Nucleobase Protection of Deoxyribo- and UNIT 2.1 Ribonucleosides SALIENT FEATURES OF of synthesis. For example, the exocyclic amino OLIGONUCLEOTIDE SYNTHESIS groups of nucleobases, the 2′-hydroxy groups DNA and RNA are biopolymers comprised of sugars (UNIT 2.2), and the internucleotidic of nucleotide units. The synthesis of DNA and phosphodiester functions, are protected using RNA as segments consisting of several nucleo- persistent protecting groups. tides each, referred to as oligonucleotides, is The present commentary addresses issues quite a challenging endeavor, requiring the related to the persistent protection of nucleo- rapid and quantitative coupling of nucleosidic bases. The commentary is not a comprehensive units to form internucleotidic linkages. In order review of published literature but is intended to to achieve high efficiency in the coupling reac- highlight the salient features of nucleobase pro- tions, it is necessary to direct the reaction to the tection. Only selected references are cited, and desired site in the nucleoside. This is achieved readers should refer to recent reviews (Beau- by using appropriate protecting group strate- cage and Iyer, 1992; Iyer and Beaucage, 1999) gies. and primary literature for more comprehensive To date, five methods of oligonucleotide citations. synthesis have been reported, all of which use protected nucleosides: (1) The H-phosphonate GENERAL ASPECTS OF approach (Michelson and Todd, 1955; Hall et NUCLEOBASE PROTECTION al., 1957; Froehler et al., 1986, and references The choice of nucleobase protecting groups therein; Garegg et al., 1986, and references and deprotection protocols is of paramount therein; UNIT 3.4); (2) the phosphodiester ap- importance in oligonucleotide synthesis, and is proach, pioneered by Khorana and co-workers dictated by many considerations. The protect- (Gilham and Khorana, 1958; Khorana, 1979); ing groups for nucleosides should be designed (3) the phosphotriester approach, which her- on the basis of several criteria, which have been alded a new era in nucleic acid synthesis by summarized by Reese (1978). (1) It should be enabling more rapid synthesis and purification possible to introduce the protecting group using (Letsinger and Ogilvie, 1969, and references a stable reagent that is readily obtainable. (2) therein; Reese, 1978); (4) the phosphite strat- The group should be achiral. (3) The nuclear egy (Letsinger and Lunsford, 1976, and refer- magnetic resonance (NMR) spectrum of the ences therein; Matteucci and Caruthers, 1980); protected nucleoside should be simple to inter- and (5) the phosphoramidite approach, pres- pret. (4) The protecting group should be readily ently the most popular method for oligonu- installed on the nucleoside. (5) The group cleotide synthesis (Beaucage and Caruthers, should enhance the solubility of the nucleoside 1981; UNIT 3.3). Over the years, improvements in organic solvents so that it can be adapted for in coupling chemistries, along with advances coupling reactions. (6) The group should be in methods of solid-phase assembly, have revo- stable to reagents employed in oligonucleotide lutionized the art of oligonucleotide synthesis. synthesis. (7) The group should not cause other In the synthesis of oligonucleotides, persist- structural changes in the nucleoside during its ent as well as transient protection of amino, installation or removal, or during oligonu- imido, and hydroxy groups of nucleosides are cleotide assembly. employed. Transient protecting groups are used A number of nucleobase protecting groups to temporarily block one of the hydroxy groups. have been reported. Improved procedures for For example, the 4,4′-dimethoxy trityl group their installation and removal have been pub- (DMTr) is used for transient protection of the lished. Nevertheless, unforeseen problems 5′ hydroxyl of a nucleoside (UNIT 2.3). The tran- have been reported when certain protecting sient protecting group is removed at the begin- groups are employed in oligonucleotide syn- ning of each coupling cycle. This enables the thesis. For example, N-acylated purine nucleo- coupling reaction to be directed to the desired sides, particularly N-acylated deoxyadenosine, hydroxy group during the synthesis cycle. On are more predisposed to depurination than their the other hand, persistent protecting groups are unprotected counterparts, making the growing Protection of those that remain on the nucleoside throughout oligonucleotide chain susceptible to cleavage Nucleosides for Oligonucleotide chain assembly and are only removed at the end during the synthesis cycle. In fact, each pro- Synthesis Contributed by Radhakrishnan P. Iyer 2.1.1 Current Protocols in Nucleic Acid Chemistry (2000) 2.1.1-2.1.17 Copyright © 2000 by John Wiley & Sons, Inc. tected purine and pyrimidine nucleobase exhib- Table 2.1.1 pKa Values of Nucleoside its different patterns of reactivity compared Nucleobases with the unprotected nucleoside. Certain pro- Nucleobase (site of protonation/ pK tecting groups are also sensitive to reagents deprotonation)a a used in oligonucleotide synthesis, resulting ′ either in their premature removal or in produc- 2 -Deoxythymidine (N3) 9.93 Guanosine (N1) 9.42 tion of base-modified products (reviewed by Uridine (N3) 9.38 Beaucage and Iyer, 1992). Cytidine (N3) 4.17 The sections that follow attempt to highlight Adenosine (N1) 3.52 the structural basis for the nucleophilicity and aFor numbering of nucleoside positions, see Figure 2.1.1. reactivity of nucleosides. An overview of the For additional pKa values, see Clauwaert and Stockx strategies employed for nucleobase protection (1986); Dunn and Hall (1975). is also provided. NUCLEOSIDE TAUTOMERISM tions, the tautomeric equilibrium could shift to AND pK VALUES where both imino and enol forms could exist a (Saenger, 1984). In turn, these factors influence The nucleophilicity of nucleobases (Fig. the reactivity of the nucleosides. 2.1.1) is dictated by the pKa of the amino and amido functions and their tautomeric forms. Table 2.1.1 lists the pKa values of nucleobases. REACTIVITY OF NUCLEOSIDES The amide-like nitrogens (N3 of uridine and Nucleosides participate in electrophilic and N1 of guanosine) are acidic in character, nucleophilic substitution reactions as well as whereas the ring nitrogens are basic. Therefore, addition reactions (Shabarova and Bogdanov, at strongly alkaline pH, the proton at N3 of 1994). Quite clearly, protecting groups and the uridine and thymidine and that at N1 of guanos- protocols for their installation and removal ine are removed. Under acidic conditions (at should be designed to avoid various side reac- pH ∼3), the sites of protonation are N1 of tions. adenosine and N3 of cytidine. At more acidic Nucleobases undergo substitution reactions pH, the N7 of guanosine and adenosine and O4 with electrophilic reagents. For example, both of uridine are protonated. Thus, all the bases N- and O-alkylation of the imide and lactam remain mostly uncharged in the physiological groups occur with alkylating agents. The N7 range of pH 5 to 9 (Saenger, 1984). position of purines is also a potential site for It is noteworthy that each of the nucleosides electrophilic attack (Fig. 2.1.5). Because of A, C, and G becomes protonated at one of the these competing reactions, simple alkylation of ring nitrogens rather than on the exocyclic exocyclic amino function is not a viable pro- amino group. Thus, the electron pair of the tection strategy for nucleobases. On the other amino group can delocalize into the heteroaro- hand, it is possible to chemoselectively acylate matic ring. Indeed the C–NH2 bonds of A, C, the exocyclic amino group. Thus, acyl-type and G are ∼1.34 Å long and have 40% to 50% protecting groups are widely used for the pro- double-bond character (Saenger, 1984). The tection of the exocyclic amino groups of nu- charge on a nucleobase, its tautomeric struc- cleosides (Fig. 2.1.7). tures, and its ability to form and accept hydro- The imide/lactam NH of thymidine, uridine gen bonds are also determined by its pKa values (pKa, 9.38), and guanosine (pKa, 9.42) is as well as by the pH of the medium. At physi- weakly acidic and can deprotonate under basic ological pH, the major nucleobases exist almost conditions. The resulting nucleophilic anion exclusively in the amino and keto tautomeric can react with a variety of reagents such as forms. However, under appropriate pH condi- activated phosphates, dicyclohexylcarbodi- O NH O NH2 O 2 4 7 6 7 H C 5 3 3 N 5 1 N 5 1 3 4 NH 5 3 5 4 N 6 NH 5'1 5'2 N 5'1 5'2 NH 8 8 2 2 2 6 1 H H 1 2 H H 1 9 2 9 6 6 HO N 4 HO N HO N O HO N O N O N 4 N NH2 HO 5' O 3 O O 5' O O 3 1' 1' 4' 3' 2' 4' 3' 2' HO HO HO HO H HO OH Nucleobase 2'1 Protection of deoxyadenosine deoxyguanosine deoxythymidine deoxycytosine uridine Deoxyribo- and Ribonucleosides Figure 2.1.1 Structures and numbering of nucleosides. 2.1.2 Current Protocols in Nucleic Acid Chemistry imide (DCC), mesitylene sulfonyl chloride, 1- Tri-O-acetyluridine (S.1) reacts with MSNT (mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole to produce the triazolo derivative S.2 (Fig. (MSNT), acid chlorides, phosphitylating re- 2.1.2; Reese and Ubasawa, 1980). During de- agents, and electrophilic reagents that are em- protection with ammonium hydroxide, S.2 ployed during coupling reactions. These side gives cytidine (S.3). Interestingly, during de- reactions result in nucleobase-derived N- and protection with either the tetramethyl- O-products. guanidinium salt of syn-4-nitrobenzaldoxime Nucleosides also react with a variety of or tetrabutylammonium fluoride (TBAF), S.2 nucleophilic reagents. For example, cytosine reverts to uridine (den Hartog et al., 1982). reacts with hydroxylamine at neutral and acidic The triazolo derivative S.2 is also known to pH to give the corresponding hydroxylamine react with other nucleophiles.