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# An RNA Chemical Phosphorylation Reagent Based on the “DMT-On” Strategy

Coelio Vallée, Jawad Belkheir, Nina Gonnet, Rémy Lartia

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Coelio Vallée, Jawad Belkheir, Nina Gonnet, Rémy Lartia. An RNA Chemical Phosphorylation Reagent Based on the “DMT-On” Strategy. Organic Letters, 2025, 27, pp.6233 - 6237. 10.1021/acs.orglett.5c01932 . hal-05131643

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# An RNA Chemical Phosphorylation Reagent Based on the "DMT-On" Strategy

Coelio Vallée, Jawad Belkheir, Nina Gonnet, and Rémy Lartia*

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Cite This: *Org. Lett.* 2025, 27, 6233–6237

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Supporting Information

**ABSTRACT:** We present a new phosphorylation reagent built around a dihydroxyacetone core. DNA and, more interestingly, RNA can be easily phosphorylated by using a protocol similar to that routinely employed in the synthesis of unmodified nucleic acids. The well-known advantages associated with the dimethoxytrityl group (on-resin monitoring by trityl cation assay, delayed retention on HPLC, simple deprotection steps, and the use of equipment commonly found in oligo laboratories) are thus maintained.

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The automated synthesis of unmodified oligodeoxyribonucleotides (ODN) and oligoribonucleotides (ORN) is a well-established procedure routinely performed in laboratories. One salient feature is the retention of a lipophilic handle, generally dimethoxytrityl group (DMTr), at the very end of the synthesis, enabling trityl cation assay, easy separation of the desired sequence from truncated ones by RP-HPLC purification, and easy recovery of the expected ODN by a subsequent acidic treatment. A ca. 10 min treatment in 80% aqueous AcOH is now considered a standard.¹,² This strategy has been successfully extended to 5'-modification of ODN by amine since the '80s.³⁻⁵

Conversely, the incorporation of a phosphate residue at the 5'-end of an ODN or ORN is less straightforward than one might think. So far, at least 15 reagents or methods have been designed over the last 40 years.⁶⁻¹⁷ This quest was driven by the biological significance of phosphorylated oligonucleotides, as they find applications in various molecular techniques (linkers, cloning, ligase chain reaction, etc.).¹⁸⁻²⁰

Unfortunately, none of these reagents are fully user-friendly. The pioneering reagent A⁷ (see Figure 1) lacks the lipophilic trityl handle and does not allow RP-HPLC purification. Similarly, the commercially available reagent B¹⁴ loses it upon final basic deprotection, leading to the same drawbacks. In contrast, reagent C¹⁵ is a second-generation phosphorylation reagent and allows HPLC purification. However, the release of phosphorylated nucleic acids requires an ORN-incompatible final basic treatment. Very recently, synthesis of 5'-phosphorylated ORN made a leap forward with the development of the o-nitrobenzylated reagent D²¹,²² and 2-pyridinyl reagent E.²³ After isolation of the ORN protected by D, the desired 5'-phosphorylated ORN is released upon UV irradiation at 365 nm. Similarly, the newly developed reagent E allows for the phosphorylation of the ODN and ORN 5'-phosphorylation through microwave irradiation. However, this constitutes a bottleneck in the synthesis process, as irradiation lamps (or microwave oven) are not routinely present in nucleic

<figure><img src="image_8.png" /><figcaption>**Figure 1.** Chemical structures of some commercial (A–C) or described (D, E) phosphorylation reagents.</figcaption></figure>

acid synthesis laboratories. Moreover neither D nor E enables phosphorylation yield estimation by the well-known DMTr⁺ cation assay.²⁴

In search of a new phosphorylation reagent based on the "DMT-On" strategy, we investigate the instability of 1,3-dihydroxyacetone monophosphate (DHAP) in aqueous media.²⁵ DHAP is known to decompose spontaneously into a mineral phosphate anion via an enediol intermediate.²⁶,²⁷ For

Received: May 13, 2025

Revised: May 28, 2025

Accepted: June 2, 2025

Published: June 3, 2025

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biological purposes, fresh DHAP is often prepared from its dimethyl acetal derivative by gently heating it in pure water. However, DHAP is so unstable that a substantial fraction of the product decomposes directly into phosphate once obtained.²⁸,²⁹

Consequently, we assumed that a reagent with an embedded DHAP moiety, such as **1** (Scheme 1), would be well-suited to

<figure><img src="image_10.png" /><figcaption>**Scheme 1.** Structures of Phosphoramidites 1a–d and Synthesis of Phosphoramidite 1a from DHA and 1d from TRISᵃ</figcaption></figure>

ᵃReagents and conditions: (a) CH(OMe)₃, APTS, MeOH, o/n, 80%; (b) DMTrCl, DMAP, Pyr/CH₂Cl₂; (c) iPr₂NP(Cl)OCNE, CH₂Cl₂, 1 h, 70–83%; (d) PhCH(OMe)₂, APTS, o/n, 63%; (e) NaIO₄, KH₂PO₄, o/n, 82%; (f) CH(OiPr)₃, CSA, iPrOH, 1 h, 41%; (g) H₂ (20 bar), Pd(OH)₂, iPrOH, 61%.

perform the 5'-phosphorylation of ODN/ORN. In our first attempts, the carbonyl group of the dihydroxyacetone (DHA) was protected in the form of a methyl acetal. DHA was reacted directly with trimethylorthoformate to afford alcohol 2a,³⁰ which in turn was trytilated and phosphorylated into the reagent **1a**.

The CPG-supported model of ODN **8** (ON = T₆; see Scheme 2) was reacted with **1a** using the classical coupling

<figure><img src="image_11.png" /><figcaption>**Scheme 2.** Synthesis of 5'-Phosphorylated ON 10ᵃ</figcaption></figure>

ᵃReagents and conditions: (a) (i) ON elongation, (ii) **1a–d**, 5-ethylthiotetrazole, 5 min; (b) 40% aq. CH₃NH₂, 10 min, 60 °C; (c) AcOH/H₂O (4:1), 10 min; (d) 100 mM phosphate, pH = 7, 80 °C, 1 h. ON = ODN or ORN. R = Me (a), Et (b), n-Pr (c), or i-Pr (d).

procedure (coupling time of 5 min; 96% yield). It is important to note at this stage that reagent **1a** enables loading to be easily determined by the DMTr cation assay. Following deprotection from the support and purification, the 5'-protected ODN **9a** underwent 80% AcOH deprotection. Unfortunately, the acetal moiety in **9a** was found to be particularly reluctant to

acidolysis.³¹,³² After 60 min reaction time, only 32% of expected 5'-phosphate ODN **10** was released (8% after 10 min), whereas **11a** was detected as the main product.

DHA, protected with bulkier substituents, is known to exhibit enhanced acid sensitivity.³³ Thus, ethylated (**1b**) and *n*-propylated (**1c**) phosphorylation reagents were synthesized from *bis*-acetoxy-DHA **4**.³⁴ However, the corresponding 5'-protected ODN **9b** and **9c** (ON = T₆) were revealed to be only slightly more sensitive toward acidolysis (respectively 40% and 50% deprotection yields after 10 min, 85% and 93% after 1 h; see Figure SI-1). In both cases, **11b–c** and, in to a minor extent, **12** were obtained as byproducts according to UPLC-MS.

We finally turned toward the introduction of bulkier isopropyl groups. Isopropyl ketalization of **4** or DHA using CH(OiPr)₃ and sulfuric acid, cerium trifluorosulfonate,³⁵ or APTS as a catalyst was unsuccessful. We reasoned that a cyclic ketone such as **6** would be an interesting intermediate because it would enhance the reactivity of the carbonyl group while protecting the hydroxyl groups of DHA. Commercially available TRIS was converted into 1,3-dioxane **5**, which in turn was oxidized to the cyclic ketone **6**.³⁶ The ketone was conveniently acetalized in **7** and subjected to hydrogenation and tritylation, leading to alcohol **3d**. Classical procedures afforded isopropylated phosphoramidite reagent **1d**.

The model on-support ODN **8** (See Scheme 2, ON = T₁₂) was reacted with **1d** leading, after deprotection, to form the expected 4144.9 amu ODN **9d** with 96% coupling yield. Due to the lipophilic nature of the isopropyl acetal, the resulting ODN **9d** was particularly well resolved from the failure sequence. This ODN was then collected, lyophilized overnight, desalted on an SEC disposable cartridge, and lyophilized again without substantial loss of the acetal moiety (Figures 2a and SI-5). Finally, dried **9d** (50 nmol) was taken up in 80% aqueous AcOH solution (250 µL, 10 min, rt) and evaporated to dryness. RP-HPLC analysis showed the disappearance of the main peak and the appearance of two faster-eluting peaks (10/12, see Figure 2b) in ca. a 1:2 ratio; product **11d** was not detected. The major product corresponded to the desired phosphorylated product **10** with the expected 3667.6 amu mass. The minor product displayed +72 amu mass and was assumed to be partially deprotected **12**.

Full conversion of this mixture to the desired phosphorylated ODN **10** was achieved by heating at neutral pH (100 mM phosphate buffer, pH = 7) at 80 °C for 90 min (see Figure 2c; see Figures SI-1–SI-3 for further consideration of the hydrolysis of ODN **9a–d**; see SI 1.4 for consideration of the **12** to **10** conversion).

Using this protocol, a series on-support ODN **8** was conveniently phosphorylated with a >95% yield, then cleaved, purified by RP-HPLC, and deprotected to yield the 5'-phosphorylated ODN (see Table 1, entries 1–6). We next turned toward synthesis of the more challenging 5'-phosphorylated ORN. A series of various ORN was synthesized on-support using 2'-tBDMS protecting groups and then reacted with reagent **1d**. Deprotection following classical procedures (40% aq. methylamine, 10 min at 60 °C, and DMSO/TEA/TEA·3HF treatments) led to the expected 5'-protected ORN **9d** (Table 1, entries 7–13). After RP-HPLC purification and desalting steps, the ORN were treated with 80% aq. AcOH solution (250 µL, 10 min, RT) and evaporated to dryness, thus producing a totally (**10**) and partially (**12**) deprotected phosphorylated ORN mixture in a
<figure><img src="image_12.png" /><figcaption>**Figure 2.** RP-HPLC analysis of ON 10d, 11, and 12 (ON = T₁₂). RP-HPLC analysis of ODN 9d (ON = T₁₂) after RP-HPLC purification (a) after 10 min. RP-HPLC analysis following AcOH 80% treatment of ODN 10 and 12 (b) and after 90 min of heating in 100 mM phosphate buffer pH = 7.0 (c).</figcaption></figure>

ca. 1:2 to 2:1 ratio. Heating in 100 mM phosphate buffer (pH = 7.0) at 80 °C for 90 min led to the expected phosphorylated ORN 10 (see Table 1, and Figure SI-4 for the representative UPLC trace).

RNA is known to be more susceptible to chemical degradation than DNA. Although RNA instability at high temperatures has mainly been reported for sequences that are significantly longer than those obtained by chemical synthesis,³⁷,³⁸ we set out to determine the impact of treatment on our sequences. Other authors have reported that heating short RNA sequences at 90 °C does not cause significant degradation.²³ PAGE analysis of a 20-mer RNA showed

negligible degradation after 2 h of heating in 100 mM phosphate buffer (pH = 7, 80 °C). However, the RNA chain was found to degrade slightly with a longer heating time (see Figure SI-6).

This newly developed reagent is a user-friendly alternative for the synthesis of 5'-phosphorylated nucleic acids, particularly ORN, as it is the only one based on the well-established DMT strategy. The yield of the final coupling can be quantified by a trityl cation assay of the oligonucleotide still anchored to the solid support. Failure sequences can be easily removed by RP-HPLC purification, and the expected 5'-phosphorylated ORN 10 can be released using methods, reagents and devices that are used daily in ON synthesis. We believe that this new reagent will be a breakthrough in the straightforward production of 5'-phosphate oligonucleotides. Among all the reagents developed in the last 40 years, the 1d reagent is the one whose processing is closest to that of natural oligonucleotides.

## ■ ASSOCIATED CONTENT

### Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

**SI** Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.orglett.5c01932.

¹H, ¹³C, and ³¹P NMR spectra for compounds **1a-d**, **2d**, **3d**, and **5-8**; ESI-MS spectra for ODN and ORN **10d** and **13**; experimental procedures for compounds **1d**, **2d**, **3d**, and **5-8**; additional comments on the conversion of **12** into **10**; enlarged version of Figure 2; and gel analysis of RNA heated at 80 °C (PDF)

## ■ AUTHOR INFORMATION

### Corresponding Author

Rémy Lartia - Département de Chimie Moléculaire, Université Grenoble Alpes, 38000 Grenoble, France; orcid.org/0000-0002-0049-6726; Email: remy.lartia@univ-grenoble-alpes.fr

<table><caption>**Table 1.** Sequence and Molecular Masses of Protected (9d) and Phosphorylated (10) ODN (Entries 1–6) and ORN (Entries 7–13)</caption><thead><tr><th rowspan="2">entry</th><th rowspan="2">ON</th><th rowspan="2">sequence of supported ON 8</th><th colspan="2">molecular weight of 5'-protected ODN or ORN 9d</th><th colspan="2">molecular weight of 5'-phosphorylated ODN or ORN 10</th></tr><tr><th>experimental</th><th>calculated</th><th>experimental</th><th>calculated</th></tr></thead><tbody><tr><td>1</td><td>ODN</td><td>TTT TTT</td><td>2319.2</td><td>2318.6</td><td>1842.7</td><td>1843.2</td></tr><tr><td>2</td><td>ODN</td><td>TTT TTT TTT TTT</td><td>4144.9</td><td>4144.0</td><td>3667.6</td><td>3666.6</td></tr><tr><td>3</td><td>ODN</td><td>AAC CGA CCA AGA GTT</td><td>5124.1</td><td>5124.1</td><td>4647.6</td><td>4647.8</td></tr><tr><td>4</td><td>ODN</td><td>TCA AGT CAG TTA GC</td><td>4816.9</td><td>4817.0</td><td>4340.5</td><td>4340.7</td></tr><tr><td>5</td><td>ODN</td><td>CAC GTC GAG CCG AT CGA GCT GGA T</td><td>7937.8</td><td>7935.4</td><td>7460.9</td><td>7458.8</td></tr><tr><td>6</td><td>ODN</td><td>CGA CGA CGA CGA</td><td>4218.8</td><td>4218.9</td><td>3742.4</td><td>3742.6</td></tr><tr><td>7</td><td>ORN</td><td>UUU GGG AGU CUU ACA AUU GG</td><td>6945.0</td><td>6942.4</td><td>6464.5</td><td>6465.8</td></tr><tr><td>8</td><td>ORN</td><td>GUG UGU GUG U</td><td>3752.7</td><td>3751.5</td><td>3275.1</td><td>3274.9</td></tr><tr><td>9</td><td>ORN</td><td>UAG CGG UGG A</td><td>3797.8</td><td>3796.6</td><td>3320.3</td><td>3320.0</td></tr><tr><td>10</td><td>ORN</td><td>UUU UUU UUU UUU UUU</td><td>5086.9</td><td>5087.1</td><td>4609.8</td><td>4610.5</td></tr><tr><td>11</td><td>ORN</td><td>UCA AAC ACC AAG AAU</td><td>5305.2</td><td>5306.1</td><td>4830.4</td><td>4830.0</td></tr><tr><td>12</td><td>ORN</td><td>AAC UUC C</td><td>2679.2</td><td>2679.6</td><td>2204.2</td><td>2204.3</td></tr><tr><td>13</td><td>ORN</td><td>UUU UUU UU</td><td>2468.3</td><td>2467.4</td><td>2468.3</td><td>2467.4</td></tr></tbody></table>
## Authors

Coelio Vallée – Département de Chimie Moléculaire,
Université Grenoble Alpes, 38000 Grenoble, France

Jawad Belkheir – Département de Chimie Moléculaire,
Université Grenoble Alpes, 38000 Grenoble, France

Nina Gonnet – Département de Chimie Moléculaire,
Université Grenoble Alpes, 38000 Grenoble, France

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.5c01932

## Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript

## Notes

The authors declare no competing financial interest.

## ACKNOWLEDGMENTS

The Nanobio-ICMG platform (UAR 2607) is acknowledged for providing facilities for the synthesis and purification of oligonucleotides and mass spectrometry analyses. The authors thank R. Guéret and L. Fort for running the ESI-MS analysis and B. Darses and M. Fayolle (Département de Chimie Moléculaire, Université Grenoble Alpes, Grenoble, France) for technical assistance. This work was partially supported by the Labex ARCANE (ANR-16-CE11-0006-01), CBH-EUR-GS (ANR-17-EURE-0003), and the région Auvergne-Rhône-Alpes.

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