Design, Synthesis, Antiviral Activity, and Pre-Formulation Development of Poly- L -Arginine-Fatty Acyl Derivatives of Nucleoside Reverse Transcriptase Inhibitors

Article abstract of DOI:10.1080/15257770.2014.945649

The objective of this work was to design conjugates of anti-HIV nucleosides conjugated with fatty acids and cell-penetrating poly-L-arginine (polyArg) peptides. Three conjugates of polyArg cell-penetrating peptides with fatty acyl derivatives of alovudine

Full text of DOI:10.1080/15257770.2014.945649

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Design, Synthesis, Antiviral Activity,
and Pre-Formulation Development of
Poly-L-Arginine-Fatty Acyl Derivatives
of Nucleoside Reverse Transcriptase
Inhibitors
Bhanu P. Pemmarajua, Swapnil Malekara, Hitesh K. Agarwala, Rakesh
K. Tiwariab, Donghoon Oha, Gustavo F. Donceld, David R. Worthena &
Keykavous Parangabc
a Department of Biomedical and Pharmaceutical Sciences, College of
Pharmacy, University of Rhode Island, Kingston, RI, United States
Click for updates
b Chapman University, School of Pharmacy, Irvine, CA, United States
c Chao Family Comprehensice Cancer Center, School of Medicine,
University of California, Orange, CA, United States
d CONRAD, Department of Obstetrics and Gynecology, Eastern
Virginia Medical School, Norfolk, VA, United States
Published online: 16 Dec 2014.
To cite this article: Bhanu P. Pemmaraju, Swapnil Malekar, Hitesh K. Agarwal, Rakesh K. Tiwari,
Donghoon Oh, Gustavo F. Doncel, David R. Worthen & Keykavous Parang (2015) Design, Synthesis,
Antiviral Activity, and Pre-Formulation Development of Poly-L-Arginine-Fatty Acyl Derivatives of
Nucleoside Reverse Transcriptase Inhibitors, Nucleosides, Nucleotides and Nucleic Acids, 34:1, 1-15,
DOI: 10.1080/15257770.2014.945649
To link to this article: http://dx.doi.org/10.1080/15257770.2014.945649
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Nucleosides, Nucleotides and Nucleic Acids, 34:1–15, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2014.945649
DESIGN, SYNTHESIS, ANTIVIRAL ACTIVITY,
AND PRE-FORMULATION DEVELOPMENT OF POLY-L-
ARGININE-FATTY ACYL DERIVATIVES OF NUCLEOSIDE
REVERSE TRANSCRIPTASE INHIBITORS
Bhanu P. Pemmaraju,1 Swapnil Malekar,1 Hitesh K. Agarwal,1
Rakesh K. Tiwari,1,2 Donghoon Oh,1 Gustavo F. Doncel,4 David R. Worthen,1
and Keykavous Parang1,2,3
1Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of
Rhode Island, Kingston, RI, United States
2Chapman University, School of Pharmacy, Irvine, CA, United States
3Chao Family Comprehensice Cancer Center, School of Medicine, University of California,
Irvine, Shanbrom Hall, Orange, CA, United States
4CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School,
Norfolk, VA, United States
GRAPHICAL ABSTRACT
Cell-Penetraꢁng
Poly-Arg Pepꢁde
Nucleoside
Analogue
Faꢀy acid
FLT, 3TC, FTC 1,12-Dodecanedicarboxylic
acid
R7
The objective of this work was to design conjugates of anti-HIV nucleosides conjugated with
2
fatty acids and cell-penetrating poly-L-arginine (polyArg) peptides. Three conjugates of polyArg cell-
penetrating peptides with fatty acyl derivatives of alovudine (FLT), lamivudine (3TC), and emtric-
itabine (FTC) were synthesized. In general, the compounds exhibited anti-HIV activity against X4
and R5 cell-free virus with EC50 values of 1.5–16.6 μM. FLT-CO-(CH2)12-CO-(Arg)7 exhibited
EC50 values of 2.9 μM and 3.1 μM against X4 and R5 cell-free virus, respectively. The FLT con-
jugate was selected for further preformulation studies by determination of solution state degradation
and lipid solubility. The compound was found to be stable in neutral and oxidative conditions and
moderately stable in heated conditions.
Received 16 June 2014; accepted 13 July 2014.
The views expressed by the authors do not necessarily reflect the views of USAID or CONRAD.
Address correspondence to K. Parang: Chao Family Comprehensice Cancer Center, School of
Medicine, University of California, Irvine, Shanbrom Hall, 101 The City Drive, Orange, CA 92868.
E-mail: parang@chapman.edu
1

2
B. P. Pemmaraju et al.
Keywords anti-HIV agents; fatty acids; lipophilicity; nucleosides; polyarginine; reverse
transcriptase
INTRODUCTION
According to UNAIDS estimation, 35.3 million people are living with
Acquired Immunodeficiency Syndrome (AIDS) as of 2013. High Activity
Antiretroviral Therapy (HAART) involves the use of a minimum of three an-
tiretroviral drugs including protease inhibitors, non-nucleoside reverse tran-
scriptase inhibitors, and nucleoside reverse transcriptase inhibitors. HAART
has increased the lifespan of HIV-infected patients and has converted this
terminal illness into a chronic and manageable disease.[1] Although the
FDA-approved anti-HIV nucleosides zidovudine (AZT), lamivudine (3TC),
and emtricitabine (FTC) have reduced the mortality rate in patients, their
use is still complicated because of toxicity, chronic dosage, development
of resistant virus, and incomplete elimination of the viral reservoirs. More-
over, these anti-HIV nucleosides have limited cellular uptake due to their
hydrophilic nature. Upon intracellular uptake, they need to undergo three
phosphorylation steps in order to become active nucleoside triphosphate
analogues.[1] Thus, novel drug-delivery systems are urgently needed in or-
der to generate nucleoside conjugates with better anti-HIV profile, such as
higher cellular uptake and anti-HIV activity.
The primary objective of this work was to design conjugates of anti-
HIV nucleosides conjugated with fatty acids and cell-penetrating polyArg
peptides. Herein, we designed polyArg-fatty acyl derivatives of anti-HIV nu-
cleosides with the expectation to overcome their limited cellular uptake. The
objective was to design multifunctional anti-HIV drug conjugates where:
1. Nucleosides act as reverse transcriptase inhibitors (NRTIs);
2. Long-chain fatty acyl component acts as an N -myristoyl transferase (NMT)
inhibitor and also improves cellular uptake; and
3. The poly-L-arginine component plays a crucial role as a cell-penetrating
peptide.
Long-Chain Carboxylic Acids as NMT Inhibitors
Long-chain carboxylic acids are known to inhibit NMT, which is re-
sponsible for the myristoylation of various HIV proteins, such as P17 capsid
protein, Pr55gag, Pr160gag-pol, and p27nef in the virus-infected host cells.[2]
Myristoylation of viral proteins allows the viral protein components to be-
come more hydrophobic.[3] Certain heteroatom-containing myristic acid
analogues, such as 12-thioethyldodecanoic acid, 4-oxatetradecanoic acid,
and 2-methoxydodecanoic acid derivatives, inhibit HIV-1 replication in
acutely infected T-lymphocytes.[4,5] 12-Thioethyldodecanoic acid was found

Poly-L-Arginine-Fatty Acyl Derivatives of Nucleosides
3
to be moderately active against HIV-infected T4 lymphocytes with an EC50
value of 9.4 μM.[6]
Various 5ꢀ-O-fatty acyl derivatives of nucleoside reverse transcrip-
tase inhibitors (3ꢀ-azido-3ꢀ-deoxythymidine (zidovudine, AZT), 3ꢀ-fluoro-3ꢀ-
deoxythymidine (alovudine, FLT), (-)-2ꢀ,3ꢀ-dideoxy-3ꢀ-thiacytidine (lamivu-
dine, 3TC), 5-fluoro-(-)-2ꢀ,3ꢀ-dideoxy-3ꢀ-thiacytidine (emtricitabine, FTC),
2ꢀ,3ꢀ-didehydro-2ꢀ,3ꢀ-dideoxythymidine (stavudine, d4T) were found to have
enhanced anti-HIV activity profiles as compared with their parent nucleo-
sides against X4, R5 strains, and cell-associated virus, presumably due to in-
creased cellular uptake caused by their high lipophilic profile.[6–14] These es-
ter conjugates and others reported by other researchers[15,16] were expected
to act as bifunctional agents through intracellular hydrolysis by esterases to
release nucleoside reverse transcriptase inhibitors and fatty acids.
Poly-L-Arginine as a Cell-Penetrating Peptide
An important subclass of these molecular transporters are CPPs, which
contain guanidinium-rich transporters (GRTs). The positively charged
guanidinium groups on the arginine side chains are responsible for its pen-
etrating ability through interaction with the phospholipid bilayer. The de-
velopment of these polyarginine transporter molecules was inspired by the
lead HIV-1 transcription transactivator protein (Tat) made up of repeated
arginine and lysine residues.[14] This transporter is highly polar, readily sol-
uble in water, and, unlike other polar drugs, travels across the lipid bilayer
membrane.[17] Studies have shown that peptides with 6–20 arginine residues
pass through the membrane, showing rapid uptake across the membrane
and into the nucleus without any signs of acute toxicity.[17] Molecular trans-
porters, when attached to poorly bioavailable drugs, could enable them to
pass through the biological membrane as shown previously.[18]
Herein, we report the design, synthesis, and biological activities of three
poly-L-arginine-linked (poly-Arg) 1,12-dodecanedicarboxylate derivatives of
anti-HIV nucleosides (FLT, 3TC, and FTC; Figure 1) and their application
Cell-Penetraꢁng
Poly-Arg Pepꢁde
NucleosideAnalogue
Faꢀy acid
-
1,12 Dodecanedicarboxylic acid
R7
FIGURE 1 Poly-L-arginine-linked fatty acylated nucleosides. (Color figure available online).

4
B. P. Pemmaraju et al.
as multifunctional anti-HIV agents. It was expected that the conjugation of
nucleosides to the poly-L-arginine-fatty acyl residue could result in the de-
velopment of anti-HIV agents with enhanced efficacy, and/or higher uptake
into infected cells.
RESULTS AND DISCUSSION
FLT (1), N4-amino-protected 3TC (8), N4-amino-protected FTC (9),
and 1,12-dodecandicarboxylate-polyarginine resin were used as the building
blocks. N4-DMTr-3TC (8) and N4-DMTr-FTC (9) were synthesized according
to a previously reported procedure.[12] First, tert-butyldimethylsilyl chloride
(TBDMS–Cl) was reacted with 3TC (2) or FTC (3) in the presence of im-
idazole to afford 5ꢀ-O-TBDMS-3TC (4) or 5ꢀ-O-TBDMS-FTC (5). Next, the
N4–amino group of 4 and 5 was protected with 4,4ꢀ-dimethoxytrityl (DMTr)
protecting group, by reaction with DMTr-Cl in the presence of pyridine to
yield 6 and 7, respectively. Finally, TBDMS was removed by using tetrabuty-
lammonium fluoride (TBAF) to yield N4-DMTr-3TC (8) and N4-DMTr-FTC
(9) (Scheme 1).
SCHEME 1 Synthesis of N4-DMTr protected of FTC and 3TC nucleosides.[12]
Second, the polyarginine peptide (R7) was manually synthesized by agi-
tation of resin using nitrogen gas with a solid-phase Fmoc/tBu strategy. The
preloaded Fmoc-L-arginine(Pbf)-Wang resin (10) was swelled in DMF, and
this was followed by deprotection of Fmoc group by using 20% piperidine
in DMF.
The resin was washed with DMF and coupled with Fmoc-Arg(Pbf)-
OH in the presence of coupling reagents 1,1,3,3-tetramethyluronium hex-
afluorophosphate (HBTU) and N ,N -diisopropylethylamine (DIPEA) as a
base, respectively, in DMF at room temperature under nitrogen. The de-
protection and coupling cycles were repeated 5 more times, followed by
the final N -terminal Fmoc de-protection using 20% piperidine in DMF
to yield NH2-polyarginine-Wang resin 11. A small amount of resin cleav-
age confirmed the assembly of polyarginine peptide on Wang resin. 1,12-
Dodecanedicarboxylic acid was coupled to NH2-polyarginine Wang resin 11

Poly-L-Arginine-Fatty Acyl Derivatives of Nucleosides
5
to yield 1,12-dodecanedicarboxylate-polyarginine resin 12 in the presence
of a combination of coupling reagents (HBTU, HOAt, and DIC; Scheme 2).
SCHEME 2 Synthesis of hepta-L-arginyl-1,12-dodecanedicarboxylate-nucleoside conjugates 13-15.
Peptidyl polyarginine dodecandicarboxylate resin 12 was conjugated
with the nucleoside analogues (1, 8, or 9) in the presence of HBTU, HOAt
DIPEA, and DIC in anhydrous DMF (Scheme 2). The resin was cleaved
along with the deprotection of Pbf and DMTr protective groups from the
side chain of arginine and the corresponding nucleosides, respectively, using
reagent R for 4 hours at room temperature. The conjugates were purified
by preparative reverse-phase HPLC. The purity of final products (>95%)
was confirmed by analytical HPLC. The chemical structures of compounds
were determined by a SELDI-TOF mass spectrometer on a Ciphergen pro-
tein chip instrument using α-cyano-4-hydroxycinnamic as a matrix and by
1H and 13C NMR.
The anti-HIV activity of the compounds was evaluated according to a pre-
viously reported procedure.[11,19–21] Table 1 shows the anti-HIV activities of
the conjugates compared to their parent analogues. No cellular cytotoxicity

6
B. P. Pemmaraju et al.
TABLE 1 Anti-HIV activity of dicarboxylic acid ester conjugates of nucleoside conjugates (1–7)
Cell-Associated
Cytotoxicity
Cell-Free Virus
Virus
CTSa EC50 X4c EC50 R5d EC50
CTCe EC50
(μM)
Compound
Chemical Name
b(μM)
(μM)
(μM)
FLT (1)
3TC (2)
FTC (3)
13
14
15
3ꢀ-Fluoro-2ꢀ,3ꢀ-deoxythymidine
(-)-2ꢀ,3ꢀ-Dideoxy-3ꢀ-thiacytidine
(-) 2ꢀ,3ꢀ–Dideoxy-5-fluoro-3ꢀ-thiacytidine
FLT-CO-(CH2)12-CO-R7
>100
>100
>100
>30
>30
>30
0.2
7.5
0.48
2.9
8.7
3.0
0.1
2.6
0.18
3.1
16.6
1.5
>100
18.4
21.9
>30
>30
>30
3TC-CO-(CH2)12-CO-R7
FTC-CO-(CH2)12-CO-R7
aCytotoxicity assay (MTS); b50% Effective concentration; cSingle-round infection assay (lymphocy-
totropic strain, X4); dSingle-round infection assay (monocytotropic strain, R5); eCell-associated transmis-
sion assay (X4).
was observed up to the highest tested concentration for the conjugates (EC50
> 30 μM). The conjugates showed no significant anti-HIV activity against
cell-associated virus (EC50 > 30 μM). All conjugates exhibited less anti-HIV
activity when compared with the parent nucleoside analogues against cell-
free virus. FLT conjugate 13 (EC50 = 2.9–3.1 μM) showed lesser potency
than its parent nucleoside (EC50 = 0.1–0.2 μM) against X4 and R5 cell-free
virus. FTC conjugate 15 (EC50 = 1.5–3.0 μM) showed less anti-HIV-1 activity
when compared to that of FTC (EC50 = 0.18–0.48 μM) against X4 and R5
cell-free virus. The 3TC conjugate 14 not only showed approximately 2-fold
higher inhibition against the X4 virus (EC50 = 8.7 μM) than against the
R5 virus strain (EC50 = 16.6 μM), but also exhibited less potency than that
of 3TC (EC50 = 2.6–7.5 μM). The conjugates were less potent than their
parent structures possibly due to the limited uptake. Different strains of HIV
are known to have different gp120 V3 loops. Both the X4 and R5 strains of
HIV possess a high positive charge density on their V3 loop,[22,23] suggesting
that the presence of positive charge on the conjugates could block the inter-
action with gp120. Thus, these conjugates exhibited lesser anti-HIV activity
when compared with their parent analogues because of the limited cellular
uptake.
To better understand the physicochemical properties of these com-
pounds in designing more optimized compounds, we further explored the
stability and lipid solubility of these compounds. As the FLT-conjugate con-
tains several potentially labile bonds, forced degradation of an aqueous
solution of 13 at elevated temperature, in acid, base, and under oxidizing
conditions, were conducted. Compound 13 was selected for further evalu-
ation in solution-state degradation studies and determination of lipid solu-
bility (partition coefficient) Thus, it was necessary to determine the relative
stability of these compounds under different conditions. Furthermore, these

Poly-L-Arginine-Fatty Acyl Derivatives of Nucleosides
7
FIGURE 2 Degradation studies of FLT conjugate 13. A: Standard 13, Retention time 9.7 minutes; B:
Heat: 1 mL stock + 1 mL methanol at 40◦C; C: Acid: 1 mL stock + 1 mL 1N HCl at 40◦C; D: Base: 1 mL
stock + 1 mL 1N NaOH at 40◦C; E: Oxidation: 1 mL stock + 1 mL H2O2 at 40◦C; F: Water: 1 mL stock
+ 1 mL H2O at 40◦C. (Color figure available online).
conjugates were designed to be used in areas where shipping and storage
conditions are not ideal in terms of temperature and humidity.
At heated (panel B) conditions, significant degradation of the com-
pound 13 was observed resulting in a second HPLC peak at 9 minutes.
Under both acidic and alkaline conditions, the compound degraded to a
great extent resulting in a different peak at 7 minutes (panels C and D).
Minimal degradation of the drug was noted at neutral (panel E) and ox-
idative (H2O2; panel F) conditions (Figure 2). Thus, the FLT conjugate was
relatively stable in neutral and oxidative conditions, but less stable in heated,
acidic, and alkaline conditions.
The Log P of the compound was determined by distributing the com-
pound between an equal volume of n-octanol (organic) and pH 4 acetate

8
B. P. Pemmaraju et al.
buffer (aqueous) with stirring at room temperature for 2 days, followed by
HPLC studies. The Log P of the compound was found to be −0.34, indicat-
ing that the compound is hydrophilic, most likely due to the presence of
several guanidinium moieties from hepta-L-arginine. FLT has a Log P value
of −0.52. These data suggest that the compound is still very hydrophilic
despite the presence of the long 1,12-dodecane dicarboxylic acid linker.
Various gel formulations of the compound were manufactured using
non-ionic (HPC-SL) and anionic (Carbopol) polymers, with or without the
inclusion of the thermo-reversible gelling (Pluronic F-127) polymer. The
derivative was used for dissolution studies using four different gels, with
or without the thermogelling polymer. The HPC-SL formulation consisted
of 2.25% w/v in water whereas Carbopol consisted of 0.2% w/v. For the
thermogelling formulations, the HPC-SL and Carbopol gels were mixed with
20% w/v solution of Pluronic F-127(3:1 v/v ratio) in water. The formulations
were then sealed in dialysis tubes, and the rate and extent of compound
dissolution were determined in simulated vaginal fluid at 37◦C. There was
no observed drug release that could be identified by UV spectrophotometry
at 220 and 256 nm, suggesting that the compound was unstable in these
formulations.
CONCLUSIONS
In conclusion, three poly-L-arginyl-1,12-dodecanedicarboxylate nucleo-
side conjugates of FLT, 3TC, and FTC were designed and synthesized using
solid-phase chemistry. The structures of the compounds were confirmed
by NMR and mass spectroscopy. The compounds were evaluated for their
anti-HIV activity against cell-free and cell-associated virus. FLT conjugate
13 showed EC50 values of 2.9–3.1 μM against X4 and R5 virus. The com-
pound was relatively stable in neutral and oxidative conditions, and un-
stable in heated, acidic, and alkaline conditions. The FLT conjugate was
hydrophilic, as the Log P was found to be −0.34. The derivative was eval-
uated in dissolution studies using four different hydrogels gels with and
without a thermogelling polymer. Gel formulations of the compound were
manufactured using non-ionic (HPC-SL) and anionic (Carbopol) polymers
with or without the inclusion of a thermo-reversible gelling (Pluronic F-127)
polymer. The compound was unstable or did not undergo release from the
tested hydrogel formulations. These data indicate that the presence of posi-
tively charged CPP could impede the interactions between positively charged
V3 loop in gp120 and the conjugates. Further optimization of conjugated
CPP–fatty acid–nucleoside conjugates is required to generate compounds
with improved anti-HIV activity and optimized stability and formulation
performance.

Poly-L-Arginine-Fatty Acyl Derivatives of Nucleosides
EXPERIMENTAL
9
Materials and Methods
Materials
Nucleosides (1-3) were purchased from Euro Asia Trans Continen-
tal (Bombay, India) for the nucleoside ester conjugate synthesis. [2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate]
(HBTU), N,N -diisopropylethylamine (DIPEA), and 1,12-dodecanedicarbox-
ylic acid were bought from Sigma Aldrich Chemical Co. Solvents and all
other reagents were purchased from Fisher Scientific. The products were
purified using a Phenomenex-Gemini C18 column (10 μm, 250 × 21.2 mm)
with Hitachi HPLC system using a gradient system at a constant flow rate of
10 mL/minute (Table 2). The purity of the final products was confirmed by
using a Hitachi analytical HPLC system on a C18 column using a gradient
system (water:acetonitrile 30:70 v/v) at a constant flow rate of 1 mL/minute
with UV detection at 220 and 265 nm. The chemical structures of the
final products were determined by NMR spectrometry (1H NMR and 13C
NMR) on a Bruker NMR spectrometer (400 MHz) and confirmed by a
SELDI-TOF mass spectrometer on a Ciphergen protein chip instrument
using α-cyano-4-hydroxycinnamic acid as a matrix.
Chemistry
Synthesis of Polyarginine (11). The peptide synthesis was carried out
in Bio-Rad polypropylene columns by shaking and mixing using a Glass
Col small tube rotator or on a PS3 automated peptide synthesizer (Rainin
Instrument Co., Inc.) at room temperature unless otherwise stated. Fmoc-
Arg(Pbf)-Wang resin (10, 1.1 mmole, 2.97 g, 0.37 mmole/g) was placed in
a manual peptide synthesis container (250 mL) equipped with a three-way
stopper. Resin was swelled in N ,N -dimethylformamide (DMF, 150 mL) with
constant N2 bubbling for 30 minutes (2 times). The Fmoc group was removed
TABLE 2 HPLC method used for purification of the final compounds
Water Concentration
A (%)
Acetonitrile Concentration
B (%)
Flow Rate
(mL/minute)
Time (minute)
0.00
1.00
5.0
25.0
35.0
60.0
65.0
70.0
100.0
100.0
90.0
70.0
60.0
0.0
0.0
0.0
1.0
10.0
10.0
10.0
10.0
10.0
10.0
1.0
10.0
30.0
40.0
100.0
100.0
0.0
0.0
100.0

10
B. P. Pemmaraju et al.
from the resin in the presence of piperidine (20% v/v in DMF, 2 × 50 mL)
for 15 minutes. The resin was washed with DMF (5 × 50 mL). Fmoc-Arg(Pbf)-
OH (3 equiv, 3.3 mmol, 2.14 g) and HBTU (3 equiv, 1.25 g, 3.3 mmol) were
added to the reaction vessel, followed by the addition of DMF (25 mL) and
DIPEA (6 equiv, 6.6 mmol, 1.15 mL) (Scheme 2) and bubbled with N2 for
2.5 hours. The resin was washed with DMF (5 × 25 mL) to remove unreacted
starting materials, impurity, and reagents. The deprotection and coupling
cycles were repeated 5 more times to add a total of seven arginine residues
on the resin. N -Terminal Fmoc was deprotected using piperidine (20% v/v
in DMF) to afford resin 11. Resin 11 was washed with DMF (5 × 100 mL).
The structure of 11 was confirmed by cleaving a small amount of the resin
in the presence of reagent R (TFA/thioanisole/1,2-ethanedithiol/anisole,
90:5:3:2 v/v/v/v, 2 mL) for 4 hours. The crude peptide was precipitated
in the presence of cold diethyl ether (Et2O, 150 mL), and separated and
washed by centrifugation (3 × 15 mL) at 4000 rpm for 5 minutes. The
crude peptide was dissolved in water (0.1%, TFA). The molecular weight of
poly-L-arginine (R7) was confirmed by a SELDI-TOF mass spectrometer on a
Ciphergen protein chip instrument. MS (SELDI-TOF) (m/z): C42H86N28O8,
calcd, 1111.3; found, 1111.1 [M]+.
Synthesis of Polyarginine 1,12-Dodecanedicarboxylate (12). 1,2-
Dodecanedicarboxylic acid (1.43 g, 5.5 mmol), HBTU (2.09 g, 5.5 mmol),
and 1-hydroxy-7-azabenzotriazole (HOAt, 0.74 g, 5.5 mmol) were added
to the reaction vessel containing polyarginine resin 11 in anhydrous DMF
(200 mL), followed by the addition of DIPEA (8.1 mmol, 1.42 mL) and N,N ꢀ-
diisopropylcarbodiimide (DIC, 9.0 mmol, 1.4 mL; Scheme 2). The peptidtyl
resin was bubbled with N2 for 7 hours and then washed with DMF (5 ×
100 mL). Resin 12 was finally washed with DMF (5 × 150 mL). A small
amount of the resin was cleaved using reagent R (TFA/thioanisole/1,2-
ethanedithiol/anisole, 90:5:3:2 v/v/v/v, 2 mL) for 4 hours, precipitated
with cold diethyl ether (200 mL), and finally centrifuged at 4000 rpm for 5
minutes. The residue was dissolved in water, and the formation of product
12 was confirmed with a SELDI-TOF mass spectrometer. MS (SELDI-TOF)
(m/z): C56H110N28O11, calcd, 1351.7; found, 1352.0 [M + H]+.
General Procedure for the Synthesis of Polyarginine-1,12-
Dodecanedicarboxylate Acylated Nucleoside Analogues (13-15). N4-4,4ꢀ-
Dimethoxytrityl (DMTr)-3TC (8) and N4-DMTr-FTC (9) were synthesized as
described in the previously published procedure.[12,13] Peptidyl polyarginine
dodecandicarboxylate resin 12 (0.7 g), the nucleoside analogues (1, 8, or
9, 1.1 mmol), HBTU (417 mg, 1.1 mmol), and HOAt (148 mg, 1.1 mmol)
were dissolved in anhydrous DMF (25 mL) in a round-bottomed flask
(100 mL) followed by the addition of DIPEA (1.95 mmol, 342 μL) and DIC
(2.5 mmol, 383 μL) (Scheme 2). The reaction vessels were flushed with N2
and placed on a shaker to mix the reagents.

Poly-L-Arginine-Fatty Acyl Derivatives of Nucleosides
11
The reaction mixture was filtered off, and the resin was washed with
DMF (3 × 20 mL), methanol (3 × 20 mL), and dichloromethane (DCM,
3 × 20 mL). The resin was cleaved together with the deprotection of Pbf and
DMTr protective groups from the side chain of arginine and correspond-
ing nucleosides, respectively, using reagent R (20 mL) for 4 hours at room
temperature. The resin was filtered, and the filtrate was added drop-wise
to cold diethyl ether (50 mL) for precipitation followed by centrifugation
at 4000 rpm. The precipitates were washed with diethyl ether (2 × 50 mL)
to afford solid crude peptide–nucleoside conjugates (13–15). The precipi-
tates were dried and dissolved in 50% acetonitrile and water. The conjugates
were purified by preparative reverse-phase HPLC (Shimadzu LC-8A prepara-
tive liquid chromatograph) on a Phenomenex-Gemini C18 column (10 μm,
250 × 21.2 mm) at 10.0 mL/minute flow rate using a gradient of 10%
acetonitrile (0.1% TFA) in water (0.1% TFA) to 100% acetonitrile (0.1%
TFA) (Table 2). Chromatograms were recorded at 220 and 265 nm using
a UV detector. The purity of final products (>95%) was confirmed by an-
alytical HPLC. The chemical structures of compounds were determined by
a SELDI-TOF mass spectrometer on a Ciphergen protein chip instrument
using α-cyano-4-hydroxycinnamic as a matrix and by 1H and 13C NMR.
1-[5ꢀ-O-(3ꢀ-Fluoro-2ꢀ,3ꢀ-dideoxythymidinyl)]tetradecan-1,14-dioate conju-
gate of PolyArg (FLT-OCO(CH2)12CONH-RRRRRRR-OH, 13). Yield
1
(210 mg, 60.5%). H NMR (400 MHz, CD3OD, δ ppm): 7.30 (s, 1H, H-6),
6.12 (dd, J = 5.7 and 8.6 Hz, 1H, H-1ꢀ), 5.15 (dd, J = 4.3 and 53.2 Hz, 1H, H-
3ꢀ), 4.04–4.26 (m, 7H, NH CH CO), 3.90–4.00 (m, 1H, H-4ꢀ), 3.52–3.66
(m, 2H, H-5ꢀ and H-5ꢀꢀ), 3.26–3.35 (m, 2H, = NH CH2), 2.95–3.10 (m,
10H, = NH CH2), 2.70–2.90 (m, 2H, = NH CH2), 2.40–2.55 (m, 1H, H-
2ꢀꢀ), 2.05–2.25 (m, 5H, CH2O and H-2ꢀ), 1.84–1.90 (br s, 7H, 5-CH3 and
CH2CH2CO), 1.35–1.80 (m, 32H, = NH CH2 CH2), 1.08–1.20 (br s, 16H,
methylene protons). 13C NMR (CD3CN, 100 MHz, δ ppm): 175.87, 173.26,
172.79, 172.69, 156.36, 135.79, 118.26, 114.78, 84.89, 82.22, 63.19, 52.82,
51.83, 48.74, 40.22, 37.14, 33.16, 27.49, 24.07, 19.98, 19.25, 13.83, 11.43,
0.73, 0.52, 0.31, 0.11, 0.10, 0.36.MS (SELDI-TOF) (m/z): C66H121FN30O14,
calcd, 1576.97; found, 1577.5 [M + H]+.
1-[(−)-2ꢀ,3ꢀ-Dideoxy-3ꢀ-thiacytidine]tetradecan-1,14-dioate conjugate of
PolyArg (3TC-OCO(CH2)12CONH-RRRRRRR-OH, 14). Yield (180 mg,
1
52.3%). H NMR (400 MHz, CD3OD, δ ppm): 8.27 (d, J = 7.8 Hz,
1H, H-6), 6.42–6.48 (m, 1H, H-1ꢀ), 6.34 (d, J = 7.5 Hz, 1H, H-5),
5.60–5.80 (m, 1H, H-4ꢀ), 4.58–4.68 (m, 1H, NH CH CO), 4.40–4.50 (br
s, 5H, NH CH CO), 4.30–4.40 (m 1H, NH CH CO), 3.78 (dd, J =
12.8 and 5.4 Hz, 1H, H-5ꢀꢀ), 3.61 (d, J = 12.8 Hz, 1H, H-5ꢀ), 3.42–3.52
(m, 1H, H-2ꢀꢀ), 3.22–3.40 (br s, 15H, H-2ꢀ, = NH-CH2), 2.52–5.64 (m, 2H,
CH2CONH ), 2.36–2.44 (m, 2H, CH2COO), 1.65–2.10 (m, 32H, = NH-
CH2-CH2), 1.30−1.50 (br s, 16H, methylene protons). 13C NMR (CD3CN,

12
B. P. Pemmaraju et al.
TABLE 3 HPLC method used for stability and degradation studies
Water Concentration
A (%)
Acetonitrile Concentration
Flow Rate
(mL/minute)
Time (minute)
B (%)
0.00
1.00
20.0
25.0
30.0
35.0
100.0
100.0
0.0
0.0
100.0
100.0
0.0
0.0
100.0
100.0
0.0
1.0
1.0
1.0
1.0
1.0
1.0
0.0
100 MHz, δ ppm): 176.50, 176.46, 174.91, 173.47, 172.76, 172.69, 172.65,
172.59, 172.48, 162.08, 161.74, 159.40, 156.29, 148.20, 143.62, 94.00, 86.78,
84.18, 63.21, 53.17, 52.83, 52.77, 52.43, 40.17, 36.95, 35.03, 33.33, 28.51,
28.38, 28.30, 28.11, 28.02, 27.91, 27.67, 27.57, 24.96, 24.21, 24.07, 24.01,
23.92. MS (SELDI-TOF) (m/z): C64H119N31O13S, calcd, 1561.93; found,
1562.70 [M + H]+.
1-[(−)-2ꢀ,3ꢀ-Dideoxy-5-fluoro-3ꢀ-thiacytidine]tetradecan-1,14-dioate con-
jugate of PolyArg (FTC-OCO(CH2)12CONH-RRRRRRR-OH, 15). Yield
(130 mg, 37.5%); 1H NMR (400 MHz, CD3OD, δ ppm): 8.11 (d, J = 6.7 Hz,
1H, H-6), 6.22–6.27 (m, 1H, H-1ꢀ), 5.44 (dd, J = 2.9 and 4.3 Hz, 1H, H-
4ꢀ), 4.68 (dd, J = 12.6 and 4.4 Hz, 1H, H-5ꢀꢀ), 4.30–4.48 (m, 7H, H-5ꢀ,
NH CH CO), 4.20–4.30 (m, 1H, NH CH CO), 3.45–4.65 (m, 1H,
H-2ꢀꢀ), 3.15–3.28 (m, 15H, H-2ꢀ, CH2NH), 2.13–2.38 (m, 4H, CH2COO
and CH2CONH), 1.40–1.90 (m, 32H, methylene protons), 1.10–1.30 (br
s, 16H, methylene proton); 13C NMR (CD3CN, 100 MHz, δ ppm): 172.88,
172.41, 171.79, 171.21, 159.47, 158.84, 158.52, 156.76, 124.80, 99.43, 86.63,
82.02, 63.77, 52.02, 40.04, 39.83, 39.62, 39.41, 39.20, 38.99, 38.78, 36.06,
35.05, 33.16, 28.87, 28.79, 28.35, 28.26.25.15, 24.86, 24.29. MS (MALDI-
TOF) (m/z): C64H118FN31O13S, calcd, 1580.91; found, 1581.5 [M + H]+.
Stability and Degradation Studies. The stability of 13 was evaluated by
using HPLC. All degradation studies were carried out at a drug concentration
of 1 mg/mL. The solution stability studies were conducted by using the stock
solution of compound 13 in the presence of room temperature (25◦C),
heat (40◦C), neutral (water), acidic (1N HCl), alkaline (1N NaOH) and
oxidation (3% H2O2) conditions at 40◦C. The compound was incubated in
the above mentioned solutions for 24 hours. All the samples were kept at
room temperature for 1 hour, and analytical HPLC was run for 35 minutes.
The solvent system used was water:acetonitrile (0.1% trifluoroacetic acid),
and the HPLC was run at a flow rate of 1 mL/minute at 220 nm and 256 nm
wavelengths (Table 3).
Partition Coefficient (Log P). Log P HPLC studies were carried out by
distributing 21.9 mg of compound 13 in 250 μL each of n-octanol (organic)
and pH 4 acetate buffer (aqueous). The mixture was stirred for 2 days at
room temperature. The analytical HPLC was run using water:acetonitrile

Poly-L-Arginine-Fatty Acyl Derivatives of Nucleosides
13
TABLE 4 Dissolution media composition for (6 liters)
Acetic
Lactic
Potassium
Calcium
Sodium
Chemicals
Acid Urea Glucose Acid Glycerol Hydroxide Hydroxide Chloride pH
Weight (grams)
6
24
30
12
0.96
8.4
1.3
21
4.2
(0.1% TFA) as a solvent system at a flow rate of 1 mL/minute at 220 and
256 nm wavelengths for each collected fraction (Table 3).
Gel Formulations. Vaginal gel formulations of compound 13 were man-
ufactured using non-ionic (HPC-SL) and anionic (Carbopol) polymers, with
or without the inclusion of thermo-reversible gelling (Pluronic F-127) poly-
mer. The HPC-SL formulation consisted of 2.25% w/v in water whereas
Carbopol consisted of 0.2% w/v. For thermogelling formulations, the afore-
mentioned HPC-SL and Carbopol gels were mixed with a 20% solution of
Pluronic F-127 (3:1 v/v ratio). A drug load comprising 1.6% (w/v) of FLT
conjugate was loaded in the gels and was sealed in SpectraPor dialysis tubing
with MWCO of 3500 Da. The tubes were suspended in 70 mL of dissolution
media (Table 4) held at 37 0.5 ◦C with a stirring speed of 75 rpm using a
0.5-inch stir bar. The dissolution media consisted of simulated vaginal fluid
(Table 4). These dissolution samples were transferred to UV 96-well plates
(CostarꢁR ,) and were then analyzed using a SpectraMax M2 UV detector
(Molecular Devices, PA, USA).
Anti-HIV Assays. The anti-HIV activity of the compounds was evaluated
according to the previously reported procedure.11,19–21 Compound anti-HIV
activity was evaluated in single-round (MAGI) infection assays using X4 (IIIB)
and R5 (BaL) HIV-1 and P4R5 cells expressing CD4 and co-receptors. In sum-
mary, P4R5MAGI cells were cultured at a density of 1.2 × 104 cells/well in a
96-well plate approximately 18 hours prior to infection. Cells were incubated
for 2 hours at 37◦C with purified, cell-free HIV-1 laboratory strains IIIB or
BaL (Advanced Biotechnologies, Inc., Columbia, MD), in the absence or
presence of each agent. After 2 hours, cells were washed, cultured for an
additional 46 hours, and subsequently assayed for HIV-1 infection using the
Galacto-Star β-Galactosidase Reporter Gene Assay System for Mammalian
Cells (Applied Biosystems, Bedford, MA). Reductions in infection were cal-
culated as a percentage relative to the level of infection in the absence of
agents, and 50% inhibitory concentrations (EC50) were derived from regres-
sion analysis. Each compound concentration was tested in triplicate wells.
Cell toxicity was evaluated using the same experimental design but without
the addition of virus. The impact of compounds on cell viability was assessed
using an MTT (reduction of tetrazolium salts) assay (Invitrogen, Carlsbad,
CA).

14
B. P. Pemmaraju et al.
FUNDING
Support for this subproject (MSA-03-367) was provided by CONRAD,
Eastern Virginia Medical School under a Cooperative Agreement (HRN-
A-00-98-00020-00) with the United States Agency for International Devel-
opment (USAID). We also acknowledge National Center for Research
Resources, NIH, and Grant Number 8 P20 GM103430-12 for sponsoring
the core facility.
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