[batty2279]

Hello all... new poster here!  Thanks in advance for any helpful suggestions!  I am a biochemist attempting to synthesize molecular probes, so sometimes I bite off more than I can chew

I started with 200 mg of inosine monophosphate:




The goal was to acetate-protect the two hydroxyl groups so that this molecule would be soluble in organic solvents (mainly DCM) for purification purposes.  I reacted the IMP in DCM with DMAP (nucleophilic catalyst) and acetic anhydride to complete the estrification at the hydroxyl groups.  I know the reaction worked because of the absence of a suspenion; all of the IMP is now miscible in the DCM.

The problem I'm having now stems from the work up I suppose.  I did a standard DCM/water wash several times, dried the solvent layer with sodium bicarbonate anhydrous sodium sulfate, and then dried down the product on the roto-vap.  Only problem is I only garnered about 5 mg of product!  I have no idea where the loss is.  It can't be in the water layer since the product is now soluble in the DCM... so is it possible that while washing, the phosphate group was deprotonated, and it somehow formed a salt with the sodium sulfate?  Any insight would be massive!  I have no experience working with phosphate chemistry  


:edited for lack of coffee:

[discodermolide]

Hello all... new poster here!  Thanks in advance for any helpful suggestions!  I am a biochemist attempting to synthesize molecular probes, so sometimes I bite off more than I can chew

I started with 200 mg of adenosine monophosphate:  http://upload.wikimedia.org/wikipedia/commons/thumb/f/fb/AMP_structure.svg/200px-AMP_structure.svg.png


The goal was to acetate-protect the two hydroxyl groups so that this molecule would be soluble in organic solvents (mainly DCM) for purification purposes.  I reacted the AMP in DCM with DMAP (nucleophilic catalyst) and acetic anhydride to complete the estrification at the hydroxyl groups.  I know the reaction worked because of the absence of a suspenion; all of the AMP is now miscible in the DCM.

The problem I'm having now stems from the work up I suppose.  I did a standard DCM/water wash several times, dried the solvent layer with sodium bicarbonate, and then dried down the product on the roto-vap.  Only problem is I only garnered about 5 mg of product!  I have no idea where the loss is.  It can't be in the water layer since the product is now soluble in the DCM... so is it possible that while washing, the phosphate group was deprotonated, and it somehow formed a salt with the sodium bicarb?  Any insight would be massive!  I have no experience working with phosphate chemistry 

Dried with sodium bicarbonate? You usually use sodium sulfate anhydrous or magnesium sulfate.
Look in the water phase, if you actually used bicarbonate then you made the Na salt of the phosphate. Run a TLC or HPLC of the H₂O phase and the DCM phases.

[batty2279]

hahaha oops yes, anhydrous sodium sulfate is indeed what I used.  Haven't had my coffee yet 

[discodermolide]

hahaha oops yes, anhydrous sodium sulfate is indeed what I used.  Haven't had my coffee yet 

Well have a coffee and check the aqueous phase!

[batty2279]

It is hard to see if there is any product in the water layer because the residual DMAP is in there, so it kind of washes out any UV activity from the product.  I am running over now to check the LC-MS of the 5 mg of product I did recover to ensure it is the right product, but clearly I have a loss somewhere... either in the water layer, or in the beaker with the dried sodium sulfate (as a precipitated phosphate salt).  Grrr... 

Thanks for replies!  Any other ideas are welcomed!

[discodermolide]

It is hard to see if there is any product in the water layer because the residual DMAP is in there, so it kind of washes out any UV activity from the product.  I am running over now to check the LC-MS of the 5 mg of product I did recover to ensure it is the right product, but clearly I have a loss somewhere... either in the water layer, or in the beaker with the dried sodium sulfate (as a precipitated phosphate salt).  Grrr... 

Thanks for replies!  Any other ideas are welcomed!

Take the water layer with you to the LC-MS

[Doc Oc]

Lots of potential issues with this chemistry.

1) How old is the acetic anhydride?  Was it sealed well?  If it's old and people haven't been careful with it (ie; left it open on the bench for a long time) then a lot of it could be hydrolyzed.  Also, how many equivalents are you adding?  For acetylations, I generally added 3 per group I was trying to acetylate, unless the bottle was brand spanking new.

2) There are more things besides the hydroxyls that can and will react with an acylating agent.  The phosphate for one (though they are weak nucleophiles, in the right conditions they can act like a carboxylate and be much more reactive) as well as the aniline (also not a great nucleophile, but good enough to be a problem).  The mass spec might tell you how many things have acylated, but knowing where they've acylated will take some detective work, and it won't necessarily be straight forward.

3) DMAP needs to be washed out under acidic conditions, neutral will not be enough.

One of the more common ways to protect the hydroxyl groups of nucleosides is to make the acetonide.  I would explore that rather than acylation, this seems like more of a headache than it's worth.

[batty2279]

waiting on MS results now...  thanks for great reply Doc Oc.

To answer 1, yes it is a fairly new bottle.  I too usually add 3 eqs per hydroxyl, so i started with 6 eqs total.  But after 24 hours, it was almost all still in a suspension.  I found some old literature of guys in Russia acetylating nucleosides with up to 8 eqs per hydroxyl, so I tried this and within an hour it worked (seemingly). 

I will explore your suggestions in #2, and thanks for the DMAP removal idea.  It was around pH = 8, so I'll try to take it down lower.  And yes, I had considered possible use of an acetonide, so it's funny you mentioned that.  The estrification had worked so well on my previous probes, I figured it was worth a shot first. 

Let's see what this MS tells me.  Thanks guys!

[batty2279]

So it appears as though discodermolide was correct in his assumption.  The acetylated IMP is showing up in the water layer when running the negative mode MS.  So it seems that the synthesis worked, however my hope that the compound would remain in the organic layer failed miserably.  So I'm guessing it is miscible in both layers, since it was completely dissolved in the DCM at the conclusion of the reaction, but potentially has a great affinity for the water layer during work up.  I guess HPLC could be used to separate it out after the reaction, but that defeats the entire purpose of trying to protect it to get it into organic layers.   

[discodermolide]

So it appears as though discodermolide was correct in his assumption.  The acetylated IMP is showing up in the water layer when running the negative mode MS.  So it seems that the synthesis worked, however my hope that the compound would remain in the organic layer failed miserably.  So I'm guessing it is miscible in both layers, since it was completely dissolved in the DCM at the conclusion of the reaction, but potentially has a great affinity for the water layer during work up.  I guess HPLC could be used to separate it out after the reaction, but that defeats the entire purpose of trying to protect it to get it into organic layers.   

You could try and make a tetra alkyl ammonium salt. This may be organic soluble.

[OC pro]

At pH of 8 it will be of course in the water layer. You formed sodium phosphonate being water-soluble. Try to go down a bit with the pH (~2-3). Then try again extracting with ethyl acetate.   

[batty2279]

Yea that's what I was afraid of.  I did a sodium bicarbonate wash on the first work-up to neutralize any remaining acetic anhydride, but at that pH, I figured the product formed a water soluble salt.  Should I skip the Ac2O neutralization?  Or can I use a basic wash first, followed by an acidic wash to re-protonate the phosphate so it is again soluble in the organic solvent?  Thanks again OC pro!

[synthon]

Maybe you could also protect the amine?  I'm having a similar problem with a nitrogen heavy ring-system with a primary amine. 

I'm also curious what the impurities are that you'd like to remove.  Most workups for deprotection involve aqueous washes, so when you deprotect you might have the same issue with impurities, only from a different source.