CN103450040A - Synthesis method of D-threonine - Google Patents
Synthesis method of D-threonine Download PDFInfo
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Abstract
The invention relates to a synthesis method of D-threonine (I). The method comprises the following steps: preparing D-allothreonine (V) from L-threonine (VI) serving as a raw material through racemization of amino acid racemase, action of L-threonine deaminase and purification sequentially; performing esterification on D-allothreonine (V) serving as a starting material to obtain an intermediate (IV); protecting amino by using benzoyl to obtain a compound of a formula (III); performing intramolecular cyclization on the compound of the formula (III) in the presence of sulfoxide chloride and turning over the spatial configuration of hydroxyl to obtain an oxazoline intermediate (II); performing ring opening on the intermediate (II) under the action of acid and removing a protecting group to obtain D-threonine (I). The synthesis method of D-threonine (I) is smart in design, the starting materials are cheap and readily available, the process flow is simple and practical, and a new method is provided for producing D-threonine on a large scale.
Description
Technical field
The present invention relates to the synthetic technical field of amino acid, particularly the synthetic method of alpha-non-natural amino acid D-Thr.
Background technology
D-Thr (formula (I)) is the optical isomer of natural amino acid L-threonine, is a kind of important alpha-non-natural amino acid.It is mainly used in the fields such as chiral drug, chiral additives, chiral auxiliary(reagent), the chiral source synthetic as chirality at pharmaceutical industry.As a kind of optically active organic acid, there is irreplaceable effect in the asymmetric synthesis process of some chipal compounds, mainly for the production of the Threonine protective material of New-type wide-spectrum microbiotic, D-Soviet Union ammonia alcohol, polypeptide building-up process.
About synthesizing of D-Thr, the method that can find at present is very limited, basically in conceptual phase.As document Tetrahedron:Asymmetry, 2010, (21) 2956 – 2965 and Tetrahedron:Asymmetry, 2006, (17) 455 – 467 have reported take the example of nickelous as the synthetic D-Thr of basic chiral coordination compound, but the selectivity of chiral ligand complex structure, reaction and yield are not high, be not suitable for preparation; Document Tetrahedron Letters in addition, 1997,38 (39), 6953-6956 also mentioned with the azepine ternary of chirality assistant agent not ring expansion become the method for Wu Yuan oxazoline ring, but the same with the reason of top method, be not suitable for preparing D-Thr.
The industrial process of D-Thr is not seen in report, and the method for most convenient production D-Thr is (being following synthetic method A) as shown in Figure 1.Take glycine as raw material, first with bivalent cupric ion, generate cupric glycinate complex compound, then react with acetaldehyde, obtain the copper-threonine complex compound; Generally this complex compound is the mixture of four isomer, and due to the difference of solubleness, the ratio of DL-Threonine and DL-allothreonine is about 4:1～3:1; Remove DL-allothreonine isomer by periodic crystallisation copper-threonine complex compound, then decopper(ing), just can obtain the DL-Threonine.The DL-Threonine is again by the protection of the ethanoyl amino, and selective hydrolysis obtains D-Thr under the effect of PAF-AH.
Synthetic route A:
The weak point of aforesaid method is, in order to remove DL-allothreonine isomer, it is on the low side that periodic crystallisation copper-threonine complex compound obtains the yield of DL-Threonine; And to use hydrogen sulfide or resin, inconvenient operation during decopper(ing); The DL-Threonine obtained in addition must further split and just can obtain single D-Thr with enzyme, and the intermediate utilization ratio only has 50%.This method complex operation, total recovery is low, and cost is high.
Therefore, need to provide a kind of preparation method of new D-Thr, raw material is easy to get, technical process is simple, just can reduce production costs, and is conducive to the industrialization of product.
Summary of the invention
In order to have overcome above-mentioned shortcoming of the prior art, the invention provides a kind of industrial production process of D-Thr, the method starting raw material cheaply is easy to get, technical process is simple, and do not need complicated operation, can greatly reduce production costs, be conducive to suitability for industrialized production, be suitable for large-scale promotion application.
As shown in synthetic route B (being the synthetic route shown in Fig. 2), it is starting raw material that L-threonine (VI) is take in the present invention, by the racemization of amino acid racemase and the effect successively of L-threonine desaminase, and obtains D-allothreonine (V) by purifying.
Synthetic route B:
For another example shown in synthetic route C (synthetic route) as shown in Figure 3; D-allothreonine (V) obtains intermediate (IV) by esterification; obtain again the compound of formula (III) with benzoyl protection amino; then formula (III) intramolecular cyclization under the effect of sulfur oxychloride; and the steric configuration get of upset hydroxyl is Dao oxazoline intermediate (II), Hou oxazoline intermediate (II) open loop under sour effect, sloughs protecting group and obtain D-Thr (I).
Synthetic route C:
Wherein, R is C
1-4alkyl, benzyl.
The industrial preparative method of above-mentioned D-Thr of the present invention (I) (synthetic route as shown in Fig. 2-Fig. 4) specifically comprises the following steps:
(a) L-threonine (VI) is raw material, in the aqueous solution, by the racemization of amino acid racemase, obtains the mixed solution of L-threonine (VI) and D-allothreonine (V);
(b) by pyroprocessing by the amino acid racemase inactivation, then add the L-threonine desaminase, by the effect of L-threonine desaminase, L-threonine (VI) is converted into to batanone acid and ammonium ion, so that purifying;
(c) obtain D-allothreonine (V) by simple purification;
(d) with D-allothreonine (V) starting raw material, by with C
1-4alcohol or benzylalcohol esterification obtain intermediate (IV);
(e) intermediate (IV) obtains the compound of formula (III) again with benzoyl protection amino;
(f) compound of formula (III) intramolecular cyclization under the existence of sulfur oxychloride, and the steric configuration of upset hydroxyl, get is Dao oxazoline intermediate (II);
(g) oxazoline intermediate (II) open loop under sour effect, slough protecting group and obtain described D-Thr (I).
Preferably, C in step (d)
1-4alcohol be preferably methyl alcohol or ethanol; The condition of esterification is with corresponding C
1-4alcohol or benzylalcohol be solvent, acid or thionyl chloride catalysis, the reaction times is 2～48h, temperature is 0 ℃～100 ℃
Preferably, in step (e), intermediate (IV) is dissolved in certain solvent, take triethylamine as alkali, and add Benzoyl chloride to prepare the compound of formula (III); Reaction solvent is preferably methylene dichloride or methyl alcohol.
Preferably, in step (f), the compound of formula (III) is dissolved in certain solvent, then drips sulfur oxychloride and prepare formula (II) oxazoline intermediate; Reaction solvent is preferably methylene dichloride or toluene.
Preferably, in step (g), by formula, (II) oxazoline intermediate adds in aqueous hydrochloric acid, reflux, and regulate acid-basicity and obtain described D-Thr (I).
At first the present invention provides the industrial preparative method of a kind of D-Thr (I), and the preparation method that next provides the derivative formula (III) of a kind of D-allothreonine (V) provides the method for a kind of D-of preparation allothreonine (V) again.
The industrial preparative method of a kind of D-Thr (I), derivative formula (III) intramolecular cyclization under the existence of sulfur oxychloride by D-allothreonine (V), and the steric configuration get of upset hydroxyl (II) ， oxazoline intermediate (II) open loop under sour effect, sloughs protecting group and obtains described D-Thr (I) Dao the oxazoline intermediate; As follows:
Wherein, R is C
1-4alkyl or benzyl.
Preferably, the compound of formula (III) is dissolved in certain solvent, then drips sulfur oxychloride and prepare formula (II) oxazoline intermediate; Reaction solvent is preferably methylene dichloride or toluene.
Preferably, by formula, (II) oxazoline intermediate adds in aqueous hydrochloric acid, reflux, and regulate acid-basicity and obtain D-Thr (I).
The preparation method of the derivative formula (III) of above-mentioned D-allothreonine (V), with D-allothreonine (V) starting raw material, by with C
1-4alcohol or benzylalcohol esterification obtain intermediate (IV); Intermediate (IV) obtains formula (III) with benzoyl protection amino again; As follows:
Wherein, R is C
1-4alkyl or benzyl.
Preferably, C
1-4alcohol be preferably methyl alcohol or ethanol; The condition of esterification is with corresponding C
1-4alcohol or benzylalcohol be solvent, acid or sulfur oxychloride are catalyzer, under 0 ℃～100 ℃, react 2～48h.
Preferably, intermediate (IV) is dissolved in certain solvent, take triethylamine as alkali, and add Benzoyl chloride to prepare the compound of formula (III); Reaction solvent is preferably methylene dichloride or methyl alcohol.
A kind of method for preparing D-allothreonine (V), the L-threonine (VI) of take is raw material, first by its amino racemization, obtains the mixed solution of L-threonine and D-allothreonine; By the effect of L-threonine desaminase, make remaining L-threonine be converted into the material of easily removing again; Finally by simple purification, obtain D-allothreonine (V); As shown in synthetic route B.
Preferably, the method for amino racemization is to adopt the amino acid racemase effect, makes L-threonine (VI) become the mixture of L-threonine and D-allothreonine.
Preferably, the remaining L-threonine of enzymatic conversion be first by pyroprocessing by the amino acid racemase inactivation, add the L-threonine desaminase to be transformed; The material of easily removing that L-threonine changes into is batanone acid and ammonium ion.
Preferably, simple purification refers to by decolorizing with activated carbon, concentrated, and the method for crystallization obtains D-allothreonine (V).
Beneficial effect of the present invention specifically is:
1, raw material is easy to get.L-threonine is by fermentative Production at present, and fermentation costs is low, low price, and industrial a large amount of preparations, therefore adopt the with low cost of this processing method.
2, easy and simple to handle.Preparation method of the present invention designs ingenious, first with enzyme process, take L-threonine as raw material acquisition D-allothreonine, by 4 conventional step chemical reactions, the sulfur oxychloride of usining upset hydroxyl, as committed step, does not need complicated operation just can obtain easily D-Thr (I) again; The invention provides good D-Thr industrialized preparing process, be suitable for large-scale promotion application.
The accompanying drawing explanation
Fig. 1 is the existing one-tenth route A for preparing D-Thr.
Fig. 2 is the synthetic route B of preparation D-allothreonine (V).
Fig. 3 is for preparing the synthetic route C of D-Thr (I).
Fig. 4 is for preparing total synthetic route of D-Thr (I).
Fig. 5 is amino acid racemase expression plasmid collection of illustrative plates.
Fig. 6 is L-threonine desaminase expression plasmid collection of illustrative plates.
Embodiment
Content for a better understanding of the present invention, be described further below in conjunction with specific embodiment.
Embodiment 1: the racemization of amino acid racemase and L-threonine desaminase are expressed the structure of bacterial classification
Main agents: restriction enzyme and Taq archaeal dna polymerase (Fermentas), T4DNA ligase enzyme and alkaline phosphatase CIAP(TAKARA), KOD neo plus archaeal dna polymerase (TOYOBO), plasmid extraction test kit and a small amount of glue reclaim test kit (Axygen), kantlex (Shanghai Sheng Gong bio-engineering corporation).The synthetic subclone that reaches of synthetic, the full gene of primer all entrusts Nanjing Genscript Biotechnology Co., Ltd. to complete.
Amino acid racemase is expressed the structure of bacterial classification:
Gene order (NCBI accession number: NC_002947) according to the Pseudomonas putida KT2440 source amino acid racemase alr reported, synthetic this sequence of full gene, two ends design restriction enzyme site NdeI and BamHI, be subcloned into carrier pET24a upper, obtain recombinant plasmid pET24a-alr.The recombinant plasmid pET24a-alr built is transformed to escherichia coli expression host BL21 (DE3) by Calcium Chloride Method, obtain amino acid racemase and express bacterial classification BL21 (DE3)/pET24a-alr.Its amino acid racemase expression plasmid collection of illustrative plates as shown in Figure 5.
The L-threonine desaminase is expressed the structure of bacterial classification:
Gene order (the NCBI accession number: AP009048) of the L-threonine desaminase ilvA originated according to the intestinal bacteria W3110 reported, the design primer is as follows: ilvA-F:5 '-CATGGTCTCCCATGGCTGACTCGCAACCCCTGTC-3 ' (Eco31I), ilvA-R:5 '-CGGGATCCCTAACCCGCCAAAAAGAAC-3 ' (BamHI).The PCR reaction system comprises: each 50pmol of ilvA-F and ilvA-R, W3110 bacterium liquid 1 μ L, 1X KOD plus buffer, 0.2mM dNTP, 25mM MgSO
4, KOD plus2U, moisturizing is to total system 50 μ L.The pcr amplification condition is: 95 ℃ of 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 68 ℃ of 1.5min, repeat 30 circulations, 68 ℃ of 10min.After the PCR reaction finishes, with agarose gel electrophoresis, analyzed, the specific band of an about 1.5kb detected, for required.Glue reclaims test kit and reclaims pcr amplification product in a small amount, uses Eco31I and BamHI in 37 ℃ of double digestion 3-6 hour.PET28b uses NcoI and BamHI in 37 ℃ of double digestion 3-6 hour, reclaims respectively enzyme and cuts product.Utilize Eco31I to be connected with the NcoI isocaudarner, these two fragments are spent the night in 16 ℃ of connections with the T4DNA ligase enzyme, Transformed E .coli DH5 α competent cell, coating is containing the solid LB substratum (peptone: 10g/L of kantlex, yeast extract: 5g/L, NaCl:10g/L, agar: 20g/L, kantlex: 100 μ g/mL) in 37 ℃ of overnight incubation.The picking transformant carries out sequence verification, finally obtains recombinant plasmid pET28b-ilvA, transforms escherichia coli expression host BL21 (DE3) and is expressed, and obtains the L-threonine desaminase and expresses bacterial classification BL21 (DE3)/pET28b-ilvA.Its L-threonine desaminase expression plasmid collection of illustrative plates as shown in Figure 6.
The preparation of embodiment 2:D-allothreonine
The shake flask fermentation of the expression bacterial classification of two enzymes: preparation substratum TB(12g/L peptone, 24g/L yeast extract, 5g/L glycerine, 2.13g/L KH
2pO
4, 16.43g/L K
2hPO
43H
2o).Substratum TB is sub-packed in 500mL triangle shaking flask, liquid amount 100mL, then in high-pressure sterilizing pot in 121 ℃ of heat sterilization 20min.After cooling, in sterilizing TB substratum, add the kantlex of final concentration 100 μ g/mL (to be made in advance the high density mother liquor, and filtration sterilization), so the expression bacterial classification of latter two enzyme is seeded to respectively in substratum, in 37 ℃, 220rpm shaking table, is cultured to OD600=5-6, add 0.2mM IPTG to induce, temperature is reduced to 28 ℃, continues to be cultured to the 24h left and right, and fermented liquid 8000rpm is centrifugal, remove supernatant, collect thalline standby.
The preparation of two enzyme crude enzyme liquids: it is resuspended that the above-mentioned centrifugal thalline obtained adds purified water by 200g/L, then ultrasonic disruption (voltage 400W, ultrasonic time 3s, interval time 5s, work times 80 times, in ultrasonic procedure, the suspension thalline is cooling with ice bath), obtain crude enzyme liquid after fragmentation.
Enzyme catalysis: the L-threonine that takes 200g, add suitable quantity of water to dissolve, adjust PH to 7.0 with 10% ammoniacal liquor, be warming up to 30 ℃, add 200ml racemase crude enzyme liquid, add water to final volume 2000ml, stirring velocity 200rpm, controlling 30 ℃ of temperature is transformed, be converted into L-threonine content and drop to half left and right, and while not descending, be heated to 70 ℃, insulation 30min, then be cooled to 37 ℃, adjust pH8.0 with 10% ammoniacal liquor, add L-threonine desaminase crude enzyme liquid 100ml, stirring velocity 200rpm, temperature is controlled 37 ℃ and is transformed, react to L-threonine and be less than the 1g/L stopped reaction.
Purifying: above-mentioned conversion fluid is warming up to 60 ℃, adds 0.2% gac, stir decolouring 30min, then filter after adding 1% diatomite, collect filtrate and carry out concentrating under reduced pressure, stop concentrating when having a large amount of crystal to separate out (the 2000mL feed liquid is concentrated into the 400mL left and right), more than being cooled to crystallizing at room temperature 4h, suction filtration is collected crystal, with 95% alcohol drip washing, obtains crystal again with 95% alcohol making beating, suction filtration obtains crystal, 60 ℃ of forced air dryings, obtain the D-allothreonine of 80.5g
1h NMR (400MHz, D
2o) δ 4.39 – 4.32 (m, 1H), 3.83 (d, J=3.8Hz, 1H), 1.19 (d, J=6.6Hz, 3H); MS (ESI) m/z=120 (M
++ 1), ee%>99%, total recovery 40.3%.
Embodiment 3:
1, the preparation of D-allothreonine methyl esters (3-2)
D-allothreonine (3-1) 6.0g is dissolved in the anhydrous methanol of 36mL, and solution is cooled to 0 ℃, drips sulfur oxychloride 11.8g, dropwise, 0 ℃ of reaction 48h, TLC monitoring reaction is complete, concentrated white solid D-allothreonine methyl ester hydrochloride (3-2) 8.5g, the yield 99.5% of obtaining.
1H?NMR(400MHz,MeOD)δ4.26(d,J=6.6Hz,1H),4.09(d,J=3.5Hz,1H),3.85(s,3H),1.27(d,J=6.6Hz,3H);MS(ESI)m/z=134(M
++1).
2, the preparation of N-benzoyl-D-allothreonine methyl esters (3-3)
Under nitrogen protection; D-allothreonine methyl ester hydrochloride (3-2) 7.0g is dissolved in to the 35mL anhydrous methanol; add again triethylamine 11.5g; system is cooled to 0 ℃; slowly drip Benzoyl chloride 5.8g, dropwise rear insulation 2h, the TLC monitoring reaction is complete; remove methyl alcohol under reduced pressure; add water 50ml, system is extracted with ethyl acetate three times, and the organic phase after merging washes with water successively; the saturated common salt washing; anhydrous magnesium sulfate drying, filter, the concentrated white solid that obtains; oven dry obtains N-benzoyl-D-allothreonine methyl esters (3-3) 8.8g, yield 89.9%.
1H?NMR(400MHz,CDCl
3)δ7.92–7.76(m,2H),7.54(s,1H),7.46(d,J=7.8Hz,2H),7.16(d,J=6.3Hz,1H),4.88(s,1H),4.29(d,J=3.1Hz,1H),3.83(s,3H),3.59(d,J=5.5Hz,1H),1.24(d,J=6.5Hz,3H);MS(EI)m/z=238(M
++1).
3, (4R, 5S)-5-methyl-2-phenyl-4, the preparation of 5-dihydro-oxazole-4-carboxylate methyl ester (3-4)
Under the protection of nitrogen, N-benzoyl-D-allothreonine methyl esters (3-3) 6.0g is dissolved with the 44mL methylene dichloride, room temperature drips sulfur oxychloride 6.0g, add rear backflow 2h, TLC detection reaction terminal, be cooled to room temperature, remove methylene dichloride under reduced pressure, add cold water 50ml, be adjusted to weakly alkaline with sodium bicarbonate solid, system is extracted with ethyl acetate three times, organic phase after merging washes with water successively, the saturated common salt washing, anhydrous magnesium sulfate drying, filter, concentrate and obtain faint yellow oily matter (4R, 5S)-5-methyl-2-phenyl-4, 5-dihydro-oxazole-4-carboxylate methyl ester (3-4) 5.5g, yield 95%.
1H?NMR(400MHz,CDCl
3)δ8.06–7.90(m,2H),7.56–7.46(m,1H),7.46–7.34(m,2H),5.14–5.02(m,1H),4.99(d,J=10.2Hz,1H),3.77(d,J=7.8Hz,3H),1.39(d,J=6.3Hz,3H);MS(EI)m/z=220(M
++1).
4, the preparation of D-Thr (3-5)
By (4R, 5S)-5-methyl-2-phenyl-4,5-dihydro-oxazole-4-carboxylate methyl ester (3-4) 5.5g adds in the aqueous hydrochloric acid of 6mol/L of 55ml, reflux 2h, TLC detection reaction terminal, be cooled to room temperature, filtration, the concentrated crude product safran oily matter 4.0g that obtains of filtrate, crude product is with after 55mL ethanol heating for dissolving, drip the 4.5g pyridine in backflow, separate out a large amount of white powder solids, cooling, filter, dry and obtain D-Thr (3-5) 2.32g, yield 77.6%.
1H?NMR(400MHz,D
2O)δ4.30(dd,J=6.6,4.0Hz,1H),3.78(d,J=3.8Hz,1H),1.15(d,J=6.6Hz,3H);MS(ESI)m/z=120(M
++1),ee%>99%.
Embodiment 4:
1, the preparation of D-allothreonine ethyl ester (4-1)
D-allothreonine (3-1) 15.0g is dissolved in the dehydrated alcohol of 60mL, solution is cooled to 0 ℃, pass into hydrogen chloride gas to saturated, then be heated to 80 ℃ of back flow reaction 2h, TLC monitoring reaction is complete, concentrated 18.5g white solid D-allothreonine carbethoxy hydrochloride (4-1), the yield 99.8% of obtaining.
1H?NMR(400MHz,MeOD)δ4.26(d,J=6.6Hz,1H),4.09(d,J=3.5Hz,1H),4.10(q,J=6.5Hz,2H),1.27(d,J=6.6Hz,3H),1.24(t,J=6.5Hz,3H);MS(ESI)m/z=148(M
++1).
2, the preparation of N-benzoyl-D-allothreonine ethyl ester (4-2)
Under nitrogen protection; D-allothreonine methyl ester hydrochloride (4-1) 18.0g is dissolved in to the 90mL dehydrated alcohol; add again triethylamine 30.0g; system is cooled to 0 ℃; slowly drip Benzoyl chloride 15.0g, dropwise rear insulation 2h, the TLC monitoring reaction is complete; remove ethanol under reduced pressure; add water 120ml, system is extracted with ethyl acetate three times, and the organic phase after merging washes with water successively; the saturated common salt washing; anhydrous magnesium sulfate drying, filter, the concentrated white solid that obtains; oven dry obtains N-benzoyl-D-allothreonine methyl esters (4-2) 28.4g, yield 92.3%.
1H?NMR(400MHz,CDCl
3)δ7.92–7.76(m,2H),7.54(s,1H),7.46(d,J=7.8Hz,2H),7.16(d,J=6.3Hz,1H),4.88(s,1H),4.29(d,J=3.1Hz,1H),4.12(q,J=6.5Hz,2H),3.59(d,J=5.5Hz,1H),1.24(d,J=6.5Hz,3H),1.22(t,J=6.5Hz,3H);MS(EI)m/z=252(M
++1).
3, (4R, 5S)-5-methyl-2-phenyl-4, the preparation of 5-dihydro-oxazole-4-carboxylic acid, ethyl ester (4-3)
Under the protection of nitrogen, N-benzoyl-D-allothreonine ethyl ester (4-2) 28.4g is dissolved with 200mL toluene, room temperature drips sulfur oxychloride 28.0g, add rear backflow 2h, TLC detection reaction terminal, be cooled to room temperature, remove toluene under reduced pressure, add cold water 200ml, be adjusted to weakly alkaline with sodium bicarbonate solid, toluene extraction three times for system, organic phase after merging washes with water successively, the saturated common salt washing, anhydrous magnesium sulfate drying, filter, concentrate and obtain faint yellow oily matter (4R, 5S)-5-methyl-2-phenyl-4, 5-dihydro-oxazole-4-carboxylic acid, ethyl ester (4-3) 24.2g, yield 91.8%.
1H?NMR(400MHz,CDCl
3)δ8.06–7.90(m,2H),7.56–7.46(m,1H),7.46–7.34(m,2H),5.14–5.02(m,1H),4.99(d,J=10.2Hz,1H),4.07(q,J=6.5Hz,2H),1.39(d,J=6.3Hz,3H),1.26(t,J=6.5Hz,3H);MS(EI)m/z=234(M
++1).
4, the preparation of D-Thr (3-5)
By (4R, 5S)-5-methyl-2-phenyl-4,5-dihydro-oxazole-4-carboxylic acid, ethyl ester (7-1) 24.2g adds in the aqueous hydrochloric acid of 6mol/L of 200mL, reflux 2h, TLC detection reaction terminal, be cooled to room temperature, filtration, the concentrated crude product safran oily matter 18.0g that obtains of filtrate, crude product is with after 200mL ethanol heating for dissolving, drip the 20g pyridine in backflow, separate out a large amount of white powder solids, cooling, filter, dry and obtain D-Thr (4-1) 10.2g, yield 82.3%.
1H?NMR(400MHz,D
2O)δ4.30(dd,J=6.6,4.0Hz,1H),3.78(d,J=3.8Hz,1H),1.15(d,J=6.6Hz,3H);MS(ESI)m/z=120(M
++1),ee%>99%.
Embodiment 5:
1, the preparation of D-allothreonine benzyl ester (5-1)
D-allothreonine (3-1) 10.0g is dissolved in the benzylalcohol of 50mL, solution is cooled to 0 ℃, drip sulfur oxychloride 30.0g, dropwise, be heated to 100 ℃ of reaction 4h, TLC monitoring reaction is complete, reaction solution dilutes with isopropyl ether, separate out a large amount of white solids, filtering drying obtains D-allothreonine benzyl ester hydrochloride (5-1) 17.0g, yield 96.7%.
1H?NMR(400MHz,MeOD)δ7.37–7.30(m,5H),5.09(s,2H),4.26(d,J=6.6Hz,1H),4.09(d,J=3.5Hz,1H),1.27(d,J=6.6Hz,3H);MS(ESI)m/z=210(M
++1).
2, the preparation of N-benzoyl-D-allothreonine benzyl ester (5-2)
Under nitrogen protection; D-allothreonine benzyl ester hydrochloride (5-1) 17.0g is dissolved in to the 85mL anhydrous methanol; add again triethylamine 28.0g; system is cooled to 0 ℃; slowly drip Benzoyl chloride 14.1g, dropwise rear insulation 2h, the TLC monitoring reaction is complete; remove methyl alcohol under reduced pressure; add water 120ml, system is extracted with ethyl acetate three times, and the organic phase after merging washes with water successively; the saturated common salt washing; anhydrous magnesium sulfate drying, filter, the concentrated white solid that obtains; oven dry obtains N-benzoyl-D-allothreonine benzyl ester (5-2) 21.7g, yield 85.3%.
1H?NMR(400MHz,CDCl
3)δ7.92–7.76(m,2H),7.54(s,1H),7.46(d,J=7.8Hz,2H),7.39-7.32(m,5H),7.16(d,J=6.3Hz,1H),5.10(s,2H),4.88(s,1H),4.29(d,J=3.1Hz,1H),3.59(d,J=5.5Hz,1H),1.24(d,J=6.5Hz,3H);MS(EI)m/z=314(M
++1).
3, (4R, 5S)-5-methyl-2-phenyl-4, the preparation of 5-dihydro-oxazole-4-benzyl carboxylate (5-3)
Under the protection of nitrogen, N-benzoyl-D-allothreonine benzyl ester (2-1) 21.7g is dissolved with the 130mL methylene dichloride, room temperature drips sulfur oxychloride 18.6g, add rear backflow 2h, TLC detection reaction terminal, be cooled to room temperature, remove methylene dichloride under reduced pressure, add cold water 150ml, be adjusted to weakly alkaline with sodium bicarbonate solid, system is extracted with ethyl acetate three times, organic phase after merging washes with water successively, the saturated common salt washing, anhydrous magnesium sulfate drying, filter, concentrate and obtain faint yellow oily matter (4R, 5S)-5-methyl-2-phenyl-4, 5-dihydro-oxazole-4-benzyl carboxylate (11-1) 18.0g, yield 87.8%.
1H?NMR(400MHz,CDCl
3)δ8.06–7.90(m,2H),7.56–7.46(m,1H),7.46–7.34(m,2H),7.38–7.31(m,5H),5.16(s,2H),5.14–5.02(m,1H),4.99(d,J=10.2Hz,1H),1.39(d,J=6.3Hz,3H);MS(EI)m/z=296(M
++1).
4, the preparation of D-Thr (3-5)
By (4R, 5S)-5-methyl-2-phenyl-4,5-dihydro-oxazole-4-benzyl carboxylate (5-3) 18.0g adds in the aqueous hydrochloric acid of 6mol/L of 180mL, reflux 2h, TLC detection reaction terminal, be cooled to room temperature, filtration, the concentrated crude product safran oily matter 6.4g that obtains of filtrate, crude product is with after 55mL ethanol heating for dissolving, drip the 7.3g pyridine in backflow, separate out a large amount of white powder solids, cooling, filter, dry and obtain D-Thr (3-5) 5.3g, yield 73.2%.
1H?NMR(400MHz,D
2O)δ4.30(dd,J=6.6,4.0Hz,1H),3.78(d,J=3.8Hz,1H),1.15(d,J=6.6Hz,3H);MS(ESI)m/z=120(M
++1),ee%>99%.
The above; it is only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the premise of not departing from the present invention, also can make some improvement and supplement, these improvement and supplement and also should be considered as falling into protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change of making when utilizing the disclosed above technology contents, the equivalent variations of modifying and developing, be equivalent embodiment of the present invention; Simultaneously, the change of any equivalent variations that all foundations essence technology of the present invention is done above-described embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1. the synthetic method of a D-Thr (I), it is characterized in that, derivative formula (III) intramolecular cyclization under the existence of sulfur oxychloride by D-allothreonine (V), and the steric configuration get of upset hydroxyl is Dao oxazoline intermediate (II), by obtaining oxazoline intermediate (II) open loop under sour effect, slough protecting group and obtain described D-Thr (I);
Wherein, R is C
1-4alkyl or benzyl.
2. synthetic method according to claim 1, it is characterized in that, the compound of described formula (III) is dissolved in solvent, dripping sulfur oxychloride reaction makes described formula (II) oxazoline intermediate, wherein the reaction solvent of preparation formula (II) compound is methylene dichloride or toluene again.
3. synthetic method according to claim 1, is characterized in that, by described formula, (II) oxazoline intermediate adds in aqueous hydrochloric acid, reflux, and regulate acid-basicity and obtain described D-Thr (I).
4. synthetic method according to claim 1, is characterized in that, the preparation method of the derivative formula (III) of described D-allothreonine (V) is, with D-allothreonine (V) starting raw material, by with C
1-4alcohol or benzylalcohol esterification obtain intermediate (IV); Described intermediate (IV) obtains described formula (III) compound with benzoyl protection amino again;
Wherein, R is C
1-4alkyl or benzyl.
5. synthetic method according to claim 4, is characterized in that, described C
1-4alcohol be methyl alcohol or ethanol; The reaction conditions of described esterification is: with corresponding C
1-4alcohol or benzylalcohol be solvent, take acid or sulfur oxychloride as catalyzer, react 2-48h under 0 ℃～100 ℃.
6. synthetic method according to claim 4, it is characterized in that, intermediate (IV) is dissolved in solvent, take triethylamine as alkali, and add Benzoyl chloride to prepare the compound of described formula (III), wherein the reaction solvent of preparation formula (III) compound is methylene dichloride or methyl alcohol.
7. a method for preparing D-allothreonine (V), is characterized in that, the L-threonine (VI) of take is raw material, first by its amino racemization, obtains the mixed solution of L-threonine (VI) and D-allothreonine (V); By the effect of L-threonine desaminase, make remaining L-threonine (V) be converted into the material of easily removing again; Finally by simple purification, obtain described D-allothreonine (V);
8. method according to claim 7, is characterized in that, the method for described amino racemization is to adopt the amino acid racemase effect, makes L-threonine (VI) become the mixture of L-threonine (VI) and D-allothreonine (V).
9. method according to claim 7, is characterized in that, the remaining L-threonine of enzymatic conversion be first by pyroprocessing by the amino acid racemase inactivation, then add the L-threonine desaminase to be transformed; The material of easily removing that L-threonine changes into is batanone acid and ammonium ion.
10. method according to claim 7, is characterized in that, described simple purification refers to by decolorizing with activated carbon, concentrated, and the method for crystallization obtains described D-allothreonine (V).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103772224A (en) * | 2014-02-25 | 2014-05-07 | 成都百事兴科技实业有限公司 | Preparation method of D-threonine |
| CN105001098A (en) * | 2015-07-26 | 2015-10-28 | 嵊州市油脂化工有限公司 | Method for preparing 3(R)/(S)-amidogen-1-butanol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103772224A (en) * | 2014-02-25 | 2014-05-07 | 成都百事兴科技实业有限公司 | Preparation method of D-threonine |
| CN103772224B (en) * | 2014-02-25 | 2015-04-15 | 成都百事兴科技实业有限公司 | Preparation method of D-threonine |
| CN105001098A (en) * | 2015-07-26 | 2015-10-28 | 嵊州市油脂化工有限公司 | Method for preparing 3(R)/(S)-amidogen-1-butanol |
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