[ollie]

I have been trying to coupling cellulose acetate (see diagram) with an amine terminated molecule for some time but failed.

The way I have been going about this is to deacetylate the cellulose acetate to get a regenerated cellulose which I then activate with epichlorohydrin (Epoxide activation) or Sodium Periodate (Aldehyde activation) before coupling with my amine terminated linker (see diagram).

I was hoping someone could help be by suggesting an alternative method, or by advising on the current method that I am using. I do also have the possibility of using a hydroxyl terminated linker (see diagram) if that helps.

Any advice is greatly appreciated!



For reference I have attached the protocols that I have been using below:

EPOXIDE

1.   Deacteylize CA membrane by treating with 0.1M NaOH (4mg/mL) in H2O/EtOH 2:1 for 24 h to get a regenerated cellulose membrane

2.   React with Epichlorohydrin (7mL in 100mL 1.1M NaOH) at rt for 4hours

3.   Stop the reaction decanting the reaction mixture and washing the membrane in 10 volumes of distilled water

4.   Wash with DI H2O Thoroughly

5.   Immerse in 50mM amine terminated linker at rt for 12 h

6.   Deactivate remaining aldehyde groups by the addition of 1M ethanolamine pH9 (in PBS) 24h

7.   Convert initially formed carbinolamine into more stable amine be adding sodium cyanoborohydride (NaBH3CN 62.84 g/mol) 5mg in 1mL borate buffer. Stirred for 30 mins @ rt. Add additional 5mg and stir for 30 mins.

8.   Wash in PBS & DI H2O for 2 h


ALDEHYDE

1.   Deacteylize CA membrane by treating with 0.1M NaOH (4mg/mL) in H2O/EtOH 2:1 for 24 h to get a regenerated cellulose membrane

2.   Oxidize in 0.05M sodium periodate (NaIO4) to produce dialdehyde groups for 9 h in Darkness

3.   Cease this reaction by the addition of ethylene glycol after 9 hours (5mL) stir for 30 mins

4.   Wash with DI H2O Thoroughly

5.   Immerse in 50mM amine terminated linker at rt for 12 h

6.   Deactivate remaining aldehyde groups by the addition of 1M ethanolamine pH9 (in PBS) 24h

7.   Convert initially formed carbinolamine into more stable amine be adding sodium cyanoborohydride (NaBH3CN 62.84 g/mol) 5mg in 1mL borate buffer. Stirred for 30 mins @ rt. Add additional 5mg and stir for 30 mins.

8.   Wash in PBS & DI H2O for 2 h

[Doc Oc]

Where are you trying to attach the amine?

[ollie]

In the deacetylated form to each of the 3 hydroxyl groups that replace the acetate.

Epoxide example is attached. I would expect the same from the aldehyde reaction.

But if there is a way to bind either the H2N- or -OH terminus of the linker to part of the acetate group before deacetylation then that may work too.

[orgopete]

Not knowing where these recipes came from nor what they were used with (cellulose?), I would suggest repeating a known reaction to verify your reagents and technique. Since steps 6 and 7 of the epoxide method should not apply, we cannot judge the suitability of this procedure to this problem.

[ollie]

Is there any other way of covalently binding the amine or hydroxyl terminus of the linker to the acetate groups?

[Doc Oc]

The wording you're using is still a bit ambiguous.  Are you trying to replace the hydroxyl groups entirely?  In your diagram you've highlighted the acetyl protecting groups in red, but the hydroxyls are still in black.  Do they stay or do you want the whole thing gone and the sulfonamine there instead?

Orgopete is right, you should try to find an established method for this, that is absolutely the best possible thing you can do for yourself.  I see a large number of challenges with what you're trying to do.  I don't see how the epoxide method could possibly work since you don't have an epoxide to start with.  The oxidation/reductive amination looks more reasonable, but you run the risk of overoxidizing the primary alcohol to a carboxylic acid with sodium periodate.  Where did you get these procedures?

[ollie]

Thanks for your help.

The methods are from various books are journals involved with membrane activation (example references below).


The Epichlorohydrin is used to create an epoxide group on the cellulose material (after deacetylating the cellulose acetate)


Chemists from Pierce (www.piercenet.com) also seemed to favour the aldehyde partial oxidation method, though i am aware that in industry they prefer Epoxide activation as it gives better activity.
The industry standard used to be CNBr activation but this is considered out of date practise.


All of these methods come from protein immobilization technology.
They should just work but they the one i have tried aren't, and i cant figure out why.
A flaw with my process is that i am determining the attachment of the linker using an indirect method: Namely the capture of lysozyme by the 3-amino-1-proanesulfonic acid.


http://books.google.co.uk/books?id=3QaUuTg6L1cC&pg=PA29&lpg=PA29&dq=ligand+coupling+to+cellulose+acetate&source=bl&ots=1gKLej5wr0&sig=5ELUDzodo7T_LMCK20h9eHuRy0Y&hl=en&ei=svgMTJPYNJKI0wTMhq1l&sa=X&oi=book_result&ct=result&resnum=1&ved=0CBkQ6AEwAA#v=snippet&q=periodate&f=false

Journal of Membrane Science 182 (2001) 227–234

[orgopete]

While the addition of more information is useful, it does not resolve the problem. For example, why start with acetyl cellulose and hydrolyze the acetyl groups? Why not just start with cellulose? If the O-alkylation with epichlorohydrin and link to polymer succeeded, how or why should there be a reaction with the aminosulfonic acid? How does lysozyme capture the cellulose with an aminosulfonic acid?

[ollie]

Sorry if this is becoming less clear, feel free to stop trying to advise if you think there is no hope.

I start with Cellulose acetate because i need a cellulose derivative which is soluble in commonly available solvents, such as DMF, acetone.
This is then processed into a membrane form.

I was then deacetylating since the only methods that i am aware of to attach amine groups to this membrane structure is via the protein immobilization methods i described previously.

Epoxide groups are known to react with NH2 over a period of a few hours to yield a covalently bound product: HO-CH-CH2NH


The indirect method of activity detection works since the Sulfopropyl group which has bound to the regenerated cellulose membrane captures lysozyme under acidic (pH 5) conditions. The lysozyme can then be removed by high salt concentration elution buffer and measured via UV spec 280nm (Ion exchange chromatography)


Apart from the epoxide and aldehyde activation attempts i have tried to create COOH groups by oxidation with 30% hydrogen peroxide, which i can then use EDC NHS coupling chemistry to bind NH2- terminus molecules. The degradation to the membranes by the hydrogen peroxide proved a problem here.

[Doc Oc]

Jeez, this is actually getting more convoluted.  It sounds like you need to be collaborating with someone that has a solid organic chemistry background, there are many issues with the syntheses you have tried that an organic chemist would have spotted right away and saved you the trouble.

Let me ask you, the diagram I've attached, is this along the lines of what you're looking for?  It matters because you talked about oxidizing the hydroxyl groups to carboxylic acids (this route has fundamental flaws, both in the design and the materials used, but that's another story for another time) and then coupling them to the amine using EDC.  Thing is, that would give you an amide, not the amine, and that would change the properties of your molecule.  You also suggest the epoxide route, but that would give you yet a different product.

So I guess my main question is, what exactly is it that you're trying to do?  You've tried several different things and they all seem to lead to different product (some of the synthetic routes are the equivalent of driving into a brick wall).  So it seems like you're just concerned with attaching the sulfonamine, but simply doing that will not guarantee your success in this experiment nor will it gain you any good understanding of what's going on unless you account for the potential variables (ie; what will the reactivity difference be between a sulfonamide and sulfonamine?)

[ollie]

Sorry, i have spent many months trying many different things to get this to work.

The basis of what i am trying to do is bind a molecule via its amine terminus (in this case 3-amino-1-proanesulfonic acid)
to polymeric cellulose.
I dont mind how it is done but every similar example i can find uses regenerated cellulose rather than cellulose acetate - hence the reason for my deacetylation.
I have to start out with polymeric cellulose acetate so can either deacetylate that membrane to give a regenerated cellulose membrane for which i have to then activate to bind to my amine terminus molecule.
Or if there is a way of using the acetate groups help bind my amine terminus molecule i can do that.

The aim is to have enough sulfopropyl groups covalently bound to the polymer cellulose membrane so that it can capture and (under elution condition) remove lysozyme.

[Doc Oc]

Okay great, now I see what you're trying.  That is basically the diagram that I posted, so we're on the same page.  That being the case, I think your best option is to remove the acetate groups (although I feel that NaOH is maybe a bit too harsh of a condition and that you could use something milder like MeOH/K2CO3), then the reductive amination.  The problem with that route is that all of the stereocenters will get scrambled, so if that's important then you will need to re-design the route.  But if you're just trying to get the sulfonamines on there, that should work.  Keep in mind there's a pH issue as well and that you will probably need a basic condition for the amine to react, otherwise it will probably be protonated and less apt to do what you want.  But you won't need to worry about that until after the oxidation.

[ollie]

OK, Thanks for your help, i will give that a go.

What is the difference between 3-Amino-1-propanesulfonic acid & 3-Amino-1-propanesulfonic acid sodium salt, apart from the obvious? I.e which one do i want?

http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=3-amino-1-propanesulfonic+acid&D7=0&D10=3-amino-1-propanesulfonic+acid&N1=S_ID&ST=RS&N25=0&F=PR

[Doc Oc]

I don't think it will make too much difference which you choose (I could very well be wrong though).

If you pick the acid you will need to get rid of that acidic proton, otherwise it will deactivate your amine (as I said earlier, you will probably need to run the reaction in basic conditions to account for this).

If one is significantly cheaper than the other (can't imagine that they are) then I'd get that one, but for convenience I'd pick the sodium salt.

[orgopete]

If you plan to use the aminopropanesulfonic acid, then you should use your aldehyde procedure. Replace the amine linking with the sulfonic acid and reduce with cyanoborohydride. That should give your aminopropanesulfonic acid modified cellulose. If that is required for your lysozyme binding, then this could work.