Efficient preparation method of Fmoc-Lys (Boc)-OH

Abstract

The invention discloses an efficient preparation method of Fmoc-Lys (Boc)-OH, which comprises the following steps: A, grinding and uniformly mixing lithium hydroxide and L-lysine hydrochloride in proportion to obtain mixed powder; b, benzaldehyde is added into water, the mixed powder is added in a stirring state, and after the reaction is finished, centrifugation, pulping, re-centrifugation and drying are performed to obtain H-Lys (Bal)-OH; c, the H-Lys (Bal)-OH and 9-fluorenyl methyl-N-succinimido carbonic ester are subjected to a reaction, and Fmoc-Lys-OH is obtained after hydrolysis; and D, Fmoc-Lys-OH and di-tert-butyl dicarbonate are subjected to a reaction, and the product is obtained. By innovating the preparation process of the target product, under the condition of ensuring low cost and efficient preparation of the target product, no heavy metal reagent is used, the treatment difficulty of the obtained waste liquid is greatly reduced, the product quality is obviously improved, and the method is very suitable for industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical intermediate preparation, and in particular to a high-efficiency preparation method of Fmoc-Lys(Boc)-OH. Background Art

[0002] Fmoc-Lys(Boc)-OH is a protected amino acid, an organic synthesis intermediate and a pharmaceutical intermediate. It can be used in biochemical research, organic synthesis and pharmaceutical research and development. Its structural formula is shown below:

[0003]

[0004] Since the key starting material L-lysine has two amino groups in its molecular structure and has almost the same chemical properties, a highly selective synthesis method is required. Liu Meiyan, Zeng Youlin, Yang Pengbiao and others reported in the journal "Fine Chemical Intermediates" that copper salts were used to form complexes with carboxyl and α-amino groups, first protecting the Boc group on the side chain, then removing the copper ion, and adding the Fmoc protecting group to the α-amino group. The advantages of this method are high selectivity, and the reagents used are all common reagents and low cost. The disadvantage is that a large amount of heavy metal salts (copper salts) are used, which not only increases the difficulty of wastewater treatment, but also the samples are prone to excessive heavy metals (or ignition residues) and unqualified properties (color), which is not suitable for industrial mass production. Summary of the invention

[0005] The purpose of the present invention is to provide a simple, efficient and environmentally friendly Fmoc-Lys(Boc)-OH preparation method for the above-mentioned problems. The present invention is summarized through experimental research. The starting material is mixed with lithium hydroxide and then reacted with benzaldehyde, and then the target product is prepared by a conventional method. Under the condition of ensuring low cost and efficient preparation of the target product, heavy metal reagents do not need to be used, the difficulty of treating the obtained waste liquid is greatly reduced, the product quality is significantly improved, and the quality stability is good. It is very suitable for industrial-scale production and overcomes the shortcomings of the existing process.

[0006] The technical solution adopted by the present invention is as follows: an efficient preparation method of Fmoc-Lys(Boc)-OH, comprising the following steps:

[0007] A. Grind and mix solid powdered lithium hydroxide (preferably without sodium hydroxide, which is too alkaline and easily leads to amino acid racemization) and solid powdered L-lysine hydrochloride in proportion to obtain a mixed powder;

[0008] B. Add benzaldehyde to water, stir for a period of time, add mixed powder to the mixed solution of benzaldehyde and water at a certain speed under stirring, control the temperature at 20-30° C. to react, centrifuge after the reaction, beat, centrifuge again, and dry to obtain H-Lys(Bal)-OH;

[0009] C. reacting the obtained H-Lys(Bal)-OH with 9-fluorenylmethyl-N-succinimidyl carbonate to obtain Fmoc-Lys-OH after hydrolysis;

[0010] D. The obtained Fmoc-Lys-OH is reacted with di-tert-butyl dicarbonate to obtain Fmoc-Lys(Boc)-OH.

[0011] In the above preparation method, the chemical reaction formula involved is as follows:

[0012]

[0013] Furthermore, the molar ratio of solid powdered lithium hydroxide to solid powdered L-lysine hydrochloride is 0.8-1.3:1, for example, it can be 0.8:1, 0.9:1, 1:1, 1.05:1, 1.1:1, 1.3:1, etc.

[0014] Further, the molar ratio of benzaldehyde to L-lysine hydrochloride is 1.02-1.1:1, for example, it can be 1.02:1, 1.03:1, 1.05:1, 1.08:1, 1.09:1, 1.1:1, etc.

[0015] Furthermore, the mass ratio of benzaldehyde to water is 1:4-6, for example, it can be 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0016] Furthermore, the adding speed of the mixed powder is 0.5-2 kg / min, for example, it can be 0.5 kg / min, 0.8 kg / min, 1.0 kg / min, 1.2 kg / min, 1.5 kg / min, 2 kg / min, etc.

[0017] Furthermore, in step B, the solvent used for pulping is selected from one or more of ethanol, n-propanol, isopropanol, and tert-butanol.

[0018] Furthermore, in step C, the molar ratio of 9-fluorenylmethyl-N-succinimidyl carbonate to H-Lys(Bal)-OH is 0.9-1.0:1, for example, 0.9:1, 0.93:1, 0.95:1, 0.98:1, 1.0:1, etc.

[0019] Further, in step C, H-Lys(Bal)-OH reacts with 9-fluorenylmethyl-N-succinimidyl carbonate in an organic solvent, and the organic solvent is selected from one or more of methanol, ethanol, n-propanol, and isopropanol.

[0020] Further, in step D, the molar ratio of di-tert-butyl dicarbonate to Fmoc-Lys-OH is 1.1-1.5:1, for example, 1.1:1, 1.15:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, etc.

[0021] In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention has found through experimental research that L-lysine hydrochloride is used as a starting material, which is mixed with lithium hydroxide and then reacted with benzaldehyde to obtain H-Lys(Bal)-OH, H-Lys(Bal)-OH reacts with 9-fluorenylmethyl-N-succinimidyl carbonate and hydrolyzes to obtain Fmoc-Lys-OH, and Fmoc-Lys-OH reacts with di-tert-butyl dicarbonate to obtain the target product. The present invention mixes the starting material with lithium hydroxide and then reacts with benzaldehyde, and then prepares the target product by a conventional method. This method does not use heavy metal reagents while ensuring low cost and high efficiency in preparing the target product, and the difficulty of treating the obtained waste liquid is greatly reduced, the product quality is significantly improved, and the quality stability is good, which is very suitable for industrial-scale production and overcomes the shortcomings of the existing process. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The following terms or definitions are provided merely to aid understanding of the present invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0024] Unless otherwise defined hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the present invention are intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0025] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0026] Example 1

[0027] (1) Preparation of H-Lys(Bal)-OH:

[0028] 12 kg of solid powder lithium hydroxide and 91 kg of solid powder L-lysine hydrochloride (molecular weight 182.65) were ground and mixed uniformly to obtain a mixed powder; 250 kg of water was added to a 500 L glass-lined reactor, 55 kg of benzaldehyde (molecular weight 106.12) was added to the water, and after stirring and mixing, the mixed powder was added to the mixed solution of benzaldehyde and water at a rate of 1.0 kg / min under stirring (50 Hz), and the temperature was controlled at 20-30° C. to react for 1 hour. After the reaction was completed, the mixture was centrifuged, and 182 kg of ethanol was used for pulping for 1 hour. The mixture was centrifuged and dried to obtain 102 kg of white solid (H-Lys(Bal)-OH, molecular weight 234.29), with a yield of 87.4%;

[0029] (2) Preparation of Fmoc-Lys-OH:

[0030] Add 306 kg methanol, 102 kg H-Lys(Bal)-OH, and 132 kg triethylamine (molecular weight 101.19) to a 1000 L glass-lined reactor, control the temperature at 20-30° C., add 137 kg 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSu) (molecular weight 337.33) in batches, and control the temperature at 20-30° C. to react; after the reaction is completed, concentrate to dryness, add 306 kg water and 102 kg ethyl acetate, adjust the pH value to 1-2 with hydrochloric acid, stir for 1 hour, discard the organic phase, adjust the pH value of the aqueous phase to 6-7, centrifuge, and dry to obtain 133 kg white solid (Fmoc-Lys-OH, molecular weight 368.43), yield: 82.9%;

[0031] (3) Preparation of Fmoc-Lys(Boc)-OH:

[0032] To a 3000L glass-lined reactor, 1330kg of water, 266kg of ethanol, 133kg of Fmoc-Lys-OH and 76kg of sodium carbonate were added, and 95kg of di-tert-butyl dicarbonate (molecular weight 218.25) was added dropwise at a temperature of 20-30°C. After the addition was completed, the temperature was controlled at 20-30°C for reaction. After the reaction was completed, 665kg of ethyl acetate was added, and the pH value of the aqueous phase was adjusted to 2-3 with a citric acid aqueous solution. The aqueous phase was discarded, and the organic phase was washed with a sodium chloride aqueous solution to a pH value of 6-7. After concentration, 665kg of petroleum ether was added for dispersion, and 139kg of white solid (Fmoc-Lys(Boc)-OH, molecular weight 468.54) was obtained after centrifugation and drying. The purity detected by HPLC was 99.5%, the yield was 82.2%, and the total yield was 60.0%.

[0033] Example 2

[0034] (1) Preparation of H-Lys(Bal)-OH:

[0035] 12 kg of solid powder lithium hydroxide and 91 kg of solid powder L-lysine hydrochloride (molecular weight 182.65) were ground and mixed uniformly to obtain a mixed powder; 250 kg of water was added to a 500 L glass-lined reactor, 57.3 kg of benzaldehyde (molecular weight 106.12) was added to the water, and after stirring and mixing, the mixed powder was added to the mixed solution of benzaldehyde and water at a rate of 1.2 kg / min under stirring (50 Hz), and the temperature was controlled at 20-30° C. to react for 1 hour. After the reaction was completed, the mixture was centrifuged, and 182 kg of isopropanol was used for pulping for 1 hour. The mixture was centrifuged and dried to obtain 106 kg of white solid (H-Lys(Bal)-OH, molecular weight 234.29), with a yield of 90.8%;

[0036] (2) Preparation of Fmoc-Lys-OH:

[0037] Add 318 kg of ethanol, 106 kg of H-Lys(Bal)-OH, and 160 kg of triethylamine (molecular weight 101.19) to a 1000 L glass-lined reactor, control the temperature at 20-30° C., add 142 kg of 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSu) (molecular weight 337.33) in batches, and control the temperature at 20-30° C. to react after the addition; after the reaction is completed, concentrate to dryness, add 318 kg of water and 106 kg of ethyl acetate, adjust the pH value to 1-2 with hydrochloric acid, stir for 1 hour, discard the organic phase, adjust the pH value of the aqueous phase to 6-7, centrifuge, and dry to obtain 141 kg of white solid (Fmoc-Lys-OH, molecular weight 368.43), yield: 84.6%;

[0038] (3) Preparation of Fmoc-Lys(Boc)-OH:

[0039] To a 3000L glass-lined reactor, 1410kg of water, 282kg of ethanol, 141kg of Fmoc-Lys-OH and 80kg of sodium carbonate were added, and 108kg of di-tert-butyl dicarbonate (molecular weight 218.25) was added dropwise at a temperature of 20-30°C. After the addition was completed, the temperature was controlled at 20-30°C for reaction. After the reaction was completed, 705kg of ethyl acetate was added, and the pH value of the aqueous phase was adjusted to 2-3 with a citric acid aqueous solution. The aqueous phase was discarded, and the organic phase was washed with a sodium chloride aqueous solution to a pH value of 6-7. After concentration, 705kg of petroleum ether was added for dispersion, and 152kg of white solid (Fmoc-Lys(Boc)-OH, molecular weight 468.54) was obtained after centrifugation and drying. The purity detected by HPLC was 99.6%, the yield was 84.8%, and the total yield was 65.1%.

[0040] Example 3

[0041] (1) Preparation of H-Lys(Bal)-OH:

[0042] 12 kg of solid powder lithium hydroxide and 91 kg of solid powder L-lysine hydrochloride (molecular weight 182.65) were ground and mixed uniformly to obtain a mixed powder; 250 kg of water was added to a 500 L glass-lined reactor, 60 kg of benzaldehyde (molecular weight 106.12) was added to the water, and after stirring and mixing, the mixed powder was added to the mixed solution of benzaldehyde and water at a rate of 0.8 kg / min under stirring (50 Hz), and the temperature was controlled at 20-30° C. to react for 1 hour. After the reaction was completed, centrifugation was performed, and 182 kg of ethanol was used for pulping for 1 hour. Centrifugation and drying were performed to obtain 108 kg of white solid (H-Lys(Bal)-OH, molecular weight 234.29), with a yield of 92.5%;

[0043] (2) Preparation of Fmoc-Lys-OH:

[0044] Add 324 kg methanol, 108 kg H-Lys(Bal)-OH, and 163 kg triethylamine (molecular weight 101.19) to a 1000 L glass-lined reactor, control the temperature at 20-30° C., add 145 kg 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSu) (molecular weight 337.33) in batches, and control the temperature at 20-30° C. to react; after the reaction is completed, concentrate to dryness, add 324 kg water and 108 kg ethyl acetate, adjust the pH value to 1-2 with hydrochloric acid, stir for 1 hour, discard the organic phase, adjust the pH value of the aqueous phase to 6-7, centrifuge, and dry to obtain 147 kg white solid (Fmoc-Lys-OH, molecular weight 368.43), yield: 86.6%;

[0045] (3) Preparation of Fmoc-Lys(Boc)-OH:

[0046] To a 3000L glass-lined reactor, 1470kg of water, 294kg of ethanol, 147kg of Fmoc-Lys-OH and 84kg of sodium carbonate were added, and 104kg of di-tert-butyl dicarbonate (molecular weight 218.25) was added dropwise at a temperature of 20-30°C. After the addition was completed, the temperature was controlled at 20-30°C for reaction. After the reaction was completed, 735kg of ethyl acetate was added, and the pH value of the aqueous phase was adjusted to 2-3 with a citric acid aqueous solution. The aqueous phase was discarded, and the organic phase was washed with a sodium chloride aqueous solution to a pH value of 6-7. After concentration, 735kg of petroleum ether was added for dispersion, and 156kg of white solid (Fmoc-Lys(Boc)-OH, molecular weight 468.54) was obtained after centrifugation and drying. The purity detected by HPLC was 99.7%, the yield was 83.4%, and the total yield was 66.8%.

[0047] Comparative Example 1

[0048] This example was prepared according to the literature method:

[0049] (1) Preparation of [Lys(Boc)]2Cu:

[0050] Add 1200 kg of water to a 3000 L glass-lined reactor, add 91 kg of L-lysine hydrochloride and 152 kg of basic copper carbonate under stirring (50 Hz), react at 90-100 ° C for 30 min, cool to about 60 ° C, filter, transfer the filtrate to another 3000 L glass-lined reactor, add 76 kg of sodium bicarbonate and 243 kg of ethanol, control the temperature at 20-30 ° C, add 152 kg of di-tert-butyl dicarbonate; after addition, react for 12 hours, centrifuge to obtain a solid, use 1200 kg of water each time to beat until no lysine remains, and the obtained solid ([Lys(Boc)]2Cu) is directly put into the next step;

[0051] (2) Preparation of Fmoc-Lys(Boc)-OH:

[0052] To a 3000L glass-lined reactor, 600kg of water, 100kg of sodium carbonate, [Lys(Boc)]2Cu solid, 100kg of EDTA-2Na, and then 300kg of ethyl acetate were added, and 100kg of 9-fluorenylmethyl-N-succinimidyl carbonate was added in batches under stirring. The temperature was controlled at 15-25°C to complete the reaction; after post-treatment and drying, 112kg of off-white solid was obtained, with a total yield of 48.0% (molecular weight 468.54), properties: off-white solid (slightly blue), and the purity detected by HPLC was: 99.3%.

[0053] From the results of comparative document 1, it can be seen that this method not only has a too long preparation time (the preparation time exceeds 12 hours, while the preparation time of the present invention does not exceed 8 hours), the total yield is about 48%, the purity is only 99.3%, and the property is blue, indicating that it obviously contains copper impurities. The preparation method of the present invention has a total yield of more than 60%, a product purity of more than 99.5%, does not use heavy metal reagents, and has obvious technical advantages.

[0054] Comparative Example 2

[0055] Referring to the preparation method of Example 1, lithium hydroxide and L-lysine hydrochloride were not pre-mixed when preparing H-Lys(Bal)-OH, and the preparation of other Fmoc-Lys-OH and Fmoc-Lys(Boc)-OH was the same as in Example 1, specifically:

[0056] (1) Preparation of H-Lys(Bal)-OH:

[0057] Add 250 kg of water and 12 kg of lithium hydroxide to a 500 L glass-lined reactor, stir until completely dissolved, then add 91 kg of L-lysine hydrochloride (molecular weight 182.65), add 55 kg of benzaldehyde (molecular weight 106.12) after complete dissolution, control the temperature at 20-30 ° C to react for 1 hour, centrifuge, and then beat with 182 kg of ethanol for 1 hour, centrifuge and dry to obtain 89 kg of white solid (H-Lys(Bal)-OH, molecular weight 234.29), yield: 76.3%;

[0058] (2) Preparation of Fmoc-Lys-OH:

[0059] Same as Example 1;

[0060] (3) Preparation of Fmoc-Lys(Boc)-OH:

[0061] Same as Example 1.

[0062] From the results of Comparative Example 2, it can be seen that the conventional feeding method has a great influence on the yield of preparing H-Lys(Bal)-OH, which is reduced from the original 87.4% to 76.3%, a large decrease, indicating that the feeding method of the present application can significantly improve the yield of H-Lys(Bal)-OH.

[0063] Comparative Example 3

[0064] In order to verify whether the feeding method of the present invention can improve the yield by itself, the feeding method of the present invention is introduced into Comparative Example 1 with reference to the preparation method of Comparative Example 1, specifically:

[0065] (1) Preparation of [Lys(Boc)]2Cu:

[0066] 91 kg of solid powder L-lysine hydrochloride and 152 kg of solid powder basic copper carbonate were ground and mixed uniformly to obtain a mixed powder; 1200 kg of water was added to a 3000 L glass-lined reactor, and the mixed powder was added at a rate of 1.0 kg / min under stirring (50 Hz), reacted at 90-100° C. for 30 min, cooled to about 60° C., filtered, and the filtrate was transferred to another 3000 L glass-lined reactor, and then 76 kg of sodium bicarbonate and 243 kg of ethanol were added, and 152 kg of di-tert-butyl dicarbonate was added at a temperature of 20-30° C.; after the addition was completed, the reaction was continued for 12 hours, and then the solid was centrifuged to obtain a solid, and 1200 kg of water was used each time to beat the slurry until no lysine remained, and the obtained solid ([Lys(Boc)]2Cu) was directly put into the next step;

[0067] (2) Preparation of Fmoc-Lys(Boc)-OH:

[0068] To a 3000L glass-lined reactor, 600kg of water, 100kg of sodium carbonate, [Lys(Boc)]2Cu solid, 100kg of EDTA-2Na, and then 300kg of ethyl acetate were added, and 100kg of 9-fluorenylmethyl-N-succinimidyl carbonate was added in batches under stirring. The temperature was controlled at 15-25°C to complete the reaction; after post-treatment and drying, 109kg of off-white solid was obtained, with a total yield of 47.0% (molecular weight 468.54), properties: off-white solid (slightly blue), and the purity detected by HPLC was: 99.3%.

[0069] It can be seen that when the feeding method of the present invention is applied to Comparative Example 1, the yield of the target product is not significantly improved, indicating that the feeding method of the present invention does not produce a positive effect alone when preparing H-Lys(Bal)-OH, but has a synergistic effect with the present invention using benzaldehyde to prepare H-Lys(Bal)-OH, thereby improving the yield.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An efficient preparation method of Fmoc-Lys(Boc)-OH, characterized in that: The steps include: A. Grind and mix solid powdered lithium hydroxide and solid powdered L-lysine hydrochloride in proportion to obtain a mixed powder; B. Add benzaldehyde to water, stir for a period of time, add mixed powder to the mixed solution of benzaldehyde and water at a certain speed under stirring, control the temperature at 20-30° C. to react, centrifuge after the reaction, beat, centrifuge again, and dry to obtain H-Lys(Bal)-OH; C. reacting the obtained H-Lys(Bal)-OH with 9-fluorenylmethyl-N-succinimidyl carbonate to obtain Fmoc-Lys-OH after hydrolysis; D. The obtained Fmoc-Lys-OH is reacted with di-tert-butyl dicarbonate to obtain Fmoc-Lys(Boc)-OH.

2. The efficient preparation method according to claim 1, characterized in that: The molar ratio of solid powder lithium hydroxide to solid powder L-lysine hydrochloride is 0.8-1.3:

1.

3. The efficient preparation method according to claim 1, characterized in that: The molar ratio of benzaldehyde to L-lysine hydrochloride is 1.02-1.1:

1.

4. The efficient preparation method according to claim 1, characterized in that: The mass ratio of benzaldehyde to water is 1:4-6.

5. The efficient preparation method according to claim 1, characterized in that: The adding speed of the mixed powder is 0.5-2kg / min.

6. The efficient preparation method according to any one of claims 1 to 5, characterized in that: In step B, the solvent used for pulping is selected from one or more of ethanol, n-propanol, isopropanol, and tert-butanol.

7. The efficient preparation method according to claim 6, characterized in that: In step C, the molar ratio of 9-fluorenylmethyl-N-succinimidyl carbonate to H-Lys(Bal)-OH is 0.9-1.0:

1.

8. The efficient preparation method according to claim 7, characterized in that: In step C, H-Lys(Bal)-OH reacts with 9-fluorenylmethyl-N-succinimidyl carbonate in an organic solvent, wherein the organic solvent is selected from one or more of methanol, ethanol, n-propanol, and isopropanol.

9. The efficient preparation method according to claim 8, characterized in that: In step D, the molar ratio of di-tert-butyl dicarbonate to Fmoc-Lys-OH is 1.1-1.5:1.

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