Sauron
International Hazard
Posts: 5351
Registered: 22-12-2006
Location: Barad-Dur, Mordor
Member Is Offline
Mood: metastable
|
|
Enzymatic resolution of Racemic Norleucine
I am preparing racemic Norleucine (an unnatural amino acid) and need to subsequently resolve it into the l-isomer.
There are basically three possible ways to do this:
1. combine the racemate with an isomer of an optically active alkaloid. Seperate the two isomers by repeated crytallizations. Laborious and tedious
and the alkaloids of choice can be expensive.
2. Chiral preparative HPLC chromatography. Beyond my skills and budget.
3. Enzymatic resolution. My choice.
The enzyme is papain, and the principle is as follows:
a. Prepare the anilide of the Nle racemate.
b. Activate the papain.
c. React the substrate with the racemate anilide. Only the l-isomer will be hydrolyzed and thus can be readily seperated from the d-Nle anilide.
This is a literature procedure from J.Biol.Chem. I can dig up the reference and the full text if anyone is interested. That journal is free online,
the life sciences boys apparently have a different ethic than the rest of us, very refreshing.
I am soliciting comments, advice, suggestions on this procedure.
l-Nle is not commonly available commercially and when it is tends to be very expensive.
The synthetic part is very straightforward. Caproic acid is alpha-brominated by one of several means, and the product is reacted with concentrated
ammonium hydroxide to form the alpha-amino acid. All that is a cakewalk. The fun is in the resolution of the isomers.
I will use the l-Nle in a peptide synthesis:
Ac-Nle-cyclo(Asp-His-D-Phe-Arg-Trp-Lys)-OH
Bremelanotide, aka PT-141
All the other AAs are OTC. As usual the decisionmaking re protection strategy and tactics is tricky and the final cyclization is not very efficient.
The cyclizing agent is DPPA (diphenyl phosporyl azide) which is prepared from the corresponding chloride (available) and NaN3.
I am using classical methods, rather than SPPS as I want to obtain more than a smear at the bottom of a sample vial. I am setting up a pair of Waters
PrepLC 4000 HPLC systems with 47mm x 300 mm radial compression modules for RP to purify the product. These systems can handle multiple grams per
injection. I am running Millenium32 V3.2 chromatography manager on a busLAC/E card under Windows to control each system. What fun.
I do method development and analysis on a Waters 600 using 8mm RCM colums from which I can scale directly to 25, 40 and 47mm prep columns
[Edited on 26-12-2006 by Sauron]
[Edited on 26-12-2006 by Sauron]
|
|
|
quino
Hazard to Self
Posts: 91
Registered: 19-11-2006
Member Is Offline
Mood: No Mood
|
|
http://www.jbc.org/cgi/reprint/182/2/451
J. Biol. Chem. Greenstein et al. 182 (2): 451. (395K)
|
|
|
Sauron
International Hazard
Posts: 5351
Registered: 22-12-2006
Location: Barad-Dur, Mordor
Member Is Offline
Mood: metastable
|
|
Thanks, I have that article but now everyone can have it and I dont have to bother to post it.
It is nice that JBC gives their content away while the ACS makes me pay $25 per journal article.
|
|
|
![[*]](images/xpblue/default_icon.gif){kind=icon}
