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Nucleoside Di- and Triphosphates As a New Generation of Anti-HIV Pronucleotides

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Nucleoside Di- and Triphosphates As a New Generation of Anti-HIV Pronucleotides applied sciences Review Nucleoside Di- and Triphosphates as a New Generation of Anti-HIV Pronucleotides. Chemical and Biological Aspects Marta Rachwalak 1,2,* , Joanna Romanowska 1, Michal Sobkowski 1 and Jacek Stawinski 1,3,* 1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna´n,Poland; [email protected] (J.R.); [email protected] (M.S.) 2 Mellon College of Science, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA 3 Arrhenius Laboratory, Department of Organic Chemistry, Stockholm University, S-10691 Stockholm, Sweden * Correspondence: [email protected] (M.R.); [email protected] (J.S.); Tel.: +14-12-268-5453 (M.R.); +48-61-852-8502 (ext. 1549) (J.S.) Abstract: This review provides a short account of the chemical synthesis of nucleoside di- and triphosphates on a historical background, together with the use of this class of compounds as potential pronucleotides in anti-HIV therapy. Keywords: nucleoside di- and triphosphates; anti-HIV pronucleotides; synthetic strategies; biologi- cal activity 1. Introduction Citation: Rachwalak, M.; Phosphorylated nucleosides, collectively called nucleotides, play pivotal roles in Romanowska, J.; Sobkowski, M.; various biological processes. Naturally occurring deoxy- and ribonucleoside triphosphates Stawinski, J. Nucleoside Di- and are basic building blocks for the enzymatic synthesis of DNA and RNA and participate Triphosphates as a New Generation also in energy transfer processes, intracellular signaling, and the regulation of proteins’ of Anti-HIV Pronucleotides. biological activity [1]. Scientists have long been interested in synthetic nucleotide analogs Chemical and Biological Aspects. because they allow the study of complex biological systems and themselves have potential Appl. Sci. 2021, 11, 2248. https:// therapeutic and diagnostic value [2,3]. doi.org/10.3390/app11052248 Di- and triphosphates of modified nucleosides are often used as compounds of po- tential antiviral (e.g., anti-HIV) activity. The core of this class of compounds consists Academic Editor: Qi-Huang Zheng of 20,30-dideoxynucleoside 50-triphosphates (ddN, e.g., AZT, d4T, ddI, ddC, 3TC, FTC), which are inhibitors of reverse transcription (RT) during HIV replication [4,5]. However, Received: 2 February 2021 direct administration of such compounds to patients is pointless, because as negatively Accepted: 26 February 2021 charged molecules they are not effectively transported through the cell membrane [6], Published: 4 March 2021 which practically precludes their bioavailability [7–9]. The simplest solution would be to administer their prodrugs, dideoxynucleosides, which in vivo would be phosphorylated Publisher’s Note: MDPI stays neutral to the corresponding triphosphates. Although this approach works in many cases (e.g., with regard to jurisdictional claims in AZT), it turned out that some dideoxynucleosides (e.g., ddU), which are precursors of the published maps and institutional affil- very potent anti-HIV compounds (e.g., ddU 50-triphosphate), are practically not phospho- iations. rylated to nucleoside 50-monophospate (NMP) in the cell. Such dideoxynucleosides, when administered in the form of free nucleosides, usually showed no anti-HIV activity [4,5,7,8]. Moreover, AZT also suffers several drawbacks associated with the unfavorable kinetics of its successive in vivo phosphorylations (leading in consequence to serious adverse effects Copyright: © 2021 by the authors. of the AZT therapy) [10] and the existence of the thymidine kinase-deficient cells, in which Licensee MDPI, Basel, Switzerland. AZT is not phosphorylated and remains as an inactive prodrug prior to clearance (such This article is an open access article cells become reservoirs of viruses inaccessible for the therapy) [11]. Therefore, knowing distributed under the terms and the limitations of the use of 20,30-dideoxynucleosides as a potential anti-HIV therapeutics, conditions of the Creative Commons the pronucleotide idea was born. Attribution (CC BY) license (https:// Pronucleotides in their basic concept are electrically neutral phosphate derivatives creativecommons.org/licenses/by/ of biologically active nucleosides, in which protective groups on the phosphate residues 4.0/). Appl. Sci. 2021, 11, 2248. https://doi.org/10.3390/app11052248 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 30 Appl. Sci. 2021, 11, 2248 2 of 28 Pronucleotides in their basic concept are electrically neutral phosphate derivatives of biologically active nucleosides, in which protective groups on the phosphate residues neu- neutralize electrical charges. In such form, phosphorylated nucleosides can easily pen- tralize electricaletrate charges. cell membranes.In such form, Inside phosphorylated the cell, the nucleosides masking groups can easily are removedpenetrate via enzymatic cell membranes.and/or Inside chemical the cell, hydrolysis,the masking which groups lead are to removed the release via of enzymatic the corresponding and/or nucleoside chemical hydrolysis,50-monophosphate which lead to (ddNMP). the release Then, of the cellular corresponding kinases in nucleoside a stepwise 5 manner′-mono- phosphorylate phosphate (ddNMP).ddNMP Then, ultimately cellular to kinases its 20,30 in-dideoxynucleoside a stepwise manner 5 phosphorylate0-triphosphate (ddNTP),ddNMP an anti-HIV ultimately to itsactive 2′,3′-dideoxynucleoside species (Scheme1)[ 95,′12-triphosphate,13]. (ddNTP), an anti-HIV active spe- cies (Scheme 1) [9,12,13]. Scheme 1. A general principle of the monophosphate pronucleotides mode of action. Scheme 1. A general principle of the monophosphate pronucleotides mode of action. Initially, whenInitially, developing when the developing concept of thepronucleotides, concept of pronucleotides, it was assumed it as was an assumedabso- as an abso- lutely necessarylutely condition necessary that condition the electric that charges the electric in the charges prodrug in the molecule prodrug moleculewere com- were completely pletely masked.masked. Following Following this paradigm, this paradigm, various various pronucleotide pronucleotide strategies strategies were were pro- proposed, which posed, which werewere based based mainly mainly on on esters esters or or amides amides as aspotential potential protective protective groups groups for for5′- 50-phosphate phosphate moietymoiety of ddNMP of ddNMP (e.g., (e.g., studies studies of McGuigan of McGuigan [11,14,15], [11,14 Imbach,15], Imbach [16–19], [16 Meier–19], Meier [20,21], [20,21], KraszewskiKraszewski [22,23], [ 22and,23 others], and othersgroups). groups). However, However, over the over years, the it years, has been it has ob- been observed served that certainthat certaintypes of types pronucleotides of pronucleotides endowed endowed with negative with negative charges charges could also could ef- also effectively fectively penetratepenetrate cell membranes, cell membranes, and showed and showed high biological high biological activity activity (e.g., the (e.g., research the research of the of the WagnerWagner [24–28] and [24– 28Kraszewski] and Kraszewski groups [2 groups3,29,30]). [23 This,29,30 challenged]). This challenged the validity the of validity of the the generally acceptedgenerally postulate accepted of postulate electrical of neutrality electrical as neutrality the basic as criterion the basic for criterion the effec- for the effective tive transport transportof pronucleotides of pronucleotides through cell through membranes, cell membranes, but at the same but at time the sameit opened time it opened up up new possibilitiesnew possibilities for extending for the extending spectrum the of spectrum the designed of the pronucleotides designed pronucleotides to include to include new new ionic structuralionic structural features. features. The pronucleotideThe pronucleotidestrategy for delivering strategy monophosphorylated for delivering monophosphorylated antiviral nucleosides antiviral nucleosides to the cell did not,to the however, cell did completely not, however, solve completely the problem solve of the the effective problem in of vivo the genera- effective in vivo gen- tion of an activeeration metabolite, of an activeddNTP. metabolite, Sometimes ddNTP. the enzymatic Sometimes phosphorylation the enzymatic of phosphorylation 2′,3′- of dideoxynucleoside20,30-dideoxynucleoside monophosphates to monophosphates di- and ultimately to to di- triphosphates and ultimately is very to triphosphates diffi- is very cult or has undesirabledifficult or kinetics. has undesirable Typically, kinetics. such problems Typically, arise such during problems the arise conversion during theof conversion of a monophosphorylateda monophosphorylated nucleoside into nucleoside its corresponding into its corresponding diphosphate. For diphosphate. example, AZT For example, AZT is a good substrateis a good for thymidine substrate forkinase, thymidine similarly kinase, to natural similarly thymidine, to natural but thymidine, thymidylate but thymidylate kinase convertskinase AZTMP converts into AZTDP AZTMP much into AZTDPless efficiently much lessthan efficiently TMP into than TDP TMP [10]. intoThus, TDP [10]. Thus, in such situationsin such it would situations be advantageous it would be advantageous to deliver the to pronucleotides deliver the pronucleotides to the cells toin the cells in the the form of suitablyform of protected suitably protecteddiphosphates diphosphates of antiviral of antiviralnucleosides nucleosides
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