Inosine | CAS 58-63-9 | ≥98%

Technical Specifications

Product Description & Scientific Applications

Inosine (hypoxanthine 9-β-D-ribofuranoside; Ino) is a naturally occurring purine ribonucleoside formed by hypoxanthine linked to D-ribofuranose through a β-N9-glycosidic bond. Within mammalian purine metabolism, inosine sits at a central junction: it is generated by irreversible hydrolytic deamination of adenosine by adenosine deaminase (ADA; EC 3.5.4.4) and is itself phosphorolytically cleaved by purine nucleoside phosphorylase (PNP; EC 2.4.2.1) to hypoxanthine and α-D-ribose-1-phosphate, which hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) then converts to IMP — the common gateway feeding both AMP and GMP nucleotide pools. Inosine is also the enzymatic product of adenosine-to-inosine RNA editing by ADAR-family deaminases on double-stranded RNA, and of A34-to-I34 deamination at the tRNA wobble position by the ADAT2/ADAT3 heterodimer. Because inosine's hypoxanthine base lacks the C6-amino group of adenine and the C2-amino group of guanine, it shows a UV maximum near 248–250 nm (blue-shifted relative to adenosine) and a context-dependent base-pairing pattern that underlies its use in degenerate oligonucleotide design and in epitranscriptomic mapping workflows.

Adenosine-to-Inosine RNA Editing (ADAR Family)

A-to-I editing is one of the most abundant post-transcriptional modifications in the mammalian transcriptome and is catalysed by ADAR-family enzymes through hydrolytic C6 deamination of adenosines in double-stranded RNA. Mammals encode three ADAR genes: ADAR1 (ADAR), ADAR2 (ADARB1), and ADAR3 (ADARB2). ADAR1 is expressed as two isoforms — constitutively expressed, primarily nuclear ADAR1p110, and interferon-inducible, predominantly cytoplasmic ADAR1p150, the latter carrying an additional Zα domain and a nuclear export signal. ADAR2 is nuclear, with brain-enriched expression, and is the enzyme responsible for the canonical Q/R recoding of glutamate-receptor (GRIA2) pre-mRNA. ADAR3 is brain-restricted and catalytically inactive in vivo, with proposed regulatory roles. Because inosine is read as guanosine by the translational machinery and during reverse transcription, A-to-I editing can recode amino acids, alter splice-site recognition, modulate microRNA biogenesis and targeting, and contribute to the marking of endogenous double-stranded RNA as “self” relative to MDA5-mediated cytosolic dsRNA sensing. Inosine is the canonical analytical target in epitranscriptomic mapping workflows including inosine chemical erasing sequencing (ICE-seq, based on acrylonitrile-mediated N1-cyanoethylation of inosine, which disrupts inosine base-pairing and arrests reverse transcription at edited sites), Endonuclease V-mediated inosine-specific RNA cleavage, and high-resolution LC-MS/MS detection.

Wobble I34 Decoding by ADAT2/ADAT3

At position 34 (the wobble position) of eukaryotic tRNA anticodons, adenosine is enzymatically deaminated to inosine by an essential ADAT heterodimer composed of catalytic ADAT2 and structurally indispensable ADAT3, whose pseudoactive site replaces the catalytic glutamate with an inactive residue such as valine. Both subunits adopt the cytidine deaminase (CDA) superfamily fold with the C/H…PCXXC zinc-binding signature, structurally linking tRNA editing chemistry to the broader CDA mechanistic family. A single tRNA carrying I34 can decode three synonymous codons (NNU, NNC, NNA), expanding codon-recognition capacity and compressing the required tRNA repertoire. The bacterial homologue TadA is a homodimer that has been adapted as the deaminase module of adenine base editors (ABEs) for programmable A-to-G genome editing, providing an additional research vector connecting inosine chemistry to nucleic-acid engineering. Inosine is therefore central to research in tRNA biology, translation fidelity, codon-usage bias, ribosome decoding, base-editing tool development, and molecular evolution of the genetic code.

Purine Salvage and PNP Substrate Specificity

Mammalian purine nucleoside phosphorylase (PNP; EC 2.4.2.1) is a homotrimeric enzyme of ~31 kDa subunits that is highly specific for 6-oxopurine nucleosides: in human PNP, the catalytic efficiency (kcat/KM) for inosine is approximately 350,000-fold greater than for adenosine, reflecting active-site recognition of the 6-oxopurine base by specificity residues including Asn243 and Glu201. This specificity is the mechanistic reason that mammalian adenosine catabolism proceeds efficiently through the sequential ADA → inosine → PNP route rather than direct PNP phosphorolysis of adenosine. PNP catalyses reversible phosphorolysis of inosine to hypoxanthine and α-D-ribose-1-phosphate; hypoxanthine is then condensed with PRPP by HGPRT to give IMP, which feeds both AMP (via adenylosuccinate synthase/lyase) and GMP (via IMP dehydrogenase and GMP synthase) nucleotide pools. The reversibility of the PNP reaction also makes inosine a valuable starting material in chemoenzymatic synthesis: trans-glycosylation through the α-D-ribose-1-phosphate intermediate enables transfer of the sugar to modified purine bases for preparation of purine nucleoside analogues, while pyrimidine analogues generally require coupled pyrimidine-nucleoside-phosphorylase cascades.

Degenerate Oligonucleotide Design

Inosine and 2'-deoxyinosine pair with all four canonical bases, with a context-dependent design heuristic often given for deoxyinosine-containing DNA duplexes as I:C > I:A > I:T ≈ I:G. This near-universal but non-equivalent pairing makes inosine and deoxyinosine useful as “wobble positions” in degenerate primer and probe design, reducing the number of separate oligonucleotides needed to cover sequence variants while maintaining productive hybridisation. Practical applications include consensus primer design for amplification of homologous genes across divergent species, conserved-region targeting in expression analysis, probe rescue for partially divergent targets, and screening of mixed templates. Because inosine pairing is context-dependent rather than truly universal, design discipline is required: inosine bases are most useful at positions of true sequence ambiguity within otherwise specific oligonucleotides, and are not a substitute for full sequence specificity in single-mismatch-sensitive applications.

Spectroscopic and Analytical Reference

Inosine has a UV absorption maximum near 248–250 nm with ε ≈ 12,000 M−1cm−1 at neutral pH, clearly blue-shifted from adenosine (λmax ≈ 260 nm) and distinguishable from guanosine by UV spectral shape and its 15-Da lower mass at the nucleoside level (inosine 268.08 Da vs guanosine 283.09 Da, monoisotopic). Reverse-phase C18 HPLC and ion-pair LC-MS protocols use inosine as a primary retention and quantitation standard for purine-pool analysis, alongside adenosine, guanosine, hypoxanthine, xanthine, and uric acid. Inosine is recognised and cleaved 3' to its position by RNase T1 alongside guanosine, providing an enzymatic readout of inosine-containing RNA fragments. 1H NMR signatures are diagnostic: the H1' anomeric proton at δ ≈ 5.9–6.1 ppm in D2O, and the hypoxanthine H8 and H2 singlets near δ 8.2–8.3 ppm.

Other Applications

• Reference standard for HPLC, UHPLC, and LC-MS analysis of purine nucleosides and nucleotides
• Substrate for PNP assays and product/reference standard in ADA, ADAR, and ADAT activity and inhibition workflows
• Analytical target in epitranscriptomic mapping of A-to-I RNA editing, including ICE-seq, Endonuclease V-mediated cleavage assays, RNase T1 analysis of inosine-containing RNA fragments, and mass spectrometry
• Building block and reference material for inosine- and 2'-deoxyinosine-modified oligonucleotides in degenerate primer/probe design
• Starting material for chemoenzymatic synthesis of purine nucleoside analogues via reversible PNP-mediated trans-glycosylation
• Standard purine riboside in 1H, 13C, and 15N NMR studies of nucleoside conformation, glycosidic-bond geometry, and tautomeric chemistry.

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