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Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: Babcock_Hall on February 24, 2022, 06:13:28 PM

Title: FMOC deprotection and work-up
Post by: Babcock_Hall on February 24, 2022, 06:13:28 PM
Our functional groups are tertiary-butylester, vinyl sulfone and FMOC-protecting group on nitrogen.  Our goal is to remove the FMOC group and then couple with an amino acid, derivative as the N-hydroxysuccinimide ester.  We plan to use diethyl amine to remove the FMOC group, which two groups have used in the presence of a vinyl sulfone.*  One of the groups purified by HPLC but did not provide the column, the loading, or the solvent.  The other group removed the volatiles but did not do any further purification before moving on to a coupling reaction.  I have to wonder whether or not the fulvene adduct with diethyl amine will be volatile enough to remove.  I don't have any experience with purifying amines over silica, but I have read that sometimes triethylamine is used in the solvent.  Does anyone have any suggestions on the best way to proceed?
*there is one paper that indicated when an electron-withdrawing group is bound to the sulfur of the sulfone, that addition into the vinyl group can happen.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on February 25, 2022, 04:55:55 AM
No, the fulvene is to large. Yes 1-5% TEA in the eluent is usefull. Pack the column without the TEA. Is there a risk of 1,4-addition of diethyl amine to the vinylsulphone? Maybe use tertiary amine instead? DBU?
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on February 25, 2022, 10:06:34 AM
I have also heard that dibenzofulvene can polymerize; therefore, one should not let the mixture sit around indefinitely.  I think that the addition is more of a problem when one has a compound with a S-pyridyl group.
EDT
I have also heard that toluene can help with the removal of the remaining diethyl amine in rotary evaporation.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on February 25, 2022, 02:30:49 PM
Diethyl amine has low bp. No problem. If you use cromatoghraphy these things solve themselves.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 01, 2022, 02:58:48 PM
We used diethyl amine and followed by TLC.  Then we removed the volatiles.  The solvent we were going to use to move dibenzofulvene through the silica column is 40/60 ethyl acetate and hexanes.  The product is insoluble in it.  The product can be suspended in DCM, but I would not call it a true solution.  Any thoughts about the best way to proceed?
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 01, 2022, 03:25:24 PM
Use DCM/MeOH
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 01, 2022, 08:44:52 PM
When we used TLC, we initially thought that 99:1 EtOAc/TEA was sufficient to move the product.  DCM/MeOH was more successful; thank you.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 03, 2022, 03:09:14 PM
Is there any reason why chloroform might work better than DCM in terms of solubilizing the amine?  I have run columns to purify phosphonates in both solvents previously.
Title: Re: FMOC deprotection and work-up
Post by: wildfyr on March 04, 2022, 12:07:02 PM
No, they are quite similar, though DCM is a bit more polar. I seldom have met small molecules where it dissolved in one but not the other.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 04, 2022, 01:17:34 PM
You have EtOH as stabilizer in chloroform, this can be a problem if to polar. I think they are similar as wildfyr says. Maybe you need to test this in small scale.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 16, 2022, 10:02:08 AM
From the silica column we obtained two small pools of products, neither one of which is our compound of interest.  We cannot account for all of the mass that we put onto the column.  Back to the drawing board.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 16, 2022, 05:05:45 PM
Do you see any ethyl groups that could come from 1,4-addition? Do you see double bond?
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 16, 2022, 05:31:26 PM
I did not see a double bond.  I also did not see the CH3SO2 group.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 16, 2022, 11:58:44 PM
Maybe you did get 1,4-addition and this product is so polar that it did not elute.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 17, 2022, 05:27:47 AM
I suggest you try 1eq. DBU att 0°C. Its a poor nuchleophile and strong base. Also run in a NMR-tube, you will get signals from DBU but the double-bond should be visible and the deprotection possible to monitor.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 17, 2022, 09:26:53 AM
We tried flushing the column with 5% methanol, 1% TEA, and 94% DCM, but I don't know the results yet.  Two other groups deprotected FMOC in the presence of a vinyl sulfone; that does not preclude our getting addition, but that is why we chose the route that we did.  Also even if we did get addition, the S-methyl group should still be somewhere.
EDT
One group purified using RP-HPLC at this stage in their synthesis and obtained 23 mg in 54% yield.  Our scale is larger, and we do not have a column.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 17, 2022, 09:55:44 AM
Yes, something else is happening. Try DBU is my suggestion. Dont use excess.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 17, 2022, 02:22:23 PM
We used up all of our material.  However, we can use a small portion of the corresponding Weinreb amide as a model and deprotect that, if we just need conditions that will move the deprotected product chromatographically.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 18, 2022, 01:20:48 AM
OK.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 21, 2022, 06:19:08 PM
We double-checked the H-1 NMR of the first fraction, the one that eluted in 40:60 EtOAc/hexanes.  This looks like our product in low yield, although it contains two almost equal populations of two similar things, plus impurities.  I don't have an explanation for the existence of two closely related molecules, in that both substances seem to have a double bond in the trans-configuration.  Another puzzle is why it eluted relatively early.  We will continue to think about it.

We could synthesize the dipeptide by deprotecting FMOC at the stage of having a Weinreb amide (not the route that we first tried), which makes a small-scale deprotection marginally more interesting.  However, then we face the question of how best to perform the reduction in the presence of the second residue and the peptide bond joining it (a solvable problem, I am sure).
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 22, 2022, 03:44:39 PM
They can not be amines then, they are far to non-polar. Even with pure EtOAc they would move slowly on silica. How do they look on TLC with Ninhydrine?
If you can get LC-MS running you would be much better off, maybe you can use another departments resources? You can save lot of time with that.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 22, 2022, 04:18:42 PM
I just now looked at the alpha-CH signal, and it is the only signal for which there is a large change in chemical shift, about 0.7 ppm difference between the two forms.  In contrast, the two H3C-S signals are very similar in chemical shift; the two tertiary-butyl ester signals are very similar in chemical shift, and the two vinyl hydrogens are fairly close to each other.  I am starting to think that almost half of the molecule is in the form of an adduct on nitrogen.  Our solvent was acetonitrile, and diethyl amine was of course also present.  However, I do not see any extraneous methyl groups by H-1 NMR.  LC/MS is starting to look appealing.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 23, 2022, 05:48:52 AM
Maybe one of the compounds are a product of aminolysis of the ester? That would give different alpha-proton shift. So you have a diethylamide of half of the material. Or as you suggest, a ring-closed lactam.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 23, 2022, 06:30:45 AM
You could avoid this by using Boc on the amine and a different group on the carboxyl. This way you get the amine as TFA-salt and it does not attack the ester.
I am not sure what group is best on the carboxyl, maybe a 2-nitro benzyl, this is deprotected by reduction, it forms a lactam when it cleaves off. Reduction can be made by many reagents, iron/acetic acid is one. Sodium sulfide is also used I think. I beleive you can find neutral conditions that does not affect the Boc or double-bond.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 23, 2022, 09:09:02 AM
I would have to use reduction deprotection conditions that did not affect a vinyl sulfone.  Two groups were able to remove the FMOC group in the presence of a vinyl sulfone; I think that we lack experience.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 23, 2022, 01:00:26 PM
Yes, I know, but you need to avoid free amine as base, att least for prolonged time. If it can form a 5 or 6-membered lactam you have trouble. Its not surprising that you have some issues, you have many functional groups, its not like the textbook anymore, a lot can happen. You can probably find reduction conditions that not affect the vinylsulfone but it takes some work.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 23, 2022, 02:20:33 PM
The tertiary-butylester is still intact, as judged by integration of the H-1 NMR signals, but there are two populations.  A hypothesis consistent with the present information is that there is an adduct on nitrogen, but not one that gives rise to observable H-1 NMR signals.  We will do a model deprotection next, but we will check the H-1 NMR prior to silica gel chromatography.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 24, 2022, 01:09:53 AM
That sounds non-logic, where is nitrogen coming from? Must be from diethyl amine or the amino acid nitrogen. If it has not added to the double bond or has attacked the ester its hard to understand.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on March 28, 2022, 08:44:14 AM
There are two populations of many signals in the H-1 NMR, and one has to explain this in some fashion.  I was not trying to propose any specific chemistry, which I admit would be difficult to do.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on March 28, 2022, 09:14:53 AM
OK. So pure material is the way to go then to solve the NMR-mystery. Sometimes its really hard to interpret NMR, when you have a mixture it can be very confusing.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 04, 2022, 08:25:42 AM
Yes, something else is happening. Try DBU is my suggestion. Dont use excess.
DBU will probably not form an adduct with the dibenzofulvene.  Is there an advantage to not forming this adduct?
Title: Re: FMOC deprotection and work-up
Post by: rolnor on April 04, 2022, 04:31:40 PM
I dont know. But with DBU you dont need excess and the reaction is probably fast at low temp. DBU can not add to the double-bond or attack the ester. I guess you had diethyl amine in large excess?
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 07, 2022, 03:58:39 PM
I wanted to come back to the H-1 NMR of the deprotection once more.  There were two populations of signals for the S-CH= vinyl hydrogen, the tertiary-butyl ester, and the HC(alpha).  Of these, only HC(alpha) showed a big difference in chemical shifts.  Only one of the two signals was around 3.5 ppm, which is where I would expect it to be for a free amine, based on Kurt Wuthrich's 1975 table of chemical shifts of amino acids and tetra peptides.  The other was roughly 0.7 ppm downfield from the more upfield signal.  I don't have a good candidate for what group might be present on nitrogen to cause this downfield shift.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 07, 2022, 04:04:22 PM
I started a SciFinder survey of reaction conditions in which the FMOC group was removed in the presence of a tertiary-butylester.  I was surprised at the number of times someone said that the material was used without further purification for the next step of the synthesis.  I plan to continue this exercise until I get sick of it.  On the one hand, I don't like the idea not purifying on general principles; on the other, I don't want to ignore the experience of other workers.  I would presently rank the alternatives (lowest being the most preferred) as 1. Silica in the presence of TEA.  2 or 3.  No purification at this step.  2 or 3. Basic alumina.  4. solid phase extraction or other reverse phase techniques.  5. Amine-conjugated silica.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on April 08, 2022, 12:23:17 AM
If the reaction work reasonable well, say 75%yield, then there will be no problem with assigning NMR of the crude material and you will fell less need for purification. As for now, something is wrong. To use the chemistry you have now and just push it through all the steps without chromatography seems not very satisfying. It could work but I think that you need to spend more time on the first step and use LC-MS. The problem can be easy to fix. I would not spend to much time assigning NMR-signals on a mixture, this can be very confusing. You can run LC-MS on the NMR-sample as a start.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 16, 2022, 10:24:06 AM
We were able to purify the smaller portion of the amine bearing the Weinreb amide and an ester-protected carboxylic acid using silica and 1% TEA.  So far, we have not seen the problem of having two populations by NMR (I ordered some basic alumina to have as a possible future alternative).  We plan to use this material to test out coupling conditions.  We will also purify our larger sample of amine.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 20, 2022, 10:58:47 AM
I found a copy of Chapter 2, by Gregg B. Fields that deals with FMOC chemistry (Methods in Molecular Biology, Volume 35).  There is a paragraph dealing with removal of the FMOC group in solution, and it had a couple of interesting ideas.  One is to use a reagent that forms an adduct with dibenzofulvene that can be extracted into pH 5.5 phosphate buffer:  "Such an adduct is obtained when either 4-(aminomethyl)piperidine (44) or tris(2-aminoethyl)amine is used for Fmoc removal (IO)."  There are two related problems that I can foresee.  One is that the free amine will have some water solubility, and the other is that at this pH, the amine will protonate.  I have not yet tracked down the references that he gives.

The other idea looks slightly more attractive, which is to use a polymeric amine to capture dibenzofulvene and filter it away.  "Polymeric-bound amines, such as piperazine-PS (2.4 mEq/g) (45) and a copolymer of styrene, 2,4,5-trichlorophenyl acrylate, and N,N’-dimethyl-N,N’-bisacryloylhexamethylene diamine, with subsequent replacement of activated ester groups by l-(2aminoethyl)piperazine (3.3 mEq/g) (46), also efficiently remove the Fmoc group in solution-phase syntheses. The use of polymeric-bound amines allows for the isolation of the free amino component by simple filtration of the resin, since the polymer traps the dibenzofulvene (45,46)."

We tried to run a column on a deprotected free amine yesterday, and there was a great deal of precipitate.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 20, 2022, 05:37:03 PM
In our most recent deprotection, there was a great deal of white precipitate that sat on top of the silica column, but we have not finished evaluating the experiment; therefore, I cannot say whether or not the precipitate is associated with a good or bad result.  The price of polystyrene-piperazine is moderately high, but not prohibitive.  I like the idea of resin-supported piperazine in some respects, but possibly a cheaper alternative would be to use centrifugation in Corex glass centrifugation tubes if the precipitate forms and if one is certain that all of the desired product is in solution.  Corex tubes can be spun more quickly than simple glass test tubes.

Here are the four references within the Chapter written by Fields, from my previous message:

10. Carpino, L. A., Sadat-Aalaee, D., and Beyermann, M. (1990) Tris(2-ammoethyl) amine as a substitute for 4-(aminomethyl)piperidine in the FMOC/polyamine approach to rapid peptide synthesis. J. Org. Chem. 55, 1673-1675.  https://pubs.acs.org/doi/10.1021/jo00292a050

44. Beyermann, M, Bienert, M., Niedrich, H., Carpino, L. A., and Sadat-Aalaee, D (1990) Rapid continuous peptide synthesis via FMOC amino acid chloride coupling and 4-(aminomethyl)piperidine deblocking. J. Org. Chem. 55,721-728.  https://pubs.acs.org/doi/abs/10.1021/jo00289a056

45. Carpino, L. A. and Williams, J. R. (1978) Polymeric de-blocking agents for the
fluoren-9-ylmethoxycarbonyl (FMOC) amino-protecting group. J. Chem Soc, Chem. Communications 450, 451.  DOI   https://doi.org/10.1039/C39780000450

46 Arshady, R., Atherton, E., and Sheppard, R. C. (1979) Basic polymers for the cleavage
of fluorenylmethoxycarbonyl ammo-protecting groups n peptide synthesis Tetrahedron Lett., 1521-1524.

Supra Sciences makes a couple of solid phase versions of piperazine.  http://suprasciences.com/wp-content/uploads/2014/07/Piperazine1.pdf
Title: Re: FMOC deprotection and work-up
Post by: rolnor on April 21, 2022, 05:15:58 AM
I think you complicate matters a lot. Keep it simple, monitor the reaction with LC-MS.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 21, 2022, 02:29:04 PM
Centrifugation using Corex tubes is something with which we have experience.  Also, we are augmenting detection by PMA staining with detection using ninhydrin, and it is helpful.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on April 22, 2022, 12:53:13 AM
Yes, ninhydrin is usefull.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 26, 2022, 01:23:06 PM
The NMR spectra of the second deprotected, purified Weinreb amide look good. Our next step will be to try a small-scale coupling.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on April 27, 2022, 02:00:38 AM
Great news!
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on April 30, 2022, 02:43:54 PM
On another discussion board it was suggested to use 4-methylpiperidine.  It should react in a similar way to piperidine, but it is not a potential precursor to controlled substances.  It also came up that piperidine is better at forming an adduct with dibenzofulvene than dimethylamine is.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on April 30, 2022, 05:16:17 PM
I think its a bit silly that piperidine is controlled, then methyl amine should also be controlled, a lot of compounds should be. But the idéa is good, Use 4-methyl instead.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on June 30, 2022, 10:08:20 AM
I reread this thread and one I started at ChemPros.  I think that we will try 4-methylpiperidine in either DMF or acetonitrile and try to remove the adduct via silica gel chromatography in the presence of 1% TEA.  If that gives problems, we will try DBU, as per Rolnor's suggestion.  One person at ChemPros also suggested DBU, and he or she also said that he went ahead with his or her coupling without purifying.  I see advantages and disadvantages to the no-purification approach.  After our first try (which did not go so well), we have had some success purifying at the amine stage; therefore, I am reluctant to change at the moment.  That having been said, I also contacted a group that made a similar vinyl sulfone dipeptide, and they did not purify after removing FMOC.  There are many roads to the top of the mountain...

I tried cleaning some glassware with a white precipitate that might have been the polymer, and it was very difficult.  The white mass wanted to adhere to the glass.  Just an anecdote.
Title: Re: FMOC deprotection and work-up
Post by: Babcock_Hall on June 30, 2022, 03:43:21 PM
We tried 4-methylpiperidine in DMF, and we removed much of the white precipitate using centrifugation in Corex tubes.  Most of the DMF and 4-MP came off via rotary evaporation; we recently bought a DryFast Ultra pump which is capable of higher vacuum than our other pump (a colleague pointed out that if the rotary evaporator is leaky, then some of this potentially higher vacuum will not be realized).  We are using high vacuum now.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on June 30, 2022, 05:08:39 PM
Great, we often put the products on the freezedryer over night. Thats good vacuum.
Title: Re: FMOC deprotection and work-up
Post by: rolnor on July 01, 2022, 07:57:58 AM
You can allså heat gently with a hair dryer+vacuum to remove traces further.