nucleoside inhibitors of reverse transcriptase. The azi- dothymidine molecule is derivatized more often at the. 5'-hydroxy group [1, 2]. To provide efficient delivery ...
ISSN 0012-5008, Doklady Chemistry, 2008, Vol. 419, Part 2, pp. 95–97. © Pleiades Publishing, Ltd., 2008. Original Russian Text © I.Yu. Ponedel’kina, V.N. Odinokov, E.A. Saitgalina, E.S. Lukina, U.M. Dzhemilev, 2008, published in Doklady Akademii Nauk, 2008, Vol. 419, No. 4, pp. 512–514.
CHEMISTRY
Conjugation of 3'-Azido-3'-deoxythymidine with Heparin I. Yu. Ponedel’kina, V. N. Odinokov, E. A. Saitgalina, E. S. Lukina, and Corresponding Member of the RAS U. M. Dzhemilev Received October 26, 2007
DOI: 10.1134/S0012500808040046
of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) in aqueous solution (the concentration of heparin VI was 0.015 mmol/mL, molar ratio VI : VII : EDC = 1 : 25 : 4, pH 4.7–4.8, 20–25°C, 24 h). Since there is no reaction of heparin with D,L-lysine acetylated at the α-amino group under the same conditions, we can conclude that the conjugation of heparin proceeds at the α-amino group of D,L-lysine. Lysine-modified heparin VIII was purified by reprecipitation three times from water with methanol. The positive ninhydrin test indicates the presence of primary amino groups in conjugate VIII; its 1H NMR spectrum (the ratio of normalized signal intensities of the (CH2)3 group of lysine (δ 1.0–2.0 ppm) and anomeric protons of the heparin moiety (δ 5.1–5.5 ppm)) indicates that all carboxy groups of heparin are transformed into ω-aminoamide groups.
Conjugation at the carboxy groups of heparin and the 5'-hydroxy groups of azidothymidine was accomplished through a D,L-lysine–succinic spacer. The conjugate is consistent with 90% conversion of heparin carboxy groups into corresponding amide groups. 3'-Azido-3'-deoxythymidine (azidothymidine) is one of the widely used antiviral drugs from the class of nucleoside inhibitors of reverse transcriptase. The azidothymidine molecule is derivatized more often at the 5'-hydroxy group [1, 2]. To provide efficient delivery to certain kinds of cells, decrease toxicity, and prolong action, azidothymidine is covalently bound to polymeric transport systems of protein nature [3–6] and sulfated oligo- [7] and polysaccharides [8–10]. A synergism of the antiviral action of conjugates was observed in some cases [10]. As a matrix for azidothymidine, we used heparin, an available natural polysulfated glycosaminoglycan showing an affinity to the V3 domain of glycoprotein 120 (gp 120), one of the proteins of the HIV virion capsid, and an inhibiting effect on the growth and replication of the virus [11–13]. For the conjugation of azidothymidine with heparin, we developed the following way of their transformations. The reaction of azidothymidine (I) with succinic anhydride (II) in dimethylformamide (DMF) in the presence of p-(dimethylamino)pyridine (DMAP) (90°C, 1 h) led to azidothymidine hemisuccinate (III) identical to the compound reported earlier [14]. To activate the carboxy group, hemisuccinate III was transformed into corresponding N-hydroxysuccinimide ester V via reaction with an equimolar amount of N-hydroxysuccinimide (IV) and dicyclohexylcarbodiimide (DCC) in CH2Cl2 (the concentration of hemisuccinate III was 0.1–0.2 mmol/mL, 0–20°C, 10–20 min). Heparin VI was modified at the carboxy groups by reaction with D,L -lysine ( VII ) in the presence
Target conjugate IX was obtained by the coupling of N-hydroxysuccinimide ester V and lysine-modified heparin VIII in a 1 : 1 mixture of a 0.1 M aqueous solution of NaHCO3 and dimethylformamide (the concentration of VIII was 0.02 mmol/mL, V : VIII = 4.5 : 1, pH 8.5, 20–25°C, 18–24 h). Conjugate IX thus obtained was purified by reprecipitation three times from water with methanol. The ratio of normalized intensities of the signal at δ 1.98 ppm, corresponding to the protons of the methyl group at the C-5 atom of the azidothymidine residue, and the signals at δ 5.1– 5.5 ppm of the anomeric protons of the carbohydrate scaffold of heparin indicates that, in the molecule of conjugate IX, azidothymidine is covalently bound to 90% of the heparin carboxy groups. UV spectrum of conjugate IX (H2O, λmax, nm): 270. 1H NMR (D2O, δ, ppm): 1.98 (s, 3H, H3C–C-5), 1.0–2.0 (m, 6H, (CH2)3), 2.64 (m, 2H, H2C-2'), 2.75 (m, 4H, CO(CH2)2CO), 5.1– 5.5 (m, 2H (anomeric protons in the heparin moiety)), 6.27 (m, 1H, HC-1'), 7.61 (s, 1H, HC-6).
Institute of Petrochemistry and Catalysis, Ufa Scientific Center, Russian Academy of Sciences, pr. Oktyabrya 141, Ufa, 450075 Bashkortostan, Russia 95
96
PONEDEL’KINA et al. O H3C
NH
O
HO
O H3C
N
O
O +
O
N3
DMAP
N
O
HOOC(CH2)2COO
O
O +
DMF
H NON O
N3
O
I
NH
II
III
IV O
H3C
NH
O DCC
O
N OCO(CH2)2COO
CH2Cl2
O
N
O
N3 V
O
O HO OSO3H COOH VI
O HO
OSO3H O NHR
O n
+ NH2CH(COOH)(CH2)3CH2NH2
VII
EDC –H2O
OSO3H O
O
O O HO NHR n OSO3H CONHCH(COOH)(CH2)3CH2NH2 VIII
O HO
R = SO3H or Ac; SO3H/Ac = 70/30
V + VIII
O
O
OSO3H O
O O HO n n NHR OSO3H CONHCH(COOH)(CH2)3CH2NHCO(CH2)2COO
O HO
H3C 5'
4 NH 5 3 6 1 2
N
O
O
1'
4' 3'
2'
N3 IX Scheme 1.
Thus, we accomplished for the first time the conjugation of azidothymidine with heparin. The coupling of N-hydroxysuccinimide ester of azidothymidine hemisuccinate with D,L-lysine-modified heparin results in a conjugate containing 90% of azidothymidine residues per disaccharide unit of heparin. UV spectra were obtained on a Specord M-40 spectrophotometer; 1H NMR spectra were recorded in D2O solutions on a Bruker AMX-300 spectrometer operating at 300.13 MHz using sodium 3-(trimethylsilyl)-1propanesulfonate as an internal reference. TLC was carried out using Silufol UV-254 plates (ethyl acetate, anisaldehyde as the developer).
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CONJUGATION OF 3'-AZIDO-3'-DEOXYTHYMIDINE WITH HEPARIN 7. Gao, Y., Katsuraya, K., Kaneko, Y., Mimura, T., Nakashima, H., and Uryu, T., Polym. J., 1998, vol. 30, pp. 243– 248. 8. De Clercq, E., J. Med. Chem., 1986, vol. 26, pp. 1561– 1569. 9. Gao, Y., Katsuraya, K., Kaneko, Y., Mimura, T., Nakashima, H., and Uryu, T., Polymer J., 1998, vol. 30, pp. 31– 36. 10. Vlighe, P., Clerc, T., Pannecouque, C., Witvrouw, M. De Clercq, E., Salles, J.P., and Kraus, J. L., J. Med. Chem., 2002, vol. 45, no. 6, pp. 1275–1283.
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