Transcript Biocatalysis in Organic Synthesis

Biocatalysis in Organic Synthesis
References
Biotranformation In Organic Chemistry
Kurt Faber, 4th Edition
Springer-Verlag
 Nature Insight, Biocatalysis
Nature, 2001, 409

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.
Tetrahedral Structure of
Methane
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sp3 orbitals on C overlap with 1s orbitals on 4
H atom to form four identical C-H bonds
Each C–H bond has a strength of 438 kJ/mol
and length of 110 pm
Bond angle: each H–C–H is 109.5°, the
tetrahedral angle.
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
Amino Acids
Building 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.
The assignment of (R) and (S) notation for glyceraldehyde and
L-alanine .
Zwitterion

Amino acid exists as a dipolar ion.
-COOH loses H+, -NH2 gains H+.
•
Actual structure depends on pH.

=>
Structure and pH
=>
The Acidic Residue of the
Proteins
Sodium Glutamate

味精的化學名稱是麩氨酸鈉﹐麩氨酸是
一種氨基酸﹐是一種非必需的氨基酸。
和食鹽一樣﹐味精也是鈉的來源之一。
味精含有13%的鈉（食鹽為39%）﹐
因此高血壓患者及必需控制鈉鹽攝取的
人﹐不但要控制食鹽﹐也要限制味精攝
取。
味精的發酵



味精的化學名稱是麩酸鈉(MONOSODIUM GLUTAMATE)，因
具有強烈鮮味，世界各國普遍作為食品加工及膳食調味之用，而
成為重要民生日用品之一。
味精是麩酸的納鹽，所謂麩酸，是一種有益人體的氨基酸。公元
1866年德國人(RITTHAUSEN)博士從研究麵筋分解中，首先發
現了麩酸鈉。經過42年之後，始經日本池田博士繼續研究於
1908年試驗成功，獲得專利權，同年由德國人GROF氏獲得，惟
其使用原料乳酪，再經共同研究結果，得知除麵筋、乳酪外，凡
含有蛋白質較豐富的肉品如牛肉、魚、黃豆等，皆可提製味精。
麩酸鈉之成為工業產品，始于日本鈴木商店，即味之素株式會社
前身，其所生產味精，以味之素名稱開始問世後，迄今已有80多
年的歷史。
最初日本味之素係利用小麥為原料，採用蛋白質分解法製造，這
雖製造方法繼續半個世紀之久，直到1956年，日本協和公司發
明醱酵法製造味精以後，包括我國在內的世界製造味精國家亦先
後迎頭趕上，於是舊的蛋白質分解法宣告淘汰。
味精的發酵

生產味精的主要原料西元1958年利用微
生物生產味精的醱酵技術開發成功，主
要是利用葡萄糖、果糖或蔗糖為糖源，
經特別篩選的味精生產菌種吸收代謝後，
合成大量的麩胺酸，是屬於生物合成的
天然胺基酸。這些特別篩選的微生物會
將糖蜜中的糖轉變成麩胺酸。每消耗一
公斤的糖，約可產生0.5公斤的麩胺酸，
生產效率非常高。
D-Glucose can cyclize in two ways, forming
either furanose or pyranose structures.
Figure 19.4
The tricarboxylic acid cycle.
Figure 25.9
The glutamate dehydrogenase reaction.
Amino Acids Can Join Via Peptide Bonds
Figure 4.2
The -COOH and NH3+ groups of two
amino acids can
react with the
resulting loss of a
water molecule to
form a covalent
amide bond.
蛋白質的四級構造
一
級
構
造
二
級
構
造
三
級
構
造
四
級
構
造
Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.129
Figure 15.22
The 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
() 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)
Chemical Conversion by Use
of Proteins
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.
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
Reactions of Amides
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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
Amino Acids Can Join Via Peptide Bonds
Figure 4.2
The -COOH and NH3+ groups of two
amino acids can
react with the
resulting loss of a
water molecule to
form a covalent
amide bond.
The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA
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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
The catalytic triad
of chymotrypsin .
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.
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.
Ligation of the Peptides,
Subtiligase
知識問題
洗衣粉添加酵素真的有用嗎?

洗衣粉的添加劑包括：酶製劑、漂白劑、螢光增白劑、香料等物
質。洗衣粉通過添加不同的添加劑使其具有不同的洗滌功效。比
如有些洗衣粉內含有藍色、藍綠色或紅色的顆粒，一般來說，有
這些色粒就說明這種洗衣粉中加有酶製劑。酶製劑就是俗稱的
「酵素」，包括：蛋白酶、脂肪酶、澱粉酶、纖維酶和複合酶等。
蛋白酶可去除 蛋白污漬；脂肪酶可去除各種不同的污漬；澱粉酶
去除澱粉污漬；纖維酶可使容易起“球”的纖維平整光亮；還有
複合酶，它可以起到幾種酶所起的共同作用。其他的添加劑，如
漂白劑可除去能被氧化的污漬；螢光增白劑能將紫外光轉變為可
見藍色光，使織物外觀亮麗；香料能掩蓋污垢怪味等等。
Figure 9.2
Several spontaneously formed lipid structures.
Enzymes display three major
types of selectivities:
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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 Lamino 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.
The Chemoselectivities of the Enzymes
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.
Biocatalysis in Green Chemistry
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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.
The use of biocatalysis to obtain
enantiopure compounds
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Kinetic resolution of a racemic
mixture
Biocatalysed asymmetric synthesis
Kinetic resolution of a racemic
mixture
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.
dynamic resolution
If it is possible to perform such
resolutions under conditions where the
two substrate-enantiomers are
racemizing continuously, all substrate
may in theory be converted into
enantiopure product.
Deracemization Reaction
monoamine oxidase isolated from Aspergillus niger
Biocatalysed asymmetric
synthesis
Reduction of Carbonyl
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Reduction of aldehyde yields 1º alcohol.
Reduction of ketone yields 2º alcohol.
Reagents:
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Sodium borohydride, NaBH4
Lithium aluminum hydride, LiAlH4
Raney nickel
=>
Sodium Borohydride
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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.
O
C
H
H
H
C
H
O
+
H
H3O
O H
C
H
=>
Lithium Aluminum Hydride

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Stronger reducing agent than sodium
borohydride, but dangerous to work with.
Converts esters and acids to 1º alcohols.
O
C
OCH3
H
LAH
H3O+
C
O H
H
=>
Catalytic Hydrogenation
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Add H2 with Raney nickel catalyst.
Also reduces any C=C bonds.
OH
O
NaBH4
OH
H2, Raney Ni
=>
Figure 17.10
Hydrogen 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.
Biocatalysed asymmetric synthesis
for carbonyl reduction
Yeast is a biocatalyst for the enantioselective reduction of ketones.
Baeyer-Villiger oxidation
The Mechanism
example
Baeyer-Villiger oxidation using
Whole Cell Catalysis
Baeyer-Villiger monooxygenase or BVMO
cofactors such as NADPH
Drug Manufacture and Discovery by
Microbial Bioconversion
-The industrial conversion and
biosynthesis of Penicillin
Penicillin 青黴素
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.
佛來明發現青黴素
Fleming
Chain
Florey
(1945)
牛頓 (1985) no. 25 p. 122
佛來明（Alexander Fleming）於1928年發現黴菌（青
黴菌Penicillium）抑制細菌（金黃葡萄球菌
Staphylococcus）的現象；佛羅瑞（Howard Florey）
和芡恩（Ernst Chain）則於1939年從青黴菌分離青黴
素（penicillin）證明其治療細菌感染的功效。其後
（1940到1945年）在美國發展出大量生產青黴素的技
術，這是結合微生物學家、生化學家、和遺傳學家篩
選並改造出能在沉浸液體培養（submerged liquid
culture）中大量累積與菌體生長沒有直接關聯的二級
代謝產物（secondary metabolites）且進行好氧
（aerobic）生長的絲狀真菌，並透過工程師的努力以
通氣攪拌醱酵槽大量生產的結果。佛來明、佛羅瑞、
芡恩於1945年獲得諾貝爾獎。
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.
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.
The
Biosynthetic
Pathway of
b-lactam
Analogs
Central Dogma
複製
Replication
DNA
轉 錄 Transcription
Reverse
逆轉錄 Transcription
RNA
轉 譯 Translation
Protein
Juang RH (2004) BCbasics
人類胰島素
Human Insulin
目標基因剪接
Kleismith & Kish (1995) Cell and Molecular Biology, p. 115
基因接入載體
Stryer (1995) Biochemistry, p. 119
送入宿主細菌
挑出所要群落
探針 Probe
互補 DNA
專一性抗體
得到純系轉殖菌株
大量培養生產
The Gene Clusters of Penicillin
Biosynthesis