Reaction Mechanism and Kinetics of the Traceless Staudinger Ligation

Abstract

The traceless Staudinger ligation enables the formation of an amide bond between a phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here, the coupling of peptides by this reaction was characterized in detail. Experiments with [(18)O]H(2)O indicated that the reaction mediated by (diphenylphosphino)methanethiol proceeded by S-->N acyl transfer of the iminophosphorane intermediate to form an amidophosphonium salt, rather than by an aza-Wittig reaction and subsequent hydrolysis of the resulting thioimidate. A continuous (13)C NMR-based assay revealed that the rate-determining step in the Staudinger ligation of glycyl residues mediated by (diphenylphosphino)methanethiol was the formation of the initial phosphazide intermediate. Less efficacious coupling reagents and reaction conditions led to the accumulation of an amine byproduct (which resulted from a Staudinger reduction) or phosphonamide byproduct (which resulted from an aza-Wittig reaction). The Staudinger ligation mediated by (diphenylphosphino)methanethiol proceeded with a second-order rate constant (7.7 x 10(-3) M(-1) s(-1)) and yield (95%) that was unchanged by the addition of exogenous nucleophiles. Ligations mediated by phosphinoalcohols had lower rate constants or less chemoselectivity. Accordingly, (diphenylphosphino)methanethiol was judged to be the most efficacious known reagent for effecting the traceless Staudinger ligation.

Water-Soluble Phosphinothiols for Traceless Staudinger Ligation and Integration with Expressed Protein Ligation

“…This Ala + Gly coupling was found to proceed with a second-order rate constant of k 2 = 7.4 × 10 −3 M −1 s −1 in water. This rate constant, which is similar to that reported previously for a Gly + Gly coupling mediated by phosphinothiol 1 in DMF/D 2 O (6:1) (k 2 = 7.7 × 10 −3 M −1 s −1 ), 17 corresponds to t 1/2 = 10 min for a reaction with 0.16 M azide.…”

“…The utility of phosphinothiol 9 was determined by monitoring its ability to mediate a traceless Staudinger ligation in water with a 13 C NMR assay developed in our laboratory. 17 The major product of the reaction of phosphinothioester 14 with azide 15 was the amine byproduct, glycinamide, which is formed by the reduction of the azido group. The yield of the desired product, amide 16, was strongly dependent on the pH of the reaction mixture, increasing at high pH (Figure 1).…”

“…26 We tested our hypothesis by using intramolecular Coulombic interactions to modulate the acidity of the iminophosphorane intermediate. 27 Specifically, we synthesized bis(pdimethylaminoethylphenyl)phosphinomethanethiol (17), which has dimethyl amino groups that could not only impart water solubility but also (when protonated) serve to discourage protonation of the iminophosphorane nitrogen. We chose tertiary amino groups, rather than primary or secondary ones, to obviate intramolecular S→N acyl transfer in ensuing thioesters.…”

“…Spectral data. 1 17 ; 207 mg, 0.62 mmol) and [2-13 C]-2-azido-acetamide (15) (63 mg, 0.62 mmol) were dissolved in THF (6.5 mL). The resulting solution was allowed to stir for 12 h. The white precipitate that formed was isolated by filtration to give AcGly[ 13 C α ]GlyNH 2 (27) as a white solid in 61% yield.…”

“…The resulting reaction mixtures were stirred for 16 h and the reaction was monitored with our 13 C NMR assay. 17 Production of Met(−1)RNase A-Gly-mxe Intein-Chitin-binding Domain Plasmid pJK01, which directs the expression of the Met(−1)RNase A-Gly-mxe intein-chitinbinding domain fusion protein, 34 was transformed into E. coli BL21(DE3) cells. Luria-Bertani (LB) medium (5 mL) containing ampicillin (0.10 mg/mL) was inoculated with a single colony, and the resulting culture was grown for 16 h at 37 °C.…”