Preparation method of Fmoc-L-lysine

Abstract

The invention provides a preparation method of Fmoc-L-lysine, which comprises the following steps: S1, reacting L-lysine hydrochloride with aromatic aldehyde to generate an imine intermediate; and S2, enabling the imine intermediate to react with an Fmoc protection reagent, and hydrolyzing to generate Fmoc-L-lysine. According to the preparation method disclosed by the embodiment of the invention, an orthogonal protection method is adopted, firstly, aromatic aldehyde reacts with epsilon-NH2 to obtain an imine intermediate to play a protection role, then, an Fmoc protecting group is introduced into alpha-NH2, finally, the epsilon-NH2 is exposed again by utilizing the acid instability of the imine intermediate to obtain the Fmoc-L-lysine, the feasibility of the whole synthesis route is strong, and the synthesis process is simple and convenient. The total yield is high, the used raw materials are cheap and easy to obtain, and the synthesis cost is low.

Description

Technical Field

[0001] The invention belongs to the technical field of compound preparation, and more specifically, relates to a method for preparing Fmoc-L-lysine. Background Art

[0002] Peptide synthesis is a process in which different types of amino acids are connected by peptide bonds in a certain order. Some of the amino acids involved in the synthesis contain multiple active groups. In order to obtain a peptide in a specific order, these active groups must be protected first, and then the protective groups are removed after the peptide bonds are formed. Therefore, protected amino acids are important raw materials for peptide synthesis. It is of great practical value to study the synthesis process of protected amino acids and reduce production costs.

[0003] Among them, Fmoc-L-lysine (as shown in the following formula (1)) is widely used in peptide synthesis and can be used to prepare antibody-drug conjugates and synthesize PROTAC drug small molecules.

[0004]

[0005] At present, there are few methods for synthesizing Fmoc-L-lysine at home and abroad. The main method is to use L-lysine as the starting material and directly react with Fmoc-Osu or Fmoc-Cl to introduce the Fmoc protecting group. However, this method has poor selectivity and is prone to form a large amount of ε-NH2 protected byproduct N'-Fmoc-L-lysine (as shown in the following formula (2)). There are problems such as low yield and low purity of Fmoc-L-lysine.

[0006] Summary of the invention

[0007] In view of this, the object of the present invention is to provide a method for preparing Fmoc-L-lysine with a short synthetic route, high total yield, strong route feasibility, cheap and readily available raw materials, and low synthetic cost.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The method for preparing Fmoc-L-lysine according to an embodiment of the present invention comprises the following steps:

[0010] Step S1, reacting L-lysine hydrochloride with an aromatic aldehyde to generate an imine intermediate;

[0011] Step S2, reacting the imine intermediate with an Fmoc protecting agent and hydrolyzing the imine intermediate to generate Fmoc-L-lysine.

[0012] Furthermore, the step S1 comprises:

[0013] L-lysine hydrochloride is reacted with the aromatic aldehyde in a first solvent under the action of a first base to generate the imine intermediate.

[0014] Furthermore, the aromatic aldehyde is benzaldehyde, p-methoxybenzaldehyde, or a mixture thereof, the first base is any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, and sodium carbonate, and the first solvent is any one or more of water, methanol, ethanol, and tetrahydrofuran.

[0015] Furthermore, in the step S1, the molar ratio of L-lysine hydrochloride: first base: aromatic aldehyde is 1:(0.8-1.5):(0.8-1.2), the reaction temperature is -5-40°C, and the reaction time is 3-12h.

[0016] Furthermore, in the step S1, after the reaction is completed, the step further includes: filtering with suction, and slurrying the filter cake to obtain the imine intermediate.

[0017] Furthermore, the step S2 comprises:

[0018] The imine intermediate is reacted with the Fmoc protecting agent in a second solvent under the action of a second base, and dilute hydrochloric acid is added to adjust the pH to 1-2 to perform a hydrolysis reaction to generate the Fmoc-L-lysine.

[0019] Furthermore, the Fmoc protecting agent is selected from any one or more of Fmoc-Cl, Fmoc-Osu, and Fmoc-Amox.

[0020] Furthermore, the second base is sodium hydroxide, sodium carbonate, or a mixture thereof, and the second solvent is any one or more of water, methanol, ethanol, and tetrahydrofuran.

[0021] Furthermore, in the step S2, the molar ratio of the imine intermediate: the second base: the Fmoc-protecting agent is 1:1.0-3.5:1.0-1.5; the reaction temperature is -20-30°C; and the reaction time is 3-10 hours.

[0022] Furthermore, in the step S2, after the reaction is completed, the method further comprises: after the hydrolysis reaction is completed, adding sodium hydroxide solution to adjust to weak acidity, concentrating the organic reagent under reduced pressure, and beating the mixture to obtain the Fmoc-L-lysine.

[0023] The above technical solution of the present invention has at least one of the following beneficial effects:

[0024] According to the preparation method of the embodiment of the present invention, an orthogonal protection method is adopted. First, an aromatic aldehyde is reacted with ε-NH2 to obtain an imine intermediate for protection. Then, an Fmoc protecting group is introduced into α-NH2. Finally, the acid instability of the imine intermediate is utilized to re-expose ε-NH2 to obtain Fmoc-L-lysine. The feasibility of the entire synthetic route is strong, the total yield is high, the raw materials used are cheap and easily available, and the synthesis cost is low. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0026] The following first describes in detail the method for preparing Fmoc-L-lysine according to an embodiment of the present invention.

[0027] The method for preparing Fmoc-L-lysine according to an embodiment of the present invention comprises the following steps:

[0028] Step S1, reacting L-lysine hydrochloride with an aromatic aldehyde to generate an imine intermediate;

[0029] Step S2, reacting the imine intermediate with an Fmoc protecting agent and hydrolyzing the imine intermediate to generate Fmoc-L-lysine.

[0030] That is to say, the preparation method according to the embodiment of the present invention adopts an orthogonal protection method, that is, firstly, an aromatic aldehyde is reacted with ε-NH2 to obtain an imine intermediate for protection, then an Fmoc protecting group is introduced into α-NH2, and finally, the acid instability of the imine intermediate is utilized to re-expose ε-NH2 to obtain Fmoc-L-lysine. The feasibility of the entire synthetic route is strong, the total yield is high, the raw materials used are cheap and easily available, and the synthesis cost is low.

[0031] The following describes step S1 and step S2 respectively.

[0032] According to some embodiments of the present invention, step S1 comprises:

[0033] L-lysine hydrochloride is reacted with the aromatic aldehyde in a first solvent under the action of a first base to generate the imine intermediate.

[0034] Under the action of a base (ie, the first base), L-lysine hydrochloride is liberated to obtain a free state (ie, L-lysine radical), and the free state undergoes a condensation reaction with an aromatic aldehyde to obtain an imine intermediate.

[0035] Here, the aromatic aldehyde may be, for example, benzaldehyde, p-anisaldehyde, or a mixture thereof.

[0036] When the aromatic aldehyde is p-tolualdehyde, the reaction process is shown in the following formula (3) to obtain an imine intermediate (I):

[0037]

[0038] When the aromatic aldehyde is benzaldehyde, the reaction process is shown in the following formula (4) to obtain an imine intermediate (II):

[0039]

[0040] Wherein, as the first alkali, it can be any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, and sodium carbonate. Among them, a mixed alkali of sodium hydroxide and lithium hydroxide is preferred, which can ensure the complete release of L-lysine hydrochloride and provide a weakly alkaline environment for the system.

[0041] The first solvent may be any one or more of water, methanol, ethanol, and tetrahydrofuran, preferably water, as L-lysine hydrochloride and inorganic base have better solubility in water, which is convenient for preparing the solution and carrying out the reaction.

[0042] Furthermore, in step S1, the molar ratio of L-lysine hydrochloride: first base: aromatic aldehyde is 1:(0.8-1.5):(0.8-1.2), and a slight excess of base can ensure that L-lysine hydrochloride is completely free and provide a weak alkaline environment. Preferably, the molar ratio of L-lysine hydrochloride: first base: aromatic aldehyde is 1:1.05:0.9.

[0043] The reaction temperature is -5 to 40°C, preferably 0 to 5°C, at which the stability of the imine intermediate can be maintained and the operation is convenient. In addition, the reaction time is 3 to 12 hours, preferably 6 hours.

[0044] Furthermore, in the step S1, after the reaction is completed, the step further comprises: suction filtration and slurrying the filter cake to obtain the imine intermediate. That is, after the reaction is completed, the imine intermediate can be obtained by simple separation.

[0045] Furthermore, the step S2 comprises:

[0046] The imine intermediate is reacted with the Fmoc protecting agent in a second solvent under the action of a second base, and dilute hydrochloric acid is added to adjust the pH to 1-2 to perform a hydrolysis reaction to generate the Fmoc-L-lysine.

[0047] When the aromatic aldehyde is p-tolualdehyde, that is, the imine intermediate is imine intermediate (I), the reaction process is shown in the following formula (5):

[0048]

[0049] When the aromatic aldehyde is benzaldehyde, that is, the imine intermediate is imine intermediate (II), the reaction process is shown in the following formula (6):

[0050]

[0051] Furthermore, the Fmoc protecting agent is selected from any one or more of Fmoc-Cl, Fmoc-Osu, and Fmoc-Amox, preferably Fmoc-Cl, which has a higher atomic utilization rate.

[0052] Furthermore, the second alkali is sodium hydroxide, sodium carbonate, or a mixture thereof, wherein sodium carbonate is preferred, which can act as an acid-binding agent and provide an alkaline buffer environment to improve product stability.

[0053] The second solvent is any one or more of water, methanol, ethanol, and tetrahydrofuran, wherein a mixed solvent of ethanol, tetrahydrofuran and water is preferred, and dissolving the Fmoc protecting agent in tetrahydrofuran can effectively reduce the occurrence of side reactions.

[0054] Furthermore, in step S2, the molar ratio of the imine intermediate: the second base: the Fmoc-protecting agent is 1: 1.0 to 3.5: 1.0 to 1.5. Preferably, the molar ratio of the imine intermediate: the second base: the Fmoc-protecting agent is 1: 3.0: 1.2. The reaction temperature is -20 to 30°C, preferably -15 to -10°C; the reaction time is 3 to 10 hours.

[0055] Furthermore, in the step S2, after the reaction is completed, the method further comprises: after the hydrolysis reaction is completed, adding sodium hydroxide solution to adjust to weak acidity, concentrating the organic reagent under reduced pressure, and beating the mixture to obtain the Fmoc-L-lysine.

[0056] In summary, the preparation method of Fmoc-L-lysine provided by the present invention is simple and effective, and the synthetic route only requires two steps, the route is short, the total yield is high, the route is more feasible, and the synthetic cost is low.

[0057] The following is a detailed description of the method for preparing Fmoc-L-lysine of the present invention through specific examples.

[0058] Example 1

[0059] (1) Preparation of imine intermediate (I)

[0060] In a 2L round-bottom flask, add 500mL of water and 100.0g of L-lysine hydrochloride, cool to 0-5°C, add 200mL of sodium hydroxide aqueous solution (19.7g of sodium hydroxide) and 50mL of lithium hydroxide aqueous solution (2.0g of lithium hydroxide) dropwise in sequence, maintain the system temperature at 0-5°C, add 67.1g of p-methoxybenzaldehyde dropwise, and keep the reaction warm for 6h.

[0061] The filter cake was filtered and washed with 200 mL of water, and then slurried twice with 400 mL of acetonitrile to obtain 130.5 g of imine intermediate (I) with a yield of 90.2%.

[0062] (2) Preparation of Fmoc-L-lysine

[0063] In a 2L round-bottom flask, add 300mL of ethanol and 50.0g of imine intermediate (I), cool to -15 to -10°C, drop 150mL of sodium carbonate aqueous solution (24.1g of sodium carbonate), then drop 140mL of tetrahydrofuran solution containing 58.7g of Fmoc-Cl and 200mL of sodium carbonate aqueous solution (36.1g of sodium carbonate) at the same time, keep warm and react for 6h after the addition is completed. Add 6M HCl to adjust the pH to 1 and stir for 20min.

[0064] The pH was adjusted to weak acidity (6.0) with sodium hydroxide solution, and the organic solvent was removed by concentration under reduced pressure. 200 mL of water was added and slurried twice to obtain 59.2 g of Fmoc-L-lysine with a yield of 85.0%.

[0065] Example 2

[0066] (1) Preparation of imine intermediate (I)

[0067] In a 2L round-bottom flask, add 500mL of water and 100.0g of L-lysine hydrochloride, cool to 10-15°C, add 200mL of sodium hydroxide aqueous solution (19.7g of sodium hydroxide) and 50mL of lithium hydroxide aqueous solution (2.0g of lithium hydroxide) dropwise in sequence, maintain the system temperature at 10-15°C, add 67.1g of p-methoxybenzaldehyde dropwise, and keep the reaction warm for 4h.

[0068] The mixture was filtered, and the filter cake was washed with 200 mL of water and then slurried twice with 400 mL of acetonitrile to obtain 121.7 g of imine intermediate (I) with a yield of 84.1%.

[0069] (2) Preparation of Fmoc-L-lysine

[0070] In a 2L round-bottom flask, add 300mL of ethanol and 50.0g of the imine intermediate (I), cool to -15 to -10°C, drop 150mL of sodium carbonate aqueous solution (24.1g of sodium carbonate), then drop 190mL of tetrahydrofuran solution containing 76.6g of Fmoc-Osu and 200mL of sodium carbonate aqueous solution (36.1g of sodium carbonate) at the same time, keep warm and react for 6h after the addition is completed. Add 6M HCl to adjust the pH to 1 and stir for 20min.

[0071] The pH was adjusted to weak acidity (6.0) with sodium hydroxide solution, and the organic solvent was removed by concentration under reduced pressure. 200 mL of water was added to slurry twice to obtain 57.6 g of Fmoc-L-lysine with a yield of 82.6%.

[0072] Example 3

[0073] (1) Preparation of imine intermediate (II)

[0074] In a 2L round-bottom flask, add 500mL of water and 100.0g of L-lysine hydrochloride, cool to 0-5°C, add 200mL of sodium hydroxide aqueous solution (19.7g of sodium hydroxide) and 50mL of lithium hydroxide aqueous solution (2.0g of lithium hydroxide) dropwise in sequence, maintain the system temperature at 0-5°C, add 52.3g of benzaldehyde dropwise, and keep the reaction warm for 5h.

[0075] The mixture was filtered, and the filter cake was washed with 200 mL of water and then slurried twice with 400 mL of acetonitrile to obtain 112.2 g of imine intermediate (II) with a yield of 87.5%.

[0076] (2) Preparation of Fmoc-L-lysine

[0077] In a 2L round-bottom flask, add 300mL of ethanol and 50.0g of imine intermediate (II), cool to -15 to -10°C, drop 65mL of sodium hydroxide aqueous solution (10.2g of sodium hydroxide), then drop 150mL of tetrahydrofuran solution containing 66.2g of Fmoc-Cl and 100mL of sodium hydroxide aqueous solution (17.1g of sodium hydroxide), respectively, and keep warm for 6h after the addition is completed. Add 6M HCl to adjust the pH to 1.5 and stir for 20min.

[0078] The pH was adjusted to weak acidity (6.0) with sodium hydroxide solution, and the organic solvent was removed by concentration under reduced pressure. 200 mL of water was added to slurry the mixture twice to obtain 61.5 g of Fmoc-L-lysine with a yield of 78.2%.

[0079] Example 4

[0080] (1) Preparation of imine intermediate (II)

[0081] In a 2L round-bottom flask, add 500mL of water and 100.0g of L-lysine hydrochloride, cool to 0-5°C, add dropwise 300mL of sodium carbonate aqueous solution (58.0g of sodium carbonate), maintain the system temperature at 0-5°C, add dropwise 52.3g of benzaldehyde, and react at this temperature for 5h.

[0082] The mixture was filtered, and the filter cake was washed with 200 mL of water and then slurried twice with 400 mL of acetonitrile to obtain 108.8 g of imine intermediate (II) with a yield of 84.8%.

[0083] (2) Preparation of Fmoc-L-lysine

[0084] In a 2L round-bottom flask, add 300mL of ethanol and 50.0g of imine intermediate (II), cool to -5-0°C, drop 170mL of sodium carbonate aqueous solution (27.1g of sodium carbonate), then drop 150mL of tetrahydrofuran solution containing 66.2g of Fmoc-Cl and 230mL of sodium carbonate aqueous solution (40.7g of sodium carbonate) at the same time, keep warm and react for 4h after the addition is completed. Add 6M HCl to adjust the pH to 2 and stir for 20min.

[0085] The pH was adjusted to weak acidity (6.0) with sodium hydroxide solution, and the organic solvent was removed by concentration under reduced pressure. 200 mL of water was added for slurrying twice to obtain 63.8 g of Fmoc-L-lysine with a yield of 81.2%.

[0086] In summary, the method for preparing Fmoc-L-lysine of the present invention has the advantages of short synthesis route, low cost and high yield.

[0087] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Fmoc-L-lysine, characterized in that: The steps include: Step S1, reacting L-lysine hydrochloride with an aromatic aldehyde to generate an imine intermediate; Step S2, reacting the imine intermediate with an Fmoc protecting agent and hydrolyzing the imine intermediate to generate Fmoc-L-lysine.

2. The method according to claim 1, characterized in that The step S1 comprises: L-lysine hydrochloride is reacted with the aromatic aldehyde in a first solvent under the action of a first base to generate the imine intermediate.

3. The method according to claim 2, characterized in that The aromatic aldehyde is benzaldehyde, p-methoxybenzaldehyde, or a mixture thereof; the first base is any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, and sodium carbonate; and the first solvent is any one or more of water, methanol, ethanol, and tetrahydrofuran.

4. The method according to claim 2, characterized in that: In the step S1, the molar ratio of L-lysine hydrochloride: first base: aromatic aldehyde is 1:(0.8-1.5):(0.8-1.2), the reaction temperature is -5-40°C, and the reaction time is 3-12h.

5. The method according to claim 2, characterized in that: In the step S1, after the reaction is completed, the step further includes: filtering with suction and slurrying the filter cake to obtain the imine intermediate.

6. The method according to claim 1, characterized in that The step S2 comprises: The imine intermediate is reacted with the Fmoc protecting agent in a second solvent under the action of a second base, and dilute hydrochloric acid is added to adjust the pH to 1-2 to perform a hydrolysis reaction to generate the Fmoc-L-lysine.

7. The method according to claim 6, characterized in that The Fmoc protecting agent is selected from any one or more of Fmoc-Cl, Fmoc-Osu, and Fmoc-Amox.

8. The method according to claim 6, characterized in that The second base is sodium hydroxide, sodium carbonate, or a mixture thereof, and the second solvent is any one or more of water, methanol, ethanol, and tetrahydrofuran.

9. The method according to claim 6, characterized in that In the step S2, the molar ratio of the imine intermediate: the second base: the Fmoc-protecting agent is 1:(1.0-3.5):(1.0-1.5); the reaction temperature is -20-30°C; and the reaction time is 3-10 hours.

10. The method according to claim 6, characterized in that In the step S2, after the reaction is completed, the step further includes: after the hydrolysis reaction is completed, adding sodium hydroxide solution to adjust to weak acidity, concentrating the organic reagent under reduced pressure, and beating the pulp to obtain the Fmoc-L-lysine.

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