A method for efficient asymmetric synthesis of arylpropionic acid drug molecules

Abstract

This invention discloses a highly efficient asymmetric synthesis method for aryl propionic acid drug molecules. The method uses racemic aldehydes as substrates and thiamine pyrophosphate-dependent enzymes as biocatalysts. Electrolysis is performed in a diaphragm-free electrolytic cell under conditions of an external redox dielectric and constant current, in a buffer salt solution and nitrogen atmosphere. This method achieves dynamic kinetic oxidation of aldehydes via bioelectrocatalysis, enabling the high-efficiency and high enantioselectivity synthesis of aryl propionic acid drug molecules. The method eliminates the need for redox agents and additives, is simple to operate, operates under mild reaction conditions, and is green and sustainable. This invention applies bioelectrocatalysis to asymmetric synthesis, providing a green and efficient new enzymatic synthetic route for the asymmetric synthesis of aryl propionic acid drug molecules, which has significant application prospects in the pharmaceutical industry.

Description

Technical Field

[0001] This invention relates to the field of bioelectrocatalysis technology, and in particular to a method for the efficient asymmetric synthesis of arylpropionic acid drug molecules. Background Technology

[0002] Arylpropionic acids are a large class of nonsteroidal anti-inflammatory drugs (NSAIDs) with strong anti-inflammatory, antipyretic, and analgesic effects and low toxicity. These compounds contain a chiral carbon atom in their molecular structure and exist as a pair of optical isomers, with the S-configuration typically exhibiting higher reactivity than the R-configuration. Currently, the chemical synthesis of enantiomeric arylpropionic acids usually requires expensive chiral ligands and complex synthetic steps, which does not align with the principles and requirements of green chemistry. Biocatalysis, due to its green, mild, efficient, and highly selective nature, is widely used in the synthesis of bioactive molecules and pharmaceutical intermediates. Similarly, electrocatalysis, utilizing renewable electrical energy, uses electrons as a cheap and pollution-free reagent to replace exogenous oxidants and reductants, and is also considered a powerful and green synthetic method. Bioelectrocatalysis integrates the advantages of both and is widely used in the construction of biosensors and biofuel cells; however, its application in asymmetric synthesis still faces many challenges, such as the incompatibility between biocatalysts and electrochemical conditions and difficulties in heterogeneous electron transfer. Currently, the combination of biocatalysis and electrocatalysis is mainly limited to two modes: one is to use electrical energy to realize the cyclic regeneration of cofactors in oxidoreductases; the other is the cascade of electrocatalysis and biocatalysis. In this mode, the intermediates or products generated by electrocatalysis participate in the subsequent biocatalytic process. Both modes only realize the natural reaction of biocatalysis.

[0003] Thiamine pyrophosphate (ThDP)-dependent enzymes are a class of stable and diverse biocatalysts that catalyze C–C bonding or C–C cleavage reactions through mechanisms such as the formation of Breslow intermediates with substrates and polarity reversal. Studies have shown that these enzymes are compatible under certain electrochemical conditions. Combining thiamine pyrophosphate (ThDP)-dependent enzymes with electrochemistry to unlock more asymmetric, non-natural transformations is of great significance for the green and efficient synthesis of enantiomeric active molecules and drug intermediates, and holds promise for opening up new directions in bioelectrocatalysis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for the efficient asymmetric synthesis of aryl propionic acid drug molecules. This invention develops an electroenzymatically catalyzed method for the efficient asymmetric synthesis of aryl propionic acid drug molecules. This method is green, environmentally friendly, sustainable, and has low processing costs, making it potentially significant in the pharmaceutical industry.

[0005] The technical solution of the present invention is as follows:

[0006] A method for the efficient asymmetric synthesis of arylpropionic acid drug molecules, wherein the method uses racemic aldehydes as substrates and thiamine pyrophosphate-dependent enzymes as biocatalysts, and is carried out in a diaphragm-free electrolytic cell under the conditions of an external redox dielectric and a constant current, in a buffer salt solution and nitrogen atmosphere; the general reaction formula is as follows:

[0007]

[0008] in:

[0009] R 1 Selected from heteroaryl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl;

[0010] R 2 Selected from methyl, ethyl, propyl, isopropyl, allyl, and pentenyl;

[0011] R 3 Hydrogen atoms or halogen atoms;

[0012] The substituents of the phenyl group are selected from halogen atoms, methoxy groups, methylthio groups, trifluoromethyl groups, isopentyl groups, thiophene-2-ylformyl groups, benzoyl groups, cyclopentanone-2-ylethyl groups, ester groups, or cyano groups;

[0013] The substituents of naphthyl are methoxy and cyclohexyl;

[0014] The substituents of the biphenyl group are halogen atoms;

[0015] The heteroaryl group is selected from benzofuranyl, benzothiopheneyl, carbazoyl, pyrazolyl or pyridinyl with halogen atom substitution.

[0016] Further, the substrate is selected from 2-(4-isobutylphenyl)propanal and its derivatives, 2-(6-methoxynaphthyl-2-yl)propanal and its derivatives, 2-(2-fluoro-4-biphenyl)propanal and its derivatives, 2-(4-benzoylphenyl)propanal and its derivatives, 2-(4-(2-oxocyclopentyl)methyl)phenyl)propanal and its derivatives, 2-(5H-benzopyrano[2,3-b]pyridin-7-yl)propanal and its derivatives, 2-(4-cyclohexyl-1-naphthyl)propanal and its derivatives, 2-(4-thiophene-2-carbonyl)phenylpropanal and its derivatives, 2-(6-chloro-9H-carbazole-2-yl)propanal and its derivatives, and 2-(10-oxo-10,11-dihydrodibenzo[b,f]thiophene-2-yl)propanal and its derivatives.

[0017] Further, the thiamine pyrophosphate-dependent enzyme is selected from LlKdcA or an LlKdcA mutant, PfBAL or a PfBAL mutant; LlKdcA is a branched-chain keto acid decarboxylase derived from Lactococcus lactis; PfBAL is a benzaldehyde lyase derived from Pseudomonas fluorescens; the thiamine pyrophosphate-dependent enzyme is obtained by induction expression of Escherichia coli BL21(DE3); the thiamine pyrophosphate-dependent enzyme catalyzes the reaction in the form of a pure enzyme or whole cells; when the thiamine pyrophosphate-dependent enzyme is a pure enzyme, the amount of pure enzyme relative to racemic aldehyde is 0.05-2 mol%; when the thiamine pyrophosphate-dependent enzyme is a whole cell, the amount of whole cell is OD. 600 =20-50.

[0018] Further, the amino acid sequence of LlKdcA is shown in SEQ ID NO.1; the LlKdcA mutant is one in which at least one of the following mutations exists in the amino acid sequence corresponding to SEQ ID NO.1: H at position 112 is mutated to A, L or F; H at position 113 is mutated to A, L or F; F at position 382 is mutated to A, L or W; V at position 461 is mutated to A, L or F; E at position 462 is mutated to A, L or F; I at position 465 is mutated to A, L or F; and F at position 542 is mutated to A, L or W.

[0019] Further, the amino acid sequence of the PfBAL is shown in SEQ ID NO.2; the PfBAL mutant is one in which at least one of the following mutations exists in the amino acid sequence corresponding to SEQ ID NO.2: A at position 480 is mutated to G, L or F; T at position 481 is mutated to A, L or F; F at position 484 is mutated to A, L or W.

[0020] Further, the redox dielectric is selected from any one of ferrocene methanol, α-ferrocene ethanol, ferrocene dimethanol, ferrocene carboxylic acid, acetylferrocene, ferrocene, and 2,2,6,6-tetramethylpiperidine oxide; the molar ratio of the redox dielectric to the racemic aldehyde is 0.25-1.5:1;

[0021] Furthermore, the constant current is 0.2-5.0 mA, and the charge is 4-8 F / mol; the electrodes in the electrolytic cell include a positive electrode and a negative electrode, wherein the positive electrode is any one of a graphite electrode, a platinum electrode, and a BDD electrode, and the negative electrode is a graphite electrode or a platinum electrode.

[0022] Further, the buffer salt solution is selected from any one of 3-morpholine propanesulfonic acid buffer, phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, N-tris(hydroxymethyl)methylglycine buffer, imidazole buffer; hydroxyethylpiperazine ethylthiosulfate or tris(hydroxymethyl)methylglycine salt buffer; the concentration of the buffer salt solution is 25-200 mmol / L, and the pH is 6.0-8.0.

[0023] Furthermore, the method is specifically as follows:

[0024] In a nitrogen atmosphere, a buffer salt solution is added to a diaphragmless electrolytic cell with a magnetic stirrer, followed by the sequential addition of thiamine pyrophosphate-dependent enzyme, racemic aldehyde, and redox dielectric. Electrodes are then inserted and a constant current is applied to initiate the electrolysis reaction. After the reaction is complete, acetonitrile is added to the reaction solution to quench the reaction.

[0025] Furthermore, the rotational speed of the magnetic stirrer is 400-800 rpm.

[0026] The beneficial technical effects of this invention are as follows:

[0027] This invention combines electrochemical synthesis with a thiamine pyrophosphate (ThDP)-dependent enzyme biocatalysis system to achieve dynamic kinetic oxidation of racemic aldehydes, providing a new strategy for the efficient asymmetric synthesis of aryl propionic acid drugs. Specifically, the active pocket of the thiamine pyrophosphate (ThDP)-dependent enzyme allows for specific recognition of S-configured substrates, and the enzyme's own basic amino acid residues accelerate substrate racemization. Mechanistically, S-configured substrates preferentially react with the cofactor ThDP to form a stable Breslow intermediate. This intermediate undergoes two single-electron oxidations in the presence of a redox medium to form an acylammonium cation intermediate, which is subsequently attacked by hydrophilic nucleophiles, forming stereoselective aryl propionic acid products and releasing the ThDP cofactor. Through screening a ThDP-dependent enzyme mutant library (based on molecular docking to determine amino acid residues near the active site) and optimizing reaction conditions, this invention achieves high-yield and highly enantioselective synthesis of aryl propionic acid drugs. This invention combines biocatalysis with electrosynthesis, expanding the catalytic range of ThDP-dependent enzymes and developing a novel bioelectrocatalyst for asymmetric catalysis. This invention utilizes readily available and inexpensive graphite electrodes as both positive and negative electrodes, employs whole cells as catalysts, and reduces the amount of pure enzyme used to as low as 0.05 mol%, offering simplicity and strong practicality. The racemization of the racemic aldehyde substrate in this invention is promoted by the enzyme's own basic amino acid residues, avoiding the introduction of racemic reagents and exhibiting excellent atom economy. In summary, this invention provides a green and efficient new electroenzymatic synthesis route for the asymmetric synthesis of arylpropionic acid compounds, with significant application prospects in the pharmaceutical industry.

[0028] The method for synthesizing chiral drug molecules by bioelectrocatalysis described in this invention is green, environmentally friendly, and sustainable; it requires no redox agents or chiral ligands and has low process costs.

[0029] The device described in this invention is an undivided electrolytic cell, which is convenient for feeding and simple for post-processing.

[0030] The reaction conditions of this invention are mild, can be carried out at room temperature and pressure, and have a wide range of applicable substrates.

[0031] This invention can use inexpensive and readily available graphite electrodes as positive and negative electrodes, and the amount of biocatalyst can be reduced to 0.05 mol%. Whole-cell catalysis can also be used, and the reaction can be scaled up to 1 mmol with the same yield and enantioselectivity, making it highly practical.

[0032] This invention develops a method for the efficient asymmetric synthesis of aryl propionic acid drug molecules via electroenzyme catalysis, which has potential significant implications for the pharmaceutical industry. Attached Figure Description

[0033] Figure 1 This is the chiral HPLC chromatogram of the racemic standard flurbiprofen.

[0034] Figure 2 The image shows the chiral HPLC chromatogram of the enantiomeric enriched product (S)-flurbiprofen prepared in Example 4 of this invention.

[0035] Figure 3 Racemic standard (S)-flurbiprofen 1 H-NMR spectrum.

[0036] Figure 4 For racemic standard (S)-flurbiprofen 13 C-NMR spectrum.

[0037] Figure 5 This is the chiral HPLC chromatogram of the racemic standard sulprofen.

[0038] Figure 6 The image shows the chiral HPLC chromatogram of the enantiomer enriched product (S)-sulprofen prepared in Example 5 of this invention.

[0039] Figure 7 Racemic standard (S) - Sulprofen 1 H-NMR spectrum.

[0040] Figure 8 For racemic standard (S) - sulprofen 13 C-NMR spectrum. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0042] The sources and amino acid sequences of the thiamine pyrophosphate (ThDP)-dependent enzymes used in the following examples / comparative examples are as follows:

[0043] LlKdcA enzyme is a branched-chain keto acid decarboxylase derived from Lactococcus lactis, and its amino acid sequence is shown in SEQ ID NO. 1.

[0044] SEQ ID NO.1:

[0045] MGYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISHKDMKWVGNANELNASYMA

[0046] DGYARTKKAAAFLTTFGVGELSAVNGLAGSYAENLPVVEIVGSPTSKVQNEGKFVHHTLAD

[0047] GDFKHFMKMHEPVTAARTLLTAENATVEIDRVLSALLKERKPVYINLPVDVAAAKAEKPSL

[0048] PLKKENSTSNTSDQEILNKIQESLKNAKKPIVITGHEIISFGLEKTVTQFISKTKLPITTLNFGK

[0049] SSVDEALPSFLGIYNGTLSEPNLKEFVESADFILMLGVKLTDSSTGAFTHHLNENKMISLNID

[0050] EGKIFNERIQNFDFFESLISSLLDLSEIEYKGKYIDKKQEDFVPSNALLSQDRLWQAVENLTQS

[0051] NETIVAEQGTSFFGASSIFLKSKSHFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDGSLQL

[0052] TVQELGLAIREKINPICFIINNDGYTVEREIHGPNQSYNDIPMWNYSKLPESFGATEDRVVSKI

[0053] VRTENEFVSVMKEAQADPNRMYWIELILAKEGAPKVLKKMGKLFAEQNKSLEHHHHHH

[0054] The PfBAL enzyme is a benzaldehyde lyase derived from Pseudomonas fluorescens, and its amino acid sequence is shown in SEQ ID NO.2;

[0055] SEQ ID NO.2:

[0056] MAMITGGELVVRTLIKAGVEHLFGLHGAHIDTIFQACLDHDVPIIDTRHEAAAGHAAEG

[0057] YARAGAKLGVALVTAGGGFTNAVTPIANAWLDRTPVLFLTGSGALRDDETNTLQAGIDQVA

[0058] MAAPITKWAHRVMATEHIPRLVMQAIRAALSAPRGPVLLDLPWDILMNQIDEDSVIIPDLVL

[0059] SAHGARPDPADLDQALALLRKAERPVIVLGSEASRTARKTALSAFVAATGVPVFADYEGLS

[0060] MLSGLPDAMRGGLVQNLYSFAKADAAPDLVLMLGARFGLNTGHGSGQLIPHSAQVIQVDP

[0061] DACELGRLQGIALGIVADVGGTIEALAQATAQDAAWPDRGDWCAKVTDLAQERYASIAAK

[0062] SSSEHALHPFHASQVIAKHVDAGVTVVADGALTYLWLSEVMSRVKPGGFLCHGYLGSMGV

[0063] GFGTALGAQVADLEAGRRTILVTGDGSVGYSIGEFDTLVRKQLPLIVIIMNNQSWGATLHFQ

[0064] QLAVGPNRVTGTRLENGSYHGVAAAFGADGYHVDSVESFSAALAQALAHNRPACINVAVA

[0065] LDPIPPEELILIGMDPFALE

[0066] PpBFD enzyme is a benzoylformate decarboxylase derived from Pseudomonas putida, with the amino acid sequence shown in SEQ ID NO.3;

[0067] SEQ ID NO.3:

[0068] MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQ

[0069] ASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEALLTNVDAANLP

[0070] RPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

[0071] VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAP

[0072] RCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISVTC

[0073] DPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDT

[0074] LNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEPERQ

[0075] VIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVPGIDF

[0076] RALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

[0077] PaBAL enzyme is a benzaldehyde lyase derived from Polymorphobacter arshaanensis, with the amino acid sequence shown in SEQ ID NO.4;

[0078] SEQ ID NO.4:

[0079] MSSPEARYTGGDLLAQTLHDAGVTKIFALHGGHHEALFKGCIDQGIDLIDFRHEAAAG

[0080] HAADAYARTTGKLGVCIITAGPGFTNAISAIANAQLDASPVLFLIGAPPLREVETNPLQGGID

[0081] QIAMARPAAKWALSIPSTERVRDLTAMAIRKAMTGRKGPVVLEIPIDILHMSVTGAQATPSA

[0082] GLAVRPQPAPAPEEVAALAELLLRAERPVIVAGLESASAATAVALRALVAKLPLPVFAKPQAY

[0083] GLLPAGHACDAGAAGNLAVLPIIGAGAPDLVILLGARLGLMLGGRSGALVPHDAHVVQIYS

[0084] DASEIGRLRDIDLPIAADCAQTLTALTKALAAVDLPDTSAWTARAAGAKALAASAWPDAEV

[0085] AGGIHPYHAAKAVANAAGQDAAYVFDGGESSSWGTATVAVDAPARVLSHGYLGCLGIGPG

[0086] FAIGMQIAHPDRRVVQVTGDGAMGFHIQEFDTMVRHRLPIVTVILNNQVWGMSIHGQQM

[0087] MYGANYNVITKLGSTQYASIAAAFGCHAERVTAFAEIAPAMARAFASGKPALVEIMTDADV

[0088] VHPATVAMLGQLAEGSRDIMIPYYENIAAS

[0089] The CDH enzyme is cyclohexane-1,2-dione hydrolase derived from Azoarcus sp., and its amino acid sequence is shown in SEQ ID NO.5;

[0090] SEQ ID NO.5:

[0091] MAIKRGADLIVEALEEYGTEQVVGFIGHTSHFVADAFSKSHLGKRVINPATELGGAWM

[0092] VNGYNYVKDRSAAVGAWHCVGNLLLHAAMQEARTGRIPAVHIGLNSDGRLAGRSEAAQQ

[0093] VPWQSFTPIARSTQRVERLDKVGEAIHEAFRVAEGHPAGPAYVDIPFDLTADQIDDKALVPRG

[0094] ATRAKSVLHAPNEDVREAAAQLVAAKNPVILAGGGVARSGGSEALLKLAEMVGVPVVTTS

[0095] TGAGVFPETHALAMGSAGFCGWKSANDMMAAADFVLVLGSRLSDWGIAQGYITKMPKF

[0096] VHVDTDPAVLGTFYFPLLSVVADAKTFMEQLIEVLPGTSGFKAVRYQERENFRQATEFRAA

[0097] WDGWVREQESGDGMPASMFRAMAEVRKVQRPEDIIVTDIGNHTLPMFGGAILQRPRRLVT

[0098] SMAEGILGCGFPMALGAQLAEPNSRVFLGTGDGALYYHFNEFRVAVEHKLPVITMVFTNES

[0099] YGANWTLMNHQFGQNNWTEFMNPDWVGIAKAFGAYGESVRETGDIAGALQRAIDSGKPA

[0100] LIEIPVSKTQGLASDPVGGVGPNLLLKGREIPVDTGGSMYPGENLLHLKSLE

[0101] The EcMenD enzyme is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase derived from Escherichia coli, and its amino acid sequence is as shown in SEQ ID NO.6;

[0102] SEQ ID NO.6:

[0103] MSVSAFNRRWAAVILEALTRHGVRHICIAPGSRSTPLTLAAAENSAFIHHTHFDERGLGHLALGLAKVSKQPVAVIVTSGTAVANLYPALIEAGLTGEKLILLTADRPPELIDCGANQAIRQPGMFASHPTHSISLPRPTQDIPARWLVSTIDHALGTLHAGGVHINCPFAEPLYGEMDDTGLSWQQRLGDWWQDDKPWLREAPRLESEKQRDWFFWRQKRGVVVAGRMSAEEGKKVALWAQTLGWPLIGDVLSQTGQPLPCADLWLGNAKATSELQQAQIVVQLGSSLTGKRLLQWQASCEPEEYWIVDDIEGRLDPAHHRGRRLIANIADWLELHPAEKRQPWCVEIPRLAEQAMQAVIARRDAFGEAQLAHRICDYLPEQGQLFVGNSLVVRLIDALSQLPAGYPVYSNRGASGIDGLLSTAAGVQRASGKPTLAIVGDLSALYDLNALALLRQVSAPLVLIVVNNNGGQIFSLLPTPQSERERFYLMPQNVHFEHAAAMFELKYHRPQNWQELETAFADAWRTPTTTVIEMVVNDTDGAQTLQQLLAQVSHL

[0104] The PO enzyme is pyruvate oxidase, and its amino acid sequence is shown in SEQ ID NO.7;

[0105] SEQ ID NO.7:

[0106] MSDNKINIGLAVMKILESWGADTIYGIPSGTLSSLMDAMGEEENNVKFLQVKHEEVGAMAAVMQSKFGGNLGVTVGSGGPGASHLINGLYDAAMDNIPVVAILGSRPQRELNMDAFQELNQNPMYDHIAVYNRRVAYAEQLPKLVDEA ARMAIAKRGVAVLEVPGDFAKVEIDNDQWYSSANSLRKYEPIAPAAQDIDAAVELLNNSKRPVIYAGIGTMGHGPAVQELARKIKAPVITTGKNFETFEWDFEALTGSTYRVGWKPANETILEADTVLFAGSNFPFSEVEGTFRNVDN FIQIDIDPAMLGKRHHADVAILGDAGLAIDEILNKVDAVEESAWWTANLKNIANWREYINMLETKEEGDLQFYQVYNAINNHADEDAIYSIDVGNSTQTSIRHLHMTPKNMWRTSPLFATMGIAIPGGLGAKNTYPDRQVWNIIGDGA FSMTYPDVVTNVRYNMPVINVVFSNTEYAFIKNKYEDTNKNLFGVDFTDVDYAKIAEAQGAKGFTVSRIEDMDRVMAEAVAANKAGHTVVIDCKITQDRPIPVETLKLDSKLYSEDEIKAYKERYEAANLVPFREYLEAEGLESKYIK

[0107] In the examples below, the PfBAL-T481L mutant indicates that the amino acid residue (threonine, T) at position 481 of the amino acid sequence SEQ ID NO.2 of PfBAL has been changed to leucine (L).

[0108] Example 1

[0109] The preparation of thiamine pyrophosphate (ThDP)-dependent enzymes with different functions and sources is detailed below:

[0110] (1) Introduce a recombinant plasmid containing the target gene into competent cells.

[0111] The amino acid sequences (SEQ ID NO.1 to SEQ ID NO.7) of thiamine pyrophosphate-dependent enzymes from different sources were sent to the gene department of Sangon Biotech (Shanghai) Co., Ltd. for synthesis. After codon optimization, the genes were synthesized and recombined into the pET28a(+) vector to construct recombinant plasmids. The LlKdcA mutant and PfBAL mutant were constructed by point mutation. The amino acid sequences after point mutation were sent to Sangon Biotech (Shanghai) Co., Ltd., and after codon optimization, the genes were synthesized and ligated into the pET28a(+) vector to construct recombinant plasmids of the mutants.

[0112] (2) Introducing the recombinant plasmid into competent cells

[0113] The recombinant plasmid was in lyophilized powder form. Centrifugation was used to allow the lyophilized powder to settle to the bottom. 40 μL of sterile water was added to dissolve the lyophilized powder, and centrifugation was repeated. Then, BL21(DE3) competent cells were slowly thawed on ice. 1 μL of the thawed recombinant plasmid was added to 100 μL of BL21(DE3) competent cells, gently tapped, and placed on ice for 30 min. Then, the cells were heat-shocked in a 42°C water bath for 90 s, and immediately incubated on ice for 5 min. Next, 300 μL of fresh, antibiotic-free liquid LB medium was added, and the cells were incubated at 37°C and 220 rpm for 40 min. The incubation was then spread onto solid LB agar plates containing 50 μg / mL kanamycin sulfate and incubated at 37°C for 12 h. Single colonies were picked and inoculated into 6 mL of liquid LB medium to obtain a pre-activated seed culture for subsequent sequencing and scale-up culture.

[0114] (3) Induced expression of thiamine pyrophosphate-dependent enzyme

[0115] The pre-activated 6 mL seed culture was inoculated into 800 mL of TB liquid medium containing kanamycin sulfate at a final concentration of 50 μg / mL. After culturing at 37℃ and 220 rpm for 4-5 h, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM was added to induce protein expression for 16 h.

[0116] The cells were collected by centrifugation at 7000 rpm for 10 min, and whole cells of ThDP enzymes from different sources were obtained. After separation and purification of the whole cells of ThDP enzymes from different sources, pure ThDP enzymes from different sources were obtained.

[0117] Example 2

[0118] The preparation of (S)-ibuprofen is carried out by the following reaction:

[0119]

[0120] Its preparation method is as follows:

[0121] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(4-isobutylphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0122] After the reaction was complete, the electrolytic cell was removed from the glove box, and 2 mL of acetonitrile was added to quench the reaction. 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was 61% when analyzed by ultra-high performance liquid chromatography (UHPLC), and the enantiomeric excess of the product was 96% when analyzed by chiral UHPLC. The racemic standard characterization data are as follows:

[0123] 1 H NMR (400MHz, CDCl3) δ7.21(d,J=8.1Hz,2H),7.09(d,J=8.1Hz,2H),3.70(q,J=7.2Hz,1H) ,2.44(d,J=7.1Hz,2H),1.91-1.78(m,1H),1.49(d,J=7.2Hz,3H),0.89(d,J=6.6Hz,6H).

[0124] 13 C NMR (100MHz, CDCl3) δ180.9,140.8,136.9,129.4,127.3,45.0,44.9,30.1,22.4,18.1.

[0125] Example 3

[0126] The preparation of (S)-ibuprofen is carried out by the following reaction:

[0127]

[0128] Its preparation method is as follows:

[0129] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(4-isobutylphenyl)propanal, and 15 μmol of the redox electrolyte α-ferrocene ethanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0130] After the reaction was complete, the electrolytic cell was removed from the glove box, and 2 mL of acetonitrile was added to quench the reaction. 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min. 1 mL of the mixture was collected and filtered through a 0.22 μm organic filter. The yield was quantified using ultra-high performance liquid chromatography (UHPLC) at 56%, and the enantiomeric excess of the product was 96% using chiral HPLC. The racemic standard characterization data are as follows:

[0131] 1 H NMR (400MHz, CDCl3) δ7.21(d,J=8.1Hz,2H),7.09(d,J=8.1Hz,2H),3.70(q,J=7.2Hz,1H) ,2.44(d,J=7.1Hz,2H),1.91-1.78(m,1H),1.49(d,J=7.2Hz,3H),0.89(d,J=6.6Hz,6H).

[0132] 13 C NMR (100MHz, CDCl3) δ180.9,140.8,136.9,129.4,127.3,45.0,44.9,30.1,22.4,18.1.

[0133] Example 4

[0134] The preparation of (S)-flurbiprofen is carried out by the following reaction:

[0135]

[0136] Its preparation method is as follows:

[0137] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0138] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was 67% when quantitatively analyzed by ultra-high performance liquid chromatography (UHPLC), and the enantiomeric excess of the product was 96% when analyzed by chiral UHPLC. The racemic standard characterization data are as follows:

[0139] 1 H NMR (400MHz, CDCl3) δ7.55-7.50(m,2H),7.46-7.33(m,4H),7.20-7.12(m,2H),3.78(q,J=7.2Hz,1H),1.56(d,J=7.2Hz,3H).

[0140] 13 C NMR (100MHz, CDCl3) δ180.0, 159.7 (d, J = 247.2Hz), 140.9 (d, J = 7.6Hz), 135.4, 130.9 (d, J = 3.9Hz), 128. 9(d,J=2.9Hz),128.4,128.1(d,J=13.5Hz),127.7,123.7(d,J=3.3Hz),115.4(d,J=23.6Hz),44.8,18.0.

[0141] Example 5

[0142] The preparation of (S)-sulprofen is carried out by the following reaction:

[0143]

[0144] Its preparation method is as follows:

[0145] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(4-thiophene-2-carbonyl)phenylpropanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0146] After the reaction was complete, the electrolytic cell was removed from the glove box, and 2 mL of acetonitrile was added to quench the reaction. 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was quantified using ultra-high performance liquid chromatography (UHPLC) at 55%, and the enantiomeric excess of the product was 99% using chiral HPLC. The racemic standard characterization data are as follows:

[0147] 1 H NMR (400MHz, CDCl3) δ7.88-7.82(m,2H),7.72(d,J=5.0Hz,1H),7.65(d,J=3.8Hz,1H),7.5 0-7.43(m,2H),7.16(dd,J=5.0,3.8Hz,1H),3.85(q,J=7.2Hz,1H),1.57(d,J=7.1Hz,3H).

[0148] 13 C NMR (100MHz, CDCl3) δ187.7,179.6,144.1,143.5,137.2,134.8,134.3,129.6,128.0,127.8,45.3,18.0.

[0149] Example 6

[0150] The preparation of (S)-sulprofen is carried out by the following reaction:

[0151]

[0152] Its preparation method is as follows:

[0153] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) was added to a 5 mL electrolyzer in a glove box. Then, at 400 rpm, whole-cell OD of the PfBAL-T481L mutant was added sequentially. 600 = 50, 10 μmol of 2-(4-thiophene-2-carbonyl)phenylpropanal, and 15 μmol of the redox dielectric ferrocene methanol. A graphite electrode was inserted, and the electrochemical parameters were set as follows: cross-current 0.4 mA, charge 5 F / mol, and the electrolysis reaction was initiated.

[0154] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was 77% when analyzed by ultra-high performance liquid chromatography (UHPLC), and the enantiomeric excess of the product was 83% when analyzed by chiral UHPLC. The racemic standard characterization data are as follows:

[0155] 1 H NMR (400MHz, CDCl3) δ7.88-7.82(m,2H),7.72(d,J=5.0Hz,1H),7.65(d,J=3.8Hz,1H),7.5 0-7.43(m,2H),7.16(dd,J=5.0,3.8Hz,1H),3.85(q,J=7.2Hz,1H),1.57(d,J=7.1Hz,3H).

[0156] 13 C NMR (100MHz, CDCl3) δ187.7,179.6,144.1,143.5,137.2,134.8,134.3,129.6,128.0,127.8,45.3,18.0.

[0157] Example 7

[0158] The preparation of (S)-naproxen is carried out by the following reaction formula:

[0159]

[0160] Its preparation method is as follows:

[0161] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 700 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(6-methoxynaphthyl-2-yl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0162] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The yield was quantified by ultra-high performance liquid chromatography (UHPLC) at 33%, and the enantiomeric excess of the product was 90% using chiral HPLC. The racemic standard characterization data are as follows:

[0163] 1 H NMR (400MHz, CDCl3) δ7.68 (d, J = 9.2Hz, 3H), 7.40 (dd, J = 8.5, 1.9Hz, 1H), 7.16-7.06 (m, 2H), 3.90 (s, 3H), 3.86 (q, J = 7.1Hz, 1H), 1.58 (d, J = 7.1Hz, 3H).

[0164] 13 C NMR (100MHz, CDCl3) δ180.6,157.7,134.8,133.8,129.3,128.9,127.2,126.2,126.1,119.0,105.6,55.3,45.2,18.1.

[0165] Example 8

[0166] The preparation of (S)-ketoprofen is carried out by the following reaction:

[0167]

[0168] Its preparation method is as follows:

[0169] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 600 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(4-benzoylphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: 0.4 mA current and 5 F / mol charge, and begin the electrolysis reaction.

[0170] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was quantified using ultra-high performance liquid chromatography (UHPLC) at 59%, and the enantiomeric excess of the product was 78% using chiral HPLC. The racemic standard characterization data are as follows:

[0171] 1 H NMR (400MHz, CDCl3) δ7.83-7.73 (m, 3H), 7.69 (dt, J = 7.7, 1.4Hz, 1H), 7.62-7 .53(m,2H),7.50-7.41(m,3H),3.83(q,J=7.2Hz,1H),1.56(d,J=7.2Hz,3H).

[0172] 13 C NMR (100MHz, CDCl3) δ196.4,179.8,140.1,137.9,137.4,132.5,131.6,130.1,129.3,129.2,128.6,128.3,45.2,18.1

[0173] Example 9

[0174] The preparation of (S)-vidaprofen is carried out by the following reaction:

[0175]

[0176] Its preparation method is as follows:

[0177] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(4-cyclohexyl-1-naphthyl)propionaldehyde, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0178] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was 73% when quantitatively analyzed by ultra-high performance liquid chromatography (UHPLC), and the enantiomeric excess of the product was 99% when analyzed by chiral UHPLC. The racemic standard characterization data are as follows:

[0179] 1 H NMR (400MHz, CDCl3) δ8.19-8.13(m,1H),8.13-8.17(m,1H),7.54-7.48(m,2H),7.44(d,J=7.6Hz,1H),7.36(d,J=7.6Hz,1H),4.50(q,J=7.0 Hz,1H),3.37-3.25(m,1H),2.03-1.97(m,2H),1.95-1.89(m,2H),1.86-1.79(m,1H),1.65(d,J=7.1Hz,3H),1.58-1.49(m,4H),1.34(m,1H).

[0180] 13 C NMR (100MHz, CDCl3) δ180.9,143.5,133.6,131.8,131.6,125.7,125.4,124.4,124.0,123.8,122.1,41.0,39.3,34.3,34.1,27.3,26.5,17.8.

[0181] Example 10

[0182] The preparation of (S)-loxoprofen is carried out by the following reaction:

[0183]

[0184] Its preparation method is as follows:

[0185] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(4-(2-oxocyclopentyl)methyl)phenyl)propanal, and 15 μmol of the redox dielectric ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: 0.4 mA current and 5 F / mol charge, and begin the electrolysis reaction.

[0186] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was 63% when quantitatively analyzed by ultra-high performance liquid chromatography (UHPLC). The enantiomeric excess values ​​of the product were 94% and 96% when analyzed by chiral UHPLC. The characterization data of the racemic standard are as follows:

[0187] 1 H NMR (400MHz, CDCl3) δ7.23(d,J=8.1Hz,2H),7.12(d,J=8.2Hz,2H),3.70(q,J=7.2Hz,1H),3.12(dd,J=13.8,4.1Hz,1H),2.55-2. 46(m,1H),2.41-2.27(m,2H),2.16-2.02(m,2H),2.02-1.89(m,1H),1.81-1.64(m,1H),1.60-1.51(m,1H),1.49(d,J=7.2Hz,3H).

[0188] 13 C NMR (100MHz, CDCl3) δ220.3,180.3,139.1,137.6,129.2,127.6,51.0,44.9,38.1,35.2,29.2,20.5,18.1.

[0189] Example 11

[0190] The preparation of (S)-pranoprofen is carried out by the following reaction:

[0191]

[0192] Its preparation method is as follows:

[0193] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(5H-benzopyrano[2,3-b]pyridin-7-yl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0194] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was 63% when quantitatively analyzed by ultra-high performance liquid chromatography (UHPLC). The enantiomeric excess values ​​of the product were 94% and 96% when analyzed by chiral UHPLC. The characterization data of the racemic standard are as follows:

[0195] 1 H NMR(400MHz,DMSO-d6)δ12.31(s,1H),8.22-8.07(m,1H),7.81-7.61(m,1H),7.20-7.14 (m,3H),7.08(d,J=8Hz,1H),4.12(s,2H),3.66(q,J=7.1Hz,1H),1.37(d,J=7.1Hz,3H).

[0196] 13 C NMR (100MHz, DMSO-d6) δ175.8,158.3,150.5,146.7,139.3,137.2,128.2,127.5,120.7,120.5,116.9,116.1,44.4,27.7,19.0.

[0197] Example 12

[0198] The preparation of (S)-carbofen is carried out by the following reaction:

[0199]

[0200] Its preparation method is as follows:

[0201] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. Then, at 800 rpm, add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(6-chloro-9H-carbazol-2-yl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: crosscurrent 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0202] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was quantified using ultra-high performance liquid chromatography (UHPLC) at 44%, and the enantiomeric excess of the product was 92% using chiral HPLC. The racemic standard characterization data are as follows:

[0203] 1 H NMR (400MHz, DMSO-d6) δ12.29(s,1H),11.36(s,1H),8.17(d,J=2.1Hz,1H),8.09(d,J=8.1Hz,1H),7.49(d,J=8.6Hz,1H ),7.41(s,1H),7.36(dd,J=8.6,2.2Hz,1H),7.11(dd,J=8.2,1.5Hz,1H),3.84(q,J=7.0Hz,1H),1.45(d,J=7.1Hz,3H).

[0204] 13 C NMR (100MHz, DMSO-d6) δ176.0,141.0,140.2,138.8,125.6,124.1,123.3,121.1,120.9,120.1,119.2,112.8,110.3,45.6,19.4.

[0205] Example 13

[0206] The preparation of (S)-zaltoprofen is carried out by the following reaction:

[0207]

[0208] Its preparation method is as follows:

[0209] First, in a glove box, add 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 5 mL electrolytic cell. At 800 rpm, sequentially add 0.1 μmol of LlKdcA purified enzyme, 10 μmol of 2-(10-oxo-10,11-dihydrodibenzo[b,f]thiophene-2-yl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: cross-current 0.4 mA, charge 5 F / mol, and begin the electrolysis reaction.

[0210] After the reaction was complete, the electrolytic cell was removed from the glove box. 2 mL of acetonitrile was added to the electrolytic cell to quench the reaction, and 100 μL (2 mg / mL) of 1,3,5-tribromobenzene was added as an internal standard. After vigorous shaking, the mixture was centrifuged at 10,000 rpm for 5 min, and 1 mL was collected and filtered through a 0.22 μm organic filter. The reaction yield was quantified by ultra-high performance liquid chromatography (UHPLC) to be 30%, and the enantiomeric excess of the product was 97% using chiral HPLC. The racemic standard characterization data are as follows:

[0211] 1 H NMR (400MHz, CDCl3) δ8.19 (dd, J=8.0, 1.6Hz, 1H), 7.62-7.56 (m, 2H), 7.44-7.38 (m, 2H), 7.33-7. 28(m,1H),7.16(dd,J=8.0,2.0Hz,1H),4.36(s,2H),3.73(q,J=7.2Hz,1H),1.49(d,J=7.1Hz,3H).

[0212] 13 C NMR (100MHz, CDCl3) δ191.4,179.5,141.9,140.1,137.9,136.1,133.6,132.5,131.53,131.50,130.9,128.7,126.8,126.5,51.0,44.9,18.1.

[0213] Example 14

[0214] The scaled-up preparation of (S)-flurbiprofen is shown in the following reaction formula:

[0215]

[0216] Its preparation method is as follows:

[0217] First, in a glove box, add 80 mL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) to a 300 mL electrolytic cell. Then, at 800 rpm, add 5 μmol of LlKdcA purified enzyme, 1 mmol of 2-(6-chloro-9H-carbazol-2-yl)propanal, and 1.5 mmol of the redox electrolyte ferrocene methanol. Insert the graphite electrode and set the electrochemical parameters to: 5 mA current, 5 F / mol charge, and begin the electrolysis reaction.

[0218] After the reaction was complete, the electrolytic cell was removed from the glove box. The reaction solution was extracted three times with 100 mL of ethyl acetate, the organic phases were combined, and dried over anhydrous sodium sulfate. The solution was purified using a silica gel column and the yield was calculated to be 55%.

[0219] Comparative Example 1

[0220] In the experimental conditions of Example 4, pyruvate oxidase PO was used to replace LlKdcA enzyme, while the rest remained unchanged. This served as Comparative Example 1, as follows:

[0221] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 0.1 μmol of pyruvate oxidase (PO), 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol were added sequentially. A graphite electrode was inserted, and the electrochemical parameters were set to: crosscurrent 0.4 mA, charge 5 F / mol, and the electrolysis reaction was started. (S)-Fluorbiprofen could not be obtained.

[0222] Comparative Example 2

[0223] Under the experimental conditions of Example 3, PpBFD enzyme was used to replace LlKdcA enzyme, while all other conditions remained unchanged. This serves as Comparative Example 2, as detailed below:

[0224] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 0.1 μmol of PpBFD enzyme, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol were added sequentially. A graphite electrode was inserted, and the electrochemical parameters were set to: 0.4 mA current and 5 F / mol charge. The electrolysis reaction was then initiated. (S)-Fluorbiprofen could not be obtained.

[0225] Comparative Example 3

[0226] In the experimental conditions of Example 3, the CDH enzyme was used to replace the KdcA enzyme, while the rest remained unchanged. This serves as Comparative Example 3, as detailed below:

[0227] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 0.1 μmol of CDH enzyme, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol were added sequentially. A graphite electrode was inserted, and the electrochemical parameters were set to: 0.4 mA current and 5 F / mol charge. The electrolysis reaction was then initiated. (S)-Fluorbiprofen could not be obtained.

[0228] Comparative Example 4

[0229] Under the experimental conditions of Example 3, the EcMenD enzyme was used to replace the LlKdcA enzyme, while the rest remained unchanged. This serves as Comparative Example 4, as detailed below:

[0230] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 0.1 μmol of EcMenD enzyme, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol were added sequentially. A graphite electrode was inserted, and the electrochemical parameters were set to: crosscurrent 0.4 mA, charge 5 F / mol, and the electrolysis reaction was started. (S)-Fluorbiprofen could not be obtained.

[0231] Comparative Example 5

[0232] Under the experimental conditions of Example 3, the PaBAL enzyme was used to replace the LlKdcA enzyme, while the rest remained unchanged. This serves as Comparative Example 5, as detailed below:

[0233] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 0.1 μmol of PaBAL enzyme, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol were added sequentially. A graphite electrode was inserted, and the electrochemical parameters were set to: crosscurrent 0.4 mA, charge 5 F / mol, and the electrolysis reaction was started. (S)-Fluorbiprofen could not be obtained.

[0234] Comparative Example 6

[0235] Under the experimental conditions of Example 3, no current was applied, and everything else remained unchanged. As Comparative Example 5, the details are as follows:

[0236] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 0.1 μmol of KdcA enzyme, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal, and 15 μmol of the redox electrolyte ferrocene methanol were added sequentially without applying electricity. (S)-Fluorbiprofen could not be obtained.

[0237] Comparative Example 7

[0238] Under the experimental conditions of Example 3, LlKdcA enzyme was not added, and everything else remained unchanged, as Comparative Example 7, as follows:

[0239] First, 1800 μL of 3-morpholinopropanesulfonic acid buffer (50 mM, pH = 6.5) was added to a 5 mL electrolytic cell in a glove box. Then, at 800 rpm, 10 μmol of 2-(2-fluoro-4-biphenyl)propanal and 15 μmol of the redox dielectric ferrocene methanol were added sequentially. A graphite electrode was inserted, and the electrochemical parameters were set as follows: crosscurrent 0.4 mA, charge 5 F / mol, and the electrolysis reaction was initiated. (S)-Fluorbiprofen could not be obtained.

[0240] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for the efficient asymmetric synthesis of arylpropionic acid drug molecules, characterized in that, The method uses racemic aldehydes as substrates and thiamine pyrophosphate-dependent enzymes as biocatalysts. The process involves electrolysis in a diaphragm-free electrolytic cell under conditions of external redox dielectric and constant current, in a buffer salt solution and nitrogen atmosphere. The general reaction formula is as follows: in: R 1 Selected from heteroaryl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl; R 2 Selected from methyl, ethyl, propyl, isopropyl, allyl, and pentenyl; R 3 Hydrogen atoms or halogen atoms; The substituents of the phenyl group are selected from halogen atoms, methoxy groups, methylthio groups, trifluoromethyl groups, isopentyl groups, thiophene-2-ylformyl groups, benzoyl groups, cyclopentanone-2-ylethyl groups, ester groups, or cyano groups; The substituents of naphthyl are methoxy and cyclohexyl; The substituents of the biphenyl group are halogen atoms; The heteroaryl group is selected from benzofuranyl, benzothiopheneyl, carbazoyl, pyrazolyl or pyridinyl with halogen atom substitution.

2. The method according to claim 1, characterized in that, The substrates are selected from 2-(4-isobutylphenyl)propanal and its derivatives, 2-(6-methoxynaphthyl-2-yl)propanal and its derivatives, 2-(2-fluoro-4-biphenyl)propanal and its derivatives, 2-(4-benzoylphenyl)propanal and its derivatives, 2-(4-(2-oxocyclopentyl)methyl)phenyl)propanal and its derivatives, 2-(5H-benzopyrano[2,3-b]pyridin-7-yl)propanal and its derivatives, 2-(4-cyclohexyl-1-naphthyl)propanal and its derivatives, 2-(4-thiophene-2-carbonyl)phenylpropanal and its derivatives, 2-(6-chloro-9H-carbazole-2-yl)propanal and its derivatives, and 2-(10-oxo-10,11-dihydrodibenzo[b,f]thiophene-2-yl)propanal and its derivatives.

3. The method according to claim 1, characterized in that, The thiamine pyrophosphate-dependent enzyme is selected from LlKdcA or its mutant, PfBAL or its mutant; LlKdcA is a branched-chain keto acid decarboxylase derived from Lactococcus lactis; PfBAL is a benzaldehyde lyase derived from Pseudomonas fluorescens; the thiamine pyrophosphate-dependent enzyme is obtained by induction expression in Escherichia coli BL21(DE3); the thiamine pyrophosphate-dependent enzyme catalyzes the reaction in the form of a pure enzyme or whole cells; when the thiamine pyrophosphate-dependent enzyme is a pure enzyme, the amount of pure enzyme relative to racemic aldehyde is 0.05-2 mol%; when the thiamine pyrophosphate-dependent enzyme is a whole cell, the amount of whole cell is OD. 600 =20-50.

4. The method according to claim 3, characterized in that, The amino acid sequence of LlKdcA is shown in SEQ ID NO.1; the LlKdcA mutant is an amino acid sequence corresponding to SEQ ID NO.1 with at least one of the following mutations: H at position 112 is mutated to A, L or F; H at position 113 is mutated to A, L or F; F at position 382 is mutated to A, L or W; V at position 461 is mutated to A, L or F; E at position 462 is mutated to A, L or F; I at position 465 is mutated to A, L or F; F at position 542 is mutated to A, L or W.

5. The method according to claim 3, characterized in that, The amino acid sequence of the PfBAL is shown in SEQ ID NO.2; the PfBAL mutant is a mutant with at least one of the following mutations in the amino acid sequence corresponding to SEQ ID NO.2: A at position 480 is mutated to G, L or F; T at position 481 is mutated to A, L or F; F at position 484 is mutated to A, L or W.

6. The method according to claim 1, characterized in that, The redox dielectric is selected from any one of ferrocene methanol, α-ferrocene ethanol, ferrocene dimethanol, ferrocene carboxylic acid, acetylferrocene, ferrocene, and 2,2,6,6-tetramethylpiperidine oxide; the molar ratio of the redox dielectric to the racemic aldehyde is 0.25-1.5:

1.

7. The method according to claim 1, characterized in that, The constant current is 0.2-5.0 mA, and the charge is 4-8 F / mol; the electrodes in the electrolytic cell include a positive electrode and a negative electrode, wherein the positive electrode is any one of a graphite electrode, a platinum electrode, and a BDD electrode, and the negative electrode is a graphite electrode or a platinum electrode.

8. The method according to claim 1, characterized in that, The buffer salt solution is selected from any one of 3-morpholine propanesulfonic acid buffer, phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, N-tris(hydroxymethyl)methylglycine buffer, imidazole buffer; hydroxyethylpiperazine ethylthiosulfate or tris(hydroxymethyl)methylglycine buffer; the concentration of the buffer salt solution is 25-200 mmol / L, and the pH is 6.0-8.

0.

9. The method according to claim 1, characterized in that, The method is as follows: In a nitrogen atmosphere, a buffer salt solution is added to a diaphragmless electrolytic cell with a magnetic stirrer, followed by the sequential addition of thiamine pyrophosphate-dependent enzyme, racemic aldehyde, and redox dielectric. Electrodes are then inserted and a constant current is applied to initiate the electrolysis reaction. After the reaction is complete, acetonitrile is added to the reaction solution to quench the reaction.

10. The method according to claim 9, characterized in that, The magnetic stir bar rotates at a speed of 400-800 rpm.

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