Name: synthesis of masarimycin, a small molecule inhibitor of gram-positive bacterial growth
Uploaded: 2022-01-07T14:30:00
Description: Watch this Scientific Journal Video about Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth at JoVE.com

Research was supported by the National Science Foundation under grant number 2009522. NMR analysis of masarimycin was supported by the National Science Foundation major research instrumentation program award under grant number 1919644. Any opinions, findings, and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

1. General methods
NOTE: All compounds were purchased from standard suppliers and used without further purification.
<ol>
	<li>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.</li>
	<li>Record all nuclear magnetic resonance (NMR) spectra on a 400 MHz spectrometer. 
	NOTE: 1H- NMR and 13...

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...

Peptidoglycan (PG) is a mesh-like polymer that delineates cell shape and structure in both Gram-positive and Gram-negative bacteria1,2. This heteropolymer is a matrix of amino sugars cross-linked by short peptides3,4,5,6 with a backbone composed of &#946;-(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 metabolism of PG involves a tightly coordinated system of biosynthetic and degradative enzymes to incorporate new material into the cell wall7,8. Degradation of PG is carried out by enzymes collectively referred to as autolysins9 and further classified based on the specificity of the bond cleaved. Autolysins participate in many cellular processes including cell growth, cell division, motility, PG maturation, chemotaxis, protein secretion, genetic competence, differentiation, and pathogenicity10,11. Unraveling the specific biological functions of individual autolysins can be daunting, due in part to functional redundancy. However, recent biophysical8,12,13 and computational studies12 have provided new insight into their roles in PG metabolism. In addition, recent reports have provided further insight into the synthesis14 and membrane-mediated15,16,17 steps in PG metabolism. A thorough understanding of the relationship between degradative and synthetic pathways of PG metabolism could give rise to previously untapped antibiotic targets.
While there have been significant advances in methodology to study glycobiology in eukaryotes, bacterial glycobiology and, in particular, PG metabolism has not advanced at a similar rate. Current chemical approaches to study PG metabolism include fluorescently labeled antibiotics18, fluorescent probes19,20, and metabolic labeling21,22,23,24. These new approaches are providing new ways to interrogate bacterial cell wall metabolism. While some of these strategies are capable of labeling PG in vivo, they can be species-specific19, or only work in strains lacking a particular autolysin25. Many PG labeling strategies are intended for use with isolated cell walls26 or with in vitro reconstituted PG biosynthesis pathways20,27,28. The use of fluorescently labeled antibiotics is currently limited to biosynthetic steps and transpeptidation18.
The current knowledge of bacterial autolysins and their role in cell wall metabolism comes from genetic and in vitro biochemical analysis11,29,30,31,32. While these approaches have provided a wealth of information on this important class of enzymes, deciphering their biological role can be challenging. For instance, due to functional redundancy33, deletion of an autolysin in most cases does not result in halting bacterial growth. This is despite their implied role in cell growth and division7,12. Another complication is that genetic deletion of bacterial autolysins can give rise to meta-phenotypes34. Meta-phenotypes arise from the complex interplay between the pathway affected by the genetic deletion and other interconnected pathways. For instance, a meta-phenotype can arise via a direct effect such as the lack of an enzyme, or an indirect effect such as a disruption of regulators.
Currently, there are only a few inhibitors of glycosidase autolysins such as N-acetylglucosaminidases (GlcNAcase) and N-acetylmuramidases, which can be used as chemical probes to study the degradation of PG. To address this, the diamide masarimycin (previously termed as fgkc) has been identified and characterized35 as a bacteriostatic inhibitor of Bacillus subtilis growth that targets the GlcNAcase LytG32 (Figure 1). LytG is an exo-acting GlcNAcase36, a member of cluster 2 within glycosyl hydrolase family 73 (GH73). It is the major active GlcNAcase during vegetative growth32. To our knowledge, masarimycin is the first inhibitor of a PG-acting GlcNAcase that inhibits cellular growth. Additional studies of masarimycin with Streptococcus pneumoniae found that masarimycin likely inhibits cell wall metabolism in this organism37. Here, the preparation of masarimycin is reported for use as a chemical biology probe to study physiology in the Gram-positive organisms B. subtilis, and S. pneumoniae. Examples of morphological analysis of sub-minimum inhibitory concentration treatment with masarimycin, as well as a synergy/antagonism assay are presented. Synergy and antagonism assays using antibiotics with well-defined modes of action can be a useful way to explore connections between cellular processes38,39,40.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Iodobenzoic acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>I7675-25G</td>
			<td>corrosive, irritant, light yellow to orange-brown powder</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>109827-4L</td>
			<td>flammable, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>SIGMA-ALDRICH</td>
			<td>34851-4L</td>
			<td>flammable, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>Aluminum backed silica plates</td>
			<td>Sorbtech</td>
			<td>4434126</td>
			<td>silica gel XG F254 on aluminum backed plates</td>
		</tr>
		<tr>
			<td>chloroform-d</td>
			<td>SIGMA-ALDRICH</td>
			<td>151823-50G</td>
			<td>solvent for NMR</td>
		</tr>
		<tr>
			<td>Compact Mass Spectrometer</td>
			<td>Advion-Interchim</td>
			<td>Advion CMS</td>
			<td>compact mass spectrometer equiped with APCI source and atmospheric solids analysis probe</td>
		</tr>
		<tr>
			<td>Corning Costar 96 well flat bottom plates-sterile</td>
			<td>fisher chemical</td>
			<td>07-200-90</td>
			<td>for synergy/antagonism assays</td>
		</tr>
		<tr>
			<td>cover slips</td>
			<td>fisher chemical</td>
			<td>12-547</td>
			<td>for microscopy</td>
		</tr>
		<tr>
			<td>Cyclohexanecarboxaldehyde</td>
			<td>CHEM-IMPEX INT&#39;L INC.</td>
			<td>24451</td>
			<td>flammable, irritant, colorless to pink liquid</td>
		</tr>
		<tr>
			<td>Cyclohexyl isocyanide</td>
			<td>SIGMA-ALDRICH</td>
			<td>133302-5G</td>
			<td>irritant, colorless liquid, extremly unpleasant odor</td>
		</tr>
		<tr>
			<td>Cyclohexylamine</td>
			<td>SIGMA-ALDRICH</td>
			<td>240648-100ML</td>
			<td>corrosive, flammable, irritant, colorless liquid unless contaminated</td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>SIGMA-ALDRICH</td>
			<td>537446-4L</td>
			<td>flammable, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>flash silica cartridge (12g)</td>
			<td>Advion-Interchim</td>
			<td>PF-50SIHP-F0012</td>
			<td>pack of flash silica columns (12g) for purification of masarimycin</td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>F8775-25ML</td>
			<td>fixing agent for microscopy</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA-ALDRICH</td>
			<td>H8651-25G</td>
			<td>buffer for microscopy fixing solution</td>
		</tr>
		<tr>
			<td>Hexane, mixture of isomers</td>
			<td>SIGMA-ALDRICH</td>
			<td>178918-4L</td>
			<td>environmentally damaging, flammable, irritant, health hazard, colorless liquid</td>
		</tr>
		<tr>
			<td>High performance compact mass spectrometer</td>
			<td>Advion</td>
			<td>expression</td>
			<td>Atmospheric Solids Analysis Probe (ASAP), low resolution</td>
		</tr>
		<tr>
			<td>High Vac</td>
			<td>eppendorf</td>
			<td>Vacufuge plus</td>
			<td>vacuum aided by centrifugal force and temperature</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>258148-2.5L</td>
			<td>corrosive, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>320331-2.5L</td>
			<td>strong acid</td>
		</tr>
		<tr>
			<td>immersion oil</td>
			<td>fisher chemical</td>
			<td>12-365-19</td>
			<td>for microscopy</td>
		</tr>
		<tr>
			<td>Iodine, resublimed crystals</td>
			<td>Alfa Aesar</td>
			<td>41955</td>
			<td>environmentally damaging, irritant, health hazard, dark grey/purple crystals</td>
		</tr>
		<tr>
			<td>Mestre Mnova</td>
			<td>MestreLab Research</td>
			<td></td>
			<td>software for processing NMR spectra</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>439193-4L</td>
			<td>flammable, toxic, health hazard, colorless liquid</td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>SIGMA-ALDRICH</td>
			<td>M9140-25G</td>
			<td>microscopy stain for staining cell walls</td>
		</tr>
		<tr>
			<td>meuller-hinton agar plates + 5% sheep blood</td>
			<td>fisher chemical</td>
			<td>B21176X</td>
			<td>growth media for Streptococcus pneumoniae</td>
		</tr>
		<tr>
			<td>meuller-hinton broth</td>
			<td>fisher chemical</td>
			<td>DF0757-17-6</td>
			<td>growth media for Streptococcus pneumoniae</td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>fisher chemical</td>
			<td>22-310397</td>
			<td>for microscopy</td>
		</tr>
		<tr>
			<td>Microwave Synthesis Labstation</td>
			<td>MILESTONE</td>
			<td>START SYNTH</td>
			<td>device that requires the ventilation of a fume hood, equipped with synthesis carousel</td>
		</tr>
		<tr>
			<td>NMR tubes</td>
			<td>SIGMA-ALDRICH</td>
			<td>Z562769-5EA</td>
			<td>5mm NMR tubes 600 MHz</td>
		</tr>
		<tr>
			<td>Nuclear Magnetic Resonance (NMR)</td>
			<td>Bruker</td>
			<td>Ascend 400</td>
			<td>large superconducting magnet (400MHz)</td>
		</tr>
		<tr>
			<td>optochin</td>
			<td>fisher chemical</td>
			<td>AAB21627MC</td>
			<td>ethylhydrocupreine hydrochloride</td>
		</tr>
		<tr>
			<td>petrie plates</td>
			<td>Celltreat</td>
			<td>229695</td>
			<td>for preparing agar plates for bacterial growth</td>
		</tr>
		<tr>
			<td>Primo Star Bright field/Phase contrast Microscope with ERc5s camera</td>
			<td>Zeiss</td>
			<td></td>
			<td>for morphology studies</td>
		</tr>
		<tr>
			<td>puriFlash</td>
			<td>interchim</td>
			<td>XS520plus</td>
			<td>flash chromatography purification system</td>
		</tr>
		<tr>
			<td>resazurin</td>
			<td>SIGMA-ALDRICH</td>
			<td>R7017-1G</td>
			<td>for synergy/antagonism assays</td>
		</tr>
		<tr>
			<td>Rotary Evaporator</td>
			<td>Heidolph</td>
			<td>Hei-VAP Value &#34;The Collegiate&#34;</td>
			<td>solvent evaporator</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>SIGMA-ALDRICH</td>
			<td>S6014-500G</td>
			<td>irritant, white powder</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>fisher chemical</td>
			<td>S271-1</td>
			<td>crystalline, colorless</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SIGMA-ALDRICH</td>
			<td>S5886-500G</td>
			<td>for growth of B.subtilis and preparation of LB media</td>
		</tr>
		<tr>
			<td>Sodium sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>7985592-500G</td>
			<td>anhydrous, granular, white</td>
		</tr>
		<tr>
			<td>tryptone</td>
			<td>fisher chemical</td>
			<td>BP1421-500</td>
			<td>for growth of B.subtilis and preparation of LB media</td>
		</tr>
		<tr>
			<td>Whitney DG250 Workstation</td>
			<td>Microbiology International</td>
			<td>DG250</td>
			<td>anaerobic workstation. Anaerobic gas mixture used: 5% hydrogen, 10% carbon dioxide, balance nitrogen</td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>fisher chemical</td>
			<td>BP1422-500</td>
			<td>for growth of B.subtilis and preparation of LB media</td>
		</tr>
		<tr>
			<td>Zen Lite (blue) software</td>
			<td>Zeiss</td>
			<td></td>
			<td>for acquiring micrographs</td>
		</tr>
	</tbody>
</table>

synthesis of masarimycin, a small molecule inhibitor of gram-positive bacterial growth

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.

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. subtilis35,37 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...

Watch this Scientific Journal Video about Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth at JoVE.com

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

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.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...

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