Following this guide, I get the impression that EDC/NHS activation is used to link to primary amines to carboxyls. Wouldn't it also activate secondary amines on arginine and other amino acids? If not, how does it distinguish between primary amines and secondary amines? Thanks.
https://www.lifetechnologies.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chemistry-crosslinking.html
Are these less accessible or something? I read that lysines are positive so tend to face outward on proteins. Almost all resources I read talk about conjugating to primary amines instead of just amines. Any idea why?
EDC NHS can be used on secondary amines, however they are exceedingly uncommon in proteins. The only true example would be in the rare case of an N-terminal proline residue that is solvent accessible.
The other amino acids don't really contain any secondary amines. Arginine doesn't really contain a secondary amine, it contains a very basic guanidyl group. Likewise the R2NH groups of histidine and tryptophan are part of an aromatic system that significantly attenuates their nucleophilicity, so they do not behave like secondary amines either.
Reaction of active esters like NHS esters with secondary amines can be slow compared to reaction with primary amines, but it depends on the details.
The important thing for Adam is that Eric is correct. Primary amines are essentially the only nucleophiles available that will react, if you are working with proteins rather than doing general organic chemistry. Just remember that the N-terminal amino group is also a primary amine, though its pK is rather different than that of lysine (lysine is more basic). So, you might form an amide at the N-terminus and at the lysine epsilon amino groups. It depends on how accessible the N-terminus is.
You get better control of this site-selectivity, if you care, with reductive amination, which allows you in some cases to react selectively at the N-terminus under the slightly acidic conditions required to form the imine intermediate. Under those conditions, lysines are protonated and don't react with aldehydes or ketones to form imines (Schiff bases). You may need to optimize the boron hydride reagent. Much literature is available to guide you. Good luck.
Just want to add my favorite classical examples of weird highly selective acylations of lysine.
(i) Lys dissolved in neat TFA (sic!) is acylated selectively at N-alfa with equivalent of Tfac2O at 0o =>Tfac-Lys
(ii) Lys dissolved in basic aqueous buffer pH=8.5 (if memory works) when treated with Tfac-SEt is trifluoroacetylated selectively at side chain NH2=>Lys(Tfac)
for details see ol good Grinstein-Wintz manual
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