Chemical mutation method from dehydroalanine to asparaginic acid

Abstract

The invention relates to a method for realizing chemical mutation from dehydroalanine to aspartic acid (Asp) through a Michael addition reaction of a photocatalytic dehydroalanine (Dha) compound and a carbon dioxide free radical anion (CO2 <.->) generated by ammonium formate. According to the invention, ammonium formate which is cheap and easy to operate and store is used as a carbon source, so that a noble metal catalyst and traditional carbon sources which are difficult to store and operate, such as carbon dioxide and carbon monoxide, are not used, and the method has the advantages of light regeneration and no pollution; according to the present invention, the method has advantages of mild reaction conditions, general room temperature, good selectivity, wide substrate application range and the like, can achieve the hydrocarboxylation modification of the dehydroalanine (Dha) and the short peptide containing the dehydroalanine (Dah), and can chemically mutate the dehydroalanine (Dha) into the aspartic acid (Asp), such that the green path is provided for the aspartic acid synthesis; the future work can focus on mechanism deepening, stereoselectivity improvement and complex biomolecule application, and is expected to become an important tool for precise modification of protein.

Description

Technical Field

[0001] This invention relates to a photocatalytic method for the hydrocarboxylation of dehydroalanine compounds. The method achieves efficient conversion to aspartic acid derivatives through a Michael addition reaction of dehydroalanine derivatives with carbon dioxide radical anions generated by ammonium formate under photocatalytic conditions. This technology provides a novel strategy for the synthesis of β-amino acids and their derivatives, and has significant application value in peptide drug modification and bioprobe preparation. Compared to traditional malonate alkylation or Arndt-Eistert reactions, this method is simpler and more suitable for the later-stage modification of complex molecules. Background Technology

[0002] Dehydroalanine (Dha) is a naturally occurring, non-genetically encoded amino acid. In vivo, it is generally obtained through the dehydration of tryptophan (Ser) or the elimination of phosphorylated tryptophan (pSer). It can also be obtained in vitro through chemical methods by eliminating Ser or cysteine ​​(Cys). Its unique α,β-unsaturated carbonyl structure provides specific reaction sites for the modification of peptides and proteins. Various chemical transformations can be used to add multiple modifying groups to peptides and proteins, thereby achieving peptide and protein functionalization. With the rapid development of organic photochemistry, utilizing visible light photocatalysis to activate and transform various chemical bonds has become an important synthetic strategy and method in organic chemistry. This strategy has the advantages of mild conditions, high chemoselectivity, good functional group tolerance, and a broad substrate range, making it particularly advantageous for the chemical modification of peptides and proteins, especially for the modification of Dha. In 2017, Jui's research group first reported the photocatalytic radical conjugation addition reaction between halopyridine and dehydroalanine. Under the conditions of [Ir(ppy)2(dtbbpy)]PF6 as a photocatalyst and stoichiometric hans ester as a reducing agent, pyridine free radicals can be generated using halopyridine as a free radical precursor, which then undergo free radical addition with dehydroalanine. The resulting intermediate is then reduced by hans ester and subsequently protonated to obtain the final product (Chemical Science, 2017, 8(12): 7998-8003). In 2018, this research group used tertiary amines as free radical precursors to generate carbon free radicals under photocatalysis, which were then added to Dha to achieve alkylation modification of amino acids and peptides. The reaction conditions using organic solvents limited its application in protein modification (ACS Catalysis, 2018, 8(10): 9115-9). In order to further achieve the modification of Dha-like compounds in a green, mild and efficient manner. In 2022, Wang Xiaolei's research group used hansyl ester as a carbamoyl radical precursor to achieve amidation modification with Dha radical under photocatalysis, and also successfully achieved amidation on the natural antimicrobial peptide nisin, synthesizing a protein post-translational modification analog (The Journal of Organic Chemistry, 2022, 87(10):6852-9). In 2023, Sun Wangsheng's research group synthesized a chiral β-acyl alanine derivative by in-situ generation of acyl radicals from aryl carboxylic acids under photocatalysis and addition with Dha (Organic Letters, 2023, 25(46):8338-43). However, a method for achieving mutation of Dha-Asp analogs through hydrogenation and carboxylation of Dha-like compounds has not yet been reported. Therefore, there is a need to provide a photocatalytic system with broad substrate applicability at room temperature and pressure for the mutation reaction of dehydroalanine derivatives to aspartic acid compounds. Summary of the Invention

[0003] This invention provides a chemical mutation method for converting dehydroalanine derivatives into aspartic acid compounds via photocatalysis. The method utilizes the photocatalysis of 4CzlPN under light irradiation, primarily leveraging the Michael addition reaction between the carbon dioxide radical anions generated from ammonium formate and dehydroalanine compounds, thus completing the mutation from dehydroalanine compounds to aspartic acid compounds under mild conditions. The chemical reaction equation of the described technical solution is as follows:

[0004] To achieve the above objectives, the present invention adopts the following technical solution.

[0005] A method for chemical mutation from dehydroalanine to aspartic acid, comprising the following steps.

[0006] Dehydroalanine compounds, formate, photocatalyst, hydrogen atom transfer reagent, and magnetic stir bar were added to a dry reaction tube. After purging with argon three times, a solvent was added to prepare solution A.

[0007] When solution A is irradiated with a light source, dehydroalanine undergoes a hydrogen carboxylation reaction with formate and a hydrogen atom transfer reagent, resulting in a chemical mutation from dehydroalanine to aspartic acid.

[0008] After the reaction was complete, the solvent was removed, and the mixture was separated and purified using a rapid silica gel column chromatography.

[0009] It should be noted that the solvent used in step 1 is one or more of dimethyl sulfoxide, N,N-dimethylformamide, and dimethyl sulfoxide / N,N-dimethylformamide, and the solvent has undergone deoxygenation treatment and purged with argon gas at least three times, with each argon purging lasting at least ten minutes. Dimethyl sulfoxide is preferred.

[0010] In step 1, the dehydroalanine compounds are derivatives of dehydroalanine with protected amino and carboxyl groups, and at least one dipeptide compound containing dehydroalanine.

[0011] The photocatalyst screened in step 1 is one or more of Ir[dF(CF3)ppy]2(dtbbpy)PF6, 4DPAIPN, RFTA, [Ru(bpy)3](PF6)2, EosinY, and 4CzlPN. 4CzlPN is preferred.

[0012] In step 1, the formate is at least one of sodium formate dihydrate, potassium formate, cesium formate, and ammonium formate. Ammonium formate is preferred.

[0013] In step 1, the HAT reagent is at least one of p-Thiocresol, 1-adamanthiol (1-AdSH), triethylenediamine (DABCO), and quinuclidine. Quinuclidine is preferred.

[0014] The light source in step 2 is a 40W blue LED lamp, and the reaction is carried out at room temperature for 12 hours.

[0015] In step 3, after the reaction is complete, the mixed solvent is directly placed in a lyophilizer to freeze-dry the solvent, and then separated and purified using a rapid silica gel column. The eluent used is petroleum ether / ethyl acetate = 1 / 1, and 1‰ acetic acid is added to separate and obtain the product.

[0016] The beneficial effects of this invention are as follows:

[0017] For the first time, a photo-oxidation-reduction strategy was used to activate formate, and the free radical species generated by it were used to add to Dha, chemically mutating Dha to Asp, thus achieving specific chemical modification of the peptide.

[0018] The method of this invention utilizes quinine rings ( versus SCEin MeCN)

[166] and the oxidation potential of 4CzIPN ( Matching with SCE, a quinine ring was used to quench the excited-state catalyst 4CzIPN via single-electron transfer to obtain an active tertiary amine-based hydrogen atom transfer (HAT) reagent. Literature data shows that the bond dissociation energy of the CH bond in formate (BDE = 86 kcal / mol) is significantly lower than that of the NH bond in the quinine ring cation (BDE = 100 kcal / mol), making a HAT process between formate and the resulting radical cation possible. This allows for the HAT process under photoredox catalysis, where formate undergoes a HAT process to extract hydrogen, generating a carbon dioxide anion radical which then undergoes a radical addition reaction with the Dha substrate, followed by a SET process and protonation to yield the hydrocarboxylated product.

[0019] The method of this invention features mild reaction conditions and requires no metal catalyst, thereby improving the safety and environmental friendliness of the reaction. Furthermore, this method exhibits broad substrate applicability, providing a powerful tool for synthesizing peptides and proteins containing Asp. Utilizing safe, reliable, and easily manipulated formate as a carbon source, it enables the chemical mutation of Dha into Asp, further achieving specific chemical modification of peptides. Attached Figure Description

[0020] Figure 1 NMR spectrum of compound 1

[0021] Figure 2NMR spectrum of compound 2

[0022] Figure 3 NMR spectrum of compound 3

[0023] Figure 4 NMR spectrum of compound 4

[0024] Figure 5 NMR spectrum of compound 5

[0025] Figure 6 NMR spectrum of compound 6

[0026] Figure 7 NMR spectrum of compound 7

[0027] Figure 8 NMR spectrum of compound 8

[0028] Figure 9 NMR spectrum of compound 9

[0029] Figure 10 NMR spectrum of compound 10

[0030] Figure 11 NMR spectrum of compound 11

[0031] Figure 12 NMR spectrum of compound 12

[0032] Figure 13 NMR spectrum of compound 13

[0033] Figure 14 NMR spectrum of compound 14

[0034] Figure 15 NMR spectrum of compound 15

[0035] Figure 16 NMR spectrum of compound 16 Detailed Implementation

[0036] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. These specific details are provided for better explanation, and therefore the scope of protection of the present invention is not limited to the examples given. All raw materials used in this invention are known compounds, which can be commercially available or synthesized using methods known in the art.

[0037] The present invention provides a method for chemical mutation from dehydroalanine to aspartic acid, comprising the following steps. It should be noted that the following preferred embodiments are all performed according to the following steps.

[0038] Dha substrate (0.2 mmol), 4CzlPN (7.89 mg, 5 mol%), quinine ring (4.45 mg, 0.04 mmol), ammonium formate (63.06 mg, 1 mmol) and magnetic flux were added to a dry 10 mL reaction tube. After sealing the reaction tube, it was purged three times with argon gas, and then ultra-dry dimethyl sulfoxide (2 mL) was added.

[0039] At room temperature, the reaction system was placed under a 40W blue LED lamp and reacted for 12 hours.

[0040] After completion, the dimethyl sulfoxide in the system was lyophilized directly, and then separated and purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent, and 1‰ acetic acid was added.

[0041] Example 1: Preparation of compound 13-acetamido-4-methoxy-4-oxobutyric acid:

[0042] The Dha substrate methyl 2-acetamidoacrylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A pale yellow oily substance was obtained, with a yield of 82%.

[0043] The structure and NMR data of the product obtained in Example 1 are as follows: 1 H NMR (400MHz, CDCl3): δ6.87(d,J=8.3Hz,1H),4.80(dt,J=8.6,4.4Hz,1H),3.70(s,3H),3.04–2.93(m,1H),2.77(dd,J=17.5,4.4Hz,1H),2.00(s,3H). 13 CNMR (100MHz, CDCl3): δ173.42,171.36,170.73,52.79,48.48,36.10,22.96.

[0044] Example 2: Preparation of compound 23-((S)-2-((tert-Butoxycarbonyl)amino)propamido)-4-methoxy-4-oxobutyric acid:

[0045] Dha substrate methyl(S)-2-(2-((tert-Butoxycarbonyl)amino)propamido)acrylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and a magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 41%.

[0046] The structure and NMR data of the product obtained in Example 2 are as follows: 1 H NMR (600MHz, CDCl3) δ7.54 (dd, J=42.4, 8.4Hz, 1H), 5.54–5.26 (m, 1H), 4.86 (dq, J=11.0, 6.5, 5.5Hz, 1H), 4.36 (dt, J= 40.3,7.5Hz,1H),3.74(d,J=8.9Hz,3H),3.08(ddd,J=14.9,10.2,4.5Hz,1H),2.88–2.79(m,1H),1.70–1.18(m,12H). 13 C NMR (150MHz, CDCl3) δ173.91,173.29,171.34,156.01,80.75,52.96,49.91,48.47,36.10,28.39,18.90,18.56.

[0047] Example 3: Preparation of compound 34-(((S)6-(((benzyloxy)carbonyl)amino)1-(tert-butoxy)1-oxohexanoic acid-2-)amino)3-((tert-butoxycarbonyl)amino)4-oxobutyric acid:

[0048] Dha substrate tert-butyl N 6 -(benzooxy)carboyl)-N 2-(2-(tert-butyl)amino)acryloyl)-L-lysine (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and a magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A pale yellow oily substance was obtained, with a yield of 82%.

[0049] The structure and NMR data of the product obtained in Example 3 are as follows: 1 H NMR(400MHz, CDCl3)δ7.70(dd,J=38.2,8.5Hz,1H),7.44–7.30(m,5H),5.84( dd,J=39.1,9.4Hz,1H),5.32–4.92(m,2H),4.87(ddd,J=16.9,8.7,4.5Hz,1H) ,4.44–4.16(m,1H),3.71(d,J=16.4Hz,3H),3.12(ddd,J=21.4,17.7,4.4Hz,1 H),2.82(ddd,J=17.8,5.9,4.3Hz,1H),2.10–1.95(m,1H),1.07–0.79(m,6H). 13 CNMR(100MHz, CDCl3)δ174.18,171.83,171.63,171.09,170.96,157.05,156.90,136.10,136.02,128.66,128.37, 128.17,67.48,67.41,60.04,59.84,52.91,52.78,48.44,48.11,35.94,31.84,31.49,19.12,19.04,18.23,18.13.

[0050] Example 4: Preparation of compound 43-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamide)-4-methoxy-4-oxobutyric acid:

[0051] Dha substrate tert-butyl(S)-2-((3-methoxy-3-oxoprop-1-en-2-yl)carbamoyl)pyrrolidine-1-carboxylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and a magnetic flux were added to a dry 10 mL reaction tube. The tube was sealed and purged three times with argon gas. Ultra-dry dimethyl sulfoxide (2 mL) was then added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the dimethyl sulfoxide in the system was lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 eluent and 1‰ acetic acid added. A yellow oily substance was obtained, with a yield of 54%.

[0052] The structure and NMR data of the product obtained in Example 4 are as follows: 1 H NMR(400MHz, CDCl3)δ7.80–7.39(m,1H),4.78(d,J=49.4Hz,1H),4.43–4.22(m,1H),3.73 (s,2H),3.58–3.28(m,2H),3.18–2.75(m,2H),2.29–1.75(m,3H),1.43(d,J=5.3Hz,6H). 13 C NMR (150MHz, CDCl3) δ175.61,174.17,172.72,154.89,80.92,61.04,52.77,48.50,47.29,36.37,31.18,28.46,24.62.

[0053] Example 5: Preparation of compound 53-benzamide-4-((carboxymethyl)amino)-4-oxobutyric acid:

[0054] Dha substrate (2-benzoylacryloyl), glycerol (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and a magnetic flux were added to a dry 10 mL reaction tube. The tube was sealed and purged three times with argon gas. Ultra-dry dimethyl sulfoxide (2 mL) was then added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the dimethyl sulfoxide in the system was lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 47%.

[0055] The structure and NMR data of the product obtained in Example 5 are as follows: 1 H NMR (400MHz, CDCl3) δ7.44–7.23(m,7H),5.27–5.15(m,2H),4.84–4.71(m,1H),4.67–4.57(m,1H),3.68(d, J=2.1Hz,3H),3.09–2.85(m,1H),2.77–2.61(m,2H),2.43(ddd,J=17.3,9.3,3.3Hz,1H),2.32–2.06(m,2H). 13 C NMR (101MHz, CDCl3) δ174.00,173.35,171.12,170.88,151.21,135.14,128.63,128.47,128.13,68.34,59.75,52.84,48.45,35.80.

[0056] Example 6: Preparation of compound 6,3-benzamide-4-(((S)-1-methoxy-1-oxo-3-phenylpropane-2-yl)amino)-4-oxobutyric acid:

[0057] The Dha substrate methyl(2-benzamidoacryloyl)-L-phenylalanine (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 83%.

[0058] The structure and NMR data of the product obtained in Example 6 are as follows: 1H NMR (400MHz, CDCl3) δ7.58(d,J=8.5Hz,1H),5.35(dd,J=36.6,9.2Hz,1H),4.86(dt,J=15.6,5.2Hz,1H),4.15(dt,J=48.8,8.1Hz ,1H),3.70(d,J=3.5Hz,3H),3.11–2.99(m,1H),2.76(t,J=14.3Hz,1H),1.76(s,1H),1.40(d,J=5.4Hz,11H),0.95–0.82(m,6H). 13 C NMR (100MHz, CDCl3) δ171.96,171.42,156.28,80.43,58.48,52.75,48.50,37.94,37.49,28.37,24.80,15.46,11.35.

[0059] Example 7: Preparation of compound 73-benzamide-4-(((S)-1-methoxy-1-oxopropyl-2-yl)amino)-4-oxobutyric acid:

[0060] The Dha substrate methyl(2-benzamidoacryloyl)-L-alanine (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid was added. A yellow oily substance was obtained, with a yield of 71%.

[0061] The structure and NMR data of the product obtained in Example 7 are as follows: 1 H NMR(600MHz, CDCl3)δ7.34(d,J=13.7Hz,1H),5.78(d,J=30.7Hz,1H),4.69–4.66(m,1H),4.54(s, 1H),3.75(s,3H),3.11–2.69(m,2H),2.50(q,J=7.4,6.0Hz,2H),2.26–1.96(m,5H),1.46(m,9H). 13C NMR (100MHz, CDCl3) δ173.94,172.03,170.99,156.12,80.92,53.20,52.93,48.58,35.99,32.75,30.08,28.38,15.41.

[0062] Example 8: Preparation of compound 83-(di(tert-butoxycarbonyl)amino)-4-(((S)-1-methoxy-4-(methylthio)-1-oxobut-2-yl)amino)-4-oxobutyric acid:

[0063] The Dha substrate methyl (2-(di(tert-butyloxycarbonyl)amino)acryloyl)-L-methionine hydrochloride (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 42%.

[0064] The structure and NMR data of the product obtained in Example 8 are as follows: 1 H NMR(600MHz, CDCl3) δ6.79(dd,J=13.7,7.7Hz,1H),5.17(dt,J=9.1,6.7Hz,1H),4.71–4.64(m,1H),3.73(d,J=17.7Hz,3H),3.37( ddd,J=17.3,6.5,3.1Hz,1H),2.84(ddd,J=17.3,6.9,5.0Hz,1H),2.59–2.36(m,2H),2.23–1.91(m,5H),1.49(d,J=10.6Hz,18H). 13 C NMR (150MHz, CDCl3) δ176.37,172.01,169.27,152.02,84.29,55.83,52.64,51.90,34.92,31.71,29.92,28.03,15.57.

[0065] Example 9: Preparation of compound 93-(2(tert-butoxycarbonyl)amino)4-(((S)1,5-dimethoxy-1,5-dioxopentan-2)amino)4-oxobutyric acid:

[0066] The Dha substrate dimethyl(2-(bis-tert-butoxycarbonyl)aminoacryloyl)-L-glutamic acid (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as eluent and 1‰ acetic acid added. A white solid was obtained, with a yield of 64%.

[0067] The structure and NMR data of the product obtained in Example 9 are as follows: 1 H NMR(400MHz, Methanol-d4)δ7.81(ddd,J=8.6,4.1,1.4Hz,2H),7.63–7.39(m,3H),7.30–7.07(m,5H),4.97(td,J=7.5,6.1Hz,1H), 4.70(ddd,J=7.9,6.8,5.4Hz,1H),3.68(d,J=9.6Hz,3H),3.15(ddd,J=13.8,5.5,4.2Hz,1H),3.08–2.88(m,1H),2.85–2.66(m,2H). 13 C NMR(100MHz,MeOD)δ164.48,163.56,163.32,160.69,128.28,125.53,123.50 ,120.85,120.06,119.10,118.48,118.37,45.61,43.27,42.18,28.84,27.06.

[0068] Example 10: Preparation of compound 103-(di(tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutyric acid:

[0069] The Dha substrate methyl 2-(di-tert-butoxycarbonyl)aminoacrylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 40%.

[0070] The structure and NMR data of the product obtained in Example 10 are as follows: 1 H NMR (600MHz, CDCl3) δ7.87–7.68(m,3H),7.60–7.36(m,4H),5.13–5.05(m,1H),4.52(h,J=7.3Hz,1H),3.68( d,J=31.0Hz,3H),3.05(ddd,J=17.0,7.8,4.6Hz,1H),2.82(ddd,J=17.0,6.8,2.7Hz,1H),1.51–1.36(m,3H). 13 C NMR (150MHz, CDCl3) δ174.82,173.10,170.64,167.95,133.22,132.29,128.80,127.43,52.69,49.70,48.62,36.26,17.97.

[0071] Example 11: Preparation of compound 113-((S)-2-(((benzyloxy)carbonyl)amino)propionamido)-4-methoxy-4-oxobutyric acid:

[0072] Dha substrate methyl(S)-2-(2-(((benzyloxy)carbonyl)amino)propamido)acrylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and a magnetic flux were added to a dry 10 mL reaction tube. The tube was sealed and purged three times with argon gas. Ultra-dry dimethyl sulfoxide (2 mL) was then added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the dimethyl sulfoxide was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 eluent and 1‰ acetic acid added. A white solid was obtained, with a yield of 51%.

[0073] The structure and NMR data of the product obtained in Example 11 are as follows: 1 H NMR (400MHz, Methanol-d4) δ7.90–7.83(m,2H),7.67–7.37(m,3H),5.04(dd,J=7.9,5.7Hz, 1H),3.97(s,2H),3.71(s,3H),2.99(dd,J=16.7,5.7Hz,1H),2.84(dd,J=16.7,7.9Hz,1H). 13 C NMR (100MHz, MeOD) δ174.11,173.67,171.63,170.19,135.08,132.95,129.52,128.60,52.63,51.72,42.02,36.67.

[0074] Example 12: Preparation of compound 123-((S)-2-(((benzyloxy)carbonyl)amino)-3-methylbutamido)-4-((S)-2-(methoxycarbonyl)pyrrolidine-1-yl)-4-oxobutyric acid:

[0075] Dha substrate methyl(2-((S)-2-((benzyloxy)carbonyl)amino-3-methylbutamido)acryloyl)-L-proline (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 59%.

[0076] The structure and NMR data of the product obtained in Example 12 are as follows: 1 H NMR (400MHz, CDCl3) δ6.85(dd,J=7.7,3.3Hz,1H),5.13(t,J=6.6Hz,1H),4.50(dtd,J=13.0,7.7,5.3Hz,1H),3.75–3.61(m,3H),3.59(d,J=4.2H z,3H),2.71(ddd,J=16.6,9.2,6.6Hz,1H),2.38–2.23(m,3H),2.11(dddd,J=15.3,12.2,7.6,2.2Hz,1H),1.98–1.85(m,1H),1.56–1.34(m,18H). 13 C NMR (100MHz, CDCl3) δ174.17,173.15,171.79,169.41,151.89,83.77,52.48,52.39,51.99,51.74,29.77,27.81,27.41,27.25.

[0077] Example 13: Preparation of compound 133-((S)-2-(((benzyloxy)carbonyl)amino)-3-methylbutamido)-4-methoxy-4-oxobutyric acid:

[0078] The Dha substrate methyl(S)-2-(2-(((benzyloxy)carbonyl)amino)-3-methylbutamido)acrylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as eluent and 1‰ acetic acid added. A pale yellow oily substance was obtained, with a yield of 32%.

[0079] The structure and NMR data of the product obtained in Example 13 are as follows: 1 H NMR (400MHz, CDCl3) δ8.15–7.20(m,12H),5.42(d,J=23.5Hz,1H),5.22–4.93(m,3H),4.41(d,J=23.6Hz,1H),3 .36–3.01(m,2H),2.94(d,J=25.7Hz,1H),2.75(d,J=18.9Hz,1H),1.69(d,J=65.1Hz,2H),1.54–1.27(m,13H). 13 C NMR(150MHz,MeOD)δ174.02,173.07,172.98,170.20,158.92,138.45,135.08,132.98,129.56,129. 44,128.91,128.59,128.55,82.94,67.33,54.55,51.86,36.65,32.29,30.33,28.24,23.82,20.85.

[0080] Example 14: Preparation of compound 143-(S)-2-benzoylpropane-4-methoxy-4-oxobutyric acid:

[0081] The Dha substrate methyl(S)-2-(2-benzamidopropamido)acrylic acid (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A pale yellow oily substance was obtained, with a yield of 83%.

[0082] The structure and NMR data of the product obtained in Example 14 are as follows: 1 H NMR (600MHz, CDCl3) δ7.13 (t, J=9.4Hz, 1H), 5.74 (dd, J=14.3, 8.1Hz, 1H), 4.85 (dq, J=8.5, 5.2, 4.4Hz, 1H), 4.37 (dq, J=12.6, 5.1, 4.5Hz, 1H), 3.74 (s, 3H), 3.22–2.76 (m, 4H), 1.44 (dd, J=15.9, 3.3Hz, 18H). 13 C NMR (150MHz, CDCl3) δ174.26,171.29,171.23,170.72,170.32,156.00,82. 57,80.22,52.96,52.93,51.26,48.40,37.89,36.14,28.44,28.01,27.98.

[0083] Example 15: Preparation of compound 153-benzamido-4-(((S)6-(((benzyloxy)carbonyl)amino)1-(tert-butoxy)1-oxohexanoic acid)amino)4-oxobutyric acid:

[0084] Dha substrate tributylN 2 -(2-Anilineacryloyl)-N 6-(benzyloxy)carboyl)-L-lysine ester (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and a magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as the eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 35%.

[0085] The structure and NMR data of the product obtained in Example 15 are as follows: 1 H NMR (400MHz, CDCl3) δ7.34(q,J=3.2Hz,5H),7.09(d,J=7.6Hz,1H),5.50(d,J=8 .6Hz,1H),5.10(dd,J=12.3,9.0Hz,2H),4.87(dt,J=8.2,5.1Hz,1H),4.52(ddd ,J=30.9,8.6,4.3Hz,1H),4.14–4.01(m,1H),3.80–3.67(m,5H),2.96–2.87(m, 1H),2.17(ddq,J=32.6,13.2,7.0Hz,2H),2.05-1.95(m,4H),0.99–0.83(m,6H). 13 C NMR (100MHz, CDCl3) δ172.90,172.08,171.58,169.29,156.52,136.35,128.65,128.23 ,128.23,67.19,63.76,60.37,59.10,52.74,47.39,31.29,28.98,25.02,19.32,17.69.

[0086] Example 16: Preparation of compound 163-((S)-1-((benzyloxy)carbonyl)pyrrolidine-2-carboxamide)-4-methoxy-4-oxobutyric acid:

[0087] Dha substrate benzyl(S)-2-((3-methoxy-3-oxoprop-1-en-2-yl)carbamoyl)pyrrolidine-1-carboxylate (0.2 mmol), photocatalyst 4CzlPN (7.89 mg, 5 mol%), HAT reagent quinine ring (4.45 mg, 0.04 mmol), carboxylic acid source ammonium formate (63.06 mg, 1 mmol), and magnetic flux were added to a dry 10 mL reaction tube. After sealing the tube, the reaction mixture was purged three times with argon gas, and then 2 mL of ultra-dry dimethyl sulfoxide (DMSO) was added. The reaction system was placed under a 40 W blue LED lamp at room temperature and reacted for 12 hours. After the reaction, the DMSO in the system was directly lyophilized and then purified by rapid silica gel column chromatography with petroleum ether / ethyl acetate = 1 / 1 as eluent and 1‰ acetic acid added. A colorless oily substance was obtained, with a yield of 32%.

[0088] The structure and NMR data of the product obtained in Example 16 are as follows: 1 H NMR(400MHz, CDCl3)δ7.73(s,1H),7.34–7.26(m,5H),6.15(d,J=7.9Hz,1H),5.16–5.00(m,2H),4.86–4.71(m,1H),4.59–4.36 (m,1H),4.28(dd,J=17.5,6.8Hz,1H),4.07–3.34(m,5H),2.42–2.15(m,2H),2.04–1.78(m,4H),1.38(dd,J=17.2,7.1Hz,3H). 13 CNMR (100MHz, CDCl3) δ172.55,170.32,169.30,162.55,155.99,136.34,128.65,128 .40,128.21,67.11,64.27,58.96,54.36,49.92,45.41,28.55,24.97,22.43,18.61.

[0089] In summary, the beneficial effects of this invention are as follows: Formate can generate carbon dioxide radical anions under photo-oxidation-reduction catalysis, avoiding the use of toxic, flammable, and difficult-to-handle CO and CO2 as carboxyl sources for hydrogen carboxylation reactions. Dha, as a unique reaction site in polypeptide and protein structures, offers the possibility of specific chemical modification due to its unique chemical properties. This invention utilizes ammonium formate as an ideal 1C synthon, successfully developing a photo-oxidation-reduction method using 4CzlPN as a photocatalyst and quinine ring as a HAT reagent, which can achieve hydrogen carboxylation modification of Dha and short peptides containing Dha, chemically mutating Dha to Aspartic acid (Asp). Furthermore, the reaction system conditions of this invention are mild and the operation is simple, thereby improving the safety and environmental friendliness of the reaction. The mechanism of this reaction has also been studied, and a possible reaction pathway has been proposed based on literature. In addition, this invention demonstrates broad substrate applicability, providing a powerful tool for the synthesis of polypeptides and proteins containing Aspartic acid.

Claims

1. A chemical mutation method for converting dehydroalanine to aspartic acid, characterized in that: Dehydroalanine compound (I) reacts with formate (II), photocatalyst (III), hydrogen atom transfer reagent (IIII) in a suitable organic solvent and at a specific temperature to give aspartic acid compound (IIIII). The chemical reaction equation is as follows: The structure of the dehydroalanine compound (I) mutated into aspartic acid is as follows: a derivative of dehydroalanine with protected amino and carboxyl groups, or at least one dipeptide compound containing dehydroalanine, as shown below:

2. The formate is at least one of sodium formate dihydrate, potassium formate, cesium formate, and ammonium formate.

3. The photocatalyst is at least one of Ir[dF(CF3)ppy]2(dtbbpy)PF6, 4DPAIPN, RFTA, [Ru(bpy)3](PF6)2, EosinY, 4CzlPN, and the hydrogen atom transfer reagent is a quinine ring.

4. The method according to claim 1, characterized in that, The solvent used was dimethyl sulfoxide (DMSO), the concentration of dehydroalanine compounds was 0.1 mmol / mL, the concentration of ammonium formate was 0.5 mmol / mL, the concentration of photocatalyst 4CzlPN was 25 mmol / mL, and the concentration of hydrogen atom transfer reagent quinine ring was 0.02 mmol / mL. The reaction tube was placed in a reaction tube, sealed, and then purged three times with argon gas.

5. The method according to claim 1, characterized in that, The light source used at room temperature is a 40W blue LED lamp, and the reaction time is 12 hours.

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