THE JOURNAI, OF BIOLOGICAL CHEMISTRYVol. 241, No. 13, Issue of July 10, pp. 3028-3035, 1966

Printed in U.S.A.

The Stereochemistry of the Conversion of D and L

1,2-Propanediols to Propionaldehyde*

(Received for publication, December 20, 1965)

B. ZAGALAK,t PERRY A. FREY,§ G. L. KARABATSOS, AND ROBERT H. ABELES

From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154, and the

Department of Chemistry, Michigan State University, East Lansing, Michigan

SUMMARY

When D-1,2-propanediol is converted to propionaldehydein the presence of dioldehydrase and dimethylbenzimidazolyl-cobamide coenzyme, 1 of 2 C-1 hydrogens of the substrateis stereospecifically transferred to position C-2. The hydro-gen transferred is the hydrogen introduced into D-1,2-propanediol by horse liver alcohol dehydrogenase whenD-lactaldehyde is reduced to D-1,2-propanediol. WhenL-1 ,2-propanediol reacts, the other C-1 hydrogen exclusivelyis transferred. The displacement of the secondary hydroxylgroup of propanediol by hydrogen, which takes place whenD- or L-1,2-propanediol is converted to propionaldehyde,occurs with inversion of configuration at C-2. These resultssuggest that the removal of the secondary hydroxyl groupmay proceed through a displacement reaction in which thehydride functions as displacing nucleophile.

It was also shown that the reduction of D- and L-lactalde-hyde with horse liver alcohol dehydrogenase and a-deu-terated reduced diphosphopyridine nucleotide leads toD- and L-propanediol of identical configuration at C-1. There-fore, the stereochemical course of the reduction by horseliver alcohol dehydrogenase is not influenced by the con-figuration of the carbon adjacent to the center which isreduced.

hyde by dioldehydrase (propanediol hydro-lyase, EC 9.2.1.28)(1). This reaction formally involves the displacement of thesecondary hydroxyl group of propanediol by 1 of the C-1 hydro-gens of 1, 2-propanediol. This hydrogen transfer occurs withoutexchange with the hydrogen of the solvent (2). The detailedmechanism of this hydrogen transfer is unknown. Any mecha-nism that has been, or will be, proposed must be consistent withthe stereochemistry of the reaction. We have, therefore, in-vestigated several aspects of the stereochemistry.

Experiments were carried out to establish whether 1 of the2 C-1 hydrogens is selectively transferred in the conversionof propanediol to propionaldehyde. This was done by preparingstereospecifically labeled D- and L-1,2-propanediol by the reduc-tion of D- and L-lactaldehyde with horse liver alcohol dehy-drogenase and a-deuterated reduced diphosphopyridine nucleo-tide, and converting the resulting diols to propionaldehyde withdioldehydrase and 5, 6-dimethylbenzimidazolylcobamide coen-zyme. The reactions that were carried out and the possibleresults are illustrated by the following equations.

CH3-CHOH-CHO + DPND alcohol dehydrogenase

CH3-CHOH-CHDOH + DPN

dioldehydraseCH,-CHOH-CHDOH DBC cozyme

DC C coHnzyme

CH3-CHD--CHO or CH3--CH2--CDO

(1)

(2)

One of the reactions in which a cobamide coenzyme is involvedis the conversion of D- and -1,2-propanediol to propionalde-

* Publication No. 429 from the Graduate Department of Bio-chemistry. A part of this work was carried out at the Departmentof Biological Chemistry, University of Michigan, Ann Arbor,Michigan. This work was supported in part by Public HealthService Research Grant GM-012633 (to R. H. A.) from the Na-tional Institute of General Medical Sciences. A preliminaryreport of portions of this work has appeared (Biochem. Biophys.Res. Commun., 18, 551 (1965)). Inquiries regarding this workshould be addressed to R. H. Abeles, Graduate Department ofBiochemistry, Brandeis University, Waltham, Massachusetts02154.

tPresent address, Department of Biochemistry, College ofAgriculture, Poznan, Poland.

§ Public Health Service Predoctoral Fellow (National Instituteof General Medical Science Fellowship 7-FI-GM-20,226-02).

If the reaction is stereospecific, either propionaldehyde-2-d orpropionaldehyde-l-dl will be formed. If it is not stereospecific,a mixture of the two aldehydes will be obtained.

The second problem investigated was whether the displacement of the secondary hydroxyl group of D- and -1,2-propane-diol proceeds with retention or inversion of configuration. Theproblem is illustrated by the following equation, in which Fisherprojections are used. Structure I results if the reaction proceeds

It* 901 C

H*--C--OH CH

H--COH -, H*-C-H or

CH, CH,

I

0C/

II

(3)

with inversion, and Structure II results if it proceeds with reten

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tion of configuration at C-2. To distinguish between thesetwo possibilities, the reaction was carried out with a substratecontaining deuterium, (i.e. H* = D), so that optically activea,euteropropionaldehyde was obtained. The configurationof this aldehyde was established by converting it to a-deutero-propionic acid and comparing the optical rotation with that ofsynthetically prepared a-deuteropropionic acid of known con-figuration.

EXPERIMENTAL PROCEDURE

Substrates: D- and L-1 ,2-Propanediol--d-D- and L-1,2-propanediol, stereospecifically labeled with deuterium at C-1,were prepared by the enzymatic reduction of D- and L-lactalde-hyde with DPND.' D- and L-Lactaldehyde were prepared by amodification of a published procedure (5) from L- and D-threo-nine, respectively. D- or L-Threonine, 25 mmoles (Sigma), 9.1 gof ninhydrin, and 600 ml of 0.05 M sodium citrate buffer, pH 5.4,were combined and boiled for 15 min with vigorous stirring.After it was cooled, the mixture was filtered, and the filtratewas treated batchwise with sufficient Dowex 1-X8 resin in thebicarbonate form to raise the pH to 6.5. After it was stirredfor 2 to 3 hours, the resin was filtered and the filtrate was ad-justed to pH 4 with Dowex 50 resin in the hydrogen ion form.The resin was filtered, and the filtrate was concentrated to avolume of 50 to 100 ml on a rotary evaporator. The concentratewas again treated with resin as above and further concentratedto 20 to 30 ml. Resin treatments were repeated until the solu-tion was colorless at pH 7.5 and did not cause the anion resin todarken after stirring at pH 7 to 7.5 for 2 hours. Usually threeor four resin treatments were required. The solution was madeup to 25.0 ml and analyzed for lactaldehyde by first reducingan aliquot with a 10-fold excess of KBH4 and then analyzingfor 1,2-propanediol. No 1,2-propanediol or hydroxyacetonecould be detected prior to KBH 4 reduction, although, in the testsemployed, as little as 1% would have been easily detected. Theyields of D- and L-lactaldehyde were 10 and 9.5 mmoles, respec-tively.

Lactaldehyde was then reduced with horse liver alcohol de-hydrogenase and DPND. To avoid addition of large quantitiesof DPND, the reduction was carried out in a coupled systemin which CH3CD 20H was used to reduce DPN. The followingreactions were employed.

alcohol dehydrogenaseCH3-CD2OH DPN (4)(4)

CH--CDO + DPND

withdrawing aliquots and analyzing for 1,2-propanediol. Thereduction of the L isomer was 95% complete after 30 min. Thereduction of the D isomer proceeded more slowly, and an addi-tional 7 units of enzyme were added after 30 min. The reactionwas allowed to continue until the reduction was essentiallycomplete. The reaction mixtures were concentrated to 10 to 20ml on a rotary evaporator, and were freed of protein and saltsby passage through a column (17 X 250 mm) of Dowex 50 inthe H+ form and then a column (16 X 360 mm) of Dowex 1-X8in the OH- form. Fractions containing diol were pooled, con-centrated to 5 to 10 ml, and again passed through the deionizingcolumns. Fractions of 10 ml each were collected and analyzedfor 1,2-propanediol. In each case, a single fraction containedmost of the diol. These fractions of L- and D-1,2-propanediol-1-dl had specific optical rotations ([a]25) of +23.3° and -21.3° ,respectively, and no lactaldehyde was detected in the analysisfor carbonyl compounds as 2,4-dinitrophenylhydrazones, al-though as little as 0.5% could have been detected. The yieldsof D- and L-1,2-propanediol obtained were 4.6 and 5.0 mmoles,respectively. The [a], 5' for L-1,2-propanediol of +20.8 ° (7.5g/100 ml) has been reported (4).

Preparation of 2-p-Nitrophenyl-4-methyl-1 ,3-dioxolanes-Amodification of the procedure of Salmi (7) and of Fieser (8) wasused for synthesizing these acetals. p-Nitrobenzaldehyde(Matheson, Coleman, and Bell) was purified by charcoal treat-ment and recrystallized from water. Twice recrystallized mate-rial melted at 103-1040 (uncorrected). Acetals were preparedfrom dl-1,2-propanediol. dl-1 ,2-propanediol-l-d 1, and stereo-specifically labeled D- and L-1,2-propanediol-l-d1. dl-1,2-Propanediol-l-d, was prepared by LiA1D4 reduction of dl-lactaldehyde. The procedure described below was used toprepare acetals of stereospecifically labeled D- and L-1,2-pro-panediol-l-d,, obtained as aqueous deionized solutions afterhorse liver alcohol dehydrogenase reduction. An aliquot con-taining 2.5 mmoles of diol was concentrated on a rotary evapora-tor until an oil remained. This was dissolved in 10 ml of ethylacetate, dried over anhydrous sodium sulfate, and, after filtra-tion, concentrated to 5 ml. This solution was added to 380 mgof p-nitrobenzaldehyde, 60 ml of benzene, and a few crystals ofp-toluenesulfonic acid, and the mixture was refluxed under acondenser so constructed that condensate could be removedduring reflux. The mixture was refluxed 2 hours, during which50 ml of condensate were removed at a slow rate and discarded.The reaction mixture was combined with 50 ml of ethyl acetateand extracted with 20 ml of water and 20 ml of saturated sodium

L- or D-CH2--CHOH-CHO + DPND alcohol dehydrogenase L- or D-CH-CHOH-CHDOH + DPN

CHCD20OH + L- or D-CH--CHOH-CHO -* L- or D-CH--CHOH-CDHOH + CH3-CDO

The reaction mixture consisted of 6.5 ml of 0.1 M potassiumPhosphate buffer (pH 7.5), 30 mg of horse liver alcohol dehy-drogenase (Worthington, 45 units) (6), 1.5 ml of ethanol-l-d2(New England Nuclear), and 6 mmoles of D- or L-lactaldehyde.The reaction mixture was then adjusted to pH 8.0. The reac-tion was started by the addition of 5.0 mg of DPN and allowedto proceed at 30°. The course of the reaction was followed by

'The abbreviations used are: DPND, a-DPNH4-D; DBCcoenzyme, 5,6-dimethylbenzimidazolylcobamide coenzyme. The1Dand L as well as R and S nomenclature (3) are used in this paper;)()-1,2-propanediol = 1,2(R)-propanediol (4).

bisulfite; it was then extracted twice more with 20 ml of water.The organic layer was dried over anhydrous sodium sulfate andconcentrated to an oil on a rotary evaporator. The productwas crystallized by dissolving it in 5 ml of anhydrous methanoland cooling it for 24 hours in a freezer at -15 °. The crystalswere filtered in the cold, transferred to a vial, and immediatelydried over P205 in a vacuum desiccator. When it was warmedto room temperature, the product was a mixture of oil andcrystals. The product from dl-1,2-propanediol was crystallineand melted at 53-54 ° (uncorrected). Products were analyzedby determining the formaldehyde formed after acid hydrolysis

(5)

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Stereochemistry of Conversion of 1, 2-Propanediol to Propionaldehyde

OH TsCI OTs CN [H] CHO [o COOHI Ts I CN- I [H I i

D-C-H -- D-C-H - H-C-D -- H-C-D -- H-C-DI I I ICH3 CHCH3 CH3 CH3 CH3 CH3

I (S)-deutero-ethanol

2 (R)-deutero-propionic acid

FIG. 1. Reaction sequence employed for the synthesis of 2(R)-deuteropropionic acid

to 1,2-propanediol and subsequent periodate cleavage. In allcases, 95 to 105% of the calculated amount of formaldehyde wasreleased. Comparison of the infrared spectra of the productsresulting from stereospecifically labeled D- and L-1,2-propanediol-1-dl with that obtained from authentic d-1,2-propanediol con-firmed their structures.

L-1,2-Propanediol-l-dl (Nonstereospecifically Labeled)--Thiscompound was prepared by reducing L-lactaldehyde with NaBD 4.An aqueous solution of NaBD4 (740 mg/2 ml) was added grad-ually to a solution of 35 mmoles of lactaldehyde in 25 ml of water.The solution was kept in an ice bath throughout the addition.NaBD 4 was added until all carbonyl compound, as assayedcolorimetrically with 2,4-DNPH, had reacted. The pH wasadjusted to 3 to destroy excess NaBD 4. The solution was thendesalted by passage through Dowex 1-HCO3- and Dowex50-H +. The fractions containing diol were concentrated to 50ml. The diol content was assayed by the NaIO 4 (9) method.The solution of diol so obtained was used in subsequent experi-ments. In control experiments, dl-propanediol was preparedfrom dl-lactaldehyde. The diol so obtained behaved identicallywith a solution of authentic 1,2-propanediol when used as asubstrate for dioldehydrase. The [a]2 of L-1,2-propanediolprepared by the above procedure was 4-24.6 (3.2 g/100 ml).

Synthesis of 2(R)-Propionic Acid-2-d -The reaction sequenceemployed in this synthesis is shown in Fig. 1. The startingmaterial, ethanol-l-dl, was obtained by the reduction of acetalde-hyde-l-di with DPNH and horse liver alcohol dehydrogenase(10). This alcohol is known to have the S configuration (11).The only step in the reaction sequence that directly involves theasymmetrical center is the displacement of the tosyl group bycyanide. The nature of the reactants assures that this reactionproceeds predominantly with inversion of configuration. Thepossibility was considered that racemization of the nitrile oraldehyde might occur. However, the ae-deuteropropionic acidobtained from this reaction sequence contained 0.93 atom ofdeuterium per molecule. Therefore, extensive racemizationwould not have occurred. The optical rotatory dispersion curveof 2(R)-propionic-2-dl acid is shown in Fig. 6.

The deuteroethyltosylate used in the sequence of reactionswas prepared according to published procedures (12). Deutero-ethyltosylate was converted to propionitrile by a modificationof the method of Rodionov (13). The reaction was carried outin a distilling flask so that the propionitrile could be removed bydistillation continuously as it was formed. Deuteroethyl-tosylate (10 g) was added to 3.4 g of potassium cyanide in 4.5ml of water. The reaction mixture was then carefully heatedto initiate the reaction. Propionitrile was continuously removedby distillation at atmospheric pressure as it was formed. Thereaction was allowed to proceed for about 50 min. The distillate,which consisted of two layers, water and propionitrile, was cooledin an ice bath, and 0.5 ml of 20 % HC1 was added. The solution

was shaken in order to extract the organic phase with the aqueousphase. The aqueous phase was saturated with CaC12, and theorganic layer was removed. The water layer was then extractedwith 5 ml of ether, and the ether and organic layers were com-bined. After it was dried with potassium carbonate, the etherwas removed and the propionitrile was distilled at atmosphericpressure. The fraction distilling between 90 and 97° was col-lected. The yield of propionitrile was 57 %.

The propionitrile was then reduced to propionaldehyde withlithium-aluminum-triethoxyhydride (14). A typical preparationwas carried out as follows. LiAlH 4, 1057 mg (112 mmoles ofhydrogen) (95% purity), was dissolved in 27 ml of anhydrousether. This process required about 45 min. The reactionflask was cooled in an ice bath, and 3.9 ml of ethyl acetate (39.8mmoles) were added with stirring over a period of 15 min.Propionitrile, 1.5 g (26.5 mmoles), was then added over a 5-minperiod. The reaction was allowed to proceed for an additional50 min. It was stopped by the addition of 5 to 10 ml of H20.Sufficient 15% H2SO4 was added to dissolve the aluminumhydroxide. The pH was adjusted to 4 to 5, and the ether layercontaining the propionaldehyde was removed by distillationwithout prior separation from the water layer. The propionalde-hyde distilled with the ether. The ether solution of propionalde-hyde obtained in this way, 3 mmoles in 15 to 17 ml, was added to20 ml of 0.02 M potassium phosphate buffer, pH 7.5. The solu-tion was cooled in an ice bath, and 6 ml of 6% KMn0 4 wereadded; the oxidation and subsequent isolation of propionic acidwere carried out as described for the preparation of propionicacid from enzymatically formed propionaldehyde.

Preparation of 2,4-Dinitrophenylhydrazones of PropionaldehydeEnzymatically Formed from Stereospecifically Labeled - andL-l ,2-Propanediol-1 -dl--The reaction mixtures for conversionof the stereospecifically labeled diols to propionaldehyde con-sisted of 0.04 M potassium phosphate buffer (pH 8.0), 0.52 mmoleof diol, 1.0 ml of DBC coenzyme (100 ug per ml), and purifieddioldehydrase dialyzed free of 1,2-propanediol. The finalvolume was 10 ml. Because of the large deuterium isotope rateeffect, 130 units of enzyme were used for conversion of D-1,2-propanediol-l-di, and 17 units were used for conversion of theL isomer. The reaction mixtures were contained in test tubes,22 X 150 mm, that were placed inside 1-liter Erlenmeyer flaskscontaining 250 ml of 2,4-dinitrophenylhydrazine (0.1% 2,4dinitrophenylhydrazone in 0.05 M H2S04). The flasks weretightly stoppered and maintained at 37° . After 40 min, thereaction was stopped by tipping the flask to mix the 2,4-dinitro-phenylhydrazine with the enzyme reaction mixture. The2,4-dinitrophenylhydrazone precipitated immediately, and wasfiltered and dried after stirring for 15 min. The dry crudeproduct was dissolved in benzene and passed through a column4.5 X 1.1 cm, of neutral alumina (Fisher adsorption aluminato remove excess 2,4-dinitrophenylhydrazine. The yello,

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effluent was evaporated to dryness on a rotary evaporator.The residue was crystallized from absolute ethanol. Fromstereospecifically labeled D-1,2-propanediol-1-dl, 91.5 mg of2,4-dinitrophenylhydrazone (74%), m.p. 152-153 ° , were ob-tained, and from the corresponding L isomer 112 mg (90%),in.p. 153-154°, were obtained. Literature value is 154 ° (15).

Propionic Acid from D- and L-1,2-Propanediol-l-dl-D-1,2-propanediol-l-dl (3 mmoles) was added to 16 ml of 0.2 M potas-sium phosphate buffer, pH 8, containing 60 mg of bovine serumalbumin, 100 /ug of DBC coenzyme, and 1200 units of diolde-hydrase. The reaction mixture was maintained for 45 min at370 in the dark in a stoppered flask. At the end of the reaction,the propionaldehyde was removed by distillation at 100 atatmospheric pressure. The distillate (15 ml) was collected inan ice bath. During the distillation of the aldehyde, some lossof deuterium from the a position of propionaldehyde occurred,as well as some racemization. However, distillation greatlyfacilitates subsequent reactions. The loss of deuterium in noway influences the results, since it cannot alter the sign of therotation of the monodeuteropropionic acid that is finally isolated.All subsequent conclusions are based on the sign of rotation onlyand not its absolute magnitude.

To the distillate containing the aldehyde, 0.2 ml of 1 M potas-sium phosphate buffer, pH 7.5, was added. The reaction mix-ture was kept in an ice bath. To the ice-cold reaction mixture,6.0 ml of 6% KMnO 4 were added with continuous stirring.During the addition of KMnO 4, the pH was maintained at 7.5by the addition of NaOH. After the addition of KMnO4 wascompleted, the reaction mixture was maintained at 0° for anadditional 15 min. The excess KMnO 4 was destroyed withNaHSO 3. The precipitate was then removed by centrifugationand washed with a few milliliters of water, which were added tothe supernatant fluid. The supernatant fluid was adjusted topH 10 with NaOH and brought to dryness at 30° . The residuewas taken up in 1.0 ml of 0.5 M H2S0 4, and the propionic acidwas purified by chromatography on a silicic acid column (16).

Equal volumes of water were added to each of the fractionsfrom the column which contained propionic acid. The fractionswere then titrated with NaOH with constant stirring. Noindicator was used, and the pH of the aqueous phase was meas-ured directly. To avoid addition of excess NaOH, the fractionswere titrated to pH 5.6. The aqueous layers containing sodiumpropionate were combined and concentrated to 0.5 ml. To theconcentrate, 30 ml of acetone were added slowly with stirring.The resulting precipitate of sodium propionate was filtered offand washed with acetone and ether. The sodium propionatewas recrystallized from ethanol-acetone. The infrared spectrumof the sample was taken. In trial runs with nondeuteratedpropanediol, the infrared spectrum of the sodium propionateso obtained was identical with that of an authentic sample ofsodium propionate. The deuterated compound showed charac-teristic differences in certain regions 'of the spectrum. Thedeuterium content of the sodium propionate obtained from thedeutero-l,2-propanediol was 0.62 atom per molecule.

The conversion of L-1,2-propanediol-1-dl (nonstereospecifi-eally labeled) to propionic acid was carried out in the samemanner as for D-1,2-propanediol-l-d. The deuterium contentof the resulting sodium propionate was 0.34 atom per molecule.The lower deuterium content is expected since the diol waschemically reduced with NaBD4. This reduction is nonstereo-

specific; only 50% of the deuterium can be transferred to C-2,so that a maximum deuterium content of 0.5 atom is expected.This will be further reduced by deuterium loss during isolation.

Enzymes-Dioldehydrase was prepared as previously de-scribed (1). The specific activity of the enzymes used wasbetween 45 and 60. Horse liver alcohol dehydrogenase waspurchased from Worthington.

Analytical Procedures-Dioldehydrase (1) and horse liveralcohol dehydrogenase (6) were assayed by described procedures.Propanediol was assayed by the periodate method used for thedetermination of serine (9) and formaldehyde was determinedby the chromotropic acid method (9). Optical rotatory dis-persion curves were determined on a Bendix-Polarimatic 460Bpolarimeter. Deuterium analyses were performed by the fallingdrop method by a commercial laboratory (Joseph Nemeth,Urbana, Illinois). NMR (nuclear magnetic resonance) spectrawere obtained with a Varian A-60 spectrometer.

RESULTS

Stereospecificity of Hydrogen Transfer-The purpose of theseexperiments was to establish whether the enzyme distinguishesbetween the 2 C-1 hydrogens of D- and L-1,2-propanediol.For this purpose, D- and L-1 ,2-propanediol-l-dl were synthesizedby the reduction of D- and L-lactaldehyde with horse liver alcoholdehydrogenase and DPND. Horse liver alcohol dehydrogenase-catalyzed reactions are known to be stereospecific (17); i.e.the reduction produces exclusively one of the two possibleisomers. It has, however, not been established that the reduc-tion of all substrates leads to identical configurations. In thisparticular reaction, the possibility must be considered that theconfiguration at C-2 of lactaldehyde affects the steric courseof the reduction at C-1, so that D- and L-1,2-propanediol-l-dlderived from D- and L-lactaldehyde may not have the sameconfiguration at C-1. The clarification of this point is essentialfor subsequent arguments. Therefore, the NMR spectra of thep-nitrobenzaldehyde acetals of the two diols were examined inorder to establish whether the two isomeric C-l-deuterodiolshave identical configurations at C-1. These acetals can havethe configurations shown in Equations 6 and 7. If D- and L-

FromD-lactaldehyde --

FromL-lactaldehyde -4

H H

CHC6H4Ni

A

,H 3 H

HHD

0 0

C \C 8H4N(

B

H D

CHKo0 o (6)

CHCH 4N0O2

A'

CH, D

O (7)

C02 6 H 4NO2

B'

propanediol-l-di have the same configuration at C-1, StructuresA and B or A' and B' would be obtained. The spectrum ofA compared to B and that of A' compared to B' will be differentsince the vicinal protons are trans in B and cis in A. If the con-figurations at C-1 are opposite, A and B' or A' and B would

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result. The spectrum of A compared to B' and that of A' com-pared to B will be identical. The results obtained are shownin Fig. 2. Since the spectra are clearly different, it can be con-cluded that the reduction of D- and L-lactaldehyde by horse liver

D

C

B

CH0 O

CHD- CH - CH,

1N. O,(p)

CH -CH,

NO, ()

- CH,

i)

Hs

-r = 5.75 6.43

Fro. 2. NMR spectra of p-nitrobenzaldehyde acetals of iso-meric 1,2-propanediols. A, dl-1,2-propanediol; B, dl-1,2-pro-panediol prepared by LiAID 4 reduction of dl-lactaldehyde; C,D-1,2-propanediol-l-dl enzymatically from D-lactaldehyde; D,L-1,2-propanediol-dl enzymatically from L-lactaldehyde. Spec-tra were taken in carbon tetrachloride.

6

oI

, 3

E

2

o0 I 2 3 4 5 6 7 8 9 10

Time (min)

FIG. 3. Relative rates of dehydration of deuterated and non-deuterated substrates by dioldehydrase. Reaction mixtures con-sisted of the following: dioldehydrase, 1 unit; potassium phosphatebuffer, pH 8.0, 40 moles; DBC coenzyme, 100 ig; substrate, 20#moles. Total volume, 1.0 ml, 37° . The reactions were startedby addition of coenzyme. Aliquots of 0.20 ml were withdrawn attime intervals, and the reaction was stopped by pipetting themdirectly into the 2,4-dinitrophenylhydrazine used for determina-tion of propionaldehyde. , D-1,2-propanediol; O, D-1,2-pro-panediol-l-di; , L-1,2-propanediol; O, L-1,2-propanediol-l-d,.

alcohol dehydrogenase leads to D- and L-propanediol with iden-tical configurations at C-1.

The two labeled isomeric diols, obtained from the enzymaticreduction of D- and L-lactaldehyde, were then separately con-verted to labeled propionaldehyde in the presence of dioldehy-drase and DBC coenzyme. The product of this reaction canbe either CHCHDCHO or CH3CH 2CDO or a mixture of thetwo compounds. If a-deuteropropionaldehyde or deutero-propionaldehyde-C-1 is formed, it can be concluded that theenzyme stereospecifically selects 1 of the 2 C-1 hydrogen atoms,while a mixture of the two aldehydes indicates lack of stereo-specificity. Two criteria were applied to distinguish betweenthese two possibilities. First, the rate of propionaldehyde forma-tion was examined. It was previously shown that the conversionof dl-1 ,2-propanediol-l-d 2 to propionaldehyde proceeds atone-tenth the rate of the nonisotopic species (18). It is expected,therefore, that if the reaction proceeds with deuterium abstrac'tion, i.e. results in the formation of a-deuteropropionaldehyde,then VH/VD = 10. If, on the other hand, deuteropropionalde-hyde-C-1 is formed, which does not involve deuterium transferno isotope effect, or perhaps only a small secondary deuteriuisotope effect, will be found. An intermediate isotope effectwill be expected if both hydrogen and deuterium are transferredThe kinetics of the formation of propionaldehyde from the twisomeric diols is shown in Fig. 3. The conversion ofD-propanediol-l-dl to propionaldehyde proceeds approximateone-tenth as fast as that of the nonisotopic substrate, while theconversion of L-propanediol to propionaldehyde shows no isotope effect. It can be concluded, therefore, that deuterium 5transferred when D-propanediol-l-dl reacts and that hydrogenis transferred when L-propanediol-l-d 1 reacts.2

2 The argument presented here is based on the assumption thathe conversion of both D- and L-1,2-propanediol-1-di will showdetectable isotope effect (VaI/VD = 1.2 can be detected) if deut'rium is transferred. The experiments described here show t$sthe conversion of D-1,2-propanediol-l-di shows a significant i(

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HH - CH ,CH,

H4

N4

0-'~ ' '" ICHD- C

H 4H - q,u,

BHS N,

I I I

"' 0.98 1.70 2.08 2.35

FIG. 4. NMR spectra of 2,4-dinitrophenylhydrazones derivedfrom propionaldehyde (A), from propionaldehyde produced enzy-aatically from D-1,2-propanediol-l-dl (B), and from proiponalde-

hyde produced enzymatically from L-1,2-propanediol-l-di (C).

tope effect. The question remains, then, whether the conversionOf L-1,2-propanediol-l-di, if it proceeds with deuterium transfer,ill show a detectable isotope effect. There appears to be little

doubt that an appreciable isotope effect would occur, since Vu/Pi ~ 10 (18) for dl-1,2-propanediol-l-d2, and Km (L-1,2-propane-iiol) < Km (D-1,2-propanediol) (P. A. Frey and R. H. Abeles, un-Published data). Clearly, the kinetic arguments for or againstdeuterium transfer would be invalid if the reaction of L-1,2-pro-

These results were confirmed by the NMR spectra of the 2,4-dinitrophenylhydrazone derivatives of propionaldehyde formedfrom the two diols. The spectra are shown in Fig. 4. SpectrumB is the spectrum of propionaldehyde 2,4-dinitrophenylhydra-zone derived from D-propanediol-l-dl, and Spectrum C is thatderived from L-propanediol-l-dl. The triplet at r = 2.35 inA becomes a broadened doublet in B and is absent in C. Fromthis, we conclude that the deuterium is at the methylene groupin B and at C-1 in C. The propionaldehyde formed from D-1,2-propanediol-l-di, therefore, has the structure CH3-CHD--CHO, and that from L-propanediol-l-dl has the structure CH3-CH2-CDO. The hydrogen transfer is sterospecific, therefore,but I of the 2 hydrogens of C-1 of propanediol is transferredwhen L-propanediol is the substrate, and the other is transferredwhen D-propanediol is the substrate.

Stereochemistry of Displacement of Secondary Hydroxyl of D-

and L-1 ,2-Propanediol--The formation of propionaldehyde from1,2-propanediol involves the displacement of the secondaryhydroxyl group of propanediol by hydrogen. In this displace-ment, the hydrogen can approach from the same side as thehydroxyl group, in which case the configuration at C-2 is main-tained, or it can approach from the opposite side, in which caseinversion of configuration at C-2 occurs. If the reaction iscarried out so that deuterium rather than hydrogen is transferred,optically active propionaldehyde will result. From the con-figuration of the propionaldehyde the stereochemistry of thereaction can be determined. The configuration of propionalde-hyde derived from D-1, 2-propanediol-l-d, which will be obtainedif the reaction proceeds either by retention or by inversion, isshown in Fig. 5. The configuration of the propionic acids de-rived from each aldehyde is also shown. If the reaction pro-ceeds by inversion, 2(S)-deuteropropionaldehyde will beobtained, and, if it proceeds by retention, 2(R)-deuteropropion-aldehyde will result. From L-1,2-propanediol-l-dl, 2(R)-deu-teropropionaldehyde will be obtained, if inversion occurs, and2(S)-deuteropropionaldehyde will be obtained if the reactionproceeds with retention.

The configuration of the propionaldehyde obtained from D-

and L-propanediol was established by conversion to propionicacid and by comparison of the optical rotatory dispersion withthat of propionic acid of known configuration. The opticalrotatory dispersion curves for the sodium propionate derivedfrom D- and L-1,2-propanediol-l-dl, as well as for syntheticsodium 2(R)-deuteropropionate, are shown in Fig. 6. Theseresults show that the propionic acid obtained from D-propanediolhas the S configuration, whereas that from L-propanediol hasthe R configuration. It can be concluded, therefore, that theconversion of 1,2-propanediol to propionaldehyde proceedswith inversion of configuration at C-2. The rotation of thesodium propionate derived from aldehydes which were preparedenzymatically from propanediol is lower than that of the synthe-sized sodium propionate. This is due to the lower deuteriumcontent of these acid salts. Sodium propionate obtained fromD-propanediol contains 0.62 atom of deuterium per molecule, andthat obtained from L-propanediol has 0.34 atom per molecule,whereas the synthetic material contains 0.94 atom per molecule.The reason for the lower deuterium content in the enzymatically

panediol-l-di proceeds without isotope effect when deuteriumtransfer occurs. This possibility, however, appears highly im-probable.

Issae of July 10, 1966 3033

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Stereochemistry of Conversion of 1, 2-Propanediol to Propionaldehyde

CHO

H-C-D

CH3

CHDOH ' 2 (R)-deutero-H-C-OH propionlidehyde

CH3

-propanediol- I - d-proponediol-i-di CHO

D-C-H

CH3

2 (S) -deutero-propionaldehyde

COOH

H-C-DICH3

2 (R)-deutero-propionic acid

COOH

D-C-H

CH3

2(S)-deutero-propionic acid

FIG. 5. Possible configurations of -deuteropropionic acid and a-deuteropropionaldehyde which can be obtained from D-l,2-pro-panediol-l-d.

+

20

15

10 oI0

05 0O

12~~~~~~~~0

20 · ~ ~ ~ ~ ~ 0 _

220 230 240 250 260 270 280 290

mF

FiG. 6. Optical rotatory dispersion of sodium -deuteropro-pionate. , synthetic sodium 2(R)-deuteropropionate; A, enzy-matically formed from L-1,2-propanediol-l-d; 0, enzymaticallyformed D-1,2-propanediol-l-d.

derived material has already been discussed under "PropionicAcid from D- and L-1,2-Propanediol-l-dl."

DISCUSSION

When D- and L-1,2-propanediol are converted to propionalde-hyde, 1 of the 2 C-1 hydrogens of the diol replaces the ORgroup at C-2. This hydrogen shift occurs without exchangewith the hydrogen of the solvent (2), and 1 of the 2 C-1 hydro-gens is exclusively selected for transfer, as is the case in all en-zymatic reactions in which a carbon-hydrogen bond is broken.The novel aspect of this reaction is that the C-1 hydrogen whichis transferred when D-1,2-propanediol reacts is not the samehydrogen as that which is transferred when L-1,2-propanediolreacts. It must be concluded, therefore, that the configuration

at C-2 determines which of the 2 C-1 hydrogens is transferredduring the course of the reaction. This control of the stericcourse of the reaction by the configuration at C-2 can readilybe understood if it is assumed that the following conditionsapply to the enzymatic reactions. (a) An identical mechanismis operative in the reaction of D- and L-propanediol. Conse-quently, the stereochemistry of the reaction of both isomers willbe identical. (b) D- and L-Propanediol interact through 3identical atoms or groups of atoms at three binding sites of theenzyme. The geometry of the three binding sites of the enzymeis relatively rigid.

The conformational restrictions of the molecule, resultingfrom the second condition, force the two isomers to assumeconformations such that the position of the C-2 hydroxyl grouprelative to a specific C-1 hydrogen is not identical in the twoisomers. The conformations that the two isomers of propanediolmust assume in order to interact with identical binding sitesof the enzyme and the consequent spatial relation of the C-2hydroxyl group relative to the C-1 hydrogens are shown in Fig.7 by three-dimensional models. The three-dimensional modelsshow that the C-1 hydrogen trans to the secondary hydroxylgroup is not identical in both isomers. A deuterium atom istrans in Structure 1, and a hydrogen atom is trans in Structure 2.

In the foregoing discussion, it was arbitrarily assumed thatthe points of interaction between enzyme and substrate are thetwo hydroxyl groups and the methyl group. The absoluterequirement for two hydroxyl groups in both substrates (1)and probably also in inhibitors (18) suggests that these mayin fact, be two points of interaction. Whether the third poinlof interaction is the methyl group or the C-2 hydrogen cannot bedecided.

It has also been established that the conversion of propanedi0to propionaldehyde proceeds with inversion of configuration aC-2. This suggests the possibility that the hydroxyl group Iremoved in a displacement reaction, and, since the reactioninvolves a hydrogen transfer, a hydride ion could function aithe displacing nucleophile. A displacement in which a hydrid!ion participates can occur either by way of an intramoleculw1,2-shift or by a two-step process, in which the hydrogen first transferred to a hydrogen acceptor, possibly the coenzymeand then to C-2 of the substrate. Support for the role of thlcoenzyme as hydride acceptor is provided by the reaction

1,2 (R)-D-1,2-

retention

inversion

I

3034 Vol. 241, No. 13

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B. Zagalak, Perry A. Frey, G. L. Karabatsos and Robert H. AbelesThe Stereochemistry of the Conversion of D and L 1,2-Propanediols to Propionaldehyde

1966, 241:3028-3035.J. Biol. Chem. 

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