Question
Asked 19th Dec, 2017
Mol ratio of EDC/NHS is based on the mol of the carboxyl group or amine group?
I'd like to use EDC/NHS in an excess platform to conjugate an amine group to the carboxyl group of a polysaccharide. Considering this fact that i don't have the optimize mol ratio for my components i need to assume 2,3,5,...mol of EDC,NHS to the mol of one of these two components and i'm not sure which one would be the better choice.
Most recent answer
We used a 1:1 molar ratio of EDC:NHS for our application but that was for large-scale cross-linking of two proteins:
Popular answers (1)
Bundesanstalt für Materialforschung und -prüfung
NHS can not do any harm to your molecules, therefore a higher access can be used and lead to a faster reaction and a higher yield, particularly, if you can not calculate the concentration of your carboxylic groups properly. It is not really advisable to calculate the reagent relative to the amino groups, because the reaction between the carboxylic group and EDC is always the first step (see lower link). Therefore, two-step protocols are usually preferable. In this case, you can also use a larger excess of amine in the second step, if it is not too expensive.
If you want to read more about bioconjugation, the book of Greg Hermanson can be highly recommended:
4 Recommendations
All Answers (8)
Bundesanstalt für Materialforschung und -prüfung
I do not know your specific system. In general, you may use a significant excess of NHS (e.g. 2x of carboxyl) and an equimolar amount (e.g. 1x of carboxyl) of EDC. Too much EDC will lead to byproducts, particularly with the amino groups. Please note that EDC is extremely sensitive to hydrolysis. Any open vial might be inactive.
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I have some personal experience with polysaccharides containing carboxyl groups and attempts to functionalize them using carbodiimide chemistry. One thing that I have learned is that the typical equivalent ratio that you would use for conjugation of small molecules carrying a carboxyl/amino pair respectively is usually too small when dealing with a usually very large polysaccharide.
Several factors come into play here - the size of the polysaccharide, the sterical availability of carboxyl groups not only in the context of 1 monosaccharide unit but also in terms of how the entire chain is folded up and some of the monosaccharide residues might not be that accessible.
My suggestion is to consider longer reaction times and a higher excess of reagents. I've used up to 50-fold of carbodiimide per molar equivalent of carboxyl group in a polysaccharide sometimes and then 100 eq of NHS. And reactions overnight.
Michael G. Weller is right in saying that too much EDC can cause significant side-products (especially formation of guanidinium-adducts with your amine which can totally destroy your attempts for functionalization). You can avoid this by functionalizing the polysaccharide with EDC/NHS first and then adding the amine later.
If I am not mistaken, the presence of NHS alone will reduce the amounts of guanidinium adducts.
The problem in figuring out how much it is actually going to be is rooted in the molecular weight of the polysaccharide which in some cases can be measured by SEC or something similar - but in many times you'll have to just guess.
If I don't have data - I normally say - okay - let's do the calculation as if the polysaccharide was 50 kDa in MW and I start from there and do the reaction and see if it worked. If I got too much incorporation - then it's easy to go down with the reagents to get your perfect degree of substitution.
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Concordia University Montreal
Thank you Stjepan Krešimir Kračun for your answer, totally useful
As you said the molecule carrying the amine group is really small in comparison with the polysaccharide and my calculation end up with a really small quantity of this molecule so I started to guess instead.
as I get , you suggested an overnight reaction including the reagent and the polysaccharide and later adding the amine molecule due to the by products problem, is that right?
Concordia University Montreal
Thank you Michael G. Weller for your answer
just wondering why is the mol ratio of NHS is different from EDC?
I have many references that these two reagents are used in the same mol ratio.
And is it possible to calculate them in comparison with the Amine group? (e.g. 5x Amine)
1 Recommendation
Bundesanstalt für Materialforschung und -prüfung
NHS can not do any harm to your molecules, therefore a higher access can be used and lead to a faster reaction and a higher yield, particularly, if you can not calculate the concentration of your carboxylic groups properly. It is not really advisable to calculate the reagent relative to the amino groups, because the reaction between the carboxylic group and EDC is always the first step (see lower link). Therefore, two-step protocols are usually preferable. In this case, you can also use a larger excess of amine in the second step, if it is not too expensive.
If you want to read more about bioconjugation, the book of Greg Hermanson can be highly recommended:
4 Recommendations
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