Summary of Study ST001995
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001267. The data can be accessed directly via it's Project DOI: 10.21228/M8XX3Q This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001995 |
| Study Title | Mutasynthetic production and antimicrobial characterisation of Darobactin darobactin analogs (MS analysis) |
| Study Summary | There is great need for therapeutics against multi-drug resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer-membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding for the synthesis of darobactin A can also be found in other γ-proteobacterial families. Therein, the precursor peptides DarB-F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The generated analogs were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for co-crystallization with the target BamA, revealing an identical binding site to darobactin A. Besides its potency, darobactin B did not exhibit cytotoxicity and was slightly more active against Acinetobacter baumanii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotics class, which is likely to expand to several promising therapeutic candidates. |
| Institute | Justus-Liebig-University Giessen |
| Laboratory | Schäberle Laboratory |
| Last Name | Mettal |
| First Name | Ute |
| Address | Ohlebergsweg 12, Gießen, Hesse, 35392, Germany |
| Ute.Mettal@chemie.uni-giessen.de | |
| Phone | +49 641 97219 142 |
| Submit Date | 2021-11-18 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML, d |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-11-21 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Combined analysis:
| Analysis ID | AN003252 | AN003253 | AN003254 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 | Agilent 1290 Infinity | Agilent 1290 Infinity |
| Column | Macherey-Nagel EC 100/2 Nucleoshell C18 2.7μm | Waters Acquity BEH C18 (100 x 2mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | QTOF | QTOF | QTOF |
| MS instrument name | Bruker micrOTOFQ II | Bruker micrOTOFQ II | Bruker maXis II |
| Ion Mode | POSITIVE | POSITIVE | POSITIVE |
| Units | N/A (structure elucidation) | N/A (structure elucidation) | N/A (structure elucidation) |
Chromatography:
| Chromatography ID: | CH002396 |
| Chromatography Summary: | Standard LCMS measurements were performed on a Dionex Ultimate3000 (Thermo Scientific, Darmstadt, Germany) using an EC 100/2 Nucleoshell C18 2.7μm column (Macherey-Nagel, Düren, Germany) HPLC coupled to a micrOTOFQ II (Bruker Daltonics, Bremen, Germany) ESI-qTOF-HRMS. The LC system was run in the following gradient (A:H2O; B: MeOH, ; Flow: 200 μL/min): 0 min: 90% A, 5 min: 90% A, 35min: 0% A, 50min: 0% A, 51min: 90% A, 60 min: 90% A. The injection volume was 5 µL. |
| Methods Filename: | Chromatography Protocol.docx |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Macherey-Nagel EC 100/2 Nucleoshell C18 2.7μm |
| Flow Gradient: | 0 min: 90% A, 5 min: 90% A, 35min: 0% A, 50min: 0% A, 51min: 90% A, 60 min: 90% A |
| Flow Rate: | 200 μL/min |
| Solvent A: | 100% water |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002397 |
| Chromatography Summary: | Alternatively, a setup consisting of an Agilent Infinity 1290 UPLC system (Agilent, Santa Clara, CA, USA) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 100 mm) column and an Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column (2.1 × 5 mm; both columns purchased from Waters, Eschborn, Germany) coupled to a DAD detector and a micrOTOFQ II mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization source was employed. The LC system was operated using a gradient (A: H2O, 0.1% formic acid; B: MeCN, 0.1% formic acid; flow: 600 μL/min): 0 min: 95% A; 0.80 min: 95% A; 18.70 min: 4.75% A; 18.80 min: 0% A; 23.00 min: 0% A; 23.10 min: 95% A; 25.00 min: 95% A and the column oven temperature was set to 45 °C. The injection volume was 5 µL. |
| Methods Filename: | Chromatography Protocol.docx |
| Instrument Name: | Agilent 1290 Infinity |
| Column Name: | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| Column Temperature: | 45 °C |
| Flow Gradient: | 0 min: 95% A; 0.80 min: 95% A; 18.70 min: 4.75% A; 18.80 min: 0% A; 23.00 min: 0% A; 23.10 min: 95% A; 25.00 min: 95% A |
| Flow Rate: | 600 μL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002398 |
| Chromatography Summary: | High accuracy measurements were performed on a 1290 UHPLC system (Agilent, Santa Clara, CA, USA) equipped with DAD, ELSD and maXis II™ (Bruker, Billerica, MA, USA) ESI-qTOF-UHRMS with the following gradient: 0 min: 95% A; 0.30 min: 95% A; 18.00 min: 4.75% A; 18.10 min: 0% A; 22.50 min: 0% A; 22.60 min: 95% A; 25.00 min: 95% A (A: H2O, 0.1% formic acid; B: acetonitrile, 0.1% formic acid; flow: 600 μL/min). The employed column was an Acquity UPLC BEH C18 1.7 μm (2.1 × 100 mm) column with an Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column (2.1 × 5 mm). The column oven temperature was maintained at 45°C. The injection volume was either 1 or 2 µL. |
| Methods Filename: | Chromatography Protocol.docx |
| Instrument Name: | Agilent 1290 Infinity |
| Column Name: | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| Column Temperature: | 45 °C |
| Flow Rate: | 600 μL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
