Malonic acid

Whole plants of GJ were purchased from Tongling Hetian Chinese Medicine Pieces Co. Ltd. (Anhui, China) and smashed into powder with a disintegrator (Tianjin Tester Instrument Co, Ltd., Tianjin, China). Analytical grade reagents including choline chloride (≥98% purity), betaine (≥98% purity), oxalic acid (≥98% purity), DL-malic acid (≥98% purity), citric acid (≥98% purity), and malonic acid (≥98% purity) were purchased from Sigma-Aldrich (Shanghai, China). Analytical pure methanol, ethanol, and acetone were purchased from Tianjin Tianli Chemical Reagent Co. Ltd. (Tianjin, China). EA (≥98% purity) was purchased from Sichuan Vicky Biotechnology Co., Ltd. (Sichuan, China). DPPH (≥98% purity) was purchased from Diyibio Biotechnology Co., Ltd. (Shanghai, China). The chromatographic grade methanol was purchased from Honeywell (Arizona, USA). The chromatographic grade phosphoric acid (≥98% purity) was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd (Tianjin, China). Ultra-pure water produced by Milli-Q IQ7000 (Millipore Corp, USA) was used throughout the experiment.

Oligonucleotide primers were
synthesized by Eurofins Genomics Germany GmbH (Ebersberg, Germany)
or Integrated DNA Technologies (Coralville, IA, USA). GeneJET Gel
Extraction and Plasmid Miniprep kits were used for DNA purification
(Thermo Fisher Scientific, Waltham, MA, USA). The Gibson assembly
master mix was purchased from New England Biolabs (Ipswich, MA, USA).
DNA fragments for plasmid construction were amplified by PCR using
Phusion HF (New England Biolabs, Ipswich, MA, USA) or PrimeStar HS
DNA polymerase (Takara Bio, Kusatsu, Shiga, Japan). DreamTaq DNA polymerase
(Thermo Fisher Scientific, Waltham, MA, USA) was used for colony PCR. CouR, RtMatB, and SpMae1 gene sequences were codon-optimized and synthesized by Doulix (Explora,
Venice, Italy). 4CL from Arabidopsis
thaliana was amplified from pCfB854.^{58 (link)} The genes CHS from Rhododendron
simsii and CHI from Paeonia suffruticosa were codon-optimized and synthesized
by GenScript Biotech (Piscataway Township, NJ, USA). Analytical standards
of naringenin (≥95%, TLC), malonic acid (≥98.5%, GC),
phloretic acid (≥97.5%, HPLC), and p-coumaric
acid (≥98%, HPLC) were obtained from Sigma-Aldrich/Merck KGaA
(Darmstadt, Germany). Phloretin (≥99%, HPLC) was obtained from
Extrasynthese (Lyon, France).

Vinyl pyrrolidone (VP), iron (III) chloride hexahydrate (FeCl₃·6H₂O), sodium acetate, malonic acid (MA), and molybdenum disulfide (MoS₂) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetic acid and ethylene glycol were procured from Duskan Pure Chemical (Kyungki-do, Korea). Methanol and ethanol were acquired from Fisher Scientific (Seoul, Korea). Ammonium persulfate (APS), 3-methacryloxypropyltrimethoxysilane (MPS), Ethylene glycol dimethacrylate (EGDMA), and 2-methylpropionitrile (AIBN) were purchased from Daejung Chemicals & Metals (Gyonggido, Korea). The standard chemicals of (+)-catechin (C, ≥98%), (−)-epicatechin (EC, ≥90%), (−)-epigallocatechin (EGC, ≥95%), (−)-epicatechin gallate (ECG, ≥95%), and (−)-epigallocatechin gallate (EGCG, ≥98%) were supplied by Tokyo Chemical Industry (Tokyo, Japan). All other solvents used in the experiment were either of HPLC or analytical grade.

All chemicals utilized were of the greatest analytical purity. They included 2-(amino methyl imidazole) = (AMI), oxalic acid, and malonic acid that Sigma-Aldrich supplied. The metal salts of CuCl₂·2H₂O (Sigma-Aldrich) and K₂PdCl₄(Merck) were used. Organic solvents such as ethyl alcohol (99%) and dimethylformamide (DMF), in addition to dimethyl sulfoxide (DMSO), were used. All preparations are usually made use of de-ionized water collected from glass equipment.

The tested choline ILs were synthesized by mixing choline bicarbonate (Sigma-Aldrich, St. Louis, USA, 80% in H₂O) with carboxylic acids (acetic acid, lactic acid, malonic acid, isovaleric acid, isobutyric acid, 2-methylbutyric acid; all from Sigma-Aldrich, used as received) in aqueous solutions [34 (link)]. The chemical identity of the obtained ILs (for structures, names and abbreviations, see Table 1) was confirmed by ¹H-NMR spectroscopy (Bruker Avance II 400 MHz, Bruker Corporation, Billerica, MA, USA) using D₂O as a solvent. The water content was determined by Karl Fischer’s coulometric titration (Metrohm 756 KF Coulometer, Metrohm AG, Herisau, Switzerland). All ILs were dried under high vacuum and stored in a glovebox under an argon atmosphere (water content below 0.5 ppm) prior to use.

Malonic acid, 2-¹³C-Malonic acid, 4-coumaric acid, 2-methylthiazole-5-carboxylic acid, resveratrol, p-coumaric acid, 7-phenyl heptanoic acid, 6-phenyl hexanoic acid, 5-(4-flurophenyl)valeric acid, 4-biphenylacetic acid, N-acetyl-L-cysteine, benzoic acid adenosine triphosphate (ATP), coenzyme A trilithium salt (CoA), magnesium chloride, dimethyl sulfoxide (DMSO) hexahydrate, phospho(enol) pyruvate potassium salt, PEG 4000, NaCl, potassium and sodium phosphate, IPTG, kanamycin, penicillin, streptomycin, were purchased from Sigma-Aldrich. 2-Chloro-1,3-thiazole-5-carboxylic acid and 4-methylthiazole-5-carboxylic acid were obtained from Matrix Scientific.