Total Synthesis and Evaluation of [Ψ[CH₂NH]Tpg⁴]Vancomycin Aglycon:  Reengineering Vancomycin for Dual d-Ala-d-Ala and d-Ala-d-Lac Binding

Abstract

An effective synthesis of [Ψ[CH 2 NH]Tpg 4 ]vancomycin aglycon (5) is detailed in which the residue 4 amide carbonyl of vancomycin aglycon has been replaced with a methylene. This removal of a single atom was conducted to enhance binding to D-Ala-D-Lac countering resistance endowed to bacteria that remodel their D-Ala-D-Ala peptidoglycan cell wall precursor by a similar single atom change (ester O for amide NH). Key elements of the approach include a synthesis of the modified vancomycin ABCD ring system featuring a reductive amination coupling of residues 4 and 5 for installation of the deep-seated amide modification, the first of two diaryl ether closures for formation of the modified CD ring system (76%, 2.5-3:1 kinetic atropodiastereoselectivity), a Suzuki coupling for installation of the hindered AB biaryl bond (90%) on which the atropisomer stereochemistry could be thermally adjusted, and a macrolactamization closure of the AB ring system (70%). Subsequent DE ring system introduction enlisted a room temperature aromatic nucleophilic substitution reaction for formation of the remaining diaryl ether (86%, 6-7:1 kinetic atropodiastereoselectivity) completing the carbon skeleton of 5. Consistent with expectations and relative to the vancomycin aglycon, 5 exhibited a 40-fold increase in affinity for D-Ala-D-Lac (K a = 5.2 × 10 3 M −1 ) and a 35-fold reduction in affinity for D-Ala-D-Ala (K a = 4.8 × 10 3 M −1 ) providing a glycopeptide analogue with balanced, dual binding characteristics. Beautifully, 5 exhibited antimicrobial activity (MIC = 31 μg/mL) against a VanA resistant organism that remodels its D-Ala-D-Ala cell wall precursor to D-Ala-D-Lac upon glycopeptide antibiotic challenge displaying a potency that reflects these binding characteristics.The most common strains of enterococci resistant to vancomycin (1), VanA and VanB, possess an inducible resistance pathway in which the terminal dipeptide of the cell wall peptidoglycan precursor is modified from D-Ala-D-Ala to D-Ala-D-Lac. 1 Binding of the antibiotic to this modified ligand is reduced 1000-fold leading to a 1000-fold drop in antimicrobial activity. 1d,k In a recent disclosure, 2 we provided the first experimental study on the origin of this loss in binding affinity, partitioning the effect into lost H-bond and repulsive lone pair contributions, Figure 1. Thus, the binding affinity of vancomycin for 3, which incorporates a methylene (CH 2 ) in place of the linking amide NH of Ac 2 -L-Lys-D-Ala-D-Ala, was compared with that of Ac 2 -L-Lys-D-Ala-D-Ala (2) and Ac 2 -L-Lys-D-Ala-D-Lac (4). The vancomycin affinity for 3 was approximately 10-fold less than that of 2, but 100-fold greater than that of 4. This indicated that the reduced binding affinity of 4 (4.1 kcal/mol) may be attributed to both the loss of a key H-bond and a destabilizing lone pair/lone pair interaction introduced with the ester oxygen of 4 (2.6 kcal/mol) with the latter, not the H-bond, being responsible for the greater share (100-fold) of the 1000-fold binding reduction. The...

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

“…In a series of studies, we reported the first vancomycin analogs that contain changes at a key single-atom site in its target binding pocket (residue 4 carbonyl O → S, NH, H 2 ), the latter two of which were designed to directly address this underlying molecular basis of resistance to vancomycin ( Fig. 1) (43)(44)(45)(46)(47)(48)(49)(50). These rationally designed binding pocket modifications reinstated binding to the altered target D-Ala-D-Lac and maintained binding affinity for the unaltered target D-Ala-D-Ala.…”

The race for antimicrobials in the multidrug resistance era

“…The conversion of D ‐Ala‐ D ‐Ala to D ‐Ala‐ D ‐Lac in the peptidoglycan termini is one of the mutations that provide resistance to native vancomycin, however, chemical modifications directed to key single‐atom sites in the binding pocket of vancomycin seem to enable the antibiotic to bind both the modified and the unaltered targets in the peptidoglycan (Crowley and Boger, 2006; Xie et al ., 2012; Okano et al ., 2015). Peripheral structural modifications, such as a (4‐chlorobiphenyl) methyl (CBP) group, increased the antimicrobial potency of vancomycin and provided, with multiple synergistic mechanisms of action, a delay in the emergence of resistance (Okano et al ., 2014, 2015).…”

Importance of the Structure of Vancomycin Binding Pocket in Designing Compounds Active Against Vancomycin-resistant Enterococci (VRE)

“…Fig. 1) to avoid the unfavorable electrostatic interaction with the modified peptidoglycan terminal D-Ala-D-Lac [20,21]; b) the Nterminal was elongated in order to introduce an additional hydrogen-bonding with D-Ala-D-Lac [22]. In order to explore the possible conformational effect, hence the relative three-dimensional orientation of NHs, compound 2 and 3 having D-Leucine (found in vancomycin), and D-leucine, respectively, were synthesized; the glycine was attached to leucine terminal in order to incorporate depicted in the Fig.…”