Use of a Dihydroxyacetone Derivative as Protecting Reagent to Phosphorylate Oligonucleotides

Abstract

We present here a new reagent enabling the supported synthesis of oligodeoxynucleotides (ODNs) and oligoribonucleotides (ORNs) containing a phosphate group at the 5′‐terminal position after complete deprotection. This reagent, derived from a dihydroxyacetone core, contains a dimethoxytrityl (DMTr) group. The procedure for final deprotection is very similar to that routinely used in the synthesis of unmodified ODNs or ORNs. In particular, it preserves the advantages of the “trityl‐on” method, namely facile purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), short treatment with an acetic acid solution in water, the possibility of on‐resin monitoring by trityl cation assay, and the use of equipment commonly founded in chemical laboratories. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Phosphorylation of oligodeoxyribonucleotides (ODNs)

Alternate Protocol: Phosphorylation of oligoribonucleotides (ORNs)

Support Protocol: Preparation of phosphorylation reagent 1

Introduction

Phosphorylated oligonucleotides in both DNA and RNA sequences find applications in various molecular techniques—conjugation (Chu et al., 1983), linkers (Zhelkovsky & McReynolds, 2011), cloning (Schilder & Görke, 2023), PCR (Barany, 1991), and gene construction (Wosnick et al., 1987).

The 5′‐monophosphorylation of oligonucleotides can often be carried out enzymatically, with a kinase transferring a monophosphate group from adenosine triphosphate (ATP) to the free 5′‐hydroxyl group of the oligonucleotide (Chaconas & van de Sande, 1980). This process can be easily performed on a small scale. However, it is tedious to scale up. Furthermore, the efficiency of the conversion may depend on the sequence and the enzyme batch. Consequently, the development of reagents enabling the chemical phosphorylation of nucleic acids has been the subject of intense research for nearly 40 years.

The ideal reagent would allow the synthesis of 5′‐phosphate oligonucleotides as easily as that of unmodified oligonucleotides. It should therefore combine the following advantages: chemical coupling by phosphoramidite chemistry, RNA compatibility, a lipophilic group to facilitate purification, the ability to perform assays on a support, and the ability to be deprotected with common reagents and readily available equipment.

None of the existing reagents combined all these advantages. Historical reagent A (Uhlmann & Engels, 1986; see Fig. 1) lacks a lipophilic trityl group and cannot be purified by reverse‐phase high‐performance liquid chromatography (RP‐HPLC). Similarly, reagent B (Horn & Urdea, 1986), which is commercially available, loses its trityl group during the final basic deprotection, resulting in the same disadvantages. Reagent C (Guzaev et al., 1995) belongs to the second generation of phosphorylation reagents and can be purified by HPLC. However, releasing phosphorylated nucleic acids requires a final basic treatment incompatible with oligoribonucleotides (ORNs). Progress has been made in the synthesis of phosphorylated RNA, as evidenced by the development of reagent D (Ototake et al., 2024; Pradère et al., 2017). Unfortunately, this reagent cannot be used to estimate the phosphorylation yield with the well‐known DMTr⁺ cation test (Olah et al., 1966). Additionally, generating the phosphorylated oligonucleotide requires an irradiation lamp, a device that may not be available in all laboratories dedicated to nucleic acid chemistry.

Figure 1.

Structures of phosphorylation reagent 1 along with older phosphorylation reagents. DMTr, 4,4′‐dimethoxytriphenylmethyl.

The strategy presented here is closely related to the well‐known DMT‐on strategy widely used for synthesis and purification of natural oligodeoxynucleotides (ODNs; Beaucage & Caruthers, 2001). It relies on a phosphorylation reagent we have developed, reagent 1 (see Fig. 1), whose synthesis is described in the Support Protocol (see Fig. 2). Its use in the phosphorylation of nucleic acids is identical to that of unmodified oligonucleotides, with the sole exception that additional heating (pH 7, 80°C, 90 min) must be carried out after the well‐known acetic acid treatment. Its use in the phosphorylation of ODNs (Basic Protocol) and ORNs (Alternate Protocol) is presented.

Figure 2.

Preparation of phosphorylation reagent 1 by chemical synthesis.

CAUTION: All reactions must be run in a suitable fume hood with efficient ventilation. Many of the reactions in this article are highly exothermic; safety glasses and reagent‐impermeable protective gloves should be worn.

PHOSPHORYLATION OF OLIGODEOXYRIBONUCLEOTIDES (ODNs)

This protocol describes the use of reagent 1 to obtain oligonucleotides with a phosphate group at the 5′ position. It comprises four steps, which are illustrated in Figure 3: (1) reaction between reagent 1 and the oligonucleotide on the support ODN1 to produce 5′‐modified ODN2 still anchored on the support, (2) partial deprotection and cleavage from the support leading to on‐solution ODN3 containing a dimethoxytrityl group at the 5′ end to facilitate purification, (3) separation of the oligonucleotide ODN3 of interest from the truncated oligonucleotides, and (4) final deprotection of the phosphate group at the 5′ end in two steps: mildly acidic treatment leading to expected product ODN5 together with partially deprotected ODN4, followed by heating in a PBS buffer in order to fully convert remaining ODN4 into ODN5.

Figure 3.

Preparation, purification, and deprotection of the phosphorylated nucleic acids. CNE, 2‐cyanoethyl; ODN, oligodeoxynucleotide; ORN, oligoribonucleotide. The gray ball denotes the CPG solid support.

Materials

• Phosphorylation reagent 1 (Support Protocol)

• Dichloromethane (DCM)

• Acetonitrile (MeCN)

• 0.25 M ethylthiotetrazole (ETT) in acetonitrile (Glen Research)

• 0.2 M iodine in 7:2:1 (v/v/v) tetrahydrofuran (THF)/pyridine/water (Glen Research)

• 3% (w/v) trichloroacetic acid (TCA) in DCN (Glen Research)

• 1:1:8 (v/v/v) pyridine/acetic anhydride/THF

• 10% (v/v) N‐methylimidazole in THF

• 40% (w/v) methylamine (Sigma‐Aldrich, cat. no. 426466)

• Triethylammonium acetate (TEAE)

• N,N‐Diisopropylethylamine (DIEA), 99% (CAS no. 7087‐68‐5)

• 80% (v/v) aqueous acetic acid

• 100 mM sodium phosphate buffer, pH 7.0 (see recipe)

• Oligonucleotide synthesizer (we used a discontinued 3400 model from Applied Biosystem)

• Heating block

• Centrifugal evaporator

• Reverse‐phase chromatographic chain

• Lyophilizer

• NAP25 disposable column for size‐exclusion chromatography (Fisher Scientific, cat. no. 10186464)

• 1Dissolve reagent 1 in a dry mixture of 3:2 (v/v) DCM/MeCN at 0.1 M final concentration.

  Unlike most commercially available amidites, 1 is not soluble in pure acetonitrile.

• 2Couple the phosphoramidite reagent 1 using a 5‐min time in the final step of oligonucleotide synthesis. Do not perform final detritylation.

  We perform this synthesis using the following: 0.25 M ethylthiotetrazole (ETT) in MeCN as activator, 0.02 M iodine in 7:2:1 (v/v/v) THF/water/pyridine as oxidizer, 1:1:8 (v/v/v) pyridine/acetic anhydride/THF and 10% (v/v) N‐methylimidazole in THF as cap A and B reagents, 3% (w/v) TCA in DCM as deblock reagent, and acetonitrile (<30 ppm water) as washing solvent; a preloaded 1000‐Å controlled‐pore glass (CPG) support; and ^AcdC, ^dmfdG, ^BzdA, and T phosphoramidites.

  The exact programming of the synthesizer will differ depending on the model used.

  The synthesis was performed at the 200‐nmol scale (∼6.0 mg; loading: 30 mmol/g).

• 3Remove the support from the reactor and transfer it into a vial.
• 4Add 0.5 ml of 40% (w/v) methylamine, close the vial tightly, and heat it at 60°C for 10 min.
• 5Cool the vial at room temperature and add ∼5 µl DIEA.

  Do not open the vial too early, as the excess pressure generated by heating may cause hot methylamine to spray onto your face.

• 6Evaporate until the volume is reduced by about half (to ∼0.2 ml). Collect the supernatant, wash the resin three times with water using 500 µl each time, and collect the aqueous phases.

• 7Purify the 5′‐DMTr‐containing ODN3 by using preparative RP‐HPLC (see Andrus & Kuimelis, 2001) with the following recommended conditions: prepacked C18 column (length 250 mm, diameter 10 mm, particle size 7 µm, porosity 100 Å) at 4 ml/min flow; eluent A, 50 mM TEAA, pH 7, in 95:5 (v/v) water/MeCN; eluent B, 9:1 (v/v) MeCN/water. Analyses were monitored at 260 nm. Tritylated ODN3 was purified using the following gradient: 0% B for 2 min, then 0% to 45% B in 23 min.
• 8Collect the ODN‐containing fraction in a 50‐ml plastic centrifuge tube or similar.
• 9Remove the TEAA salts as follows: freeze the sample; lyophilize once; take it up in 1 ml water by adding water, stirring, and pipetting out; desalt on a disposable size‐exclusion column following the manufacturer's recommendations (practical details are given in Basic Protocol 3 of Leuck & Wolter, 2003); and lyophilize again in a 2.0‐ml microcentrifuge tube.

  At the end of this step, the oligonucleotide obtained will have a high retention time due to the presence of two lipophilic groups: DMTr and isopropyl acetal. Figure 4A shows a 24‐mer ODN3 (sequence: CAC GTC GAG CCG ATC GAG CTG GAT) purified in this way. Figure 4C shows the mass spectrum of this modified ODN (7937.8 amu) versus 7935.4 (calculated).

Figure 4.

RP‐UPLC traces and ESI‐MS analysis results for a 24‐mer oligonucleotide (sequence: CAC GTC GAG CCG ATC GAG CTG GAT) phosphorylated by reagent 1. (A and B) RP‐HPLC results for purified ODN3 (A) and after complete deprotection of the ODN (ODN5). (C and D) ESI‐MS analyses of ODN3 (MW(exp) = 7937.8; MW(calcd) = 7935.4; C) and ODN5 (MW(exp) = 7460.9; MW(calcd) = 7458.8; D).

• 10Dissolve the sample contained in the microcentrifuge tube in 400 µl of 80% (v/v) aqueous acetic acid for 10 min at room temperature. Evaporate the mixture to dryness.

  A centrifugal evaporator is the most suitable device for this operation because it prevents the sample from heating up and therefore minimizes the rate of depurination.

  It is essential to remove all acetic acid to ensure that the pH of the solution is 7.0 during the next operation.

  At the end of this stage, the DMTr group will have been removed in its entirety, and the mixture will contain the expected ODN5 at a level of approximately 30% to 70%. The remainder of the mixture will consist of ODN4, whose 5′ end is partially unprotected.

  For short sequences, it is possible to distinguish between ODN4 and ODN5 by reverse‐phase ultra‐performance liquid chromatography (RP‐UPLC; see Fig. 5). For longer sequences, the two peaks are indistinguishable, but mass spectrometry will reveal a peak at +72 amu in addition to the expected mass peak of the phosphorylated oligonucleotide ODN5.

Figure 5.

RP‐UPLC traces for a mixture of a short‐sequence ODN4 and ODN5 (sequence: TTT TTT TTT TTT), after phosphorylation by reagent 1 and acid acetic treatment but before heating in PBS buffer. ODN4 (peak a) eluted slightly earlier than final expected product ODN5 (peak b).

• 11Add 500 µl of 100 mM sodium phosphate buffer (pH 7.0) to the residue, stir, pipet out, and heat for 90 min at 80°C.

  This step converts remaining ODN4 into the expected ODN5 by thermolysis.

  It is essential that the pH of the solution be 7.0 and that the temperature be 80°C.

  In fact, we found that at a slightly lower pH (pH = 6) or lower temperature (60°C), the reaction was still incomplete (<80%) after 2 hr. When the temperature is too low and the pH is slightly acidic at the same time, the reaction stops progressing altogether.

PHOSPHORYLATION OF OLIGORIBONUCLEOTIDE (ORN)

This alternate protocol describes the use of reagent 1 to obtain 5′‐phosphorylated oligoribonucleotides in the RNA series. The introduction of 1 into the oligonucleotide chain and, at the end, the release of the phosphate group by acid treatment are carried out in the same way as described in the basic protocol for the DNA series. However, for clarity, as RNA synthesis itself differs from DNA synthesis, this protocol is presented to avoid any confusion.

Additional Materials (also see Basic Protocol)

• Dimethylsulfoxide (DMSO), 99% (CAS no. 67‐68‐5)

• Triethylamine (TEA), 99% (CAS no. 121‐44‐8)

• Triethylamine trihydrofluoride (TEA∙3HF), 97% (CAS no. 73602‐61‐6)

• Quenching buffer (Glen Research, cat. no. 60‐4210‐80)

• 1Dissolve reagent 1 in a dry mixture of 3:2 (v/v) DCM/MeCN at 0.1 M final concentration.

  Unlike most amidites, 1 is not soluble in pure acetonitrile.

• 2Couple phosphoramidite reagent 1 using a 5‐min time in the final step of oligonucleotide synthesis.

  We perform this synthesis using the following: 0.25 M ethylthiotetrazole (ETT) in MeCN as activator, 0.02 M iodine in THF/water/pyridine as oxidizer, acetic anhydride in THF and 16% N‐methylimidazole in THF as cap A and B reagents, 3% TCA in DCM as deblock reagent, and acetonitrile (<30 ppm water) as washing solvent; a preloaded 1000‐Å CPG support; and ^AcC, ^dmfG, ^BzA, and U phosphoramidites. 2′ positions were protected by t‐butyldimethylsilyl (tBDMS) groups.

  The exact programming of the synthesizer differs depending on the model used.

  Synthesis was performed at the 200‐nmol scale (∼6.0 mg; loading: 30 mmol/g)

• 3Remove the support from the reactor and transfer it into a vial.
• 4Add 0.5 ml of 40% (w/v) methylamine solution, close the vial tightly, and heat it at 60°C for 10 min.
• 5Cool the vial at room temperature.

  Do not open the vial too early, as the excess pressure generated by heating may cause hot methylamine to spray onto your face.

• 6Evaporate until the volume is reduced by about half (to ∼0.2 ml). Collect the supernatant, wash the resin three times with water using 500 µl per wash, and collect the aqueous phases.
• 7Freeze and lyophilize the samples.
• 8Successively add 115 µl DMSO, 60 µl TEA, and 75 µl TEA∙3HF. Heat at 65°C, vortex, and heat at 65°C for 2 hr 30 min.
• 9Add 1 ml quenching buffer.
• 10Desalt the sample on a disposable size‐exclusion column following the manufacturer's recommendations (practical details are given in Basic Protocol 3 of Leuck & Wolter, 2003) and lyophilize again.

  The analysis of the crude reaction mixture of an ORN3 20‐mer (UUU GGG AGU CUU ACA AUU GG) is shown in Figure 6A. It can be seen that the strong lipophilicity of the group added by reagent 1 significantly delays elution and easily separates it from truncated sequences.

Figure 6.

RP‐UPLC analysis of a crude deprotection mixture of a protected 20‐mer ORN3 (sequence: UUU GGG AGU CUU ACA AUU GG) at different stages. (A) After treatment with 40% methylamine, 60°C, 10 min followed by 115 µl DMSO, 60 µl TEA, and 75 µl TEA∙3HF, 60°C, 2 hr. (B) After subsequent RP‐HPLC purification and treatment with 80% acetic acid, room temperature, 10 min, and 100 mM sodium phosphate buffer, pH 7, 80°C, 90 min.

• 11Purify the 5′‐DMTr‐containing ORN3 by preparative RP‐HPLC (see Andrus & Kuimelis, 2001) using the following recommended conditions: prepacked C18 column (length 250 mm, diameter 10 mm, particle size 7 µm, porosity 100 Å) at 4 ml/min flow; eluent A, 50 mM TEAA, pH 7, in 95:5 (v/v) water/MeCN; eluent B, 9:1 (v/v) MeCN/water. Monitor analyses at 260 nm. Purify tritylated ODN3 using 0% B for 2 min followed by a gradient of 0%‐45% B over 23 min.
• 12Collect the ORN‐containing fractions.
• 13Remove the TEAA salts by following the steps below: freeze the sample, lyophilize once, dissolve in 1 ml water, desalt on a disposable size‐exclusion column following the manufacturer's recommendations (practical details are given in Basic Protocol 3 of Leuck & Wolter, 2003), and lyophilize again.

• 14Dissolve the sample in 400 µl of 80% (v/v) aqueous acetic acid for 10 min at room temperature. Evaporate the mixture to dryness.

  A centrifugal evaporator is the most suitable device for this operation because it prevents the sample from heating up and therefore minimizes the rate of depurination.

  It is essential to remove all acetic acid to ensure that the pH of the solution is 7.0 during the next operation.

  At the end of this stage, the DMTr group will have been removed in its entirety, and the mixture will contain the expected ODN at a level of ∼30%‐70%. The remainder of the mixture will consist of ORN4 whose 5′ end is partially unprotected.

• 15Add 100 mM sodium phosphate buffer (pH 7.0) to the residue, stir, pipet out, and heat for 90 min at 80°C.

  This step converts remaining ORN4 into the expected ORN5 by thermolysis.

  It is essential that the pH of the solution be 7.0 and that the temperature be 80°C. In fact, we found that at a slightly lower pH (pH = 6) or lower temperature (60°C), the reaction remained incomplete (<80%) after 2 hr. When the temperature is too low and the pH is slightly acidic at the same time, the reaction stops progressing altogether.

  The analysis of an ORN5 (UUU GGG AGU CUU ACA AUU GG) obtained at the end of these treatments is presented in Figure 6B.

PREPARATION OF THE PHOSPHORYLATION REAGENT 1

Initially, several reagents were developed that differed only in the nature of the acetal group. Only the presence of a bis‐isopropyl acetal group allows rapid deprotection of the corresponding functionalized ODN/ORN in an 80% aqueous acetic acid solution. The other acetal groups tested (methyl, ethyl, n‐propyl) led to 5′‐modified ODN/ORN displaying a too long deprotection time. Here, we present the multistep synthesis of reagent 1, which is also depicted in Figure 2.

Materials

• Dimethylformamide (DMF)

• Tris∙Cl, 99% (CAS no. 1185‐53‐1)

• Benzaldehyde dimethyl acetal, 95% (CAS no. 1125‐88‐8)

• p‐Toluene sulfonic acid (PTSA; CAS no. 919‐30‐2)

• Ethyl acetate (AcOEt)

• Dichloromethane

• Triethylamine (TEA)

• Silica, particle size 63‐200 µm

• Methanol

• Potassium phosphate monobasic (KH₂PO₄), 98% (CAS no. 7778‐77‐0)

• Sodium metaperiodate, 99% (CAS no. 7790‐28‐5)

• Sodium thiosulfate, 98% (CAS no. 7772‐98‐7)

• Sodium or magnesium sulfate

• NaCl‐saturated water solution (brine)

• Isopropanol, technical‐grade

• Triisopropylorthoformate, 97% (CAS no. 4447‐60‐3)

• (1S)‐(+)‐10‐Camphorsulfonic acid (CSA), 98% no. CAS no. 3144‐16‐9)

• 5% (w/v) sodium bicarbonate (NaHCO₃) in water

• Pentane

• Cyclohexane

• Acetone

• Isopropanol, technical grade

• Palladium hydroxide (Pd(OH)₂), 10%‐20% on charcoal, moist (Sigma‐Aldrich cat. no. 76063)

• Celite 545 (Carl Roth cat. no. 0011.2)

• Pyridine

• 4‐(Dimethylamino)pyridine (DMAP), 95% (CAS no. 1122‐58‐3)

• Dimethoxytrityl chloride (DMTrCl), 97% (CAS no. 40615‐36‐9)

• N,N‐Diisopropylethylamine (DIEA), 99% (CAS no. 7087‐68‐5), dried on KOH

• 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite, 95% (CAS no. 89992‐70‐1)

• 10‐, 50‐, and 250‐ml round‐bottomed flasks

• Hot plate and magnetic stirrer

• 10‐, 50‐, and 250‐ml graduated cylinders

• Rotary evaporator, capable of reaching 10 mbar or less

• Vacuum pump, capable of reaching 0.5 mbar or less

• Sintering funnel, at least 30 mm in diameter

• TLC plate

• Filter funnel

• Separatory funnel

• Hydrogen generator able to reach 20‐bar pressure

• Dropping funnel

• 1‐ml gas‐tight syringe

• 1Pour into a 250‐ml round‐bottomed flask, in the following order: 160 ml DMF, 20.0 g Tris∙Cl (127 mmol; 1 eq.), 22 ml benzaldehyde dimethylacetal (147 mmol; 1.16 eq.), and 1.2 g PTSA (7 mmol; 5%).
• 2Stir overnight at room temperature.
• 3Add 1 ml triethylamine and stir an additional 10 min.
• 4Evaporate solvent until a final volume of 50 ml is reached.
• 5Add 14 ml triethylamine and stir the resulting mixture for 15 min.

  A white powder will precipitate after ∼5 min.

• 6Add 500 ml ethyl acetate and stir for an additional 10 min. Filter the resulting heterogeneous mixture. Collect the filtrate and evaporate it.
• 7Add a minimal amount of 99:1 (v/v) dichloromethane/triethylamine to the residue and stir until it dissolves. Pour the resulting solution carefully into a sintering funnel (≥30 cm diameter) containing a short pad of silica gel (2 cm height). Wash the pad with 99:1 (v/v) dichloromethane/triethylamine until the excess benzaldehyde in the filtrate has been completely removed, as observed by TLC.

  Follow the filtration process by TLC using 90:10:1 (v/v/v) AcOEt/MeOH/TEA as eluent. The impurities to be removed display Rf values of >0.9, whereas the expected product has an Rf of 0.39.

• 8Wash the silica pad with 90:10:1 (v/v/v) dichloromethane/methanol/triethylamine until the desired product is recovered, as observed by TLC. Evaporate the filtrate to dryness.
• 9Product 2 is obtained as a white powder. Yields are typically in the 60%‐70% range.

  NMR characterizations are as follow: NMR ¹³C (CDCl₃: 137.3; 129.1; 128.3; 126.1; 103.0; 73.8; 63.9; 51.4. NMR ¹H (CDCl₃): 7.25‐7.41 (5H; mult); 5.31 (1H,s); 3.86 (2H; d; J = 11 Hz); 3.73 (2H; d; J = 11 Hz); 3.57 (3H; br); 3.32 (2H; s).

  All NMR data (¹H, ¹³C and ³¹P) for compounds 1–6 are available in the Supporting Information.

• 10Pour into a 250‐ml round‐bottomed flask, in the following order: 80 ml water, 4.6 g compound 2 (22.0 mmol; 1 eq.), and 4.6 g KH₂PO₄ (22.0 mmol; 1 eq.). Stir vigorously for 20 min at 4°C.
• 11Add 3.0 g sodium periodate (22.0 mmol; 1 eq.) portionwise. Stir the reaction mixture for 3 hr at 4°C and then for 16 hr at room temperature.

  The initially white slurry will turn milky during course of the reaction.

• 12Add 5.4 g sodium thiosulfate portionwise, followed by 100 ml water. Extract the aqueous mixture with dichloromethane using 15 washes of 50 ml each.

  The decantation time is longer during the first extractions. The aqueous phase becomes clearer with each extraction.

• 13Wash the combined organic phases with 20 ml brine. Dry the mixture on MgSO₄, filter out the magnesium sulfate, and evaporate the filtrate to dryness.
• 14Product 3 is obtained as a white powder. Yields are typically in the 72%‐82% range.

  NMR characterizations are as follows: NMR ¹³C (DMSO‐d₆): 205.1; 138.2; 129.5; 128.7; 126.6; 99.7; 73.5. NMR ¹H (DMSO‐d₆): 7.40‐7.51 (5H; mult.); 6.04 (1H; s); 4.64 (2H; d; J = 18 Hz); 4.50 (2H; d; J = 18 Hz).

• 15Pour into a 50‐ml round‐bottomed flask, in the following order: 6 ml isopropanol, 1.0 g compound 3 (5.61 mmol; 1 eq.), 1.7 ml triisopropylorthoformate (7.30 mmol; 1.3 eq.), and 65 mg CSA (280 µmol; 5%). Stir for 1 hr at room temperature.

  The white suspension became clearer after ∼30 min of stirring at room temperature.

• 16Add 20 ml dichloromethane and 20 ml 5% NaHCO₃. Stir for an additional 5 min.
• 17Remove aqueous phase. Wash organic phase three times with 20 ml of 5% NaHCO₃ solution each time and then once with 20 ml brine. Dry organic phase on magnesium sulfate, filter, and evaporate the filtrate to dryness.
• 18Purify the oily residue by column chromatography using 8:1 (v/v) pentane/ethyl acetate as eluent.

  By TLC using 3:1 (v/v) cyclohexane/acetone, the expected product displays a Rf of 0.70 (the starting material has an Rf of 0.20).

• 19Product 4 is obtained as a colorless to yellowish oil. Yields are typically in the 41%‐70% range.

  NMR characterizations are as follow: NMR ¹³C (pyr‐d₅): 138.9; 128.9; 128.2; 126.8; 101.4; 93.1; 63.7; 62.6; 24.5; 24.0. NMR ¹H (pyr‐d₅): 7.73 (2H; d; J = 7 Hz); 7.33‐7.40 (3H; mult); 5.62 (1H; s); 4.28 (2H; d; J = 11.5 Hz); 4.14 (2H; heptuplet; J = 6 Hz); 4.09 (2H; heptuplet; J = 6 Hz); 3.83 (2H; d; J = 11 Hz); 1.28 (6H; d; J = 6.5 hz); 1.10 (2H; d; J = 6 Hz).

• 20Pour into a 50‐ml round‐bottomed flask, in the following order: 10 ml technical‐grade isopropanol, 1.085 g compound 4 (3.87 mmol), and 196 mg powdered Pd(OH)₂ on charcoal (0.28 mmol; 7%).

  Use of anhydrous isopropanol seems to be detrimental.

  The reproducibility of the reaction appears to be dependent on the palladium batches used.

  Use of another alcohol as solvent leads to acetal exchange

• 21Establish a hydrogen atmosphere in the flask by evacuating it and then filling it with hydrogen (three times).

  CAUTION: Hydrogen is a highly flammable gas. The use of a hydrogen cylinder, hydrogen generator, or hydrogen pressure system should be carried out by qualified personnel.

• 22Stir under hydrogen atmosphere at 20 bar for 8 hr.
• 23Filter the black solution by transferring it into a sintering funnel (≥30 mm diameter) containing a short pad of Celite (2 cm height). Evaporate the filtrate to dryness.
• 24Purify the residue by column chromatography using 3:1 (v/v) cyclohexane/acetone as eluent.

  By TLC using 3:1 (v/v) cyclohexane/acetone as eluent, the expected product displays a Rf = 0.20 (the starting material has an Rf of 0.75).

• 25Product 5 is obtained as a colorless oil. Yields are typically in the 42%‐61% range.

  NMR characterizations are as follow: NMR ¹³C (DMSO‐d₆): 102.6; 62.6; 60.5; 24.6; NMR ¹H (DMSO‐d₆): 4.40 (2H; t; J = 5 Hz; OH); 4.02 (2H; heptuplet; J = 6 Hz); 3.36 (4H; d; J = 5 Hz); 1.09 (12H; d; J = 6 Hz).

• 26Pour in a 10 ml round‐bottomed flask, in the following order: 4 ml of 1:1 (v/v) pyridine/dichloromethane, 440 mg compound 5 (2.29 mmol; 1 eq.), and 45 mg DMAP (0.37 mmol; 16%).
• 27Add a solution of 700 mg dimethoxytrityl chloride (2.07 mmol; 0.9 eq.) in 20 ml dichloromethane dropwise over the course of 20 min. Stir for 3 hr at room temperature.
• 28Dilute reaction mixture with 20 ml dichloromethane.
• 29Washed three times with 5% NaHCO₃ solution, 20 ml each time, and once with 20 ml brine. Dry organic phase on sodium sulfate, filter, and evaporate to dryness.
• 30Purify by column chromatography using 10:1:0.05 (v/v/v) until the DmTrCl disappears (as observed by TLC), and then 8:1:0.05 (v/v/v) pentane/acetone/triethylamine. Evaporate to dryness.

  By TLC using 3:1 (v/v) cyclohexane/acetone, the expected product will display an Rf of 0.43 (the starting material has an Rf of 0.20 and DMTrCl has an Rf of 0.33).

• 31Product 6 is obtained as a colorless oil. Yields are typically in the 30%‐55% range.

  NMR characterizations are as follow: NMR ¹³C (acetone‐d₆): 159.6; 146.0; 131.2; 129.2; 128.4; 127.5; 113.7; 102.6; 86.8; 64.0; 63.5; 63.4; 55.4; 24.8; 24.7. NMR ¹H (acetone‐d₆): 7.75 (2H; d; J = 8 Hz); 7.39 (4H; J = 9 Hz; 7.29‐7.33 (3H; mult); 6.88 (4H; d; J = 9 Hz); 4.05 (2H; heptuplet; J = 6 Hz); 3.79 (2H; s); 3.78 (6H; s); 3.18 (2H; s); 1.11 (6H; d; J = 6 Hz); 1.00 (6H; d; J = 6 Hz).

• 32Pour into a 10‐ml round‐bottomed flask, in the following order: 5 ml dry dichloromethane, 450 mg compound 6 (910 µmol; 1 eq.), and 332 µl dry DIEA (1.91 mmol; 2 eq.). Stir for 5 min.

  To ensure that there is no water in the assembly, the balloon and syringes used should first be heated in an oven and then cooled under vacuum.

• 33Inject by syringe 284 µl of chloro(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite (1.27 mmol; 1.4 eq.). Stir the resulting mixture for 2 hr at room temperature.
• 34Dilute crude solution with 20 ml dichloromethane.
• 35Wash the organic phase three times with 20 ml of 5% NaHCO₃ solution each time and then once with 20 ml brine.
• 36Dry on sodium sulfate, filter, and evaporated to dryness.
• 37Purify the oily residue by column chromatography using 10:1:0.05 (v/v/v) pentane/ethyl acetate/triethylamine as solvent.

  By TLC, the expected product will display a Rf of 0.42 (starting material: Rf 0.33) in 10:1 (v/v) pentane/ethyl acetate.

• 38Product 1 is obtained as a white foam. Yields are typically in the 51%‐72% range.

  NMR characterizations are as follow: NMR ³¹P (acetone‐d₆): 147.4; NMR ¹³C (acetone‐d₆): 159.6; 146.2; 136.8; 131.2; 129.3; 128.4; 127.5; 118.9; 113.7; 102.3; 86.9; 64.4; 64.3; 64.2; 64.1; 59,7; 59.6; 55.5; 43.9; 43.8; 25.04; 25.00; 24.97; 24.93; 24.90; NMR ¹H (acetone‐d₆): 7.55 (2H; d; J = 7.5 Hz); 7.41 (4H; J = 7 Hz); 7.30‐7.33 (2H; mult); 7.21‐7.24 (1H; mult); 6.88 (4H; d; J = 7 Hz); 4.06 (2H; heptuplet; J = 6 Hz); 3.90 (1H; mult); 3.83 (2H; mult); 3.79 (6H, s); 3.76 (1H; mult); 3.65 (2H; mult); 3.21 (2H; mult); 2.67 (2H; t; J = 6 Hz; 1.20 (6H; d; J = 6.5 Hz); 1.14 (6H; d; J = 6.5 Hz); 1.12 (3H; d; J = 6 Hz); 1.10 (3H; d; J = 6 Hz); 1.05 (3H; d; J = 6 Hz); 1.02 (3H; d; J = 6 Hz).

REAGENTS AND SOLUTIONS

Sodium phosphate, pH 7, 100 mM

• 5.77 ml of 1 M sodium phosphate dibasic (Na₂HPO₄) solution

• 4.23 ml of 1 M sodium phosphate monobasic monohydrate (NaH₂PO₄∙H₂O) solution

• ∼80 ml distilled water

• Adjust pH to 7.0 with 10 M NaOH

• Add distilled water to 100 ml

• Store up to 1 year at room temperature

COMMENTARY

Critical Parameters

As in any synthesis involving modified amidites, it is essential to use the driest possible solution of reagent 1. The addition of a molecular sieve desiccant is recommended.

After deprotection in a strongly basic medium, the reduction in total volume must not lead to total evaporation of the solvent, as in that case a substantial part of the product would lose its trityl group, making it impossible to separate by HPLC.

The acetic acid must be completely evaporated during the conversion of ODN3 to ODN4 + ODN5. Traces of residual acids would alter the pH during the next step and significantly slow the thermolysis reaction of ODN4 to ODN5.

Also, monitor the temperature of 80°C during the thermolysis reaction; a lower effective temperature slows down the reaction.

Troubleshooting

Table 1 lists problems that may arise with this procedure along with their possible causes and solutions.

Table 1.

Troubleshooting Guide for Synthesis of ODN5/ORN5

Problem	Possible cause	Solution
Low coupling of reagent 1	Traces of moisture	Thoroughly dry reagent 1
Slight decrease in ODN4 in spite of a 90‐ min heating time.	Thermolysis conditions not optimal	Check effective pH and temperature; extend heating time
Presence of shorter sequences after thermolysis	Strand break occurred	Check effective pH and temperature; reduce heating time

Understanding Results

At the end of the deprotection reaction, a phosphorylated oligonucleotide should be obtained, which is indicated by a single peak in RP‐HPLC.

Two examples of such oligonucleotides are provided in Figure 7 for DNA (sequence: AAC CGA CCA AGA GTT, Fig. 7A) and RNA (sequence: UAG GAA AAA GAG GAU, Fig. 7B).

Figure 7.

UPLC analyses of phosphorylated ODN5 and ORN5. (A) Phosphorylated ODN5 (sequence: AAC CGA CCA AGA GTT); (B) phosphorylated ORN5 (sequence: UAG GAA AAA GAG GAU).

Time Considerations

The synthesis of reagent 1 requires ∼10 days of work. Synthesis of the ODN or ORN on a support, 5′ phosphorylation, and deprotection in a strongly basic medium require 1 day of work. The purification and lyophilization steps require 1 day of work. Deprotection of the 5′ end group and its conversion to a 5′‐phosphate ODN require an additional day.

Author Contributions

Rémy Lartia: Conceptualization; data curation; formal analysis; resources; supervision; validation; writing—original draft; writing—review and editing.

Conflict of Interest

The author declares no conflict of interest.

Supporting information

Full NMR data (¹H, ¹³C, and ³¹P) for compounds 1–6.

Lartia, R. (2025). Use of a dihydroxyacetone derivative as protecting reagent to phosphorylate oligonucleotides. Current Protocols, 5, e70215. doi: 10.1002/cpz1.70215

Data Availability Statement

The data that support this protocol are available in the supplementary material of this article.