CN118909030A - Preparation method and application of glutathione and derivatives thereof - Google Patents

Abstract

The invention discloses a preparation method and application of glutathione and its derivatives, and belongs to the technical field of fine chemicals. The preparation method of glutathione and its derivatives comprises the following steps: 1) mixing a compound shown in formula 4, a compound shown in formula 5, an organic base and a condensing agent to react to obtain a compound shown in formula 6; 2) mixing the compound shown in formula 6, a maleimide or derivative protecting group removing reagent, a 9-borane heterobicyclo [3,3,1] nonane protecting group removing reagent, an organic base and an organic solvent to react to obtain glutathione and its derivatives shown in formula a; wherein R ₁ is selected from one or more of H, C ₁ -C ₁₀ alkyl, C ₃ -C ₆ cycloalkyl; R is selected from Br or H. The preparation method has mild reaction conditions, is environmentally friendly, is suitable for scaled-up production, and the protection and deprotection of cysteine and glutamic acid in the preparation process are more efficient, and the amount of the protection or deprotection reagent is low, and the total yield calculated with glutamic acid as the starting material is improved.

Description

A preparation method and application of glutathione and its derivatives

Technical Field

The present invention relates to the technical field of fine chemical industry, and in particular to a preparation method and application of glutathione and its derivatives.

Background Art

Polypeptide is also referred to as peptide. Peptide is a compound formed by α-amino acids connected by peptide bonds. It is an intermediate product of protein hydrolysis in the body and a direct executor of life activities. Active polypeptides have all or part of the functions and characteristics of proteins. The most important thing is that they can be mass-produced by chemical synthesis. Since the solid-phase synthesis method for producing peptides in the 1990s, the industrialization of polypeptides has been rapid, and polypeptides are increasingly widely used in industries such as medicine, health products and cosmetics.

Glutathione (L-Glutathione), chemical name: N-(N-L-γ- lutamylL-cysteinyl))glycine, that is, N-(N-L-γ-glutamyl-L-cysteinyl)glycine, is divided into oxidized and reduced forms: reduced glutathione is the main active state (accounting for about 95%), it is a tripeptide composed of glutamic acid, cysteine and glycine, among which glutamic acid forms a peptide bond with cysteine by γ-carboxyl group. Because it contains free -SH group, it is often represented by GSH, as shown in Figure 1; oxidized glutathione (oxidized glutathione) is represented by GSSG, which is formed by the oxidation of two GSH molecules and the loss of H and then the transformation and combination.

Figure 1 Glutathione

The reduced glutathione shown in Figure 1 plays an important role in the body. Reduced glutathione is an endogenous active peptide that is widely present in the human body. It participates in all aspects of melanin production and can scavenge free radicals, resist oxidation, whiten the skin, lighten spots, and resist aging.

In addition, glutathione is compounded with different ingredients to enhance the efficacy synergistically, such as glabridin, glutathione, carnosine and vitamin C, or glabridin, glutathione, carnosine and vitamin C derivatives, which significantly improve the whitening effect. Microneedle is a new transdermal drug delivery system. Through hollow microneedles, glutathione and hyaluronic acid are injected subcutaneously to form a new, efficient and odorless transdermal delivery method. The prepared glutathione-loaded microneedle array (GSH-MN) contains 10% glutathione polymer prepolymer, which has good uniformity and suitable mechanical properties. In terms of loading rate, 17.4% glutathione can be loaded in the microneedle; in terms of release behavior, GSH-MN dissolves and releases the loaded GSH within 10 minutes after insertion into the pig skin without being oxidized, which can significantly and efficiently inhibit melanin production.

There are four main methods for preparing glutathione GSH: direct extraction, chemical synthesis, fermentation and enzyme conversion. Among them, fermentation is the most promising method for producing GSH. The earliest patent for preparing GSH from yeast was produced in 1938. It has been continuously improved and enhanced for decades. At present, fermentation has become the main method for producing GSH. Japan began to study the production of GSH by fermentation very early, among which yeast fermentation has been successfully used in Japan for industrial production of GSH. The domestic market currently lacks reliable products, and imported products are expensive. The biosynthesis method can efficiently synthesize glutathione in large quantities, but it has strict requirements on the production environment (CN107090483B), and the price of production raw materials and equipment is relatively high. The chemical synthesis method is more stable and has low equipment cost. The solid phase synthesis method in the chemical synthesis method is suitable for the preparation of long-chain polypeptides, but the production cost is high, and the condensation agent and the fixing of the protecting group lack selectivity; the liquid phase synthesis method is suitable for the preparation of short peptides, with low cost, less non-specific products, mild reaction conditions, and the disadvantage is that it is easy to produce side reactions after deprotection.

Glutathione is a short-chain tripeptide suitable for liquid phase synthesis. Patent CN113880910A uses the traditional protecting group benzyl chloride to protect cysteine. Deprotection requires hydrogen bromide-acetic acid, which is not only toxic but also has a strong pungent odor. The total yield of glutathione is low, only about 8%; Patent CN 102093468 A uses thionyl chloride to prepare glycine benzyl ester hydrochloride. The total yield of synthesizing reduced glutathione from cysteine is 12.7%. The yield is low and thionyl chloride is toxic, which is not suitable for large-scale production; Patent CN107573402A uses L-cystine to prepare acyl chloride through the protection of phthalic anhydride. The water separation temperature is 80-180°C, the solvent is thionyl chloride, and the reaction temperature is 40-80°C. The conditions are relatively severe, the reaction reagents are toxic and irritating, and the amino acid is easily racemized, which is not suitable for large-scale industrial production.

Therefore, it is of great significance to study and develop a new and efficient method for preparing glutathione.

Summary of the invention

In view of this, the technical problem to be solved by the present invention is to provide a preparation method of glutathione and its derivatives and their application. The preparation method has mild reaction conditions, fast reaction speed, and a high total yield of the product calculated based on cysteine as the starting material.

In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

The present invention provides a method for preparing glutathione and its derivatives, comprising the following steps:

1) mixing the compound represented by Formula 4, the compound represented by Formula 5, an organic base and a condensing agent to react to obtain the compound represented by Formula 6;

2) Mixing the compound represented by formula 6, a reagent for removing the protecting group of maleimide or its derivative, a reagent for removing the protecting group of 9-borahertabicyclo[3,3,1]nonane, an organic base and an organic solvent to obtain glutathione and its derivatives represented by formula a;

, , ,

Formula 4 Formula 5 Formula 6

Formula a;

Wherein, R ₁ is selected from one or more of H, C ₁ -C ₁₀ alkyl, C ₃ -C ₆ cycloalkyl;

R is selected from Br or H.

The preparation method of the present invention adopts maleimide or its derivatives to protect cysteine, adopts 9-borahertabicyclo[3,3,1]nonane protecting group reagent to selectively protect the α-carboxyl group of glutamic acid, and the two are combined to prepare glutathione and its derivatives shown in formula a under mild reaction conditions.

The glutathione can be directly prepared by the above method.

When R ₁ is selected from H, the formula a is glutathione.

Alternatively, the glutathione can also be prepared by hydrolyzing the glutathione derivative prepared by the above method.

The hydrolysis agent is selected from acids.

The acid includes but is not limited to hydrochloric acid and the like.

The C ₁ -C ₁₀ alkyl group includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl and the like.

The C ₃ -C ₆ cycloalkyl group includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

In the present invention, preferably, R ₁ is selected from one or more of H, C ₁ -C ₆ alkyl, C ₅ -C ₆ cycloalkyl;

Preferably, said R is selected from Br.

The C ₁ -C ₆ alkyl group includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, neopentyl, hexyl and the like.

The C ₅ -C ₆ cycloalkyl group includes, but is not limited to, cyclopentyl and cyclohexyl.

More preferably, the C ₁ -C ₆ alkyl group further contains a substituent;

The _R1 is selected from fluorenylmethyl or adamantylmethyl.

The structures of the above-mentioned fluorenylmethyl group or adamantylmethyl group are as follows:

.

The dash in the above fluorenylmethyl group or adamantylmethyl group merely indicates the connection position.

Preferably, in the step 1), the organic base is selected from one or more of triethylamine (TEA), pyridine, 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), N,N-dimethylaniline, and N,N-diisopropylethylamine (DIEA); more preferably, it is one or more of 1-hydroxybenzotriazole (HOBT), N,N-diisopropylethylamine, and triethylamine (TEA); further preferably, it is a mixture of 1-hydroxybenzotriazole and triethylamine or N,N-diisopropylethylamine.

The condensing agent in the step 1) is selected from one or more of N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate (PyAOP), and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU). More preferred are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) or N,N′-dicyclohexylcarbodiimide (DCC).

The organic base mentioned above plays a basic role in step 1) and also has a certain catalytic effect.

In some specific embodiments of the present invention, preferably, in step 1), the organic base is selected from a mixture of HOBT and TEA, and the condensing agent is selected from EDCI; or, the organic base is selected from N,N-diisopropylethylamine, and the condensing agent is selected from HATU;

Alternatively, the organic base is selected from a mixture of HOBT and TEA, and the condensing agent is selected from DCC.

Preferably, in the step 2), the protecting group reagent for removing maleimidyl or its derivative is selected from one or more of dithiothreitol (DTT), triphenylphosphine and tributylphosphine; more preferably, dithiothreitol.

In the present invention, the DTT can reduce the "-S-" in the compound shown in Formula 6 to a thiol "-SH" and then remove the maleimide or its derivative protecting group.

Preferably, in the step 2), the reagent for removing the protecting group of 9-borabicyclo[3,3,1]nonane (abbreviated as 9-BBN) is selected from one or more of tetrabutylammonium fluoride (TBAF), ammonium fluoride, tetrafluoroboric acid-diethyl ether complex, and hydrogen fluoride-pyridine complex; more preferably, it is tetrabutylammonium fluoride or ammonium fluoride; further preferably, it is tetrabutylammonium fluoride.

In the present invention, the tetrabutylammonium fluoride can cleave (S)-3-(5'-oxo-9λ ⁴ -boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxazaborolidine]-4'-yl)propanoic acid (abbreviated as 9-BBN-glutamic acid) to remove the 9-BBN protecting group.

Preferably, the organic base in step 2) is selected from one or more of triethylamine, pyridine, and 4-dimethylaminopyridine; more preferably, it is triethylamine or pyridine; and further preferably, it is triethylamine.

In the above step 1), preferably, the molar ratio of the compound represented by formula 4 and the organic base is 1:(1-1.2); in some specific embodiments of the present invention, it is preferably 1:1 or 1:1.1.

Preferably, the molar ratio of the compound represented by Formula 4 and the condensing agent is 1:(1-1.5); in some specific embodiments of the present invention, it is preferably 1:1.1, 1:1.2.

Preferably, the molar ratio of the compound represented by Formula 4 to the compound represented by Formula 5 is 1:(1-1.2). In some specific embodiments of the present invention, it is preferably 1:1.

In the above step 2), preferably, the molar ratio of the compound represented by formula 6 to the organic base is 1:(0.1-0.5). In some specific embodiments of the present invention, it is preferably 1:0.2, 1:0.3, 1:0.4.

Preferably, the molar ratio of the compound represented by Formula 6 to the maleimide-free or derivative protecting group reagent is 1:(3-10). In some specific embodiments of the present invention, the molar ratio is preferably 1:3.3, 1:4.4, or 1:5.5.

Preferably, the molar ratio of the compound represented by Formula 6 to the reagent for removing the 9-BBN protecting group is 1:(1-5). In some specific embodiments of the present invention, the molar ratio is preferably 1:2.0, 1:3.0, or 1:4.0.

In the preferred embodiment of the present invention, the method for preparing glutathione and its derivatives shown in formula a comprises the following steps:

1) The compound represented by formula 6 and a 9-BBN protecting group removal agent are mixed and reacted to obtain system S1;

2) Adding a maleimide-free or derivative protecting group reagent and an organic base to the above system S1 to react, thereby preparing glutathione and its derivatives shown in formula a.

The preparation method of glutathione and its derivatives shown in the above formula a first removes the 9-BBN protecting group through step 1) and then removes the protecting group of maleimide or its derivatives through step 2), so that the reaction rate and efficiency are better.

Preferably, the method for preparing the compound represented by formula 4 comprises the following steps:

1) The compound represented by Formula 2 and the glycine and its derivatives represented by Formula 7 react with a condensing agent and an organic base to obtain the compound represented by Formula 3;

2) reacting the compound represented by formula 3 with an acid to remove the tert-butyloxycarbonyl (Boc) group to obtain the compound represented by formula 4;

, ,

Formula 2 Formula 3 Formula 7.

Preferably, in the method for preparing the compound represented by formula 4, the condensing agent in step 1) is selected from one or more of N,N'-dicyclohexylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate, and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; more preferably, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N,N'-dicyclohexylcarbodiimide (DCC) or O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

Preferably, in the preparation method of the compound represented by Formula 4, the organic base is selected from one or more of triethylamine, pyridine, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, N,N-dimethylaniline, and N,N-diisopropylethylamine; more preferably, it is one or more of triethylamine, pyridine, 4-dimethylaminopyridine, and 1-hydroxybenzotriazole (HOBT); further preferably, it is 4-dimethylaminopyridine or 1-hydroxybenzotriazole.

In some specific embodiments of the present invention, preferably, in the method for preparing the compound represented by formula 4, the organic base in step 1) is selected from HOBT, and the condensing agent is selected from EDCl;

Alternatively, the organic base is selected from N,N-diisopropylethylamine, and the condensing agent is selected from HATU;

Alternatively, the organic base is selected from a mixture of HOBT and TEA, and the condensing agent is selected from DCC.

Preferably, in the method for preparing the compound of formula 4, the acid in step 2) is selected from one or more of hydrochloric acid, trifluoroacetic acid, and phosphoric acid. In some specific embodiments of the present invention, hydrochloric acid is preferred.

Preferably, in the method for preparing the compound represented by formula 4, the solvent for the reaction in step 2) is selected from one or more of methanol, ethanol, dioxane and ethyl acetate.

In the above step 1), preferably, the molar ratio of the compound represented by Formula 2 to the glycine and its derivatives represented by Formula 7 is 1:(1.0-1.3); in some specific embodiments of the present invention, it is preferably 1:1.1.

Preferably, the molar ratio of the compound represented by Formula 2 to the condensing agent is 1:(1.1-1.5); in some specific embodiments of the present invention, it is preferably 1:1.2 or 1:1.3.

Preferably, the molar ratio of the compound represented by Formula 2 to the organic base is 1:(1.1-2.0); in some specific embodiments of the present invention, it is preferably 1:1.2 or 1:1.5.

In the above step 2), preferably, the molar ratio of the compound represented by formula 3 to the acid is 1:(5-10). In some specific embodiments of the present invention, it is preferably 1:5, 1:6, or 1:8.

In the above preparation method, the compound represented by formula 2 is prepared by mixing maleimide or its derivatives with L-cysteine and di-tert-butyl dicarbonate under alkaline conditions. The compound represented by formula 5 is prepared by mixing glutamic acid, 9-BBN-glutamic acid and an organic solvent;

Among them, the molar ratio of glutamic acid to 9-BBN-glutamic acid is 1:(1.1-1.5).

The present invention also provides application of the above preparation method in preparing S-lactylglutathione, S-nitrosoglutathione or S-acetyl-L-glutathione.

By adopting the preparation method of the present invention, glutathione and its derivatives shown in formula a are first prepared, and then the sulfhydryl groups therein are further reacted to prepare S-lactylglutathione, S-nitrosoglutathione or S-acetyl-L-glutathione, so that the overall process of the preparation method of S-lactylglutathione, S-nitrosoglutathione or S-acetyl-L-glutathione is simple and efficient.

Compared with the prior art, the preparation method of glutathione and its derivatives provided by the present invention comprises the following steps: 1) mixing the compound shown in formula 4, the compound shown in formula 5, an organic base and a condensing agent to react to obtain the compound shown in formula 6; 2) mixing the compound shown in formula 6, a reagent for removing the protecting group of maleimide or its derivative, a reagent for removing the protecting group of 9-borahertabicyclo[3,3,1]nonane, an organic base and an organic solvent to react to obtain the glutathione and its derivatives shown in formula a; wherein R1 is selected from one or more of H, C1-C10 alkyl, and C3-C6 cycloalkyl; and R is selected from Br or H. The reaction conditions of the preparation method are mild, environmentally friendly, suitable for large-scale production, and the protection and deprotection of cysteine and glutamic acid in the preparation process are more efficient, and the amount of the protecting or deprotecting reagent is low, and the total yield calculated with glutamic acid as the starting material is improved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the nuclear magnetic spectrum of glutathione prepared in Example 1 ¹ H NMR (400 MHz, DMSO-δ ₆ ).

DETAILED DESCRIPTION

In order to further illustrate the present invention, the preparation method and application of glutathione and its derivatives provided by the present invention are described in detail below in conjunction with the embodiments.

The numbers below the compound structures in the following embodiment equations represent the compound numbers. For example, the product of the reaction equation in step 1 of embodiment 1 is compound 1. The other embodiments also have compound numbers. Among them, compound 4, compound 6, compound a, compound 2, compound 3, and compound 7 are the specific compound structures under the general formulas of formula 4, formula 6, formula a, formula 2, formula 3, and formula 7, respectively.

Example 1

Step 1. Synthesis of N-Boc-L-cysteine (1)

L-cysteine (6.01 kg, 50.0 mol) was dissolved in 0.6 N sodium hydroxide aqueous solution (50 L). After it was fully dissolved, 30.0 L of dioxane was added, and di-tert-butyl dicarbonate Boc ₂ O (15.26 kg, 70.0 mol) was added dropwise at a uniform rate while stirring at 0℃～5℃. After the addition was completed, the mixture was stirred at room temperature overnight. The dioxane was removed by rotary evaporation, and the remaining Boc ₂ O was washed away with ethyl acetate. The aqueous layer was adjusted to pH 1-2 with 0.1 N dilute hydrochloric acid, and then extracted with ethyl acetate for 3 times. The organic layers were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a colorless syrupy product 1 (9.94 kg, yield 90.0%). 1H NMR (400 MHz, DMSO-d ₆ ): 12.83(s, 2H), 7.21 (d, J = 8.4 Hz, 2H), 4.20–4.14 (m, 2H), 3.12–3.08 (m, 2H), 2.91–2.85 (m, 2H), 1.38 (s, 9H);

Step 2: Synthesis of S-maleimide-N-Boc-L-cysteine (2)

3-Bromomaleimide (7.87 kg, 45.0 mol) and N-Boc-L-cysteine (9.94 kg, 45.0 mol) were dissolved in 100 L tetrahydrofuran, and triethylamine (13.75 kg, 135.0 mol) was added. The mixture was reacted at room temperature overnight, and the reaction end point was detected by HPLC. The excess solvent was removed by rotary evaporation, and the mixture was dissolved in ethyl acetate, and washed three times with distilled water. The organic phase was extracted and retained, and washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate. The filtrate was filtered and retained, and the solvent was removed by rotary evaporation. The mixture was dried in vacuo to obtain 12.75 kg of light yellow solid with a yield of 89.7%. 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 6.43 (d, 1H), 6.09 (s, 1H), 4.42 (dt, 1H), 3.51–3.37 (m, 2H), 1.42 (s, 9H).

Step 3. Synthesis of S-maleimide-N-Boc-L-cysteinyl-glycine ethyl ester (3)

N-maleimide-N-Boc-L-cysteine (12.75 kg, 40.4 mol) and glycine ethyl ester (4.10 kg, 46.1 mol) were dissolved in 200 L tetrahydrofuran, and EDCI (9.29 kg, 48.5 mol), HOBT (7.09 kg, 52.5 mol) and TEA (6.13 kg, 60.6 mol) were added and stirred at room temperature overnight. THF was removed by rotary evaporation, and the mixture was washed with ethyl acetate/water three times. The organic phases were extracted and combined, filtered and evaporated to dryness. The product was precipitated in a cold solution of methyl tert-butyl ether, and 12.47 kg of white solid (3) was obtained by slurrying and filtration. The yield was 77%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.18 (t, 1H), 6.95 (s, 1H), 6.68 (d, 1H), 4.50 (dt, 1H), 4.18 (q, 2H), 3.96 (dd,2H), 3.26 (t, 2H), 1.41(s, 9H), 1.21(t, 3H).

Step 4. Synthesis of S-maleimide-L-cysteinyl-glycine ethyl ester (4)

The dipeptide compound 3 (12.47 kg, 31.1 mol) obtained in step 3 was dissolved in 77.75 L HCl-EtOH (2M), stirred at room temperature for 4 h, and after HPLC detection, the solvent was removed by rotary evaporation to obtain product 4, 8.56 kg, with a yield of 91.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (t, 1H), 6.97 (s, 1H), 4.15 (q, 2H), 3.97 (td, 1H),3.91 (dd, 2H), 3.82 (dd, 1H), 3.65 (dd, 1H), 3.34 – 3.23 (m, 2H), 1.24 (t, J= 6.6 Hz, 3H).

Step 5. Synthesis of (S)-3-(5'-oxo-9λ4-boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxazaborolidine]-4'-yl)propanoic acid (abbreviated as 9-BBN-glutamic acid, 5)

Glutamic acid (1.47 kg, 10.0 mol) and 9-BBN-OMe (1.67 kg, 11.0 mol) were dissolved in 15 L methanol. The reaction was monitored by HPCL. Hexane was added for washing (3 times, 2 L each time). The filter cake was filtered and retained, dissolved in 2.5 L ethyl acetate solution, washed 3 times with 0.5 M hydrochloric acid solution, 3 times with 0.5 M NaOH solution, and 3 times with saturated NaCl solution. The organic phases were combined, the solvent was dried over anhydrous sodium sulfate, the filtrate was filtered and retained, and concentrated to obtain 1.64 kg of light yellow viscous liquid with a yield of 61.4%. 1H NMR(400 MHz, DMSO-d6) δ 6.53 (dd, 1H), 5.87 (dd, 1H), 3.61 (qd, 1H), 2.10-2.01(m, 1H), 1.80-1.62 (m, 6H), 1.69–1.52 (m, 5H), 1.55–1.3 0 (m, 2H) ,0.51(s,1H), 0.46(s,1H).

Step 6. Synthesis of S-maleimide-L-cysteinyl-ethyl ester glycyl-9-BBN-glutamic acid (6)

Compound 4 (8.56 kg, 28.5 mol), compound 5 (7.61 kg, 28.5 mol), EDCI (6.01 kg, 31.3 mol), HOBT (1-hydroxybenzotriazole) (4.62 kg, 34.2 mol) were dissolved in 30 L THF, and TEA (2.88 kg, 28.5 mol) was added. After reacting at room temperature overnight, the organic phase was washed with ethyl acetate/water and evaporated to obtain 18.34 kg of crude product.

Step 7: Synthesis of Glutathione

The crude product of step 6 was dissolved in 120 L THF, and TBAF (tetrabutylammonium fluoride, 17.98 kg, 57.0 mol) was added to remove the protecting group 9-BBN. After spot plate detection, dithiothreitol DTT (14.43 kg, 93.5 mol) and TEA (0.66 kg, 6.56 mol) were added to remove the maleimide protecting group. The reaction solution was concentrated to remove the organic solvent and then dissolved in ethyl acetate. After washing with water, the separated liquid was dehydrated by anhydrous sodium sulfate, and the filtrate was retained after filtration. The organic solvent was concentrated after washing with a silica gel column to obtain 6.31 kg of glutathione ethyl ester with a yield of 66%. Calculated based on cysteine as the starting material, the total yield of glutathione ethyl ester in Example 1 was 37.5%.

The obtained compound a (glutathione ethyl ester) was dissolved in 0.4M dilute hydrochloric acid and glutathione was obtained by rotary evaporation at 60°C with a yield of 92.1%. The hydrogen spectrum data of glutathione are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.34 (s, 1H), 4.34 (s, 1H), 3.69 (s, 2H), 3.38 (s, 1H), 2.81 (s, 1H), 2.69(s, 1H), 2.35 (s, 2H), 1.94 (s, 2H).

Example 2

Step 1. Synthesis of N-Boc-L-cysteine (1)

L-cysteine (6.0 kg, 50.0 mol) was dissolved in saturated potassium bicarbonate aqueous solution (50 L). After it was fully dissolved, 30 L of dioxane was added, and di-tert-butyl dicarbonate Boc ₂ O (17.0 kg, 70.0 mol) was added dropwise at a uniform rate while stirring at 0℃～5℃. After the addition was completed, it was stirred at room temperature overnight. The dioxane was removed by rotary evaporation, and the remaining Boc2O was washed with ethyl acetate. The aqueous layer was adjusted to pH 1-2 with 0.1 N dilute hydrochloric acid, and then extracted with ethyl acetate for 3 times. The organic layers were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a colorless syrupy product 1 (7.85 kg, 72%).

Step 2: Synthesis of S-maleimide-N-Boc-L-cysteine (2)

Maleimide (6.21 kg, 35.5 mol) and N-Boc-L-cysteine (7.85 kg, 35.5 mol) were dissolved in 100 L tetrahydrofuran, and triethylamine (10.9 L, 108.1 mol) was added. The mixture was reacted at room temperature overnight, and the reaction end point was detected by HPLC. The excess solvent was removed by rotary evaporation, and the mixture was dissolved in ethyl acetate, washed three times with distilled water, and the organic phase was retained by extraction, washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate. The filtrate was filtered and retained, and the solvent was removed by rotary evaporation. The mixture was dried in vacuo to obtain 9.87 kg of a light yellow solid with a yield of 88%.

Step 3. Synthesis of S-maleimide-N-Boc-L-cysteinyl-glycine ethyl ester (3)

S-maleimide-N-Boc-L-cysteine (9.87 kg, 31.2 mol) and glycine ethyl ester (3.19 kg, 35.8 mol) were dissolved in 350 L of tetrahydrofuran, and N,N-diisopropylethylamine (4.82 kg, 37.3 mol) and HATU (15.34 kg, 40.3 mol) were added. The mixture was stirred at room temperature overnight, and THF was removed by rotary evaporation. The mixture was washed with ethyl acetate/water three times, and the organic phases were extracted and combined, filtered, and evaporated to dryness. The product was precipitated in a cold solution of methyl tert-butyl ether, and 9.14 kg of white solid (3) was obtained by slurrying and filtration. The yield was 73%.

Step 4. Synthesis of S-maleimide-L-cysteinyl-glycine ethyl ester (4)

The S-maleimide-N-Boc-L-cysteinyl-glycine ethyl ester (9.14 kg, 22.8 mol) obtained in step 3 was dissolved in 93.60 L HCl-AcOEt (2M), stirred at room temperature for 4 h, and after HPLC detection, the solvent was removed by rotary evaporation to obtain the product S-maleimide-L-cysteinyl-glycine ethyl ester without Boc protecting group, 6.16 kg, with a yield of 89.7%.

Step 5. Synthesis of 9-BBN-glutamic acid (5)

Glutamic acid (1.47 kg, 10.0 mol) and 9-BBN-OMe (1.83 kg, 12 mol) were dissolved in 15 L methanol. The reaction was monitored by HPCL. Hexane was added for washing (3 times, 2 L each time). The filter cake was filtered and retained, dissolved in 2.5 L ethyl acetate solution, washed 3 times with 0.5 M hydrochloric acid solution, 3 times with 0.5 M NaOH solution, and 3 times with saturated NaCl solution. The organic phases were combined, the solvent was dried over anhydrous sodium sulfate, the filtrate was filtered and retained, and concentrated to obtain 1.83 kg of light yellow viscous liquid with a yield of 68.5%.

Step 6. Synthesis of S-maleimide-L-cysteinyl-ethyl ester glycyl-9-BBN-glutamic acid (6)

Compound 4 (6.16 kg, 20.4 mol), compound 5 (5.45 kg, 20.4 mol), diisopropylethylamine (2.91 kg, 22.49 mol), and HATU (9.34 kg, 24.58 mol) were dissolved in 160 L THF and reacted at room temperature overnight. The solvent was evaporated to obtain 9.89 kg of crude product.

Step 7: Synthesis of Glutathione

The crude product of step 6 was dissolved in 2 L THF, and tetrabutylammonium fluoride TBAF (21.34 kg, 81.6 mol) was added in sequence. After spot plate detection, dithiothreitol DTT (17.19 kg, 111.42 mol) and triethylamine 0.83 kg (8.16 mol) were added. The mixture was treated and purified according to the method in Example 1 to obtain 3.43 kg of glutathione ethyl ester with a yield of 50.1% (the yield was calculated by combining steps 6 and 7). The total yield of glutathione ethyl ester in Example 2 was 20.4% using cysteine as the starting material.

The compound a (glutathione ethyl ester) obtained above was dissolved in 0.4 M dilute hydrochloric acid and subjected to rotary evaporation at 60° C. to obtain glutathione with a yield of 95.2%.

Example 3

Step 1. Synthesis of N-Boc-L-cysteine (1)

L-cysteine (6.0 kg, 50.0 mol) was dissolved in 0.6 N sodium hydroxide aqueous solution (50 L). After it was fully dissolved, 30 L of dioxane was added, and di-tert-butyl dicarbonate Boc ₂ O (17.0 kg, 70.0 mol) was added dropwise at a uniform rate while stirring at 0℃～5℃. After the addition was completed, the mixture was stirred overnight at room temperature. The dioxane was removed by rotary evaporation, and the remaining Boc ₂ O was washed away by ethyl acetate. The aqueous layer was adjusted to pH 1-2 with 0.1 N dilute hydrochloric acid, and then extracted with ethyl acetate for 3 times. The organic layers were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a colorless syrupy product 1 (7.66 kg, 70%).

Step 2: Synthesis of S-maleimide-N-Boc-L-cysteine (2)

3-Bromomaleimide (6.12 kg, 35.0 mol) and N-Boc-L-cysteine (7.74 kg, 35.0 mol) were dissolved in 100 L tetrahydrofuran, and pyridine (8.43 kg, 106.56 mol) was added. The mixture was reacted at room temperature overnight, and the reaction end point was detected by HPLC. The excess solvent was removed by rotary evaporation, and the mixture was dissolved in ethyl acetate, and washed three times with distilled water. The organic phase was extracted and retained, and washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate. The filtrate was filtered and retained, and the solvent was removed by rotary evaporation. The mixture was dried in vacuo to obtain 9.07 kg of a light yellow solid with a yield of 82%.

Step 3. Synthesis of S-maleimide-N-Boc-L-cysteinyl-glycine ethyl ester (3)

S-maleimide-N-Boc-L-cysteine (9.07 kg, 28.7 mol), glycine ethyl ester (2.93 kg, 32.9 mol), and TEA (4.39 kg, 43.4 mol) were dissolved in 120 L tetrahydrofuran, and DCC (7.08 kg, 34.3 mol) and HOBT (5.01 kg, 37.1 mol) were added. The mixture was stirred at room temperature overnight, and THF was removed by rotary evaporation. The mixture was washed with ethyl acetate/water three times, and the organic phases were extracted and combined, filtered, and evaporated to dryness. The product was precipitated in a cold solution of methyl tert-butyl ether, and 8.86 kg of white solid was obtained by slurrying and filtration. The yield was 77%.

Step 4. Synthesis of S-maleimide-L-cysteinyl-glycine ethyl ester (4)

The dipeptide compound S-maleimide-L-cysteinyl-glycine ethyl ester (8.86 kg, 22.09 mol) obtained in step 3 was dissolved in 88.36 L HCl-MeOH (2M) and stirred at room temperature for 4 h. After HPLC detection, the solvent was removed by rotary evaporation to obtain the product 4 without the Boc protecting group, 6.27 kg, with a yield of 94.3%.

Step 5. Synthesis of 9-BBN-glutamic acid (5)

Glutamic acid (1.47 kg, 10.0 mol) and 9-BBN-OMe (2.23 kg, 15.0 mol) were dissolved in 15 L methanol. The reaction was monitored by HPCL. Hexane was added for washing (3 times, 2 L each time). The filter cake was retained by filtration and dissolved in 2.5 L ethyl acetate solution. The mixture was washed 3 times with 0.5 M hydrochloric acid solution, 3 times with 0.5 M NaOH solution, and 3 times with saturated NaCl solution. The organic phases were combined, the solvent was dried over anhydrous sodium sulfate, and filtered to obtain the product (2.29 kg, 8.57 mol) with a yield of 85.7%.

Step 6. Synthesis of S-maleimide-L-cysteinyl-ethyl ester glycyl-9-BBN-glutamic acid (6)

Compound 4 (6.26 kg, 20.83 mol), compound 5 (5.56 kg, 20.83 mol), DCC (4.74 kg, 23.0 mol), and HOBT (3.39 kg, 25.10 mol) were dissolved in 20 L THF, and TEA (2.11 kg, 20.83 mol) was added. The reaction was carried out at room temperature overnight, and the solvent was evaporated to remove the solvent. The condensation agent was washed with water and evaporated to dryness to obtain 11.6 kg of crude product.

Step 7: Synthesis of Glutathione

The crude product of step 6 was dissolved in 80 L THF, and TBAF (16.34 kg, 62.5 mol), DTT (9.64 kg, 62.5 mol), and TEA (0.67 kg, 6.67 mol) were added in sequence to obtain 4.11 kg of glutathione ethyl ester with a yield of 59% (the yield of steps 6 and 7 was calculated together). Taking cysteine as the starting material, the total yield of glutathione ethyl ester in Example 3 was 24.5%.

The compound a (glutathione ethyl ester) obtained above was dissolved in 0.4 M dilute hydrochloric acid and subjected to rotary evaporation at 60° C. to obtain glutathione with a yield of 93.1%.

The above embodiments are only used to help understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims (10)

1. A method for preparing glutathione and its derivatives, characterized in that it comprises the following steps: 1) mixing the compound represented by Formula 4, the compound represented by Formula 5, an organic base and a condensing agent to react to obtain the compound represented by Formula 6; 2) Mixing the compound represented by formula 6, a reagent for removing the protecting group of maleimide or its derivative, a reagent for removing the protecting group of 9-borahertabicyclo[3,3,1]nonane, an organic base and an organic solvent to obtain glutathione and its derivatives represented by formula a; , , , Formula 4 Formula 5 Formula 6 Formula a; Wherein, R ₁ is selected from one or more of H, C ₁ -C ₁₀ alkyl, C ₃ -C ₆ cycloalkyl; R is selected from Br or H. 2. The preparation method according to claim 1, characterized in that _R1 is selected from one or more of H, C1 _{- C6} alkyl, _C5 - _C6 cycloalkyl; The R is selected from Br. 3. The preparation method according to claim 2, characterized in that the C ₁ -C ₆ alkyl group further contains a substituent; The _R1 is selected from fluorenylmethyl or adamantylmethyl. 4. The preparation method according to claim 1, characterized in that the organic base in step 1) is selected from one or more of triethylamine, pyridine, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, N,N-dimethylaniline, and N,N-diisopropylethylamine. 5. The preparation method according to claim 1, characterized in that the condensing agent in step 1) is selected from one or more of N,N'-dicyclohexylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate, and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. 6. The preparation method according to claim 1, characterized in that the maleimide removal or its derivative protecting group reagent in step 2) is selected from one or more of dithiothreitol, triphenylphosphine, and tributylphosphine; In the step 2), the reagent for removing the 9-borahertabicyclo[3,3,1]nonane protecting group is selected from one or more of tetrabutylammonium fluoride, ammonium fluoride, tetrafluoroboric acid-diethyl ether complex, and hydrogen fluoride-pyridine complex; In the step 2), the organic base is selected from one or more of triethylamine, pyridine and 4-dimethylaminopyridine. 7. The preparation method according to any one of claims 1 to 6, characterized in that the preparation method of the compound represented by formula 4 comprises the following steps: 1) The compound represented by Formula 2 and the glycine and its derivatives represented by Formula 7 react with a condensing agent and an organic base to obtain the compound represented by Formula 3; 2) reacting the compound represented by formula 3 with an acid to remove the tert-butyloxycarbonyl group to obtain the compound represented by formula 4; , , Formula 2 Formula 3 Formula 7. 8. The preparation method according to claim 7, characterized in that the condensing agent in step 1) is selected from one or more of N,N'-dicyclohexylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate, and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; The organic base is selected from one or more of triethylamine, pyridine, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, N,N-dimethylaniline, and N,N-diisopropylethylamine. 9. The preparation method according to claim 7, characterized in that the acid in step 2) is selected from one or more of hydrochloric acid, trifluoroacetic acid, and phosphoric acid; The solvent for the reaction in step 2) is selected from one or more of methanol, ethanol, dioxane and ethyl acetate. 10. Use of the preparation method according to any one of claims 1 to 9 in the preparation of S-lactylglutathione, S-nitrosoglutathione or S-acetyl-L-glutathione.