Sulfonate derived phosphoramidates as active intermediates in the enzymatic primer-extension of DNA

Abstract

Novel unnatural 5'-phosphoramidate nucleosides, capable of being processed as substrates by DNA polymerases for multiple nucleotide incorporations, have been designed. The mimics feature metabolites such as taurine and a broad range of aliphatic sulfonates coupled through a P-N bond to the 5'-phosphate position of deoxynucleotides, to allow binding interactions in the enzyme active site. The utility of all of the analogues as pyrophosphate mimics was demonstrated for the chain elongation of DNA, using both thermophilic and mesophilic microbial polymerases.

Syntheses of 5′‐Nucleoside Monophosphate Derivatives with Unique Aminal, Hemiaminal, and Hemithioaminal Functionalities: A New Class of 5′‐Peptidyl Nucleotides

“…Steady‐state kinetic analysis (Table ) indicated a comparable V max value, a K M that was 991‐fold higher, and a final V max / K M ratio for compound 2 a that was 994‐fold lower than the natural substrate. This result falls within the range of kinetic values that have been previously reported for other leaving groups in our laboratory …”

“…Azido compound 16 was obtained in good yield from glycine derivative 5 by using the DPPA/DBU method, and it was then converted into its amino form 17 by classical reduction in the presence of PPh 3 . The ensuing phosphoramidate coupling was carried out under standard DCC coupling conditions to afford a diastereoisomeric mixture of compounds 18 a and 18 b in good yield. Our initial efforts to separate isomers 18 a, b after the final debenzylation step turned out to be problematic; however, the protected diastereoisomers could be successfully separated by preparative RP‐HPLC to yield pure 18 a and 18 b in an approximate 1:1 ratio.…”

“…This result falls within the range of kinetic values that have been previously reported for other leaving groups in our laboratory. [24] Conclusion An umber of useful strategies wered eveloped and effectively refined to generated ifferent phosphodiester and mixed (N/O, S/O) phosphoester linker moieties in the preparation of an ovel class of 5'-modified conjugateso fT MP with the dipeptide l-Ala-Gly.T he considerable challenges posed by the sensitive nature of the derived phosphoaminal, -hemiaminal or -hemithioaminal functionalities demanded as ystematic elaboration of specifically tailored synthetic technologies. The introductiono fa na mino group at the a-position of ag lycine residue successfully activated the corresponding dipeptide for attack at the phosphorus atom of TMP,w hich led to the requisite phosphoaminal ester conjugates.…”

“…The collected fractions were freezedried repeatedly until the mass remained constant to obtain ad iastereoisomeric mixture of compound 23 (158 mg, 85 %, white solid). 1 N-Carbobenzyloxy-l-alanyl-d,l-2-mercaptoethanolglycine benzylester (24) Et 3 N( 0.55 mL, 3.92 mmol) was added dropwise to as tirring solution of compound 12 (1.4 g, 3.27 mmol) and 2-mercaptoethanol (0.25 mL, 3.59 mmol) in dry DMF (12 mL) at 0 8C. The reaction mixture was slowly warmed to room temperature and left stirring for 16 h. The resulting mixture was concentrated in vacuo.…”

Cascade aryne insertion/vinylogous aldol reaction of vinyl-substituted β-keto/enol carbonyls

“…With this assumption, cycloheptenone 5 15 was subjected to carbomethoxylation with NaH as a base, and dimethyl carbonate ( 6 ) as an electrophile in anticipation of the formation of 4 . 16 With 1 H NMR and mass spectroscopic analysis revealing the introduction of a –CO 2 Me group, the Stoltz protocol was initiated with aryne 3a in the presence of CsF.…”