I'm trying to conjugate carboxylic groups terminated nanoparticles with amine terminated polyethylene glycol, I'm using EDC/NHS for the conjugation and DMF as the solvent. The particles after conjugation are not settling down but after purification and redispersion in water they are settling down!! Can you tell me why this is happening? I need my particles to be dispersible in aqueous media.
Hi Varun,
Have you characterized the nanoparticle for amide bonds?
If reaction is not working, then try the reaction in basic condition (by adding diisopropyl ethylamine or triethyl amine) in anhydrous DMF as a solvent. Then check for dispersion in water.
Hello Manohar,
I've not characterized the nanoparticles for presence of amide bond yet, the particles are somewhat dispersible in DMF but not in water. On leaving the particles overnight only a part settled down but in the case of water the particles are settling down within few minutes.
I think it is better to confirm amide bond first and then try water or DMF in basic conditions.
Hi Varun, if you are simply coupling -COO to -NH by EDC, then the NHS step is unnecessary.
The NHS is unstable and if you can avoid it-please avoid it.
If you have nanoparticles (NP) with COO groups, dissolve them in 10 mM MES pH 5.5, add EDC (5-10 molar excess). Don't use DMF. NPs and PEG have to be in amine-free buffers.
If you cant use MES in H2O, solubilize NPs in DMSO (10X) before solubilizing in 10mM MES, pH 5.5.
Add the N-terminal PEG dropwise to the NP solution with stirring or swirling. You will get precipitation.
Hello Steingrimur, DMF has two methyl groups bonded to nitrogen atom, not two hydrogen for amide bond formation. Can you explain why I should't use DMF? DMF I'm using is not anhydrous so I'm gonna check it's pH. I'll do the reaction in buffers today and will update about the results.
Hello,
DMF is mildly unstable to air/moisture, which is especially noticable with bottles that have been opened some time prior to use. A degradation product of DMF is dimethylamine, which can also participate in the coupling reactions as an amine. Especially with older bottles of DMF, it is possible to smell the dimethylamine as a fishy smell. The dimethylamine causes problems when doing very dilute reactions (which you are probably doing) as the amount of amine becomes significant compared with the amine you wanted to couple. In critical cases, open a fresh bottle or indeed, avoid DMF. Checking the pH will usually not work as traditional pH measurements do not work (well) in water-free systems, although adding a drop of water to pH paper wetted with the reaction mixture can give you an idea of acidity. Using a mixed system of buffer/organic solvent sounds like a good alternative indeed.
Hi Varun, I did not say that you should not use DMF.
DMF has amide bonds that are way less reactive than primary amines. As Steffen said, there might be issues with old DMF.
I only suggested DMSO because it will never have COO or NH2 groups that could interfere with EDC coupling.
I did the FTIR of the products obtained after doing reaction in water (pH 8) and in DMF. Both are showing the formation of amide bond (peak at 1631 cm-1) and C-N bond ( 1380 cm-1), and other common peaks, but there are additional peaks in the DMF one at 1734 cm-1 (COOH group, it shouldn't be present but the peak is very small) and two peaks at around 1212 cm-1 and 1156 cm-1. I don't know why these additional peaks are coming.
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