Orfamide D – G

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Introduction

Orfamides D through G are cyclic lipodepsipeptides produced by non-ribosomal peptide synthetases. They were first extracted as a minor compound from Pseudomonas sp. CMR5c which produces orfamide B as major compound. The orfamides D – G belong to a group of structurally similar CLiPs called the Orfamide group. This group also contains orfamide A (Gross, 2007), orfamide B (Ma, 2016), orfamide H (Ma, 2019), orfamides J – M (De Roo, 2022), poaeamides A and B (Zachow, 2015, Nguyen, 2016) and the PPZPMs (Weisshoff, 2014).

Original publicationOrfamide D – E: D’Aes, 2014
Orfamide F – G: Ma, 2016
Original sourceOrfamide D – E: Pseudomonas sessilinigenes CMR12a (minor compounds)
Orfamide D – G: Pseudomonas aestus CMR5c (minor compounds)
Other known sources (non-putative)n.a.
Stereochemistry determined byn.a.

Chemical properties

CASn.a.
Molecular formulaOrfamide D: C61H108N10O17
Orfamide E: C63H110N10O17
Orfamide F: C65H114N10O17
Orfamide G: C65H116N10O17
Molecular weightOrfamide D: 1253.6 g/mol
Orfamide E: 1279.6 g/mol
Orfamide F: 1307.7 g/mol
Orfamide G: 1309.7 g/mol
Mono-isotopic massOrfamide D: 1252.7894 Da
Orfamide E: 1278.8050 Da
Orfamide F: 1306.8363 Da
Orfamide G: 1308.8520 Da
SolubilitySoluble in methanol, ethanol, DMF, DMSO, acetonitrile
CMCn.a.
3D conformationn.a.
NMR data available in literatureDMF-d7 (De Roo, 2022)

Chemical structure

Analysis of the structures of orfamide D – G was performed by NMR spectroscopy and mass spectrometry. (D’aes, 2014; Ma, 2016) The structure of orfamide D is 3-OH C12:0 – Leu1 – Glu2 – Thr3 – Val4 – Leu5 – Ser6 – Leu7 – Leu8 – Ser9 – Val10 and is cyclized by means of an ester bond between the C-terminal carbonyl and the hydroxyl side chain of Thr3. Orfamide E and F both feature an unsaturation in their fatty acid tail, having a 3-OH C14:1 or 3-OH C16:1 fatty acid. Orfamide G features a 3-OH C16:0 fatty acid tail. Consequently, these CLiPs all differ from orfamide B only at their fatty acid tail.

Schematic sequences of orfamides D through G, where shapes indicate amino acid configuration (circles = L-AA, squares = D-AA) and colors indicate amino acid polarity (green = hydrophobic, red = polar)

NMR fingerprint data

Recently, it was established that the planar structure and stereochemistry of CLiPs can be assessed by simple comparison to a reference. (De Roo, 2022) More specifically, by matching NMR spectra of a CLiP from a newly isolated bacterial source with those of existing (reference) CLiPs, one can determine whether they are identical or not. A detailed explanation on what NMR fingerprint matching is, and how to use it, can be found here.

Below, we provide the reference NMR data of orfamides D – G in various formats. This data is recorded in acetonitrile-d3 at room temperature, and can be used to asses similarities of newly isolated CLiPs to orfamide D – G. ATTENTION: The solvent of choice for NMR reference spectra is DMF-d7. However, since no spectra are available in this solvent, we provide spectra in acetonitrile-d3. If you wish to compare spectra of your own CLiP to those of orfamides D – G, please use identical conditions!

Please note that orfamides D – G have an identical peptide portion compared to orfamide B. So, be sure to check the identity of the fatty acid moiety. Moreover, since orfamides D – G only differ in the fatty acid tail, the same reference spectra can be used for all. Indeed, the length and saturation of the fatty acid does not impact the 1H or 13C chemical shifts.

References

D’Aes, et al. “To settle or to move? The interplay between two classes of cyclic lipopeptides in the biocontrol strain Pseudomonas CMR12a.” Environmental Microbiology 16, 7 (2014): https://dx.doi.org/10.1111/1462-2920.12462.

De Roo, et al. “An nuclear magnetic resonance fingerprint matching approach for the identification and structural re-evaluation of Pseudomonas lipopeptides.” Microbiology Spectrum 0, 0
https://dx.doi.org/doi:10.1128/spectrum.01261-22.

Gross, et al. “The genomisotopic approach: a systematic method to isolate products of orphan biosynthetic gene clusters.” Chemistry & Biology14, 1 (2007): https://dx.doi.org/10.1016/j.chembiol.2006.11.007.

Ma, et al. “Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species.” Frontiers in Microbiology7 (2016): https://dx.doi.org/10.3389/fmicb.2016.00382.

Ma, Z., S. Zhang, J. Liang, K. Sun, and J. Hu. 2019. ‘Isolation and characterization of a new cyclic lipopeptide orfamide H from Pseudomonas protegens CHA0’, Journal of Antibiotics.

Nguyen, et al. “Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides.” Nature Microbiology2 (2016): https://dx.doi.org/10.1038/nmicrobiol.2016.197.

Weisshoff, et al. “PPZPMs – a novel group of cyclic lipodepsipeptides produced by the Phytophtora alni associated strain Pseudomonas sp. JX090307 – the missing link between the viscosin and amphisin group.” Natural Products Communications9, 7 (2014).

Zachow, et al. “The novel lipopeptide poaeamide of the endophyte Pseudomonas poae RE*1-1-14 is involved in pathogen suppression and root colonization.” Molecular Plant-Microbe Interactions28, 7 (2015): https://dx.doi.org/10.1094/MPMI-12-14-0406-R.

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