[guccimayne]

Hello all

My first post. I've been having issues with, what appears to be, a very easy chemical reaction. And yet, I'm struggling with it. I'm trying to couple a carboxylic acid onto a polypeptide that I synthesized containing an alcohol (Serine) to create an ester bond. I was pointed to Steglich esterification which uses DCC and DMAP to move this process along.

See: https://www.organic-chemistry.org/namedreactions/steglich-esterification.shtm

I'm basically modeling my reaction off a paper that used this same reaction, save for molar amounts.  I carry this process out as two steps.

STEP 1 (see attachment) involves forming an anhydride of my acid using DCC in DCM, stirring 60 min for 35 degrees. I then rotovap off the DCM. I notice a white, cloudy mixture appears, which I'm assuming is supposed to be the anhydride and the O-acylsourea intermediate. This hangs around after rotovap.


STEP2 (see attachment) involves adding my Fmoc-protected polypeptide dissolved in 20mL DMF (it's not soluble in DCM) plus a couple of DMAP crystals to the now-dried anhydride/O-acylisourea mixture to start the formation of the ester. I let this go O/N at room temp. The next day I deprotect the Fmoc by adding piperidine (up to 20%) to this reaction for 20 mins and then I ether crash before running HPLC to purify for mass spec and lyophilization.

For whatever reason, I always end up with a TON of my de-protected peptide on its own and very little peptide coupled via an ester bond on the serine. I'm unclear where in this process I am going wrong as this reaction seems straightforward, but clearly I'm missing something. Am I adding too little DMAP? I understand the ester formation is slow without it. Are my temperatures off? The paper I referenced did the same thing I am doing, but the link I provided above had things at 0 degrees C.

In any case, any help you can provide would be greatly appreciated. Oh and in case anyone is wondering, the serine has an -amide terminus, but it comes off looking like -NH2 on my drawing software. You can ignore that.

Thank you

[rolnor]

This kind of anhydride is not very reactive, you can make a acid chloride with thionyl chloride instead. Then you should try to monitor the reaction with HPLC. You should then not use DMF as solvent for the coupling because the acid chloride can react with it. I am not sure what solvent would be OK, sulpholane? It melts at 27°C; https://en.wikipedia.org/wiki/Sulfolane

[kriggy]

Well, first of all, most likely you wont get any ester at all with that free amino group. You definitely need to protect the amine (experienced that myself with aminoalcohols: full conversion to amide even if more sterically hindered or benzyl protected)

Second, in my experience, adding additional base helps a lot, I used 1equivalent of DMAP and it worked like a charm.

Third, I dont understand why you are pre-forming the anhydride when you can just mix it all together and stir overnight?

Try other solvents, I never used DMF for this reaction but DCM or acetonitrile worked well for me. Especially acetonitrile since you can quench the DCC with oxalic acid afterwards which is more difficult to do in DCM.

Anyway, my conditions that I  used for esterifications or amidations:

a) acid (1eq) and aclohol (1 eq) were dissolved in solvent (DCM or ACN), DMAP (1eq) was added followed by DCC (1eq). Stirred overnight at RT, quench with oxalic acid (if in ACN) filter, extract, collumn

b) acid (1 eq) and amine (1eq) dissolved in DMF. HOBt (2 eq) was added, followed by EDCl (2eq). Stirred until completion (aprox. 2hrs), diluted with EtOAc, extracted with K2CO3 and HCl. Purify if needed by column

[rolnor]

I thought it is serincarboxamide, not terminal -NH2? The amine-end of the peptide is Fmoc-protected? I agree, you need 1 eq. base.

[guccimayne]

Hi,

Thanks for the replies. Sorry for the confusion, the -NH2 on serine is not an amine, but rather an amide C-terminus from the resin I synthesized the peptide on. It just looks funny on MarvinSketch. The only amine is the Fmoc protected N terminus.

Regarding the anhydride, that was what was suggested to me by a postdoc in the lab. From my understanding, it will form when an excess of my acid reacts with the O-acylisourea intermediate. By lowering the molar equivalent of the acid I would be left with only the acylisourea intermediate (and urea) and that should still react with DMAP, yeah?

I see the point that the pH may play a factor and I may have to add base to this reaction. I brought this up to my PI and they suggested Pyridine or Triethylamine. I’ll try at 1:1 mol equivalent as you’ve suggested.

[kriggy]

Yeah you can use other bases but you need a DMAP as a nucleophilic catalyst, I just used DMAP as both to make the setup bit easier.

Yes the anhydride will form but its just easier to do the direct acylation IMO. Maybe the acylurea intermediate is more reactive than the anhydride

[guccimayne]

So I gave this reaction another try. I'm doing everything in one go instead of a separate anhydride step and still no luck

My conditions were:

•   0.5 mmol (71uL) 4Azidopropionic acid + 5% mol equiv DMAP crystal + 0.5mmol DCC (105mg) dissolved in 10mL DMF stirring 10 min
•   Adding 0.5mmol NET3 (70uL)+ 0.5 mmol (352 mg) FmocGGGGRS dissolved in 10mL DMF, stirring O/N at RT
•   Filter to remove urea, add 5mL piperidine to make 20% solution in DMF to deprotect for 20 min. Ether crash and HPLC


Once again, I'm getting a massive peak around 5.5 mins and according to MALDI that's just the peptide without Fmoc and no acid. A teeny tiny peak around 16 minutes pops up and that's actually my coupled peptide-acid conjugate, by weight. So even adding a base at 1:1 molar equiv, I'm having trouble forming this ester. (See attachment 1)

I dunno what else I could be missing. My PI thinks maybe some TFA from the peptide cleavage solution off the resin could be interfering with the reaction, so he suggested I HPLC purify the peptide first, then try again. Is there anything else jumping out at you all that I'm doing wrong? Is there a certain order I'm supposed to add these reagents or a temperature requirement? 

[rolnor]

Have you good NMR on the azido-acid? Also, kan the guanidine group in R be a problem?

[kriggy]

I suggest doing the acylation without the deprotection step to see if it works or not. It might be piperidine cleaving your ester. Its unlikely but who knows, its 5% solution but 5ml is huge excess.

I would also increase the amount of DCC and DMAP, two equivalents or somehing like that. Residual TFA might cause a problems (residual acetic did to my wife doing those kind of acylations). It might be scavenging your DCC but the TFA-ester gets decomposed by piperidine in the workup.

What I could suggest from my experience, add DCC last. I would try dissolve acid , add peptide, DMAP and then DCC. Possibly in solvent like acetonitrile or DCM. I never used DCC in DMF and dont know anyone who did.
Furthermore, you might get lucky with other activators such as DIC/EDCl/HATU/COMU...

[guccimayne]

Have you good NMR on the azido-acid? Also, kan the guanidine group in R be a problem?

I haven't done NMR on the azidoacids themselves. I purchase them from a vendor, so I simply trust that it's correct. Maybe that's my fault. (https://synthonix.com/1941.html) As far as the Fmoc-peptide goes, I put it on MALDI and confirm that it has the appropriate weight (711Da).

I suggest doing the acylation without the deprotection step to see if it works or not. It might be piperidine cleaving your ester. Its unlikely but who knows, its 5% solution but 5ml is huge excess.

The ester is known to be pH sensitive, so if there's too much acid (TFA) or base (Piper+DMAP+NET3) that could definitely affect things. I only add 5mL to the 20mL solution to make a 20% solution (25mL total) but I could try scaling it back to a 10% solution, maybe for 10 mins instead of 20.

What I could suggest from my experience, add DCC last. I would try dissolve acid , add peptide, DMAP and then DCC. Possibly in solvent like acetonitrile or DCM. I never used DCC in DMF and dont know anyone who did.
Furthermore, you might get lucky with other activators such as DIC/EDCl/HATU/COMU...

I'll try upping the DCC and Base molar equivalents. I could try replacing NET3 with DMAP entirely, as well. And I'll add DCC afterwards when the other species have a chance to react for about 10-15 min.

I don't use DCM for this process because the Fmoc group on the peptide makes it poorly solvable. I've tried it before and while I do have some better results in terms of generating my coupled peptide, the Fmoc-peptide mostly precipitates out of solution and forms a large lump in my flask. The Fmoc-peptide can dissolve in Acetonitrile if you think that'll be a better option than DMF...

[rolnor]

Is it OK to make acylations in presence of a arginine? If you buy the azidoacid it should be OK. I agree that the deprotection can cleave the ester.

[kriggy]

I wonder if you could go even lower with the mount of piperidine. Its not about what is the % of the cleaving coctail but also how much you add.  I mean, adding 10ml of 10% solution is same amount of piperidine as 1 ml of neat.
In 20% piperidine, the half life of depotection is about 5 or 10 seconds IIRC. So you can try to do the deprotection rally quick. Maybe 20 minuts is too long for the ester to survive but maybe, it will survive only 1 minute or 30 seconds while fmoc drops off.

[guccimayne]

I think in the interest of making as few changes as possible so I can know where things are going wrong/right, I will adjust two things on the next go-around. First I will HPLC purify the Fmoc-peptide just to get any TFA out of the way as a potential confounder. Then I will adjust the deprotection step.

From what I've read about it in the link below, it takes about 6 seconds for 50% Fmoc removal at 20% piperidine in DMF so I'll do maybe 30 seconds just to be sure, then ether crash.
(https://link.springer.com/protocol/10.1385%2F0-89603-273-6%3A17)

The only reason why I am doing it so long now is because in the past, I was doing 20% piperidine for 10 minutes and I was actually getting a ton of my peptide-acid conjugate at the end, but it still had Fmoc on it. I wasn't ether crashing back then, I was just rotovapping the DMF off and running HPLC on the dry product. So it's like I fixed one thing, broke another thing, ha.

[rolnor]

It would be better with another PG like Boc but then you have to redo the whole thing I guess?