edited by
ARDEN P. ZIPP
the microscale loboratory
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 opti-
cally active pharmaceutical agents ( I ) and agrochemicals
(2)are associated to a given absolute configuration. Conse-
quently, there is interest in the development of methodol-
ogy 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 demonstr-
ate the efficiency and enantiospecificity of microbiallen-
zyme-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 (4-
hydroxy-3-methoxybenzaldehyde(1))is reduced to vanillyl
alcohol (4-hydroxy-3-methoxybenzylalcohol (2)) by whole
yeast suspended in a n aqueous sucrose solution (6,7). The
reaction is catalyzed by aldehyde dehydrogenase that uses
nicotinamide adenine dinucleotide (NADH) and Hi. Re-
duction of a carbonyl group by this enzyme involves trans-
fer of thepro-R hydride of NADH to the re face (8) of the n
svstem to vield (S) alcohols (9).Because vanillvl alcohol
doe&not contain a stereocenter, it fails to illustrate the en-
antioselecti\it\~asuect of the reaction. Therefore, we have
reduced vanillin b; yeast in deuterium oxide (heavy water)
Figure 1. The reaction to give a-deuteriovanillylalcohol.
SUNY-CoRland
c0-nd.Nyi3M5
Figure 2. Conformationalanalysis of the diastereomeric Mosher's es-
ters 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 con-
firmed 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 prod-
uct 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 con-
v 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
-
sienals fur the !S.Sr3 . .
. . and tS.Kr3 diaatereomers of eaual
intensity. For the optically active sample of 2, only oneset
of signals was observed, thus suggesting that the enantio-
meric excess was 97 3%.*
..
The absolute confirmration of the onticallv active almhol
was determined by analjhis of the ' ~ i - ~ ~ ~ " s ~ eofc itstrum
hlosher'sester t12, The Fisher pmjections dep~rtedin Fig-
ure 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 ac-
tive alcohol 2 (see Fig. ZB),only one vanillyl-OCH3 signal
was observed a t 3.52ppm, thereby suggesting that the ab-
solute configuration of the alcohol must be R.
Conclusions
This experiment demonstrates the effkiency and en-
antiospecificity of yeast-mediated reduction of vanillin to
give a-deuteriovanillinyl alcohol. Furthermore, it also
(Continued on nezt page)
Volume 70 Number 6 June 1993 A155
the microscale laboratory
607, 1575, 1376, 1236, 1033,
984, 836, 724, 573 cm-'; 'H-
NMR (CDCI4 6 6.89 (m. 3H.
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 eal-
umnl retention time 6.67 min;
MS(E1)d z (rel.Intensity) 155
(MI,95, CsH9DO), 138(Mf-
-LA
OH, 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 mo-
lecular sieves 3A) in a 25-
mLflask. Chill the solution
in an ice bath, then add a
freshly weighed-out sam-
ple 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 succes-
sively 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. Concen-
ration 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 ob-
Experimental tained by the students were 80-116 mg (52-75% yield).
Reduction of Vaniiiin in DzO M.p., TLC, IR, 'H-NMR, and GC-MS data are similar to
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)-(-1-
plug the flask with a piece of cotton, and stir the suspen- methoxytrifluoropheny1 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 mix-
then extract the supernatant with ether (100 mL each) ture using a water aspirator. This will give the acid chlor-
three times. Combine the ether extracts and dry it with ide (MTPA-C1) a s a n oil. Dissolve alcohol 2, DMAP (N,N-
sodium 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 re- flask and let i t stand a t room temperature for 1 h, then
main. Wash the crvstals with a solution of 20% ethvl ace- transfer it into a n NMR tube. Dilute it to about 0.4 mL
tate: 80% ether (30-60°, 2 mL). Remove the sol- 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,
The yields obtained by students were 77-168 mg (3843% 2200 (C-D), 1747 (ester), 1643, 1600, 1556, 1442 cm-I; 'H-
yield). M.p. 108-11O0C;Silica gel TLC (1:lethyl acetate:tolu- NMR (CDCl?)6 for (S,R)isomer: 3.52 (6, vanillyl-OCHa);5.32
ene) R,=0.50: IR (KBr) v 3445,3200(broad),2920,2150(C-Dl, (s. vanillvl-OH):(SS) isomer: 3.61 (s vanillyl-OCHJ, 5.31 (s,

A156 Journal of Chemical Education

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,
...,
. .
.. .
...
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.
The Microscale Separation of Lycopene
and p-Carotene from Tomato Paste
James Goodrich. Chris Parker. and Ruff Phelos
Lou~s~anaState ~ 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 instruc-
tional 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 chromatog-
raphy (for separation) and UV-VIS spectrometry (for anal-
ysis).
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 op-
tional 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. Pos-
itive 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
Acknowledgment
Support from NSF-ILIP and NSF-Young Scholars grants
(USE-8851427 and RCD-9055029) are gratefully acknowl-
edged.
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: h-
ington, 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
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. How-
ever, many of the procedures-found in laborkary manuals
are both tedious and comdicated for students in a beein-
ning 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 non-
toxic dves. The exneriment is suitable for laree
" laboratom
sections because it requires inexpensive and easily dispos-
able eaui~mentand cheniicals. In addition. the results can
be qu&&ied by measuring the collected &ethylene blue
spectrophotometrically.
Procedure .
Asmall uniform plug of glass wool is placed into the bot-
tom 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% eth-
anol 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 alu-
mina 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 experi-
ment 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 col-
umn, 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
'Author to whom correspondence shoud be addressed.