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Topic: Determining the optical purity for a family of new amino acid derivatives  (Read 3101 times)

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marywilkinson

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #15 on: January 29, 2025, 02:46:37 AM »
Good question! There are a few ways to check for racemization. Since your compounds are LC-soluble, chiral HPLC or UPLC would be a solid first step to see if enantiomers separate cleanly. If that doesn’t work, derivatization with Mosher’s reagent is a great approach—though solubility might be a challenge.

Chiral NMR could work if you find a compatible solvent, maybe CDCl₃ with an additive. Another option is circular dichroism (CD) spectroscopy, but that depends on whether your compounds show optical activity.

Offline Borek

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #16 on: January 29, 2025, 03:00:09 AM »
Good question! There are a few ways to check for racemization. Since your compounds are LC-soluble, chiral HPLC or UPLC would be a solid first step to see if enantiomers separate cleanly. If that doesn’t work, derivatization with Mosher’s reagent is a great approach—though solubility might be a challenge.

Chiral NMR could work if you find a compatible solvent, maybe CDCl₃ with an additive. Another option is circular dichroism (CD) spectroscopy, but that depends on whether your compounds show optical activity.

Probably good moment to let you all know: lately we have an influx of newcomers posting - out of nowhere - what looks like technical answers to sophisticated questions. It is never clear if it is really someone with expertise, who decided to register to help (in my experience forum almost never works this way), or if it is some bot using AI to generate an answer.

Please report, especially if it is clear to you the answer is off. I believe I am able to catch most of these posts, but - when they go esoteric - they are regularly beyond my level of expertise, and it is not easy to decide whether to ban, or not to ban.
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Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #17 on: January 29, 2025, 09:46:45 AM »
I checked different suppliers, and it was $400 for a larger quantity of the acid chloride. I  should have checked Sigma-Aldrich before I ordered the free acid (thanks for the tip).  At least on paper, Marfey's reagent looks like the simplest approach, and the separation between the derivatives (which are diastereomers) can be quite good using a moderately shallow gradient.*  This is basically Sanger's reagent, fluorodinitrobenzene with an alanine amide group bound to the aromatic ring.  The amine group of an amino acid or peptide displaces fluorine in a nucleophilic aromatic substitution.  I just ordered a small quantity of this reagent also, proving that I am not particularly good at managing money.
*The gradient is roughly 1% change in acetonitrile per minute.  There is a "reagent related peak" in some of the chromatograms, which I just now noticed.  I will have to look into how to recognize this peak (probably by its parent ion value in MS), so as not to confuse it with a peak from a putative diastereomer.
EDT
In the Katritzki paper, they reflux with thionyl chloride for 50 hours, which seems like a long time.  This strikes me as possibly problematic, because of the small scale of the reaction.
« Last Edit: January 29, 2025, 10:59:20 AM by Babcock_Hall »

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #18 on: January 29, 2025, 09:47:00 PM »
I cAt least on paper, Marfey's reagent looks like the simplest approach, and the separation between the derivatives (which are diastereomers) can be quite good using a moderately shallow gradient.*
SNP
EDT
In the Katritzki paper, they reflux with thionyl chloride for 50 hours, which seems like a long time.  This strikes me as possibly problematic, because of the small scale of the reaction.
The Marfey chemistry looks simpler on paper (one step versus two and a half steps) and shorter in time of reaction, but there would be time spent on developing a gradient.  In contrast the the formation of the Mosher amide leads to the simpler analysis (via F-19 and possibly C-13 and H-1 NMR).  It is odd that Katritzki and collaborators, who used three equivalents of BtH, did not remove the excess BtH by an extraction.  I trust that the column that they employed will do so.

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #19 on: January 30, 2025, 12:29:15 PM »
Just as I suspected, marywilkinson (author of yesterdays post) is no longer with us after another post containing unrelated link to some laundry related site.

Sorry to hijack the thread.
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Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #20 on: January 30, 2025, 03:06:39 PM »
I did not see any errors in her reply.  The distillation of SOCl2 from quinoline gave a clear liquid with constant boiling point.  Mosher's acid is a viscous liquid that makes handling it challenging.  I am a little worried that I will slowly lose the thionyl chloride over the 50 hours of reflux.
« Last Edit: January 30, 2025, 05:02:29 PM by Babcock_Hall »

Offline rolnor

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #21 on: January 31, 2025, 07:56:07 PM »

To make an acid chloride you don't need 50 hours of reflux, this is simply wrong, 10min is more than enough, you can try this on a model compound.

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #22 on: February 01, 2025, 09:42:13 AM »
General Procedure for Synthesis of 1: 3,3,3-Trifluoro-2-
methoxy-2-phenylpropionic acid (MTPA) (0.75 g, 3.2 mmol) and
thionyl chloride (3 mL) were refluxed together for 50 h. After excess
thionyl chloride was removed by vacuum evaporation at rt, the
residual oil was dissolved in dry dichloromethane (20 mL) and 3
equiv of 1H-benzotriazole (BtH) (1.14 g, 9.6 mmol) was added to
it and stirred at rt for 12 h. The volatiles were evaporated.
Chloroform (5 mL) was added to the residue and then purified by
silica gel column (20 cm   2 cm) chromatography using chloroform
(100 mL) as an eluent to give 1-benzotriazol-1-yl-3,3,3-trifluoro-
2-methoxy-2-phenylpropan-1-one.

I agree that it is very atypical, yet that is what the protocol above specified.  I am also concerned about whether the chromatography will remove the excess BtH, but maybe it will be fine.  I tried contacting one of the coauthors to ask some questions, but he is retired.  In retrospect I should have followed this reaction using F-19 NMR.  My bad.
« Last Edit: February 01, 2025, 09:52:59 AM by Babcock_Hall »

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #23 on: February 01, 2025, 04:59:42 PM »
I added 3 equivalents of BtH, and a precipitate is forming with time.  I presume this to be the hydrochloride salt of BtH, but I am not sure.

Offline rolnor

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #24 on: February 02, 2025, 04:34:14 AM »

It seems like a good reagent. You could make the acylazide of the trifluoroacid with diphenylphosphorylazid/ TEA, they can also be chromatographed without hydrolyzing. You have a lot of options, it would be interesting to test if you can just boil your amino acid with hexamethyldisilazane to give the TMS-ester of the carboxyl on the amino acid, evaporate and then just couple this with DCC and the trifluoro acid, then add water to get rid of the TMS, this would simplify a lot. I really miss practical chemistry, I even dreamt of it two nights ago.

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #25 on: February 02, 2025, 10:31:40 AM »
The protocol does not indicate performing a filtration, and there is no extraction prior to chromatography.  If I filter, there is less chance that the column will clog up, but I am not 100% sure of the identity of the precipitate, which has increased over time.

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #26 on: February 03, 2025, 11:24:17 AM »
Update:  We spun down the precipitate in a Corex centrifuge tube (we saved the solid for now) and removed the DCM.  In chloroform (Katritzki's solvent), BtH stayed at the baseline, and the presumed product has an Rf > 0.6.  We will purify tomorrow and perform the derivatization the day after.
« Last Edit: February 03, 2025, 01:28:38 PM by Babcock_Hall »

Offline rolnor

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #27 on: February 03, 2025, 02:44:09 PM »

With this type of "amide" it feels like it should be highly crystalline I guess. Nice that it seems OK! The Mosher-amide will be an acid so it will be tailing on TLC, but it should be possible to determine anyway if it's a success, maybe you are planning to monitor the reaction with NMR?

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #28 on: February 03, 2025, 04:34:52 PM »
Probably F-19 NMR could be used to monitor the progress of the reaction between MTP-Bt and an amino acid, which forms a Mosher amide.  We plan to use one more equivalent of base than was specified in the original protocol, because our amino acids are in the form of a hydrochloride salt and the original protocol used amino acids that are presumably not hydrochloride salts.    Katritzki and Collaborators did not perform a column afterwards; they perform an extraction (protocol below).  I have occasionally purified molecules bearing carboxylic acid groups over silica, but my recollection is that the solvent was quite polar.  We don't plan to run a column; we don't have much experience working on such a small scale, and we don't have a reason to believe that a column is necessary.

General Procedure for Synthesis of 2a-d: Benzotriazol-1-yl-
3,3,3-trifluoro-2-methoxy-2-phenylpropan-1-one (0.2 g, 0.6 mmol)
was added to amino acid, di-, or tripeptide (0.6 mmol) in CH33CN/
water (2:1, 15 mL). Triethyl amine (0.12 mL, 0.9 mmol) was added
dropwise to the above reaction mixture and stirred at rt for 12 h.
The volatiles were removed, and the residue was added to
ethylacetate (50 mL) and washed with 4 N HCl (10 mL); the organic
layer was separated and dried over Na2SO4, filtered, and evaporated
to give the products 2a-d.

« Last Edit: February 03, 2025, 05:53:19 PM by Babcock_Hall »

Offline rolnor

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #29 on: February 05, 2025, 04:22:51 AM »
OK, it seems very straightforward. A little bit like Donald's road to the abyss...

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