Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Summary

A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

Abstract

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

Introduction

Peptidoglycan (PG) is a mesh-like polymer that delineates cell shape and structure in both Gram-positive and Gram-negative bacteria^1,2. This heteropolymer is a matrix of amino sugars cross-linked by short peptides^3,4,5,6 with a backbone composed of β-(1,4)-linked alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues (Figure 1)¹. Attached to the C-3 lactyl moiety of MurNAc is the stem peptide. The....

Protocol

1. General methods

NOTE: All compounds were purchased from standard suppliers and used without further purification.

1. Carry out thin-layer chromatography (TLC) on an aluminum plate precoated with silica gel XG F254. Detect spots under a UV lamp, by immersion in p-anisaldehyde stain, or by exposing to I₂ vapor.
2. Record all nuclear magnetic resonance (NMR) spectra on a 400 MHz spectrometer.
   NOTE: ¹H- NMR and ¹³C-NMR spectra were referenced to residual solvent peaks. Coupling constants are given in [Hz] and chemical shifts in [ppm].
3. Record atmospheric....

Results

Masarimycin is a small molecule bacteriostatic inhibitor of B. subtilis and S. pneumoniae and has been shown to inhibit the exo-acting GlcNAcase LytG in B. subtilis^35,37 and target the cell wall in S. pneumoniae³⁷. Masarimycin can be efficiently prepared either by the classical or microwave-assisted organic synthesis with yields in the 55%-70% range. Microwave-assisted synthesis has the advantage of a si.......

Discussion

Masarimycin is a single micromolar bacteriostatic inhibitor of B. subtilis³⁵ and S. pneumoniae³⁷ growth. In B. subtilis, masarimycin has been shown to inhibit the GlcNAcase LytG³⁵, while the precise molecular target in the cell wall of S. pneumoniae has not been identified³⁷. Synthesis of masarimycin using either the classical organic synthesis or microwave procedure provides the inhibitor in good yi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Iodobenzoic acid	SIGMA-ALDRICH	I7675-25G	corrosive, irritant, light yellow to orange-brown powder
2-Propanol	SIGMA-ALDRICH	109827-4L	flammable, irritant, colorless liquid
Acetonitrile	SIGMA-ALDRICH	34851-4L	flammable, irritant, colorless liquid
Aluminum backed silica plates	Sorbtech	4434126	silica gel XG F254 on aluminum backed plates
chloroform-d	SIGMA-ALDRICH	151823-50G	solvent for NMR
Compact Mass Spectrometer	Advion-Interchim	Advion CMS	compact mass spectrometer equiped with APCI source and atmospheric solids analysis probe
Corning Costar 96 well flat bottom plates-sterile	fisher chemical	07-200-90	for synergy/antagonism assays
cover slips	fisher chemical	12-547	for microscopy
Cyclohexanecarboxaldehyde	CHEM-IMPEX INT'L INC.	24451	flammable, irritant, colorless to pink liquid
Cyclohexyl isocyanide	SIGMA-ALDRICH	133302-5G	irritant, colorless liquid, extremly unpleasant odor
Cyclohexylamine	SIGMA-ALDRICH	240648-100ML	corrosive, flammable, irritant, colorless liquid unless contaminated
Ethyl acetate	SIGMA-ALDRICH	537446-4L	flammable, irritant, colorless liquid
flash silica cartridge (12g)	Advion-Interchim	PF-50SIHP-F0012	pack of flash silica columns (12g) for purification of masarimycin
formaldehyde	SIGMA-ALDRICH	F8775-25ML	fixing agent for microscopy
HEPES	SIGMA-ALDRICH	H8651-25G	buffer for microscopy fixing solution
Hexane, mixture of isomers	SIGMA-ALDRICH	178918-4L	environmentally damaging, flammable, irritant, health hazard, colorless liquid
High performance compact mass spectrometer	Advion	expression	Atmospheric Solids Analysis Probe (ASAP), low resolution
High Vac	eppendorf	Vacufuge plus	vacuum aided by centrifugal force and temperature
Hydrochloric acid	SIGMA-ALDRICH	258148-2.5L	corrosive, irritant, colorless liquid
hydrochloric acid	SIGMA-ALDRICH	320331-2.5L	strong acid
immersion oil	fisher chemical	12-365-19	for microscopy
Iodine, resublimed crystals	Alfa Aesar	41955	environmentally damaging, irritant, health hazard, dark grey/purple crystals
Mestre Mnova	MestreLab Research		software for processing NMR spectra
Methanol	SIGMA-ALDRICH	439193-4L	flammable, toxic, health hazard, colorless liquid
methylene blue	SIGMA-ALDRICH	M9140-25G	microscopy stain for staining cell walls
meuller-hinton agar plates + 5% sheep blood	fisher chemical	B21176X	growth media for Streptococcus pneumoniae
meuller-hinton broth	fisher chemical	DF0757-17-6	growth media for Streptococcus pneumoniae
microscope slides	fisher chemical	22-310397	for microscopy
Microwave Synthesis Labstation	MILESTONE	START SYNTH	device that requires the ventilation of a fume hood, equipped with synthesis carousel
NMR tubes	SIGMA-ALDRICH	Z562769-5EA	5mm NMR tubes 600 MHz
Nuclear Magnetic Resonance (NMR)	Bruker	Ascend 400	large superconducting magnet (400MHz)
optochin	fisher chemical	AAB21627MC	ethylhydrocupreine hydrochloride
petrie plates	Celltreat	229695	for preparing agar plates for bacterial growth
Primo Star Bright field/Phase contrast Microscope with ERc5s camera	Zeiss		for morphology studies
puriFlash	interchim	XS520plus	flash chromatography purification system
resazurin	SIGMA-ALDRICH	R7017-1G	for synergy/antagonism assays
Rotary Evaporator	Heidolph	Hei-VAP Value "The Collegiate"	solvent evaporator
Sodium bicarbonate	SIGMA-ALDRICH	S6014-500G	irritant, white powder
Sodium chloride	fisher chemical	S271-1	crystalline, colorless
Sodium chloride	SIGMA-ALDRICH	S5886-500G	for growth of B.subtilis and preparation of LB media
Sodium sulfate	SIGMA-ALDRICH	7985592-500G	anhydrous, granular, white
tryptone	fisher chemical	BP1421-500	for growth of B.subtilis and preparation of LB media
Whitney DG250 Workstation	Microbiology International	DG250	anaerobic workstation. Anaerobic gas mixture used: 5% hydrogen, 10% carbon dioxide, balance nitrogen
yeast extract	fisher chemical	BP1422-500	for growth of B.subtilis and preparation of LB media
Zen Lite (blue) software	Zeiss		for acquiring micrographs