Synthetic Strategies and Parameters UNIT 3.3 Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method

The study of nucleic acids has, over the years, strategies for the preparation of oligodeoxyri- driven the development of fundamental method- bonucleotides. Milestones that led to the dis- ologies necessary for the examination of their covery of the phosphoramidite method for oli- structure and chemistry. The ability to produce godeoxyribonucleotide synthesis are chrono- substantial quantities of sequence-defined syn- logically reported. Alternate strategies to the thetic DNA has been invaluable to nucleic acid preparation of deoxyribonucleoside phos- research. Synthetic oligonucleotides have facili- phoramidites are then described to underscore tated structural investigations and provided a bet- the versatility with which these synthons can ter understanding of the interactions between be obtained. The mechanisms of deoxyribonu- nucleic acids and, for example, binding and/or cleoside phosphoramidite activation and the modifying proteins. In addition, synthetic oli- factors affecting condensation rates are dis- godeoxyribonucleotides have been extensively cussed along with the importance of “capping” applied to the preparation of primers for enzy- and oxidation reactions toward defining opti- matic synthesis, amplification, and DNA se- mal performance conditions for oligonu- quencing. They have also been commonly used cleotide synthesis. Finally, alternate phos- in site-directed mutagenesis experiments and as phoramidite methods to oligodeoxyribonu- hybridization probes for diagnostic purposes. cleotide synthesis and additional deprotection More recently, modified synthetic oligonu- strategies are presented to demonstrate facile cleotides have been targeted to cellular messenger access to synthetic oligonucleotides for a vari- RNAs in an attempt to control gene expression ety of applications. This unit is intended to and develop therapeutic agents against various complement APPENDIX 3C on the synthesis and types of cancer and human infectious diseases purification of oligonucleotides. (Beaucage and Iyer, 1993; Crooke and Bennett, 1996). On this basis, the availability of synthetic ACCOUNTS OF CHEMICAL oligonucleotides has undoubtedly paved the way RESEARCH IN DNA to the biotechnology revolution. OLIGONUCLEOTIDE SYNTHESIS The basic strategy in oligonucleotide syn- A major advance in the chemical synthesis thesis resembles that of the stepwise synthesis of oligodeoxyribonucleotides was accom- of polypeptides. Typically, a functionally pro- plished in the mid-1970s through the develop- tected nucleotide monomer is linked to a grow- ment of the “phosphite triester” method ing oligonucleotide chain that is then chemose- (Letsinger and Lunsford, 1976). This approach lectively deprotected and allowed to couple relied on the use of P(III) chemistry and en- with the next nucleotide monomer of the de- tailed the reaction of 5′-O-phenoxyacetyl sired sequence. Although the coupling of nu- thymidine S.1 with 2,2,2-trichloroethyl phos- cleotide monomers was traditionally carried phorodichloridite S.2 to generate the corre- out in solution according to the phosphodiester sponding deoxyribonucleoside-3′-O-phos- (Khorana, 1968) and phosphotriester (Letsin- phorochloridite S.3 and variable amounts of ger and Ogilvie, 1969) methods, these strate- (3′→3′)-dinucleoside phosphite triester S.4 gies ultimately culminated in the development (Fig. 3.3.1). Addition of 3′-O-mono- of modern automated techniques using the methoxytrityl thymidine to the reaction mix- phosphoramidite method (Beaucage and ture gave, after treatment with aqueous iodine, Caruthers, 1981; Caruthers et al., 1987a). the dinucleoside triester S.5 along Given the major impact the phos- with the symmetrical (3′→3′)- and (5′→5′)- phoramidite approach has had on the synthesis dinucleoside phosphate triesters S.6 and S.7, of oligonucleotides since its inception in the respectively. The formation of S.7 presumably early 1980s, this commentary provides an over- resulted from the reaction of 3′-O- Synthesis of view of the parameters affecting the perform- monomethoxytrityl thymidine with unreacted Unmodified ance of the method and a number of compatible S.2. It should be noted that the phosphite triester Oligonucleotides

Contributed by Serge L. Beaucage and Marvin H. Caruthers 3.3.1 Current Protocols in Nucleic Acid Chemistry (2000) 3.3.1-3.3.20 Copyright © 2000 by John Wiley & Sons, Inc. PhOCH COO Thy 2 O Thy Thy PhOCH2COO Cl CCH OPCl PhOCH COO O 3 2 2 2 O 2 O P OH 2,6-lutidine/THF O Cl3CCH2O O P 1 Cl OCH CCl 2 3 O PhOCH2COO Thy 3

4

Thy PhOCH COO Thy Thy MMTrO HO 2 PhOCH2COO O O O 1. O O MMTrO O O Thy O POCH2CCl3 O P OCH2CCl3 O P OCH2CCl3 2. I /H O O Thy O O Thy 2 2 O O

O MMTrO PhOCH2COO Thy MMTrO

MMTr, 4-methoxytrityl 567 Ph, phenyl

Figure 3.3.1 The phosphite triester method to oligodeoxyribonucleotide synthesis.

approach to oligonucleotide synthesis was re- and inert atmosphere. Although nucleoside markably rapid; the preparation of S.5 was chlorophosphites/tetrazolides led to the rapid complete within 1 hr. In the mid-1970s, the and efficient preparation of DNA oligonu- formation of internucleotide linkages with such cleotides on silica supports, the technical hard- kinetics was unmatched. ship associated with handling these synthons Later, the deoxyribonucleoside chloro- impeded automation of oligodeoxyribonu- phosphite S.8 (Fig. 3.3.2) was synthesized and cleotide syntheses. reacted with deoxyribonucleosides covalently These problems vanished when deoxyri- attached to a silica support (S.9) through a bonucleoside phosphoramidites were devel- 3′-O-succinate linkage (Matteucci and Caruth- oped in the early 1980s (Beaucage and Caruth- ers, 1981; see also UNITS 3.1 and 3.2). The ers, 1981). These synthons were stable to air dinucleoside phosphate triester S.10 was pro- oxidation and hydrolysis under normal labora- duced in yields > 90%. Addition of 1H-tetrazole tory conditions but readily activated under mild to chlorophosphite S.8 significantly improved acidic conditions to form internucleotide link- condensation rates and coupling yields. The ages in near-quantitative yields. Because of application of nucleoside chlorophosphites or these properties, deoxyribonucleoside phos- the corresponding tetrazolides to solid-phase phoramidites have been conducive to auto- synthesis of oligonucleotides was, however, mated oligonucleotide synthesis on polymeric tedious. Because of inherent sensitivity to supports and are still the preferred synthons for moisture and air oxidation, the preparation of such syntheses. The preparation of these re- these reagents from reactive bifunctional agents was straightforward and involved the phosphitylating agents had to be performed at reaction of protected deoxyribonucleosides, −78°C under rigorously anhydrous conditions such as S.11, with chloro-(N,N-dimethy-

DMTrO B O

B DMTrO HO B O O O I2/H2O O O POMe O O O B N P O P H Cl OMe O O O 8 9 N P O H Oligodeoxyribo- DMTr, 4,4'-dimethoxytrityl B, Thy or any N-protected nucleobase 10 nucleotide Me, methyl Synthesis Using P, solid support the Phosphor- amidite Method Figure 3.3.2 Application of the phosphite triester approach to solid-phase DNA synthesis. 3.3.2

Current Protocols in Nucleic Acid Chemistry Cl DNA synthesis (Adams et al., 1983; McBride P DMTrO B Me N OMe O DMTrO B 2 and Caruthers, 1983). For instance, the de- O 12 O oxyribonucleoside phosphoramidite S.16 (Fig. OH i-Pr2NEt P 3.3.5), unlike S.13, survived silica gel purifica- Me2N OMe

11 13 tion and, as a result, consistently showed sta- Et, ethyl bility in acetonitrile solutions for several days i-Pr, isopropyl without significant decomposition (Adams et al., 1983). This class of phosphoramidites en- Figure 3.3.3 Preparation of deoxyribonu- abled reliable oligodeoxyribonucleotide syn- cleoside phosphoramidite monomers. theses (51-mers) on controlled-pore glass (CPG). In the early 1980s, these 51-mers were lamino)methoxyphosphine S.12 and N,N-di- the largest DNA segments ever chemically syn- isopropylethylamine (Fig. 3.3.3). The rapid re- thesized. action afforded deoxyribonucleoside phos- One drawback to the use of S.16 in auto- phoramidites S.13 without the wasteful forma- mated oligonucleotide synthesis is the noxious tion of (3′→3′)-dinucleoside phosphite thiophenolate treatment required for postsyn- triesters. The phosphoramidites S.13 were iso- thesis removal of the methyl phosphate protect- lated by conventional laboratory techniques ing groups (Daub and van Tamelen, 1977). and stored as dry powders (Beaucage and Although demethylation of methyl phosphotri- Caruthers, 1981). esters can be effected by 2-mercaptobenzothia- The phosphoramidite method is distinctive zole (S.17; Andrus and Beaucage, 1988) or in that it enables the conversion of relatively disodium 2-carbamoyl-2-cyanoethylene-1,1- stable deoxyribonucleoside phosphoramidite dithiolate (S.18; Dahl et al., 1990) under odor- derivatives into highly reactive intermediates less conditions, this deprotection step was in- suitable for DNA oligonucleotide synthesis. convenient because it added to the time and For example, addition of 1H-tetrazole to the reagents needed for oligonucleotide processing phosphoramidite S.13 and 3′-O-levulinyl (Fig. 3.3.5). In an effort to simplify and shorten thymidine S.14 in dry acetonitrile generated the postsynthesis deprotection protocols, the de- dinucleoside phosphite triester S.15 (Fig. 3.3.4) oxyribonucleoside phosphoramidite S.19 in almost quantitative yields within a few min- (Sinha et al., 1984) was prepared under condi- utes, according to 31P-NMR spectroscopy tions similar to those originally reported by (Beaucage and Caruthers, 1981). This method- Beaucage and Caruthers (Fig. 3.3.5). This ology has been successfully applied to the phosphoramidite derivative was more stable solid-phase synthesis of oligodeoxyribonu- than the methyl phosphoramidite S.16 in wet cleotides of varying chain lengths (Caruthers et acetonitrile (Zon et al., 1985). Furthermore, al., 1982; Josephson et al., 1984). The sensitiv- phosphoramidite S.19 generated oligonu- ity of phosphoramidite S.13 to a slightly acidic cleotides from which the 2-cyanoethyl phos- environment, however, precluded silica gel pu- phate protecting groups are cleaved under the rification. Consequently, the chemical stability basic conditions required for deprotection of of crude S.13 in acetonitrile was variable, and the nucleobase protecting groups (Tener, 1961; the use of this type of phosphoramidite in auto- Letsinger and Mahadevan, 1966; Letsinger and mated systems not always reliable. Thus de- Olgilvie, 1969). Such a convenient “single oxyribonucleoside phosphoramidites with step–single reagent” postsynthesis deprotec- N,N-dialkylamino groups different from N,N- tion protocol led to the widespread acceptance dimethylamino were investigated as potential of phosphoramidite S.19 for automated solid- alternatives to S.13 in automated solid-phase phase oligonucleotide synthesis.

DMTrO B O

HO Thy O 1H-tetrazole O 13 POCH3 MeCN LevO O Thy O 14 LevO

Lev, levulinyl 15 Synthesis of Unmodified Oligonucleotides Figure 3.3.4 Activation of deoxyribonucleoside phosphoramidites toward oligonucleotide synthesis. 3.3.3

Current Protocols in Nucleic Acid Chemistry B DMTrO B − + DMTrO O NC S Na O N SH − + Na O S O S O P NH2 P i-Pr2N OMe i-Pr2N OCH2CH2CN 17 18 16 19

Figure 3.3.5 Deoxyribonucleoside phosphoramidite monomers with improved stability properties and reagents for the deprotection of methyl phosphate triesters.

ALTERNATE STRATEGIES TO dichloroimidazole produced only a small THE PREPARATION OF amount of (3′→3′)-dinucleoside phosphite tri- DEOXYRIBONUCLEOSIDE ester (< 10%). Thus, as soon as it was generated, PHOSPHORAMIDITES phosphoramidite S.21 was immediately acti- The basicity and/or nucleophilicity of ami- vated by addition of 1H-tetrazole and used in nophosphine derivatives such as S.20 depends the manual synthesis of an oligodeoxyribonu- on the nature of the functional groups bound to cleotide (22-mer). This strategy eliminated the phosphorus and on the interactions each of tedious isolation and purification of deoxyri- these groups might have with the vacant d bonucleoside phosphoramidites and problems orbital of the phosphorus atom through pπ-dπ associated with the stability of phos- overlap (Boudjebel et al., 1975). The bis-(dial- phoramidite solutions. Despite these appealing kylamino) S.20 (Fig. 3.3.6) would, features, this novel approach to the synthesis of therefore, be more prone to protonation by a deoxyribonucleoside phosphoramidites and weak acid than would the deoxyribonucleoside oligonucleotides has never been automated, phosphoramidite S.21, because it has been perhaps because of engineering limitations. demonstrated that an alkoxy group contributes In this context, it has been shown that reaction less to pπ-dπ interactions than does a dialky- of the deoxyribonucleoside S.11 with bis-(N,N- lamino group (Mathis et al., 1974). This con- diisopropylamino)methoxyphosphine S.22 (Fig. cept was convincingly tested when the deoxyri- 3.3.7) and limiting amounts of 1H-tetrazole or its bonucleoside S.11 was reacted with bis-(pyr- N,N-diisopropylammonium salt afforded the cor- rolidino)methoxyphosphine S.20 and the weak responding deoxyribonucleoside phos- acid 4,5-dichloroimidazole (Beaucage, 1984; phoramidite S.16 in isolated yields varying be- Moore and Beaucage, 1985) to generate the tween 82% and 92% (Barone et al., 1984; Lee deoxyribonucleoside phosphoramidite S.21 and Moon, 1984). This method is definitely within 10 min, in yields exceeding 86% (Fig. recommended for the preparation of a variety 3.3.6). Presumably because of weaker pπ-dπ of 2-cyanoethyl deoxyribonucleoside phos- interactions, further activation of S.21 by 4,5- phoramidite derivatives (like S.19) from phos-

DMTrO B O N 4,5-dichloroimidazole 11 P O N OMe N-methylpyrrolidone P N OMe

20 21

Figure 3.3.6 Chemoselective preparation of deoxyribonucleoside phosphoramidites in situ using bis-(pyrrolidino)methoxyphosphine and 4,5-dichloroimidazole.

DMTrO B O i-Pr2N N,N-diisopropylammonium tetrazolide 11 P i-Pr2N OR or 1H-tetrazole O P i-Pr2N OR 22 R = CH3 23 R = CH2CH2CN 16 R = CH3 Oligodeoxyribo- 19 R = CH2CH2CN nucleotide Synthesis Using Figure 3.3.7 Chemoselective preparation of deoxyribonucleoside phosphoramidites using bis- the Phosphor- (N,N-diisopropyl)alkoxyphosphine and limiting amounts of 1H-tetrazole or its N,N-diisopropylam- amidite Method monium salt. 3.3.4

Current Protocols in Nucleic Acid Chemistry NEt2 P B B Et2N NEt2 DMTrO DMTrO O O 24 CF3CONH(CH2)4OH 11 N,N-diethylammonium O N,N-diethylammonium O tetrazolide P tetrazolide P Et2N NEt2 Et2N O(CH2)4NHCOCF3

25 26

Figure 3.3.8 Preparation of deoxyribonucleoside phosphoramidites using hexaethylphosphorus triamide and limiting amounts of N,N-diethylammonium tetrazolide. phorodiamidite S.23 (Fig. 3.3.7) because of the automated oligodeoxyribonucleotide synthesis mildness and high chemoselectivity of the because anhydrous conditions are absolutely phosphinylation reaction. necessary for optimum coupling reactions. In- Along similar lines, reaction of S.11 with stead, the nonhygroscopic weak acid 1H-tetra- hexaethylphosphorus triamide S.24 (Fig. 3.3.8) zole has been and is still extensively used as an and an equimolar amount of the N,N-diethyl- activator for deoxyribonucleoside phos- ammonium salt of 1H-tetrazole cleanly gave phoramidites. In an incisive experiment, the nucleoside phosphorodiamidite S.25 Seliger and Gupta (1985) demonstrated that (Yamana et al., 1989). Addition of, for example, activation of the solid-phase-linked deoxyri- 4-(N-trifluoroacetylamino)butan-1-ol to the re- bonucleoside phosphoramidite S.28 with 1H- action mixture produced the deoxyribonu- tetrazole generated the putative phosphoro- cleoside phosphoramidite S.26 (Fig. 3.3.8) in tetrazolide derivative S.29 (Fig. 3.3.10). Imme- isolated yields exceeding 90% (Wilk et al., diate condensation of S.29 with a 1997). The versatility of this procedure allows deoxyribonucleoside covalently attached to a facile access to phosphoramidites different solid support (such as S.9) gave the dinu- from the conventional methyl or 2-cyanoethyl cleoside phosphite triester S.30 in yields deoxyribonucleoside phosphoramidites. greater than 95%. The generation of S.29 during activation of deoxyribonucleoside phosphoramidites by 1H- ACTIVATION OF tetrazole was further investigated using diethoxy- DEOXYRIBONUCLEOSIDE (N,N-diisopropylamino)phosphine S.31 and PHOSPHORAMIDITES diethoxy-(N-tetrazolyl)phosphine S.32 (Fig. The elegance of the phosphoramidite ap- 3.3.11) as models (Berner et al., 1989). 31P-NMR proach to oligodeoxyribonucleotide synthesis spectrum of S.32 shows a resonance at 126 ppm. emanates from the transformation of relatively This resonance is also apparent when S.31 or stable deoxyribonucleoside phosphoramidite phosphoramidite S.16 is mixed with 1H-tetrazole derivatives to highly reactive intermediates for in acetonitrile (McBride and Caruthers, 1983). rapid and efficient chain extension reactions. It should be noted that P-diastereomerically Such a conversion is catalyzed by weak acids. pure S.16 (Sp or Rp) rapidly epimerized at For example, activation of phosphoramidite phosphorus upon activation with 1H-tetrazole S.13 with N,N-dimethylaniline hydrochloride and produced a diastereomeric deoxyribonu- ≈ (pKa 5.15) generated the deoxyribonu- cleoside phosphite triester when reacted with cleoside chlorophosphite S.27 (Fig. 3.3.9) in ethanol (Stec and Zon, 1984). These data indi- 31 quantitative yield according to P-NMR spec- cate that activation of deoxyribonucleoside troscopy (Beaucage and Caruthers, 1981). Be- phosphoramidites with 1H-tetrazole occurs cause most tertiary amine hydrochlorides are through a rapid and reversible protonation fol- hygroscopic, these could not be used in routine lowed by a slower and reversible formation of the phosphorotetrazolide intermediate S.29. Paradoxically, activation of the nucleoside bi- DMTrO B O N,N-dimethylaniline hydrochloride cyclic phosphoramidites S.33-S.35 (Fig. 13 O CHCl3 3.3.12) with 1H-tetrazole proceeded with low P Cl OMe epimerization at phosphorus and led to highly 27 stereoselective syntheses of oligonucleoside phosphorothioates (Guo et al., 1998; Iyer et al., Figure 3.3.9 Activation of deoxyribonucleoside 1998). A higher energy barrier to pseudorota- Synthesis of phosphoramidites with N,N-dimethylaniline hy- tion is apparently responsible for the reduced Unmodified Oligonucleotides drochloride. P-epimerization of these oxazaphospholidi- 3.3.5

Current Protocols in Nucleic Acid Chemistry B DMTrO B DMTrO O O DMTrO B O O O 1H-tetrazole 9 P Et O P MeO N MeO O B MeCN P O CH2 MeO N N O N N O 29 N P P O H

28 30

Figure 3.3.10 Mechanism of the activation of deoxyribonucleoside phosphoramidites by 1H- tetrazole during solid-phase oligonucleotide synthesis.

nes during activation (Guo et al., 1998). It is (Xin and Just, 1996); benzotriazole and 5-chlo- somehow surprising that, although the confor- robenzotriazoles (Xin and Just, 1996); and 4,5- mationally restricted bicyclic oxazaphos- dichloro-, 2-bromo-4,5-dicyano-, and 4,5-di- pholidines S.33 and S.34 allowed highly cyano-imidazoles (Xin and Just, 1996). 5- stereoselective syntheses of dinucleoside Ethylthio- and 5-benzylthio-1H-tetrazoles are phosphorothioates (SP:RP > 9:1), the bicyclic also potent in the activation of phos- phosphoramidite S.36 produced the same phoramidites; these activators have been par- dinucleotides with only moderate stereose- ticularly useful in RNA synthesis (Wincott et lectivity (RP:SP ≈ 3:1) (Guo et al., 1998). The al., 1995; Wu and Pitsch, 1998). It is important bicyclic phosphoramidites S.33-S.35 are to note, however, that 1H-tetrazole and those nonetheless promising candidates for the more acidic activators (pKa < 4.8) can cleave preparation of P-diastereomerically enriched the 5′-dimethoxytrityl (DMTr) group of de- oligonucleoside phosphorothioates. oxyribonucleoside phosphoramidites to a small Reagents other than 1H-tetrazole have also extent and trigger the formation of activated been used to activate deoxyribonucleoside dimers. The coupling of these dimers during phosphoramidites, including N-methylimida- chain extension produced oligonucleotides zolium trifluoromethanesulfonate (Arnold et longer (n + 1) than the expected size (n) (Krotz al., 1989); N-methylimidazole hydrochloride et al., 1997a). The rates of DMTr deprotection (Hering et al., 1985); pyridinium tetrafluorobo- by an activator depend on its acidity, exposure rate (Brill et al., 1991); pyridinium chloride, time, and nature of the nucleobase carrying the pyridinium bromide, and pyridinium 4-methyl- DMTr group (purines deprotect faster than benzinesulfonate (Beier and Pfleiderer, 1999); pyrimidines). Typically, when the activation of N-methylanilinium trifluoroacetate (Fourrey deoxyribonucleoside phosphoramidites is per- and Varenne, 1984); N-methylanilinium formed by 1H-tetrazole for a period of 100 sec, trichloroacetate (Fourrey et al., 1987); benzimi- ∼0.3%–0.9% of (n + 1)-mers is observed; how- dazolium triflate (Hayakawa et al., 1996); imi- ever, when 1H-tetrazole is replaced by 5- dazolium triflate (Hayakawa and Kataoka, ethylthio-1H-tetrazole under similar condi- 1998); pyridine hydrochloride/imidazole tions, ∼1.0%–4.3% of (n + 1)-mers are gener- (Gryaznov and Letsinger, 1992); 5-tri- ated. It should, therefore, be understood that fluoromethyl-1H-tetrazole (Hering et al., extended coupling times and the use of more 1985); 5-(4-nitrophenyl)-1H-tetrazole (Froe- acidic activators during solid-phase oligode- hler and Matteucci, 1983); 5-(2-nitrophenyl)- 1H-tetrazole (Pon, 1987; Montserrat et al., DMTrO B DMTrO B 1994); 1-hydroxybenzotriazole (Claesen et al., O O 1984); 2,4,5-tribromo- and 2-nitro-imidazoles O X O X P P ON ON

S R OEt OEt N i-Pr NP NP 33 X = H 35 X = H 2 N 34 X = OMe 36 X = OMe OEt N OEt

Oligodeoxyribo- 31 32 nucleotide Figure 3.3.12 Nucleoside bicyclic phos- Synthesis Using Figure 3.3.11 Model compounds used in the phoramidites for the preparation of P-dias- the Phosphor- study of phosphoramidite activation by 1H- tereomerically enriched oligonucleoside amidite Method tetrazole. phosphorothioates. 3.3.6

Current Protocols in Nucleic Acid Chemistry oxyribonucleotide synthesis will result in lower pling yields averaging 45% on a CPG support recovery of full-length oligomers, because (Sekine et al., 1986). Under similar conditions, longer than full-length oligonucleotides will be activated S.38 required a coupling time of 60 produced. These observations prompted an ex- min to produce yields of 65%–70% (Casale and tensive search for less-acidic, more-nucleophilic McLaughlin, 1990). Such coupling yields are activators. It has been reported that 4,5-dicy- far below those obtained with conventional anoimidazole (Xin and Just, 1996) is less acidic deoxyribonucleoside phosphoramidites (pK 5.2), more soluble, and more nucleophilic a (∼99%) and thus underscore the importance of than 1H-tetrazole (Vargeese et al., 1998). More- steric hindrance when designing nucleobase over, the usefulness of 4,5-dicyanoimidazole protecting groups and/or modified nucleobases relates not only to efficient synthesis of oli- gonucleotides but also to the preparation of toward the synthesis of oligodeoxyribonu- nucleoside phosphoramidites from phos- cleotides and their analogues. One effective phorodiamidite S.23. Even though 1H-tetra- approach to lessening steric interferences is to zole is still very popular as an activator for increase the distance between the bulky entity deoxyribonucleoside phosphoramidites, 4,5- and the phosphoramidite function by the use of dicyanoimidazole is an attractive option for the flexible linkers. For example, unlike S.38, the activation of deoxyribonucleoside and ribonu- deoxyribonucleoside phosphoramidite S.39 cleoside phosphoramites in solid-phase oligonu- (Fig. 3.3.13) has been efficiently incorporated cleotide synthesis. into oligonucleotides under the conditions used for standard 2-cyanoethyl deoxyribonu- cleoside phosphoramidites (Bergstrom and FACTORS AFFECTING THE Gerry, 1994). CONDENSATION RATES OF Other factors influencing the coupling rates DEOXYRIBONUCLEOSIDE of activated deoxyribonucleoside phos- PHOSPHORAMIDITES phoramidites have been investigated by Dahl The steric bulk of specific guanine N-2 func- and his colleagues (1987). In a systematic tional groups has been shown to affect signifi- cantly condensation rates and coupling effi- study, it was observed that condensation rates ciency of these deoxyribonucleoside phos- varied with the nature of phosphoramidite O- phoramidite derivatives. Two classic examples alkyl and N,N-dialkylamino groups. Typically, illustrating this fact are the activation of phos- coupling rates decreased according to the fol- phoramidites S.37 and S.38 with 1H-tetrazole lowing order: O-methyl > O-(2-cyanoethyl) > (Fig. 3.3.13). In the case of activated S.37, a O-(1-methyl-2-cyanoethyl) > O-(1,1-di- condensation time of 10 min generated cou- methyl-2-cyanoethyl) and N,N-diethylamino >

O O N N NH NH OBz DMTrO N N NH PixO N N NH O O

O BzO O P P NOMe OBz i-Pr NOCHCH CN O 2 2 2

37 38

4-NO2PhCH2CH2O

N N

DMTrO N N N O N O H N

O P OCH CH CN i-Pr2N 2 2

Bz, benzoyl 39 4-NO2Ph, 4-nitrophenyl Pix, 9-phenylxanthen-9-yl Synthesis of Figure 3.3.13 Unmodified Deoxyribonucleoside phosphoramidites functionalized with nucleobase bulky Oligonucleotides groups. 3.3.7

Current Protocols in Nucleic Acid Chemistry N,N-diisopropylamino > N- > N- DMTrO B O methylanilino.

i-Pr2N OOO The effect of steric hindrance on coupling P Si rates is further illustrated by activation of the NCCH2CH2O 2′-substituted nucleoside phosphoramidites 46 S.40-S.43 (Fig. 3.3.14) and their competitive DMTrO B condensations with thymidine covalently at- O O N tached to a solid support (Kierzek et al., 1987). 2 O OO Dimer formation was quantitated and corre- P i-Pr N OCH CH CN lated with the condensation rates of S.40-S.43. 2 2 2 47 The amount of dimers formed decreased when the groups at C-2′ increased in sizes; thus 2′-H ′ ′ ′ Figure 3.3.15 Efficient ribonucleoside phos- > 2 -O-methyl > 2 -O-tetrahydropyranyl > 2 - phoramidites for solid-phase RNA synthesis. O-tert-butyldimethylsilyl (Kierzek et al., 1987). In agreement with these findings, the deoxyribonucleoside phosphoramidites S.44 steric bulk near the nucleosidic phos- (Polushin, 1996) and S.45 (Jørgensen et al., phoramidite moiety are likely to interfere with 1994) have also exhibited significantly lower coupling rates and should be given considera- condensation rates and coupling efficiency be- tion when developing novel phosphoramidite cause of steric factors (Fig. 3.3.14). Like steri- monomers. cally hindered groups attached to nucleobases (vide supra), it is also possible to decrease the SIGNIFICANCE OF THE steric demand of 2′-O-bulky protecting groups “CAPPING” REACTION IN THE by increasing the distance between these CHEMICAL SYNTHESIS OF groups and the phosphoramidite function. Spe- OLIGODEOXYRIBONUCLEOTIDES cifically, the ribonucleoside 2′-O-triiso- propylsilyloxymethyl phosphoramidite S.46 The phosphoramidite approach to oligode- (Weiss, 1998; Wu and Pitsch, 1998; Fig. oxyribonucleotide synthesis is renowned for its 3.3.15) allows much faster coupling reactions high coupling efficiency. Nonetheless, oli- (2 min) in solid-phase oligoribonucleotide gonucleotide chain extension does not occur ′ synthesis than do ribonucleoside 2 -O-tert- quantitatively even under optimum conditions. butyldimethysilyl phosphoramidites (5–8 As a result, the desired n-mer oligodeoxyri- min) under essentially identical conditions bonucleotide is contaminated in the final prod- (see UNIT 3.4). Similarly, coupling reactions of uct with a population of shorter (n − 1)-oligom- the ribonucleoside 2′-O-(o-nitrobenzy- ers. Separation of the n-mer oligonucleotide loxymethyl) phosphor- amidite S.47 (Fig. from (n − 1)-mers can be challenging; however, 3.3.15) are faster (2 min) than those effected this problem is almost completely eliminated by ribonucleoside 2′-O-(o-nitrobenzyl) by acetylation of the remaining unphosphity- phosphoramidites (10 min) under the same lated oligonucleotides after each condensation conditions (deBear et al., 1987; Schwartz et step. This “capping” reaction terminates the al., 1992). Thus functional groups generating elongation of the unphosphitylated oligomers

NHBz

N

DMTrO N O DMTrO Ura DMTrO Thy O O TBDMSO O O O R i-Pr N OHN O 2 POMe P P i-Pr2NOCH2CH2CN NCCH2CH2O O i-Pr2NOCH2CH2CN

40 R = H 44 45 41 R = OMe 42 R = OThp 43 R = OTBDMS

Oligodeoxyribo- Thp, tetrahydropyran-2-yl nucleotide TBDMS, tert-butyldimethylsilyl Synthesis Using the Phosphor- Figure 3.3.14 Nucleoside phosphoramidites functionalized with 2′- or 3′-sterically demanding amidite Method groups. 3.3.8

Current Protocols in Nucleic Acid Chemistry that would otherwise occur during the next agent in solid-phase oligonucleotide synthesis coupling step. A very effective capping reagent has allowed facile separation of capped failure is a solution of acetic anhydride, 2,6-lutidine, sequences from trityl-off full-length oligonu- and N-methylimidazole in tetrahydrofuran cleotides by reverse-phase HPLC (RP-HPLC; (Farrance et al., 1989). Such a capping formu- Natt and Häner, 1997). This capping method lation not only prevents extension of un- simplified RP-HPLC purification of synthetic phosphitylated oligomers but also efficiently oligonucleotides and resulted in higher isolated reduces the concentration of O6-phosphitylated yields. Phosphoramidite capping reagents may, guanine residues that are generated during the however, like nucleoside phosphoramidites, condensation step (Pon et al., 1986; Eadie and phosphitylate nucleobases (especially at O-6 of Davidson, 1987). The capping formulation is guanosines) and eventually lead to the forma- rich in acetate ions; these nucleophiles effi- tion of, for example, 2,6-diaminopurine resi- ciently cleave O6-phosphitylated guanine ad- dues (Eadie and Davidson, 1987). This poten- ducts by attacking tricoordinated “enol tial problem has not, as yet, been thoroughly phosphites” (like S.48; Fig. 3.3.16) and releas- investigated. Until this issue is resolved, use of the standard and well-studied acetic anhy- ing unmodified guanine residues. Adducts dride/N-methylimidazole/2,6-lutidine capping such as S.48 or its oxidized form S.49, if not formulation during solid-phase oligonu- destroyed, can serve as secondary sites for cleotide synthesis is recommended. oligonucleotide synthesis and lead to the for- mation of a complex mixture of branched oli- godeoxyribonucleotides. The generation of THE OXIDATION REACTION IN these adducts is most efficiently minimized THE SYNTHESIS OF when the capping reaction is performed before OLIGODEOXYRIBONUCLEOTIDES the oxidation step. ACCORDING TO THE O,O-Diethyl-N,N-diisopropyl phosphora- PHOSPHORAMIDITE METHOD midite has also been reported as an improved Oxidation of a newly generated phosphite capping reagent in oligonucleotide synthesis triester linkage to the corresponding phosphate (Yu et al., 1994). On the basis of the data triester function is an essential step in auto- presented, this phosphoramidite exhibited a mated synthesis of oligodeoxyribonucleotides capping efficiency that was only modestly su- by the phosphoramidite approach. Without oxi- perior to that of the standard acetic anhy- dation, an internucleoside phosphite triester dride/N-methylimidazole/2,6-lutidine capping function decomposes under the acidic condi- formulation. The phosphoramidite capping re- tions required for cleavage of the 5′-di- agent was also claimed to not produce nucleo- methoxytrityl group (Matteucci and Caruthers, base modification; supporting data were, how- 1981). Thus oxidation of phosphite triesters is ever, not shown. Interestingly, recent use of the absolutely necessary to ensure consistent high- lipophilic O-(2-cyanoethyl),O-octyl-N,N-di- yielding oligodeoxyribonucleotide syntheses. isopropyl phosphoramidite as a capping re- An aqueous solution of iodine (0.05–0.1 M)

DMTrO B DMTrO B O O

O DMTrO B DMTrO B O O O OOCHP 2CH2CN P O OCH2CH2CN O O O N N N N O POR O POR O N N NHi-Bu O N N NHi-Bu O O

O O

PP

48 R = CH2CH2CN 49 R = CH2CH2CN

i-Bu, isobutyryl Synthesis of 6 Unmodified Figure 3.3.16 Postulated O -guanine adducts generated during the chain extension step of the Oligonucleotides synthesis cycle according to the phosphoramidite method. 3.3.9

Current Protocols in Nucleic Acid Chemistry and 2,6-lutidine (Letsinger and Lunsford, tion of this modified 2′-deoxyguanosine into 1976) or pyridine (Usman et al., 1985) in tetra- oligonucleotides via the phosphoramidite ap- hydrofuran is generally used for this task; this proach is sensitive to iodine-containing solu- formulation is stable and provides rapid-reac- tions regardless of iodine concentration tion kinetics, usually without formation of side (Anonymous, 1996). products. When the N-2 amino function of In this context, it should be noted that when guanine is protected with a N,N-dimethylfor- applied to oligonucleotide synthesis, the ben- mamidine group (Zemlicka and Holy, 1967; zylic deoxyribonucleoside phosphoramidite McBride et al., 1986; Vu et al., 1990) during S.51 (Fig. 3.3.17) generated internucleoside automated oligonucleotide synthesis, however, o-methylbenzyl phosphite esters that were sen- use of the traditional iodine formulation as sitive to aqueous iodine oxidation. This sensi- oxidant led to cyanation of guanine at N-2 tivity to iodine resulted in the loss of benzylic (Mullah et al., 1995). It has also been shown phosphate protection (Caruthers et al., 1987b). that this side reaction is completely eliminated The absence of phosphate protecting groups did by the use of a lower-concentration (0.02 M) not, however, impair subsequent additions of iodine oxidation reagent without losing speed S.51 to the DNA chain. In fact, an and efficiency in the conversion of internu- oligothymidylic acid (20-mer) was prepared by cleoside phosphite triesters to phosphate tri- the iterative incorporation of S.51 with an av- esters (Mullah et al., 1995). Thus the latter erage coupling efficiency of 96%. It was specu- aqueous iodine formulation is recommended lated that phosphate-phosphite mixed anhy- for standard oligonucleotide synthesis. drides could have been generated from the For specific applications, however, interaction of phosphate diesters with activated nonaqueous oxidizing reagents may advanta- S.51 and then cleaved by excess 1H-tetrazole geously offer an alternative to aqueous iodine to regenerate the deoxyribonucleoside phos- for the oxidation of oligodeoxyribonucleoside phorotetrazolide intermediates needed for phosphite triesters. For example, m-chloroper- chain extension. benzoic acid (Tanaka and Letsinger, 1982); Because of the inherent hazards involved iodobenzene diacetate and tetra-n-butylam- with handling peroxides, the use of oxaziridine monium periodate (Fourrey and Varenne, S.50 is, therefore, recommended for the oxida- 1985); tert-butyl hydroperoxide (Hayakawa et tion of phosphite triesters of those modified al., 1986; Hayakawa and Kataoka, 1998); di- oligonucleotides that are reactive to iodine tert-butyl hydroperoxide; cumene hydroperox- and/or necessitate rigorously anhydrous condi- ide; hydrogen peroxide; bis-trimethylsilyl per- tions. Moreover, the use of S.50 in oligonu- oxide, and catalytic amounts of trimethylsilyl cleotide synthesis does not lead to detectable triflate (Hayakawa et al., 1986); dinitrogen nucleobase modifications (Anonymous, 1996). tetroxide and molecular oxygen in the presence It has also been shown that oxidation of the of 2,2′-azobis(2-methylpropionitrile) under dinucleoside 2-cyano-1,1-dimethylethyl thermal or photochemical conditions (Ben- phosphite triester S.52 (Fig. 3.3.18) with iodine trude et al., 1989); and (1S)-(+)-(10-camphor- in the presence of water, alcohols, and amines sulfonyl) oxaziridine (S.50; Ugi et al., 1988) produced the corresponding dinucleoside have been effective. The oxaziridine S.50 (Fig. phosphate S.54, phosphate triester S.55, and 3.3.17) is particularly useful for the synthesis S.56, respectively (Nielsen of oligonucleotide containing multiple 7- and Caruthers, 1988). It is postulated that under deaza-2′-deoxyguanosine residues. Incorpora- these oxidative Arbuzov-type conditions, elimination of the 2-cyano-1,1-dimethylethyl group led to the dinucleoside phosphoryl iodide

DMTrO Thy intermediate S.53. The formation of S.53 is O supported by 31P-NMR data and thus provides N O CH3 a versatile pathway to the synthesis of oligode- S O P i-Pr N O O O 2 oxyribonucleotide analogues from deoxynu- 50 51 cleoside 3′-O-(2-cyano-1,1-dimethylethyl) or o-methylbenzyl phosphoramidites. Figure 3.3.17 An oxaziridine derivative as a Another example of the importance of P(III) Oligodeoxyribo- useful oxidant in the synthesis of oligonu- nucleotide cleotides containing iodine sensitive residues, oxidation in oligodeoxyribonucleotide synthe- Synthesis Using and a benzylic deoxyribonucleoside phos- sis according to the phosphoramidite approach the Phosphor- phoramidite suitable for the preparation of oli- is the incorporation of internucleotide phos- amidite Method gonucleotide analogues. phorothioates linkages into these bio- 3.3.10

Current Protocols in Nucleic Acid Chemistry Thy DMTrO Thy DMTrO DMTrO Thy O O O

O O O I2 H2O O PI O POR P Thy NCCH2C(CH3)2O O THF O Thy or ROH O Thy O O O

AcO AcO AcO

52 53 54 R = H 55 R = CH3

R'NH2

DMTrO Thy O

O O PNHR' O Thy O

AcO

Ac, acetyl 56 R' = n-butyl

Figure 3.3.18 Access to oligodeoxyribonucleotide analogues from deoxynucleoside (2-cyano- 1,1-dimethylethyl) phosphoramidites. molecules. Oligonucleotides carrying internu- phorothioate groups (Iyer et al., 1990). Given cleotide phosphorothioate diesters display en- the biological significance of oligonucleoside hanced resistance to hydrolysis catalyzed by phosphorothioates, application of S.58 to the nucleases (Eckstein, 1985). Because of this synthesis of these modified oligonucleotides property, oligodeoxyribonucleoside phos- has spurred interest in the development of ad- phorothioates have been extensively used as ditional sulfurizing reagents. The most notable antisense molecules in the inhibition of gene sulfur-transfer agents that have been reported expression. Automated synthesis of these during this decade include N,N,N′,N′- modified oligonucleotides via the phos- tetraethylthiuram disulfide (Vu and phoramidite method consists of replacing the Hirschbein, 1991), dibenzoyl tetrasulfide (Rao aqueous iodine oxidation step by a sulfuriza- et al., 1992), bis-(O,O-diisopropoxyphosphi- tion reaction that had originally been effected nothioyl) disulfide (Stec et al., 1993), benzyl- by elemental sulfur. Given the poor solubility triethylammonium tetrathiomolybdate (Rao of elemental sulfur in organic solvents, its use and Macfarlane, 1994), bis(p-toluenesul- in automated systems has been difficult. This fonyl)disulfide (Efimov et al., 1995), 3-ethoxy- problem was eliminated when phenylacetyl di- 1,2,4-dithiazoline-5-one (Xu et al., 1996), thi- sulfide (S.57; Kamer et al. 1989; Roelen et al., iranes (Arterburn and Perry, 1997), 1991) and 3H-1,2-benzodithiol-3-one 1,1-di- bis(ethoxythiocarbonyl)tetrasulfide (Zhang et oxide (S.58; Iyer et al., 1990; Regan et al., al., 1998), and 3-methyl-1,2,4-dithiazoline-5- 1992) were employed as sulfurization reagents one (Zhang et al., 1999). Out of these sulfur- (Fig. 3.3.19). These compounds are soluble in transfer reagents, 3H-1,2-benzodithiol-3-one organic solvents and produce efficient and 1,1-dioxide and 3-ethoxy-1,2,4-dithiazoline-5- rapid sulfurization kinetics. For example, S.58 one are currently the most extensively used in converted the dinucleoside phosphite triester solid-phase synthesis of oligonucleoside phos- S.59 to the corresponding phosphorothioate phorothioates. dimer S.60 in yields exceeding 99% within 30 sec at 25°C (Iyer et al., 1990; Regan et al., STRATEGIES IN THE 1992). Deprotection of S.60 afforded the dinu- DEPROTECTION OF SYNTHETIC cleoside phosphorothioate S.61 (Fig. 3.3.19). OLIGODEOXYRIBONUCLEOTIDES Thus the sulfur-transfer reagent S.58 has en- The efficiency of the phosphoramidite abled reliable automated synthesis of phos- method for solid-phase synthesis of oligode- Synthesis of phorothioated oligomers carrying either exclu- oxyribonucleotides is such that oligonu- Unmodified Oligonucleotides sively or a predetermined number of phos- cleotides up to 50 bases long can be synthesized 3.3.11

Current Protocols in Nucleic Acid Chemistry O O S S S O S O O 57 58

B DMTrO B DMTrO O O

O O O O O S POCHCH CN S POCH2CH2CN − + 2 2 S S B O B O O O O O O

58 O O

P P

59

B HO DMTrO B O O

O O − deprotection O PS S POCH2CH2CN O B O B O O

OH O

61 P

60

Figure 3.3.19 Preparation of oligodeoxyribonucleoside phosphorothioates according to the solid- phase phosphoramidite method.

within a few hours. While the cleavage of these solution of methylamine and ammonium hy- oligonucleotides from solid supports is nor- droxide has been employed for the deprotection mally accomplished by treatment with concen- of oligonucleotides carrying N-acetyl cyto- trated ammonium hydroxide for ∼1 hr at ambi- sines, N-benzoyl adenines, and N-isobutyryl ent temperature, it will take ∼10 hr at elevated guanines (Reddy et al., 1994). With this re- temperature (55°C) to deprotect the N-is- agent, oligonucleotides were cleaved from obutyryl group of guanines, and N-benzoyl solid supports in 5 min at ambient temperature, group of cytosines and adenines (see UNIT 2.1). and complete deprotection was accomplished This time-consuming deprotection step clashed in 5 min at 65°C. It should, however, be em- with the urgent demand for synthetic oligode- phasized that an aqueous solution of methyl- oxyribonucleotides and thus provided an incen- amine and ammonium hydroxide cannot be tive to improve the chemistry involved with used for the deprotection of oligonucleotides postsynthesis oligonucleotide processing. Spe- bearing conventional N-benzoyl cytosines be- cifically, methods for rapid removal of oligonu- cause primary amines have been reported to cleotide protecting groups have attracted con- attack N4-anisoyl- or N4-benzoyl-2′-deoxy- siderable attention and motivated the develop- cytidine at C-4 to produce N4-alkylated 2′-de- ment of novel base-labile blocking groups for oxycytidine derivatives (Weber and Khorana, nucleobases (Schulhof et al., 1987; Uznanski 1972; Reddy et al., 1997). et al., 1989; Kuijpers et al., 1990; Vu et al., Gaseous amines such as ammonia or methyl- 1990; Beaucage and Iyer, 1992; Sinha et al., amine have also been employed under pressure Oligodeoxyribo- nucleotide 1993; Iyer et al., 1997; see also UNIT 2.1). Con- to achieve mild and rapid deprotection condi- Synthesis Using centrated solutions of ammonia in water, etha- tions (Boal et al., 1996). For example, oligode- the Phosphor- nol, or methanol have been used for the cleav- oxyribonucleotides having cytosines, ade- amidite Method age of these groups. Alternatively, an aqueous nines, and guanines N-protected with a tert- 3.3.12

Current Protocols in Nucleic Acid Chemistry butylphenoxyacetyl group were released from thesis of oligonucleotides bearing base-sensi- CPG supports and fully deprotected at 25°C by tive functional groups because treatment with pressurized ammonia or methylamine within concentrated ammonium hydroxide at elevated 35 or 2 min, respectively. It has also been shown temperature will no longer be required for oli- that when the N-benzoyl group is used for gonucleotide deprotection. Furthermore, depu- protection of cytosines and adenosines, and rination of adenine and guanine residues under N-isobutyryl for guanines, complete deprotec- the acidic conditions required for the removal tion of oligodeoxyribonucleotides by ammonia of the 5′-O-DMTr group will become even less gas will take ∼7 hr at 25°C. At that temperature, likely. More data are still needed to assess it would take ∼36 hr for concentrated aqueous whether the synthesis of oligodeoxyribonu- ammonium hydroxide to accomplish the same cleotides according to the phosphoramidite task (Boal et al., 1996). method without nucleobase protection is The use of ammonia or methylamine gas trouble-free. The method is promising in that it allows the simultaneous deprotection of a large may significantly expedite the production of number of oligodeoxyribonucleotides. In fact, synthetic oligonucleotides by shortening post- the number of oligonucleotides or CPG col- synthesis oligonucleotide processing time. umns that can be deprotected is limited only by Another strategy toward the preparation of the size of the pressure vessel employed. Be- oligodeoxyribonucleotides entails the stepwise cause no water is present during deprotection, condensation of dinucleotide phosphoramidite fully deblocked oligonucleotides remain ad- blocks such as S.62-S.65 (Fig. 3.3.20) instead sorbed to CPG and thus prevent cross-contami- of conventional monomeric deoxyribonu- nation between columns. Oligonucleotides can cleoside phosphoramidites for chain extension. then be eluted from individual columns with a Activation of S.62 with 1H-tetrazole produced minimum amount of water for further purifica- coupling yields (∼99%) similar to those gener- tion, if desired. This deprotection procedure ated by monomeric phosphoramidites (Kumar eliminates hazards inherent to the handling and and Poonian, 1984). The incorporation of S.63 heating of aqueous amine solutions in glass into oligonucleotides allowed syntheses of ran- vials and, more important, the time-consuming domized DNA sequences containing the 20 evaporation of these solutions. The gas-phase codons corresponding to all natural amino ac- deprotection methodology is recommended ids (Neuner et al., 1998). The efficiency of when oligonucleotides carrying base-sensitive dinucleotide phosphoramidites to solid-phase nucleobases demand mild deprotection condi- oligonucleotide synthesis has been further tions or when rapid deprotection is needed to demonstrated by the preparation of a large oli- accelerate the production of synthetic oligonu- gomer (101-mer) through repetitive condensa- cleotides. tions of the dimeric phosphoramidite S.64 (Wolter et al., 1986). Furthermore, the impurity ALTERNATE STRATEGIES TO profile of oligonucleoside phosphorothioates THE SYNTHESIS OF synthesized by iterative coupling of the OLIGODEOXYRIBONUCLEOTIDES thioated dinucleotide phosphoramidite S.65 ACCORDING TO THE (Krotz et al., 1997b) showed at least 70% re- PHOSPHORAMIDITE METHOD duction of the (n − 1)-mers and a ∼50% reduc- The versatility of the phosphoramidite ap- tion of phosphodiester formation when com- proach to oligodeoxyribonucleotide synthesis pared to profiles obtained by standard mono- has been further demonstrated by the use of mer phosphoramidite couplings. deoxyribonucleoside phosphoramidites with The use of dimeric phosphoramidites in the unprotected nucleobases. The success of this synthesis of unmodified oligodeoxyribonu- strategy depends on a modified synthesis cycle cleotides has not been widely adopted, prob- protocol that involves treatment of the solid ably because a library of up to 16 combinatorial support with an equimolar solution (0.1 M) of dimers had to be prepared to accomplish the pyridine hydrochloride and aniline in acetoni- synthesis of one oligonucleotide. Conversely, trile (Gryaznov and Letsinger, 1991) or the application of dimeric phosphoramidites to benzimidazolium triflate in methanol (Hayak- oligonucleotide analogue synthesis has been awa and Kataoka, 1998) immediately after each popular especially for the incorporation of condensation reaction. This treatment destroys modified internucleotide bridges. For example, nucleobase adducts that are forming on the the dimeric 5′-–linked thymidine Synthesis of oligonucleotidic chain during each coupling phosphoramidite S.66 (Zhao and Caruthers, Unmodified Oligonucleotides step. This procedure should facilitate the syn- 1996) and S.67 (Kofoed and Caruthers, 1996) 3.3.13

Current Protocols in Nucleic Acid Chemistry Thy DMTrO B DMTrO O O

O O

O POMe X POCH2CH2CN O B O Thy O O

O O P P R2N OR' i-Pr2N OCH2CH2CN

62 R = R' = CH3 64 X = O 63 R = CH(CH3)2, R' = CH2CH2CN 65 X = S

Figure 3.3.20 Solid-phase oligonucleotide synthesis using dinucleotide phosphoramidite deriva- tives.

have been prepared and incorporated into oli- and Shortle, 1992; Virnekäs et al., 1994), S.70 godeoxyribonucleotides to assess the physico- (Lyttle et al., 1995), S.71 (Ono et al., 1995; chemical and biochemical properties imparted Kayushin et al., 1996; Zehl et al., 1996), and by such modifications (Fig. 3.3.21). For similar S.72 (Gaytán et al., 1998; Fig. 3.3.22) repre- purposes, and given the growing interest in the senting the codons for all 20 amino acids has development of therapeutic oligonucleotides, a been achieved. The incorporation of S.69 into plethoric number of dimeric phosphoramidites oligonucleotides was accomplished by allow- structurally related to S.66-S.68 have been pre- ing a coupling time of 1 min and performing pared in recent years. Because of the intense the trinucleotide condensation step twice. Un- activity in this area of research, only a fraction der these conditions, coupling yields averaged of the work has, so far, been reviewed (see 96%–98.5% (Virnekäs et al., 1994). Consider- Beaucage and Iyer, 1993; Sanghvi and Cook, ing that each trinucleotide condensation adds 1994; Agrawal and Iyer, 1995; Iyer et al., 1999). three nucleobases to the growing oligonu- Oligodeoxyribonucleotides have also been cleotide chain, these coupling yields are prepared by the condensation of trinucleotide equivalent to three individual monomeric phos- phosphoramidite blocks to enable oligonu- phoramidite condensations, each with a cou- cleotide-directed mutagenesis. More and more, pling efficiency of 98%–99.5%. Incorporation oligonucleotides of mixed composition are be- of the trinucleotide phosphoramidites S.71 and ing used to generate combinatorial libraries of S.72 into oligonucleotides via automated solid- variants in the search for peptides and proteins phase synthesis occurred in yields that varied with improved properties. The most direct route with the sequence of the trinucleotide block to controlled mutagenesis is indeed the use of used. Nonetheless, the incorporation of these trinucleotide synthons that correspond to the trinucleotide phosphoramidite blocks into syn- amino acid codons needed. The synthesis of thetic DNA in the controlled, codon-by-codon trinucleotide phosphoramidites S.69 (Sondek construction of combinatorial libraries of struc-

DMTrO B O DMTrO Thy DMTrO Thy O O O1 O 2 O O POPhCl-2 O O P Thy O3 O Thy O BnO O4 B O O O P i-Pr N OCH CH CN P O 2 2 2 i-Pr N OCH CH CN 2 2 2 P i-Pr2N OCH2CH2CN

66 67 68

Bn, benzyl Oligodeoxyribo- PhCl-2, o-chlorophenyl nucleotide On , atom or group of atoms Synthesis Using the Phosphor- Figure 3.3.21 Solid-phase synthesis of oligonucleotide analogues from dimeric phosphoramidites amidite Method carrying modified internucleotidic linkages. 3.3.14

Current Protocols in Nucleic Acid Chemistry tural genes will be invaluable in creating mo- LITERATURE CITED lecular diversity by mutagenesis. Adams, S.P., Kavka, K.S., Wykes, E.J., Holder, S.B., and Galluppi, G.R. 1983. Hindered dialkylamino nucleoside phosphite reagents in the synthesis of CONCLUDING REMARKS two DNA 51-mers. J. Am. Chem. Soc. 105:661- Owing to the high performance of the phos- 663. phoramidite method, synthetic oligodeoxyri- Agrawal, S. and Iyer, R.P. 1995. Modified oligonu- bonucleotides became readily available and fu- cleotides as therapeutic and diagnostic agents. eled the biotechnology revolution that has irre- Curr. Opin. Biotechnol. 6:12-19. versibly changed biomedical research and the Andrus, A. and Beaucage, S.L. 1988. 2-Mercap- pharmaceutical industry. For example, without tobenzothiazole—An improved reagent for the the ability to rapidly and efficiently synthesize removal of methyl phosphate protecting groups DNA oligonucleotides, the development of the from oligodeoxynucleotide phosphotriesters. polymerase chain reaction (PCR) and its mul- Tetrahedron Lett. 29:5479-5482. tiple applications would have been difficult, if Anonymous. 1996. Non-aqueous oxidation with 10- not impossible, because this technology com- camphorsulfonyl-oxaziridine. The Glen Report pletely depends on the use of DNA primers. 9:8-9. Similarly, the phosphoramidite method has Arnold, L., Tocik, Z., Bradkova, E., Hostomsky, Z., been instrumental in the development of auto- Paces, V., and Smrt, J. 1989. Automated chlo- mated DNA sequencing, which also requires ridite and amidite synthesis of oligodeoxyri- rapid and efficient synthesis of fluorescent bonucleotides on a long chain support using amidine protected purine nucleosides. Collect. DNA primers. Another important biological Czech. Chem. Commun. 54:523-532. application for oligodeoxyribonucleotides generated by the phosphoramidite method re- Arterburn, J.B. and Perry, M.C. 1997. Rhenium catalyzed sulfurization of phosphorus(III) com- lates to site-specific mutagenesis of protein pounds with thiiranes: New reagents for phos- genes. Mutagenesis of this type has been used phorothioate ester synthesis. Tetrahedron Lett. to study protein structure-function relation- 38:7701-7704. ships and to alter the therapeutic spectrum of Barone, A.D., Tang, J.-Y., and Caruthers, M.H. pharmaceutically active proteins. In addition, 1984. In situ activation of bis-dialkylamino- the phosphoramidite method has been particu- —A new method for synthesizing de- larly useful in the synthesis of modified oli- oxyoligonucleotides on polymer supports. Nucl. gonucleotides for diagnostic applications and Acids Res. 12:4051-4061. as potential therapeutic drugs. Although the Beaucage, S.L. 1984. A simple and efficient prepa- latter research area is relatively new, several ration of deoxynucleoside phosphoramidites in oligonucleotide-based drugs have already situ. Tetrahedron Lett. 25:375-378. reached the clinic, and others are under pre- Beaucage, S.L. and Caruthers, M.H. 1981. De- clinical investigation to benefit public health oxynucleoside phosphoramidites—A new class and push further the frontiers of knowledge. of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859-1862. Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phos- B DMTrO R'O B O O phoramidite approach. Tetrahedron 48:2223- 2311. O O O POR O POPhCl-2 Beaucage, S.L. and Iyer, R.P. 1993. The synthesis of B O O B modified oligonucleotides by the phos- O O phoramidite approach and their applications. O O Tetrahedron 49:6123-6194. O POR O POPhCl-2 Beier, M. and Pfleiderer, W. 1999. Pyridinium O B O B O O salts–An effective class of catalysts for oligonu- cleotide synthesis. Helv. Chim. Acta 82:879- O O 887. P P i-Pr2N OR i-Pr2N OCH2CH2CN Bentrude, W.G., Sopchik, A.E., and Gajda, T. 1989. 69 R = CH3 71 R' = DMTr Stereo- and regiochemistries of the oxidations of 70 R = CH CH CN 72 R' = Fmoc 2 2 2-methoxy-5-tert-butyl-1,3,2-dioxaphosphori Fmoc, 9-fluorenylmethoxycarbonyl nanes and the cyclic methyl 3′,5′-phosphite of PhCl-2, o-chlorophenyl thymidine by H2O/I2 and O2/AIBN to P-chiral . 17O NMR assignment of phospho- Figure 3.3.22 Trinucleotide phosphoramidite rus configuration to the diastereomeric thymid- Synthesis of ine cyclic methyl 3′,5′-monophosphates. J. Am. Unmodified blocks for the controlled, codon-by-codon, con- Oligonucleotides struction of combinatorial gene libraries. Chem. Soc. 111:3981-3987. 3.3.15

Current Protocols in Nucleic Acid Chemistry Bergstrom, D.E. and Gerry, N. 1994. Precision se- Dahl, B.H., Nielsen, J., and Dahl, O. 1987. Mecha- quence-specific cleavage of a nucleic acid by a nistic studies on the phosphoramidite coupling minor-groove-directed metal-binding ligand reaction in oligonucleotide synthesis. I. Evi- linked through N-2 of deoxyguanosine. J. Am. dence for nucleophilic catalysis by tetrazole and Chem. Soc. 116:12067-12068. rate variations with the phosphorus substituents. Nucl. Acids Res. 15:1729-1743. Berner, S., Mühlegger, K., and Seliger, H. 1989. Studies on the role of tetrazole in the activation Dahl, B.H., Bjergårde, K., Henriksen, L., and Dahl, of phosphoramidites. Nucl. Acids Res. 17:853- O. 1990. A highly reactive, odourless substitute 864. for thiophenol/triethylamine as a deprotection reagent in the synthesis of oligonucleotides and Boal, J.H., Wilk, A., Harindranath, N., Max, E.E., their analogues. Acta Chem. Scand. 44:639-641. Kempe, T., and Beaucage, S.L. 1996. Cleavage of oligodeoxyribonucleotides from controlled- Daub, G.W. and van Tamelen, E.E. 1977. Synthesis pore glass supports and their rapid deprotection of oligoribonucleotides based on the facile cleav- by gaseous amines. Nucl. Acids Res. 24:3115- age of methyl phosphotriester intermediates. J. 3117. Am. Chem. Soc. 99:3526-3528. Boudjebel, H., Gonçalves, H., and Mathis, F. 1975. deBear, J.S., Hayes, J.A., Koleck, M.P., and Gough, Étude de la liaison P—N dans le motif S P— G.R. 1987. A universal glass support for oligonu- 2 cleotide synthesis. Nucleosides Nucleotides NMe3 en résonance magnétique nucléaire et par la réaction d’échange avec le trifluoroacétate de 6:821-830. méthyle. Bull. Chem. Soc. Chim. France 628- Eadie, J.S. and Davidson, D.S. 1987. Guanine modi- 634. fication during chemical DNA synthesis. Nucl. Acids Res. 15:8333-8349. Brill, W.K.-D., Nielsen, J., and Caruthers, M.H. 1991. Synthesis of deoxydinucleoside phos- Eckstein, F. 1985. Nucleoside phosphorothioates. phorodithioates. J. Am. Chem. Soc. 113:3972- Annu. Rev. Biochem. 54:367-402. 3980. Efimov, V.A., Kalinkina, A.L., Chakhmakhcheva, Caruthers, M.H., Beaucage, S.L., Becker, C., Ef- O.G., Schmaltz Hill, T. and Jayaraman, K. 1995. cavitch, W., Fisher, E.F., Galluppi, G., Goldman, New efficient sulfurizing reagents for the prepa- R., deHaseth, P., Martin, F., Matteucci, M., and ration of oligodeoxyribonucleotide phos- Stabinsky, Y. 1982. New methods for synthesiz- phorothioate analogues. Nucl. Acids Res. ing deoxyoligonucleotides. In Genetic Engineer- 23:4029-4033. ing: Principles and Methods, Vol. 4 (J.K. Setlow Farrance, I.K., Eadie, J.S., and Ivarie, R. 1989. Im- and A. Hollaender, eds.) pp. 1-17. Plenum, New proved chemistry for oligodeoxyribonucleotide York. synthesis substantially improves restriction en- Caruthers, M.H., Barone, A.D., Beaucage, S.L., zyme cleavage of a synthetic 35 mer. Nucl. 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Current Protocols in Nucleic Acid Chemistry Gryaznov, S.M. and Letsinger, R.L. 1992. Selective Kamer, P.C.J., Roelen, H.C.P.F., van den Elst, H., O-phosphitilation with nucleoside phos- van der Marel, G.A., and van Boom, J.H. 1989. phoramidite reagents. Nucl. Acids Res. 20:1879- An efficient approach toward the synthesis of 1882. phosphorothioate diesters via the Schönberg re- action. Tetrahedron Lett. 30:6757-6760. Guo, M., Yu, D., Iyer, R.P., and Agrawal, S. 1998. Solid-phase stereoselective synthesis of 2′-O- Kayushin, A.L., Korosteleva, M.D., Miroshnikov, methyl oligoribonucleoside phosphorothioates A.I., Kosch, W., Zubov, D., and Piel, N. 1996. A using nucleoside oxazaphospholidines. Bioorg. convenient approach to the synthesis of trinu- Med. Chem. Lett. 8:2539-2544. cleotide phosphoramidites—Synthons for the generation of oligonucleotide/peptide libraries. Hayakawa, Y. and Kataoka, M. 1998. Facile synthe- Nucl. 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Current Protocols in Nucleic Acid Chemistry Mathis, R., Lafaille, L., and Burgada, R. 1974. Rao, M.V. and Macfarlane, K. 1994. Solid phase Fréquence d’absorption de la liaison P-N dans synthesis of phosphorothioate oligonucleotides des composés du phosphore tricoordonné. Spec- using benzyltriethylammonium tetrathio- trochim. Acta, Part A 30:357-370. molybdate as a rapid sulfur transfer reagent. Tetrahedron Lett. 35:6741-6744. Matteucci, M.D. and Caruthers, M.H. 1981. Synthe- sis of deoxyoligonucleotides on a polymer sup- Rao, M.V., Reese, C.B., and Zhengyun, Z. 1992. port. J. Am. Chem. Soc. 103:3185-3191. Dibenzoyl tetrasulphide—A rapid sulphur trans- fer agent in the synthesis of phosphorothioate McBride, L.J. and Caruthers, M.H. 1983. An inves- analogues of oligonucleotides. Tetrahedron Lett. tigation of several deoxynucleoside phos- 33:4839-4842. phoramidites useful for synthesizing deoxyoli- gonucleotides. Tetrahedron Lett. 24:245-248. 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Current Protocols in Nucleic Acid Chemistry Zemlicka, J. and Holy, A. 1967. Preparation of N-di- Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Sum- methylaminomethylene derivatives—A new mers, M.F., and Byrd, R.A. 1985. Analytical method of a selective substitution of nucleoside studies of ‘mixed sequence’ oligodeoxyribonu- amino groups. Coll. Czech. Chem. Commun. cleotides synthesized by competitive coupling of 32:3159-3168. either methyl- or β-cyanoethyl-N,N-diisopropy- lamino phosphoramidite reagents, including 2′- Zhang, Z., Nichols, A., Alsbeti, M., Tang, J.X., and deoxyinosine. Nucl. Acids Res. 13:8181-8196. Tang, J.Y. 1998. Solid phase synthesis of oli- gonucleotide phosphorothioate analogues using bis(ethoxythiocarbonyl)tetrasulfide as a new sul- fur-transfer reagent. Tetrahedron Lett. 39:2467- Contributed by Serge L. Beaucage 2470. Center for Biologics Evaluation and Research Zhang, Z., Nichols, A., Tang, J.X., Han, Y., and Tang, Food and Drug Administration J.Y. 1999. Solid phase synthesis of oligonu- Bethesda, Maryland cleotide phosphorothioate analogues using 3- methyl-1,2,4-dithiazolin-5-one (MEDITH) as a Marvin H. Caruthers new sulfur-transfer reagent. Tetrahedron Lett. University of Colorado 40:2095-2098. Boulder, Colorado Zhao, Z. and Caruthers, M.H. 1996. Synthesis and preliminary biochemical studies with 5′-deoxy- 5′-methylidyne phosphonate linked thymidine oligonucleotides. Tetrahedron Lett. 37:6239- 6242.

Oligodeoxyribo- nucleotide Synthesis Using the Phosphor- amidite Method 3.3.20

Current Protocols in Nucleic Acid Chemistry