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

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.

Keywords: 8-azapurines; enzymatic ribosylation; fluorescent nucleosides; purine nucleoside phosphorylase.

Figure 1

Ribosylation of 8-azaguanine with α-d-ribose-1-phosphate: possible reaction products. The purine numbering is maintained for simplicity. Only the major tautomeric form of 8-azaguanine (N(9)H) is shown.

Figure 2

The HPLC elution profile for the mixture obtained from the ribosylation of 8-azaguanine with α-d-ribose-1-phosphate as a ribose donor, catalyzed by the N243D mutant of the calf PNP. The Kromasil C8 column (5 µm × 250 × 10 mm) was used, and the reaction mixture eluted with water (10 min) followed by 0%–30% methanol gradient. The first peak (~6 min retention time) was identified as unreacted 8-azaguanine.

Figure 3

Fluorescence changes during the incubation of 16 µM N7-ribosyl-8-azaDaPur in 1000-diluted blood in the presence of 25 mM phosphate. Spectra, excited at 300 nm, were recorded every 5 min, and after 60 min an aliquot of the purified calf PNP was added, and fluorescence recorded after next 3 min (dotted line, 5-fold diminished relative to remaining curves). Note that the first (lowest) curve reflects blood fluorescence background, and minimum at 415 nm is due to the re-absorption by hemoglobin.