Enzymatic Transglycosylation Features in Synthesis of 8-Aza-7-Deazapurine Fleximer Nucleosides by Recombinant E. coli PNP: Synthesis and Structure Determination of Minor Products

Abstract

Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of "non-typical" minor products of the reaction. In addition to "typical" N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2'-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2'-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this "upside down" arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products.

Keywords: catalytic site; fleximer base; molecular modeling; recombinant E. coli PNP.

Figure 1

The structures of modified bases and nucleosides that E. coli PNP accepts as substrates.

Figure 2

Modified heterocyclic bases with different glycosylation sites (highlighted in blue—normal and red—unusual).

Figure 3

8-Aza-7-deazapurine fleximer bases.

Figure 4

Structural formulas with the numbering of atoms in cycles. A—pyrazole cycle, B—pyridine cycle, C—the first ribose/2-deoxyribose residue, D—the second one.

Figure 5

Enzymatic transglycosylation of fleximer base 12 with the formation of isomer products (highlighted with a red border). The nitrogen atoms involved in the glycosylation reaction are shown in red.

Figure 6

(A)—Conversion of base 12 to ribosides 15, 16, and bis-riboside 17, (B)—Conversion of base 12 to 2′-deoxyriboside 18 and bis-2′-deoxyriboside 19.

Figure 7

Conversion of fleximer base 12 to pyrazole riboside 15, aminopyridinium riboside 16, and bis-riboside 17 at various pH. (A)—15 (pyrazole riboside), (B)—16 (pyridine riboside), (C)—17 (bis-riboside), (D)—18 (pyrazole 2′-deoxyriboside), (E)—pyridine 2′-deoxynucleoside was not found in the reaction, (F)—19 (bis-2′-deoxyriboside).

Figure 8

Possible structures of fleximer riboside 16 and comparison of proton chemical shifts in the NMR spectra of nicotinamidriboside [39] and clitidine [53]. Fragments ¹H NMR spectrum of nucleoside 16. The ¹H chemical shifts are shown in black, the ¹³C in blue and the ¹⁵N in red. Turquoise arrows depict the interactions between the protons and the nitrogen atom of pyridine cycle, and blue arrows are the interactions of the protons and the first carbon atom of ribose.

Figure 9

Structures of minor fleximer nucleosides.

Figure 10

Model of inosine interaction in the PNP active site.

Figure 11

Interactions between fleximer base 12 (A) and riboside 15 (B) in the PNP active site.

Figure 12

Interactions between fleximer base 12 (A) and riboside 16 (B) in the PNP active site.

Figure 13

Interactions of the fleximer riboside 16 in the PNP active site, “alternative position”.