[Babcock_Hall]

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?

[rolnor]

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?

[Babcock_Hall]

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.

[Babcock_Hall]

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.

[rolnor]

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.

[Babcock_Hall]

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.

[Babcock_Hall]

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.

[Babcock_Hall]

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

[rolnor]

I think you complicate matters a lot. Keep it simple, monitor the reaction with LC-MS.

[Babcock_Hall]

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.

[rolnor]

Yes, ninhydrin is usefull.

[Babcock_Hall]

The NMR spectra of the second deprotected, purified Weinreb amide look good. Our next step will be to try a small-scale coupling.

[rolnor]

Great news!

[Babcock_Hall]

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.

[rolnor]

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.