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Author: Subject: bioreduction of ketones to chiral alcohols with carrot slices
karlos³
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[*] posted on 13-10-2019 at 14:38
bioreduction of ketones to chiral alcohols with carrot slices


So as I am a fan of chiral bioreductions using yeast, I have sooner or later(actually at least three years ago) stumbled upon yeast reductions using daucus carota, i.e. carrots.
I am just right now in the process of running a reduction of an azidoketone(the azide of 2-acetonaphthone) using carrot slices, leading to an (R)-azidoalcohol.
Usually, it will give an (S)-alcohol, just not in the case of azidoketones.
Attached are a few interesting papers about this certain case of reduction.

I have 1,2l of tap water with 200g of carrot slices up on the stirrer, and dropped 2g of azidoacetonaphthone dissolved in 30ml ethanol in it, causing a milky appearance of the mixture.

I think that has quite some advantage over yeast, since you won't end up with more cells than you started with, and thus have an easier work-up without the slime of yeast trapping product.
I just hope the workup will be as easy.

Already bought thousand seeds of the wild carrot type, since their hairy root culture is said to work much better here.

Attachment: carrot bioreduction 2.pdf (293kB)
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Attachment: ketone reduction carrots.pdf (46kB)
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Attachment: ketone reduction carrots compendium.pdf (47kB)
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Metacelsus
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[*] posted on 14-10-2019 at 03:56


Wow! I never would have thought that carrot could do this. I'm excited to see if it worked for you.



As below, so above.
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karlos³
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[*] posted on 14-10-2019 at 09:48


We will see over the course of the week :)
I am sure some crossed fingers will be beneficiary :D
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[*] posted on 15-10-2019 at 11:28


Nice input thanks very much!
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karlos³
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[*] posted on 16-10-2019 at 11:40


Ok so I filtered the carrot slices out after almost four days of vigorous stirring, washed them with 3x80ml of water and then put the combined soup without furthers treatment into a large sep funnel.
I extracted the liquid with 3x80ml of DCM, washed that with water and brine, and dried it over Na2SO4.
Then I distilled the solvent off and was left with 1,4g of a yellow oil, which tested negative on ketones with DNP.
So I assume it is the alcohol(maybe with some organic crap in there too).

Next time I will use a julienne cutter to get a maximum surface area instead of thin carrot slices, I guess it will be easier to stir then too.
It was really easier to get rid of the biocatalyst compared to yeast, actually I used a sieve to remove the carrot slices and afterwards during the extraction no emulsion occured like it can easily happen with yeast.
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[*] posted on 16-10-2019 at 11:56


Just as long as nobody mistakes it for soup. :D

Have you assessed the chirality of the product at all?
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karlos³
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[*] posted on 16-10-2019 at 12:48


Mmhhm, organic azide soup, tastes good, but only once :D

No I haven't checked the chirality, and I don't plan to do it, lacking the instruments.

I will straight up continue and reduce it to the amine with zinc and ammonium formate and then put that amino alcohol to nefarious uses not suitable for the SM board :P

E: oh, and the theoretical yield from 9,4mmol of azidoacetonaphthone to the produced alcohol amounted to 6,5mmol or 69,5%(assumed it is all the product).
I don't even plan any further purification, I will just use it as it is for the reduction.

[Edited on 16-10-2019 by karlos³]
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Metacelsus
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[*] posted on 17-10-2019 at 05:13


Quote: Originally posted by karlos³  

Then I distilled the solvent off and was left with 1,4g of a yellow oil, which tested negative on ketones with DNP.
So I assume it is the alcohol(maybe with some organic crap in there too).


Carotenes, perhaps? :D




As below, so above.
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[*] posted on 17-10-2019 at 07:35




Carrot reduction meme.png - 1.1MB




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[*] posted on 17-10-2019 at 08:37


Two can play at that game.

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karlos³
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[*] posted on 17-10-2019 at 09:24


Lamb sauce? Not without carrots!
How sweet of you, you made memes for this :)

I thought the yellow colour could be beta carotenes too, but then again, the azidoketone was already a somewhat yellow liquid.
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[*] posted on 17-10-2019 at 10:19


If surface area is important, is there some reason not to just grate them?

Coarse carrot gratings like they put in potato salad might be stirrable and present greater surface area.

Carrot peelings too. They're even thinner.


If you'd like a richer, more complex flavor for your reaction mixture you could always throw in some celery and 1/2 an onion along with a bouquet garni.
(I'm just pointing this out before Happyfoodance does.)






Do you want red P?
Eat lots of beets
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karlos³
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[*] posted on 17-10-2019 at 12:23


Gratings will work even better, for sure, and thus I will use them the next time.
Plants have the advantage that they don't propagate and result in a slime of cell mass, trapping product and all.
I just used slices because that one paper said that, next time I will use something even better like gratings.

Oh no celery will provide the opposite enantiomer(at least for normal ketones, instead of (S)- it will lead to (R)-alcohols)!
I wanted the (R)-alcohol.
But I remember that celery the next time I cook the residual carrots up in a stew :P

I don't remember which enantiomer the onions result in, but these also work... I guess it sounds to someone uneducated like were talking soup recepts here :D
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[*] posted on 5-11-2019 at 07:19
bioreduction of ketones to chiral alcohols with celery gratings!


Ok so I was thinking long and hard about which ketone to reduce next with plant matter, and then I remembered the attached japanese document about the yeast reduction of masked primary aminoketones(e.g. N-alkyl phthalimides, succinimides, saccharines).
Initially I wanted to use phenacyl saccharin because saccharines gave the best yield with yeast, but I would have to prepare it first from bromoacetophenone.
But then I remembered that I already had some phthalimidoacetophenone ready, made years earlier.

So I grated 200g celery, put them in a 2l widemouth flask with a large stirbar, added 1,2l tap water and a bit of citrate buffer to reach a pH of 6,5.
Then added the 2,14g phenacylphthalimide(8mmol) dissolved in 100ml of ethanol dropwise, causing a milky appearance as it suspended in the mixture.
The whole lot was heated to 30°C in the water bath.
Now we'll wait...

Attachment: hefereduktion phthalimidoketone.pdf (7.7MB)
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karlos³
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[*] posted on 11-11-2019 at 07:28


I have to say, on the second day I added 5g polysorbate, tween 80, to the celery based reduction to help the reaction as some kind of PTC substitute.
PEG-400 would probably work likewise or even better but I don't have that.
On the fifth day I filtered the celery out, and washed the residual celery three times with 50ml water, and then I pressed the fluids out of the mush.
Then extracted the milky fluid with 3x80ml DCM, pooled that, and washed it once with water and brine, then dried it over sodium sulfate.
Distilled the DCM off and was left with a bit of a yellow oil, which I filled into a vial and weighed it.
It amounted to 0,53g, which corresponds to 24,7% of the theory(1,98mmol).
Surprisingly, it crystallised a few minutes later into small sugar-like yellow crystals, which still appear to look a bit moist.
I have not yet attempted to purify it further via recrystallisation I have to add, but so far it looks good, I can probably take a mp test in the future after I recrystallised that stuff.

Edit: I have to add, I did not run a TLC on the product as it is likely not that pure(even has a faint hint of celery odour), I have only checked for the presence of ketones with DNP to confirm it has reacted.

Gratings really work well and these plant matter based reductions sure are a lot of fun!
Something very unusual, but I really start to like this method.
I believe the low yield in this case could be due to the phthalimide substitution, as the japanese paper suggest, since phthalimides gave the lowest yield of them all.
Once I did a bioreduction with yeast on phenacyl succinimide, that gave a higher yield as could be expected than this bioreduction with celery.
I really need to try it again on phenacyl saccharin with celery or carrots to satisfy my curiosity.

[Edited on 11-11-2019 by karlos³]
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[*] posted on 12-12-2019 at 04:55


I did it again, but this time on a ketone called benzylmethyl ketone, using celery.
200g celery, 1,2l of tap water and citrate buffer(pH 6,5), and dropped the ketone in 100ml ethanol with 3g polysorbate 80 in.
Five days later, the usual workup, and the obtained 1,09g of (R)-1-Phenyl-2-propanol were ketone free tested with DNP(which is a yield of 53,7% of the theory).
However, the smell is very interesting, as the racemic 1-phenyl-2-propanol smells like roses as everyone who worked with it knows, but the (R)-enantiomer smells citric/wooden with a hint of mayflowers.
The (S)-enantiomer, obtained by yeast reduction smells different, a hint of roses, but mostly earthy.
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[*] posted on 12-12-2019 at 05:45


In order to easify the workup, it would be prudent to try cold pressed carrot or celery juice (prefer the latter because it mostly lacks carotenes).
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karlos³
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[*] posted on 12-12-2019 at 06:44


I don't know if there are enough cells left?
But compared with yeast, the veggie cells don't reproduce, so the workup is pretty easy and not even close as prone to forming emulsions.

I will form the tosylate of the obtained (R)-alcohol next, this will be helpful to indicate if the chirality is right, as the tosylates of (S)- and (R)-enantiomer differ according to the attached document in their melting points for around 5°C.

[Edited on 12-12-2019 by karlos³]

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[*] posted on 12-12-2019 at 11:12


Can regular liquid Propylene Glycol be substituted for the PEG-400?

Also, are yeast cells that hard to break apart to release the wanted product?

[Edited on 12-12-2019 by G-Coupled]
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karlos³
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[*] posted on 12-12-2019 at 11:42


I haven't used PEG-400, I used tween 80.
The product with yeast cells isn't in the cells, it is produced outside of course.
How would it even be incorporated?
Yeast release enzymes in the solution, where the substrate is, and the enzymes and the substrate react together.
The cells can trap the product, but not inside their cells, but in the cell mass in between.

But yeast cells aren't hard to break apart if you ever want to make yeast extract to flavour your food, they simply hydrolyze with acid.
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karlos³
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[*] posted on 20-12-2019 at 14:27


Coconut water! Wow!
See this here! https://pdfs.semanticscholar.org/aa98/5b25ba836f8c88d2dd70e7...
And check this paper here! :o
https://sci-hub.se/10.1016/j.molcatb.2008.06.022

So steve was somewhat looking in the right direction with his suggestion of using juice :)

That is an enzyme brew that is not comparable for ease of workup with any cell suspension, now I really have to try this one out, how cool!


[Edited on 20-12-2019 by karlos³]
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karlos³
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[*] posted on 10-1-2020 at 15:04


Oh man, the hunt for green coconuts in the middle of winter in europe is way too hard!
I bought three of the ripened variety, but could only collect 140ml of the filtered coconut water.
And the stuff sold in tetra paks is heat treated, so the enzymes are likely denaturized.

However, I found something that sounds likewise good, and which is probably better to store(i.e., doesn't need pasteurisation to be conserved).
Sugar cane juice!
Likewise without any plant matter unlike the veggie reductions, and containing only conserving agents like benzyl alcohol etc, no heat treatment destroying the enzymatic action of the stuff.

Here are the related papers:

Attachment: Edible_catalysts_for_clean_chemical_reactions_Bior.pdf (184kB)
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Attachment: 10.1016@j.molcatb.2008.01.004.pdf (356kB)
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Attachment: bauer1946.pdf (781kB)
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[Edited on 10-1-2020 by karlos³]
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