biomolecules Article Radical Dehalogenation and Purine Nucleoside Phosphorylase E. coli: How Does an Admixture of 20,30-Anhydroinosine Hinder 2-fluoro-cordycepin Synthesis Alexey L. Kayushin 1, Julia A. Tokunova 1, Ilja V. Fateev 1 , Alexandra O. Arnautova 1, Maria Ya. Berzina 1, Alexander S. Paramonov 1 , Olga I. Lutonina 1, Elena V. Dorofeeva 1, Konstantin V. Antonov 1, Roman S. Esipov 1, Igor A. Mikhailopulo 2 , Anatoly I. Miroshnikov 1 and Irina D. Konstantinova 1,* 1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 GSP, B-437 Moscow, Russia; [email protected] (A.L.K.); [email protected] (J.A.T.); [email protected] (I.V.F.); [email protected] (A.O.A.); [email protected] (M.Y.B.); [email protected] (A.S.P.); [email protected] (O.I.L.); [email protected] (E.V.D.); [email protected] (K.V.A.); [email protected] (R.S.E.); [email protected] (A.I.M.) 2 Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus; [email protected] * Correspondence: [email protected]; Tel.: +7-905-791-17-19 Abstract: During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and Citation: Kayushin, A.L.; Tokunova, 30-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reac- J.A.; Fateev, I.V.; Arnautova, A.O.; tion and decrease of yield down to 5% were encountered. An unknown nucleoside was found Berzina, M.Y.; Paramonov, A.S.; Lutonina, O.I.; Dorofeeva, E.V.; in the reaction mixture and its structure was established. This nucleoside is formed from the 0 0 0 Antonov, K.V.; Esipov, R.S.; et al. admixture of 2 ,3 -anhydroinosine, a byproduct in the preparation of 3- deoxyinosine. More- 0 0 0 0 0 Radical Dehalogenation and Purine over, 2 ,3 -anhydroinosine forms during radical dehalogenation of 9-(2 ,5 -di-O-acetyl-3 -bromo- 0 0 Nucleoside Phosphorylase E. coli: -3 -deoxyxylofuranosyl)hypoxanthine, a precursor of 3 -deoxyinosine in chemical synthesis. The 0 0 How does an Admixture of products of 2 ,3 -anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during 20,30-Anhydroinosine Hinder the synthesis of 2-fluorocordycepin. The progress of 20,30-anhydroinosine hydrolysis was investi- 2-fluoro-cordycepin Synthesis. gated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate Biomolecules 2021, 11, 539. https:// substances in sufficient quantities. Two intermediates were isolated and their structures were con- doi.org/10.3390/biom11040539 0 0 firmed by mass and NMR spectroscopy. A mechanism of 2 ,3 -anhydroinosine hydrolysis in D2O is fully determined for the first time. Academic Editors: Anthony J. Berdis and Jesús Fernandez Lucas Keywords: purine nucleoside phosphorylase; biocatalyze; 30-deoxyinosine; 2-fluorocordycepin; deuterium oxide Received: 14 January 2021 Accepted: 12 March 2021 Published: 7 April 2021 1. Introduction Publisher’s Note: MDPI stays neutral 0 with regard to jurisdictional claims in Among the modified nucleosides, 3 -deoxyribonucleosides remain very promising 0 published maps and institutional affil- objects to study. The series of their research began with cordycepin—3 -deoxyadenosine iations. (1)—the natural antimetabolite of adenosine (2) (Figure1). Cordycepin was isolated from extracts of Cordyceps millitaris tissues in 1950 year [1,2]. This drug is an antioxidant and has a pronounced immunomodulatory effect. In the medicine of Southeast Asia (China, Japan, and Korea), Cordyceps extract has been used since ancient times as a remedy for tumors, Copyright: © 2021 by the authors. viral and bacterial infections [3–7]. Currently, cordycepin is produced commercially in Licensee MDPI, Basel, Switzerland. China using the Chinensa militaris [4,8]. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Biomolecules 2021, 11, 539. https://doi.org/10.3390/biom11040539 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 17 The introduction of a chlorine or fluorine atom into a heterocycle of a nucleoside cardinally changes the biological properties of the molecule: pharmacokinetic prop- Biomolecules 2021, 11, 539 2 of 17 erties, tissue distribution, metabolic pathway, and rate, as well as pharmacodynam- ics and cytotoxicity [11–14]. Figure 1. Adenosine, cordycepin, and their synthetic analogs. Figure 1. Adenosine, cordycepin, and their synthetic analogs. Synthetic C2-substituted analogs of cordycepin (3, 4) have a very interesting biological activityRecent that may studies be applied have inshown medicine. that One2-fluorocordycepin of the best-known ( analogs3) is more of cordycepin efficient than is 2- fluorocordycepincordycepin, in the (2-F-Cord, treatment3) (Figure of human1)[ 9 ,10African]. This trypanosomiasis compound is currently [9,10]. beingHence, actively creat- studieding effective and tested ways as to an synthesize antitumor and2-fluorocordycepin antiviral drug. The (3) introduction is a good direction of a chlorine of re- or fluorinesearch. atom into a heterocycle of a nucleoside cardinally changes the biological properties of theIt molecule: is known pharmacokinetic that 3′-deoxynucleosides properties, tissue can distribution,be synthesized metabolic using pathway, nucleoside and rate,phosphorylases as well as pharmacodynamics (transglycosylation and reaction). cytotoxicity In [ 11our–14 laboratory,]. an efficient method for obtainingRecent studies of purine have nucleoside shown that phosphorylase 2-fluorocordycepin was (3developed) is more efficient [15], and than we cordy- used cepin,this enzyme in the treatment in transglycosylation of human African reactions. trypanosomiasis [9,10]. Hence, creating effective waysHowever, to synthesize the 2-fluorocordycepin problem of a possible (3) is a goodsour directionce of 3-deoxyribose of research. arises [16,17]. Therefore,It is known it is necessary that 30-deoxynucleosides to determine the can optimal be synthesized 3-deoxyribose using nucleoside donor among phospho- pu- rylasesrine nucleosides. (transglycosylation Recent industrial reaction). In developments our laboratory, have an efficient made cordycepin method for obtainingavailable offor purine use as nucleoside a source of phosphorylase 3-deoxyribose. was However, developed 3′-deoxyinosine [15], and we used (3′-dIno, this enzyme9) is likely in transglycosylationto perform better reactions.as a substrate in the synthesis of new nucleosides. However,To test which the problem compound of a possiblecould be source a better of 3-deoxyribosedonor of 3-deoxyribose, arises [16,17 ].we Therefore, recently itimplemented is necessary to a determinemodified chemical the optimal method 3-deoxyribose for 3′-deoxyinosine donor among synthesis purine nucleosides. from com- Recentmercially industrial available developments inosine (5) have (Scheme made 1) cordycepin [18]. The availabledetails of for this use synthesis as a source will of be 3- 0 0 deoxyribose.discussed later. However, 3 -deoxyinosine (3 -dIno, 9) is likely to perform better as a substrate in the synthesis of new nucleosides. To test which compound could be a better donor of 3-deoxyribose, we recently im- plemented a modified chemical method for 30-deoxyinosine synthesis from commercially available inosine (5) (Scheme1)[18]. The details of this synthesis will be discussed later. The first trial syntheses of 2-fluorocordycepin using purine nucleoside phosphorylase (PNP) (Scheme2) yielded encouraging results: the conversion of 2-fluoroadenosine into the target product reached 67% after 7 days from the start of the reaction. In this synthesis, 2-fluoroadenosine (2-F-Ado) rather than 2-fluoroadenine (2-F-Ade) was used as the base donor. This choice was dictated by the extremely low solubility of 2-fluoroadenine in water (less than 0.5 mM at 50 ◦C) [18]. Biomolecules 2021, 11, x FOR PEER REVIEW 3 of 17 Biomolecules 2021, 11, 539 3 of 17 Biomolecules 2021, 11, x FOR PEER REVIEW 3 of 17 Scheme 1. The synthesis of 3′-deoxyinosine (9). The first trial syntheses of 2-fluorocordycepin using purine nucleoside phos- phorylase (PNP) (Scheme 2) yielded encouraging results: the conversion of 2- fluoroadenosine into the target product reached 67% after 7 days from the start of the reaction. In this synthesis, 2-fluoroadenosine (2-F-Ado) rather than 2-fluoroad- enine (2-F-Ade) was used as the base donor. This choice was dictated by the ex- SchemetremelyScheme 1. 1.lowThe The synthesissolubility synthesis of of 30-deoxyinosine 32-fluoroadenine′-deoxyinosine (9). (9 ).in water (less than 0.5 mM at 50° C) [18]. The first trial syntheses of 2-fluorocordycepin using purine nucleoside phos- phorylase (PNP) (Scheme 2) yielded encouraging results: the conversion of 2- fluoroadenosine into the target product reached 67% after 7 days from the start of the reaction. In this synthesis, 2-fluoroadenosine (2-F-Ado) rather than 2-fluoroad- enine (2-F-Ade) was used as the base donor. This choice was dictated by the ex- tremely low solubility of 2-fluoroadenine in water (less than 0.5 mM at 50° C) [18]. Scheme 2. The synthesis of 2-fluorocordycepin (3). Scheme 2. The synthesis of 2-fluorocordycepin (3). However, when we scaled the synthesis up to hundreds of milligrams of 2-fluorocordycepin, an unexpected slowdown of the reaction and a decrease in the conversion of 2-F-Ado into 2-fluorocordycepin down to 5% were encountered. Although the PNP worked normally, the mixture contained an unexpectedly small quantity of 3-deoxyribose phosphate (judging by the quantity of hypoxanthine, 5.45%), while 2-fluoroadenine (2-F-Ade) was the main product of the reaction (Figure S1). In addition, another nucleoside was found in the reaction mixture (FigureScheme S1, 2. RTThe = synthesis 4.802 min), of [M2-fluorocordycepin + H]+ = 241.0923 (Figure(3). S2). Biomolecules 2021, 11, 539 4 of 17 Therefore, it was crucial to determine what disrupts the normal course of the transgly- cosylation reaction, what is the nature of the byproduct, and whether it is associated with the work of PNP.