Tyr(SO4)
residues, and two versus three Tyr(SO4) or Tyr(CH2SO3) residues.
Results from these studies demonstrated that tyrosine sulfonate
can function as a sulfo-tyrosine isostere, allowing us to construct
a non-hydrolyzable, sulfonated CCR5 Nt peptide that was used to
screen for inhibitors to the CCR5 Nt sulfo-tyrosine binding site
on gp120. In addition to increased stability, incorporation of an
unnatural amino acid bearing an azido moiety and a tri-ethylene
glycol segment (ate-Ala) increased solubility of these peptides to
levels approaching 100 mg per mL. The orthogonal azido group
facilitated late-stage biotinylation through [2 + 3] Hüisgen cycliza-
tion without side reactions from the other functionalities. We
anticipate that sulfonated, chemically stable CCR5 Nt peptide ana-
power (49 dB on our probe); a T1q filter (30 ms) was incorporated
to suppress protein resonances. A minimum of 512 scans and 4000
points were used to ensure high quality data with good signal-to-
noise. On- and off-resonance spectra were processed indepen-
dently, and subtracted to provide a difference spectrum. For all
complexes, the signal corresponding to 15e was most strongly
enhanced; thus spectra were normalized to this signal.
3.3. Production of 412d-antigen-binding fragment (Fab)
Monoclonal antibody 412d was purified from mouse–human
hybridomas by staphylococcal protein A chromatography. 412d
Fab was generated by endoproteinase Lys-C digestion of the 412d
IgG and purified as described previously.27 Briefly, the 412d anti-
body was cleaved by Lys-C protease (Roche) to produce Fab, with

S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
10119
affinity (Protein A), size-exclusion (Superdex S200), and anion-ex-
3.6. N-Boc azido tris-(ethylenoxy)
L-alanine (10)
change (Mono-Q) chromatography used to separate the most cat-
ionic (double-sulfated) fragment.
To a cooled, 0 °C, solution of Boc
L-Ser (29.7 g, 145 mmol) in
DMF (400 mL) was added NaH (12.8 g, 319 mmol, 60% dispersion
in mineral oil). The cloudy suspension was allowed to stir for an
additional 15 min at rt. Azido bromo PEG14 (23.0 g, 96.6 mmol) in
DMF (100 mL) was added dropwise and the mixture was stirred
overnight, 15 h. The solution was concentrated in vacuo and puri-
fied by Si gel chromatography (10% MeOH/CH2Cl2) to afford 10
(26.0 g, 75% yield). 1H NMR (CD3OD, 300 MHz) d 4.27 (br, 1 H),
3.88 (dd, 1H, J = 4.5 Hz, 9.3 Hz), 3.74 (d, 1H, J = 3.3 Hz), 3.66 (m,
8H), 3.38 (t, 2H, J = 4.8 Hz), 1.45 (s, 11H). 13C NMR (CD3OD,
75 MHz) d 158.1, 101.9, 80.8, 80.0, 72.1, 71.9, 71.8, 71.6, 71.3,
51.9, 28.6. ATR-FTIR (cmꢀ1): 3697, 2974, 2923, 2871, 2104, 1709,
1393, 1367, 1165, 1119, 1056, 1033. HRMS (TOF-MS-ES) m/z
361.1696, (361.1723 Calcd for C14H26N4O7, M-H).
3.4. Surface plasmon resonance
Surface plasmon resonance experiments were performed using
a Biacore T100 instrument and accompanying software. Following
optimization by pH scouting, CD4 (0.5 M in 20 mM acetate, pH 5.5)
was immobilized on a CM5 chip with HBS-EP as immobilization
buffer to a response level of 500 RUs, and remaining activated sur-
face was capped with 1 M ethanolamine (pH 8.0). Immobilization
buffer was replaced and the chip equilibrated with PBS as the run-
ning buffer for binding and kinetics analyses.
For analyses of peptides’ binding to immobilized protein, solu-
tions of CCR5 Nt peptides were prepared in PBS at concentrations
of 30–50 mM, and appropriate twofold serial dilutions were em-
ployed for binding studies. After capture of soluble HIV-1 gp120
3.7. N-Fluorenylmethylcarbonyl azido tris-(ethylenoxy) L-ala
(YU2 strain, 1
lM in PBS) onto the CD4 flow cell (FC) to a response
nine (11)
of 2000 RUs, peptide solutions were injected with association and
dissociation times of 120 and 180 s, respectively. Binding analyses
were performed at 25 °C at a flow rate of 30 lL/min using serially
arranged control (FC1) and active flow (FC2) cells. The CD4 surface
was regenerated using a short pulse (8 s) of regeneration solution
N-Boc-protected amino acid 10 (1.4 g, 3.9 mmol) was dissolved
in CH2Cl2 (10 mL) and cooled to 0 °C. Following the addition of tri-
isopropylsilane (0.8 mL, 3.9 mmol), trifluoroacetic acid (2 mL) was
syringed into the stirring mixture. The reaction was allowed to
warm to rt overnight (15 h) and concentrated to dryness in vacuo.
TOF-MS-ES revealed complete conversion to free amino acid
[HRMS-ESI m/z 261.1206 [M-H] (C9H17N4O5 calculated
261.1199)] which was dissolved in H2O:dioxane (10:10 mL) and
adjusted to pH 8 with DIPEA (2.5 mL, 15 mmol). To the cooled mix-
ture (0 °C) was added a solution of FmocCl (1.2 g, 4.7 mmol) in
dioxane (5 mL). The solution was allowed to warm to rt overnight
(16 h) and concentrated to dryness in vacuo. The crude mixture
was purified using silica gel column chromatography with a gradi-
ent of 5–10% MeOH:CH2Cl2 over 10 column volumes to provide 11
(1.26 g, 67% over 2 steps). 1H NMR (DMSO-d6, 300 MHz) d 7.89 (d,
2H, J = 7.5 Hz), 7.73 (d, 2H, J = 7.2 Hz), 7.42 (t, 2H, J = 7.2 Hz), 7.32
(t, 2H, J = 7.5 Hz), 4.26 (m, 3H), 4.09 (m, 1H), 3.68 (d, 1H,
J = 4.8 Hz), 3.57 (t, 2H, J = 4.8 Hz), 3.55 (t, 2H, J = 5.1 Hz), 3.52 (s,
4H, 2.07), 3.38 (t, 2H, J = 6.3 Hz), 3.36 (t, 2H, J = 4.8 Hz), 1.12 (t,
(50 mM NaOH, 1 M NaCl) at a flow rate of 60 lL/min. Using the
T100 BiaEvaluation software, all data were doubly referenced to
control flow cell and zero concentration data curves prior to curve
fitting using a 1:1 binding model.
To analyze proteins and complexes binding to immobilized pep-
tides, a Biacore SA (streptavidin) chip was docked onto a Biacore
T100 with PBS as the running buffer at 5 lL/min. All flow cells
(FCs) were conditioned with injections (3 ꢃ 60 s) of 50 mM NaOH,
1 M NaCl. Biotinylated peptide 14 (50 nM in PBS) was immobilized
onto FC2 to a response level of ꢁ300 RUs. All proteins samples
were purified on a Superdex 200 16/60 gel filtration column in
PBS, and quantified by A280 measurements prior to use. Serially di-
luted solutions of proteins in PBS (gp120, CD4, CD4–gp120 com-
plex) were prepared and injected sequentially through FC1 and
FC2. All protein binding analyses were performed at 25 °C at a flow
rate of 20
short pulse (8 s) of 50 mM NaOH, 1 M NaCl using a flow rate of
60 L/min. Data were processed using T100 BiaEvaluation software
l
L/min. Regeneration of the surface was achieved with a
2H, J = 7.2 Hz). 1H NMR (CD3OD, 300 MHz)
d 7.90 (d, 2H,
J = 6.9 Hz), 7.79 (t, 2H, J = 6.2 Hz), 7.49 (t, 2H, J = 7.5 Hz), 7.42 (dt,
2H, J = 1.2 Hz, 7.5 Hz), 4.46 (d, 2H, J = 6.3 Hz), 4.34 (m, 2H), 3.96
(dd, 1H, J = 5.6 Hz, 9.8 Hz), 3.87 (dd, 1H, J = 5.6 Hz, 9.8 Hz), 3.72
(m, 8H), 3.43 (m, 2H), 1.00 (t, 2H, J = 3.9 Hz). 13C NMR (DMSO-d6,
75 MHz) d171.9, 155.9, 143.9, 140.7, 127.6, 127.1, 125.3, 120.1,
70.4, 69.7, 69.6, 69.3, 65.7, 60.4, 54.9, 50.0, 48.6, 46.7, 45.1, 40.3.
ATR-FTIR (cmꢀ1) 3310, 3066, 2884, 2105, 1717, 1450. HRMS
(TOF-MS-ES) m/z 485.2041 (485.2036 Calcd for C24H29N4O7, M+H).
l
where data from the control flow cell (FC1) was subtracted from
the binding/peptide flow cell (FC2). These binding curves were ref-
erenced to a negative control (buffer injection) giving double refer-
enced binding data.
3.5. CCR5 Nt peptide ELISAs
Neutravidin-coated high binding capacity, pre-blocked ELISA
plates (Pierce) were washed three times with assay buffer (Dul-
becco’s phosphate buffered saline, 0.05% P20, 1% fetal bovine ser-
um, and 2% bovine serum albumin). Plates were incubated with
biotinylated CCR5 Nt peptide 14 (500 nM) for 1 h at rt, then
washed three times with assay buffer. A complex made of 60 nM
3.8. Solid phase peptide synthesis of CCR5 Nt TYSN analog 12
CLEARTM amide resin was swelled in NMP for 10 min with gentle
agitation by a flow of dry N2 (g) and washed with copious amounts
of CH2Cl2 and NMP. Removal of the Fmoc protecting group was
accomplished through treatment with 20% piperidine for 20 min.
Following deprotection, the resin was washed with CH2Cl2, MeOH,
and NMP successively prior to coupling of a subsequent amino
acid. Each coupling step required exposure of the resin with 4
equiv each of Fmoc amino acid, PyBOP, and DIPEA for 2–4 h. Each
coupling was followed by wash cycles of CH2Cl2 and MeOH fol-
lowed by acetylation with 20% Ac2O in CH2Cl2 for 20 min. A double
coupling using Fmoc-TYSN was required for TYS3 and TYS10.
Treatment of the cooled resin with TFA/H2O/TIS (95:2.5:2.5) at
4 °C for 2 h effectively cleaved the peptide from the resin with con-
comitant global deprotection, after which the peptide was precip-
itated by addition of cold MTBE (5 ꢃ volume of TFA). Peptides were
YU2DV1V2 gp120, 120 nM sCD4 and 60 nM 2.2c IgG in assay buf-
fer was incubated for 1 h with or without addition of inhibitor
(either a CCR5 Nt analog or 412 d IgG). Antibody 2.2c is an A32-like
antibody that binds to the C1–C4 region of monomeric gp120
(James Robinson, personal communication). The complex was then
added to an ELISA plate with immobilized 14 and incubated for 1 h.
Plates were washed three times with assay buffer, and HRP-conju-
gated goat anti-human IgG was used to detect the presence of
bound gp120–CD4–2.2c IgG complex. The plates were developed
using the TMB Peroxidase EIA Substrate Kit (Bio-Rad).

10120
S. N. Lam et al. / Bioorg. Med. Chem. 16 (2008) 10113–10120
purified using RP-HPLC with a YMC-Pack ODS-AQ column using
10 mM NH4CHO2 and MeOH as eluents. Lyophilization provided
12 as a white powder. 1H NMR (D2O, 500 MHz) d7.27 (d, 2H,
J = 7.7 Hz), 7.23 (d, 2H, J = 7.7 Hz), 7.20 (d, 2H, J = 7.7 Hz), 7.13 (t,
4H, J = 7.7 Hz), 7.05 (d, 2H, J = 8.2 Hz), 6.94 (d, 2H, J = 7.7 Hz),
6.75 (d, 2H, J = 8.2 Hz), 4.55 (m, 1H), 4.40 (m, 2H), 4.34–4.23 (m,
5H), 4.14 (m, 2H), 4.08–3.93 (m, 17H), 3.88 (d, 4H, J = 5.9 Hz),
3.84–3.68 (m, 21H), 3.59 (br s, 21H), 3.39 (t, 2H, J = 4.7 Hz), 3.11–
3.00 (m, 4H), 2.93–2.90 (m, 6H), 2.79 (dd, 1H, J = 6.9 Hz, 17 Hz),
2.68–2.50 (m, 6H), 2.35 (t, 3H, J = 8.1 Hz), 2.18 (t, 2H, J = 7.6 Hz),
2.11–1.87 (m, 14H), 1.76 (m, 1H), 1.70–1.64 (m, 3H). 1.33 (m,
4H), 1.11 (br d, 7H, J = 6.2 Hz), 1.01 (m, 2H), 0.86 (d, 10H,
J = 6.7 Hz), 0.79 (t, 6H, J = 7.3 Hz), 0.75–0.70 (m, 8H), 0.66 (d, 3H,
J = 6.7 Hz). 13C NMR (D2O, 150 MHz) d178.53, 178.32, 178.25,
174.89, 174.73, 174.51, 174.16, 174.07, 173.99, 173.92, 173.61,
173.52, 173.40, 173.28, 173.14, 172.59, 172.53, 172.01, 169.20,
155.25, 136.55, 136.43, 131.26, 130.02, 129.90, 129.83, 129.21,
128.81, 128.11, 126.15, 125.95, 125.11, 118.21, 117.60, 116.25,
115.17, 114.31, 111.35, 110.51, 107.39, 105.36, 102.91, 102.68,
100.77, 100.70, 100.64, 98.01, 70.77, 70.32, 70.26, 70.22, 69.91,
67.72, 64.39, 61.78, 61.55, 61.35, 60.15, 59.80, 59.30, 57.23,
56.61, 56.25, 56.01, 55.24, 54.26, 54.15, 54.05, 53.64, 51.55,
51.13, 50.98, 50.86, 48.80, 43.21, 37.35, 37.06, 36.65, 31.68,
31.16, 30.87, 30.03, 27.42, 26.87, 25.48, 25.32, 22.44, 19.39,
19.12, 18.37, 15.47, 15.43, 11.26, 10.45. HRMS (TOF-MS-ES) m/z
2633.0002 [M+H], 1315.9836 [Mꢀ2H], 876.6529 [Mꢀ3H],
(2633.0006 Calcd for C109H158N25O45S3 [M+H]).
tion of gp120 and antibodies, and the NIH AIDS Research and Ref-
erence Regent Program for soluble CD4. This work was supported
by the NIDDK (C.A.B.) and NIAID (P.D.K. and R.W.) Intramural Re-
search Programs, a grant from the Bill and Melinda Gates Founda-
tion Grand Challenges in Global Health Initiative (P.D.K. and R.W.),
and the Intramural AIDS Targeted Antiviral Program (IATAP) of the
Office of the Director, NIH (C.A.B.).
Supplementary data
Spectral data for protected amino acids 10 and 11, peptides 12
and 13, and complete 1H and 13C assignments for peptides 2–4, 6, 7
and 14. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2008.10.005.
References and notes
1. Wyatt, R.; Sodroski, J. Science 1998, 280, 1884–1888.
2. Berger, E. A.; Murphy, P. M.; Farber, J. M. Annu. Rev. Immunol. 1999, 17, 657–
700.
3. Farzan, M.; Mirzabekov, T.; Kolchinsky, P.; Wyatt, R.; Cayabyab, M.; Gerard, N.
P.; Sodroski, J.; Choe, H. Cell 1999, 96, 667–676.
4. Farzan, M.; Chung, S.; Li, W.; Vasilieva, N.; Wright, P. L.; Schnitzler, C. E.;
Marchione, R. J.; Gerard, C.; Gerard, N. P.; Sodroski, J.; Choe, H. J. Biol. Chem.
2002, 277, 40397.
5. Cormier, E. G.; Persuh, M.; Thompson, D. A. D.; Lin, S. W.; Sakmar, T. P.; Olson,
W. C.; Dragic, T. Proc. Natl. Acad. Sci. 2000, 97, 5762–5767.
6. Farzan, M.; Vasilieva, N.; Schnitzler, C. E.; Chung, S.; Robinson, J.; Gerard, C.;
Choe, H.; Sodroski, J. J. Biol. Chem. 2000, 275, 33516–33521.
7. Huang, C.-C.; Lam, S. N.; Acharya, P.; Shahzad-ul-Hussan, S.; Stanfield, R. L.;
Tang, M.; Xiang, S.-H.; Robinson, J.; Sodroski, J.; Wilson, I.; Wyatt, R.; Bewley, C.
A.; Kwong, P. D. Science 2007, 317, 1930–1934.
8. Kitagawa, K.; Aida, C.; Fujiwara, H.; Yagami, T.; Futaki, S.; Kogire, M.; Ida, J.;
Inoue, K. J. Org. Chem. 2002, 66, 1–10.
9. Mayer, M.; Meyer, B. Angew. Chem. Int. Ed. 1999, 38, 1784–1788.
3.9. Reduction of 12 to amine 13
Azide 12 (10 mg, 3.8 lmol) was dissolved in MeOH/H2O/HOAc
10. An alternate explanation may in part be attributed to the absence of
a
(1:4:0.1, 5 mL) and Pd/C (5 mg) was added. The flask was evacu-
ated and charged with H2 (g); this was repeated twice. The mixture
was stirred under H2 (g) at atmospheric pressure for 2 h after
which the Pd/C was removed by filtering through a pad of Celite
and the solvent removed in vacuo. HRMS data revealed complete
reduction of azide 12 to amine 13. HRMS (TOF-MS-ES) m/z
1302.4938 [Mꢀ2H], 867.9971 [Mꢀ3H] (calcd for C109H160N23O45S3
[MꢀH] 2604.9944).
hydrogen bond acceptor (carboxylate) in the TYS binding site should
monobasic phosphate be present Quiocho, F. A. Kidney Int. 1996, 49, 943–946.
11. Gonzalez-Muniz, R.; Cornille, F.; Bergeron, F.; Ficheux, D.; Pothier, J.; Durieux,
C.; Roques, B. P. Int. J. Pept. Prot. Res. 1991, 37, 331–340.
12. Miranda, M. T. M.; Liddle, R. A.; Rivier, J. E. J. Med. Chem. 1993, 36, 1681–1688.
13. Seibert, C.; Cadene, M.; Sanfiz, A.; Chait, B. T.; Sakmar, T. P. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 11031–11036.
14. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004–
2021.
15. For a recent review of reactions involving organic azides Bräse, S.; Gil, C.;
Knepper, K.; Zimmermann, V. Angew. Chem. Int. Ed. 2005, 44, 5188–5240.
16. Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 2141–2143.
17. Kohn, M.; Breinbauer, R. Angew. Chem. Int. Ed. 2004, 43, 3106–3116.
18. Iyer, S. S.; Anderson, A. S.; Reed, S.; Swanson, B.; Schmidt, J. G. Tetrahedron Lett.
2004, 45, 4285–4288.
19. Roy, B. C.; Santos, M.; Mallik, S.; Campiglia, A. D. J. Org. Chem. 2003, 68, 3999–
4007.
20. Hwang, J.; Deming, T. J. Biomacromolecules 2001, 2, 17–21.
21. An eight step synthesis (overall yield of 11%) of the free amine of 11, starting
3.10. Synthesis of biotin conjugated 14
To a solution of azide 12 (14.7 mg, 5.6
alcohol (1:1, v/v, 700 L) were added CuSO4 (50 mM, 4
ascorbate (100 mM, 180 L), and propargyl biotin (5.0 mg,
18 mol) and the mixture was stirred overnight at rt. Complete
lmol) in PBS/tert- butyl
l
lL), sodium
l
l
conversion to conjugate 14 was verified by LCMS; MS (ES) m/z
1457.5 [Mꢀ2H], 970.5 [Mꢀ3H] (2915.12 Calcd for C122H176N28
O47S4, M). Purification was accomplished using RP-HPLC using a
Waters Symmetry-Prep C8 column with 20 mM NH4CHO2 and
CH3CN as mobile phases. Lyophylization gave 14 as white powder.
Complete 1H and 13C NMR assignments are provided in the Sup-
porting Information.
from an L-serine derived aziridine followed by nucleophilic ring opening, has
been reported Larsson, U.; Carlson, R. Acta Chem. Scand. 1994, 48, 511–516.
22. Attempts to couple amine 13 to a carboxymethyl chip using a variety of
conditions such as varying pH and salt concentration were unsuccessful, likely
owing to the negative charge of both the peptide and surface.
23. http://pymol.sourceforge.net/.
24. Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Prot. Eng. 1995, 8, 127–134.
25. Wu, L.; Gerard, N. P.; Wyatt, R.; Choe, H.; Parolin, C.; Ruffing, N.; Borsetti, A.;
Cardoso, A. A.; Desjardin, E.; Newman, W.; Gerard, C.; Sodroski, J. Nature 1996,
384, 179–183.
26. Wyatt, R.; Sullivan, N.; Thali, M.; Repke, H.; Ho, D.; Robinson, J.; Posner, M.;
Sodroski, J. J. Virol. 1993, 67, 4557–4565.
Acknowledgments
27. Huang, C.-C.; Venturi, M.; Majeed, S.; Moore, M. J.; Phogat, S.; Zhang, M. Y.;
Dimitrov, D. S.; Hendrickson, W. A.; Robinson, J.; Sodroski, J.; Wyatt, R.; Choe,
H.; Farzan, M.; Kwong, P. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2706–2711.
We thank J. Lloyd for HRMS data, J. Robinson for 412d and 2.2c
antibodies, C.-C. Huang for assistance with expression and purifica-