UJIAN AKHIR SEMESTER

COURSE: CHEMISTRY OF NATURAL MATERIALS

LECTURER: Dr. Syamsurizal, M.Si

TIME: 22-29 DECEMBER 2012

1. Explain the triterpenoid biosynthetic pathway, identify the factors that are very important to determine the quantity produced triterpenoid very much!

Answer : 

         Triterpenoid  is a compound that carbon skeleton derived from six isoprene units and reduced biosynthesis of acyclic hydrocarbons C-30, which skualena, these compounds are colorless, crystalline, high melting point and is optically active (Harborne, 1987).

Triterpenoid compounds can be divided into four groups, namely: real triterpenes, saponins, steroids and cardiac glycosides.

Triterpenoids is one class of compounds terpenoid natural ingredients.

Terpenoid biosynthetic pathway begins with three basic reactions, namely;

The first basic reaction : is a formation of active isoprene derived from acetic acid via the mevalonic acid pathway.

Acetic acid did Claisen type condensation produces Asetoasetil Ko-A after being activated by coenzyme A. then the compound will also ni aldol type condensation resulting branched carbon chains such findings in mevalonic acid pathway with acetyl Co-A.

The second reaction is the reaction joining two unit head and the tail of compounds that form terpenoida isoprene (mono-, di-, seskui-, Sester-and poly-)

The reaction continued after the formation of mevalonic acid is phosphorylated, elimination of phosphoric acid, and decarboxylation which then produces IPP (isopentenyl Pyrophosphate) which will then be berisomerasi with DMAPP (Dimethyl Allyl Pyrophosphate) with the help of isomerase enzymes to be joined from head to tail. This merger occurs because electrons attack the double bond of the isopentenyl Pyrophosphate carbon atom at the electron-deficient DMAPP and then generate geranil Pyrophosphate (GPP) in the presence of pyrophosphate ion exclusion. IPP and GPP units will combine to form famesil Pyrophosphate (FPP) with the same flow mechanism and is one seskuiterpenoida compounds.

Then diterpenoida compounds derived from geranil - geranil pyrophosphate (GGPP) with the same mekansme through unit PP and GPP.

The third is tail joining reaction of unit C-15 and C-20 which then produces triterpenoida and steroida.

Triterpenoida and tetraterpenoida derived from dimerization of C-15 and C-20 which is not a continuous polymerase unt C-5. Dimerization FPP into skualena which is the basis and source triterpenoda steroida.

Based triterpenoid biosynthetic pathway, it can be seen that the factor - an important factor that determines the quantity produced abnyak triterpenoids are:

• Merging head and tail isoprene unit and the tail of the C-15 and C-20 are produced triterpenoids and steroids.
• The addition of enzyme to be involved in triterpenoid biosynthesis pathway.
• Optimizing producing pathway by inhibiting triterpenoid work mevalonic acid pathway.
• Cyclization process squalen compounds that can form triterpenoids.

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

Answer : 

Infrared spectroscopy is a method of observing the interaction of molecules with electromagnetic radiation whose wavelength is in the region 0.75 - 1,000 μm or wave number 13000-10 cm-1.

The infrared spectrum of a compound provides an overview of the functional group in an organic molecule.

For the identification of the IR spectrum, the first step is the identification of the main group (C = O, O - H, N - H, C - O, C = C, C = N, and NO) goal is to compare it with the literature that are known compounds.

Nuclear magnetic resonance spectroscopy (NMR) provides an overview of the types of atoms, number, and the hydrogen atom (1H NMR) and (carbon (13C NMR).

NMR spectroscopy is based on absorption of radio waves by certain nuclei in organic molecules, when the molecule is in a strong magnetic field.

For example, the flavonoid compounds:

Flavonoid Structure

Test physical properties of flavonoids conducted on the isolated compound in the form of solids.

Another important analysis using IR spectrophotometer to determine the functional groups in a compound, followed by GC-MS analysis of the spectra to determine the structure of these compounds. The results of the analysis with a spectrophotometer UV and IR showed that only f2, f4 and f9 which are isoflavones. Suspicion that the active compounds in the rhizome Intersection ireng are isoflavones, the identification of further structure only at fraction f2, f4 and f9.

IR spectrophotometer to determine the functional gugusgugus compounds are in fraction f2.

and for NMR spectrume of flavnoid compounds :

This was obtained as yellow crystals, 13 mg, R_f = 0.76, mp. 250 °C. UV–Vis λ_max in MeOH: (nm) 248, 281 sh, 307 sh 318 sh 362; (AlCl₃) 269 sh, 299, 319 sh, 423; (AlCl₃/HCl) 264, 296 sh, 320, 458; (NaOAc) 252 sh, 292, 331, 379; (NaOAc/H₃BO₃) 263 sh, 315, 381; (NaOMe) 252, 299, 331, 361, 410. IR (KBr), ν = 605, 700 (C–H, Ar), 1097 (C–O, ethe), 1512, 1570 (CC, Ar), 1604 (CO) and 3354 cm⁻¹ (OH). ¹H-NMR (DMSO-d₆), Fig. 1, δ = 6.89 (d, J = 8.8 Hz, 2H, H-6, H-8), 7.54 (dd, J = 2.2 Hz, J = 8.8 Hz, H5′, H-6′), 7.69 (d,J = 2.20 Hz, H-2′), 7.92 (d, J = 9.5 Hz, 1H, H-5), 10–14 ppm (broad singlet, 4-OH)

Figure 1. The ¹H-NMR chemical shift of flavonoid compound 1 in DMSO-d₆

Figure 2. The ¹³C-NMR chemical shift of flavonoid compound 1 in DMSO-d₆

Compound 1 substituted in position −7 as indicated by its UV–Vis spectra upon addition of diagnostic shift reagents (NaOAc). When we added boric acid (H₃BO₃) to methanolic sodium acetate bands 1 shifted (+19) nm indicating B-ring catechol system. However, flavonoids with 3′,4′ – hydroxylation pattern give two peaks in the UV–Vis spectrum band II has two peaks, the B-ring catechol moiety is probably located at C-3′ and C-4′. Further evidence in favour of the above structure accumulated from the mass spectrum (Fig. 4) where the molecular ion M⁺ 286.

Figure 4. Mass spectrum of flavonoid compound 1.

              

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

Answer :

Alkaloids are a class of compounds which mostly heterocyclic nitrogenous bases and contained in plants (but this does not exclude the compounds derived from animals).

Amino acids, peptides, proteins, nukleotid, nucleic acids, amino sugars, and antibiotics are usually not classified as alkaloids. And the same principle, which is a neutral compound biogenetics related alkaloids belong to this group.

Alkaloids are usually classified according to the origin of molecular similarity (precursors), based on the metabolic pathway (metabolic pathway) that is used to form a molecule. If the biosynthesis of an unknown alkaloids, alkaloid compounds are classified according to their names, including the name of the compound that does not contain nitrogen (due to their molecular structure present in the final product.

Example: opium alkaloid sometimes called "phenanthrenes"), or under the name of a plant or animal in which the compound was isolated. If after alkaloid was studied, an alkaloid modified classification according to the assessment, it usually takes the name-the-amine important biological striking in the process of synthesis.

Therefore, isolation of alkaloids can be drawn in the form of salts with alcohol in an acidic environment.

Alkaloid salts already in the base form and then withdrawn by the organic solvent.

Classification alkaloids are:

• Heterocyclic alkaloids
• Alkaloids with nitrogen and amine eksosiklik alifatis
• Alkaloid putreskin, spermidin, and sperin
• Peptide alkaloids
• Terpenes and steroidal alkaloids (Sudarmin, 1999)

Alkaloids consist of  carbon, hydrogen, nitrogen, and usually contains many alkaloids oksigen. Compounds contained in the roots, seeds, wood and leaves from plants  and also of animals.

Metabolisme of  alkaloid compounds is the result of plant and used as a backup for the synthesis of the plant alkaloid protein. Kegunaan is as protective of pests, plants and regulator amplifier hormones work. Alkaloids have physiological effects.

Source is a flowering plant alkaloid, angiosperms, animals, insects, marine organisms and microorganisms.  Included alkaloid plant families that are Liliaceae, solanaceae, Rubiaceae, and papaveraceae (Tobing, 1989).

4. Explain the relationship between the biosynthesis, isolation methods and the establishment of the structure of compounds of natural ingredients. Give an example?

Answer : 

Biosynthesis is the formation of a naturally occurring molecule in the cells of other molecules that are less complicated structure, through endeorganik reaction.

While the biosynthetic pathway can be defined as a sequence or a process in which consists of the stages of formation of simple compounds into complex compounds. Biosynthesis process will take place very complex, depending on the available enzymes that similar plants growing in different areas it is possible to have a certain metabolite formation paths are not identical (the phenomenon of "vikarias: Chemical-Ras).

The reason why the biosynthetic pathway to be studied are:

1. Precursors can turn into a more useful new compounds with the aid of cell suspension
2. Based biosynthesis, secondary metabolites can be fed with a precursor to a product faster with cell suspension cultures
3. Changing certain compounds into other compounds to replace the reaction with cell suspension cultures

How to determine the biosynthetic pathway in tissue culture are:

1. With the analysis of complex compounds that can be detected constituent building blocks that can lead us to the original compounds and biosynthesis pathways
2.  Labeling with radioisotopes.

Usefulness know biosynthetic pathway is able to perform derivatization. Once we know the biosynthesis pathway, and it branched biosynthesis pathway, we can do the blocking on one of the branches. With the blocking is so we can improve secondary metabolite biosynthetic pathways we want from us is not blocking.

Method engineering biosynthetic pathways :

1. With the addition of substrates, precursors, or enzymes that play a role.

One approach that can be used to increase the production of secondary metabolites in vitro culture is the addition of precursors. The addition of precursor into the culture medium to stimulate the activity of certain enzymes involved in the biosynthesis pathway, so as to increase the production of secondary metabolites.

One study conducted by Zakiah, et al (2003) showed that the addition of squalen as precursors to increase production in callus cultures Azadirahtin (Azadirachta indica A.Juss) with the addition of plant growth regulators 2,4-D and BAP. Addition skualen done after callus cell suspensions from 6 day provide azadirahtin levels in the cell while being increased. Giving azadirahtin done with 3 concentration is 10 μM, 100 μM and 1000 μM. The research proves that azdirahtin production increased rapidly on day 4 after the addition azadirahtin the 10-day-old culture with high levels of 0.0076 ± 0.006 g / g DM or increased to 85, 366% over azadirahtin content was highest in the control (0.041 g / g BK).

2. Activation of enzymes involved in the biosynthesis pathway.

3. Environmental factors engineering.

The addition of precursors intended to streamline the process of biosynthesis or in other words to increase the production of secondary metabolites. Besides the addition of precursors, giving "stress" in the culture can also affect the production of secondary metabolites. Events that may arise because of the treatment is likely to be the formation of new compounds that are not found in plant origin (de novo synthesis), but generally provide a favorable outcome. This type of "stress" are common, such as lack of water (draft), the lack of light, lack of nutrients (minerals), the temperature is above or below the optimum.

Generally in a growth phase, secondary metabolite biosynthesis takes place very slowly and often not started. After the growth phase ends, the phases of the production or secondary metabolite biosynthesis underway. The addition of precursors intended to streamline the process of biosynthesis or in other words to increase the production of secondary metabolites.

In order to increase the precursor biosynthesis pathway in the production of secondary metabolites:

1. To compound the desired amount can be increased by manipulating the media and with the addition of precursor compounds / precursors, stimulate the activity of certain enzymes involved in the biosynthesis pathway, thus increasing the production of secondary metabolites, such as the addition of skualen a significant effect in improving the content on the cell azadirahtin according to research conducted by Zakiah (2003)
2. Getting secondary metabolism which is a form of differentiation of plant cells
3. To obtain the content of secondary metabolites that are higher than its parent.