North of Cincinnati Ohio.

Dude had an attitude. 🐢

I feel better.

Anton……

Argh I can’t get the last one.

Will look at those thanks.

It’s a thought. Thanks.

Dull rubber adapters.

They should make these in funky colors and designs. 🧪 #chemsky

Bizarre. Reminder to self: don’t piss off my lab mates.

Ugh. I meant 2.1 g imidazole.

I think it’s 2.1 g MgCl2. Not 21 g.

Oh so you print it yourself?

Wot

I need that!

Will do. I'm not convinced I will figure this out. Maybe. I'm now also working on a totally synthetic method that may turn out to be better/cheaper anyway. But I'd still like to figure this out.

Whoa that's different. I'll check this out. Thanks.

I have found only one paper where someone does this at any scale: Wu, J. Biol. Chem. Volume 299, Issue 10, 2023, 105142. Similar conditions. He just uses excess ATP instead of trying to regenerate it. I tried this with no better success than our process.

In the past chemists have gotten yields varying from 0-60%, with most ~20-40%. I'm doing even worse, getting ~10% or so.

41.2 g (224 mmol) acetyl phosphate LiK added over first 24 hr w/2nd addition of same between 24-48 hr. 2M NaOH to control pH at 6.8. Total volume 8L Millipore water. 37 deg C. Glass rxn vessel w/paddle, stirring slowly. Enzymes added by using a small amt of rxn sol'n, dissolving enzymes, and adding.

Our recipe (developed a couple of decades ago?): 47.3 g (309 mmol) glutamic acid, 21.4 g (400 mmol) NH4Cl, 45.7 g (83 mmol) ATP, 21 g (31 mmol) imidazole, 38.5 g (190 mmol) MgCl2-6H2O, 3000 U glutamine synthetase, 6000 units acetate kinase,

I’m just using NaOH and a pH controller to keep the pH steady. Although in fact so little reaction happens that the pH barely changes anyway.

Will look. Thanks.

Yes.

Yes. www.sigmaaldrich.com/US/en/produc...

You've lost me here. FWIW, I'm just an organic chemist, not well-practiced in using enzymes. Thus my troubles.

DTT?

37 deg C, pH 6.8. MgCl2 also added.

I’m making stable-labeled versions of Gln. For example, using 15N-labeled NH4Cl.

Yes but apparently it shouldn’t to this extent. So does the ADP produced so we recycle that back to ATP.

Anything you could find would be great!