07-08-04 17:51
No 518285
Haven't seen this posted yet and only two hits for R-PAC in TFSE®
Also ScottyDog check this link.
http://www.swsbm.com/Abstracts/Ephedra-A
I went to reply to your request in another thread and the post was deleted.
I dug up more links in addition...just hollar!
Review of Enhanced Production of R-Phenylacetylcarbinol (R-PAC) Through Enzymatic Biotransformation
University of New South Wales
School of Biotechnology and Biomolecular Sciences
Sydney, NSW 2052, Australia
The pharmaceutical intermediate R-Phenylacetylcarbinol (R-PAC), used in the manufacture of the decongestants ephedrine and pseudo-ephedrine can be produced enzymatically by the condensation of benzaldehyde and decarboxylated pyruvate using partially purified pyruvate decarboxylase (PDC). The traditional yeast-based fermentation method of production involves concentrations of 10-22 g/l R-PAC and yields of 55-65% on benzaldehyde have been reported with significant added benzaldehyde converted to benzyl alcohol due to the presence of alcohol dehydrogenase (ADH) or other oxido-reductase activity in the yeast (Rogers et al, 1997).
An enzymatic process based on partially purified PDC overcomes this problem of benzyl alcohol production. Up to 28 g/l R-PAC with improved yields have been reported by our group for an enzyme-based process using partially purified Candida utilis PDC (Shin and Rogers, 1996).
Screening of PDC from 14 ethanol producing filamentous fungi has also demonstrated their potential for R-PAC production( Rosche et al, 2001). Under optimised conditions of improved buffering and enzyme stabilization, partially purified Rhizopus javanicus and C. utilis PDC both produced up to 50 g/l R-PAC with good yields (Rosche et al, 2002a).
Based on knowledge of optimised biotransformation conditions and inactivating and inhibitory influences of substrates and products, an aqueous/organic two-phase reaction system was designed to maximize R-PAC production. The toxic substrate benzaldehyde which partitioned away from the enzyme into the organic phase (octanol) was delivered continuously in soluble concentrations to the aqueous phase containing pyruvate and PDC. R-PAC and by-products acetoin and acetaldehyde were continuously extracted into the organic phase. Under optimised conditions 141 g/l R-PAC was achieved in the organic phase with additional 19 g/l formed in the aqueous phase in 50 hours using Candida utilis PDC. Molar yields on consumed benzaldehyde and pyruvate were 90% and 73% respectively. The results demonstrate the potential of two-phase enzymatic biotransformation processes to increase product concentrations and enzyme efficiencies (product / U enzyme ) (Rosche et al, 2002b).
P.L. Rogers, H.S. Shin, B.Wang, Adv. Biochem. Eng. 1997, 56, 33-59
H.S. Shin, P.L. Rogers, Biotech. Bioeng. 1996, 49, 52-62
B. Rosche, V. Sandford, M. Breuer, B. Hauer, P. Rogers, Appl. Microbiol. Biotechnol. 2001, 57, 309- 315
B. Rosche, N. Leksawasdi, V. Sandford, M. Breuer, B. Hauer, P. Rogers. Appl. Microbiol. Biotechnol. 2002a, 64, 94-100
B. Rosche, V. Sandford, M. Breuer, B. Hauer, P. Rogers, J. Mol. Cat B: Enzymatic, 2002b, 19-20, 109-115
Remember, it's not,
"Hi, how are you?" it's
"How high are you?"
(Hive Bee)
07-08-04 19:31
No 518310
Nice work ware, I haven't seen actual indepth information about a bi-phase system before.
benzaldehyde converted to benzyl alcohol due to the presence of alcohol dehydrogenase (ADH) or other oxido-reductase activity in the yeast (Rogers et al, 1997)
I've read on the WWW, a post by ORG on another board that, the addition of EtOH remedies this problem to some extent, because alcohol is already present, and so it causes the responsible yeast elements to not produce benzyl alcohol.
That's why alcohol producers sometimes need to use triple fermentations to produce very strong alcohol, for consumtion.
Producing an excellent yeast culture seems to bee the hardest part, and bringing it all together.
But foxy said in the biosynth thread that, a high diacetyl brewers strain was a possible goer!
There's also some details about producing a suitable yeast using rotten apricots, on Rhodiums site, but the yields are going to bee low for a homebased prep using this yeast, I think.
(Hive Addict)
07-08-04 20:34
No 518321
Here's more of some relevance I think...
Active site mutants of pyruvate decarboxylase from Zymomonas mobilis
A site-directed mutagenesis study of L112, I472, I476, E473 and N482
Martina POHL1, P. SIEGERT1, K. MESCH1, H. BRUHN1 and J. GRÖTZINGER2
1 Institut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, Germany
2 Institut für Biochemie der RWTH Aachen, Klinikum, Germany
(Received 18 May/6 August 1998) 2 EJB 98 0670/4
The homotetrameric pyruvate decarboxylase (PDC) from Zymomonas mobilis requires the cofactors
thiamin diphosphate and Mg21 for catalytic activity. We have investigated the role of various amino acid
residues in the direct environment of the active site. The role of residue E473 in the catalytic activity and
stability of the enzyme was probed by several mutations. All mutant enzymes were either inactive or
failed to give any recombinant protein. The close interaction of E473 and N482, which can be deduced
from the X-ray structure, has been probed by mutagenesis of N482 to D. This mutation has a significant
influence especially on the carboligation reaction of PDC, whereas the binding of the cofactors and the
thermostability were not affected. These data suggest a specific interaction of N482 and E473 which is
essential for coordinating the second aldehyde molecule during carboligation.
Three hydrophobic residues (L112, I472 and I476) in the vicinity of the active centre have been
investigated with respect to their potential influence on the transition states during catalysis. In contrast
to L112, I472 and I476 influence the decarboxylation and carboligation reactions. The enlarged substratebinding
site of PDCI472A allows the decarboxylation of longer aliphatic 2-keto acids (C4-C6) as well as
aromatic 2-keto acids besides pyruvate. Carboligations using PDCI472A as a catalyst yielded 2-
hydroxypropiophenone, benzoin and phenylacetylcarbinol. The enantioselectivity of PAC formation is
impaired by mutations of both I472 and I476. The stereochemistry is most significantly affected with the
mutant enzyme PDCI476E, which catalyses predominantly the synthesis of (S)-phenylacetylcarbinol.
Keywords: 2-hydroxy ketone; pyruvate decarboxylase ; site-directed mutagenesis ; thiamin diphosphate;
phenylacetylcarbinol.
Remember, it's not,
"Hi, how are you?" it's
"How high are you?"
(Hive Bee)
07-09-04 01:00
No 518351
swibb has also been following the idea thread of exploring the viability of psuedo synthesis. in his studies swibb has found a reference to a method published in the rsc.com website. here's a link to the sample reference http://www.rsc.org/pdf/molecularworld/al
section 4.4 is the section of interest. swibbs in depth knowledge isn't there so he's unsure of how feasible this method even is, but does anyone have access to or already have any of these publications?
108
Reaction 4.13 reviews our progress so far; we have a three-stage route leading from
Z-1-phenylprop-1-ene, via a bromoalcohol and an oxirane, to pseudoephedrine. Yet
Planning the synthesis of pseudoephedrine
(4.13)
H2
H H H H
C C C C
catalyst surface
Figure 4.1
Schematic view of the catalytic
hydrogenation of an alkyne to an
alkene.
C C
C6H5 CH3
H H
CH C6H5 C 3 + H2
Pd/BaSO4/quinoline
C
again, unfortunately, Z-1-phenylprop-1-ene is not commercially available, so we
have to go still further back and devise a way of making the alkene:
You should already know of a method of making Z-alkenes from alkynes. What
was the procedure?
Addition of hydrogen to an alkyne using Lindlar’s catalyst produces a Z-alkene.
The Z-alkene is obtained because the mechanism of hydrogenation involves the
delivery of both hydrogen atoms from the metal surface to the same side of the
alkyne (Figure 4.1). A poisoned catalyst is used to inhibit the hydrogenation of the
product alkene, thus allowing the alkene to be isolated (Part 1, Section 2.1).
What alkyne should we use to prepare Z-1-phenylprop-1-ene?
1-Phenylpropyne, C6H5C~CCH3.
The equation for the reaction is:
(4.14)
You will be pleased to learn that 1-phenylpropyne is commercially available, so we
have now completed the planning of our synthetic route to pseudoephedrine from a
readily available starting material. The four-stage route is shown in Scheme 4.2
(overleaf).
You may have wondered why we didn’t treat a bromoalcohol with methylamine and
go straight to pseudoephedrine, rather than via the oxirane. This route may well
work, but, because methylamine is basic, it would probably proceed via formation
of an oxirane intermediate anyway. So, simply to keep track of the chemistry, we
shall stick with the present route: isolation of the oxirane, followed by reaction with
methylamine.
Nevertheless, the important message here is that our knowledge of organic reactions,
and in particular their mechanisms, has allowed us to control the way in
which groups are added. We have predicted that the \OH and \
plus its enantiomer
HOBr
C C
HO
C6H5
H
H
Br
CH3NH2
Z-1-phenylprop-1-ene
base C C
HO
C6H5
H
H
NHCH3 4.1
C C
C6H5 CH3
H H
O
CH3 CH3
C C
C6H5 CH3
H H
plus its enantiomer plus its enantiomer
(Chief Bee)
07-09-04 09:10
No 518396
(Rated as: good read)
Production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde using partially purified pyruvate decarboxylase (PDC)
Hyoun S. Shin, Peter L. Rogers
Biotechnology and Bioengineering, 49(1), 52-62 (2000) (../rhodium/pdf /l-pac.partia
Abstract
Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) as a key intermediate for L-ephedrine synthesis has been evaluated using pyruvate decarboxylase (PDC) partially purified from Candida utilis. PDC activity was enhanced by controlled fermentative metabolism and pulse feeding of glucose prior to the enzyme purification. With partially purified PDC, several enzymatic reactions occurred simultaneously and gave rise to by-products (acetaldehyde and acetoin) as well as L-PAC production. Optimal reaction conditions were determined for temperature, pH, addition of ethanol, PDC activity, benzaldehyde, and pyruvate:benzaldehyde ratio to maximize L-PAC, and minimize by-products. The highest L-PAC concentration of 28.6 g/L (190.6 mM) was achieved at 7 U/mL PDC activity and 200 mM benzaldehyde with 2.0 molar ratio of pyruvate to benzaldehyde in 40 mM potassium phosphate buffer (pH 7.0) containing 2.0 M ethanol at 4°C.
The Hive - Clandestine Chemists Without Borders
07-16-04 05:47
(Rated as: insignificant)