jwisse
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amide coupling reagents
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.
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chemoleo
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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.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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jwisse
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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 
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chemoleo
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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 
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]
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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chemoleo
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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.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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jwisse
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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.
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