Synthesis and Purification of Diastereomers: Sodium Borohydride Reduction of a Chiral Ketone

Procedure

Pre-lab

Two of the reagents in this experiment, NaBH4 and MgSO4, are hygroscopic, they absorb water from the air. Please keep their reagent bottles capped except when it is absolutely necessary to open them.

You may have trouble finding hazard information on the ketone (1) starting material, but you can assume that it is volatile and flammable (this is the case for most organic compounds), and you should protect yourself accordingly.

You may also have trouble finding the physical properties of your compounds. Some of these properties are listed below. As always, you need to complete this table and enter it in your lab notebook before coming to lab.

CompoundMol Wt(g/mol)mp(oC)bp(oCmmHg)d(g/mL)

Amount Desired

ketone 1
?
-10
189760 7210
0.887
2 g ? mmol
trans alcohol 2
?
56
189760 7610  
cis alcohol 3
?
37
2017509212  
NaBH4
?
   1 g ? mmol
2-propanol ????

Molecular modeling (Week 1)

A set of molecular modeling activities will be distributed in lab. These will demonstrate the modeling procedures and the calculations of conformer ratios required for a conformational analysis of 2 and 3. The molecules in the activities are slightly different from your products, but practicing with them will show you how to conduct a conformational analysis of 2 and 3. Once you have finished your “practice” session, do the following and record your results in your lab notebook.

Using Spartan’20 (Chemistry computer lab), build (do not sketch) a model of 2a [NOTE]Select Rings: Cyclohexane to introduce the ring.
Free rotation can occur around the CO single bond. This means each “chair alcohol conformer” is really a family of conformers that 1) share the same chair conformation, while 2) possessing different and unique OH orientations. You are interested only in the most stable member of each family. That is, you will take the strain energy of 2a to be the strain energy of the OH conformer of 2a that places the OH group in its most stable orientation. You will do the same for 2e, 3a, and 3e. (This is exactly analogous to the way you treated the ethyl group in 1-ethyl-3-methylcyclohexane in the molecular modeling activity.) The best OH orientation must be identified by trial-and-error. You can, if you want, 1) build the same chair conformer with three different OH orientations (three is the magic number because there are three different ways to stagger the OH relative to the ring), 2) minimize the strain energy of each model, and 3) identify the OH orientation that creates the lowest strain energy and record its energy. The other, less stable, family members should be ignored. Building hint #1: Rather than build three separate models with different OH orientations, just build a single model, minimize its energy and note the OH bond orientation. Then use internal rotation to re-orient the OH bond to a different staggered conformation (see hint #2), and minimize the energy again. Repeat for the third OH orientation. Remember, you are interested only in the OH orientation that gives the lowest strain energy. All of these operations can be performed in the Edit Build window Building hint #2: To change the orientation of the OH group by internal rotation, you need to be looking at the model in the Edit Build window. Select the bond that you want to rotate (a red arrow encircles the active bond). Then move the cursor into the ‘internal rotate’ strip on the left side of the window, press the LEFT mouse button and move the cursor up and down inside this zone to reorient the OH group.
. Click on the minimize button to calculate your model’s equilibrium geometry and strain energy.

Record the strain energy in the energy units given by the program. Also inspect the model’s structure carefully. Does it correspond to the conformation that you started with? How would you describe this conformation? Minimally, this means considering the following: Did the ring adopt a chair conformation? What are the orientation(s) of rotatable substituent(s)? Are any of the atoms positioned in ways that significantly distort the “chair” or “staggered” arrangements one normally expects?

Analyze 2e, 3a, and 3e in the same way.

Once you have collected your four strain energies, identify the preferred conformations of 2 and 3, and also calculate the difference in strain energies between alcohol conformers, i.e., between 2a and 2e, and between 3a and 3e. Use this energy difference to estimate the equilibrium constant for each conformational equilibrium at room temperature (assume ΔG = difference in strain energies; see the molecular modeling activities handout for helpful formulas connecting strain energy and equilibrium constant). Do your results support the qualitative conformational analysis (and subsequent coupling constant analysis) given in the Background?

Reaction (Week 2)

Weigh and transfer NaBH4 (~1 g) to a capped round bottom flask, and add sufficient isopropanol so that the ketone will be roughly 0.5 M. To this solution, add 3,3,5-trimethylcyclohexanone (2 g), recap the flask, and stir.

Follow the progress of the reaction by TLC using a 4:1 mixture (v/v) of hexane:ethyl acetate as your eluting solvent [NOTE]The positions of 1-3 on the TLC plate cannot be established using a UV lamp because these compounds do not absorb the lamp’s UV radiation. You will visualize the plates using a chemical detection method, that is, you will treat your plate with a reagent that converts 1-3 into colored substances. The necessary reagent (a mixture of 2.5% 4-anisaldehyde, 1% acetic acid, and 3.5% sulfuric acid in 95% ethanol) will be provided to you and its use will be demonstrated in class. HAZARD: the reagent is highly corrosive and should only be used on a suitable surface and in a fume hood..

Workup (Week 2/3)

When the reaction is finished, pour the mixture into a separatory funnel containing 20 mL of saturated brine, 10 mL of deionized water, and 40 mL of ether [NOTE]Use extraction-grade ether from the red cans.. Separate the layers, and extract the aqueous layer with two additional 25 mL portions of ether. Dry the combined organic extracts over MgSO4, gravity filter, and remove the ether and isopropanol from the filtrate by rotary evaporation.

After the ether and isopropanol have been removed, distill the residue under vacuum using short-path distillation head and a cow connected to the ‘dry vac pump’ [NOTE]The vapor from the boiling liquid may not make the apparatus hot enough for a successful distillation, that is, the vapor may condense in the flask without entering the distillation head. If this happens, there are a few options you can try. Once you notice that boiling has ‘stalled’ inside the flask, try: raising the fume sash a bit (this will change how much cool air flows over your apparatus; downside this reduces the protection you receive from the fume sash), constructing a small ‘tent’ around the apparatus using cotton, foil, or a towel, to shield the apparatus from the cool air flowing into the hood (downside: you won’t be able to see inside your apparatus), using a heat gun to heat all of the upper flask and the distillation head (HAZARD: heat guns become extremely hot; do not heat flammable material or material that might melt).. Collect any compounds that distill below 100 oC  into one “udder,” then when the temperature begins rising past 100 oC rotate the cow to an empty udder. Collect a few drops, then rotate the cow again, and collect the main fraction (record the temperature range). If any of your udders get filled, rotate to the next one and continue collecting the fraction. Once your distillation is complete, disconnect the vacuum, then turn the pump off. Weigh your sample then obtain its IR and 1H NMR spectra. Prepare a full NMR spectrum, and include an expansion of the region from 3.5-4.5 ppm. For the expansion, use “peak picking” to record the frequencies, not the chemical shifts of each peak in the expanded region. Integrate the expanded region to find the cis/trans ratio.

Chromatography (Week 3/4)

To determine the components of the distilled product, begin by making a dilute solution that you can use to spot your TLC plate. Dissolve 1-2 drops of the distilled product in 1 mL of ethyl acetate then spot this solution on a TLC plate. You should also spot the starting material standard and make a co-spot. Develop the TLC plate using a 4:1 volume by volume (v/v) mixture of hexane:ethyl acetate as your eluting solvent and visualize your plate using the Anisaldehyde dip reagent provided. The two diastereomers are resolvable (but close!) by TLC. Identify the two spots that correspond to the products, then prepare for your column.

Separate the cis and trans isomers present in a 400 mg sample utilizing dry-column flash chromatography. For this part of the procedure, choose someone to partner with and run the column together.

Prepare and apply sample mixture. To start you will need to prepare the column. Please refer back to the steam distillation of an essential oil for a complete set of instructions on how to prep your silica column. Once your column is ready, dissolve 400 mg of your distilled sample in 3-4 mL of hexanes. Remove the filter paper from your column, then carefully apply this solution evenly to the top of your silica column using a pipet. Once you are finished, replace the filter paper.

Elute the column. Refer back to the essential oil procedure for the basic sequence of eluting the column. For this experiment you will use a different mixture of solvents then we used purifying clove oil, which will achieve a more gradual solvent gradient. Please refer to the standard fractions portion of this experiment for a complete list of the solvent combinations you will use. The following Hexanes:Ethyl Acetate solvent mixtures will be provided to you in lab: 95:5, 90:10, 85:15, 80:20, and 70:30. Pure hexanes and ethyl acetate will also be available.

Analyze the fractions.

Round 1. Once you collect 24 fractions, test all of them by applying a small sample of each to 1-2 fluorescent TLC plate(s) (label your spot locations in pencil!). You can divide the plate into a “grid” if you like. You will not be developing the plate in your TLC chamber, only viewing it.

The fractions that contain your compounds are very dilute. Therefore, apply all of the liquid in your capillary tube to the TLC plate each time you spot a fraction. You can make sure that the spot stays very small as follows: quickly touch the capillary tube to the plate, blow on the plate to evaporate the solvent, then touch the capillary to the same spot to add more sample. Always let the solvent evaporate before applying more sample.

After you have spotted all of your fractions (clean the capillary between fractions by blotting it dry!), stain the plate using the Anisaldehyde reagent (do not elute the plate!) and heat to see which fractions contain a compound. Record these data in your notebook.

Round 2. Once you know which fractions test positive for a compound, elute (using 80:20, Hexanes:Ethyl Acetate) all of the “positive” fractions on fresh TLC plates to see what these fractions contain. It is best to apply consecutive “positive” fractions to a plate e.g. Fractions 12, 13, and 14. You should also spot a 5% solution of a standard sample to compare your “unknowns” (fractions) against a “known.” This will help you identify the compounds present.

After TLC analysis, combine all fractions that are either pure or enriched in one of the two diastereomers into a weighed round bottom flask. Then combine the fractions that are either pure or enriched in the other diastereomer into another round bottom flask. Each partner will rotovap one of the diatereomers and weigh the purified product. Each of you will prepare an 1H NMR sample for each alcohol. You should have one sample for the cis alcohol, and a second NMR sample for the trans alcohol. Expand the 3.5-4.5  ppm region as you did previously do analyze your final spectra.