Enzymatic Glycosylation of Vancomycin Aglycon: Completion of a Total Synthesis of Vancomycin and N- and C-Terminus Substituent Effects of the Aglycon Substrate

Abstract

Studies on the further development of the sequential glycosylations of the vancomycin aglycon catalyzed by the glycosyltransferases GtfE and GtfD and the observation of unusual, perhaps unexpected, aglycon substrate substituent effects on the rate and efficiency of the initial glycosylation reaction are reported.

Total Syntheses and Initial Evaluation of [Ψ[C(═S)NH]Tpg4]vancomycin, [Ψ[C(═NH)NH]Tpg4]vancomycin, [Ψ[CH2NH]Tpg4]vancomycin, and Their (4-Chlorobiphenyl)methyl Derivatives: Synergistic Binding Pocket and Peripheral Modifications for the Glycopeptide Antibiotics

“…As a consequence, the sequential glycosylations of the modified aglycon derivatives were examined alongside the vancomycin aglycon and its C-terminus hydroxymethyl derivative using the enzymatic approach. 47 The recombinant glycosyltranferases GtfE and GtfD from a vancomycin producing strain of A. orientalis (ATCC19795) were expressed in E. coli from the corresponding constructs 43a and were purified to homogeneity (His 6 tag). Notably and although the endogenous glycosyl donors for both enzymes are the TDP-sugars, 43 UDP-sugars have been shown to be as effective co-substrates for both enzymes.…”

“…47 The UDP-vancosamine, possessing the required β-anomer stereochemistry, was prepared by a procedure described by Kahne 49 to access TDP-vancosamine with modifications to the synthetic route that incorporate uridine versus thymidine. 47 With use of the purified enzymes and the synthetic glycosyl donors UDP-glucose 50 (for GtfE) and UDP-vancosamine 47 (for GtfD), conditions were optimized for the two sequential glycosylations of vancomycin aglycon ( 2 ) as well as its C-terminus hydroxymethyl derivative 15 . 47 Of the two glycosylation reactions, the initial GtfE-catalyzed incorporation of glucose using UDP-glucose exhibited the greatest aglycon substrate sensitivity and those bearing a C-terminus hydroxymethyl group were established to be much less effective than the corresponding carboxylic acids.…”

“…47 With use of the purified enzymes and the synthetic glycosyl donors UDP-glucose 50 (for GtfE) and UDP-vancosamine 47 (for GtfD), conditions were optimized for the two sequential glycosylations of vancomycin aglycon ( 2 ) as well as its C-terminus hydroxymethyl derivative 15 . 47 Of the two glycosylation reactions, the initial GtfE-catalyzed incorporation of glucose using UDP-glucose exhibited the greatest aglycon substrate sensitivity and those bearing a C-terminus hydroxymethyl group were established to be much less effective than the corresponding carboxylic acids. Our previously reported optimization efforts focused on this glycosylation reaction and examined simultaneously both the vancomycin aglycon ( 2 ) and substrate 15 (37 °C).…”

Peripheral modifications of [Ψ[CH 2 NH]Tpg 4 ]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics

“…The approach used herein, which we suggest represents a case of durable antibiotic discovery by design, relied on the total synthesis of the candidate antibiotics (58)(59)(60) to obtain the previously inaccessible compounds. Although not highlighted in the preceding discussion, the total synthesis of the starting pocketmodified aglycon(s) (26 steps) (48), enzymatic installation of the disaccharide (2 steps) (58), and subsequent addition of the two peripheral modifications (2 steps) represent remarkable accomplishments in their own right.…”

“…Although not highlighted in the preceding discussion, the total synthesis of the starting pocketmodified aglycon(s) (26 steps) (48), enzymatic installation of the disaccharide (2 steps) (58), and subsequent addition of the two peripheral modifications (2 steps) represent remarkable accomplishments in their own right. Finally, the work herein was conducted with the aminomethylene analog of vancomycin, in which the residue 4 amide carbonyl was removed.…”