Sciencemadness Discussion Board

Extracting/Precipitating LPAC from octanol

zimaman - 15-5-2009 at 18:27

There are several papers about the biosynthesis of LPAC (l-phenylacetylcarbinol) by fermenting yeast in a two-phase system of octanol:H2O. In the papers, the researchers obtain this alpha-hydroxy ketone (in a ~100g/L octanol yield) by extractions with a 200-fold volume of water. That's a lot of water if you're planning to do a 1/2 to 1L biosynth.

I'm thinking there must be a better way to extract this compound from the octanol layer. Would there be a way to precipitate it from the organic layer? I know Organikum said that the bisulfite adduct formation is no good in extracting LPAC from water and that the use of hydroxylamine to form the oxime was better. Would this work in octanol?

Nicodem - 16-5-2009 at 10:18

Are we supposed to be guessing or what? How is anybody to answer when you gave not a single reference for any of the "several papers about the biosynthesis of LPAC (l-phenylacetylcarbinol) by fermenting yeast in a two-phase system of octanol:H2O"?

Lately I feel like a broken record by continuously saying this, but anyway: Open thread without providing references only in the Beginnings section. Besides, what was the point in opening yet another thread on this topic?

zimaman - 16-5-2009 at 11:59

References:

1. Cells of Candida utilis for in vitro (R)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor
Bettina Rosche1, Michael Breuer, Bernhard Hauer & Peter L. Rogers
Published in: Biotechnology Letters (2005) 27: 575-581

2. Improved (R)-phenylacetylcarbinol production with Candida utilis pyruvate decarboxylase at decreased organic to aqueous phase volume ratios
Cindy Gunawan, Michael Breuer, Bernhard Hauer, Peter L. Rogers, Bettina Rosche.
Published in: Biotechnology Letters (2008) 30:281–286

3. Enhanced production of R-phenylacetylcarbinol (R-PAC) through enzymatic biotransformation
Bettina Rosche, Vanessa Sandford, Michael Breuer, Bernhard Hauer and Peter L. Rogers.
Published in: Journal of Molecular Catalysis B: Enzymatic
Volumes 19-20, 2 December 2002, Pages 109-115.

4. (R)-phenylacetylcarbinol production in aqueous/organic two-phase systems using partially purified pyruvate decarboxylase from Candida utilis
Vanessa Sandford, Michael Breuer, Bernhard Hauer, Peter Rogers, Bettina Rosche.
Published in: Biotechnology and Bioengineering
Volume 91 Issue 2, February 2005, Pages 190 - 198

zimaman - 11-7-2009 at 18:52

I've uploaded the two most relevant files I referenced earlier. Is it possible to have this thread moved back to the Organic Chemistry section?

Attachment: RoscheBCellsOfCandidaUtilisforinvitroRPhenylacetylcarbinolProductionInAnAqueousOctanolTwoPhaseReactor2005.pdf (400kB)
This file has been downloaded 1414 times

Attachment: GunawanCImprovedRphenylacetylcarbinolProductionWithCandidaUtilisPyruvateDecarboxylaseAtDecreasedOrganicToAqueousPhaseVol (309kB)
This file has been downloaded 1407 times

birdman - 24-7-2009 at 12:12

Problem formulation: You've got to have the phenylacetylcarbinol in the solution before you can extract it, and I've been told by someone who keeps up with alot of people who've tried that, in his words, "I know of nobody who has had much success with this method".
According to him, the problem is the toxicity of benzaldehyde and L-PAC itself. I read in one of the papers (don't remember which) that specialized strains were developed. My friend as well, said the yeast is the problem. Industry has developed strains that tolerate the toxins but without the specialized strains and a way to control respiratory quotient, you aren't likely to get much in the way of L-PAC. More likely, benzyl alcohol.

JohnWW - 24-7-2009 at 19:05

Biochemical/microbiological methods of synthesis of phenylacetylcarbinol, (C6H5)(CH3C(=O))CH(OH), being highly stereospecific, would result in either the L or the R enantiomer, while organic chemical synthesis of it would tend to result in a mixture which is racemic or nearly so. This raises another question: are there any references regarding the rate of racemization (which can be measured by the optical rotatory dispersion of plane-polarized light) in solution of these enantiomers, which is likely at elevated temperatures? It is also to be noted that a racemic mixture, if it could be crystallized from solution, would probably form separate crystals of each enantiomer, which may be large enough to allow visual separation.

zimaman - 24-7-2009 at 21:48

The point of the octanol:H2O two-phase system is to keep the toxicity of the benzaldehyde and L-PAC from affecting the cells. Both of these chemicals partition preferentially in the organic octanol layer. The substrate and product toxicity is far less relevant in this system. The papers that I've posted seem to suggest that strict respiratory control isn't wholly necessary. They are all from the same research group at the Uni of New South Wales and people have cast doubt upon whether they're results are honest or reproducible.

The problem is how would one go about separating the L-PAC from the octanol without doing the 200-fold volume of water extraction. Their boiling points are quite near to each other: Octanol is 195C and L-PAC is 205-207C. Maybe distillation with a strong pulling vacuum applied? Any thoughts?

JohnWWW, I don't know the rate at which the racemization would occur but if heated, I would guess pretty quickly. I'll post a more recent article on the synthesis in water. Some researchers in Australia reproduced the successful results of some crafty-yet-busted speed cooks from Adelaide and tested the product. They mention that the PAC produced was 99% laevo.



Attachment: Manufacturing by-products from, and stereochemical outcomes of the biotranformation of benzaldehyde used in the synthesi (637kB)
This file has been downloaded 2834 times

not_important - 24-7-2009 at 22:48

Quote:
The problem is how would one go about separating the L-PAC from the octanol without doing the 200-fold volume of water extraction. Their boiling points are quite near to each other: Octanol is 195C and L-PAC is 205-207C. Maybe distillation with a strong pulling vacuum applied? Any thoughts?

Vacuum doesn't make it easier to separate, all it does is lower the boiling points. A 10 C difference at ~200 C and 1 bar is going to take a pretty good fractionation column with high reflux.

Counter current extraction might reduce the total volume of water. However a quick reading of the documents you provided seems to show that the water extraction is for analytical purposes, not production.

I don't know how you phrased your question re bisulfite, but methyl ketones including 2-propanones do form addition compounds with bisulfite of proper concentration. You need not form a solid addition compound, instead use ether or EtOAc to help extract the octanol away from the bisulfite addition compound in water. You would need some decent solubility refs to work out details.

Alternatively the people busted in Aus may have added EtOH-water to the crude octanol, and then MeNH2 followed by NaBH4, forming the target aminoalcohol. After that a biphase extraction with aqueous acid and ether or DCM should separate the aminoalcohol from the octanol.

zimaman - 27-7-2009 at 12:43

The busted Australians actually did the reaction in a one-phase water system, the way it's usually done in industry. There was no octanol used so the l-pac could just be normally extracted with their favorite non water-miscible solvent. But like in the usual one-phase biotransformations, the benzaldehyde and l-pac are toxic to the yeast and the yields end up at a maximum of ~20g/L of water. In a octanol-water two-phase system, the yield can be as high as 212 g/L of octanol (which isn't prohibitively expensive).

About the bisulfite adduct, I've read that some ketones are resistant to bisulfite addition due to steric hindrance caused by adjacent groups (in this case the adjacent hydroxy group). I'll post a link below:
http://designer-drugs.com/pte/12.162.180.114/dcd/chemistry/e...

Unfortunately, solubility refs on l-pac are probably impossible to find. I'll probably give the reaction a shot on a 100-ml scale and see if the bisulfite adduct works with this particular ketone.

not_important - 27-7-2009 at 19:04

Bisulfite addition vs ketone structure., see one of my posts in:
http://www.sciencemadness.org/talk/viewthread.php?tid=7689

Remember what you are attempting to do is make the ketone less soluble in the organic phase. Big excess of bisulfite, even have some solid, to force the equilibrium in favor of the addition product. Neutral solution, acids and bases undo the addition product.

zimaman - 28-7-2009 at 11:14

I read your post and I was thinking: if only 1% of acetophenone forms the addition product with equivalent amounts of bisulfite added that probably doesn't bode well for something like 1-hydroxy-1-phenyl-2-propanone (l-pac).

Are you suggesting, that the bisulfite addition will work because, with most of the pac in the octanol layer, the pac concentration in the water will be low enough that the addition product will form readily? Would I need to keep adding bisulfite to keep the concentration high as it pulls the pac out of the octanol layer? Thanks for you help.

How to clean the L-PAC after hydrogenation.

XuTai10 - 8-9-2011 at 08:51

Hello everyone. I want you all to ask for advice on how to clean the L-PAC by hydrogenation according to U.S. Patent No. 7,176,332 B2.

L-PAC is usable again, but it seems to me that it is not entirely clear. The view is colored in black, and its yield in the final product is smaller.

Thanks for any advice. Sorry for my English, not perfect.