Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the generation of the sulfurising agent from phenylacetyl disulfide (PADS)

Abstract

The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has an optimal combination of properties. This is best achieved by an initial 'ageing' of PADS for 48 h in acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting step is formation of the disulfide anion from the carbanion.

Synthesis of Fused Indoline‐Cyclobutanone Derivatives via an Intramolecular [2+2] Cycloaddition

“…Reaction progress at room temperature was monitored by 1 H NMR spectroscopy. Within two hours, 55 % conversion to known cyclobutanone 37 was observed, indicative of a [2+2] cycloaddition mechanism involving a ketene. Although the alternate pathways cannot be fully discounted for the formation of the indoline‐cyclobutanone fused products, this experiment does indicate the feasibility of Path A .…”

Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the sulfurisation of phosphites by phenylacetyl disulfide (PADS)

“…Solutions of PADS in acetonitrile with 3-picoline slowly degrade to give polysulfides. 14 However, when using 'fresh' solutions of PADS for the sulfurisation of phosphites, the active sulfurising reagent must be the intact diacyl disulfide (3) as the rate of sulfurisation of phosphite esters by 'fresh' PADS are two orders of magnitude faster than the rate of degradation of PADS under similar conditions.…”

“…This reagent is unusual in that it is best 'aged' in a basic acetonitrile solution to obtain optimal sulfurisation activity 13 , often using 3-picoline as the base. We have recently shown that this is due to the formation of diacyl polysulfides (4) by an unusual elimination E1cB mechanism 14 . Although both 'fresh' and 'aged' PADS convert phosphite esters to the corresponding thiophosphate, 'ageing' improves the rate and efficiency of sulfurisation.…”

“…We have recently shown that this is due to the formation of diacyl polysulfides (4) by an unusual elimination E1cB mechanism. 14 Although both 'fresh' and 'aged' PADS convert phosphite esters to the corresponding thiophosphate, 'ageing' improves the rate and efficiency of sulfurisation. Herein we address the reasons for this difference and report the kinetics and mechanism of both sulfurisation reactions.…”

Catalytic Asymmetric and Stereodivergent Oligonucleotide Synthesis

“…Notably, by controlling the stereochemistry at P(III), we can access a variety of P(V) products. For example, treatment of the intermediate phosphite with t-BuOOH affords the phosphate 3, while sulfurization using phenylacetyl disulfide (PADS) affords the phosphorothioate 4 (25). Notably, the loading of (S)-B7 could be reduced to 5 mol% to produce (SP)-4 in 57% yield at 98:2 dr (26) by increasing the reaction concentration to 0.40 M. As methods for the synthesis of selenophosphate ( 27 ), boranophosphate ( 28), phosphoramidite ( 29), and methyl phosphonate ( 30 ) from the P(III) oxidation state have all been reported, this new catalytic protocol enables access to a wide range of functionalized P(V) products (Fig 1B).…”