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 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.

Molecules that have one carbon with 4 different substituents have a nonsuperimposable mirror image – enantiomer

Build molecular models to see this

Enantiomers and the Tetrahedral Carbon

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.

Amino AcidsBuilding Blocks of Proteins

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 19.4The tricarboxylic acid cycle.

Figure 25.9The glutamate dehydrogenase reaction.

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.

Amino Acids Can Join Via Peptide Bonds

一級構造

蛋白質的四級構造

四級構造

二級構造

三級構造

Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.129

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() and two of 146 residues (); mass = 64.45 kD. Each polypeptide has a heme; the Hb tetramer binds four O2. (Illustr

ation: 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 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

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.

Amino Acids Can Join Via Peptide Bonds

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

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 9.2Several spontaneously formed lipid structures.

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.

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

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 Another important advantage of biocatalyst

s 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

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.

monoamine oxidase isolated from Aspergillus niger

Deracemization Reaction

Biocatalysed asymmetric synthesis

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

=>

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.

HC

O

HC

H

OHC

H

OH HH3O+

=>

Lithium Aluminum Hydride

Stronger reducing agent than sodium borohydride, but dangerous to work with.

Converts esters and acids to 1º alcohols.C

O

OCH3C

OH H

HH3O+

LAH =>

Catalytic Hydrogenation

Add H2 with Raney nickel catalyst. Also reduces any C=C bonds.

O

H2, Raney Ni

OH

NaBH4

OH

=>

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.

Biocatalysed asymmetric synthesisfor 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

牛頓 (1985) no. 25 p. 122

FlemingChainFlorey(1945)

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 st

ainless steel fermenters, which have a capacity of around 10000 dm3.

                                                                                                                                                                                                                                                      

Fermentation of Penicillin The fermenter is sterilised by steam and then load

ed 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 -lactam Analogs

RNA

DNA

Protein

轉 錄

轉 譯

Transcription

Translation

Replication複 製

逆轉錄 ReverseTranscription

Central Dogma

Juang RH (2004) BCbasics

挑出所要群落

得到純系轉殖菌株 大量培養生產

人類胰島素Human Insulin

探針 Probe

互補 DNA

專一性抗體

送入宿主細菌

基因接入載體

目標基因剪接

Stryer (1995) Biochemistry, p. 119

Kleismith & Kish (1995) Cell and Molecular Biology, p. 115

The Gene Clusters of Penicillin Biosynthesis