Total Synthesis of Mutanobactins A, B from the Human Microbiome: Macrocyclization and Thiazepanone Assembly in a Single Step

Abstract We report the first total syntheses of tricyclic mutanobactins A and B, lipopeptides incorporating a thiazepanone, isolated from Streptococcus mutans, a member of the human oral microbiome. A rapid, solid‐phase peptide synthesis (SPPS) based route delivers these natural products from a cascade of cyclization reactions. This versatile process was also employed in a streamlined synthesis of mutanobactin D. Additionally, we provide an independent synthesis of a truncated mutanobactin A analog, utilizing a novel thiazepanone amino acid building block.

(R)-2-(benzoylthio)-4-(tert-butoxy)-4-oxobutanoic acid (7) To a suspension of K2CO3 (5.46 g, 39.5 mmol, 0.57 equiv.) in methanol (80 mL) was added thiobenzoic acid (9.33 mL, 79.0 mmol, 1.14 equiv.) at a rate that sustains gentle bubbling. After bubbling has subsided (60 min) the resulting yellow solution was carefully concentrated at reduced pressure. The solid residue was dissolved in DMF (80 mL) and cooled to -40 °C. A solution of bromide 6 (17.5 g, 69.2 mmol, 1.00 equiv.) in DMF (40 mL) was added dropwise. The reaction was allowed to slowly reach room temperature and stirred for two days. After concentration at 60 °C the residue was dissolved in ethyl acetate and water, the phases separated, and the aqueous one extracted twice with additional ethyl acetate. The combined organic phases were washed twice with water and once with sat. brine, dried over MgSO4 and concentrated to give the title compound (21.9 g, quant.) as a yellow syrup, which was used without further purification.
This oil was dissolved in ethanol (9 mL) and hydrazine hydrate (0.105 mL, 2.15 mmol, 2.30 equiv.) was added. After refluxing for 3.5 h and cooling to -20 °C, white precipitate was filtered off and washed with cold ethanol. Concentration of the filtrate at reduced pressure gave 493 mg light-yellow solid.
After stirring for 18 h, LC-MS indicated full consumption of starting material. 10% citric acid and ethyl acetate were added, and the layers separated. The aqueous layer was extracted twice more with ethyl acetate, the combined organic layers dried over MgSO4, and concentrated at reduced pressure to give 55.0 mg of a colorless solid.
The solid was dissolved in acetic acid (2 mL). 0.4 mL 30% HBr in acetic acid was added dropwise. After 90 min, the reaction mixture was added dropwise to 100 mL ice-cold ether. After aging at -20 °C for 1 h and centrifugation (4000 rpm, 10 min), the ether layer was decanted to leave a brown solid (64.7 mg).

Standard Fmoc-SPPS-protocol
Fmoc-Loading determination: According to Dong et al [2] , x mg (typically 12 -18 mg) of washed and thoroughly dried resin were treated with 2 mL of 2 vol% DBU in DMF and the suspension shaken for 15 min. Afterwards, 48 μL of the supernatant were diluted with 2952 μL acetonitrile. The relative absorbance Abs304 of this solution was measured at 304 nm against a blank sample (48 μL of 2 vol% DBU in DMF diluted with 2952 μL acetonitrile). The Fmoc loading was determined according to: Fmoc cleavage: The resin was treated with a mixture of 20% piperidine in DMF (1 × 5 min, 1 x 10 min). Subsequently, it was washed with DMF, CH2Cl2, and ether.
HATU coupling: The corresponding carboxylic acid (3.0 equiv), HATU (3.0 equiv) and DIPEA (4.0 equiv) were dissolved in DMF. The solution was aged for 3 min and then added to the resin. The suspension was shaken for 20 min (40 min in case of D-Ala), after which the solution was drained. The resin was washed thoroughly with DMF, CH2Cl2, and ether. Completion of coupling was monitored with the Kaiser-test.
Cleavage from resin: The thoroughly washed and dried resin was treated with 20 mL per g resin of either cocktail A (TFA-waterthioanisole, 95:4:1 by volume) or cocktail B (TFA-water, 95:5 by volume) for 15 min. Then, the suspension was drained, and the cleavage cocktail treatment was repeated twice more.