[OmgImDiene]

Hello,

I performed a chemienzymatic reaction directly from this Nature protocol:

http://www.nature.com/nprot/journal/v5/n4/full/nprot.2010.3.html

Judging by the TLC, the reaction worked beautifully, the monosaccharide starting material disappeared and I see a spot with a lower Rf than the monosaccharide standard, which is consistent w/ phosphorylation as described in the protocol.  I followed their procedure EXACTLY:

*Mod edit: removed excess quote*

My problem is that during purification I no longer see a spot coming out of the column that is consistent by TLC with the spot I saw from my reaction.  The Rf has increased and is now consistent w/ the starting material.  I rotavaped everything and in terms of the mass obtained back, it matches closely to the amount of starting material I used.  I'm concerned that the phosphate group is rather unstable and degraded during column chromatography giving back my starting material product.  Is there some other safer way of purfying the material so that I can remove ATP?  I really am in a tight bind here and need to figure this out ASAP. 

[Dan]

What sort of Rf difference are we talking here? You should co-spot the material that came off the column against an authentic sample of starting material to check and/or compare the NMR spectra.

[OmgImDiene]

What sort of Rf difference are we talking here? You should co-spot the material that came off the column against an authentic sample of starting material to check and/or compare the NMR spectra.

Here's the TLC:

https://imgur.com/a/cTwDx

The spot in the middle burns like a carbohydrate should (5% sulfuric in MeOH--->heat).  The spot also has a similar movement exactly according to the Nature protocol paper linked and the byproducts/ATP left over look exactly like the Nature protocols figures too.  When I was doing the column, however, my suspected product spot it no where to be found, and what reappears is the spot at the top.  I did spot the material obtained from my column w/ my starting monosaccharide and they were on top of each other, so I'm fairly certain it is unphosphorylated monosaccharide I obtained.  I'm using standard silica, which I know is slightly acidic.  I was worried about that initially, but the Nature paper didn't mention neutralizing it or anything and loaded the column with straight chloroform.  Is the acidity the problem?

[Babcock_Hall]

I only have time for a brief, very general comment right now. Typical phosphomonoesters are generally not very labile, but around pH 4, they do go through a maximum in the pH rate profile).  However, phosphomonoesters at the anomeric carbon are more labile.

[Babcock_Hall]

I don't have access to this particular journal.*  What purification method are you using presently?  A second general comment:  A well-trodden way to separate phosphate esters and anhydrides is using classical anion-exchange chromatography on Sephadex (A-25?), in the presence of a gradient of triethylammonium bicarbonate (the pH is in the ballpark of 7.6).  The ATP will elute much later in the gradient than a monophosphate.  Making 1 M stock triethylammonium bicarbonate is relatively easy; you just use a sparging tube connected to a side-arm flask with solid carbon dioxide.  The gas is bubbled through a solution of (preferably distilled) TEA and water.  Then the TEAB solution is tightly stoppered.  Look for papers by Richard and Frey or Sheu and Frey in the 1970s for some examples.  My guess is a gradient of maybe 10 mM to 500 mM TEAB would be appropriate.  The TEAB is removed by repeated rotary evaporation.  Unless the phosphate ester is very, very labile, I would not predict that the rotary evaporation step would lead to decomposition.
EDT
*I have looked over the purification protocol now.

[rolnor]

I wonder, if you use silica, what mobilephase do you use, is it acidic? There is reagent that are specific for phosphates, long time since i made it, I think its molybden and perchloric acid, acetone, maby you can find the recipe.
If you use reversed-phase silica you can use basic mobilephase, methanol water triethylamine, I suppose the product would be more stable then using acidic mobile/stationery-phase

[rolnor]

I have worked with nucleoside monophosphates and both sephadex/trietylaminehydrogencarbonate and RP-18 metanol/water/triethylamine works fine.

[Babcock_Hall]

I only have time for a brief, very general comment right now. Typical phosphomonoesters are generally not very labile, but around pH 4, they do go through a maximum in the pH rate profile).  However, phosphomonoesters at the anomeric carbon are more labile.

Glucose 1-phosphate is more labile than typical monophosphates at pH values near 0-1, because of a C-O bond cleaving mechanism.  See Figure 5-2 in Bioorganic Mechanisms, Volume 2, Bruice and Benkovic.

Is the problem in the preparative chromatography or in the TLC?

[Babcock_Hall]

I have worked with nucleoside monophosphates and both sephadex/trietylaminehydrogencarbonate and RP-18 metanol/water/triethylamine works fine.

Do you have a reference to the RP-18 protocol?  I just read over the protocol from the Nature paper in question.  I do not recall seeing this kind of purification (a step gradient of ammonium bicarbonate in methanol in dichloromethane).  In previous discussions of using silica to purify polar compounds, I don't recall having seen anything similar.  Is this a fairly common technique?  Does some silica dissolve?

[Dan]

I'm using standard silica, which I know is slightly acidic.  I was worried about that initially, but the Nature paper didn't mention neutralizing it or anything and loaded the column with straight chloroform.  Is the acidity the problem?

You can check your product's stability on silica by running a 2D TLC (e.g. see half way down this page). I normally wait 15-30 mins between the two runs.

[rolnor]

No, I dont have a reference, sorry. It was a method I developed but never published.