Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

Abstract

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.

Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System

“…The application of nucleosides as substrates for the synthesis of nucleotides together with purified biocatalysts offers significant advantages over the approaches described above. Natural nucleosides are generally cheaply available as commercial reagents and modified nucleosides can be accessed enzymatically via known methods in high yield ( Kamel et al, 2019 ; Kaspar et al, 2020 ; Yehia et al, 2020 ). The use of purified enzymes minimizes side reactions and simplifies reaction workup.…”

Reverse C-glycosidase reaction provides C-nucleotide building blocks of xenobiotic nucleic acids

“…Natural enzymes of C-glycoside biosynthesis, such as tRNA pseudouridine synthase 27,28,51,52 and C-glycosynthase 15,[18][19][20][21]53,54 have complex substrate requirements, leading to perceived limitations on their applicability. Therefore, unlike N-nucleosides for which N-nucleoside phosphorylases present excellent tools of glycoside synthesis [55][56][57][58][59][60] , biocatalytic methods are lacking for C-nucleoside synthetic chemistry.…”

Spectral Unmixing‐Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases

“…Nucleoside phosphorylases (NPs), for instance, catalyze the reversible phosphorolytic cleavage of nucleosides into the corresponding free nucleobase and pentose-1-phosphate (Scheme 1) and are widely applied for the preparation of modified nucleosides. [1][2][3][4][5][6][7][8][9][10][11] Consequently, their kinetic and thermodynamic characterization has attracted increased interest and demanded the development of efficient analytical tools. [12,13] Recently, we reported a UV/Vis spectroscopy-based method for the monitoring of these reactions that largely eliminated the need for HPLC.…”