Semester Exam

Name                 : Tiara

Student Number    : RSA1C110003

1.       Explain the triterpenoid biosynthetic pathway, identify important factors that determine the quantities produced of many triterpenoids.

There are at least 4000 known triterpenes. They are derived from mevalonic acid. Triterpenes are precursors to steroids in both plants and animals. Many triterpenes occur free, but others occur as glycosides (saponins) or in special combined forms.

 

 

Triterpenes are derived from a triterpene squalene that is formed by condensation of two units of farnesyl pyrophosphate. Squalene is then converted to 2,3-squalene epoxide and cyclized by two different pathways to make most triterpenes.

squalene ----> 2,3-squalene epoxide ----> cyclization

Terpenoids are built up from C5 units, isopentenyl diphosphate (IPP). IPP is supplied from the cytosolic mevalonic acid (MVA) pathway and the plastidal methylerythritol phosphate (MEP) pathway. Triterpenoids and sesquiterpenoids are biosynthesized via the MVA pathway. The first diversifying step in triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene catalyzed by oxidosqualene cyclase (OSC). After the cyclization of 2,3-oxidosqualene catalyzed by OSC, a triterpenoid undergoes various modifications including P450-catalyzed oxidation and UGT-catalyzed glycosylation. Blue arrows, OSC-catalyzed steps; red arrows, P450-catalyzed steps; green arrows, additional modifications including UGT-catalyzed steps.

some important factors are:

·        squalene synthetase (enzyme, , carries out all the steps from FPP to squalene and also there are two catalytic sites involved.

·        oxidosqualene cyclase as a catalyse in cyclization

The two most important types of triterpenes are derived by orientation of the precursor squalene epoxide in two distinct arrangements followed by condensation. The other main type leads to pentacyclic triterpenes. Pentacyclic triterpenes are produced by arrangement of squalene epoxide in a chair-chair-chair-boat arrangement followed by condensation. These compounds are also extremely common and are found in most plants. Common representatives of this group are α-amyrin, β-amyrin, friedelin, oleanolic acid and taraxerane. (see the picture above)

The other image for more understand

 

2.      Describe the structure determination of flavonoids, specificity and intensity of absorption signal by using IR    and NMR spectra. Give the example, at least two different structures.

Determining the Structure of an Organic Compound

• The analysis of the outcome of a reaction requires that we know the full structure of the products as well as the reactants

• In the 19^th and early 20^th centuries, structures were determined by synthesis and chemical degradation that related compounds to each other

• Physical methods now permit structures to be determined directly. We will examine:

-     mass spectrometry (MS)

- infrared (IR) spectroscopy

- nuclear magnetic resonance spectroscopy (NMR)

- ultraviolet-visible spectroscopy (VIS)

 

 

IR Spectroscopy

An important tool of the organic chemist is Infrared Spectroscopy, or "IR". IR spectra are acquired on a special instrument, called an IR spectrometer. IR is used to gather information about compound's structure, assess its purity, and sometimes to identify it.

Infrared radiation is that part of the electromagnetic spectrum between the visible and microwave regions. Infrared radiation is absorbed by organic molecules and converted into energy of molecular vibration, either stretching or bending. Different types of bonds, and thus different functional groups, absorb infrared radiation of different wavelengths. A IR spectrum is a plot of wavenumber (X-axis) vs percent transmittance (Y-axis). (Note: wavelength can be used instead of wavenumber and absorbance instead of percent transmittance)

           

• IR region lower energy than visible light (< red – produces heating as with a heat lamp)

• 2.5 ´ 10^-6 m to 2.5 ´ 10^-5 m region used by organic chemists for structural analysis

• IR energy in a spectrum is usually measured as wavenumber (cm^-1), the inverse of wavelength and proportional to frequency

• Specific IR absorbed by organic molecule related to its structure

• IR energy absorption corresponds to atomic movements, such as vibrations and rotations from bending and stretching of bonds between groups of atoms

• Energy is characteristic of the bonding of atoms in a functional group

• Bond stretching dominates higher energy modes

• Light objects connected to heavy objects vibrate fastest: C-H, N-H, O-H

Infrared Spectra: Functional Groups

Characteristic higher energy IR absorptions used to confirm the existence of the presence of functional groups

NMR Spectroscopy

Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained. It relies on the phenomenon of nuclear magnetic resonance and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules.

Chemical shift          : The location of different nmr resonance signals is dependent on both the external magnetic field strength and the rf frequency. Since no two magnets will have exactly the same field, resonance frequencies will vary accordingly and an alternative method for characterizing and specifying the location of nmr signals is needed.

Signal Strength             : The magnitude or intensity of nmr resonance signals is displayed along the vertical axis of a spectrum, and is proportional to the molar concentration of the sample. Thus, a small or dilute sample will give a weak signal, and doubling or tripling the sample concentration increases the signal strength proportionally.

π-Electron Functions   : Pi-electrons are more polarizable than are sigma-bond electrons, as addition reactions of electrophilic reagents to alkenes testify. Therefore, we should not be surprised to find that field induced pi-electron movement produces strong secondary fields that perturb nearby nuclei.

Solvent Effects : Chloroform-d (CDCl₃) is the most common solvent for nmr measurements

Catechin

IR Spectra

 

The IR spectra of (+)-catechin has a broad band around 3400-2600 cm-1 region corresponding to aliphatic and aromatic C-H, phenolic and alcoholic O-H stretching. A band at 1620 cm-1 observed may be due to aromatic C=C stretching. Other stretchings were comparable with IR spectra of authentic (+)-catechin.

NMR spectra

Catechin molecule contains 15 carbons. In the 13C-NMR spectra, signals appeared at δ 27.7, 66.3, 80.9, 93.9, 95.1, 114.5, 115.1, 118.4 due to C-4, C-3, C-2, C-8, C-2́, C-5́, C-6́ carbons respectively and others aromatic carbons showed peaks at δ 99.1, 130.6, 144.6, 144.8, 155.3, 156.1 and 156.4.

Quercetin

IR spectra

 

IR spectrum of the isolated compounds provide information regarding the absorption peak wavenumber hydroxyl group at 3369 cm-1. Hydroxyl group is an OH stretch-bonded (bonded to hydrogen), OH bound to look at the wave numbers 3450-3200 cm-1 which form a broad band with a strong intensity. The existence of the hydroxyl group is also strengthened by the emergence of a wave-CO-at 2956 cm. showed a stretch - CH aliphatic and reinforced by the emergence of absorption at 1498-1359 cm, indicating a stalling - CH. The existence of strain-C = O carbonyl indicated by absorption at wave numbers 1658 cm-1. Wavenumber absorption band at 1606 cm-1 indicate the presence of C = C stretch Absorption band at wavenumber 1574 cm. Regional absorption at wave number 821 cm-1 indicate the presence of two neighboring H in the aromatic ring.

NMR spectra

3.   In isolation of alkaloids, in initial stages required acid or base conditions. Explain the basis of the use that reagents, and give examples of at least three kinds of alkaloids.

The use of thet reagent is to liberate the salt bond into Free Alkaloidal. It has been observed that the alkaloids invariably occur in the plant sources as the salt of acids, such as: oxalates, tannates etc. Therefore, when the plant substance is exposed to an alkaline medium, the alkaloidal salts are readily converted to the corresponding alkaloid bases.

Choice of Alkali Indeed, the choice of a suitable mineral base (alkali) for the ease of liberation of the alkaloid from the salts is not only very vital but also equally significant and largely depend on the following factors, namely:

(a) Natural state of the alkaloids: It has been observed that the salt of a strongly basic alkaloid with a mineral acid usually tends to undergo cleavage under the influences of a stronger base. Likewise, the corresponding salt of a weakly basic alkaloid and a relatively weak organic acid shall require a rather weaker base for its cleavage.

(b) Chemical characteristics of the alkaloidal base: The usage of strong alkali e.g., NaOH or KOH should be avoided as far as possible by virtue of the fact that certain alkaloids undergo hydrolysis on prolonged contact with a strong base.

Example :

1.         Nicotine

Alkaloid extracted from the leaves of plants, flowers, fruit, bark, and roots are dried and then crushed. Alkaloid extracted with solvents, eg ethanol, and then evaporated. Then, Extracts were obtained inorganic acids to produce a quaternary ammonium salt and then extracted again. Quaternary ammonium salt obtained was treated with sodium carbonate to produce these alkaloids were then extracted with a solvent-free such as ether and chloroform. A mixture of alkaloids obtained finally separated in various ways, such as chromatographic methods.

2.       Caffeine

Caffeine extraction from tea powder, the solubility of caffeine in water is 22mg/ml at 25°C, 180mg/ml at 80°C, and 670mg/ml at 100°C. Here the organic solvent Dichloromethane is used to extract caffeine from aqueous extract of tea powder because caffeine is more soluble in dichloromethane (140mg/ml) than it is in water (22mg/ml).The dichloromethane - caffeine mixture can then be separated on the basis of the different densities of dichloromethane and water because dichloromethane is much denser than water and insoluble in it. Residual water is separated from dichloromethane by drain out the dichloromethane through separating funnel, thus dichloromethane passed through the funnel while polar solvents such as water is still remains in the funnel. Water and dichloromethane is slightly soluble in each other. So, after separating the solvents, residual water will remain the organic layer. Mainly anhydrous sodium sulfite is used for the removal of water from organic layer. Anhydrous sodium sulfite is an insoluble inorganic solid which will absorb water, thus drying it.

3.       Morphine

Extract morphine from poppy straw. In the opium poppy the alkaloids are bound to meconic acid. The method is to extract from the crushed plant with diluted sulfuric acid, which is a stronger acid than meconic acid, but not so strong to react with alkaloid molecules. The extraction is performed in many steps (one amount of crushed plant is at least six to ten times extracted, so practically every alkaloid goes into the solution). From the solution obtained at the last extraction step, the alkaloids are precipitated by either ammonium hydroxide or sodium carbonate. The last step is purifying and separating morphine from other opium alkaloids. Opium poppy contains at least 40 different alkaloids, but most of them are of very low concentration. Morphine is the principal alkaloid in raw opium and constitutes ~8-19% of opium by dry weight (depending on growing conditions)

4.   Describe the relationship between biosynthesis, methods of isolation and structural determination of natural product compounds. Give an example.

The relationship between  biosynthesis, isolation and structural determination were biosynthesis conducted for the formation of natural products compound with some specific molecules using basic reaction,  after knowing the result of the natural compound than ​​isolate it to test the component of natural product in the sample to be tested. Isolation is done by separating some compounds present in the sample using a solvent to produce a single compound structure to be determined in several ways. Identification cab be use IR or  NMR spectra.

Example : Nicotine

 

Nicotine (C10H14N2) is a naturally occurring liquid alkaloid. An alkaloid is an organic compound made out of carbon, hydrogen, nitrogen and sometimes oxygen. These chemicals have potent effects on the human body.

Nicotine biosynthesis

 

The biosynthetic pathway of nicotine involves a coupling reaction between the two cyclic structures that compose nicotine. Metabolic studies show that the pyridine ring of nicotine is derived from nicotinic acid while the pyrrolidone is derived from N-methyl-Δ¹-pyrrollidium cation. Biosynthesis of the two component structures proceeds via two independent syntheses, the NAD pathway for nicotinic acid and the tropane pathway for N-methyl-Δ¹-pyrrollidium cation.

The NAD pathway in the genus nicotiana begins with the oxidation of aspartic acid into α-imino succinate by aspartate oxidase (AO) (an enzyme that catalyzes the chemical reaction). This is followed by a condensation with glyceraldehyde-3-phosphate and a cyclization catalyzed by quinolinate synthase (QS) to give quinolinic acid. Quinolinic acid then reacts with phosphoriboxyl pyrophosphate catalyzed by quinolinic acid phosphoribosyl transferase (QPT) to form nicotinic acid mononucleotide (NaMN). The reaction now proceeds via the NAD salvage cycle to produce nicotinic acid via the conversion of nicotinamide by the enzyme nicotinamidase.

The N-methyl-Δ¹-pyrrollidium cation used in the synthesis of nicotine is an intermediate in the synthesis of tropane-derived alkaloids. Biosynthesis begins with decarboxylation of ornithine by ornithine decarboxylase (ODC) to produce putrescine. Putrescine is then converted into N-methyl putrescine via methylation by SAM catalyzed by putrescine N-methyltransferase (PMT). N-methylputrescine then undergoes deamination into 4-methylaminobutanal by the N-methylputrescine oxidase (MPO) enzyme, 4-methylaminobutanal then spontaneously cyclize into N-methyl-Δ¹-pyrrollidium cation.

The final step in the synthesis of nicotine is the coupling between N-methyl-Δ¹-pyrrollidium cation and nicotinic acid. Although studies conclude some form of coupling between the two component structures, the definite process and mechanism remains undetermined. The current agreed theory involves the conversion of nicotinic acid into 2,5-dihydropyridine through 3,6-dihydronicotinic acid. The 2,5-dihydropyridine intermediate would then react with N-methyl-Δ¹-pyrrollidium cation to form enantiomerically pure (–)-nicotine

Isolation

Alkaloid extracted from the leaves of plants, flowers, fruit, bark, and roots are dried and then crushed. Alkaloid extracted with solvents, eg ethanol, and then evaporated. Then, Extracts were obtained inorganic acids to produce a quaternary ammonium salt and then extracted again. Quaternary ammonium salt obtained was treated with sodium carbonate to produce these alkaloids were then extracted with a solvent-free such as ether and chloroform. A mixture of alkaloids obtained finally separated in various ways, such as chromatographic methods.

Structural Determination using IR spectra

 

On this spectrum, we can notice several peaks, which characterise the different chemical functions of nicotine:

·    Around 3400 cm^-1, we can see the large peak of water (it deals with a liquid film).

·    Between 2970 and 2780 cm^-1 : C-H stretching.

·    The peak at 1677 cm^-1 : aromatic C=N double bond stretching.

·    The peak at 1691 cm^-1 : aromatic C=C double bond stretching.

·  The peaks at 717 cm^-1 and 904 cm^-1 correspond to the out of plane bending of the C-H bond of the monosubstituted pyridinic cycle.