Sulfotyrosine dipeptide: Synthesis and evaluation as HIV-entry inhibitor

Article abstract of DOI:10.1016/j.bioorg.2016.07.012

Human immunodeficiency virus type 1 (HIV-1) is responsible for the worldwide AIDS pandemic. Due to the lack of prophylactic HIV-1 vaccine, drug treatment of the infected patients becomes essential to reduce the viral load and to slow down progression of the disease. Because of drug resistance, finding new antiviral agents is necessary for AIDS drug therapies. The interaction of gp120 and co-receptor (CCR5/CXCR4) mediates the entry of HIV-1 into host cells, which has been increasingly exploited in recent years as the target for new antiviral agents. A conserved co-receptor binding site on gp120 that recognizes sulfotyrosine (sTyr) residues represents a structural target to design novel HIV entry inhibitors. In this work, we developed an efficient synthesis of sulfotyrosine dipeptide and evaluated it as an HIV-1 entry inhibitor.

Full text of DOI:10.1016/j.bioorg.2016.07.012

Bioorganic Chemistry 68 (2016) 105–111
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Sulfotyrosine dipeptide: Synthesis and evaluation as HIV-entry inhibitor
a,
Tong Ju a,1, Duoyi Hu b,1, Shi-Hua Xiang b, , Jiantao Guo
⇑
⇑
a Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, United States
b Nebraska Center for Virology, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Human immunodeficiency virus type 1 (HIV-1) is responsible for the worldwide AIDS pandemic. Due to
the lack of prophylactic HIV-1 vaccine, drug treatment of the infected patients becomes essential to
reduce the viral load and to slow down progression of the disease. Because of drug resistance, finding
new antiviral agents is necessary for AIDS drug therapies. The interaction of gp120 and co-receptor
(CCR5/CXCR4) mediates the entry of HIV-1 into host cells, which has been increasingly exploited in
recent years as the target for new antiviral agents. A conserved co-receptor binding site on gp120 that
recognizes sulfotyrosine (sTyr) residues represents a structural target to design novel HIV entry inhibi-
tors. In this work, we developed an efficient synthesis of sulfotyrosine dipeptide and evaluated it as an
HIV-1 entry inhibitor.
Received 18 May 2016
Revised 19 July 2016
Accepted 25 July 2016
Available online 25 July 2016
Keywords:
HIV entry inhibitor
Sulfotyrosine
Sulfotyrosine dipeptide
Sulfopeptide
Ó 2016 Elsevier Inc. All rights reserved.
gp120
Antiviral therapy
Protein sulfation
1. Introduction
over drugs targeting HIV enzymes: (1) limited cell toxicity due to
their extracellular nature; and (2) beneficial effects upstream to
Human immunodeficiency virus type 1 (HIV-1) has evolved into
one of the serious global public health crisis over the past thirty
years. It is estimated that HIV/AIDS pandemic has already claimed
AIDS-related deaths of 40 million people, and 35 million people are
living with this virus, and approximately 2.0 million people are
newly infected by HIV-1 each year according to UNAIDS [1]. There
is currently no cure and no effective prevention methods available
[2,3]. Drug treatment for controlling viral loads and for prolonging
patients’ lives is the main therapy for HIV-1 infections. A number
of different antiviral agents are currently available, such as reverse
transcriptase (RT) inhibitors, protease inhibitors, and integrase
inhibitors [4]. However, HIV-1 drug resistance posts a serious
problem in these antiviral therapies due to the high mutation rate
of the virus. Therefore, searching for additional and especially new
classes of antiviral drugs is still essential to fight against HIV/AIDS.
To this end, efforts have been continuously made to develop new
antiviral drugs including maturation inhibitors, entry inhibitors,
and fusion inhibitors. Among them, antiviral agents that can block
HIV-1 entry (entry inhibitors) are considered as an effective class of
drug and has been increasingly exploited in recent years but is still
scarce [5]. The entry inhibitors would have two major advantages
damages that would occur to the cell later in the viral life cycle.
HIV-1 entry into the target cells requires two receptors: a
primary receptor CD4 and a co-receptor CCR5 (or CXCR4). Upon
binding to the primary receptor CD4, gp120 undergoes a large con-
formational change and exposes its binding site for co-receptor
CCR5 or CXCR4 [6,7]. The binding of co-receptor causes further
conformational change and leads to membrane fusion between
the virus and the target cell, which allows the viral RNA genome
to enter the target cell. Since viral entry involves a multi-stage pro-
cess, such as viral attachment (CD4-engagement), co-receptor
binding, membrane fusion (pre- or post-fusion), HIV-1 entry inhi-
bitors can be classified into different stage inhibitors [8]. Currently,
only two entry inhibition drugs are approved by FDA for clinical
uses. One is a membrane fusion inhibitor, which is a 38-amino acid
peptide drug derived from the HIV-1 gp41 glycoprotein sequence
and is marketed under the trade name Fuzeon (T20, Enfuvirtide)
[9–11]. This drug prevents membrane fusion through blocking
the formation of the post-fusion structure [9–11]. The success of
Fuzeon demonstrates that peptides can be used as drugs against
HIV-1 infection. The other FDA-approved drug is Maraviroc
(MVC; trade name Selzentry) [12–15], which targets the HIV-1 cel-
lular factor, co-receptor CCR5, and inhibits HIV-1 entry by blocking
gp120-CCR5 interaction. Besides the above two FDA-approved
entry inhibitors, there are several small molecule drugs, such as
BMS-663068 and cenicriviroc (TBR-652), and antibody-based
⇑
Corresponding authors.
E-mail addresses: sxiang2@unl.edu (S.-H. Xiang), jguo4@unl.edu (J. Guo).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bioorg.2016.07.012
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

106
T. Ju et al. / Bioorganic Chemistry 68 (2016) 105–111
Table 1
drugs, such as Ibalizumab, and peptide-based drugs, such as Sifu-
virtide, are currently under clinical trials. It is apparently that
blocking viral entry is an effective strategy to prevent HIV-1 infec-
tions [5,8,16,17].
Computational analyses of sulfopeptides.a
Entry Peptides
Cdoc
energy energy
Initial
CHARMm Potential
energy
energy
Sulfotyrosine (sTyr) residues on the N-terminal region of cell
surface co-receptor (CCR5 or CXCR4) are the key recognition ele-
ment of gp120 [18,19]. It was reported previously that sulfopep-
tides exhibited activity to block the association of gp120 with
CCR5 co-receptor and inhibited the viral entry [20]. An antibody
(412d) that binds to the co-receptor CCR5 binding site was found
to be sulfated in its heavy chain CDR3 loop [21]. A recent report
in Nature showed that an AAV-expressed eCD4-Ig, a fusion of
CD4-Ig with a small CCR5-mimetic sulfopeptide, could provide
durable protection against multiple SHIV infections in the non-
human primate model [22]. These results indicated that the
CCR5-mimetic sulfopeptide could inhibit HIV entry by competing
for the co-receptor binding site. In the present work, we seek to
synthesize and evaluate a sulfotyrosine dipeptide as an HIV-1
entry inhibitor by targeting viral envelope glycoprotein gp120.
According to our computational analysis, sulfotyrosine dipeptide
is likely a minimum active peptide sequence to bind to gp120. Sul-
fotyrosine dipeptide or its derivatives can be potentially converted
into small drug-like organic molecules for therapeutic applications.
Comparing to tyrosine-sulfated gp120 antibodies (e.g., 412d [23])
and peptides as HIV entry inhibitors, sulfopeptide-derived drug-
like organic molecules should possess advantages in cost-
effectiveness, conformational restriction, metabolic stability, and
oral bioavailability.
1
2
3
4
sTyr-Asp-Ile-Asn-sTyr 58.42
21446.60 À276.15
22083.00 À186.74
21390.70 À225.04
20507.00 À255.83
À276.15
À186.74
À225.04
À255.83
sTyr-Asp-Ile-Asp-sTyr 70.94
sTyr-Ile-Asp-Asn-sTyr 62.13
sTyr-Asn-Ile-Asn-sTyr 58.84
5
6
7
8
sTyr-Asp-Asn-sTyr
sTyr-Asp-Asp-sTyr
sTyr-Asn-Asp-sTyr-
sTyr-Ile-Asp-sTyr
sTyr-Asp-Ile-sTyr
sTyr-Ser-Ser-sTyr
sTyr-Asn-Asn-sTyr
sTyr-Ile-Asn-sTyr
sTyr-Asn-Ile-sTyr
sTyr-Ser-Asn-sTyr
sTyr-Ile-Ile-sTyr
61.05
69.17
54.93
53.41
51.33
40.15
48.65
41.27
39.27
41.81
33.75
20935.60 À203.78
21557.30 À188.33
20557.50 À200.32
20753.50 À141.32
20945.30 À157.43
20089.90 À144.26
20010.40 À234.48
20160.80 À180.89
19943.20 À194.14
19891.70 À180.58
20083.30 À138.22
À203.78
À188.33
À200.32
À141.32
À157.43
À144.26
À234.48
À180.89
À194.14
À180.58
À138.22
9
10
11
12
13
14
15
16
17
18
19
sTyr-Asp-sTyr
sTyr-Asn-sTyr
V-Ile-sTyr
47.04
41.56
41.02
35.79
20747.20 À203.13
20133.50 À186.00
20360.70 À135.52
20681.20 À139.00
À203.13
À186.00
À135.52
À139.00
sTyr-Ser-sTyr
20
sTyr-sTyr
33.28
22192.50 À123.73
À123.73
a
A full set of data can be found in Table S1.
residues during solid-phase peptide syntheses often suffers low
yield. This is mainly due to the extensive sTyr hydrolysis under
the reaction conditions, the incomplete deprotection of protected
sTyr precursors, and/or the incomplete hydrolysis of peptide pro-
duct from the resin under mild conditions [25–28]. To overcome
this difficulty, a global sulfation approach was developed. In this
approach, sulfation of tyrosine residues was conducted after the
peptide was fully assembled. Different types of protection/depro-
tection strategy [31,32] or new protecting groups [33] have been
developed to minimize the desulfation or achieve the selective sul-
fation of certain tyrosine residues in the peptide sequence.
In this study, we focused on the solution-based global sulfation
approach to synthesize the desirable sulfotyrosine dipeptide. Our
first synthetic scheme involved the direct sulfation of unprotected
tyrosine dipeptide (NH2-Tyr-Tyr-COOH) using either concentrated
sulfuric acid [34] or chlorosulfuric acid [35]. We have shown that
both reagents could be used to synthesize sTyr with excellent
yield [36]. Unfortunately, incomplete sulfation of the unprotected
NH2-Tyr-Tyr-COOH was observed. Only mono-sulfated dipeptide
was obtained with yields in the range of 20–47% (Table 2). This
result indicated that concentrated sulfuric acid or chlorosulfuric
acid only have limited sulfating power. The formation of a small
amount (<5%; entries 3 and 4, Table 1) of sulfotyrosine dipeptide
(NH2-sTyr-sTyr-COOH) could be observed in two cases when
chlorosulfuric acid was used as sulfating reagent. Longer reaction
time, higher temperature, and more equivalences of sulfating
agents did not improve the yield of NH2-sTyr-sTyr-COOH (data
not shown). The very low yield of di-sulfated product might be
resulted from a potential strong repulsion of the two negatively
charged sulfate groups.
2. Results and discussion
2.1. Computational analysis and rationale
Recently acquired structural information on the interaction
between co-receptor CCR5 and gp120 [19] and the interaction
between CD4-induced antibody [23] and gp120 has revealed sev-
eral important sites for drug discovery. One of them is a conserved
CCR5-binding site of gp120 [24], which is mainly located at the
base of the gp120 V3-loop (Fig. S1). This site features a small, deep,
and positively charged pocket for the binding of negatively charged
sTyr residues on either the N-terminus of CCR5 [19] or the 412d
antibody. The V3-loop of gp120 represents a very attractive site
for drug development (Fig. S1). A sequence alignment (Fig. S1B)
suggests that an acidic and tyrosine-rich peptide sequence, espe-
cially in the presence of sTyr residues, is the critical recognition
element of the gp120 V3-loop.
In the present study, we sought to identify CCR5 N-terminus-
like or 412d CDR3-loop-like short sulfopeptides that have similar
or better binding affinity towards the gp120 V3-loop. Based on
the sequences of the N-terminus and the CDR3 loop of 412d anti-
body, a series of peptides were designed and their docking energies
towards the gp120 V3-loop were shown in Table 1. Our calcula-
tions indicated that the docking (Cdoc) and the binding (Potential
energy) energies of sulfotyrosine dipeptide were not significantly
reduced although this dipeptide has a largely reduced size. There-
fore, we decided to examine sulfotyrosine dipeptide as an HIV-1
entry inhibitor.
Next, a couple of stronger sulfating agents, SO3/pyridine and
SO3/DMF, were examined. Since these two reagents are reactive
towards amine [34], the free amino group of the substrate,
NH2-Tyr-Tyr-COOH, was protected with Boc group to yield
Boc-Tyr-Tyr-COOH. As shown in Table 3, the desired Boc-sTyr-
sTyr-COOH product could be obtained by using either SO3/Pyr or
SO3/DMF as sulfating reagent. SO3/DMF was apparently a more
powerful sulfating reagent than SO3/Pyr, which is consistent with
the literature [37]. After an optimization of reaction time and
solvent, the desired product could be obtained in 71% yield when
the best set of reaction conditions was used (entry 4, Table 3).
2.2. Synthesis of sulfotyrosine dipeptide
Sulfopeptides can be chemically synthesized either by direct
incorporation of sTyr residues into a growing peptide chain, or
by sulfating Tyr residues in a peptide post-assembly [25–28].
Regardless of recent progress [29,30], the preparation of sTyr-
containing peptides is still challenging. Direct incorporation of sTyr

T. Ju et al. / Bioorganic Chemistry 68 (2016) 105–111
107
Table 2
Sulfation of NH2-Tyr-Tyr-COOH dipeptide using sulfuric acid or chlorosulfuric acid.
Entry
NH2-Tyr-Tyr-COOH (eq.)
Sulfating agent
Reaction condition
Outcomes
1
2
3
1
1
1
Con. H2SO4 (6 eq.)
HSO3Cl (1.25 eq.)
HSO3Cl (1.25 eq.)
À5 °C, 40 min
À10 °C, 5 min
À5 °C, 30 min
20% mono-sulfated
30% mono-sulfated
45% mono-sulfated
<5% disulfated
4
5
1
1
HSO3Cl (3 eq.)
HSO3Cl (5 eq.)
0 °C, 1 h
0 °C, 2 h
47% mono-sulfated
<5% disulfated
47% mono-sulfated
Table 3
Sulfation of NH2-Tyr-Tyr-COOH dipeptide using SO3/Pyr or SO3/DMF.
Entry
1
Sulfating agent
Time (h)
12
Temperature (°C)
Solvent
DMF
Isolated yield (%)
20
SO3/Pyr
(10 eq.)
SO3/Pyr
(24 eq.)
SO3/DMF
(24 eq.)
SO3/DMF
(24 eq.)
SO3/DMF
(24 eq.)
SO3/DMF
(24 eq.)
30
30
70
25
25
70
2
3
4
5
6
24
12
24
48
24
DMF/Pyr (4:1)
37
25
71
67
65
DMF
DMF/Pyr (4:1)
DMF/Pyr (4:1)
DMF/Pyr (4:1)
In the final step of synthesis, the Boc protecting group was
removed by using TFA/H2O (90%) at 0 °C [38]. The reaction was
monitored by HPLC using an analytical C18 RP column. The depro-
tection could be complete in 40 min with 95% yield. The overall
synthetic scheme was shown in Scheme 1.
However, severe degradation of the final product (NH2-sTyr-
sTyr-COOH) was observed during a large-scale purification using
a prep-RP column. Three additional peaks were found and identi-
fied to contain NH2-sTyr-Tyr-COOH, NH2-Tyr-sTyr-COOH, and
NH2-Tyr-Tyr-COOH, respectively (Fig. S2). We next examined a
Scheme 1. (a) DCC, TEA, 4:1 CH3CN:DMF; (b) LiOH, r.t.; (c) SO3/DMF, 4:1 DMF:pyridine; (d) 9:1 TFA:water, 0 °C.

108
T. Ju et al. / Bioorganic Chemistry 68 (2016) 105–111
purification procedure using anion-exchange chromatography. We
were able to obtain NH2-sTyr-sTyr-COOH in 90% yield with higher
than 96% purity (estimated by analytical RP-HPLC). The final
product was confirmed by both NMR (Figs. S3 and S4) and mass
spectrometry (Fig. S5 and Table S2).
the sulfotyrosine dipeptide could happen during the assay (48 h)
at 37 °C.
3. Conclusions
We developed a facile procedure to efficiently synthesize sulfo-
a
2.3. Biological evaluation
tyrosine dipeptide by using N -protected tyrosine dipeptide as the
starting material and SO3/DMF complex as the sulfating agent. To
our best knowledge, this is the first report for the synthesis of this
compound. Although sulfotyrosine dipeptide did not show any
inhibitory effect against HIV-1 entry, our optimized synthetic pro-
cedure could be applied to the synthesis of longer peptides that
contain multiple sTyr residues. This will facilitate the synthesis
and screening of a large library of sulfopeptides as HIV-1 entry
inhibitors.
To test if sulfotyrosine dipeptide could act as an HIV-entry inhi-
bition, we performed in vitro neutralization assay by using our pre-
viously reported method [39]. This assay measures the
neutralization as a function of the reduction in Tat-regulated Luc
reporter gene expression after a single round infection of TZM-bl
cell (a HeLa cell clone expressing both CD4 and CCR5) [40]. The
expression of the two reporter genes, firefly luciferase and Escher-
ichia coli b-galactosidase, were under the control of an HIV long-
terminal repeat sequence. Env-pseudotyped viruses, which are
deficient of env gene in the genome of the mature virions, were
used to infect TZM-bl cells. The infection by Env-pseudotyped
viruses activated the expression of luciferase reporter gene. The
luciferase activity should be directly proportional to the number
of infectious virus particles present in the initial inoculum. In the
presence of an HIV-entry inhibitor, a lower infection rate would
lead to a decrease in the relative luminescence unit (RLU) of the
emitted light from the luciferase reaction.
4. Experimental section
4.1. Materials and general methods
All starting materials and reagents unless otherwise noted were
obtained from Sigma-Aldrich Chemicals Company (St. Louis, MO).
All reactions were carried out under argon with freshly distilled
solvents. CH2Cl2 was distilled from calcium hydride under argon.
DMF was dried and distilled over calcium hydride under vacuum
and stored over 4 Å sieves under argon. Flash chromatography
was performed using silica gel 60 Å (234–400 mesh) obtained from
Sigma-Aldrich. 1H NMR spectra were obtained using a Bruker
Avance III 300 MHz or a Bruker Avance III 400 MHz spectrometer.
Chemical shifts (d) for 1H NMR spectra run in CDCl3 were reported
in parts per million (ppm) relative to the internal standard tetram-
ethylsilane (TMS). Chemical shifts (d) for 1H NMR spectra run in
CD3OD were reported in ppm relative to residual solvent protons
(d 3.30). Chemical shifts (d) for 1H NMR spectra run in D2O were
reported in ppm relative to sodium 3-trimethylsilylpropionate-2,
2,3,3-d4 (TSP). TLC was performed by using Analtech TLC Uni-
platesTM developed in a solvent system containing n-butanol:
acetic acid:water = 3.5:0.5:1. TLC plates were stained by Ninhydrin
stain solution (0.1% Ninhydrin with 0.5% acetic acid in acetone) or
visualized under 254 nm UV light. Analytical RP-HPLC was per-
In the neutralization assay, the pseudotyped viruses were
mixed with each of the serial diluted compounds (ranging from
0 lM to 960 lM), including tyrosine dipeptide (NH2-Tyr-Tyr-
COOH), sTyr, mono-sulfated tyrosine didpeptide (a mixture of
NH2-sTyr-Tyr-COOH and NH2-Tyr-sTyr-COOH), and sulfotyrosine
dipeptide (NH2-sTyr-sTyr-COOH). As shown in Fig. 1, no obvious
inhibition was observed for the sulfotyrosine dipeptide. Moderate
neutralization effects were observed for sTyr and mono-sulfated
tyrosine didpeptide at a very high concentration. As shown in
Fig. S1, the CCR5-binding pocket of gp120 contains two sTyr bind-
ing sites for sTyr10 and sTyr14 of CCR5 [19]. At very high concen-
trations, two sTyr (or mono-sulfated tyrosine dipeptides) might
bind with gp120, which impaired the function of gp120 and led
to the observed inhibitory effects. The inhibition assay with sulfo-
tyrosine dipeptide showed large standard deviations. It is not
uncommon for an inactive drug to display large variations in bio-
logical assays. This observation might also be a result of the low
stability of sulfotyrosine dipeptide. Unpredictable hydrolysis of
formed with
a
Poroshell 120 EC-C18 column (2.7 lm,
4.6 mm  50 mm) by using Agilent 1260 Infinity Quaternary pump
equipped with a 1260 Infinity Variable Wavelength Detector
(VWD). Positive and negative ion electrospray (ESI) experiments
were performed with a Waters Q-TOF Ultima mass spectrometer.
1:1 CH3CN/H2O is used as a solvent for +ve ion analysis. Analytical
HPLC was performed using a linear gradient of CH3CN (0–25%; in
0.1% TFA) at 1 mL/min over 15 min with the UV detector set to
260 nm. Prep-HPLC was conducted with YMC-AA1205 C18 column
(3
l
m, 4.6 mm  250 mm) using a linear gradient of CH3CN (5–
35%; in 0.1% TFA) at 1 mL/min over 60 min, with UV detector set
to 230 nm and 260 nm.
4.2. Molecular docking
The docking was preformed using Discovery Studio Client ver-
sion 4.0 (BIOVIA, San Diego, CA). The ligand docking program
CDOCKER [41] was used for all the docking simulations with the
CHARMm force field. CDOCKER is a grid-based program in which
the receptor is held rigid but the ligand is allowed the flexibility
for binding. The structure of gp120 (PDB: 3QAD) in complex with
CD4 (two domains) and a CD4-induced antibody 412d was used
as the receptor, which was cleaned and prepared by adding hydro-
gens, and typed with the CHARMm force field. The CCR5-N-termus
structure (PDB: 2RLL) was used as a reference. All peptide ligands
Fig. 1. Neutralization assays. Four compounds were examined in the assay,
including sTyr, tyrosine dipeptide (NH2-Tyr-Tyr-COOH), mono-sulfated tyrosine
dipeptide (a mixture of NH2-sTyr-Tyr-COOH and NH2-Tyr-sTyr-COOH), and sulfo-
tyrosine dipeptide (NH2-sTyr-sTyr-COOH). The relative luminescence unit (RLU) of
the emitted light from the luciferase reaction was measured in an Illuminometer.
Each data point is the average of multiple measurements with standard deviation.

T. Ju et al. / Bioorganic Chemistry 68 (2016) 105–111
109
were constructed using Sketch and typed with the same force field.
The ligand-biding site was defined based on the binding site of
412d antibody (or the CCR5 N-terminus) (19). Docking parameters
were set up as the following: top hits numbers 10, conformation
dynamics steps 1000, annealing heating steps 2000, target temper-
ature cooling 5000. The best poses from each ligand were selected
based on the Cdocking energy scores and listed in Table 1.
which was directly used in the next synthetic step without further
purification.
a
4.6. N -[tert-butoxycarbonyl]-
L
-(O-sulfate)-tyrosyl-L-(O-sulfate)-
tyrosine di-ammonium salt (4)
To a solution of 3 (450 mg, 1 mmol) in a mixture of anhydrous
DMF (20 mL) and anhydrous pyridine (5 mL), SO3/DMF complex
(3.7 g, 24 mmol) was added. Under the protection of argon, the
reaction was allowed to stir at 25 °C for 24 h. After the removal
of solvent, a colorless residue was obtained. While being cooled
on an ice-water bath, 30 mL of water was added on to the reaction
vessel, followed by the addition of 20 mL of saturated NaHCO3 until
pH reached 4–5. Precipitation was then filtered out, and the filtrate
was reduced to an appropriate volume. The product was purified
on the diethylaminoethyl cellulose column by a gradient elution
of 1 L NH4HCO3 buffer (0–0.8 M, pH ꢀ 7.0, pH was adjusted by
dry ice) in chromatography chamber at 4 °C. The collected fractions
(8 mL in each tube) were analyzed by UV–vis spectroscopy. The
fractions with strong UV absorbance at 265 nm (corresponding to
0.3–0.6 M NH4HCO3 fractions) were combined. The solvent of the
combined pool was evaporated under reduced pressure. To the
residues, 10–20 mL of frozen-dry MeOH was added to extract the
desired compound. Evaporation of MeOH afforded a colorless solid
of diammonium salt of 4 (453 mg, 0.71 mmol, 71%). 1H NMR
(MeOD, 300 MHz): d 7.22–7.15 (m, 8H), 4.68–4.63 (dd, J = 8.0 Hz,
1H), 4.02–3.99 (dd, J = 8.0 Hz, 1H), 3.20–3.18 (m, 2H), 3.00–2.91
4.3. -Tyrosine methyl ester (5)
L
This compound was synthesized according to a procedure by
Rudolf [42]. Thionyl chloride (4 mL, 55.0 mmol) was added drop-
wise to a suspension of L-tyrosine (5.45 g, 30.1 mmol) in anhydrous
MeOH (100 mL, 247 mmol) at 0 °C under argon protection. When
half amount of thionyl chloride was added, the solution turned
clear. The reaction mixture was allowed to reach room tempera-
ture and stirred overnight. After removing the solvent by rotary
evaporation, the residue was washed with diethyl ether
(2 Â 50 mL). Compound 5 was obtained as a white solid (5.8 g,
29.2 mmol, 97%). 1H NMR (300 MHz, D2O): d 7.13 (d, J = 8.7 Hz,
2H), 6.87 (d, J = 8.7 Hz, 2H), 4.36 (dd, J = 5.7, 7.2 Hz, 1H), 3.82 (s,
3H), 3.24 (dd, J = 5.7, 14.7 Hz, 1H), 3.13 (dd, J = 7.2, 14.7 Hz, 1H).
a
4.4. N -[tert-butoxycarbonyl]-
L-tyrosine (6) [43]
L-Tyrosine (2 g, 11 mmol) was dissolved in a mixture of diox-
ane/water (50 mL/25 mL), followed by the addition of 25 mL of
NaOH (1 M). Di-tert-butyl dicarbonate (2.64 g, 12.1 mmol) was
then added and the reaction mixture was allowed to stir at room
temperature for 2 h. The reaction mixture was extracted by EtOAc
(3 Â 20 mL). The combined extraction was dried over Na2SO4. After
removing the solvent by rotary evaporation, the compound 6 was
obtained as a white solid (3 g, 10 mmol, 90%). 1H NMR (300 MHz,
CDCl3): d 7.10 (d, J = 8.3 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 5.25 (d,
J = 6.9 Hz, 1H), 4.52 (s, 1H, OH), 4.32–4.30 (m, 1H), 3.00–2.96 (m,
2H), 1.38 (s, 9H).
(m, 2H), 1.40 (s, 9H). ESI MS(+):
C23H28N2O13S2, theoreti-
cal = 604.10, [M+H]+ = 605.10.
4.7. L-(O-sulfate)-tyrosyl-L-(O-sulfate)-tyrosine di-ammonium salt (2)
Compound 4 (453 mg, 0.71 mmol) was dissolved in the cold
solution of TFA/water (9:1; 16 mL). The resulting reaction mixture
was stirred at 0 °C for about 40 min, until all the starting material
was completely consumed (monitored by TLC). Upon removal of
TFA by rotary evaporation, the residue, as a foaming white solid,
was washed twice by Et2O and evaporated again to further remove
TFA. To the residue, 4 mL of 0.05 M NH4HCO3 buffer (pH = 7) was
added, and a diluted ammonium hydroxide solution was applied
to adjust pH to 6–7. The product was purified by DEAE column
with a gradient elution of NH4HCO3 buffer (0–0.8 M; 1 L). The
desired compound was found in the 0.3–0.6 M NH4HCO3 fractions.
The combined fractions were evaporated by under reduced pres-
sure. To the residue, 10–20 mL of ice-cold dry MeOH was added
to extract the desired compound. Evaporation of MeOH afforded
the diammonium salt of 2 (343.8 mg, 0.64 mmol, 90%) as a color-
less solid. 1H NMR (D2O, 400 MHz): d 7.35–7.12 (m, 8H), 4.56–
4.47 (dd, J = 8.0 Hz, 1H), 4.30–4.20 (dd, J = 8.0 Hz, 1H), 3.34–3.23
(m, 2H), 3.12–2.89 (m, 2H); 13C NMR (D2O, 175 MHz): d 177.10,
168.05, 150.66, 149.81, 135.53, 131.63, 130.78, 130.39, 122.08,
121.46, 56.74, 54.02, 36.79, 36.01. HR-ESI MS(+): C18H20N2O11S2,
theoretical = 504.0508, [M+H]+ = 505.0599.
a
4.5. N -(tert-butoxycarbonyl)-
L-tyrosyl-L-tyrosine (3) [44]
To a mixture of 5 (0.7 g, 3.55 mmol) and 6 (1 g, 3.55 mmol) in
CH3CN/DMF (40 mL/10 mL), triethylamine (0.48 mL, 3.6 mmol)
was added while the reaction vessel was cooled on an ice bath.
DCC (0.95 g, 4.6 mmol) was subsequently added and the reaction
mixture was allowed to stir at room temperature for 3 h. The pre-
cipitated DCU was removed by filtration and the solvent from the
filtrate was evaporated. The residue was partitioned between
EtOAc (100 mL) and 1 M HCl (100 mL). The aqueous phase was
extracted with EtOAc (3 Â 100 mL). The combined organic frac-
tions were washed with saturated NaHCO3 (100 mL), brine
(100 mL), and dried (MgSO4). The solvent was removed under
reduced pressure and the residue was purified by flash chromatog-
raphy (2:1 EtOAc:hexane) to afford Boc-Tyr-Tyr-OMe as a white
solid (1.3 g, 3.0 mmol, 84%). 1H NMR (300 MHz, CDCl3): d 7.37
(br, 2 H), 6.91 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 2H), 6.68 (d,
J = 8.0 Hz, 4H), 6.55 (1H, buried amide NH), 5.27 (br, 1H), 4.72
(m, 1H), 4.29 (m, 1H), 3.64 (s, 3H), 2.96–2.89 (m, 4H), 1.40 (s, 9H).
To a solution of Boc-Tyr-Tyr-OMe (500 mg, 1.1 mmol) in THF
(12.5 mL), an aqueous solution (12.5 mL) of LiOHÁH2O (92 mg,
2.2 mmol) was added dropwise while the reaction vessel was
cooled on an ice bath. The resulting reaction mixture was subse-
quently stirred at room temperature for 4 h until all the starting
material was consumed (monitored by TLC). Acetic Acid was added
to adjust pH to 5. The reaction mixture was then extracted by
EtOAc (3 Â 15 mL). The combined organic phase was dried over
MgSO4. Evaporation of the solvent afforded 3 as a white solid,
4.8. Generation and titration of viral stocks
293T cells were grown at 37 °C with 5% CO2, and maintained in
DMEM medium with 4.5 g/L D-glucose and 584 mg/L -Glutamine
(Gibco), supplemented with 10% FBS and 1ÂL penicillin-
streptomycin. Twenty-four hours prior to the transfection,
1.5 Â 106 cells were plated in 10 cm dishes coated with 0.01%
Poly-L-lysine (Sigma-Aldrich). Cells were transfected by adding
6
l
g of replication-deficient plasmid that encodes the genome of
the pseudotyped HIV (pCMV-Gag-Pol), 600 ng of HIV envelope
construct (pSVIIIenv), and 18 L of FuGENE 6 (Promega) diluted
in un-supplemented DMEM. Supernatants of cell cultures were
l

110
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Acknowledgement
This research was supported by the New Faculty Startup Fund
(to J.G.), ARD Strategic Funding (to S.X.), and an Interdisciplinary
Grant (to J.G. and S.X.) from the University of Nebraska-Lincoln.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.07.
012.
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