[tovarich kirov]

Dear Colleagues,

Please help me! I need to make NHS ester of N-Acetyl-L-Tyrosine. Could you write me the reaction condition? 

P.S. Sorry but I am not a chemist...if it is possible please write more clear

[discodermolide]

Why do you not search for the structures and try and write the reaction equation. You can also search for the conditions.
Then you can post them here.

[tovarich kirov]

I have done it. And I tried to make it by a standart condition for coupling (equamolar amount (40 μmol of each), of NHS, DCC and N-Acetyl-L-Tyrosine in DMF). But when I used it for research it did not work. So I thought that maybe anybody has experiences in this type of coupling reaction and can help me.

[discodermolide]

I take it that everything was dry?
NHS esters are very labile and on a μmol level traces of water will hurt.

[tovarich kirov]

When I take higher concentration of members of the reaction obtained NHS-N-acetyl-L-Tyrosine gives a white precipitate when I add it to Sodium bicarbonate (130mM, ph 8.2) solution for further coupling of tyrosine to protein. Do you know what it can be?

[discodermolide]

Well adding it to a pH 8.2 solution is probably not such a good idea!
I guess you may hydrolyse the NHS ester and precipitate NHS or it could be the Na salt of the amino acid or it may be DCC-urea.
Keep it away from water and solutions of pH 8.2.
Do the coupling in dry DMF.

[tovarich kirov]

It is a standart protocol for coupling of NHS ester by Sodium Bicarbonate Buffer (pH 8.2). So I do not think that this is the reason of precipitation.

[discodermolide]

What do you think it is then?
If it's a standard protocol where did you go wrong?

[Babcock_Hall]

tovarich kirov, With which protein are you working?  Has it been chemically modified previously (in the literature)?

discodermolide, It might be possible to prepare the reagent in an organic solvent (prior to coupling), but the coupling itself will probably have to be done in aqueous solution, unless I am missing something.

[discodermolide]

Surely you can use DMF for the coupling?
Or DMF/water?

[Babcock_Hall]

tovarich, Are you trying to couple to a peptide (meaning something that might be 10-20 amino acids long), or are you trying to couple to a protein?  Can you practice your coupling reaction with a commercially available NHS-ester?  You would obviously need some sort of assay to decide whether or not coupling had occurred.

discodermolide, When I suggested reacting my protein with an electrophilic reagent and using DMSO with a solvent, my advisor threatened me with bodily harm.  Maybe if I had suggested DMF, it would have been OK with him.  More seriously, I know that a few proteins tolerate some organic solvents, but to the best of my knowledge, it is very situation-dependent.

[discodermolide]

LOL, I would have used it anyway. If the boss says no to your ideas then he must be wrong and you right

[tovarich kirov]

Thank you for answers!

Yes now we are thinking to check the reaction by measurement the protein mass (or peptide) before and after coupling. So I hope it will clarify something..

Unfortunatelly no NHS-N-acetyl-L-tyrosine are comercially available((
So maybe I have to find any another reagents...like this.

What are the differences between DMF and DMSO as so solvents for NHS esters coupling?

[discodermolide]

Solubility?

[Babcock_Hall]

tovarich, NHS-biotin (and its sulfo-derivative) are commercially available, and there are books and articles on how to perform this sort of work.  Some of this background information may be transferrable to your project.  With respect to the possibility of using a commercially available NHS-ester as a practice reagent, I don't know of a quantitative assay for a biotinylated protein directly (maybe there is one that uses antibody-based technology), but one might devise an assay based on capturing the biotinylated protein or peptide with avidin, then quantitating it.  There is also an NHS ester of fluorescein that may be commercially available, and some reactive dyes are (or were) also commercially available as NHS esters.

This link mentions the use of both DMF and DMSO to make the NHS-biotin soluble prior to coupling.  It also discusses the competing hydrolysis reaction, which lowers yield:
http://www.piercenet.com/browse.cfm?fldID=F330F14F-EBCC-97DB-7F6E-9664D3ACE886#nhsester

These links mention that the DMF (or DMSO) must be free of amines:
http://www.lumiprobe.com/protocols/nhs-ester-labeling
http://www.atto-tec.com/fileadmin/user_upload/Katalog_Flyer_Support/Procedures.pdf