Hi!
So I am looking forward to couple drugs, peptides and proteins to amine decorated nanoparticles. These nanoparticles have primary amines on their surface as they are covered by APTMS and or APTES. Therefore, I thought to do cross linking reaction of carboxyl groups on molecules of interest (listed above).
A crosslinker between carboxyl and amines widely use is EDC. As my molecules of interest and nanoparticles are all water soluble, EDC sounds like the right choice, since it is also water soluble.
One of my big questions is pH and reactivity: the buffer I have chosen is 20mM MES pH 5. Has no phosphates and right pH for carboxyl and EDC reaction. But what about the amines. At pH 5, they will be protonated. I am guessing that they won't as reactive as in a basic pH (~9).
To make carboxyl-EDC-activated intermediates more stable, sulfo-NHS can be added. Now, again, I read that amines readily react over NHS esthers at pH 8-9.
So... should I start the reaction at pH 5. Then after a couple of hours, add my amine-nanoparticles with some TEA and make the pH basic?
I have also read and remembered, the first step of the reaction (activation of carboxyl groups by EDC) is recommended to be conducted at 0˚C.
Last but not least: how much -COOH:EDC:sulfoNHS:-NH2 is recommended?
Any thoughts, ideas and recommendations are more than welcomed!
Thanks!!!
Well, ideal case is the other way round. COOh at surface and NH2 in liquid. So your concerns are actually right, because the activation of COOH in liquid required lower pH and therefore amine would be protonated when the carboxyl are activated. However you can still try to do it. In my case it worked at room temperature in acetic buffer and of course you should have enough EDC/NHS as it is consumed during reaction, i.e. a small excess is required. The efficiency/kinetic will be lower/slower compared to COOH functionalized NPs
I definitely second Anton's post, but the reaction will stay yield quite OK if you go for Sulfo-HNS at a pH between 6.5 and 7.5; yes, not ideal but it should work. I went that route when I was forced to couple -NH2 liposomes to the C-terminus of antibodies. The reaction is a bit dirty, since you will have quite some excess of NHS, but you should be able to get rid of it quite easily. Don't try to first activate and separate the activated moieties, that won't be stable enough to survive the purification steps.
If you want to go the EDC way, it is still feasible but slightly trickier. I was working on amide bonds formation in organic solvent and throwing EDC and TEA in was very easy and did the job. In an aqueous environment it won't be that easy I believe.
So, let me make both your ideas/suggestions clear:
1) From Anton: do EDC/NHS in buffer at pH (...?) with a slight excess of EDC/NHS as it is consumed/hydrolized during the reaction at RT.
2) From Marco: try Sulfo-NHS at pH 6.5-7.5 without purifying intermediate products. What carbodiimide do you recommend if not EDC for water-based reactions Marco?
As far as I am aware. Only sulfo-NHS is soluble in water and not NHS. Same as EDC vs DCC or DIC. Then I am dependant on making sulfo-NHS/EDC work.
Then, question: what woould be the better conditions?
1) nature of buffer
2) pH
3) temperature
4) amine : carboxylic reagents ratio
Thanks.
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