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Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: chysce on July 18, 2017, 04:41:00 AM

Title: Amino acid racemization during peptide synthesis
Post by: chysce on July 18, 2017, 04:41:00 AM: Hi there. I am doing some solution phase peptide synthesis. I'm using a standard coupling procedure:
1 eq of amino acid ester, 1,1 eq of NBoc protected amino acid, 1,5 eq of HBTU, 1,5 eq of HOBt and 2 eq of DIPEA or Et3N, at room temperature overnight. I'm conducting reaction in DCM rather than DMF for easier workup...

However I am almost always getting a mixtures of diastereoisomers which I can't separate. I know it's a common issue in peptide synthesis and hopefuly some of you have experience with this.

Thanks
Title: Re: Amino acid racemization during peptide synthesis
Post by: rolnor on July 18, 2017, 01:58:40 PM: This all seems strange, racemization is more a problem if you make long peptidechains.
How do you analyze your product, If you use NMR perhaps you see different conformations, it depends on what amino acids you use?
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce on July 18, 2017, 03:37:12 PM: I made esters of various amino acids with a chiral alcohol (all are enantiopure compounds) and now I'm trying to add some more amino acids to the peptide... and every time i get diastereoisomers (even tho compounds have only 2 amino acids and a chiral alcohol on one end).

I use NMR for characterization.. and almost all of the signals are in something like 8:2 ratio or so. C13 also gives double signals. I thought those could be rotamers but even when I remove boc group... double signals remain.

I used leucine, glycine, tryptophane, methionine, phenylalanine and proline.
Title: Re: Amino acid racemization during peptide synthesis
Post by: rolnor on July 19, 2017, 05:04:08 AM: The only thing I can think of is maby the alcohol reacts when you put on more amino acids? Not a very good explanation.
Title: Re: Amino acid racemization during peptide synthesis
Post by: kriggy on July 19, 2017, 12:08:38 PM: Did you try different coupling conditions?
Title: Re: Amino acid racemization during peptide synthesis
Post by: TheUnassuming on July 19, 2017, 01:41:44 PM: To piggy back on kriggy, it might take you screening a few different coupling conditions before you find one that is best for your needs. It wasn't an amino acid, but for one coupling I did that tended to racimize I found using propylphosphonic acid cyclic anhydride gave the best results.

Is you free acid coupling partner the same in all the reactions you have tried?
Title: Re: Amino acid racemization during peptide synthesis
Post by: kriggy on July 19, 2017, 08:10:50 PM: Yeah, thats what I ment. My friends had a great experience with t3p, I usualy make acyl chloride but my acid is aromatic so it cant racemize via ketene formation. Also we do a lot of solid phase chemistry that relies on amino acid amide synthesis, usualy they use HOBT, DMAP, DIC (1eq) in DCM/DMF mixture.

Also, did you try the reaction in DMF? It seems to me that you are following lit procedure but you changed the solvent so it might be the reason why its not working
Title: Re: Amino acid racemization during peptide synthesis
Post by: phth on July 20, 2017, 02:40:03 AM: Quote from: kriggy on July 19, 2017, 08:10:50 PM
Yeah, thats what I ment. My friends had a great experience with t3p, I usualy make acyl chloride but my acid is aromatic so it cant racemize via ketene formation. Also we do a lot of solid phase chemistry that relies on amino acid amide synthesis, usualy they use HOBT, DMAP, DIC (1eq) in DCM/DMF mixture.

Also, did you try the reaction in DMF? It seems to me that you are following lit procedure but you changed the solvent so it might be the reason why its not working

T3P works best with pyridine. The side product is ketene formation 1-2 % run at 0º. 20 % epimerization seems like much. OP I would suggest learning about things like HOBt. DMF doesn't matter if you extract correctly because it is a water soluble impurity. A common mistake is to evaporate DMF. Dilute 4x DMF volume EtOAc, and extract 4x DMF volume water, and you will get an emulsion the first time.
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce on July 20, 2017, 03:05:04 AM: Thank you for advices.

I don't have T3P in my lab. I have EDCI though and will try couple of test reactions. One in DMF with previously mentioned conditions and one in DCM with EDCI. Those are my options at the moment.

I'll report back with the results :)
Title: Re: Amino acid racemization during peptide synthesis
Post by: clarkstill on July 20, 2017, 03:47:35 AM: From your description, it sounds like you're doing a coupling on something like this:

I don't know the numbers, but I would guess that, once the carboxylic acid is activated the alpha hydrogen is considerably more acidic here than in a normal polypeptide, due to the adjacent electronegative oxygen? So your molecule might be especially prone to epimerization via ketene formation. Or maybe I'm incorrect in the sort of structure you are looking at, in which case never mind!

A slight aside, but there is a very simple NMR method to check whether pairs of peaks are due to rotamer formation or a mixture of non-interconverting species. If they are indeed rotamers, then if you do a 1D gradient nOe experiment and irradiate a particular peak, the corresponding peak in the other rotamer gives a peak of the same phase. Much quicker and simpler than running VT experiments etc.

Steve Ley talks about it in JOC 2012, 5198.
Title: Re: Amino acid racemization during peptide synthesis
Post by: rolnor on July 20, 2017, 04:06:06 AM: Is it perhaps possible to replace TEA with pyridine to avoid basic conditions?
Title: Re: Amino acid racemization during peptide synthesis
Post by: clarkstill on July 20, 2017, 05:45:34 AM: Someone with much more experience of peptide chemistry can probably advise, but you could definitely try it out.

Also, I realized about 5 mins after posting that what I said/the structure I gave was stupid - you're probably using an Fmoc or Boc strategy and growing from the N- not the C-terminus?

I need some coffee this morning :S
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce on July 20, 2017, 05:50:08 AM: Just to avoid confusion..

Here's what I'm working with: (https://www.chemicalforums.com/proxy.php?request=http%3A%2F%2Fimgur.com%2F54kUyYG&hash=04da9c80455e3e1090591dd11105c207aa8e2d30)

(I can't see the image for some reason so here's the link http://imgur.com/54kUyYG )

P.S. Thanks clarkstill for that JACS reference.. it will help a lot :)
Title: Re: Amino acid racemization during peptide synthesis
Post by: clarkstill on July 20, 2017, 06:59:58 AM: It doesn't look especially vulnerable to racemization... aapptec have a decent guide to avoiding such problems here: https://www.aapptec.com/aggregation-racemization-side-reactions-i-494.html (primarily oxazolone formation) but it looks like the main suggestion is to add HOBt, which you're already doing.
Title: Re: Amino acid racemization during peptide synthesis
Post by: BobfromNC on July 20, 2017, 10:13:45 AM: Esters are easier to racemize. I would try a faster coupling agent, like HATU, and then run the reaction as short a time as possible, then work it up and get it away from the base as fast a possible. Letting it stir overnight with any base is a risk, most amide couplings are done in a few minutes if done right, so leaving it to stir overnight will lead to racemization, even a slow process can go a long way overnight.
Title: Re: Amino acid racemization during peptide synthesis
Post by: kriggy 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
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce 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.
Title: Re: Amino acid racemization during peptide synthesis
Post by: TheUnassuming 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?
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce 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.
Title: Re: Amino acid racemization during peptide synthesis
Post by: TheUnassuming 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.
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce 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??
Title: Re: Amino acid racemization during peptide synthesis
Post by: BobfromNC 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.
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce 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.
Title: Re: Amino acid racemization during peptide synthesis
Post by: TheUnassuming 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!
Title: Re: Amino acid racemization during peptide synthesis
Post by: chysce 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...
Title: Re: Amino acid racemization during peptide synthesis
Post by: TheUnassuming 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.