edited by
the microscale loboratory
ARDEN P. ZIPP SUNY-CoRland c0-nd.Nyi3M5
Microscale Yeast Mediated Enantiospecific Reduction of Vanillin, and the Absolute Configuration of (-)-(R)-a-~euteriovanill~l Alcohol A Bioorganic Chemistry Experiment Moses Lee Furman University Greenville. SC 29613
I t is well recognized that the biological activity of optically active pharmaceutical agents ( I ) and agrochemicals (2)are associated to a given absolute configuration. Consequently, there is interest in the development of methodology for the preparation of enantiomerically pure products (3). Enzymatic and microbial transformations [such a s yeast (4,511 are known to possess high enantioselectivity. Therefore, a s part of our sophomore organic chemistry course we wanted to introduce a n experiment to demonstrate the efficiency and enantiospecificity of microbiallenzyme-mediated reactions and the use of NMR methods in determining the e.e. and absolute configuration of chiral alcohols. Synthesis of a-Deuteriovanillyl Alcohol 2 I n a published undergraduate experiment, vanillin (4hydroxy-3-methoxybenzaldehyde(1))is reduced to vanillyl alcohol (4-hydroxy-3-methoxybenzylalcohol (2)) by whole The yeast suspended in a n aqueous sucrose solution (6,7). reaction is catalyzed by aldehyde dehydrogenase that uses nicotinamide adenine dinucleotide (NADH) and Hi. Reduction of a carbonyl group by this enzyme involves transfer of thepro-R hydride of NADH to the re face (8) of the n svstem to vield (S) alcohols (9).Because vanillvl alcohol doe¬ contain a stereocenter, it fails to illustrate the enantioselecti\it\~asuect of the reaction. Therefore, we have reduced vanillin b; yeast in deuterium oxide (heavy water)
Figure 2. Conformationalanalysis of the diastereomeric Mosher's esters 3. to give n-deuteriovanillyl alcohol 2 (see Fig. I),in 3 6 8 3 % yield, which has a stereocenter (10). Similarly, reduction of vanillin with sodium borodeuteride also gave racemic 2 in good yeld. The presence ot'a deuterium atom in 2 was confirmed by GC-MS studies in which the GC peak at 6.67 min gave a ~ o l e c u l a rion signal a t 155 amu-for the formula C8H9D03.In addition, a weak IR absorption band a t 2150 em-' for the C-D stretch was observed. The racemic product gave no optical activity, but the yeast product gave a specific rotation, [alDZz,of -0.29'. This demonstrates that enzyme-catalyzed reactions are enantioselective because the energies of the diastereotopic enzyme-substrate [E(S) or E(R)I transition states are different. Determination of the e.e and Absolute Configurationof the Yeast Derived Product The racemic and optically active alcohols 2 were conv e r t e d to t h e i r Mosher's e s t e r 3 w i t h (S)-(-)methoxytrifluorophenylacetyl chloride (MFTA-C1) using a microscale Drocedure (11). The 'H-NMR s ~ e c t r u mof the ester derived from the raccmic alcohol 2 showed two sets of diaatereomers of eaual sienals fur the !S.Sr3 . . and tS.Kr3 . . intensity. For the optically active sample of 2, only oneset of signals was observed, thus suggesting that the enantiomeric excess was 97 3%. The absolute confirmration of the onticallv active almhol .. was determined by analjhis of the ' ~ i - ~ ~ ~ " s ~ eofc its trum hlosher'sester t12, The Fisher pmjections dep~rtedin Figure 2 ol'the diastereomers, 3(S,S! iind 3tS,R,, show that in the latter isomer. the ~anillvl-methoxvsrouDis shielded in comparison to that for the ~ ( s , s )dia&eoker. As shown in Figure 3A the two vanillyl-OCH3 signals derived from the racemic alcohol 2 a t 3.61 and 3.52 ppm were assigned to the 3(S,S) and 3(S,R) diastereomers, respectively. I n the 'H-NMR spectrum of 3 synthesized from the optically active alcohol 2 (see Fig. ZB),only one vanillyl-OCH3 signal was observed a t 3.52ppm, thereby suggesting that the absolute configuration of the alcohol must be R.
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Figure 1. The reaction to give a-deuteriovanillylalcohol.
Conclusions This experiment demonstrates the effkiency and enantiospecificity of yeast-mediated reduction of vanillin to give a-deuteriovanillinyl alcohol. Furthermore, it also (Continued on nezt page)
Volume 70 Number 6 June 1993
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the microscale laboratory 607, 1575, 1376, 1236, 1033, 984, 836, 724, 573 cm-'; 'HNMR (CDCI4 6 6.89 (m. 3H.
-LA
nol), 4.60 (s hr, lH, HI, 35% (s, 3H, OCH,), 2.00 ( s br, lH, OH); GC-MS [HP5890A, 12m x 0.2mm Hp-l cross-linked methyl silicone (film thickness 0.33 mm) capillary GC ealumnl retention time 6.67 min; MS(E1)d z (rel.Intensity) 155 (MI,95, CsH9DO), 138(MfOH, 501, 137(84). 123(53Jd 108(55),107(68),66(99):[ale (ethanol) = -0.29"(c= 0.021 dmL). Reduction of Vanillin with NaBD4
Dissolve vanillin (152 m g , 1.00 mmol) i n dry methanol (dried over molecular sieves 3A) in a 25mLflask. Chill the solution in an ice bath, then add a freshly weighed-out sample of NaBD, (83 mg, 2.0 mmol). S t i r t h e solution under a drying tube a t O°C for 1 5 min, then a t room temperature for 90 min. F ~ g ~ 3r e 'h-NMRspectra of the d~asrereomerlcMosher s esters 3 (A) and (BI, ester 3 aerwea from the Remove the solvent using a racemlc ano opt ca ly act ve u de~ler~ovan~lly alcohols 2, respect vely (MTPA 1s Mosher s aclO anhydrloe rotary evaporator, dissolve The ' h NMR spectra were recorded on a Var an VXR 300s spectrometer, ana TMS was Lsed as the lnlernal the oily residue in ether (50 standard of S = 0 ppm mL) then wash it successively with water (5 mL) and brine (5 mL). Dry the shows how the enantiomeric cxccss and absolute c o n f i e ether extract with sodium sulfate then filter it. Concenration of alcohols can he e~tablishcdby 'H-NMK studies. trate the filtrate in a rotary evaporator and crystallize the product using the aformentioned procedure. The yields obExperimental tained by the students were 80-116 mg (52-75% yield). M.p., TLC, IR, 'H-NMR, and GC-MS data are similar to Reduction of Vaniiiin in DzO those of the optically active product, except [al~'' (ethanol) I n a 250-mL Erlenmever flask add 99.8% deuterium = V (c=0.025 g/mL). oxide (100 ~ L Isucrose , tiable sugar, 10 g,, vanillin (0.20 g, 1.3 mmol~,and a Tcflon-coated mametic stirring bar. Stir Preparation of Mosher's Ester 3 for a f e w m i n u t e s to dissolve tGe vanillin, t h e n add Fleischmann's "active dry" yeast (7 g, one packet). Loosely I n a dry 25-mL round-bottomed flask, a d d (S)-(-1plug the flask with a piece of cotton, and stir the suspenmethoxytrifluoropheny1 acetic acid (MTPA, 2.8 mg, 0.012 sion a t mom temperature for 16 h. At that time add more mmol), dry DMF (0.9 pL, 0.012 mmol), hexane (0.5 mL) yeast (about 3.5 g) and continue to stir for another 24 h. and oxdyl chloride (5 pL, 0.057 mmol). Stopper the flask, Centrifuge the fermentation broth a t 2000 rpm for 10 rnin, and after 1h a t room temperature, concentrate the mixthen extract the supernatant with ether (100 mL each) ture using a water aspirator. This will give the acid chlorthree times. Combine the ether extracts and dry it with ide (MTPA-C1) a s a n oil. Dissolve alcohol 2, DMAP (N,Nsodium sulfate, then mncentrate it in a tared flask using a dimethylamiuopyridine, one crystal, -1 mg) i n d r y rotary evaporator. An oily residue will result. Dissolve it in triethylamine (4 pL, 0.03 mmol) and CDCl3(0.1 mL), then CH&L - - (. 1 mL). then remove the solvent with a -gentle add this solution to the above acid chloride. Stopper the stream of nitrogen. A white crystalline product will reflask and let i t stand a t room temperature for 1 h, then main. Wash the crvstals with a solution of 20% ethvl acetransfer it into a n NMR tube. Dilute it to about 0.4 mL ether (30-60°, 2 mL). Remove the soltate: 80% with CDC13 containing 0.1% TMS, and record a 'H-NMR vent with a Pasteur pipet and leave the product to dry spectrum. overnight. TLC (1:lethyl acetate: toluene)R p 0.83; IR(CHC13)v3600, 2200 (C-D), 1747 (ester), 1643, 1600, 1556, 1442 cm-I; 'HThe yields obtained by students were 77-168 mg (3843% NMR (CDCl?)6 for (S,R)isomer: 3.52 (6, vanillyl-OCHa);5.32 yield). M.p. 108-11O0C;Silica gel TLC (1:lethyl acetate:tolu(s. vanillvl-OH):(SS) isomer: 3.61 (s vanillyl-OCHJ, 5.31 (s, ene) R,=0.50: IR (KBr) v 3445,3200(broad),2920,2150(C-Dl,
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the microscale laboratory vanillyl-OH).GC-MS: 7.79 min; no M+ ion was observed under our conditions. Acknowledgment The Experimental Techniques 1Class of Winter 1992 is acknowledged. Literature Cited 1. Gmas, M A n n u o l Rep.Med Chem. 1990,25,32&231. 2. Remos T o m b G.M.: Bellus, D.Anpelu Chem. Inl. Ed. E n d 1993.30, 1193-1215. 5. Csuk,R.; Glanzer, B. I. Chem Re". 1991,31,41C97. 6 . Nimitz J. S. Er~rrimpnfsin O w n i c Chemistry : Rentice-Hall: Endewood Clifi,
. ...,
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7 . Banesbv,A.R.:Stauton, J.;Wfitshire,H.R.J C S . Perkin 1 1 W h 11561162
11. Ward, D.; Rhee, C. K lbhohedmn Lett 1991,32,7165-7166. 12. Yamaguehi, S. hkymmtric Synthosla;Mamison, J. D., Ed.:Academic Reas, Ine.: New York. 1983: Val. 1,pp 125-152.
Acknowledgment Support from NSF-ILIP and NSF-Young Scholars grants (USE-8851427 and RCD-9055029) are gratefully acknowledged. Literature Cited 1. Lehman, J. W. Opmtioionol O~gonicChemlafry;A l l p andBsmn: Bostrm, 1981. 2. Ranman, P J Chem. Edrrc. 1985,62,640. 3. Wiltiaman, K L. Mocmsmlaond Microscale OgonicExprimntn, D.C. Heath: hington, MA, 1989. 4. Fieser, L.F.:Williamson, K L. organic Experiments, 6thed.;D.C.Heath: Lexingtrm, M A 1987 5 . McKone, H . T . J Chem.Edue. 1973,56,6'76.
6 . Tantillo, M.J . Chem. Edue. IS-, 65,254. 7 . p s n a , D. L.; L-pman, G. M.; and k e , 0 . S. Infmdudion to Organic Iabomtory Tmhniguss; Q.B. Saunders Ca.:Philadslphia,PA, 1976. 8 . Sadier, G.;Davis, J.; Derman, D.J. FoodSci. 1990, hi, 1460.
Separation of Methylene Blue and Fluorescein: A Microscale Undergraduate Experiment in Column Chromatography
The Microscale Separation of Lycopene and p-Carotene from Tomato Paste James Goodrich. Chris Parker. and Ruff Phelos Lou~s~ana State ~ n ~ ; e r sIn~~hreve~orl t~ Shrevepoll. LA 71 115
Experiments for the isolation and analysis of carotenoids from carrot andlor tomato pastes are popular in instructional chemistry laboratories (Id)but oRen require 3.5-5 h. We have developed an experiment for the isolation and separation of p-carotene and lycopene from tomato paste on the microscale level that affords the isolation of two compounds from a single common foodstuff and requires less than 3 h to complete. The compounds can be rapidly analyzed. It provides experience with column chromatography (for separation) and UV-VIS spectrometry (for analysis). The column is prepared with insertion of a glass plug into the tip of a 5.75 in. Pasteur pipet. Alayer of alumina (80-200 mesh) (dry-packed or slurried in 99:l petroleum ether-acetone) ( 6 ) is added to a height of -8 an.An optional layer of sand (3 mm) may be added to complete the column. The use of approximately 3 g of tomato paste provides enough lycopene extract for spectral analysis. The tomato paste is extracted with petroleum ethedacetone (50:50) (3 x 10 mL) and filtered. The combined extracts are washed with saturated aqueous sodium chloride (25 mL), 10% aqueous potassium carbonate (25 mL), water (25 mL), and dried with sodium sulfate. After concentrating the volume in vacuo to several milliliters, it is placed on the column for separation. The C)-carotene1s eluted with petroleum ether acetone (99:11andthe lycopene ~selutedwitha 9W10 mlxture. Positive oressure noolied with a ruhber bulh will soeedelution but can cause band spreading, particularly with the lycopene. Eluants were analyzed spectrophotometrically using (90:lO) petroleum ether-acetone as a reference. The characteristic absomtions of lvco~enewere observed near 504, 473, and 446 i m ; whereas, 'p-carotene occurs at 476 and 450 nm (7).Quantitative vields can be calculated using the molar absorptivity at 470 nm (1.85 x 105) (8). A158
Journal of Chemical Education
Paris ~voronos'and Edward Sarlo Queensborough College of the City University of New Yorh Bayside. NY 11364
Often column chromatography (CC) is used to separate mixtures and/ or purify compounds. The procedure is so commonly employed that the technique usually is included in the first semester of an oreanic chemistrv course. However, many of the procedures-found in laborkary manuals are both tedious and comdicated for students in a beeinning organic labllratory c k w ctable,. We offer a simple microscale CC experiment for separatin~and isolating the componentsofa 5Q50 methylme hlue-fluorescein mixture that uses a disp~~sahle pipet, two nontoxic solvents (water and ethanol.. and aluminn and that allows the student to see the clean separation of the blue and yellow colored nontoxic dves. The exneriment is suitable for laree " laboratom sections because it requires inexpensive and easily disposable eaui~mentand cheniicals. In addition. the results can be qu&&ied by measuring the collected ðylene blue spectrophotometrically.
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Procedure Asmall uniform plug of glass wool is placed into the bottom of a disposable pipet with the aid of a thin glass rod, wire or "unfolded" paper clip. Alumina is added until it forms a 3-in. high column. The pipet is filled with 95% ethanol and, with a finger placed on top, inverted and shaken vigorously to ensure that the alumina is evenly suspended in the solvent and that all air bubbles are eliminated. The pipet is turned right-side up and secured over a test tube using a clamped, one-hole rubber stopper. Traces of alumina adhering to the glass may be washed down with a few drops of ethanol. The solvent level must be kept above the top of the column of alumina throughout the experiment by continuously adding ethanol. Two to three drops of a green dye solution composed of 5%methylene blue and 5% fluorescein in 95% ethanol is added directly to the column, which is then eluted using 95% ethanol. The first component, methylene blue, is collected in a test tube placed under the tip of the pipet. When the eluate is no
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