Arnaud Lanoue et al- Kinetic Study of Littorine Rearrangement in Datura innoxia Hairy Roots by ^13-C NMR Spectroscopy

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K i n e t i c S t u d y o f L i t t o r i n e R e a r r a n g e m e n t i n D a t u r a i n n o x i a H a i r y R o o t s b y 13 C

N M R S p e c t r o s c o p y

Arnau d Lanoue,†,§ Michele Boite l-Cont i,*,† Jean-Charles Portais,‡,⊥ Jean-Claude Laberche,† J ea n-Noel Bar botin, ‡

Philippe Christen,§ and Brigitte Sangwan-Norreel†

Laboratoire A nd rogenese et Biotechn ologie, L aboratoire d e Genie Cellulaire, Universite de Picardie Jules Verne,

33 R ue S aint Leu, F-80039 Am iens Cedex 01, France, and Laboratoire de Chim ie Analytique Pharm aceutique, Un iversite d e

Geneve, 20 Bd d ’Yvoy, 1211 Geneva 4, S witzerland

Received November 30, 2001

The kin etics of tropane a lkaloid biosynt hesis, par ticularly the isomerization of littorine in to hyoscyamine,were studied by analyzing the kinetics of carbon-13 (13C) in metabolites of Datura innoxia hairy rootcultures fed with labeled tropoyl moiety precursors. Both littorine and hyoscyamine were the majoralkaloids accumulated, while scopolamine was never detected. Feeding root cultures with ( R S )-phenyl-[1,3-13C2]lactic acid led to 13C spin -spin coupling detected on C-1′ and C-2′ of the hyoscyamine skeleton,which validated the intram olecular rearr angement of littorine into hyoscyamine. Label from phenyl[1-13C]alanine or ( R S )-phenyl[1,3-13C2]lactic acid was incorporated at higher levels in littorine than inhyoscyamine. Initially, the a ppar ent h yoscyamine biosynthesized rat e (vap ph yo ) 0.9 µm ol 13C‚flask -1‚d -1)was lower th an littorine format ion (vap plitto ) 1.8 µm ol 13C‚flask -1‚d -1), suggesting th at th e isomerizationreaction could be rate limiting. The results obtained for the kinetics of littorine biosynthesis were inagreement with t he r ole of this compound as a direct precursor of hyoscyamine biosynthesis.

The tr opane alkaloids hyoscyamine a nd scopolamine ar e

secondary metabolites synthesized in several solanaceousplants of the genera Atropa, Datura, Duboisia , a n d Hyos-

cyamus. These natural products are anticholinergic agents

used in medicine as ant ispasmodics, and in ophth almology.

In plants, h yoscyamine a nd scopolamine ar e synth esized

i n t he root s a nd a re t ra nsl oc a t e d i nt o t he a e ri a l pa rt s .Transgenic root cultures obtained by transformation via

Agrobacterium rhizogenes1-3 provide plant material with

a h igh growth rat e on hormone-free media 4,5 and alkaloid

yields similar to or higher than in untransformed plants.

Recently, tran sformed root cultur es were u sed to investi-gate t he biosynthesis of tropane a lkaloids. This biosynth e-

sis involves the conversion of ornithine into tropine in six

steps6 a nd of phe nyl a la ni ne i nt o phe nyll a ct a t e i n t wosteps.7-9 Tropine and phenyllactate (Figure 1) then con-

de nse i nt o l i t t ori ne , a s re port e d by Robi ns e t a l .10 Th ebiosynthesis of hyoscyamine involves the molecular rear-

ran gement of littorine. H owever, t he reaction mechan ism

is still uncharacterized.11 -14 Alth ough significant p rogress

allows a better u ndersta nding of tropane a lkaloid biosyn-

thesis, l i ttle is known about its regulation. If hairy rootcul t ure provide s a l a rge bi oma ss,15 t he ma i n proble m

resides in the low accumu lation of metabolites. Conse-

quently, some st rat egies ha ve to be developed t o enhan ce

productivity; one of these is to explore th e limiting steps

in hyoscyamine biosynthesis. Therefore, the activities of several enzymes involved in tr opane alk aloid biosynt hesis

were investigated in order to determ ine the factors limitingthe meta bolic flux.2,16 However, en zymatic activities do not

give informa tion about flux rat es between biosyntheticintermediat es. A kinetic study of hyoscyamine biosynth esis

was t her efore conducted t o explore possible meta bolic ratelimitations.

In recent years, 13C-labeling str ategies have been u sedto provide furth er insights into biosynthetic path ways of

* To whom correspondence sh ould be a ddressed. Tel: 33(0)3.22.82.79.56.Fax: 33(0)3.22.8276.12. E -mail: [email protected].

† Labora toire Andr ogenese et Biotechnologie, Univer site de P icardie J ulesVerne.

‡ Laborat oire de Ge nie Cellula ire, Un iversite de Picardie J ules Vern e.⊥ C u rre n t a d d re ss: C e n tre d e B ioin gen ie rie Gilb e rt Du ra n d , INS A

Toulouse, 135 av Rangueil, 31077 Toulouse Cedex 4, France.§ Laboratoire de Chimie Analytique Phar maceutique, Universite de

Gen ev e.

F i g u re 1 . Schematic pathwa y of h yoscyamine and scopolaminebiosynthesis. [1′,3′-13C]littorin e is converted int o [1′,2′-13C]hyoscyamine.Arrows only represent chemical origins.

1131 J. Nat. Prod. 2002, 65 , 1131-1135

10.1021/np010612c CCC: $22.00 © 2002 American Chemical Society and American Society of Pha rma cognosyPublished on Web 07/11/2002



tropane alkaloids and have played a key role in demon-

strating t he intr amolecular rearra ngement of littorine into

hyoscyamine.7,10,17 Th e 13C-labeling technique is powerfuli n providing ki ne t ic da t a , i ncl uding me t a bol ic fl uxe s,

although th is has n ot been applied to th e tropane a lkaloid

pathway. The present work reports on a kinetic study of

littorine rearrangement into hyoscyamine using labeled

precursors, e.g., phenyl[1-13C]alanine and ( R S )-phenyl[1,3-13C2]lactic acid.

R e s u l t s a n d D i s c u s s i o n

21-Day-old t ran sformed roots were subcultured in n ew

medium containing labeled or unlabeled phenylalanine (4

mM) a nd i ncuba t e d for va rious pe ri ods of t i me . The se

conditions were used to stu dy tr opane alkaloid biosynthesis

with a minimum root growth. After harvesting the roots,phenylalanine was extracted from the tissues as well as

from the medium and its concentration was determined

(Figure 2). The medium content decreased linearly from

day 0 to day 2 with a rat e of 160 µm ol‚flask -1‚d -1. During

t he sa me pe ri od t he phe nyl a l a ni ne a c c umul a t i on i n t he

roots was 8 µmol‚flask -1‚d-1, indicating that ph enylalanine

utilization (difference between consumption and accumula-

tion) was 152 µm ol‚flask -1‚d -1. The rate of consumption

decreased from day 2 to day 5; during this period pheny-

lalanine was no longer detected in the medium. The rate

of phenylalanine accumulation in the roots decreased to

0.8 µm ol‚flask -1‚d -1 during this period. Accumu lation of

phenylalanine in the roots occurred up to day 10. Then,

the phenylalanine root content decreased up to day 20 ata r at e of 2.8 µmol‚flask -1‚d-1. Phenylalanine was efficientlyt a ken u p by D. i nnox ia h a i r y r o ot s a n d w a s in d e edconsumed within 5 days. These results agree with thoseobtained by Kitamura et al.18 for Duboisia leichhardtii.

Transformed roots of solanaceous species generally ac-cumu late large amounts of hyoscyamine an d minor quan ti-ties of scopolamine.19 Tropane alkaloids were extra cted andanalyzed by HPLC. At first, hyoscyamine (36.5 µmol‚flask -1)and littorine (35.1 µm ol‚flask -1) were th e ma jor alkaloidsde t ect e d. The ha i ry root s of D. i nnox ia s t u d ie d h e r eaccumu late hyoscyamine (25 µmol/g dr y weight) to a largere xt e nt t h a n D. stramonium (20 µmol/g dry weight)14 a n d

Atropa belladonn a (6 µmol/g dry weight).20 D u r i n g t h estudied period (20 days), roots did not accumu late either

scopolamine or 6 β-hydroxyhyoscyamine, the other deriva-tives of hyoscyamine. In contr ast, D. innoxia hairy rootsaccumulated more littorine (21 µmol/g dry weight) thanother species (3-10 µmol/g dry weight).14,20,21 The littorine/ hyoscyamine ra tio was near ly 1 in D. innoxia hairy roots,whereas it was in the r ange 0.2-0.5 for other transformedroots. Further investigation is in progress to determinewhe t he r t he hi ghe r l it t orine a nd hyoscya mine cont e nt scould be correlated to the absence of scopolamine.

As previously r eported by Robins et al.,2,22 a decrease intropane alkaloid content was observed a fter subculturing(Figure 3). Here, the decrease represented 18.4% and 25.6%of the initial conten ts of hyoscyamine and littorine, respec-tively. Then, th e alka loid concentra tion increased from da y2 to day 5 to almost reach the initial content, suggestingthe occurrence of alkaloid synthesis. Finally, the alkaloidcontent decreased again from day 5 to day 20. Losses of alkaloids were 20% and 40% for hyoscyamine a nd littorine,re spect i ve ly. The re wa s no a l ka loi da l r e le a se i nt o t hemedium du ring the incubation period. It is noteworthy tha ttropic acid, a u sual hydrolysis pr oduct of tropan e alkaloids,as well as scopolamine or other known alkaloid derivatives,w er e n ot d e te ct e d e i t h er i n t h e r oot s or i n t h e l iq u idmedium.

The conversion of exogenous ph enylalanine in to tropan ealkaloids was monitored by stable isotopic experiments (13CNMR). First, r oots were incubated for 5 days with 4 mMphenylalanine. 13C N M R s p ect r a r e su l t in g fr om t h e secontrol experiments (Figure 4A) exhibited two regions of

F ig u r e 2 . Profile of phenylalanine in the medium (b) and in the roots(O) during feeding with 4 mM phenylalanine.

F i g u r e 3 . Profile of total littorine and hyoscyamine contained intransformed root cultures during feeding with 4 mM phenylalanine:(O) littorine in the r oots, (0) littorine in the medium, (b) hyoscyamin ein the roots, (9) hyoscyamine in the medium. Data points representthe mean values of four experiments.

F i g u r e 4 . Expansion at 120-180 ppm of 13 C NMR spectrum of thealkaloidal extract from root cultures of Datura innoxia fed withunlabeled (A) or phenyl[1-13 C]alanine (B) showing enrichment inhyoscyamin e C-1′ (171.8 ppm) and littorine C-1′ (173.2 ppm).

1132 J ou rn al of N atu ral Prod u cts, 2002, Vol. 65, N o. 8 L an ou e et al.



interest, one conta ining the carbons from the a romatic ring(120-140 ppm) a nd t he ot he r cont a i ni ng t he ca rbonylgroups (C-1′ of hyoscyam ine ) 171.8 ppm, C-1′ of litt orine) 173.2 ppm). In agreement with HPLC analyses, hyos-cyamine a nd littorine were the major alkaloids detected,whereas neither scopolamine nor 6 β-hydroxyhyoscyaminewa s de t ect e d. The a bsenc e of t he l a t t e r a l ka loi ds wa sdefinitively confirmed by capillar y electr ophoresis (data notshown). In a second experiment, roots were incubated for5 da ys wi t h 4 mM phe nyl [1-13C]alanine. By compar isonwith the control spectrum, signals from C-1 ′ of hyoscyamin ea nd l it t orine (Fi gure 4B) ha d muc h hi ghe r i nt e nsit i es,whereas t he signal from other carbons (including t hose of the aromatic ring) remained constant. This clearly indi-ca t e d t h a t 13C was incorporat ed from th e exogenous ph e-nylalanine. Fractional enrichment s were 18% and 8% for

littorine and hyoscyamine, respectively. Moreover, someextra peak s corresponding to 13C e nri c hme nt we re a l soobserved. Their chemical shifts (172.7-167.9 ppm) couldnot be correlated to known alkaloids by comparison withcommercial standards or literature data.

To ascertain that littorine conversion into hyoscyaminein D. innoxia occurred via the intramolecular rearrange-ment depicted in D. stramonium ,10 root cultures were fedwi t h 4 mM ( R S )-phenyl[1,3-13 C2]lactic acid for 24 h. The13C incorporation in littorine and hyoscyamine was deter-mined a s described in the Experimental Section. In corpo-ra tion could be observed in both C-1′ (173.2 ppm ) and C-3′(40.5 ppm) of l it t orine , i ndica t i ng t ha t t he e xogenousphenyllactate was efficiently incorporated and metabolizedby roots (Figure 5). Furthermore, two satellite peaks weredetected for h yoscyamine C-1′ (171.8 ppm , J ≈ 55 Hz) andC-2 ′ (54 ppm, J ≈ 55 Hz) in addition to the singlets. Aspreviously reported in D. stramonium root cultures,10 t h eoccurren ce of such sat ellites ind icated th at th e two labeledcarbons becam e adjacent du ring t he conversion of littorineinto h yoscyamine (Figure 1). The fractional enrichmentswere 11.8% and 2.1% in littorine and hyoscyamine, respec-tively.

Kinetics of hyoscyamin e synth esis from extern al phen y-lalanine was investigated. Two 21-day-old series of hairyroots were subcultured in new liquid medium fed with 4mM phenyl[1-13C]alanine for various periods of time (rang-i ng from 0 t o 20 da ys), a n d t he i sot opi c pa t t e rn of rootalkaloids was examined. To obtain accura te qu ant itative

data, all feeding experiments were performed in pa rallelwith either labeled or unlabeled precursors. All 13C NMRspectra were produced with the same acquisition param-eters, allowing direct comparison of spectra from labeledand unlabeled experiments. Advant age was also taken fromt he de t e c t i on of na t ura l l y a bunda nt 13C c a r b o n s i n t h earomatic ring, which offered intramolecular references inall spectra. This strategy enabled an unambiguous quan-tification of label incorporation at each incubation time.

Kitamura et al.18 reported the conversion of 14C-labeledphen ylalanine int o tropane alka loids as a fun ction of time,but the radioactivity was recovered in th e total a lkaloidalfra c t i on. In t he pre se nt work, s t udi e s usi ng 13C-labeledsubstra tes were designed to evaluate t he kinetics of tropanealkaloid biosynth esis. The kinet ics of alkaloid labeling ar egiven i n F i gure 6. 13 C wa s i ncorpora t e d i nt o l i t t ori nebetween da y 2 and day 5, which a greed with the increasein littorine content mentioned above (see Figure 3). Theappar ent rat e of incorporation into littorine (vap plitto) was1.8 µm ol13C‚flask -1‚d -1. The highest enr ichment found inl it t orine (16%) wa s observe d on da y 5. Aft e r t ha t , t hea mount of l a bel ed l it t orine de cre a se d (v ap plitto ) -0.4

µmol13C‚flask -1‚d-1), indicatin g compoun d consu mpt ion forhyoscya mine biosynt he si s. Thi s i s consi st ent wit h t hedecrease in littorine content observed in Figure 3. 13C was

F ig u r e 5 . 13 C NMR spectrum of the alkaloidal extract from transformed root cultures of Datura innoxia fed with ( R S )-phenyl[1,3-13C]lactic acid(A) or ( R S )-phen yllactic acid (B). H: Hyoscyamin e, L: Littorine, J 1,2 : carbon spin-spin coupling (≈55 Hz). All regions are displayed with thesame vertical scale.

F ig u r e 6 . Profile of [1′-13 C]littorine a nd [1′-13C]hyoscyamine content sin transformed root cultures during feeding with 4 mM phenyl[1-13C]-

a l a ni n e: (O) [1′-13 C]littorine, (b) [1′-13 C]hyoscyamine. Data pointsrepresent the mean values of duplicate experiments; standard errorwas less than 4% of the mean.

L ittorin e R earran gem en t in Datu ra J ou rn al of N atu ral Prod u cts, 2002, Vol. 65, N o. 8 1133



incorporat ed into h yoscyamine between da y 2 and day 5,although at a lower rate (vapphyo ) 0.9 µmol 13C‚flask -1‚d-1).After day 5, the amount of labeled hyoscyamine tended toa plateau (vap ph yo ) 0.09 µm ol 13C‚flask -1‚d -1). As a resu lt,the fractional enrichment in h yoscyamine increased from8% on da y 5 to 17% on da y 20. The latter value was similarto the highest fractional enrichment observed in littorine,suggesting that no dilution posterior to littorine occurred.The highest en richment foun d in littorine was observed onday 5, whereas it was on day 20 for hyoscyamine. These

results indicate that the labeling transfer occurred untilday 20. The apparent rates reported for the biosynthesisof tropane alkaloids su ggest tha t the isomerization reactioncould be rate limiting. Considering the complex mechanismof littorine isomerization,23 i t i s sugge st e d t ha t t hi s i s alimiting step for hyoscyamine biosynthesis.

Incorporation of ( R S )-phenyl[1,3-13C2]lactic acid in hyos-cyamine wa s lower th an tha t of littorine after 24 h . Similarresults were observed with phenyl[1-13C]alanine on day 5,when maximal labeling of littorine was observed. After 5days, with the latter substrate, labeling of hyoscyamineincreased, whereas that of littorine decreased. All theseresults are consistent with the role of littorine as interme-diate in the biosynthesis of hyoscyamine in D. innoxia.

Whereas the maximum rate of phenylalanine consump-tion occurred very early (see Figure 2), incorporation of 13Cinto tropane alkaloid was important after day 2, i.e., whenthe intracellular pool of phenylalanine had significantlyincreased (by a factor 2). Labeled hyoscyamine, 4 µm ol13C‚flask -1, represented approximately 1% of the initiall a be le d phe nyl a la ni ne fe d i n t he me dium a nd 10% of phenylalanine observed in the roots, indicating that themajority of phenylalanine is used for other processes. Thesedata suggest tha t th e competition between t ropane alkaloidsynthesis and other phenylalanine utilizing pathways iswidely in favor of the latter.

The inten sities of unassigned peak s corr esponding to 13Cenrichment already observed in Figure 4B increased un tilday 5, further decreased u p to day 10, an d were n o longer

detected thereafter. No signal from apoatropine, 6 β-hy-droxyhyoscyamine, or scopolamine was detected in thecourse of the experiments. Moreover, losses of alkaloidswe re obse rve d i n Fi gure 3. In t he whol e pl a nt , t ropa nea l ka loi ds a re norma l ly synt he si ze d i n t he root s a nd a rethen translocated into the aerial parts. In contrast, trans-forme d root s c onst i t ut e bot h t he synt he si s a nd st ora georgan. Th us, a possible tu rn over could avoid a toxic alka loidaccumu lation for t he cell. It has been foun d th at exogenoustropic acid is converted into glucose esters in D. innoxia

hairy roots.24 A possible recycling of tropane alkaloids in Duboisia root cultures h as been discussed by Kitamur a etal .25 These auth ors suggest a h ypothetical pathway of tropicacid recycling origina ting in hyoscyamin e. However, fur th er

investigations with labeled substra tes ar e still required t obet t e r unde rst a nd t ropa ne a l ka l oi d me t a boli sm i n ha i ryroot s. Al so, t he e nzyme s i nvol ve d i n t he l a st s t e ps of alkaloid biosynthesis must be chara cterized.

E x p e r i m e n t a l S e c t i o n

G e n e r a l E x p e r i m e n t a l P r o c e d u r e s . NMR spectra wereobtained in CDCl3 at 3 0 0 MH z u s i n g a Bru k er 3 0 0 MH zspectrometer. Phenyl[1-13C]alanine was provided by Euriso-Top (St. Aubin, Fran ce) (99% specific enr ichment). ( R S )-Phenyl[1,3-13C2]lactic acid (80.9% specific enrichment) waskindly pr ovided by J . G. Woolley (Faculty of Applied Sciences,De Monfort University, Leicester, UK).

H a i ry R o o t C u l t u r e s . Transformed root cultures of D.

innoxia Mill. were obtained and maintained as described by

Boitel-Cont i et al.26 They were cultured in 250 mL Erlenmeyerflasks conta ining 100 mL of Gamborg’s B5 culture medium 27

supplemented with 30 g/L sucrose for 21 days. H airy rootswere grown on a rotary shaker (80 rpm) at 27 ( 1 °C undercool light fluorescent lamps (35 µE ‚m -2‚s-1), with a 16 h light/8h dark period. The culture conditions were determined in orderto study tropane alkaloid biosynthesis with a minimum rootgrowth. This was obtained by subculturing a 21-day-old entireroot culture in new B5 medium. The roots occupied all theavailable volume in the flask and, under such conditions,remained in a stationary phase. Solutions for feeding experi-ments were prepar ed from neutr al a queous st ock solutions (13mg/mL) at appr opriate concentr ations an d were filter-sterilizeddirectly into the flasks. Transformed roots were subculturedi n n ew med ia con t ai n in g 4 mM of l ab eled or u n l abeledphenylalanine.

H P L C A l k a l o i d A n a l y s i s . To analyze the alkaloids, th eroots were dried at 60 °C overnight. The powdered, driedma ter ial (40 mg) was soaked in 4 m L of met ha nol for 5 h. Afterevaporation, the residue was dissolved in an appropriatevolume of mobile phase, filtered through a Millex 0.22 µm(Millipore, Bedford, MA) and analyzed by HPLC (Waters 510appar atu s). The volume injected was 20 µL (Rheodyne, RohnertPark, CA). Hyoscyamine and littorine were quantified withan ODS-AQ (YMC, Kyoto, Japan) column (5 µm, 250 × 4.6mm) at ambient temperature with UV detection at 204 nm

(Wat ers 484 UV detector; Milford, MA). The m obile pha se wasH 2O-CH 3CN -H 3PO4 (89:10.8:0.2) with 1 g/L KH 2P O4 supple-mented with triethylamine a t pH 3.1. The flow ra te was 0.8mL/min. Quant ification was achieved by reference to externa lstan dards of hyoscyamine, littorine, scopolamine, tropic acid,phenyllactic acid, and phenylpyruvic acid (Sigma-Aldrich, St.Louis, MO) an d expressed in µm ol‚flask -1. Phenylalanineanalyses were carried out on a Beckman 6300 amino acidanalyzer.

S p e c t r o s c o p i c A n a l y s i s . For NMR alkaloid analysis, 1 gof dried plant material was extracted overnight in 500 mL of MeOH -CHCl3-28% NH 4OH (50:50:1.4). This extract wasfiltered, evaporated under reduced pressure, and rinsed threetimes with 20 mL of 0.1 N HCl. After addition of 3 mL of 28%NH 4OH, alkaloids were extracted with 3 × 30 mL of CHCl3.The organic phase was reduced to dryness, and the residue

was collected in CDCl3. NMR a nalyses were acquired u singacquisition pa ram eters a s follows: spectral width, 15 000 Hz;flip a ngle 90°; dat a s ize, 16 K; nu mber of scans, 10 000; protondecoupling was carried out during acquisition (1.08 s). A 3 Hzl in e b road en in g w as ap p li ed p rior t o Fou ri er t ran s form.Chemical shifts were expressed a s ppm relative to CDCl3. Theassignments of NMR r esonances were m ade on the basis of l it erat u re d at a 9,28,29 an d w ere con fi rmed b y s p ik i n g rootextracts with reference samples.

S p e c i f ic E n r i c h m e n t C a lc u l a t i on s . Th e 13C s ig n alsdetected in samples from unlabeled experiments correspondedto the 1.1% 13C naturally occurring in each carbon position.In samples from labeled experiments, 13C signals correspondedto the naturally occurring level plus the level incorporated(when occurring) from the exogenous labeled precursor. Then,

the differences in intens ity observed for a given signal bet weenspectra from labeled/unlabeled experiments represented theincorporat ion r ate of the labeled precursor in t he corr espondingcarbon position. Because the precursors were labeled in theC-1 ′-C-3 ′ positions only, no label could be incorporated in thephenyl moiety or in the tropane moiety of tropane alkaloids.T h u s , s i gn al s from t h e aro mat ic moi et y cou l d b e u s ed a sintramolecular references to estimate the natur al abunda nceof 13C. Quantitative analysis of label incorporation was per-formed as described below: C i fractional enrichmen t (%) ) [( I ci / I ar ‚c)labeled/( I ci / I ar ‚c)unlabeled]× 1.1, where I ci is the intensityof the signal from “i” carbon (C-1′ to C-3′) in the alkaloidincluding satellite peaks, I ar ‚c is the intensity of the signal fromthe aromatic carbon, and 1.1% is the correction for the 13Cnatural abundance. This method compensates for small in-strumenta l variations.

1134 J ou rn al of N atu ral Prod u cts, 2002, Vol. 65, N o. 8 L an ou e et al.



A c k n o w l e d g m e n t . We tha nk J. G. Woolley (Faculty of Applied Sciences, De Monfort University, Leicester, UK) forkindly providing ( R S )-phenyl[1,3-13C2]lactic acid.

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