Biocatalysis in Organic Synthesis

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Biocatalysis in Organic Synthesis
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References

• Biotranformation In Organic Chemistry
• Kurt Faber, 4th Edition
• Springer-Verlag
• Nature Insight, Biocatalysis
• Nature, 2001, 409

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Advantages of Biocatalysis

• The key word for organic synthesis is
  selectivity which is necessary to obtain a high
  yield of a specific product. There are a large
  range of selective organic reactions available
  for most synthetic needs. However, there is
  still one area where organic chemists are
  struggling, and that is when chirality is
  involved, although considerable progress in
  chiral synthesis has been achieved in recent
  years.

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Tetrahedral Structure of Methane

• sp3 orbitals on C overlap with 1s orbitals on 4 H
  atom to form four identical C-H bonds
• Each CH bond has a strength of 438 kJ/mol and
  length of 110 pm
• Bond angle each HCH is 109.5, the tetrahedral
  angle.

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Enantiomers and the Tetrahedral Carbon

• Molecules that have one carbon with 4 different
  substituents have a nonsuperimposable mirror
  image enantiomer
• Build molecular models to see this

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Amino AcidsBuilding Blocks of Proteins
Anatomy of an amino acid. Except for proline and
its derivatives, all of the amino acids commonly
found in proteins possess this type of structure.
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The assignment of (R) and (S) notation for
glyceraldehyde and L-alanine .
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Zwitterion

• Amino acid exists as a dipolar ion.
• -COOH loses H, -NH2 gains H.

• Actual structure depends on pH.
  
  gt

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Structure and pH
gt
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The Acidic Residue of the Proteins
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Sodium Glutamate

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  ?????13??(???39)????????????????????????????????
  ?????   

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• ???????????(MONOSODIUM GLUTAMATE),???????,????????
  ???????????,?????????????
• ????????,????,??????????????1866????(RITTHAUSEN)??
  ????????,???????????42???,?????????????1908?????,?
  ????,??????GROF???,????????,????????,?????????,???
  ??????????????????,???????
• ??????????,????????,??????????,??????,???????????,
  ????80?????????????????????,??????????,??????????
  ????,??1956?,?????????????????,???????????????????
  ???,???????????????

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• ???????????1958???????????????????,???????????????
  ??,?????????????????,????????,????????????????????
  ??????????????????????????,????0.5??????,????????

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D-Glucose can cyclize in two ways, forming either
furanose or pyranose structures.
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Figure 19.4The tricarboxylic acid cycle.
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Figure 25.9The glutamate dehydrogenase reaction.
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Amino Acids Can Join Via Peptide Bonds
Figure 4.2The ?-COOH and ?-NH3 groups of two
amino acids can react with the resulting loss of
a water molecule to form a covalent amide bond.
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Nelson Cox (2000) Lehninger Principles of
Biochemistry (3e) p.129
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Figure 15.22The myoglobin and hemoglobin
molecules. Myoglobin (sperm whale) one
polypeptide chain of 153 amino acid residues
(mass 17.2 kD) has one heme (mass 652 D) and
binds one O2. Hemoglobin (human) four
polypeptide chains, two of 141 amino acid
residues(a) and two of 146 residues (b) mass
64.45 kD. Each polypeptide has a heme the Hb
tetramer binds four O2. (Illustration Irving
Geis Rights owned by Howard Hughes Medical
Institute. Not to be reproduced without
permission)
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Chemical Conversion by Use of Proteins
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Figure 14.4 Formation of the ES complex results
in a loss of entropy. Prior to binding, E and S
are free to undergo translational and rotational
motion. By comparison, the ES complex is a more
highly ordered, low-entropy complex.
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Hydrolysis Conversion of Esters into Carboxylic
Acids

• An ester is hydrolyzed by aqueous base or aqueous
  acid to yield a carboxylic acid plus an alcohol

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Reactions of Amides

• Heating in either aqueous acid or aqueous base
  produces a carboxylic acid and amine
• Acidic hydrolysis by nucleophilic addition of
  water to the protonated amide, followed by loss
  of ammonia

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Amino Acids Can Join Via Peptide Bonds
Figure 4.2The ?-COOH and ?-NH3 groups of two
amino acids can react with the resulting loss of
a water molecule to form a covalent amide bond.
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The Serine Proteases

• Trypsin, chymotrypsin, elastase, thrombin,
  subtilisin, plasmin, TPA
• All involve a serine in catalysis - thus the name
  
• Ser is part of a "catalytic triad" of Ser, His,
  Asp
• Serine proteases are homologous, but locations of
  the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
  always as His-57, Asp-102, Ser-195

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The catalytic triad of chymotrypsin .
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Structure of chymotrypsin (white) in a complex
with eglin C (blue ribbon structure), a target
protein. The residues of the catalytic triad
(His57, Asp102, and Ser195) are highlighted.
His57 (blue) is flanked above by Asp102 (red) and
on the right by Ser195 (yellow). The catalytic
site is filled by a peptide segment of eglin.
Note how close Ser195 is to the peptide that
would be cleaved in a chymotrypsin reaction.
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Crystal structure of Subtilisin
(serine endopeptidase) is a proteolytic enzyme
initially obtained from Bacillus subtilis. It is
secreted in large amounts from many Bacillus
species.
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Ligation of the Peptides, Subtiligase
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Figure 9.2Several spontaneously formed lipid
structures.
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Enzymes display three major types of
selectivities

• Chemoselectivity Since the purpose of an enzyme
  is to act on a single type of functional group,
  other sensitive functionalities, which would
  normally react to a certain extent under chemical
  catalysis, survive. As a result, biocatalytic
  reactions tend to be "cleaner" and laborious
  purification of product(s) from impurities
  emerging through side-reactions can largely be
  omitted.
• Regioselectivity and Diastereoselectivity Due to
  their complex three-dimensional structure,
  enzymes may distinguish between functional groups
  which are chemically situated in different
  regions of the substrate molecule.
• Enantioselectivity Since almost all enzymes are
  made from L-amino acids, enzymes are chiral
  catalysts. As a consequence, any type of
  chirality present in the substrate molecule is
  "recognized" upon the formation of the
  enzyme-substrate complex. Thus a prochiral
  substrate may be transformed into an optically
  active product and both enantiomers of a racemic
  substrate may react at different rates.

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The Chemoselectivities of the Enzymes
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Artificial substrates used in studies of the
mechanism of chymotrypsin
The Hydrolases of Esterase and Lipase are
uptaking the similar triad mechanisms of serine
protease such as chymotrypsin
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• These reasons, and especially the latter, are the
  major reasons why synthetic chemists have become
  interested in biocatalysis.
• This interest in turn is mainly due to the need
  to synthesise enantiopure compounds as chiral
  building blocks for drugs and agrochemicals.

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Biocatalysis in Green Chemistry

• Another important advantage of biocatalysts are
  that they are environmentally acceptable, being
  completely degraded in the environment.
• Furthermore the enzymes act under mild
  conditions, which minimizes problems of undesired
  side-reactions such as decomposition,
  isomerization, racemization and rearrangement,
  which often plague traditional methodology.

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The use of biocatalysis to obtain enantiopure
compounds

• Kinetic resolution of a racemic mixture
• Biocatalysed asymmetric synthesis

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Kinetic resolution of a racemic mixture
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Kinetic resolution of a racemic mixture
the presence of a chiral object (the enzyme)
converts one of the enantiomers into product at a
greater reaction rate than the other enantiomer.
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dynamic resolution

• If it is possible to perform such resolutions
  under conditions where the two substrate-enantiome
  rs are racemizing continuously, all substrate may
  in theory be converted into enantiopure product.

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Deracemization Reaction
monoamine oxidase isolated from Aspergillus niger
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Biocatalysed asymmetric synthesis
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Reduction of Carbonyl

• Reduction of aldehyde yields 1º alcohol.
• Reduction of ketone yields 2º alcohol.
• Reagents
• Sodium borohydride, NaBH4
• Lithium aluminum hydride, LiAlH4
• Raney nickel
  gt

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Sodium Borohydride

• Hydride ion, H-, attacks the carbonyl carbon,
  forming an alkoxide ion.
• Then the alkoxide ion is protonated by dilute
  acid.
• Only reacts with carbonyl of aldehyde or ketone,
  not with carbonyls of esters or carboxylic acids.

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Lithium Aluminum Hydride

• Stronger reducing agent than sodium borohydride,
  but dangerous to work with.
• Converts esters and acids to 1º alcohols.

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Catalytic Hydrogenation

• Add H2 with Raney nickel catalyst.
• Also reduces any CC bonds.

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Figure 17.10Hydrogen and electrons released in
the course of oxidative catabolism are
transferred as hydride ions to the pyridine
nucleotide, NAD, to form NADH H in
dehydrogenase reactions of the type AH2 NAD
? A NADH H The reaction shown is catalyzed
by alcohol dehydrogenase.
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Biocatalysed asymmetric synthesisfor carbonyl
reduction
Yeast is a biocatalyst for the enantioselective
reduction of ketones.
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Baeyer-Villiger oxidation
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The Mechanism
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example
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Baeyer-Villiger oxidation using Whole Cell
Catalysis
Baeyer-Villiger monooxygenase or BVMO
cofactors such as NADPH
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• Drug Manufacture and Discovery by Microbial
  Bioconversion
• -The industrial conversion and biosynthesis of
  Penicillin

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Penicillin ???
Fleming, at his laboratory in St. Mary's Hospital
(now one of Imperial College teaching hospitals)
in London, noticed a halo of inhibition of
bacterial growth around a contaminant blue-green
mold on a Staphylococcus plate culture.
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Fleming Chain Florey (1945)
?? (1985) no. 25 p. 122
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???(Alexander Fleming)?1928?????(???Penicillium)??
??(??????Staphylococcus)??????(Howard
Florey)???(Ernst Chain)??1939??????????(penicillin
)???????????????(1940?1945?)????????????????,?????
?????????????????????????????(submerged liquid
culture)???????????????????????(secondary
metabolites)?????(aerobic)???????,????????????????
????????????????????1945????????
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This antibiotic is manufactured commercially
using Penicillium chrysogenum.  The penicillin is
produced in large stainless steel fermenters,
which have a capacity of around 10000 dm3.
                                                  
                                                  
                                                  
                                                  
                                              
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Fermentation of Penicillin

• The fermenter is sterilised by steam and then
  loaded with a sterilised growth medium for the
  Penicillium chrysogenum to feed on, containing
  lactose, amino acids and mineral salts amongst
  other things. The temperature and pH are
  monitored constantly to ensure that the
  conditions in the fermenter are optimum for the
  bacteria. The fermenter is also continuously
  stirred and sterile air fed in.
• The penicillin in the resultant broth after 160
  200 hours of fermenting is obtained by solvent
  extraction.

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The Biosynthetic Pathway of b-lactam Analogs
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Central Dogma
DNA
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Replication
Reverse Transcription
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Transcription
RNA
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Translation
Protein
Juang RH (2004) BCbasics
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????? Human Insulin
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Kleismith Kish (1995) Cell and Molecular
Biology, p. 115
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Stryer (1995) Biochemistry, p. 119
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The Gene Clusters of Penicillin Biosynthesis