Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies

Article abstract of DOI:10.1016/j.bmcl.2009.12.053

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) t

Full text of DOI:10.1016/j.bmcl.2009.12.053

Bioorganic & Medicinal Chemistry Letters 20 (2010) 987–990
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and studies of triphosphate analogues for the production
of anti AZT-TP antibodies
b
a
Camille Roucairol a, Stéphane Azoulay a, , Marie-Claire Nevers , Jérôme Golebiowski ,
*
Christophe Créminon b, Jacques Grassi b, Alain Burger a, , Danièle Duval
a
*
a Laboratoire de Chimie des Molécules Bioactives et Arômes, UMR 6001 CNRS, Institut de Chimie de Nice, Université de Nice-Sophia Antipolis, Nice, France
b CEA, iBiTec-S, Service de Pharmacologie et d’Immunologie, Saclay, F- 91191 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in
HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular
modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of devel-
oping an immunoassay for therapeutic drug monitoring.
Received 3 November 2009
Revised 3 December 2009
Accepted 13 December 2009
Available online 21 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Triphosphate analogues
Antibodies
AZT
Nucleoside triphosphates are important metabolites in many
cellular processes, and analogues of these compounds have major
therapeutic applications in HIV. The triphosphate metabolites
(NRTI-TP) of the antiviral analogues are the active chemical spe-
cies. They compete into infected cells with endogenous nucleotides
for incorporation into the replicating HIV DNA, which results in
termination of viral replication. Great interindividual variability
in the production of phosphorylated metabolites, as well as poor
correlation between plasma drug levels and therapeutic outcome,
emphasize the need for easy routine quantification methods of tri-
phosphate metabolites. The ability to detect and quantitate the
NRTI-TP concentrations in infected cells will lead to a better under-
standing of the pharmacology of these agents and facilitate the dis-
covery of improved therapeutic approaches.
techniques present the valuable advantage to be directly and easily
applicable to clinical routine analysis of large series of samples.
Herein we described our study for the production of antibodies di-
rected against the triphosphorylated form (AZT-TP) of the most
widely used nucleoside inhibitor in AIDS therapy, AZT ( Fig. 1).
The synthesis of several triphosphorylated analogues of AZT and
the characterization of the corresponding antibodies are presented.
Previous attempts to generate specific and sensitive pAb against
triphosphate nucleosides using direct linkage to the triphosphate
or isostere were not fully successful.7,8 Therefore, we tried to eval-
uate a systematical approach which combines both novel strate-
gies and adapted concepts that had been successful for the
synthesis of triphosphate analogues in previously reported
studies.9,10
However the monitoring of nucleoside metabolites in treated
patients requires highly sensitive analytical methods and so far
few methods have been published using mainly direct LC–MS/MS
detection techniques1,2 or indirect dephosphorylation methods.3,4
Despite the advantages of LC–MS/MS, this method is hindered by
high technical requirement and cost of use. In our aim to develop
antiviral enzyme immunoassays5,6 (EIA), we were interested in
the development of a general strategy for direct measurement of
triphosphorylated analogues. The objective is to establish compet-
itive EIA based on the use of specific rabbit polyclonal antibodies
(pAb). Indeed EIA are potentially very sensitive, and these
* Corresponding authors. Tel.: +33 4 92 07 61 15/53; fax: +33 4 92 07 61 51 (S.A.).
E-mail addresses: azoulay@unice.fr (S. Azoulay), burger@unice.fr (A. Burger).
Figure 1. AZT, AZT-TP and AZT-TP analogues.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.053

988
C. Roucairol et al. / Bioorg. Med. Chem. Lett. 20 (2010) 987–990
One of the crucial steps of the EIA elaboration process is the pro-
and 4.5 Å for AZT-TP, 1, 2, 3 and 4, respectively) suggest that all
isosteres are less globular than AZT-TP. Compounds 1 and 4
showed however shapes closer to that of AZT-TP while 2 and 3
exhibited slightly more elongated structures, probably due to the
duction of antibodies allowing a very specific detection associated
with high sensitivity. As small molecules, AZT-TP analogues are
haptens, not immunogenic per se. Thus generating pAb against
small haptenic molecules requires their covalent linkage to a larger
immunogenic protein carrier by the intermediary of a spacer arm
in order to trigger off the immune response. The mode of attach-
ment should be such that the relevant structural determinants of
the analyte are free to interact with the immune system. Following
successful protocol in the obtention of specific monophosphorylat-
ed AZT antibodies11, we decided to introduce the spacer arm on the
nucleobase (Fig. 1). Furthermore in the case of the triphosphorylat-
ed form, another major difficulty raises from both the chemical and
the enzymatic instability of pyrophosphate bridges leading to a ra-
pid hydrolysis of AZT-TP into AZT in recipient animals. This strong
instability could impair the achieving of specific pAb directed
against the AZT-TP. To avoid this problem, we decided to synthe-
size different isosteric and isoelectronic AZT-TP analogues. Several
groups have previously reported the synthesis of triphosphate
mimics in which one or two bridging oxygen of triphosphate is re-
placed by methylene, halomethylene, or imido groups.9,10,12 As ex-
pected, a single bridge modification on a triphosphate moiety can
enhance the stability of theses analogues to dephosphorylating en-
CF2 group between the
trary, the lack of CF2 group naturally makes 1 behave like the ori-
ginal AZT-TP, with a closer distance between the unit in the
a and b phosphates groups. On the con-
c
phosphate tail and the thymine residue. 4 recovers however a clo-
ser interaction between these two moieties suggesting that a CF2
group between the b and
c units of the triphosphate tail does
not strongly affect the triphosphate orientation with respect to
the thymine base.
The results presented above prompted us to synthesize the tar-
get haptens 1–4. The spacer arm on AZT was introduced first to
give a common intermediate 5.11 Beside the fact that the four tri-
phosphate analogues can be prepared from 5, introduction in an
earlier step of the lipophilic side chain is advantageous since it
makes the AZT derivatives more soluble in organic solvents and
thus easier to handle and to purify.
The triphosphate analogues 11–13 required for the preparation
of haptens 1–3 were synthesized in three steps starting from 5
according to a modified procedure adapted from Blackburn13
(Scheme 1).
zymes.9 The substitution of the
c-phosphate by a
c-methylphos-
The 50-hydroxyl group of 5 was first activated by a tosylate
group and then reacted with the tristetrabutylammonium salt of
pyrophosphate14 or difluoromethylenediphosphonate10 to give
after ion-exchange chromatography the diphosphate analogues 9
and 10 in 43% and 38% yield, respectively. The reaction was per-
formed with an excess of the pyrophosphate and diphosphonate
salts (5–7 equiv) to limit the formation of the dimer and at 60 °C
to increase the reaction rate (1 h 30 min at 60 °C, 5 days at room
temperature). The triphosphate derivatives 11 and 12 were ob-
phonate as well as a bridging oxygen by a CF2 group on AZT
greatly improved the half-life in human blood serum.10 Based on
the literature data, four different mimics in which the
c-phosphate
has been replaced by a -methylphosphonate and/or the first or
c
second bridging oxygen by a CF2 group have been designed (Fig. 1).
Moreover, antigen–antibody recognition is based on steric crite-
ria and interactions resulting from the electronic properties of the
molecules. The triphosphate mimics should preserve as much as
possible the electronic distribution and the spatial conformation of
the target compound AZT-TP. Previous reports suggest that the ste-
ric and pKa between difluoromethylphosphonates and phosphates is
quite similar. In order to support the above observations in terms of
molecular conformation and charge distributions, a molecular mod-
elling study of the compound 1 to 4 without the spacer arm (R = H)
was preliminary carried out (see Supplementary data).
tained by a procedure used for the preparation of nucleoside
c-
substituted triphosphates. Reaction of 9 and 10 with methylphos-
phonic dichloride, triazole and triethylamine gave after ion-ex-
change chromatography compounds 11 and 12 in 49% and 43%
yield, respectively. The same procedure was applied for 13 (29%
yield) using phosphorus oxychloride instead of methylphosphonic
dichloride. The connection between the phosphates of 11 and 12
was further confirmed by 2D 31P–1H NMR which showed correla-
A structural analysis of the five structures illustrated in Figure 2
was performed. The average gyration radius data (4.4, 4.8, 6.1, 5.4
tions between a c-P and terminal CH3.
-P and H-50, and
The triphosphate analogue 14 required for the preparation of
hapten 4 was obtained in 47 % in a one pot synthesis starting from
5 according to the procedure described by Wang.10
The final haptens 1–4 were obtained after ammonolysis of com-
pounds 11–14 at 40 °C for 3 h in 90–97% yield. In order to avoid
side reactions, the progress of the reaction was monitored by 19F
NMR spectroscopy. A characteristic upfield shift was observed be-
tween ꢀ76.3 ppm and ꢀ76.0 ppm upon the cleavage of the trifluo-
roacetyl group protecting the terminal NH2 of the spacer arm. After
3 h, removal of the protecting group was completed. Upon these
conditions, the integrity of the triphosphate group was unaffected
as checked by 31P and 19F NMR spectroscopy. The structures of
haptens 1–4 were ascertained from 31P, 19F, 1H NMR and MS anal-
ysis (see Supplementary data).
In order to prepare the immunogens, the haptens 1 to 4 were con-
jugated to BSA with glutaraldehyde to afford IMU-1 to 4, respec-
tively. BSA was preferred to keyhole limphet hemocyamin because
of its solubility in various aqueous buffer and the possibility to carry
out mass spectra analysis on the synthesised conjugates. The molec-
ular weights of the immunogen IMU-1 to 4 were determined by Mal-
di-TOF and found to be 78,171 Da, 79,078 Da, 76,958 Da and
78,828 Da, respectively, which represent an average coupling of 17
molecules of hapten per BSA for IMU-1, 2 and 4 and 14 for IMU-3.
The tracer T1 to T4 were obtained by covalently coupling,
respectively, the hapten 1 to 4, previously activated with N-succin-
Figure 2. Typical 3D-structures of AZT-TP and 1,2,3,4 compounds. The atom colour
is as follows: C, grey; O, red; N, blue; H, white; P, orange and Cl, green.

C. Roucairol et al. / Bioorg. Med. Chem. Lett. 20 (2010) 987–990
989
Scheme 1. Syntheses of AZT analogues. Reagents: (i) TsCl, DMAP, CH2Cl2; (ii) HOP(O)2XP(O)2O3ꢀ, 3Bu4N+, DMF; (iii) CH3POCl2, triazole, Et3N, DMF; (iv) POCl3, triazole, Et3N,
DMF; (v) 2-chloro-4H-1,3,2 benzodioxa phosphorin-4-one, pyridine, DMF; (vi) HOP(O)2CF2P(O)2O3ꢀ, 3Bu4N+, DMF; (vii) I2, pyridine, H2O.
imidyl S-acetylthioacetate, to acetylcholine esterase (AChE) using
the heterobifunctional reagent succinimidyl 4-(N-maleimidometh-
yl)cyclohexane-1-carboxylate (SMCC) (see Supplementary data).
Each immunogen (IMU-1–4) was used to immunize two rabbits
to provide the desired pAb-1 to 4. A marked immune response was
observed with the rabbits after the first booster injection providing
high titer antisera (1/10,000 to 1/500,000). The competitive immu-
noassays were performed in 96 well microtiter plates coated with
an anti-rabbit mouse monoclonal antibody (see Supplementary
data). For each individual bleeding, standard curves were estab-
lished using either the original hapten or AZT-TP as standard. How-
ever, for clarity reason, only the antiserum exhibiting the best
result for each molecule was presented in Table 1.pAb-1, 2 and 4
showed very good sensitivity ranging from 0.6 to 1.8 ng/mL in
homologous system against the original hapten, while pAb-3 ap-
peared less efficient with a sensitivity of 68 ng/mL. The use of
AZT-TP as standard with the combination of the pAb-1, 3 and 4
and its corresponding tracer resulted in an average of 100-fold de-
crease in the sensitivity which now ranged from 66 to 105 (diago-
nal of Table 1, section B), whereas surprisingly AZT-TP fails to
compete with the association pAb-2/T2. The chemical structure
of the enzyme tracer plays a crucial role in the sensitivity and spec-
ificity of the EIA.15,16 Therefore, the screening of all possible pAb/
tracer combination was performed using AZT-TP as competitor
analyte. Sensitivity ranged from 84 to 564 ng/mL and no improve-
ment could be noticed. T3 and T4 were effective in all combination
while T3 showed worst sensitivity. T2 was not able to compete
with AZT-TP in any case. These results are in accordance with
molecular modelling, indicating that introduction of CF2 group in-
stead of an oxygen induced a more hindered triphosphate part and
thus difficulty to compete for a smaller analogue as AZT-TP in the
paratope pocket. However despite a similar shape to AZT-TP, T1
was not effective in heterologous system suggesting that terminal
hydroxyl should play a critical role in the recognition process.
Interestingly, the use of a tracer synthesized for a previous study
(TG4)7 increased dramatically the sensitivity of the assay (55–
440 times). Indeed, in that case, the AChE enzyme was coupled di-
rectly on the triphosphate disfavouring the pAb /TG4 complex.
Specificity studies were consecutively performed by preparing
the calibration curves with several structurally related compounds
(AZT, monophosphorylated AZT (AZT-MP) and diphosphorylated
AZT (AZT-DP)) and measuring them with the same assay
(Table 2). Excepted in the case of the combination pAb-3/T4, AZT
was never recognized by the pAb. For AZT-MP and AZT-DP, results
Table 1
IC50 values in homologous and heterologous system in ng/mL
pAb-1
pAb-2
pAb-3
pAb-4
1.8
A/Homologous system
Hapten 1/T1
Hapten 2/T2
Hapten 3/T3
Hapten 4/T4
0.6
0.3
68
B/Heterologous system
AZT-TP/T1
a
66
—
—
—
AZT-TP/T2
—
—
—
—
AZT-TP/T3
AZT-TP/T4
AZT-TP/TG4
369
177
0.15
84
101
—
105
564
—
560
85
1.2
a
IC50 >1 lg/mL

990
C. Roucairol et al. / Bioorg. Med. Chem. Lett. 20 (2010) 987–990
Table 2
die des Professions Liberales. C. Roucairol is a recipient of a Ph.D.
fellowship from the CNRS and region PACA.
Cross-reactivity of related metabolites
Antibody
Tracer
Cross-reactivity (%)
AZT-MP
AZT
AZT-DP
Supplementary data
a
pAb-1
pAb-1
pAb-1
pAb-1
T1
T3
T4
TG4
—
6
174
—
48
300
40
—
—
—
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.053.
12
716
pAb-2
pAb-2
T3
T4
—
—
—
—
16
110
References and notes
pAb-3
pAb-3
pAb-3
T3
T4
TG4
—
5
—
5
5
57
10
132
950
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T4
TG4
—
—
82
5
1400
46
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a slight
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In our case, all synthesized analogues showed good in vivo sta-
bility since excellent immunological responses were recorded. In
conclusion, the approach presented here allowed us to obtain sen-
sitive and quite specific antibodies against AZT-TP. Our results
showed that isostere strategy should be considered more in detail
when production of antibodies of chemically or enzymatically sen-
sitive compounds is needed.
Acknowledgements
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This work was supported in part by Ensemble contre le Sida
(SIDACTION), the Conseil General 06 and Caisse d’Assurance Mala-