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Overview of the Synthesis of UNIT 13.1 and

Phosphorylated play a domi- ity to the synthesis. Side reactions can occur, nant role in . Primary , such as of the nucleoside, phos- DNA replication and repair, RNA synthesis, phorylation of the , as well as chemi- synthesis, , polysac- cal alteration of nucleobase analogs. Due to charide , and 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 use lenging. 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 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 . densely packed functionality present in phos- In spite of the many potential difficulties phorylated nucleosides. This poses a formida- associated with nucleoside 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- , 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 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 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 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 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 , 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 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 .

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 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. and belong to CHEMICAL REACTIVITY OF group I, whereas , , and NUCLEOSIDES 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 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 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 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 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- Mitsunobu reaction cleoside mono-, di-, and triphosphates by dis- The Mitsunobu reaction (Fig. 13.1.3) exem- placement of the tosylate leaving group (Dav- plifies in-situ electrophilic activation of an al- isson et al., 1987; Wu et al., 2003; also see UNIT cohol functional group (Kimura et al., 1979). 13.2). Displacement of tosylate by bis(tetra-n- Activation is achieved by conversion of butylammonium) phosphate or tris(tetra-n- S.8 to the corresponding alkoxyphosphonium butylammonium) pyrophosphate (S.1) gives salt (S.9). In-situ displacement of the alk- the corresponding nucleoside 5′-phosphate or oxyphosphonium salt by dibenzylphosphate 5′-diphosphate (S.3-S.6; Fig. 13.1.2), respec- gives triester S.10 and triphenyl- tively. Similarly, displacement of tosylate by phosphine oxide (S.11). This reaction is driven tetrakis(tetra-n-butylammonium) triphosphate by the formation of triphenylphosphine oxide (S.2) gives the nucleoside 5′-triphosphate. and a strong phosphate ester bond. Deprotec- However, this latter reaction has only been tion via hydrogenolysis of the benzyl protect- useful for the synthesis of ATP (S.7; Burgess ing groups affords the nucleoside monophos- and Cook, 2000). phate. Protected nucleosides can be To successfully execute a displacement re- phosphorylated in dioxane or tetrahydrofuran action, care must be exercised in preparation (THF), usually at an elevated temperature and handling of the hygroscopic tetra-n-butyl- (60°C). However, phosphorylation of unpro- phosphate salts. One nice feature tected pyrimidine nucleosides is usually con- of displacement reactions is that either pro- ducted at room temperature in polar aprotic tected or unprotected nucleosides can be phos- such as N,N-dimethylformide (DMF) phorylated. Ketal-protected nucleosides com- or hexamethylphosphorous triamide (HMPT), bine with tris(tetra-n-butylammonium) pyro- in yields exceeding 75% (Kimura et al., 1979). Nucleoside Phosphorylation phosphate much more rapidly than their Syntheses involving nucleosides in and Related unprotected counterparts. Reactions with pro- polar aprotic solvents are complicated by intra- Modifications 13.1.3

Current Protocols in Nucleic Acid Chemistry Supplement 15 O O

EtO NN OEt Ph O O H HO B Ph P O B BnO P O B O O PPh3 O (BnO)2 P O O Ph OBn + Ph P Ph DMF (B = U, C, T) Ph OH OH (H) or OH OH (H) OH OH (H) Pyr (B = A, G) 891011

Figure 13.1.3 Electrophilic activation of nucleoside alcohols. The Mitsunobu phosphorylation. Bn, benzyl; DMF, N,N-di- methylformamide.

NH2

N N O N N (BnO)2 P O O

OH OH

12

Figure 13.1.4 The N3,5′-cyclonucleoside salt of adenosine, the product of the Mitsunobu reaction in polar aprotic . Bn, benzyl.

molecular cyclization, forming N3,5′-cyclonu- and reaction conditions dictate the site of alky- cleoside salts (Fig. 13.1.4). Formation of the lation. These criteria are especially important purine cyclonucleosides can be avoided by con- for three-coordinate compounds of phospho- ducting the phosphorylation reaction in anhy- rus, which will be considered first. drous pyridine (Saady et al., 1995a). Phospho- Phosphites are three-coordinate (σ3) phos- rylation of nucleosides with modified purine phorus species containing three total bonds (λ3) nucleobases (e.g., N6-chloro- and N6-azido- and are exceptionally useful for nucleoside ) are successful in pyridine. However, chemistry. Phosphites exist as either a configu- anhydrous pyridine is not a suitable solvent for rationally stable structure or a pair or phosphorylation of unprotected pyrimidine nu- tautomeric structures, depending upon the ex- cleosides, as low yields are observed (Kimura tent of esterification (Fig. 13.1.5). Trialkyl et al., 1979). Finally, the Mitsunobu reaction is phosphites are configurationally stable struc- unsuitable for nucleoside 3′-phosphorylation tures characterized by a nucleophilic phospho- due to competing intramolecular cyclization, rus and are incapable of tautomerizing to an resulting in the formation of anhydronu- H-phosphonate form. These species are easily cleosides. oxidized by electrophilic oxidants and are sub- ject to air oxidation as well. In contrast, both CHEMICAL REACTIVITY OF dialkyl phosphites (S.15) and monoalkyl PHOSPHORUS phosphites (S.16) exist primarily in the four- coordinate H-phosphonate (σ4,λ5) . Phosphorus as a Nucleophile The H-phosphonate tautomer is resistant to Overview of the Phosphorous acids and ethers are ambident oxidation. Therefore, oxidation of four-coordi- Synthesis of nucleophiles (Fig. 13.1.5), reactive on phos- nate H-phosphonates proceeds by way of the Nucleoside Phosphates and phorus or . Accordingly, the phosphorus three-coordinate phosphite tautomer. In con- Polyphosphates coordination number, degree of esterification, 13.1.4

Supplement 15 Current Protocols in Nucleic Acid Chemistry ambident nucleophiles

O O [Ox] O Pyr, AcCl OR RO P OR′′ RO P OR′′ RO P R′′ OP OR′ OR′ OR′ OR′

13 R = R′= alkyl; R′′ = H 14 R = R′= alkyl; R′′ = H 15 R = R′ = alkyl; R′′= H 17 R = R′= alkyl 16 R = alkyl; R′ = R′′ = H

Figure 13.1.5 The principle reactive species of phosphites.

phosphoramidite ( σ3, λ3) phosphoramidate (σ4, λ5)

RO O N PN RO P N RO OR

18 R = alkyl 19

Figure 13.1.6 Examples of electrophilic amino-phosphorus compounds important in nucleoside chemistry. trast to oxidation, O-alkylation occurs primar- nucleotide synthesis (see Nucleosides via sta- ily through the H-phosphonate tautomer. ble intermediates). Acylation of H-phosphonates is affected by the strength of the base used. In the presence of Phosphorus as an Electrophile weak bases such as pyridine, mono- and dialkyl There are two coordination states common phosphites are typically O-acylated forming to electrophilic phosphorus, the phosphite O-acylphosphite ester intermediates (S.17). (three-coordinate) and phosphate (four-coordi- Transesterification of O-acylphosphite esters nate) states. Two important amine-containing readily occurs in alcoholic solvents. However, phosphorus compounds that can function as in the presence of a strong base, competitive electrophiles are phosphoramidites (σ3,λ3) and P-acylation of the phosphonates can occur, phosphoramidates (σ4,λ5) (Fig. 13.1.6). Phos- forming acylphosphonates (UNIT 3.4). Acylphos- phoramidites are three-coordinate species with phonates readily degrade without undergoing sigma bonds to two alkoxy groups and an amino transesterification. functionality. Phosphoramidates are four-coor- Another phosphorus nucleophile is the four- dinate species containing two alkoxy groups coordinate, five-bond (σ4,λ5) phosphoric acid. and an amino group bound to a phosphoryl Unlike H-phosphonates, phosphoric acid is ex- group. The amino moiety of these electrophilic clusively O-alkylated. However, O-alkylation phosphorus species has a P-N bond that is of phosphoric acid has found only modest ap- considerably weaker than the P-O bonds (70 plication in the synthesis of nucleoside phos- versus 86 kcal/mol; Corbridge, 1995). Further, phates, due to the limited solubility of phospho- the amino moieties retain their basicity due to ric acid in organic solvents. Biphasic solvent weak pπ-dπ bonding interactions. The amino systems and phase-transfer catalysts have been moieties can become activated under acidic used to facilitate phosphorylation. Alkylam- conditions to become good leaving groups. Nucleoside monium salts of phosphoric acid are quite sol- However, the basicity of the amino group can Phosphorylation and Related uble in organic solvents and are often used in be affected by delocalization of the Modifications 13.1.5

Current Protocols in Nucleic Acid Chemistry Supplement 15 N N HN N NN NN RO RO N RO N 20a N PN PNH 20b PN N RO RO NN path a RO N

18 21 22

Nuc OH Nuc OH path b RO PONuc RO

23

Figure 13.1.7 Mechanistic rationale of N,N-diisopropyl dialkyl phosphoramidite coupling with a nucleoside.

O O O O R′OH R′OH RO P OPOR RO P N RO P OR′ OR OR N OR OR

24 25 26

Figure 13.1.8 Tetrakis(alkyl) break down in pyridine to active phosphorylating agents.

O O O O O O RO P OH Nuc P O B RO P OPO B RO P Nuc HO P O B + O O + O Nuc Pα Nuc Pβ OR O OR O OR O

OH OH OH

28 pKa ≅ 2 29 27 30 31 pKa ≅ 7

Figure 13.1.9 Selectivity in nucleophilic attack upon tris(substituted) nucleoside pyrophosphates.

Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates 13.1.6

Supplement 15 Current Protocols in Nucleic Acid Chemistry lone pair into aryl substitution on the amine, by nucleophiles in tris(substituted) nucleoside engendering stability under acidic conditions. pyrophosphates (Fig. 13.1.9). Accordingly, aliphatic amines are essential. Di- In principle, phosphates such as phosphoric isopropylamine is widely used owing to its ease acid and its cyclic-ester trimer (trimetaphos- and purity in preparation, as well as the balance phate) contain electrophilic phosphoryl centers between its chemical stability and reactivity and should undergo transesterification reac- (McBride and Caruthers, 1983). In phos- tions with alcohols. In practice, this reaction phoramidate chemistry, the amino moiety is finds limited application. However, the reaction often morpholine or imidazole. of trimetaphosphate with unprotected ribonu- Phosphoramidates can directly combine cleosides is noteworthy. Monophosphorylation with nucleophiles, but do so very sluggishly. In of either the 2′- or 3′-hydroxyl group of unpro- the presence of catalysts such as acidic azoles tected ribonucleosides (A, C, G, U) can be or divalent metals, phosphoramidates react achieved in aqueous base. The reaction requires much more readily with nucleophiles. Azoles elevated temperatures if sodium trimetaphos- such as imidazole, tetrazole, 1-methylimida- phate is used (Tsuhako et al., 1984), but can be zole, 4,5-dicyanoimidazole, and other nitro- conducted at room temperature with tris(tetra- substituted variations are useful as acidic cata- methylammonium) trimetaphosphate (Saffhill, lysts. Unlike phosphoramidates (σ4,λ5), phos- 1970). Regardless of which trimetaphosphate phoramidites (σ3,λ3) require activation by an is used, prolonged reaction times are required acidic azole (e.g., 1H-tetrazole S.20a, pKa = (up to 4 days). Aside from the prolonged reac- 4.76) to form two electrophilic intermediates: tion times, the reaction suffers from a lack of protonated phosphoramidite S.21 and tetra- regioselectivity. Mixtures of 2′-monophos- zolide S.22 (Fig. 13.1.7). Protonated phos- phate and 3′-monophosphate nucleosides are phoramidite S.21 can react with tetrazole anion obtained. Interestingly, S.20b to form a tetrazolide intermediate (path are not phosphorylated by trimetaphosphate. a) or can directly capture a nucleoside (path b). Interestingly, breakdown of phosphoramidite REAGENTS USED IN SYNTHETIC S.18 is zero order in alcohol and second order NUCLEOTIDE SYNTHESIS in tetrazole (Nurminen et al., 1998). Other common electrophilic phosphorus Phosphorus Oxychloride motifs are phosphorus halides and phosphate Phosphorylation of nucleosides with phos- anhydrides. Unlike phosphorus amines, phos- phorus oxychloride (POCl3) initially yields a phorous halides and phosphate anhydrides do nucleoside phosphorodichloridate. Phos- not require activation for rapid reaction with phorodichloridates such as S.32 (Fig. 13.1.10) nucleophiles. The general structure of a can be hydrolyzed, providing the nucleoside tetrakis(alkyl) pyrophosphate S.24 is shown in monophosphate (not shown). The reaction is Figure 13.1.8. Because the reaction of an alco- conducted in a trialkylphosphate solvent with hol with tetrakis(alkyl) pyrophosphates is slug- an excess of POCl3 at a low temperature (−5°C). gish, a base is generally used to reduce the time However, phosphorylation under these condi- of reaction. Pyridine cleaves tetrakis(alkyl) py- tions leads to mixtures of 5′- and 2′(3′)-nucleo- rophosphates at either phosphoryl center, form- side phosphates. Protection of the vicinal diols ing dialkyl phosphate and the phosphoryl- in ribonucleosides alleviates regioselectivity pyridinium species S.25, an active phosphory- problems. Thus, phosphorylation of ketal-pro- lating agent. tected nucleosides proceeds in high yield Unlike tetrakis(alkyl) pyrophosphate, in (Yoshikawa et al., 1967). Unprotected nucleo- tris(substituted) nucleoside pyrophosphates sides can be regioselectively 5′-phosphorylated (S.27; Fig. 13.1.9) either phosphoryl center (Pα by modifying the reaction conditions described or Pβ) can be attacked by a nucleophile. The above. The addition of to the phosphory- selectivity is based upon which phosphoryl lating reagent results in selective 5′-phospho- center is the better leaving group. A phosphoryl rylation of unprotected nucleosides in moder- center becomes a better leaving group when it ate to high yield (Yoshikawa et al., 1967). is part of the more acidic phosphate (e.g., S.28 Despite the acidity of the reaction medium, versus S.31). Increased electron withdrawing depurination has not been reported to be a characteristics of the alkyl substituents will significant side-reaction. make a phosphate more acidic and, hence, a Phosphorus oxychloride is also used to syn- Nucleoside Phosphorylation better leaving group. Therefore, the phosphoryl thesize nucleoside triphosphates. However, and Related center of the weaker acid (i.e., S.29) is attacked construction of nucleoside diphosphates has Modifications 13.1.7

Current Protocols in Nucleic Acid Chemistry Supplement 15 O O O O O HO P O O HO P O P O O O O HO P OPO B Cl P O B HO P O P OPO B O O O O O O O O Cl O O O HOP O OH OH OH OH OH OH O 33 32 34

Cl Cl Cl N Cl N H O H 35 O P O O 35 O PO B O O PO O OH OH 36

H2O

NTP

Figure 13.1.10 Synthesis of nucleoside triphosphates from phosphorodichloridates via trimetaphosphate intermediates.

not been reported with this reagent. To under- promoted by the acidity of the reaction me- stand why, it is beneficial to understand the dium. Unexpectedly, the addition of the mechanistic rationale behind the synthesis of sponge reduced the reaction time for phospho- nucleoside triphosphates using POCl3. rylation from 12 hours to 2 hours (Kovacs and As shown in Figure 13.1.10, nucleoside Otvos, 1988). Finally, the POCl3 methodology triphosphates are synthesized by adding a is not particularly suitable for selectively intro- tributylamine/DMF solution of either inorganic ducing radiolabels at the Pβ or Pγ phosphoryl ortho-phosphate (Mishra and Broom, 1991) or of nucleoside triphosphates. This is because pyrophosphate (Ludwig, 1987) to the nucleo- of trimetaphosphate S.36 is nonse- side phosphorodichloridate S.32. ortho-Phos- lective. Hydrolysis can occur at either the Pβ or phate or pyrophosphate displaces chloride from Pβ′ position, leading to a mixture of products. nucleoside phosphorodichloridate S.32, form- ing intermediate S.33 or S.34, respectively. Phosphorochloridites and Excess POCl3 present in the reaction medium Phosphorochloridates combines with DMF to form the Vilsmeier In general, both phosphorochloridite reagent (S.35), which promotes the dehydra- [(RO)2PCl] and phosphorochloridate tion of intermediates S.33 and S.34, resulting [(RO)2POCl] reagents are O-protected. There in the formation of nucleoside 5′-trimetaphos- are many protecting groups that have been phate (S.36; Glonek et al., 1974). Hydrolysis developed. In the case of phosphorochloridite of the trimetaphosphate intermediate under reagents, the protecting groups are removed buffered conditions gives the NTP as a stable after oxidation of the phosphite triester. There- product. fore, protecting groups need to be stable to Phosphorylation of nucleosides containing oxidants such as peroxides, organic peracids, non-natural nucleobases is possible with the and molecular halogens. Protecting groups POCl3/trialkylphosphate reagent combination have been developed that are labile to acid, (Nairne et al., 2002). However, modifications base, or alternate conditions (e.g., reductive of the nucleobase that are sensitive to acid (e.g., fission or photolysis). The functionality inher- Overview of the alkynyl substituents) are not always compatible ent in the nucleoside will determine which Synthesis of with the POCl3 reagent. Addition of the proton is most suitable for any given Nucleoside Phosphates and sponge 1,8-bis(dimethylamino)naphthalene application. Nucleosides with modifications in Polyphosphates has been reported to mitigate side-reactions the nucleobase require the most judicious 13.1.8

Supplement 15 Current Protocols in Nucleic Acid Chemistry B B B PGO O (EtO)2PCl PGO O I2/H2O PGO O

37 38 39 OH OPGO OPG O OPG P EtO PO EtO OEt OEt

I2

PGO B 1) B O O HO O

40 41 O OPG 2) Br2 O OH 3) base O P I 4) deprotection HO PO OEt O

Br

Figure 13.1.11 Phosphitylation as a method for NMP synthesis. PG, protecting group. choice of protecting group, as the common triester is purified by silica-gel chromatogra- nucleobases (A, C, T, G, U) are fairly compat- phy. Including triethylamine (1% to 2%) in the ible with many deprotection conditions. eluent, however, usually prevents significant Phosphorochloridite reagents can be used to decomposition of the phosphite triester during synthesize nucleoside monophosphates. The purification. Last, phosphite triesters can air- general strategy is outlined in Figure 13.1.11. oxidize, which is essentially an irreversible Phosphitylation of protected nucleoside S.37 chemical modification. with diethyl phosphorochloridite produces the Phosphorochloridites can be used to synthe- phosphite triester S.38. Oxidation of the size nucleoside triphosphates as well. In Figure phosphite triester is usually effected by perox- 13.1.12, phosphitylation of a protected nucleo- ide, organic peracid, or aqueous . Follow- side (S.42) with salicyl phosphorochloridite ing oxidation, the trialkyl phosphate S.39 is (S.43) is carried out in pyridine/dioxane or then deprotected to provide the nucleoside mo- pyridine/DMF. The phosphite triesters pro- nophosphate (not shown). However, phosphite duced are diastereomers and are not isolated; intermediate S.38 can be oxidized with iodine rather, transesterification of the salicyl moiety in situ to give halophosphate S.40. Various is effected by adding a solution of bis(tri-n- nucleophiles can be trapped by S.40, and ap- butylammonium) pyrophosphate/tributyl- propriate deprotection then affords the nucleo- amine in DMF. Complete transesterification side diester S.41. This dual phosphityla- leads to the formation of the cyclic phosphite tion/phosphorylation strategy is useful for species S.45. Oxidation of phosphite S.45 with preparation of nucleoside diesters that are in- aqueous iodine produces nucleoside 5′-trimeta- accessible by standard phosphoramidite meth- phosphate. Concomitant hydrolysis of the nu- odology, such as nucleoside- diester S.41 cleoside 5′-trimetaphosphate yields protected (Stowell and Widlanski, 1995). nucleoside triphosphates as the product. The The synthesis of nucleoside monophos- ester protecting groups used to protect the nu- phates via phosphorochloridites is conceptu- cleoside are removed by aminolysis. By this ally straightforward, but can be experimentally process, TTP was synthesized from the corre- challenging. First, phosphorochloridites are sponding protected nucleoside in 72% yield air-sensitive reagents that are usually prepared (Ludwig and Eckstein, 1989). in low yield and purified by vacuum distillation. Phosphorochloridite reagents are known to Nucleoside Second, phosphite triesters are labile towards phosphitylate nucleobases at ambient tempera- Phosphorylation − ° and Related acid. This can be problematic if the phosphite tures but not at low temperatures ( 78 C; Imai Modifications 13.1.9

Current Protocols in Nucleic Acid Chemistry Supplement 15 O

O O O O O P O O P O O Cl HO P OPOH 1) I /H O HO T P O T O PO T 2 2 O 43 O O O O 2) NH3 O O PO TTP O OAc OAc OAc 45 42 44

Figure 13.1.12 Salicyl phosphorochloridite. A useful reagent for NTP synthesis.

and Torrence, 1981). Given that temperature is phorochloridates can be sluggish, requiring a factor in nucleobase phosphitylation, the ex- prolonged reaction times. The reaction time can perimental conditions under which salicyl be reduced through use of a nucleophilic cata- phosphorochloridite is used may promote nu- lyst such as 1-methylimidazole or iodide cleobase phosphitylation. The extent to which (Stromberg and Stawinski, 1987). These cata- salicyl phosphorochloridite may phosphitylate lysts function by forming a more electrophilic nucleobases has not been thoroughly investi- phosphorylating agent. However, increasing gated. Experimental data in the original paper the electrophilicity of the phosphorochloridate do not conclusively rule out nucleobase also results in increased nucleobase phospho- phosphitylation as a source of reduced yield. rylation. Protecting groups that are strongly As an aside, N-protected nucleobases are rec- electron withdrawing also increase the electro- ommended during construction of nucleoside philicity of the phosphorochloridate. H-phosphonates using salicyl phosphorochlo- Unlike their three-coordinate counterparts, ridite (UNIT 2.6). four-coordinate phosphorochloridates can be Nucleobase phosphitylation can be of par- used to synthesize nucleoside 3′- or 5′-mono- ticular concern when constructing nucleotides phosphates from N-unprotected nucleosides. that have nucleobase modifications. Typical Chemo- and regioselective phosphorylation yields for phosphorylation of nucleosides with can be affected through “functional group acti- modified nucleobases are <45%, with yields in vation” (Uchiyama et al., 1993). Treatment of the 15% to 30% range being common (Jurczyk an appropriate hydroxyl-protected, N-unpro- et al., 1999; Trevisiol et al., 2000). Nucleobase tected nucleoside with tert-butylmagnesium phosphitylation may also be a concern when chloride results in the formation of a nucleoside using salicyl phosphorochloridite with oli- alkoxide (Fig. 13.1.13). Group I nucleosides godeoxyribonucleotides. Recent application of (adenosine and cytidine) require only one the salicyl phosphorochloridite methodology equivalent of strong base, whereas group II on solid support provided a low yield (15% to nucleosides (guanosine, thymidine, uridine) re- 30%) of the desired 5′-triphosphate-capped oli- quire two equivalents of base. Upon treatment gonucleotide (Lebedev et al., 2001). Finally, of the alkoxide with phosphorochloridate, the due to the intermediacy of the nucleoside 5′- nucleoside-dialkyl-phosphate is obtained in trimetaphosphate, this methodology is unlikely high yield. Competition experiments demon- to be used for selective radiolabeling of nucleo- strate the preferential deprotonation of the hy- side triphosphates at the Pβ or Pγ positions. droxyl functionality over nucleobase function- Phosphorochloridates share many similari- ality for group I nucleosides. This deprotona- ties with phosphorochloridites. For example, tion is expected because the sugar hydroxyl is many of the same protecting groups are used more acidic than the nucleobase functionality. Overview of the and similar side reactions are observed. How- Chemoselectivity for group II nucleosides Synthesis of ever, phosphorochloridates provide direct ac- is not explicable based upon acidity. The 3′- Nucleoside cess to nucleoside dialkyl-phosphates without alkoxide bis-protected thymidine Phosphates and Polyphosphates the need for oxidation. Reactions with phos- S.54 (Fig. 13.1.14) is preferentially phospho- 13.1.10

Supplement 15 Current Protocols in Nucleic Acid Chemistry Group I: adenosine and cytidine O O A t BuMgCl (1 eq) A A HO O ClMgO O (EtO)2 P Cl (EtO)2 P O O THF OO OO OO

46 47 48

Group II: thymidine, uridine and guanosine

T T O T TBSO O TBSO O TBSO O tBuMgCl (1 eq) (EtO)2 P Cl

THF 49 OH 50 OMgCl 53 O

(EtO)2 P O

O MgCl O TBSO T TBSO T P (OEt)2 O (EtO)2 P Cl O

51 OH 52 OH

Figure 13.1.13 Grignard-promoted chemoselective based upon acidity (group I) or reversibility (group II). TBS, tert-butyldimethylsilyl.

O O MgCl O CH CH3 N 3 N N O N O N O N O TBSO TBSO (EtO)2 P Cl TBSO O O O + (0.1 eq)

OMgCl OTBS (75%) O EtO P O 1 eq 50 eq OEt 54 55 56

Figure 13.1.14 Competition experiment establishing alkoxide as the more reactive phosphorochloridate acceptor. TBS, tert-butyldimethylsilyl. rylated by a limiting amount of diethyl phos- version provides exclusively the O-phosphory- phorochloridate in the presence of a 50-fold lated product S.53 after 96 hr. excess of N5-magnesium iminoxide bis-pro- tected thymidine S.55. This result corresponds Phosphoramidites and to the more basic magnesium alkoxide being Phosphoramidates phosphorylated in preference to the weaker Phosphoramidites (σ3,λ3) can be used for magnesium iminoxide base. Interestingly, the synthesis of NMPs, although their most treatment of 5′-protected thymidine S.49 with popular application is found in the synthesis of one equivalent of base results in phosphoryla- oligodeoxyribonucleotides (UNIT 3.3). Unlike tion of the iminoxide functionality (Fig. phosphoramidates (σ4,λ5), the construction of Nucleoside 13.1.13). However, formation of phosphorami- a nucleotide from phosphoramidites requires a Phosphorylation and Related date S.52 is reversible. Intermolecular intercon- minimum of two synthetic steps. The first step Modifications 13.1.11

Current Protocols in Nucleic Acid Chemistry Supplement 15 Classic phosphorimidazolidate approachÐsuitable for dNTP synthesis O O O O O P OPOH T N T HO P O O CDI N P O O O O O O TTP

57 OH 58 OH

Reverse approachÐsuitable for NTP synthesis

O N 61 H3CO P N O O O O O G G O G HO P O CDI RO P O H3CO P O P O P O O O ZnCl2 O O O OOO R= H O 60 O OOH59 OH R= P O 62 OH OH OH O

Figure 13.1.15 Phosphorimidazolidates as a phosphorylation strategy. CDI, carbonyldiimidazole.

O NDP OPOH O

O O O O HO P O B PhO P OPO B O (PhO)2 P Cl O O OPh O

OH OH OH OH O O O P OPOH 63 64 O O

NTP

Figure 13.1.16 The anion exchange strategy for synthesis of NDPs and NTPs.

is the coupling of a phosphoramidite to a nu- of the nucleobase is not observed when mild cleoside, forming a nucleoside-dialkyl- oxidants are used (e.g., I2/H2O or tBuOOH). phosphite, followed by oxidation to form the Phosphoramidate NMPs are finding recent nucleoside-dialkyl-phosphate. A nucleophilic application as NMP prodrugs (Zemlicka, functionality in the nucleobase may be oxidized 2002), as well as precursors for constructing under such conditions. The N1 and N3 posi- NDPs and NTPs. A classic method for the tions of adenosine and cytidine, respectively, synthesis of dNTPs via nucleoside phos- Overview of the Synthesis of are examples of nucleobase moieties prone to phoramidates involves the activation of an Nucleoside oxidation. Fortunately, appreciable oxidation NMP with carbonyldiimidazole (CDI) to give Phosphates and the nucleoside phosphorimidazolidate S.58 Polyphosphates 13.1.12

Supplement 15 Current Protocols in Nucleic Acid Chemistry U HO O

HO U O O O O OTBS O O O O HO P O P O P S HO PO HO P O P OPS A I B 67 O O O O O S O OTBS O O O HO PO B = Ade; R = H B = Uri; R = OH S U OH OH R O

66 65 OH OH

68

Figure 13.1.17 Synthesis of nucleoside phosphorothiol esters by the displacement strategy. TBS, tert-butyldimethylsilyl.

(Fig. 13.1.15). Addition of acidic pyrophos- phoanhydride (S.64) is exposed to the alkylam- phate activates the phosphorimidazolidate to- monium salt of phosphate or pyrophosphate in ward nucleophilic displacement by pyrophos- pyridine solution to give the NDP or NTP, phate, giving the dNTP (Hoard and Ott, 1965). respectively. The reaction is quite selective for This method is rarely used for the synthesis of the displacement of diphenylphosphate from triphosphates. Ribonucleosides the mixed phosphoanhydride S.64. The pres- with unprotected vicinal diols (S.59) readily ence of the anionic oxygen on the Pα of S.64 form cyclic carbonates (S.60) when treated renders the nucleoside phosphate a weaker acid with CDI. Additionally, nucleoside phos- than diphenylphosphate. The product pyro- phorimidazolidates can react sluggishly with phosphate is resistant to cleavage because the phosphate or pyrophosphate, requiring pro- product possesses relatively poor leaving longed reaction times or the use of catalysts groups. such as 1-methylimidazole or divalent cations (MnII, CdII). Reversal of the general strategy, NUCLEOTIDE ANALOGS such that the NDP is the nucleophile and the Analogs may be derived by modification of phosphorimidazolidate (e.g., S.61) is the elec- the sugar, base, or phosphorus component of a trophile, results in a high yield of the desired nucleotide. Phosphate analogs have great util- NTP without the need for long reaction times. ity for studying the mechanism and stereospe- This strategy has been used with success for the cificity of enzyme-catalyzed reactions and for synthesis of Pγ-methyl-capped guanosine 5′- investigating biochemical pathways. Sugar and triphosphates (e.g., S.62) in the presence of nucleobase analogs are also used widely as ZnCl2 (Kadokura et al., 1997). therapeutic agents and mechanistic probes. A method for synthesis of the triphosphate of Di- and Triphosphates from modified nucleosides by a variation of the Monophosphates: Use of Phosphorus Yoshikawa procedure is presented in UNIT 1.5. Anhydrides The following section will focus on method- Michelson (1964) described a general ologies and issues associated with the synthesis method termed “anion exchange” for the syn- of analogs of the phosphorus component of thesis of NDP, NTP, and P1-nucleoside-5′-P2- nucleotides. . The method is compatible with unpro- tected nucleotides and gives good yields. In Thio-Phosphorus Derivatives short, an alkylammonium NMP is phosphory- Synthesis of thio-phosphorus derivatives of Nucleoside Phosphorylation lated with diphenyl phosphorochloridate (Fig. nucleotides are particularly problematic. The and Related 13.1.16). The resultant nucleoside mixed phos- can occupy either a bridging (phos- Modifications 13.1.13

Current Protocols in Nucleic Acid Chemistry Supplement 15 Cl N P B OCE B B MMTrO O MMTrO O HO O

SH 69 S 70S 71 P HO PO N OCE OH

Figure 13.1.18 A phosphoramidite approach toward the synthesis of phosphorothiol esters. CE, cyanoethyl; MMTr, 4-monomethoxytrityl.

phorothiol ester) or a non-bridging (phos- ing phosphoramidite methodology and dis- phorothioate) position in the phosphate com- placement reactions at electrophilic 5′-. ponent of the nucleotide. To appreciate the However, the majority of bridging phos- difficulties associated with thio-phosphorus phorothiol esters are synthesized in oligonu- nucleotide synthesis, a brief discussion of the cleotides. Nevertheless, the methods should be properties of thio-phosphorus acids is war- applicable to the synthesis of nucleosides con- ranted. taining a phosphorothiol ester linkage. Thio-phosphorus acids have similar acidity Both nucleoside 5′-tosylates and 5′-iodides to the corresponding phosphate. For example, have been displaced with phosphorothioates. diethyl phosphate and O,O-diethyl phos- The reactions are usually conducted in DMF phorothioate have pKa’s of 1.37 and 1.49, re- and require prolonged reaction times (24 to 48 spectively (Quin, 2000). There are several com- hr). Both purine and pyrimidine bases have peting factors that affect the acidity of a phos- been used with or without base protection. phorothioate. Although the sulfur atom is larger Syntheses of 5′-thiotriphosphates of uridine and should stabilize the conjugate base better and adenosine have been described. Unlike the than oxygen, competing effects such as solva- synthesis of ATP, synthesis of 2′,5′-dide- tion, substituent effects, and bond strengths oxyadenosine-5′-thiotriphosphate (S.66; Fig. also affect the acidity of thio-phosphorus acids. 13.1.17) by displacement of the 5′-iodide of nucleoside S.65 was accomplished in poor Synthesis of nucleoside phosphorothiol yield (21%). A similar yield was reported for esters 5′--5′-thiotriphosphate (19%; Bridging phosphorothiol esters are attrac- Patel and Eckstein, 1997). Construction of tive as phosphate surrogates because they are dinucleotides containing a 5′- linkage achiral, thus reducing molecular complexity (e.g., S.68) have also been carried out by dis- and easing purification. Several methods have placement reactions. Yields are similar for dis- been used to access these compounds, includ- placement of 5′-tosylates or 5′-iodides by de-

O

S S O O

Overview of the 72 Synthesis of Nucleoside Phosphates and Figure 13.1.19 3H-1,2-Benzodithiol-3-one 1,1-dioxide. A soluble alternative to elemental sulfur. Polyphosphates 13.1.14

Supplement 15 Current Protocols in Nucleic Acid Chemistry O TMSO X H P O B P O B HO P O B O TMSCl O S8 O OH TMSO SH 75 X = O OH OH OH 73 74

1) I2, H2O 1) S8 2) deprotection or 2) deprotection 75 X = O 79 X = S

S S B O B HO O OPOH OPO O (EtO) P Cl 2 H + H OPG OPG

76 77 78

Figure 13.1.20 Synthesis of phosphorothioate nucleosides from H-phosphonate precursors. PG, protecting group; TMS, trimethylsilyl.

HS O HS O O O O O SH NDP O O O P P or P HO P O P OOR + HO P O P OOR HO P O P OOR OH OH OH OH OH OH

80 P(R) 81 P(S) 80 P(R)

A O R=

OH OH

Figure 13.1.21 Selective enzymatic degradation of contaminating diastereomers is a common method to separate mixtures of P(R) and P(S) triphosphorothioates. oxyribo- and ribonucleoside 3′-phos- cleotides, although this avenue has not been phorothioates, respectively (Chladek and fully explored. Nagyvary, 1972; Thomson et al., 1996). The phosphoramidite approach appears to Synthesis of nucleoside phosphorothioates be a more viable entry into 3′- or 5′-thionu- Nucleoside phosphorothioates are com- cleotides. However, most applications using monly accessed either by oxidation of trialkyl phosphoramidites are relevant to the construc- phosphites with sulfur, or by condensation with tion of containing bridging protected thiophosphoryl agents. Hydrolysis of phosphorothiol esters and not 5′-thionu- the alkyl protecting groups produces the nu- cleotides. One approach to obtain bridging cleotide phosphorothioate. phosphorothiol esters is outlined in Figure Nucleoside trialkylphosphites are com- 13.1.18 (Cosstick and Vyle, 1990). Modifica- monly accessed via phosphoramidite method- Nucleoside tion of this approach should be suitable for ology (see Phosphoramidites and Phos- Phosphorylation ′ ′ and Related high-yield syntheses of 3 - or 5 -thionu- phoramidates). Oxidation of the trialkyl Modifications 13.1.15

Current Protocols in Nucleic Acid Chemistry Supplement 15 O S S O O O P O S HO B Cl P Cl B HO P O P O O PO B O Cl P O O O Cl Cl O O O PO DMF O OH OH (H)(RO)3 P O OH OH (H) OH OH (H) 82 83 84

H2O

O O O O O O O O O HO P O P O B B HO P O (PhO)2 P OPO B HO P O P OPO O (PhO)2 P Cl O O O O S S O O S

OH OH OH OH (H) OH OH (H) 86 87 85

Figure 13.1.22 Synthesis of 5′-(1-thio)triphosphates via anion exchange or through thiophosphorochloridates.

O O O O O O O O S O O P O S 1) CEO P OPOH 1) HO P OPOH CEO P O P OPO T P O T O P O T O S O 89 O O O O S O O O PO 2 2) S8 2) S8 O OAc OAc OAc 92 90 88

1) Li S HO 2 2) NH3

O O O O O S T T HO P O P OPO O HO P O P OPO O S O S O O S

OH OH 91 93

Figure 13.1.23 Salicyl phosphorochloridite provides versatility. Chiral and achiral thio-phosphorus analogs can be synthe- sized from a common precursor. CE, cyanoethyl.

Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates 13.1.16

Supplement 15 Current Protocols in Nucleic Acid Chemistry O HO P O A O 18 O A O O O P 18 1) O O HS 18 SH O OO 1) NaIO4 O 18 A O P O P O A (PhO)2 P O P O O 95 OH OH 2) H+ O OMe HO P O HO O S 3) HO O A OH OH 2) chromatography O OH OH 94 96 P(S) 97 P(S) OO

OMe

Figure 13.1.24 Synthesis of terminal chiral phosphates of known stereochemical configuration. phosphite can be carried out with elemental An alternate procedure to access 5′-phos- sulfur. However, oxidation with elemental sul- phorothioates makes use of 9-fluorenemethyl fur is sluggish due to poor solubility of the H-phosphonothioate S.77. This reagent con- sulfur oxidant. Alternative sulfur oxidants such denses readily (<3 min) with protected nucleo- as 3H-1,2-benzodithiol-3-one 1,1,-dioxide sides in the presence of diethyl phosphorochlo- (S.72; Fig. 13.1.19) have been introduced to ridate to give nucleoside H-phosphonothioate eliminate the solubility problems associated diester S.78 (Jankowska et al., 1997). The sali- with elemental sulfur (Iyer et al., 1990). Unlike ent feature of this method is that phos- elemental sulfur, S.72 is soluble in acetonitrile, phorothioates can be accessed via aqueous io- oxidizes phosphites readily, and does not mod- dine oxidation of H-phosphonothioate S.78, or ify nucleobases on prolonged exposure (Regan phosphorodithioates (S.79) can be obtained by et al., 1992). However, the full scope and utility sulfur oxidation of S.78. The method is suitable of oxidant S.72 has yet to be assessed. It is not for many deoxyribonucleosides and gives good known, for example, whether unprotected nu- to high yields of the phosphorothioates and cleosides are compatible with this reagent. phosphorodithioates (Jankowska et al., 1998). Another method to access nucleoside phos- Although nucleoside 5′-phosphorothioates phorothioates is by oxidation of H-phosphon- are more complex to synthesize than nucleoside ates (Fig. 13.1.20). Thus, phosphorous acid can 5′-phosphates, the synthesis of nucleoside 5′- be condensed with an unprotected nucleoside di- and 5′-triphosphorothioates is even more in the presence of N,N′-ditolylcarbodiimide to complicated. Esterification of a nucleoside 5′- give predominately the nucleoside 5′-H-phos- phosphorothioate results in the formation of a phonate S.73. Silylation of the nucleoside 5′- new chiral center. The majority of chemical H-phosphonate with trimethylsilyl chloride methods available to access nucleoside 5′-di- (TMSCl) forms the intermediate silylphosphite and 5′-triphosphorothioates are nonstereose- S.74. Oxidation with elemental sulfur in pyri- lective. Therefore, a mixture of P(S) and P(R) dine then gives the nucleoside 5′-phos- diastereomers are produced during the synthe- phorothioate S.75 (Chen and Benkovic, 1983). sis. Separation of the diastereomers by conven- Although no protecting groups are necessary, tional chromatography is non-trivial. One this method is limited by the long reaction times popular strategy for obtaining a pure dias- needed for the condensation (∼3 days) and the tereomer is to enzymatically degrade the con- low yields of the nucleoside 5′-H-phosphonates taminating isomer (Fig. 13.1.21). For example, produced (30% to 64%). Additionally, some both nucleoside diphosphate kinase and hexo- Nucleoside nucleoside di-H-phosphonates are produced as kinase selectively hydrolyze the P(S) isomer of Phosphorylation byproducts. and Related Modifications 13.1.17

Current Protocols in Nucleic Acid Chemistry Supplement 15 OO OOO RO PPOH RO P P P OH OR OR OR OR OR

98 R = Bu4N 99 R = Bu4N 100 R = Bn 101 R = Bn

Figure 13.1.25 Structural variations of phosphonates make them suitable for use with either displacement strategies or Mitsunobu reaction. Bn, benzyl; Bu, butyl.

ATPαS leaving the pure P(R) isomer of ATPαS For construction of nucleoside 5′-(1,1- untouched (Eckstein, 1979). dithio)triphosphates, transesterification of nu- Despite being nonstereoselective, many cleoside phosphite triester S.88 with pyrophos- chemical methods used to synthesize nucleo- phate followed by oxidation with elemental side triphosphates are applicable to the con- sulfur leads to the formation of thiotrimeta- struction of nucleoside 5′-(1-thio)triphos- phosphate S.92. Selective cleavage of phates. Unprotected nucleosides can be mixed thiotrimetaphosphate S.92 at Pα occurs in DMF with thiophosphoryl trichloride in a trialkyl- with lithium sulfide. Selectivity is not observed phosphate solvent to form the corresponding when pyridine/dioxane is used as the solvent. thiophosphorodichloridate (S.83; Fig. Aminolysis of the protecting groups then pro- 13.1.22). Introduction of pyrophosphate to the vides thymidine 5′-(1,1-dithio)triphosphate reaction mixture results in formation of the S.93. Although the method produces S.93 in nucleoside thio-trimetaphosphate S.84. Hy- very low yield (15%), S.93 may serve as a good drolysis of thio-trimetaphosphate S.84 pro- achiral phosphorothioate derivative. duces the nucleoside 5′-(1-thio)triphosphate Frey has introduced a very elegant method S.85. This method has been applied to the for the synthesis of ATP with chiral α, β, or γ synthesis of several nucleosides, including dA, terminal phosphates of known configuration. dG, T, and U. Although it is an expedient route The phosphates are rendered chiral by contain- to nucleoside 5′-(1-thio)triphosphates, yields ing sulfur and heavy oxygen (18O). Thus, are typically low (Arabshahi and Frey, 1994). adenosine 5′-(1-thio,1-18O)diphosphorothio- Michelson’s technique of anion exchange ate, adenosine 5′-(2-thio,2-18O)diphosphoro- has also been applied to the synthesis of nucleo- thioate, and adenosine 5′-(3-thio,3-18O)tri- side 5′-(1-thio)triphosphates (Fig. 13.1.22). phosphorothioate have been synthesized (Rich- Thus, activation of AMPS and GMPS (S.86) ard et al., 1978). The basic strategy is to with diphenyl phosphorochloridate followed selectively thiophosphorylate adenosine with by displacement with pyrophosphate gives the thiophosphoryl chloride followed by hydroly- 18 ATPαS and GTPαS in reasonable yield (Chen sis with H2 O, yielding S.94 (Fig. 13.1.24). et al., 1983). Michelson’s anion-exchange reaction is used to The salicyl phosphorochloridite methodol- displace diphenylphosphate with 2′,3′- ogy has been applied to the synthesis of nucleo- methoxy-methylidene AMP (S.95), providing side 5′-(1,3-bisthio)triphosphates and nucleo- diadenosine S.96. Separation of the dias- side 5′-(1,1-dithio)triphosphates (Fig. 13.1.23; tereomers at this point ensures configurational Ludwig and Eckstein, 1991). Transesterifica- purity of the phosphorus stereocenter in the tion of nucleoside phosphite triester S.88 with product. Sodium periodate oxidation of the excess of P1-O-(cyanoethyl)-P1-thiopyrophos- unprotected vicinal diols of S.96 cascades into phate (S.89) followed by oxidation with ele- complete degradation of the adenosine moiety, mental sulfur gives the branched pentaphos- providing (2-thio)diphosphorothioate S.97. phate S.90. Selective hydrolysis along with Similar methodology is used to obtain adeno- concomitant deprotection of the cyanoethyl sine 5′-(1-thio,1-18O)diphosphorothioate and protecting group occurs under basic conditions adenosine 5′-(3-thio,3-18O)triphosphorothio- to give thymidine 5′-(1,3-bisthio)triphosphate ate. Overview of the Synthesis of S.91. Selectivity for the hydrolysis follows the Nucleoside principles outlined earlier (see Phosphorus as Phosphates and an Electrophile). Polyphosphates 13.1.18

Supplement 15 Current Protocols in Nucleic Acid Chemistry Michaelis-Arbuzov chemistry

(BnO)2POMe O OO KCN OO 103 BnO P Cl BnO PPOMe BnO PPOH OBn OBn OBn OBn OBn

102 104 105

Selenophosphonate anion chemistry

Se SeN Se 1) BnOH, tetrazole OOO Cl2PNMe2 MeO P Li MeO P P P OMe 2) mCPBA MeO P P P OMe OBn OBn OBn OBnOBn OBn

106 107 108

Figure 13.1.26 Strategies to access phosphonates. mCPBA, meta-chloroperoxybenzoic acid.

Phosphonate Derivatives rophosphonates in good yield (Dixit et al., Replacing a bridging oxygen with a 1984). Similarly, displacement of 5′-tosylates methylene functionality in pyrophosphate pro- with bis-methylene triphosphonates (S.99) duces a pyrophosphonate (Fig. 13.1.25). The give the nucleoside analogs with α-β and β-γ bond dissociation for a C-P bond is 65 bridging methylenes (Stock, 1979). Signifi- kcal/mol, which is considerably less than the cantly better yields are obtained for the dis- P-O bond dissociation energy (86 kcal/mol; placement reaction when performed in acetoni- Corbridge, 1995). Despite this, pyrophosphon- trile at room temperature, as opposed to heating ates have considerably more thermal stability in DMF, although acetonitrile requires longer than phosphoanhydrides. In addition, pyro- reaction times. The nice feature about accessing phosphonates are more resistant to hydrolysis, nucleoside phosphonates in this fashion is that a consequence arising from the reduced elec- no protecting groups are needed on either the trophilicity of the phosphoryl group when nucleoside or the phosphonate. bonded to the methylene unit. However, pyro- The Mitsunobu reaction has also been used phosphonate is neither a geometric nor an elec- in the construction of nucleoside phosphonates. tronic isostere of pyrophosphate. The oxygen- Unlike the displacement methodology, pro- oxygen nonbonded distance in a pyrophos- tected nucleosides as well as protected phos- phonate is ∼16% greater than in a phonates are usually used under Mitsunobu pyrophosphate. Pyrophosphonate is not capa- conditions. The reaction has been perfected ble of accepting a bond to the bridg- such that both purine and pyrimidine nucleo- ing atom like pyrophosphate can. Despite these sides can be used without significant competing differences, nucleoside pyrophosphonates are cyclization to the anhydro-nucleoside. Phos- used as enzymatically stable, nonhydrolyzable phorylation of protected nucleosides via the surrogates for nucleoside pyrophosphates Mitsunobu reaction gives higher yields with (Engel, 1977). tris(benzyl) pyrophosphonate (S.100) than Nucleoside di- and triphosphonates have with tetrakis(benzyl) bis-methylene triphos- been prepared either with a single bridging phonate (S.101). The yields for the phosphory- methylene unit replacing the bridging α-β or lation step with tris(benzyl) pyrophosphonate β-γ , or with total oxygen replacement are comparable to the displacement method on in these bridging positions. Direct displace- comparable nucleosides. Slightly better yields ment reactions conducted on nucleoside 5′-to- for the phosphorylation step with tetrakis(ben- Nucleoside sylates with tris(tetrabutylammonium) pyro- zyl) bis-methylene triphosphonate are ob- Phosphorylation and Related phosphonate (S.98) give the nucleoside 5′-py- served for the Mitsunobu reaction than with the Modifications 13.1.19

Current Protocols in Nucleic Acid Chemistry Supplement 15 O O O O O H O O O O O P N P OH H HO P O A (PhO) P OPO A HO P N P OPO A O (PhO)2 P Cl 2 O O O O O O O O O

OH OH OH OH OH OH

109 110 111 APPNP

O O H O O O O O O P N P OH H H A A kinase B TsO O O O HO P N P O O phosphoenol pyruvate HO P O P N P O O O O O O O

OH OH OH OH OH OH

112 113 114 APNPP

O Cl Cl P NPCl O Cl O O Cl Cl H HO A Cl P NPO A HO P N P O A O 116 O TEA-bicarb buffer O Cl Cl O O (MeO)3 PO OH OH OH OH OH OH

115 117 118

Figure 13.1.27 The common strategies used to access imidophosphate derivatives of adenosine. TEA, triethylamine.

displacement method. Deprotection of the nu- diacid form of triphosphonate S.108 is revealed cleoside benzyl-protected phosphonates is rou- upon selective demethylation with tinely performed with trimethylsilyl bromide . or catalytic hydrogenolysis. Either method seems to work equally well. Imidophosphate Derivatives Protected pyrophosphonates can be con- The chemistry of the imidodiphosphate and structed by the Michaelis-Arbuzov reaction diimidotriphosphate functional groups closely (Saady et al., 1995b) or by phosphorylation of mirrors that of the phosphoramidate functional methane selenophosphonate anions (Eymery et group (see Phosphoramidites and Phos- al., 1999). The Michaelis-Arbuzov reaction al- phoramidates). Thus, imidodiphosphates and lows ready access to tris(benzyl) pyrophos- diimidotriphosphates are stable in alkaline me- phonate (S.105; Fig. 13.1.26). However, ex- dia, but hydrolyze in acidic media (Tomasz et perimental reproducibility of the Michaelis- al., 1988). Despite its acid lability, the imido- Arbuzov reaction can be tricky. Recently, phosphate functional group has been used in selenophosphonate anion chemistry has been nucleotides as a phosphate analog. When either developed that allows efficient access to bis- the α/β or β/γ oxygen of ATP is replaced with methylene triphosphonates (Klein et al., 2002). nitrogen, the result is adenosine imidotriphos- Methaneselenophosphonate anion S.106 cou- phate, abbreviated APNPP and APPNP, respec- ples with dichlorophosphoroamidite producing tively. If both the α/β and β/γ oxygens are selenophosphoramidite S.107. Owing to the replaced by nitrogen, the result is adenosine reduced basicity of the selenophosphoryl moi- diimidotriphosphate, APNPNP. ety, competing deprotonation of the seleno- Adenosine imidotriphosphate mimics ATP Overview of the phosphoramidite intermediates is not observed. for many applications. This mimicry may arise Synthesis of Tetrazole-promoted esterification of phos- because the physical properties of the imi- Nucleoside phoramidite S.107 is followed by oxidation to dodiphosphate and pyrophosphate functional Phosphates and Polyphosphates give bis-methylene triphosphonate S.108. The groups are quite similar (Larsen et al., 1969). 13.1.20

Supplement 15 Current Protocols in Nucleic Acid Chemistry Although imidodiphosphate is not a geometric reagent, however, and yields nucleoside 5′- isostere in the truest sense, the bond angle and chlorides as byproducts. Many purine and bond lengths are very close to those of pyro- pyrimidine nucleoside-α,β-imidodiphos- phosphate. For example, the P-N-P and P-O-P phates obtained through imidophosphorylation bond angles are 127.2° and 128.6°, respec- have been converted to their triphosphate coun- tively. The bond lengths are also very similar: terparts using creatine kinase (Li et al., 1996). 1.68 Å (P-N) and 1.63 Å (P-O). Unlike a phos- Access to nucleoside-α,β-β,γ-diimido- phate ester linkage, an imidophosphate can, in triphosphates is very challenging; more so than principle, accept or donate a H-bond. However, imidotriphosphates. In fact, no satisfactory the shortened P-N bond length suggests partial method is yet available. Adenosine-α,β-β,γ-di- P=N characteristic of the imidophosphate, imidotriphosphate has been accessed by reac- which mitigates the H-bond donating and ac- tion between adenosine 5′-tosylate and the cepting abilities. Various nucleosides with the tetrabutylammonium salt of diimidotriphos- imidophosphate are inhibitors phate in acetonitrile (Ma et al., 1990). The of HIV-RT1 (Li et al., 1996) and ATP/GTP desired product was obtained in poor yield (Batra et al., 1987), but are sub- (7%) along with another diimidotriphosphate, strates for E. coli alkaline (Yount AP(NP)2 (6%). et al., 1971). Several imidophosphate derivatives of Nucleoside imidophosphates are widely adenosine and guanosine are commercially used in their triphosphate form. As a result, available. However, use of commercial prepa- synthetic methods have focused principally on rations requires HPLC purification prior to use the triphosphate form as the target. There are to remove the contaminating phosphoramidate several approaches to synthesize nucleoside APPN (Penningroth et al., 1980; Batra et al., imidophosphates, and no single approach is 1987). APPN does not reappear after prolonged superior. All methods give similar, yet variable, storage (8 months) of repurified APPNP, sug- yields for attachment of the imidophosphate gesting the phosphoramidate contaminant is a functionality to the nucleoside. byproduct of preparation. Synthesis of nucleoside-β,γ-imidotriphos- phates can be accomplished by Michelson’s 32P-Radiolabeled Derivatives anion-displacement methodology (Yount et al., This section will focus on radiolabeled com- 1971). Activation of AMP with diphenyl phos- pounds containing 32P modifications to the phorochloridate followed by displacement of phosphate component of nucleotides. There are diphenyl phosphate with imidodiphosphate several important provisos to consider when gives APPNP (S.111; Fig. 13.1.27). This choosing a method to synthesize radiolabeled method has been used successfully to incorpo- derivatives of nucleotides. First is the location rate heavy nuclides of oxygen and nitrogen into of the radiolabel. Second is the availability of the phosphate component by the labeling of reagents in labeled form. Third is the time imidodiphosphate (Reynolds et al., 1983). required for synthesis to be complete. Because The preparation of nucleoside-α,β-imi- the half- of 32P is 14 days, prolonged syn- dotriphosphates is generally carried out in a theses reduce the specific activity of the labeled two-step procedure. The nucleoside-α,β-imi- nucleotide. Taken together, these provisos dodiphosphate is chemically synthesized and place severe restrictions on the methodologies then enzymatically phosphorylated. Displace- that are available to synthesize radiolabeled ment of the tosylate leaving group in adenosine nucleotides. 5′-tosylate S.112 by tris(tetrabutylammonium) Nucleotides with radiolabeled phosphates imidodiphosphate gives access to adenosine- are commercially available for common nu- α,β-imidodiphosphate S.113 (Ma et al., 1988). cleoside mono- and triphosphates (C, G, T, dA, Enzymatic phosphorylation of S.113 by creat- and others). The nucleotide triphosphates are ine kinase using phosphoenol pyruvate as the widely available with 32Pα and 32Pγ labels. phosphoryl donor provides access to APNPP However, there is a conspicuous lack of 32Pβ- (S.114). An alternate strategy uses imidophos- labeled triphosphates. Therefore, synthetic phorylation of unprotected nucleosides with methods are necessary to obtain 32Pβ-labeled trichloro-[(dichlorophosphoryl)imido]phos nucleotides. Synthetic methods are also neces- phorane (S.116; Tomasz et al., 1988). Both sary to obtain radiolabeled mono-, di-, and ribo- and deoxyribonucleosides have been imi- triphosphates of nucleosides with sugar and/or Nucleoside Phosphorylation dophosphorylated using phosphorane S.116. nucleobase modifications. There are many and Related Phosphorane S.116 is also a good chlorinating methods available for the synthesis of nucleo- Modifications 13.1.21

Current Protocols in Nucleic Acid Chemistry Supplement 15 O O O O 1) Cl P OPCl HO A HO P O A HO P O A O Cl Cl O O OH DCC OH ∆ OO 119 OH OH 2) H2O, pH= 7 120 OO 121 P OOH HOP O OH

TEA-SO3

O O O O bovine spleen O O A ribonuclease-T2 A phosphodiesterase II A HO S O P O O HO S O P O O HO S O P O O O OH O OH O OH

123 OOH 122 OO 124 OH O HO P O P O POH OOH OH OH

Figure 13.1.28 Combination methodology for the synthesis of PAPS. DCC, dicyclohexylcarbodiimide; TEA-SO3, triethylamine-N-sulfonic acid.

O

N

NN H

125

Figure 13.1.29 4-Morpholine-N,N′-dicyclohexyl carboxamidine. A useful base for increasing solu- bility of NMPs.

tides with 31P that cannot be used with 32P, tion of the mixed anhydride with 32P-ortho- because 32P is not available in as many coordi- phosphoric acid produces the 32P-labeled nu- nation states as 31P. The most readily available cleoside triphosphate. This method is compat- form of 32P is an aqueous solution of ortho- ible with various NDPs (A, C, G, U) for the phosphoric acid. Consequently, many of the construction of 32Pγ-labeled NTPs. The reac- phosphorylation reactions with 32P are con- tion is complete in a short period of time, but ducted in dimethyl sulfoxide (DMSO), di- requires extensive handling of the 32P reagent methylformamide (DMF), or water. (Janecka et al., 1980). This method does not Nucleoside phosphate mixed anhydrides allow for the construction of 32Pβ-labeled and nucleoside phosphoramidates are electro- NDPs, as the NMP mixed anhydride does not philic phosphorus species that are suitable for react to an appreciable extent with phosphoric Overview of the 32 Synthesis of use with P-ortho-phosphoric acid. Treatment acid (Michelson, 1964). Nucleoside of NDPs with ethylchloroformate produces a The phosphoramidate approach relies upon Phosphates and nucleoside phosphate mixed anhydride. Reac- ready access to the nucleoside phosphormor- Polyphosphates 13.1.22

Supplement 15 Current Protocols in Nucleic Acid Chemistry pholidate. Condensation of AMP or ADP with complete enzyme-mediated process has been morpholine in the presence of dicyclohexylcar- developed. The enzymatic process has the ad- bodiimide (DCC) gives the corresponding nu- vantages that the reaction is performed in the cleoside phosphormorpholidates. 32Pβ- or 32Pγ- container that the 32P is shipped in and the labeled ATP can be obtained from AMP- and intermediates do not need to be purified. A ADP-morpholidate, respectively. Reaction of consequence, though, is that the final purifica- AMP-morpholidate with 32Pβ-ρ-nitrobenzyl tion can be difficult (Walseth and Johnson, diphosphate gives 32Pβ-labeled ATP in low 1979; Walseth et al., 1991). yield. Reaction between 32P-ortho-phosphoric As with any enzymatic synthesis of nucleo- acid and ADP-morpholidate gives 32Pγ-labeled tides, if the phosphoryl donor is ATP, contami- ATP in good yield. The nice feature of this nation of the product NTPs with ATP is possi- method is that commercially available unpro- ble. This is particularly problematic with 32P- tected nucleosides can be used. A drawback of labeled ATP, as the specific activity can be the method is the vast excess of reagents used reduced significantly (Furuichi and Shatkin, throughout the process. Both DCC and 32P-or- 1977). This can be addressed by use of alternate tho-phosphoric acid are used in excess. This is phosphoryl donors such as phosphoenol pyru- disadvantageous because the urea byproduct of vate or acetyl phosphate. Enzyme systems have DCC can be difficult to separate from nucleo- been developed for the synthesis of 32Pβ-NTPs tides, and excess 32P reagent contributes to from phosphorylation of NMPs (Furuichi et al., additional radioactive waste. Additionally, the 1977) and by with reaction requires prolonged reaction times, polynucleotide (Kaufmann et which results in reduced activity of the 32P-la- al., 1980). beled nucleotides (Werhli et al., 1965). Introduction of the 32P label at the Pα of NDP 3′-PHOSPHOADENOSINE-5′-PHOS- or NTP is possible by condensation of a 2′,3′- PHOSULFATE (PAPS) isopropylidene ribonucleoside with 32P-ortho- 3′-Phosphoadenosine-5′-phosphosulfate phosphoric acid in the presence of trichloroace- (PAPS) is the source used by sulfotrans- tonitrile and triethylamine. Following the re- ferases for the sulfation of , drugs, pro- moval of the protecting group, the 32P-labeled teins, and saccharides (Klaassen et al., 1997). monophosphate can be phosphorylated with PAPS has been synthesized for direct use as a (2-cyanoethyl)phosphoryl- or (2-cyano- and to study biological processes. ethyl)pyrophosphoryl imidazolidate, provid- Methodologies developed for the synthesis of ing 32Pα-NDPs and 32Pα-NTPs, respectively PAPS reflect the application. Methods combin- (Symons, 1970). Extension of this methodol- ing both and enzymes have ogy with unprotected nucleosides is possible, been detailed. Synthetic methods typically although the first condensation step produces a have fallen short of providing PAPS in pure mixture of 2′(3′)- and 5′-nucleoside monophos- form, as both the 2′- and 3′-phosphate regioi- phates. This mixture can be selectively phos- somers are produced. Thus, synthesis of PAPS phorylated with enzymes to yield the desired by pure synthetic methods will not be ad- 32Pα-NTPs or 32Pα-dNTPs (Symons, 1974). dressed. In addition to the purely chemical and mixed chemical/enzymatic syntheses of 32Pα-NTPs, a

Ph

Ph O 1) OMe O P OMe HO B OMe HO B O 127 O

126 OH OH 2) H O 128 OO 2 P OOH

Nucleoside Figure 13.1.30 Selective transesterification of unprotected nucleosides by oxyphosphorane Phosphorylation leads to regioselective formation of 2′,3′-cyclic phosphates. and Related Modifications 13.1.23

Current Protocols in Nucleic Acid Chemistry Supplement 15 Enzymatic Methods obtain these cyclic phosphates: cyclization by Pure enzymatic synthesis of PAPS is par- dehydration or cyclization by nucleophilic at- ticularly suitable when PAPS is a cofactor used tack on electrophilic phosphorus. The latter is in sulfation reactions. In these instances a large often promoted by adding base to the reaction quantity of PAPS is not necessary. Rather, what medium. is needed is a means to reconvert PAP back to Cyclization by dehydration is commonly the coenzyme PAPS. Systems have been devel- affected with DCC, although chloroformate oped that directly convert PAP to PAPS reagents and trifluoroacetic anhydride have (Burkart et al., 1999) or that accomplish this also been used. Preparation of cAMP, cCMP, conversion through several enzymatic steps cGMP, and cUMP have been carried out by (i.e., PAP → AP → APS → PAPS; Lin et al., heating the corresponding NMP-4-mor- 1995). The one real disadvantage of this meth- pholine-N,N′-dicyclohexylcarboxamidinium odology is its level of sophistication. Although salts in pyridine/DCC (Smith et al., 1961). very elegant, multienzymatic regeneration of Unlike tri-n-butylamine or tri-n-octylamine, PAPS requires construction of a plasmid, as the base 4-morpholine-N,N′-dicyclohexylcar- well as overexpression and purification of sev- boxamidine S.125 (Fig. 13.1.29) imparts better eral enzymes (Burkart et al., 1999). These pro- solubility upon nucleoside monophosphates at cedures are routine in molecular labo- high temperatures. Despite the better solubility ratories, but may be intimidating to the syn- of the carboxamidinium salts, guanosine and thetic chemist. cytidine need N-benzoyl protecting groups for full dissolution. The yields for this reaction are Combination Methods generally good. Figure 13.1.28 summarizes methods to ob- DCC-promoted dehydration can also be tain PAPS by combination methodology. The used for the synthesis of cyclic-2′,3′-phos- key to several combination methods is the avail- phates. Cyclization of a commercial mixture of ability of 2′,3′-cyclic phosphoadenosine 5′- 2′,5′- and 3′,5′-bis(phospho)adenosine with phosphosulfate (S.122). Several approaches DCC at elevated temperature provides cyclic- are available to access this intermediate. 2′,3′-phosphoadenosine-5′-phosphate (S.120; Adenosine (S.119) can be phosphorylated with Fig. 13.1.28; Sekura, 1981). Similarly, reaction pyrophosphoryl chloride followed by hydroly- of unprotected nucleosides (A, C, G, U) with sis to give 2′,3′-cyclic phosphoadenosine 5′- pyrophosphoryl chloride followed by buffered phosphate (S.120; Horwitz et al., 1977). Alter- hydrolysis (pH = 7) results in the formation of nately, cyclic phosphate S.120 can be obtained cyclic-2′,3′-phosphonucleoside-5′-phosphates by cyclizing a commercial mixture of 2′,5′- and (Fig. 13.1.28; Simoncsits and Tomasz, 1975). 3′,5′-bis(phospho)adenosine (S.121) with This reaction is successful for adenosine, cytid- DCC at elevated temperature (Sekura, 1981). ine, and guanosine. However, cyclic-2′,3′- Sulfation of cyclic phosphate S.120 is most phosphouridine-5′-phosphate is obtained in commonly effected with triethylamine-N-sul- low yield via this procedure. These reactions fonic acid (Horwitz et al., 1980; Sekura, 1981). have the inherent drawback that the bis-phos- 5′-Phosphosulfate nucleosides are quite prone phate is produced instead of pure cyclic-2′,3′- to hydrolysis and decomposition. Therefore, phosphate. This limitation has been overcome yields for the sulfation step tend to vary (46% by the use of the five-coordinate oxyphos- to 75%). Cyclic phosphate S.122 can be cleaved phorane S.127 (σ5,λ5) instead of pyrophospho- by either bovine spleen phosphodiesterase II or ryl chloride. Treatment of an unprotected nu- ribonuclease-T2 to provide 2′- or 3′-phos- cleoside with oxyphosphorane S.127 in pyri- phoadenoside-5′-phosphosulfate, respectively dine followed by hydrolysis provides the (Horwitz et al., 1980; Sekura, 1981). The en- nucleoside cyclic-2′,3′-phosphate (S.128; Fig. zymatic cleavage of cyclic phosphates is toler- 13.1.30). In this manner, both adenosine and ant to minor structural modifications of the cytidine cyclic-2′,3′-phosphates have been re- nucleobase (Horwitz et al., 1981). gioselectively synthesized in high yield (Chen et al., 1997). CYCLIC NUCLEOSIDE Entrée into 3′,5′-cyclic phosphates is possi- PHOSPHATES AND ble through base-promoted cyclization of phos- Overview of the PHOSPHOROTHIOATES phorodichloridates. Treatment of an unpro- Synthesis of Two important types of nucleoside cyclic tected nucleoside with POCl3 produces a phos- Nucleoside ′ ′ ′ ′ Phosphates and phosphates are 2 ,3 - and 3 ,5 -cyclic phos- phorodichloridate that is hydrolyzed with an Polyphosphates phates. There are two common approaches to aqueous solution of KOH in acetonitrile at 0°C 13.1.24

Supplement 15 Current Protocols in Nucleic Acid Chemistry O S S S KOH t-BuOK B P P B Cl P O S O O O O2N OPO O O O B O O B O Cl (P(R) major) (P(S) major) 2 HO HO OH OH OH OH

129 130 P(R) 131 P(S) 132

1) K, CS2 2) NaOH 3) NH3, MeOH

Ph O 1) Ph3P-CCl4 O NH Ph P 2) PhNH2 P P O O 3) chromatography N O O O O O B H O O B O O B

HO HO HO

133 134 135

Figure 13.1.31 Synthesis of cyclic phosphorothioates. to produce the cyclic phosphate in low yield The synthesis of cAMPS and other cyclic (22% to 49%). This procedure varies slightly phosphorothioates is difficult because the phos- from the Yoshikawa procedure (Fig. 13.1.10) phorothioate functional group is part of a six- in that water is omitted from the reaction milieu membered ring. The phosphorus center is during formation of the phosphorodichloridate. therefore stereogenic. Both the P(R) and P(S) Omission of water during the initial phospho- stereoisomers are configurationally stable. As rylation step furnishes mixtures of nucleoside- shown in Figure 13.1.31, cyclic-3′,5′-phos- 2′(3′)-5′-diphosphates (Yoshikawa et al., phorothioates can be accessed by base-pro- 1967). Therefore, the reduced yields obtained moted cyclization of phosphorothiodichlori- for this cyclization reaction may be due to lack dates (S.129; Genieser et al., 1988) or nucleo- of regio-control in the initial phosphorylation side 5′-bis(ρ-nitrophenyl)phosphorothioates step. (S.132; Eckstein and Kutzke, 1986). Cyclic As secondary messengers, nucleoside cy- phosphorothioates synthesized by these meth- clic-3′,5′-phosphates are important in many ods are mixtures of phosphorus stereoisomers. biological pathways. Investigation of the path- Interestingly, ester displacement reactions give ways could benefit from the ready availability predominately the P(S) stereoisomer, whereas of nucleoside cyclic phosphate analogs that chloride displacement results in primarily the have increased enzymatic and hydrolytic sta- P(R) stereoisomer (Fig. 13.1.31). Resolution of bility. Phosphorothioate derivatives of cyclic- the individual stereoisomers from these reac- 3′,5′-phosphates such as cAMPS are, in fact, tions is possible, but the process is tedious and hydrolyzed more slowly than the parent phos- the yields are generally poor. phate (Eckstein, 1979). Additionally, the ste- An alternate method to access the individual reochemical course of enzyme-catalyzed reac- stereoisomers is through the stereospecific tions has been determined by using nucleoside Horner-Wadsworth-Emmons reaction of nu- cyclic-2′,3′-phosphorothioates (Usher et al., cleoside phosphoranilidates (S.134 or S.135) Nucleoside 1970). with potassium reduced carbon (Fig. Phosphorylation and Related 13.1.31; Stec, 1983). This approach has the Modifications 13.1.25

Current Protocols in Nucleic Acid Chemistry Supplement 15 nucleoside nucleoside HO B O nucleoside kinase monophosphate kinase diphosphate kinase NMP NDP NTP (base-specific) (base-specific) OH OH

Figure 13.1.32 Enzyme-catalyzed phosphorylation of nucleosides proceeds in a stepwise fashion.

advantage that commercially available nucleo- can be used as phosphate donors. Phosphoenol side cyclic phosphates can be used. The reac- pyruvate is more stable in solution than acetyl tion sequence is outlined in Figure 13.1.31 and phosphate and is used extensively for the phos- has been applied to adenosine, cytidine, phorylation of NDPs. Hydrolytically stable guanosine, and uridine. Additionally, the sepa- phosphate donors are especially important ration of the nucleoside phosphoranilidate when the nucleoside is phosphorylated at slow stereoisomers is easier than separation of the rates (such as the case with modified nucleo- nucleoside cyclic phosphorothioates. However, sides). the chemical synthesis of the nucleoside phos- Nucleosides containing modified nucleo- phoranilidates proceeds in low yield. bases cannot always be phosphorylated using synthetic methods due to incompatibility with ENZYMATIC NUCLEOTIDE the reaction conditions. Enzymatic phosphory- SYNTHESIS lation offers an alternative to synthetic methods In principle, enzymes can be used to synthe- because the reaction conditions are generally size NMPs, NDPs, or NTPs. However, -free mild and phosphorylations are regio- and che- enzyme-catalyzed reactions are most routinely moselective. However, there has been limited used for the synthesis of NTPs. Enzymes cata- success for enzyme-mediated phosphorylation lyze the phosphorylation of nucleosides to NTP of modified nucleosides. Unnatural purine- and in a stepwise fashion by the successive action pyrimidine-like nucleobases are phosphory- of nucleoside kinase, nucleoside monophos- lated with reduced efficiency (kcat/Km) by D. phate kinase, and nucleoside diphosphate ki- melanogaster (by a nase (Fig. 13.1.32). The first two enzymes in factor of 102 to 104; Wu et al., 2002). this pathway are nucleoside specific, whereas, Phosphorylation of sugar-modified nucleo- nucleoside diphosphate kinase is not nucleotide sides can also be problematic. The that specific. phosphorylate AZT to AZT-TP do so with re- Nucleoside kinase, nucleoside monophos- duced efficiency (Van Rompay et al., 2000). For phate kinase, and nucleoside diphosphate ki- example, NDP kinase phosphorylates AZT-DP nase primarily use ATP as the phosphate donor. with reduced efficiency (a factor of 103 to 104; Therefore, ATP has to be supplied or regener- Schneider et al., 2001). This is interesting be- ated, depending upon scale, during the nucleo- cause NDP kinase has little specificity for either tide syntheses (Chenault et al., 1988). Due to the nucleobase or the or of the use of ATP as phosphate donor, pure nu- natural NDPs. However, critical H-bonds are cleotide product can be difficult to obtain. Pure provided by the 3′-hydroxyl group to stabilize NTP product (where N = C, T, U, G) can be a conformation of the nucleotide that facilitates especially difficult to separate from ATP. phosphorylation. Thus, a minor modification in Purity of product NTPs can be increased by the architecture of the nucleoside (3′-OH to several modifications of the general enzymatic 3′-N3) results in the destabilization of the active scheme outlined in Figure 13.1.32. Use of com- conformation of the nucleotide within the ki- mercially available NMPs and regeneration of nase. catalytic amounts of ATP are two approaches In contrast to the problems associated with to reduce contamination by ATP/ADP. Another enzymatic phosphorylation of AZT-DP, NDP way is to use alternate enzymes/phosphate do- kinase has been used successfully to phospho- Overview of the nors to effect the phosphorylation. For exam- rylate nucleoside diphosphates containing non- Synthesis of ple, by choosing conditions that favor the back- natural nucleobases (Wu et al., 2003). Diphos- Nucleoside wards reactions of pyruvate or acetate kinase, phates of azole carboxamide deoxyribonu- Phosphates and Polyphosphates phosphoenol pyruvate and acetyl phosphate cleosides (or azole carboxamide 13.1.26

Supplement 15 Current Protocols in Nucleic Acid Chemistry ribonucleosides) are phosphorylated by NDP tide synthesis has yet to be satisfactorily met. kinase using ATP as the phosphate donor (see Nominally, an ideal method permits the regio- UNIT 13.2). Good to high yields of the desired and chemoselective phosphorylation of all nu- azole carboxamide nucleoside triphosphates cleosides and nucleoside analogs (base or sugar are obtained. Precursor diphosphates are ob- modifications alike). Additionally, an ideal tained by way of Poulter’s tosylate displace- method should facilitate the synthesis of any ment methodology. This combination syn- phosphate-chain analog (thio-, borano-, imido-, thetic/enzymatic method is notable for several and others) from common intermediates or an reasons. First, the method is of interest for its appropriate reagent system. potential as a general means to phosphorylate This overview has presented some common nucleoside diphosphates containing non-natu- methods and reagents used to construct nucleo- ral nucleobases. Second, the method addresses tides. Despite the elegant design of the many contamination of the desired nucleoside methods available, it is clear that no single triphosphate product with ATP/ADP. Use of an method is “ideal” under the criteria outlined ATP regeneration system involving phos- above. This is due in part to the variation in phoenol pyruvate and mini- chemical properties of the nucleobases. Che- mizes the amount of ATP in the reaction milieu. moselectivity may be improved by transiently Nevertheless, contaminating ATP/ADP is con- masking the nucleophilic moieties of the nu- veniently removed by retention on a boronate cleobases. Alternately, finding conditions that affinity gel column. The boronate affinity gel selectively degrade any phosphorylated forms a complex with the vicinal diols of ri- (phosphitylated) nucleobase moieties shows bonucleotides; thus, azole carboxamide de- potential for a general solution. Progress has oxyribonucleotide products conveniently pass been made on chemoselective phosphitylation through the gel. of N-unprotected nucleosides using phos- There are two issues unique to enzyme-cata- phoramidite reagents. Both transient protection lyzed phosphorylations that are not encoun- via protonation of basic nucleobases (Sekine et tered during chemical syntheses: enzyme sta- al., 2003) and selective cleavage of phosphity- bility and product inhibition. The source of the lated nucleobases (Gryaznov and Letsinger, enzyme can greatly affect the required reaction 1991) have been successfully employed in de- conditions. For example, acetate kinase from oxyoligonucleotide syntheses. Modification of E. coli contains a thiol group that can be oxi- these methods may ultimately be useful for the dized. To prevent inactivation due to oxidation, synthesis of nucleoside mono-, di-, and triphos- reactions are often conducted in the presence phates. However, a general solution for the of a reducing agent (DTT) or under an inert regio- and chemoselective phosphorylation of atmosphere. Interestingly, acetate kinase from nucleosides and analogs without invoking the B. stearothermophilus cannot be autooxidized use of protecting groups is a very difficult because it does not contain a thiol group (Kim problem that awaits a general solution. and Whitesides, 1987). Aside from enzyme inactivation, inhibition LITERATURE CITED of the enzymes can also cause a decrease in the Albert, A. 1973. Ionization constants of . The phosphorylation reactions and . In Synthetic Procedures in Nucleic catalyzed by NMP and NDP kinases are revers- Acid Chemistry (W.W. Zorbach and R.S. Tipson, eds.) pp. 1-46. Wiley-Interscience, New York. ible. The consequence of this reversibility is that a large buildup of NDP can inhibit NMP Arabshahi, A. and Frey, P.A. 1994. A simplified procedure for synthesizing nucleoside 1- kinase. Thus, the rate at which NMP kinase thiotriphosphate: dATPαS, dGTPαS, UTPαS, produces NDP product slows dramatically. and dTTPαS. Biochem. Biophys. Res. Commun. Product inhibition is another complicating fac- 204:150-155. tor when designing enzyme-catalyzed phos- Batra, J.K., Lin, C.M., and Hamel, E. 1987. Nucleo- phorylations. tide interconversions in protein preparations, a significant complication for ac- curate measurement of GTP hydrolysis in the STATE OF THE ART presence of adenosine 5′-(β,γ-imidotriphos- In a 1958 Nobel Prize speech, Sir Alexander phate). Biochemistry 26:5925-5931. Todd made the following observation, “we are Burgess, K. and Cook, D. 2000. Syntheses of nu- thus still seeking an ideal method for unsym- cleoside triphosphates. Chem. Rev. 100:2047- metrical pyrophosphate synthesis” (Todd, 2059. Nucleoside Phosphorylation 1958). Despite the passage of almost 50 years, and Related the challenge of an “ideal method” for nucleo- Modifications 13.1.27

Current Protocols in Nucleic Acid Chemistry Supplement 15 Burkart, M.D., Izumi, M., and Wong, C.-H. 1999. Gryaznov, S.M. and Letsinger, R.L. 1991. Synthesis Enzymatic regeneration of 3′-phosphoadenos- of oligonucleotides via with unpro- ine-5′-phosphosulfate using aryl sulfotrans- tected bases. J. Am. Chem. Soc. 113:5876-5877. ferase for the preparative enzymatic synthesis of Hoard, D.E. and Ott, D.G. 1965. Conversion of sulfated . Angew. Chem. Int. Ed. mono- and oligodeoxyribonucleotides to 5′- Engl. 38:2747-2750. triphosphates. J. Am. Chem. Soc. 87:1785-1788. Chen, J.-T. and Benkovic, S.J. 1983. Synthesis and Horwitz, J.P., Neenan, J.P., Misra, R.S., Rozhin, J., separation of diastereomers of deoxynucleoside ′ Huo, A., and Philips, K.D. 1977. Studies on 5 -O-(1-thio)triphosphates. Nucl. Acids Res. bovine adrenal sulfotransferase III. 11:3737-3751. Facile synthesis of 3′-phospho- and 2′-phos- Chen, X., Zhang, N.-J., Li, Y.-M., Jiang, Y., Zhang, phoadenosine 5′-phosphosulfate. Biochim. Bio- X., and Zhao, Y.-F. 1997. Direct phosphorylation phys. Acta 480:376-381. of nucleosides by oxyphosphorane. Tetrahedron Horwitz, J.P., Misra, R.S., Rozhin, J., Helmer, S., Lett. 38:1615-1618. Bhuta, A., and Brooks, S.C. 1980. Studies on Chenault, H.K., Simon, E.S., and Whitesides, G.M. bovine adrenal estrogen sulfotransferase. V. Syn- 1988. Cofactor regeneration for enzyme-cata- thesis and assay of analogs of 3′-phosphoade- lysed synthesis. In and Genetic nosine 5′-phosphosulfate as cosubstrates for es- Engineering Reviews (G.E. Russell, ed.) pp. trogen sulfurylation. Biochim. Biophys. Acta 221-270. Intercept, Wimborne, Dorset, England. 613:85-94. Chladek, S. and Nagyvary, J. 1972. Nucleophilic Horwitz, J.P., Neenan, J.P., Misra, R.S., Rozhin, J., reactions of some nucleoside phosphorothioates. Huo, A., and Philips, K.D. May 1981.U.S. patent J. Am. Chem. Soc. 94:2079-2085. 4,266,048. Synthesis of Analogs of 3′-Phos- ′ Corbridge, D.E.C. 1995. Phosphorous. An Outline phoadenosine 5 -Phosphosulfate (PAPS). of its Chemistry, Biochemistry and Uses. El- USPTO Patent Full-Text and Image Database. sevier, Amsterdam. USA, The United States of America as repre- sented by the Department of Health. Cosstick, R. and Vyle, J.S. 1990. Synthesis and properties of dithymidine phosphate analogues Imai, J. and Torrence, P.F. 1981. Bis(2,2,2-trichlo- containing 3′-thiothymidine. Nucl. Acids Res. roethyl) phosphorochloridite as a reagent for the 18:829-835. phosphorylation of oligonucleotides: Prepara- tion of 5′-phosphorylated 2′,5′-oligoadenylates. Davisson, V.J., Davis, D.R, Dixit, V.M., and Poulter, J. Org. Chem. 46:4015-4021. C.D. 1987. Synthesis of nucleotide 5′-diphos- phates from 5′-O-tosyl nucleosides. J. Org. Iyer, R., Phillips, L.R., Egan, W., Regan, J.B., and Chem. 52:1794-1801. Beaucage, S.L. 1990. The automated synthesis of sulfur-containing oligodeoxyribonucleotides Dixit, V.M. and Poulter, C.D. 1984. Convenient using 3H-1,2-benzodithiol-3-one 1,1-dioxide as syntheses of adenosine 5′-diphosphate, adeno- a sulfur-transfer reagent. J. Org. Chem. 55:4693- sine 5′-methylenediphosphonate, and adenosine 4699. 5′-triphosphate. Tetrahedron Lett. 25:4055- 4058. Janecka, A., Panusz, H., Pankowski, J., and Koz- iolkiewicz, W. 1980. Chemical synthesis of nu- Eckstein, F. 1979. Phosphorothioate analogues of cleoside-γ-[32P]triphosphates of high specific nucleotides. Acc. Chem. Res. 12:204-210. activity. Prep. Biochem. 10:27-35. Eckstein, F. and Kutzke, U. 1986. Synthesis of nu- Jankowska, J., Cieslak, J., and Kraszewski, A. 1997. cleoside 3′,5′-cyclic phosphorothioates. Tetrahe- 9-Fluorenemethyl H-phosphonothioate, a versa- dron Lett. 27:1657-1660. tile reagent for the preparation of H-phos- Engel, R. 1977. Phosphonates as analogues of natu- phonothioate, phosphorothioate, and phos- ral phosphates. Chem. Rev. 77:349-367. phorodithioate monoesters. Tetrahedron Lett. 38:2007-2010. Eymery, F., Iorga, B., and Savignac, P. 1999. Syn- thesis of phosphonates by nucleophilic substitu- Jankowska, J., Sobkowska, A., Cieslak, J., Sobk- tion at phosphorus: The SN P(V) reaction. Tetra- owski, M., Krazewski, A., Stawinski, J., and hedron 55:13109-13150. Shugar, D. 1998. Nucleoside H-phosphonates. 18. Synthesis of unprotected nucleoside 5′-H- Furuichi, Y. and Shatkin, A.J. 1977. A simple method ′ β 32 phosphonates and nucleoside 5 -H-phos- for the preparation of [ - P]purine nucleoside phonothioates and their conversion into the 5′- triphosphates. Nucl. Acids Res. 4:3341-3355. phosphorothioate and 5′-phosphorodithioate Genieser, H.-G., Dostmann, W., Bottin, U., Butt, E., monoesters. J. Org. Chem. 63:8150-8156. and Jastorff, B. 1988. Synthesis of nucleoside- ′ ′ Jurczyk, S.C., Kodra, J.T., Park, J.-H., Benner, S.A., 3 ,5 -cyclic phosphorothioates by cyclothio- and Battersby, T.R. 1999. Synthesis of 2′-deoxy- phosphorylation of unprotected nucleosides. 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Supplement 15 Current Protocols in Nucleic Acid Chemistry Kadokura, M., Wada, T., Urashima, C., and Sekine, Ludwig, J. and Eckstein, F. 1989. Rapid and efficient M. 1997. Efficient synthesis of γ-methyl-capped synthesis of nucleoside 5′-O-(1-thiotriphos- guanosine 5′-triphosphate as a 5′-terminal phates), 5′-triphosphates and 2′,3′-cyclophos- unique structure of U6 RNA via a new triphos- phorothioates using 2-chloro-4H-1,3,2-benzodi- phate bond formation involving activation of oxaphosphorin-4-one. J. Org. Chem. 54:631- methyl phosphorimidazolidate using ZnCl2 as a 635. catalyst in DMF under anhydrous conditions. Ludwig, J. and Eckstein, F. 1991. Synthesis of nu- Tetrahedron Lett. 38:8359-8362. cleoside 5′-O-(1,3-dithiotriphosphates) and 5′- Kaufmann, G., Choder, M., and Groner, Y. 1980. O-(1,1-dithiotriphosphates). J. Org. Chem. Synthesis of carrier-free β-32P-nucleoside- 56:1777-1783. triphosphate in almost quantitative yields. Anal. Ma, Q.-F., Babbitt, P.C., and Kenyon, G.L. 1988. 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Current Protocols in Nucleic Acid Chemistry Supplement 15 Saady, M., Lebeau, L., and Mioskowski, C. 1995a. Todd, A. 1958. Synthesis in the study of nucleotides. Synthesis of adenosine-5′-phosphates and 5′-al- Science 127:787-792. kylphosphonates via the Mitsunobu reaction. Tomasz, J., Vaghefi, M.M., Ratsep, P.C., Willis, Tetrahedron Lett. 36:2239-2242. R.C., and Robins, R.K. 1988. Nucleoside imi- Saady, M., Lebeau, L., and Mioskowski, C. 1995b. dodiphosphate synthesis and biological activi- Synthesis of di- and triphosphate ester analogs ties. Nucl. Acids Res. 16:8645-8664. via a modified Michaelis-Arbuzov reaction. Tet- Trevisiol, E., Defrancq, E., Lhomme, J., Laayoun, rahedron Lett. 36:5183-5186. A., and Cros, P. 2000. Synthesis of nucleoside Saffhill, R. 1970. Selective phosphorylation of the triphosphates that contain an aminooxy function cis-2′,3′-diol of unprotected ribonucleosides for “Post-Amplification Labeling”. Eur. J. Org. with trimetaphosphate in aqueous solution. J. 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[53] Adenosine 3′-phosphate ′ Van Rompay, A.R., Johansson, M., and Karlsson, A. 5 -phosphosulfate. In Detoxification and Drug 2000. Phosphorylation of nucleosides and nu- Metabolism: Conjugation and Related Systems cleoside analogs by mammalian nucleoside mo- (W.B. Jakoby, ed.) pp. 413-415. Academic Press, nophosphate kinases. Pharmacol. Ther. 87:189- New York. 198. Simoncsits, A. and Tomasz, J. 1975. Simple one- Verheyden, J.P.H. and Moffatt, J.G. 1970. Halo step synthesis of ribonucleoside 2′,3′-cyclic ′ sugar nucleosides. I. Iodination of the primary phosphate 5 -phosphates. Biochim. Biophys. hydroxyl groups of nucleosides with methyl- Acta 395:74-79. triphenoxyphosphonium iodide. J. Org. Chem. Smith, M., Drummond, G.I., and Khorana, H.G. 35:2319-2326. 1961. Cyclic phosphates. IV. Ribonucleoside- ′ ′ Walseth, T.F. and Johnson, R.A. 1979. The enzy- 3 ,5 cyclic phosphates. A general method of matic preparation of [α-32P]nucleoside triphos- synthesis and some properties. J. Am. Chem. Soc. phates, cyclic [32P]AMP, and cyclic [32P]GMP. 83:698-706. Biochim. Biophys. Acta 526:11-31. Stec, W.J. 1983. Wadsworth-Emmons reaction re- Walseth, T.F., Yuen, P.S.T., and Moos, M.C. Jr. 1991. visited. Acc. Chem. Res. 16:411-417. Preparation of α-32P-labeled nucleoside triphos- Stock, J.A. 1979. Synthesis of phosphonate ana- phates, adenine dinucleotide, and logues of thymidine di- and triphosphate from cyclic nucleotides for use in determining 5′-O-toluenesulfonylthymidine. J. Org. Chem. adenylyl and guanylyl cyclases and cyclic nu- 44:3997-4000. cleotide phosphodiesterase. Methods Enzymol. Stowell, J.K. and Widlanski, T.S. 1995. Mechanism- 195:29-44. based inactiviation of ribonuclease A. J. Org. Werhli, W.E., Verheydden, D.L.M., and Moffatt, Chem. 60:6930-6936. J.G. 1965. Dismutation reactions of nucleoside polyphosphates. II. Specific chemical syntheses Stromberg, R. and Stawinski, J. 1987. Iodide and α β γ 32 ′ iodine catalysed phosphorylation of nucleosides of -, -, and - P-nucleoside 5 -triphosphates. by phosphorodiester derivatives. Nucleosides & J. Am. Chem. Soc. 87:2265-2277. Nucleotides 6:815-820. Wu, Y., Fa, M., Tae, E.L., Schultz, P.G., and Romes- Symons, R.H. 1970. Practical methods for the rou- berg, F.E. 2002. Enzymatic phosphorylation of tine chemical synthesis of 32P-labelled nucleo- unnatural nucleosides. J. Am. Chem. Soc. side di- and triphosphates. Biochim. Biophys. 124:14626-14630. Acta 209:296-305. Wu, W., Bergstrom, D.E., and Davisson, V.J. 2003. Symons, R.H. 1974. Synthesis of [α-32P]ribo- and A combination chemical and enzymatic ap- 5′-triphosphates. Methods proach for the preparation of azole carboxamide Enzymol. 29:102-115. . J. Org. Chem. 68:3860- 3865. Thomson, J.B., Patel, B.K., Jimenez, V., Eckart, K., and Eckstein, F. 1996. Synthesis and properties Yoshikawa, M., Kato, T., and Takenishi, T. 1967. 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Supplement 15 Current Protocols in Nucleic Acid Chemistry Yount, R.G., Babcock, D., Ballantyne, W., and Ojala, D. 1971. Adenylyl imidodiphosphate, an analog containing a P-N- P linkage. Biochemistry 10:2484-2489. Zemlicka, J. 2002. Lipophilic phosphoramidates as antiviral pronucleotides. Biochim. Biophys. Acta 1587:276-286.

Contributed by David C. Johnson II and Theodore S. Widlanski Indiana University Bloomington, Indiana

Nucleoside Phosphorylation and Related Modifications 13.1.31

Current Protocols in Nucleic Acid Chemistry Supplement 15