Carbon-13 Fourier transform nuclear magnetic resonance spectroscopy. II. Pattern of biosynthetic incorporation of [1-13C]- and [2-13C]acetate into prodigiosin

The prodiginine family of bacterial alkaloids is a diverse set of heterocyclic natural products that have likely been known to man since antiquity. In more recent times, these alkaloids have been discovered to span a wide range of chemical structures that possess a number of interesting biological activities. This review provides a comprehensive overview of research undertaken toward the isolation and structural elucidation of the prodiginine family of natural products. Additionally, research toward chemical synthesis of the prodiginine alkaloids over the last several decades is extensively reviewed. Finally, the current, evidence-based understanding of the various biosynthetic pathways employed by bacteria to produce prodiginine alkaloids is summarized.

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“…However, this proposal was subsequently revised in light of further experimental data. 141,142 (Scheme 27). …”

Section: Biosynthesismentioning

confidence: 99%

Structure, Chemical Synthesis, and Biosynthesis of Prodiginine Natural Products

et al. 2016

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“…l g 2 This communication presents the results of the incorporation of [lJ3C] acetate into the key metabolite ergosterol.…”

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confidence: 99%

¹³C Fourier transform n.m.r.: XIII—reassignment of the ¹³C spectrum of ergosterol

Cushley

Filipenko

1976

Org. Magn. Reson.

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Abstract-The complete labeling pattern of ergosterol isolated from(Merck, Sharpe and Dohnie, Canada). The yeasts were incubated for 20 h at 30 "C with constant aeration. A 500 ml culture of yeast was harvested by centrifuging to a final wet weight of 14g. The procedure for the isolation and purification of ergosterol was modified from that followed by Fryberg et ~1 .~ The 13C n.m.r. spectrum was determined on twice chromatographed (t.1.c. silica gel G ) ergosterol acetate (18 mg). G.1.p.c.: (98% pure), 0.25 mm i.d. x 40m column, coated with 3 % Silar-IOC on Gas-Chrom Q 100/120; temperature, 220°C; flow rate, 40 ml/min He. Relative retention time 1.6, relative to cholestanol acetate; Amax: 261.5,271, 281.5,293.5; Amax (authentic): 262, 271.5, 282, 294, envelopes were identical; m/e 378 ( M -CH,COOH), 367, 334, 312, 252. The spectrum was identical with authentic ergosterol acetate except for isotope multiplets, e.g. m/e 378 + (1,2,3, . . . The n.m.r. system consists of a Varian XL-100-15 spectrometer fitted with a TTlOO FT attachment. Conditions were: data set = 8 K; digitizing rate = 10 KHz; pulse width = 25 ,us; pulse delay = 2 s. Figure 1 (A) displays the proton noise-decoupled I3C n.m.r. spectrum of ergosterol acetate in CDCI,. The assignment of the individual carbons are indicated on the spectrum and the chemical shifts are contained in Table 1. The assignments are essentially those of Reich et al. ,5 however (see below). carbons are substantially enhanced over natural abundance. The biosynthetically enriched carbons correspond with the well-established pathway to steroids from acetyl CoA viz. acetate + mevalonate + isopentenyl pyrophosphate + farnesyl pyrophosphate 4 squalene -+ Of particular importance in the assignments of the enriched carbons are the pair of AB doublets due to 13C-l I-13C-12 and l3C-8-I3C-14, created by the head to head condensation of two farnesyl moieties and the subsequent cyclization step of squalene by cyclase. The assignments are confirmed by the carbon-carbon coupling constants, J(I 1-12) = 33 = 45 Hz.The characteristic triplet for C-12 in the enriched spectrum [ Fig. 1 (B

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“…We used two different media, NA and PDA, to demonstrate the efficacy of our method with bacteria growing on two different types of agar, which can further be adopted to other solid media. Using HRMS coupled to MALDI-imaging, the incorporation of 15 N could be distinguished from the natural 13 C-monoisotopic mass with the ± 2 ppm ion filter. Labeling with [1-13 C]-Lproline was confirmed by the high ion intensities that clearly differentiated with the natural 13 C abundance (approximately 22%).…”

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confidence: 99%

“…Using HRMS coupled to MALDI-imaging, the incorporation of 15 N could be distinguished from the natural 13 C-monoisotopic mass with the ± 2 ppm ion filter. Labeling with [1-13 C]-Lproline was confirmed by the high ion intensities that clearly differentiated with the natural 13 C abundance (approximately 22%). Furthermore, incorporation of the D 3 -methyl group of [methyl-D 3 ]-L-methionine into prodigiosin (3) was confirmed with a mass shift of 3 Da that excluded interference and mixing with natural M + 1-and M + 2-13 C-monoisotopic masses.…”

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confidence: 99%

“…We provide a proof-of-concept of our established methodology using an endophytic bacterium we discovered recently; a S. marcescens strain that is capable of biosynthesizing the red-pigmented prodiginines as well as the biosurfactants serratamolides (see Figure S1 in the Supporting Information for their chemical structures, 1 – 31 ) . We used stable isotope labeling approaches in conjunction with earlier studies not only to deduce the biosynthetic pathway of the compounds but also to pinpoint the incorporation sites of the labels 15 NH 4 Cl, [1- 13 C]- l -proline, [methyl-D 3 ]- l -methionine, and [ 15 N]- l -serine into prodiginines and serratamolides. − Supplement concentrations of 1 or 2 mg/mL gave sufficient incorporation rates during fermentations in liquid nutrient broth (NB) …”

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confidence: 99%

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Stable Isotope Labeling of Prodiginines and Serratamolides Produced by Serratia marcescens Directly on Agar and Simultaneous Visualization by Matrix-Assisted Laser Desorption/Ionization Imaging High-Resolution Mass Spectrometry

2018

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Matrix-assisted laser desorption/ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) is an important technique for visualizing the spatial distribution of compounds directly on the surface of organisms such as microorganisms, insects, plants, animals, and human tissues. However, MALDI-imaging-HRMS and the stable isotope labeling approach have never been combined for the detection and simultaneous visualization of labeled and unlabeled compounds, their analogues and derivatives, as well as their precursors. Herein, we present a methodology that labels microbial secondary metabolites directly on agar with stable isotopes and allows concurrent spatial distribution analyses by MALDIimaging-HRMS. Using a thin film of labeled agar supplemented with [1-13 C]-L-proline, [methyl-D 3 ]-L-methionine, 15 NH 4 Cl, or [ 15 N]-L-serine overlaid on unlabeled agar, we demonstrate the incorporation of labeled precursors into prodiginines and serratamolides produced by an endophytic bacterium, Serratia marcescens, by MALDI-imaging-HRMS and HPLC−HRMS. Further, we show the incorporation of CD 3 into prodigiosin as well as its characteristic fragments directly by MALDI-imaging-HRMS 2 . Our methodology has several advantages over currently existing techniques. First, both labeled and unlabeled compounds can be visualized simultaneously in high spatial resolution along with their labeled and unlabeled precursors. Second, by using a thin film of labeled agar, we utilize minimum amounts of expensive labeled compounds (1−3 mg) ensuring a cost-effective method for investigating biosynthetic pathways. Finally, our method allows in situ visualization and identification of target and nontarget compounds without the need of isolating the compounds. This is important for compounds that are produced by microorganisms in low, physiologically, or ecologically relevant concentrations.

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Carbon-13 Fourier transform nuclear magnetic resonance spectroscopy. II. Pattern of biosynthetic incorporation of [1-13C]- and [2-13C]acetate into prodigiosin

Cited by 22 publications

References 1 publication

Structure, Chemical Synthesis, and Biosynthesis of Prodiginine Natural Products

Structure, Chemical Synthesis, and Biosynthesis of Prodiginine Natural Products

¹³C Fourier transform n.m.r.: XIII—reassignment of the ¹³C spectrum of ergosterol

Stable Isotope Labeling of Prodiginines and Serratamolides Produced by Serratia marcescens Directly on Agar and Simultaneous Visualization by Matrix-Assisted Laser Desorption/Ionization Imaging High-Resolution Mass Spectrometry

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Carbon-13 Fourier transform nuclear magnetic resonance spectroscopy. II. Pattern of biosynthetic incorporation of [1-13C]- and [2-13C]acetate into prodigiosin

Cited by 22 publications

References 1 publication

Structure, Chemical Synthesis, and Biosynthesis of Prodiginine Natural Products

Structure, Chemical Synthesis, and Biosynthesis of Prodiginine Natural Products

13C Fourier transform n.m.r.: XIII—reassignment of the 13C spectrum of ergosterol

Stable Isotope Labeling of Prodiginines and Serratamolides Produced by Serratia marcescens Directly on Agar and Simultaneous Visualization by Matrix-Assisted Laser Desorption/Ionization Imaging High-Resolution Mass Spectrometry

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¹³C Fourier transform n.m.r.: XIII—reassignment of the ¹³C spectrum of ergosterol