Click chemistry in water

Hi everyone!

I'm very new to SPR and only had a couple of weeks with a previous employee to learn how to use the instrument before I took over. The instrument is new here in the group and only me and a PhD student know how to use it, neither of us being very experienced. I'm an organic chemist.

I need to immobilize peptides containing propargylglycine (terminal alkyne) on an azide-modified chip using CuSO4, sodium ascorbate and a THPTA ligand. I have carried out the same click reaction in water with another azide and it works fine. The person who ran SPR before me tried to do the same on a gold surface in a buffer (PBS) but the CuSO4 precipitates and causes problems. She then ran the same in milli-Q water and got some good results. When we tried to reproduce this, the baseline drifted very dramatically down. I contacted the producer of the chip and he recommended not to run the experiments in water, due to electrostatic interactions with anything that might be in the water. He said running with a medium ionic strenght buffer would be better. I tried this and got a much better baseline, but needed to change to water for the immobilization due to the copper solubility issues and again got dramatic baseline drifts and even large negative signal when injecting the first peptide.

My question is: Can I perform the click reaction where I dissolve all reagents in water but keep the running buffer as PBS? Is there a more suitable buffer for this?

The perfect solution would of course be using a strained alkyne like DBCO instead of a terminal alkyne, which would mean no copper catalyst. But we're buying the peptides from abroad and they're expensive enough with the propargylglycine modification. And DBCO is big enough that it might cause problems due to steric effects.

Hi,
As you encountered Cu and phosphate will precipitate.
First option is to change from running buffer. HEPES or TRIS are good alternatives provided that they are compatible with your chemistry. We use 10 mM Hepes with 150 mM NaCl as buffer for most reactions / kinetic measurments.
This will give you a buffer (constant pH) and the salt will maintain proper ionic strength (~fysiological). Both will add to a better stable baseline.
In case of immobilization (I am not familiar with click chemistry) we use HBS as running and the ligand can be in a different buffer as long as all components are compatible (no precipitation of side chemistry).
Compare: www.sprpages.nl/immobilization/immobilization-procedures/amine
In the end: use a compatible flow buffer and dissolve your ligand preferably in a buffer or in water.

kind regards
Arnoud

Thank you for the reply, Arnoud!

The previous person did encounter problems with Cu precipitating in HEPES buffer, but I will try it out too. I have, however, found numerous articles and commercial products using CuSO4/ascorbate chemistry in phosphate buffer and no mention of the precipitation. I am curious to know if I can premix the THPTA ligand with the CuSO4 in water (the complexation should stabilize the copper in aqueous buffers, according to literature) and add to my buffer, hopefully minimizing the precipitation. Does that sound right?

Again, I'm new to this kind of chemistry and have run all my previous click reactions in water and tBuOH or DMSO, and therefore never had to use buffers before. So I'm definitely not sure about anything!

Since I am not a chemist I cannot give you a definite answer. Still any solution with phosphate and Cu can give precipitates but I can imagine that the low pH of the ascorbate will lower the risk.
if you are not seeking immobilisation on one channel you can perform the click chemistry outside the instrument on the whole sensor chip. This will prevent precipitates inside the instrument. Also you can perform the click chemistry as usual in water. However, by immobilising outside the instrument you cannot follow the immobilization level.

Arnoud