Dear Charles,

First of all, depends which reaction you would like to do. Your cytidine.HCl salt is of course not soluble in dry organic solvent and your reaction would not work well. I already worked with salts in reaction and from my side I made the reaction using 1 equivalent of dry triethylamine for 1 equivalent of salt in dry organic solvent (your compound without HCl should be soluble in organic solvent). Indeed, by this way you can solubilize your compound in situ by making the free base. Concerning the workup, you can remove the salt formed (Et3N.HCl) just by filtration on Celite under reduced pressure. Then after evaporation of the organic solvent under vacuo, you can get your expected molecule.

Best regards and good luck ;-)

What is the pH of a aqueous solution? Try putting it into warm water at a near saturated concentration and then neutralising the solution with sodium bicarbonate (sensitive molecules don't appreciate the use of NaOH unless under strong stiring at the time of addition). Upon cooling of the solution on ice, your product may crash out of solution. If it doesn't you could try extracting it from the salt using ethyl acetate or other suitable non-aqueous-soluble organic solvent. After all material is in the organic layer, dry it it saturated brine solution and anhydrous (i.e. kept in oven) magnesium sulfate.

Alternatively you might try desalting it on a C18 Sepak, but I have always had better results from the first method as the Sepaks can be quite low yielding for small samples.

Try to dissolve the Cytidine*HCl analogue (I assume it is solid compound) in AcOEt and add some NaHCO3. CO2 formation will move the equilibrium in order to have a full conversion of HCl so I expect to have an organic solution with an organic compound and NaCl. Try to filetr the NaCl from the organic solution and remove (dry) AcOEt: I expect to have cytidine free base.

please let me know

use any organic solvent that may dissolve your organic compound

Dear Charles,

First of all, depends which reaction you would like to do. Your cytidine.HCl salt is of course not soluble in dry organic solvent and your reaction would not work well. I already worked with salts in reaction and from my side I made the reaction using 1 equivalent of dry triethylamine for 1 equivalent of salt in dry organic solvent (your compound without HCl should be soluble in organic solvent). Indeed, by this way you can solubilize your compound in situ by making the free base. Concerning the workup, you can remove the salt formed (Et3N.HCl) just by filtration on Celite under reduced pressure. Then after evaporation of the organic solvent under vacuo, you can get your expected molecule.

Best regards and good luck ;-)

Suspend your compound in a solvent where the cytidine will be soluble. Bubble dry NH3 into the solution (prepare by dropping NH4OH on KOH). The ammonium chloride is then filtered from the solution.

Use NaHCO3 in MeOH, once that the neutralization occurs try to solubilize your released compound in absolute EtOH to avoid NaCl solubilization, then filter and evaporate. Then you should have the desired compound without the HCl. Hope this helps.

Use silver carbonate instead of nitrate.

Or an anion-exchange resin in hydroxide or carbonate(bicarbonate) form.

Thank you to everyone for taking the time to answer. I totally blanked on using bicarbonate, that sounds like the best approach, however the triethyl amine also caught my eye. Good to have options. @Farid, Ag2CO3 is insoluble in water, most heavy metal carbonates are insoluble.

Use Sephadex-LH 50.

May be neutralize it in water with NaOH or Na2CO3 and then extraction with dichloromethane.

For example with Gel Filtration like G-25 Gel.

If I had to neutralize this, I'd go with bicarb as well. Alternatively, you could just do the next step in the presence of 1 eq. tea in a polar solvent as this would be less time consuming, but this really depends of what you want to do with it.

Another option would be to use reverse phase HPLC with acetonitrile/ (1% NH4OAc in water). The salts that comes through can generally be liophilized quite easily.

I think it could be neutralised by NaOMe in MeOH and the resulted salt could be removed by Gel filtration using G-10 column with H2O/MeOH solvent system.

BOL

I second the suggestion offered by Gilles. Dissolving your ammonium chloride salt in aqueous base and then extracting the amine into a volatile organic solvent (e.g. EtOAc, Et2O, CH2Cl2, etc.) is likely the most straightforward and broadly applicable approach described in this thread.

One occasional problem with this approach is that the amine might still be more soluble in the aqueous phase than in convenient organic solvents. This can be addressed by continuous extraction of the aqueous phase using a modified Dean-Stark setup.

Masking and demasking protocol or column chromatography with silica gel stationery

phase

yes i agree with Mr. Zach Erno

Dear Charles,

If you are doing your subsequent reaction in any alcohol then first you prepare equimolar amount sodium alkoxide by taking 1 equivalent of sodium with respect to your substrate cytidine•HCl in appropriate alcohol then add cytidine•HCl after stirring at room temperature check the pH of that reaction mixture. After neutralization completed you can go further. If you want to use any solvent other than alcohol, after formation of sodium alkoxide you can remove the solvent through evaporation then add appropriate solvent you want to use. After addition of cytidine•HCl, if precipitate comes out decant the organic layer, as the ppt may be NaCl and then perform your desired reaction.

Ion exchange resins with amine or quaternary ammonium functional groups work well. I've used purified versions of Dowex-1 or Amberlite IRA-400 in the hydroxide form. It's been a while so I don't remember the exact resin. Very clean versions are available (Biorad) but you may want to get the chloride form and cycle it to the hydroxide to be sure it is clean.

1) Add calc. EtONa/MeONa (from Na+EtOH or MeOH), filter NaCl, EtOH/MeOH remove by evaporation.

or

2) add calc. Tetramethylguanidine - its strong enough to abstract HCl from any usual organic bases. Addition of Hunig base EtN(i-Pr)2 (in excess) may by also useful

Neutralize it in water. Then do liophilization. Then just dissolve your compound in organic solvent of choose.

Neutralize with NaOH (aq) and extract with your desired organic solvent. Use separatory funnel.

Organic solvent must form two phases with aqueous salt solution. For example, CH2Cl2, toluene, ether....

Dear Charles,

Sorry by this observation, but are you 100% sure that you can not perform the subsequent reaction in water? You would be amazed by number of reactions that where supposed to not work in water and they do, sometimes even better than in organic solvents.

Good luck, you have very nice suggestions above.

One can make Cytidine.HCl react with activated metal powders like zinc dust or iron in an organic solvents. After stirring for 30 min to 1 h, you can filter the reaction mixture. Filtrate will have your required compound. It worked for many amino acid esters hydrochloride salts. and hence it is worth trying.

In chromatography, the salts do not move, that is, stays at the starting point. By a simple column chromatography, you can eluate free ligand from your salt.

Ion exchange chromatography has alreday been mentionned. Otherwise, if your molecuel is big enough, dialysis can be an option. Finally, selective precipitation of one of your compound (salt or target cpnd) by addition of a water miscible co-solvant (alcool, THF, DMF...) can also be envisionned, but the outcome is generally more difficult to anticiptate...

A continuous liquid-liquid extractor is often excellent for this. There are two types of apparatus for this one is for organics more dense than water the other is for organics less dense than water. Good luck!

Try dean's stark app it may solve ur problem

Try dean's stark app it may solve ur problem

Try to neutralize with Tributylamine. The resulting Tributylamine HCl salt is soluble in many organic solvents. You can also try non-basic HCL scavengers such as epoxides (epichlorohydrin if it does not harm your compound) or unsaturated alkenes (limonine or alpha-pinene).

Stir it with sodium hydroxide on alumina; subsequent filtration will leave your free base in a dry solution. Solvent has to be stable to the NaOH -- toluene, ethers, and NMP are safe choices. NaF on alumina should also work; it's a good proton sponge and should permit you to use EtOAc, DMF, or DMSO.

Alternatively, if the proton adduct isn't a problem, then you can lipophilize it: e.g. Add 1 eqv of NaBArF [http://en.wikipedia.org/wiki/Non-coordinating_anion]. NaCl would precipitate out and you would now have [cytidine•H]BArF.

Use Saturated solution of of NaHCO3

hi charles - if you can give us any information regarding the reaction you intend to perform on your cytidine•HCl analog it would be much easier to suggest appropriate solvents and strategies for obtaining salt-free product... in any case, my first suggestion would be to use DMF as solvent and pottassium carbonate as base to neutralize the HCl in addition, you could add 10 mol% N(Bu)4HSO4 to the mixture as a phase transefer catalyst - I've used these conditions many times for performing reactions on compounds that are difficult to dissolve in more conventional solvents.

Few systems which I handled and made free from salt:

1) Serine methyl ester HCl. Dissolved in dry CHCl3, added 1.2 eqv Et3N then a clear solution was obtained. Added dry EtOAc to ppt out the Et3NHCl, filtered through sintered funnel, solution was removed to get HCl free serine methyl ester.

2) We made morpholino from ribonucleosides, in the procedure a huge amount salt was present. I dissolved in water in presence of 1.3 equivalent NaHCO3 sometimes MeOH was added to get a clear solution. Passed through Dowex resin cation exchange. Washed with water once more. Then eluted with 10% ammonia or 10% ammonia in water and MeOH mixture. Eluent was checked TLC, ninhydrin as our morpholino nitrogen was ninhydrin active.

3) We purify slight bigger molecule (cation oligomer, MWt ~ 1500) by Qsepharose column. By UV detection, collected the sample. Sometimes, we used 600 cut off dialysis bag.

We handled many such samples and some cases standardized, some cases we still need further standardization for getting salt free samples.

4) Many amino acid synthesis papers, reported the procedure using cation exchange or anion exchange resin then elution with buffer either ammonia solution or Et3NHOAc buffer (pH can be adjusted), evaporates in liophiliser.

The ammonium bicarbonate salt can be made by passage through an ion exchange column. Then you rotovap it dry.

So make the cytosine carbonate salt. Look up how ATP analogues such as ATP gamma S are produced. I guess see above. Answer is there from Dr. Sinha.

You can use exchange resin, easy for all

using required amount of NaOH solution you get cytidine analog and NaCl. But cytidine must be soluble in an orgaic solvent such that water and NaCl are insoluble ..then by using continuous extraction apparatus the compound can be extracted into the organic solvent slowly but continuously....then dry the organic extract, eaporate the solvent and you get the product.

Just dissolve your product in water, add a small excess of NaOH (if your product is stable) or TEA and then pass it through preparative reverse phase HPLC or MPLC (salts and NaOH run with the solvent front). Then lyophilize and get your product as free amine. Also multiple extraction from alcaline 30% NaOH solution should work if you find an organic solvent non soluble in water in which cytidine free amine is soluble...

Andrea's answer seems the easiest. But wouldn't you want to add a large excess to make sure all the cytidine is in the right salt form?

In my opinion adding an aqueous base and do an extraction is maybe not the right choice, because I think cytidine itself is quite good soluble in water. On the other hand its solubility in organic solvents is only poor. So I think it would be hard to find a solent that is suitable for dissolving the unprotonated form while the salt precipitates...CH2Cl2 or AcOEt might already be to apolar to dissolve the cytidine. As already mentioned above, I would also suggest to dissolve or suspend the HCl salt in DMF, NMP, DME or something like that and deprotonate the compound in situ using TEA or DIPEA. If you have to get rid of the proton I would add equimolar amounts of NaH as the base. This will deprotonate your hydrochloride and leave only NaCl. And I would assume that NaCl is compatible with most reactions.

I don't think extraction will work either, but reverse phase HPLC or ion exchange would work nicely. I have used Dowx resin in the past to make adenosine analogues and we used dry ice to make probably triethylammonium carbonate, ie something that can be rotovapped away. One can probably buy the triethylammonium carbonate, already made today, or some other carbonate/amine. For example, even pyridine carbonate would work. Just take amine, dissolve it in water, and put dry ice in a erlenmyer with a stopper on top, and attach a tube with a frit to where the vacuum attachment is on the and put the frit in the container with the water/ amine. Check the pH and then sti

On the other hand, if someone already has a reverse phase column and HPLC, then Andre's method would also work. Sodium hydride seems extreme to me. Could it reduce the cytidine as well?

Stop adding CO2 when you have the desired pH, ie around 7. Equilibrate the column with low concentrations of buffer, ie 10mM, and then wash with 10mM. Then elute off with high concentrations of buffer, ie 0.5 M. One can even buy spin columns today with ion exchange resins, and do this in five minutes.

Sorry about the typing. iPads not the easiest thing to use.

NaH is not that extreme and this procedure has already been applied for a similar problem http://dx.doi.org/10.1016/j.tetlet.2008.07.090.

Thank you for the clarification Daniel.

Try this:

Dissolve your compound in aqueos ammonia to neutralize the HCl salt and then pass the solution through an SPE reverse phase column or cartidge, like Waters Sep-Pak c-18 cartridge (for small amounts) or PoraPak RXn rp (for higher quantities). Wash the cartridge or column with an small amount of diluted ammonia solution and then elute your basic compound with methanol (or other suitable organic solvent, e.g. ethanol). If water is undesirable in your reaction, dry with MgSO4, filter and remove solvent (and ammonia) under reduced pressure.

Dissolve your salt in aq. medium, add water immiscible solvent in which cytidine base should dissolve. Add appropriate base and make pH alkaline and separate solvents. Dry organic layer and proceed for your further work.

The most convenient procedure is probably to use the hydrochloride as is, in a polar solvent (eg. DMF), and to add triethylamine to the mixture to solubilize the salt. Of course, depending on the type of reaction, this may or may not be a problem.

If this is not an option, you will indeed need to resort to some form of reverse phase chromatography.

If you need to do a multi-step synthesis on the molecule, it may be desirable to use some sort of solid support, so that product isolation can be done by simply filtering your support out of the reaction mixture and washing with appropriate solvents.

Good luck!

I had a very similar problem when I was working with my RNA analogs some time ago. I offer you two possible solutions.

(1) Dissolve your cytidine-HCl analog in a minimal amount of water then add excess pyridine to neutralize the acid. Rotovap off the excess pyridine, then dissolve the compound in EtOAc and pass through flash chromatography using a MeOH/CH2Cl2 eluent.

(2) Consider adding ammonium bicarbonate to your aqueous cytidine-HCl analog solution. The salt is fairly volatile and thermally unstable so gentle warming while rotovaping should remove any excess salt that may linger.

Try out recrystallization from mixture isopropyl alcohol-water.

Or for obtaining of your compounds in form of free base use an application of anion-exchanger resin in OH – form. For more detailed information you can me write: [email protected].

Cryoscopic technique is thr best for removing salts from org compds

Depending on the cytidine derivative you're working with, the free base might not be sufficiently soluble in organic solvents either. This is especially true if the hydroxyl groups are unprotected. I would follow up on above suggestions if your chemistry allows it. Use DMF (or DMA) as the organic solvent in combination with Et3N (or DiPEA) to generate the free base of your cytidine derivative in situ. Typically, DMF is a decent solvent for (modified) nucleosides and its removal is still achievable under high vacuum.

Good luck!

If it is base dissolve in dry acetone. Most of the inorganic salt are insoluble in this. Just filter and concentrate the filtrate under vacuum and isolate back the pure materiel

I agree with the contributors above that in situ generation of the free base in a solvent like DMF using bases like TEA or DIPEA is the way to proceed.

The best method is extraction process. You may use water and chloroform as an extract. The water soluble organic compounds will be more stable in chloroform. Try it.

You can try to solubilise in CCl4 for org compunds

Dissolve your salt in an equimolar solution of sodium ethoxide. Rotavap, dissolve the residue in EtOAc, and you may remove NaCl washing this EtOAc solution with water.

The best method, extraction with petrolium ether, and evaporate the extract under vacuum to seaparate the product

C. Robertson

I use RO (Reverse osmosis) in ordert to desalt NOM (natural organiv matter) in surface water!

Egil Gjessing

1.electrophoresis is a solution

2. pass through a C18 prep LC column like flash chromatography to remove the salts.

You should try to dissolve your salt in organic solvent by adding triethylamine or Dipea (equimolar range) in the reactional suspension! After that, you may filter the misture in sílica (or Celite) or proceed to the next step performing in situ reaction!

Good luck!

Best regards!

My preferred method of working with similar molecules is to dissolve/suspend it in a suitable solvent(dichloromethane works well in most cases). Add 1.5 eq TEA. Stir for some time. Filter through a silica/alumina bed. Altenatively, dissolve in DMF, add organic base and use as such for next reaction.

Dissolve in water, neutralize with a slight excess of NaOH, and de-salt by passing through a mixed-bed ion exchange resin. Lyophilization will leave the free base. If it's a hydrate, you can remove residual water by azeotropic distillation with ethanol.

Another solution I use regularly is to neutralise with NaHCO3 in aqueous solution then, if the cytidine derivative has any lipophilic tendencies, absorb onto silanised silica (a relatively cheap reverse phase stationary phase) and wash the silica with water to remove all salts. Finally, desorb the desired compound from the silica with water-MeCN; the more MeCN it is soluble in the easier it is to get rid of water, but do not exceed about 90% MeCN or residual exposed SiOH will retain the product on the silica. Rotavap down the filtrate, re-evaporating two or three times to remove residual water.

First u convert corbonyl to oxime then further convert to keto

Use ammonium hydroxide to neutrilize acid (HCl) than mildly heat the product. Because by this reaction water and ammonium chloride will form and NH4Cl can easily remove via simple heating (sublimation reaction).

Rahul has the obvious solution (please forgive the pun).

Even NaOH should work. You simply collect the free base in a non-polar solvent. DCM, ether, etc. All the salts stay in the aqueous layer. We do this all the time. Basic O chem (oh dear another pun)

Actually Roberto nailed it first. Typically you would run the pH up to 11 with a base, add an organic solvent, add a saturated NaCl solution to push the organic base into the solvent and using the same solvent extract the aqueous layer a couple of more times. Since the base seems to have appreciable solubility in water DO NOT wash the organic layer with H2O but rather run it on a flash column or as Roberto said use celite or silica for the stationary medium.

You could use a basic resin, such as SAX. Dissolve the compound in methanol and pass down the SAX (or any basic resin). Evaporate the filtrate.

If cytidine is soluble in hexane or ether, you could firslty neutralize HCl using base, then concentrate mother liquid and disolve residue in hexane or ether. at low Tm, NaCl can precipitate

You can use resin.

See method here https://www.researchgate.net/publication/274779930_How_to_successfully_convert_a_water-soluble_organic_onium_salt_into_its_free_base_or_change_its_anion_by_using_an_anion-exchange_resin 

Greetings. I think that  you should be able to use sodium bicarbonate in absolute EtOH or MeOH and add dry Na2SO4, or Et3N in THF, since these reagents would give you the chlorine free organic compound soluble in the employed solvent. And the generated salts (NaCl or Et3HN+Cl-) should be removed through filtration, separating also the hydrated Na2SO4 when used. Hope this is helpful for your research. Best regards. 

There are several "Non-basic" acid scavengers to convert acid salt to free base. Epoxides are one such type. Try 1,2-epoxy-butane, propylene oxide (low boiling liquid), ethylene oxide (gas),,even epichlorohydrin. These have been used in literature, especially ipatents, provided your molecule do not interact.. You can even try stirring with barium hydroxide powder.

Best of Lucks.

Dissolve cytidineHCl in water, add in a column pack with cation exchange resin say Dowex resin. Cytidine will bind to the dowex sulphonic acid resin column. Elute with water once then with 10 to 30% aqueous ammonium hydroxide (33% strength) in methanol. To collect the solution and lyophilized it.  (see the supporting information of Nature Chemical Biology. 2007, 3, 650 – 651)

Enrico can I do same method for gemifloxacin mesylate to remove mesylate salt and how will remove byproducts

Hi Asia,

I just sent to you a message. Do you want to perform additional steps on your Gemifloxacin? Or just get it as free base?

Regards

Pass the solution through a ion exchange resin

Since your compound has a primary amine functionality, you could try the following method which I've successfully used for a number of highly water soluble aryl piperazine salts. Charge a round bottom flask with a portion of your salt, then add some 2M ammonia in methanol and stir for 20 minutes. You'll see a white precipitate form which is ammonium chloride (poorly soluble in methanol). Vac off the methanol and excess ammonia, then to the resulting solid add a small amount of cold DMSO (ammonium chloride is insoluble in DMSO at 25C and below). Filter, then to remove the DMSO run through an SCX-2 cation exchange cartridge (hence the need for an amine functional group). Vac down again and you should have your solid freebase.

You should be able to do this for non-amine containing compounds, but it will just require a strong rotary evaporator to pull of the DMSO in the final step.

As all the formula described by authors are quite impressive. which formula can be used industrially as economical as well as  short time productivity.

Please give own opinions. 

You can extract free base from your cytidine.HCl after washing with saturated solution of sodium bicarbonate...Dissolve salt form in water than add bicarbonate solution.. than add organic solvent (you can check the good solvent at small scale using apendrof)..Wash the aqueous phase several time with organic solvent..Collect it ..Dry it over Na2So4 and evaporate the solvent to get free base.

We can remove dissolved salts from water by tartarazine then dissolved in DMSO

when evaporate approximately all dissolved

could be removed

you can add butanol or dcm to make them precipitate out.

or lyophilise the mixture and after concentration you can extract your compound with other organic solvent.

I encountered the same issue with proflavine. HCl. As proflavine its self have good solubiltit in water. I tried most of suitable methods as mentioned above but they do not work. suggest some others.

You neutralize with NaOH and remove of water by vacuum distillation. Resolved in ethanol, NaCl dissolved 0% in ethanol.