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 pKa values of the thiol and the amine, however.
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 pKa values of the thiol and the amine, however.
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.
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.
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.
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.
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.
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.
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.
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.
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)?
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)?
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.
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.
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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.
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.
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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.
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.
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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.
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.
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Can you do this in reverse order? The NHS ester won't react with the sulfhydryl (thiol) group.
Can you do this in reverse order? The NHS ester won't react with the sulfhydryl (thiol) group.
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Thank you for the suggestion!
Thank you for the suggestion!
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Are you able to successfully label the peptide with SMCC (without doing the DNA conjugation first)?
Are you able to successfully label the peptide with SMCC (without doing the DNA conjugation first)?
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