I am doing synthesis to get catechin gallate protected by benzyl chloride. After the esterification process, I am doing TLC preparative with eluent hexane:ethyl acetate to separate spots (compounds). Then I re-extract with chloroform and store it at room temperature until the extract dries. On the morning of the first day after re-extraction, I monitor the chloroform extract by TLC and there is one spot. However, in the afternoon, I check again and there are four spots. What's happened with the extract storage in chloroform?
your compound may contaminant , purify and see if improve or make GC or do NMR, change solvent system
Chloroform always contains traces of HCl and is not a good long term solvent. Better to evaporate using vacuum at low temperature and store in a freezer. You can try dichloromethane as alternative solvent.
Are you sure of the complete benzoylation of all the 7 phenolic hydroxyls present in the 3-O-galloyl-catechin ? If not, you can have intramolecular transesterification from one position to another ... explaining the multiplicity of the compounds after a while ...
If yes, then it could be some "conformers" generated around the O-heterocycle ("C2-sofa" or "C3-sofa", ...) because of the two very bulky substituents at the two vicinal positions C2 and C3 in a trans relationship! The energetic barrier between the conformations could be sufficient to behave as diastereoisomers.
I suspect you may be cleaving some of, not all of esters as a result of trace HCl found in chloroform. Try using a more inert solvent than chloroform i.e dichloromethane (not sure if your compound will dissolve). You can also store your chloroform over KOH to reduce acid formation. One way to tell for sure is to do a time course NMR with chloroform as the solvent (take a fresh small early and then take NMR every 2 hours). You observe that formation of new peaks from the partial cleavage of the protecting group.
Dear Novi, did you check the pH of your chloroform? it is very often acidic (4-5). So any PG sensitive to acid is in "danger". I would recommand NOT to leave a compound in solution in Chloroform. Do you have access to any evaporation device? evaporator?...
I fully agree with Teuns van Ree and Laurent Bornaghi. Use a vacuum evaporator and store in a freezer. Stephen Chaterpaul has also devised a good measure to track the problem. I think this will solve your problem.
I fully agree with Robert and Joseph and as a solution with Alok. Freeze it and freeze dry it.
There are a lot of possibilities, first could be chemical transformation, also that the chemical structure went into a more polar or apolar conformation, hence different r.f. in TLC, so that depending on the structure of your molecule the plausible possibilities are more evident.
Robert Gostl: i'm using benzyl chloride for synthesis from Merck with CAT number 8.01809.1000, not benzoyl chloride. Next time i'll try to store in methylene chloride.
Adam Seliman: i'm not so sure if the compound contaminated, i always wash my vial or other glass apparatus with water, then methanol and acetone, wait until dry before use it. May be change the solvent is a good idea.
Teuns van Ree: thank you for your explanations Sir, it's very helpful. I think evaporate using vacuum in low temperature is better but my sample is very tiny, and the smallest sample place in our evaporator apparatus is 100 mL. I'll try to store in dichloromethane.
Joseph Vercauteren: I'm doing synthesis catechin gallate from starting materials catechin and methyl gallate. For the first time i'm protecting catechin with benzyl chloride, with hope all the hydroxyl group of catechin (except hydroxyl attached to C3 position) will be protected. After that i'm protecting methyl gallate with benzyl chloride and also with hope all hydroxyl group from methyl gallate will be protected. Then, i'm doing hydrolisis process to change protected methyl gallate into protected gallic acid. After hydrolisis process, i'm doing esterification process, to combine protected catechin with protected gallic acid into catechin gallate using chloroform as solvent for esterification process.
Stephen Chaterpaul: Ok, Sir. I'll try to changed the solvent with dichloromethane and also try to store chloroform over KOH. May be for NMR solvent i'll use deuterium methanol. If it possible to observe that formation of new peaks from the partial cleavage using LC-MS/MS sir? I have little problems to analyze using NMR, because i must send my sample to another institution due to my lab haven't NMR apparatus.
Laurent Bornaghi: i already check the chloroform pH and its pH is 5. Thank you for your information about dangerous store PG in chloroform, it's very helpful. Yes, i have evaporator apparatus but my sample is very tiny so it's difficult for me due to the smallest sample place in evaporator is 100 mL, i haven't smaller than it.
Alok Nahata: If i could do the evaporation with my limited apparatus, Sir..
Valeriano dal Cin: Thank you for the suggestion, Sir. But, i'm not so sure if my freeze dryer can freezing chloroform.
Hiram Beltran: Thank you for your opinion, Sir. I'll study more.
As mentioned before, I think you have hydrolysis of your ester bond with acidic CHCl3. I would not try to store it over KOH though as KOH is strong enough to deprotonate chlorinated solvents (strong base and CHCl3/CH2Cl2 are not compatible...).
In general, it is not a good thing to keep chemicals in solution for a long period of time. In your case, there clearly is decomposition, so I would try to minimize the time in solution.
The idea of switching to DCM is a good one, especially because it's ebullition point is much lower than chloroform's, so it would take less time to evaporate. If you can, I would try and blow nitrogen over your extracts. This way it prevents oxygen and moisture from entering in solution with your product for a prolonged period of time. Also, try to avoid exposure to light as there's a lot of UV-sensitive groups on your molecure (7 benzyls!). Not sure if it's a part of the problem, but since you saw decomposition happening in the day-time, and none overnight, maybe it's part of the problem. But really, the cause of your problem does not matter in this case: Try to shorten the time in solution and you'll likely solve your problem.
On another note, why do you use prep-TLC to purify your product? this should be a quite clean reaction. I think I would try to triturate the compound in Ethanol/water. Benzyl chloride is a liquid, so it's not going to crash out and I don't know what your base was, but it's likely liquid or water soluble...
Robert Gostl: thanks for your suggestion. If you don't mind, please explain to me how it could be, Sir.. Thank you..
Pierre-Andre Fournier: Thanks for your information, it's very helpful. Yes, Sir.. i agree with minimizing time in solutions, for small amount of sample we can use the nitrogen to make it dry, but stock of nitrogen in our lab is limited at that time and i'm in a hurry. Oh...my apologize to inform that I already doing partition chloroform-water 3 times after esterification and take the chloroform phase (because the compound is in the chloroform phase), adding Magnesium sulfate anhydrous, filtering and evaporating in evaporator. And then doing prep-TLC because i think it's the quickest way to get the compund to analyze.. Please correct me if i'm wrong. Thank you very much.
What esterification do you mention? If you use catechin gallate as starting material, there is no esterification stage...
However, if you are sure in the structure of Bn- protected catechin gallate, the only reason for this problem can be the acidic hydrolysis of 1 ester bond...
I also would not suggest using KOH in chlorofom to reduce it's acidity - very reactive dichlorocarbenes can be formed..
To advice something - is it possible to publish the synthetic procedure you use?
Thank you for your suggestion, Sir.
As I mentioned above, esterification between protected catechin and protected gallic acid. I use starting material catechin and methyl gallate. Please find synthesis procedure attached along this comment...
Thank you, Novi,
Do you have any analysis of compounds A, C and D? Are you sure in their structures?
Possibly you can provide the NMR FID files?
In practice, a mixture products benzylation are obtained, not a single product as mentioned in the procedure. A mixture product of benzylation are further separated using column chromatographic. From the NMR spectrum known that hydroxyl groups of catechin aren't fully protected.
What is your suggest about this result?
Are the reagent not suitable?
Or the time of synthesis should be longer? (i did approx. 70 hours for catechin benzylation and approx 40 hours for methyl gallate benzylation).
Whether it is normal condition considering the bulky structure?
Thank you very much.
I find your conditions alright, although I would heat it up a bit. For the methyl gallate, I would heat it a bit (maybe 70°C, 18h rxn time should be enough) with at least 3.5 equivalents of BnCl, 4 eq. of K2CO3 and a small amount of potassium iodide (forms a benzyl iodide in situ, better electrophile). I would use potassium carbonate instead of sodium since it is more soluble in DMF. If you have some, you can add a catalytic amount of cesium carbonate too since Cs-alkoxide are even more soluble. If you don't have full conversion to the desired product,make sure you still have benzyl chloride/iodide in there since some water present in the reaction can cause it to decompose. If there is no more BnCl, add another equivalent since nothing wrong can happen in this case. Once all product is converted to the desired one, I would add about 4x the volume of DMF you had (if 100mL of DMF, I would add 400 mL water). I bet you a white precipitate will form: filter it and wash with water.
For the catechin benzylation, I would do the same (70°C, 18h, 4.2 eq. BnCl, KI, K2CO3). The only difference is that you cannot push it as hard because of the secondary alcohol.
I think there is no reaction with chloroform. The molecule could be light sensitive and get oxidized in presence of oxygen (dissolved oxygen !!). Your prepared ester itself may be existing as a pair of enantiomers. The quinonoid structure that could arise from the oxidation product could be coloured and the separation into the two more enantiomers may be responsible for the appearance of more spots. Try to avoid any dissolved oxygen and the results could improve.
Many of the respondents have misunderstood the question. Questioner is asking that the already preparated gallate ester is now being protected by benzylation using benzyl halide.
You are not right, Narayan. Indeed, the question has changed from the very beginning. Now, Novi is asking about the loss of purity of the ester (gallate) prepared as shown in her own scheme (using coupling agent, such as DCC), from perbenzylated (on the phenolic functions) catechin and gallic acid.
Anyway, none of the enantiomers you supposed is able to explain the 4 spots appearing on TLC after a while. TLC are "classical" support (not stated to be chiral) and therefore, cannot differentiate enantiomers.
Completely agree with Joseph. Enantiomers can not cause different spots on TLC unless you use a special support.
Dear Novi, it seems that your first problem is that most of your benzylations are not complete. Follow Pierre-Andre advices, it should solve that problem.
Pierre-Andre Fournier: thank you for your great suggestions, Sir. I'll try to do your guidance.
Narayan Garimella: Thank you for your suggestions, Sir. But from a journal that i had read, known that products of this synthesis should be rasemic because of the partial benzylations. And I already did separation by prep-TLC and take 1 major spot, then re-extracted with chloroform, put it in the vial and exposure it in room temperature until dry, but after a while I do TLC control, the major spot comes into 4 spots. I agree with Joseph and Leonid that enantiomers of compound can't appears as different spots on TLC.
Joseph Vercauteren & Leonid Voskressensky: I do agree, Sir. Thank you for the suggestions.
Laurent Bornaghi: Ok, Sir. I'll try to follow the guidance. Thank you.
The structure of the major compound isolated by Prep. TLC may be provided for further discussion.
May I know the colour ( brown probably )of the impurities that are developing after exposoure etc.
Many of these poly-phenols are both air and light sensitive. It may be decomposing. Also the spots on TLC may not necessarily be representative of the amount of material present. The new spots may be just a small amount of the total mass.
I agree with Mr William Harter about the sensitivity of Polyphenols specially in the cases of catechins which are light sensitive.are u sure that u have obtained Catechin gallate in chloroform coz these compounds are mostly not eluted by chloroform and remain soluble in more polar solvent like methanol.
the reason of your identification of more spots is the concentration of spot on the plate.sometimes when the concentration is low the other spots are not visible and with the increased concentration the spots appear.
you can dry the obtained fraction and make a mg/ml solution in methanol to be sure about is and HPLC analysis can be done using that.
The appearance of four spots (partial esterified products) may not be new, they were actually present in the original mixture which were not visible in the first attempt while doing TLC prep as dilute solution was used. They reappeared as the so called separated product as mixture of four spots because this time the concentrated solution was used for TLC.
The additional spots development can be due to :
1.Incomplete esterification ( benzoylation of polyhydroxy phenols /catechins need to be done with care....pl.check that the reaction is done in totally moisture free atmosphere and continuously monitor the reaction mixture by TLC for completion of esterification)
2.Ensure that the CHCl3 used for extraction is free from moisture/or may be try to extract with other solvents like Hexane to get single ester with out degraded products
.
- Badges
- Science topic