Lipase Catalysed Synthesis Of Amides Biology Essay

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Amide is the organic compound which contains the acyl function group connected to nitrogen atom from the carbon atom the single bond which links the carbonyl atom with nitrogen atom is called an amide linkage. (Specer et al, 2004).

The general of amide is (R-CONH2) which is considered one derivative of ammonia that is one hydrogen atom has been replaced by acyl group, amides can be derived from primary amines (R-NH2) to give a primary amides and from secondary amines to give a secondary amides, also amides are regarded as derivatives of carboxylic acids in which the hydroxyl group has replaced by amine. (Specer et al, 2004).

1.3. Physical properties:

At the room temperature, all the amides are solids expect formamide because it is unsubstituted amide, unsubstituted amides can form a network of intermolecular hydrogen bonds and this character makes melting points for these substances so high. However, the substitution of alkyl or aromatic group on nitrogen reduces the number of inter molecular hydrogen bond, consequently the melting point will decrease, so disubstituted amide often have lower melting and boiling points than monosubstituted and unsubstituted amides. Amides are rather water soluble especially which contains less than six carbons and this is due to ability of amides to form hydrogen bond with water even disubstituted amides can do this because of the presence of carbonyl oxygen. . (Specer et al, 2004).

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Fig. 2: The intermolecular hydrogen bonds of amides

1.4. Chemical properties:

1.4.1. Neutrality:

Amides are neither basic nor acidic, they are neutral because the carbonyl group bonded to the nitrogen has decreased the basicity of the original amine

1.4.2. Amide hydrolysis:

Amide hydrolysis is the reaction between amide and water in the presence of acid or base with heat to produce carboxylic acid and amine. In fact, amide hydrolysis is very important reaction in biochemistry because it is a center reaction in the digestion of proteins and the breakdown of proteins with cells. (Specer et al, 2004).

1.5. Applications:

1.5.1. Polymers:

Amides are pervasive in nature and technology as structural materials. The best known of all synthetic amides is the fiber known as nylon. In 1931, the American chemist Wallace Hume Carothers discovered a process for making one of the first synthetic fibers. He found that the addition of adipic acid to hexamethylene diamine resulted in the formation of a strong, fiber-like product to which he gave the name Nylon 66. The 66 part of the name reflects the fact that adipic acid and hexamethylene diamine each contain six carbon atoms in their molecules. The reaction between these two substances results in the formation of a long polymer, somewhat similar to the structure of natural protein. As in protein, the subunits of nylon are joined by amide bonds. For this reason, both protein and nylon can be thought of as polyamides, compounds in which a large number of amide units are joined to each other in a long chain. Other types of nylon were also developed at later dates. One form, known as Nylon 6, is produced by the polymerization of a single kind of molecule, 6-aminohexanoic acid. The bonding between sub-units in Nylon-6-amide bonds is the same as it is in Nylon 66. In all types of nylon, the fiber obtains its strength from hydrogen bonding that occurs between oxygen and hydrogen atoms on adjacent chains of the material. Another type of polymer is formed when two of the simplest organic compounds, urea and formaldehyde, react with each other. In this reaction, amide bonds form between alternate urea and formaldehyde molecules, resulting in a very long polyamide chain. Urea formaldehyde polymers are in great demand by industry, where they are used as molding compounds, in the treatment of paper and textiles, and as a binder in particle board, to mention but a few uses.

(Raymond Seymour et al, 1986)

1.5.2. Drugs:

1.5.2.1. General drugs:

Amides have physiological properties, a number of amides play valuable rules in medicine, for example the amide acetaminophen marked under name of Tylenol which is considered as amide and used as pain reliever represented in fig 2

Fig. 2: Acetaminophen

Table 1 below shows a few important amides and their uses.

Table .1: Some important amides in medicine (Specer et al, 2004).

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Structure

Trade name

Use

Thiopental

(Pentothal)

Intravenous

anesthesia

Amobarbital

(Amytal)

Treatment of Insomnia

Diazepam

(Valim)

Tranquilizer

Ampicillin

(Polycillin)

Antibiotic

1.5.2.2. Anticancer drugs:

One of the most important applications of amides as anticancer agent for many cancer types, for example, the amide alkolides (1-3) are evaluated for their anticancer activity againist colon (HT-29(, breast (MCF-7) and lung(A549) human cancer cell lines (Srinivas et al,2009).

Fig. 3: Examples of the amide alkolides

Moreover, N-substituted amides of 3-(3-ethylthio-1, 2, 4-triazole-S-yl) propenoic acid were found to be biologically active and effective in vitro against lung cell line. (Anna Pachuta et al, 2009).

1- 2- 3- 4-

Fig. 4: Examples of the amide 3-(3-ethylthio-1, 2, 4-triazole-S-yl) propenoic acid

1.6. Synthesis:

Amides are commonly formed via reactions of a carboxylic acid with an amine. Many methods are known for driving the unfavorable equilibrium to the right:

RCO2H + R'R"NH RC(O)NR'R" + H2O

For the most part, these reactions involve "activating" the carboxylic acid and the best known method, the Schotten-Baumann reaction, which involves conversion of the acid to the acid chlorides: (Michael B. Smith et al, 2001)

Table .2: Some important reactions for synthesis of amides

Reaction name  

Substrate  

Comment

Beckmann rearrangement

cyclic ketone

reagent: hydroxylamineand acid

Schmidt reaction

ketones

reagent: hydrazoic acid

nitrile hydrolysis

nitrile

reagent: water; acid catalyst

Willgerodt-Kindler reaction

aryl alkyl ketones

sulfur and morpholine

Passerini reaction

carboxylic acid, ketone or aldehyde

Ugi reaction

isocyanide, carboxylic acid, ketone, primary amine

Bodroux reaction

carboxylic acid, Grignard reagentwith an anilinederivative ArNHR'

2-Lipase

2.1. Definition:

A lipase is a water soluble enzyme of considerable physiological significance and industrial potential properties which catalyzes the hydrolysis and synthesis of ester bonds formed from glycerol and long chain of fatty acids. (Monica Fernandez and Cristina Otero, 2001).

lipases are involved in many stages of lipid metabolism including fat digestion, reconstitution, absorption and lipoprotein metabolism, many lipases are very active in organic solvents, they catalyze a number of a useful reactions such as: esterification, transesterification, regioselective acylation of glycol and synthesis of peptides, commercially useful lipases are usually obtained from microorganisms. (Rohit Sharma et al, 2001)

2.2. Applications:

Lipases occur widely in nature, but only microbial lipases are commercially significant, lipases have many applications including organic synthesis, hydrolysis of fats and oils, resolution of racemic mixture, detergents, food processing, synthesis of fine chemicals, production of cosmetics and pharmaceutical chemistry, major applications of lipases are summarized in table.3 (Rohit Sharma et al, 2001)

Table.3: Industrial applications of Microbial lipases

Industry Action Product or application

Detergents Hydrolysis of fats Removal of oil stains from fabrics

Dairy foods Hydrolysis of milk fat Development of flavoring agents in milk

Bakery foods Flavor improvement Shelf-life prolongation

Beverages Improved aroma Beverages

Food dressings Quality improvement Mayonnaise, dressings, and whippings

Health foods Transesterification Health foods

Meat and fish Flavor development Meat and fish products; fat removal

Fats and oils Transesterification; hydrolysis Cocoa butter, margarine, fatty acids

Chemicals Enantioselectivity, synthesis Chiral building blocks, chemicals

Pharmaceuticals Transesterification, hydrolysis Specialty lipids, digestive aids

Cosmetics Synthesis Emulsifiers, moisturizers

Leather Hydrolysis Leather products

Paper Hydrolysis Paper with improved quality

Cleaning Hydrolysis Removal of fats

Literature review

Natural products which contain amide group are biologically interesting molecules and play an important role in modern drug discovery, especially in cancer treatment (ch srinivas et al, 2009).

There are many plants in the world contain so important natural products which have cytotoxic activity a against human cancer cells. One of the most important plants is Litsea (L. auraceae) which comprises nearly 200 species, which are widely found in tropical and subtropical Asia, North America, and subtropical South America. The Litsea plants have been reported to have a significant cytotoxic activity against human tumor cells, including human breast adenocarcinoma, non-small-cell lung cancer, and glioblastoma cell lines . (Hitoshi Tanaka et al, 2009).

Hitoshi Tanaka described the isolation and structural elucidation of a new amide, N-trans-sinapoylmethoxytyr amine figure (1), along with three known amides (2-4).

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Figer.5: The four amides extracted from litsea

After making full review for the literatures, it was concluded that compound (1) has never been synthesized and there is no any suggested method for its synthesis, compound (2) has only one method for synthesis (Jae Park et al, 2003), with a low yield (55.0)%, compound (3) has been prepared by (Hitoshi Tanaka et al, 1989), also with a low yield (55.0)%, compound (4) was prepared by reacting amino ethyl phenol with chloro carbonyl vinyl methoxy ester, (Esaku Nomura et al,2003) , although this method gave good yield (94.0)% but it needs hydrazine as a reagent which considered dangerous chemical . Also (Jae Park et al, 2003) have synthesized compound (4) with a low yield (55.0) %.

In addition to them, Yoshimitsu have synthesized compound (4) was synthesized by the coupling of ferulic acid with tyramine in t-BuOH using (DCC) (Yoshimitsu Yamazaki et al, 2008).

Nowadays, many catalysts used to synthesize amides, but biocatalysts enzymes such as lipases catalysts are considered the best catalysts because of its high stability in organic media and their ability to accept a great variety of substrates (Monica Fernandez and Cristina Otero, 2001).

Problem of the research

Litsea plants are considered a major source for natural products which have a significant cytotoxtic activity against human tumor cells, therefore four amides (1),(2),(3) and (4) which are components of litsea plants are so valuable and important compounds to be studied.

A new amide N-trans-sinapoylmethoxytyr amine (1) which was discovered by (Hitoshi Tanaka et al, 2009), who has isolated compound (1) from the leaves and twigs of L. auriculata. However, there is no any suggested method for the synthesis of compound N-trans-sinapoylmethoxytyr amine compound (1), compound (2) has only one method for synthesis (Jae Park et al, 2003), with a low yield (55.0)%, compound (3) has been prepared by (Hitoshi Tanaka et al, 1989), also with a low yield (55.0)%, compound (4) was prepared by (Esaku Nomura et al,2003) , although this method gave good yield (94.0)% but it needs hydrazine as a reagent which considered as a dangerous chemical . Also (Jae Park et al, 2003) have synthesized compound (4) with a low yield (55.0) %. In addition to them, (Yoshimitsu Yamazaki et al, 2008) have synthesized compound (4) was synthesized by the coupling of ferulic acid with tyramine in t-BuOH using (DCC).

The yield of the extraction method from the plant is too poor (each 10 kg of the plant is required to extract only 60 mg of the compound (1), 133 mg of compound (2), 28.9 mg of compound (3) and 47 mg of compound (4). Therefore, it is important to find a new direct method to synthesis compounds (1), (2), (3) and (4).

Main goals

The first goal of this research is to synthesize the amides, N-trans-sinapoylmethoxytyr amine compound (1), N-trans-sinapoyltyr amine compound (2), N-trans-feruloylmethoxytyr amine compound (3) and N-trans-feruloyltyr amine compound (4).

To optimize the best synthesis conditions for each compound.

Statistical (ANOVA) test and (RSM) analysis for the data will be calculated.

To study kinetics of the reactions for each compound.

To study the anticancer activity for each compound.

Methodology

Synthesis:

Compound (1) N-trans-sinapoylmethoxytyr amine will be synthesized by reacting (3, 5-Dimethoxy-4-hydroxycinnamic acid) with (3-O-Methyldopamine hydrochloride) in the presence of Lipase catalyst.

Compound (2) N-trans-sinapoyltyr amine will be synthesized by reacting reacting (3, 5-Dimethoxy-4-hydroxycinnamic acid) with (Tyramine hydrochloride) in the presence of Lipase catalyst.

Compound (3) N-trans-feruloylmethoxytyr amine will be synthesized by reacting reacting (4-Hydroxy-3-methoxycinnamic acid) with (3-O-Methyldopamine hydrochloride) in the presence of Lipase catalyst.

Compound (4) N-trans-feruloyltyr amine will be synthesized reacting (4-Hydroxy-3-methoxycinnamic acid) with (Tyramine hydrochloride) in the presence of Lipase catalyst.

The equation below represents the scheme of synthesis:

Identification of the new compounds using IR, NMR and test the purity of it using HPLC.

Studying the cytotoxic activity and bioactivity of the four amide compounds against cancer.

Optimize the best conditions for the reaction by studying:

Effect of time on the reaction

Effect of temperature on the reaction

Effect of the organic solvent on the reaction

Effect of molar ratio on the reaction

Effect of lipase amount on the reaction

Expected Results

Four amides will be prepared using direct synthesis in presence of lipase catalyst, N-trans-sinapoylmethoxytyr amine compound (1) which is new amide, N-trans-sinapoyltyr amine compound (2), N-trans-feruloylmethoxytyr amine compound (3) and N-trans-feruloyltyr amine compound (4).

The yield of the products for the synthesis method expected to be good.

One amide at least expected to be bioactive against cancer cells.

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