DOI: 10.1002/adsc.201100505 Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila Daniela Ubiali, a,d Carla D. Serra, b Immacolata Serra, a Carlo F. Morelli, b Marco Terreni, a,d Alessandra M. Albertini, c Paolo Manitto, b,d, * and Giovanna Speranza b,d,e, * a Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, via Taramelli 12, I-27100 Pavia, Italy b Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, via Venezian 21, I-20133 Milano, Italy Fax: (+ 39)-02-5031-4072; phone: (+ 39)-02-5031-4097; e-mail: giovanna.speranza@unimi.it c Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, via Ferrata 1, I-27100 Pavia, Italy d Italian Biocatalysis Center, via Taramelli 12, I-27100 Pavia, Italy e Istituto di Scienze e Tecnologie Molecolari, CNR, via Golgi 19, I-20133 Milano, Italy Received: June 29, 2011; Published online: January 12, 2012 Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/adsc.201100505. Abstract: Purine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over-expressed in Escherichia coli, pu- rified, characterized about its substrate specificity and used for the preparative synthesis of some 6-sub- stituted purine-9-ribosides. Substrate specificity to- wards natural nucleosides showed that this PNP cata- lyzes the phosphorolysis of both 6-oxo- and 6-amino- purine (deoxy)ribonucleosides. A library of nucleo- side analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay re- vealed that 1-, 2-, 6- and 7-modified nucleosides are accepted as substrates, whereas 8-substituted nucleo- sides are not. A few transglycosylation reactions were carried out using 7-methylguanosine iodide (4) as a d-ribose donor and 6-substituted purines as ac- ceptor. In particular, following this approach, 2- amino-6-chloropurine-9-riboside (2c), 6-methoxypur- ine-9-riboside (2d) and 2-amino-6-(methylthio)pur- ine-9-riboside (2g) were synthesized in very high yield and purity. Keywords: Aeromonas hydrophila ; biotransforma- tions; chemoenzymatic synthesis; glycosylation; nu- cleoside phosphorylases; nucleosides Introduction Nucleoside phosphorylases (NPs; E.C. 2.4.2) catalyze the reversible cleavage of the glycosidic bond of (de- oxy)ribonucleosides in the presence of inorganic or- thophosphate (P i ) as a second substrate to generate the nucleobase and a-d-(deoxy)ribose-1-phosphate [(d)R-1-P)] [Eq. (1)]. [1,2] If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result. Thus, NPs provide a potentially suitable tool for che- moenzymatic synthesis of both natural and unnatural nucleosides. To obtain the b-oriented glycoside bond their use appears to be a convenient alternative to chemical methods which suffer from low stereoselec- tivity, multi-step procedures and modest total yields. [3] Several procedures based on the “transglycosylation” reaction (Scheme 1) have been employed (using both isolated enzymes and whole cells) to prepare a wide variety of nucleosides. [4–11] Structural analysis revealed that there are two dis- tinct families of NPs: NPI and NPII. Members of the NPI family show either a trimeric or a hexameric qua- ternary structure. Enzymes belonging to this large family accept a wide range of purine nucleosides (PNPs) and uridine (UP). Members of the NPII family display a dimeric structure and are specific for pyrimidine nucleosides. [1] With regard to purine nucleoside phosphorylases (PNPs), the most studied member of NPI family, Bzowska et al. [12] proposed a classification based on molecular mass (mm), subunit composition and sub- 96 # 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Adv. Synth. Catal. 2012, 354, 96 – 104 FULL PAPERS