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Difficult Sequences

Difficult Sequences - Peptide Forum

Difficult Sequences - Peptide Forum. Ask and discuss questions on peptide protocols, custom peptide synthesis, peptide identification and peptide sequencing.


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  #1  
Old 04-04-2008, 02:06 AM
Pipette Filler
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Default Difficult Sequences



How can i overcome the incomplete coupling and deprotection during solid phase peptide synthesis concerning the difficult sequence?
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Old 04-04-2008, 05:45 PM
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Default Re: Difficult Sequences

Hello,

Microwave-assisted SPPS is proven to increase efficiency of coupling and deprotection (thus higher yields) in difficult sequences. For a good recent paper see International Journal of Peptide Research and Therapeutics, Vol. 13, Nos. 1–2, June 2007 pp. 203–208.

Cheers,
John

Last edited by peppy; 04-04-2008 at 05:48 PM.
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Old 04-05-2008, 01:49 AM
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Default Re: Difficult Sequences

Quote:
Originally Posted by peppy View Post
Hello,

Microwave-assisted SPPS is proven to increase efficiency of coupling and deprotection (thus higher yields) in difficult sequences. For a good recent paper see International Journal of Peptide Research and Therapeutics, Vol. 13, Nos. 12, June 2007 pp. 203208.

Cheers,
John
Hello,
Thx for your reply and suggestion~
I have one more question on the topic, how about using the mixed-solvent system such as DMSO/DMF (v:v=1:1 or other proportions) which can serve as the electron donor solvent?
Or addition of chaotropic salts such as Lithium chloride to the reaction solvent?
As for microwave-assisted SPPS, specific apparatus should be used in order to confirm the reproducibility of SPPS. Microwave oven will not safe and cannot reach good reproducibility for our synthesis.
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Old 04-18-2008, 12:00 AM
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Default Re: Difficult Sequences

What do you call a "difficult sequence"? To my mind there are two types:

(a) Peptides that are difficult to synthesize due to aggregation during chain elongation - these are most of the difficult sequences we see. In that case:

As John already mentioned I would suggest microwave assisted peptide synthesis. It produces much better results than conventional methods.
In case you do not have access to microwave system (we use Discover / Liberty from CEM) you can try:
-use a good coupling reagent (e.g. Pybop)
-use a good resin (e.g. ChemMatrix)
To my experience the use of chaotropic salts, special solvents (magix mixture), ... does not help a lot ...

(b) Peptides that are difficult to synthesize due to sterical problems. In that case:

Use HATU as coupling reagent. In case you think it is too expensive - maybe using capping at the crucial steps might help you.

Andreas
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Old 04-18-2008, 06:12 AM
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Default Re: Difficult Sequences

THX Andres!

You have helped me a lot!

Good luck with you and i will try microwave assisted synthesis someday!
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Old 04-18-2008, 06:34 PM
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Default Re: Difficult Sequences

I agree with AplaGen. Capping agents and pseudo-prolines increase the success and the yield of difficult sequences greatly. I had a 42 resiudes long peptide with which I always got tens of peaks at the HPLC. When I used a capping agent and psuedo-prolines at certain positions, all problems were solved.
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Old 04-19-2008, 12:08 AM
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Default Re: Difficult Sequences

Quote:
Originally Posted by musgun View Post
I agree with AplaGen. Capping agents and pseudo-prolines increase the success and the yield of difficult sequences greatly. I had a 42 resiudes long peptide with which I always got tens of peaks at the HPLC. When I used a capping agent and psuedo-prolines at certain positions, all problems were solved.
Excuse me~
Capping agents or coupling agents?

What coupling agents do you always use?

We often use DIC/HOBt(2.5eq), for the Fmoc-AA-OH difficult to couple,we usually use PyBop(2.5eq)/HOBt(2.5eq)/DIPEA(5eq) for recoupling. I donot know if this will help to the final product. The Kaiser Test showed the completeness of coupling.
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Old 05-01-2008, 05:49 PM
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Default Re: Difficult Sequences

Quote:
Originally Posted by yuanhenglidlut View Post
Excuse me~
Capping agents or coupling agents?

What coupling agents do you always use?

We often use DIC/HOBt(2.5eq), for the Fmoc-AA-OH difficult to couple,we usually use PyBop(2.5eq)/HOBt(2.5eq)/DIPEA(5eq) for recoupling. I donot know if this will help to the final product. The Kaiser Test showed the completeness of coupling.
No it is capping (or terminating) agent, such as N-(2-Chlorobenzyloxycarbonyloxy)succinimide. It terminates the unreacted amine groups.

But I think pseudo-prolines might help better than capping agents in your case. Try putting pseudo-proline di-peptides at the positions where you think the coupling is failing. They help by acting like a proline and preventing the peptide to fold during the coupling. You can find more info at [Only registered users see links. ]

Last edited by musgun; 05-01-2008 at 05:55 PM.
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