Phosphorothioate substitution

A phosphorothioate substitution replaces an oxygen of a phosphate group with sulfur. In a nucleic acid backbone, the usual experimental substitution replaces a non-bridging oxygen, while retaining the oxygen bridges connecting neighboring nucleotides. It changes backbone chemistry without deleting a nucleotide or changing its nucleobase sequence. The altered group is a phosphate analogue rather than a disulfide bond.

The two non-bridging positions matter. For a phosphate joining different nucleotide environments, replacing one oxygen or the other can give different configurations at phosphorus. Such phosphorothioate diastereomers can interact differently with an RNA-folding environment or a metal ion. The 2013 U6 metal rescue experiment tested both $R_P$ and $S_P$ substitutions at selected sites, rather than treating every sulfur replacement as equivalent.^[ref]

An original counting example shows the extra information required. At three independently stereogenic, substituted phosphates, two possible configurations at each site give $2^3=8$ configuration combinations. All eight can have the same nucleotide sequence and elemental formula. Measuring molecular mass alone therefore cannot tell which configuration combination was supplied. If every oxygen-to-sulfur replacement is made once at a selected site, each adds approximately $32-16=16$ unified atomic mass units; three add about $48$, regardless of those configuration choices. These rounded atomic masses are illustrative, not exact isotope masses.

Replacing oxygen with sulfur also changes metal–ligand interactions. A loss of RNA activity in magnesium followed by preferential rescue with a different metal can test whether the original oxygen helped position a catalytic ion. It can also reflect altered folding or assembly, so the comparison needs controls. The substitution is a targeted chemical perturbation whose interpretation depends on position, configuration and the reaction being measured.

A phosphorothioate diastereomer is a stereochemical form of a phosphorothioate substitution that differs in configuration without changing connectivity. In a nucleic-acid phosphate joining different nucleotide environments, replacing a non-bridging oxygen with sulfur can make phosphorus a stereogenic center. The resulting $R_P$ and $S_P$ forms have the same elemental composition but a different spatial arrangement at that center.

They are diastereomers in the usual nucleic-acid context because the rest of the RNA already contains fixed stereochemistry, including its ribose units. Changing one phosphorus configuration while preserving those other centers does not produce the mirror image of the entire RNA. It is therefore misleading to call such a pair enantiomers merely because one phosphorus label changes from R to S. Assignment of the labels uses the Cahn-Ingold-Prelog priority rules, not the order in which a nucleotide sequence is written.

Two independently stereogenic substituted phosphates give four combinations: $(R_P,R_P)$, $(R_P,S_P)$, $(S_P,R_P)$ and $(S_P,S_P)$. Their molecular masses are the same. A mass measurement alone cannot determine either the configuration at one site or the proportions in an unresolved mixture.

A simple activity model shows why mixture composition can matter. Suppose equal amounts of two purified forms separately give product fractions $0.8$ and $0.2$ in a stipulated assay. If molecules act independently and the assay response is linear, a mixture containing fraction $f$ of the first form gives $p=0.8f+0.2(1-f)$. At $f=1/2$, $p=0.5$. These invented numbers illustrate averaging; competition for limiting components can invalidate that model.

The U6 metal rescue experiment supplies an actual chemical distinction. Sulfur substitutions with opposite phosphorus configurations at the same U6 position showed differing metal-rescue behavior, including differences between branching and exon ligation.^[ref] Position, configuration and measured reaction therefore belong in the description of a phosphorothioate reagent.