cz United States Patent Smallridge et al. (10) Patent No.: (45) Date of Patent: US 6,271,008 BL Aug. 7, 2001 (5) YEAST-BASED PROCESS FOR PRODUCTION OF L-PAC (75). Ieventors: Andrew John Smallridge; Maurice ‘Arthur Trewhella; Margaret Mary Del Guidiee, all of Fooseray (AU) (73). Assignees: Vietoria University of Technology: Polychip Pharmaceuticals PTY LTD, both of Victoria (AU) (©) Notice: Subject to any disclaimer, the term ofthis patent is extended or adjusted under 35 USC. 154) by 0 das. (21) Appl. Nos 097701,704 (2) PCT Filed: Jun. 3, 1999 (86) PCTNo: — PCT/AU99/00433 §371 Date: Feb,2, 2001 §§102(¢) Date: Feb.2, 2001 (87) PCT Pub. No: WO99/63103 PCT Pub. Date: Dee. 9, 1999 (30) Foruign Application Priority Data Jan. 3,198 (AU) Pe 3682 (1) Ime (2) US.CL ©12P 726, C12P 728 BS/LAK; 435/150, 435/156; 4359942, 435/148, 150, 435/156, 942 (58) Field of Search (66) References Cited US. PATENT DOCUMENTS. SA7SAI3 121992. Coughlin FOREIGN PATENT DOCUMENTS 5004630 5/1990 (WO) OTHER PUBLICATIONS. P. Nikolovs et a ‘o-phase systems effect of solvent on product formation and cell structure", Journal of Industrial Microbiilogy, 210-169-177 (1992 ‘Whole cell yeast biotransformations in M. Takemoto etal, “Synthesis of optically active aphe- aylpyridylmethanols with Baker's Yeast", Chom. Pharm Bull, 42:802-805 (1994), Primary ExaminerHetbert 3 Lilling (74) Anorney, Agen, or Firm—Fulbright & Jaworski, LLP 6 ABSTRAC This invention relates to yeastmediated eaalysisin organic solvens, and in particular the yeast-mediated eondensaton between pyruvate and a substituted aromatic aldchyde 10 yield the corresponding. acyloin (hydroxy ketone) com- Pound, The invention provides « method of synthesis of a substituted earinol compound, comprising the sep of sub- jcting the coresponding substituted aromatic aldebyde to seyloin condensation mediated by a yeast ia an organic solvent under non-fermenting conditions. Preferably the yeast is Saccharomyces cerevisiae. Tn & preferred ‘embodiment, the reaction is that hetween pyruvate and benzaldehyde to yield phenylacetylearinol, the precursor to ephedrine, to yield a product of high enantiomeric purty. 30 Claims, No Drawingscz United States Patent Smallridge et al. (10) Patent No.: (45) Date of Patent: US 6,271,008 BL Aug. 7, 2001 (5) YEAST-BASED PROCESS FOR PRODUCTION OF L-PAC (75). Ieventors: Andrew John Smallridge; Maurice ‘Arthur Trewhella; Margaret Mary Del Guidiee, all of Fooseray (AU) (73). Assignees: Vietoria University of Technology: Polychip Pharmaceuticals PTY LTD, both of Victoria (AU) (©) Notice: Subject to any disclaimer, the term ofthis patent is extended or adjusted under 35 USC. 154) by 0 das. (21) Appl. Nos 097701,704 (2) PCT Filed: Jun. 3, 1999 (86) PCTNo: — PCT/AU99/00433 §371 Date: Feb,2, 2001 §§102(¢) Date: Feb.2, 2001 (87) PCT Pub. No: WO99/63103 PCT Pub. Date: Dee. 9, 1999 (30) Foruign Application Priority Data Jan. 3,198 (AU) Pe 3682 (1) Ime (2) US.CL ©12P 726, C12P 728 BS/LAK; 435/150, 435/156; 4359942, 435/148, 150, 435/156, 942 (58) Field of Search (66) References Cited US. PATENT DOCUMENTS. SA7SAI3 121992. Coughlin FOREIGN PATENT DOCUMENTS 5004630 5/1990 (WO) OTHER PUBLICATIONS. P. Nikolovs et a ‘o-phase systems effect of solvent on product formation and cell structure", Journal of Industrial Microbiilogy, 210-169-177 (1992 ‘Whole cell yeast biotransformations in M. Takemoto etal, “Synthesis of optically active aphe- aylpyridylmethanols with Baker's Yeast", Chom. Pharm Bull, 42:802-805 (1994), Primary ExaminerHetbert 3 Lilling (74) Anorney, Agen, or Firm—Fulbright & Jaworski, LLP 6 ABSTRAC This invention relates to yeastmediated eaalysisin organic solvens, and in particular the yeast-mediated eondensaton between pyruvate and a substituted aromatic aldchyde 10 yield the corresponding. acyloin (hydroxy ketone) com- Pound, The invention provides « method of synthesis of a substituted earinol compound, comprising the sep of sub- jcting the coresponding substituted aromatic aldebyde to seyloin condensation mediated by a yeast ia an organic solvent under non-fermenting conditions. Preferably the yeast is Saccharomyces cerevisiae. Tn & preferred ‘embodiment, the reaction is that hetween pyruvate and benzaldehyde to yield phenylacetylearinol, the precursor to ephedrine, to yield a product of high enantiomeric purty. 30 Claims, No DrawingsUS 6,271,008 BI 1 YEAST-BASED PROCESS FOR PRODUCTION OF L-PAC ‘This invention relates to organie compounds useful 35, precursors Tor the Syathesis of a-variety of produc, par ticulaey tor synthesis of compounds useful as E pharma ‘ceutical agents, The method ofthe invention wlises yeast- mediated catalysis inorganic solvents, and in panicular the yeast-medisied condensation hebwoen pyruvate and a sib- Suted aromatic aldehyde to yield the corresponding acy- Toin (hydroxy Ketone) compound. In preferred ‘embodiment, the reaction is that between pyruvate and benzaldehyde o yield phenylacetylearhino, the precursor to ‘ephedrine, in high enantiomerie purity BACKGROUND OF THE INVENTION Physicochemical method for production of enantiomer cally pure compounds usally involve multi-sep syatbesis ‘orporating one ot more steps which ate asymmetric, and laborious purification procedures. Such methods are not ‘only tedious, but frequenly provide relatively poor yields Alteratively enantiomerically-pure saring materials can be used, together with enantioselective reaction steps; however, such pure starting materials ae available only for Avery limited number of desired compounds Tn an atempt to overcome the diliculies of using tad tional organic chemical methods, biological systems have heen intensively investigated. Such sysems show a very high degre of steeascletvity in thie reactions and there fore microbiological, enzymatic or chemoenaymatic rea tions for achieving spociic rection steps with «varity of reagents have beet attempted. For example, microorganisms. ‘ofa number of genera have Been proposed for synthesis of optically active a-substituted derivatives of Shydoxypropionic acid for use as intermediates in the synthesis of compounds such as c-tocopherol, muscanes and pharmaceutical, insecticidal and agricultural chemical agents (US, Pat, No. 4,734.67 by Hoflman-La Roche, Inc). Most such procedures use whole-cell fermentation sysiems in agueots media, or isolated enzymes wilh & Specie desired. activity: However, fermentation systems present the disudvantage that puriication of the desired Product can be dificult, ad yieks tend to be low; while the Yield snd convenience of the resetion ean be improved by Utilising immobilised cells, or cells which have been selected or genetically modified, this adds significantly t0 the cost of the process, The se of purified enzymes is normally peohiitively expensive, and again without the use ‘of immobilised enzyme the yield tends to be low and purification difficult In recent years intense efforts have been directed rowards development of mets which ae highly selective, provide 2 good rate of transformation, and enable exsy, non ‘chromatographic separation and purification of the product, would be particularly desirable if reactions could be ‘carried out in organi solvents, since these are particularly ‘convenient for large scale reactions and purifications. Tihs been shown that dry baker's yeas is able to effect non-fermentatve reduction of a-Keto esters inorganic sol ‘ens sch as hexane or benzene, o produce the eotrespand= ing. ahydroxy esters with good yiekd and. selectivity (Nakamura et sl, 1988; Nakamura tal, 1990; Nakamura et al, 199]; Nakamira et al, 1993); reduction of P-keto esters in petroleum ether, ciety! ether, toluene, carbon tetrachlo- ride and petol has also heen demonsrated (ayasinghe eal, 1903; Jayasinghe etal, 1994; North, 199). Although ini- 2 reaction involves te yeast-induced condensation of benz 2 ly it was thought that immobilisation of yeast, for ‘example in polyurethane, was essential in order maintain Stability of cell membrane-bound coenzymes for the dehy Aogenases and reductases. which eaalyse the reaction (Nakamura et al, 1988; Nakamura ct al, 1990), it was subsequently found that the addition of a very small pro portion of water to the exganie syslem wou avoid the teed for immobilisation (Nakamura etal, 1991) Ephedrine (c-{1-{methylamino)ethylpenzene-methanol), originally isolate from plants of the genus Ephedra, osc as the naturally-oceueting isomers. I-ephedrine and ‘pseudoephedrine, and other pharmacologealy etve so mers include c-ephedrine and L-pseudoepbedrine. These ‘compounds are adteoersie sympathomimetic agents and hhave antiistamine activity; ephedrine is widely used asa bronchodiatr, while d-pseudoephedrne is widely used asa ‘decongestant, Compounds of these groups are present in & very wide rage of prescription and over-the-counter phar- ‘maceutical Formulations, ‘The production of Lphenylaceylearbino}, a precusor of LLephedrne, by catalysis using whole baker's yeast cells in aqueous medium was one ofthe frst microbial bioransfor- mation processes to be used commercially (Neuberg and Hirsch, 1921; soc als Hildebrandt snd Klavehn, 193), This ‘dehyde with acelyloveneyme A. The rection fas beea ‘widely investigated, and has been shown to be mediated by the enzyme pyruvate decarboxylase (Groger Sehmander snd Motes, 1966), It has also been shawn tht the reaction has a telatively broad specitiity for he subsea, enabling variety of substituted aromatic aldehydes ta be converted to the corresponding subsitutedoptially-active phenylaee= ‘ylearbinols (Long, James and Ward, 1589) Although this yeast-catalysed system has been widely exploited this bas nommally tlised aqueous systems, which are inconvenient for large-scale extraction and purification, ‘which require organic sevens. Additionally, fermentation systems present the disadvantage thit purification of the ‘desired product can be difficly, and yields tend to be low; ‘while the yield and convenience of the reaction cin be improved by uilising immobilised cells, or ces which have ben selected or genetically modified this adds significantly 10 the east of the process. The use of purified enzymes is formally prohibitively expensive, and again without the use of immobilised enzyme the yield tends to be low and purification dificult We have now surprisingly found that yesst-mediated acyloin condensation of benzaldehyde ean be achieved in an bomganic solvent using non-fermenting yeast, and that add on of «small proportion of ethanol tothe reaction mixture suppresses formation of undesired side-products, Even more surprisingly, by performing the reaction at reduced temperature, an even greater eduction of side-reactionsc be achieved, without less of catalytic activity. The ellct of reduction in temperature appears to be generally applicable {0 both aqueous and non-aqueous systems utilising a non- fermenting yeas Although Ward and co-workers have earrod out inves ations using whole cell yeast biotransformation in two~ phase organic systems with a water content of atleast 10% (Nikolova and Ward, 1991; 19924; 1992b; Ward, 1995), the Yields of phenylacetylearbinol were low, and the levels of Sldeprodcts wore unacceptably high ‘The first description of the symthesis of Lephedrine was contained in a patent by Hildebrandt and Klavehn (1934) and made use of the discovery by Neuburg and HirschUS 6,271,008 BI 3 (2921) that fermenting strains of Saccharomyces cerevisae fn agueous systems would convert benzaldehyde to pen) lacetylerbinol. The yield of the earbinol was typically about 18%, and significant amounts of bath benzyl aleobol and henzhie acid were abtained as side-prodcts. Ina preferred embodiment, yield of around 24% with the almost total absence of side-pducts were obtained using, the method ofthe invention, SUMMARY OF THE INVENTION Ta a fest aspect, the invention provides method of synthesis of a carbinol compound, comprising the step of subjecting the corresponding aromatic aldehyde 10 acyloin condensation mediated by a yeast in an organic solveat under nos-fermenting conditions, in the presence of an Aliphatic alcohol or aliphatic ak ‘Any yeast capable ofellctng reduction may be used. It is economically advantageous to use the cheapest yeast available, and ordinary baker's yeast, Saccharomryees ‘erevisiag, is prefered. Stains of yeast adapted olbet Purposes, inluding brewing yeast and wine o shery yeasts ‘ould also be employed. Stains specifically adapted to ‘organic solvent eavironment or Tor eninced relict tellicency may be used; sch strains include conventionally- ‘selected and geactically modified strains. For maximum ‘ficiency of reaction, itis advisable to pesca the maximum Surface area of yeast for contact with the reactants. This can be effected by using “active” cried yeas, which is readi ‘commercially available a8 “instant dry yeas”, anc may be ‘Stored at room temperature. Alleratively, well-pulversed ‘ry baker's yeast may be used. Other yeas, sueh 38 those ‘described in U.S. Pat. No. 4,734,367, oF fungi such a8 those ‘sched in Chénevert etal (1992) may also be used, The person skilled in the at will readily be able to test whether fay specific organism will function for the purposes of the invention, using the methods described herein. Preferably the aliphatic alcohol or aliphatic aldehyde is ‘ethanol or actaldehd, suitably 0.1 ml per @ yeas. This resuls in significant increase inthe yield of earbino, and Feduces the amount of aromatic alcohols produced a a Side-reaction. Ethanol is prefered, since this results in ‘superior conversion of the aromatic aldehydes tothe desired ‘atbinol, und Tower yield of undesired reduction product Without wishing tobe bound by any particular theory, its helioved that the ethan or acetaldede provides an alter- native substrate for the eedictase enzymes this inhibiting, the formation @ F side products such as benzyl aleobol, ‘Therefor, it is predicted that other aliphatic aleobols or aliphatic akletydes could perform the same funtion Although the reaction can be performed at ambient temperature, suitably 16-24" C., preferably 20° C, we have ‘suprisingly found that signiicantly better results are ‘oblained at lower temperatures, inthe range 0-S2° C. The feason forthe improved performance and further reduction ‘of side-resctions which is observed isnot presently under- ‘stood however, we have observed thatthe activity ofthe yeast at these reduced temperatures i comparable to tht Ambient temperature. This result & particularly suepisig, hecause it wauld normally be expected that a yeastmediated reaction would demonsirate a temperature optimum at amb ‘eat or sighly elevated temperature, although Shiu and Rogers (1996), have shown thut isolated pyruvate decarboxylase, the enzyme involved inthe acyloin conden ‘sion ection, exhibits increased activity at 4° C. ‘The solvent may be any sitable organic solvent of low ot moderate polarity, such as petroleum ether, carbon 4 tetrachloride, hexane and other hydrocarbons, diethyl ether, toluene, or enaene. We have shawn that redsetion of ethyl acetoaceiate (another yeast-mediated reaction) can be Schioved in good yields and with high enantiscletivity in range of mixed organic solvent syslems, including 2ethoxyethanolidicthy ether, pyedine\earbon tetrachloride, cthloroformypetroleum ether and ethyl accatetoluene. We have found that petoleum ether i especially suitable, and bus the advantage of low cost. We have found tha in the yeast-mediated reduction of ey acetoacetate, baker's Yeas Fetans its reducing a activity when 1 to 30% vi of a polar Solvent is add to a non-polar onganie solvent and this is also expected to be the ease in he current invention, because the erucal factor inthe choice of the solvent is whether the yeast remains active which isnot depensent onthe particular feaction concerned, Preferably 1 40 3% of polar solvent is Added, The polar solvent is preferably. chloroform, Aichloromethune, methyl ethyl Ketone, or methyl abut ketone; the best resuls for mixed solvents were obtained with chloroform or dichloromethane in poioleum ether. In genera, a single solvent is prefered. 11 is known that in onder to preserve functioning of an enzyme in an organic solvent environment its necessary for the enzyme tobe Tully hydated by being surtounded by 4 few layers of water molecules. Ths requirement i sted 2% by providing a ratio of 0.6 10 1.2 ml waterig of yeas, preferably 1.0 ml waterig of yeast, This resulls in a single phase cngunic system as al ofthe water is absorbed into the yetst. A two-phase system reduces the yield ofthe product and makes isolation ofthe product considerably more dil- ful. ‘Once the yeast mediated reaction has been completed, the yeast can readily he separated from te reaction mixture by frat and washing. The reaction mixture, comprising pxlict, unreacted sacting. material, solvent and_ minor Impurities, 8 subjected to conventional purilation, for ‘example by lash distillation, 10 yield the purified product. Optionally the yeast ean be extracted with an organie solvent sich as ethyl acelae to yield 2 marginal amount of further precuc. In 2 prefered embadiment, the invention provides a method for yeastsmediated conversion of benzaldehyde 10 phenylacetyearbinol, according to the following reaction: sce 11 willbe cleanly understood thatthe benzaldehyde, the pyruvic aid, or both may optionally be substituted, and that pyruvate, for example sodium pyruvate, may be used as an lleritive to pyruvic acid. However, preferably pyruvic cid is used, since the sodium pyruvate is more expensive, nd we have found that one quarter as much pyrovi acid is ‘required compared to pyruvate. Aromatic aldehydes subst ‘uted with alkyl, ary, balo, nit, hydroxy, alkoxy, amino, «atbonyl, thioxy or thioslkoxy grovps.or composites ofthese troups may’ also he used stead of benzaldehyde For either sodium pyruvate or pyruvic acid, the pH ofthe pyruvatefcitate buler solution is preferably berween 5 and 6, more preferably pH 5.45. Between 0 6 and 12 ml bulferia, fof yeas should preferably be used for optimal results.US 6,271,008 BI 5 While the ratio of yeast to substrate will vary depending ‘on the individual system, and is readily determined exper- mentally using routine rial and error methods, we have feud tat for the conversion of benzaldehyde o phenylace Iylearbinol the optimum ratio is 5g yeastmmol benzalde- Hyder increasing the amount of yeas ests only sna, inerease in conversion, ax lower amounts of yeast provide lower conversion, Similatly, the optimum reaction time may readily be determined and forthe benzaldehsde-phenylacetycarbinol ‘system we have investigated reaction times from 12 (0 72 Thou, and have found that when the reaction & continued for longer than 24 hours there i very litle improvement in ‘conversion, and that there is an increase in production of by-products Tn a particularly preferred embodiment, production of dese side-peoduets i reduced by performing the cally sis reaction st helowe ambien! temperature. Prefershly the temperature is 0-5° C. Ina second aspect, the invention provides » method of synthesis of a substtued eainol compound comprising the ‘Slep of subjecting the corresponding substiuted aromatic aldehyde to aevloin condensation mediated by a yeast in aa ‘organic solvent under non-fermenting conditions, in which the reaction mixture comprises water Sufiient to activate the yeast but not to form a two phase System, the invention provides.» metho! of synthesis of a substituted earinol compound comprising the ‘Step of subjecting the corresponding substituted aromatic aldehyde to aeyloin condensation mediated by a yeast in ‘organic solvent under non-fementing conditions in which the reaction is performed at O-10? C. For the purposes of this specification it understood tht the word comprising, meat not limited 10 ad that the word “comprise sponding meaning, DETAILED DESCRIPTION OF THE INVENTION “The invention will now be described in detail by way of reference only to the following non-limiting examples EXAMPLE 1 Yeast-Mediated Acyloin Condensation of Benzaldchyde Using Pyevvis Acid Pyrovic acd uller was prepared hy dissolving pyavie acid (1044, 119 mmo} in 100 al of 005 M Sain ‘ae, Ammonium acsate was added 1 give a pH of 5.45 Beacaidehy (108 mg, 1 mmol, peoleum eter (0 ma) anol (05 ml}, bakes yeast (5g) and the pyruvic acd Buller (S al) were sie a 0" for 24h, The mixture Was then Blicred and the yeast wanbod wilh dist ether “Th combined manic layers were thn Waxed with 10% sedi carbonate. Aer emeval af the solvent in Yc the product was pure by Mash distin 200" C1 ex) to Sve phensloeslearbino! GO mg, 20% el). Gas coma. tography (GC) of the product showed pure PAC (LL.SS tin) Chizal GC show! aio of 9535, 00% ce [llye-262.6 (oa 745, CHCL). "HNMR (CDCI) & 738-752, M, Ph; 8512, CHE 2.06, CH EXAMPLE 2 YeastMediated Acyloin Condensation of Benzaldehyde Using Sodium Pyruvate 9.5 mlof 0 Mcittic acid and 40.5 ml of 0.1 M t-sadiuen citrate were diluted to 100 ml to give a pH 6 citrate baller ‘Solution. (Ref: Butlers for pH and Metal fon Conte, D.D. Persia and B. Dempsey, Publ. John Wiley and Sons, pg, 103). 6 Petco spirit (40 ml), ethanol (05 ml), the pH 6 citrate buffer (5 mi) and sodium pyruvate 25g, 28 mmol) were stir at room temperature for Ih. Benzaldehyde (127 me, 1.2 mol and baker's yeast (5 g) were then add snd the reaction sired at 5° C. for 24 h. The mixture was then fered and yeast washed with diethyl ether. After removal of te solvent in vacuo the produet was purified by lash Aisillation (200° C1 mm) to give phenylacetylearbinol (44 img, 24%). The GC ofthe product showed pure PAC (11.88. ‘min. Chiral GC showed & ratio of 97.5:2:5, 95% ee, [elo=-3758 (c-1.6, CHCL). "NMR (CDC 733-732, M, Ph; 8 5.12 s, Cli; 8 2.06, 8, CH TH will be apparent to the person skied i the art that ‘while the invention has heen described in some detail forthe purposes of clarity and understanding, various modifications nd alterations to the embodiments and methods described herein may be made without departing from the seopeof the inventive concept disclosed in this specification, References cited herein ae listed on the following pages, and are incorporated herein by this reference Ww be clearly understood tat, although a mumber of prior art publications are refered to herein, this reference toes aot constitute an admission that any ofthese documents forms part of the common general Knowledge in the, ia yo 25 Australia or in any other country. REFERENCES, (Chenevert,R. Fortier, G. and Rhlid, R.B. Tetrahedron, 1992. 48 6769-6776 (Couk, R. sd Glanzer, B.1. Chom, Rev., 1991 91 49-57 Groger, D,, Schmander, HP. aad Motbes, K. Z. All Mikrobo., 1966 6.275 ildebranek, G. and Klavehn, W. 1934 US. Pat No. 1,956, 950 Hudlicky, T, Gillman, G. apd Andersen, C. Tetrahedron ‘Asymmetry, 1992 3 281 Jayasinghe, L. ¥, Smallrdge, A, J and Thewhella, M. A “Teirahedion Leters, 1993 34 3949. Jayasinghe, L. ¥, Kedituwakkv, D., Smallrdge, A.J. and “Trewhella, M.A. Bull. Chem. Soe. Ipn. 1998 67 2528 Kaviai, A, Asano, T and Ina, ¥. Bull. Chem, Soe. Jpa., 1988 61 3014 Long, A., James, P. and Ward, 0. P. Biotechnol. Bioeng, 1989 33 637-860, Nakamura, K, Inoue, K., Ushio, K. Oka, 8. and Ohno, A. J. On. Chein., 1988 58 2589" 2503 Nakamura, K, Miya, Inoue, K., Kawasaki, S., Oka, S. ‘nd Ohno, A. Bioesalyss, 1990'3 17-24 Nakamura, K Kondo, 8, Kawai, ¥. and Ohno, A. Terahe= “doo Leters, 1991 32 7075 Nakamura, K, Kondo, 8., Kaa, ¥. and Ohno, A. Bull Chem. Soe. Jpn, 1998 66 2738 Neubers, Cand Hirsch, J. Biochem. Z.,1921 115 282-310. Nikolova, and Ward O. P. Biotechnol, Bioeng, 1991 38 493-498 Nikoiova, P. and Ward, ©. P. J. Industrial Microbiology, 19028 10 169-177 Nikolova, P. and Ward, 0. P. Biocatalysis in Non- Conventional Meda, edited by J."Tramper et al, 19925 Elsevier Science Publishers B. V. 675-680 North, M. Tetahedson Leters, 1996 37 1699-1702 Sakaki, 1, Kobayashi, 8 Sato, M. and Kaneko, C. Chem, Pharm Bull, 1989'37 2952"2961 Servi, S. Synthesis, 1990 1-25 Shiv, H.S. and Rogers, PL. Biotechool Bioeng, 1996 49 52-62 Ward, O. P. Can. J. Bot, 1995 73 S1043-S1048,US 6,271,008 BI 7 What is claimed is: 1. A method of synthesis of carbinol compound, com= prising the step of subjecting the corresponding aromatic ldehyde wo an aeyloin condensation reaction mediated by & yeast in an organic solvent under non-fermenting conditions, inthe presence ofan aliphatic alobal a aliphatic aldehyde, in whieh the reaction mixture comprises water saliceat (0 activate the yeast, but not sullicent to form a two phase ‘system. 2A method according to claim 1, in which the yeast is Saccharomyces cerevisiae ‘3. Amethod according o claim 1, n which the yeas is of «strain specifically adapted to an organic solvent environ= ment or for enhanced reduction efficiency. “4A method according to claim 1, in Which the yeast is active dried yeas. A method scootding elsim 4, ia which the aliphatic aleobiol or aliphatic aldehyde is ethanol or acetaldehyde respectively 6A method according 1o claim Sin which the proportion ‘of ethanol or actaldehyde is 0.1 mils yeast 7_Amethod according to claim 8, in which the aliphatic aleoliol or aliphatic aldehyde is ethanol '8.A method according to claim 1, in which the reaction is performed at ambient temperature 5° meta! according to claim I, in which the retin is performed at 16-28" C. 10. Amethod according 1 claim 9, in which the reaction 's performed a 20° C THA method accoading to ela 1, in which the reaction is performed at 0-10° C. 12. A method according to claim 1, wherein the orpanic solvent selected from the group consisting of petroleum ther, carbon tetrachloride, hexane, other hydrocubors, ‘ctl eer, toluene, and benzene ‘A method according to eluim 1, wherein the organic solvent is petroleum ether M4. A method according to claim 1, wherein the organic solve isa mixed organic solvent sytem, 15.A method according to ela 14, in which the mix ‘organic solvent system is selected from the group consisting ‘of 2-ethoxyethanol/diethyl ether, pyridine/earbon tetrachloride, cloroform/petroleum ether and ethyl acetate) toluene 416. A method acconding to ela 15, in which the mixed ‘organic solvent system comprises 1 to 30% wiv of « polar ‘organic solvent aided to 2 non-polar organic solvent 17. A method aecording 10 claim 16, in which the mixed ‘organic solvent system comprises 1 to 5% viv of polar ‘organic solvent aided to 4 non-polar cnganic solvent. 8 18. A method according to claim 17, in which the polar organic solvent is selected from the group consisting of chloroform, dichloromethane, methyl ethyl Ketone, and methyl isobuty ketone 19. Amethod according to claim 18, in which the reaction {s performed in chloroform or dichloromethane ia petoleum eter. 20. method according 0 claim 1, in which the reaction mixture comprises water at 3 ratio of 6 to 1.2 ml water of yeast 21. A method according to claim 1, in which the reaction is conversion ofan optionally-substitited aromatic aldehyde ‘© tho comesponding acyloin compound by condensation ‘with an optionaly-substituted pycuvat compound. 22. Amethod according to claim 21, in which te aromatic aldehyde substituted with one of more groups selected fe the group consisting of alkyl, ary, hal, nitro, bydroxy, alkoxy, amino, carbonyl, thiory or thioalkoxy substituted with alkyl, aryl, halo, nitro, hydroxy, alkoxy, amino, catbonyl, thioxy of thoalkoxy alkyl, aryl, halo, itr, hydroxy, alkoxy, amino, carbonyl, thioxy and thoalkoxy. 23. Amethod according to claim 21, in which te reaction is conversion of benzaldehyde to phenyleetyearinol 24, Amethod according to claim 21, in which te pyruvate ‘compound is pyrvie acid 25. Amethod according to claim 24, in which the restion is performed at pl of between 5 and 6. 26.Amethod according to claim 2, in whieh the pyruvate compound is sodium pyrwte 27. Amethod according 0 claim 26, in which the reaction is performed at a plf of between 5 and 8. 28. A method according 1a elaan 1, in which the reaction time is 12 t 24 hours 29A method of synthesis of substitvied earbinol compound, comprising the step of subjecting the coer sponding substituted aromatic aldehyde (© acyl conden Sation mediated by a yeast in an onganie solvent unwer 1non-fermenting condition, in which the reaction mixture comprises water sufficient to activate the yeast but not Saicient to form a t¥0 phase sytem, ‘30. A method of synthesis of a substituted carbinol compound, comprising the slep of subjecting the corte ‘ponding substituted aromatic aldehyde (© acyloin conden Sition mediated hy & Yeast in an organic solvent under fon-fermenting conditions, ia which the reaction is per: Formed at 0-10" C,