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14 pages, 816 KiB

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Chemo-Enzymatic Generation of Highly Fluorescent Nucleoside Analogs Using Purine-Nucleoside Phosphorylase

by Alicja Stachelska-Wierzchowska and Jacek Wierzchowski

Biomolecules 2024, 14(6), 701; https://doi.org/10.3390/biom14060701 - 14 Jun 2024

Cited by 2 | Viewed by 1958

Chemo-enzymatic syntheses of strongly fluorescent nucleoside analogs, potentially applicable in analytical biochemistry and cell biology are reviewed. The syntheses and properties of fluorescent ribofuranosides of several purine, 8-azapurine, and etheno-purine derivatives, obtained using various types of purine nucleoside phosphorylase (PNP) as catalysts, as well as α-ribose-1-phosphate (r1P) as a second substrate, are described. In several instances, the ribosylation sites are different to the canonical purine N9. Some of the obtained ribosides show fluorescence yields close to 100%. Possible applications of the new analogs include assays of PNP, nucleoside hydrolases, and other enzyme activities both in vitro and within living cells using fluorescence microscopy. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides—2nd Edition)

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11 pages, 2565 KiB

Open AccessArticle

Excited-State Proton Transfer in 8-Azapurines I: A Kinetic Analysis of 8-Azaxanthine Fluorescence

by Jacek Wierzchowski and Bogdan Smyk

Molecules 2020, 25(12), 2740; https://doi.org/10.3390/molecules25122740 - 12 Jun 2020

Cited by 7 | Viewed by 2763

Abstract

A super-continuum white laser with a half-pulse width of ~75 ps was used to observe the kinetics of a postulated excited-state proton transfer in 8-azaxanthine and its 8-methyl derivative. Both compounds exhibited dual emissions in weakly acidified alcoholic media, but only one band was present in aqueous solutions, exhibiting an abnormal Stokes shift (>12,000 cm⁻¹). It was shown that long-wavelength emissions were delayed relative to the excitation pulse within alcoholic media. The rise time was calculated to be 0.4–0.5 ns in both methanol and deuterated methanol. This is equal to the main component of the fluorescence decay in the short-wavelength band (340 nm). Time-resolved emission spectra (TRES) indicated a two-state photo-transformation model in both compounds. Global analysis of the time dependence revealed three exponential components in each compound, one of which had an identical rise-time, with the second attributed to a long-wavelength band decay (6.4 ns for aza-xanthine and 8.3 ns for its 8-methyl derivative). The origin of the third, intermediate decay time (1.41 ns for aza-xanthine and 0.87 ns for 8-methyl-azaxanthine) is uncertain, but decay-associated spectra (DAS) containing both bands suggest the participation of a contact ion pair. These results confirm the model of phototautomerism proposed earlier, but the question of the anomalous isotope effect remains unsolved. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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13 pages, 983 KiB

Open AccessArticle

Fused Heterocyclic Systems with an s-Triazine Ring. 34. Development of a Practical Approach for the Synthesis of 5-Aza-isoguanines

by Ahmad Junaid, Felicia Phei Lin Lim, Yvonne Peijun Zhou, Wai Keung Chui and Anton V. Dolzhenko

Molecules 2019, 24(8), 1453; https://doi.org/10.3390/molecules24081453 - 12 Apr 2019

Cited by 5 | Viewed by 3942

Abstract

Purine isosteres present excellent opportunities in drug design and development. Using isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were explored to establish a robust general protocol for the preparation of these compounds. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents. Full article

(This article belongs to the Special Issue Non-Natural Multi-Heteroatom Heterocycles: New Chemical Space)

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10 pages, 870 KiB

Open AccessArticle

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

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

Molecules 2016, 21(1), 44; https://doi.org/10.3390/molecules21010044 - 28 Dec 2015

Cited by 13 | Viewed by 5406

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. Full article

(This article belongs to the Special Issue Nucleoside Modifications)

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12 pages, 414 KiB

Open AccessArticle

Enzymatic Synthesis of Highly Fluorescent 8-Azapurine Ribosides Using a Purine Nucleoside Phosphorylase Reverse Reaction: Variable Ribosylation Sites

by Alicja Stachelska-Wierzchowska, Jacek Wierzchowski, Beata Wielgus-Kutrowska and Goran Mikleušević

Molecules 2013, 18(10), 12587-12598; https://doi.org/10.3390/molecules181012587 - 11 Oct 2013

Cited by 19 | Viewed by 6740

Abstract

Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-β-d-riboside (λ_max 365 nm), while for N8-β-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7. Full article

(This article belongs to the Special Issue Synthesis of Nucleosides, Nucleotides and Their Derivatives)

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9 pages, 372 KiB

Open AccessArticle

Molecular and Crystal Structure of 7,7-Dimethyl-2-pyridin-4-yl-6,7-dihydro-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-amine [1]

by Anton V. Dolzhenko, Geok Kheng Tan, Lip Lin Koh, Anna V. Dolzhenko and Wai Keung Chui

Crystals 2011, 1(3), 136-144; https://doi.org/10.3390/cryst1030136 - 11 Aug 2011

Cited by 2 | Viewed by 7407

Abstract

When crystallized from ethanol, 7,7-dimethyl-2-pyridin-4-yl-6,7-dihydro-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-amine forms crystals which have monoclinic (P2₁/n) symmetry with unit cell dimensions a = 7.3326(5) Å, b = 19.4897(14) Å, c = 8.6586(6) Å, α = 90°, β = 106.069(2)°, γ = 90°, V = 1189.06(14) Å³, Z = 4. The triazine ring in the molecule has a flattened boat conformation with gem-dimethyl groups as flagpole and bowsprit at the bow. The puckering parameters for the ring are: Q = 0.2996(14) Å, θ = 111.7(3)° and φ = 124.1(3)°. In the crystal, molecules are arranged in the three types of chains generated by the intermolecular NH···N hydrogen bonds. The extended chains with the C(11) graph-set motif running along a [010] axis are formed by the amino group hydrogen atom and the pyridine nitrogen atom of another molecule. The C(4)C(6) chains with the R²₂(8) binary graph-set motif running along a [101] direction are formed by linking the amino group hydrogen atom and the hydrogen atom at the triazine nitrogen atom with the triazole and triazine nitrogen atoms of another molecule, respectively. The centrosymmetric inverted dimers are formed via the C-H···π interactions between the methyl group hydrogen and the pyridine ring of the pair molecule. Full article

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