I recently made an esters of cholesterol on 40 mg scale (not acetate). Using DCC+DMAP conditions followed by 10% K2CO3 and 10% HCl washes to get rid of DMAP and unreacted acid and I obtained pure product in 78% and 64% yields after chromatography.

Im makind some more esters using same conditions but with 10% NaOH wash instead of carbonate because it seems carbonate is not enough to was the acid im using into aq. layer. I observed no hydrolysis and obtained my esters in about 60-80% yields after chromatography.

I think water wash is fine even with some acid or base added. If you are worried you can get rid of pyridine with aq. CuSO4 wash and acetic acid can be azeotroped with solvents like toluene or heptane or freeze dried.

Salam Mathew: I'm not sure i got your point , can you sent me your schematic diagram of your reaction equation ? may be i can help.

Warm Regard

You can use Steglich esterification as Michal mentioned or you remove the HOAc azeotropically with Toluene or n-Heptane after the first evaporation.

Alcohols are sufficiently reactive and can react even in absence of a base. You can try the acylation with Ac2O by stirring the compound with excess (say 1.5 equiv) of acetic anhydride for a certain period followed by removal of unreacted acetic anhydride and acetic acid by product under vaccum (if necessary with flow of N2 or Argon). If there is any solubility problem arises during reaction then anhyd. dichloromethane can be used as solvent. After removal of the solvent the pure acetate derivative should be obtained.

As for the reaction: Gandhi is right to use neat Ac2O only, but you may use (dried) NaOAc or very minimal, catalytic amount of FeCl3*6H2O. For your nanoreaction 1-2 very small crystal is enough. Using this latter Lewis acid catalyst the reaction is very fast, 2-5 min at room temperature.

As for the workup: You can use extraction efficiently but

a) use bicarbonate in the aq, phase and avoid the use carbonates, because the carbonates pH unlike HCO3 is enough high to initialize the hydrolysis;

b) work as fast as possible.

To avoid the use of base in workup, you can extract the the reaction mixture with 0.5-2 N (as you like it) NaHSO4 solution, approy 2-3x more molar fold for the pyridine (if use use DMAP, but for this reaction it has no advantage, you need to calculate also with that). It can be assumed that your product is soluble in apolar solvents, hexanes/heptane can be used as solvent, but you know it better than me. For the drying after the extraction you obviously use Na2SO4, but to remove the eventual acid residue, you can add a small amount of drying K2CO3 - it can be used for drying purposes (because it is not soluble in organic solvents, the hydrolysis cannot occur).

Regarding the N2 flow to completely remove AcOH/py is not a really suitable method because of their high boiling points. After removal of the reagent/py excesses the more volatile solvent is considerably better.

Good luck

The method as mentioned by Laszlo Jicsinszky is quite good but for large scale preparations only. Since here Matthew D. Wolhowe is using substrate of conc 1 microgram level it is better not to use any solid reagents like DMAP, or Fecl3 or NaOAc or anything that can produce any solid biproduct. These things will be difficult to remove from the produt as these are not volatile. Conventional extraction followed by washing with water in a separatory funnel is also not possible with such low concentration of substrate. That's why it is better to use Ac2O directly and as only reagent. Both unreacted Ac2O and AcOH produced after reaction can be removed completely under high vacuum and as the acetate derivative produced must be a solid it will not be volatile and will not go out under the condition used.. Hope there will be no problem and the acetate produced will be almost pure. Heating at high temperature is also not desirable as it may lead to little bit of alkene via elimination reaction of acetates if there is any beta hydrogen present.

Gandhi: you are right with the amounts but the on nanoscale the dilution with 2-5 ml solvents and extraction is not a challenge. Although I do not like (more exactly, I hate) those reactions but here the students are regularly do 10-50 mg scale reactions with extraction.

Edward: In theory it works but in the real life you will be never able to completely remove pyridine from an organic phase with mild acidic water - except of large number of extractions.

The problem is that many times the pyridine/carboxylic acids are soluble also in organic solvents. Even the formic acid salts. Yes, I know what the partition coefficient is. Additionally, the AcOH pKa is ~4.8, so the difference is practically negligible, i.e. if the AcOH is equivalent, the pyridine is surely not completely ionized. Neither the Py.

As I told you, the mild acid aq. extraction works ... in theory. For the real removal of pyridine, you need to use a real acidic (pH ~2) medium for extraction.

In what we agree: the simple water wash is surely not enough.

You can remove acetic anhydride and pyridine by first aqueous/organic workup using carbonate/bicarbonate solution and MDC or ethyl acetate and then washing the organic layer with aqueous copper-sulphate solution to remove pyridine. Quantity of copper sulphate depends on quantity of pyridine used. You can wash organic layer multiple times with copper sulphate solution. This is indicated process as once pyridine will be removed there will be no or minimal color change observed in copper sulphate solution.

Hey all. Sorry for the slow reply, but thank you so much for all your input on the workups. Ghandi, your suggestions are, essentially, what we always used to to. It always worked before. However, for several months now we've been having horrible acetylation yields off sterols. We can acetylate a sterol standard, compare it to a silylated batch, and get 30% yield vs 100% yield. Acetylating an n-alcohol works just fine.

We've tried changing out for new reagents for fear of water, we've tried different glassware or teflon reaction vessels, and now I've tried phase separations instead of evaporation. I tried triethylamine instead of pyridine. I tried acetyl chloride instead of the anhydride. I tried all sorts of ratios of the anhydride, pyradine, and toluene as an additional solvent. Nothing has worked.

Right now I'm trying 1:1 anhydride:pyridine with DMAP and with n-methylimidazole as catalysts. I'll have to do the 'hated' microscale phase separation.

Does anyone have any ideas for why I might have such difficulty acetylating sterols?

Also: my reactions I tried with TEA turned deep yellow, and even after phase separation I got massive quantities of residue. Any ideas what this is? Oxidation products? I'm concerned because my reactions with DMAP are currently turning yellow as well.

Dear Mathew David Wolhowe, is it possible to disclose the structure of the sterol? Because sometime due to some stereo chemical orientation of the -OH group, acylation may be difficult. It can only be predicted/ explained once the structure is known.

Best work-up for acetic anhydride/pyradine acetylation is that remove unreacted material by distillation under reduced pressure.The treat the mixture with water and extract in an ether.