Gram-scale total synthesis of teixobactin promoting binding mode study and discovery of more potent antibiotics

Abstract

Teixobactin represents a new class of antibiotics with novel structure and excellent activity against Gram-positive pathogens and Mycobacterium tuberculosis. Herein, we report a one-pot reaction to conveniently construct the key building block L-allo-Enduracidine in 30-gram scale in just one hour and a convergent strategy (3 + 2 + 6) to accomplish a gram-scale total synthesis of teixobactin. Several analogs are described, with 20 and 26 identified as the most efficacious analogs with 3~8-fold and 2~4-fold greater potency against vancomycin resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus respectively in comparison with teixobactin. In addition, they show high efficiency in Streptococcus pneumoniae septicemia mouse model and neutropenic mouse thigh infection model using methicillin-resistant Staphylococcus aureus. We also propose that the antiparallel β-sheet of teixobactin is important for its bioactivity and an antiparallel dimer of teixobactin is the minimal binding unit for lipid II via key amino acids variations and molecular docking.

Fig. 1

Current challenges and retrosynthetic analysis of teixobactin. There are three challenges in scalable total synthesis of teixobactin. Teixobactin could be synthesized via the convergent strategy (3 + 2 + 6)

Fig. 2

Synthetic strategy to l-allo-enduracididine. There are three reported routes to provide End. Our synthetic route just takes 1 h via a one-pot reaction to synthesize desired End

Fig. 3

¹H NMR spectrum of intermediates 5. a NOE and ¹H NMR for intermediates 5a. The red arrow indicates the peaks of intermediate 5a-1. The black arrow indicates the peaks of intermediate 5a-2. The blue arrow indicates the peaks of intermediate 5a-3. b MS data for intermediates 5a (LC–MS analysis using MeOH as a solvent)

Fig. 4

Gram-scale total synthesis of teixobactin. a (1) 4 equiv. I₂, 5 equiv. NaHCO₃, 0 °C, 1 h; (2) MeOH/AcOH (9:1), 30 °C, 15 min, 66% for 2 steps; b (1) 3 equiv. LiOH, THF/H₂O; (2) Pd(OH)₂/C, H₂, MeOH/AcOH (9:1). c 3 equiv. CbzOSu, 4 equiv. DIEA, DCM, 30 °C, 4 h, 80%; d (1) 30 % TFA, 30 °C, 30 min; (2) 1.2 equiv. Boc-Ser(tBu)-OH, 1.2 equiv. HATU, 3 equiv. DIEA, DCM/DMF, 30 °C, 3 h; e (1) 1.5 equiv. Fmoc-Ile-OH, 1.5 equiv. EDCI, 0.2 equiv. DMAP, DCM, 12 h; (2) 33% Et₂NH, 30 °C, 15 min; 79%; f (1) 33% Et₂NH, r.t., 10 min; (2) 3 equiv. Alloc-Ala-OH, 3 equiv. HATU, 3 equiv. DIEA, DCM/DMF, 30 °C, 3 h, 71%; g 2 equiv. LiOH, THF/H₂O (3:1), 0 °C, 4 min; h 2.5 equiv. compound 12, 2 equiv. DEPBT, 2.5 equiv. DIEA, THF/DMF, 30 °C, overnight, 63%; i (1) 0.3 equiv. Pd(PPh₃)₄, 2 equiv. 1,3-dimethylbarbituric acid, DCM, 30 °C, 1 h; (2) 4 equiv. HATU, 4 equiv. HOAT, 8 equiv. DIEA, DCM/DMF, 30 °C, 24 h, 58%; j (1) 3 M HCl, 15 min; (2) 1 equiv. compound 17, 1.2 equiv. DEPBT, 1.2 equiv. DIEA, DMF,12 h; k (1) Pd(OH)₂/C, H₂, MeOH/HCOOH, 1 h; (2) TFA:TIPS:H₂O: 95:2.5:2.5. 31% from compound 16

Fig. 5

MIC (µg ml⁻¹) for VRE TH4938. a Teixobactin was provided by Novobiotic Pharmaceuticals. b Teixobactin was synthesized via 5 + 6 strategy. This assay was conducted three times (n = 3). Source data are provided as a Source Data file

Fig. 6

Time-dependent killing assay and the in vivo assay of Compounds 20 and 26. a Time-dependent killing of VRE ATCC 29212 by teixobactin and Compound 20. This assay was done twice to confirm the results (n = 2). Data represent two independent experiments ± s.d. Source data are provided as a Source Data file. b Single dose treatment (i.v., 1 h post infection, six female mice per group) in septicemia protection model using S. pneumoniae D39. Survival was depicted 48 h after infection. ***P < 0.001 (determined by nonparametric log-rank test). Source data are provided as a Source Data file. c BALB/c female mice were infected with bioluminescent S. pneumoniae Xen-10 (A66). At left, two mice were treated with compound 20 (2 mg kg⁻¹) and the right two mice were negative control. After 48 h, four mice were using IVIS Lumina II. d Single dose (i.v., 2 h post infection, three mice per group) treatment with compound 20 and vancomycin in neutropenic mouse thigh infection model using MRSA 33591. For drug-treated animals, thigh colony-forming units (c.f.u.) were determined at 26 h post infection. For controls, c.f.u. in thighs was determined at 0, 2, and 26 h post infection. Source data are provided as a Source Data file. Source data are provided as a Source Data file

Fig. 7

Exploring the relationship between antiparallel β-sheet of teixobactin and its bioactivity. a Model of b sheet formation of teixobactin and site mutations; b site variations to abolish intermolecular H-bond interaction. c CD spectrum of teixobactin and analogs 27 and 28; d MIC for MRSA (BAA 1695)

Fig. 8

Docking structure of compound 26 and truncated lipid II. a Structure of lipid I, lipid II, C55-PP, and truncated lipid II; b binding model of teixobactin assembly and lipid II; c detailed binding mode between dimer of analog 26 and truncated lipid II; step 1: protein preparation (dimer of compound 26); step 2: ligand preparation (truncated lipid II); step 3: receptor grid generation; step 4: ligand docking with Schrödinger Suites Release 2017-1. d Summary of SAR of teixobactin analog 26