nucleoside analogues: pH/conc.to dissolve??

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Sat Feb 21 14:02:06 EST 1998


In article
<Pine.A32.3.91.980220102016.17228A-100000 at mail.med.cornell.edu>,
hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) wrote:

> Greetings!
>         I am using AZT, ddI, and ddG in an attempt to inhibit a RT-like 
> enzyme. I purchased these reagents in powdered form from 3 different 
> companies, all of whom did not supply a data sheet nor could they really 
> help me over the phone. Most papers I've read say that they dissolved 
> them in water or DMSO. For my experiments, I need them in water or cell 
> media at fairly high concentrations (500micromolar), but I the first time 
> I prepared them in water (at 10mM stocks), they were difficult to 
> dissolve (thus i had to heat them) and became inactive.

Posted and mailed.

I cannot comment on AZT or ddI but we have found that ddG is notorious for
its poor solubility in aqueous solutions. Heating is a standard procedure
for getting stock solutions (10 mg/ml or roughly 40 mM) of ddG to
dissolve. Place your tube/bottle/flask of ddG suspension in a waterbath at
70-75°C and continue to swirl the suspension until it dissolves (5-10
minutes).

An additional note - ddG is very labile to acid environments; it tends to
break down into the sugar and free base. I would advise not making your
stock in water but rather in PBS (pH 7.2-7.4) or in a weak Tris-HCl
solution (10 mM at pH 7.4-7.5). This will aid in preventing breakdown of
your ddG.

I'm curious - how do you know that the ddG became inactive? From the
sounds of it you were doing tissue cultue and it is known that certain
cell lines, for example, are notoriously poor transporters/metabolizers of
certain nucleosides.

Cheers

Karl the hepB guy



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