"Chemistry of Natural Products Test"

SEMESTER FINAL EXAM MATERIALS CHEMISTRY CULTURE

Name                          : Chitra Karina Dewi

NIM                            : RSA1C110006

Course                        : Chemistry of Natural Materials

Credits                        : 2

Lecturer                     : Dr. Syamsurizal, M.Si

Time                           : 22-29 December 2012

INSTRUCTIONS: This exam open book. But no cheating allowed, if it is found, then you otherwise FAIL. Your answers posted bolg respectively.

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

Answer:

Triterpenoid Biosynthesis

            Triterpenoids including steroids are a highly diverse group of natural products widely distributed in plants. Plants often accumulate these compounds in their glycosylated form – saponin. Saponins comprise hydrophobic triterpenoid aglycones called sapogenin and one or more hydrophilic sugar moieties.

            Triterpenoids have a very bitter taste, especially present in Rutaceae plants, Meliceae and Simaroubeaceae as limonin in citrus fruits (also classified as a sense of bitter alkaloids) and kukurbitasin D in Cucurbitaceae plants and diosgonin. In the form of triterpenoids found in plant sap of Euphorbia and Havea.

Triterpenoid Biosynthesis

            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, whereas monoterpenoids, diterpenoid, and tetraterpenoids are biosynthesized via the MEP pathway. The first diversifying step in triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene catalyzed by oxidosqualene cyclase .

 Figure 1. Triterpenoid biosynthetic pathway. 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.

            After an OSC constructs the basic triterpenoid skeleton, the skeleton is modified to a hydrophobic aglycone called sapogenin. The first modification is oxidation catalyzed by cytochrome P450 monooxygenase (P450), and this step enables further modifications such O-glycosylation. P450 is highly diverse and catalyzes several kinds of chemical reactions committed to the secondary metabolism .

Glycosylation is essential for saponin biosynthesis. Glycosylation increases the water solubility and changes the biological activity of triterpenoid. Uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) recognize a wide range of natural products as acceptor molecules.P450 species and UGTs belong to multigene families and are the key factors for explosive diversification of other natural products in plants. In the case of reported P450 species in saponin biosynthesis, those CYP families vary respecting not only the carbon skeletons of the triterpenoid substrates but also the target positions of the reactions. The diversity of these enzymes makes identification of the genes for saponin biosynthesis difficult. The genes involved in triterpenoid biosynthesis identified in plants to date are presented as follows.

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

            Infrared spectrophotometry is more widely used for identification of a compound through the group functions. For the purpose of structure elucidation, the wavenumber region 1400 - 4000 cm-1 which is at the left-ir spectrum, an area that is particularly useful for the identification of functional groups, which is the absorption of the stretching vibration.
 

            Nuclear Magnetic Resonance (NMR) is one of the easiest methods of analysis used in modern chemistry. NMR is used to determine the structure of natural and synthetic components are new, the purity of the components, and the direction of chemical reactions in solution as well as the relationship of components that can undergo chemical reactions.

            In the determination of the structure of flavonoid compounds by using IR spectroscopy can be seen that the structure of flavonoids that have the carbon - carbon aromatic, carbon single bonds and oxygen, carbon single bonds - hydrogen and oxygen-hydrogen single bond in which the data obtained carbon double bond - Carbon C = C: has a catchment area of ​​absorption of light at 1500 - 1600 cm-1 with medium intensity absorption and sharp. Double bond carbon - oxygen C = O: is one of the very useful absorption, which can be found in the area around 1705 - 1725 cm-1 with a strong and sharp absorption intensity. Single bond carbon - oxygen C-O: having absorption in the 'fingerprint region', which can be found in the surrounding area between 1000 - 1300cm-1, with a weak and broad absorption intensity. Carbon single bonds - hydrogen C - H: having light absorption occurs in the region 3050-3150 cm-1 absorption, the absorption intensity due to the range of weak and sharp C - H aromatic. Oxygen single bonds - Hydrogen O - H: absorb light differently, depending on environmental conditions. Bond O - H will be very easy to spot in a sour because it will result in absorption intensity width or valley that is very knowledgeable on the area around 3200-3500 cm-1.

            And the NMR spectrum of the 1 H-NMR spectroscopy, showing signs of the H atom bound and develop a molecular signal H atom bound to O atoms in benzene is shown first then you will see the signal from H atoms bound to C atoms in benzene and the latter signal appears the H atoms bound to O atoms in the benzene that have double bonds with other O atoms. All signals that appear to have such a sharp intensity.

Examples of flavonoid compounds that can be produced through spectroscopic IR and NMR:

a) -> IR spectra of antochianin

         In Cluster-OH is around 3700-3100 cm -1 absorption bands induce a strong signal. and in group C = O is the absorption at 2100 cm-1 regions have a fairly small signals. on the C = C absorption is shown by region 1600 cm-1 with the signal being. And on the CO absorption region showed approximately 1080-1300 cm -1 with a small signal absorption.

   ->NMR spectrum antochianin

     At resonance anthocyanin is 6-7 minutes with a very strong signal and shows a very high retention, while resonances were also detected at the OH group. With about 9-10 minutes resonance region that has a strong signal and there is a high retention.

b)  Catechins
Determination of the wavelength of maximum absorption of catechins in the solvent water and acetate buffer showed the same maximum wavelength is 279 nm, as shown below:

 Figure 2. The wavelength of maximum absorption of catechins in acetate buffer pH 5.2.

Catechins have dikompleks with β-cyclodextrin provide maximum absorption wavelength equal to 279 nm, namely catechins. β-cyclodextrin is a cyclic oligosaccharide consisting of 6-8 glucose units, has no chromophore groups so that no absorption by UV-Vis spectrophotometer (Yuwono, 1999). So absorption spectrophotometer was read at the absorption of catechins.

In determining the levels of catechins that are dissolved in the water used standard curve by spectrophotometry at a wavelength of maximum absorption. From this calibration curve, the calibration curve obtained by the equation y = 11.589 x + 0.0035 with r = 0.9999.

Figure 3. Catechin calibration curve in water solvent.

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

Answer:

                 Alkaloids in the form of solid crystals with a melting point or have a certain range of decomposition. Can also take the form of amorphous and some like nicotine and Konini liquid.
            Most alkaloids colorless, but some species of aromatic compounds colored complex. In general, only the free base alkaloids soluble in organic solvents although some pseudoalakaloid and protoalkaloida soluble in water. Quaterner alkaloids alkaloids and salts are very soluble in water.

                 Alkaloids are alkaline-dependent electron pair on nitrogen that is alkaloids is base. If the functional group adjacent to the nitrogen release electrons are electrons in the availability of nitrogen compounds are more up and pull electrons decreases the availability of the electron pair and the effect caused alkaloids can be neutral or even slightly acid.
Basicity alkaloids causing compounds are very susceptible to decomposition by heat and light, especially in the presence of oxygen. The reaction product is often in the form of N-oxide. Decomposition olakloida during or after isolation can cause problems if the storage lasts a long time. Formation of salts with organic or inorganic compounds often prevent decomposition.

Reaction Compounds Alkaloids

The general reaction for alkaloid

1. The precipitation reaction for alkaloid

Reaction Mayer: HgI2

· How to: + reactant substances arising Mayer precipitate yellow or clear yellow solution → + alakohol sediment dissolves. Reactions were performed in glass and crystal objects can be seen in the microscope. If done in a test tube and then transferred, Crystals can be damaged. Not all of the alkaloids precipitated by reaction mayer. Deposition occurs due to the reaction depends on the formula mayer alkoloidnya wake.

Reaction Bouchardat

· How to: sample + reagent substances Bouchardat → red brown, + alcohol → precipitate dissolved.

2. Color reaction

    * With strong acid: concentrated H₂SO₄ and HNO₃ concentrated (generally produces yellow or red)

    * Marquis Reagent

          o Substance + 4 drops of formalin + 1 ml of concentrated H₂SO₄ (through the wall of the tube, it slowly) → color.

          o Forhde Reagent: a solution of 1% NH₄ molybdate in concentrated H₂SO₄

§ Substance + tawny reagent Forhde → tawny

§ Substance diazo + A (4 parts) + diazo B (1 part) + NaOH until alkaline → intense red color.

§ Reaction Nelzer substances in absolute alcohol solution + 1 drop of CuSO₄ and CS₂ à brown like oil.

§ Mandelin reaction: H₂SO₄ + substance + FeCl₃  

§ Reaction Roux: 1 tts tts KMnO4 NaOH + 1 + 20 tts shake à à Na nitroprusside solution and precipitate, the solution was taken.

§ Reaction Serulas & Lefort: a solution of substance in dilute H₂SO₄ + KI + CHCl₃ à shaken; CHCl₃ layer will be colored.

§ Reaction Huseman: + substances concentrated H2SO4 à heated over a flame to produce apomorphine + 65% HNO₃ + KNO₃ à solid color.

§ Reaction Bosman: solution of the substance in dilute H₂SO₄ + KMnO₄ à shaken with CHCl₃; CHCl₃ layer will be colored violet then brown precipitate formed.

§ Reaction Zwikker: +1 ml Pyridines Substance + CuSO₄ à 10% long stem colorless, non-specific crystal and glass objects created.

§ The reaction of ammonium vanadate Mandelin ½% in water + concentrated H₂SO₄.

§ Reaction Murexide: Substance + 1 drop 3% H₂O₂ or solid KClO₃ + 1 drop 25% HCl, heated in a water bath to dry à little orange; + NH₄OH à Color Purple

§ Reaction Parri: Substance + Co (NO₃) ₂, then + NH₄OH vapor purple.

                      # Vitally reaction: HNO₃ + smoky substance, evaporated on the water bath until dry, spir + / alkali purple, hold in acetone

                            * Apomorphine: red

                            * Strychnine: purple red

                            * Veratrin: chocolate orange

§ Reaction Lieberrman: concentrated H₂SO₄ + HNO₃ concentrated

§ Sanchez reaction: p +-nitrodiabendazol substance (p-nitoanilin + NaNO₂ + NaOH) à à purple orange.

§ Pesez reaction: H₂SO₄ + + lar substances. KBr, heat on the water bath à green, blue à drawn with CHCl3 green.

§ Reaction Thalleiochin: solution of substance in dilute acetic acid + 1 drop of bromine aqua emerald berlebihàhijau NH₄OH + + kloroformàdifloresensi

§ Reaction Erytrochin: solution of substance in dilute HCl + aqua bromine (to yellow) + CHCl₃ + + potassium ferrocyanida NH₄OH, shake homogeneous → CHCl₃ layer red.

§ Reaction Sanchez. (Reagent: p-nitronilin saturated solution in 1% H₂SO₄ + NaNO₂). Substance H₂SO₄ + 75% + 1 drop of reagent + NaOH → blue, acidify with H₂SO₄ → orange.

§ Reaction Feigel: 5 drops of H₂SO₄ pkt + + bit yohimbine ad soluble crystalline chloral hydrate heat in red → blue WB stable, + water → color disappeared.

§ esterification reaction: Substance + alcohol + H₂SO₄ conc. → Heat the distinctive smell.

§ The reaction of isonitriles: Substance + spirit + KOH → heat → → CHCl₃ plus heat again → iso smell nitrile (soon soured as toxic smell / foul)

§ Reaction Runge: Heated with 25% HCl plus NaOH → cool → → weak base ad dirty purple

§ indophenol reaction: HCl → cool Heat with diluted chlorine + water + phenol → → look dirty purple plus excess NH₄OH + HNO₃ → à blue not yellow.

§ Ehrlich reaction: Solids reagent p-DAB + HCl → canary yellow

§ Reaction Wassicky: substance p-DAB + + concentrated H₂SO₄ à purple

§ Reaction matches: HCl + substance and matchstick dipped à orange / yellow.

3. Reaction Crystals:

   1. Reactions Crystal dragendorf

In the glass object, substance HCl + stir, then drops dragendorf at the edges and do not shaken, let stand 1 minute crystals dragendorf

2. 2. The reaction of Fe-Cu-complex and complex:

On the object glass, spilled gas with Fe-Cu-complex compleks and then cover with a cover glass heat briefly, then look at the crystal formed.

1. In glass objects, substances and acid + sprinkled with powdered sublimat spatel, slightly shaken on it à Kristal looks.

2. Iodoform reaction: NaOH until alkaline substances spilled + sol. Iodii then heated to yellow (forming iodoform), then see the cherry blossoms in the microscope crystals.

3. Reaction Herapatiet. (Reagent: methylated + water + vinegar + a little prickly and aqua iodine H₂SO₄ until slightly yellow on the object glass). Substance + 1 drop of reagent → crystal plate (brown / violet)

Identification of Compounds Alkaloids

1. Alkaloid Derivatives Phenyl Alanine

1.1 Alkaloids Amin

1.1.1 Ephedrine HCl

Origin (ephedrine): Ephedra vulgaris

Organoleptic: fine white powder, odorless, bitter taste

Solubility: soluble in approximately 4 parts water

Reaction Identification:

1. Substances in the solution of PbSO₄ + NaOH + water violet.

2. Substances in the solution of 0.1 N NaOH + water + 3 ml CCl₄ shaken, allowed to separate the organic layer less copper + à à whipped then cloudy precipitate is formed.

3. Oxidation by KMnO₄ benzaldehid smell.

4. Iodoform reaction (+)

5. Reaction Nelzer: substance in absolute alcohol solution + 1 drop of CuSO₄ and CS₂ brown oil.

6. Substance + NaOH + sulfanilic red.

7. Substances in the water solution of HCl +, H₂O₂ + NaCl + NaOH + 6 drops of red-violet.

8. Substances in water solution + AgNO₃ precipitate (AgCl), washed with water, + NH₄OH precipitate will dissolve back.

1.2 Alkaloids Benzyl Isokuinolon

1.2.1 Morphine

Origin: Papaver somniferum

Synonyms: Dionin

Organoleptic: white crystal

Solubility: soluble in 12 parts of water

Reaction Identification:

1. Reaction KING, SANCHEZ, and FESEZ (+)

2. Substance FeCl₃ + H₂SO₄ + boiled + HNO₃ berwrna blue red / dark red brown.

1. Iodoform reaction (+)

2. Reaction FROHDE: yellow green.

3. Reaction MANDELIN: yellow green.

4. Reaction MARQUIS: purple for a long time.

5. Substances in the solution of I2 + HCl à precipitate soluble in spirit.

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

 answer:

                  We know that in the presence of a compound biosynthesis, methods of isolation and structural determination of compounds. Which for the scientist, the determination of the structure of a compound is a primary requirement. It was as if they were looking for a structure such as a mother who is looking for his lost son. Previously (before 1900 AD), there is no modern tools (such as IR, NMR, MS, etc.) to determine the structure, but whose name scientists still trying to figure out the structure of a particular compound. Because there is no tool, once scientists can only make comparisons between the structures of unknown compounds structurally similar to compounds whose structures are known, by comparing the physical and chemical properties. If the properties are the same, the same compounds. In addition, once the scientists also rely on chemical reactions in determining the structure, if you have the same reaction to the response with specific reagents, the structure of these compounds are assumed to be equal. For example, compounds A and B are equally treated with Fehling reagent, that both compounds are equally produce brick red precipitate, it is assumed that the same compounds.

            In fact, it is far from the truth, because the compounds are different but equally the aldehyde group, reaction with Fehling reagent will produce a precipitate mostly red brick. Thus, the compound acetaldehyde with butanaldehid together will produce a brick red precipitate when reacted with Fehling reagent. While acetaldehyde and butanaldehid are different compounds, the nature and function differently.

            The need to define the structure to be increased since the scientists are trying to isolate or synthesize a compound. Isolation is the process of extracting (take) a particular chemical compound contained in a material (such as leaves, stems, etc.) with a specific intent. Generally, a compound isolated because it is known that the compound has the activity to treat the disease. Obviously, compounds isolated structure needs to be determined in order to know what the actual compound obtained from the isolation. Besides isolation, scientists often perform synthesis, which is making a desired compound from other compounds. Compounds synthesized structure need to be determined to prove the success of the synthesis has been done.

            The increasing age of the age, technology is growing. In the mid-20th century, began to appear modern tools for the determination of the structure of chemical compounds. Ultraviolet Spectroscopy (UV) was introduced in the 1930's, Infrared Spectroscopy (IR) in the 1940s, Mass Spectroscopy (MS) in the 1950s.

One example is:

Biosynthesis pathway Auron

            Auron is a plant flavonoid that produce yellow flowers on some specific makhkota. Auron does not have the core structure of flavonoids, but biosynthesis is directly derived from kalkon which is a very important precursor of all flavonoids. Higher efficiency in the formation of 6-glucoside of Auron when compared with the same aglycone allow that kalkon-4-glucoside may be the substrate for the biosynthesis of Auron in real interest rates. It has been demonstrated in Antirrhinum magus by isolating kalkon 4'-O-glucosyltransferase that catalyzes the form of tetra-and pentahydroxychalcone 4'-O-glucosides. Intermediate 4'-O-glucoside was converted into 6-O-glucosides by auresidin synthesis. This enzyme is produced and accumulated in the cytoplasm.

            Of particular interest is the synthesis auresidin, (the oxidation of polyphenols (PPO), which catalyzes the change of the tetra-and pentahydroxychalcone be Auron aureusidin and bracteatin,) has placed well in the vacuole lumen. In contrast to all PPOs in other plants associated with plastids. In the present invention discussed the molecular form of the enzyme characteristics such as low pH optimum for catalytic activity.

            4'-glucosilasi kalkon occurs in the cytoplasm. 4'-glycosides kalkon transferred into the vacuole where final changes to Auron aureusidin and bracteatin catalyzed. Competition between multiple synthesis pathway (anthocyanins, flavonoids and aurone biosynthesis) requires the same catalyst. Kalkon contained in Torenia hybrid transgenic plant that has 4'glokosiltransferase kalkon and sintasis aureusidin of Antirrhinum magus that can increase the production of anthocyanin simultaneously aurone which is regulated by RNA.

 Isolation Aurone

• Auron extracted with alcohol or alcohol-water mixture. Glycosides in Auron increased solubility in water so that the alcohol-water mixture can be used to extract.
• column chromatography with eluent n-hexane, chloroform, methanol, ammonium hydroxide.
Sample Isolation Auron:

1. Algae washed with water, dried for 5 days and soaked with methanol for 1 week.
2. Methanol extracts of algae in the filter and in uapkan under vacuum conditions.
3. The residue in the distillation with water and successively fractionated with n-hexane, Chloroform, EtOAc and n-butanol.

4. Extracts from EtOAc entered into a column containing silica gel
5. Dieluasi with a mixture of n-hexane - Chloroform and Chloroform-methanol
6. Results eluasi on preparative TLC with solvents with Chloroform-methanol-ammonium hydroxide (9.5: 0.5: 3 drops)