Download Enzymes are Pure Chemistry Emil Fischer The first

E. Fischer:
“Um ein Bild zu gebrauchen, will ich sagen,
Enzymes are Pure Chemistry
Ulf Hanefeld
Catalysis, An Integrated Approach
Schiermonnikoog
November 29 – December 04, 2009
dass Enzym und Glucosid wie Schloss und
Schlüssel zu einander passen müssen, um
eine chemische Wirkung auf einander
ausüben zu können. Diese Vorstellung hat
jedenfalls an Wahrscheinlichkeit und an Werth
für die stereochemische Forschung
gewonnen, nachdem die Erscheinung selbst
aus dem biologischen auf das rein chemische
Gebiet verlegt ist.”
Ber. Dtsch. Chem. Ges., 1894, 27, 2985-2993.
Emil Fischer
To use a picture I would like to say, that
enzyme and glycoside have to match each
other like lock and key in order to potentially
have a chemical effect on each other. This
concept has become more likely and has
gained value for the investigation for
stereochemical research, now that it is part
of the field of pure chemistry rather than
biology.
Ber. Dtsch. Chem. Ges., 1894, 27, 2985-2993.
The first stereoselective synthesis
M. North,
Tetrahedron: Asymmetry, 2003, 14, 147-176.
J. -M. Brunel, I. P. Holmes,
Angew. Chem., Int. Ed. 2004, 43, 2752–2778.
J. Holt, U. Hanefeld,
Curr. Org. Syn. 2009, 6, 15-37.
Prunus amygdalus Hydroxynitrile lyase
~61 000 Da
78x52x46 Å
• highly R-selective
• readily available
• Prunus amygdalus = Almond
• R-mandelonitrile is natural substrate
L. Rosenthaler, Biochem. Z. 1908, 14, 238-253.
1
• highly S-selective
• readily available
• dimer in solution
• acetone cyanohydrin is natural substrate
Prunus amygdalus • highly R-selective
Hydroxynitrile lyase • readily available
~61 000 Da
78x52x46 Å
Hevea brasiliensis
Hydroxynitrile lyase (HbHNL)
29 229 Da, 30x38x48 Å
Results for one
HNL cannot be
directly compared
with those for
other HNL’s.
Hevea brasiliensis Hydroxynitrile lyase (HbHNL)
C1347H2066O384N326S8; 29 229 Da
30x38x48 Å
Enzyme in synthesis
Chemical
Amount
Price
(S)-(-)-/(R)-(+)1,1’Bi(2-naphthol)
10 g
121.60 €
TMSCN
25 g
96.80 €
KCN
25 g
19.50 €
PaHNL
1000 U
131.50 €
MeHNL
1 ml
(3000 U)
103.00 €
PaHNL-CLEA/
MeHNL-CLEA
50 mg
125.00 €
It has to be taken
into account that
they are
structurally not
related!!!
• highly S-selective
• readily available
• dimer in solution
Which conclusions can be drawn?
•
•
•
•
Did the enzymes make or break a bond?
Are enzymes difficult to obtain?
Do they cost too much?
In which unit (gram, mol, etc.) are
enzymes used?
• Why are they actually so huge?
J. Holt, U. Hanefeld,
Curr. Org. Syn. 2009, 6, 15-37.
What types of catalysts are there?
• Heterogeneous catalysts
• Homogeneous catalysts
• Enzymes (homogeneous or
heterogeneous)
What do they have in common?
• All work as catalysts
• All follow the rules of kinetics (Michaelis
and Menten, Lindeman-Hinshelwood,
Eley-Rideal mechanism and LangmuirHinshelwood, Arrhenius)
• For many reactions either of the three
types of catalysts can be applied
• Each type of catalyst has advantages
and disadvantages
2
What is different?
What is different?
• Homogeneous catalysts and enzymes
have one active site
• Heterogeneous catalysts have different
active sites on the surface, at the edges
and corners
• Diffusion, adsorption, and particle size
are very important parameters in
heterogeneous catalysis but are rather
unimportant for homogeneous catalysts
and enzymes
• Enzymes are huge, homogeneous
catalysts are much smaller (H+)
• Enzymes are used in units U and these
have to be measured beforehand
• Homogeneous catalysts are highly
defined and are added in moles
• Heterogeneous catalysts have different
active sites and are added in grams
Classification of Enzymes
Which type of enzyme is used?
EC numberClass
Reaction
1
2
3
Oxidoreductases
Transferases
Hydrolases
4
5
6
Lyases
Isomerases
Ligases
(synthetases)
Electron transfer
Group-transfer
Hydrolysis
(transfer of functional groups to water)
Double bond additions
Shuffling groups within a molecule
Formation of C-C, C-S, C-O, etc. by
condensations at the expense of ATP
Why are enzymes so huge?
• Degrading enzymes, such as lipases,
esterases and proteases but also alcohol
dehydrogenase and lyases have the
advantage that they are selective for one
functional group but not selective for a
specific substrate
• Synthetic enzymes (anabolism) tend to be
very substrate specific, are therefore less
flexible and can not be used so widely
Why are enzymes so huge?
No substrate bound
Hevea brasiliensis Hydroxynitrile lyase (HbHNL)
C1347H2066O384N326S8; 29 229 Da
30x38x48 Å
Transition state bound
When the transition state is reached the enzyme is much more ordered,
more hydrogen bonds exist, more stable beta sheets and alpha helixes
3
Benefit in enthalpy (ΔΔH#) of some enzymecatalysed reactions relative to the reactions in
free solution
Enzymes align substrates
D. H. Williams, E. Stephens and M. Zhou, Chem. Commun, 2003, 1973–1976.
Enzyme
Rate Acceleration
ΔΔH#, kJ mol-1 (s-1) due to ΔΔH#
Chorismate dismutase
Chymotrypsin
Staphylococcal nuclease
Bacterial α-glucosidase
Urease
Yeast OMP decarboxylase
233
266
263
280
293
2143
106
1012
1011
1014
1016
1025
• Enzymes are huge, the scaffold fixes the
substrate and aligns it with the binding sites of
the enzyme
• Due to the perfect alignment the acid base
catalysis that occurs has its charges much more
distributed than in normal acid base catalysis
Enzymes align substrates
Enzymes align substrates
• The enzyme basically encompasses the
substrates
• This is only possible due to its size
• The aligned substrate can now react
with the relative weak acids and bases
in the enzyme
• What is the entropy here and in the
homogeneous reaction?
H. M. Weiss, J. Chem. Edu. 2007, 84, 440-442.
Mechanism of a D-2-deoxyribose-5-phosphate aldolase.
Intramolecular versus intermolecular
Classification of Enzymes
EC numberClass
Reaction
1
2
3
Oxidoreductases
Transferases
Hydrolases
4
5
6
Lyases
Isomerases
Ligases
(synthetases)
Electron transfer
Group-transfer
Hydrolysis
(transfer of functional groups to water)
Double bond additions
Shuffling groups within a molecule
Formation of C-C, C-S, C-O, etc. by
condensations at the expense of ATP
Entropically favoured by fixing conformation
4
What is a (Serine) Hydrolase?
• Serine hydrolases are enzymes that
hydrolyse an ester, thioester or amide bond
• Hydrolases are enzymes that hydrolyse
other C-O or C-N or C-S bonds
• Serine Hydrolases have a serine in their
active site
• Hydrolases catalyse hydrolysis reactions
(enantio)selectively under mild conditions
How to deprotonate?
How does this Hydrolase work?
• Serine acts as a nucleophile that attacks
the substrate
• It is the alcohol group of the serine that
does the trick
• This alcohol needs to be deprotonated to
obtain an alcoholate => the nucleophile
• How is this achieved?
Chymotrypsin
• Localised charge
• Strong base necessary
• Charge delocalisation => weak base
O
Asp
O
His
H N
N
H
O
Ser
O
O
Chymotrypsin Catalysis
Asp102
His57
Ser195
Step 2, Formation of first Michaelis Complex
Catalytic “triad’’
Step 1
Substrate diffuses
into active site
Positioning group
(hydrophobic)
5
Step 3, Formation of Tetrahedral Intermediate
Catalytic “triad’’
Step 4, Release of first product (amine)
Catalytic “triad’’
“Oxy-anion hole”
Acyl-enzyme
“TI1”
Product-1
Step 5, Water approaches (2nd substrate)
Catalytic “triad’’
Step 6, Formation of Tetrahedral Intermediate
Catalytic “triad’’
Acyl-enzyme
Oxy-anion hole
“TI2”
Substrate-2
Nucleophilic
addition of water
Step 7, Formation of Michaelis Complex
Step 8, Release of second product
Catalytic “triad’’
Catalytic “triad’’
Repulsion
Dissociation
of acid
Product-2
6
Catalytic triad: Charge relay
Bi-bi Ping-pong kinetics
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
The tetrahedral intermediate
Attack of the nucleophile
Bi-bi Ping-pong kinetics
Is this really reversible?
J. H. Kastle, A. S. Loevenhart,
Am. Chem. J. 1900, 24, 491-525.
7
Attack of the nucleophile
Difference between ester and amide
Why use a hydrolase?
What are hydrolases used for?
• Hydrolysis with acids and bases often
require harsh reaction conditions
• A lot of waste is generated
• Lipases, esterases and proteases catalyse
hydrolysis reactions (enantio)selectively
under mild conditions
• All this makes enzymatic catalysis very
clean and green
• As degrading enzymes they are selective
for a functional group but not for a single
molecule
• Hydrolases are virtually never used for
what they were evolved for: hydrolysis
• Lipases, esterases and proteases catalyse
hydrolysis reactions enantioselectively
under mild conditions
• Hydrolases are therefore used to obtain
enantiopure compounds from racemates
Rule of Kazlauskas
Rule of Kazlauskas
Secondary alcohols
Secondary alcohols and primary amines
R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, L. A. Cuccia, J. Org. Chem., 1991,
56, 2656-2665. C. K. Savile, R. J. Kazlauskas, J. Am. Chem. Soc., 2005, 127, 12228-12229.
R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, L. A. Cuccia, J. Org. Chem., 1991,
56, 2656-2665. C. K. Savile, R. J. Kazlauskas, J. Am. Chem. Soc., 2005, 127, 12228-12229.
8
Rule of Kazlauskas
What about tert. alcohols?
primary alcohols
• Very few enzymes accept these bulky
substrates
• Few examples and even less
enantioselective examples
• Limited scope but very interesting
Burkholderia cepacia lipase (formerly called Pseudomonas cepacia lipase, PCL)
preferably catalyses the hydrolysis/acylation of only one of the depicted enantiomers
of the chiral primary alcohol (ester). The selectivity is low if oxygen is bound to
the chiral carbon. L: largest substituent, M: medium sized substituent.
A. N. E. Weissfloch, R. J. Kazlauskas, J. Org. Chem., 1995, 60, 6959-6969.
Chymotrypsin Catalysis
Asp102
His57
Ser195
What about chiral acids?
What about chiral acids?
Step 1
Substrate diffuses
into active site
H
L
COOH
M
Preferred enantiomer in Candida rugosa lipase (CRL)-catalysed hydrolysis and
esterification reactions of chiral acids. L: largest substituent, M: medium sized
substituent.
Positioning group
(hydrophobic)
Differences between the enzymes?
M. C. R. Franssen, H. Jongejan, H. Kooijman, A. L. Spek, N. L. F.L. Camacho Mondril,
P. M. A. C. Boavida dos Santos, A. de Groot, Tetrahedron Asymmetry, 1996, 7, 497-510.
Interfacial activation
• Lipases are commonly interfacially
activated. Their natural function is the
hydrolysis of fat. They can be used in
water and apolar organic solvents (MeOH,
EtOH, DMSO tend to be bad)
CMC = critical micellar concentration
K. Faber, Biotransformations in Organic Chemistry, 5th edition, Springer, 2004.
9
Lid of lipase from Thermomyces
lanuginosus
U. Hanefeld,
L. Gardossi and
E. Magner
Chem. Soc. Rev.
2009, 38, 453–468.
Slight difference in catalytic triad
catalytic triade
His
O
N
N
H
H
Glu
O
oxyanion
Ser
O
hole
-RCOOR'
+RCOOR'
-RCOOH +RCOOH
H
His
N
H
O
Glu
N H
O
O
N
H
His
H
O
N
N
R
O
Ser
O
oxyanion
hole
+R'OH
-H2O
O
R
O
O
Glu
hole
O
Ser
oxyanion
O
Glu
N H
O
Ser
+H2O
R'
His
R
O
-R'OH
O
oxyanion
hole
Difference between ester and amide
CAL B surface
A. Basso, P. Braiuca, S. Cantone, C. Ebert, P. Linda, P. Spizzo,P. Caimi, U. Hanefeld, Giuliano
Degrassi and L. Gardossi, Adv. Synth. Catal. 2007, 349, 877 – 886.
Differences between the enzymes?
• Esterases show no interfacial activation,
they were evolved to hydrolyse esters and
they tend to be sensitive to organic
solvents. Mostly used for hydrolysis
reaction in water (+ polar solvent).
• Proteases were evolved to hydrolyse
proteins. Since esters are less stable than
proteins they can easily hydrolyse them.
Many are stable in apolar organic
solvents. No interfacial activation
How to select the right hydrolase?
• Hydrolases are degrading enzymes,
therefore they are not substrate specific
• They are selective for esters, amides and
thioesters
• Lipases and esterases were evolved to
hydrolyse esters
• Proteases were evolved to hydrolyse
proteins
• Although we understand a lot and can
predict much the best approach is:
10
Reactions in water
Summary
• Almost hydrolysis only – but if amides
need to be synthesised this can be done in
water.
• Either high buffer concentration or pH-stat
(automatic burette).
• Often low concentrations.
• Often difficult separations – water is
difficult to remove.
• Serine Hydrolases can hydrolyse and
synthesize esters and amides
• Water, alcohols and amines can be
nucleophiles
• They are enantioselective
• For secondary alcohols enzymes for both
enantiomers exist
• For primary alcohols stereo differentiation is
also possible
• Chiral acids can also be resolved
Reactions in Organic Solvents
Kinetic resolution
A: in water
• Substrates dissolve better
• Higher substrate concentration
• Enzyme does not dissolve → easy work
up
• Different reactions become possible
• Hydrolases can be used to make esters
• Hydrolases can be used to make amides
• First described in 1913
(S)-Y
+YH
R
C
R'
X
C
enzyme-catalysed
(S)-Y
acylation, fast
B: in dry organic solvent
R'
C
not catalysed,
very slow
XH
X = O, NH; Y = C or heteroatom
enzyme-catalysed
(S)-Y
acylation, fast
C
C
Xacyl
R'
R
XH
not catalysed,
very slow
R
(R)-Y
C
Xacyl
R'
Dynamic kinetic resolution
R
C
Xacyl
+YH
R
X = O, NH
(R)-Y
C
R
XH
R'
R
XH
R
(R)-Y
R
(R)-Y
XH
R'
R'
dynamic racemisation
C
not catalysed,
very slow
Xacyl
R'
R'
R
(R)-Y
C
(S)-Y
C
R'
R
XH
R'
dynamic
+YH
C
R
(R)-Y
Dynamic kinetic resolution
R
R
enzyme-catalysed
Xacyl
(S)-Y
hydrolysis, fast
R'
M. Bourquelot, M. Bridel, Ann. Chim. Phys. 1913, 145.
(S)-Y
X = O, NH; Y = C or heteroatom
R
C
R'
C
R'
Xacyl
X
(S)-Y
C
enzyme-catalysed
(S)-Y
acylation, fast
R'
C
X = O, NH
R
(R)-Y
C
R'
Xacyl
R'
dynamic racemisation
dynamic
+YH
R
XH
R
XH
not catalysed,
very slow
(R)-Y
C
Xacyl
R'
Kinetic resolutions give
only 50 % yield!!!
11
Dynamic kinetic resolution
R
(S)-Y
+YH
R
C
R'
C
R
XH
enzyme-catalysed
(S)-Y
acylation, fast
R'
C
X = O, NH
R
+YH
(R)-Y
C
Xacyl
R'
dynamic racemisation
dynamic
X
Desymmetrisation
Hydrolases desymmetrise symmetrical compounds.
R
XH
not catalysed,
very slow
(R)-Y
R'
C
Xacyl
R'
The “meso-trick”
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
Organic solvents
•
•
•
•
Apolar, aprotic (alkanes, petrol ether)
Polar, aprotic (DMSO, DMF)
Protic (Alcohols)
Water miscible (DMSO, DMF, THF,
dioxane)
• Water immiscible (toluene, many ethers,
alkanes, esters)
Degree of polarity
solvent
log p
DMSO
Dioxane
DMF
Methanol
Actone
Ethylacetate
Diethylether
Diisopropylether
Toluene
Hexane
Octane
-1.3
-1.1
-1.0
-0.76
-0.23
0.68
0.85
1.9
2.5
3.5
4.5
Degree of polarity
• When working with enzymes log P is used
• Log P is the log of the partition coefficient
of a solvent between 1-octanol and water
log P
H2O miscible
-2.5 to 0
yes
Reaction in water with a little solvent
(10-50 %), otherwise enzyme †
0 to 1.5
partially
often bad for enzymes, only with very
stable enzymes
1.5 to 2.0
low
Works often but not always,
unpredictable
>2.0
no
Enzymes are very stable in these
solvents
Effect on enzyme
Organic solvents versus water
• In water the enzyme is dissolved
• In a polar protic (methanol) or polar
aprotic (DMSO) solvent the enzyme can
be dissolved
• In a water immiscible solvent the enzyme
does not dissolve
• Does an enzyme need to move?
• How does an enzyme deactivate?
12
Why are enzymes so huge?
Benefit in enthalpy (ΔΔH#) of some enzymecatalysed reactions relative to the reactions in
free solution
D. H. Williams, E. Stephens and M. Zhou, Chem. Commun, 2003, 1973–1976.
No substrate bound
Transition state bound
When the transition state is reached the enzyme is much more ordered,
more hydrogen bonds exist, more stable beta sheets and alpha helixes
Enzyme
Rate Acceleration
ΔΔH#, kJ mol-1 (s-1) due to ΔΔH#
Chorismate dismutase
Chymotrypsin
Staphylococcal nuclease
Bacterial α-glucosidase
Urease
Yeast OMP decarboxylase
233
266
263
280
293
2143
106
1012
1011
1014
1016
1025
Enzymes need to move a little to accept the
substrate and to facilitate the transition state.
Water acts as lubricant
Organic solvents versus water
Organic solvents versus water
How does an enzyme deactivate?
Hydrolysis of the peptide chain
Unfolding
Asparagine and Glutamine amide might
hydrolyse
• All of this is caused or aided by water
• So in organic solvent the enzyme should
be more stable
• But the enzyme has to stay flexible
• What about the pH? Enzymes have pH
optima in water
• In one phase + organic solvent nothing
changes
• In two phase systems there is a water
layer, nothing changes
• Only organic solvent (one phase) – there
is no pH.
•
•
•
•
Organic solvents versus water
Organic solvents and water
• Only organic solvent (one phase) – there is
no pH.
• An organic buffer pair can be used, but
inconvenient
• Approach of choice: make the enzyme
preparation at the ideal pH of the enyzme.
Lyophilise (freeze-dry) or immobilise at pH
optimum
• Enzyme has memory effect, since it is rigid
in org sol is stays in optimal conformation.
• The enzyme needs some flexibility
• Most of the water is bulk water and if it is
gone it does not matter
• But a little water needs to be on the
enzyme as structural water or molecular
lubricant
• Really dry enzymes tend to be less active
• But too much water causes enzymes to
lump together – unless they are
immobilised (think of diffusion)
13
Organic solvents and water
Reactions in organic solvents
• How much water does the enzyme need?
• If water saturated solvent is used enzymes
tend to work well but it can cause many
side reactions
• Control water concentration i.e. water
acitivity aw
• Salt pairs can be used for this
• Not all hydrolases are stable in dry organic
solvents. Lipases that were evolved for
hydrophobic substrates perform best,
esterases worst.
• Apolar solvents are better than water miscible
ones
• Immobilisation can help
• Small amounts of water might be added, too
• Additives might help
aw
Salt pair
CaCl2 xH2O / 2 H2O
0.037
Na2HPO4/Na2HPO4•2H2O
0.16
Na2HPO4•2H2O/Na2HPO4•7H2O
0.57
Na2HPO4•7H2O/Na2HPO4•12H2O
0.80
Reactions in organic solvents
How to synthesise an ester
• If all the parameters discussed are taken care
of enzymes can display similar activity in
organic solvent as in water
• Reactions performed: esterification,
transesterification, amide synthesis
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
How to synthesise an ester
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
How to synthesise an ester
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
14
Dynamic kinetic resolution
H. Pellissier, Tetrahedron 2008, 64, 1563-1601
How to synthesise an amide
Dynamic kinetic resolution
H. Pellissier, Tetrahedron 2008, 64, 1563-1601
How to synthesise an amide
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
Industrial application
U. Hanefeld, Org. Biomol. Chem. 2003, 1, 2405 – 2415.
Review: F. van Rantwijk, R.A. Sheldon, Tetrahedron 2004, 60, 501
dkr
Y.K. Choi, M.J. Kim, Y. Ahn and M.J. Kim, Org. Lett., 2001, 3, 4099-4101.
15
Conclusions
dkr
Martijn A. J. Veld, Karl Hult, Anja R. A. Palmans, E. W. Meijer, Eur. J. Org.
Chem. 2007, 5416–5421.
kr for chiral intermediates
AcO
HO
AcO
+
NH
Burkholdia cepacia lipase
(formerly called
Pseudomonas cepacia
lipase)
NH
O
O
racemate
O
Cl
OMe
Cl
ee > 99 %
OH
O
OMe
+
Cl
O
Cl
NH
O
O
CCL
V. H. M. Elferink, J. G. T. Kierkels, M. Kloosterman,
J. H. Roskam (Stamicarbon B.V.), EP 369553, 1990.
N
captopril
OH
OH
O
O
DSM Andeno process
Z. Liu, R. Weis, A. Glieder,
Food Technol. Biotechnol.,
2004, 42, 237–249.
OH
OH
O
kr for chiral intermediates
COOH
lactonase
O
O
R. N. Patel, J. Howell, R. Chidambaram,
S. Benoit and J. Kant,
Tetrahedron: Asymmetry, 2003, 14, 3673–3677.
AcO
+
NH
O
• Hydrolases are versatile catalysts for the
clean and green synthesis of enantiopure
compounds
• Synthetic dynamic kinetic resolutions enable
hydrolase-“catalysed” bond syntheses
• Given the great promiscuity of the
hydrolases, in particular the lipases, new
nucleophiles other than water can be
introduced
• Reverse reaction in Organic Solvent
possible
+
COOH
HO
HO
D-pantoic acid
rac-pantolactone
O
N
J. Ogawa, S. Shimizu,
Curr. Opin. Biotechnol., 2002, 13, 367–375.
OH
Vitamin B5
H.-J. Gais, C. Griebel, H. Buschmann,
Tetrahedron: Asymmetry, 2000, 11, 917–928.
chemical racemisation
O
O
N
N
H
O
N
O
Bacillus lentus protease
OEt
N
O
N
N
H
O
O
human rhinovirus
protease inhibitors
OH
O
+
O
N
5 % DBU, rt, 1 h
C. A. Martinez, D. R. Yazbeck, J. Tao,
Tetrahedron, 2004, 60, 759–764.
O
N
N
H
O
OEt
O
Clean-up dkr for chiral compounds
O
S
CF3
in situ
racemisation with
trioctylamine
O
S
CF3
O
COOH
Candida rugosa lipase
O
O
Desymmetrisation
C.-Y. Chen, Y.-C. Cheng, S.-W. Tsai,
J. Chem. Technol. Biotechnol., 2002,
77, 699-705.
S-fenoprofen
OEt
OEt
in situ
racemisation with
NaOH
O
O
COOH
Candida rugosa lipase
O
O
H. Fazlena, A. H. Kamaruddin, M. M. D. Zulkali,
Bioprocess Biosyst. Eng., 2006, 28, 227–233.
S-ibuprofen
S
S
O
O
Pseudomonas cepacia lipase
O
O
N
NC
N
NC
SPr
NC
SPr
O
SPr
O
O
O
O
N
NC
OH
O
trimethylamine
O
N
O
N
NC
N
NC
roxifiban
J. A. Pesti, J. Yin, L.-H. Zhang, L. Anzalone,
J. Am. Chem. Soc., 2001, 123, 11075-11076.
J. A. Pesti, J. Yin, L.-H. Zhang, L. Anzalone,
R. E. Waltermire, P. Ma, E. Gorko,
P. N. Confalone, J. Fortunak, C. Silverman,
J. Blackwell, J. C. Chung, M. D. Hrytsak,
M. Cooke, L. Powell, C. Ray,
Org. Proc. Res. Dev., 2004, 8, 22-27.
R. N. Patel, A. Banerjee, L. Chu, D. Brozozowski, V. Nanduri, L. J. Szarka,
J. Am. Oil Chem. Soc., 1998, 75, 1473-1482.
R. Öhrlein, G. Baisch, Adv. Synth. Catal., 2003, 345, 713 – 715.
16
“meso” trick
O
O
AcO
O
Pseudomonas fluorenscens
lipase (PFL)
OAc
O
AcO
yield = 98 %
ee > 98 %
OH
meso
O
O
O
PFL
OAc
AcO
HO
O
OAc
yield = 79 %
ee = 96 %
meso
C. Bonini, R. Racioppi, L. Viggiani, G. Righi, L. Rossi, Tetrahedron: Asymmetry, 1993, 4, 793-805.
Z.-F. Xie, H. Suemune, K. Sakai, Tetrahedron: Asymmetry, 1993, 4, 973-980.
U. Zutter, H. Iding, P. Spurr, and B. Wirz, J. Org. Chem. 2008, 73, 4895–4902.
17