Copyright
Enzymatic
Reprinted from The Journal of Organic Chemistry, Vol. 52, page 2608, June 12, 1987 O 1987 by the American Chemical Society and reprinted by permission of the copyright owner.
Routes to Enantiomerically 1-Butene Oxider
Enriched
H. Keith Chenault, Mahn-Joo Kim, Alan Akiyama, Toshifumi Mivazawa.Ethan S. Simon. and Geoige M. Whitesides*
Depar t ment of ChemLsrt o si t 1''c ambri dge' : I ; #:;i :r' r[t,zti " Receiued Nouember 20, 1986 This paper comparesseveralroutes to enantiomerically enriched l-butene oxide (1) in which resolution is achieved by using an enzymatic reaction (SchemeI). This research had two objectives: to enumerateseveralgeneralstrategies that can be followed in practical, enzvme-basedroutes to enantiomerically enriched epoxides and to compare the utility of these routes in the particular caseof compound 1. In general, we restrict our account to reactions that proceedwith high valuesof enantiomeric excess(ee) and that have the potential for preparing 50-g quantities of product. Enantiomerically enriched epoxidesare useful synthons in chiral synthesis. General routes are now available to only epoxy alcohols and analoguesof these substances.2'3 All reactions were conducted on scalesthat generated 1-5 g of I as product. The chemical yields reported in SchemeI are oueroll yields for conversionof the substrate for the enzymatic reaction to 1; these yields are not optimized. We established the optical purity of I by 1H NMR analysisin the presenceof Eu(hfc)r.a With careful calibration, this method can detect a 1To enantiomeric i mpur it y ( i. e. ,98% e e ). Acylase I (EC 3.5.1.14)is commercially available, inexpensive, and stable. It hydrolyzes a range of Nacyl-a-aminoacidswith high (>99% ee) enantioselectivity.s Both enantiomers are easily recovered,and the reaction can be run virtually to completion. In resolutions of 2aminobutanoic acid, the reaction stops spontaneouslyafter hydrolysis of the S enantiomer is complete,and lossesof material occur entirely in the recoveryand purification of product. Conversionof the 2-amino acid via the corresponding 2-chloro acid and chlorohydrin to the epoxide preserveschirality well.6 (1) Supported by the National Institutes of Health, Grant GM-3036?, and Naval Air Systems Command, MDA g03-86-M-0b0b.
0022-3263 I 87I 1952-2608$01.50/00
Lipase (porcinepancreas,EC 3.1.1.3)and cholesterol esterase (CE, EC 3.1.1.13)are broad-specificityenzymes showingsubstrate-dependentenantioselectivity.3'7For the hydrolysis of 2-bromobutyl butyrate, without extensive optimization,the best ee was obtained with CE and was 80% at 7i Voc<>nversion of the racemic starting material. Although this number could, in principle, be improved by carrying the reactionto higher conversion,and probably also by varying temperatureand pH, we have not done so sincealternative proceduresseemedpreferable. We note, however,that high valuesof ee can be obtained in kinetic resolutionsof other halohydrins (e.g.2-bromopropylbutyrate: >98% ee at 60Toconversion)and that this method providesa good route to certain optically active epoxides. t -Lactate dehydrogenase(L-LD H , E C 1.1 . 1. 27) and o-lactate dehydrogenase (D-LDH, EC 1.1.1.28)are both highly enantioselective.8The r, enzyme acceptsa broad range of small and medium-sizeunhindered a-keto acids; the n enzyme is more restrictive toward its substrates.e The LDH enzynes are interesting in asymmetric slmthesis becausethey comprise one of the few systems in which enzymes catalyzing reactions having opposite enantioselectivities are both commercially available. Unfortunately, the range of products available by this method in both o and I forms is limited by the range of substratesaccepted by both enzymes. The LDH-catalyzed reactions (coupled with formate/formate dehydrogenase(FDH) for in situ regenerationof NADHIO) can be run to completion with high enantioselectivities (>99Vo ee) and good isolated yields (84-9980). ( 2 ) S h a r p l e s sK , . B . C h e m .B r . 1 9 8 6 , 2 2 , 3 8 - 4 4 . (3) Ladner, W. E.; Whitesides,G. M. J. Am. Chem. Soc. 1g84,106, 7250-7251. (4) Chiral shift reagents: Fraser, P. R. In Asymmetric Synthesis; Morrison, J. D., Ed.; Academic: New York, 1983;Vol. 1, pp 173-196. (5) Birnbaum, S. M.; Levintow, L.; Kingsley, R.B.; Greenstein,J. P. J . B i o l . C h e m . 1 9 5 2 , 1 9 4 , 4 5 5 - 4 7 0G . r e e n s t e i nJ, . P . M e t h o d s E n z y m o l . 1957.3.554-570. (6) Fu, S. C.; Birnbaum, S. M.; Greenstein,J. P. J. Am. Chem. Soc. 1954,76,6054-€058.Koppenhoeffer,B.; Weber, R.; Schurig, V. Synthesis 1 9 8 2 ,3 1 6 - 3 1 8 . (7) Lavagre, J.; Verrier, J.; Baratti, J. Biotech. Bioeng. 1982,24, 2175-2188. (8) Hirschbein,B. L.; Whitesides,G. M. J. Am. Chem. Soc.1982.104. 4458-4460. (9) Kim, M.-J.;Simon, E. S.; Whitesides,G. M., unpublished results. ( 1 0 ) S h a k e d , Z . ; W h i t e s i d e s ,G . M . J . A m . C h e m . S o c . 1 9 8 0 . 1 0 2 . 7 1 0 5 - 7 1 0 7 .W i c h m a n n ,R . ; W a n d r e y ,C . ; B r i c k m a n n ,A . F . ; K u l a , M . - R . Biot ech. Bioeng. 1981,23, 2789-2802. 1987 American
Chemical Societv
J . O r u . ( ' h e , m . .\ : o l . 5 2 , . \ r i 1 2 , 1 9 8 7 2 6 0 9
Notes
S c h e m eI . E n z y m a t i c R o u t e st o E n a n t i o m e r i c a l l yE n r i c h e d l - [ J u t e n eO x i d e " (chemical yreld, ee)
cH3coNH
!co,,
cH3coNH
fL-ao,n /'
,r/
Et'
-7r
ococaH 7 CE 20%a
"acorn
E|I
ooH
\--l
c o, H
NADH NAD
c ------>
Br.
O C O C a H7
\___-/
o -P
(19,95)
Et/
o /\
_'-
(7. 76)
Er' HO o
--:-----
OH
',, ',
/
l
h'i -------->
Er'
El'
trt>'"^
'>-"'
Er'
/\
-->
Et/
-"-cozH
(16,96)
*t'
d,€
D.LDH
GIyDH
/ \
Et'
"--co2H Et'
EI'
H Otr. L-LDH
o
ct,e
cl
HzN
40"/oa
o{ \/
cl \.o"" / Er
y*
o
,,P
(55, >98)
o
'''-
l\
(48, >98)
Et'+
o
l\
(28, >98)
Er'
or)eenr nl i' mer is 50o/.. b2 N HCl, 100'C,2h(90%) '6N " Sincethis reaction is a kineticresolution,the maximumtheoreticalyield ol'r(';Hr|oK. dBH3.THF .25 (90%). "xoH. :10 c,H,,OH. room temperature(ll:}%). rtsH:r.THF, HCl. NaNOr, 10.C (70%). "c {75%). 0 " C . 2 0 h ( 8 1 - 9 1 % )h. 3 0 %H R r A c O H , 1 5 ' 2 5 . C , 3 h ( 6 ; 9 0 % ) . ' C . H l l O K , ( ' r , H , i O 1 l , r ( , o m l e n l p e r a t ul rl le%( i)0.
Glycerol dehydrogenase (GIyDH, EC 1.1.1.6)reduces 1-hydroxy-2-butanoneand a number of small a-keto alcoholswith good enantioselectivity(94-98% ee).tl It also requiresin situ NADH regeneration. A number of other enzymesare also, in principle, applicable to the synthesisof enantiomericallyenriched epand various oxides. Epoxide hydratase (EC 4.2.L.64)r2 are particularly interesting. These oxidative enzymes13 enzymesoperate,however,with variable degreesof enantioselectivity. Further, none is commercially available,and the oxidative enzymesare too difficult to manipulate for routine laboratory use in synthesis. We conclude that enzymatic catalysis offers a number of practical routes for the preparation of epoxideswith high enantiomericpurities. For the synthesisof 1, methods based on LDH have the advantageof high enantiomeric purity and good chemical yield. Methods basedon acylase I have advantages of low cost of enzyme and starting material and of operational simplicity. For other epoxides, the best route will depend upon the substancebeing synthesized. AcylaseI acceptshindered side chains and has very high enantioselectivity; lipases are active with water-insolublesubstrates;routes basedon LDH or GIyDH may be favored if starting materials are padicularly readily available. Experimental Section Materials and Methods. Enzymesfrom Sigmawereused I (porcinekidney,lyophilized withoutI'urtherpurification:acylase
powder, grade I), CFI (porcine pancreas,lyophilized powder), r,-LDH (rabbit muscle,crystallinesuspensionin 2.1 M (NH1)2S04, type II), I)-LDII (l,actohacillusleichmannli,crystallinesuspension sp.,lyophilized and GIyDH (Cellulornonos in ll.2 M (NH{)2SOa), powder). FDH (yeast, Iyophilized powder) frorn Boehringer Mannheim was usedwithout further purification. tr-Bromobutyryl bromide came from Fluka. Butyryl chloride came from Aldrich. from BASF was distilled three times before 1-HydroxSr-2-butanone use. All other chemicalsand biochemicalscame from Sigma and were used without further purification. Water wasdistilled twice, the secondtime from a Corning AG-lb glassstill. Argon (welding grade) was used without further purification. THF was freshly distilled from Na/benzophenone. lH NMR spectrawere recordedon Bruker AM-300 and AM-250 instruments with peaksrei'erencedto MenSi (CDCl.r,acetone-d6) or DSS (D2O). A Chemtrix 45AR pH controllerequippedwith an LKB 2120 peristaltic pump afforded pH control of certain reactions. Enzyme Assays. Acylase I was assayedwith racemic Nacid (40 mM) in 0.1 M potassiumphosphate acetyl-2-aminobuty'ric buffer, pH 7.5,40 'C. The rate of releaseof free amino acid was determined by removing aliquots, precipitating the protein by adding an equal volume of L4% HCIO4,and allowing a portion of eachacidifiedaliquot to react with ninhydrin.la l- and o-lactate were assayedwith 2-oxobutanoicacid following dehydrogenases the literature method.rs Glycerol dehydrogenaseand formate dehydrogenasewere assayedby known procedures.16Enzymes immobilized on PAN gel were assayedby using a procedure analogousto that for the solubleenzyrnesexcept that the cuvettes were inverted every 10 s to keep the gel suspended.lT One unit
( 1 4 ) R o s e n ,H . A r c h . B i o c h e m .B i o p h y s . 1 9 5 7 , 6 7 , 1 0 - 1 5 . (15) Methods of EnzS'matic Analltsis, 3rd ed.; Bergmeyer, H. fJ., B e r g m e y e r .J . , G r a s s l ,M . , E d s . ; V e r l a g C h e m i e : W e i n h e i m , G e r m a n y , 1983;Vol. 2, pp 232-233. ( 1 6 ) M e t h o d s o f E n z y m a t i c A n a l l ' s r . s3, r d e d . ; B e r g m e y e r ,H . L I . , B e r g m e y e r .J . , G r a s s l ,M . , E d s . ; V e r l a g C h e m i e : W e i n h e i m , G e r m a n y , 1 9 8 3 ;V o l . 2 , p p 2 1 l i - 2 1 5 ( g l y c e r o ld e h y d r o g e n a s e )1, 8 3 - 1 8 4 ( f o r m a t e dehydrogenase).
2 6 1 0 J . 0r g. Che m .,Vo l . 5 2 , N o . 1 2 , 1 9 8 7
Notes
of enz5rmerefers to that amount of enzymewhich produces1 pmol min.l of product under assayconditions. Determination of Enantiomeric Excesses. Enantiomeric excesses of (fi)- and (S)-2-aminobutanoic acidsweredetermined b y ' H N N { R a n a l y s i so f t h e ( + ) - M T P A a m i c l e s rosf t h e c o r r e spondingamino acid methyl esters.re (,lalibrationshowedthat 'fhe a 0.5% diastereomericimpuritS'could be detected. enantiomeric excesses of (R)- and (S)-2-h1'clroxybutanoic acidswere likewise measured by tH NMR anaiysis of the O-(+)-MTPA derivativeslsof the corresp
mL ol ether. The etherealextractswere washedwith H2O,dried ((laCl2),and evaporatedunder reduced pressureto give 8.3 g acid: bp a6-50 'C (0.25torr) ilit.z3 00%) of (r?)-2-chlorobutanoic o C t H ( i 6 N M R ( C D C I 3 6) 1 . 0 5l t , J = i . 3 H z , torr)l; bp 99-101 : l H , C H r ) .2 . 0 2( m , 2 H , C H 2 ) ,4 . 2 6( d d .J = 5 . 8 ,7. 6 H z , I H , C H ) ; I R ( n e a t ): 1 5 0 02 5 0 0( O H ) , 1 7 2 0( C : O ) c m ' . (S)-2-Aminobutanoic acid wasconvertedto (S)-2-chlorobutanoic a c i d ( 7 0 %) i n a s i m i l a r m a n n e r . Conversionof (,R)-and (S)-2-ChlorobutanoicAcids to (,R)and (S)-2-Chloro-l-butanol. BH3.THF2areducedthe crude R chlorohydrins(90%): tH and S chloroacidsto the corresponding r )1 . 0 2( t , J = 7 . 4 H 2 , 3 H , C H I ) ,1 . 6 1 1 . 8 8( m , 2 NMR (CDC].6 . H), H , C H 3 C H ) , 2 . 3 4( b r s , I H , O H ) , 3 . 5 8 - 3 . 7(7m , 3 H , C H 2 O C i n a g r e e m e nw t i t h l i t . 2 5I;R ( n e a t )3 3 7 0 ( b r , O H ) c m - r . Conversion of (Il)- and (S)-2-Chloro-l-butanolto (S)- and (l?)-l-Butene C)xide.6Finely,'ground, dry KOH (6.8g, 122mmol) w a s a d d e ds l o w l yt o t h e c h i l l e d ( - 3 0 " C ) ( R ) - c h l o r o h y d r i n( 6 . 6 g, 61 rnmol). KCI precipitated. After the addition of KOH, (S)-1-huteneoxide was distilled from the reaction through a short-path distillation head to give i).3g (75%) of product: 96% ee;bp 59 S1 oC;rH NI\{R (CDCIJ 6 0.98(t, J = i.5H2,3 H, CH.r). 1 . 5 5( m , 2 H , C H T C H 2 ) , 2 . 4( m 5 , I H ) a n d 2 . 7 1( m , 1 H ) ( C H 2 O ) , 2 . 8 7( m . 1 H . C H ) . Similar reactionof the (S)-chkrrohydringave(R)-1-buteneoxide (i5% yield,9STo ee). Cholesterol Esterase Catalyzed Hydrolysis of 2-Bromobutyl Butyrate. 2-Bromobutyl butyrate (30.9g, 138 mmol), obtained by the lithium aluminum hydride reduction26of 2bromobutyryl bromide and subsequent esterification of 2was mixed with 40 mL bromo-1-butanolwith butyryl chloride,2? of 0.05N{ pot,assiumphosphatebuffer, pH 7.0,and 40 mL of HrO in a 250-mL three-neckround-bottomedflask equippedwith a stir bar and pH electrode.Addition of 5 N NaOH adjustedthe pH to 7.0. CE (0.15g in 1 mL of buffer) was added. Addition of 5 N NaOH using a pH controllerkept the reactionat pH 7.0; the volume of baseused indicated the extent of reaction. After 27 mL of basehad been added (777o conversion),the reaction was poured into 400 mL of ether. The layers were separated,and the aqueouslayer was extracted with 4 x 100 mL of ether. The combinedethereallayerswere evaporatedunder reducedpressure. The residuewas dissolvedin 250 mL of pentane and washedwith 8 X 211mL of iSVo aqueousMeOH to remove the alcohol. The organic laver was dried over Na2SOa,and the pentane was evaporatedunder reducedpressure.Kugelrohr distillation of the residualoil gave(R)-2-bromobutylbutyrate (6.1g, 84% yeld based on iiTo conversion):80Vo ee;bp 44-47 'C (0.20torr). Conversion of (R)-2-Bromobutyl Butyrate to (S)-l-Butene Oxide. (E)-2-Bromobutylbutyrate (3.0g, 80% ee) and 6 mL of freshly distilled 1-pentanolwere combinedin a 100 mL roundbottomed flask equipped with a dropping funnel and Vigreaux column leading to a condenserand receivingflask. The condenser was connected to a circulating cold bath set at 0 oC, and the receivingflask was cooledin an ice-EtOH bath. A 1.2 M solution o f C 5 H 1 1 O Ki n l - p e n t a n o l ( 1 1 . 2 m L , 1 3 . 5 m m o l ) w a s a d d e d dropwise to the mixture. After the solution had stirred for approximately 15 min, it was heated in an oil bath. When the temperature of the oil bath reached 170 oC, the epoxide began distilling. Its yield was 0.32 g (.33%): 76Toee; bp 56-58 oC; the lH NMR spectrum (CDCI3)agreedwith that of 1 obtained from 2-chloro-1-butanol. (-R)- and (S)-2-Hydroxybutanoic Acids. To a 1-L, threeneck round-bottomedflask equipped with a stir bar, pH electrode, and septawere transferred 2-oxobutanoicacid (15.3g, 150 mmol), sodium formate (11.7g, 170 mmol), 2-mercaptoethanol(50 rrl, 0.75 mmol), and Tris-HCl (0.45g, 3.8 mmol) in 300-400mL of H2O. ConcentratedNaOH adjustedthe pH to 7.5. The solution was degassedby bubbling Ar through it for 15 min. NAD (0.75
( 1 7 ) P o l l a k , A . ; B l u m e n f e l d ,H . ; W a x , M . ; B a u g h n , R . L . ; W h i t e s i d e s , G. M. J. Am. Chem. Soc. 1980,102,6324-6336. (18) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973,95, 512-5L9. ( 1 9 ) U h l e , F . C . ; H a r r i s , L . S . J . A m . C h e m . S o c . 1 9 5 6 ,7 8 , 3 8 1 - 3 8 4 . (20) Methyl estersformed by reaction with CH2N2. ( 2 1 ) R a o , K . R . ; B i r n b a u m , S . M . ; K i n g s l e y ,R . B . ; G r e e n s t e i n J, . P . J. Biol. Chem.1952,198,507-524. ( 2 2 ) G r e e n s t e i nJ, . P . ; G i l b e r t ,J . B . ; F o d o r , P . J . J . B i o l . C h e m . 1 9 5 0 , 182,45t-456.
(23) Reeve, W.; Steckel, T. F. Can. J. Chem. 1980, 58, 2784-2788. (24) Yoon, N.M.; Pak, C. S.; Brown, H. C.; Krishnamurthy, S.; Stocky, T. P. J. Org. Chem.1973, 38, 2786-2792. (25) Nakajima,T.; Nakamoto,Y.; Suga,S. BuLl.Chem. Soc.Jpn.1975, 'r8, 960-965. ( 2 6 ) F i c k e t t , W . ; G a r n e r ,H . K . ; L u c a s ,H . J . J . A m . C h e m .S o c . 1 9 5 1 , 73,5063-5067.St.ewart,C. A.; Vanderwerf,C. A. J. Am. Chem.Soc. 1954, 76,7259-1264. ( 2 7 )S o n n t a g N , . O . Y . C h e m .f i e o . 1 9 5 3 5 , 2,231 '116.
2 6 1I mmol) was added. Then, L- or D-LDH (150-400 U) and FDH (40-50 U) immobilized on PAN gelt?were added as a suspension in 10O-200mL of degassedH2O. The flask was capped with septa, and Ar bubbled through the solution. HCI (2.56N) added by a pH controllermaintained the reactionnear pH 7.5. The reaction progresswas followed b5'measuringthe volume of HCI consumed' Within 5 days the reaction was complete. The enzl'rnecontaining gel was isolatedby centrifugation and washedwith degassedH2O. The aqueouslayers were combined and concentratedby rotary evaporationto 70-80 m[,, acidified to pH 2.0 with 6 N HCl, and extracted with 4 x 1?0 mL of ether. The ethereal layers were combined, dried over MgSO4,and evaporatedunder reduced pressureto give (S)-2-hydroxybutanoicacid (r--LDH used) (15.0 5 C d e c ( l i t . 2 8m p 5 2 . 7 - 5 3 . 5 ' C ) ; g , 9 5 7 a )[ > 9 9 % e e ;m p 5 4 . 5 - 5 5 . o (lit.28 (c [a]o16+6.4o (c 11.03,CHCI3)); [o]o" +?.15o 8.13,CHCI3) l H N M R ( C D C I 3 )6 1 . 0 0( t , J = 7 . 4 H 2 , 3 H , C H 3 ) ,1 . 7 5a n d 1 . 8 8 ( m , 1 H e a c h ,C H 2 ), 4 . 2 4 ( d d ,J = 4 . 5 , 6 . 9H z , 1 H , C H ) , 6 . 7 2( b r , OH); IR (Nujol) 3500-2650(OH), 1730 (C:O) c--'l or (R)-2hydroxybutanoicacid (o-LDH used) (13.9g, 89%) l>99% eermp 53-55 oc dec; [.]n'o -5.6o (c 3.?1,cHcl3)l; the lH NMR and IR spectra were in agreementwith those for the S enantiomer. Conversion of (,8)- and (S)-2-Hvdroxybutanoic Acids to (R)- and (S)-Butane-1,2-diol. BH3'THF2areducedthe R and S hydroxy acids to (R)-butane-1,2-diol(9.7 g,81%) lbp 122-125 lH NMR (CDCI:r)6 'c (30 torr); [*]o" +12.6" (c 3.23,EIOH); 0 . 9 0( t , J = 7 . 5 H 2 , 3 H , C H 3 ) ,1 . 4 1( m , 2 H , C H 3 C H 2 )3, . 3 ?( m . ( b r s ,2 H , 2 x O H ) ;I R ( n e a t ) 1 H , C H ) , 3 . 5 8( m , 2 H , C H 2 O ) , 3 . 6 7 (11.7g' 3350(br, OH), 1045(C-O) cm-rl and (S)-butane-1,2-diol 9 t % ) [ b p 9 a - 9 6 ' c ( 9 t o r r ) ; l n ] n ' 2- 1 5 . 3 5 o( c 2 . 6 0 ,E t O H ) l ; t h e lH NMR and IR spectra were in agreementwith those of the R enantiomer. Analytical data for both enantiomers agreedwith the literature values.2e Conversion of (.R)- and (S)-Butane-1,2-diol to (-R)- and (S)-2-Acetoxy-1-bromobutane. Reaction of the diols with 30% (17.3g, 82%) HBr-AcOHD's gave (R)-2-acetoxy-1-bromobutane lH NMR 'C (21 torr); [o]o2t +17.8o (c 2.73,ether); [bp 87-91 (CDCI3) 6 0.90 (:t, J = 7.4 Hz, 3 H, CH3CH2),1.69 (m, 2 H. C H y C H ) , 2 . 0 7 ( s , 3 H , C H 3 C O ) ,3 . 4 5( m , 2 H , C H 2 O ) , 4 . 9 1( m , 1 H, CH); IR (neat) 1?35 (C:O) cm-l1 and (S)-2-acetoxy-1bromobutane(22.3g,9lTo) [bp 68-70'C (9 torr); [.]n" -23.16" (c 4.14,ether)]; lH NMR and IR spectrawere in agreementwith those for the R enantiomer. Analy'tical data for both enantiomers agreed with the literature values.2e'3olH NMR spectroscopy indicated that the products contained approximately 7% lac etoxy -2-brom ob utane. Conversion of (,R)- and (S)-Z-Acetoxy-l-bromobutane to (,R)- and (S)-l-Butene Oxide. Treatment of (R)- and (S)-2Acetoxybromobutanes(5 M in dry l-pentanol) with 1 equiv of
1 . 1 8M C b H r l O K i n l - p e n t a n o l( a d d e do v e r 3 0 - 6 0 m i n a t 0 ' C ) followed by distillation of the product through a 15-cmVigreaux column equippedwith a condensercooledto -10 "C gaye(R)-1oC; b u t e n eo * i d " 6 . 2 g , 8 l % ) l > 9 8 % e e ;b p 5 9 - 6 2 [ o ] n 2 2+ 1 4 . 8 0 ( c 1 . 1 8 ,e t h e r ) ( l i t . 2 e1 a l , - , 2+11 3 . 6 " ( c 1 . 1 3 5 ,e t h e r ) ) ;t H N M R spectrum in agreementwith that of 1 obtained from 2-chloro-1oxide(5.86g,7lTo) [>98% ee;bp 5W2 butanolland (S)-1-butene - 1 2 . 2 5( c 6 , d i oC; [ . ] 2 2 n- 1 2 . 0 0 o( c 4 . 9 0 ,d i o x a n e )( l i t . 3 1[ . t ] 1 6 r , oxane));lH NMR spectrum in agreementwith that for the S enantiomerl. Enzymatic Preparation of (.R)-Butane-1,2-diol. A threenecked, 500-mL, round-bottomedflask was chargedwith am(4.56 g,60 mmol), 1-hydroxy-2-butanone monium formate (11.78 g, 50 mmol), Tris-HCl (47 mg, 0.5 mmol), and 50 mL of water. The pH was adjustedto 7.5with 1 N KOH. The flask rvassealed with septa and fitted with an Ar inlet and outlet, a pH probe, and an inlet for 2.1 N HCl. The solution was degassedb1-bubbling, Ar through it for t h and NAD (0.15mmol) was added. FDFi (67 tf) and GDH (100U) immobilizedon PAN gell7rvereadded as a suspensionin 50 mt, of H2O. A pH controller maintained t h e p H a t i . 7 * 0 . 1 b v a d d i n g 2 . 1 N H C I : A r b u b b l e c ti h r o t r g h the reaction. After 14 davs, the enzyme-containinggels were removedby centrifugation(51 U of FDH and 44 U of (iDH were recovered).The aqueousportion was continuouslyextrac'tedwith with K2COr.and extractedwith il x ether for 3 davs.sat,uratecl 1 0 0 m [ , o f e t h e r . C o n c e n t r a t i o no f t h e e t h e r e a lp o r t i o n sa f t e r dn'ing overK'COqvieldeda paleyellowliqtrid(3.5g). Distillation oC through a short-pathcolumn [122-125 (l]0 torr)l vieldedthe d i o l ( 2 . 8 4g , 6 4 % ) . i d e n t i f i e db y ' H N M R s p e c t r o s c o p r ' . Conversion of (,R)-Butane-1,2-diolto (R)-l-Butene Oxide. (ft)-Butane-1,2-diol(from the GDH-catall'zed reaction) rvas convertedto (R)-1-buteneoxide by the same two-stepmethod from the LDH-catalyzedreactions.The usedwith butane-1,2-diol y i e l d w a s 3 . 9 0g A T V c f r o m t h e d i o l ) : > 9 8 % e e ; [ a l p 2 r+ 1 3 ' 3 8 0 , ther). k 1 . 2 2 5e H.K.C. and A.A. acknowledge Acknowledgment. support from NIH Training Grant 5-T32-GM-07598 (1984-1985 and 1985-1986,respectively).
