Construction Of Photo-Enzymatic Cascade System For The Chiral Synthesis Of (R)-1-[3,5-Bis(Trifluoromethyl)Phenyl] Ethanol

In the process of cancer treatment,chemotherapy is a common choice,but nausea and vomiting are also a common side effect of chemotherapy.Aprepitant,as a NK-1 receptor antagonist,is also widely combined with other methods to prevent nausea and vomiting clinically.（R）-1-[3,5-Bis（trifluoromethyl）phenyl]ethanol is the key chiral intermediate for the synthesis of aprepitant.Now,more and more attentions have been given to the problems in synthesizing this chiral compound such as organic solvents pollution and sustainability.Even the biocatalysis with mild reaction conditions,high selectivity and strong sustainability,enzyme immobilization and the regeneration of expensive cofactor NADPH always restricted the development of the synthesis method.For example,the tradditional chemical linkage in immobilization often results in the destruction of enzyme active centers and a significant decrease in activity due to the unwanted and random chemical linkage.Regeneration of NADPH often use triethanolamine or glucose as the hydrogen donor,which increases the complexity and cost of the downstream process,and the sustainability needs to be further improved.First of all,in order to overcome the unwanted and random chemical linkage in traditional covalent immobilization with destruction of enzyme active centers,unnatural amino acids were incorporated into the peptides of enzyme protein,and the reconstructed enzyme was expressed and prepared with bio-orthogonal chemical reaction groups.To this end,alcohol dehydrogenase（ADH）,which has the ability to catalyze the reverse reaction,was used as the model enzyme,and 156Y,208Y and229Y were replaced with p-propargyloxy-L-phenylalanine（p-Pa F）and expressed in MG1655,respectively.Then,the obtained enzyme proteins of wild-type,single-point mutant such as ADH-156Y,ADH-208Q,ADH-229Y,and double-point mutant ADH-156Y-229Y were all tested for their catalytic properties.The expression level of the wild type is 20 mg·L^-1,and the enzyme activity is 0.185U·mg^-1,while the expression level of the single-point mutant type is 18 mg·L^-1,and the enzyme activity is 0.174 U·mg^-1.The expression level of the double-point mutant was 8 mg·L^-1,and the enzyme activity was 0.170 U·mg^-1.The results showed that the expression level and specific activity of the single-point insertion modification were similar to those of the wild-type.As the number of mutated sites gradually increased,compared with single-point mutation,the enzyme expression level would decrease while maintaining the activity.Then,rapidly and precisely bio-orthogonal cross-linked enzymes（CLEs）were prepared from cell lysate using bifunctional cross-linking agents under microwave radiation at ultra-low temperature.Depending on the incorporation of two unnatural amino acids（UAA）in the peptide of every enzyme protein molecule,cell lysate was used to prepare the CLEs under consective microwave radiation by clicking reaction using Cu I catalyst.The effect of the dosage of Cu I on the enzyme activity of ADH-156Y-229Y CLEs was first studied through a simple preliminary experiment.It was found that 0.4 equivalent of Cu I was used as a catalyst to ensure the catalytic activity and complete the cross-linking at the same time.In the bio-orthogonal clicking reaction,the intermolecular crosslinking of the above-mentioned ADH double-point mutants was achieved using bis-PEG3-azide by cycloaddition reaction.The obtained CLEs was characterized using scanning electron microscopy,confocal fluorescence microscopy,mass spectrometry and other detection methods.The precisely chemically crosslinkage in CLEs of the two-point mutation ADH was also confirmed using acidolysis and LC-MS.Finally,a photo-enzymatic cascade system was preliminarily constructed and applied in the coupled chiral catalytic synthesis of（R）-1-[3,5-bis（trifluoromethyl）]phenylethanol.Prior to this,three different Ti O₂ catalytic materials（60nm nano Ti O₂,100nm nano Ti O₂ and Ti O₂ nanotubes）were explored for the catalytic NADPH regeneration under simulated light.It was found that the yield of NADPH using Ti O₂ nanotubes was up to 59%and the concentration of the obtained NADPH was2.36 mg/m L.To confirm the source of hydrogen,the electron spin resonance（ESR）was used to detect the generated hydroxyl radicals and electrons,and a four-line spectrum with a relative intensity of 1:2:2:1 can be clearly observed.In addition,the ESR spectrum showed a stable signal with three peaks with an intensity of 1:1:1,and the signal intensity would decrease continuously,which confirmed the generation of hydrogen and electrons.This also provides strong evidence for the smooth progress of the subsequent photo-enzyme coupling system catalytic reaction.By combing photochemical regeneration of NADPH using H₂O as hydrogen donor,the reverse catalytic reduction by alcohol dehydrogenase CLEs was used to syntheisize the aprepitant intermediate（R）-1-[3,5-bis（trifluoromethyl）]phenylethanol.Detected by HPLC spectroscopy,it was observed that more and more（R）-1-[3,5-bis（trifluoromethyl）]phenylethanol was synthesized as reaction progress’eprogressed and the yield of target product reached 41%after 24 hours and ee value was higher than 99.99%.At the same time,in order to ensure that the Ti O₂nanotubes and ADH CLEs can be reused,we have also carried out 5 rounds of batch reuse.The reaction time for each rerecycle was 12 hours and all the yields reached about 30%,which showed that the catalytic stability of this type of enzyme aggregates and the feasibility of photocatalytic regeneration of NADPH with Ti O₂nanotubes using water as the hydrogen donor.In conclusion,in this work,unnatural amino acid p-propargyloxy-L-phenylalanine（p-Pa F）was inserted in the peptide chain of alcohol dehydrogenase to prepare precise cross-linked enzyme aggregates,which effectively avoided the unwated and random chemical cross-linking in traditional methods and protected the enzyme active center and catalytic activity.Additionally,using water as the hydrogen donor to regenerate NADPH under Ti O₂ nanotube photocatalysis,photo-enzymatic cascade system was constructed and applied in the coupled chiral reduction to prepare the chiral（R）-1-[3,5-bis（trifluoro）Methyl)]phenethyl alcohol.The system presented good reuse stability in this chiral alcohol synthesis depended on the bio-orthogonal cross-linked enzymes（CLEs）and efficient Ti O₂ nanotube catalyst.H₂O was used as hydrogen donor,which avoided the pollution and unsustainability resulted from other hydrogen donors such as triethanolamine（TEA）and ethylene diamine tetraacetic acid（EDTA）.Therefore,this photo-enzymatic coupled chiral reduction method,combining Ti O₂ nanotubes for photocatalytic regeneration of NADPH with precisely cross-linked enzyme aggregates for catalytic reduction,can be further extended to other enzyme-catalyzed reduction reactions,and is expected to promote the green and sustainable synthesis of fine chemicals and pharmaceutical intermediates.