oligonucleotide synthesis volume 288 || di- and oligonucleotide synthesis using h-phosphonate...

Di- and Oligonucleotide Synthesis 81

81

From: Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and ApplicationsEdited by: P. Herdewijn © Humana Press Inc., Totowa, NJ

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Di- and Oligonucleotide Synthesis Using H-PhosphonateChemistry

Jacek Stawinski and Roger Strömberg

SummaryIn this chapter, a concise account of the synthesis of oligonucleotides using the

H-phosphonate methodology is given. It includes various methods for the preparation of thestarting material, nucleoside 3'-H-phosphonate monoesters, their conversion into dinucleo-side H-phosphonate diesters, oxidative transformations of dinucleoside H-phosphonates intothe corresponding phosphate and phosphorothioate derivatives, and protocols for the synthesisof oligonucleotides and their phosphorothio analogs.

Key Words: H-phosphonate approach; H-phosphonate monoesters; PCl3/imidazole,H-pyrophosphonate, dinucleoside H-phosphonate diesters, H-Phosphonate diesters, oxidation,sulfurization, oligonucleotide synthesis, phosphorothioate oligonucleotide, diphenyl H-phos-phonate.

1. IntroductionAlthough the most common method today for the synthesis of oligonucle-

otides and their analogs on solid support is the phosphoramidite approach (1),the H-phosphonate methodology (2–7) can often be a viable alternative. Thelatter is not only well suited to solid phase synthesis but it appears to be apreferred means for the preparation of dinucleotides (8,9) and oligonucleotides(10,11) in solution.

Formation of an internucleotide bond via the H-phosphonate approachconsists of condensation of protected nucleoside H-phosphonate monoesterswith a nucleoside in the presence of a coupling agent to produce the corre-sponding dinucleoside H-phosphonate diesters, which can be subsequentlyoxidized to the target phosphodiester linkage (2,3,12) (Scheme 1). TheH-phosphonate and the phosphoramidite methods are making use of a reactive

82 Stawinski and Strömberg

P(III) intermediate to get fast and efficient condensation with the 5'-OH groupof a nucleoside moiety. The main difference between these approaches is thatin the H-phosphonate method reactive P(III) intermediates are tetracoordinatedspecies and are generated from stable H-phosphonate monoesters using acondensing agent, whereas in the phosphoramidite method P(III) intermedi-ates are tricoordinated and are generated via acid-promoted activation of oftenunstable, trivalent nucleoside phosphoramidites. In the latter approach, prod-ucts of the condensation are phosphite triesters bearing a phosphate-protectinggroup that has to be removed after oxidation

The most important advantages of the H-phosphonate method (13–15) arethat the starting materials (nucleoside H-phosphonate monoesters) are hydro-lytically stable, easy to prepare and handle, and because excess of a condens-ing agent is usually used, the synthesis is less sensitive to adventitious water.The latter aspect is particularly important for solution-phase synthesis and inpreparations in which only a small excess of building blocks is used.

Scheme. 1. Synthesis of oligonucleotides via H-phopshonate and phosphoramiditemethods.


The condensation step of the elongation cycle, that is, the formation of aninternucleotide H-phosphonate linkage between a nucleoside 3'-H-phosphonatemonoester and the support-bound 5'-hydroxylic component, is usually carriedout in pyridine–acetonitrile (MeCN) mixtures. Out of various condensingagents initially tested (13–15) pivaloyl chloride (PvCl ) gave the best results inautomated solid support synthesis of oligonucleotides, and it is still the mostfrequently used reagent. The reaction in pyridine or MeCN–pyridine mixturesusing two to five equivalents of PvCl is usually fast and goes to completion inless than 1 min.

A large number of other condensing agents are available and can, in someinstances, be superior to PvCl, especially for solution synthesis. Most notableof these are 5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphinane (DMOCP)(16), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (OXP) (16) and adamantanecarbonyl chloride (AdCl) (17) (Fig. 1).

Because both H-phosphonate and phosphoramidite methods are based onP(III) phosphorus compounds, they also provide an easy access to various phos-phate analogs, or P(V) compounds, by introducing the appropriate changes tothe oxidation step (15,18).

In this chapter, preparation of nucleoside H-phosphonate monoesters (seeSubheadings 3.1.1.– 3.1.3.), synthesis of dinucleoside H-phosphonate diesters(see Subheading 3.2.1.) and their oxidative transformations to the correspond-ing phosphate (see Subheading 3.2.2.) and thiophosphate (see Subheading3.2.3.) derivates, and synthesis of oligonucleotides (see Subheading 3.3.1.)via the H-phosphonate approach are presented.

2. Materials1. 10% Aqueous sodium thiosulfate (Na2S2O3).2. 2% I2 in pyridine–water (98:2, v/v).3. 5% Aqueous sodium bicarbonate (NaHCO3).4. DMOCP or OXP.5. MeCN, anhydrous, kept over 3Å sieves.6. Dichloroacetic acid (DCA) or trichloroacetic acid.7. Diethyl ether, anhydrous.8. Diphenyl H-phosphonate (DPHP).

Fig. 1. Examples of the most common condensing agents in H-phosphonate couplings.


9. Hexane.10. Imidazole.11. Methanol.12. Methylene chloride (CH2Cl2), freshly distilled from CaH2.13. Phosphonic acid (H3PO3).14. Phosphorus trichloride (PCl3), freshly distilled.15. PvCl, freshly distilled.16. Pyridine, anhydrous, kept over 4Å sieves.17. Suitably protected nucleosides.18. Sulfur.19. Silica gel (Merck silica gel 60).20. Triethylamine, distilled and stored over CaH2.21. 0.5–2M triethylammonium bicarbonate (TEAB, pH = 7.5); prepared by passing

CO2 through a suspension of water containing the appropriate amount of triethy-lamine, until the pH value has reached 7.5.

22. Polystyrene support (or controlled pore glass support) from Applied Biosystemsloaded (20– 30 μmol/g) with a 5'-DMT or MMT nucleoside succinate (or equiva-lent nucleoside loaded solid support); typical scale 1–2 μmol (ca. 50 mg sup-port).

23. Dichloroethane (CH2ClCH2Cl), anhydrous, kept over 4Å sieves.24. Protected nucleoside 3'-H-phosphonate building blocks (see Subheadings 3.1.1.–

3.1.3.) 15 μmol/coupling and 15 μmol extra for margins and priming of solutions.25. Concentrated (28–32%) aqueous ammonia– ethanol (3:1) or concentrated ammo-

nia (aqueous) or methanol saturated with ammonia.

3. Methods3.1. Synthesis of Nucleoside 3'-H-Phosphonate Monoesters(see Scheme 2)

Nucleoside 3'-H-phosphonate monoesters (triethylammonium [TEAH+] or1,8-diazabicyclo[5.4.0]undec-7-ene [DBUH+] salts) are stable solids, resistantto air oxidation and to hydrolysis by air moisture. In this respect, they offerdistinct advantages over nucleoside phosphoramidites as precursors in the syn-thesis of nucleotides and their analogs (13,15,19,20). Nucleoside H-phosphonate monoesters can be stored for several months at ambienttemperature or in a refrigerator without noticeable decomposition.

Phosphonylation with the PCl3/imidazole reagent system (see Subheading3.1.1.) is a straightforward procedure, applicable for the preparation of bothdeoxyribo- and ribonucleoside 3'-H-phosphonates. There are several variantsof this protocol that differ in the kinds of azolides, external bases, or solventsused for the reaction (3,6,8,21). Formation of symmetrical H-phosphonatediesters under the reaction conditions is negligible. If present, these uncharged


species are easily removed during chromatography. A possible inconvenienceof using this reagent system is that it must be generated in situ prior to synthesis.

Pyridinium H-pyrophosphonate (HPP) (see Subheading 3.1.2.) (22) is aconvenient phosphonylating reagent. It can be generated in situ fromphosphonic acid and a suitable condensing agent or prepared and stored as astock solution in pyridine. The moderate reactivity of HPP makes it possible toleave the reaction mixtures containing a nucleoside and five equivalents ofpyrophosphonate overnight without the danger of side reactions occurring withthe heterocyclic base residues. Because the condensing agent is completelyconsumed in the activation process of phosphonic acid and does not participatein the reaction of a nucleoside with H-pyrophosphonate, its chemical nature isof minor importance for the final yield of the H-phosphonate monoesters.Indeed, practically the same results are obtained using DHOCP (23) as con-densing agents. Mild reaction conditions, reasonably high yields, and simpleexperimental procedure make this method convenient both for small- and large-scale preparations. Another advantage of this method is that starting materialsare stable, cheap, and commercially available. Because HPP reacts very slowlywith 2'-O-protected ribonucleosides, this reagent is less useful for the prepara-tion of ribonucleoside 3'-H-phosphonates. Other methods for the preparationof nucleoside H-phosphonate monoesters, which make use of phosphonic acidand a condensing agent, have also been reported (24,25).

Subheading 3.1.3., which involves transesterification of diphenyl H-phosphonate (DPHP) with suitable protected nucleosides (26), probably repre-sents the most convenient approach for the preparation of both deoxyribo- andribonucleoside H-phosphonate monoesters. DPHP is an inexpensive, commer-cially available, stable, and easy to handle reagent that affords H-phosphonatemonoesters of purity usually better than 95% even without column chromatog-raphy (26). No side reactions involving the heterocyclic bases were detectedafter treatment of fully protected nucleosides with 20 molar excess of diphenylH-phosphonate during 8 h in pyridine. Although the reagent in pyridine under-goes disproportionation (27), this process is much slower than the trans-esterification reaction, and the side products formed (phenyl H-phosphonateand triphenyl phosphite) are easily removed during a regular work up. For alarge-scale synthesis it is possible to significantly reduce the excess of diphe-nyl H-phosphonate used (to 2–3 equivalents) by adding the reagent in adropwise manner to the solution of a nucleoside.

Other reagents useful in the preparation of nucleoside H-phosphonatesinclude, 2-cyanoethyl (N-isopropylamino)chlorophosphine (2), salicylchloro-phosphite (28), bis(N,N-di-isopropylamino)chlorophosphine (28), 2-cyano-ethyl H-phosphonate (29), and aryl H-phosphonate monoesters (30).


3.1.1. Synthesis of Nucleoside 3'-H-Phosphonate Monoesters 2Using PCl3/Imidazole/Triethylamine Reagent System (PCl3/Im)

Imidazole (5.8 g, 86 mmol) (see Note 1) is dissolved in 200 mL CH2Cl2 andthe solution kept on an ice-water bath (see Note 2). Under vigorous stirring,(2.45 mL, 28 mmol PCl3) is added, followed by 12.5 mL, 90 mmol triethy-lamine dissolved in 10 mL CH2Cl2. The mixture is stirred for 15 min and thena solution of a suitably protected nucleoside (see Note 3) (8 mmol) in 200 mLCH2Cl2 is added dropwise for 30 min. After the addition is completed, thecooling bath is removed, and the mixture is kept at room temperature withcontinuous stirring for about 1 h (thin-layer chromatography [TLC] analysis)(see Note 4). The reaction mixture is quenched by the addition of 20 mL 0.1MTEAB, pH 7.5, and extracted with 2M TEAB, pH 7.5, (3× 200 mL). The organiclayer is separated, dried over Na2SO4, and after evaporation of the solvent thecrude product is purified by a short column chromatography using a stepwisegradient of MeOH in CH2Cl2 (containing 0.1% Et3N) (0–10%). The fractionscontaining the desired product are combined, concentrated, and after dissolv-ing in a small amount of CH2Cl2, precipitated from hexane–diethyl ether mix-ture (1:1, v/v). The protected nucleoside 3' -H-phosphonate (triethylammoniumsalts) (see Note 5) are obtained as a white, microcrystalline powder. Yields70–95%. 31P nuclear magnetic resonance (NMR) (in pyridine): P = 1–4 ppm(1JPH = 600– 630 Hz).

3.1.2. Synthesis of Nucleoside 3'-H-Phosphonate MonoestersUsing H-Pyrophosphonate (HPP)

A suitably protected nucleoside 1 (2 mmol) is rendered anhydrous by evapo-ration of added pyridine and the residue is dissolved in 10 mL of 2M stocksolution of phosphonic acid (20 mmol) in pyridine (see Note 6). To the stirredreaction mixture, 11 mmol DMOCP, or 11 mmol PvCl is added (see Notes 7and 8), and progress of the reaction is monitored by TLC (see Note 9). Afterapprox 3 h, the reaction mixture is quenched with 2 mL 1M TEAB and thenpartitioned between 75 mL CH2Cl2 and 50 mL 2M TEAB buffer, pH 7.5. Theorganic layer is evaporated, the residue dissolved in CH2Cl2 and purified byflash column chromatography on silica gel using a stepwise gradient of MeOHin CH2Cl2 (containing 1% of pyridine) (0–10%, v/v). After chromatography,the product is again partitioned between dichloromethane and 0.05M TEABbuffer to remove inorganic salts, if present (see Note 10). After precipitationfrom hexane-diethyl ether (1:1, v/v), white solids of nucleoside H-phospho-nates and triethylammonium salts (see Note 11) are obtained. Yield 85–95%.31P NMR (in pyridine): P = 1–4 ppm (1JPH = 600–630 Hz).


3.1.3. Synthesis of Nucleoside 3'-H-Phosphonate MonoestersUsing DPHP

To the solution of a suitably protected nucleoside 1 (1 mmol) (see Note 12)in 5 mL pyridine, DPHP (7 mmol, 1.34 mL) is added (see Notes 13–15). After15 min (TLC analysis) (see Note 16) the reaction mixture is quenched by theaddition of 2 mL H2O (see Note 17) and is left standing for 15 min. The solventis evaporated and the residue is partitioned between 20 mL CH2Cl2 and 20 mL5% aqueous NaHCO3. The organic layer is extracted two times with 20 mL 5%aqueous NaHCO3 (see Note 18), dried over Na2SO4, and evaporated to an oil.The residue is dissolved in 3 mL methylene chloride and purified (see Note 19)by chromatography on silica gel using a stepwise gradient of methanol(0–10%) in methylene chloride (containing 0.5% of triethylamine). The frac-tions containing product are combined, concentrated, and after dissolving in asmall amount of CH2Cl2, precipitated from hexane–diethyl ether mixture (1:1,v/v). The protected nucleoside 3'-H-phosphonate (triethylammonium salts) (seeNote 20) is obtained as a white, microcrystalline powder. Yields (26) 80–90%.31P NMR (in pyridine): P = 1–4 ppm (1JPH = 600– 630 Hz).

3.2. Synthesis of Dinucleoside H-Phosphonate Diesters and TheirConversion Into Phosphate and Thiophosphate Derivatives(see Scheme 3)

The formation of dinucleoside H-phosphonates of type 4 via a condensingagent-promoted coupling of suitably protected nucleoside H-phosphonates 2with nucleosidic components 3 (Scheme 2) is a reliable and usually a highyielding reaction. The produced H-phosphonate diesters 4 are in general stableenough to be purified by a silica gel chromatography, but for most syntheticapplications, they can be transformed into various phosphate analogs withoutisolation. Owing to the chirality of the phosphorus center, dinucleoside H-phosphonates exist as pairs of RP and SP diastereomers, which can be separatedby silica gel column chromatography and subjected to various stereospecifictransformations (13,15).

To ensure fast and efficient coupling to H-phosphonate diesters, condensa-tions are usually performed in pyridine or in a mixture of pyridine and anothersolvent, which most commonly is MeCN. Because preactivation of H-phosphonate monoesters 2 with a condensing agent results in formation of ter-valent phosphorus species (31), which may give rise to side products formation,it is important to add the condensing agent as the last component to the reac-tion mixture (31) (see Subheading 3.2.1.).


Scheme. 2. Synthesis of nucleoside H-phosphonate monoesters.

Scheme. 3. Formation of an internucleotide bond via the H-phosphonate approach.

Side reactions are usually negligible during H-phosphonate diester forma-tion (32). Condensations of H-phosphonate monoesters 2 with a nucleosidiccomponent 3 to produce the corresponding H-phosphonate diesters 4 are rapid,and thus possible competing reactions of condensing agents with the hydroxy-lic component are not able to compete significantly. However, in the presenceof excess of a coupling agent, some subsequent reactions involving the P-Hbond (e.g., P-acylation) or reactive heteroaromatic lactam systems (e.g., in theguanine residue), can occur (32). For this reason unnecessary excess of con-


densing agent should be avoided, and the reaction mixture is best worked upsoon after the condensation is complete.

H-phosphonate diesters (e.g., 4) are more susceptible to oxidation thanH-phosphonate monoesters (e.g., 2), and oxidative transformations of thesecompounds (e.g., oxidation, sulfurization) that lead to the formation of the cor-responding phosphate diesters or their analogs are usually quantitative and easyto perform (see Subheadings 3.2.1.2. and 3.2.2.). The conversion ofH-phosphonate diesters into the corresponding phosphate derivatives is mostconveniently performed using iodine in aqueous pyridine (33,34). A tertiarybase, or a basic solvent (e.g., pyridine), is an indispensable component of thereaction mixture, or else the oxidation is slow. Because oxidation ofH-phosphonate diesters with iodine involves the corresponding iodophosphatesas reactive intermediates, to secure clean formation of phosphate derivatives oftype 5a water has to be present in the reaction mixture to rapidly hydrolyze theproduced iodophosphates. With limited amounts of water present, iodo-phosphates may react with other nucleophilic species present in the reactionmixture—for example, phosphate diesters—and produce relatively stablepyrophosphates as side products. If oxidation according to Subheading 3.2.2.would be inefficient for any reason, one can add trimethylsilyl chloride (33,35)to the reaction mixture before treatment with iodine. This transformsH-phosphonate diesters of type 4 into tricoordinated silyl derivatives that arerapidly oxidized with iodine in aqueous pyridine (33).

Sulfurization of H-phosphonate diesters 4 to produce thiophosphatederivatives 5b is a straightforward transformation (7,9,36). However, becausesulfurization in pyridine is a slow reaction (a few hours), one should secure anhy-drous reaction conditions to avoid competing hydrolysis of H-phosphonatediesters 4 by adventitious water. If hydrolysis of the starting material 4 wouldbecome an issue, one can add to the reaction mixture one to two equivalents oftrimethylsilyl chloride. This would secure an anhydrous reaction condition andalso facilitate sulfurization owing to formation of tricoordinated silyl deriva-tives from 4. Sulfurization of dinucleoside H-phosphonates is a stereospecificreaction (37–39). To produce separate diastereomers of thiophosphate 5a,usually the starting H-phosphonate diesters 4 are separated into diastereomers,and these are subjected separately to sulfurization (9,37). For other types ofoxidative transformations of H-phosphonate diesters, for review see refs.13,15,19, and 20.

3.2.1. Synthesis of Dinucleoside-(3'– 5')-H-Phosphonates 4

Nucleoside H-phosphonate monoester 2 (1.1 mmol) (see Note 21) and asuitably protected nucleoside 3 (1 mmol) are rendered anhydrous by repeatedevaporation of added pyridine (3 × 5 mL). The gummy residue is dissolved in


10 mL pyridine and while stirring 3 mmol PvCl (see Note 22) is added in oneportion. When TLC analysis indicates that the reaction is over (see Note 23), 1 mL2M TEAB is added. The reaction mixture is concentrated and the residue par-titioned between 30 mL methylene chloride and 20 mL 0.5M TEAB, pH 7.5.The organic layer is collected, dried over anhydrous Na2SO4, and evaporated.The crude reaction mixture containing H-phosphonates 4 (see Note 24) is thenpurified on a silica gel column using a stepwise gradient of ethyl acetate intoluene (30–50%) as eluent (see Note 25). The fractions containing product arecombined, concentrated, and after dissolving in a small amount of CH2Cl2,precipitated from hexane–diethyl ether mixture (1:1, v/v). The protecteddinucleoside H-phosphonate diesters 4 are obtained as a white, microcrystallinepowder. Yields 85– 95%. 31P (in pyridine): P = 7–10 ppm (1JPH = 700–720 Hz).

3.2.2. Synthesis of Dinucleoside-(3'– 5')-Phosphates 5a

Dinucleoside H-phosphonate 4 (1 mmol) (see Note 26) is dissolved in 10 mLpyridine, and a 2% solution of iodine in pyridine/water (96:4, v/v; 10 mL) isadded (see Note 27). After 5 min (see Note 28) the reaction mixture is dilutedwith 30 mL methylene chloride, washed with 10 mL 10% aqueous Na2S2O3,and then with 10 mL 1.0M TEAB. The organic layer is separated, solventremoved by evaporation under vacuum, and the residue is chromatographed ona silica gel column using a stepwise gradient of methanol (1–10%) in CH2Cl2

containing 0.1% triethylamine. After evaporation of the desired fractions, com-pound 5a (TEAH+ salt) is obtained as a white solid. Yields: >90%. 31P NMR(in pyridine): P = –1–3 ppm.

3.2.3. Synthesis of Dinucleoside-(3'–5')-Thiophosphate 5b

Dinucleoside H-phosphonate diester 4 (0.5 mmol) (see Note 29) is dissolvedin 20 mL pyridine, and 4 equivalents of elemental sulfur are added. The reac-tion mixture is stirred for 2–3 h (TLC analysis, see Note 28), pyridine is evapo-rated, and the residue passed through a short silica gel column using a stepwisegradient of methanol (0–10%) in CH2Cl2 containing 0.1% of triethylamine.Yields >90%. 31P NMR (in pyridine): P = 59–63 ppm.

3.3. Synthesis of Oligonucleotides

Oligonucleotide synthesis employing H-phosphonates is considerably sim-pler than when using the phosphotriester or phosphoramidite procedures. Theelongation cycle includes only two chemical steps: deprotection of the termi-nal 5'-OH function of the support-bound oligonucleotide and its coupling witha nucleoside 3'-H-phosphonate in the presence of a condensing agent. Aftercompletion of the desired number of elongation cycles (i.e., assembly of theoligomeric chain), a single oxidation cycle is performed (whereas this is per-


formed in each elongation cycle for the phosphoramidite method) to convertthe internucleoside H-phosphonate functions to phosphodiesters (or some ana-log such as phosphorothioates). Thus, because H-phosphonate functions in theoligomeric chain remain intact during all steps in the elongation cycle, the oxi-dation can be carried out in a single step after assembly of the oligomeric chainhas been completed. This can be especially advantageous for the preparation ofoligonucleotide analogs—for example, synthesis of phosphorothioates. In thisinstance the less reactive but inexpensive elemental sulfur can be used and thesulfurization reaction can be carried out outside the machine in a separate reac-tion vessel.

The efficiency of each elongation step in oligonucleotide synthesis on solidsupport is high (at least 98–99%), but the procedure may need some adjust-ment for each particular machine. The most important aspects are (1) time ofpreactivation of the H-phosphonate before it reaches the solid support, (2) con-centration of the condensing agent used, and (3) the proportion of pyridine inthe solvent mixture.

Because preactivation of H-phosphonate monoesters with PvCl (see above)gives bis-acyl phosphites (31), this must occur to some extent also when acondensing agent and an H-phosphonate are mixed before entering the columnin a synthesizer. No side products that could arise from this species have so farbeen detected during solid-phase synthesis, but it has been shown that too longpreactivation substantially slows down the coupling reaction. This can alsogive some capping of 5'-OH functions (21). Even if condensation with nucleo-side bis-acyl phosphites gives the correct product on a solid support, thereaction with a nucleoside is substantially slower (40) than that of anH-phosphonate-carboxylic acid anhydride. Although some formation of bis-acyl phosphite is tolerable (because excess of H-phosphonate to alcohol is nor-mally used), a clear conclusion is that the preactivation time, before the reactionmixture reaches the column, is best kept at a minimum. This also puts restric-tions on the machine design. Apart from minimizing preactivation time onecan also adjust the performance and control the extent of preactivation by theamount of condensing agent (which is usually used in excess) and the solventcomposition used for the reaction.

To obtain high-purity crude oligonucleotides, the oxidation must be virtu-ally quantitative. Any remaining H-phosphonate functions are instantaneouslycleaved in the subsequent step of ammonia treatment (41). Incomplete oxida-tion will produce shorter oligomers (with dangling H-phosphonate functions)rather than modified oligomers, and this will reduce the yield and purity of theproduct. For oligomer synthesis it is therefore advisable to use a longer oxida-tion time than that used for oxidation of dinucleoside H-phosphonates (lessthan 2 min) (33). For example, an oxidation time of 10 min with 2% iodine in


pyridine–water (98:2) seems to be sufficient for synthesis of 20 mers (4,5), butbecause oxidation needs to be carried out only once during the synthesis, to beon safe side one can extend the oxidation time even further (20–30 min) withonly a marginal increase in the total synthesis time.

An interesting feature of H-phosphonate-based ribonucleic acid (RNA)synthesis is that the condensation is stereoselective (42,43). This has beenexploited (together with the stereospecific sulfurization (9) and nuclease P1treatment) for synthesis of phosphorothioate RNA fragments that contain onlyRp-thiophosphate linkages (42,43).

3.3.1.Machine-Assisted Oligonucleotide Synthesis

Charge the synthesizer with solvents and detritylation solution (these canusually be kept on the synthesizer until consumed). Dissolve the protectednucleoside 3'-H-phosphonates (TEAH+ or DBUH+ salts) in pyridine, andevaporate the solvent twice under reduced pressure. Dissolve the residue inCH3CN-pyridine (3:1, v/v) to a concentration of 50 mM, and transfer to appro-priate vessels for attachment to the synthesizer (see Note 31). Prepare a PvClsolution (225 mM in CH3CN-pyridine [3:1, v/v], 0.2 mL/coupling and a littleextra for margins—for example, approx 0.2 mL) directly in the vessel that is tobe attached to the synthesizer (see Note 32). Prime the solutions and connectthe column/cartridge filled with nucleoside loaded support (approx 50 mg,1–2 μmol). The iodine solution is prepared by dissolving 0.4 g of iodine in20 mL pyridine– water 98:2 just before the oxidation step (see Note 33).Machine-assisted synthesis is then carried out according to the elongation cycle(see Table 1). If ordinary phosphodiester linkages are required, then the oxida-tion cycle (see Table 2) is performed immediately after completion of the elon-gation cycles. If a phosphorothioate oligonucleotide is desired, then thesulfurization protocol in Subheading 3.3.1.1. is followed instead of the iodine–water oxidation cycle.

3.3.2. The Sulfurization Step (in Synthesis of OligonucleosidePhosphorothioates)

For synthesis of oligonucleotides with all linkages converted intophosphorothioates, the following sulfurization step is performed instead of theoxidation in Table 2.

After completion of the machine-assisted elongation cycles, the cartridge/column is removed from the synthesizer and the support is washed with diethylether and air-dried using a syringe with a luer fitting. The support is thenremoved from the cartridge, transferred into a 2-mL cryovial (with screw cap)and treated with a solution of elemental sulfur in pyridine (1 mL 0.055M S8) atroom temperature for 18–20 h. The support is then washed with pyridine (3 × 2


Table 2The Final Oxidation Cyclea

Step Reagent/solvent Time Flow rate

1. Wash DCE 1.5 min 2 mL/min2. Detritylation 3% DCA in DCE 2.5 min3. Wash DCE, 2 min

MeCN, 1 min 3 min4. Oxidation 2% I2 in Pyridine-H2O (98:2) 30 min 1 mL/min5. Wash Pyr/MeCN, 4 min 8 min 2 mL/min

MeCN, 4 min

aPerformed once after all elongation cycles are completed.

mL), MeCN (2 × 2 mL), and dichloromethane (3 × 2 mL). After drying underreduced pressure the support is then subjected to ammonolysis as described inthe next section.

Table 1The Elongation Cycles

Step Reagent/solvent Time Flow rate

1. Wash Dichloroethane (DCE) 1.5 min 2 mL/min

2. Detritylation 3.5% DCA in DCE 2.5 min(see Note 34)

3. Wash DCE, 2 min 5 minAcetonitrile (MeCN) 1.5 minMeCN/pyridine 3:1, 1.5 min

4. Coupling 50 mM H-phosphonate, 0.1 min 2.1 min 1 mL/min(see Note 35) 225 mM PvCl, 0.1 min

50 mM H-phosphonate, 0.1 min225 mM PvCl, 0.1 min50 mM H-phosphonate, 0.1 min

pushing forward the segments 2 mL/minwith pyridine/MeCN, 0.1 min.

Recycling in a closed loop, 1.5 min.

5. Wash MeCN/Pyridine 3:1, 2 min 3 min 2 mL/minMeCN, 1 min

Total time for one elongation cycle: 14.1 min

Abbr: Dichloroethane,DCE; dichloroacetic acid, DCA.


3.3.3. Cleavage From Support After Completion of Synthesisand Base Deprotection

After completion of the machine-assisted synthesis, the cartridge/column isremoved from the synthesizer, and the support is washed with diethyl ether andair-dried using a syringe with a luer fitting. The support is then removed fromthe cartridge and transferred into a 1.5-mL cryovial (with screw cap) to whichthe ammonolysis solution is added (about 1 mL for up to a 1-mmol scale). Theammonolysis conditions for deoxyribonucleic acid (DNA) or 2'-O-alkyl RNAsynthesis are usually concentrated aqueous ammonia at 60°C for 16–20 h (fordeprotection and cleavage from support, the reaction time can vary dependingon the kind of base protecting groups used). In RNA synthesis with 2'-O-TBDMS protection and N2-phenoxyacetyl protection for guanosine, N6-butyrylfor adenosine, and N4-propionyl for cytidine, ammonolysis is done with con-centrated ammonia (aqueous)-ethanol (3:1) at 20–25°C for 8–16 h (longer timefor longer sequences, e.g., 50–60 mers).

The support is then filtered off, washed with one portion of concentratedaqueous ammonia or concentrated aqueous ammonia–ethanol (3:1), concen-trated and purified by ion exchange, and reversed-phase high-pressure liquidchromatography (for oligoribonucleotide synthesis with 2'-O-TBDMS protec-tion an additional deprotection step is done first) (see Note 36).

4. Notes1. Imidazole was made anhydrous by repeated evaporation of added anhydrous

MeCN.2. For guanosine derivatives the reaction is preferably carried out at lower

temperature (to avoid side reactions on the base, which lower the yield)—forexample, –40 or –78°C. The solution is not brought to room temperature, butinstead is kept for 1 h at the low temperature after addition of all reagents. Thenthe cold reaction mixture is poured directly into the TEAB solution in a separat-ing funnel.

3. The nucleoside was rendered anhydrous by consecutive evaporation of addedpyridine and MeCN. For the preparation of suitably protected deoxy- and ribo-nucleoside, see Chapters refs. 8–11.

4. Formation of a base-line material (TLC on silica gel plates, solvent: chloroform–methanol 9:1, v/v).

5. On some occasions, DBUH+ salts (6) of H-phosphonate monoesters can be moreuseful than TEAH+ salts. The transformation can be effected by washing a solu-tion of nucleoside H-phosphonate monoester (triethylammonium salt) in CH2Cl2

with 0.2M DBU bicarbonate buffer (pH 8.5, prepared analogously to TEABbuffer).

6. Stock solution of 2M phosphonic acid in pyridine was prepared by evaporation ofadded pyridine to the appropriate amount of phosphonic acid and dissolving theresidue in anhydrous pyridine.


7. One should not exceed this amount of a condensing agent, otherwise more reac-tive species can be generated from phosphonic acid (44).

8. Instead of generating in situ H-pyrophosphonate from phosphonic acid and a con-densing agent in pyridine, one can prepare a 1M stock solution of thisphosphonylating reagent by adding 0.5 equivalents of PvCl or DMOCP to 2Mstock solution of phosphonic acid in pyridine. The stock solution of HPP in pyri-dine is stable for several months at room temperature.

9. Formation of a trityl positive baseline material (TLC on silica gel plates, solvent:chloroform–methanol 9:1, v/v).

10. This, in principle, may happen when DMOCP is used as a condensing agent.11. See Note 5 in Subheading 3.1.1.12. The nucleoside was rendered anhydrous by evaporation of added pyridine.13. Diphenyl H-phosphonate was a commercial grade from Aldrich and contained

ca. 10% of phenol.14. An excess of phosphonylating agents is necessary to avoid the formation of sym-

metrical dinucleoside H-phosphonate diesters.15. In the instance of 2'-O-protected ribonucleosides, the amount of diphenyl

H-phosphonate can be reduced to 3-mol equivalents.16. Disappearance of the starting nucleosidic material (TLC on silica gel plates,

solvent: chloroform–methanol 9:1, v/v).17. In the case of ribonucleosides, a mixture of water–triethylamine (1:1 v/v, 2 mL)

was used to speed up hydrolysis of the intermediate nucleoside phenylH-phosphonate.

18. This extraction removes most phenol and phenyl H-phosphonate formed duringthe reaction and from hydrolysis of diphenyl H-phosphonate.

19. Precipitation of crude reaction products (after extraction with aqueous NaHCO3)from CH2Cl2 into hexane–diethyl ether (1:1, v/v) usually afforded nucleosideH-phosphonates of purity better than 95%.

20. See Note 5 in Subheading 3.1.1..21. H-phosphonate monoesters can be used as a TEAH+ or DBUH+ salt.22. Instead of PvCl, other condensing agents—for example, DMOCP or OXP—can

be used.23. Reaction is usually over within 1 min, but to make sure we suggest waiting for

3–5 min before the first TLC analysis. A baseline material (H-phosphonate 2) isconverted into a compound with a higher Rf value (TLC on silica gel plates,solvent: chloroform–methanol 9:1, v/v). In the instance of using DMOCP or OXPas coupling agents, the reaction is usually completed within 40 and 20 min,respectively, but can in some cases be up to 2 h.

24. Owing to the chirality at the phosphorus center, H-phosphonate diesters of type 4are formed as a mixture of RP and SP diastereomers.

25. This solvent system usually permits separation of H-phosphonates 4 diastere-omers, if so desired.

26. Synthesis of a dinucleoside H-phosphonate 4 and its oxidation by iodine to pro-duce dinucleoside phosphate 5a, can be performed as a one-pot reaction withoutisolation of 4. The aqueous iodine solution is then simply poured directly into the


coupling reaction mixture in Subheading 3.2.2. as soon as TLC analysis revealscomplete condensation.

27. During oxidation, water has to be present in the reaction mixture at the momentof addition of iodine, otherwise significant amounts of stable pyrophosphatederivatives can be formed.

28. Formation of a baseline material (TLC on silica gel plates, solvent: chloroform–methanol 9:1, v/v).

29. If a single H-phosphonate diastereomer (see Notes 4 and 5 in Subheading 3.2.1.)is used, then only one isomer of the phosphorothioate is obtained because thesulfurization is stereospecific. Synthesis of a dinucleoside H-phosphonate 4 andits oxidation with elemental sulfur to produce dinucleoside thiophosphate 5b canbe performed as a one-pot reaction without isolation of 4. The latter proceduregives a mixture of the two phosphorothioate stereoisomers.

30. Formation of a baseline material (TLC on silica gel plates, solvent: chloroform–methanol 9:1, v/v).

31. When preparing and transferring the H-phosphonate solutions it is important forhigh yields in coupling that moisture be avoided as much as possible. It is advis-able to transfer under a flow of dry gas (nitrogen or argon) or by syringes throughsepta for the best results. To some extent this can be compensated by use of ahigher concentration of PvCl. For some modified H-phosphonate building blocksthere may be solubility problems, but for these several alternative solvent mix-tures, MeCN/pyridine 1:1 (v/v), neat pyridine, and THF/pyridine 1:1 (v/v), canbe used without compromising the coupling yield too much.

32. The quality of the PvCl is important for the outcome of the synthesis. Becausethe reagent decomposes with time, it should be distilled regularly (e.g., once every1–2 mo). The distilled material, divided into a number of smaller vials (to avoidtoo frequent opening and closing of the same bottle), can be safely stored in afreezer. When preparing and transferring the PvCl solutions it is crucial thatmoisture be avoided as much as possible. It is advisable to transfer under a flowof dry gas (nitrogen or argon) or by syringes through septa for the best results. Itis also advisable to prepare a fresh PvCl solution for each synthesis, but if onewishes to keep the solution for a couple of days on the synthesizer it is best toomit the pyridine in the PvCl solution and compensate it by increasing the pyri-dine content in the H-phosphonate solutions. If there are problems with moistureone can to some extent compensate by using a higher concentration of PvCl (e.g.,300 mM, 4 equivalents, or 375 mM, 5 equivalents) but usually 225 mM and verydry conditions gives slightly better results.

33. The oxidation solution is usually prepared while the elongation cycles are run-ning to have this as fresh as possible because basic aqueous iodine solutions aredisproportionate, which can give somewhat slower oxidation. Although there isprobably enough of a time margin in this protocol, it may be better to play it safe.

34. 1% trifluoroacetic acid can be used instead of dichloroacetic acid in synthesis ofRNA fragments or for sequences not containing deoxypurine nucleosides (e.g.,dG and dA). Trichloroacetic acid 2–3% is also commonly used.


35. In the coupling step, H-phosphonate and PvCl are taken up in alternatingsegments of 100 μL. The volume of the tubing between valve and column(including the pump hose of the peristaltic pump through which the reagents flow)is 0.4 mL, which means that the front of the condensation mixture reaches thecolumn when the last segment is taken up from the reagent bottles. The segmentsare then pushed into a loop that includes the column, the loop is closed, and thecondensation mixture is recycled, giving a total condensation cycle time of 2.1 min(effective condensation time ca. 1.6 min). The effective time of preactivation forthe nucleoside H-phosphonate when it first reaches the column is about 0.4 min,and at the end of condensation it totals to 2.0 min. An alternative setup is possibleif the machine allows for reversed flow. The segments are then passed throughthe column at 1 mL/min for 0.4 min, the flow is lowered to 0.5 mL/min, and thedirection reversed for 1 min, giving a total condensation cycle time of 1.9 min(effective condensation time 1.5 min). The effective time of preactivation of thenucleoside H-phosphonate when it first reaches the column is about 0.3 min forthis setup, and at the end of condensation it totals to 1.8 min.

36. After filtration the solution is lyophilized and, to remove the 2'-O-TBDMS, theresidue is dissolved in triethylamine trihydrofluoride (0.3 mL, neat), and the mix-ture is left for 8 h (or up to 16 h for convenience) at room temperature; 30 μLH2O and 1 mL n-butanol is added, the mixture is left at –20°C for 1 h and theprecipitate is collected by centrifugation.

AcknowledgmentsFinancial support from the Swedish Research Council is gratefully acknowl-

edged.

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