[tksubbu]

Can anyone suggest a good procedure for synthesizing NHS esters of Amino acids wherein the alpha-amine is a secondary amine conjugated to a dye?

subbu

[russellm72]

What kind of Dye do you have? Cy Dye, Rhodamine derivative???

R.

[tksubbu]

I basically have tamra through a 5-(6-) amide linkage and another with nitrobenzodioxazole through a secondary amine linkage with the alpha animo group.

subbu

[Yggdrasil]

I've done some labeling of proteins with NHS esters of fluorescein.  Basically, I carry out the reaction in 100mM potassium phosphate buffer, pH 7.0 (this allows the dye to react selectively with the N-terminal amine group, if you don't have to worry about reacting with side chain amines, you can carry the reaction out at pH 8.2 or 8.5, just remember to use a buffer like borate that does not contain amines).

I exchange my protein into the labeling buffer by either dialysis or using a desalting column and, dissolve the dye in DMF or DMSO (do this right before adding it to the protein solution) add enough dye solution to get a 5:1 molar ratio of dye:protein.  I let the reaction run for about an hour at 37^o (don't forget to use dark tubes so that the dye doesn't photobleach).

The instructions say to quench the reacting by adding 1M tris, but I just run the mixture through a desalting column to remove the excess dye.

[GSK]

I have a problem isolating the NHS ester of 5-hydroxy compound; it really wants to lactonize anytime DCC is added (it would also happen if the NHS ester could be formed). Would the use of a buffer help? I have no success in organic solvents, and my compound is very acid sensitive.

[Yggdrasil]

If you need to prevent acidification of your reaction mixture, then you should probably use a buffer (but, is acid formed during the reaction in the first place?  If not, then you won't need a buffer).  I use a buffer for my labeling reactions because the substrate is a protein which is extremely pH sensitive and will denature at the wrong pH or ionic strength.

[GSK]

the only reason i was thinking buffer is because I want to avoid lactonization, which seems to be rapid in organic solvents even at 5C. I could make the NHS ester in a buffered solution and then do the subsequent protein conjugation all in one pot.