Millie,
I completely agree with Steve, but I'll add 3 bits of personal observation.
First, in my hands, HATU (or TFFH) is almost never better than HBTU. Those
rare cases include very difficult couplings, especially with unusual AA and
synthons that really aren't even AA.
That said, coupling the unusual AA biocytin (and its eCA extended cousins,
such as Lys[eCA-biotin]-OH) onto a linker is really tough. I definitely
recommend that you couple overnight. I also definitely suggest double
coupling. And I even recommend gentle heat. 40-50 degrees won't hurt, as
long as you have HOBt included to suppress racemization. And my guess is
that you don't really care if your C-terminal AA racemizes, anyway, since
it's probably just a tag.
Finally, when you're starting with a hindered C-terminal AA, it helps to use
a solid phase with a low substitution level (<0.2 mmol/g for resins). Your
usual supplier can make them for you as a special order, if they're not
offered in the catalog. Ask them.
Whenever I have to make a peptide with biocytin at the C-ter, I do all of
these things, and I still expect bad results. I just suck up my pride, take
the hit in yield, and plan to purify the product. At least the deletion
products usually separate out pretty well. (They run early in RPHPLC, as
you'd expect.)
Best of luck!
Mark
-----Original Message-----
From: Steven Johnson [mailto:labswine@yahoo.com]
Sent: Wednesday, January 24, 2001 8:49 PM
To: Recipients of ABRF List
Subject: Re: HATU vs HBTU
Sure Millie, you could do that...but, I'd also add an
equivalent amount (with respect to the AA
stoichiometry) HOBt to minimize the potential of
racimization.
Something else you might try is to add the
AA/Activators to the deprotected resin in DMF/DCM
(9:1), then let shake for about 5 minutes, THEN, add
your DIEA. Other things you might try are
TFFH...makes the acid flouride in-situ, even a double
coupling may be better using your first initial
conditions than trying a 'hotter' reagent that might
cause you problems down the road. I trust you are
doing a Kaiser test (ninhydrin) for completeness of
coupling prior to moving on (absence of blueness).
My $0.02 ($0.03 Canadian)
Regards,
Steven Johnson, B.S. Chem.
Research Associate
Process Development, Chemistry
Biomeasure, Inc. Milford, MA, USA
--- mcada002@mc.duke.edu wrote:
>
>
> Anyone having experience with both activators:
>
> Please respond per your experience in terms of
> completeness of coupling. I
> understand that HATU is "faster" in coupling than
> HBTU. But...is it better in
> that you get a higher percentage of sites coupled
> with the amino acid in
> question.
>
> I have experienced incomplete coupling of the first
> amino acid onto
> Fmoc-L-Lys-(e-BiotinLC)0OH to Rink amide MBHA resin.
> This is the first time I
> have used this resin and I am told the coupling may
> be difficult. So, I'm
> thinking I should couple the first amino acid onto
> the resin overnite. And I'm
> wondering if I would be better off using HATU
> instead of HBTU?
>
> Thanks in advance,
>
> Millie McAdams
> HHMI Biopolymer Facility @ DUMC
>
>
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