Sciencemadness Discussion Board

amide coupling reagents

jwisse - 19-4-2005 at 06:29

Hi, i'm looking for the differences in the amide coupling reagents like TBTU, HBTU, HATU, BOP and PyBop.
I don't mean the differences in chemical structure, but the differnences like the anion (BF4 vs PF6).
Or the effect of the difference of the BENZOTRIAZOL-1-YL (TBTU, HBTU) and 7-AZABENZOTRIAZOL-1-YL (HATU).
Anyone who knows a webside or a book let me know.

chemoleo - 19-4-2005 at 07:30

Peptide synthesis, huh. Guess what, that's what I am stuck with right now.
Anyways, check the Novabiochem catalogue and website, it sells all sorts of different reagents and explains what advantage of each is.
HATU is most commonly used, but PyBob is even stronger, but has other problems. Stick with HATU if you can. It's better than HoBT and all these other ones.

jwisse - 19-4-2005 at 23:38

Thanks! As stated on this website "Comparative experiments between HBTU and TBTU have shown that the counter ion has no influence on coupling rates or levels of enantiomerization [1].


[1] R. Knorr, et al. (1989) Tetrahedron Lett., 30, 1927


So that an answer to one of my questions.

But why use HATU instead of TBTU? HATU is more expensive and as the counter ion has no influence it must be the Azabenzotriazol-1-yl in HATU that makes a difference. Why?
You say PyBob is a stronger coupling reagent than HATU. Can you explain why? My guess is that it is not the forming of the active ester is the slow step but the attack of the nucleophilic amine.

I'm currently doing solution phase TBTU couplings using DiPEA as base. It works well most of the times, but I like to expend my knowledge about coupling reagents.

If you tell your problems in peptide sythesis maybe i can help you :)

chemoleo - 20-4-2005 at 15:32

A few refs on HATU (the standalone coupling reagent) and HoAT (HoBT) (that can be used in conjuction with active ester (OPfp, ODhbt) and carbodiimide (DCC, DIPCDI, EDC). Maybe you can dig out more chemistry there.

I don't know *why* the HATU is supposed to be better than HBTU/TBTU, but there are references that deal with it. It has an additional OH (O-) on the triazole ring, and is linked to a pyridine instead of a benzene, though. I imagine this makes it more polar. See the below for HATU

Apparently it causes 50% less racemisation, and greater coupling efficiency. I once found a site that dealt with the chemical reactions in detail, but I can't find it anymore :(
While HBTU/TBTU is differs only with regards to its counterion, see the structure below:

HBTU above.

TBTU above.


PyBop - again it has this benzotriazole strucure
. I imagine the entity forming the active ester is chemically just a lot more reactive than the carbodiimide ester, so yes, the formation of the amide is the problem, less so than the active ester. But that's almost always the problem afaik. What use would an activated amino acid ester be if it had trouble forming in the first place?

My problems?
1) The peptides are long, betw 50-60 aa.
2) I get a *bloody* deletion, of unknown identity (formerly I thought it was a proline, but no more so), which I can't purify away by reverse phase. I thought I could remove this problem by introducing a capping step (with DIPEA/acetic anhydride) after each extended coupling reaction, so as to irreversibly block any unreacted/uncoupled material from any further couplings. Thus I should get smaller fragments stopping at the former deletion site, which I remove by HPLC.
Sadly, I still get the deletion, however, despite numerous double couplings, extended cycle times and consistent acetic anhydride capping throughout the synthesis. I don't really understand why, a.a. is a small molecule (much smaller than the active amino acid esters), it should reach even the most hidden and aggregated free NH2's. Yet it doesn't, while further couplings with large amino acid esters seems no problem. Totally illogical :o

Anyway, I foudn now that after a.a. 40, the mass is fine, I don't get a deletion. Hence the problem is in the segment 40-60. Am trying to pinpoint the site of trouble. It's solid phase synth. btw.

Out of curiosity, re. solution phase synthesis, how do you purify the intermediates, after each coupling step (I know next to nothing about it), or rather, how do you remove the reactants from the peptide after each coupling? I can see the advantages of it though, at least you are less likely to get aggregation on the support matrix!

[Edited on 21-4-2005 by chemoleo]

chemoleo - 21-4-2005 at 04:41

Scrap the above. It looks like it's the phophothreonine causing the problem. Somehow it undergoes elimination of the phosphogroup, producing the split peak :(
All other steps seem fine.

jwisse - 21-4-2005 at 04:57

I'm not an expert on solid phase peptide coupling, so i can help you there.
Neighter am i an expert on solution phase peptid build up as i'm couling 1 peptide (3 aa's) to an hetrocycle via a amid bound forming.

But i can tell you the priciples of the solution phase synthesis of peptides. It's a patented method called DioRassp (Diosynth Rapid Solution Synthesis of Peptides.) You can find it here : Organic Process Research & Development (2005), 9(1), 98-101


It works as followed.:

Coupling with: EDC/HoBt/EtOAc/30-60 min

Z-AA2-OH(excess) + H-AA1-O-tBu ---> Z-AA2-AA1-O-tBu + Z-AA2-OH + Z-AA2-OBt (active esther)

Quence with H-(B)Ala-OBzl.p-Tos/NMM that gives:

Z-AA2-AA1-O-tBu + Z-AA2-OH + Z-AA2-(B)Ala-OBzl

Do an Acidic washing (removes the excess H-(B)Ala-OBzl, EDU and NMM)
Do an Basic washing (removes the excess HOBt and p-TosOH

Than a Hydrogenation with AcOH/NMM, 10% Pd/C in H2O gives:


H-AA2-AA1-O-tBu + H-AA2-OH + H-AA2-(B)Ala-OH

A neutral washing removes the NMM

A Basic washing removes H-AA2-OH, H-AA2-(B)Ala-OH, AcOH

Gives pure H-AA2-AA1-O-tBu in EtOAc

Don't know why the hydrogenation is don in Water.. I would say that this would be preformed in Ethylacetate as well.