CA1225956A - Process for preparing l-carnitine and chemical intermediates employed therein - Google Patents

Abstract

ABSTRACT

A process for preparing L-carnitine which comprises exposing .gamma.-substituted acetoacetic acid esters or amides to the fermentative enzymatic action of a microorganism which elaborates L- .beta. -hydroxyacyl CoA dehydrogenase [EC
1.1.1.35] , recovering the resulting, optically active, corresponding .gamma.-substituted- .beta. -hydroxybutyric acid deriva-tive and converting said derivative to L-carnitine. An improvement in the process is also disclosed which com-prises reacting a 4-chloro-3(R)-hydroxybutyrate with sodium iodide or bromide to produce the corresponding 4-iodo- or 4-bromo-3(R)-hydroxybutyrate, converting the 4-iodo or 4-bromo-3(R)-hydroxybutyrate to the trimethyl-amino-3(R)-hydroxybutyrate salt, then converting the trimethylamino-3(R)-hydroxybutyrate salt into L-carnitine inner salt. Novel chemical intermediates prepared in the processes are also disclosed.

Description

`` ~L;~2~956 ,, Process for ~reparing L-Carnitine and Chemical Intermediates Employed Therein The present invention relates to processes for producing L-carnitine.;jSpecifically, it relates to a process for microbiologically reducing ~-substltuted-acetoacetic esters or amides into their respective -~L- ~ -hydroxy- y -substitùted-butyric acid derivatives, which derivatives can be readily converted into L-carnitine chloride. It also relates to novel chemical intermediates employed in the process.
As is well known, carnitine ( ~-hydroxy- y -trimethyl-amino butyr~c acid) contains a center of asymmetry and therefore~f carnitine exists in two stereoisomeric forms, the D and the L forms.
L-carnitine lS normally present in the body where it functlons~to~carry activated long-chain free fatty acids through the mitochondrial membrane. Since the mitochon-drial membrane is impermeable to acyl CoA derivatives, long-chain free fatty acids can enter only when esterifi-. :

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cation with L-carnitine has taken plac~. The carrier function of L-carnitine is exerted bo~h by transporting active long-chain fatty acids from the sites of their bio-synthesis, for the example the ~icrosomes, to the mitochondria where they are oxidized, and by transportin~
acetyl CoA from the mitochondria, wherein it is formed, to the extramitochondrial site~ wher~ the synthesis of long-chain fatty acids occurs, e.g., in th2 micro~omes wherein acetyl CoA can be utilized ~or synthesizing cholesterol and fatty acids.
~ hile it has ~een established that the laevorotatory isomer (L~carnitine~ exclusively i~ the biolosic form (D-carnitine has never been detected so far in mammalian tissues), the D,L-carnitine racemate has be~n used for a number of year~ for different indications. For examp}e, D,L-carnitine i5 sold in Europe as an appetite stimula~t, and it has boen reported thak the material ha~ an efect on the ~rowth rate of children; see e.g., Borniche et al., Clinica Chemica Acta, 5, 171-176, 1960 and Alexander et al., ~Protides in the Biological Fluids", 6th Colloquim, Bruges, 1958, 306-310. U.S. Patent No. 3,830,931 deso cribes improvements in myocardial contractility and systolic rhythm in congestive heart failure which can often be obtained through administration of D,L-carni-tine. U.S. Patent No. 3,968,241 describes the use of D,L-carnitine in cardiac arrhythmias. U.S. ~atent No. 3,810,994 discloses the use of D,L-carnitine in the treatment of obesity.
Recently, however, there has been an increasing emphasis on the importance of utilizing exclusively ~he carnitine laevorotatory isomer for at l~ast some thera-peutic applications. It has, in fact, been shown that D-carnitine is a competitive inhibitor of carnitine-linked enzymes such as carnitine acetyl trans~erase (CAT) and carnitine palmityl transferase (PTC). Morsover,

2~gs6 recent evidence suggests that D-carnitine can deplete L-carnitine from the heart tissue. Consequently, it is essenti~l that L-carnitine exclusively be administered to patients under medical treatment for heart diseases or lowering of blood lipids.
Several processes have been proposed for producing carnitine on an industrial scale. The chemical synthesis i of carnitine unavoidably leads, however, to a racemic mixture of the D and L isomers. Consequently, resolution methods have to be employed to obtain the separate opti-cal antipodes from the racemate. These resolution methods are, however, cumbersome and expensive.
It is an object of this invention to produce L-carnitine in good yleld through a combination of microbiological and chemical processes.
An object of the present invention is to provide an improved process for synthesizing L-carnitine from readily available moderate cost raw materials.
Another object of the present invention is to disclose i the preparation of novel, useful optically-active interme-, diates for the synthesis of L-carnitine and its salts or i esters.
Another objéct~ of the present invention is to provide processes for preparing L-carnitine via the trimethylamine displacement of the~halo group of a ~-halo-3~R)-hydroxybutyrate.
Still another ~object of the present invention is to provlde a process for producing 4-iodo or 4-bromo-3(R)-hydroxybutyrates from 4-chloro-3(R)-hydroxybutyrates.
These and other objects of the invention will become more apparent as the description thereof proceeds.

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~L2;;~5~56 The advantages of the present invention will be apparent those skilled in the art from the following detailed description.
That the ~-keto function in the 3-position in the y -substituted-acetoacetic acid derivatives can be reduced by hydrogenation over Pt/C is known (e.g., U.S. Patent No. 3,969,406). However, the hydroxy compound resulting from such method is racemic. In contrast, by employing the fermentative action of a microorganism in accordance with the process of the present invention, the hydrogen-i ation of the oxo-function at the 3-position can be accom-plished stereoselectively to yield optically active y--substituted ~-hydroxybutyric acid derivatives.
' In particular, upon suitable selection (as hereinbelow descrlbed) of the substrate to be exposed to the fermentative action of the microorganisms in accordance with the process of thls inventlon, the 3(R) or L epimeric configuration is obtained. This coniguration is required for the convexsion 'i lnto~the natural L-carnitlne.
¦ ~ ;Broadly,~ this invention comprises the use of the microbial ¦ reductase enzyme, L- ~ -hydroxyacyl CoA dehydrogenase [EC
! 1 .1 .1 . 35] , to catalyze stereoselective hydrogenation of ~ -substituted acetoacetic acid derivatives as hereinbelow defined.
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~ Therefore, in,accordance with its broadest aspect, the process of the present invention for preparing optically : acitive y-substituted ~-hydroxybutyric acid derivatives having the fomula ~ ~ V

~`` :l~ZS9~i6 wherein X is selected from Cl, Br, I and OH and R is a radical in straight chain, branched chain or cyclic configuration selected from ; the class consisting of alkoxy radicals having ~rom 1 to about 15 carbon atoms; '~
; alkylamino radicals having from abou~ 5 to about 15 carbon atoms; --:: :
~ ~c~ycloalkoxy radicals and cycloalkylamino :
radicals having from about 5 to about 12 carbon atoms;
phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms;
phenylamlno and phenylalkylamino radlcals hav~ng~the f~rmu}~

N-0-A~ and~ -N-~CH-0-A ~where Y and Z-are selected~ rom H,~an~alkyl~group having from to about~8 carbon `atoms,~;phenyl or benzyl and A~is~lselected~from~H, GH3, Cl and Br from correspondlng ~y-substltuted~acetoacetic acid esters or amides, comprises subjectlng~sald y -substituted acetoacetlc acid esters or~amldes to the~fermenta~tive~enzymatic action~of~a microorganlsm which elaborates L- ~-hydroxyacyl CoA dehydrogenase ~EC 1 1 1 35~, and : , , -eco~verlng the~desired optically active y-substituted- ~-hydroxybutyric acid derivatives , .: ~ , ;

.L~Z59S6 In particular, in order to prepare optically active y-substituted 3(~)-hydroxybutyric acid derivatives having the formula and 3(R) configuration OH H
X ~ R .

the process comprises subjecting compounds having the formula XCH2~C-CH2CR

wherein X and R have the above-identified meanings provlded that if R is an alkoxy radical it has from 5 to about 15 carbon atoms, to the fermentative enzymatic action of a microorganlsm which elaborates L- ~-hydroxyacyl CoA dehydrogen:ase [EC 1.:1.1.35] . , and:
recovering ~the~desired optically active 4-substituted 3(R)--hydroxybutyric acid derivatives from the fermentative reaction mixture.
It has been found that any microorganism which produces the desired enzyme is capable of functioning to catalyze the stereo`selective reduction. Particularly suitable are those microorganisms of the class Ascomycetes, the orders Endomycetales, Mucorales, Monlliales and Eurotiales, and the genus Saccharomyces. Particularly preferred is Saccha-romyces~Cerevisiae.

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,~ ' ~9S6 To prepare optically-active 4-substituted 3(R)--hydroxybutyrate esters containing 1-4 carbon atoms, it is necessary to use purified L- ~ -hydroxyacyl CoA dehydrogenase , [EC 1.1.1.35~ such as that of porcine heart, because intact microorganism possess interfering oxido-reductases of opposing configuration. Hence, microbial reduction of e.g. 4-chloro~
'. acetoacetic esters of 1-4 carbons produce 4-chloro-3-hydroxy-. butyrates of unsatisfactory optical purities.
. Therefore, the present invention also provides a process for preparing optically active y-substituted 3(R) hydroxy-l butyric acid derivatives hauing the formula and 3(R) config-¦ uration , G l~

l ~ .
wherein X is Cl, Br, I or OH and R is an alkoxy radical ! ~
'1 having from 1 to 4 carbon atoms whlch comprises . subjecting~compounds having the formula .

~C~C~2C~R
wherein X and R have the above-identified meaning to the enzymatic actlon of L- ~-hydroxyacyl CoA dehydrogenase ~EC 1.1.1.353 in purified form, and ~
~recovering the desired optically active y -substituted

3(R)-hydroxybutyrlc acid derivatives from the enzymatic ; ~ ~ reaction mlxture.
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- - 8 - 1~59S6 , The present invention thus provides compounds ~ having the formula and 3(R) configuration :: . . OH o x~ ~l~

. wherein X is selected from Cl, Br, I and OH and R is a radical in straight chain, branched chain or cyclic configuration selected from ~he class consisting of , ~ alkoxy radicals having from 1 to about 15 : carbon atoms;
,i ; : I alkylamino radicals having from about 5 to about 15 carbon atoms;
cycloalkoxy radicals and cycloalkylamino radlcals having from about 5 to about 12 carbon atoms ~ :
; phenoxy and phenylalkoxy radicals having f,rom 7 to about 14 carbon atoms ~ : ~ phenylamlno and~phenylalkylamino radicals ; having the formulas : ~ ~ y y z N-0-A; and -N-CH-0-A vhere~ Y and Z are :
: selected~from H, an alkyl group having from ;; - 1 to abou:t 8 carbon atoms, phenyl or benzyl and A lS selected from H, CH3, Cl and ~r.
~, ~:

, -` 9 ~ 259~;6 The optically-active y-substituted-L- ~-hydroxybutyric acid derivatives may then be reacted with trimethylamine to yield the corresponding ~ -trimethylammonium-L- ~-hydroxy-butyric acid derivative, which can be readily converted i into L-carnitine by treatment with aqueous acids. The following is a schematic o~ the reaction steps of this process.

X ~ ~ or~a~i~m >
or L-~-hydroxyacyl I Co~ dehydrogenase II
X~Cl, Br, I,: OH f~3 c~3-7 ! ~ , Ca3 3C- ~ C2E ~ ~3C-N
X~ C~ X C~3 : .

~ IV III
¦ L-Carnit~ne It has been found that the foregoing reactlon I ~
takes placè more easily i~ X = Cl. However, since the subse-quent reaction II --~ III occurs with better yields when X = iodine or bromine, it is preferred to prepare ~irst the Cl-derivative and then convert it into the correspondillg I- or~Br-derivative.
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- lo~ 259~6 The present invention also relates to an improved process which comprises first converting 4~chloro-3(R)--hydroxybutyrate ester to the corresponding 4-iodo- or

4-bromo-3(R)-hydroxybutyrates. For the sake of semplicity, reference will be hereinbelow made to the I-derivative. The iodohydrin (V) may be reacted smoothly with trimethylamine at room temperature to yield VI
which is readily converted to L-carnitine according to . . the following reaction sequence:
i Cl ~`~R ~ I ~R

R ~ ~ or este~
. 1~3:
3 MeOH l C~3 . : a ~ r c~3 N~ ~ i3 ~OR
~3 ~ OH fonn I C~3 L-carnitine ! :

The foregoing process as exemplified by the equation :
is subject to numerous variations. Regardless of which form is then made available, the~ester is reacted with sodium iodide in a suitable solvent such as 2-butanone, ~ acetonY, butanol, etc. The princlpal reaction desired at :
- ~ , 59~6 , .

this point in t~e reaction wit~ sodilL~ iodide i~ a dis-- placement reaction which orms the iodohydrin V without di~t~rbing the chiral c~nt~r on t~e ad~ac~nt carhon atom.
For thi~ reaction at l~as~ enough sodilLm iod~do i~ ~e-guirod to di~plac~ all chloride ~xom II. Ger.erally ~peaXing, a light oxc~ of sodium iodide i~ u~ed.
Th~ reaction o~ V with trimethyla~ine can bo carried otlt at mild tempexatur~ (e.g., 25C) (See S. G. ~oot~ and M. R. Boot3, J. Pharm. Sci., 64, 1262, 1~75), in a variety of ~olvent3 such a~ metha~ol ar ethanol containing an exce~s o~ trimethylamino. It i3 notowort~y that depend-ing on the alcoholic ~olvent used, there i3 e~tor-ex-,~ change t~king place. Eor exa~ple, wh~n methanol i use~
`,J as soLvent, ~-carnitine methyL e~ter i~ obtained in ~he~
reaction. ~hi~exchange reac~ion i~ adva~tagoou~ ~ecaus~
~; it i~ know~ that L-ca~itine methyl e ter can be tran3-~ formed directLy to the free-base form o~ ~-carnitine by ~i passi~g through an ion-~xchange-:column (0~ ) Esee E.
;~j Stzae~and J.~orenz, J~ PhYsiol. Chem.~ ~1966) 344j 276].
. , ~ :It;can~b- s-~n from~the~descr~ption of th~ foregoing I processes that a number of n~w and highly usefuI optically-active intermediate~are formed. E~pecially useuI are th- 4-iodo~3(R)-hydroxybutyric acid alXyl esters where . the alkyL groups ha~e from~six to:ten car~on atoms each.
i ~he octyl e~ter i~ particularly pref~rred.
Microorgani~ms which have t~e desired oxido-reductase activity are w~ known in the microbiological art and .~ any o~ such ~icroorganisms can be employed in conducting the process of the~prese~t invention (See,~K. Kieslicht Microbial Transor~ations of Non-Steroid Cyclic Com-pounds" (Georg Thieme Publi~her~, Stuttgart, 1975)) wi~th any of ~he genera of microorganisms specifically - - described herein being particularly applicable. Readily available nd in~xpensive microorganisms of ~he genus , .

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Saccharomyce~, e.g., brewer' 3 yeast, baker' 3 yeast a~dwinemaker'~ yeast (Saccharomyces v~ni) have been ound ts produce the L-~hydroxylacyl CoA dehydro~enase [EL 1.1.1.351 a~d to be eminently ad~antage~us in ~
carrying out the proce3s of the inventlon. ~he è~zyme d~cribed by S.J. Wakil and E.M. Barne~ Jr. i~
Comprehen~ive iochemistry Vo. 185(1971) p 57-104.
The 4-s~bstituted-acetoacet~c sub~tr~te can bo corporated i~ a n~trient medium of standard compocition in which such organi.m~ are cultivated and the usual condition3 of fermentation can then b~ ~mployed to effect the reductive tra~sfor~ation. Alt~rnati~ely, the active principle can be removed ~rom th~ growing culture of the microor~anis~, ~or~in~tancc ~y ly~i~ o~ the coll~ to relea~e th~ enzyme~, or by su~pension of the resting c~
in~ ai fre h~aq~eou~ sys~em. I~ any of the~e technigues the ~-keto function wilL be seIectively reduc~d, 50 ~on~
a~ the active enzyme~elaborated ~y the microorganisms is pre3ent in th~:medium. Of course, ~ho temperature,.
time and~pressur- conditions under which the co~tact:~f the 4-~ub~titu~-d-acetoacetic deri~ativ~ with ~he reduc-tive enzyme i carried out are i~terdepoAde~t as:will be apparent to tho~e skilIed in the art. Eor instance, with ge~tle heating and at atmos~heric pre~sure the t~me reguired to effect the roductive con~ersion will be les5 than if it progse~se at room temperature under condi-tions otherwi3e the same. Of course, neither tempera-turè, nor pressure, nor time, ~ould be ~o great that it results in the substrate bei~g degraded. ~Where a growing `culture of the or~anism i5 bein~ used, the process condi-tions s~ould also be sufficiently gentle CO the organism i5 not killed before it elaborates suf~icient hydrolytic enzymes to permit ~he reaction to proceed. Generally, at atmospheric pressure, '~he temperature can range .rom .

X5gS~
_ 13 -about 10C to about 35C, and the time from about 12 hour~ to a~ou~ 10 day~.
:. In the following exa~pl~s which a~e pre~ented to .i. illustrat~ thi3 i~vention a~d ar~ not to ~e con3trued as ; - limiting the cop~ of the appended ~laim3, the y~halo ~ ac~toacetic acid derivatiYe ~ub~trat~ to be subjected ~o -' mlcrobiolog~cal reduction w~r~ pr~pared from diketene ~ . ~ccordi~g to the gsneral method o~ C. D. ~u~d and ~. L.
:. Abernethy (J. Am. Chem. Soc., 62, 1147, 1940) for ~he chloro-ac~toacetic derlvatives and F. Chick, N. T. ~.
, WiI more~lJ. Chem. Soc., 1978 ~191~0)] for th~
1~ y-~romo-acetoacetic dcrivatives via thc followinq re-:1, action ~egue~ce:

=c~ ~2 _2 > XC~2C-C~
: ~ O--C=O- /
: ;' ~
iketene ~ RNK OR
~ ` ;R ~ ' 1I R
',' ,,, XC~2CC~2C-N~ ' XC~i2C ~2COR
'. :~ where X = Cl or Br ~. : . Y =~ or alXyl : ~ a~= as d-fined previously : - Alternatively, if desired, the y-halo acetoacetic! ` ac~d derivatives ca~ ba prepared from y-halo acetic es~ers via:a conventional Grignard reaction. For ex-ample, Y-chloro acetoacetic octyl èster was readily ~ prepared by refluxing:y-chloro octyl ester with two :~ equivalents of magne~ium in ether for 48 hour After removal:of the solvent the acetoacetic octyI ester was recovered in about 70~ yield.

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~., ~. . . . . .

S9S~, ?

y-~ydroxy acetoacetic acid derivatives were prepar~,d from their correspondint~ y-~romoacetoacetic acid deriva-tives by s'cirring in a dioxane-wa'c~r ( l: l ) solutaon containing CaCO3 at 25C ~or 12 hour~.
- Eac~ of the products produced in accordance with ~e ~;1 following example~ was identified as to ~tructure through :' ~he U8~ 0~ nuclear magn~tlc re~onanc~ (nmr), infrared . spectra, and by thin layer chromatographic mobilitie~.
Th~ optical purity and the ab~olute configuraton o~ the .. products were established by their co~ver~io~ into 7. L-carniti~e a well a~ by conver io~ into their est~rs ¦, whi~h a~e~readily analyzed by nmr spectrometry, and ,., optical rotation.
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Exam~le 1 !Yeas~_~
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(~)4-Chloro-3(R)-hydroxybutyric acid octyl ester was pre-1 paret~ as~follows:
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~ C~o .1 .
I r, A. Fermentation. Surface growth from a one week old agar slant of Can,dida ke~fr. NRRL Y-329, grown on agar of the following composit~on:
Gms Agar............................ 20 Gluco~e......................... 10 YeaQt extract................... .2.5 K2HP'04.......................
Distilled water, q.s. 1 1~ ter ~St~riLized 15 min at 20 p,~,1.

2S9S~

was su~pended ~n S ml of an 0. 85% ~alino 901utlo~.
One ml portion~ o~ thi~ sucpen io~ wero u~ed to inoculat~ a 250 ml Erlen~y~r fla~ (F-l ~tag~) ~: co~taini~g 50 ~1 o~ th~ ~ollowing ~Q~iUm (Vogel'~
~diu~):
., ~ Gm~
: : Y~a~t ~xt~act................... 5 ~ Ca~a~ino ~cids.................. 5 : Dext~os-........................ 40 Na3-citsat~-5 1/2 ~ O........... 3 g 2P04~ ....... "..... , 5 g . N~ N03......... ----------- 2 g `! cacl2.2~20.................... o.l ~
M~S04.7~2~ . O.2 g Trace elament solution........ 0.1 ~1 : Di~tilled water, ~.~. 1 liter : ; p~ 5.6 (steril~ze~ for 15 min at 30 p.s,i.

Trace element solution Gm/100 ~1 Citric acid-1~20................... 5 : : ZnS4-7~20- ------- ............... 7 (N~4)2(54)2 6~2-uS04-5~20------.-................. 0.25 M~S04.1~20......................... 0.05 B03-----------------------. 0.05 NaH2M4-2~2---------............. o.05 ~ Tho flask was incubated at 25C on a rotary shaker `~. (250 cycles/min - 2" radius) for 24 ~ours, afte~
which ~ 10% by volume transfer was made to ano~cr 250 ml Erlenmeyer flask (F-2 stage) containing 50 ml ~: of Vogel's medium. A'tor 24 hours o' incubation on a rotary ~haker, 150 mg of y-chloroacetoacetic ac_d octyl ester in 0.1 ml of 10,~ ~.ween 80 was ad~ed.

* Trademark ~ A

, 3L2Z595~
, - 16 --- ..

The E-2 stage ~la~k was then incubated ~or an ad-dltional ~4 hour~ under the conditions u~ed in the incubation of th9 Fol stage flask~
;. 3. I~olation. Twe~ty-four ~curs after the addition o~ the . y-chloroacetoa~cotic acid octyl ~tor, th~ cell3 were j ~ removed ~y cent~i~ugatio~. Tha supernatant was . exhaustively ~xtracted with 50 ml of ~thyl acetat~
¦ thr~ time~. ~he ~thyl acetate wa3 d~ied over , . ~ Na2S04 and -Yaporat~ to aford a~ oily r~sidue (186 ! mg)~ he residus was dis~ol~ed in 0.5 ~1 of th~
j ~obilc phasc and added onto a column (l x 2S cm) of ¦,. ilica ~el ( ~ -kie3elgel. 60). TSe column wa~ ~.luted:
:wit SXelly B-~t~yl ace~ate (8:1) .an~ 14 ml frac .; tionR were collected. Fractions 6 and 7 containin~
. : the desired pxod~ct were pool~d and concentrat~d to dryne s~yi~Iding 120 m~ of crystall~ne re~idu~.
: : Recrystallization fro~:ethyI acetatei-hexane:afforded : : ~107;mg~:o 4-:chloro-3(R)-hydroxybutyric acid octyl , : ~ etster, [a~] ~ ~13-3 (c, 4-45)~ (~C~C13); pmr~(8CDC13) . : 0.88.~[3~, tr. distortional,. C-~3-(C~2)~-]; 1-28 [10~, ~ C~2:)~5~ 1-65 (2~ m~ C-~2-C~2-~ );
¦. : (2~,~d, J = 6 ~2, - ~ -C~2-COOR); 3!Z2 (1~ br., OH
I -0~); 3.60 (2~1, d, J = 6 Elz, ClCE2-C~-R~; 4.20 (3H, 1}~
, ~2 ~ C~2 and -~ -C~2C~2) Anal. Calcd for t ~ ~ O
C12X2303Cl:~: C, ~57.47; ~I, 9.25. Found: C, 57.52;
9.07. [TLC Rf - 0.5, 3rinkma~n silica gel plate, 0.25 cm EM; Skelly B:ethyl acetate ~5:1).]

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, ~2595~

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Exam~le 2 Restin~ Cells. One hundred grams o~ commercial _esh baker' 8 yeast Sacc~aromyce~cerevisiae (Red Star) was suspended in 250 ~1 of tap watar to which was added 10 g - of 3ucrose and 3.6 g o~ y chloroacetoacetic octyl ester.
After the contents wer~ incubatcd at 2SC on a rotary haker (250 cycl~s/~inute - 2" ra~iu3) for ~4 hours, an additional 10 g o suGrose wa~ added tc the fla~k and the reaction wa allowed to proceed for another 24 ho~r~. -I - 2h- cells wero then removed by filtration thrau~h a pa~I of celite. The c~ wcre washed with wate~ and ethyl acetate~.~ The washings were combined wi+~h th~ fil~rate I and exhaustively extracted with ethyl acetate. ~he ethyl ; acetate layer wa~ dried over MgSO4 an~ evaparated to giYe a~ oily residue, which was chromatographed over a silica ~el column to yield 2.52 g of 4-chloro-3(R)-hydroxy~utyric : acid octyl estar, as a low mel~ing solid; ~a]23+13.2 ~c, 4.0, C~C13~)- ~
:, ' ~xamDle 3 .
(~4-C~loro-3~R)hydroxybutyrie acid benzyl ester was prepared as follows:
;.;, Cl ~ - > Cl ~

` A. Fermentation. Surfaca growth from~a_one week old , ~ agar slant of Gliocladium virens A~C 13362, grown on agar of the following composition:

~'`" .

`

x~

i -`;
Gms . M21t extract.......................20 , Gluco3e............................20 Pepto~e.......................
: Aga~........................... ~ 20 Distilled watsr, q.~. l liter j (St~rilized 15 min at 2a .
va~ suspended in S ~1 oX an 0.85X saline solution.
1~ O~e ~l po~tions of thi~ suspensio~ were used to ;1 inocu~late a 250 ml E~lenmeyor fla3k (F-l stage) .~ containing SO~ml of the following medium (Soybean ! dext~o3e~medium):
, Soybean meal................... 5 j ' ~ D~xt~ose....................... 20 g NaCl........................... 5 g . ~ K~2~Po4~ --,-------............ 5 g . ~ Yeast.... :............. :....................... 5 g. ;~ Water.... ~..................................... 1 1 . .
. ~ : : p~ adju3ted to 7.0:
., .:
. ~ Autoclava~ at ~15 p. 5. i. for 15 minutes . 1 : : : ~ : , .
: ~ Th-~flas~ wàs~incubated~at 25~C on a rotary shaker , ~ (250~cycle~Jmin~- 2n radius~ for 24 hours, after - , :which~a 10%:~by volume transfe~ was mad~ to another :250:ml:~Erlenmeyer flask (F-2 stage) containin~
I : 50~ml of soyb~an dextrose~me~ium. Aft~r 24 hours of lncubation~on:a rotary sha~er,~ l~0 ~g o~
: Y-chloroacetoa etic benzyl est`er in 0.~1 ml of :1 . lQ~. Tween~:80 was added.~ The F-2 stage flask~was then incubated or an addit~onal 24 hours under : ehe conditions~used in the incubation of the F-l stage ~lasXs.
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!356 .

B. Isolation. Twenty-four hour~ at~r the addition o~ the - Y-chloroacetoacetic benzyl ester, the mycelia were removed by filtration. Th~ filtxate wa3 exh~ustlvely ~xt~acted with 50 ml o~ ethyl ac¢tat~ three time~.
Th~ e~hyl ac~tate lay~r wa~ dried over MgS04 and concentrat~d in vacuo ~o yi~ld a ~idue (16Q mg).
The~ res~due wa$ chromatographe~ ovor a $il~ca gel : (MN-~ie~elgel 60) column ~1 x 25 c~). ThQ column .... .
" was eluted with Skelly B and ethyl acetate (lO:l) i . ~ ~nd 12 ml f~action~ wer~ collecte~. Fractio~ 16 containi~g the desired product wor~ pooled and co~cent~ated to dryness to afford 115 mg of 4-c~l~oro-3(R)-hydroxybutyric acid~benzyL ester, I ~a]D3~7 (c, 5.26; C~C13);: pmr (~ CDC13) 2-65 (2H, 'I ~, J =-6 Hk, ~-C~2Coor),. 3.20 (~, br, -0~); 3.54 ! : o~
I : (2~,~ d, J - 6 ~z, Cl-C~21 )i 4.20 (lH, =, -C~2- ~ -C~2-~, S l2 ~2a, s, -~-o-C_2C6~5); 1.31 ~5F' s, fi~e aromatic i : proton-~). Anal.calcd.for C~ 303Cl: C, 57.77; ~, : ~ . ~5.73-. Found::~ C,~57.64; H, 5.67. [Tr silica gel EM 3rlnkmann p~late, 0.25 cm, R~ = 0.43, Skelly B-ethyl ace.tate (5~:1? -1 !` ' - ~
I : ExamDles 4-23 `~ m e procedure of Example 1 was repeatad with each of the organi s listed in Table:l except that -chloro-~ acetoacetic~acid octyl ester was added at a concentration : ~ of ~1 mg/ml~. Con~ersion to the desired product (~)4-chloro-3(R)-hydroxybutyric acid octyl ester was obta~ed. The procedures of these Examples were repeated by continuously adding the : substrate to the yeast media. The weight ratio substrate/yeast was about 1:1.5 with excellent conversion into the desired product.

~ 25956 ~.' i - ExamDle~ 24-4~

; The procedure of Example 3 wa repeated with each of the or~ani~s li~ted in Table 2 axcept that y-chlQr~o acetoac~ti~ -octyl ester (1 mg/ml) was used. Tran~-~- formation to th~ desir~d compound (~)4-chloro-3(~)-hydroxy~uty~ic ;. acid octyl ~er wa~ o~tained.

,~, Exa~Dle~ 4~-6B -~
!; ' .
i She p~o~dure o~ Ex~mple 1 was repea~ed with each of .. th~ organisms ~i3t~d in Tabl~ 1 excep~ that y-chloro-acetoac2tic acid ben2yl: sster ~1 mg/~1) wa~ u3ed as the ,- ~ubstrate. Con~orsion to the de~ired pro~uct.(+)4-chloro-,i 3(R)-hydroxy~utyric acld benzyl e3t~r was ohtai~d.
.
' : Exam~le 6~-~3 ,~ ' , ' The~proc~dure of Example 3 wa ~repeated with:each o~
t ` ~the organisms listod i~ Table 2 usi~g ~-chLoroacetoacetic . ~ acid benzyl~e~ter (1 ~ 1) a~ substrate. Transfo ~ation . ,~ to~t~e~de~ired~co:mpound~ )4-chloro-3(R)-hydroxybutyric - ;ac~d benzyl ~ster was obtaine~.
. ~ ~
7 ExamDle g4 1, . (+)4-Chloro-3(Rj-hydroxybutyric acid anilide wa3 prepared in accordance wi~h th~ procedure o~ Example 2 axcept that 4-chloroacetoacetanilide was used at a con-centration o 1~mg/ml.

Cl ~ ~ ~ Cl ., ... . , ........ . ~ ... .......
. . ~ .

2~95~

~: ;
, i a~ the substrate for ~he conversion into ~he desired optically-ac~i~e product, m.p. 110-111C; ta]23~17.5 (c, 3 0 f~ ' , C~C13 ~; pmr ( ~ CD3CCD3 ~ 2 - 67 ( 2~, d, J = 6 }~z O~C--~2-~ONHR), 3 . 66 (2~, d, J = 6 Ez, clca2cao~-R), 4. 43 ( 18, ~, ~CH 2-C~OE~-C~2- ), 7 . 03-7 . 44 ( 3a, m, aromatic : protons, meta and para), 1.69 (2~, d, ;J = 6 ~z, aromatic proton3, o~tho~, 9.24 ~1~, br, ~-N-~). hnal. calcd for , Cl0~12NOzCl Cj 56.21; ~, 5.66. Found: C, 56.17; ~,

5 . 47 .
'I "
Exam~les ~5-114 ,'' :
T~e proc~dure of Example 1 wa3 repeated with each of th~ organism~ li ted in Tabla 1 except that Y-chloro-I acetoacetanilide was added at a concentration of 1 mg/ml.
,' ~In.~a11~cases con.version to the desired produc., :)4-c~loro-3(R)-hydroxybutyric acid anilide was obtained.
. 1 . .
.1 Exam21es llS-139 . .
~; ~he:procedure ~of Example 3 was repeated wi~h the . :: organism listed in Table~2. Y-Chloroacetoacetanilide was introduced at a concentration of 1 ~gjml. In these cases, conversion to~the desired (~)4-chloro-3~R)-hydroxybutyric acid anilide was achieved.

Exam~les 140-lS9 ~ ~ The procedure:of Example 1 wa5 repeated with the : organisms listed in Table 1 except that Y-bromoaceto-acetic acid octyl ester (l mg/ml) was used as ~he sub-str~te. Conversion to the desired produc~, (l)4-bromo-3(~)-hydroxybuty-ic ~cid oc~yl ester was obta-ned.

,~ .

L22595fi EXamD1eg 1 60- 184 ~ he procedure of Example 3 was repeatsd with the organisms li~ted in Table 2 except th~t y-bromoaceto-acetic acid octyl o~ter (1 mg/ml) was us~d. Conversion :~ to the desired product, (~)4-~romo-3(R)-hydroxy~utyric ' acid octyl est~r wa~ obtained.

j Exam~les 185-204 ~, ~, Tha procedure o~ Exa~ple 1 was repeat~d wi~ th~
oryani~m~ listed in Table 1 except that Y-~romoaceto-~, acetic acid benzyl e~tex (1 mgjml) wa~ u ~d a~ the sub-'. strate. Conversion to the desir¢d product, (~)4-~romo-3~R)~-hydroxybutyric acid be~zyl ester was obtained.
1,, EXam~1eg 2_5-229 The procedure o~Example 3 wa repeated with the ~ organisms licted:i~ TabLs 2 except that Y-bromoacetoacatic !` acid~ benzyl ester (1 mg/ml)~was used.~ Conversion to the : desirod:product, (~+j4-bromo-3~R~-hydroxybutyric acid enzyl~ester was obtained.

Examl~les 230-249 , The procedure of Example 1 was repeated with ~he organi3ms llsted in Table 1 except that y-~romoacetanilide (1 mg/ml) was used as the substrate. ~Conyersion to the desired (+)4-bromo-3(R)-hydroxybutyric acid anilide was obtained, `: :

.

.

gs~

Exam~le3 250-274 , The proceduxe of Example 3 was repeated with the - organisms listed in Table 2 except that y-bromoacetanilide ( 1 mg/ml) waY u~ed a~ the substsate. ~on~ersion to the ^ d~sired (~4-bro~o-3(R)-hydroxybutyric acid anilide wa3 obtained.
.... .

Exam~les 275-294 , ~he procedure of Example 1 wa~ repeatod with the -¦ organism li~te~ i~ Tabl~ 1 exczpt that Y-hyd~oxyace-toacetic oc~yl ester (1 mg/ml ) wa~ used a~ the sub~tra.~e.
ConverYion to the desired 4-hy~roxy-3(R)-hydroxyb~tyric acid octyl ester wa obtained.
,. , ! xam~les 295-319 !-! ~ ~he procedure of Example 3 was repeated with the ,j; organisms~ listed in Table 2 exeept that Y-hydroxya~e--; toacetic~octyl ester (1 mg/ml) wa5~ us~d as the substrata.
Conversion to the desired 4-hydroxy-3(R)-hydroxybutyric j acid octyl ester~was obtainet.
.
Exam~les 320-339 ;
The procedure of Example 1 was rapeatad with the organi~ms llsted in $ab1e 1 excapt that Y-hydroxyace-toacetanilide (1 mg/ml) was used as the substrate.
; ` ~onversion ~to the desired 4-hydroxy~3-~ hydroxybutyric acid anllide was obtained.

~ .
.

-~2~i9S~

~ " ' .

: . Exam~les 340-364 The procedure of Example 3 was repeated with the organisms listed in Table 2 except that y-hy~roxyace-toaceta~il$de (1 mg/ml~ was ~sed a~ the substrate.
Convers~on to t~ de~irod 4-hydroxy-3(R)-hydroxybutyric . -~ acid anilide wa3 obtained.

~, Y~ ExamPle 365 ': :
. Methvl-4-chloro-_1R)-hvdroxybutYrat~
: :..

C ~ ~ C ~ ~3 ;!
~i:
: VII VIII
j.
,:~
1: :
~, Yethyl-4-chloroaeetoac-tate~(VII) ~100 mg) wa5 . I incubated with`2 9 uni~5 of porcine heart (EC 1.1.1.35), ~` ~ B-hydroxyacylcoA dehydrogenase (S$sma, ~4626), and 1` ~ 1.36 ~g of NADH. (Sigma, 90%~ in 30 ml of 0.1 M sodlum -`!' phosphate ~u~fer, pH 6.5.
¦ : ~Ator 30 hours at 25C, the reaction mixture was ex-I ~ tracted four ~ime~ with 30 ml of ethyl acetate. The organic layer was dried over sodium sulfate a~.d ~as evaporated to dryness under reduced pressure. The resi-due (90:mg) was chromatographed ove~ a silica gel (12 g) column~(l.3 x 34 cm). The column was eluted with a solvent system consisting 0f SXelly 3-ethyl acetate ~8:1) and 20 ml fr~ct~ons were collected. Fractions 9-11 contained the desired .~ethyl-4^chIoro-3(~)-hydroxy~utyrate (VIII) as : ~ r~vealed by T~C, ;a]23 ~ 23.5~ (c, 5.2 C~C13) were pooled.

. `
.

---` ~2~9s6 E~am~le 366 The procedure of example 365 was re~eated using ~thyl-4-chloroacetoacatate a3 ~he subst~ate to a~ord ~ . e~hyl-4-chloro-3~R)-hydroxybutyrate, 1c]23 ~ 22.7 ~c, :. 4.7 C~C13).
, ,~, ~l~ Examle 367 1, . , The procedure of axa~ple 365 wa~ repeated usin~
n-pxopyl-4-chloroacetoaeetate a~ the ~ubstrate to afford n-propyl-4-chloro-3(R)-hydroxy~utyrate, ~~3 ~ 21.5~ (c, ! s.~ ~Cl3).
~1~ . Exam~le 362 ,.; :
,. The p~ocedure of example 36~ was repeated using 'i` n-butyl-4~chloroacetoacetate as the substra~e to afford n-butyL-~-c~ioro-3(g)-hydroxybutyrate, ~a]23 + 20.1 (c, . 3.1, CaC13).
, ~ ~
General ~ro-~e ~ re f~r t~ e~n er-lo~ o~ 4~halo-3(RL-hYdroxu-butYrlc:-sters and amides ~nto ~Carnitine.
,. . ~.
: `~. ~ Example 369 `!`` ' ~ A mix~ture of 4-chloro-3(R)-hydroxyl,utysic acid oc~yl :` ester (1.5 g), ethanol (3 ml) and trimethyla~ne (25 wt %
~` 301ution) in~water (3 ml) was heated at 80-90C for about 2 hours. The solvents and excess trimethyl~ne were evaporated to dryness in vacuo to yield 1.8 g of c~ude ' , .

2~g~

.

residue. The crude product (1 g) was heated at 80-90C
in a solution of 10% ~Cl (7 ml) for 1.5 hours. After evaporation o~ the sol~ents unde~ reduced pressure, the crude product wa~ axtracted twice with ab~olute ethanol (10 ml3 and ths ~thanol was evaporated in vacuo. ~he cry~tallin~ residue wa~ dissolved in a ~maLl quantity o~
ethanol and th~ L-carnitine chloride was precipitated by the addition o~ ether in good yield (320 mg~, m.p. 142 (dec.); ~] - 23.7 (c, 4 5~ ~2)' The L-carnitine chloride can be readily converted to.
. .
the pharmaceutically pre~err~d L-carnitin~ inner salt by ion exchange mean3 as is well known in the art.
' Example 370 Oct~1-4-iodo-3(R~-hvdroxYbut~rate (IX~

., , j.:. ~ ~ ~ O

-':. ~ X ~17 ~ ~ ~ ~17 II IX
~, .
~` ., A mixture of octyl-4-chloro-3(R)-hydroxybutyrate (II) (1.~26 g), and anhydrous NaI (1.2g) in 15 ml of methyl ethyl ketone was refluxed for 24 hours. The mixture was rotor evaporated and reacted with ether (100 ml) and:water (50 ml). The organic phase was separated and washed with 10% scdium thiosul~ate ~olution (lSa ml), brine (150 ml) and drled over anhydrous sodium suL'ate.
The solvent was evaporated under reduced pressure to ar~or~ 1.762 g of IX as a pale-yellow oil; I~ (thln Z~;~56 o film) 3460 c~ 1 (o~) ~nd 1730 c~ 1 (est~r C=0); ~M~
(CDC13) _ 3.93-4.27 (~, 3~), 3.17 (d, 2~), 2.50 (d, 2~), 1.50-1.87 (~, 2~, 1.30 (b~, 12~), 0.~3 ~m, 3~).
o Transformation of oct~l-4-iodo-3(R)-h~droxY~tYr~te (IX~
~nto ~-earnitine ~ o N-C~3 ; I~`OC8El~
. T C~3 C~3 IX : X

~ 3 ~ 0 ,...... . C~3 1~ ~

. C~3 o-L-Car~itine To ~ olutlon of lX (1.593 ~ $A ~etha~ol (15 m'~
was added at 25X olution of aqueous trimet~ylamine (8 ml). The mixture wa~ ~tirred at 27C for 20 hours. The ~olvents and t~e exc~s~ trimethylamine were evaporated off under reduced pressure to ~f~ord a semi-crystalli~e soli~, X. Thi5 r~sidue wa washed wi'lh ~mall amounts of ether to remov~ the octanol a~d the~ di3solved in wat-r and passed over a Dowex l-x4 ~0~ for~ - 50-100 mesh, column volume (2.5 x 15 cm). The column was washed w~th di~tilled water. Removal of the 501ve~.~ i3 vacuo * Trademark G l` ' .
-: from the first 200 ~1 of ~he eluate save ~-carnitine as a white cry~talline solid (490 mg, 6~ yield) ~a~23 _ 2g 29 (c, 6.5 ~2)' ,, .
i Exam~le 371 ¦i ~ $he pro~dur~ of ex~mple 370 was r-peated u~i~g : hexyl-4-chloro-3(R~-hyd~oxy~utyrate to yield hexyl-4-~iodo-3(R)-hydroxy~utyrate, which wa then con~rted to , L-carnitine.
'``
ExamDle_372 i~
I ~ ~ me~: procedure of exa~pl~ 370 was repeat¢d u~in~.
! hepty~-4-chloro-3(R)-hyd~oxybutyrate to yield heptyl-4-: ~ : iodo-3(a)-hydroxybutyrate, *hich was then conver.ted ~o ; ; ~ L-carnitine.
,.
; ~ am~1~ 373 : ~ Th-~procedur-~of ampl-:370 wa~ rep-ated using .~ decyl-4-`chl;oro-3~(;R)-hydroxybutyrate to yield~decyl-4-i: : ~iodo-~3(R:)-hydroxy~utyrate, whcih was then;co~verted to i;~ : L-carnitine. ` : ~ :
. ~
. ~ ; EXamD1e 374 :
~,. Th¢~procedure of~oxampIe 370 was repeated using ¦` methyl-4-chloro-3~(R)-hydroxybutyrate (VIIIj to~i~e methyl-4-iodo-3(R)-hydroxybutyrat2, which was then con~erted into L-carnitine.
.

~:: :

.

:~ :

.

~2~i9S~i;

, :
Exam~le 37~

The procadure of example 370 was repeated u~ingethyl-4-cAloro-3~R)-hydroxybutyrat~ to give ethyl-4-iodo-3(R)-hydroxybutyrate, which wa3 then conve2ted into L-carnitin~.
~.
, . EXa~P1~; 3?6 ,;
i The procedure of exampl~ 370 wa rspeated using n-propyl-4chloro~3(R)~hydxoxybutyrate to gi~e n-propyl~
4-io~o-3(~)-hydroxybutyrate, w~1ch was then converted ! ~ into L-carnitine.
, ~
, Exam~le 377 The procedur~ of exampl- 370 w~s repeated using : :n-~utyl-4-chloro-3(R~-hydroxybutyrate to gi~e n-butyl-4-iodo-3(R)-hydroxybutyrate, which was then converted . ~ into L-carn1tine.
Representative:yeast~ that produc~ the desi.red :~ enzymc:are listed in Tablo. 1 and representative fungi are 11`Jted in~Table 2.

1:

;

: ~

lX259Sf~

able 1 ~Yeast~ ?

1. Candida lipol~tica NRRL Y-1095 2. Ca~dida ~seudotro~icalis NRRL Y-1264 3. MYcoderma cerevi_iae N~RL Y-1615 4. Torula lactosa NRRL Y-32g 5. Geotrichum_candidum 'NR~L7 Y-552 : 6. Hansen~la anom~ala NRRL Y-366 : 7. an~enula sub~elliculosa NRRL Y-1683 ~, 8. Pichia alcoho-oe~ NRRL Y-2026 ,' 9. Saccharomvces cere~i iae NRRL Y-12,532 1 10. Saccharomvces lactis NRRL Y-1140 ¦ 11. 2y~osaccharomYces ~riorianu~ NRRL Y-12,624 12.
I Saccharomvces acidifacien ~ NRRL Y-7253 . 13. Rloe~kera cortici3 ATCC 20109 -- 14. CrvDtococus masceran-~ ATCC:24194-15. o~otorula s~. ATCC 20254 : ' l~. Cand~da~albicans ATC~ 7~2 ' ~ 17. ~Dipodascus albidus :ATCC 12934 . 'i7 ~ 18. Saccharomvces~cerevis~ae (comm'ercial. Red Star) 19. Rhodo~orula~rubra~ NRR~ Y-lS92 . 1. 20. `Oosora:lacti3 ~:ATCC 14318 ¦ NRRL Northern~Regional Research Lab. at Peoria, Illinois.
: . ATCC:- American Type ulture Collection at Rockville, Maryland.

: :

, .

\

, ~ - 31 -.. .

.. Table 2 (F~nc 1. Gliocladium viren3 ATCC 13362 : 2. Caldariom~ce3 fumaqo ATC~ 16373 3. Linder~na ~enni30~0ra ~TC~ 12442 4. er~illu ochraceus NRRL 405 5. Trichoderma ligno~um ATCC ~678 , .

6. ete~oce~halum autantiacum ATCC 16328 I: 7. Entomophthora coronata NRRL 1912 ¦ . 8. ScoPulariopsi~ co~s~antini NRRL 1860 , 9. 2yqorhYnchu~heteroamus ATC~ 6743 . 10. Sco~ularioDsis~bre~icauli~ NRRL 2157 i ~ .
, 11. P~izo~us ar~hizu~ ; 2286 . 12 . Pen~ ei~lLium~thomia NRR~ 2077 ' 13. Mu~or h~emalis (-) NRRL 4088 j ~ 14.: 8~ochlamY~ nivea A~CC 12550 ~j 15~. Penici~llium ~atulum N~RL I952 16~. : etarrhiz~um anIsopliae A~CC 24942 17:~. PeniciIlium i~landic~m: ATCC I0127 . j ~ 1~3.~Cunnin~amella~ele~an~~ ATCC 10028a l9.~:Cunnin~ham~lla ~echlnulata ATCC 11585a . . 20. As~er~illus fumi~atus~ ATCC 16907 : ~ ~ 21. As~er~ilIus amstel;odami N~R$ 90 22~.~ Gliocladium ro~e~m ~ATCC 10521 ~u~ ~ : 23. As~erqillus ~i~anteus ATCC lOOS9 I'. 24. Absidia:blaX~le~ana AT~C l0148b Ij~ 25. Penicillium rocueforti; NRR 849a ' ~ .

:
, ' ' .

Claims (24)

WHAT IS CLAIMED:
1 . A process for preparing optically active .gamma.-substituted .beta.-hydroxybutyric acid derivatives having the formula wherein X is solected from Cl, Br, I and OH and R is a radical in straight chain, branched chain or cyclic configuration selected from the class consisting of alkoxy radicals having from 1 to about 15 carbon atoms;
alkylamino radicals having from about 5 to about 15 carbon atoms;
cycloalkoxy radicals and cycloalkylamino radicals having from about 5 to about 12 carbon atoms phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms phenylamino and phenylalkylamino radicals having the formulas where Y and Z are selected from H, an alkyl group having from

1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH3, Cl and Br from corresponding .gamma.-substituted acetoacetic acid esters or amides, which comprises subjecting said .gamma.-substituted acetoacetic acid esters or amides to the fermentative enzymatic action of a microorganism which elaborates L- .beta.-hydroxyacyl CoA dehydrogenase [EC 1.1.1.35], and recovering the desired optically active .gamma.-substituted- .beta. -hydroxybutyric acid derivatives.

2. The process of claim 1 for preparing optically active .gamma.-substituted 3(R)-hydroxybutyric acid derivatives having the formula and 3(R) configuration wherein X; and R have the above-identified meanings, provided that if R is an alkoxy radical it has from 5 to about 15 carbon atoms, which comprises subjecting compounds having the formula wherein X and R have the above-identified meanings to the fermentative enzymatic action of a microorganism which elaborates L- .beta. -hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] , and recovering the desired optically active 4-substituted 3-(R)-hydroxybutyric acid derivatives from the ferment-ative reaction mixture.

3. The method of claim 2 wherein the microorganism is selected from the class Ascomycetes.

4. The method of claim 2 wherein the microorganism is selected from the orders Endomycetales, Mucorales, Monillales or Eurotiales.

5. The method of claim 2 wherein the microorganism is selected from the genus Saccharomyces.

6. The method of claim 2 wherein the microorganism is Saccharomyces cerevisiae.

7. The method of claim 2 wherein the .gamma.-substituted acetoacetic acid derivative subjected to fermentative enzymatic action is .gamma.-chloro-acetoacetic acid octyl ester.

8. The method of claim 2 wherein the .gamma.-substituted aceto-acetic acid derivative subjected to fermentative enzymatic action is .gamma.-chloro-acetoacetic acid benzyl ester.

9. The method of claim 2 wherein the .gamma.-substituted aceto-acetic acid derivative subjected to fermentative enzymatic action is .gamma.-chloro-acetoacetanilide.

10. The method of claim 7 wherein the microorganism is Saccharomices cerevisiae.

11. The method of claim 8 wherein the microorganism is Saccharomyces cerevisiae.

12. The method of claim 9 wherein the microorganism is Saccharomyces cerevisiae.

13. The process of claim 1 for preparing optically active .gamma.-substituted 3(R) hydroxybutyric acid deriv-atives having the formula and 3(R) configuration wherein X has the above-identified meanings and R is an alkoxy radical having from 1 to 4 carbon atoms which comprises subjecting compounds having the formula wherein X and R have the above-identified meaning to the enzymatic action of L- .beta. -hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] in purified form, and recovering the desired optically active .gamma.-substituted 3(R)-hydroxybutyric acid derivatives from the enzymatic reaction mixture.

14. The process of claim 13 wherein said L- .beta. -hydroxyacyl CoA dehydrogenase [EC 1.1.1.35] in purified form is that isolated from porcine heart.

15. Compounds having the formula and 3(R) configuration wherein X is selected from Cl, Br, I and OH and R is a radical in straight chain, branched chain or cyclic configuration selected from the class consisting of alkoxy radicals having from 1 to about 15 carbon atoms;
alkylamino radicals having from about 5 to about 15 carbon atoms;
cycloalkoxy radicals and cycloalkylamino radicals having from about 5 to about 12 carbon atoms phenoxy and phenylalkoxy radicals having from 7 to about 14 carbon atoms phenylamino and phenylalkylamino radicals having the formulas where Y and Z are selected from H, an alkyl group having from 1 to about 8 carbon atoms, phenyl or benzyl and A is selected from H, CH3, Cl and Br whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

16. The compound of claim 15 wherein R is a straight-chain alkoxy radical having from 1 to 10 carbon atoms whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

17. The compound of claim 15 wherein R is OC10H21 whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

18. The compound of claim 15 wherein R is OC8H17 whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

19. The compound of claim 15 wherein R is OC7H15 whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

20. The compound of claim 15 wherein R is OC6H13 whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

21. The compound of claim 15 wherein R is a lower alkoxy radical having from 1 to 4 carbon atoms whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

22. The compound of claim 15 wherein R is selected between phenoxy and phenylalkoxy substituted with lower alkyl, halo and nitro group whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

23. The compound of claim 15 wherein R is benzyloxy and X is selected from C1 and Br whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.

24. The compound of claim 15 wherein R is phenylamino and X is selected from Cl and Br whenever prepared or produced by the process of claim 2 or by any obvious chemical equivalent thereof.