CN1243880A - Process for synthesizing optically active cyanohydrin compound by oxynitrile cracking and enzyme catalysis of new enzyme source - Google Patents
Process for synthesizing optically active cyanohydrin compound by oxynitrile cracking and enzyme catalysis of new enzyme source Download PDFInfo
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Abstract
The present invention relates to a process for preparing chira (R)-cyanohydrin compound by the reaction of oxynitrilase catalyzed carbonyl compound on cyanogen or nitrile compound. Said oxynitrilase uses peach kernel, loquat kernel or the seed of Dacao vegetable as enzyme source.
Description
The present invention relates to a kind of oxynitrile cracking enzyme, is that employing peach kernel, loquat or Herba Viviae Sativae seed are the thick oxynitrile cracking enzyme that new enzyme source makes, but this enzyme catalysis synthesis of optically active cyanohydrin compound.
Cyanohydrin compound is important organic synthesis intermediate, can change into compounds such as alpha-alcohol ketone, alpha hydroxy acid, beta-amido alcohol, amido nitrile and aziridine easily.Chemosynthesis product is racemic cyanohydrin compound under the no enzyme catalysis condition, because enzyme develops into enzyme catalysis synthesis of optically active cyanohydrin compound from chemosynthesis to height chemistry, zone and spatial specificity and the enzyme reaction mild condition of reaction.Almond (Almond) is an enzyme source easily, the R-cyanalcohol lyase that is made by the almond source [(R)-oxynitrilase] catalysis synthesis of optically active cyanohydrin compound has carried out studying (Griengl, H etc., J.Chem.Soc., Chem.Commun.1997,1933), with existing (Kiljunen, the E etc. of reporting of the R-cyanalcohol lyase catalyzed reaction of rough enzyme and pure enzyme, Tetrahedron, Asymmetry, 1996,7.1105; 1997,8,1225; 1997,8,1551; Effenberger, F.Angew., Chem.Int.Ed.Engl., 1994,33,1555; Enantiomer., 1996,1,359).Usually the water damping fluid is as the satisfied reaction conditions (Brussee, J etc., Tetrahedron, 1990,46,979) and the water-organic solvent two-phase reaction system of development afterwards.Because carbonyl compound and enzyme have lipophilicity and wetting ability respectively, reaction is inhomogeneous in the profit two phase reaction, and aqueous phase easily produces the racemization product of chemical addition and causes product ee value low, purification difficult is difficult to suitability for industrialized production, and people such as woods Guoqiang adopt almond to make thick oxynitrile cracking enzyme as the enzyme source, and with this enzyme at organic solvent or contain catalysis synthesis of optically active cyanohydrin compound in the organic solvent system of little water, develop continuous flow phase enzymic catalytic reaction technology, be more suitable for need of industrial production (CN98110818.0), except that Semen Armeniacae Amarum, also report jowar rice shoot (Tetrahedron Lett, 1990,31,1249), rubber tree (Tetrahedron Lett., 1993,34,4769), cassava (Angew.Chem.Int.Ed.Engl., 1996,35,4), apple, Lee and cherry seed (TetrahedronAsymmetry, 1997,8,1551; 1997,8,1225) as the oxynitrile cracking enzymic synthesis chiral cyanohydrin in enzyme source.Though the research in oxynitrile cracking enzyme catalysis synthesis of chiral cyanalcohol field is awfully hot, it is to be solved that the production that is used for chiral cyanohydrin also has many problems to have, and also needs constantly to explore the method for new enzyme source and suitability for industrialized production thereof.
The purpose of this invention is to provide a kind of oxynitrile cracking enzyme that obtains from new enzyme source.
Another object of the present invention provides a kind of purposes of the oxynitrile cracking enzyme that is obtained by new enzyme source, i.e. synthesis of chiral
Another object of the present invention provides a kind of purposes of the oxynitrile cracking enzyme that is obtained by new enzyme source, i.e. synthesis of chiral cyanohydrin compound and method thereof.
Has this feature of toxicity according to traditional Chinese medicine and pharmacy theory and cyanogen glucoside, to nearly hundred kinds toxic or have antibechic but the vegetable cell of phlegm efficacy effect investigate and sound out, and therefrom selected the several plant cell to carry out The effect as the enzyme source, obtain several new enzyme sources that can be used as the oxynitrile cracking enzyme.
The seed of oxynitrile cracking enzyme employing peach kernel Peach (Prunus persica L.), loquat Loquat (Eriobotrya L.) of system of the present invention or Herba Viviae Sativae (Vida Sativa L.) obtains the oxynitrile cracking enzyme as new enzyme source after broken, organic solvent take off ester.Also can be through broken, water-soluble solvent leaching or extraction, leach liquor or extracting solution through concentrating, perhaps leach liquor or extracting solution reconcentration and making behind reconcentration or organic solvent extraction after dialysis or the column chromatography for separation.The oxynitrile cracking enzyme that is obtained can be thick product.
Described peach kernel, Pi handle or Herba Viviae Sativae seed adopt historrhexis's machine to pulverize usually through cleaning, dry, allowancing for bark, and the organic solvent of doubly measuring with 1-3 removes lipid matter 1-3 time, obtains white or the thick enzyme powder of little yellow through the 50-200 mesh sieve.This enzyme powder drying is handled common moisture 8-10%, places refrigerator to store.
Perhaps after fragmentation, with water-soluble solvent such as phosphorus ring-sodium citrate buffer solution, acetic acid-vinegar ring sodium damping fluid, in imidazoles-salt ring damping fluid in 0-20 ℃ of following homogenizing homogenate, filtration.Leaching or extracting solution are through centrifugal treating, and leaching or extracting solution concentrate; Perhaps leaching or extracting solution reconcentration after dialysis ion-exchange chromatography or column chromatography gel separation can make the oxynitrile cracking enzyme.
Described organic solvent is fat, ether, alcohol, the aromatic hydrocarbons of low carbon chain, substituted arene, alkane, haloalkane or dimethyl formamide etc.
Described water-soluble solvent can be inorganic salt or the organic salt aqueous solution or the damping fluid of pH3-6.5, or the mixed solution of above-mentioned solution and water-miscible organic solvent.
Adopting of the present invention is pure cyanogen lyase energy catalyzed carbon based compound and the cyanogen or the nitrile compound reaction generation optical active cyanalcohol compound in enzyme source with peach kernel, Radix Eriobotryae or Herba Viviae Sativae seed, promptly is R with the molecular formula
1COR
2Oxy-compound, molecular formula is R
3The prussiate of CN, in organic solvent and 0-50 ℃ the time, pure cyanogen lyase catalyzed reaction 0.01-120 hour, generates the optical active cyanalcohol compound at 0.05-48 hour recommendation response time.R wherein
1=R
4C
6H
4, R
4OC
6H
4, R
4OCH
2C
6H
4,
C
6H
4CH=CH, R
5CH=CH,
R
2=H, C
1-3Alkyl, R
3=H, (CH
3)
2COH or HCOH, R
4=H, C
1-4Alkyl, R
5=C
1-8Alkyl, R
6=H, C
1-4Alkyl,
Or CH
3OCH
2-.The mol ratio of described carbonyl compound and cyanogen or nitrile compound is 1: 0.8-20, recommend than being 1: 1-5.Using of carbonyl compound and R-cyanalcohol lyase quantizes 0.001-100mol carbonyl compound/1 gram R-cyanalcohol lyase, recommends 0.005-20mol carbonyl compound/1 gram R-cyanalcohol lyase.Described solvent can be the solvent commonly used such as ester, ether, aromatic hydrocarbons, substituted arene, alkane, haloalkane, ketone or dimethyl formamide of low carbon chain.As methyl-formiate, ethyl formate, methyl acetate, ethyl acetate, sherwood oil, octane, hexanaphthene, ether, isopropyl ether, ethyl isobutyl ether, isoamyl oxide, methyl-phenoxide, benzene,toluene,xylene, methylene dichloride, trichloromethane, tetracol phenixin, ethylene dichloride, trichloroethane etc.
Described R-cyanalcohol lyase can adopt rough cellas powder.Above-mentioned organic solvent can contain the water of 0-10% weight.
Organic solvent described in the present invention can be the water that contains trace in the organic solvent; Also can in water, behind the prepared in reaction HCN, obtain the organic phase of minor amount of water by organic solvent extraction HCN by acetic acid or mineral acid NaCN.Usually also contain water in the rough cellas.Cyanidization agent described in the present invention can be HCN, also can be acetone cyanohydrin, formaldehyde cyanalcohol etc.
Adopt new enzyme source of the present invention to prepare the oxynitrile cracking enzyme, not only the enzyme source is abundant, and it is simple and easy to prepare the method for oxynitrile cracking enzyme, and this enzyme can be used for the synthesis of chiral cyanohydrin compound, is suitable for need of industrial production.
Following embodiment will help to understand the present invention, but not limit content of the present invention.
Embodiment 1
100 gram peach kernels, Radix Eriobotryaes or the Herba Viviae Sativae seed that shells are pulverized through organizing pulverizer, take off ester with conventional organic solvent, as stirring 6.5-2h with 100-500ml ethyl acetate, acetone or ether, filter, repeated washing is 2-4 time again, and the rough esterase powder that takes off that is obtained is that R-cyanalcohol nitrile is separated enzyme and is stored in the refrigerator standby.
Behind sodium cyanide and Glacial acetic acid or the sulfuric acid reaction, obtain to contain prussic acid organic solvent solution, anhydrous Na with solvent extraction process
2SO
4Use dry back.Perhaps directly HCN reagent is added the organic solvent that contains 0-1% weight water.
The enzyme powder (R-cyanalcohol nitrile lyase), 2.5mmol carbonyl compound and the 2.5-10mmolHCN in the 10ml solvent that are the enzyme source with the above-mentioned rough Radix Eriobotryae of 500mg stir in reaction flask; in temperature shown in the following table and reaction times reaction; the rough enzyme of filtered and recycled then; filtrate concentrates; crude product is measured productive rate after with purification by silica gel column chromatography; and after making the acetic ester or other derivative of O-protection; the asymmetric purity of cyanohydrin compound is carried out chirality high-pressure liquid phase method mensuration with the CHIRALPAKAD post, and productive rate and ee% value the results are shown in table 1.As a comparison, the results are shown in of two-phase system that adds 5% (v/v) 0.02M citric acid solution (pH5.5) in the same system with table.
Table 1 sequence number oxy-compound solvent reaction time-temperature productive rate ee
H ℃ of % %1 phenyl aldehyde isopropyl ether+damping fluid 30 76 812 isopropyl ethers 4 99 993 isopropyl ethers 30＞95＞97
It is better that the result shows that the little water reaction system that does not add damping fluid obtains the result.
Embodiment 2
With peach kernel, loquat or the peeling of Herba Viviae Sativae seed, under 0-20 ℃ at historrhexis's crusher machine, with water-soluble solution 100-200ml 50mM imidazoles-hydrochloride buffer or sodium citrate buffer (pH3-6.5), 0-10 ℃ of homogenizing homogenate, extract filters, filtrate is centrifugal through whizzer, and collecting supernatant liquor is the enzyme crude extract, concentrates and obtains thick oxynitrile cracking enzyme; Perhaps above-mentioned supernatant liquor is packed in the dialysis tubing, dialyse 1-3 time with 2L 50mM imidazoles-hydrochloride buffer, filtering and concentrating obtains thick oxynitrile cracking enzyme; The SephadexG-25 column chromatography of perhaps supernatant liquor being packed into separates the back and concentrates and obtains thick oxynitrile cracking enzyme.The also available ion-exchange chromatogram purification of above-mentioned supernatant liquor as obtaining thick oxynitrile cracking enzyme with reconcentration behind the DEAE-Cellulose52 column purification, also can intersect above-mentioned the whole bag of tricks to mix and use, to obtain purer oxynitrile cracking enzyme.
With the Radix Eriobotryae is example, and crude extract in the solution behind ion exchange chromatography, is measured oxynitrile cracking enzyme activity (J.Bio.Chem, 1979,254,23,12145) and protein content (Analytical Biochem, 1976,72,248), result such as following table
| Separation method | Volume (ml) | Total enzyme (μ) alive | Total protein (mg) | Ratio work (μ/mg) | The purifying multiple | Yield (%) |
| Crude extract | ???96 | ??265 | ????89 | ??2.9 | ??100 | |
| Ion exchange chromatography | ???96 | ??180 | ????5.3 | ??34 | ??11.7 | ??68 |
Embodiment 3
Under embodiment 1 condition, the oxynitrilase that adopts different enzymes source to obtain is that substrate is as shown in table 2 in the result of little water reaction system synthesis of chiral aromatic aldehyde with phenyl aldehyde or aubepine: product productive rate ee during the reaction of sequence number substrates enzymes source solvent comprises water amount temperature
Hour % % 1 benzaldehyde Radix Eriobotryae isoamyl ether 4 48 1-1 99 99 2 30 48 between % ℃＞12 48 100 99 6 pairs of methoxyl group Radix Eriobotryaes of 95 97 3 peach kernels, 4 48 99 99 4 30 48 97 89 5 common vetch seeds, 4 48 12 90.5 7 benzaldehydes, 30 48 20 74.5 8 peach kernels, 4 48 12 90.5 9 30 56 91 10 common vetch seeds, 12 52 94 above-claimed cpd analysis results are as follows:, (R)-, (+)-2-hydroxyl-2-phenylacetonitrile colourless oil liquid. [α]D 20+ 47.5 (c1.89, CHCl
3), e.e.＞99%;
1H NMR: δ, 3.70 (s, 1H, OH); 5.50 (s, 1H, CH); 7.50 (m, 5H, Ar-H) .IR:3414,3066,3036,2250,1495,1456,1406,1196,1043,765,701cm
-1MS:m/z:133 (M
+, 78), 132 (M
+-1,50), 116 (29), 115 (43), 106 (38), 105 (92), 91 (14), 77 (100), 57 (9), 51 (45), 43 (21). (R)-(+)-2-hydroxyl-2-(4-p-methoxy-phenyl) acetonitrile colorless solid.M.p.66-67 ℃ [α]
D 18+ 50.0 (c0.89, CHCl
3), e.e.95.3%; Lit.
54a:[α]
D 20+ 49 (c1, CHCl
3), e.e.99%.Isolated yield (after chromatography): 60%.
1H NMR: δ, 3.18 (s, 1H, OH); 3.82 (s, 3H, CH3O); 5.46 (s, 1H, CH); δ, 6.94 (d, 2H, J=8.7Hz, Ar-H); 7.43 (d, 2H, J=8.7Hz, Ar-H) .IR:3398,2248,1613,1515,1023,823cm
-1MS:m/z:(intensity): 163 (M
+, 2), 137 (11), 136 (91), 135 (100), 107 (15), 92 (14), 77 (25), 63 (9), 51 (5).
Embodiment 4
With embodiment 1, adopt
During for substrate, the oxynitrile cracking enzyme in different enzymes source is when isoamyl oxide, ether or ethanol are solvent, and reaction result is listed in table 3.Sequence number product enzyme source temperature ℃ time h productive rate % ee%1
Peach kernel 4 24 93 98.22 loquats 4 24 50 913 Herba Viviae Sativae seeds 4 96 974
The light yellow oily liquid of peach kernel 4 48 78 685 30 24 30 69.36 loquat 4 48 33 52.2 (S)-(+)-2-hydroxyl-2-(2-furyl) acetonitriles.[α]
D 21+ 50.0 (c1.60, CHCl
3), e.e.＞99%;
1H NMR: δ, 3.17 (br s, 1H, OH); 5.55 (s, 1H, CH); 6.43 (dd, 1H, J=1.9Hz, J=3.3Hz, Ar-H); 6.61 (m, 1H, Ar-H); 7.49 (m, 1H, Ar-H) .IR:3403,2256,1707,1500,1400,1269,1234,1149,1030,750cm
-1MS:m/z:123 (M
+, 53), 106 (64), 97 (27), 96 (87), 95 (100), 91 (70), 77 (28), 68 (42), 57 (76), 43 (52) (R)-(+)-the 2-hydroxy-4-phenyl-(E)-butyl-3-alkene nitrile white solid.m.p.77-78℃。[α]
D 13+26.1(c0.78，CHCl
3)，e.e.＞69.3％；
1H?NMR：δ，3.00(d，1H，J＝7Hz，OH)；5.17(t，1H，J＝5.9Hz，CH)；6.27(dd，1H，J＝5.9Hz，J＝15.9Hz，＝CH)；6.92(d，1H，J＝15.9Hz，＝CH)；7.33-7.44(m，5H，Ar-H).IR：3358，3030，2253，1654，1492，1450，1415，1300，1088，1024，976，925，756，695cm
-1MS：m/z：1590(M
+，62)，142(18)，133(96)，132(44)，131(100)，130(78)，115(35)，105(26)，104(35)，103(44)，91(35)，78(25)，77(34)，63(15)，51(24)，43(0.7).
Embodiment 5
With embodiment 1, adopt CH
3CH
2CH
2CH
2CH
2CH
2CHO,
During for substrate, with peach kernel as the oxynitrile cracking enzyme in enzyme source when isoamyl oxide or sherwood oil are solvent, reaction result is listed in the table 4.Sequence number product enzyme source temperature time h productive rate % ee%
℃ 1
Peach kernel 4 45 39 58.12
25 45 42 98.23
25 120 22 72 (R)-(+)-2-hydroxyl caprylic nitrile colourless oil liquid.[α]
D 25+ 7.5 (c0.75, CHCl
3).
1H NMR: δ, 0.88 (t, 3H, J=7Hz, CH
3); 1.23-1.62 (m, 8H, 4xCH
2); 1.84 (q, 2H, J=7Hz, CH
2); 2.98 (br s, 1H, OH); 4.47 (t, 1H, J=7Hz, CH). (R)-the own nitrile colourless oil liquid of 2-hydroxy-2-methyl.[α]
D 22+ 2.2 (c1.33, CHCl
3), e.e.98.6%.
1H NMR: δ, 0.94 (t, 3H, J=7.1,6-H); 1.24 (t, 2H, J=7Hz, 5-H); 1.35-1.51 (m, 2H, 4-H); 1.59 (s, 3H, 1-H); 1.73-1.79 (m, 2H, 3-H) .IR:3445,2961,2938,2875,2242,1465,1397,1172,1143,1063,957,887cm
-1MS:m/z:127 (M
+, 0.7), 121 (0.2), 110 (5), 102 (13), 101 (100), 94 (1), 85 (3), 83 (5), 71 (13), 68 (6), 57 (8), 43 (14). (R)-(+)-2-hydroxyl-2-phenyl propionitrile colourless oil liquid .[α]
D 17+ 4.3 (c0.47, CHCl
3), e.e.78.1%.
1H NMR: δ, 1.84 (s, 3H, CH
3); 2.97 (s, 1H, OH); 7.33-7.41 (m, 3H, Ar-H); 7.53-7.56 (m, 2H, Ar-H) .IR:3421,2927,2856,2250,1494,1451,1371,1226,1101,764,699cm
-1MS:m/z:149 (M
++ 2,9), 1480 (M
++ 1,5), 147 (M
+, 36), 132 (100), 121 (10), 105 (55), 91 (5), 77 (34), 63 (4), 51 (20), 43 (14).
Embodiment 6
With embodiment 1, when adopting the Herba Viviae Sativae seed to be the enzyme source, in the results list 5 of oxynitrile cracking enzyme catalysis synthesis of chiral cyanalcohol.Sequence number product temperature ℃ time h productive rate % ee%1
10 10-48 93 88.22
12 10-48 71 703
12 97 91 (R)-(+)-2-hydroxyl-2-(3,4-methylene-two-oxygen phenyl) acetonitrile colourless oil liquid.[α]
D 22+ 44.5 (c4.54, CHCl
3).
1H NMR:(CD
3OD/TMS): δ, 5.69 (s, 1H, CH); 6.17 (s, 2H, CH
2); 7.03 (m, 1H, Ar-H); 7.19 (m, 2H, Ar-H) .IR:3410 (w), 2907,2249 (CN), 1612,1505,1490,1448,1254,1039,933,863,815,778MS:m/z:177 (M
+, 0.1), 176 (M
+-1,0.1), 150 (M
+-HCN, 88), 149 (M
+-HCN-H, 100), 121 (31), 91 (10), 65 (12), 63 (19). (R)-and 2-hydroxyl-2-(3-pyridyl) acetonitrile white solid, m.p.85-86 ℃.
1H NMR; δ, 5.55 (br s, 1H, OH); 5.65 (s, 1H, CH); 7.45 (dd, 1H, J1=8Hz, J2=4.8Hz, Py-H); 7.98 (dd, 1H, J1=8Hz, J2=1.6Hz, Py-H); 8.56 (d, 1H, J=4.8Hz, Py-H); 8.61 (s, 1H, Py-H) .IR:3021 (w), 2820 (w), 2684 (w), 1955,1602,1585,1480,1432,1030,933,838,792,707,642.MS:m/z:135 (M
++ 1,100), 133 (M
+-1,31), 117 (M
++ 1-H
2O, 9), 108 (M
+-CN, 98), 107 (M
+-HCN, 48), 106 (M
+-HCN-H, 56), 90 (3), 78 (33), 51 (27). and ultimate analysis: C
7H
6N
2O: measured value: C:62.63%, H:4.36%, N:21.07%; Calculated value: C:62.68%, H:4.51%, N:20.88%. (R)-second hydroxyl-2-(4-fluoro phenyl) acetonitrile colourless oil liquid.[α]
D 20+36.4(c6.38，CHCl
3)，e.e.＝94.2％.
1H?NMR；δ，3.39(br，s，1H，OH)；5.52(s，1H，CH)；7.13(m，2H，Ar-H)；7.49(m，2H，Ar-H).
19F?NMR(CDCl
3/TFA，δppm)：-33.93(m，Ar-F).IR：3410(w)，2252，1606，1511，1421，1235，1038，837.MS：m/z：151(M
+，11)，133(M
+-H
2O，8)，124(M
+-HCN，85)，123(M
+-1-HCN，100)，109(11)，95(81)，75(30).HRMS：C
8H
6FNO：Found：151.0413，Cal：151.0433.
Embodiment 7
The ethyl acetate solution 10ml that adds the thick oxynitrilase powder of 0.5 gram, 10mmol aldehyde, 11-15mmnol HCN in this reaction flask, stirring reaction, filtering the thick enzyme powder that obtains can reuse.Rapid column chromatography purifying (ethyl acetate/petroleum ether, 1/6) desolventizes and obtains product, and it is as shown in the table for the result: sequence number aldehyde temperature ℃ reaction times product productive rate
*Ee
*
(hour) % %1 Radix Eriobotryae
25 84
94 782 big nest elements
30 46
90 693 peach kernels
20 96
96 40.14 Radix Eriobotryaes
25 300
90 21.45 Herba Viviae Sativaes
45 24
83 17.36 peach kernels
20 168
86 587
30 24
87 818
45 24
92 22.3
*Column chromatography chromatogram separation and purification productive rate.Productive rate calculates by the conversion thing that breaks off the base.
*The ee value splits through the chiral column high-pressure liquid phase and measures.HRMS:C
15H
16N
2O
3: measured value: 272.1179, calculated value: 272.1161. (+)-2-hydroxyl-2-(2-(N-methoxyl methyl) pyrroles) the light yellow oily liquid of acetonitrile [α]
D 20+ 104.9 (c0.667, CHCl
3), ee=81%.
1H NMR: δ, 3.30 (s, 3H, CH
3); 3.92 (br, s, 1H, OH); 5.08,5.68 (AB, 2H, J=10.9Hz, CH
2); 5.56 (s, 1H, CH); 6.13 (dd, 1H, J1=3.5Hz, J2=3.0Hz, Pyrr-H); 6.47 (dd, IH, J1=3.6Hz, J2=1.7Hz, Pyrr-H); 6.83 (dd, 1H, J1=2.9Hz, J2=1.7Hz, Pyrr-H).
13C NMR: δ, 126.38 (t-C-Pyrr); 125.59 (C-Pyrr); 118.01 (CN); 113.18 (C-Pyrr); 108.01 (C-Pyrr); 78.63 (NCH
2O); 56.55 (CHOH); 55.995 (OCH
3) .IR:3402,3117,2934,2851,2247,1486,1464,1444,1400,1277,1097,1025,732.MS:m/z (intensity %): 166 (M
+, 5), 149 (4), 139 (9), 124 (31), 108 (17), 94 (7), 80 (10), 71 (9), 57 (13), 45 (100), 41 (12) .HRMS:C
8H
10N
2O
2: measured value, 166.742, calculated value, 166.742. (R)-(+)-2-(2-(N-methyl) the pyrroles)-light yellow oily liquid of 2-hydroxyl-acetonitrile.[α]
D 20+35.7(c1.15，EtOH)，ee＝40.1％
1H?NMR：δ，2.90(br，s，1H，OH)；3.72(s，3H，CH
3)；5.50(s，1H，CH)；6.09(m，1H，Pyrr-H)；6.38(m，1H，Pyrr-H)；6.67(m，1H，Pyrr-H).
Synthesizing of embodiment 8 chirality fluorine substituted aroma aldehyde cyanalcohols
With 1-2mmol substrate aldehyde, thick oxynitrilase powder 0.2-0.5g, the ethyl acetate of the HCN of mol ratios such as 1-1.5, ether or isoamyl oxide solution 10ml, 10-20 ℃ was reacted rapid column chromatography purifying (ethyl acetate: sherwood oil=1: 4-6) 24-48 hour, obtain product, result such as following table.Product productive rate during sequence number aldehyde temperature ℃ reaction
*Ee
*
Between hour % %1 Radix Eriobotryae
4 24
36 77.32 peach kernels
20 24
96 843 peach kernels
28 24
71 84.34 Radix Eriobotryaes
4 24
40 40.15 Herba Viviae Sativae seeds
20 24
92.1 46.16 Herba Viviae Sativae seeds
12 24
70 417 Herba Viviae Sativae seeds
20 24
91 598 peach kernels
12 48
90 35
*,
*With embodiment 7.(R)-2-hydroxyl-2-(2-fluoro phenyl) acetonitrile colourless oil liquid.[α]
D 20+ 21.85 (c3.60, CHCl
3), ee=84%.
1H NMR: δ, 3.40 (br, s, 1H, OH); 5.78 (s, 1H, CH); 7.14 (m, 1H, Ar-H), 7.24 (td, 1H, J1=7.6Hz, J2=1.1Hz, Ar-H), 7.43 (m, 1H, Ar-H); 7.62 (td, 1H, J1=7.5Hz, J2=1.8Hz, Ar-H).
19F-NMR (CDCl
3/ TFA, δ ppm) :-41.02 (m, Ar-F) .IR:3412 (w), 3080,2928,2255,1618,1592,1494,1460,1234,1043,759.MS:m/z (intensity %): 151 (M
+, 6), 12 (M
+-HCN, 67), 123 (M
+-1-HCN, 100), 109 (18), 95 (50), 75 (20), 70 (8), 50 (12) .HRMS:C
8H
6FNO: measured value: 151.0417, calculated value: 151.0434. (R)-2-hydroxyl-2-(3,4-trifluoro-benzene base) acetonitrile colourless oil liquid.[α]
D 20+ 22.5 (c2.55, CHCl
3), ee=84.3%.
1H NMR: δ, 3.00 (br, s, 1H, OH); 5.54 (s, 1H, CH); 7.27 (m, 3H, Ar-H).
19F-NMR (CDCl
3/ TFA, δ ppm) :-58.3 (m, Ar-F) .IR:3412 (w), 3086,2928,2254 (CN), 1616,1522,1438,1289,1120,1042,875,825,772.MS:m/z (intensity %): 170 (M
++ 1,5), 169 (M
+, 49), 158 (3), 152 (24), 142 (74), 141 (100), 123 (26), 114 (26), 113 (67), 63 (35). ultimate analysis C
8H
5F
2NO: calculated value: C:56.81; H:2.98; N, 8.28. measured value: C:57.02; H:3.14; N:8.16. (R)-2-hydroxyl-2-(2,3-phenyl-difluoride base) acetonitrile colourless oil liquid.[α]
D 20+ 10.7 (c 3.50, CHCl
3), ee=46.1%.
1H NMR: δ, 3.25 (br, s, 1H, OH); 5.81 (s, 1H, CH); 7.17-7.23 (m, 2H, Ar-H); 7.42 (m, 1H, Ar-H).
19F-NMR (CDCl
3/ TFA, δ ppm) :-59.50 (m, 1F, Ar-F);-65.68 (m, 1F, Ar-F) .IR:3413,2930,2250,1629,1602,1494,1408,1286,1046,794,759.MS:m/z (intensity %): 169 (M
+, 53), 152 (M
+-OH, 26), 143 (M
+-CN, 100), 141 (M
+-1-HCN, 50), 123 (40), 115 (12), 114 (15), 63 (20) .HRMS:C
8H
5F
2NO-H
+: measured value: 168.0288, calculated value: 168.0261. (R)-2-hydroxyl-2-(2,5-phenyl-difluoride base) acetonitrile colourless oil liquid.[α]
D 20+ 6.7 (c1.90, CHCl
3), ee=41%.
1H NMR: δ, 3.37 (d, 1H, J=6.4Hz, OH); 5.77 (d, 1H, J=6.4Hz, CH); 7.12 (m, 2H, Ar-H); 7.35 (m, 1H, Ar-H).
19F-MR (CDCl
3/ TFA, δ ppm) :-39.34 (m, 1F, Ar-F);-46.73 (m, 1F, Ar-F) .IR:3437 (w), 3092,2910,2254,1491,1437,1404,1187,1135,1063,877,817,799,745.MS:m/z (intensity %): 170 (M
++ 1,6), 169 (M
+, 61), 1 52 (M
+-OH, 27), 149 (30), 142 (80), 141 (100), 125 (19), 123 (59), 115 (21), 114 (31), 113 (57), 95 (20), 63 (40). and ultimate analysis C
8H
5F
2NO: calculated value C, 56.81; H, 2.98, N, 8.28. measured value: C, 57.00; H, 2.96; N, 8.45.HRMS:C
8H
5F
2NO: calculated value: 169.0313, measured value: 169.0340. (R)-2-hydroxyl-2-(3,4,5-trifluoro-benzene base) acetonitrile colourless oil liquid [α]
D 20+ 28.90 (C, 1.50, CHCl
3), ee=59%
1H NMR: δ, 3.10 (br, s, 1H, OH); 5.65 (s, 1H, CH); 7.15 (s, 2H, Ar-H).
19F-NMR (CDCl
3/ TFA, δ ppm) :-58.6 (m, 2F, Ar-F);-49.5 (m, 1F, Ar-F) .IR:3415,2931,2248,1610,1545,1428,1400,1280,1042,810,796,760. ultimate analysis: C
8H
4F
3NO: measured value: C:51.08%, H:2.12%, N:7.28%; Calculated value: C:51.34%, H:2.14%, N:7.49%. (R)-2-hydroxyl-2-phenyl-pentafluoride base acetonitrile clear crystal.M.p.55-56 ℃ of .[α]
D 18+ 10 (c0.655, CHCl
3), ee=35%.
1H NMR: δ, 3.95 (s, 1H, OH); 5.83 (s, 1H, CH).
19F-NMR (CDCl
3/ TFA, δ ppm) :-82.5 (m, 2F, F-Ar);-72.3 (t, 1F, F-Ar);-64.7 (m, 2F, F-Ar) .IR:3494,2953,2266,1660,1515,1137,1052,1002,894,787,658cm
-1.MS:m/z (intensity %): 224 (M
++ 1,12), 223 (M
+, 100), 206 (78), 203 (31), 197 (43), 196 (32), 195 (43), 177 (41), 168 (20), 167 (21), 149 (10), 117 (23), 99 (19), 56 (7), 43 (0.8). ultimate analysis C
8H
2F
5NO: calculated value C:43.05; H:0.90; N:6.28. measured value: C:42.97; H:0.82; N:6.12.
Claims (8)
1. method with oxynitrile cracking enzyme catalysis aldehydes or ketones and cyanogen or nitrile compound prepared in reaction chiral cyanohydrin compound is characterized in that described oxynitrile cracking enzyme is is the enzyme source with peach kernel, Radix Eriobotryae or Herba Viviae Sativae seed.
2. the method for claim 1 is characterized in that described oxynitrile cracking enzyme is to take off ester by peach kernel, Radix Eriobotryae or Herba Viviae Sativae seed through broken, organic solvent to handle.
3. the method for claim 1, it is characterized in that described peach kernel, Radix Eriobotryae or Herba Viviae Sativae seed are through fragmentation, leach or extract reconcentration by water-soluble solvent, perhaps leach or extracting solution reconcentration and making after dialysis, gel filtration chromatography or ion-exchange separate.
4. method as claimed in claim 2 is characterized in that the described organic solvent that is used for degreasing is the ester of low carbon chain, ether, ketone or alcohol, aromatic hydrocarbons, substituted arene, alkane, haloalkane or dimethyl formamide.
5. method as claimed in claim 3 is characterized in that described water-soluble solvent can be the mixed solution of the inorganic of pH3-6.5 or the organic salt aqueous solution or above-mentioned solution and water-miscible organic solvent.
6. method as claimed in claim 5 is characterized in that described inorganic or aqueous organopolysiloxane is phosphoric acid-sodium citrate buffer solution, acetic acid-sodium-acetate buffer or imidazoles-hydrochloride buffer.
7. the method for a kind of oxynitrile cracking enzyme catalysis aldehydes or ketones as claimed in claim 1 and cyanogen compound or cyanohydrin compound prepared in reaction chiral cyanohydrin compound is characterized in that described aldehydes or ketones has following molecular formula: R
1COR
2, cyanogen compound or cyanohydrin compound have molecular formula, wherein R
1=R
4Ph
-, R
4OPh
-, R
4OCH
2Ph
-,
PhCH=CH, R
5CH=CH,
R
5,
R
2=H, C
1-3Alkyl, R
3=H, (CH
3)
2COH or HCOH, R
4=H, C
1-4Alkyl, X=O or S, R
5=C
1-8Alkyl, R
6=H, C
1-4Alkyl,
Or CH
3OCH
2-.
8. the method for a kind of oxynitrile cracking enzyme catalysis aldehydes or ketones as claimed in claim 2 and cyanogen compound or cyanohydrin compound prepared in reaction chiral cyanohydrin compound is characterized in that described aldehydes or ketones and R
3The CN mol ratio is 1: 0.8-20, and thick oxynitrile cracking enzyme is 1 with the aldehyde weight ratio: 0.001-200, in the time of 0-45 ℃, react, 0.05-48 hour, contain the water of 0-10% in the reaction system.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99113884A CN1083009C (en) | 1999-07-16 | 1999-07-16 | Process for synthesizing optically active cyanohydrin compound by oxynitrile cracking and enzyme catalysis of new enzyme source |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1203820A1 (en) * | 2000-11-01 | 2002-05-08 | Clariant GmbH | Process for the preparation of optically active cyanhydrins and secondary products |
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|---|---|---|---|---|
| JP4746084B2 (en) * | 2008-12-18 | 2011-08-10 | 三菱レイヨン株式会社 | (R) -Hydroxynitrile lyase and method for using the same |
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|---|---|---|---|---|
| US5177242A (en) * | 1991-12-17 | 1993-01-05 | Fmc Corporation | Process for preparing optically active cyanohydrins with enzymes |
| CN1093531C (en) * | 1998-05-04 | 2002-10-30 | 中国科学院上海有机化学研究所 | Catalytic synthesis of optical active cyanhydrin compound by using cyanhydrin catenase |
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