Acetylacetone

Cell growth of all strains used in this study was measured at OD₆₀₀ using a UV–vis spectrophotometer (Mecasys Co., Ltd., Korea). The dry cell weight was experimentally determined on the basis of the correlation model OD₆₀₀ 1 = 0.282 g dry cell weight (DCW)/l and used for the calculation of biomass concentration of fed-batch fermentation. Glucose concentration was estimated using a Glucose (GO) Assay Kit (Sigma-Aldrich, USA). For measurement of intracellular metabolites, 1 ml of cell pellet was harvested by centrifugation (13,000 rpm at 4 °C for 3 minutes), and the supernatant excluding the cell pellet was used for the measurement of extracellular metabolites. For the extraction of the metabolite from cells, including inter alia, heme and other porphyrins, the cell pellet was disrupted using-modified actone:HCl extraction methods described by Espinas et al.^{60 (link)}. After 1 ml of acetone:HCl (95:5) buffer was added to the cell-harvested tube, the mixture was vortexed and diluted with 1 ml of 1 M NaOH. The intracellular sample which was disrupted and supernatant was filtered using an MCE (Mixed Cellulose Ester) filter (Hyundai micro, Korea) for concentration analysis. Porphyrin concentration was determined using a high-performance liquid chromatography (HPLC) system (Waters Corporation, USA), which consists of a dual λ absorbance detector (Waters 2487), binary HPLC pump (Waters 1525), and autosampler (Waters 2707). The filtered sample was separated in a SUPELCOSIL™ LC-18-DB HPLC Column 5 μm particle size, L × I.D. 250 × 4.6 mm (Supelco Inc., USA) using a linear gradient method of 20–95% solvent A in B at 40 °C. Solvent A is a 10:90 (v/v) HPLC grade methanol:acetonitrile mixture, and solvent B is a 0.5% (v/v) trifluoroacetic acid (TFA) in HPLC grade water. The flow rate was 1 ml/min for 40 minutes, and the absorbance was determined at 400 nm. The data were estimated using Waters Empower-3 software. ALA concentration was measured using the colorimetric assay called Ehrlich’s reagent^{61 (link)}. One volume of the supernatant was chemically reacted with 0.5 volume of 1 M sodium acetate buffer (pH 4.8) and 0.25 volume of acetylacetone at 100 °C for 15 minutes. After that, the reagent mixture was cooled in an ice bath for 15 minutes. The modified Ehrlich’s reagent was added to the cooled mixture and stayed at room temperature for 30 minutes. The absorbance was determined at 554 nm using Epoch 2 microplate spectrophotometer (BioTek Instruments, USA).

Mass spectrometry measurements of phospholipid samples were performed using a 6460 Triple Quad LC-MS system equipped with a similar ACQUITY UPLC® Peptide CSH™ C18 Column as used in proteomics. However, different columns were used for each application, to prevent cross-contamination. Separation of lipids was performed using a gradient of mobile phase F (water with 0.05% ammonium hydroxide and 2 mM acetylacetone), and mobile phase G (80% 2-propanol, 20% acetonitrile, 0.05% ammonium hydroxide and 2 mM acetylacetone) at a flow rate of 300 μL min⁻¹ and a column temperature of 60 °C. To equilibrate the column, a ratio of mobile phase F to mobile phase G of 70:30 was used. Upon injection, this ratio was gradually changed to 100% mobile phase G over the course of 8 min and then kept like that for 2 min. Subsequently, over the course of 1 min, the initial 70:30 ratio of mobile phase F and G was reset, which was then used for the last 4 min of the run. The built-in autosampler of the LC-MS system was used to inject 1 μL (quantitative analysis) or 5 μL (qualitative analysis of low-abundance compounds) of sample solution.
Transitions were established based on previous work^{23 (link)}, as well as scanning measurements of purified standards. The very regular fragmentation pattern (except for LPA and CDP-DAG fragmentation always occurs at the ester linkage between an acyl chain and the glycerol) could be used to determine transitions. Synthesized phospholipids were distinguished from phospholipids present at the start of the reaction as part of the liposome matrix by incorporation of ¹³C-G3P, resulting in a 3 Da (or 6 Da for PG) mass shift.
Mass spectrometry data were analysed using the Agilent MassHunter Workstation Software Quantitative Analysis program, which automatically integrates peaks corresponding to the transitions set in the method. Integrated peak intensities were exported to MATLAB R2016b (MathWorks) for further analysis. For each transition in each sample, the average integrated counts of two injections was determined. For end products, integrated counts were converted to concentrations using linear calibration curves fitted to signals from a dilution series of standards ran before and after every mass spectrometry measurement series.

The Schiff base was prepared by mixing 1 mmol of acetylacetone dissolved in 10 ml of methanol with a 30 ml solution of cefotaxime (477 mg) in methanol for 8 h under reflux (Fig. 1).Figure 1

Schiff base synthesis from cefotaxime sodium salt and acetylacetone.

While HPLC chromatography can be used to separate the metallochelate from excess acetylacetone, in this investigation, it was used to characterize the product. After synthesis, the product was characterized by both gamma ray spectroscopy with use of a NaI(Tl) detector and reverse-phase HPLC with 40% to 60% gradient of MeCN in water mobile phase over 10 min, 1 min at 60% MeCN, decrease to 40% MeCN in 1 min, and remaining at 40% MeCN for 3 min. Other gradients and mobile phases were considered in selecting this protocol; a complete summary has been made by Broder [23 ]. TFA (volume concentration 0.1%) was added to the water phase to improve peak shape. TFA at this concentration has been shown to exhibit no absorption at 254 nm in MeCN/water [24 (link)], the wavelength at which acetylacetone was monitored spectrophotometrically. Commercially available VO(acac)₂ was dissolved in 100% EtOH at a concentration of 2 mg/mL. Acetylacetone was dissolved in EtOH at a concentration of 5.4 mg/mL. An injection volume of 5 μL corresponding to 0.01 mg VO(acac)₂ or 0.027 g acetylacetone, respectively, was used. Solutions of VO(acac)₂ were freshly prepared unless otherwise noted. In some cases, freshly prepared solutions were compared to older samples (age of preparation < 1 week) to determine possible effects of oxidation of the metallochelate on its chromatographic behavior.
A flow rate ranging from 1.0 to 1.5 mL/min was used resulting in approximately 15 min per trial chromatographic separation. The eluent was monitored optically at 254 nm for acetylacetone and at 320 nm for VO(acac)₂ unless otherwise specified [10 (link)].