ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 1, pp. 105–107. © Pleiades Publishing, Ltd., 2015. Original Russian Text © A.N. Vinnikova, V.I. Torgov, N.S. Utkina, V.V. Veselovsky, T.N. Druzhinina, S. Wang, I. Brockhausen, L.L. Danilov, 2015, published in Bioorganicheskaya Khimiya, 2015, Vol. 41, No. 1, pp. 121–123.

LETTERS TO THE EDITOR

The Synthesis of P 1[11(Anthracen9ylmethoxy)undecyl]P 2 (2Acetamido2DeoxyαDglucopyranosyl) Diphosphate and the Study of Its Acceptor Properties in the Enzymatic Reaction Catalyzed by a DRhamnosyltransferase from Pseudomonas aeruginosa1 A. N. Vinnikovaa, V. I. Torgova, N. S. Utkinaa, 2, V. V. Veselovskya, T. N. Druzhininaa, †, S. Wangb, I. Brockhausenb, and L. L. Danilova a Zelinsky

Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky pr. 47, Moscow, 119991 Russia b Department of Medicine, and Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received August 4, 2014; in final form, August 12, 2014

Abstract—P1[11(anthracen9ylmethoxy)undecyl]P2(2acetamido2deoxyαDglucopyranosyl) diphos phate, a fluorescent derivative of undecyl diphosphate 2acetamido2deoxyglucose, was chemically synthe sized. The ability of the compound to serve as acceptor substrate for the transfer of Drhamnose residue by Drhamnosyltransferase WbpZ from Pseudomonas aeruginosa PAO1 was demonstrated. Keywords: P1[11(anthracen9ylmethoxy)undecyl]P2(2acetamido2deoxyαDglucopyranosyl) diphos phate, fluorescent substrate, Drhamnosyltransferase, Pseudomonas aeruginosa PAO1 DOI: 10.1134/S106816201501015X †2 1

We have previously shown that in the study of the biosynthesis of Oantigenic polysaccharides of Salmo nella and Escherichia coli, the natural undecaprenyl diphosphate sugar acceptor substrate can be replaced by synthetic substrates that contain nonfluorescent or fluorescent chromophorecontaining lipophilic groups. Such replacement gives the opportunity to sig nificantly improve the sensitivity of detection of enzy matic reaction products, especially in the case of fluo rescently labeled compounds [1–9]. Pseudomonas aeruginosa bacteria are important opportunistic pathogens that have a Drhamnose polymer as the common Oantigen. It was recently found that Drhamnosyltransferase (WbpZ) from Pseudomonas aeruginosa PAO1 cells is capable of transferring a Drhamnose residue to phenoxyundecyl diphosphate 2acetamido2deoxyglucose [10]. In this communication, we describe the synthesis of a new fluorescent derivative, P1[11(anthracen–9 ylmethoxy)undecyl]Р2(2acetamido2deoxyαD glucopyranosyl) diphosphate (V) from 11(anthracene 9ylmethoxy)undecyl phosphate (I) and 2aceta Abbreviations: Ant, anthracen9yl

1 The article was translated by the authors. 2 Corresponding author: phone: +7 (499)

[email protected]. † Deceased.

1377570; email:

mido2deoxy3,4,6triOacetylαDglucopyrano syl phosphate (III). We showed that the synthetic compound is able to serve as an acceptor substrate for Drhamnosyltransferase (WbpZ), which catalyzes the second step in the assembly of the Pseudomonas aerug inosa PAO1 common Oantigen. WbpZ transferred DRha from GDPDRha to acceptor (V). The desired diphosphate (V) was synthesized via the phosphoroimidazolidate method [11], previously developed by us, similar to the protocol described in [6] (Scheme 1). For this purpose 11(anthracene9 ylmethoxy)undecyl phosphate (I), obtained in four steps from undec10en1ol [6, 8], was converted to the diisopropylammonium salt by treatment with an excess of diisopropylamine in abs. benzene and freeze dried. The resulting salt was converted to phosphor oimidazolidate (II) by reaction with N,N'carbonyldi imidazole (CDI) in abs. THF (20°С, 1.5 h, argon), followed by decomposing excess of activating agent with methanol and removal of the solvents. The resi due was dissolved in abs. THF and 1.4 mol equiv of diisopropylammonium 2acetamido2deoxy3,4,6 triОacetylαDglucopyranosyl phosphate (III) [1] was added. The reaction mixture was stirred (37°С, 96 h, argon) and the solvents were removed without heating. To determine the physicochemical characteristics of the protected reaction product, a small portion of the

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O

OPO3H2 (I) 1. Pr2i NH 2. CDI

O O P N O−

O (II)

OAc N

O

AcO AcO

NH OPO 2–

(III)

3

O OR RO RO

O O O NH O P O P O O− O− O

O

(IV) R = Ac (V) R = H

MeONa

Scheme 1.

reaction mixture was taken and chromatographed on a Sephadex G15 using 0.1% NH4HCO3 solution in methanol in methanol as eluent (monitored by TLC). The purified compound (IV) had Rf 0.34 (Silica Gel 60 F254; chloroform–methanol–water, 60:25:4). 1H NMR (600.13 MHz, CD3OD) δ, ppm, J, Hz: 8.45 (1H, s, arom. H), 8.33 (2H, d, J 8.4, arom. H), 7.99 (2H, d, J 8.4, arom. H), 7.52 (2H, t, J 7.2, arom. H), 7.45 (2H, t, J 7.2, arom. H), 5.57 (1H, br d, H1), 5.44 (2Н, s, ОСН2Ant), 5.29 (1H, t, J4,3 9.6, H4), 5.13 (1H, t, J3,4 9.6, H3), 4.39–4.29 (3H, m, H2, H5 and H6), 4.16 (1H, m, H6'), 3.96 (2H, m, CH2OP), 3.68 (2H, t, J 6.6, Н2С(11)), 2.15, 2.05, 2.02 and 1.97 (12H, 4s, 4COCH3), 1.63–1.59 (4H, m, Н2С(10) and Н2(С3)), 1.31–1.21 (14H, m, 7СН2). 13C NMR (150.9 MHz, СD3OD): 131.78, 129.44, 128.97, 126.85, 125.64, 95.36, 72.54, 71.46, 69.35, 69.22, 66.97, 65.53, 62.44, 31.36, 30.29, 30.14, 30.03, 26.82, 26.48, 22.69, 20.75, 19.35. 31P NMR (242.93 MHz, СD3OD): –10.4 (br d, P2), –13.3 (br d, P1). HR ESI mass spectrum: found: m/z 866.2921. Calc. for С40H54NO16P2, [M – H]–: 866.2923. The main part of the product was deacetylated without preliminary purification by addition of NaOMe solution in methanol (20°С, 2 h) followed by neutralization with conc. AcOH. After removing the solvent and dissolving the residue in water, the desired Р1[11(anthracen9ylmethoxy)undecyl]Р2(2aceta mido2deoxyαDglucopyranosyl) diphosphate (V)

was isolated with the use of the cartridges SepPak C18 (Waters, USA) by applying to them small portions of the solution and eluting with a stepwise gradient from water to methanol, and monitoring by TLC (yield 20%; Rf 0.52, chloroform–methanol–water, 10:10:3). 1H NMR (CD OD): 8.51 (1H, s, arom. H), 8.40 (2H, 3 d, J 8.4, arom. H), 8.05 (2H, d, J 8.4, arom. H), 7.55 (2H, t, J 7.8, arom. H), 7.48 (2H, t, J 7.8, arom. H), 5.55 (1H, br d, H1), 5.49 (2Н, s, ОСН2Ant), 3.98 (4H, m, СН2ОР, H2 and H5), 3.89 (1H, d, J 11.4, H6’), 3.77 (1H, t, J 9.0, H3), 3.70 (2H, t J 6.6, Н2С(11)), 3.67–3.65 (1H, m, H6), 3.38–3.34 (1H, m, H4), 2.06 (1Н, s, COCH3), 1.65–1.58 (4H, m, Н2С(10) and Н2С(3)), 1.32–1.29 (14H, m, 7СН2). 13C NMR (СD OD): 130.20, 129.48, 127.34, 126.21, 3 125.58, 96.41, 75.11, 73.24, 72.42, 71.75, 67.45, 65.87, 63.12, 60.45, 56.85, 55.62, 34.65, 32.01, 30.91, 30.80, 27.65, 27.08, 23.67, 19.90. 31P NMR (CD3OD): –10.3 (br d, P1), –12.4 (br d, P2). HR ESI mass spec trum: found: m/z 808.2203. Calc. for С34H47NO13Р2Na3, [M – 2H + 3Na]+: 808.2210. UV and fluorescence spectra (H2O): λmax, nm(ε): 247 (46400); λex 247 nm, λem 415 nm. The ability of Р1[11(anthracen9ylmethoxy)unde cyl]Р2(2acetamido2deoxyαDglucopyranosyl) diphosphate (V) to accept a Drhamnose residue from GDPDRha forming DRhaDGlcNAcα1PP (CH2)11OCH2Ant in an enzymatic reaction catalyzed by Drhamnosyltransferase (WbpZ) from Pseudomonas

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SYNTHESIS OF FLUORESCENT DERIVATIVE OF UNDECYL DIPHOSPHATE

aeruginosa PAO1 cells was examined. Enzyme assays were carried out similarly to the protocol described for Р1(11phenoxyundecyl)Р2(2acetamido2deoxy αDglucopyranosyl) diphosphate [10]. It was shown that compound (V) containing anthracenylmethoxy group had 57% of the acceptor efficiency, compared with an acceptor analogue containing a phenoxy group. Acceptor substrates and products could be sep arated by reversedphase high pressure liquid chroma tography and quantified by fluorescence measure ments. Thus, the presence of a fluorescent moiety in the acceptor eliminated the need of using radioactive nucleotide sugar for a sensitive detection of enzyme reaction product. The large anthracenyl group was well tolerated by the enzyme, and the presence of a fluorophore significantly improved the detection sen sitivity in the identification and isolation of the enzy matic reaction product. To summarize, we report the first synthesis of fluo rophorecontaining anthracen9ylmethoxyundecyl diphosphate 2acetamido2deoxyglucose and dem onstrated its ability to serve as an acceptor substrate for Drhamnosyltransferase (WbpZ) from Pseudomonas aeruginosa PAO1.

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ACKNOWLEDGMENTS The work was supported by the Russian Founda tion for Basic Research, project no. 130400358. Authors thank Prof. A.S. Shashkov for recording the NMR spectra and aid in their interpretation.

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