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Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates 13.1.6

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Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates 13.1.6 Overview of the Synthesis of Nucleoside UNIT 13.1 Phosphates and Polyphosphates Phosphorylated nucleosides play a domi- ity to the synthesis. Side reactions can occur, nant role in biochemistry. Primary metabolism, such as depurination of the nucleoside, phos- DNA replication and repair, RNA synthesis, phorylation of the nucleobase, as well as chemi- protein synthesis, signal transduction, polysac- cal alteration of nucleobase analogs. Due to charide biosynthesis, and enzyme regulation their intrinsic reactivity, the synthesis of phos- are just a handful of processes involving these phoanhydride bonds is also synthetically chal- molecules. Literally thousands of enzymes use lenging. Phosphate anhydrides are phosphory- these compounds as substrates and/or regula- lating reagents that are readily degraded under tors. The need to obtain such compounds in acidic conditions. Finally, purification of syn- both labeled and unlabeled forms, as well as a thetic nucleotides can be problematic. Ionic burgeoning need for analogs, has driven the reagents, starting materials, and mixtures of development of a myriad of chemical and en- regioisomers (2′-, 3′-, 5′-phosphates) can be zymatic synthetic approaches. As chemical en- particularly difficult to separate from the de- tities, few molecules possess the wide array of sired product. densely packed functionality present in phos- In spite of the many potential difficulties phorylated nucleosides. This poses a formida- associated with nucleoside phosphorylation ble challenge to the synthetic chemist, one that and polyphosphorylation, a certain amount of has not yet been fully overcome. This overview success has been achieved in these areas. Given will address some common methods (synthetic the wealth of phosphorylating reagents avail- and enzymatic) used to construct phosphory- able, simple phosphorylation of nucleosides at lated nucleosides. Particular emphasis is placed any of the hydroxyl groups is possible, although upon the advantages, limitations, and general- selective phosphorylation at any particular hy- ity of the methods. Additionally, the synthesis droxyl group may require specific protection of phosphorylated analogs such as phosphoryl- strategies. With sufficient attention to the na- sulfates, phosphoramidates, phosphonates, ture of the nucleobase and protecting groups, thiophosphates, and imidophosphates will be the correct choice of phosphorylating agent addressed. This overview is not intended as a should lead to a successful and high-yield syn- comprehensive review, but rather as a guide to thesis. The synthesis of nucleoside diphos- general principles associated with the synthesis phates (NDPs) can also be succesfully per- of phosphorylated nucleosides. formed in high yield. The excellent strategy introduced some time ago by Poulter for nu- ISSUES ASSOCIATED WITH cleophilic displacement of tosylates with tetra- NUCLEOSIDE PHOSPHORYLATON n-butylammonium salts of pyrophosphate con- In nature, the conversion of nucleosides to tinues to be a very useful and successful strat- mono-, di-, or triphosphates is accomplished by egy for simple preparation of diphosphates. specific enzyme-catalyzed reactions. Invari- While this is a multi-step procedure (particu- ably, a nucleoside hydroxyl group functions as larly if protected nucleosides are used), the a nucleophile in coupling with an electrophilic overall high yields, relative simplicity, and high phosphoryl donor (ATP, phosphoenol pyru- reliability of this method make it one of the vate, acetyl phosphate, and others). Binding most useful preparative strategies available for constraints in the active site of the enzyme the preparation of NDPs. guarantee high regio- and chemoselectivity in In contrast, the preparation of nucleoside the ensuing reaction. During chemical synthe- triphosphates (NTPs) and polyphosphate di- ses of nucleoside phosphates, the nucleoside esters remains somewhat less satisfactorily ad- can serve as either an electrophile or a nucleo- dressed. There is considerable hope in this area, phile. Despite this flexibility, chemical synthe- however. Properly activated nucleoside mono- ses are fundamentally more problematic than phosphates and their derivatives appear to be enzymatic ones. Phosphorylation procedures good acceptors for pyrophosphate and phos- using electrophilic phosphorus reagents gener- phate esters. The major problems here appear ally do not show high regioselectivity. Thus, it to be the highly variable yields reported for Nucleoside is often necessary to use protecting groups for specific methods. This may simply be a conse- Phosphorylation the sugar, adding yet another level of complex- quence of the idiosyncratic nature of nucleoside and Related Modifications Contributed by David C. Johnson II and Theodore S. Widlanski 13.1.1 Current Protocols in Nucleic Acid Chemistry (2003) 13.1.1-13.1.31 Copyright © 2003 by John Wiley & Sons, Inc. Supplement 15 vinyl amine nucleophiles ureido nucleophiles O O O O E E E NH H NH E NH E N N O N O N O N O aryl amine nucleophiles ureido nucleophiles E H N NH2 NH2 O N N E N [Ox] N O N O N O Figure 13.1.1 Some side reactions of nucleobases. chemistry (in that different nucleosides are in the nucleobases. Many bases also contain chemically very different), or may be related to nucleophiles such as lactam, ureido, and the inherent difficulty in the reactions them- guanidinium moieties. These moieties have dif- selves. Many laboratories that use these meth- ferent ionization characteristics. For this rea- ods are not ideally set up for organic synthesis son, Hayakawa and co-workers (Uchiyama et or have little experience in doing this kind of al., 1993) have grouped the common nucleo- chemistry. Developing robust chemistry for bases into two different categories based upon polyphosphate synthesis that can be routinely their acidity. In group I nucleosides, the sugar performed in a minimum of time, with a mini- hydroxyls are the most acidic functionality pre- mum number of steps, remains a daunting task sent. In group II nucleosides, the nucleobase for nucleoside chemists. has functionality more acidic than the sugar hydroxyls. Adenosine and cytidine belong to CHEMICAL REACTIVITY OF group I, whereas guanosine, thymidine, and NUCLEOSIDES uridine belong to group II. Unfortunately, these groupings do not directly correlate with the Nucleosides as Nucleophiles nucleophilicity of the nucleobase functionality. Both components of nucleosides, the sugar Nucleobases have different nucleophilicity to- and nucleobase, possess nucleophilic function- wards electrophiles, even under identical reac- ality. The hydroxyl groups present in the sugar tion conditions. Thus, many phosphorylation moiety of nucleosides are more acidic and less procedures rely upon protecting groups (UNIT nucleophilic than corresponding primary or 2.1) to facilitate regioselective phosphorylation. secondary alcohols in non-nucleosides. The sugar component of a nucleoside ionizes in the Nucleosides as Electrophiles pH range of 12 to 13 for ribo- and deoxyribonu- Nucleosides can be phosphorylated by a cleosides (Albert, 1973). The reduced nucleo- displacement reaction between phosphate and philicity of sugar hydroxyl groups may permit an electrophilic carbon of a nucleoside. To nucleophilic moieties in the nucleobase to com- render a carbon electrophilic, the hydroxyl pete for electrophiles. Accordingly, under- group must be converted into a leaving group standing the chemical properties of nucleo- of some kind (e.g., a halogen or sulfonate ester). bases is essential for implementing successful There are two common approaches to convert syntheses of nucleoside phosphates. the 5′-hydroxyl into an electrophile: (1) con- Overview of the The traditional nucleobases, as well as com- version to a relatively stable leaving group, or Synthesis of mon derivatives, share chemical functionality (2) in-situ activation of the 5′-hydroxyl. Nucleoside of similar reactivity (Fig. 13.1.1). Aryl and Phosphates and Polyphosphates vinyl amines are common nucleophiles present 13.1.2 Supplement 15 Current Protocols in Nucleic Acid Chemistry O O O O O O P OPOH O P OPOPOH O O O O O O O O O O B B HO P O P OPO B HO P OPO O 1 TsO O 2 O O O O O O CH3CN CH3CN OH OH OH OH OH OH 3 B = Ade 7 B = Ade 4 B = Cyt 5 B = Uri 6 B = Gua Figure 13.1.2 Nucleosides as electrophiles. Displacement of tosylate (Ts) by various phosphate species. Nucleotides via stable intermediates tected nucleosides are complete in just 2 hours, Halogenation is one common method of whereas 2 to 4 days are often necessary with activating a 5′-hydroxyl group. For example, unprotected nucleosides. It is possible that the synthesis of 5′-iodides is readily achieved with vicinal diols of ribonucleosides participate in methyltriphenoxyphosphonium iodide (MTPI; H-bonding with the pyrophosphate reagent, Verheyden and Moffatt, 1970) as well as a wide thus slowing the reaction. Additionally, the variety of other reagents. However, tosylation yields for the displacement reaction are higher of the 5′-hydroxyl with toluene sulfonylchlo- for protected nucleosides (Dixit and Poulter, ride has proven to be one of the best activation 1984), suggesting that in appropriate cases it methods. Synthesis of nucleoside 5′-tosylates may be worthwhile to use protecting groups in of adenosine, guanosine, uridine, cytidine, and spite of the added synthetic steps. thymidine has been described (Davisson et al., 1987). Nucleotides via in-situ activation: The Poulter introduced the synthesis of 5′-nu-
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