I want to start a discussion on why bioconjugation scientists choose NHS, TFP, or PFP esters for conjugating to amines. TFP and PFP esters are less susceptible to spontaneous hydrolysis in aqueous solutions than NHS esters. However, they are somewhat more hydrophobic, and their optimal pH for conjugation is slightly higher than the optimal pH for using NHS esters in bioconjugations.
If you use NHS, TFP, or PFP esters for bioconjugation to amines, what are your reasons for choosing one type of ester over another? I want to hear as many reasons for doing so as I can obtain. If you have published references that help support your reasoning, I'd love to see those.
Hi Robert Harvey Woodman. Hopefully, you already get an answer for that. I have a question related to conjugation. From my readings, I have encountered that most of the conjugation, especially when using NHS-ester chelator, DMF or DMSO is used as an organic solvent for dissolving the NHS-ester chelator. Are there any specific reasons why DMF and DMSO are frequently used?
Guobing Xiang that's a good point. I don't see too many people using Sulfo-NHS except for very hydrophobic compounds like LC-Biotin.
Hishar Hassan thanks for the question!
The reason that DMF and DMSO are so frequently used to dissolve NHS esters is that they can be made anhydrous (for example, by drying over 3 Å molecular sieves), and they are miscible in water. When a compound containing an NHS ester is dissolved in water, it starts to hydrolyze immediately, and the half-life of the ester depends on the pH. At or below pH 7, an NHS ester in water has a half-life measured in hours, but as the pH rises, the half-life gets progressively shorter. At pH 8, an NHS ester has a half-life measured in minutes. The short half-life of NHS esters in water is one reason why people look for alternative active esters such as TFP and PFP.
DMF is a bit tricky to use since it can degrade over time, forming free amines that react with the NHS ester. You should always use fresh DMF of the highest grade possible, and you should dry it over activated molecular sieves for >=48 hours before using it to dissolve your NHS ester. A good alternative to DMF is N,N'-dimethylacetamide (DMA or DMAC). It has similar properties to DMF, but it doesn't degrade over time to form free amines.
I don't prefer to use DMSO. In my experience, DMSO takes longer to dry than DMF or DMAC, and it is harder to get rid of than DMF or DMAC.
Perhaps it is interesting to know that specific grades of DMF are available for labeling and conjugation purposes (expensive, but convenient):
https://www.lumiprobe.com/p/dmf-labeling-grade?count=1&item=139
https://www.kerafast.com/productgroup/256/dmf-dimethylformamide-labeling-grade
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