Synthesis of 1,2-phenylenediamine capturing molecule for the detection of diacetyl

Here we describe the design of 1,2-phenylenediamine capturing molecule and the synthesis steps necessary for its preparation. The designed 1,2-phenylenediamine derivative is able to capture diacetyl in solution, as shown by ESIMS, forming a chemical adduct, 1-4-quinoxaline. The methyl esters of diacetyl-adduct (DAA) and pentanedione-adduct (PDA) are incorporated to the lysines in BSA and the conjugate used for antibody screening and selection. In the research article is described an enzyme-linked immunosorbent assay developed to detect and quantify diacetyl in complex media.


Raw and analyzed data Data accessibility
Data is with this article.

Value of the Data
The data explains the design of 1,2-phenylenediamine capturing molecule and could be used by others researchers.
All the steps for the synthesis of the compounds here described and the methods can be followed by other researchers.
The methods for incorporation of the diacetyl-adduct (DAA) and pentanedione-adduct (PDA) to the lysines in BSA can be used for preparation of other BSA-conjugates.

Data
The synthesis steps necessary for the preparation of the 1,2-phenylenediamine derivative (linker diamine) are described in details. The synthesis of the methyl esters of diacetyl-adduct (DAA) and pentanedione-adduct (PDA) and their incorporation in BSA to form the corresponding BSA-conjugates (BSA-DAA or BSA-PDA) are described.

General methods
Organic solvents were concentrated under reduced pressure at o 40°C. Vacuum liquid chromatography was performed on silica gel (60 H, dried in oven at 120°C) Reversed phase chromatography was performed on Sep-Pak Plus C 18 cartridge (Waters, washed with 10 mL MeCN, 10 mL 50% MeCN aq and 20 mL H 2 O before use A mixture of 1 (0.45 g, 2.0 mmol), trimethyl ortoformate (0.24 g, 0.25 mL, 2.3 mmol), methanol (14 mL) and p-toluenesulfonic acid (4 mg) was gently heated while the methyl formate formed was distilled off through a short vigreux column. When no more methyl formate was distilled off, the reaction mixture was cooled, made basic with a few drops of 2N sodium methoxide and was partitioned between ethyl acetate (50 mL) and water (50 mL). The organic phase was washed with brine (50 mL) and dried over potassium carbonate. Filtration, washing with ethyl acetate and concentration afforded the dimethyl acetate 2 (0.45 g, 83%) [2]. The crude product was used in the following step without further purification. 1  A mixture of the dimethyl acetal 2 (150 mg, 0.55 mmol), 1,2-dihydroxybenzene (72 mg, 0.65 mmol) and toluene (8 mL) was brought to reflux and part of the solvent (2 mL) was distilled through a short vigreux column. The temperature was lowered to 60°C and p-toluenesulphonic acid (3 mg) was added. Distillation of the solvent was slowly continued until pure toluene was being collected. During this operation, addition of more toluene (8 mL) was necessary to prevent that the reaction mixture become dry. Triethylamine (0.1 mL) was added to the cooled reaction mixture, which was then partitioned between ethyl acetate and water. The organic phase was washed with water (1 × 20 mL), 0.1 M sodium hydroxide (2 × 20 mL), brine (2 × 20 mL) and dried over potassium carbonate. Filtration, washing with ethyl acetate and concentration gave 3 (120 mg, 69%) [2]. The crude product was used in the following step without further purification. 1  A solution of 3 (200 mg, 0.63 mmol) in acetic acid (1.5 mL) was added drop-wise under stirring into fuming nitric acid (2 mL) on an ice bath followed by stirring at room temperature for 1 h. The reaction mixture was then poured into ice-water (5 mL) and the formed precipitate was isolated by centrifugation. The precipitate was washed with water (2 × 2 mL) and dried by freeze drying. This gave 4 (190 mg, 74%) [3]. The crude product was used in the following step without further purification. 1  A mixture of di-nitro compound 4 (81 mg, 0.20 mmol), stannous chloride di-hydrate (226 mg, 1 mmol) and 12 M HCl (0.12 mL) in ethyl acetate (12 mL) was heated at 50 o C for 2 h. When adding additional 12 M HCl (0.12 mL) and ethyl acetate (20 mL) the product solidified and was isolated by centrifugation. The solid was washed with ethyl acetate (2 × 10 mL) and dried. This gave 5 (65 mg, 78%, yellow solid) [4,5]. The crude product was used in the following step without further purification. 1  Compound 5 (10 mg, 0.024 mmol) and diacetyl (20 mg, 0.23 mol) was dissolved in water-acetic acid 2:1 (1 mL). The reaction mixture was stirred at room temperature for 1 hour. Dilution with 4 mL water followed by purification on SepPak (20-30% aq. CH 3 CN) gave the diacetyl adduct 6 (9 mg, 92%) [6]. 1  Compound 6 (9 mg, 0.022 mmol) was dissolved in a minimal volume of dioxane (0.2 mL). Water was added (0.3 mL) followed by 2 M sodium hydroxide (0.3 mL) and the reaction mixture was stirred at room temperature for 15 min. The pH was adjusted to 4.2 with 1 M and 0.1 M HCl. Purification was then directly performed by reversed phase chromatography on a C 18 SepPak cartridge. After washing with water the product was eluted with water-acetonitrile 1:1. Evaporation of the solvents gave 7 (8 mg, 95%). 1  N,N´-disuccinimidyl carbonate (1.9 mg,7.4 μmol) dissolved in dry DMF (0.1 mL) and 4-(dime-thy1amino)pyridine (1.5 mg, 12 μmol) dissolved in dry DMF (0.1 mL) were added to a solution of compound 7 (2 mg, 5.2 μmol) in dry DMF (0.4 mL) under magnetic stirring. Activation proceeded for 40 min. Dilution of reaction mixture with 0.01 M HCl to less than 10% DMF was followed by purification by reversed phase chromatography on a C 18 SepPak cartridge. After washing with wateracetonitrile 9:1 and 8:1 the product was eluted with water-acetonitrile 1:1. Evaporation of the solvents gave 8 (2.2 mg, 88%) [7]. 1  Bovine serum albumin (2 mg, 0.03 μmol) was dissolved in 0.1 M phosphate buffer pH 7.5 and added to a solution of compound 8 (0.9 mg, 2 μmol) in DMSO (100 μL) and 0.1 M phosphate buffer pH 7.5 (3 mL). The reaction mixture was gently vortexed overnight at room temperature and purified by concentration using a Millipore MW CO 30 000 filter (4000 rpm/6 min).The protein on the filter was washed with water (4 × 4 mL) and lyophilisation gave 9 [6]. MALDI-TOF-MS found m/z 70 530, that corresponds to an incorporation of~11 adducts per BSA.
Compound 5 (35 mg, 0.083 mmol) and pentanedione (35 g, μL) was dissolved in water-acetic acid 2:1 (2.5 mL). The reaction mixture was stirred at room temperature for 1 hour, and purification was then directly performed by reversed phase chromatography on a C 18 SepPak cartridge. After washing with water, water-acetonitrile 9:1 and 8:2, the product was eluted with water-acetonitrile 1:1. Eva- Compound 10 (7 mg, 0.017 mmol) was dissolved in a minimal volume of dioxane (0.2 mL). Water was added (0.3 mL) followed by 2 M sodium hydroxide (0.3 mL) and the reaction mixture was stirred at room temperature for 15 min. The pH was adjusted to 5 with 1 M and 0.1 M HCl. Purification was then directly performed by reversed phase chromatography on a C 18 SepPak cartridge. After washing with water the product was eluted with water-acetonitrile 1:1. Evaporation of the solvents gave 11 for 40 min. Dilution of reaction mixture with 0.01 M HCl to less than 10% DMF was followed by purification by reversed phase chromatography on a C 18 SepPak cartridge. After washing with wateracetonitrile 9:1 and 8:1 the product was eluted with water-acetonitrile 1:1. Evaporation of the solvents gave 12 (3.5 mg, 87%) [7]. 1  Bovine serum albumin (3.8 mg, x μmol) was dissolved in 0.1 M phosphate buffer pH 7.5 (1.6 mL) and added to a solution of compound 12 (3.5 mg, 7 μmol) in DMSO (200 μL) and 0.1 M phosphate buffer pH 7.5 (0.8 mL). The reaction mixture was gently "shaken" overnight and purified by concentration using a Millipore MW CO 30 000 filter (4000 rpm/6 min).The residual protein on the filter was washed with water (4 × 4 mL) and lyophilisation gave 13 [6]. MALDI-TOF-MS found m/z 70 380, that corresponds to an incorporation of~11 adducts per BSA.
Reaction of compound 15 with diacetyl to afford diacetyl adduct 6, analysed with ESIMS.

Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2017.09.072.