Synthetic Strategies and Parameters UNIT 3.4 Involved in the Synthesis of Oligodeoxyribo- and Oligoribonucleotides According to the H- Method

GENERAL INFORMATION AND verted to the dinucleoside or to a SYNTHETIC STRATEGIES variety of backbone-modified analogues, e.g., Although the most common method today phosphorothioates, , 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- 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) (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 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. Some recently introduced condensing labile benzoyl derivatives (Rozners et al., 1988, agents—e.g., bis(pentafluorophenyl) carbon- 1990, 1992). ate (Efimov et al., 1993) and carbonium- and The H-phosphonate approach seems to be phosphonium-based condensing agents (Wada even more suited for RNA than for DNA syn- et al., 1997)—however, seem to make the con- thesis. Potential problems—e.g., double activa- densation less sensitive to these factors. tion (i.e., the bis-acyl phosphite formation) and The reaction conditions for the removal of P-acylation—are significantly suppressed the acid-labile 5′-dimethoxytrityl or when using ribonucleoside H-phosphonates monomethoxytrityl groups (e.g., usually 1% to (Stawinski et al., 1991b). Another advantage in 2% of various haloacetic acids in an anhydrous the use of H-phosphonates for RNA synthesis chlorinated solvent) have been shown not to is that the condensation reaction is virtually as affect the integrity of the H-phosphonate link- fast as for DNA-synthesis and at least as effi- ages within a relevant time (Stawinski et al., cient, even with bulky 2′-O-protection. Synthe- 1988). The most important factors affecting the sis of oligoribonucleotides of up to 50 to 60 condensation and oxidation steps are discussed nucleotide residues in length is readily in more detail later in this unit. achieved (Agrawal and Tang, 1990, Rozners et Since the introduction of the H-phosphonate al., 1998), and the deprotection steps have been methodology for synthesis of oligonucleotides, adjusted to more base-labile N-protection that a number of improvements have been intro- gives better performance (Rozners et al., 1998). duced. These involve new condensing agents The most common choice for 2′-OH protec- and new reaction conditions. Oligodeoxynu- tion in oligoribonucleotide synthesis is the cleotide synthesis via H-phosphonates has been TBDMS group (see UNIT 3.5). Studies on the scaled up to 10 to 14 µmol using only a few stability of this group under the reaction con- equivalents of monomeric building blocks rela- ditions used for removal of N-acyl groups have Synthesis of tive to a support-bound oligomer per conden- been carried out (Stawinski et al., 1988; Wu and Unmodified Oligonucleotides sation (Gaffney and Jones, 1988). The H-phos- Ogilvie, 1990). A conclusion (relevant not only 3.4.3

Current Protocols in Nucleic Acid Chemistry to the H-phosphonate approach) was that syn- vated with a condensing agent before the addi- thesis of longer RNA oligonucleotides with tion of an alcohol, the reaction is considerably 2′-O-TBDMS protection would benefit from less efficient. This phenomenon was investi- the use of more labile N-acyl-protecting groups gated and traced back to the further activation for the heterocyclic base moieties. These were of the initially formed intermediates in the later duly introduced to the synthesis of oligori- absence of an alcohol (Garegg et al., 1987c,d,e). bonucleotides, both by the phosphoramidite Irrespective of the condensing agent used, tri- approach (Wu and Ogilvie, 1988; Chaix et al., coordinated phosphite derivatives were 1989) and the H-phosphonate method (West- formed; and upon reaction with an alcohol, man et al. 1993, 1994; Rozners et al., 1998). these species (the chemical nature of which Although Pv-Cl (Froehler et al., 1986; depends on the coupling agent used) always Garegg et al., 1986a) and perhaps adamantane gave various unwanted products in addition to carbonyl chloride (Andrus et al., 1988) are the the H-phosphonate diesters. most popular condensing agents used in solid- Preactivation of H-phosphonate monoesters phase synthesis of oligonucleotides via the H- with acyl chlorides produces bis-acyl phosphonate approach, several others that may phosphites (Garegg et al., 1987c). The conse- be considered for different applications have also quence of such preactivation is that, in solution been evaluated. These include various pivalic synthesis with near equimolar amounts of H- (Strömberg and Stawinski, 1987) or other car- phosphonate and alcohol, formation of boxylic acid derivatives (Jäger et al., 1987), phosphite triesters is unavoidable (at least to diphenyl phosphorochloridate (Hall et al., 1957; some extent). It is quite clear that such species Garegg et al., 1985), 2-chloro-5,5-dimethyl-2- must also be formed to some extent when the oxo-1,3,2-dioxaphosphinane (Strömberg and condensing agent and H-phosphonate are Stawinski, 1987), bis(2-oxo-3-oxazolid- mixed before entering the column in a synthe- inyl)phosphinic chloride (Strömberg and Staw- sizer. No side products that could arise from inski, 1987), various dialkyl phosphorochlori- this species have so far been detected in solid- dates (Strömberg and Stawinski, 1987; Staw- phase synthesis products, but it has been shown inski et al., 1988), arene sulfonic acid that too long of a preactivation substantially derivatives (Garegg et al., 1985; Jäger et al., slows down the coupling reaction, and the com- 1987; Dan’kov et al., 1988), 1,3-dimethyl-2- peting reaction of alcoholic functions with the chloro-imidazolium chloride (Sakatsume et al., condensing agent becomes more pronounced 1989, 1990), bis(pentafluorophenyl)carbon- (Gaffney and Jones, 1988). It has also been ate) (Efimov et al., 1993), and a number of shown that even if condensation with nucleo- carbonium and phosphonium derivatives side bis-acyl phosphites gave the correct prod- (Wada et al., 1997). uct on a solid support, the reaction of a bis-acyl phosphite with a nucleoside is substantially PARAMETERS OF THE slower than that of the corresponding H-phos- UNDERLYING CHEMISTRY phonate which is postulated to proceed via the mixed phosphono-carboxylic anhydride Activation of H-phosphonate (shown in Fig. 3.4.3; Garegg et al., 1987c; Monoesters Efimov and Dubey, 1990). The formation of an internucleoside linkage Because an excess of H-phosphonate to al- by the H-phosphonate approach, using acyl cohol is normally used in solid-phase synthesis, chlorides, proceeds via a mixed phosphono- some formation of bis-acyl phosphite is toler- carboxylic anhydride that reacts with the nu- able. It is clear that the preactivation time cleosidic component to produce an H-phos- should be minimized so that the mixture reach- phonate diester (Garegg et al., 1987c; Fig. ing the column has the highest ratio of mixed 3.4.3). The order in which the reactants are phosphono-carboxylic anhydride to bis-acyl added in solution synthesis with near equimolar phosphite as possible. Otherwise, the oligonu- amounts of phosphonate and nucleoside was cleotide synthesis will lose in efficiency. A found to be important for the efficiency of preactivation time that is too long can probably H-phosphonate diester formation (Garegg et not be compensated to the full extent by longer Strategies for the al., 1985, 1986c). The coupling reaction is vir- condensation time, because competing reac- Synthesis of Oligonucleotides tually quantitative when the condensing agent tions become more prominent. Apart from by the is added to a solution containing both the alco- minimizing preactivation time, it is also possi- H-Phosphonate hol and the H-phosphonate monoester. If, on ble to reduce or change the amount of condens- Method the other hand, the H-phosphonate is preacti- ing agent (which is usually used in excess) and 3.4.4

Current Protocols in Nucleic Acid Chemistry O

R1O P O H O

Pv-Cl = Cl C C(CH3)3 Pv-Cl R1= protected nucleoside-3'-yl R2= protected nucleoside-5'-yl

O O

R1O P OCC(CH3)3 mixed phosphono-carboxylic H acid anhydride

Pv-Cl R2OH

O O C C(CH ) O bis-acyl 3 3 R O P R OORP H-phosphonate diester phosphite 1 1 2 O C C(CH3)3 H O

R2OH

OR2 R1OP phosphite triester OR2

Figure 3.4.3 Activation of H-phosphonate monoesters. control the reaction by varying solvent compo- quinoline gives some side reactions with acti- sition (see below). It is convenient to use vated H-phosphonate monoesters (Stawinski et enough condensing agent to achieve conditions al., 1991a). The influence of base strength on that are not too moisture sensitive; but over a preactivation of ribonucleoside H-phosphon- certain level, additional excess of acyl chloride ates was examined, and substituted pyridines increases the level of side reactions that may that were less basic than pyridine itself were lower the coupling efficiency and overall yield. found to suppress formation of tricoordinated Particularly noteworthy was the observation species (Stawinski et al., 1991b). With these that H-phosphonate monoesters when activated less basic pyridine derivatives, the side reac- in the absence of a base (like pyridine) produce tions observed with quinoline could not be only monoactivated species (Garegg et al., detected. Steric factors are also likely to be 1987c). Efimov and co-workers found that re- important for double activation, because, for placing pyridine by the slightly weaker base example, the formation of tricoordinated spe- quinoline is sufficient to suppress the formation cies is slower for 2′-O-TBDMS-protected ri- of bis-acyl phosphite and keep its amount <5% bonucleoside H-phosphonates than for the cor- to 10% within the period of time necessary for responding 2′-deoxyribo derivatives. a condensation (Efimov and Dubey, 1990; Efi- Recently, bis(pentafluorophenyl) carbonate mov et al., 1990). This novel solvent mixture (PFPC) was advocated as a condensing agent (acetonitrile-quinoline, 4:1 v/v) was used in the for H-phosphonate diesters formation (Efimov synthesis of oligodeoxynucleotides up to 39 et al., 1993). The reagent may be as reactive as units in length (Efimov and Dubey, 1990). Al- Pv-Cl in promoting condensations and com- though the replacement of pyridine by quino- pares favorably with Pv-Cl in regard to its line in oligonucleotide synthesis via the H- reactivity toward heterocyclic bases and the Synthesis of phosphonate approach offers some advantages, 5′-hydroxyl function of a nucleosidic compo- Unmodified Oligonucleotides the rate of condensation is usually lower and nent (Efimov et al., 1993). Although the re- 3.4.5

Current Protocols in Nucleic Acid Chemistry agent produces double-activated species from P-Acylation H-phosphonate monoesters, these are as re- Another side reaction that potentially can active as the initially formed monoactivated occur when using acyl chlorides as condensing intermediate. Truncated sequences are agents in the H-phosphonate approach to oli- claimed to be significantly reduced compared gonucleotide synthesis is the formation of acyl- to the protocol that uses Pv-Cl as a condens- phosphonates (Fig. 3.4.4). When the synthesis ing agent. The utility of the reagent was dem- of an H-phosphonate diester is carried out onstrated in the synthesis of several oligode- with all reactants in solution under standard oxyribonucleotides (chain length 22 to 40) conditions (approximately equimolar amounts and oligonucleotide-phospholipid conju- of H-phosphonate and nucleosidic components gates (Efimov et al., 1993). together with 2 to 3 equiv of coupling agent), the reaction can be quenched long before any P-acylation can be detected. In solid-support Competing O-Acylation oligonucleotide synthesis, however, because of Direct reaction of a condensing agent with iterative condensation steps, reaction of H- hydroxylic functions in competition with the phosphonate functions with the condensing desired coupling reaction is another potential agent could become a problem. Acylphosphon- source of inefficiency in oligonucleotide syn- ate formation was not detected when H-phos- thesis. The suggestion that capping is important phonate diesters were exposed to 3 to 5 equiv when using the H-phosphonate approach for of Pv-Cl in pyridine for 30 min (Regberg et al., oligonucleotide synthesis points to a rather low 1988). In a study of P-acylation, however, a extent of acylation during the condensation step slow conversion of a di(deoxyribonu- (Andrus et al., 1988; Gaffney and Jones, 1988). cleoside) H-phosphonate to the correspond- Self-capping by the acyl chlorides used for ing pivaloylphosphonate went to completion in condensation was estimated to account for only the presence of 5 equiv of Pv-Cl in pyridine- ∼10% to 50% of nonphosphonylated hydroxyls acetonitrile after 15 hr at 20°C (Kuyl-Ye- (Gaffney and Jones, 1988), which represents heskiely et al., 1986). 0.1% to 0.5% of an average coupling yield of The P-acylation during a condensation re- 99%. When a fourfold excess of H-phosphon- action should in principle be less pronounced, ate to nucleoside and 5 equiv Pv-Cl were re- owing to the inhibitory effect of pyridinium acted for 1.5 min, however, the total amount of ions that are formed (Strömberg, 1987). In a 5′-O-acylation was ∼0.8% (Gaffney and Jones, condensation mixture containing 5 equiv of 1988). The extent of O-acylation depends heav- Pv-Cl (in pyridine-acetonitrile 1:1 v/v), only ∼ ily on reaction conditions, such as ratios and 15% of the produced H-phosphonate diester concentrations of reagents, and is likely to be is pivaloylated in 14 hr (Strömberg, 1987). In substantially lower when a larger excess of the presence of bases stronger than pyridine N H-phosphonate is used in conjunction with (e.g., -methylimidazole, triethylamine), the P-acylation is considerably faster (Strömberg, fewer equivalents of Pv-Cl. 1987). Note, however, that even if P-acylation The authors assert that the most important occurred to some extent during oligonucleotide reason for the occurrence of competing 5′-O- synthesis, this modification could not be easily acylation in machine-assisted synthesis is that carried through to the fully deprotected oli- the condensation is slowed down owing to gonucleotide. Oligonucleotidic chains bearing formation of bis-acyl phosphites (see above). acylphosphonate modifications will be cleaved ′ Acylation of 5 -OH functions has never been during final deprotection upon treatment with observed when the condensation reaction is aqueous ammonia, and thus only a decrease in the carried out in solution without preactivation. yield of oligomers with the desired chain length Minimizing formation of the less reactive tri- is expected. coordinated species (from preactivation) Reactions of protected ribonucleoside H- should result in higher coupling efficiency. Us- phosphonate diesters with Pv-Cl are consider- ing fewer equivalents of condensing agent rela- ably slower than those of deoxyribonucleoside tive to H-phosphonate monoester is likely to be derivatives (Stawinski et al., 1991b). The pres- Strategies for the the most efficient way to decrease 5′-O-acyla- ence of a bulky 2′-O-TBDMS-protecting group Synthesis of tion, because it will affect the rate of both Oligonucleotides vicinal to the H-phosphonate linkage in the by the O-acylation and bis-acyl phosphite formation. ribonucleoside derivatives is likely to be one H-Phosphonate Changes in solvent composition may also be reason for the observed difference. In a typical Method beneficial (e.g., less pyridine). condensation reaction (5 equiv of Pv-Cl in 3.4.6

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

O R Cl C C(CH3)3 O O R

OHP OCP C(CH3)3 O B base O B O O

O R O R

H-phosphonate diester P-acylphosphonate

R = e.g. H or protected 2'-OH, e.g., OTBDMS B = uracil, thymine, or protected adenine, cytosine, or guanine

Figure 3.4.4 P-acylation side reaction. pyridine-acetonitrile; 1:1 v/v), only a few per- H-phosphonate diesters, only traces of P-acy- cent of the produced H-phosphonate diester is lation can be detected (31P NMR) after 22 hr pivaloylated within 22 hr (Stawinski et al., when 5 equiv of Pv-Cl in quinoline-acetonitrile 1991b), which corresponds to the time required (1:3 v/v) is used. For ribonucleoside H-phos- for 880 standard condensations reactions. As a phonate diesters, this side reaction cannot be rough estimate, a 50-residue-long 2′-O- detected even after 22 hr under the same con- TBDMS-oligoribonucleoside H-phosphonate ditions (Stawinski et al., 1991b). synthesized with 5 equiv of Pv-Cl and 50% pyridine-acetonitrile (1:1 v/v) gives truncated sequences (owing to P-acylation and sub- Other Potential Side Reactions Not many studies on the extent of base sequent cleavage) to a level of 0.1%; it is further modification when using the H-phosphonate estimated that decreasing the excess Pv-Cl to 3 approach for oligonucleotide synthesis have equiv in pyridine-acetonitrile (1:3 v/v), would only produce ∼0.025% P-acylation. For oli- been reported. Error rates in oligodeoxynu- godeoxyribonucleotide synthesis, the corre- cleotides that were produced using the H-phos- sponding levels should be roughly 0.9% and phonate methodology have been estimated by 0.2% to 0.3% for a coupling time of 1 min. This sequencing (Vasser et al., 1990). The average suggests that P-acylation could well be respon- number of substitutions was found to be 1/1783 sible for a substantial part of the observation bases. These are not necessarily caused by base that DNA synthesis with H-phosphonates, in modification during synthesis, but they at least contradistinction to oligoribonucleotide syn- indicate the maximum level of modification thesis, is not quite as efficient as with phos- one may anticipate to be present in purified phoramidites (when using 5 equiv of Pv-Cl in oligomers. pyridine-acetonitrile (1:1 v/v)). It seems clear Guanosine containing dinucleoside H- that oligodeoxyribonucleotide synthesis would phosphonates has been reported to react with benefit even more than oligoribonucleotide some condensing agents (Regberg et al., 1988). synthesis from reducing the excess of coupling The guanine residue reacts with Pv-Cl to pro- agent and pyridine (and possibly the basicity duce a modification (most likely pivaloylation by using a substituted pyridine; see below). of the lactam function) that is reversed by treat- In the earlier discussion on preactivation, ment with ammonia for a much shorter time some advantages connected with using a than that used for the deprotection of oligonu- slightly weaker base than pyridine as a co-sol- cleotides. Because this reaction is also consid- vent during the condensation step were men- erably slower than the condensation, protection tioned. Such conditions are also advantageous of the heteroaromatic lactam system in guanine when considering P-acylation. The extent of seems to be unnecessary for most applications this reaction is substantially decreased when when using H-phosphonate chemistry. It may, quinoline or some substituted pyridines are however, be advisable to analyze for base modi- Synthesis of used instead of pyridine (Stawinski et al., fications if novel condensing reagents are being Unmodified Oligonucleotides 1991b). In the case of deoxyribonucleoside used. 3.4.7

Current Protocols in Nucleic Acid Chemistry Some concern has been expressed about the pletion of the synthesis. This can be advanta- possibility of a partial loss of the 4,4′-di- geous for the preparation of uniformly modi- methoxytrityl group from 5′-protected H-phos- fied oligonucleotides (e.g., elemental sulfur can phonates upon standing in vials on automated produce phosphorothioate oligomers in one synthesizers (Froehler et al., 1986; Froehler and step from H-phosphonate oligonucleotides). Matteucci, 1987). It was estimated that 5′-O- The final oxidation step in the H-phosphon- (4,4′-dimethoxytrityl)thymidine H-phosphon- ate method for oligonucleotide synthesis is usu- ate (triethylammonium salt (TEAH+)) dis- ally performed using a solution of iodine in solved in pyridine-acetonitrile (1:1 v/v) loses aqueous pyridine (Garegg et al., 1986a,b, 2% of its 5′-protection within 4 weeks (Froehler 1987b) or with iodine-water in the presence of and Matteucci, 1987). Assuming a first-order another base (triethylamine, N-methylimida- ∼ reaction, this would translate to 0.05% detrity- zole; Froehler and Matteucci, 1986; Froehler et lation per 1000 min. If fresh solutions are pre- al., 1986). Completeness of the oxidation reac- pared before synthesis, this can be considered tion is, of course, important for efficient syn- a measure of the extent to which the loss of thesis of oligonucleotides. Any oligomeric ′ 5 -protection could be for the last added nu- chain containing an H-phosphonate function cleotide unit in the synthesis of a 100-mer will be immediately cleaved in the subsequent (assuming a 10-min cycle time). The replace- ammonia treatment step (Hammond, 1962). ment of TEAH+ by 2,3,4,6,7,8,9,10,-octahy- Incomplete oxidation will produce shorter dropyrimido[1,2-a]azeponium (DBUH+) salts of than expected oligomers, thus reducing the nucleoside H-phosphonate monoesters was yield of the desired product. An oxidation time also claimed to decrease the amount of oligom- of 10 min with 2% iodine in pyridine-water ers with a chain length of n + 1 (Froehler and (98:2) seems to be sufficient for solid-phase Matteucci, 1987) owing to suppression of par- tial detritylation during the condensation step. synthesis of 20-mers (Garegg et al., 1986a,b). It is, therefore, conceivable that the loss of the Because the oxidation reaction needs to be 5′-O-(4-monomethoxy trityl group) frequently carried out only once after chain assembly, one used in RNA synthesis should probably be can extend the oxidation time (rather than using negligible under these conditions. Although the stronger bases) to ensure complete reaction detritylation step has been shown to be com- within a marginal time loss. A longer oxidation pletely compatible with H-phosphonate link- time may still be advisable for longer oligonu- ages (Stawinski et al., 1988), be aware that cleotides. using acid-labile 2′-O-protecting groups in Competitive hydrolysis of H-phosphonate H-phosphonate-based oligoribonucleotide diesters during aqueous oxidation may be con- synthesis may cause problems (Rozners et sidered a potential problem. This has not yet al., 1994). These can be alleviated by using been reported, but it may be justified to discuss the fluoride-labile 2′-O-TBDMS or base- this issue because H-phosphonate diesters are labile 2′-O-(2-chlorobenzoyl) groups. The very unstable under aqueous basic conditions former group can be recommended for the (Nylén, 1937; Westheimer et al., 1988). The synthesis of longer oligoribonucleotides, rate constant (kOH) for hydroxide-catalyzed whereas the latter can be selected for shorter oxidation with iodine was reported to be ∼1400 fragments, owing to the simplicity and cost to 4300 times larger than that for hydrolysis of of preparation of starting materials. a series of dialkyl H-phosphonates (Nylén, 1937, 1938). These values have been used to The Oxidation Step estimate that an aqueous pyridine solution con- ∼ In the phosphoramidite approach, the oxi- taining 2% iodine should give no more than 5 dation step is carried out after each coupling to 25 ppm H-phosphonate diester cleavage reaction (Beaucage and Caruthers, 1981; Mat- (Stawinski and Strömberg, 1993). This sug- teucci and Caruthers, 1981; see also UNIT 3.3). It gests that hydrolysis during oxidation is not a may be feasible to do so via the H-phosphonate serious problem in oligonucleotide synthesis method, because the condensation reaction can according to the H-phosphonate approach. be accomplished in the presence of phosphodi- The rate of oxidation of nucleoside H-phos- Strategies for the ester functions (Földesi et al., 1989; Gryaznov phonate diesters with iodine-pyridine-water Synthesis of can be increased by including a presilylation Oligonucleotides and Potapov, 1990). Given that H-phosphonate by the functions remain intact throughout the oli- step (e.g., treatment with trimethylsilyl chlo- H-Phosphonate gomeric chain assembly, the oxidation reaction ride) in the synthetic protocol (Hata and Sekine, Method can be carried out in a single step upon com- 1974; Garegg et al., 1987b). This has, however, 3.4.8

Current Protocols in Nucleic Acid Chemistry been used infrequently in oligonucleotide syn- Dan’kov, Y.V., Batchikova, N.V., Scaptsova, N.V., thesis. Besidsky, E.S., and Azhayev, A.V. 1988. H-Phos- phonate solid-phase synthesis of oligodeoxyri- bonucleotides in syringe. Bioorg. Khim. 14:615- CONCLUDING REMARKS 620. Some of the advantages of the H-phosphonate Efimov, V.A. and Dubey, I.Y. 1990. Modification of method are as follows. The starting materials are the H-phosphonate oligonucleotide synthesis on hydrolytically stable and easy to prepare and polymer support. Bioorg. Khim. 16:211-218. handle. There is no need for protection of the Efimov, V.A., Dubey, I.Y., and Chakhmakhcheva, phosphorus center. Because excess condensing O.G. 1990. NMR study and improvement of reagent is normally used, the automated syn- H-phosphonate oligonucleotide synthesis. Nu- thesis is not too moisture sensitive. The syn- cleosides Nucleotides 9:473-477. thetic procedure for building blocks and oli- Efimov, V.A., Kalinkina, A.L., and gonucleotide preparation is more time and cost Chakhmakhcheva, O.G. 1993. Dipen- effective than other methods. The activated tafluorophenyl carbonate--a reagent for the syn- thesis of oligonucleotides and their conjugates. H-phosphonates are more reactive and less sen- Nucl. Acids Res. 21:5337-5344. sitive to steric effects than the phosphoramidites; thus efficiency and rate of formation of internu- Földesi, A., Balgobin, N., Chattopadhyaya, J. 1989. cleoside linkages via ribonucleoside H-phos- Synthesis of a “branched” trinucleotide using the H-phosphonate chemistry. Tetrahedron Lett. phonates are at least as efficient (if not more so) 30:881-884. than those achieved with deoxyribonucleoside H-phosphonates. Various oligonucleoside phos- Froehler, B.C. 1986. Deoxynucleoside H-phos- phonate diester intermediates in the synthesis of phate analogues can be obtained from one pre- internucleotide phosphate analogues. Tetrahe- cursor by changing oxidation conditions. Be- dron Lett. 27:5575-5578. cause oxidation is carried out as a final synthetic step, the H-phosphonate approach can also be the Froehler, B.C. 1993. Oligodeoxynucleotide synthe- sis. H-Phosphonate approach. In Protocols for method of choice for the preparation of uniformly Oligonucleotides and Analogs (S. Agrawal, ed.) modified oligonucleotides. pp. 63-80. Humana Press, Totowa, N.J. Froehler, B.C. and Matteucci, M.D. 1986. Nucleo- LITERATURE CITED side H-phosphonates: Valuable intermediates in Agrawal, S. and Tang, J.-Y. 1990. Efficient synthesis the synthesis of deoxyoligonucleotides. Tetrahe- of oligoribonucleotide and its phosphorothioate dron Lett. 27:469-472. analogue using H-phosphonate approach. Tetra- Froehler, B.C. and Matteucci, M.D. 1987. The use hedron Lett. 31:7541-7544. of nucleoside H-phosphonates in the synthesis of Almer, H., Stawinski, J., and Strömberg, R. 1996. deoxyoligonucleotides. Nucleosides Nucleo- Solid support synthesis of all Rp-oligoribonu- tides 6:287-291. cleoside phosphorothioates. Nucl. Acids Res. Froehler, B.C., Ng, P.G., and Matteucci, M.D. 1986. 24:3811-3820. Synthesis of DNA via deoxynucleoside H-phos- Andrus, A., Efcavitch, J.W., McBride, L.J., and Gi- phonate intermediates. Nucl. Acids Res. usti, B. 1988. Novel activating and capping re- 14:5399-5407. agent for improved hydrogen-phosphonate DNA Gaffney, B.L. and Jones, R.A. 1988. Large-scale synthesis. Tetrahedron Lett. 29:861-864. oligonucleotide synthesis by the H-phosphonate Beaucage, S.L. and Caruthers, M.H. 1981. De- method. Tetrahedron Lett. 29:2619-2622. oxynucleoside phosphoramidites—a new class Garegg, P.J., Regberg, T., Stawinski, J., and Ström- of key intermediates for deoxypolynucleotide berg, R. 1985. Formation of internucleotidic synthesis. Tetrahedron Lett. 22:1859-1862. bond via phosphonate intermediates. Chem. Scr. Beaucage, S.L. and Iyer, R.P. 1993. The synthesis of 25:280-282. modified oligonucleotides by the phos- Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., phoramidite approach and their applications. Tet- Strömberg, R., and Henrichson, C. 1986a. Nu- rahedron 49:6123-6194. cleoside H-phosphonates. III. Chemical synthe- Chaix, C., Molko, D., and Teoule, R. 1989. The sis of oligodeoxyribonucleotides by the hydro- use of labile base protecting groups in oligori- genphosphonate approach. Tetrahedron Lett. bonucleotide synthesis. Tetrahedron Lett. 27:4051-4054. 30:71-74. Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., Corby, N.S., Kenner, G.W., and Todd, A.R. 1952. Strömberg, R., and Henrichson, C. 1986b. Nu- Nucleotides. Part XVI. Ribonucleoside-5′ cleoside H-phosphonates. IV. Automated solid phosphites. A new method for the preparation of phase synthesis of oligoribonucleotides by the Synthesis of mixed secondary phosphites. J. Chem. Soc. hydrogenphosphonate approach. Tetrahedron Unmodified Oligonucleotides 3669-3675. Lett. 27:4055-4058. 3.4.9

Current Protocols in Nucleic Acid Chemistry Garegg, P.J., Regberg, T., Stawinski, J., and Ström- Kung, P.-P. and Jones R.A. 1992. H-phosphonate berg, R. 1986c. Nucleoside hydrogenphosphon- DNA synthesis without amino protection. Tetra- ates in oligonucleotide synthesis. Chem. Scr. hedron Lett. 33:5869-5872. 26:59-62. Kuyl-Yeheskiely, E., Spierenberg, M., van den Elst, Garegg, P.J., Henrichson, C., Lindh, I., Regberg, T., H., Tromp, M., van der Marel, G.A., and van Stawinski, J., and Strömberg, R. 1987a. Auto- Boom, J.H. 1986. Reaction of pivaloyl chloride mated solid phase synthesis of DNA and RNA with internucleosidic H-phosphonate diesters. fragments by the hydrogenphosphonate ap- Recl. Trav. Chim. Pays-Bas 105:505-506. proach. In Biophosphates and Their Ana- logues—Synthesis, Structure, Metabolism and Marugg, J.E., Tromp, M., Kuyl-Yeheskiely, E., van Activity (K.S. Bruzik and W.J. Stec, eds.) pp. der Marel, G.A., and van Boom, J.H. 1986. A 93-98. Elsevier/North-Holland, Amsterdam. convenient and general approach to the synthesis of properly protected d-nucleoside-3′ hydrogen- Garegg, P.J., Regberg, T., Stawinski, J., and Ström- phosphonates via phosphite intermediates. Tet- berg, R. 1987b. Nucleoside H-phosphonates. rahedron Lett. 27:2661-2664. VII. Studies on the oxidation of nucleoside hy- drogenphosphonate esters. J. Chem. Soc. Perkin Matteucci, M.D. and Caruthers, M.H. 1981. Synthe- Trans. I:1269-1273. sis of deoxyoligonucleotides on a polymer sup- port. J. Am. Chem. Soc. 103:3185-3191. Garegg, P.J., Regberg, T., Stawinski, J., and Ström- berg, R. 1987c. Nucleosides H-phosphonates. V. Nylén, P. 1937. Die Kinetik der Verseifung von The mechanism of hydrogenphosphonate diester Dialkylphosphiten. II. Allgemeine Säure- und formation using acyl chlorides as coupling Basekatalyse bei der Hydrolyse von Diäthyl- agents in oligonucleotide synthesis by the hydro- phosphit. Svensk Kem. Tidskr. 49:79-96. genphosphonate approach. Nucleosides Nucleo- tides 6:655-662. Nylén, P. 1938. Die Säure- und Basekatalyse bei der Reaktion zwischen Dialkylphosphiten und Jod. Garegg, P.J., Stawinski, J., et al. 1987d. Nucleo- Z. Anorg. Allg. Chem. 235:161-182. side H-phosphonates. VI. Reaction of nucleo- side hydrogenphosphonates with arenesul- Ramzaeva, N., Mittelbach, C., and Seela, F. 1997. fonyl chlorides. J. Chem. Soc. Perkin Trans. 7-deazaguanine DNA: Oligonucleotides with II:1209-1214. hydrophobic or cationic side chains. Helv. Chim. Acta 80:1809-1822. Garegg, P.J., Stawinski, J., and Strömberg, R. 1987e. Nucleoside H-phosphonates. VIII. activation of Regberg, T., Stawinski, J., and Strömberg, R. 1988. hydrogenphosphonate monoesters by chloro- Nucleoside H-phosphonates. IX. Possible side- and aryl sulfonyl derivatives. J. Org. reactions during hydrogenphosphonate diester Chem. 52:284-287. formation. Nucleosides Nucleotides 7:23-35. Gryaznov, S.M. and Potapov, V.K. 1990. New ap- Rozners, E., Kumpins, V., Rekis, A., and Bizdena, proach in synthesis of natural and modified oli- E. 1988. Solid phase synthesis of oligoribonu- godeoxyribonucleotides by H-phosphonate cleotides by the H-phosphonate method using method. Bioorg. Khim. 16:1419-1422. 2′-O-benzoyl protective group. Bioorg. Khim. 14:1580-1582. Hall, R.H., Todd, A., and Webb, R.F. 1957. Nucleo- tides. Part XLI. Mixed anhydrides as intermedi- Rozners, E., Rekis, A., Kumpins, V., and Bizdena, ates in the synthesis of dinucleoside phosphates. E. 1990. Synthesis of oligoribonucleotides by J. Chem. Soc. 3291-3296. the H-phosphonate method using base-labile 2′- Hammond, P.R. 1962. A simple preparation of O-protecting groups. II. Some aspects of use of ′ alkyl ammonium phosphonates and some 2 -O-benzoyl and anisoyl protecting groups. comments on the reaction. J. Chem. Soc. 2521- Bioorg. Khim. 16:1531-1536. 2522. Rozners, E., Renhofa, R., Petrova, M., Popelis, J., Hata, T. and Sekine, M. 1974. Silyl phosphites. I. Kumpins, V., and Bizdena, E. 1992. Synthesis of The reaction of silyl phosphites with diphenyl oligoribonucleotides by the H-phosphonate ap- disulphides. Synthesis of S-phenyl nucleoside proach using base labile 2′-O-protecting groups. phosphorothioates. J. Am. Chem. Soc. 96:7363- 5. Recent progress in development of the 7364. method. Nucleosides Nucleotides 11:1579- 1593. Jankowska, J., Sobkowski, M., Stawinski, J., and Kraszewski, A. 1994. Studies on aryl H-phos- Rozners, E., Westman, E., and Strömberg, R. 1994. phonates. I. Efficient method for the preparation Evaluation of 2′-hydroxyl protection in RNA- of deoxyribo- and ribonucleoside 3′-H-phos- synthesis using the H-phosphonate approach. phonate monoesters by transesterification of Nucl. Acids Res. 22:94-99. diphenyl H-phosphonate. Tetrahedron Lett. Strategies for the 35:3355-3358. Rozners, E., Westman, E., Stawinski, J., Garegg, Synthesis of P.J., Bizdena, E., and Strömberg, R. 1998. Oli- Oligonucleotides Jäger, A., Charubala, R., and Pfleiderer, W. 1987. goribonucleotide synthesis with H-phosphon- by the Synthesis and characterization of deoxy and ribo ates. An improved procedure with base labile H-Phosphonate H-phosphonate dimers. Nucl. Acids Symp. Ser. N-acyl protection and two alternative 2′-O-pro- Method 18:197-200. tecting groups. Manuscript in preparation. 3.4.10

Current Protocols in Nucleic Acid Chemistry Sakatsume, O., Ohtsuka, M., Takaku, H., and Reese, Strömberg, R. and Stawinski, J. 1987. Evaluation of C.B. 1989. Solid phase synthesis of oligoribonu- some new condensing reagents for hydrogen- cleotides using the 1-[2-chloro-4- phosphonate diester formation. Nucl. Acids methyl)phenyl]-4-methoxypiperidin-4-yl Symp. Ser. 18:185-188. ′ (Ctmp) group for the protection of the 2 -hy- Tanaka, T., Tamatsukuri, S., and Ikehara, M. 1987. droxy functions and the H-phosphonate ap- Solid phase synthesis of oligoribonucleotides proach. Nucl. Acids Res. 17:3689-3696. using the o-nitrobenzyl group for 2′-hydroxyl Sakatsume, O., Yamane, H., Takaku, H., and protection and H-phosphonate chemistry. Nucl. Yamamoto, N. 1990. Use of new phosphonylat- Acids Res. 15:7235-7248. ing and coupling agents in the synthesis of oli- Vasser, M., Ng, P.G., Jhurani, P., and Bischofberger, godeoxyribonucleotides via the H-phosphonate N. 1990. Error rates in oligodeoxynucleotides approach. Nucl. Acids Res. 18:3327-3331. synthesized by the H-phosphonate method. Seela, F. and Wei, C.F. 1997. 7-Deazaisoguanine Nucl. Acids Res. 18:3089. quartets: Self-assembled oligonucleotides lack- Wada, T., Sato, Y., Honda, F., Kawahara, S., and ing the Hoogsteen motif. Chem. Commun. 1869- Sekine, M. 1997. Chemical synthesis of oligode- 1870. oxyribonucleotides using N-unprotected H- Seliger, H. and Rösch, R. 1990. Simultaneous syn- phosphonate monomers and carbonium and thesis of multiple oligonucleotides using nucleo- phosphonium condensing reagents: O-Selective side H-phosphonate intermediates. DNA Cell phosphonylation and condensation. J. Am. Biol. 9:691-696. Chem. Soc. 119:12710-12721. Stawinski, J. and Strömberg, R. 1993. H-phosphon- Westheimer, F.H., Huang, S., and Covitz, F. 1988. ates in oligonucleotide synthesis. Trends Org. Rates and mechanisms of hydrolysis of esters of Chem. 4:31-67. phosphorous acid. J. Am. Chem. Soc. 110:181- 185. Stawinski, J. and Thelin, M. 1990. Nucleoside H- phosphonates. XI. A convenient method for the Westman, E., Stawinski, J., and Strömberg, R. 1993. preparation of nucleoside H-phosphonates. Nu- RNA-synthesis using H-phosphonates. Syn- cleosides Nucleotides 9:129-135. chronizing 2′-OH and N-protection. Collect. Czech. Chem. Commun. 58:236-237. Stawinski, J., Strömberg, R., Thelin, M., and West- man, E. 1988. Studies on the t-butyldimethylsilyl Westman, E., Sigurdsson, S., Stawinski, J., and group as 2′-O-protection in oligoribonucleotide Strömberg, R. 1994. Improving the H-phos- synthesis via the H-phosphonate approach. Nucl. phonate approach to oligoribonucleotide synthe- Acids Res. 16:9285-9298. sis. Nucl. Acids Symp. Ser. 31:25. Stawinski, J., Strömberg, R., and Westman, E. Wu, T. and Ogilvie K.K. 1988. N-Phenoxyacety- 1991a. Ribonucleoside H-phosphonates. Pyri- lated guanosine and adenosine phos- dine vs quinoline--influence on condensation phoramidites in the solid phase synthesis of oli- rate. Nucleosides Nucleotides 10:519-520. goribonucleotides: synthesis of a ribozyme se- quence. Tetrahedron Lett. 29:4249-4251. Stawinski, J., Strömberg, R., and Westman, E. 1991b. Studies on reaction conditions for ribonu- Wu, T. and Ogilvie, K.K. 1990. A study on the cleotide synthesis via the H-phosphonate ap- alkylsilyl groups in oligoribonucleotide synthe- proach. Nucl. Acids Symp. Ser. 24:228. sis. J. Org. Chem. 55:4717-4724. Stein, A., Iversen, P.L., Subasinghe, C., Cohen, J.S., Stec, W.J., and Zon, G. 1990. Preparation of 35S-labelled polyphosphorothioate oligode- Contributed by Roger Strömberg oxyribonucleotides by use of hydrogenphos- Karolinska Institute phonate chemistry. Anal. Biochem. 188:11-16. Stockholm, Sweden Strömberg, R. 1987. Nucleoside H-phosphonates. Chemical studies directed towards oligonu- Jacek Stawinski cleotide synthesis. Chem. Commun. Stockholm Stockholm University Univ. 1:1-54. Stockholm, Sweden

Synthesis of Unmodified Oligonucleotides 3.4.11

Current Protocols in Nucleic Acid Chemistry