[HJay]

Hi, this is first time writing on the forum.

I have been trying to conjugate DNA with a heterobifunctional linker called SMCC (NHS ester on one end and Maleimide on the other). After I connect amine modified DNA to SMCC, I want to conjugate a peptide with the DNA-SMCC complex as you can see on the picture.

After I do all the reactions, I do HPLC and ESI-MS to confirm the product and when I read the mass, the mass I'm getting is not the final product (DNA-SMCC-Peptide). Instead, it is the mass of DNA-SMCC complex.

So I assumed that a sulfhydryl group of the peptide had a problem, but when I checked with Ellman's reagent, it was fine. So I don't know what the problem is.

Someone suggested me that the amine of the DNA actually reacted with the maleimide and NHS-ester was hydrolyzed. So that the peptide could not react with the DNA-SMCC complex.

Any thoughts?

The attached file is from Williams and Chaput Curr Protoc Nucleic Acid Chem. 2010

[wildfyr]

Yes, the amine can react with the maleimide. Maleimides are exceptionally good Michael acceptors, and can react with amines.

Can you do this in reverse order? The NHS ester won't react with the sulfhydryl (thiol) group.

[Babcock_Hall]

I agree with wildfyr and like the suggestion offered.  If changing the order is not possible, you might be able to get some selectivity by a careful choice of conditions, especially time and pH.  It may depend on the relative pK_a values of the thiol and the amine, however. 

[HJay]

Yes, the amine can react with the maleimide. Maleimides are exceptionally good Michael acceptors, and can react with amines.

Can you do this in reverse order? The NHS ester won't react with the sulfhydryl (thiol) group.

Thank you for your reply.
Hm.. If I do this in reverse order, won't NHS ester hydrolyze? I typically do maleimide and sulfhydryl reaction for overnight at room temp.

[wildfyr]

Yep. But it's as Babcock said, you may have to play with time and pH to get what you want. Nhs esters will hydrolyze more slowly under lowered temperature as well. This resource (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/amine-reactive-crosslinker-chemistry.html) says the NHS half life is about 4 hours at near 0C at neutral pH, so it seems like you could play with temp and time to get what you want. Overnight is apparently overkill anyways.

Here's an interesting idea, if you want to speed up the thio-michael reaction with maleimide, perhaps you could include a very small amount of a non-nucleophilic amine such as DBU (diazabicycloundecene). Since I suspect you're a biologist, any organic chemistry lab at your university should have some in pure form.

[Babcock_Hall]

You may have to monitor your reactions to completion and terminate them at that time.  That might prove to be challenging.  Maleimides absorb weakly (IIRC it's around 300-320 nm), and in theory this chromophore could be monitored with time.  However if there are other things which absorb in this region of the spectrum, it may not be practical to do so.
EDT
For a reaction using an inexpensive thiol, it might be possible to monitor with the DTNB reaction, but for an expensive thiol, one might want a more sensitive technique.

[wildfyr]

Could monitor it by TLC by taking aliquots and reacting them with small amounts of thiophenol in the presence of DBU. Very fast reaction. If the thiophenol is consumed, you know you still have maleimide left.

[Yggdrasil]

Maleimides are usually fairly selective for thiol groups given the correct pH conditions (~6.5-7.5) as I've definitely selectively labeled thiols on proteins which have multiple free amines available to react.  For a reaction at pH 7.2 for 3 hrs at room temp, I would not expect to see much reaction of the amines with the maleimide (unless you have a large excess of DNA over crosslinking reagent).  Maybe react DNA with an excess of SMCC, run over a desalting column to remove the unreacted SMCC, then react with a an excess of peptide.

Are you able to successfully label the peptide with SMCC (without doing the DNA conjugation first)?

[HJay]

Yep. But it's as Babcock said, you may have to play with time and pH to get what you want. Nhs esters will hydrolyze more slowly under lowered temperature as well. This resource (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/amine-reactive-crosslinker-chemistry.html) says the NHS half life is about 4 hours at near 0C at neutral pH, so it seems like you could play with temp and time to get what you want. Overnight is apparently overkill anyways.

Here's an interesting idea, if you want to speed up the thio-michael reaction with maleimide, perhaps you could include a very small amount of a non-nucleophilic amine such as DBU (diazabicycloundecene). Since I suspect you're a biologist, any organic chemistry lab at your university should have some in pure form.

Thank you for the suggestion!

[HJay]

Thank you all for kind replies. I will try the suggestions made in this post. Hope they work!