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Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According

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Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According Synthetic Strategies and Parameters UNIT 3.4 Involved in the Synthesis of Oligodeoxyribo- and Oligoribonucleotides According to the H-Phosphonate Method GENERAL INFORMATION AND verted to the dinucleoside phosphate or to a SYNTHETIC STRATEGIES variety of backbone-modified analogues, e.g., Although the most common method today phosphorothioates, phosphoramidates, etc. for synthesis of oligonucleotides and their ana- (Fig. 3.4.1). logues is the phosphoramidite approach (Beau- A number of different synthetic methods can cage and Iyer, 1993, see also UNIT 3.3), the newer be used for the synthesis of nucleoside H-phos- H-phosphonate methodology can often be a phonate building blocks. This unit discusses preferred alternative (Garegg et al., 1985, four alternative methods that are quite efficient, 1986a,b,c; Froehler and Matteucci, 1986; Froe- are experimentally simple, and make use of hler et al., 1986). The use of H-phosphonates readily available reagents. in nucleotide synthesis was pioneered by Sir The first method consists of phosphitylating Todd’s group in Cambridge, UK, who in 1952 protected nucleosides with the in situ–gener- demonstrated the formation of H-phosphonate ated tris-(1-imidazolyl)phosphine (from PCl3 diesters in a condensation reaction of H-phos- and imidazole; reagent a in Fig. 3.4.2), followed phonate monoesters with a protected nucleo- by hydrolysis of the formed nucleoside diimi- side, promoted by diphenyl phosphorochlori- dazolyl phosphite intermediate (Garegg et al., date (Corby et al., 1952; Hall et al., 1957). This 1986a,c). This method (or its variants with chemical principle was, however, not explored other azoles) is probably the most widely used further; it was rediscovered three decades later and can be recommended as a general basic (Garegg et al., 1985, 1986c) and explored for method applicable to the preparation of both oligonucleotide synthesis (Froehler and Mat- deoxyribonucleoside and ribonucleoside H- teucci, 1986; Froehler et al., 1986; Garegg et phosphonates (Froehler et al., 1986; Garegg et al., 1986a,b,c, 1987a). al., 1986a,b,c; Froehler, 1993). Imidazole from The method consists of condensing a pro- the first published procedure is preferred over tected nucleoside H-phosphonate monoester the other azoles, because it is relatively inex- with a nucleoside in the presence of a coupling pensive and forms the least reactive species. agent to produce the corresponding dinu- In the second method, pyridinium H-pyro- cleoside H-phosphonate diester. This, under phosphonate (reagent b in Fig. 3.4.2), generated various experimental conditions, can be con- in situ from phosphonic acid and a condensing R O B R O B R O B 1 O 1 O 1 O O R2 O R2 O R2 OHP CA OHP oxidation OOP O O B O B O O HO B O R2 O R O 2 or O R2 phosphotriester phosphorothioate phosphoroselenoate CA = condensing agent, e.g., pivaloyl chloride phosphoramidate methylphosphonate R1 = e.g. di- or monomethoxytrityl R2 = H or protected OH, e.g., OTBDMS B = uracil, thymine, or protected adenine, cytosine, or guanine. Figure 3.4.1 Condensation of protected nucleoside H-phosphonate monoester with a nucleoside Synthesis of and conversion to the dinucleoside phosphate or backbone-modified analog. Unmodified Oligonucleotides Contributed by Roger Strömberg and Jacek Stawinski 3.4.1 Current Protocols in Nucleic Acid Chemistry (2000) 3.4.1-3.4.11 Copyright © 2000 by John Wiley & Sons, Inc. Cl N O O O O Cl P N Cl OPOPO OPO O H H H H P O Cl a b c d R O B R O B 1 O 1 O 1. a, b, c or d HO R O R 2 2. hydrolysis 2 OHP 1 O 2 R1 = 5'-O-protecting group; R2 = H or protected OH B = uracil, thymine, or protected adenine, cytosine, or guanine. Figure 3.4.2 Reagents in the synthesis of nucleoside H-phosphonate building blocks. agent, is used as phosphonylating agent (Staw- carried out in pyridine-acetonitrile mixtures. inski and Thelin, 1990). This method seems to Out of the various condensing agents initially be most suitable for the preparation of de- tested, pivaloyl chloride (Pv-Cl) gave the best oxyribonucleoside H-phosphonates, because results in automated solid-support synthesis of phosphonylation of the 3′-hydroxy group in oligonucleotides, and it is still the most fre- protected ribonucleosides—e.g., in 2′-O-t- quently used reagent. The reaction in pyridine butyldimethylsilyl (TBDMS) protected ri- or acetonitrile-pyridine mixtures using 2 to 5 bonucleosides—occurs very slowly under equiv of Pv-Cl is usually fast and goes to com- these reaction conditions. pletion in <1 min. Nowadays, an array of other The third method is based on the transesteri- condensing agents (see below) is available; and fication of diphenyl H-phosphonate (reagent c these reagents can, in some instances, be supe- in Fig. 3.4.2) with protected nucleosides and is rior to Pv-Cl. a convenient approach to the preparation of Oligonucleotide synthesis employing H- nucleoside H-phosphonate monoesters (Jank- phosphonates is considerably simpler than syn- owska et al., 1994). Diphenyl H-phosphonate thesis using the phosphotriester or phos- is relatively inexpensive, commercially avail- phoramidite procedures. The elongation cycle able, stable, and easy to handle and gives high includes only two chemical steps: deprotection yields of nucleoside H-phosphonates. Note, of the terminal 5′-OH function of the support- however, that synthesis of ribonucleoside bound oligonucleotide and its coupling with a building blocks with this method requires more nucleoside 3′-H-phosphonate in the presence basic work-up conditions, which may prevent of a condensing agent. After completion of the its use with N-protecting groups that are quite desired number of elongation cycles (i.e., as- base labile. sembly of the oligomeric chain), a single oxi- The fourth method recommends the use of dation cycle is performed to convert the inter- 2-chloro-4H-1,3,2-benzo-dioxaphosphinan-4 nucleoside H-phosphonate functions to phos- -one (salicylchlorophosphite; reagent d in Fig. phodiesters (or some analogue, such as 3.4.2) as the phosphitylating reagent (Marugg phosphorothioates). Finally, the linkage be- et al., 1986). Because salicylchlorophosphite is tween the oligomer and the support is cleaved practically a monofunctional phosphitylating under ammonolytic conditions, which for oli- agent, it can be used in nearly stoichiometric godeoxyribonucleotide and oligoribonu- amounts. The formation of symmetrical dinu- cleotide synthesis with 2′-O-(2-chlorobenzoyl) cleoside H-phosphonate diesters is usually neg- groups, is also the final deprotection step. Pu- ligible, but removal of by-products from the rification by standard methods is then carried reagent can sometimes be a problem. out to isolate the oligonucleotides. Strategies for the The condensation step of the elongation cy- The H-phosphonate methodology became Synthesis of cles (i.e., the formation of an internucleoside commercially available (for use in automated Oligonucleotides by the H-phosphonate linkage between a nucleoside synthesizers from Applied Biosystems and H-Phosphonate 3′-H-phosphonate monoester and the support- Biosearch) soon after the initial reports on oli- Method bound 5′-hydroxylic component) is usually gonucleotide synthesis using this approach 3.4.2 Current Protocols in Nucleic Acid Chemistry were published. The research group at Applied phonate approach was used in a cartridge-based Biosystems also introduced 1-adamantanecar- procedure for simultaneous synthesis of multi- bonyl chloride as an alternative to Pv-Cl (An- ple oligodeoxynucleotides (Seliger and Rösch, drus et al., 1988). Although a capping step 1990). The H-phosphonate methodology has during oligonucleotide synthesis via H-phos- been used in the synthesis of a number of phonate intermediates initially seemed to be different oligonucleotide analogues, for exam- superfluous, its incorporation into the synthetic ple, phosphoramidates (Froehler, 1986) and protocol was shown to potentially improve the phosphorothioates (Agrawal and Tang, 1990; overall performance of the method. The proce- Stein et al., 1990), including those with all-Rp- dure simply consists of an additional conden- linkages (Almer et al., 1996). It has also been sation step using isopropyl H-phosphonate af- used in the synthesis of oligonucleotides bear- ter each nucleoside H-phosphonate coupling ing modified heterocyclic bases (Ramzaeva et (Andrus et al., 1988). The use of 2-cyanoethyl al., 1997; Seela and Wei, 1997). One particu- H-phosphonate for capping was also reported, larly interesting feature of oligonucleotide syn- although this reagent has the disadvantage of thesis when using the H-phosphonate method- being less accessible. The capping procedure ology is that it can be performed without pro- routinely used in the phosphoramidite ap- tection of the nucleobases (Kung and Jones, proach (acylation with acetic anhydride in the 1992; Wada et al., 1997). presence of nucleophilic catalysts more power- RNA synthesis is perhaps where the advan- ful than pyridine) is not compatible with H- tages of the H-phosphonate approach are most phosphonate chemistry, owing to the occur- apparent. In the first report, the t-butyldi- rence of P-acylation (see below). methylsilyl (TBDMS) group was used for pro- The efficiency of each elongation step in tection of the 2′-OH and the protocol was simi- oligonucleotide synthesis on solid support is lar to that described for DNA synthesis (Garegg usually high, but technical aspects of the pro- et al., 1986a,b). A number of subsequent reports cedure may have to be adjusted for each par- have differed in the choice of 2′-OH protection ticular machine. The most important of these (see UNITS 2.2 and 3.5). These include the use of are probably the time of preactivation of the photolabile groups (e.g., o-nitrobenzyl; Tanaka H-phosphonate before it reaches the solid sup- et al., 1987), acid-sensitive groups (e.g., 1-(2- port, the concentration of the condensing agent, chloro-4-methylphenyl)-4-methoxypiperidin- and the proportion of pyridine in the solvent 4-yl (Ctmp); Sakatsume et al., 1989), and base- mixture.
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