Conjugates of phosphorylated zalcitabine and lamivudine with SiO2nanoparticles: Synthesis by CuAAC click chemistry and preliminary assessment of anti-HIV and antiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2017.01.038

Conjugates of phosphorylated dideoxynucleoside antiviral drugs dideoxycytidine (zalcitabine) and lamivudine with SiO₂nanoparticles were obtained via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry between a nucleoside

Full text of DOI:10.1016/j.bmc.2017.01.038

Accepted Manuscript
Conjugates of phosphorylated zalcitabine and lamivudine with SiO2 nanoparti-
cles: synthesis by CuAAC click chemistry and preliminary assessment of anti-
HIV and antiproliferative activity
Svetlana V. Vasilyeva, Alexander A. Shtil, Albina S. Petrova, Sergei M.
Balakhnin, Polina Y. Achigecheva, Dmitry A. Stetsenko, Vladimir N. Silnikov
PII:
S0968-0896(16)31142-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.038
BMC 13517
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 November 2016
18 January 2017
21 January 2017
Please cite this article as: Vasilyeva, S.V., Shtil, A.A., Petrova, A.S., Balakhnin, S.M., Achigecheva, P.Y., Stetsenko,
D.A., Silnikov, V.N., Conjugates of phosphorylated zalcitabine and lamivudine with SiO2 nanoparticles: synthesis
by CuAAC click chemistry and preliminary assessment of anti-HIV and antiproliferative activity, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conjugates of phosphorylated zalcitabine and lamivudine with SiO2
nanoparticles: synthesis by CuAAC click chemistry and preliminary
assessment of anti-HIV and antiproliferative activity
Svetlana V. Vasilyeva 1a, Alexander A. Shtilb, Albina S. Petrovab, Sergei M. Balakhninc, Polina
Y. Achigechevad, Dmitry A. Stetsenkoa, Vladimir N. Silnikova
aInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russia.
bN. N. Blokhin Cancer Center, Moscow, Russia.
cInstitute of Molecular Pathology and Pathomorphology, Novosibirsk, Russia.
dState Research Centre of Virology and Biotechnology "Vector", Koltsovo, Russia
Abstract
Conjugates of phosphorylated dideoxynucleoside antiviral drugs dideoxycytidine (zalcitabine)
and lamivudine with SiO2 nanoparticles were obtained via the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) click chemistry between a nucleoside triphosphate containing an alkynyl
group at the γ-phosphate or azidothymidine triphosphate and SiO2 nanoparticles containing alkyl
azide or alkynyl groups, respectively. 4-(Prop-2-yn-1-yloxy)butylamino group has been attached
to the γ-phosphate group of dideoxycytidine (zalcitabine) and lamivudine 5’-triphosphates via
the phosphoramidate linkage. New compounds were shown to be potent killers of human colon
carcinoma cells. Anti-HIV activity of the conjugates was demonstrated as well. The conjugates
of phosphorylated lamivudine and dideoxycytidine (zalcitabine) showed higher potency than the
parent nucleosides. The conjugate of phosphorylated azidothymidine was less active against
1Corresponding author: Tel. +7(383)3635183; Fax +7(383)3635182; e-mail svetlana2001@gmail.com
1

HIV-1 than the parent nucleoside probably because of the replacement of its 3’-azido group by
1,2,3-triazole ring. These results show an opportunity for using SiO2 nanoparticles as a transport
for delivering phosphorylated nucleosides to cells in order to increase their efficiency as antiviral
and anticancer drugs.
Key words: SiO2 nanoparticles, Copper(I)-catalyzed azide-alkyne cycloaddition, Phosphorylated
dideoxynucleoside analogues, HIV-1, Colon cancer cells, Cytotoxicity.
1. Introduction
Nucleoside analogues are commonly used in HIV/AIDS therapy and as a first line therapeutics
for many tumor types. After entering through the membrane, nucleoside analogues are
phosphorylated by intracellular nucleoside kinases into active 5′-mono-, di-, and triphosphates.1
The drug resistance to nucleoside analogues refers to nucleoside transporter deficiency,2,3
reduced nucleoside kinase activity,4 over-expression of multi-drug resistance proteins (Pgp,
MDR, BCRP)5 or modifications in apoptotic pathways.6 A conceivable strategy to avoid
resistance is to use a pre-phosphorylated nucleoside directly as a drug that can thus bypass
intracellular phosphorylation. However, nucleoside triphosphates are very poorly internalized by
cells. Attempts to get around this problem by masking the phosphate groups by turning them into
phosphate esters have not been successful until recently.7 The fully substituted di- and
triphosphate esters become unstable. Difficulties in the development of lipophilic nucleoside
triphosphate (NTP) prodrugs arise due to hydrolytical instability of the pyrophosphate bond
whilst in the natural NTPs this bond is kinetically stable due to negative charges that slow down
cleavage by nucleophiles. In a recent work by Chris Meyer et al.8 membrane-permeable NTP
prodrugs have been obtained by masking the γ-phosphate group by two bio reversible benzyl
groups. However, this approach while successful requires a complicated multi-step synthesis,
and the obtained prodrugs are not soluble in water.
2

An alternative to the lipophilic prodrug approach could be in using drug delivery systems such as
liposomes, nanogels, nanoparticles etc., which could potentially solve the problem of poor cell
absorption of NTPs.1,9 Previously, we have proposed a system for delivery of 2’-
deoxyribonucleoside triphosphate (dNTP) analogues that employed SiO2 nanoparticles.10
A
simple and versatile method has been developed for the preparation of SiO2-dNTP conjugates
using a click reaction between SiO2 nanoparticles containing alkyl azide groups and dNTPs
bearing alkynyl-modified γ-phosphate group. We have shown that 2’-deoxycytidine triphosphate
in the form of SiO2-dNTP conjugate can be incorporated into the growing DNA chain.10 The rate
of polymerization differed insignificantly when either SiO2-dNTP or dNTP was processed by
HIV reverse transcriptase. Confocal microscopy has demonstrated that fluorescent SiO2-dNTP
conjugate accumulate in the cytoplasm and some of it is present in cell nuclei.10
These results provided the incentive for investigation of antiviral activity of conjugates of
dideoxynucleoside triphosphates and SiO2 nanoparticles. In addition, there are several examples
when nucleoside analogues were originally approved as antiviral agents and then it has been
shown that they display promising anticancer properties.11–13 Thus, conjugates of antiviral
phosphorylated nucleoside analogues may also possess potential antitumor activity. The aim of
this work was to prepare triphosphates of anti-HIV nucleoside drugs lamivudine and zalcitabine
with γ-alkynyl phosphoramidate group, obtain their conjugates with SiO2 nanoparicles (SiO2-
dNTP) via Cu(I)-catalyzed azide-alkyne cycloaddition and assess their antiviral and cytotoxic
properties.
2. Results and discussion
2.1. Synthesis of γ-alkynyl triphosphates of anti-HIV cytidine analogues
Synthesis of γ-alkynyl triphosphates of lamivudine (3TC) and dideoxycytidine (ddC, zalcitabine)
was carried out according to Scheme 1.
3

Scheme 1. Synthesis of γ-alkynyl triphosphates of antiviral cytidine nucleoside analogues.
A bifunctional linker 4-(prop-2-yn-1-yloxy)butylamine (L4) containing both a primary amino
group and a 1-alkynyl group was selected for the copper(I)-catalyzed azide–alkyne cycloaddition
reaction (CuAAC).14 Synthesis started from commercially available lamivudine 1 and zalcitabine
2. The nucleosides were protected by selective N-acylation of cytosine affording compounds 3
and 4.15 5’-Triphosphates 5 and 6 were obtained by the same way as in our earlier work.16 The
alkynyl linker L4 was introduced into the γ-phosphate of ddNTP then.10 Isolated yields of
alkynyl triphosphates 7 and 8 were in the range of 60-70%. Azidothymidine 5’-triphosphate 9
was prepared from azidothymidine (AZT), (Scheme 2). 17
2.2. Synthesis of conjugates of nucleoside triphosphate analogues with SiO2 nanoparticles
by CuAAC click chemistry
The procedures are outlined in Scheme 2. Nanoparticles containing alkyl azide groups 10 were
prepared by treatment of SiO2-NH2 nanoparticles with N-hydroxysuccinimide ester of 6-
azidohexanoic acid.10 Nanoparticles containing alkynyl groups 11 were prepared by treatment of
SiO2-NH2 nanoparticles with N-succinimidyl 5-(prop-2-yn-1-yloxy)-5-oxopentanoate.17 As
demonstrated earlier,10 a 3-5 fold excess of alkyne or azide triphosphate is sufficient for the
completion of CuAAC reaction with modified nanoparticles (Scheme 2).
4

Scheme 2. Synthesis of conjugates of nucleoside triphosphate analogues with SiO2 nanoparticles
via the Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC).
After the CuAAC reaction, SiO2-ddNTP conjugates were washed with 0.1 M NaCl, 10%
Na2EDTA to remove Cu ions, then with water and diethyl ether followed by air-drying. The drug
(pppLam, pppddC) content was calculated from UV absorbance of SiO2-L6-pppLam 12 and
SiO2-L6-pppddC 13 dissolved in 0.2 M NaOH using nucleoside extinction coefficient λmax/nm
(ε): 270 (7701) and λmax/nm (ε): 260 (7400). It was 0.142 µmol/mg for SiO2 conjugate of
lamivudine and 0.113 µmol/mg for SiO2 conjugate of zalcitabine, respectively. The drug content
for pppAZT was calculated from UV absorbance of SiO2-L2-pppAZT conjugate 14 dissolved in
0.2 M NaOH using AZT extinction coefficient (ε) 6650 at λmax 267 nm to give 0.117 µmol/mg.
To avoid any nonspecific adsorption of triphosphates onto SiO2-NH2 nanoparticles, experiment
was repeated under the same conditions but without Cu(I). After standard washes, UV
absorbance of obtained samples showed no nucleoside content.
5

2.3. Particle Size
Previously it was shown by small-angle X-ray scattering (SAXS) that the size of the initial SiO2–
NH2 nanoparticles (22–24 nm) differed insignificantly from that of the final conjugate SiO2–
dNTP (SiO2~pppdC and SiO2~pppdUTPFlu) (25–27 nm), that is by no more than 2–3 nm.10
Zeta-potentials (ζ) of free SiO2-NH2 and drug-loaded SiO2 nanoparticles 12-14 were measured
by dynamic light scattering using Malvern Zetasizer ZS after sonication in deionized water for 2
minutes (Table 1). The negative charge of 12-14 confirms the presence of phosphate groups on
the surface of SiO2-dNTP nanoparticles, although a significant proportion (~15%) of NH2 groups
remains hidden inside the polymer network. In addition, the mean size values and concentrations
of nanoparticles were measured using NanoSight NS300 (NTA) (Table 1).
As could be seen, the difference in particle size obtained by SAXS and NTA is significant. It
could be attributed to a relatively pronounced ability of SiO2 nanoparticles to aggregate,
especially high for SiO2-NH2 nanoparticles. The calculated and measured concentrations of the
conjugates are in reasonably good agreement.
Table 1. Average size, zeta-potential and concentration of free (SiO2-NH2) and drug-loaded SiO2
nanoparticles 12-14.
No.
Sample
Drug content,
µmol/mg of
SiO2 conjugate
–
Mean
sizea, nm
SDb, nm
ζ-potential
Concentrationc,
calc./found, particles
per mL
(DLS), mV
SiO2-NH2
150.8
25.4
102.7
46.2
24.5
1.4×108/ 1.54×108
6.87×107
–
146.3
31.6
–
–
45.1
8.3
–
–9.6
–6.2
12
13
SiO2-pppLam
SiO2-pppddC
0.142
0.113
–
1.2×108/ 5.90×107
6.06×106
–
14
SiO2-pppAZT
0.117
–34.0
aThe mean size values measured by NTA;
b SD – standard deviation calculated by the NTA software;
6

cConcentration in particles per mL as measured by NTA. Numbers represent average values
standard deviation (nꢀ=ꢀ3).
2.4. Anti-HIV-1 properties of conjugates SiO2-L6-pppLam (12), SiO2-L6-pppddC (13) and
SiO2-L2-pppAZT (14)
Antiviral activity has been determined in relation to HIV-1 subtype A. The parent nucleosides,
their triphosphates, γ-alkynyl triphosphates and conjugates 12-14 were tested. Previously, we
have found that SiO2-NH2 nanoparticles were inactive against HIV-1 and nontoxic for MT4
cells. Concentration of SiO2-NH2 nanoparticles that resulted in 50% cell death (TC50) was >500
µg/mL. The results are presented in Table 2. Conjugates SiO2-L6-pppLam 12 and SiO2-L6-
pppddC 13 were the most active against HIV-1 as their potency was higher than that of the
parent nucleosides taking into account the nucleoside content of the conjugates (concentration of
nucleotide expressed as µМ). The ratio of IC50 of parent nucleoside to IC50 of the conjugate was
10.0 for 12 and 4.5 for 13. Conjugate 13 has shown significantly lower toxicity than the parent
nucleoside at a maximum concentration investigated 400 µg/mL. Interestingly, L4-pppddC 8
was also practically nontoxic and showed better activity than the parent nucleoside: the ratio of
IC50 of the nucleoside to IC50 of L4-pppddC was 5.0. These result suggests that the attachment of
4-(prop-2-yn-1-yloxy)butylamine, linker (L4) to the γ-phosphate of a triphosphorylated
nucleoside analogue may improve anti-HIV-1 activity, possibly due to better cell uptake.
Table 2. Anti-HIV-1 activity of SiO2-ddNTP conjugates 12-14.
No.
Content
IC50, µg/mL
IC50, µМa
IC50 p.n./
TC50,
µg/mLc
>400
ISd
b
IC50
1
5
Lamivudine
pppLam
0.4 0.03
1.8 0.1
3.2 0.2
1.2 0.07
0.9 0.05
1.7 0.1
3.6 0.2
5.3 0.2
0.17 0.01
4.3 0.2
–
–
>1000
>222
>125
>333
166
>400
>400
7
L4-pppLam
SiO2-pppLam
ddC
–
12
2
10.0
–
>400
150 10
7

6
8
ddCTP
1.9 0.1
0.48 0.04
8.5 0.4
4.0 0.2
0.86 0.07
0.96 0.05
–
388 10
>400
204
>833
>47
L4-pppddC
SiO2-pppddC
5.0
4.5
13
>400
AZT
0.004 0.001
0.032 0.002
0.015 0.001
0.059 0.004
–
–
–
–
9.5 0.7
69 5
>400
2375
2162
>5
9
pppAZT
14
SiO2-pppAZT
69
3
8
0.4
SiO2-NH2
–
–
>500
–
a Concentration of the active substance (ddNTP) in the conjugate, according to drug content (see
above);
b IC50 of parent nucleoside, µМ / IC50 of conjugate, µМ;
c Maximum concentration tested was 400 µg/mL;
dIS = TC50, µg/mL / IC50, µg/mL. The data reported represent an average of three measurements.
Conjugate SiO2-L2-pppAZT 14 was less active against HIV-1 than AZT, probably because of the
replacement of 3’-azido group of the nucleoside by 1,2,3-triazole ring in the conjugate.
Moreover, at concentrations >100 µg/mL aggregation of conjugates was observed, which
manifested in the formation of a film on the bottom of culture flasks and interference with
colorimetric detection of cell viability. Other authors have also noted strong tendency for
aggregation for amino-modified silica nanoparticles, which can be reduced by adding other
functional groups such as methyl phosphonate to their surface.18 However, in our case after the
modification of SiO2 nanoparticles with triphosphate groups the aggregation of conjugates at
>100 µg/mL stayed high.
2.5. Cytotoxicity test
Antiproliferative activity of conjugates 12-14 was evaluated after a continuous 72 h treatment of
HCT116 human colon carcinoma cell line followed by MTT test. As expected, the parent
lamivudine and zalcitabine (ddC) exhibited no detectable cytotoxicity whereas their SiO2-ddNTP
conjugates 12 and 13 were highly potent with IC50 2.67
0.02 µM and 1.9
0.1 µM,
respectively (Table 3). Again, as in anti-HIV assay, the sample of L4-ppp-ddC 8 was also potent
with IC50 3.3
0.02 µM. In contrast, the compounds 13 and 8 were nontoxic against
8

nonmalignant hFB-hTERT6 skin fibroblasts. Conjugate 14 was once again less active than the
parent AZT.
Table 3. Cytotoxicity test for tumor and nonmalignant cell lines.
No.
Sample
IC50, µg/mL
IC50, µМa
НСТ116
>8.7
hFB-hTERT6
>8.7
1
5
Lamivudine
pppLam
>2.0
>2.0
>2.0
18.8
>2.0
>2.0
1.94
16.9
>4.05
>4.05
7
L4-pppLam
SiO2-pppLam
ddC
>3.3
2.67 0.02
>9.5
>3.3
>2.67
>9.5
12
2
6
ddCTP
>4.2
>4.2
8
L4-pppddC
SiO2-pppddC
3.40
>3.45
>2.26
13
1.9 0.1
AZT
0.91
1.97
>20
3.4
3.6
>7.5
>3.7
9
pppAZT
14
SiO2-pppAZT
>2.34
>2.34
a Maximum concentration for control compounds was 2 µg/ml, for conjugates 12-14 – 20 µg/ml.
Values are mean SD of three independent measurements with <10% error.
It should be noted, however, that these experiments could not unequivocally confirm that the
active component is a nucleoside triphosphate. Stability of the conjugates and compounds 7 and
8 in extracellular medium should be studied, their potential metabolites and degradation products
such as NTPs, NDPs, NMPs and nucleosides should be quantified, e.g. by HPLC or LC-MS, and
additional biological assays should be carried out to clarify the issue of stability. Taking into
account known lability of the phosphoramidate bond, it is possible that γ-phosphoramidate-
linked conjugates of NTP analogues may function as prodrugs releasing the active nucleoside or
its 5’-mono-, di- or triphosphate inside cell. There are examples of nucleoside prodrugs, which
serve essentially as a depot for nucleoside drugs such as AZT to ensure their prolonged release.19
9

Thus, our preliminary experiments have been designed firstly to ascertain the potential of SiO2
nanoparticles as cell delivery vehicles for triphosphates of nucleoside drugs in order to increase
their antiviral or/and anticancer activity.
3. Conclusions
Synthesis of γ-alkynyl triphosphates of antiviral dideoxynucleosides lamivudine and zalcitabine
(dideoxycytidine) in good yields was carried out using 4-(prop-2-yn-1-yloxy)butylamine linker.
The compounds were efficiently conjugated with SiO2 nanoparticles carrying alkyl azide groups
using CuAAC click chemistry to obtain conjugates designed to transport triphosphorylated
nucleoside analogues into cells. The conjugates potently killed human colon carcinoma cells.
Anti-HIV-1 activity of the conjugates was also detected. SiO2-ddNTP conjugates of lamivudine
and zalcitabine were the most active with the potency higher than that of the parent nucleosides.
Triphosphorylated AZT conjugate was less active against HIV-1 than AZT, probably because of
the replacement of the azido group of AZT with the triazole ring in the conjugate. These results
highlight a potential of SiO2 nanoparticles as cell delivery vehicles for nucleoside triphosphate
drugs to increase their antiviral and anticancer activity.
4. Experimental Section
4.1. General
Reagents and solvents were purchased from Sigma-Aldrich, Merck and Acros Organics and used
without further purification. Anhydrous solvents were either purchased or obtained by
distillation and stored over 3Å molecular sieves. Nucleoside analogue lamivudine was from
Fluka (Switzerland), and 2',3'-dideoxycytidine was obtained from NanoTech-S LLC
(Novosibirsk, Russia); SiO2–NH2 nanoparticles were from SkySpring Nanomaterials ( USA).
1
Mass spectra were obtained on an Agilent 6410 Triple Quadrupole LC-MS device (USA). H
and 31P NMR spectra were recorded using Bruker AV-400, AV-300, AV-600 or DRX-500 NMR
10

spectrometers. Chemical shifts (δ) are reported in ppm relative to residual solvent signals. In the
case of 31P NMR spectra 85% aq H3PO4 was used as an external standard. Coupling constants (J)
were expressed in Hertz (Hz) and spin multiples are given as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), app.t (apparent triplet) and br.s (broad singlet). UV absorption spectra
were recorded on a Shimadzu UV-1800 spectrophotometer (Japan). Preparative anion exchange
chromatography was performed on DEAE Sephadex A-25 40–120 µm (Pharmacia Fine
Chemicals, Sweden). Analytical reverse-phased (RP) HPLC was carried out on a Milichrom A-
02 chromatograph (Econova, Russia) using a 2×75 mm column packed with ProntoSIL 120-
5C18 AQ (Bischoff Leonberg, Germany). Preparative reverse-phased chromatography was
performed on Polygoprep C18, 50–100 µm (Macherey-Nagel, Germany). Thin layer
chromatography was carried out using Alufolien Kieselgel 60 F254 plates (Merck, Germany) in
appropriate solvent mixtures and was visualized by UV irradiation or spraying with ethanolic
ninhydrin for amino groups or cysteine in aq H2SO4 for nucleosides. For anti-HIV activity assay,
the following media were used: RPMI-1640 with 20 mM HEPES and RPMI-1640 with
GlutaMax (Life Technologies). Complete medium was RPMI-1640 with 20 mM HEPES buffer
(Dutch modification) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2
mM L-glutamine, 0.1% sodium bicarbonate and 20 µg/mL gentamycin. 4,5-Dimethylthiazol-2-
yl-2,5-diphenyltetrazolium bromide (MTT) from Sigma-Aldrich (USA) was dissolved in PBS,
pH 7.4 to concentration 7.5 mg/mL, filtered through 0.22 µm filter and stored at –20°C in
aliquots. A solution of 30 ml Triton X-100 (Sigma-Aldrich) with 2 ml conc. HCl in 500 ml
isopropanol was used.
The surface potential (ζ) of nanoparticles was measured on a Malvern Zetasizer Nano ZS model
ZEN3600 (Worcestershire, UK) equipped with a standard 633 nm laser. The mean size and
concentration of nanoparticles were measured using NanoSight NS300 (NTA) device kindly
provided by company "KD Systems and Equipment" (the exclusive representative of Malvern
Instruments Ltd in Russia). Nanoparticle dispersion (0.7 mg/ml) was diluted with milli-Q water
11

to achieve the desired concentration. Sample temperature (28-29º C) was fully programmable
through the Nanoparticle Tracking Analysis (NTA) software.
4.2. Synthesis of nucleoside derivatives N4-Benzoyl-1-(2’,3’-dideoxy-3’-thia-β-L-
ribofuranosyl)-cytosine (3) and N4-benzoyl-2',3'-dideoxycytidine (4)
For selective N-acylation of cytosine nucleosides (1 or 2, Scheme 1) the procedure described in15
was used. To a suspension of lamivudine (1-(2’,3’-dideoxy-3’-thia-β-L-ribofuranosyl)cytosine) 1
or zalcitabine (2',3'-dideoxycytidine) 2 (0.322 mmol, 1 equiv.) in DMF (2 mL) benzoic
anhydride (81 mg, 0.354 mmol, 1.1 equiv.) was added, and the mixture was stirred at ambient
temperature for 24 h. After removal of DMF under reduced pressure, the residue was triturated
with diethyl ether. The crystalline products were collected by filtration, washed thoroughly with
1
diethyl ether and air-dried. Yield of compound 3 was 98%. H NMR (DMSO-d6, 400 MHz, δ,
ppm): 8.48 (d, 1H, H6 (Cyt), J = 7.3 Hz); 8.01 (d, 2H, Bz, J = 7.17 Hz); 7.62 (t, 1H, Bz, J = 7.5
Hz); 7.51 (t, 2H, Bz, J = 7.5, 7.8 Hz); 7.36 (d, 1H, H5 (Cyt), J = 6.8 Hz); 6.26 (dd, 1H, H1′, J =
3.0, 5.4 Hz); 5.28 (t, 1H, H4′, J = 4.1 Hz); 3.86 (m, 2H, H5′); 3.60 (m, H, H2′); 3.26 (m, H, H2′).
Yield of compound 4 was 98%. 1H NMR (DMSO-d6, 400 MHz, δ, ppm): 8.55 (d, 1H, H6 (Cyt),
J = 7.4 Hz); 7.99 (d, 2H, Bz, J = 7.4 Hz); 7.82 (t, 1H, Bz, J = 7.4 Hz); 7.50 (t, 2H, Bz, J = 7.4,
7.9 Hz); 7.33 (d, 1H, H5 (Cyt), J = 5.6 Hz); 5.95 (dd, 1H, H1′, J = 2.1, 6.9 Hz); 4.12 (m, 1H,
H4′); 3.79-3.57 (m, 2H, H5′); 2.44-2.34 (m, H, H2′); 2.02-1.96 (m, H, H2′); 1.88-1.75 (m, 2H,
H3′).
4.3. Synthesis of nucleoside triphosphates
Compound 3 or 4 (0.1 mmol) was dissolved in anhydrous pyridine (1 mL), and the solution was
chilled on ice bath. POCl3 (0.018 mL, 0.2 mmol) was added to the solution, the mixture was
stirred for 20 min and transferred to the mixture of 0.45 M solution of bis(tetra-n-
butylammonium) pyrophosphate in acetonitrile (1 mL) and tributylamine (1 mmol, 0.23 mL)
12

under vigorous stirring. The reaction mixture was stirred for 1 h at ambient temperature, 1 M
TEAB buffer, pH 7.5 (30 mL) was added, and after 2 h the reaction mixture was evaporated to
dryness.
4.3.1. N4-Benzoyl-1-(2’,3’-dideoxy-3’-thia-β-L-ribofuranosyl)-cytosine-5’-triphosphate (5)
Crude product from the above stage was dissolved in 20% aq EtOH and purified by anion
exchange chromatography on DEAE Sephadex A-25. Elution was performed with a linear
gradient from 20% EtOH to 1 M NH4HCO3 in 20% EtOH. Appropriate fractions were collected
and evaporated. The residue was co-evaporated several times with 96% EtOH to remove traces
of the buffer. Yield of compound 5 was 37%. NMR data are in agreement with published.18
31P (D2O, 121.5 MHz, δ, ppm): -8.33 (d, 1P, Pγ, J = 19.20 Hz); -10.92 (d, 1P, Pα, J = 19.20 Hz); -
21.80 (t, 1P, Pβ, J = 19.20 Hz). 1H NMR (D2O, 400 MHz, δ, ppm): 8.48 (d, 1H, H6-(Cyt), J = 7.4
Hz); 7.89 (d, 2H, Bz, J = 7.3 Hz); 7.65 (t, 1H, Bz, J = 7.3 Hz, J = 7.7 Hz); 7.53 (t, 2H, Bz, J =
7.7 Hz); 7.39 (d, 1H, H5-(Cyt), J = 7.4 Hz); 6.29 (dd, 1H, H1′, J = 3.2 Hz, J = 5.3 Hz); 5.46 (m,
1H, H4′); 4.46-4.25 (m, 2H, H5′); 3.63 (m, H, H2’); 3.29(m, H, H2’).
4.3.2. N4-Benzoyl-2',3'-dideoxycytidine 5'-triphosphate (6)
The crude product was dissolved in 20% aq EtOH and purified by anion exchange
chromatography on DEAE Sephadex A-25 column eluted with a linear gradient from 20% EtOH
to 1 M NH4HCO3 in 20% EtOH. Appropriate fractions were collected and evaporated. The
residue was co-evaporated several times with 96% EtOH to remove traces of the buffer. Yield of
compound 6 was 35%. NMR data were consistent with published.20 31P (D2O, 121.5 MHz, δ,
ppm): –9.99 (d, 1P, Pγ, J = 19.26 Hz); –10.67 (d, 1P, Pα, J = 19.26 Hz); –21.90 (t, 1P, Pβ, J =
1
19.26 Hz). H NMR (D2O, 400 MHz, δ, ppm): 8.51 (d, 1H, H6-(Cyt), J = 7.7 Hz); 7.89 (d, 2H,
Bz, J = 7.3 Hz); 7.65 (t, 1H, Bz, J = 7.3 Hz, J = 7.7 Hz); 7.53 (t, 2H, Bz, J = 7.7 Hz); 7.46 (d,
13

1H, H5-(Cyt), J = 7.3 Hz); 6.08 (dd, 1H, H1′, J = 2.3 Hz, J = 6.7 Hz); 4.44 (m, 1H, H4′); 4.34
(m, 1H, H5′); 4.17 (m, 1H, H5′); 2.52 (m, H, H2’); 2.21 (m, H, H2’); 2.08-1.88 (m, 2H, H3’).
4.4. Attachment of the alkynylamine linker onto the γ-phosphate of ddNTP
2,2′-Dithiodipyridine (0.5 mmol) and triphenylphosphine (0.5 mmol) in DMF (0.3 mL) were
added to a solution of 2′,3′-dideoxynucleoside-5′-triphosphate (5 or 6) as a tributylammonium
salt (0.1 mmol) in DMSO (1 mL). After stirring for 30 min 4-(prop-2-yn-1-yloxy)butylamine (1
mmol) was added, the mixture was incubated at ambient temperature for 2 h and then
precipitated with 6% LiClO4 in acetone. The protected γ-modified 5′-triphosphate was then
subjected to standard N-deprotection by conc. aq. ammonia treatment. The product was purified
by anion exchange chromatography on DEAE Sephadex A-25. The elution was performed with a
linear gradient from 20% EtOH to 1 M NH4HCO3 in 20% EtOH. Fractions were collected and
evaporated. The residue was co-evaporated several times with 96% EtOH to remove traces of the
buffer and the product was precipitated with 6% LiClO4 in acetone.
4.4.1. 1-(2’,3’-Dideoxy-3’-thia-β-L-ribofuranosyl)-cytosine-5′-{N-[4-(prop-2-yn-1-
yloxy)butyl]-γ-amino}-triphosphate (7)
Yield: 62%. 31P (121 MHz, D2O, δ, ppm): –0.35 (d, J = 20.7 Hz, γ-P), –11.06 (d, J = 18.8 Hz, α-
1
P), –22.19 (t, J = 20.1 Hz, β-P). H (500, D2O, δ, ppm): 7.94 (d, 1H, H(6)-(Cyt), J = 7.5 Hz);
6.16-6.06 (app.t, 1H, H1′, J = 4.5 Hz); 5.95 (d, 1H, H(5)-(Cyt), J = 7.5 Hz), 5.31 (m, 1H, H4′);
4.25-4.10 (m, 2H, H5′); 4.03-4.00 (m, 2H, OCH2C≡CH); 3.48-3.37 (m, 3H, CH≡C,
OCH2(CH2)3); 3.08 (dd, 1H, H2′, J = 4.1, 12.2 Hz); 2.74-2.68 (m, 3H, O(CH2)3CH2NH, H2′);
13
1.47-1.31, 2.21 (m, 4H, OCH2(CH2)2CH2NH). C (125 MHz, D2O, δ, ppm): 165.4 (4-C), 156.2
(2-C), 141.9 (6-C), 95.69 (1’-C), 87.3 (5-C), 84.2 (4’-C), 84.1 (13-C), 75.4 (12-C), 70.0 (5’-C),
66.2 (11-C), 57.3 (10-C), 41.1 (7-C), 36.7 (2’-C), 27.2 (8-C), 25.7 (9-C). UV spectrum (0.2 M
14

NaOH): λmax 270 nm, ε 7,701. IR (cm-1): 3340, 3276, 2937, 2877, 1647, 1580, 1488, 1420,
1385, 1223, 1100, 902, 840, 767. ESI-MS (Agilent 6410 QQQ LC Agilent 1200), [M-H]–: calcd
for C15H24N4O12P3S 577.02, found 576.99; calcd for C15H23N4O12P3SLi 583.02, found 582.99;
C15H22N4O12P3SLi 589.02, found 589.09.
4.4.2. 2',3'-Dideoxycytidine-5′-{N-[4-(prop-2-yn-1-yloxy)butyl]-γ-amino}-triphosphate (8)
Yield: 70%. 31P (121.5, D2O, δ, ppm): –0.23 (d, 1P, Pγ,
J
=
20.8 Hz);
1
–10.59 (d, 1P, Pα, J = 18.9 Hz); –22.26 (t, 1P, Pβ, J = 19.8 Hz). H (500, D2O, δ, ppm): 7.9 (d,
1H, H(6)-(Cyt), J = 7.5 Hz); 5.98-5.94 (m, 2H, H(5)-(Cyt), H1′); 4.25 (m, 1H, H4′); 4.18-4.15
(m, 1H, H5′); 4.06-3.96 (m, 3H, H5′, OCH2C≡CH); 3.44 (t, 2H, OCH2(CH2)3NH, J = 6.5 Hz);
2.77-2.70 (m, 3H, O(CH2)3CH2NH, CH≡C); 2.34-2.27 (m, 1H, H2′); 1.97-1.93 (m, 3H, H3′, 2′);
186-1.78 (m, 1H, H3′); 1.49-1.32 (m, 4H, OCH2(CH2)2CH2NH). 13C (D2O, 125 MHz, δ, ppm):
165.52 (s, C4); 156.87 (s, C2); 141.46 (s, C6); 95.36 (s, C1’), 86.54 (s, C5); 80.31 (d, JC4’-P =
9.02 Hz, C4’); 79.03 (s, C≡CH); 75.12 (s, C≡CH); 69.69 (s, OCH2(CH2)3); 65.97 (d, J C5’-P =
5.6 Hz, C5’), 57.00 (s, OCH2C≡CH); 40.86 (s, CH2NH); 31.78 (s, C2’); 26.99 (d, J C3’-P = 9.06
Hz, C3’); 25.49 (s, CH2CH2NH); 23.88 (s, OCH2CH2). UV spectrum (0.2 M NaOH): λmax 260
nm, ε 7,400. IR (cm-1): 3360, 3276, 2973, 2877, 1692, 1647, 1524, 1488, 1385, 1223, 1100,
1057, 995, 840, 809, 767. ESI-MS (Agilent 6410 QQQ LC Agilent 1200), [M-H]– calcd for
C16H27N4O12P3 559.13, found 558.99; calcd for C16H26N4O12P3Li 565.13, found 564.99; calcd
for C16H26N4O12P3Li2 571.13, found 570.99.
4.5. Synthesis of SiO2-L-pppddN conjugates by CuAAC reaction (Scheme 2)
SiO2 nanoparticles modified with 6-azidohexanoyl groups 10 (40 mg, 17.2 µmol) were sonicated
in 200 µL H2O. A solution of γ-alkynyl triphosphates 7 or 8 (100 mM in H2O, 520 µL, 52 µmoL,
3 equiv.) was added to the suspension followed by copper(II) sulfate (172 µL of 0.5 M stock
solution in water), 1 M TEAAc buffer, pH 7 (172 µL) and sodium ascorbate (86 µL of freshly
15

prepared 1 M solution in water). The reaction mixture was purged with argon for 2 min and
stirred at ambient temperature for 12 h. Conjugates were separated by centrifugation, supernatant
was discarded. Precipitated conjugates were washed successively with 0.1 M NaCl, 10%
Na2EDTA, water, diethyl ether followed by air-drying. The nucleoside content in both products
was measured by UV absorbance of the appropriate nucleoside after dissolving in 0.2M NaOH.
4.5.1. SiO2-L6-ppp-Lam (12)
Yield: 38.5 mg. Nucleoside content: 0.142 µmol/mg of SiO2. UV spectrum (0.2 M NaOH): λmax
270 nm, ε 7,701. IR spectrum: no band of N3 at 2090 cm–1.
4.5.2. SiO2-L6-ppp-ddC (13)
Yield: 36 mg. Nucleoside content: 0.113 µmol/mg of SiO2. UV spectrum (0.2 M NaOH): λmax
260 nm, ε 7,400. IR spectrum: no band of N3 at 2090 cm–1.
4.5.3. SiO2-L2-ppp-AZT (14)
has been obtained as described earlier.17 Nucleoside content: 0.117 µmol/mg SiO2. UV spectrum
(0.2 M NaOH): λmax 267 nm, ε 6,650.
4.6. Anti-HIV-1 activity of the conjugates SiO2-L6-pppLam (12), SiO2-L6-pppddC (13) and
SiO2-L2-pppAZT (14)
4.6.1. Cells and virus
MT4 human T lymphoblastoid cell line transformed by human T lymphotropic virus type 1 was
donated by Medical Research Council AIDS Reagent Project (UK). Cell culture was grown and
maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and sub-
cultivated every 3-4 days. HIV-1 clinical isolate (CXCR4-positive subtype A) obtained from co-
culturing peripheral blood mononuclear cells from a healthy donor with the patient’s peripheral
16

blood mononuclear cells. High-titer HIV-1 subtype A stock was obtained from adapted MT-4
cells. The 50% cell culture infectious dose (CCID50) of the virus stock was determined by
endpoint titration on MT-4 cells.
4.6.2. Preparation of the solutions of test compounds
Stock solutions of the test compounds were prepared in PBS buffer, pH 7.4 at a concentration 10
mg/mL. Threefold serial dilutions of test compounds in complete medium (at 4 times final
concentrations) were performed using a 96-well V-bottom plate.
4.6.3. Cell viability and anti-HIV activity assay
The method was based on HIV-induced cytopathogenic effect (CPE) and measures the degree of
cell death after HIV infection.21 Inhibition of viral replication by anti-HIV compounds will
consequently enhance cell survival, which can be assessed spectrophotometrically via the in situ
reduction of MTT to the blue formazan product.22 Suspension of MT-4 was prepared in complete
medium, and then transferred to flat-bottomed 96-well plates at a density of 4×104 cells/well in
100 µL. Dilutions of test compounds, prepared separately, in the volume of 50 µL were added to
the wells with MT-4 cells. In the case of cytotoxicity assays, in the wells with test compounds 50
µL of complete medium was added to final volume 200 µL. In the case of anti-HIV activity
assays 50 µL of virus solution was added in constant final dosing 300 CCID50.
4.6.4. Evaluation of anti-HIV-1 activity and cytotoxicity of the compounds
After 5 days incubation, 25 µL of MTT solution warmed to 37°C was added to each well. Plates
were incubated at 37°C in a CO2 incubator for 2 h. Then, an aliquote of culture medium (180 µL)
was taken from each well without disturbing the MT-4 cell clusters containing formazan crystals.
Cell clusters, containing formazan crystals and infectious virus, were solubilized by adding 100
µL of the acidified Triton X-100 isopropanol solution to each well using the multichannel
17

pipette. Complete dissolution of formazan crystals was achieved by placing the plates on a
vibrating platform shaker for 10 min.
The absorbances were read in an eight-channel microplate reader and stacker at two wavelengths
570 and 655 nm. The absorbance at 655 nm was subtracted from the absorbance at 570 nm to
eliminate the effects of scattering by cell debris. The OD ratio, defined as ODC mock / ODC
HIV, should be at least 5. The results were processed using a SoftMaxPro software (Molecular
Devices).
4.7. Cytotoxicity for human cell lines
Compounds were reconstituted in deionized water as follows: compounds 12–14 up to 200
mg/mL, compounds 1, 2, 5, 6, 7, 8, 9 and AZT up to 20 mg/mL. From these stock solutions serial
dilutions were prepared immediately prior to experiments. Human colon carcinoma cell line
HCT116 (American Type Culture Collection, Manassas, VA) and hFB-hTERT6 skin fibroblasts
were propagated as reported by us previously.23 Cytotoxicity was assessed by the MTT test as
described previously. 23
Supporting Information
1H-NMR and 31P-NMR spectra and HPLC profiles for monomers are provided in the
Supporting Information.
File Name: Supporting_Information_File_1:
File Format: Word
Title: Supporting Information for “Conjugates of phosphorylated zalcitabine and lamivudine
with SiO2 nanoparticles: synthesis by CuAAC click chemistry and preliminary assessment of
anti-HIV and antiproliferative activity”.
Acknowledgements
The work was supported by RFBR, research project no. 16-04-00847-a.
18

References and notes
1. Senanayake, T. H.; Warren, G.; Wei, X.; Vinogradov, S.V. J. Controlled Release, 2013,
167, 200-209. DOI: 10.1016/j.jconrel.2013.01.020.
2. Gati, W. P.; Paterson, A. R.; Belch, A. R.; Chlumecky, V.; Larratt, L. M.; Mant, M. J.;
Turner, A. R. Leuk. Lymphoma, 1998, 32, 45–54. DOI: 10.3109/10428199809059245.
3. Spratlin, J.; Sangha, R.; Glubrecht, D.; Dabbagh, L.; Young, J. D.; Dumontet, C.; Cass,
C.; Lai, R.; Mackey, J. R. Clin. Cancer Res., 2004, 10, 6956–6961. DOI: 10.1158/1078-
0432.CCR-04-0224.
4. Kakihara, T.; Fukuda, T.; Tanaka, A.; Emura, I.; Kishi, K.; Asami, K.; Uchiyama, M.
Leuk. Lymphoma, 1998; 31, 405–409. DOI: 10.3109/10428199809059234.
5. Zhang, J.; Visser, F.; King, K. M.; Baldwin, S. A.; Young, J. D.; Cass, C. E. Cancer
Metastasis Rev., 2007, 26, 85–110. DOI: 10.1007/s10555-007-9044-4.
6. Galmarini, C. M.; Voorzanger, N.; Falette, N.; Jordheim, L.; Cros, E.; Puisieux, A.;
Dumontet, C. Biochem. Pharmacol., 2003, 65, 121–129. DOI: 10.1016/S0006-
2952(02)01448-X.
7. Schulz, T.; Balzarini, J.; Meier, C. ChemMedChem, 2014, 9, 762 – 775. DOI: 10.1002/c
mdc.201300500.
8. Gollnest, T.; Oliveira, T.; Rath, A,; Hauber, I.; Schols, D.; Balzarini, J.; Meier, C.
Angew. Chem. Int. Ed., 2016, 55, 5255 –5258. DOI:10.1002/anie.201511808.
9. Saiyed, Z. M.; Gandhi, N. H.; Nair, M. P. J. NeuroVirol., 2009, 15, 343-347. DOI:
10.1080/13550280903062813.
10. Vasilyeva, S. V.; Silnikov, V. N.; Shatskaya, N. V.; Levina, A. S.; Repkova, M. N.;
Zarytova,V.
F.
Bioorg.
Med.
Chem.,
2013,
21,
6101-6114.
http://dx.doi.org/10.1016/j.bmc.2012.11.057.
19

11. Suzuki, N.; Nakagawa, F.; Nukatsuka, M.; Fukushima, M. Exp. Ther. Med., 2011, 2,
393–397. DOI: 10.3892/etm.2011.244.
12. Tsai, C. Y.; Ray, A. S.; Tumas, D. B.; Keating, M. J.; Reiser, H.; Plunkett, W. Clin.
Cancer Res., 2009, 15, 3760–3769. DOI: 10.1158/1078-0432.
13. Temmink, O. H.; Bijnsdorp, I. V.; Prins, H. J.; Losekoot, N.; Adema, A. D.; Smid, K.;
Honeywell, R. J.; Ylstra, B.; Eijk, P. P.; Fukushima, M.; Peters, G. J. Mol. Cancer Ther.,
2010, 9, 1047–1057. DOI: 10.1158/1535-7163.MCT-09-0932.
14. Kupryushkin, M. S.; Konevetz, D. A.; Vasilyeva, S. V.; Kuznetsova, A. S., Stetsenko, D.
A; Pyshnyi, D. V. Nucleosides, Nucleotides Nucleic Acids, 2013, 32, 306-319. DOI:
10.1080/15257770.2013.787147.
15. Bhat, V.; Ugarkar, B. G.; Sayeed, V. A.; Grimm, K.; Kosora, N.; Domenico, P. A.;
Stocker, E. Nucleosides, Nucleotides Nucleic Acids, 1989, 8, 179-183. DOI:
10.1080/07328318908054166.
16. Abramova, T. V.; Vasileva, S. V.; Koroleva, L. S.; Kasatkina, N. S.; Silnikov, V. N.
Bioorg. Med. Chem., 2008, 16, 9127–9132. DOI: 10.1016/j.bmc.2008.09.029.
17. Vasilyeva, S. V.; Levina, A. S.; Li-Zhulanov, N. S.; Shatskaya, N. V.; Baiborodin, S. I.;
Repkova, M. N.; Zarytova, V. F.; Mazurkova, N. A.; Silnikov, V. N. Bioorg. Med.
Chem., 2015, 23, 2168-2175. http://dx.doi.org/10.1016/j.bmc.2015.02.063.
18. Bagwe, R. P.; Hilliard, L. R.; Tan, W. Langmuir, 2006, 22, 4357- 4362. DOI:
10.1021/la052797j.
19. Khandazhinskaya, A.L.; Yanvarev, D.V.; Jasko, M.V.; Shipitsin, A.V.; Khalizev, V.A.;
Shram, S.I.; Skoblov,Y.S.; Shirokova, E.A.; Kukhanova, M.K. Drug Metab. Dispos.,
2009, 3, 494-501. DOI: 10.1124/dmd.108.022269.; Khandazhinskaya, A.L.; Shirokova,
E.A.
Acta
Naturae,
2013,
5,
54-61.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848067.
20

20. Crauste, C.; Périgaud, C.; Peyrottes, S. J. Org. Chem., 2011, 76, 997-1000. DOI:
10.1021/jo1022958.
21. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyter, J.;
De Clercq, E. J. Virol. Methods, 1988, 20, 309–321. PMID: 2460479.
22. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protoc., 2008, 3, 427–434. DOI:
10.1038/nprot.2007.517.
23. Shchekotikhin, A. E.; Dezhenkova, L. G.; Tsvetkov, V. B.; Luzikov, Y. N.; Volodina, Y.
L.; Tatarskiy Jr., V. V.; Kalinina, A. A.; Treshalin, M. I.; Treshalina, H. M.; Romanenko,
V. I.; Kaluzhny, D. N.; Kubbutat, M.; Schols, D.; Pommier, Y.; Shtil, A. A.;
Preobrazhenskaya, M. N. Eur. J. Med. Chem., 2016, 112, 114-129. DOI:
10.1016/j.ejmech.2016.01.050.
21

Legends
Scheme 1. Synthesis of γ-alkynyl triphosphates of antiviral cytidine nucleoside analogues.
Scheme 2. Synthesis of conjugates of nucleoside triphosphate analogues with SiO2 nanoparticles
via the Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC).
22

Graphical abstract
23