[zelenchukta]

I have a steroid compound linked to an enzyme (See Image attached). This conjugate is stored at pH 8-8.5 with 0.1% NaN₃. The conjugate is used to test for the steroid by suppressing the enzyme with steroid specific AB.
I have noticed that after conjugation reaction is performed (via EDAC chemistry) the test works very well, however withing 24 hours I see a significant loss of specific activity (The antibody no longer binds my conjugate).

Question: Is there a reaction mechanism that may cause the ester structure (circled in red) to break down at alkaline pH?

If so how can I suppress this reaction? I can reduce pH but only to a point as it will have a negative effect on the activity of my enzyme?

[clarkstill]

Generally I'd be surprised at ester hydrolysis at pH 8 - typically one uses much higher pH to carry out basic ester hydrolysis by the BAc2 pathway (http://www.mhhe.com/physsci/chemistry/carey/student/olc/graphics/carey04oc/ref/ch20reactionsesters.html).

However, your molecule contains a free alcohol, which could potentially participate in intramolecular (6-exo-trig) acyl transfer? I guess the pH being moderate wouldn't matter so much if the reaction is intramolecular... You could probably check if this is what's going on my mass spec: if you're actually seeing hydrolysis then the mass would change over time, if its the rearrangement then your specific activity would fall, but the mass spec wouldnt change.

As for suppressing the reaction, I guess just reduce the pH as much as you can!

[zelenchukta]

So pH is the most likely parameter, can Sodium Azide (NaN₃) have any effect.

The buffer is 300mM Triss/ 10mM MgCl/ 0.1% NaN₃.

Thank you for your reply.