February 07, 2025, 12:59:07 PM
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Topic: Determining the optical purity for a family of new amino acid derivatives  (Read 1931 times)

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Offline Babcock_Hall

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A reviewer would like to know whether our novel amino acid salts RCH=CHCH2CH(NH2HCl)CO2H] racemized during the synthesis.  They are soluble in water/acetonitrile during LC-MS experiments and generally soluble in DMSO.  Only one is UV-active, which might make detection in LC more difficult.  I doubt that they would be soluble in a chiral NMR solvent, but I don't know.  What sorts of methods should we be thinking about?
EDT
One person suggested making Mosher amides.
« Last Edit: January 13, 2025, 01:07:22 PM by Babcock_Hall »

Offline rolnor

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Yes, Mosher amide would be a good idea, chiral solvents are not reliable, you might not get any separation of the peaks on NMR, a chiral column and LC would require that you have the two isomers and develop a method with a chiral column I guess.

Offline Babcock_Hall

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My concern with Mosher chemistry is that we have a free carboxylic acid in the molecule, which might react with the Mosher acyl chloride.

HPLC with a polysaccharide based stationary phase or with a chiral ion-pair reagent might work, but both of those are speculation on my part.  I am starting to search for examples.
« Last Edit: January 14, 2025, 10:21:25 AM by Babcock_Hall »

Offline rolnor

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Yes, there will be some work with either method. You could silylate the carboxylic acid maybe, then acylate, then just add water to split of the TMS-group, this way you dont have to isolate the protected amino acid first. Just dissolve the amino acid in HMDS/DMF first, evaporate?

Offline Babcock_Hall

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Katritzki et al. 2007 J Org Chem 72:4268-71.  10.1021/jo070278a
We will try this method first.  These authors made the Mosher acid chloride in situ (50 hr reflux) and added 1H Benzenetriazole.  They reacted the purified product with an amino acid or peptide and added 1.5 equivalents of TEA (some authors use 1.2 equivalents of DIPEA for amines).  Because our amino acids are hydrochloride salts, we will use an additional equivalent of base.  I can post a scheme if that would make it easier to follow.  The authors saw small differences in chemical shift using H-1, C-13, and F-19 NMR.  If we see only one signal, my interpretation is that this would be consistent with having only one isomer.  It might be possible to obtain a retention time using reverse phase LC-MS.
« Last Edit: January 20, 2025, 10:29:48 AM by Babcock_Hall »

Offline Babcock_Hall

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We are thinking of distilling the thionyl chloride from quinoline.

Offline kriggy

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My concern with Mosher chemistry is that we have a free carboxylic acid in the molecule, which might react with the Mosher acyl chloride.

HPLC with a polysaccharide based stationary phase or with a chiral ion-pair reagent might work, but both of those are speculation on my part.  I am starting to search for examples.

That is IMO not really an issue. Ive run quite a few reactions on free amino acids - be it acylations or various protections and never had issue. You can always
a) separate the byproducts by chromatography b) protect the carboxylate.

If we see only one signal, my interpretation is that this would be consistent with having only one isomer. 

That is not entirely true. Your hypothetical diastereomers might have very similar NMR signals but ofc, its quite unlikely that all of them will be same. There is also a (rather unlikely) possibility of full inversion of your acid in the synthesis so you end up with the other enantiomer.

Ive done bunch of work in this area for my PhD i can recommend few papers for you.

[1] J.M. Seco, E. Quiñoá, R. Riguera, The Assignment of Absolute Configuration by NMR, Chem. Rev. 104 (2004) 17–117. https://doi.org/10.1021/cr000665j.

[1] J.M. Seco, R. Riguera, NMR methods for the assignment of absolute stereochemistry of bioactive compounds, EMagRes 4 (2015) 1–30. https://doi.org/10.1002/9780470034590.emrstm1398.

Those two will pretty much answer all your questions on the NMR method. IMO making mosher (MTPA) amide or methoxyphenylacetic acid (MPA) amide with your amino acid is the easiest. While MPA is preffered to MTPA, I assume the fact that you can buy MTPA chloride will make the task much easier.
Make amide with both enantiomers of MTPA-chloride, make enough of if to get reliable 1H, 13C and 2D NMR to get full assignment of all signals. Dont forget that S-MTPA-chloride makes R-MTPA-amide due to change of priorities in CIP.

Offline Babcock_Hall

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Hi Kriggy,

I just finished reading your thoughtful and helpful reply, and I will read your references this morning.  The price of the Mosher acid chloride was quite high; therefore, I ordered the free acid and benzene triazole, with the intention of making an active derivative (the Mosher acid chloride is made in situ).  However, I suspect that MPA may also be made into its benzenetriazole derivative, and I need to look into this alternative.
« Last Edit: January 24, 2025, 09:04:35 AM by Babcock_Hall »

Offline rolnor

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We are thinking of distilling the thionyl chloride from quinoline.

Is this necessary? And HOBT does not seem necessary either, acid chlorides are highly reactive towards amines?

Offline rolnor

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My concern with Mosher chemistry is that we have a free carboxylic acid in the molecule, which might react with the Mosher acyl chloride.

HPLC with a polysaccharide based stationary phase or with a chiral ion-pair reagent might work, but both of those are speculation on my part.  I am starting to search for examples.

That is IMO not really an issue. Ive run quite a few reactions on free amino acids - be it acylations or various protections and never had issue. You can always
a) separate the byproducts by chromatography b) protect the carboxylate.

If we see only one signal, my interpretation is that this would be consistent with having only one isomer. 

That is not entirely true. Your hypothetical diastereomers might have very similar NMR signals but ofc, its quite unlikely that all of them will be same. There is also a (rather unlikely) possibility of full inversion of your acid in the synthesis so you end up with the other enantiomer.

Ive done bunch of work in this area for my PhD i can recommend few papers for you.

[1] J.M. Seco, E. Quiñoá, R. Riguera, The Assignment of Absolute Configuration by NMR, Chem. Rev. 104 (2004) 17–117. https://doi.org/10.1021/cr000665j.

[1] J.M. Seco, R. Riguera, NMR methods for the assignment of absolute stereochemistry of bioactive compounds, EMagRes 4 (2015) 1–30. https://doi.org/10.1002/9780470034590.emrstm1398.

Those two will pretty much answer all your questions on the NMR method. IMO making mosher (MTPA) amide or methoxyphenylacetic acid (MPA) amide with your amino acid is the easiest. While MPA is preffered to MTPA, I assume the fact that you can buy MTPA chloride will make the task much easier.
Make amide with both enantiomers of MTPA-chloride, make enough of if to get reliable 1H, 13C and 2D NMR to get full assignment of all signals. Dont forget that S-MTPA-chloride makes R-MTPA-amide due to change of priorities in CIP.

Yes, if you want to publish this work you need to have solid proof, I agree.

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #10 on: January 25, 2025, 10:52:11 AM »
Now I am confused.  The free carboxylic acid in an amino acid looks like it might also react, and this would give side products.  Surely that is why Katritzki and colleagues (10.1021/jo070278a) used BtH (see attached pdf; I also copied the protocol as a Word document if anyone wants it).  Pentafluorophenol/DCC to make the Mosher Pfp ester was also suggested to me (https://orgprepdaily.wordpress.com/2006/09/25/mosher-pfp-ester/).

Offline rolnor

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #11 on: January 25, 2025, 11:03:41 PM »
HOBT is used to make a more reactive acylating species? Or? Like DMAP? EDIT: OK, now i see, its benzenetriazole, not hydroxy benzenetriazole.

Offline Babcock_Hall

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #12 on: January 27, 2025, 08:58:56 AM »
Campbell, Jeffrey A.; Lee, Won Koo; Rapoport, Henry; J. Org. Chem.; 60; 14; 1995; 4602-4616.
A long time ago, we used to make 4-nitrophenol esters to form amide bonds to molecules having amine groups but also free carboxylic acids.  If we had that work to do over, we might use pentafluorophenyl esters or NHS esters instead.  I look upon the Katritzki method as being similar to active esters conceptually, even though this method makes an amide.  Regarding pentalfuorophenylesters, Campbell and colleagues wrote, "In the determination of the enantiomeric ratios of the target molecules, the highly lipophilic crystalline pentafluorophenyl ester of MTPA was used and had the major advantages over the acid chloride of purity, high reactivity, and simple removal of excess reagent."  I have already ordered benzenetriazole, but if it fails, the Pfp ester of MTPA sounds like a good alternative.
« Last Edit: January 27, 2025, 09:27:24 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 #13 on: January 28, 2025, 09:40:17 AM »
R Bhushan, Journal of Chromatography B, 879 (2011) 3148–3161.  doi:10.1016/j.jchromb.2011.05.058
A derivativization that may work well for LC-MS is to use 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (Marfey's Reagent), which will produce chromophoric diastereomers of a mixture.  According to ThermoFisher, the derivatives are stable for at least 48 hours.  So many good ideas, but I need more time to read up.

Offline rolnor

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Re: Determining the optical purity for a family of new amino acid derivatives
« Reply #14 on: January 29, 2025, 01:47:36 AM »

50mg Mosher-Cl is 50usd on Aldrich, is that so expensive? If you put hours in on reading, this is also a cost? I would just make the Mosher-amide, you need just 5mg product for nice NMR. When you want to acylate a free amino acid you can use NaHCO3/H2O/DCM, I dont know if this is always feasible, but it could be just this easy. You can try this with a cheap acid chloride first.

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