Hi Abhigyan,

the product is safe in acidic conditions, we routinely synthesize PEG-peptide conjugates in our group using the Michael-type addition of PEG-maleimide and cystein-functionalized peptides (see 1st Link). We use preparative HPLC for purification, where we add 0.1 vol-% TFA to the mobile phase, pH is 2-3 then.

Please be aware that the Michael-type addition does not occur at low pH, the pH should be above 4-5. Avoid basic conditions (above pH ~8), this will damage the maleimide group! See 2nd link.

Best regards,

Marcus

Btw: you could also use amino groups for this reaction. However, thiols are much faster and therefore more selective towards other functional groups (see last links).

Article Defined Polymer-Peptide Conjugates to Form Cell-Instructive ...

Article The hydrolysis of maleimide in alkaline solution

Article Relative Nucleophilic Reactivities of Amino Groups and Merca...

Article Michael addition reactions in macromolecular design for emer...

Hi Marcus,

Thank you very much. Presently I am trying to label a protein using Maleimide-Thiol reaction and purify through preparative HPLC. HPLC operating conditions are exactly same as you described. I don't see any double labeled product and had doubt about the stability of the Maleimide-Thiol adduct at low pH. Thanks for answering.

Your knowledge gives me a good extent of confidence about the stability of the adduct.

Hi Abhigyan,

how many free thiol groups do you have in your protein? You may have several products with different substitution pattern which can usually not be resolved from preparative columns - double check your product with analytical HPLC in any case!

For instance, if we add 1 equivalent peptide to 4-arm PEG Maleimide, we get a statistical mixture of 1, 2, 3, and a little bit 4-substituted species. We ccould resolve this only with an analytical column.

Best regards,

Marcus

Hi Marcus,

You are right. I am not getting separation between labeled and unlabeled protein. Both of these have identical elution time. None of the semi-preparative C18 and the analytical C4 are able to separate them. Probably analytical C18 would do the separation. This protein contains +25 charges at pH2. Possibly the high charge is creating the problem for me.

I really appreciate your suggestions.