Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.

Alicja Stachelska-Wierzchowska,Beata Wielgus-Kutrowska,Agnieszka Bzowska,Jacek Wierzchowski

doi:10.3390/molecules21010044

Alicja Stachelska-Wierzchowska, Beata Wielgus-Kutrowska + Show 2 more

Open Access

https://doi.org/10.3390/molecules21010044

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Abstract

Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

Highlights

Purine-nucleoside phosphorylase (PNP, E.C.2.4.2.1) is a key enzyme of purine metabolism, important, inter alia, for the proper activity of the immune system in mammals [1,2,3]
purine-nucleoside phosphorylase (PNP) is used as a catalyst in the gram-scale preparative ribosylation of purines and purine analogs [9,10,11], thanks to the reverse pathway of the phosphorolytic process
In the preceding papers [14,15], we have demonstrated that enzymatic ribosylation of some

Summary

Introduction

Purine-nucleoside phosphorylase (PNP, E.C.2.4.2.1) is a key enzyme of purine metabolism, important, inter alia, for the proper activity of the immune system in mammals [1,2,3]. PNP is used as a catalyst in the gram-scale preparative ribosylation of purines and purine analogs [9,10,11], thanks to the reverse (synthetic) pathway of the phosphorolytic process. We present a kinetic analysis of these processes, with application of several wild and mutated forms of PNP as catalysts. We will demonstrate a considerable selectivity of the 8-azapurine ribosylation sites with various PNP forms, and a remarkable sensitivity of the ribosylation process to point mutations at the Molecules 2016, 21, 44; doi:10.3390/molecules21010044 www.mdpi.com/journal/molecules. Molecules 2016, 21, 44 various PNP forms, and a remarkable sensitivity of the ribosylation process to point mutations at the critical critical active active site site residue. The major tautomeric form of 8-azaguanine (N(9)H) is shown

Discussion

Ribosylation

Fluorescence

Experimental Section