Uses and production methods of L-Proline

Introduction

Colorless crystals, odorless, sweet taste; easily soluble in water (solubility in water at 25°C is 162.3 g/100 ml) and ethanol, insoluble in butanol and ether, turns yellow in contact with ninhydrin test solution, and turns red after acidification with glacial acetic acid; pI 6.3, Chemicalbook decomposition point is 220-222°C; specific rotation [α]20D-85° (0.5-2.0 mg/ml, H2O), [α]20D-60.4° (0.5-2.0 mg/ml, 5 mol/L HCl).

Uses 

1. L-Proline is used as asymmetric catalysts in organic synthesis and asymmetric aldol cyclization. It is involved in the Michael addition of dimethyl malonate to alfa-beta-unsaturated aldehydes. It is a precursor of hydroxyproline in collagen. It is an active component of collagen and involved in the proper functioning of joints and tendons. It finds uses in pharmaceutical, biotechnological applications due to its osmoprotectant property. Further, it is used with ninhydrin in the chromatography.

2. L-Proline is an amino acid and precursor (with vitamin C) for collagen, the building block of the structure of tendons, ligaments, arteries, veins and muscles. It is important in wound healing.

Production methods

There are two methods for producing L-proline. One is direct fermentation, which uses glucose and a variant strain of Brevibacterium flavum or a wild-type strain of Corynebacterium glutamicum to produce L-proline through microbial fermentation. The other is chemical synthesis, which uses glutamic acid as the raw material and esterifies it with anhydrous ethanol under the catalysis of sulfuric acid. Triethanolamine is then added to liberate the aminosulfate, resulting in glutamic acid-δ-ethyl ester. This ester is then reduced with the metal reducing agent potassium borohydride to produce crude proline, which is then isolated and purified.

Planar Process

Esterification: Weigh 147g of L-glutamic acid into a three-necked flask, add 1L of anhydrous ethanol, and cool to 0°C with stirring. Then, add 80ml of H₂SO₄ dropwise. Stir and react at 0-5°C for 1 hour, then continue the reaction at room temperature for 1 hour until the reaction becomes clear. Triethylamine was added dropwise at 20°C until the pH reached 8-8.5, resulting in the precipitation of white crystals. The mixture was stirred at room temperature for an additional hour, allowed to cool to 5°C, and filtered. The crystals were washed with 95% ethanol, dried by evaporation, and vacuum-dried to yield approximately 141g of glutamic acid-δ-ethyl ester. The melting point was 178-180°C, and the yield was 80%-83%. [α]32D+29.8 (C = 1g/ml 10% HCl). Reduction: 175g of glutamic acid-δ-ethyl ester was added to a three-necked flask, along with 875ml of distilled water. Stir and cool to 5°C. Then, 3.9g of KBH4 was added portionwise over approximately 1h. The reaction was continued at room temperature for an additional hour, followed by a 3h reaction at 50°C. Cooled to 0°C, 6mol/L HCl was added to adjust the pH to 4, and the filtrate was filtered to yield the crude L-proline aqueous solution. Isolation and Purification

Ion exchange resin-alumina column chromatography: Apply a crude L-proline aqueous solution to a 732-H+ resin column at a flow rate of 4 ml/min (10 ml of resin is required for 1 g of acid). Rinse with distilled water until neutral, then elute with 1 mol/L ammonia solution. Collect the eluate containing the L-proline fragment (using silica gel G thin-layer chromatography). Concentrate the eluate to dryness under reduced pressure, dissolve it in a small amount of water, and apply it to a neutral alumina column. Elute with 60% ethanol in water (also using silica gel G thin-layer chromatography). Concentrate the collected eluate to dryness under reduced pressure, wash it several times with anhydrous ethanol, cool it slightly, add anhydrous ether, cool it, filter the crystals, and vacuum dry them to obtain L-proline. Melting point: 220-222°C (decomposition), yield: approximately 28%. [α]24D-82.4 (C = 1 g/ml, H2O). Pentachlorophenol precipitation and desorption separation method

Salt formation: Place the crude proline aqueous solution in a reaction flask. Heat to 50°C and add pentachlorophenol ethanol solution (0.111 mol/70 ml ethanol) dropwise. Stir at this temperature for 5 hours, then cool to 0°C. Filter the crystals, wash with a small amount of ice water, drain, and dry to obtain the double salt with a melting point of 240-242°C and a precipitation rate of 95%. Analysis: Place 38.4 g of the double salt in a three-necked flask, add 200 ml of distilled water and 20 ml of aqueous ammonia, stir at room temperature for 8 hours, cool to 0°C, filter, and collect the filtrate. Concentrate the filtrate under reduced pressure, add 100 ml of distilled water, filter, and decolorize with activated carbon. Extract with ether, separate the aqueous layer, and continue concentrating to dryness. Decolorize the solution several times with anhydrous ethanol, moisten with a small amount of anhydrous ethanol, add twice the amount of anhydrous ether, cool, and crystallize. Filter the crystals and vacuum dry to obtain the finished L-proline. Scale-up Production Process

Esterification: Place 15 kg of L-glutamic acid and 100 L of anhydrous ethanol into a 200 L reactor. Cool to 0°C. Add 8.1 L of concentrated H₂SO₄ dropwise with stirring. Maintain the reaction at 0°C with stirring for 1 hour. Then, maintain the reaction at 25°C with stirring for 1 hour. Add triethylamine to adjust the pH to 8.0-8.5. Stir for 1 hour until a white precipitate forms. Cool to 5°C, filter the precipitate, wash with 50 L of 95% ethanol, and vacuum dry at 50°C to obtain L-glutamic acid-δ-ethyl ester.

Reduction: Place the resulting L-glutamic acid-δ-ethyl ester into a 100 L reactor. Add 70 L of water, stir, and cool to 5°C. Add 4.3 kg of KBH₄ in portions over 1 hour. Heat to 200°C with stirring for 1 hour. Then, raise the temperature to 50°C and stir for 3-4 hours. Cool to 0°C, adjust the pH to 4.0 with 6 mol/L HCl, and filter the filtrate to obtain a crude L-proline solution. Precipitation: Place the crude L-proline solution into a 100L reaction tank, heat to 50°C, and slowly add 7L of a 1.5mol/L pentachlorophenol ethanol solution while stirring. Keep the mixture at 50°C for 5 hours, then cool to 0°C to allow crystals to precipitate. Filter the crystals and drain to obtain the complex salt.

Analysis and purification: Put the double salt into a 100L reaction tank, add 20L of 3% ammonia water, stir and react at room temperature for 7-8h, cool to 0℃, filter, wash the precipitate with a small amount of ice water, drain, combine the washing liquid and the filtrate, and then concentrate under reduced pressure to dryness. Stir and dissolve with 10L of deionized water, filter and take the filtrate, add 0.5% activated carbon, heat at 70℃ and stir to decolorize for 1h, filter and take the filtrate, let it cool to 0℃, add an equal volume of ether to extract, separate the aqueous layer, concentrate under reduced pressure to dryness, add 10L of anhydrous ethanol to dehydrate 3 times, drain, add 2L of anhydrous ethanol to the precipitate and stir evenly, then add 10L of ether, cool to 0℃, filter and take the precipitate, vacuum extract the ether, and dry at 80℃ to obtain the finished L-proline.