Method for effective enantioselective deuteration of α-hydrogen of α-amino acids without external chiral source

Abstract

The present invention relates to a method for asymmetrically deuterating the α-hydrogen of an α-amino acid derivative. Specifically, the method may comprise the steps of: preparing an α-amino acid derivative having the structure of Chemical Formula 1; treating the α-amino acid derivative with a base in a solvent to induce a deuteration reaction and produce a product; and purifying the product. [Chemical Formula 1] (R1 includes at least one selected from alkyl, alkenyl, alkynyl and hydrogen; R2 and R3 include at least one from alkyl,-C(O)alkyl, alkenyl,-(O)alkenyl, cycloalkyl,-C(O)cycloalkyl, cycloalkenyl,-C(O)cycloalkenyl, aryl,-C(O)aryl, heteroaryl,-C(O)heteroaryl, heterocyclyl, and-C(O) heterocyclyl; and R4 includes at least one from alkyl, alkenyl, alkynyl, and hydrogen)

Description

An effective and enantioselective method for deuteration of α-amino acids at the α-position without an external chiral source

[0001] The present invention relates to a method for deuterizing an amino acid.

[0002] Deuterated α-amino acids are considered important in drug development and biomedical sciences due to their kinetic isotope effect (KIE). For example, deuterated α-amino acids are used to elucidate the secondary and quaternary structures of proteins, and to investigate biochemical processes such as peptide metabolism and enzymatic mechanisms. In particular, α-amino acids with deuterium substitution at the α-position are attracting attention because they sometimes exhibit improved metabolic stability compared to standard α-amino acid moieties.

[0003] Furthermore, since the chirality of α-amino acids significantly influences their physiological activity, chiral-selective deuteration technology holds greater value than simple deuteration. However, the process of substituting α-hydrogen with deuterium can lead to the formation of double bonds and the potential loss of chirality, making high-level implementation of this technology challenging. For this reason, various methodologies have been proposed to efficiently produce chiral deuterated α-amino acids.

[0004] However, conventional techniques have several clear limitations. Methods utilizing external chiral elements (chiral metal complexes and organocatalysts) or enzyme-based techniques have a limited range of applicable substrates and poor process economics. Furthermore, methods that introduce specific protecting groups onto α-amino acids and then asymmetrically deuterate the α-site require anhydrous conditions, ultra-low temperatures (-78°C), and the use of strong bases, making them impractical for large-scale processes.

[0005] Therefore, there is an urgent need to develop a new method that can improve process convenience, maintain chirality, and effectively replace α-hydrogen with deuterium.

[0006]

[0007] The present invention aims to solve the above-described conventional problems by providing a method for chiral selectively substituting the α-position hydrogen of an α-amino acid with deuterium without introducing an external chiral source.

[0008] In addition, the present invention aims to provide a technology capable of effectively deuterizing α-site hydrogen while maintaining the chirality of amino acids without requiring extreme conditions (e.g., ultra-low temperature, anhydrous environment, strong base, etc.).

[0009]

[0010] A method for asymmetric α-deuteration of an α-amino acid derivative according to one embodiment of the present invention may include the steps of: preparing an α-amino acid derivative having a structure of chemical formula 1; treating the α-amino acid derivative with a base in a solvent to induce a deuteration reaction and generate a product; and purifying the product.

[0011] [Chemical Formula 1]

[0012]

[0013] (R1 is at least one of alkyl, alkenyl, alkynyl or hydrogen, R2 and R3 are at least one of alkyl, -C(O)alkyl, alkenyl, -C(O)alkenyl, cycloalkyl, -C(O)cycloalkyl, cycloalkenyl, -C(O)cycloalkenyl, aryl, -C(O)aryl, heteroaryl, -C(O)heteroaryl, heterocyclyl and -C(O)heterocyclyl, R4 includes at least one of alkyl, alkenyl, alkynyl or hydrogen)

[0014] According to one embodiment, the method may be to maintain the chirality of the α-position of the α-amino acid derivative and replace the hydrogen at the α-position with deuterium.

[0015] In one embodiment, the α-amino acid derivative may include an N-protecting group.

[0016] In one embodiment, the solvent is C1 to C 10 An alcohol-based solvent, comprising at least one selected from the group consisting of acetonitrile, dimethyl sulfoxide (DMSO), acetone, ethers and cyclic ethers, nitromethane, chloroform, dimethylformamide (DMF), toluene, and water (H2O), wherein the solvent may include at least one deuterium in its molecular structure to promote the deuteration reaction.

[0017] In one embodiment, the base may comprise a cation and an alkoxide (MOR).

[0018] According to one embodiment, the method further comprises a step of stopping the deuteration reaction between the step of producing the product and the step of purifying the product, wherein the step of stopping the deuteration reaction may be treating the product with a weakly acidic solution.

[0019] According to one embodiment, the step of purifying the product may be purifying the product through one or more methods selected from the group consisting of chromatography, crystallization, extraction, distillation, recrystallization, filtration, evaporation, sublimation, fractional extraction, washing, etc.

[0020] According to one embodiment, the method may further include a step of purifying the product; and then, a step of removing a protecting group (N-protecting group) of a deuterated α-amino acid derivative included in the purified product.

[0021] According to another embodiment, an α-amino acid derivative containing deuterium at the α-position has a structure represented by Chemical Formula 2, wherein Chemical Formula 2 may be prepared by an asymmetric α-deuteration method of an α-amino acid derivative according to one embodiment.

[0022] [Chemical Formula 2]

[0023]

[0024] (R1 is at least one of alkyl, alkenyl, alkynyl or hydrogen, R2 and R3 are at least one of alkyl, -C(O)alkyl, alkenyl, -C(O)alkenyl, cycloalkyl, -C(O)cycloalkyl, cycloalkenyl, -C(O)cycloalkenyl, aryl, -C(O)aryl, heteroaryl, -C(O)heteroaryl, heterocyclyl and -C(O)heterocyclyl, R4 includes at least one of alkyl, alkenyl, alkynyl or hydrogen)

[0025] According to one embodiment, the α-amino acid derivative may be a proline derivative in which the hydrogen at the α-position is deuterated.

[0026] In one embodiment, the deuteration level (DL) of the α-amino acid may be 72% or greater.

[0027] In one embodiment, the enantiomeric excess (EE) of the α-amino acid may be greater than or equal to 90%.

[0028]

[0029] The present invention has the effect of providing a method for deuterizing the α-position hydrogen of an amino acid without introducing an external chiral source and without extreme environmental conditions.

[0030] In addition, the deuteration method of the present invention has the effect of maintaining the chirality of the deuterated α-amino acid.

[0031] However, the effects of the present invention are not limited to the effects described above, and include all effects naturally implemented due to the various configurations proposed in the present invention.

[0032]

[0033] Figure 1 is a flow chart showing a method for asymmetric α-deuteration of an α-amino acid derivative according to one embodiment of the present invention.

[0034] Figure 2 is a chemical reaction formula showing a method for preparing an α-amino acid derivative comprising a structure of chemical formula 1.

[0035] Figure 3 is a chemical reaction formula showing a method for asymmetric α-deuteration of a proline derivative and a drawing showing the result.

[0036] Figure 4 is a table showing the results of performing the deuteration reaction of a proline derivative under various conditions.

[0037] Figure 5 is a formula (a) showing a plausible reaction pathway of an asymmetric α-deuteration method for proline derivatives and a graph (b) showing a free energy profile.

[0038] Figure 6 is a diagram showing the results of deuteration reactions of various proline derivatives.

[0039] Figure 7 is a diagram showing the results of a deuteration reaction of a pseudo-proline derivative.

[0040] Figure 8 is a diagram showing the results of a deuteration reaction of an acyclic α-amino acid derivative.

[0041] Figure 9 is a drawing showing a step of removing a protecting group (N-protecting group) of a deuterated α-amino acid derivative.

[0042]

[0043] The embodiments of the present invention are provided for the purpose of illustrating the technical concept of the present invention. The scope of the rights of the present invention is not limited to the embodiments presented below or the specific descriptions of these embodiments.

[0044] All technical and scientific terms used in this invention, unless otherwise defined, have the meanings commonly understood by those skilled in the art to which this invention pertains. All terms used in this invention have been selected for the purpose of more clearly explaining the invention and are not intended to limit the scope of the rights provided for in this invention.

[0045] Expressions such as “comprising,” “having,” and the like used in the present invention should be understood as open-ended terms that imply the possibility of including other embodiments, unless otherwise stated in the phrase or sentence in which the expression is included.

[0046] The singular expressions described in the present invention may include plural meanings unless otherwise stated, and this also applies to the singular expressions described in the claims.

[0047]

[0048] A method for asymmetric α-deuteration of an α-amino acid derivative according to one embodiment of the present invention may include the steps of: preparing an α-amino acid derivative having a structure of chemical formula 1; treating the α-amino acid derivative with a base in a solvent to induce a deuteration reaction and generate a product; and purifying the product.

[0049] According to one embodiment, the method may be to maintain the chirality of the α-position of the α-amino acid derivative and replace the hydrogen at the α-position with deuterium.

[0050]

[0051] Figure 1 is a flow chart showing a method for asymmetric α-deuteration of an α-amino acid derivative according to one embodiment of the present invention.

[0052] The present invention proposes a new method for obtaining an α-deuterated α-amino acid derivative by replacing a hydrogen atom at the α-position with a deuterium atom while maintaining the chirality of the α-position of the α-amino acid derivative, and consequently obtaining an α-deuterated amino acid with maintained chirality. In particular, the present invention improves process convenience by eliminating the need for extreme conditions, catalysts, etc., as in the conventional technique, and expands the scope of substrates to which the deuteration method can be applied compared to the conventional technique, so that the chirality of the α-amino acid can be maintained even after the deuteration reaction. An α-amino acid can be obtained from an amino acid derivative in which the hydrogen atom at the α-position is replaced with a deuterium atom obtained in this way.

[0053] Below, each step of the asymmetric α-deuteration method of α-amino acid derivatives is described.

[0054]

[0055] 1. Step of preparing an α-amino acid derivative having a structure of chemical formula 1 (S100)

[0056] The above method is a method of replacing hydrogen with deuterium while maintaining the chirality of the α-position of an α-amino acid derivative, and the α-amino acid derivative may include a structure of chemical formula 1.

[0057] [Chemical Formula 1]

[0058]

[0059] (R1 is at least one of alkyl, alkenyl, alkynyl or hydrogen, R2 and R3 are at least one of alkyl, -C(O)alkyl, alkenyl, -C(O)alkenyl, cycloalkyl, -C(O)cycloalkyl, cycloalkenyl, -C(O)cycloalkenyl, aryl, -C(O)aryl, heteroaryl, -C(O)heteroaryl, heterocyclyl and -C(O)heterocyclyl, R4 includes at least one of alkyl, alkenyl, alkynyl or hydrogen)

[0060]

[0061] In one embodiment, the α-amino acid derivative may include an N-protecting group.

[0062] In the case of an α-amino acid derivative that is not substituted with the above-mentioned protecting group (N-protecting group), the chirality of the α-position may not be maintained. Therefore, by replacing the N of the α-amino acid derivative with a bulky protecting group with a large steric hindrance, the chirality of the α-position can be maintained by imparting axial chirality to the double bond formed during the deuteration process.

[0063] The above protecting group (N-protecting group) may include at least one selected from the group consisting of isobutyryl, pivaloyl, and tosyl, which contain an electron withdrawing group (EWG) such as carbonyl, sulfonyl, and sulfoxide.

[0064] An α-amino acid derivative comprising the structure of the above chemical formula 1 can be prepared in a solution state in a solvent.

[0065] In one embodiment, the solvent is C1 to C 10 The solvent may include at least one selected from the group consisting of alcoholic solvents, acetonitrile, dimethyl sulfoxide (DMSO), acetone, ethers and cyclic ethers, nitromethane, chloroform, dimethylformamide (DMF), toluene, and water (H2O), and the solvent may contain deuterium. The solvent may be any solvent capable of dissolving the derivative, and is not particularly limited in the present invention.

[0066] The solvent may be included so that the α-amino acid derivative has a concentration of 0.01 mol / L to 2.0 mol / L. If the concentration exceeds 2.0 mol / L, the process efficiency may decrease, resulting in a problem of reduced yield.

[0067] In one example, the solvent may provide deuterium that can be substituted at the α-position of the α-amino acid derivative when a deuteration reaction is subsequently induced by including deuterium in the molecular structure. Through this, the deuteration reaction may proceed. The solvent is preferably C1 to C 10 It may include an alcohol-based solvent. For example, the solvent may be ethanol (EtOD) containing deuterium.

[0068]

[0069] 2. Step (S200) of treating the above α-amino acid derivative with a base under a solvent to induce a deuteration reaction and generate a product.

[0070] The step of producing the above product may be to add a base to a solution containing the α-amino acid derivative and a solvent containing deuterium and stir.

[0071] Since the above base acts to attack and remove hydrogen located at the α-position of the α-amino acid derivative, deuterium contained in the solvent can be substituted at the α-position.

[0072] In one embodiment, the base may comprise a cation and an alkoxide (MOR). As an example, the base may be, but is not limited to, sodium ethoxide (NaOEt), lithium ethoxide (LiOEt), or potassium ethoxide (KOEt).

[0073] The base may be added in an amount of 1 to 10 equivalents relative to the amino acid. If the base is included in an amount of less than 1 equivalent relative to the amino acid, the rate of deuteration may be slow, and if it is included in an amount of more than 10 equivalents, the yield may decrease due to an ester exchange reaction. Preferably, the base may be added in an amount of 1 to 5 equivalents relative to the amino acid.

[0074] The above deuteration reaction can be performed at -78.5°C to 30°C, and preferably at room temperature of 0°C to 25°C. Unlike conventional techniques, the deuteration method of the present invention can be performed at room temperature rather than ultra-low temperature conditions, thereby significantly improving process convenience.

[0075] The above deuteration reaction can be carried out for 30 minutes to 170 hours.

[0076]

[0077] According to another embodiment, an α-amino acid derivative containing deuterium at the α-position has a structure represented by Chemical Formula 2, wherein Chemical Formula 2 may be prepared by an asymmetric α-deuteration method of an α-amino acid derivative according to one embodiment.

[0078] [Chemical Formula 2]

[0079]

[0080] (R1 is at least one of alkyl, alkenyl, alkynyl or hydrogen, R2 and R3 are at least one of alkyl, -C(O)alkyl, alkenyl, -C(O)alkenyl, cycloalkyl, -C(O)cycloalkyl, cycloalkenyl, -C(O)cycloalkenyl, aryl, -C(O)aryl, heteroaryl, -C(O)heteroaryl, heterocyclyl and -C(O)heterocyclyl, R4 includes at least one of alkyl, alkenyl, alkynyl or hydrogen)

[0081] Through the asymmetric α-deuteration method of the α-amino acid derivative described above, an α-amino acid derivative in which the hydrogen at the α-position is replaced with deuterium can be obtained. As can be seen in the chemical formula 2, the hydrogen at the α-position of the α-amino acid derivative of the chemical formula 1 is replaced with deuterium in the α-amino acid derivative of the chemical formula 2, and the chirality of the α-position of the α-amino acid derivative can be maintained.

[0082] Therefore, the α-deuteration method of the present invention can produce an α-amino acid with maintained chirality more simply than with conventional techniques, and has the advantage of a wider range of substrates to which the method can be applied.

[0083]

[0084] 3. Step for stopping the above deuteration reaction (S300)

[0085] According to one embodiment, the method further comprises a step of stopping the deuteration reaction between the step of producing the product and the step of purifying the product, wherein the step of stopping the deuteration reaction may be treating the product with a weakly acidic solution.

[0086] The step of stopping the deuteration reaction is to stop the deuteration reaction after the deuteration reaction has been performed at a certain temperature and time. The weakly acidic solution can quench the deuteration reaction by neutralizing the base by providing a proton. For example, the weakly acidic solution may be, but is not limited to, a saturated aqueous solution of ammonium chloride (NH4Cl) or acetic acid (CH3COOH).

[0087] After the deuteration reaction is stopped, the solvent can be removed under reduced pressure. After adding water and an appropriate organic solvent to the solvent-removed reactant, an organic fraction can be extracted, and the organic fraction can be extracted with at least one selected from the group consisting of ethyl acetate (EtOAc), hexane, cyclohexane, toluene, benzene, acetone, acetonitrile, petroleum ether, dichloromethane (DCM), 1,2-dichloroethane (DCE), methanol (MeOH), ethanol (EtOH), isopropanol (IPA), and chloroform.

[0088]

[0089] 4. Step of purifying the above product (S400)

[0090] According to one embodiment, the step of purifying the product may be purifying the product through one or more methods selected from the group consisting of chromatography, crystallization, extraction, distillation, recrystallization, filtration, evaporation, sublimation, fractional extraction, washing, etc.

[0091] The above purifying step can be performed to remove impurities from the product obtained by the above-described method.

[0092] The extracted organic fraction can be dried and concentrated under reduced pressure. For example, the organic fraction can be treated with magnesium sulfate (MgSO4) to remove any remaining water, and the solvent can be removed under reduced pressure to dry it.

[0093] For example, the purifying step may be to purify the concentrated product through flash chromatography. After performing the purifying step, a deuterated α-amino acid derivative can be obtained.

[0094] The deuteration level of the α-position of the α-amino acid derivative obtained above is 1 It can be measured by H NMR analysis and liquid chromatography mass spectrometry (LC / MS).

[0095]

[0096] 5. Step of removing the protecting group (N-protecting group) of the deuterated α-amino acid derivative (S500)

[0097] According to one embodiment, the method may further include a step of purifying the product; and then, a step of removing a protecting group (N-protecting group) of a deuterated α-amino acid derivative included in the purified product.

[0098] A deuterated α-amino acid can be obtained by removing a protecting group (N-protecting group), such as a carbonylN-protecting group, contained in the above α-amino acid derivative.

[0099] The method for removing the protecting group (N-protecting group) of the above α-amino acid derivative is not particularly limited in the present invention, and can be performed through conventionally known reduction reactions and reactions known to be generally available. For example, the step of removing the protecting group (N-protecting group) can be performed by adding hydrochloric acid (HCl).

[0100] The method for removing the protecting group (N-protecting group) of the above α-amino acid derivative can be performed in multiple steps, and is not limited as long as it can obtain a deuterated α-amino acid as a result.

[0101] After removing the protecting group (N-protecting group) as described above, an α-amino acid containing deuterium at the α-position and maintaining chirality can be obtained.

[0102]

[0103] The present invention is described in more detail through the following examples. However, these examples are provided to illustrate some experimental methods and configurations for the purpose of illustratively explaining the present invention, and the scope of the present invention is not limited to these examples.

[0104]

[0105] Manufacturing Example 1: Manufacturing of a proline derivative comprising the structure of chemical formula 1

[0106] To prepare an α-amino acid derivative having the structure of the above-described chemical formula 1, an amino acid proline derivative was used.

[0107] Figure 2 is a chemical reaction formula showing a method for preparing an α-amino acid derivative comprising a structure of chemical formula 1.

[0108] To tert-butyl L-prolinate (10.0 g, 58.4 mmol, 1.0 equiv) in CH2Cl2 solvent (195 mL) were added Et3N (24.4 mL, 175.2 mmol, 3.0 equiv) and isobutyryl chloride (6.73 mL, 64.2 mmol, 1.1 equiv) at 0°C. The mixture was stirred at room temperature for 8 h. After stirring, the mixture was cooled to 0°C, and the reaction was stopped by adding saturated NH4Cl aqueous solution (40 mL). Then, 100 mL of water was added, and extraction was performed three times (3X100 mL) with CH2Cl2. The extracted organic fraction was dried using MgSO4 and concentrated under reduced pressure.

[0109] After concentration, the residue was purified by flash chromatography on silica gel (hexane / EtOAc=3:1, v / v), and a proline derivative tert-butyl isobutyryl-L-prolinate (4a) containing the chemical formula 1 in the form of a colorless oil was obtained (14.0 g, 99%).

[0110]

[0111] Experimental Example 1: Deuteration of Proline Derivatives

[0112] The proline derivative (4a, 0.2 mmol) obtained in Manufacturing Example 1 was treated with NaOEt (5.0 equiv) and EtOD (0.2 M) at room temperature to perform a deuteration reaction.

[0113] Figure 3 is a chemical reaction formula showing a method for asymmetric α-deuteration of a proline derivative and a drawing showing the result.

[0114] According to one embodiment, the α-amino acid derivative may be a proline derivative in which the hydrogen at the α-position is deuterated.

[0115] Through the deuteration reaction of the above chemical reaction formula, the proline derivative (4a) is transformed into a proline derivative (d-4a) in which the hydrogen at the α-position is replaced with deuterium, and it can be confirmed through HPLC that the chirality at the α-position is maintained.

[0116] In one embodiment, the deuteration level (DL) of the α-amino acid derivative may be 72% or greater.

[0117] In one embodiment, the enantiomeric excess (EE) of the α-amino acid derivative may be greater than or equal to 90%.

[0118] The deuteration reaction of the proline derivative (4a) progressed gradually with the reaction time, and after 4 hours, the yield was 95%, the deuteration level (DL) was 72%, and the enantiomeric purity was 98%. After 8 hours, the yield was 94%, the deuteration level (DL) was 98%, and the enantiomeric purity was 97%.

[0119]

[0120] To explore the optimal conditions for deuterizing the proline derivative (4a), various bases, solvents, and reaction conditions were used.

[0121] Figure 4 is a table showing the results of performing the deuteration reaction of a proline derivative under various conditions.

[0122] The concentration of the solvent EtOD did not significantly affect the reaction results (entries 2, 3), and by using the same base with only the cation changed, it was confirmed that the cation did not significantly affect the reaction results (entries 4, 5).

[0123] It was confirmed that the change in solvent composition affected the reaction results. When tetrahydrofuran (THF) was used in combination with EtOD (10:1), the yield and enantioselectivity decreased compared to when EtOD alone was used (entry 6). However, when the reaction was performed at -10℃, the yield and enantioselectivity were similar, but the reaction time was extended to 48 hours (entry 7). The result confirmed the possibility of reducing the amount of EtOD used by mixing it with a relatively inexpensive solvent (THF). When dimethylformamide (DMF), a polar solvent, was used in combination (10:1), the reaction efficiency decreased significantly (entry 8). When toluene, a non-polar solvent, was used in combination (10:1), the yield and enantioselectivity also decreased significantly (entry 9). When EtOH was mixed with EtOD (1:1), the yield and enantiomeric excess were similar to those when EtOD alone was used, but the deuteration level was significantly reduced compared to when EtOD alone was used (entry 10, 11).

[0124] The above results confirmed that using EtOD as a solvent yields high yields, deuteration levels, and enantiomeric excesses. Furthermore, the deuteration reaction was performed at room temperature, confirming that high deuteration levels and enantiomeric purity can be achieved without extreme conditions.

[0125]

[0126] Experimental Example 2: Mechanism Analysis of the Deuteration Reaction of Proline Derivatives

[0127] Based on the above results, we analyzed the mechanism of deuteration reaction of proline derivatives via chiral enolate intermediate in EtOD solvent. Density functional theory (DFT)-based computational investigation was performed using M06-2X functional with 6-311++G(d,p).

[0128] Through computational conformation analysis, it was confirmed that the most stable conformation among the 10 conformers of proline derivatives was 4a-Ⅰ, and the 4a-Ⅰ conformation was set as the starting point for computational analysis.

[0129] Figure 5 is a formula (a) showing a plausible reaction pathway of an asymmetric α-deuteration method for proline derivatives and a graph (b) showing a free energy profile.

[0130] When the proline derivative was deuterated in the conformer of 4a-Ⅰ, chiral enolate 5-Ⅰ was formed with a free energy barrier (ΔG) of 6.4 kcal / mol (TS1), and the enolate may undergo changes in chirality through several reaction pathways.

[0131] TS2 in the above 5-Ⅰ Re The free energy barrier (ΔG) of the pathway through which d-4a-Ⅰ is formed was only 5.6 kcal / mol. On the other hand, TS2 Si The energy barrier of the path through is high at 16.0 kcal / mol, and the formed ent-d-4a-Ⅱ is less stable. In addition, TS2, which has an energy barrier of 11.7 kcal / mol, racEnt-5-Ⅰ, which is formed through , is not preferred due to a higher energy barrier, even though its stability is the same as that of reactant 4a-Ⅰ. Based on the above results, TS2, which forms d-4a-Ⅰ Re It could be expected that the path of 5-I would be the most preferred among the several paths that it could take.

[0132] The entropic penalties arising from the interaction between the substrate and EtOD in 5-Ⅰ were analyzed by calculating their respective free energies. The free energy penalty of the substrate and EtOD for the deuteration reaction was 5.6 kcal / mol, and the overall free energy (ΔG) was calculated as TS2 Re 11.2, TS2 rac Ga 17.3, TS2 Si , was analyzed as 21.6 kcal / mol.

[0133] TS2 Re Wow TS2 Si When the kinetic isotope effect (KIE) was investigated, it was confirmed that the calculated reaction rate for deuterium substitution was 4.9 to 5 times slower than the reaction rate for proton substitution. Therefore, it was confirmed that the computer analysis results were consistent with the experimental results for the deuteration reaction.

[0134]

[0135] Experimental Example 3: Analysis of deuteration reactions of various proline derivatives and amino acids.

[0136] The deuteration reaction results of various proline derivatives in which various types of protecting groups were substituted on N were analyzed. Various proline derivatives were treated with 5 equivalents of NaOEt and 0.2 M EtOD at room temperature for 16 h.

[0137] Figure 6 is a diagram showing the results of deuteration reactions of various proline derivatives.

[0138] d-4b and d-4c containing propionyl or pentenoyl groups showed high yields and enantiomeric excesses. Derivative d-4e containing a phenylacetyl group showed high enantiomeric excess and deuteration levels, and in this case, almost complete deuteration substitution was confirmed up to the α-position of the amide carbonyl group.

[0139] d-4f, which contains a bulky pivaloyl group as a protecting group, had a deuteration level of only 60% after a reaction time of 16 h, but after 48 h, the deuteration level increased to 94% and the enantioselectivity also showed 96%. d-4g, which contains an N-Boc group, showed a high yield and enantioselectivity, but a low deuteration level of 24%, but after 48 h, the deuteration level increased to 56% without affecting the yield or enantioselectivity.

[0140] 4h, in which the t-butyl ester moiety of 4a was changed to ethyl ester, exhibited the same level of mirror image excess and deuteration level. On the other hand, 4i, in which the t-butyl ester moiety of 4a was changed to a dimethylamide group, did not undergo deuteration due to the decrease in acidity of the C-2 hydrogen atom. 4j (Boc- L -Val- L -Pro-O t Bu) and 4k (Boc- L -Ala- L -Pro-O t The deuteration reaction results of Bu) showed a high level of deuteration at the C-2 position with stereoselectivity. This result predicts that site-selective deuteration reaction at the C-terminal of the polypeptide will be possible.

[0141] In order to confirm the scope and limitations of the substrate, when deuteration was performed with 4m and 4n, which do not have an electron withdrawing group (EWG) in the N-protecting group, it was confirmed that the deuteration reaction did not proceed. Deuteration was also performed with 4l, which had a tosyl group as an electron withdrawing group protecting group instead of a carbonyl N-protecting group. After 16 hours of deuteration at room temperature, it showed a yield of 43%, a deuteration level of 96%, and an enantiomeric excess of 12%. To improve this, 4l was deuterated at -10℃ for 48 hours under the same reagent conditions, and it showed a yield of 84%, a deuteration level of 84%, and an enantiomeric excess of 92%. Through this, it was confirmed that an electron withdrawing group (EWG) protecting group is essential for the N-protection group in the enantioselective deuteration reaction, and a protecting group containing a double carbonyl group is the most effective.

[0142] In order to verify whether the deuteration method of the present invention can be applied to other α-amino acid derivatives, a pseudoproline skeleton (6a-d) was synthesized using serine, threonine, and cysteine ​​as starting materials, and a deuteration reaction was performed.

[0143] Figure 7 is a diagram showing the results of a deuteration reaction of a pseudoproline derivative.

[0144] Deuteration of pseudoproline proceeded faster than that of proline derivatives at room temperature, but the yield was low due to its instability. To improve this, the deuteration reaction of 6a-d was performed at temperatures lower than the basic room temperature (0℃, 4d -50℃). Serine derivatives 6a and 6b, which are analogs of proline derivatives (4a, 4h), showed similar results, and the threonine derivative 6c also showed high stereoselectivity with a diastereomer ratio of >50:1 in 88% yield. The cysteine ​​derivative 6d showed high yield and deuteration level at room temperature, but because it was almost completely racemized, the temperature was lowered (-50℃) and deuteration was performed again. This improved the results to 90% yield, 97% deuteration level, and 99% enantiomeric excess.

[0145]

[0146] The results of the deuteration reaction using acyclic α-amino acid derivatives as substrates following pseudoproline were analyzed.

[0147] Figure 8 is a diagram showing the results of a deuteration reaction of an acyclic α-amino acid derivative.

[0148] Alanine derivative 7a, in which only an isobutyryl group was introduced to the nitrogen atom, showed a high yield and deuteration level as a result of the deuteration reaction performed at 0℃, but almost complete racemization. To improve this, alanine derivative 7b was synthesized by additionally introducing a benzyl group to the nitrogen atom, and the deuteration reaction was performed. Since the deuteration reaction result at 0℃ showed a 23% enantiomeric excess, to improve this, the temperature was lowered and the reaction was performed at -40℃ for 3 days, resulting in a yield of 98%, a deuteration level of 94%, and an enantiomeric excess of 98%.

[0149] In a similar manner, lysine, phenylalanine, valine, and isoleucine derivatives (7c-f) were synthesized and deuterated at temperatures below 0°C, resulting in high yields, deuteration levels, and mirror-image excesses.

[0150]

[0151] Experimental Example 4: Preparation of deuterated α-amino acids

[0152] The protective group of the deuterated α-amino acid derivative prepared by the above method was removed to prepare a deuterated α-amino acid.

[0153] Figure 9 is a drawing showing a step of removing a protecting group (N-protecting group) of a deuterated α-amino acid derivative.

[0154] 6N HCl (1.0 mL) was added to a deuterated proline derivative (d-4a) dissolved in water at room temperature, stirred at 90°C for 48 hours, and concentrated under reduced pressure to obtain a deuterated α-amino acid (8) as a white solid (14 mg, 93%). The deuteration level (DL) of the obtained α-amino acid (8) was confirmed to be 94%, which was the same as the deuteration level of d-4a before removing the protecting group. This confirmed that even when the N-protecting group is removed from a deuterated α-amino acid derivative, a deuterated α-amino acid can be obtained while maintaining chirality without losing the deuteration level.

[0155] It was confirmed that the deuterated α-amino acid derivative (9) prepared from the identically deuterated proline derivative (d-4c), the deuterated α-amino acid derivative (10) prepared from the deuterated serine derivative (d-6a), and the deuterated α-amino acid derivative (11) prepared from the deuterated alanine derivative (d-7b) also exhibited the same deuteration level as before removal of the protecting group.

[0156] The above results confirm that the deuteration method of the present invention enables chirality-maintaining deuteration not only of single α-amino acid derivatives but also of peptide compounds comprising multiple α-amino acids. Therefore, it is expected that deuteration using the above deuteration method will also be possible for larger peptide and protein units.

[0157]

[0158] The above description is merely an illustrative example of the technical idea of ​​the present invention, and those skilled in the art will appreciate that various modifications and variations can be made without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are intended to illustrate rather than limit the technical idea of ​​the present invention, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. A step of preparing an α-amino acid derivative having a structure of chemical formula 1; A step of treating the above α-amino acid derivative with a base under a solvent to induce a deuteration reaction and produce a product; and a step of purifying the above product; comprising; A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives. [Chemical Formula 1] (R1 is at least one of alkyl, alkenyl, alkynyl or hydrogen, R2 and R3 are at least one of alkyl, -C(O)alkyl, alkenyl, -C(O)alkenyl, cycloalkyl, -C(O)cycloalkyl, cycloalkenyl, -C(O)cycloalkenyl, aryl, -C(O)aryl, heteroaryl, -C(O)heteroaryl, heterocyclyl and -C(O)heterocyclyl, R4 includes at least one of alkyl, alkenyl, alkynyl or hydrogen) 2. In paragraph 1, The above method maintains the chirality of the α-position of the α-amino acid derivative and replaces the hydrogen of the α-position with deuterium. A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

3. In paragraph 1, The above α-amino acid derivative contains a protecting group substituted at N (N-protecting group), A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

4. In paragraph 1, The above solvent is C1 to C 10 Containing at least one selected from the group consisting of alcoholic solvents, acetonitrile, dimethyl sulfoxide (DMSO), acetone, ethers and cyclic ethers, nitromethane, chloroform, dimethylformamide (DMF), toluene, and water (H2O), The solvent comprises at least one deuterium atom in its molecular structure to promote the deuteration reaction. A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

5. In paragraph 1, The above base comprises a cation and an alkoxide (MOR). A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

6. In paragraph 1, Between the step of producing the above product; and the step of purifying the above product; further comprising a step of stopping the above deuteration reaction; The step of stopping the above deuteration reaction is to treat the product with a weakly acidic solution. A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

7. In paragraph 1, The step of purifying the above product is to purify the product through one or more methods selected from the group consisting of chromatography, crystallization, extraction, distillation, recrystallization, filtration, evaporation, sublimation, fractional extraction, washing, etc. A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

8. In paragraph 1, A step of purifying the above product; thereafter, A step of removing the protecting group (N-protecting group) of the deuterated α-amino acid derivative contained in the purified product; further comprising; A method for asymmetric α-position hydrogen deuteration of α-amino acid derivatives.

9. Contains the structure of chemical formula 2, The above chemical formula 2 is prepared by the asymmetric α-deuteration method of the α-amino acid derivative of claim 1. An α-amino acid derivative containing deuterium at the α-position. [Chemical Formula 2] (R1 is at least one of alkyl, alkenyl, alkynyl or hydrogen, R2 and R3 are at least one of alkyl, -C(O)alkyl, alkenyl, -C(O)alkenyl, cycloalkyl, -C(O)cycloalkyl, cycloalkenyl, -C(O)cycloalkenyl, aryl, -C(O)aryl, heteroaryl, -C(O)heteroaryl, heterocyclyl and -C(O)heterocyclyl, R4 includes at least one of alkyl, alkenyl, alkynyl or hydrogen) 10. In paragraph 9, The above α-amino acid derivative is a proline derivative in which the hydrogen at the α-position is deuterated. An α-amino acid derivative containing deuterium at the α-position.

11. In paragraph 9, The deuteration level (DL) of the above α-amino acid derivative is 72% or more, An α-amino acid derivative containing deuterium at the α-position.

12. In paragraph 9, The enantiomeric excess (EE) of the above α-amino acid derivative is 90% or more, An α-amino acid derivative containing deuterium at the α-position.

Images