Alkyl phosphonates are organophosphorus compounds characterized by a carbon-phosphorus (C−P) bond. Alkyl phosphonates are esters or salts of phosphonic acid (H3PO3), which have the general formula RPO(OH)2, where R is an alkyl group. This structure distinguishes them from alkyl phosphates, which contain a carbon-oxygen-phosphorus (C−O−P) bond. The electron configuration of phosphorus is 1s2 2s2 2p6 3s2 3p3, and the structure of the outermost shell, which includes not only 3s and 3p but also 3d orbitals, determines its valence. Orbitals 3s and 3p have 5 electrons available for bonding with other atoms.
Alkyl phosphonates are soluble in water, especially in their deprotonated, salt form. They chelate di- and trivalent metal ions, which is particularly useful in preventing the formation of insoluble precipitates, such as mineral scale. Due to their chelating properties and ability to form protective layers on metal surfaces, they are highly effective in inhibiting scale formation and preventing corrosion.
Alkyl phosphonates are very stable under harsh conditions, including high acidity, alkalinity, and extreme temperatures. Alkyl phosphonates enable chemical modifications of the RNA backbone. In alkyl phosphonates, one of the non-bridging oxygen atoms of the phosphate group is replaced by an alkyl group, for example, a methyl group.
In a standard RNA molecule, negatively charged phosphodiester bonds link together the sugar and phosphate groups. Alkyl phosphonate modifications can replace the phosphodiester linkage with a charge-neutral alkyl phosphonate group. Alkyl phosphonate modifications used in the design of therapeutic RNA molecules enhance their performance by improving specificity, reducing toxicity, and overall safety and efficacy.
Alkyl phosphonate modifications investigated by Nikan et al. (20205)
| cHex (cyclohexyl phosphonate) | iBu (isobutyl phosphonate) | MOP (methoxypropyl phosphonate) |
|  |  |  |
| MP (methyl phosphonate) | PACE (phosphonoacetate) | Propyl (propyl phosphonate) |
| 
| 
| 
|
(Source: Nikan et al. 2025)
Alkyl phosphonate modifications are of particular interest in the development of oligonucleotide therapeutics, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASOs). Unmodified ASO and siRNA therapies are stable, can have off-target effects, and have potential toxicity. By altering the RNA backbone, alkyl phosphonate modifications can modulate interactions with cellular proteins and reduce unintended binding to non-target RNAs. For example, a single alkyl phosphonate modification in the "seed region" of an siRNA can significantly enhance its specificity and improve its therapeutic profile.
Other chemical modifications, such as phosphorothioate linkages, can lead to toxicity. Replacing these linkages with charge-neutral alkyl phosphonate linkages can reduce or eliminate toxicity in specific therapeutic oligonucleotides, making them safer for use. Charge-neutral alkyl phosphonate modifications also alter RNA's interactions with various proteins in the cell, including RNase H1, leading to modified cleavage patterns and a more favorable therapeutic outcome.
Blake et al. (1985) reported that oligodeoxyribonucleoside methylphosphonates inhibit globin synthesis in rabbit reticulocytes. Oligomers binding to the 5'-end and initiation codon regions of beta-globin mRNA inhibit both alpha- and beta-globin synthesis. In contrast, oligomers that bind to the coding region of alpha-globin mRNA or the coding region of beta-globin mRNA inhibit translation of their target mRNA in a specific manner. The effects of various oligomers on cellular globin synthesis are like those observed in the lysate system and suggest that the globin mRNA conformation is the same in both systems during translation.
Sarin et al. (1988) showed that the introduction of 18 methylphosphonate groups into a 20-mer oligonucleotide significantly increased inhibition of HIV activity. The researchers suggested that methylphosphonate-modified oligonucleotides are more stable to nuclease degradation and may allow the treatment of acquired immunodeficiency syndrome (AIDS).
Walder, J. (1988) reported that Miller and co-workers (Johns Hopkins University School of Medicine) have prepared oligonucleotide methylphosphonate derivatives further modified with psoralen to enhance their activity. Irradiation with 365-nm light results in irreversible cross-linking of the oligonucleotide to the target sequence. Such derivatives block protein synthesis very effectively. In in vitro translation systems, inhibition occurs at micromolar concentrations.
Kibler-Herzog et al. (1991) investigated the duplex stabilities of oligonucleotides modified with phosphorothioate and methylphosphonate. The study compared the stability of RNA analogs and two DNA 14-mers with their unmodified DNA counterparts. The study investigated how various modifications affect oligonucleotides' ability to form stable double-stranded structures with their complementary DNA sequences. The researchers analyzed two self-complementary DNA sequences: d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2].
By creating various phosphorothioate and methylphosphonate analogs of these oligonucleotides and varying the number, position, or chirality of the modified phosphates, Kibler-Herzog et al. found that phosphorothioate modifications destabilize the duplex, with the degree of destabilization varying depending on the modification's position. For example, modifications between adenine bases were less destabilizing than those between thymine bases in sequence D1.
Since the methylphosphonate linkage renders the phosphodiester bond resistant to nucleases, Niu et al. (2000) used this modification in primers for telomere polymerization reactions. A phosphonate modification placed at the 3’-end of a DNA primer inhibits extension of the nearby 3’-OH group by telomerase.
Soliva et al (2001) reported the solution structure of a DNA duplex with a chiral alkyl phosphonate moiety. The solved structure revealed that the presence of a neutral alkyl phosphonate unit in the middle of the helix induces a moderate bending in DNA. The DNA bends towards the major groove, whose width is reduced by ∼2 (R) and 3 (S) Å from canonical B-DNA structures, leading to the generation of a global bending in the helix, as a result, the neutralization of one phosphate can induce a non-negligible bending in the DNA structure.
Strömberg & Stawinski (2004) reported a general procedure for the preparation of oligodeoxyribo- and oligoribonucleotides using H-phosphonate monomers.
Prater & Miller (2004) reported that antisense oligo-2‘-O-methylribonucleotides and their methylphosphonate derivatives have high binding affinities for their complementary targets under physiological conditions. The two researchers observed that the methylphosphonate linkage is resistant to nuclease hydrolysis and showed that a single methylphosphonate internucleotide linkage at the 3‘-end of an oligo-2‘-O-methyl-ribo-nucleotide is sufficient to prevent degradation by the 3‘-exonuclease activity in mammalian serum. Also, complexes of the cationic lipid, Oligofectamine, with 5'-[32P]-labeled methylphosphonate modified oligo-2‘-O-methylribonucleotides were taken up by mouse L929 fibroblasts in culture.
The Tat peptide was coupled to the 5‘-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5‘-end of the oligonucleotide. Mass spectrometry confirmed the formation of the Tat peptide conjugates; however, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture."
Migawa et al. (2019) showed that fine-tuning the size, hydrophobicity, and position of chemical modifications within a PS DNA-gap region can profoundly affect ASO behavior by modulating interactions between the ASO and proteins that cause cellular toxicity. The research group systematically replaced anionic PS linkages in toxic ASOs with charge-neutral methylphosphonate (MP) linkages and found that MPs do not support RNase H1-mediated RNA cleavage near the site of incorporation into an ASO. Also, MPs incorporated into oligonucleotides using standard chemistry are more susceptible to strand cleavage under basic conditions required to deprotect oligonucleotides after solid-phase synthesis.
Replacing PS linkages with neutral alkylphosphonate linkages near the 5′-end of the DNA gap improves the therapeutic profile of toxic gapmer ASOs. The introduction of a single 2′-OMe (OMe) nucleotide at position 2 on the 5′-side of the gap also improved the therapeutic index of the ASO by reducing toxicity.
miRNAs regulate gene functions by binding to the 3′-UTRs of transcripts, utilizing nucleotides 2-8 from the 5′-end, commonly referred to as the seed region. The miRNA-like effect refers to the thermodynamic stability of the seed-mRNA duplex, determined by the melting temperature and standard free energy changes.
Arangundy-Franklin et al. (2019) described the encoded synthesis of a polymer with an uncharged backbone chemistry. Alkyl-phosphonate nucleic acids (phNA), in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl-phosphonodiester backbone enabled the enzymatic, DNA-templated synthesis of P-methyl- and P-ethyl-phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence, mixed P-methyl- / P-ethyl-phNA repertoires.
Ota et al. (2023) reported the tertiary alkylation of phosphorus atoms of phosphites bearing two 2’-deoxynucleosides. The synthesis process utilizes a carbocation generated via a light-driven radical-polar crossover mechanism, providing tertiary alkylphosphonate structures that are difficult to synthesize using other methods. The research group also confirmed the conversion of these species to oligonucleotides with charge-neutral alkylphosphonate linkages through a phosphoramidite-based approach.
Xuan et al. (2024) reviewed strategies for the preparation of organophosphate analogs, focusing on recent developments in the synthesis of alkyl phosphonates to facilitate the development of green pharmacological alkyl phosphonates. This review covers a variety of products, their specificity and relevance, and provides the underlying mechanistic rationale for their synthesis.
Nikan et al. (2025) evaluated the effect of alkyl phosphonate linkages when incorporated into the seed region of therapeutic siRNA. siRNAs modified with a single alkyl phosphonate linkage showed enhanced specificity and therapeutic profile compared to the parent siRNA. The researchers found that these modifications are most effective when positioned at the internucleotide linkage 6-7 from the 5′-end of the guide strand, revealing that siRNAs with phosphonate modification maintain robust on-target activity both in vitro and in vivo, with enhanced safety in mice.
Reference
Arangundy-Franklin S, Taylor AI, Porebski BT, Genna V, Peak-Chew S, Vaisman A, Woodgate R, Orozco M, Holliger P. A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids. Nat Chem. 2019 Jun;11(6):533-542. [PMC]
Blake, K.R., Murakami, A., Spitz, S.A., Glave, S.A., Reddy, M.P., Tso, P.O., Miller, P.S. Hybridization arrest of globin synthesis in rabbit reticulocyte lysates and cells by oligodeoxyribonucleoside methylphosphonates. Biochemistry (1985), 24: 6139-6145. [PubMed]
Kibler-Herzog, L., Zon, G., Uznanski, B., Whittier, G, Wilson, W.D. Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers. Nucleic Acids Res. (1991), 19: 2979-2986. [PMC]
Migawa MT, Shen W, Wan WB, Vasquez G, Oestergaard ME, Low A, De Hoyos CL, Gupta R, Murray S, Tanowitz M, Bell M, Nichols JG, Gaus H, Liang XH, Swayze EE, Crooke ST, Seth PP. Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins. Nucleic Acids Res. 2019 Jun 20;47(11):5465-5479. [PMC]
Nikan M, Li Q, Tanowitz M, Li H, Damle S, Annoual M, Galindo-Murillo R, Low A, Klein S, Quirk C, Vasquez G, Wan WB, Watt AT, Migawa MT, Swayze EE, Prakash TP. Single alkyl phosphonate modification of the siRNA backbone in the seed region enhances specificity and therapeutic profile. Nucleic Acids Res. 2025 Jul 19;53(14):gkaf692. [PMC]
Niu H, Xia J, Lue NF. Characterization of the interaction between the nuclease and reverse transcriptase activity of the yeast telomerase complex. Mol Cell Biol. 2000 Sep;20(18):6806-15. [PMC] -> phosphonates to study telomerase!
Ota K, Nagao K, Hata D, Sugiyama H, Segawa Y, Tokunoh R, Seki T, Miyamoto N, Sasaki Y, Ohmiya H. Synthesis of tertiary alkylphosphonate oligonucleotides through light-driven radical-polar crossover reactions. Nat Commun. 2023 Oct 31;14(1):6856. [PubMed] [PMC]
Prater CE, Miller PS. 3'-methylphosphonate-modified oligo-2'-O-methylribonucleotides and their Tat peptide conjugates: uptake and stability in mouse fibroblasts in culture. Bioconjug Chem. 2004 May-Jun;15(3):498-507. [ACS]
Sarin, P.S., Agrawal, S., Civeira, M.P., Goodchild, J., Ikeuchi, T., Zamecnik, P.C. Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates. Proc. Natl. Acad. Sci. USA (1988), 85: 7448-7451. [PNAS]
Soliva R, Monaco V, Gómez-Pinto I, Meeuwenoord NJ, Marel GA, Boom JH, González C, Orozco M. Solution structure of a DNA duplex with a chiral alkyl phosphonate moiety. Nucleic Acids Res. 2001 Jul 15;29(14):2973-85. [PMC] PDB nos 1iek, 1iey
Walder, J. Antisense DNA and RNA: progress and prospects. Genes Dev. (1988), 2: 502-504. [Genes Development]
Xuan, Ch., Zhu, Z., Li, Z., Shu, C.; 2024 Recent developments in the synthesis of pharmacological alkyl phosphonates. Advanced Agrochem 4, 1, 13-29. [Advanced Agrochem]
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