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Topic: Amino acid racemization during peptide synthesis  (Read 11661 times)

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Offline kriggy

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Re: Amino acid racemization during peptide synthesis
« Reply #15 on: July 20, 2017, 05:42:56 PM »
I second trying Py instead ot Et3N. I remember from class that pyridine is not strong enough to deprotonate the acylpyridinium intermediate but when using Et3N, the deprotonation of acyltriethylamonium intermediate can happen. Maybe this intermediate formed in your reaction and therefore the racemization. Maybe you can check it by subjecting your acid to coupling conditions but not adding any amine. Then after workup when you re-isolate the starting material and see if there was any racemization

Offline chysce

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Re: Amino acid racemization during peptide synthesis
« Reply #16 on: July 21, 2017, 06:52:19 AM »
To be honest the worst spectra were with some bulky amino acids like: Trp-Pro, Met-Pro, and Phe-Trp... which could indicate that those were mixtures of not only diastereoisomers but also rotamers... anyway...

I did 2 reactions as I mentioned before... coupling Boc-Trp-COOH and H2N-Leu-COOR under the following conditions:

1) HBTU/DMF 2eq of DIPEA
2) EDCI/DCM 2eq of DIPEA

I can't be 100% sure without C13  but it seems a bit better. Few signals suggest that it's a 9:1 mixture tho. However I'll have to wait till monday to confirm that.

@BobfromNC Sadly I don't have HATU :/ but I will try to shorten the reaction time and keep the reaction cooled.

Next week I'll definetly try a few reactions with pyridine as a base. I'm really curious about that... though I haven't seen a single example of pyridine usage in HBTU/EDCI couplings on SciFinder.

Offline TheUnassuming

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Re: Amino acid racemization during peptide synthesis
« Reply #17 on: July 21, 2017, 07:36:55 AM »
Something else I've done that you can try is to use DMAP as both your base and your catalyst.  It a can be a little bit of a pain to get rid of, but is actually a rather poor base so will just mop up free acid that is formed.

Do you have any other coupling reagents in your lab (pybop ect)?  For your EDCI reaction, did you use DMAP or HOBt? 
When in doubt, avoid the Stille coupling.

Offline chysce

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Re: Amino acid racemization during peptide synthesis
« Reply #18 on: July 21, 2017, 07:56:04 AM »
I have only EDCI, HBTU and DCC (which I honestly, don't want to use).

For my EDCI reaction i used HOBt and DIPEA.

And for DMAP workup I don't think it will be a problem since i wash my reaction with 0,5M HCl and saturated NaHCO3.

Offline TheUnassuming

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Re: Amino acid racemization during peptide synthesis
« Reply #19 on: July 21, 2017, 09:14:43 AM »
Aside from using a weaker base and playing with the coupling reagent, you could also increase the amount of HOBt/DMAP you use to decrease the amount of time your acid sits in the highly activated state. 

Also could try using an excess of your amine coupling partner to speed up the reaction since you will be doing an acid wash anyhow. 
When in doubt, avoid the Stille coupling.

Offline chysce

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Re: Amino acid racemization during peptide synthesis
« Reply #20 on: July 21, 2017, 09:32:26 AM »
Maybe I should also avoid 1h of activation of carboxylic acid with HBTU/HOBt, Et3N prior to amine addition... and just mix it all at once??

Offline BobfromNC

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Re: Amino acid racemization during peptide synthesis
« Reply #21 on: July 21, 2017, 09:44:21 AM »
Yes, that might help, once the amide is formed, the molecule is less prone to racemization.   I often only premix the acid and coupling agent for 5 minutes before adding the amine.   I would look for the disappearance of the acid/HOBT ester and once the reaction appears 95% done, I would proceed with workup, and see if that material is more optically pure.

Offline chysce

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Re: Amino acid racemization during peptide synthesis
« Reply #22 on: July 21, 2017, 09:49:01 AM »
Thanks a lot.

Now I'm really looking forward to monday :D

I'll report back next week, hopefully with some nice results.

Offline TheUnassuming

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Re: Amino acid racemization during peptide synthesis
« Reply #23 on: July 21, 2017, 01:10:38 PM »
I wouldn't let me reaction sit without an amine that long (1h), no. 

On a practical side, I've seen much of a difference in yield by not premixing my acid and coupling agent.  Adding EDCI to a stirring solution of the other reagents/starting materials works fine in most cases I've done.   I've even seen papers where people premix their EDCI/HOBt/NEt3 and add that slowly to a stirring solution of their amine and carboxylic acid with great results.  So yes, lots to try! 
When in doubt, avoid the Stille coupling.

Offline chysce

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Re: Amino acid racemization during peptide synthesis
« Reply #24 on: July 26, 2017, 03:41:47 AM »
Hi there.
On monday i did two reactions:

1) amine 1,1 eq, acid 1 eq, EDCI 1,5 eq, HOBt 1,5 eq and pyridine 2 eq in DCM
2) amine 1,1 eq, acid 1 eq, HBTU 1,5 eq, HOBt 1,5 eq and pyridine 2 eq in DMF

I premixed the acid and coupling agent for only 5 minutes before adding the amine. Both reactions were kept at 0°C for about 3 hours, and left overnight at room temp. Total reaction time ~16h.

Workup: Evaporated solvents @40°C, redisolved in EtOAc, HCl wash, NaHCO3 wash... and colum (since after extraction there was still some starting material left).

With EDCI i got diastereomeric mixture 9:1 again. With HBTU it was a bit worse.

The only thing i can try now is to reduce reaction time... but that'll have to wait since I'm going on a vacation in few days...

Offline TheUnassuming

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Re: Amino acid racemization during peptide synthesis
« Reply #25 on: July 26, 2017, 07:54:22 AM »
Hrmm... your method seems fine for those reagents.  Have you been watching the reaction to see at what intermediate its spending most of its time?  That can help you decide what you need to change to speed it up.  Also, if you can convince your PI to spend a little money on it, I would really recommend buying a different reagent to try out like t3p to see if your ratio changes. 

My SOP for EDCI couplings that you might try: EDCI (4 equiv) added to stirring solution of amine (2 equiv), base (2.5 equiv), HOBt or DMAP (2 equiv), and acid (1 equiv) in DCM (~0.5M for acid).  Quench with water, extract, wash organics with dilute HCl, dry, evaporate, column (if necessary).

I always use a large excess of EDCI because its never 100% pure and its easy to wash it away when you are done.  If I'm using DMAP I'll just leave out the extra base since it will serve that purpose as well.   

One random thing/habit I would suggest changing is your rotovap bath temp, though some might disagree.  Most of the time it won't hurt the materials to be concentrated at 40C, but I've had enough reactions that it did and now keep the bath at rt (especially if the 1st evaporation is before a workup).   As long as your pump/rotovap are well cared for, you shouldn't need to heat the evaporation flask to get standard solvents to go, room temperature is usually fine.
When in doubt, avoid the Stille coupling.

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