Semisynthesis of the Organoarsenical Antibiotic Arsinothricin

Abstract

Arsinothricin [AST (1)], a new broad-spectrum organoarsenical antibiotic, is a nonproteinogenic analogue of glutamate that effectively inhibits glutamine synthetase. We report the chemical synthesis of an intermediate in the pathway to 1, hydroxyarsinothricin [AST-OH (2)], which can be converted to 1 by enzymatic methylation catalyzed by the ArsM As­(III) S-adenosylmethionine methyltransferase. This is the first report of semisynthesis of 1, providing a source of this novel antibiotic that will be required for future clinical trials.

Chemical synthesis of the organoarsenical antibiotic arsinothricin

“…The mixtures were extracted with CH 2 Cl 2 (3 Â 70 mL) and dried over anhydrous Na 2 SO 4 . The volatiles were evaporated under reduced pressure to give 6a (12.3 g, 92%) as yellowish oil: 1 H NMR (CDCl 3 ) d 1.72 (brs, 1H), 2.04 (s, 3H), 2.50 (t, J ¼ 6.8 Hz, 2H), 4.01 (t, J ¼ 6.8 Hz, 2H); 13 mL, 20.1 g, 0.17 mol) followed by removal of volatiles under reduced pressure provided dichloro-(2-hydroxyethyl)arsine (8.7 g, 65%) as brown liquid with the 1 H and 13 C NMR spectra as reported 10 (HRMS m/z calcd for C 2 H 4 AsOCl 2 [M À H] À 188.8860, found 188.8862) instead of desired chloro(2chloroethyl)(methyl)arsine 6b.…”

“…N-[1-Carboxy-3-(hydroxyl(methyl)arsinoyl)propan-1-yl]-N,N,N-trimethylammonium hydroxide (10) (a) Reduction. AST-OH 10 (2; 50 mg, 0.22 mmol) was dissolved in the mixture of concentrated HCl and water (1 : 1, 5 mL).…”

“…Arsenic species were determined by HPLC-ICP-MS using the HPLC retention time of known standards as described previously. 10 Assay of antibiotic activity. Single colonies of E. coli W3110 were inoculated in M9 medium (47.7 mM Na 2 HPO 4 , 22 mM…”

“…Inhibition of GS activity by bAST, sAST, cAST or L-AST was analyzed by a coupled assay using GS from E. coli (Millipore-Sigma), as described previously 9 with minor modications. Briey, the GS reaction was initiated by addition of L-glutamate (5,10,20 or 50 mM) to the reaction mixture (100 mM Tris-acetate (pH 8.6), 9 mM ATP, 1 mM phosphoenolpyruvate, 60 mM Mg 2 Cl, 19 mM KCl, 45 mM NH 4 Cl, 0.25 mM NADH, 13-20 units of L-lactic dehydrogenase, 8-14 units of pyruvate kinase and 1 unit of GS) and incubated at 37 C in the absence or presence of 0.5 or 2.0 mM of bAST, sAST, cAST or L-AST and the decrease in A 340 nm was monitored to quantify oxidation of NADH to NAD + using an extinction coefficient 6230 M À1 cm À1 . Activities were corrected with the values from control assays without enzyme.…”

Fluorescent 5-Pyrimidine and 8-Purine Nucleosides Modified with an N-Unsubstituted 1,2,3-Triazol-4-yl Moiety

“…30,31 Transition-metal-catalyzed cycloaddition of TMSN 3 with alkyne has been extended into the 5-ethynyl pyrimidine nucleosides 4a-d (methods A-C, Table 2). Thus, CuI-catalyzed cycloaddition of 3′,5′-di-O-acetyl-5-ethynyl-2′-deoxycytidine 4a 32 yielded 5-(1H-1,2,3triazol-4-yl) product 5a (method A; 45%, entry 1), while unprotected 2′-deoxycytidine substrate 4b 32 provided 5-(1H-1,2,3-triazol-4-yl)-2′-deoxycyti-dine 5b entry 2). Protected 4c 33 and unprotected 5-ethynyl-2′-deoxyuridine 4d 33 gave 5-(1H-1,2,3-triazol-4yl) products 5c (65%, entry 3) and 5d 62%,entry 4).…”