Orfamide D – G

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

Chemical properties

CAS	n.a.
Molecular formula	Orfamide D: C61H108N10O17 Orfamide E: C63H110N10O17 Orfamide F: C65H114N10O17 Orfamide G: C65H116N10O17
Molecular weight	Orfamide 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 mass	Orfamide D: 1252.7894 Da Orfamide E: 1278.8050 Da Orfamide F: 1306.8363 Da Orfamide G: 1308.8520 Da
Solubility	Soluble in methanol, ethanol, DMF, DMSO, acetonitrile
CMC	n.a.
3D conformation	n.a.
NMR data available in literature	DMF-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.

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.