Method for splitting methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate

Abstract

The invention discloses an enzyme catalytic method for converting (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) methyl acetate into (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) acetic acid and a method for splitting 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) methyl acetate. The enzyme in the enzyme catalytic method includes one or more enzymes whose amino acid sequences are shown in SEQ ID NOs: 6, 7, 9 and 10. The invention adopts an enzyme catalytic splitting method to prepare (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) methyl acetate with a yield of up to 30%, and the product ee value is high, which can reach more than 93%. After purification, the ee value is as high as more than 99.5%, which is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing a non-natural amino acid derivative, in particular to a method for splitting 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester. Background Art

[0002] Chiral 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, a glycine derivative, is a non-natural amino acid and an important pharmaceutical intermediate. For example, WO2014152127A1 reported that (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester can be used as an intermediate for dipeptide or tripeptide epoxyketone protease inhibitors; European Journal of Medicinal Chemistry 45, 2010, 160-170 reported that (R) or (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester can be used as an intermediate for HIV-1 protease inhibitors.

[0003] However, the current preparation methods all obtain the racemic 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, and then resolve it to obtain the chiral 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester. The current resolution method includes HPLC separation, but the resolution efficiency is low and is not suitable for industrial production.

[0004] For example, the method disclosed in WO2014152127A1 is to catalytically hydrogenate 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester to obtain racemic 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, and then obtain the desired compound through multi-step reaction, and then use chiral HPLC to separate and obtain the desired chiral compound.

[0005]

[0006] For example, the method disclosed in European Journal of Medicinal Chemistry 45, 2010, 160-170 is that 3-oxetanone and Z-α-phosphonoglycine-trimethyl ester are subjected to Witting-Horner reaction to obtain didehydro-α-amino acid methyl ester, which is then subjected to catalytic hydrogenation and Cbz protection to obtain racemic 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, which is then separated by HPLC to obtain the desired chiral compound.

[0007]

[0008] There are also methods for chemical resolution using chiral reagents, such as II Farmaco, 2001, 56, 609-613, which discloses a method for resolving the racemic 2-amino-2-(oxetane-3-yl)acetic acid methyl ester using L-(+)-tartaric acid or D-(-)-tartaric acid, but the resolution yield is less than 20%.

[0009] Therefore, it is currently necessary to develop a method for efficiently splitting and preparing (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester. Summary of the invention

[0010] The technical problem to be solved by the present invention is to overcome the defects of low splitting efficiency and unsuitability for industrial production in the prior art method for splitting racemic 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, and provide a method for preparing non-natural amino acid derivatives. The present invention adopts an enzyme-catalyzed splitting method to prepare (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester with a yield of up to 30%, and the product has a high ee value of up to 93% or more. After purification, the ee value is as high as 99.5% or more, which is suitable for industrial production.

[0011] Substrate: Compound I, racemic 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, CAS No.: 1255717-04-4, the structural formula of Compound I is as follows:

[0012]

[0013] Product 1: Compound II (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester, CAS number: 1213783-62-0, the structural formula of compound II is as follows:

[0014]

[0015] Product 2: Compound III (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid, CAS No.: 1213783-75-5, the structure of compound III is as follows:

[0016]

[0017] Among them, product 1 is the target product.

[0018] Reaction Path:

[0019]

[0020] The technical solution of the present invention uses enzyme to catalyze the R-configuration methyl ester in the racemate to generate product acid, so as to improve the resolution rate.

[0021] The present invention mainly solves the above technical problems through the following technical solutions.

[0022] The present invention provides an enzymatic method for converting (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester into (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid, using one or more enzymes including amino acid sequences shown in SEQ ID NOs: 6, 7, 9 and 10 as catalytic enzymes.

[0023] The present invention also provides a method for splitting methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) acetate, which comprises the enzyme catalysis method as described above; wherein the methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) acetate is a racemate, or is R- or S-enriched methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl) acetate.

[0024] The method preferably comprises: mixing methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate and the catalytic enzyme and reacting them in a solvent.

[0025] Wherein, the dosage of the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester and the enzyme can be conventional in the art.

[0026] Preferably, the dosage ratio of the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester to the catalytic enzyme is 1g:1500U to 1g:25000U; for example, 1g:4680U, 1g:5276U, 1g:6645U or 1g:8920U. More preferably, the concentration of the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester in the reaction system is 15 to 50g / L; preferably 20g / L or 40g / L.

[0027] Preferably, the pH of the reaction is 6 to 8, preferably 6.5 to 7.5.

[0028] Preferably, the solvent is water or an organic solvent; the organic solvent is preferably DMSO.

[0029] In order to disperse the substrate and increase the reaction contact surface, the reaction system preferably also includes a substrate dispersant; the mass ratio of the substrate dispersant to the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester is 0.95:1 to 1.05:1, preferably 1:1.

[0030] The substrate dispersant of the present invention can be conventional in the art, preferably diatomaceous earth and / or activated carbon.

[0031] The reaction conditions described above can be conventional in the art, preferably carried out at 20-35°C.

[0032] The present invention also provides the use of an enzyme with an amino acid sequence as shown in SEQ ID NO: 6, 7, 9 or 10 in catalyzing the conversion of (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester into (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid.

[0033] The present invention also provides the use of an enzyme having an amino acid sequence as shown in SEQ ID NO: 6, 7, 9 or 10 in the splitting of 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester; preferably, the splitting is used to prepare (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester.

[0034] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0035] The reagents and raw materials used in the present invention are commercially available.

[0036] The positive and progressive effects of the present invention are:

[0037] The invention adopts an enzyme-catalyzed resolution method to prepare (S)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester with a yield of up to 30%, and the product has a high ee value of more than 93%. After purification, the ee value is as high as more than 99.5%, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the ee value detection spectrum of product 1 in the reaction solution of Example 3.

[0039] Figure 2 This is the spectrum of the substrate racemate 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester reference substance.

[0040] Figure 3 This is the spectrum of the product 2 racemate 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid reference substance.

[0041] Figure 4 This is the ee value detection spectrum of the purified product 1. DETAILED DESCRIPTION

[0042] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0043] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0044] The substrate ester racemate and product acid racemate reference substances were synthesized by our laboratory. The ester synthesis method was based on European Journal of Medicinal Chemistry 45, 2010, 160-170. Specifically, 3-oxetanone and Z-α-phosphonoglycine-trimethyl ester were used as raw materials for Witting-Horner reaction to obtain didehydro-α-amino acid methyl ester, which was then catalytically hydrogenated and Cbz protected to obtain the racemate 2-(benzyloxycarbonylamino)-2-(oxetan-3-yl)acetic acid methyl ester. The acid was prepared by chemical hydrolysis of the substrate ester.

[0045] pET21a and bugbuster protein extraction reagent were purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher; E. coli BL21 (DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0046] Phosphate buffered saline refers to PBS buffer.

[0047] TLC detection method (Thin Layer Chromatography, also known as thin layer chromatography, belongs to solid-liquid adsorption chromatography):

[0048] The developing agent is PE:EA:acetic acid = 2:1:one drop (volume ratio). UV color development.

[0049] α is the optical rotation, which is measured in a polarimeter.

[0050] The specific rotation calculation formula is:

[0051]

[0052] [α] is the specific rotation of the substance. The Chinese Pharmacopoeia (2010 edition) stipulates that the sodium spectrum D line (589.3nm) is used for measurement at a temperature of 20°C, so the specific rotation is also recorded as c is the concentration (unit: g / ml); l is the thickness of the liquid layer (unit: 1dm). When the concentration of the test sample is expressed as a percentage concentration (g / 100ml):

[0053]

[0054] Reference 4.5.4.5 Specific rotation of (S)-2-(benzyloxycarbonylamino)-2-(oxetan-3-yl acetate) in Design and synthesis of novel P2 substituents in diol-based HIV protease inhibitors, European Journal of Medicinal Chemistry, 45 (2010), 160-170 It is -19.5 (c1.0, MeOH).

[0055] Chiral HPLC Method:

[0056] Chromatographic column: Daicel Chiralpak IG (4.6mm*250mm, 5μm); mobile phase: n-hexane:ethanol=55:45; flow rate: 0.8mL / min; column temperature: 25°C; detection wavelength: 210nm, injection volume: 10μL.

[0057] The retention times of the reference ester racemates are 20.098 min and 26.072 min, see Figure 2 The retention time of the reference acid racemate is 16.617min and 18.149min, see Figure 3 .

[0058] Example 1 Preparation of lipase

[0059] According to the enzyme gene sequence shown in Table 1, the gene of the fully synthesized lipase was connected to the pET21a vector at the NdeI and HindIII sites, and the gene was successfully connected to the corresponding vector after sequencing verification. The synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (No. 211, Pubin Road, Research and Innovation Park, Jiangbei New District, Nanjing, Jiangsu Province).

[0060] The vector containing the lipase gene was transformed into the host Escherichia coli BL21 (DE3) competent cells to obtain the engineered strains containing each lipase gene. After the engineered bacteria containing the lipase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid culture medium containing 100 μg / mL ampicillin antibiotics, and cultured at 37°C for 12 hours. The inoculation amount was transferred to 150 mL of fresh LB liquid culture medium containing 100 μg / mL ampicillin antibiotics at a volume percentage of 2%, and the culture was shaken at 37°C until the OD 600 When the pH value reached about 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was induced at 25°C for 16 h. After the culture was completed, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, the cells were collected, and stored in a -20°C ultra-low temperature refrigerator for later use.

[0061] Table 1

[0062]

[0063] Example 2

[0064] 5 g of the bacterial cells collected after the cultivation in Example 1 were washed twice with 100 mM pH 7.0 phosphate buffer, then resuspended in 50 mL pH 7.0 phosphate buffer, ultrasonically disrupted, and the disrupted liquid was centrifuged to remove the precipitate, and the supernatant obtained was a crude enzyme solution containing lipase.

[0065] Add 7 mL of 100 mM pH 7.0 phosphate buffer, 200 mg of substrate, and 1 mL of DMSO to a centrifuge tube, adjust the pH to 7.0 with dilute phosphoric acid or NaOH solution, add 2 mL of crude enzyme solution, and stir at 30°C for 24 h.

[0066] After the reaction was completed, the supernatant of the reaction solution was acidified to pH 2, extracted with ethyl acetate, and the organic phase was detected by TLC. The developing solvent was PE:EA:acetic acid = 2:1:one drop, and the color was developed at a wavelength of 254 nm.

[0067] Optical rotation: adjust the pH of the reaction solution to 7.5, extract twice with ethyl acetate, take the organic phase and evaporate it, add 2 mL of methanol to dissolve it and then measure the optical rotation.

[0068] EE value detection: Take 2 mL of the reaction solution, extract with ethyl acetate, take the organic phase and perform rotary evaporation, add ethanol, and measure the ee value of the ester by HPLC. Take the aqueous phase and perform rotary evaporation to measure the ee value of the acid.

[0069] The results are shown in Table 2 below:

[0070] Table 2

[0071] Enzyme number Optical rotation value Ester ee value Acid ee value Enz.1 -0.301 98.7％ 82.6％ Enz.2 -0.283 98.5％ 76.5％ Enz.3 -0.057 88.8％ 37.6％ Enz.4 -0.256 97.8％ 88.8％ Enz.5 -0.278 97.2％ 80.2％

[0072] Determination of enzyme activity:

[0073] Enzyme activity determination method: The crude enzyme solution was diluted 5 times (100mM pH7.0 phosphate buffer) and used for enzyme activity determination. 0.2g substrate (Compound I), 2mL DMSO and 7.8mL 100mM pH7.0 phosphate buffer were added to the reaction container, preheated at 30℃ shaker 220rpm for 10min, 0.2mL diluted crude enzyme solution was added, and the reaction was terminated after 5min. The reaction solution after termination was taken for HPLC detection, and the product concentration was calculated according to the standard curve of the product, and the enzyme activity was calculated.

[0074] Enzyme activity is defined as: the amount of enzyme required to generate 1 μmol of product acid per minute at pH 7.0 and 30°C is 1 enzyme activity unit (1U).

[0075] The results are shown in Table 3 below:

[0076] Table 3

[0077] Enzyme number Enzyme activity (U / mL) Enz.1 1287 Enz.2 1435 Enz.3 306 Enz.4 2453 Enz.5 1807

[0078] Example 3

[0079] The enzyme bacteria (Enz.4) and 100 mM pH 7.0 phosphate buffer were homogenized at a ratio of 1:10 (mass to volume ratio) before use.

[0080] In the reaction system, add 55mL water, 2.75g diatomaceous earth, 2.75g substrate (Compound I), control the temperature at 25-30℃ and stir, adjust the pH at 6.5-7.5 with saturated sodium bicarbonate, add enzyme solution Enz.4 dropwise, start the reaction, and control the pH at the same time. After 1.5h of reaction, stop the reaction, and consume 10mL of homogenized enzyme solution and 4mL of saturated sodium bicarbonate. Take a small amount of reaction solution for extraction, and take the organic phase for HPLC detection. The ee value is 93%, and the detection spectrum is as follows Figure 1 .

[0081] At room temperature, 25 mL of saturated sodium bicarbonate and 50 mL of ethyl acetate were added to the remaining reaction solution, stirred for 5 min, and filtered; the organic phase was collected, washed 4 times with 15 mL of saturated sodium bicarbonate, and the organic phase was detected by bromocresol green for acid-free color development; the organic phase was spin-dried to obtain 1.1 g of a white solid, which was added to 6 mL of a mixed solvent of petroleum ether and ethyl acetate (5:1), refluxed to dissolve, cooled and crystallized, stirred at room temperature for 30 min, filtered, and dried to obtain 0.9 g of a white powder with a yield of 32.7% and an ee value of 99.5% ( Figure 4 ), specific rotation -20.

[0082] The reaction methods of other enzymes are the same as Enz.4, and the reaction test results are shown in Table 4 below:

[0083] Table 4

[0084]

[0085]

[0086] According to the above table, the ester yield of the present invention using Enz.1, Enz.2, Enz.4 and Enz.5 for catalytic reaction can reach at least 28.5%, which is much higher than the ester yield in the prior art. SEQUENCE LISTING <110> Yikelai Biotechnology (Group) Co., Ltd. <120> Method for splitting methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate <130> P20015906C <140> 2020113402272 <141> 2020-11-25 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 1174 <212> DNA <213> Uncultured bacteria <400> 1 catatgagca ttgcagatca gagtctggca aaacgtgttc agggtgttag tcagcaggca 60 attgatgaag gtcgtattgt tggtagcgtt gttctgattg cacgtcatgg tcgtgttatt 120 tatgcaaatg catcaggtta tgcagatcgt gaacagaaaa aaccgatggt tcgtgaaacc 180 cagtttcgtc tgagtagcgt tagtaaaccg tatattaccc tggcagcaat gcgtatgatt 240 gaacagcaga aactgggtct ggatgatacc gtttctcgtt ggctgccgtg gtttaccccg 300 gcactggccg atggcgttcg tccgccgatt aaaattcgtc atctgctgag tcatacagcc 360 ggtctggatt atcgtctgtc acagccggca gaaggtccgt atcatcgtct gggtattaaa 420 gatggtatgg aactgagttc actgaccctg gaacagaatc tgcgtctgct ggcccaggcg 480 gatctgctgg cagaaccggg ttcagaattt cgttatagtc tggcaattga tgttcttggt 540 gcagtgctgg aacaggttgc aggcgaaccg ctgccgcagg tttttaatca ttgggttgca 600 cagccgctgg gtctgcgtaa tacaggtttt tataccaccg atgttgataa tctggcaacc 660 gcatatcatg ataccgcagc agaaccggaa ccgattcgtg atggtatgct gctgaccctg 720 ccggaaggtt ttggttttga aattgaactg gcaccgagtc gtgcgctgga tgcacaggca 780 tatccgagcg gtggtgcagg tatggttggt gatgcagatg atgttctgca gctggttgaa 840 accctgcgca ccggtaaaga aggtattctg cagccggcaa ccgcagcact gatgcgtcag 900 gcgcatgtgg gttcacatgc agaaacccag ggtccgggtt ggggttttgg ttttggtggt 960 gcagtgctgg aagatgcaca gctggcagca accccgcagc ataatggtac actgcagtgg 1020 ggtggtgtgt atggtcatag ttggttttat gatccgcagg cagcgattag tgttgttgca 1080 ctgaccaata ccgcatttga aggtatgtca ggtcgttatc cgctgcagat tcgtgatgca 1140 gtttatggta ccaatgaacc gacccgttaa gctt 1174 <210> 2 <211> 1198 <212> DNA <213> Uncultured bacterium <400> 2 catatgagcg cagaagaact gggttttctg ccggatcgtc tggcccgtat tagcgatcat 60 attcagacca attatctgga taatggtaaa ctgccgtttg ccagcctgct gattggccgt 120 ggtgatgata ttgccctgca gtggagtagc ggtgttgccg atgatgccat ttttcgtatt 180 gcgagcatga ccaaaccggt tacctcagtt gcatttatgc agctggttga acagggtaaa 240 gttgcgctga ccgatccggt tgccaaatat attccggaat ttgcaaaact gggcgttttt 300 gttgccggtg gcggtaatat tccgtttgtg agccgtccgc cgaccatgcc gatgcgtatt 360 gttgatgtgc tgcgtcatac caccggtttt acctatagct ttcaggaacg cagcaatatt 420 gatgccgcgt atcgtaaaac cgatgttgaa agctggaccc gtagcaccag ccagagtttt 480 attgataccc tggcagaaat tccgctggaa tttgatccgg gtacccagtg gaattatagt 540 gttagcaccg atgttctggg cattctgatt gaacgtatta gcggtcagtc actgccggat 600 tattttcgtg atcatatttt tgcaccgctg ggtatggttg atacctgctt taccgttccg 660 gcagataaag cagcccgcat tccgcagtgt tttgcatttg atccggcaac caaaatgaaa 720 ctgtttgatg aagccggcgc aagtagcctg tggaccaaag gctggagctt taatagcggt 780 ggtggcggtc tggcaagcag cgttgccgat tatcatcgtt tttgccgtat gctgctgaat 840 ggcggtgccc tggatggtat tcagattatt agcccgaaaa ccctggaact gatgaccgca 900 aatcatctgc cgggtggtca ggatctgacc cagatgagca aaagcctgtt tagcgaagcc 960 gaaatggccg gtattggttt tggcctgggt tttgccacca ccattgattc tgtggcaacc 1020 ctgaccccgt gtagtaccgg tgatttttat tggggtggta tgtatagcac cgcctttttc 1080 gttgatccgg ttgaagatat tattatgatt tttatgaccc agctgatgcc gagcagcacc 1140 tatccggttc gtcgtgaaat taaaaccatg atttatagcg cactggccgc ctaagctt 1198 <210> 3 <211> 910 <212> DNA <213> Bacillus subtilis <400> 3 catatgagca atcatagcag cagcattccg gaactgagcg ataatggcat tcgttattat 60 cagacctata atgaaagcct gagcctgtgg ccggttcgct gtaaatcttt ttatattagt 120 acccgttttg gtcagaccca tgtgattgcc tctggtccgg aagatgcccc gccgctggtt 180 ctgctgcatg gtgcactgtt tagcagcacc atgtggtatc cgaatattgc cgattggagt 240 agcaaatatc gcacctatgc agtggatatt attggcgata aaaataaaag cattccggaa 300 aatctgagcg gtacccgcac cgattatgcc aattggctgc tggatgtttt tgataatctg 360 ggtattgaaa aaagccacat gattggtctg agcctgggcg gtctgcatac catgaatttt 420 ctgctgcgca tgccggaacg cgtgaaaagc gccgccattc tgagcccggc ggaaaccttt 480 ctgccgtttc atcatgatgt ttataaatat gccctgggtc tgaccgcgag taatggtgtt 540 gaaaaatgtc tgaattggat gatgaccgat cagaatgtgc tgcatccgat ttttgtgaaa 600 cagtttcagg caggtgtgat gtggcaggat ggtagccgta atccgaatcc gaaagccgat 660 ggttttccgt atgtgtttac cgatgaagaa ctgcgtagcg cccgtgtgcc gattctgctg 720 ctgctgggcg aacatgaagt gatttatgat ccgcatagcg ccctgcatcg cgccagcagc 780 tttgtgccgg atattgaagc cgaagtgatt aaaaatgccg gtcatgtgct gagcatggaa 840 cagccggcct atgtgaatga acgtgtgatg cgctttttca atgccgaaac cggtattagc 900 cgttaagctt 910 <210> 4 <211> 1243 <212> DNA <213> Trichosporon poisoning <400> 4 catatgaccg tgaccacccc gaccctgagc gccgatgcaa aaaccaaaat tgatcagatt 60 ctgcagagcg ccgttgatag caaagaaatt ccggccagca catttgcagt tgcaaccgca 120 gatctgaatg cagaaccggt ttattttaaa gcaagcggtg aacgcgtgtt tggtagcccg 180 gataaaggcc agattaatga agataccgtt ctgcagctga tgagcatgac caaactggtt 240 gttaccgttg ccgccctgca gctggttgcc gcaggtaaac tgaccctgga tgatccggaa 300 attattaata aacatctgcc ggaactgacc agcctgaaaa ttattaaatc tgccgatgat 360 gcgggtaccg atcgcaccaa accgattacc ctgcgccatc tgctgaccca tacaaatggt 420 accggttatg atgtgatggt tccgctgctg ggtgaatggg ccaaaaaaca tagccatccg 480 ggtgtgtttg caagcaatgc aaccgttcag acctttgaac tgccgctgat ttttgaaccg 540 ggtaccgcct ggaattatag cctgggtctg gattggggtg gcattctgat tgaacgtgtg 600 accggccaga gtctggatag ctattttaaa gaaaatattt ttaaaccgct gggtgcagat 660 agcctgacct ttgtgccgac cgaaaaacat tatgaacgtc tgcagcaggt tgtgacccgt 720 gatgcagaaa agaaactgac cgtgtttccg ggtattcgtg aaaccgcacc ggaaaaagtg 780 aaaggtcagg catcaggtgg tgcaggtctg tatggtaccg cacgtgatta tctgcgtttt 840 ctgcagggta ttctgcgttc tgcacagccg ggtggtattc tgccgccgga atataccgaa 900 ctgattttta gcaatcagct gccgaaagcg ccggaaggta cctttaaagg tcagtatgaa 960 tttgcagcac tgattccgca tattgatccg gcactggttg caaatggtgg tctgagccat 1020 agcctgggtg gttatctgac cacccaggat agtaaatggg gtcgtaaagc cggtagcgat 1080 ttttgggaag gtatttataa aacattttat tggatggacc cgaaaaccgg tattgccggt 1140 acctttagca cccagtgttt taattttggt gatccgaccg atccgcagga aaaagtttat 1200 aatcagctgg aacgtgccct gtatgatggt ctgcagtaag ctt 1243 <210> 5 <211> 1135 <212> DNA <213> Arthrobacter globiformis <400> 5 catatggatg cgcagaccat cgcaccgggc ttcgaatctg tggcagaact gttcggtcgt 60 ttcctgagcg aagatcgtga atattctgcg cagctggctg cgtaccaccg tggtgttaaa 120 gtactggata tctccggcgg cccgcaccgt cgtccggata gcgttaccgg tgtgttctcc 180 tgctccaaag gtgtttctgg tctggttatc gcgctgctgg ttcaggatgg cttcctggat 240 ctggatgcgg aagttgtgaa atactggccg gagttcggcg cggaaggtaa agcaaccatc 300 accgttgcac agctgctgag ccaccaggcg ggtctgctgg gcgttgaagg cggcctgacc 360 ctggcggaat acaacaactc cgaactggca gctgcgaaac tggcgcagat gcgtccgctg 420 tggaaaccgg gcaccgcttt cggctaccac gcgctgacca tcggtgtgtt catggaagaa 480 ctgtgccgtc gcatcaccgg cagcaccctg caggaaattt acgaacagcg tattcgctcc 540 gttaccggcg cgcacttctt cctgggcctg ccggaaagcg aagaaccgcg ttacgccacc 600 ctgcgttggg ctgccgatcc gtctcagccg tggatcgatc cggcgtccca cttcggtctg 660 tccgcgaact ctgcagttgg cgatatcctg gatctgccga acctgcgcga agttcgtgca 720 gcgggcctgt cctctgctgc gggcgttgcg agcgcggaag gtatggcgcg tgtttacgcg 780 gcggctctga ccggtctggc ggctaacggt gaccgcgcgg cggtggctcc gctgctgagc 840 gaagaaacca tccagaccgt gaccgcggaa caggttttcg gtattgaccg tgttttcggt 900 gaaacctctt gcttcggcac cgttttcatg aaaagccacg cgcgttcccc gtacggcagc 960 taccgtgctt tcggccacga tggcgcgagc gcgagcctgg gtttcgctga tccggtttat 1020 gaactggcgt tcggttacgt tccgcagcag gcggaaccgg gcggcgcggg ttgccgtaac 1080 ctggaactgt ccgcggcggt gcgtaaagcg gttaccgaac tggcgcagta agctt 1135 <210> 6 <211> 388 <212> PRT <213> Uncultured bacterium <400> 6 Met Ser Ile Ala Asp Gln Ser Leu Ala Lys Arg Val Gln Gly Val Ser 1 5 10 15 Gln Gln Ala Ile Asp Glu Gly Arg Ile Val Gly Ser Val Val Leu Ile 20 25 30 Ala Arg His Gly Arg Val Ile Tyr Ala Asn Ala Ser Gly Tyr Ala Asp 35 40 45 Arg Glu Gln Lys Lys Pro Met Val Arg Glu Thr Gln Phe Arg Leu Ser 50 55 60 Ser Val Ser Lys Pro Tyr Ile Thr Leu Ala Ala Met Arg Met Ile Glu 65 70 75 80 Gln Gln Lys Leu Gly Leu Asp Asp Thr Val Ser Arg Trp Leu Pro Trp 85 90 95 Phe Thr Pro Ala Leu Ala Asp Gly Val Arg Pro Pro Ile Lys Ile Arg 100 105 110 His Leu Leu Ser His Thr Ala Gly Leu Asp Tyr Arg Leu Ser Gln Pro 115 120 125 Ala Glu Gly Pro Tyr His Arg Leu Gly Ile Lys Asp Gly Met Glu Leu 130 135 140 Ser Ser Leu Thr Leu Glu Gln Asn Leu Arg Leu Leu Ala Gln Ala Asp 145 150 155 160 Leu Leu Ala Glu Pro Gly Ser Glu Phe Arg Tyr Ser Leu Ala Ile Asp 165 170 175 Val Leu Gly Ala Val Leu Glu Gln Val Ala Gly Glu Pro Leu Pro Gln 180 185 190 Val Phe Asn His Trp Val Ala Gln Pro Leu Gly Leu Arg Asn Thr Gly 195 200 205 Phe Tyr Thr Thr Asp Val Asp Asn Leu Ala Thr Ala Tyr His Asp Thr 210 215 220 Ala Ala Glu Pro Glu Pro Ile Arg Asp Gly Met Leu Leu Thr Leu Pro 225 230 235 240 Glu Gly Phe Gly Phe Glu Ile Glu Leu Ala Pro Ser Arg Ala Leu Asp 245 250 255 Ala Gln Ala Tyr Pro Ser Gly Gly Ala Gly Met Val Gly Asp Ala Asp 260 265 270 Asp Val Leu Gln Leu Val Glu Thr Leu Arg Thr Gly Lys Glu Gly Ile 275 280 285 Leu Gln Pro Ala Thr Ala Ala Leu Met Arg Gln Ala His Val Gly Ser 290 295 300 His Ala Glu Thr Gln Gly Pro Gly Trp Gly Phe Gly Phe Gly Gly Ala 305 310 315 320 Val Leu Glu Asp Ala Gln Leu Ala Ala Thr Pro Gln His Asn Gly Thr 325 330 335 Leu Gln Trp Gly Gly Val Tyr Gly His Ser Trp Phe Tyr Asp Pro Gln 340 345 350 Ala Ala Ile Ser Val Val Ala Leu Thr Asn Thr Ala Phe Glu Gly Met 355 360 365 Ser Gly Arg Tyr Pro Leu Gln Ile Arg Asp Ala Val Tyr Gly Thr Asn 370 375 380 Glu Pro Thr Arg 385 <210> 7 <211> 396 <212> PRT <213> Uncultured bacterium <400> 7 Met Ser Ala Glu Glu Leu Gly Phe Leu Pro Asp Arg Leu Ala Arg Ile 1 5 10 15 Ser Asp His Ile Gln Thr Asn Tyr Leu Asp Asn Gly Lys Leu Pro Phe 20 25 30 Ala Ser Leu Leu Ile Gly Arg Gly Asp Asp Ile Ala Leu Gln Trp Ser 35 40 45 Ser Gly Val Ala Asp Asp Ala Ile Phe Arg Ile Ala Ser Met Thr Lys 50 55 60 Pro Val Thr Ser Val Ala Phe Met Gln Leu Val Glu Gln Gly Lys Val 65 70 75 80 Ala Leu Thr Asp Pro Val Ala Lys Tyr Ile Pro Glu Phe Ala Lys Leu 85 90 95 Gly Val Phe Val Ala Gly Gly Gly Asn Ile Pro Phe Val Ser Arg Pro 100 105 110 Pro Thr Met Pro Met Arg Ile Val Asp Val Leu Arg His Thr Thr Gly 115 120 125 Phe Thr Tyr Ser Phe Gln Glu Arg Ser Asn Ile Asp Ala Ala Tyr Arg 130 135 140 Lys Thr Asp Val Glu Ser Trp Thr Arg Ser Thr Ser Gln Ser Phe Ile 145 150 155 160 Asp Thr Leu Ala Glu Ile Pro Leu Glu Phe Asp Pro Gly Thr Gln Trp 165 170 175 Asn Tyr Ser Val Ser Thr Asp Val Leu Gly Ile Leu Ile Glu Arg Ile 180 185 190 Ser Gly Gln Ser Leu Pro Asp Tyr Phe Arg Asp His Ile Phe Ala Pro 195 200 205 Leu Gly Met Val Asp Thr Cys Phe Thr Val Pro Ala Asp Lys Ala Ala 210 215 220 Arg Ile Pro Gln Cys Phe Ala Phe Asp Pro Ala Thr Lys Met Lys Leu 225 230 235 240 Phe Asp Glu Ala Gly Ala Ser Ser Leu Trp Thr Lys Gly Trp Ser Phe 245 250 255 Asn Ser Gly Gly Gly Gly Leu Ala Ser Ser Val Ala Asp Tyr His Arg 260 265 270 Phe Cys Arg Met Leu Leu Asn Gly Gly Ala Leu Asp Gly Ile Gln Ile 275 280 285 Ile Ser Pro Lys Thr Leu Glu Leu Met Thr Ala Asn His Leu Pro Gly 290 295 300 Gly Gln Asp Leu Thr Gln Met Ser Lys Ser Leu Phe Ser Glu Ala Glu 305 310 315 320 Met Ala Gly Ile Gly Phe Gly Leu Gly Phe Ala Thr Thr Ile Asp Ser 325 330 335 Val Ala Thr Leu Thr Pro Cys Ser Thr Gly Asp Phe Tyr Trp Gly Gly 340 345 350 Met Tyr Ser Thr Ala Phe Phe Val Asp Pro Val Glu Asp Ile Ile Met 355 360 365 Ile Phe Met Thr Gln Leu Met Pro Ser Ser Thr Tyr Pro Val Arg Arg 370 375 380 Glu Ile Lys Thr Met Ile Tyr Ser Ala Leu Ala Ala 385 390 395 <210> 8 <211> 300 <212> PRT <213> Bacillus subtilis <400> 8 Met Ser Asn His Ser Ser Ser Ile Pro Glu Leu Ser Asp Asn Gly Ile 1 5 10 15 Arg Tyr Tyr Gln Thr Tyr Asn Glu Ser Leu Ser Leu Trp Pro Val Arg 20 25 30 Cys Lys Ser Phe Tyr Ile Ser Thr Arg Phe Gly Gln Thr His Val Ile 35 40 45 Ala Ser Gly Pro Glu Asp Ala Pro Pro Leu Val Leu Leu His Gly Ala 50 55 60 Leu Phe Ser Ser Thr Met Trp Tyr Pro Asn Ile Ala Asp Trp Ser Ser 65 70 75 80 Lys Tyr Arg Thr Tyr Ala Val Asp Ile Ile Gly Asp Lys Asn Lys Ser 85 90 95 Ile Pro Glu Asn Leu Ser Gly Thr Arg Thr Asp Tyr Ala Asn Trp Leu 100 105 110 Leu Asp Val Phe Asp Asn Leu Gly Ile Glu Lys Ser His Met Ile Gly 115 120 125 Leu Ser Leu Gly Gly Leu His Thr Met Asn Phe Leu Leu Arg Met Pro 130 135 140 Glu Arg Val Lys Ser Ala Ala Ile Leu Ser Pro Ala Glu Thr Phe Leu 145 150 155 160 Pro Phe His His Asp Val Tyr Lys Tyr Ala Leu Gly Leu Thr Ala Ser 165 170 175 Asn Gly Val Glu Lys Cys Leu Asn Trp Met Met Thr Asp Gln Asn Val 180 185 190 Leu His Pro Ile Phe Val Lys Gln Phe Gln Ala Gly Val Met Trp Gln 195 200 205 Asp Gly Ser Arg Asn Pro Asn Pro Lys Ala Asp Gly Phe Pro Tyr Val 210 215 220 Phe Thr Asp Glu Glu Leu Arg Ser Ala Arg Val Pro Ile Leu Leu Leu 225 230 235 240 Leu Gly Glu His Glu Val Ile Tyr Asp Pro His Ser Ala Leu His Arg 245 250 255 Ala Ser Ser Phe Val Pro Asp Ile Glu Ala Glu Val Ile Lys Asn Ala 260 265 270 Gly His Val Leu Ser Met Glu Gln Pro Ala Tyr Val Asn Glu Arg Val 275 280 285 Met Arg Phe Phe Asn Ala Glu Thr Gly Ile Ser Arg 290 295 300 <210> 9 <211> 411 <212> PRT <213> Trichosporon asahii <400> 9 Met Thr Val Thr Thr Pro Thr Leu Ser Ala Asp Ala Lys Thr Lys Ile 1 5 10 15 Asp Gln Ile Leu Gln Ser Ala Val Asp Ser Lys Glu Ile Pro Ala Ser 20 25 30 Thr Phe Ala Val Ala Thr Ala Asp Leu Asn Ala Glu Pro Val Tyr Phe 35 40 45 Lys Ala Ser Gly Glu Arg Val Phe Gly Ser Pro Asp Lys Gly Gln Ile 50 55 60 Asn Glu Asp Thr Val Leu Gln Leu Met Ser Met Thr Lys Leu Val Val 65 70 75 80 Thr Val Ala Ala Leu Gln Leu Val Ala Ala Gly Lys Leu Thr Leu Asp 85 90 95 Asp Pro Glu Ile Ile Asn Lys His Leu Pro Glu Leu Thr Ser Leu Lys 100 105 110 Ile Ile Lys Ser Ala Asp Asp Ala Gly Thr Asp Arg Thr Lys Pro Ile 115 120 125 Thr Leu Arg His Leu Leu Thr His Thr Asn Gly Thr Gly Tyr Asp Val 130 135 140 Met Val Pro Leu Leu Gly Glu Trp Ala Lys Lys His Ser His Pro Gly 145 150 155 160 Val Phe Ala Ser Asn Ala Thr Val Gln Thr Phe Glu Leu Pro Leu Ile 165 170 175 Phe Glu Pro Gly Thr Ala Trp Asn Tyr Ser Leu Gly Leu Asp Trp Gly 180 185 190 Gly Ile Leu Ile Glu Arg Val Thr Gly Gln Ser Leu Asp Ser Tyr Phe 195 200 205 Lys Glu Asn Ile Phe Lys Pro Leu Gly Ala Asp Ser Leu Thr Phe Val 210 215 220 Pro Thr Glu Lys His Tyr Glu Arg Leu Gln Gln Val Val Thr Arg Asp 225 230 235 240 Ala Glu Lys Lys Leu Thr Val Phe Pro Gly Ile Arg Glu Thr Ala Pro 245 250 255 Glu Lys Val Lys Gly Gln Ala Ser Gly Gly Ala Gly Leu Tyr Gly Thr 260 265 270 Ala Arg Asp Tyr Leu Arg Phe Leu Gln Gly Ile Leu Arg Ser Ala Gln 275 280 285 Pro Gly Gly Ile Leu Pro Pro Glu Tyr Thr Glu Leu Ile Phe Ser Asn 290 295 300 Gln Leu Pro Lys Ala Pro Glu Gly Thr Phe Lys Gly Gln Tyr Glu Phe 305 310 315 320 Ala Ala Leu Ile Pro His Ile Asp Pro Ala Leu Val Ala Asn Gly Gly 325 330 335 Leu Ser His Ser Leu Gly Gly Tyr Leu Thr Thr Gln Asp Ser Lys Trp 340 345 350 Gly Arg Lys Ala Gly Ser Asp Phe Trp Glu Gly Ile Tyr Lys Thr Phe 355 360 365 Tyr Trp Met Asp Pro Lys Thr Gly Ile Ala Gly Thr Phe Ser Thr Gln 370 375 380 Cys Phe Asn Phe Gly Asp Pro Thr Asp Pro Gln Glu Lys Val Tyr Asn 385 390 395 400 Gln Leu Glu Arg Ala Leu Tyr Asp Gly Leu Gln 405 410 <210> 10 <211> 375 <212> PRT <213> Arthrobacter globiformis <400> 10 Met Asp Ala Gln Thr Ile Ala Pro Gly Phe Glu Ser Val Ala Glu Leu 1 5 10 15 Phe Gly Arg Phe Leu Ser Glu Asp Arg Glu Tyr Ser Ala Gln Leu Ala 20 25 30 Ala Tyr His Arg Gly Val Lys Val Leu Asp Ile Ser Gly Gly Pro His 35 40 45 Arg Arg Pro Asp Ser Val Thr Gly Val Phe Ser Cys Ser Lys Gly Val 50 55 60 Ser Gly Leu Val Ile Ala Leu Leu Val Gln Asp Gly Phe Leu Asp Leu 65 70 75 80 Asp Ala Glu Val Val Lys Tyr Trp Pro Glu Phe Gly Ala Glu Gly Lys 85 90 95 Ala Thr Ile Thr Val Ala Gln Leu Leu Ser His Gln Ala Gly Leu Leu 100 105 110 Gly Val Glu Gly Gly Leu Thr Leu Ala Glu Tyr Asn Asn Ser Glu Leu 115 120 125 Ala Ala Ala Lys Leu Ala Gln Met Arg Pro Leu Trp Lys Pro Gly Thr 130 135 140 Ala Phe Gly Tyr His Ala Leu Thr Ile Gly Val Phe Met Glu Glu Leu 145 150 155 160 Cys Arg Arg Ile Thr Gly Ser Thr Leu Gln Glu Ile Tyr Glu Gln Arg 165 170 175 Ile Arg Ser Val Thr Gly Ala His Phe Phe Leu Gly Leu Pro Glu Ser 180 185 190 Glu Glu Pro Arg Tyr Ala Thr Leu Arg Trp Ala Ala Asp Pro Ser Gln 195 200 205 Pro Trp Ile Asp Pro Ala Ser His Phe Gly Leu Ser Ala Asn Ser Ala 210 215 220 Val Gly Asp Ile Leu Asp Leu Pro Asn Leu Arg Glu Val Arg Ala Ala 225 230 235 240 Gly Leu Ser Ser Ala Ala Gly Val Ala Ser Ala Glu Gly Met Ala Arg 245 250 255 Val Tyr Ala Ala Ala Leu Thr Gly Leu Ala Ala Asn Gly Asp Arg Ala 260 265 270 Ala Val Ala Pro Leu Leu Ser Glu Glu Thr Ile Gln Thr Val Thr Ala 275 280 285 Glu Gln Val Phe Gly Ile Asp Arg Val Phe Gly Glu Thr Ser Cys Phe 290 295 300 Gly Thr Val Phe Met Lys Ser His Ala Arg Ser Pro Tyr Gly Ser Tyr 305 310 315 320 Arg Ala Phe Gly His Asp Gly Ala Ser Ala Ser Leu Gly Phe Ala Asp 325 330 335 Pro Val Tyr Glu Leu Ala Phe Gly Tyr Val Pro Gln Gln Ala Glu Pro 340 345 350 Gly Gly Ala Gly Cys Arg Asn Leu Glu Leu Ser Ala Ala Val Arg Lys 355 360 365 Ala Val Thr Glu Leu Ala Gln 370 375

Claims

1. An enzymatic method for converting (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester into (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid, characterized in that: As the catalytic enzyme, one or more of the enzymes whose amino acid sequences are shown in SEQ ID NOs: 6, 7, 9 and 10 are used.

2. A method for resolving methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate, comprising the enzymatic method according to claim 1; wherein: The methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate is a racemate, or R- or S-enriched methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate.

3. The method according to claim 2, characterized in that The method comprises mixing methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate and the catalytic enzyme and reacting them in a solvent.

4. The method according to claim 3, characterized in that The dosage ratio of the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester to the catalytic enzyme is 1g:1500U to 1g:25000U.

5. The method according to claim 4, characterized in that The usage ratio of the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester to the catalytic enzyme is 1g:4680U, 1g:5276U, 1g:6645U or 1g:8920U.

6. The method according to claim 4, characterized in that The concentration of the methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate in the reaction system is 15 to 50 g / L.

7. The method according to claim 6, characterized in that The concentration of the methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate in the reaction system is 20 g / L or 40 g / L.

8. The method according to claim 3, characterized in that The pH of the reaction is 6-8.

9. The method according to claim 8, characterized in that The pH of the reaction is 6.5-7.

5.

10. The method according to claim 3, characterized in that The solvent is water or an organic solvent.

11. The method according to claim 10, characterized in that The organic solvent is DMSO.

12. The method according to claim 3, characterized in that The reaction system also contains a substrate dispersant; the mass ratio of the substrate dispersant to the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester is 0.95:1 to 1.05:

1.

13. The method according to claim 12, characterized in that The mass ratio of the substrate dispersant to the 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester is 1:

1.

14. The method according to claim 12, characterized in that The substrate dispersant is diatomaceous earth and / or activated carbon.

15. The method according to any one of claims 3 to 14, characterized in that: The reaction is carried out at 20-35°C.

16. Use of an enzyme having an amino acid sequence as shown in SEQ ID NO: 6, 7, 9 or 10 in catalyzing the conversion of (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid methyl ester into (R)-2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetic acid.

17. Use of an enzyme having an amino acid sequence as shown in SEQ ID NO: 6, 7, 9 or 10 in the resolution of methyl 2-(benzyloxycarbonylamino)-2-(oxetane-3-yl)acetate.

18. The use according to claim 17, characterized in that The resolution was used to prepare methyl (S)-2-(benzyloxycarbonylamino)-2-(oxetan-3-yl)acetate.

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