I suggest chemically blocking the N-terminus by modifying it with a ketene as in J. Am. Chem. Soc. 2012, 134, 2589−2598 (see link). Then do the EDC coupling on the c-terminal Lysine as you propose.

http://pubs.acs.org/doi/pdf/10.1021/ja208009r

If you keep free -COOH groups in your peptide, you will also get di-, three- and so on -merisation as well as cyclic peptides while coupling by EDC/NHS method. I'll say: chemically protect N-terminal Lys as suggested above, then use active ester method.

Dear Andrew, as the N-terminal a-amino group is less basic it is also less nucleophilic (for electronic and steric reasons). I expect your C-terminal lysine side chain therefore being significantly more nucleophilic. It would be worth to try your envisaged coupling directly with your fragements as they are. The EDC activiation is not the most powerful which is good. It should give you additional selectivity. Regards Hans-Jörg 

RE Maxim: Thanks for your input.  Just to clarify, the COOH groups that I intend to activate and conjugate the peptide to are on a nanoparticle that I EDC/NHS activate separately.  I've found the nanoparticle-NHS ester is stable enough in my buffer to remove the excess EDC/NHS prior to the coupling so I think there's little chance of activating COOH groups on my peptide.  Thanks you both, Bruce and Hans+Jörg for your suggestions.

it will be challenging to accomplish regioselectively for the following reasons:

1. 53 aa peptide (fully protected) will be insoluble in water. Was it synthesized by SPPS?

2. If peptide is fully deprotected - inter- and intramolecular amide bond formation will be the main issue (any other amines in the sequence?)

3. If particles are activated (not the peptide) - the rate of coupling could be slow compared to the rate of active ester hydrolysis.You may also attach your peptide at multiple points (again if you have multiple amines) to the reactive surface.

The bottom line - EDC coupling is not a selective reaction, and pKa difference will not give you desired selectivity.

You may want to consider a better strategy - more orthogonal, like Staudinger ligation  or Cu catalyzed (or strain-promoted) Click chemistry. For example, you can incorporate C-teminal lysine analogue with epsilon-N3, and couple Alkyne-PEG-NH2 spacer-linker to your particles.

If you are trying to immobilize a bait for some pull-down experiment (what is you downstream application?) - you can incorporate C-terminal lysine residue with biotin on the side chain during SPPS, and use streptavidine coated magnetic beads - works great. If peptide is recombinant (not synthetic) - add a C-teminal Avi-tag (called BAP=biotin Acceptor Peptide), and use BirA ligase to biotinylate in vitro.

Good Luck

Thanks Vyacheslav.  The polypeptide is actually produced ribosomally as a fusion to a SUMO domain which I proteolytically remove and purify away.  Luckily there are no lysines in the sequence so I only have to worry about the N-terminal amine from the methionine residue coupling.

I have a new idea, actually.  I could introduce a cysteine at the N-terminus of the polypeptide.  The carboxyls on the nanoparticles would be activated with EDC/NHS as before but then I would react them with iodoacetamide.  This would then specifically couple with the thiol on the C-terminal cysteine and not the N-terminal amine at all under appropriate conditions.  Does this seem reasonable?