ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 6, pp. 783–785. © Pleiades Publishing, Ltd., 2010. Original Russian Text © N.S. Utkina, L.L. Danilov, T.N. Druzhinina, V.V. Veselovskii, 2010, published in Bioorganicheskaya Khimiya, 2010, Vol. 36, No. 6, pp. 853–855.

LETTER TO THE EDITOR

Simple Synthesis of P 1(11Phenoxyundecyl)P 2(2Acetamido 2deoxyαDgalactopyranosyl) Diphosphate N. S. Utkina1, L. L. Danilov, T. N. Druzhinina, and V. V. Veselovskii Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991, Russia Received June 11, 2010; in final form, June 16, 2010

Abstract—A simple method of the synthesis of P1(11phenoxyundecyl)P2(2acetamido2deoxyαD galactopyranosyl) diphosphate, which is a synthetic lipid acceptor for glycosyl transferases participating in the biosynthesis of Oantigenic polysaccharides of Gramnegative bacteria, is suggested. Keywords: 11phenoxyundecyl phosphate, P1(11phenoxyundecyl)P2(2acetamido2deoxyαDgalacto pyranosyl) diphosphate, 2acetamido2deoxyαDgalactopyranosyl diphosphate DOI: 10.1134/S1068162010060166

1

Cloning glycosyl transferases, which assemble repeating links of Oantigenic polysaccharides of Gramnegative bacteria with the participation of undecaprenyl diphosphate sugars, has recently become more and more widespread in biochemical practice [1]. It should be noted that among entero bacteria, which are the causative agents for many epi demiologically important enteric diseases, this bio chemical cycle often begins with the formation of lipidbound derivatives of 2acetamido2deoxyα Dglucopyranosyl or 2acetamido2deoxyαD galactopyranosyl phosphate. It was shown recently that in a number of cases artificial acceptors can be successfully used for these purposes, in which the 11 phenoxyundecyl residue replaces the natural unde caprenyl one as a lipid fragment [1–3]. A method of identifying α1,3Nacetylgalac tosaminyltransferase, which is encoded by the wbnH gene and which participates in the biosynthe sis of Oantigenic polysaccharide Escherichia coli O86:H2, is described in [3]. This enzyme catalyzes the transfer of the residue of 2acetamido2deoxy Dgalactopyranose from UDPGalNAc (substrate– donor) to the synthetic Р1(11phenoxyundecyl) Р2(2acetamido2deoxyαDgalactopyranosyl) 1 Corresponding

author; phone: (499) 1377570; email: utkin [email protected].

diphosphate ((IV), substrate–acceptor) with the formation of an α(1–3)glycoside bond. The syn thesis of the substrate–acceptor (IV) given in [3] is similar to that of a 2acetamido2deoxyαDglu copyranose analog [2] and consists of the reaction of 11phenoxyundecyl phosphoimidazolide (II) with a full acetate of 2acetamido2deoxyαD galactopyranosyl phosphate and the subsequent removal of the protective groups. Earlier, we developed a simple method of the syn thesis of 11phenoxyundecyl phosphate (I) and dem onstrated its acceptor properties in the reaction with UDPGlcNAc: polyprenylphosphateGlcNAc– phosphotransferase from Salmonella arizona O:59 [4]. Then, using the advantages of this approach, we started the synthesis of phenoxyundecyl diphosphate sugars with the aim of studying their acceptor proper ties at the next stage of assembling repeating links of Oantigenic polysaccharides. In the present report, we describe a simple method of the synthesis of Р1(11phenoxyundecyl)Р2(2 acetamido2deoxyαDgalactopyranosyl) diphos phate (IV) from 11phenoxyundecyl phosphate (I) and unprotected 2acetamido2deoxyαDgalacto pyranosyl phosphate (III).

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1. Et3N OPO3H2 2. CDI

PhO

PhO

(I)

(II)

HO

HO HO

CH2OH O

O O P N O−

N

CH2OH O

HO

O O NHAc P P O −O −O (IV) O O

OPh

NHAc 2− OPO3 (III)

Scheme.

11Phenoxyundecyl phosphate (I) was obtained by threestage synthesis from 11bromundecanoic acid [4], while 2acetamido2deoxyαDgalactopyrano syl phosphate ((III), triethylammonium salt) was obtained in two stages under the conditions of the ste reospecific synthesis of 2acetamido2deoxyαD hexopyranosylphosphates (by fusing 2acetamido2 deoxy1,3,4,6tetraОacetylDgalactopyranose in a vacuum with dioxane diphosphate with the subse quent removal of the protective acetyl groups [5]). It should be noted that in [2], the initial 11phenox yundecyl phosphate (I) was obtained by sixstage syn thesis, while the protected glucosylphosphate was obtained by fivestage synthesis. Р1(11Phenoxyundecyl)Р2(2acetamido2deoxy αDgalactopyranosyl) diphosphate (IV) was synthe sized by the phosphoroimidazolidate method devel oped earlier to obtain a polyprenylcontaining analog from unprotected galactosyl phosphate (III) [5]. For this, 11phenoxyundecyl phosphate (I) was converted into triethylammonium salt by adding the excess of triethylamine in absolute toluene and dried in the vac uum of an oil pump. The obtained salt was converted into 11phenoxyundecyl phosphoimidazolid (II) by the reaction with N,N'carbonyldiimidazole (CDI, 5.0 molequiv) in absolute THF (20°С, 18 h, argon) with the subsequent decomposition of the excess of the activating agent by methanol and the removal of the solvents. A solution of triethylammonium salt of 2acetamide2deoxyαDgalactopyranosyl phosphate ((III), 0.75 molequiv) in absolute DMSO was added to the residue dissolved in absolute THF. The reaction mixture was kept at 20°С for 96 h under argon, con centrated, and dried in the vacuum of an oil pump. To isolate compound (IV), the dry residue was dis solved in water, applied onto a SepPack C18 car

tridge, and eluted with water, a 5% solution of metha nol in water, a 10% solution of methanol in water, and methanol, controlling the elution with the help of TLC. The target product was eluted with a 10% solu tion of methanol in water. The solvents were removed in a vacuum and Р1(11phenoxyundecyl) Р2(2acetamido2deoxyαDgalactopyranosyl) diphosphate ((IV), triethylammonium salt) was obtained with a yield of 30%; Rf 0.65 (Merck Silica Gel 60 F254) in the system chloroform–methanol– water, 10 : 10 : 3. 1H NMR spectrum (600 MHz, CD3OD) δ, ppm, J, Hz: 8.78 (1 H, s, NHCOCH3), 7.5 (1 H, s, Et3N+H), 7.24 (2 H, m, Ph), 6.90 (3 H, m, Ph), 5.59 (1 H, dd, J1,2 3.5, J1, P 7.0, H1), 4.36 (1 H, dt, J2,3 10.0, H2), 4.18 (1 H, ddd, J5,6 6.2, J5,6 4.8, J5,4 1.5), 4.0–3.9 (4 H, m, CH2OPh, CH2OP), 3.92 (1 H, dd, J4,3 3.0, J4,5 1.5, H4), 3.85 (1 H, dd, J3,2 10.0, J3,4 3.0, H3), 3.78–3.68 (2 H, m, H6, H6'), 3.32 (6 H, m, N(CH2)3), 2.06 (3 H, s, NHCOCH3), 1.75 (2 H, m, CH2), 1.62 (2 H, m, CH2), 1.46 (2 H, m, CH2), 1.40– 1.28 (21 H, m, CH2, N(CH2CH3)3) (cf. [3]). 31P NMR spectrum (242.94 MHz, CD3OD) δ, ppm: –8.8 (br.s., P1), –11.0 (br.s, P2) (cf. [3]). Thus, a simple method of the synthesis of Р1(11 phenoxyundecyl)Р2(2acetamido2deoxyαD galactopyranosyl) diphosphate (IV), which is a lipid acceptor for studying bacterial glycosyltransferases, is given in this work. ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (project no. 100400197).

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REFERENCES 1. Riley, J.G., Menggad, M., MontoyaPeleaz, P.J., Sza rek, W.A., Marolda, C.L., Valvano, M.A., Schutzbach, J.S., and Brockhausen, I., Glycobiology, 2005, vol. 15, pp. 605⎯613. 2. MontoyaPeleaz, P.J., Riley, J.C., Szarek, W.A., Val vano, M.A., Schutzbach, J.S., and Brockhausen, I., Bioorg. Med. Chem. Lett., 2005, vol. 15, pp. 1205⎯1211.

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3. Yi, W., Yao, Q., Zhang, Y., Motary, E., Lin, S., and Wang, P.J., Biochem. Biophys. Res. Commun., 2006, vol. 344, pp. 631⎯639. 4. Danilov, L.L., Veselovsky, V.V., Balagurova, N.M., and Druzhinina, T.N., Bioorg. Khim., 2009, vol. 35, pp. 431⎯432 [Russ. J. Bioorg. Chem., 2009, vol. 35, pp. 301–302]. 5. Danilov, L.L., Maltsev, S.D., and Shibaev, V.N., Bioorg. Khim., 1986, vol. 12, pp. 934⎯938.

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