I have been using an oxazine dye ATTO 655 for various purposes. Based on some mass spectra evidence, I wondered if the ATTO 655-maleimide reacts to the 0H-containing solvent like water or methanol and gets hydrolyzed?
Dear Lokesh Ranjan thanks for posting this interesting technical question on RG. If you check the product information sheet for ATTO 655 you find the following statement (citation): "The product is soluble in polar solvents, e.g. dimethylformamide (DMF), dimethylsulfoxide (DMSO), or acetonitrile. However, due to their inherent reactivity, NHS-esters and maleimides must be well protected from OH-containing solvents like ethanol and, in particular, water." Thus, chances are that the dye is indeed hydroyzed in the presence of ethanol or water. The data sheet is, however, somewhat contradictory because in the first paragraph the company mentions "very good water solubility". This is clearly a bit strange.
https://www.atto-tec.com/fileadmin/user_upload/Katalog_Flyer_Support/ATTO_655.pdf
In any case I wish you good succes with your work!
NHS-esters and maleimides will decomposed/hydrolyzed with OH-containing solvents like ethanol and, in particular, water for long time storage. For labelling, reactivity of NH2(at slightly basic condition) and SH will significantly greater than OH. NHS eater may come along with a few % of acid in the product(from SM to make NHS ester or partially hydrolyzation).
Another point to add here is that you can use aqueous solvents for your maleimide reactions. While they should be stored in something like DMSO, I have performed many protein tagging reactions for MS applications of maleimide dyes in aqueous solutions.
There are two things to remember for doing this.
1. the pH range should be between 6.5 and 7.5 The closer to 6.5 the better. This favours the reaction with thiol. Above 7.5 you are going to hydrolyse.
2. If you use a disuphide bond reducing agent, make sure it is not one that contains free thiol bonds, as they are reactive to the maleimide.
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