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Lipases
H2C
HC O
H2C O
O
O
O
O
R
R
R
lipase
H2O
H2C
HC OH
H2C OH
OH
+ 3 HO
O
R
Thr
Asp/Glu
N
H
R1 O R
2
O
N
H
O
H
O
O
NNH
Gln
Ser
His
Gln Thr
Ser
His
N
H
O
R2
O
R1O
H
N
HN N
HO
O
Asp/Glu
His
Gln Thr
Ser
N
H
O
R2
O
H
N
N NH
O
O
R3OH
Asp/Glu
R1-OH
R3-OH
native enzyme
His
Gln Thr
Ser
N
H
O
R2
O
H
N
OR3
HNNH
O
O
Asp/Glu
R1O R2
OR3O R2
O
Bi-bi ping-pongmechanism !
Esterolytic vs. lipolytic activity
Act
ivit
y
0
1
2
3
5
Substrate concentration10 2 3
Act
ivit
y
0
1
2
3
5
Substrate concentration10 2 3
Esterase activity Lipase activity
Rhizomucor miehei lipase in the closed form:
nucleophilic elbow = green, lid = blue-green
Candida antarctica lipase B (no lid):
nucleophilic elbow = green (centre)
Current lipase research @ OC
*
-CO2
"strawberry aldehyde"
O COOMeRhodotorula glutinis
lipase/esterase
O COOH
+
O COOMe
* * *
O
H
Epoxide hydrolase from yeasts
Rhodotorula glutinisepoxide hydrolase
H2O
+O
RR
OH
OH
racemate
O
R
enantiomeric purity: max
yield: max
100% e.e.
50%
100% e.e.
50%
Tyr Tyr
OH
O
N
N
O
H
H
H OO
His
Asp
OH
OO
N
N HO
O
Asp
OO
His
Glu Glu
OH
O
OHO
Asp
His
N
N
H
OOH
GluGlu
His
Asp
OOH
N
N HO
O
Tyr Tyr
OH
O
Tyr Tyr
OH
O
HO
OH
O
OH
OH
Tyr Tyr
O
HO
OH
1
2b
2a
native enzyme
Reaction mechanismof epoxide hydrolase:
Present research on epoxide hydrolase
Resolution of:
…… as chiral building blocks for pharmaceuticals
More info: dr. Carel Weijers!
O
R1
R2
O
R2
OHR1
Development and integration of stable aldolases in the synthesis of
enantiopure functionalized nitrogen heterocycles
Maud CABRIÈRES, Maurice FRANSSEN
IBOS PROJECT: 5 partners
• IBOS: Integration of Biosynthesis and Organic Synthesis Program
• Dr. Franssen/Prof. Sudhölter (Org. Chem. WU)• Dr. van der Oost/Prof. de Vos (Microbiology WU)• Prof. Kieboom, University of Leiden (Bio-org. Chem.
UL)• Prof. Rutjes, University of Nijmegen (Org. Chem. KUN)
• Industrial partner: DSM
General framework of IBOS project
• Schematic overview of the project:
• Final goal: integration of new generation of Aldolases in a cascade of biocatalytic conversions, aiming at the synthesis of novel nitrogen heterocyclic compounds:
Aldolase adducts
HNL adducts
Alcohols
Aldehydes
UL
Nitrogen Heterocycles KUN WUR
Improved aldolases
DSM Improved HNL enzyme
BioRedox Project Improved oxidases
Aldehyde
Cyanohydrin
Hydroxynitrile Lyase (HNL)
N
X
OH
R1HOOC
R2
NH
R3
HOOC COOH
R4
NH
R5
( )n
OH
R6
NH
R8 R7
OH( )n
NH
O
OH
HO
C-C bond formation relevance
“ Carbon- carbon bond formation lies at the heart of many organic synthesis ”
Today’s fine chemistry challenge: forming building blocks with complete control of the stereochemistry of
stereogenic centres
Aldol reaction: one of the most powerful and famous method to enlarge carbon skeleton
Aldol condensation reaction
• Racemic mixture!
O
R1 CH2R2
O
R3
O
R1*
OH
R3
R2
B- O
R1
*
OH
R3
R2
++
Aldol condensation: Nature uses aldolases
• Specific group of lyases, very stereoselective
• Nearly 30 natural aldolases identified (2000)
• Broad range of acceptors
• Stringent requirement for donors
O
R1
O
R2
O
R1*
OH
R2
+
DonorAcceptor
Aldolase
Aldolase classification based on catalytic mechanism
• Type I Aldolase:– Donor activation
– Imine intermediate
• Type II Aldolase:– Donor activation
– Metal-dependent (Zn2+ cofactor)
OHO
P
O
-O
-O
Ser271-OHLys229
HN
Lys107
NH2+
OOPO3
2-
OH
O-
-2O3PO OH
Zn2+
O
OH
OPO32-
OH
HisHis
Tyr
Aldolases from (hyper)thermophilic microorganisms
• Archaea and bacteria with Toptimum > 80 ° C
• Deep sea smokers: Pyrococcus• Shallow marine vents: Thermotoga (DHPS)• Acidic hot spring: Sulfolobus (KDG)
Volcano• Themotoga
Pacific• Pyrococcus
Yellowstone• Sulfolobus
Relevant Aldolases involved in this project
– KDG (2-keto-3-deoxy gluconate aldolase):
– DHPS (2,3-dihydrodipicolinate synthase):
– DERA (2-deoxyribose-5-phosphate aldolase):
O
HOOC * COOH
O NH2
N*
COOHHOOC
O
HOOC*
OH*
NH2
COOH
+-H2ODHP synthase
O O
*OH
OHHO
*
*
*O
OH
HO
OH*
*OH
OH
O+ DERA aldolase
O
HOOC *OH
OH
O
*OH
OH
*
OHO
HOOCKDG aldolase+
More information on what we do:
http://www.ftns.wau.nl/oc/research/biocatalysis/biocatalysis.htm
