Synthesis of liponucleotides using bacterial phospholipase D

A series of conjugates of pharmacologically promising modified nucleosides with phospholipid was synthesized. Some liponucleotides were originally produced. Soybean lecithin served as the donor of phosphatidyl residue. Phospholipase D from strain Streptomyces netropsis BIM B-428D was engaged as biocatalyst in transphosphatidylation reaction. The yield of liponucleotide synthesis reaction varied in the range 44–95 mol% depending on the acceptor of phosphatidyl residue. 65 mg (about 68 μmoles) of pure phosphatidylclofarabine was recovered by chromatography on silica gel column, resulting in 68 mol% yield calculated from the amount of modified nucleoside supplied into the reaction mixture. K E Y W O R D S


1-Introduction
Drugs based on modified nucleosides and nucleotides play a crucial role in chemical therapy of cancer and severe viral infections [ One of the latest papers [4] described and experimentally substantiated the radically innovative approach to treatment of cancer diseases leading to the selective self-elimination of tumor cells. It is based on the ability of the enzyme telomerase to insert 6-thio-2'-deoxyguanosine (modified analog of natural 2'-deoxyguanosine) into telomerase structure. The inclusion of modified nucleoside drastically alters the composition of chromosome terminus so that it loses the capacity to be shielded by protective proteins, triggering as a consequence cancer cell apoptosis and death [5].
Recently riboside of phytohormone kinetin was proposed as the effective cancerostatic compound. It was shown that kinetin riboside displayed therapeutic efficiency in regard to acute myeloblast leucosis [6] and anti-proliferative activity toward colorectal cancer cells [7]. Cytotoxic effects of kinetin riboside are determined by the ability to cause rapid ATP exhaustion provoking genotoxic stress wherein gene CDKNIA and other shock-involved genes are activated.
Preclinical trials also revealed the potential of clofarabine to suppress growth of various solid tumors.
It is well known, however, that the majority of compounds suitable for oncotherapy suffer from side effects and limitations, like low curative index, marked toxic effects, rapid catabolism in blood vessels down to inactive substances, restraining thereby their clinical application. One of the key solutions to overcome this problem is elaboration of novel generation of pharmaceuticals (the so-called pro-drugs) by conjugating antiviral and antitumor nucleosides with phospholipids [11][12][13]. The resulting conjugates are distinguished by increased bio-accessibility, enhanced stability in blood stream, upgraded pharmacokinetic parameters, inferior toxicity. It was found, for instance, that certain dialkylglycerophosphate derivatives of clofarabine showed lower toxicity than the parent compound but preserved the elevated antitumor activity [14].
Intraperitoneal or per-oral administration of diacylglycerophosphates of clofarabine and fludarabine resulted in prolonged secretion of active nucleosides and their long-term retention in blood circulation system of test animals.
The available chemical methods of phospholipid conjugation with nucleosides are complicated and are characterized by small yields of the end products [14,15]. Earlier our team demonstrated the possibility of using relatively simple and effective 66 |2020, 3(1), 54-73 biotechnology devised by the Japanese researchers [16] for the synthesis of phospholipid derivatives of a series of natural and modified nucleosides [17,18].
This technique envisages application of bacterial phospholipase D.
The synthesized phospholipids may find use as emulsifiers, ingredients of cosmetic formulas, medicines and liposomal preparations [29].
Aim of this study -production of new phosphatidyl derivatives of pharmacologically valuable modified nucleosides using PLD from Streptomyces netropsis culture.

Materials and Methods
The

Results and Discussion
Feasibility of producing phospholipid derivatives of several modified pharmacologically significant nucleosides was evaluated. It is clear from the data summed up in Table 1 that all tested nucleosides were phosphatidylated by PLD with yields 44-95 mol% by 1-8 h of the reaction process. Noteworthy, that majority of the phosphatidyl derivatives of nucleosides shown in Table 1 (brivudine, acyclovir, gancyclovir, et al.) appear to have been prepared by us for the first time.
Likewise the similar strategy was preferred for synthesis of phosphatidyl derivatives of other nucleosides [15,30].
Preparative synthesis of liponucleotides was exemplified in this study by production of phosphatidylclofarabine. According to TLC data, product yield of the reaction reached 86 mol%.
Following recovery of the target product and its purification on silica gel column, 65 mg of phosphatidylclofarabine was produced (making the final yield 68 mol% relative to amount of nucleoside supplied into reaction mixture).
Results of NMR spectroscopy of the synthesized conjugate agree well with those provided in the reports [14] for structurally similar compounds.
Analyses of 1 H NMR spectrum revealed nucleoside

Conflict of interest
The authors clearly declared that they have no any conflict of interest.