in Baker's Yeast Reductions of Ethylacetoacetate An NMR Experiment K. 6. Lipkowitz and J. L. Mooney Indiana-Purdue University, Indianapolis, IN 46223 NMR experiments for undergraduate organic chemistry laboratories are difficult to find. In an earlier paper we presented a lahoratory exercise in which NMR spectroscopy and molecular mechanics are used in tandem for conformational analysis.' In this paper we provide a laboratory exercise in which NMR is used to monitor enantiomeric excess in asymmetric reductions. Aside from develooine a hands-on NMR exercise, this experiment was developed in response to my ohservation that students seldom have the opportunity to work with optically active material, have a poor working knowledge of stereochemical principles, have inadequate opportunities to employ techniques in stereochemistry, and have a meager vocabulary of the aforementioned science. The paucity of lahoratory exercises that focus on asymmetric centers or dissymmetric groupings is venial in light of the high cost of instrumentation needed t o measure chirovtic . .oroverties . along with the expense of purchasing chiral reagents. Nonetheless. we are ohlieed to exvose students to techniaue and methodology that is an inte& part of modern synthesis. T h r Inborators cxrrcise has been nucressfulls tauaht Revera1 times. We have a short version which can be completed in two weeks and a longer version that lasts seven weeks (see below). T h e experiment focuses on baker's yeast reduction of 8-ketoesters. This is an extension of the elegant exercise In our experiment, howoutlined by Fieser and Williams~n.~ ever, we deal directly with the alcohols rather than the dinitrobenzoate derivatives. The short version involves asymmetric reduction of only ethylacetoacetate and analysis of
'Lipkowitz, K. B. J. Chem. Educ. 1984, 61, 1051-52. Fieser. L. F.; Williamson. K. L. Organic Experiments, 5th 4.: Heath: Lexington. MA. 1983. Chapter 44. Bucdareili, M.; Fwni, A.; Moretti, I.; Twre, G. Synthesis 1983, 897-899.
enantiomeric excess. In the long version of the experiment we a t t e m ~to t understand the structure and mechanism hv which an enzyme sterrospecifically redures the ketoester. Our avvroach is tosee how strurrural modificationsoithe 3ketoeiiers influence the percent yields and the enantiomeric excess of the reduction. This type of structure-activity-relationship (SARI is commonly employed in the pharmaceutical industly for drug design, and it provides the student with the flexibility to pursue ideas gleaned from the literature or from their own imaginations. Experimental Methods
The 8-ketoesters used in the experiment were limited to those with minor structural changes in either the alcohol portion or in the acid portion of ethylacetoacetate, e.g., RCOCH&02R'where R = Me, Ph, t-hutyl and R' = Me, Et, i-vr, t-hutvl etc. Most of the reaeents are available from the idr rich c&ilog. I t is advisable Tor the students to work in groups and to combine their products so enough material for vacuum distillation and instrumental analysis is available. For the two-week version of this experiment we limit yeast reductions to ethylacetoacetate; for the extended version, research groups start by using the conditions outlined by Fieser and Williamson but quickly modify variables like time, temperature, buffers, concentrations, brand of yeast, etc. Major changes in procedure, like loading up the yeast but deleting the sugar, are found in the literature and are also a t t e m ~ t e d . ~ T o address the issue of how enantiospecificity is influenced hv ketoester structure one must orovide a method of measuring the enantigrm~rirexcess 01 the reaction. Rather than aeneratr diastereomeric adducrs of thc redurrion oroducts, we work directly with the purified enantiomeric~alcohols. Several analytical techniques including polarimetry, gas chromatography with chiral columns, chiral solvents andlor chiral shift reagents were considered as viable meth-
Volume 64
Number 11 November 1987
985
Figure 3. Spectrum of 30 p L ( f)-ethyl-3-hydroxybutanoate in 300 pL CCI, wilh 0.04 g chiral shift reagent. Sweep width = 10.00.
Figure 1.The 60-MHz proton spectrum of 30 pL (-ttethyi-3nydroxybutanoate in 300 pL CCIITMS. Sweep width = 10.00.
Figure 4. Quartets and doublns of the diastereomeric complexes in Figure 3 expanded and integrated. Sweep width = 1.00.
Figure 2. Spectrum of 30 pL (ftethyl-3hydroxybutanoate in 300 pL CCI, with 0.02 g chid shin reagent. Sweep width = 10.00.
ods to determine optical purity. Polarimetry was used when specific rotations were already known hut not many of the product optical rotations were in the literature. Hence, NMR spectroscopy with tris[3-(heptafluoropropylhydrmymethylene)-(+)-camphoratol-europiumI11 shift reagent was used to determine optical purity on a routine basis. Shift reagent studies were not performed until students demonstrated that their analyte was a t least 90% pure. This was accomplished by NMRspectroscopy or VPC analysis on 10% SE-30columns. 'To de~nmstrntethat the diastereomeric ct~mplexescan he resolved nt 6 0 hlH7 the studenrs p ~ r i m n e dketone redurtions (e.g., NaBH4 under standard conditions) to generate the corresponding racemic mixture of aln~hols.An example i i shown in figure I . This is a :W-pT. aolurim of ( f )-ethyl-3hydroxyl)utanoate in 300-pL CCI,,'l'\I5. lnrremenral addition of 0.02 g shift reagent gives rise to the series of spectra in Figures 2-3. Clearly a douhling of peaks is observed as a consequence of the two diastereomeric complexes, and upon expansion and integration (Fig. 4) one finds a 1:l ratio of enantiomers. It is mandatorv that students nrepare a racemic mixture of reduction Goduct to prove to-themselves that the NMR method can identify each enantiomer. Otherwise, they will he wondering whether the yeast has reduced 986
Journal of Chemical Education
the ketoester stereosnecificallv or whether a mixture of enantiomers has been formed tgat cannot be resolved by 60MHz NMR. For ethylacetoacetate reduction by yeast, the single set of lines observed in the shift reagent study results from reduction that has proceeded enantiospecifically. Having demonstrated that enantiomer mixtures can be quantitatively evaluated with this simple technique, the students are in a position to evaluate the optical purcty for each of the yeast reductions they perform. Summary We have nrovided a lahoratorv exercise that is safe and inexpensive, and it overcomes two deficiencies in undergraduate oreanic chemistrv laboratow curricula. First. this experiment provides a hands-on exercise in NMR spectroscoDV where the instrument is used in a robust manner: keepine ;Lck of paramagnetic shifts and full-scnlc expansions a;lotc field are reciuirrd. In addition to lenrninr how paramagnetic shift reagents can simplify spectra, eachstudent has i o understand how these Lewis acids bind t o form diastereomeric complexes that give rise to different spectra. Second, this experiment concerns fundamental concepts in stereochemistry and the vocabulary needed to describe this. I t is not beyond the grasp of sophomores to understand the difference between stereoselective and stereosnecific reactions nor is it unreasonable to expect them to understand and use vocabulary pertaining to prochiral relationships and enantiotopic faces, and, in NMR spectroscopy, understand what homoto~ic hvdrogens . . .. are. In fact. thev tend to revel in their undcrs~andingot'thesr pointsand areall too eager todiscu~s this with you. Finally, we . point
