Marcus,
I agree with everything that Steven Johnson wrote. It certainly helps a
great deal to reduce the volume of TFA before ether precipitation.
Also, if you just want to do an aqueous work-up, and you used only water for
scavenger, then definitely just dilute the TFA to about 1% in water and
lyophilize it. Don't worry if the peptide precipitates when you add water.
You'll have to get used to that.
If you really need a pellet, then you might indeed try adding hexane (some
people like to use "Petroleum Ether," which is actually a mixture of light
alkanes). TFA is miscible with almost everything.
But extremely hydrophobic peptides (especially short ones!) are known to
resist all attempts at precipitation. For those evil exceptions, desalting
via RPHPLC is appropriate. Just rotovap off all the ether you added, dilute
in water to about 0.3-1.0 mg/ml, if necessary add acetonitrile until it
dissolves (noting the final concentration of acetonitrile), load the whole
thing onto prep HPLC, and elute with a gradient as usual.
I note that you run a service laboratory. This might be a good time (if not
slightly too late) to discuss applications with your customer, especially if
it's a biologist. Anything that won't precipitate in ether is not likely to
be soluble in most aqueous solutions. Eventually you'll learn to recognize
such sequences before you make them, and discuss alternatives with your
clients. They'll learn to appreciate your expert input, and you can ask
them include you as a co-author.
Of course, just because you see 4- and 5-mer oligomers via MALDI doesn't
mean that they will be present in any given solvent system. But that may
not matter to you.
As for making Leu10, good luck! Most people find that practically any
homopolymeric peptide over about 5 or 6 amino acids in length is a
challenge. But I'd go ahead and try it if I were you. One technique that
I've used successfully in similar projects is to assemble the 10mer, and
then cleave just a small portion of resin. If MS says that your major
product is only 8-or 9-mer, then couple another Leu or two until the major
product is 10-mer. Then cleave it and deal with all the truncations in your
purification. Of course you must expect Leu10 to be twice as difficult to
precipitate in ether and dissolve in aqueous solution as Leu5.
Mark
-- . . . . . Dr. John Mark Carter MCarter@AxCellBio.com . AxCell Biosciences Corporation 826 Newtown-Yardley Road, Suite 100 Newtown, PA 18940-1721 office 267.757.1223 lab 267.757.1230 FAX 267.757.1301 www.AxCellBio.com . . Axcell Biosciences is a wholly owned subsidiary of the Cytogen Corporation. . . . . . "Ohne Analyse, keine Synthese." [Without analysis, no synthesis.] -Friedrich Engels, Anti-D¸hring, 1878-----Original Message----- From: Marcus Macht [mailto:Marcus.Macht@uni-koeln.de] Sent: Friday, October 06, 2000 7:38 AM To: Recipients of ABRF List Subject: PEPSYN: precipitation of (Leu)5?
Dear colleagues,
We are currently confronted with a precipitation problem in peptide synthesis. For a customer we synthesized a Leu-pentamer. After cleavage from the resin I acquired a MALDI spectrum of the raw product which showed a mixture of the tetra- and pentamer (approx. 1:1). When we tried to precipitate the peptide with diethylether after cleavage nothing happend. We still have a nice clear solution even after storing it at -80ƒC over night. Does anybody of you have an idea about how to precipitate the peptide? Would eventually addition of something more hydrophobic (e.g. pentane or hexane) help or will this lead to a phase separation between the organic phase and the TFA? Has anybody of you already tried to synthesize this sequence and how did you circumvent the problem? Since the synthesis itself of the sequence worked better than I expected, we will also try to sequence (Leu)10 for the same customer. Does anybody have any experience with this sequence as well? Should we maybe try to synthesize a pentamer, cleave of halve of it, protect the free peptide with Fmoc-OSu and use this as building block to finish the decamer?
Any hints are greatly appreciated! Thanks in advance.
Marcus Macht
******************************************************** Dr. Marcus Macht University of Cologne Centre for molecular medicine - Service laboratory Joseph-Stelzmann-Str. 52 50931 Cologne, Germany Tel.: +49 221 478-6995 Fax: +49 221 478-6977 e-mail: Marcus.Macht@uni-koeln.de **************************************************************************
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