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    • 专利摘要:
    The invention relates to a biocatalytic process for the preparation of p-vinylphenols, comprising a three-step one-pot reaction according to the following reaction scheme: wherein a) an optionally substituted phenol 1 by catalytic action of a tyrosine phenol lyase (TPL) and in the presence of ammonium ions with pyruvic acid (BTS b) is eliminated from the tyrosine 2 by catalytic action of a tyrosine ammonia lyase (TAL) or phenyl ammonia lyase (PAL) ammonia to give an optionally substituted p-coumaric acid 3, and c) the p-coumaric acid 3 is decarboxylated by catalytic action of a phenolic acid decarboxylase (PAD) to give the desired optionally substituted p-vinylphenol 4; d) whereby the resulting CO 2 is removed from the reaction system in order to shift the chemical equilibrium of all three reaction steps in the direction of the products.
    公开号:AT516155A4
    申请号:T130/2015
    申请日:2015-03-10
    公开日:2016-03-15
    发明作者:Wolfgang Dr Kroutil；Eduardo Dr Busto；Robert Dipl Ing Dr Fh Simon
    申请人:Universität Graz；
    IPC主号:
    专利说明:

    The invention relates to a biocatalytic process for the preparation of p-vinylphenols.
    STATE OF THE ART
    Vinylphenol derivatives serve as useful building blocks in polymer chemistry and can be used, for example, for the construction of dielectric layers in the production of chemical and biological sensors. Haiogenated derivatives are used for example in the production of flame retardants and chalcones, a known class of organic compounds with a wide range of biological activities.
    Seieective para-vinylation of unactivated phenols to para- or 4-vinylphenols is not known. Thus, direct vinylation of unactivated arenes using tin catalysts results in selective ortho-derivatization (Yamaguchi et al., J. Am. Chem. Soc., 117, 1151-1152 (1995)), while catalysis with Lewis acids, such as, for example, Lewis et al. GaCla, a mixture of ortho and para regioisomers (Yamaguchi et al., Angew Chem Chem Int 36, 1313-1315 (1997)). However, both strategies were not performed on phenol derivatives. Rather, p-vinylphenols were synthesized via Stille coupling using corresponding p-bromo-phenols and highly toxic vinyltin reagents (Littke et al., J. Am. Chem. Soc. 124, 6343-6348 (2002)). ,
    Alternatively, p-vinylphenols can be obtained from the corresponding 4-hydroxybenzaldehyde by Heck reaction using phosphonium salt catalysis (Chen et al., Tetrahedron 69, 653-657 (2013)) or by Knoevenagel condensation in the Doebner variant by catalysis with secondary amines under microwave radiation (Sinha et al., Tetrahedron 63, 960-965 (2007)).
    On the other hand, biocatalytic approaches are based on the decarboxylation of p-coumaric acids (4-hydroxycinnamic acids) using ionic liquids (Sharma et al., Adv. Synth. Catal. 350, 2910-2920 (2008)), phenolic acid decarboxylases (PADs) (PAD). Wuensch et al., Org. Lett. 14, 1974-1977 (2012)) or more particularly p-hydroxycinnamic acid or p-coumaric acid decarboxylases (pHCA-DC or pCA-DC or PDC for short), for example a PDC L. plantarum (Rodriguez et al., Proteins 78, 1662-1676 (2010) and Jung et al., Appl. Microbiol. Biotechnol. 97, 1501 (2013)), sometimes in a two-phase system (Ben-Bassat et al , Org. Process Res. Dev. 11, 278-285 (2007)):
    Usually, however, the corresponding cinnamic acid must be synthesized from the respective aldehyde, which is costly. Also, the bacterial production of p-vinylphenols from glucose using genetically manipulated bacteria co-expressing a fungal phenyl-ammonia lyase (PAL) and a bacterial p-hydroxycinnamic acid decarboxylase (PDC) by elimination of ammonia from tyrosine was reported. However, the productivities are low (0.4 g / l), the reaction is limited to this substrate, and the amounts of by-products such as phenylalanine (0.5 g / l) and cinnamic acid are significant (Qi et al., Metaboly Engin 9, 268-276 (2007)).
    Furthermore, the biocatalyzed synthesis of tyrosine derivatives starting from phenols is known:
    In Kroutil et al. Adv. Synth. Catal. 352, 731-736 (2010), a publication of the inventors' group, this reaction is used for a number of phenol derivatives, including the substituents F, CI, Br, CH3 and OCH3 at position 2 or 3 of the phenol, and using a tyrosine Phenol lyase (TPL) from Citrobacter freundii.
    Also known is the enzymatically catalyzed elimination of ammonia from tyrosine by means of tyrosine ammonia lyases (TAL) or phenyl ammonia lyases (PAL) to obtain cinnamic acid:
    See Kyndt et al., FEBS Letters 512, 240 (2002), and Louie et al., Chem. Biol. 13 (12), 1327-1338 (2006). For derivatives of tyrosine, however, this reaction has not previously been reported.
    Against this background, the object of the invention was the development of an improved biocatalyzed process for the preparation of p-vinylphenols, which enables a rapid and cost-effective production thereof.
    DISCLOSURE OF THE INVENTION
    This object is achieved by the invention by providing a biocatalytic process for the preparation of p-vinylphenols, comprising a three-step one-pot reaction according to the following reaction scheme:
    wherein a) in known manner an optionally substituted phenol 1 by catalytic action of a tyrosine phenol lyase (TPL) and in the presence of ammonium ions with pyruvic acid (BTS) to the optionally substituted tyrosine 2 is connected. b) eliminating ammonia from tyrosine 2 by catalytic action of a tyrosine ammonia lyase (TAL) or phenyl ammonia lyase (PAL) in a manner known per se, to give an optionally substituted p-coumaric acid 3, and c) in a manner known per se, the p-coumaric acid 3 is subjected to decarboxylation by catalytic action of a phenolic acid decarboxylase (PAD) to give the desired, optionally substituted p-vinylphenol 4; d) whereby the resulting CO2 is removed from the reaction system in order to shift the chemical equilibrium of all three reaction steps in the direction of the products.
    The invention is based on the combination of three per se known single reaction steps and the surprising findings that not only all three can be carried out in a one-pot reaction substantially simultaneously, without inhibiting each other to any significant extent, but that thereby consistently achieves over 90% achievable are - and also with substituted phenols as starting substrate. This was unexpected above all because of the fact that both the starting material and all products are formally phenols and thus the probability of a mutual inhibiting effect in the three individual reactions was high.
    By carrying out the three reaction steps as a one-pot reaction and shifting the chemical equilibrium of the overall reaction due to the elimination of the carbon dioxide released from the system during decarboxylation in step c), the inventors, after optimizing the reaction conditions, even succeeded in achieving overall conversions (calculated over all three stages) of consistently to achieve over 97%, as the later examples prove, which was by no means to be expected.
    The present invention thus provides a process which provides a selective, extremely rapid and almost complete conversion of phenols to p-vinyl
    Phenols in high purity allows, which was previously not possible by conventional organic-chemical way.
    The one-pot reaction according to the invention is carried out in preferred embodiments at a pH of about 8 to 9, more preferably at about pH 8, as the inventors have determined by varying the pH. Also in this aspect, the excellent overall sales are highly surprising, since the pH optimum of the first two reaction steps is more than 10, while the decarboxylation at acidic to neutral pH is significantly faster and more complete than in an alkaline medium and for the individual reaction from pH 9 hardly any turnover was observed, as the later examples prove. This result for the single reaction coincides with the literature cited at the beginning (Rodriguez et al., SO, Jung et al., Supra), where the biocatalyzed decarboxylation by means of PAD is carried out, for example, at pH 6.5 or 7.0, although Jung et al. (supra) revealed a pH optimum of the PAD they used at pH 5.8.
    However, carrying out the one-pot reaction according to the invention at pH 8 has also proved to be optimal for the reason that at this pH a non-toxic KPi buffer (potassium phosphate) can be used as the aqueous solvent, while at pH 9 more toxic reagents such as sulfonic acid salts ( eg CHES buffer: N-cyclohexyl-2-aminoethanesulfonic acid) would have to be used.
    In further preferred embodiments, a tyrosine ammonia lyase (TAL) is used as the catalyst in step b) since the inventors were hardly able to observe any conversions with the phenyl-ammonia-lyases (PAL) investigated. The tyrosine ammonia lyase (TAL) is preferably used in the form of whole cells containing the recombinant enzyme, as these show sufficiently high activity in the process according to the invention and complicated and sometimes lossy isolation of the enzyme from a cell culture can therefore be avoided ,
    The same applies to the ferulic acid decarboxylase (FAD) used as catalyst in step c) according to the present invention, which is thus also preferably used in the form of whole cells containing the recombinant enzyme.
    In further preferred embodiments, in addition to an aqueous buffer system with the appropriate pH, a water-immiscible co-solvent is used, preferably diethyl ether, more preferably 5% diethyl ether, based on the aqueous buffer system, to further increase the conversion of the overall reaction.
    The substitution pattern at the starting phenol 1 is not particularly limited as long as the para position is vacant and the substituent or substituents do not adversely affect the enzymatic reactions of steps a) to c). As shown in the later examples, the present invention works both with substituents that reduce the electron density in the aromatic ring, such as, for example, as shown in FIG. F, CI and Br, as well as with such that they increase, such. Aikyl or alkoxy. The size of the substituents should not exceed a certain level, in order not to hinder the coordination with the respective enzyme, in particular in step a), which is why hydrocarbon radicals having not more than 20 carbon atoms are to be preferred, more preferably hydrocarbon radicals having not more than 10 or not more than 6 carbon atoms. The substituent (s) at the ortho or meta position of the phenol 1 are particularly preferably selected from halogens and C 1-6 -alkyl and C 1-8 -alkoxy radicals, such as e.g. F, CI, Br or (0) CH3.
    BRIEF DESCRIPTION OF THE DRAWINGS
    The invention will be described in more detail below with reference to the attached drawings, which show in detail:
    1 shows the conversion in step a) at different pH;
    2 shows the conversion in step b) at different pH;
    3 shows the conversion in step c) at different pH;
    Fig. 4 shows the time course of the overall reaction at different pH;
    Fig. 5 shows the amounts of reactant and products at pH 7;
    FIG. 6 shows the quantities of starting material and products at pH 10; FIG.
    Figure 7 shows the conversion of the overall reaction with different co-solvents. and FIG. 8 shows the amounts of educt and products with optimum reaction.
    EXAMPLES
    There follow embodiments of the invention, which are given for illustrative purposes and are not to be understood as limiting the scope of protection.
    General procedure
    Chemical reagents were purchased from various commercial sources and used without further purification. Melting points were determined by means of samples in open capillaries and are uncorrected. ^ H and ^^ C NMR spectra were recorded using a Bruker spectrometer (^ H: 300.13 MHz, 75.5 MHz). The chemical shifts are given in ppm and the coupling constants in Hertz (Hz). The conversions of the aromatic substrates were determined by HPLC on a Shimadzu chromatograph using a Luna C18 column (25 cm x 4.6 mm O) with UV detector at different wavelengths, with turnover variations of about 2% within the systematic error limits lie. All results of all synthetic, reference, and working examples are averages of triplicates.
    Sample preparation: A MeCN / H 2 O solution (1 ml, 1: 1) containing 0.1% TFA was added to an aliquot of the reaction mixture (1 ml). The protein was removed by centrifugation and the solution was filtered through VIVAspin polyethersulfone membrane filter. The resulting solution was analyzed by HPLC under the conditions given below. The relative amounts of the compounds were calculated from the respective peak areas. Column: Luna-C18, 5 μητι; Flow rate: 1 ml / min; Temperature: 30 ° C; Gradient: from 100% H2O (0.1% TFA) to 100% MeCN (0.1% TFA) in 22 min. Wavelength: 280 nm.
    Synthesis Examples 1 to 3; Preparation of the enzyme preparations Synthesis Example 1
    Preparation of TPL as Cell-free Extract of Citrobacter freundii M379V E. coli - Clones containing the M379V TPL plasmid were propagated in LB medium obtained by sterilizing a solution (1 L) of the following components in 5 L 1-Erlenmeyer flask: tryptone (10 g / L), NaCl (5 g / L) and yeast extract (5 g / L). A preculture was prepared by inoculating 100 ml of LB medium containing ampicillin (100 mg / L). The preculture was shaken overnight at 120 rpm and 37 ° C. Thereafter, the bulbs containing ampicillin (100 mg / l) were inoculated with the preculture giving an initial ODe0 of 0.05. Subsequently, the cultures were shaken at 120 rpm and 30 ° C until an ODe0 of 0.4 to 0.6 was reached. Protein expression was induced with IPTG (0.5 mM, final concentration) and the cultures were shaken for 2 h at 20 ° C and 120 rpm. Finally, the cells were harvested by centrifugation (8000 rpm, 20 min), washed with potassium phosphate buffer (10 mM, pH 7), resuspended in KPi buffer (50 mM, 180 mM NH 4 Cl, 0.04 mM PLP, pH 8) and by means of ultrasound treatment (40% amplitude, 1 s pulse on, 2 s pulse off, 5 min) destroyed. The mixture was harvested by centrifugation (15,000 rpm, 15 minutes) and the supernatant was snap frozen in liquid nitrogen and lyophilized. The lyophilized cell-free extract was stored at 4 ° C and used unchanged in the reactions.
    Synthesis Example 2
    Preparation of TAL of Rhodobacter sphaeroides as Whole Cell Catalyst E. coli - Clones containing the TAL plasmid were propagated in LB medium obtained by sterilizing a solution (1 L) of the following components in 5 L alder - Meyer flask was prepared: tryptone (10 g / l), NaCl (5 g / l) and yeast extract (5 g / l). A preculture was prepared by inoculating 100 ml of LB medium containing kanamycin (50 mg / l). The preculture was shaken overnight at 120 rpm and 37 ° C. Thereafter, the flasks containing kanamycin (50 mg / L) were inoculated with the preculture, giving an initial ODeO of 0.05. Subsequently, the cultures were shaken at 120 rpm and 37 ° C. until an ODeO of 0.5 to 0.7 was reached Protein expression was induced with IPTG (0.5 mM, final concentration) and cultures were maintained for 24 hours Finally, the cells were harvested by centrifugation (8000 rpm, 20 min), washed with potassium phosphate buffer (10 mM, pH 8), snap frozen in liquid nitrogen and lyophilized The lyophilized cells became stored at 4 ° C and used unchanged in the reactions.
    Synthesis Example 3
    Preparation of the FAD of Enterobacter sp. E. coli - whole-cell catalyst E. coli - clones containing the FAD plasmid were propagated in LB medium prepared by sterilizing a solution (1 L) of the following components in five 1 L Erlenmeyer flasks were prepared: tryptone (10 g / L), NaCl (5 g / L) and yeast extract (5 g / L). A preculture was prepared by inoculating 100 ml of LB medium containing kanamycin (50 mg / l). The preculture was shaken overnight at 120 rpm and 37 ° C. Thereafter, the flasks containing kanamycin (50 mg / L) were inoculated with the preculture, giving an initial ODeO of 0.05. Subsequently, the cultures were shaken at 120 rpm and 37 ° C until an ODe0 of 0.5 to 0.7 was reached. Protein expression was induced with IPTG (0.5 mM, final concentration) and the cultures were shaken for 24 h at 20 ° C and 120 rpm. Finally, the cells were harvested by centrifugation (8000 rpm, 20 min), washed with potassium phosphate buffer (10 mM, pH 8), snap frozen in liquid nitrogen and lyophilized. The lyophilized cells were stored at 4 ° C and used unchanged in the reactions.
    Reference Examples 1 to 3: activity measurements
    Reference Example 1
    Activity of the TPL
    For a better comparison of the enzyme activity, the activity of TPL in the C-C coupling between phenol 1, pyruvic acid and pyruvate and ammonia was determined according to step a) by measuring the initial reaction rate by HPLC. An activity unit was defined as the amount of catalyst that catalyzed the formation of 1 pmol of tyrosine per minute under the following conditions: 30 ° C, KPi buffer 50 mM, pH 8, 850 rpm. The test mixture contained 2-chlorophenol (1b, 23mM), pyruvate (46mM), NH4Cl (180mM) and the freeze-dried cell-free extract of Synthesis Example 1 containing the overexpressed TPL (2mg). The reactions were started by the addition of the enzyme and the conversion was determined between 1 and 10 min. All measurements were performed in at least three runs. The observed reaction activities were 0.30 U per mg of cell-free extract.
    Reference Example 2
    Activity of the TAL
    For a better comparison of the enzyme activity, the activity of TAL in the reaction of tyrosine 2 to cinnamic acid derivative 3 was determined according to step b) by measuring the initial reaction rate by HPLC. One unit of activity was defined as that amount of catalyst which catalyzed the formation of 1 pmol of cinnamic acid 3 per minute under the following conditions: 30 ° C, KPi buffer 50 mM, pH 8, 850 rpm. The test mixture contained L-3-chlorotyrosine (2b, 5 mM), NH4Cl (180 mM) and E. coli total cells from Synthesis Example 2 overexpressing TAL (5 mg). The reactions were started by the addition of the enzyme and the conversion was determined between 5 and 20 minutes. All measurements were performed in at least three runs. The observed reaction activities were 9.2 mU per mg E. coli total cells.
    Reference Example 3
    Activity of the FAD
    For a better comparison of the enzyme activity, the activity of FAD in the decarboxylation of cinnamic acid derivative 3 to vinylphenol 4 in step c) was determined by measuring the initial reaction rate by HPLC. One unit of activity was defined as the amount of catalyst that catalyzed the formation of 1 μΜοΙ vinylphenol 4 per minute under the following conditions: 30 ° C, KPi buffer 50 mM, pH 8, 850 rpm. The test mixture contained 3-chloro-4-hydroxycinnamic acid (3-chloro-coumaric acid, 3b, 5 mM), NH4Cl (180 mM) and £ -co // whole cells from Synthesis Example 3 over-expressing the FAD (0.1 mg). , The reactions were started by the addition of the enzyme and the conversion was determined between 1 and 5 min. All measurements were performed in at least three runs. The observed reaction activities were 2.1 U per mg E. coli total cells.
    Reference Examples 4 to 6: Determination of pH Optimum
    Reference Example 4
    pH optimum of TPL
    The TPL-catalyzed enzymatic reaction of Reference Example 1 between 2-chloro-phenol 1b (23 mM), pyruvate (46 mM) and NH / (180 mM) using the freeze-dried cell-free extract from Synthesis Example 1 was supplemented with 0.4 mM Pyridoxalphosphat (PLP) as coenzyme with variation of the pH repeated and allowed to drain for 22 hours. Fig. 1 shows the results for pH values of 6 to 10, which show that the best conversions to L-3-Chlortyrosine 2b are achieved at a strongly alkaline pH, since in this range uncharged ammonia can take part in the reaction. Interestingly, the enzyme remains stable even in the strongly alkaline pH range, especially since even at pH 10 the best conversion has been achieved, and therefore the optimum will probably be higher.
    Reference Example 5
    pH optimum of TAL
    The TAL-catalyzed enzymatic reaction of Reference Example 2 between 3-chloro-tyrosine 2b (10 mM) and NH / (180 mM) using the whole cell preparation of Synthetic Example 2 was also supplemented with 0.4 mM pyridoxal phosphate (PLP) as coenzyme and Variation of pH repeated and allowed to drain for 6 hours. FIG. 2 again shows the results for pH values of 6 to 10, which again show that the strongly alkaline pH range above pH 10 is to be preferred. In this case, the optimum of the reaction will certainly be above pH 10, as suggested by extrapolation of a mental balance curve through the measurement points.
    Reference Example 6
    pH optimum of the FAD
    The FAD-catalyzed enzymatic reaction of Reference Example 3, i. the decarboxylation of 3-chloro-coumaric acid 3b (10 mM) using the whole cell preparation of Synthesis Example 3 to 4-vinylphenol 4b was again repeated with the addition of 0.4 mM pyridoxal phosphate (PLP) as a coenzyme and pH variation and allowed to run for 1 hour. FIG. 3 also shows the results for pH values of 6 to 10, from which it is to be stated that conversions comparable to those in the neutral range can be achieved up to pH 8, while at pH 9 scarcely any conversion occurred and practically at pH 10 no sales can be recognized. In this case, the optimum reaction will certainly be in the acidic pH range, as suggested by an extrapolation of the mental equilibrium curve and would also be expected for decarboxylation reactions for the skilled person.
    Examples 1 to 4
    One-pot reaction of steps a) to c) at varying pH
    Using the three enzymes of Synthesis Examples 1 to 3, the three-step one-pot reaction was carried out with 2-fluorophenol 1a (R = F) under the following conditions; 1a (23 mM); BTS (pyruvate) (46 mM); TPL (4 mg); TAL (20 mg); FAD (5 mg); aqueous buffer (50 mM): KPi for pH 7-8, CHES for pH 9-10; Time: 6 h; 20 ° C; Shake at 850 rpm. The pH was varied between 7 and 10 and samples were taken at time intervals and analyzed by HPLC for the content of 2-fluoro-4-vinylphenol 4a (R = F).
    Fig. 4 shows the results of these reactions. It was found that the conversions at pH 7 (Example 1, approximately 30%) or pH 10 (Example 4, approximately 45%) were markedly lower than those at pH 8 (Example 2, approximately 85%) and pH 9 80%), which in turn gave almost identical turnovers, due on the one hand to the low activity of two of the enzymes, namely TPL and TAL, at pH 7 (see Figures 1 and 2) and on the other hand to the almost Inefficiency of FAD at pH 10 (see Fig. 3) can be attributed. In view of the latter, it was surprising that at pH 10, significantly better conversions can still be achieved than at pH 7.
    In order to investigate this phenomenon, Examples 1 and 4 were repeated at pH 7 and pH 10, respectively, and the amounts of starting material 1a and of products 2a to 4a were determined by means of HPLC at time intervals.
    Figures 5 and 6 show the results of these experiments for phenol 1a (a), tyrosine 2a (◆), cinnamic acid 3a () and the desired vinylphenol 4a (x). This confirmed the above assumptions, since it was shown that at pH 7, the amount of starting material 1a decreases only very slowly and even after 6 h more than 40% of the starting phenol are present, while the total time is virtually no cinnamic acid in the reaction mixture. Thus, at pH 7, step a) is the rate-limiting partial reaction, while the decarboxylation of step c) proceeds most rapidly. At pH 10, on the other hand, the C-C coupling of step a) takes place much more rapidly, so that after 6 hours the starting material 1a had decreased to 10% of the original amount. At the same time cinnamic acid 3a accumulates steadily in the reaction mixture, which means that at pH 10, the decarboxylation is the slowest and thus the rate-limiting partial reaction.
    Due to the need to use for the adjustment of pH 9 CHES or other, much more toxic aqueous buffer than the KPi buffer for pH 8, was - despite about the same sales - of the inventors pH 8 as optimum pH for carrying out the inventive Considered method.
    Examples 5 to 8
    One-pot reaction at pH 8 with cosolvent
    The above Example 2 at pH 8 was therefore repeated, mixing varying amounts of cosolvents and determining the amounts of the final product, p-vinylphenol 4a. In Example 5, for comparative purposes, Example 2 was repeated without co-solvent, while in Example 6 DMSO as a representative of a water-miscible solvent (5 vol .-%), in Example 7 diethyl ether as a representative of a water-immiscible solvent (5 vol .-% ) and in Example 8 twice the amount of diethyl ether (10 vol .-%) were added, each based on the volume of the aqueous buffer solution.
    Fig. 7 shows the results of these experiments. It can be seen that the presence of water-miscible DMSO for conversion to product 4a is virtually nonexistent
    Difference made while water immiscible diethyl ether after 6 h reaction time in an amount of 10 vol .-%, an increase in sales of more than 10 percentage points, in an amount of 5 vol .-% but even exceeded 20 percentage points.
    Without wishing to be bound by any particular theory, the inventors believe that the increase in sales is due to the formation of two phases, with certain proportions of the four different phenols - due to their lower polarity and hydrophilicity - predominantly being proportions of the starting material 1a and the final product 4a - Are dissolved in the ether phase. This suppresses any inhibition of the individual reactions, presumably steps b) and c), and / or shifts the chemical equilibrium of the overall reaction further to the right. A factor in suppressing inhibiting effects is that with double the amount of ether, it is not possible to achieve such a high increase in conversion, because then too large amounts of the phenols - possibly of the educt - are dissolved in the organic phase, which slows down the reaction rate.
    Example 9
    One-pot reaction under optimal conditions
    After thus optimizing pH and cosolvent, the experiment of Example 7 was repeated under the following conditions: 1a (23 mM); Pyruvate (46mM): TPL (4mg); TAL (20 mg); FAD (5 mg); KPi buffer (50 mM), pH 8; 5 vol.% Et 2 O, time: 6 h; 30 ° C; Shaking at 850 rpm; and determines the amounts of the starting material 1a and the products 2a to 4a at intervals over time by means of HPLC.
    Fig. 8 shows the result of this experiment, in particular the extraordinarily rapid decrease in the amount of starting material 1a during the first minutes and the 6-hour conversion to final product 4a of more than 95%.
    Examples 10 to 20
    Production of various derivatives
    Using, or slightly varying, these optimum reaction conditions of Example 9, the substitution pattern (and sometimes also the amount of starting substrate, phenol 1) was varied as shown in Table 1 below and the conversions, i. the relative amounts after completion of the reaction were determined by HPLC after every 24 hours and 48 hours, respectively, and are also shown in the table.
    Table 1
    Note a: Double the amount of TAL Note b: Double the reaction time (48 h)
    It was found that regardless of the type (electron withdrawing or -schiebend) and the number (one or two) of the substituents R always conversions to the respective product 4 of over 97% were achieved and the starting materials of the three partial reactions each less than 1% were included.
    The person skilled in the art may assume that the reaction will also proceed in a similar manner with other substitution patterns at the starting phenol 1, as long as the substituents do not significantly hinder the enzymatic reactions.
    Work-up:
    The reactions were quenched by the addition of saturated aqueous NH 4 Cl solution and extracted with ethyl acetate (3x15 ml). The combined organic phases were dried over Na 2 SO 4 and evaporated under reduced pressure. The residue was purified by flash chromatography (20% EtOAc / hexane) and the fractions containing each vinylphenol were combined and evaporated at 100 mmHg to give the desired p-vinyl phenols throughout as colorless oils in yields of 80-90% d. Th. Revealed. The spectroscopic data of the thus prepared p-vinylphenols are shown below. 2-Fluoro-4-vinylphenol 4a: 1 H-NMR (300 MHz, MeOD): 5h [pm] = 5.10 (d, = 10.9 Hz. 1H, 2'-Hc), 5.59 ( dd, t, 2t = 0.8 Hz, = 17.6 Hz, 1H, 2'-H,), 6.59 (dd, ®Jr, 2c = 10.9 Hz,%, 2t = 17, 6 Hz, 1 H, I-H), 6.85 (dd, = 8.8 Hz, = 8.5 Hz, 1 H. 6-H), 7.02 (dd, = 2.3 Hz, = 8 , 4 Hz, 1 H, 5-H), 7.14 (dd,% = 2.1 Hz. ® J3, f = 12.4 Hz, 1 H, 3-H). '^ -NMR (75 MHz, MeOD): öc [pm] = 112.3 (C-2'), 114.1 (d, = 18.9 Hz, C-3), 118.5 (d, = 3.1 Hz. C-6), 123.7 (d, = 3.1 Hz, C-5), 131.6 (d, = 6.2 Hz, C-4), 136.9 (C). 1 '), 145.9 (d, = 13.3 Hz. C-1), 152 (d, = 240.1 Hz, C-2). (282 MHz, MeOD) 5p [pm] = -139.7 (dd, = 9.1
    Hz, = 12.5 Hz). 2-Chloro-4-vinylphenol 4b: 1 H-NMR (300 MHz, MeOD): 5 "[pm] = 5.06 (dd. = 0.7 Hz.% · Ο, ν = 10.9 Hz. 1 H, 2'-Hc), 5.56 (dd,% χ2 · ο = 0.8 Hz, = 17.6 Hz, 1H, 2'-H,), 6.55 (dd, ^ Jv, tc = 10) , 9 Hz, = 17.6 Hz, 1 H. 1'-H), 6.81 (d, = 8.4 Hz, 1 H. 6-H), 7.15 (dd,% 3 = 2.2 Hz ,% e = 8.4 Hz. 1 H, 5-H), 7.32 (d, = 2.2
    Hz. 1 H, 3-H). ^ C NMR (75 MHz, MeOD): 5c [pm] = 112.4 (C-2 '), 117.5 (C-6), 121.8 (C-2). 126.8 (C-3), 128.6 (C-5), 132.0 (C-4), 136.7 (C-1 '), 154 (C-1). 2-Bromo-4-vinylphenol 4c: 1 H-NMR (300 MHz, MeOD): δ [pm] = 5.07 (dd, = 0.6 Hz,% c, v = 11.0 Hz, 1 H. 2'- Hc), 5.57 (dd, = 0.6 Hz,% xr = 17.6 Hz. 1 H, 2'-H,), 6.55 (dd, Jr, 2c = 10.9 Hz.%, 2 't = 17.6 Hz, 1 H, I-H), 6.84 (d, = 8.4 Hz, 1 H, 6-H), 7.21 (dd = 2.1 Hz, = 8.4 Hz, 1 H, 5-H), 7.51 (d, = 2.1
    Hz, 1H, 3-H). ^ C NMR (75 MHz, MeOD): öc [pm] = 110.9 (C-2), 112.4 (C-2 '). 117.1 (C-6), 127.4 (C-5), 131.7 (C-3), 132.3 (C-4), 136.4 (C-1 '), 154.9 ( C-1). 2-Methyl-4-vinylphenol 4d: 'H NMR (300 MHz, MeOD): δ »[pm] = 2.17 (s, 3 H. CH3). 4.99 (dd, = 1.1 Hz,% cr = 10.9 Hz, 1 H, 2'-Hc), 5.53 (dd,% t, 7c = 1.1 Hz, = 17.7 Hz , 1 H, 2-H,), 6.58 (dd, = 10.9 Hz, = 17.6 Hz. 1 H.rH), 6.68 (d, = 8.2 Hz, 1 H, 6 -H), 7.06 (dd '' J5.3 = 2.2 Hz, e = 8.2 Hz, 1H, 5-H), 7.13 (i.e.,% = 2.1 Hz, 1H, 3-H). ^^ NMR (75 MHz, MeOD); 5c [pm] = 16.2 (CH3 at C-2), 110.4 (C-2 '), 115.5 (C-6), 125.5 (C-2), 125.8 (C-5) , 129.6 (C-3), 130.6 (C-4), 138.1 (C-1 '), 156.5 (C-1). 3-fluoro-4-vinylphenol 4e: 'H-NMR (300 MHz, MeOD): 5h [pm] = 5.16 (dd, = 1.4 Hz, ^ J2'c.r = 11. 3 Hz, 1 H, 2'-Hc), 5.64 (dd.% X 2'c = 1.3 Hz. = 17.8 Hz. 1 H. 2'-H,), 6.47 (dd, = 2.4 Hz, = 12.5 Hz, 1 H. 2-H). 6.57 (dd, 2 = 2.5 Hz, 5 = 8.6 Hz, 1H, 6-H), 6.74 (dd, ®Jr.2c = 11.3 Hz, ^ Jr, 2t = 17, 8 Hz, 1 H, 1'-H), 7.35 (dd,% 6 = 8.7 Hz. = 8.8 Hz. 1 H, 5-H). ^ C NMR (75 MHz, MeOD): 5c [pm] = 103.5 (d, = 25.0 Hz, C-2), 112.6 (i.e., 2.7 Hz, C-2 '). ), 113.4 (d,%, F = 4.8 Hz, C-6), 117.8 (i.e., 12.6 Hz, C-4), 128.8 (d, = 5.8 Hz, C -1 '), 130.3 (d,% F = 3.5 Hz, C-5), 159.9 (d, = 11.9 Hz, C-1), 162.3 (i.e. F = 247.4 Hz. C-3); (282 MHz,
    MeOD); 5f [pm] = -119.8 (dd "jp.s = 8.8 Hz,%, 2 = 12.6 Hz). 3-chloro-4-vinylphenol 4f: 1 H-NMR (300 MHz, MeOD): 5h [pm] = 5.18 (dd, Jzc.2t = 1.2 Hz, ^ · o, ι = 11, 0 Hz, 1 H. 2'-Hc), 5.60 (dd, "J2i, 2'c = 1.2 Hz, Xr = 17.5 Hz, 1 H, 2'-Ht), 6.71 (dd, % 2 = 2.5 Hz, ^ .5 = 8.6 Hz. 1H, 6-H), 6.79 (d, = 2.5 Hz, 1H, 2-H), 6.98 (dd ,% _2 · ο = 11.0 Hz, Vr.21 = 17.6 Hz. 1 H. I'-H), 7.47 (d, = 8.6
    Hz, 1H, 5-H). ^^ NMR (75 MHz, MeOD): 5c [pm] = 113.8 (C-2 '), 115.7 (C-6), 116.8 (C-2), 128.1 (C) 4), 128.3 (C-5), 133.7 (C-1), 134.4 (C-3), 159.2 (C-1). 2,3-Difluoro-4-vinylphenol 4g: 1 H-NMR (300 MHz, MeOD); 5h [pm] = 5.25 (dd, ^^ 2,: 21 = 1.1 Hz, = 11.4 Hz, 1 H, 2'-Hc), 5.70 (dd,% x2c = 1.1 Hz ^ J2xr = 17.8 Hz, 1 H. 2'-H,). 6.68 (ddd,% 3ρ = 2.0 Hz.% 2f = 8.0 Hz. = 8.5 Hz, 1H, 6-H), 6.72 (dd,%, 2-c = 11, 3 Hz, Vv, 2i = 17.8 Hz, 1 H. I'-H), 7.11 (ddd 5 ^ 2f = 2.3 Hz, = 8.3 Hz.% Β = 8.5 Hz, 1 H, 5-H ). '^ -NMR (75 MHz, MeOD); 5c [pm] = 113.7 (dd,% 2f = 3.2 Hz,% 3f = 3.2 Hz, C-6), 115.1 (d, = 5.1 Hz, C-2 ·), 119.2 (dd,% ^ f = 1.1 Hz, = 9.7 Hz, C-4), 122.2 (dd, ^ .3f = 4.5 Hz,% 2f = 4.5 Hz, C-5 ), 129.6 (dd, '* Jr.2F = 3.1 Hz, ®Jv, 3f = 3.3 Hz, C-1'), 141.8 (dd, ^ Jz, 3f = 14.5 Hz, ^ J2. 2f = 241.2 Hz, C-2), 147.5 (dd, = 2.8 Hz, = 10.2 Hz, C-1), 150.7 (dd,% zf = 10.9 Hz, ^ 3.3f = 248.4 Hz, C-3); 'F (282 MHz, MeOD): 5f [pm] = -146.1 (ddd, ®J3f, 6 = 2.0 Hz,' 'J3f, 5 = 8.1 Hz, ^ 3f, 2f = 18.4 Hz, 1 F, F at C-3), -165.1 (ddd,% fs = 2.4 H, = 8.0 Hz,% f_3f = 18.3 Hz, 1 F, F at C-2). 3-Chloro-2-fluoro-4-vinylphenol 4h: 'H NMR (300 MHz, MeOD): δh [pm] = 5.27 (d, J2c, r = 11.1 Hz, 1 H, 2' -Hc), 5.68 (dd, = 0.9Hz, = 17.5Hz, 1H, 2'-H,), 6.85 (dd, = 8.6Hz, = 8.6Hz, 1H , 6-H), 6.96 (dd, ®Jr, 2c = 11.0 Hz, ®Jr, 2t = 17.5 Hz, 1 H, I'-H), 7.29 (dd, = 2.0 Hz , = 8.7 Hz, 1H, 5-H). '^ -NMR (75 MHz, MeOD); 5c [pm] = 115.4 (C-2 '), 117.1 (d, = 3.1 Hz, C-6), 121.5 (d,% f = 15.1 Hz, C-3) , 122.2 (d,%, f = 4.0 Hz, C-5), 129.2 (d,% f = 1.3 Hz, C-4), 132.9 (d, "Ji-. f = 3.1 Hz, ΟΙ '), 146.8 (d,% F = 13.3 Hz, C-1), 148.9 (d, = 241.2 Hz, C-2), (282 MHz , MeOD): 5f [pm] = - 139.6 (dd,% 6 = 8.8 Hz, = 2.0 Hz).
    The present invention thus provides a one-pot process for the preparation of p-vinylphenols, according to which a variety of compounds can be prepared efficiently and inexpensively, which was not even nearly possible in the prior art.
    权利要求:
    Claims (10)
    [1]
    A biocatalytic process for the preparation of p-vinylphenols, comprising a three-step one-pot reaction according to the following reaction scheme:

    wherein a) in known manner, an optionally substituted phenol 1 is linked by catalytic action of a tyrosine phenol lyase (TPL) and in the presence of ammonium ions with pyruvic acid (BTS) to optionally substituted tyrosine 2, b) in known manner from the tyrosine 2 by catalytic action of a tyrosine ammonia lyase (TAL) or phenyl ammonia lyase (PAL) ammonia is eliminated to give an optionally substituted p-coumaric acid 3, and c) in a known per se, the p -Cumaric acid 3 is subjected to a decarboxylation by catalytic action of a phenolic acid decarboxylase (PAD) to give the desired, optionally substituted p-vinylphenol 4; d) whereby the resulting CO2 is removed from the reaction system in order to shift the chemical equilibrium of all three reaction steps in the direction of the products,
    [2]
    2. The method according to claim 1, characterized in that the reaction is carried out at a pH of about 8 to 9.
    [3]
    3. The method according to claim 2, characterized in that the reaction is carried out at a pH of about 8.
    [4]
    4. The method according to any one of claims 1 to 3, characterized in that in step b) a tyrosine ammonia lyase (TAL) is used as a catalyst.
    [5]
    5. The method according to claim 4, characterized in that the tyrosine ammonia lyase (TAL) in the form of whole cells containing the recombinant enzyme is used.
    [6]
    6. The method according to any one of claims 1 to 5, characterized in that in step c) a ferulic acid decarboxylase (FAD) is used as a catalyst.
    [7]
    7. The method according to claim 6, characterized in that the ferulic acid decarboxylase (FAD) in the form of whole cells containing the recombinant enzyme is used.
    [8]
    8. The method according to any one of claims 1 to 7, characterized in that the one-pot reaction is carried out in an aqueous buffer system in the presence of a water-immiscible co-solvent.
    [9]
    9. The method according to claim 8, characterized in that diethyl ether is used as cosolvent, preferably in an amount of 5 vol .-%, based on the aqueous buffer system.
    [10]
    10. The method according to any one of the preceding claims, characterized in that the phenol 1 is substituted in the ortho and / or meta position with one or more substituents R, preferably selected from halogens, Ci ^ alkyl and Ci-6-alkoxy are.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2003099233A2|2002-05-23|2003-12-04|E.I. Du Pont De Nemours And Company|Microbial conversion of glucose to para-hydroxystyrene|
    WO2004092344A2|2003-04-14|2004-10-28|E. I. Du Pont De Nemours And Company|A method for preparing para-hydroxystyrene by biocatalytic decarboxylation of para-hydroxycinnamic acid in a biphasic reaction medium|
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    KR20140087799A|2012-12-31|2014-07-09|삼성전자주식회사|Process of biologically producing a styrene and derivatives thereof from a aromatic carboxylic aicd|
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    JP4420719B2|2004-04-01|2010-02-24|三井化学株式会社|Method for producing L-tyrosine|CN108949840B|2018-04-19|2021-10-19|江南大学|Engineering bacterium and application thereof in production of p-hydroxycinnamic acid|
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    ATA130/2015A|AT516155B1|2015-03-10|2015-03-10|Biocatalytic process for the preparation of p-vinylphenols|ATA130/2015A| AT516155B1|2015-03-10|2015-03-10|Biocatalytic process for the preparation of p-vinylphenols|
    CN201680021349.0A| CN107532184A|2015-03-10|2016-03-03|A kind of method for preparing 4-Vinyl phenol|
    EP16718991.9A| EP3271465B1|2015-03-10|2016-03-03|Method for producing p-vinylphenols|
    JP2017547103A| JP2018507696A|2015-03-10|2016-03-03|Preparation method of P-vinylphenol|
    PCT/AT2016/050051| WO2016141397A1|2015-03-10|2016-03-03|Method for producing p -vinylphenols|
    US15/556,742| US20180057845A1|2015-03-10|2016-03-03|METHOD FOR PREPARING p-VINYL PHENOLS|
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