SEMESTER EXAM

Question

1. Explain the triterpenoid biosynthetic pathway, identify important factors that determine the quantities               produced many triterpenoids!
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!
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!
4. Explain the relationship between biosynthesis, methods of isolation and structural determination of compounds of natural ingredients. Give an example!

Answer

1. Biosynthesis of Triterpenoid

In general, the biosynthesis of terpenoids three basic reactions, namely:

• The establishment of active isoprene derived from acetic acid via mevalonic acid.
• Merging head and tail isoprene units to form mono-, seskui-, di-, Sester-, and poly-terpenoids.
• Merger tail and the tail of the unit C-15 or C-20 produces triterpenoids and steroids.

Activated acetic acid coenzyme A did produce acid asetoasetat Claisen condensation. The resulting compound is then reacted with acetyl coenzyme A did aldol condensation produces branched carbon chains as found in mevalonic acid. The reactions that followed was phosphorylated, elimination of phosphoric acid and subsequent decarboxylation produces IPP to DMAPP by the enzyme berisomerisasi isomerase. IPP as active isoprene units joined head to-tail with DMAPP and this merger is the first step of the polymerization of isoprene to produce terpenoids. This merger occurs because electrons attack the double bond carbon atoms of IPP to DMAPP a shortage of electrons followed by removal of pyrophosphate ions.

Knowledge triterpenoid biosynthetic pathways (terpenoid) allows for modification of metabolites that can be produced in greater numbers and in a shorter period of time, knowing the structure of the resulting metabolites, and then to do the synthesis to produce its derivatives. Modifications can be done in several ways, namely:

• Blocking the path to optimize the other lane. For example, inhibition of acetate-mevalonic pathway in the formation of isoprene to increase the production of isoprene on track trioses-pyruvate.
• The addition of enzymes, precursors, intermediates, or substrate (enzyme activation). The addition of these substances in the biosynthesis of measures appropriate to increase the production of metabolites. For example, the addition squalen on cell suspension cultures of neem as a precursor to the formation of azadirachtin azadirachtin done when production increases. So that needs to be made the growth curve (Zakiyah, Zulfa, et al, 2003).
• Modification of environmental growth conditions. Certain conditions can trigger cells to produce a metabolite. Based on the hypothesis that believed all along that plant secondary metabolites formed in a depressed condition, as a function of secondary metabolites is as a form of a plant response to environmental conditions to sustain life.

2. NMR SPECTRUM

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. NMR spectroscopy is a tool developed in structural biology. NMR in biology melekuler performed on the sample in the form of a solution to prior purification or extraction. Can be determined by NMR molecular structure and the changes that occur when receiving interference from outside (stimulus, disease or the addition of other substances).

nmr spectrum of flavone

nmr spectrum of quercetin

Identification by 1H NMR spectra showed the presence of five aromatic protons, hydroxyl proton, five aliphatic protons and 3 methyl protons. Based on these data showed a compound of flavonoids (quercetin), which binds to a sugar group is rhamnosil. Based on data from HMBC, rhamnosil bound to C-3 of quercetin. These compounds are known as quercetrin (quercetin-3-O-rhamnosida).

IR SPECTRUM

IR spectroscopic used to determine the functional groups. This may be due to the infrared spectra of organic compounds are typically organic, meaning its compounds will have different spectra. The use of infrared spectra in organic chemistry using the local wavenumber range 666-4000 cm-1. When the compound is passed through the infrared organic compounds, some frequencies are absorbed and others will continue. Infrared spectroscopic used to determine the structural information of organic compounds.

IR spectrum of Rimpang temu ireng

Based on the figure can be seen in the strong bands at 1714.6 cm-1 are specific to carbonyl groups. Uptake sharp 1261.4 and 1217.0 cm-1 arises from the conjugated CO group vibrations. Ribbon at 1091.6 and 1029.9 cm-1 is the absorption of the methoxy group. Ribbon at 3020.3 cm-1 comes from the = CH str supported by bands between 1600 cm-1 and 1500 cm-1 indicates the presence of an aromatic nucleus. Small bands namely weak 1652.9 cm-1 coming from the vinyl. The ribbon on the area under the 3000 cm-1 and reinforced by bands around 1450 cm-1 indicates the presence of alkyl methylene. Based on the above analysis of the IR spectrum, it can be concluded that the compounds contained aromatic group, C = O, CO, vinyl,-CH2-and methoxy.

IR spectrum of Quercetin

IR spectra of quercetrin shows the functional groups OH (3294 cm-1), CC aliphatic (2931 cm-1), C = O (1728 cm-1), C = C aromatic (1504 and 1604 cm-1) and COC ether (1064 cm-1). 13C NMR spectra showed 14 carbon aromatic, one carbonyl, and six aliphatic carbon.

3. Effectively insulating compound of natural ingredients then the selection of organic solvent to be used should be appropriate to the nature of the compound to be isolated, which will be easier to polar solvents dissolve polar compounds and otherwise non-polar compounds more soluble in non-polar solvents (Harborne, 1987).

Alkaloids are usually isolated from the plant by using the method of extraction. Solvents are used when extracting the compound mixture is acidified water molecules. This solvent will be able to dissolve the alkaloid salts, this method has been used to extract ergotamine from ergot fungus ..

Moreover, it can also alkalinize alkaloid-containing plant material by adding sodium carbonate. Bases are formed can then be extracted with an organic solvent such as chloroform or ether, it has been used to mengektraksi alkaloid compounds found in plant seeds of mahogany (Swietenia mahogany Jacq).

For alkaloids that are not heat resistant, insulation can be done using techniques alkalinize the solution concentration by first. By using this technique it will evaporate and hereinafter alkaloids can be purified. Usually used for the purification of the compound nicotine. As for the solution of the alkaloid in water that is acidic, then the solution must basified beforehand. Further alkaloids can be extracted using an organic solvent.

4. Biosynthesis, isolation and structural determination of the method of natural materials closely related compounds. By knowing the process of biosynthesis of a compound of natural materials, it will be known process / chemical reactions that occur in obtaining the desired compound. Then, to obtain the pure compound isolation process is carried out to obtain the compound of natural ingredients required / chill. In isolation, the structure can be determined. Thus, from the results of the determination of the structure can be compared with the structure of chemical compounds produced in the process of biosynthesis to match the structure.

Once known molecular structure is usually followed by a modification of the structure to obtain compounds with the desired activity and stability. Besides, with advances in biotechnology, can also be done to improve the quality of plants or organisms through tissue culture or the creation of a transgenic plant would also produce various kinds of new secondary metabolites of diverse and possibly with a different molecular structure to that found from plants initially. Thus the research opportunities in the field of natural products is also not limited. So the secondary metabolite compounds can be used for the benefit of human life.

Example is in the 1700s, chemists examine the myths that circulate in the community about the benefits of tea to stay young. Once the research is done by experts who know the biosynthesis of tea contains a variety of compounds. Then the experts to obtain the isolated compounds contained in tea. After the isolation, the experts identified strukutr compound, determining the structure of these experts know that tea contains flavonoids that are useful as antioxidants to prevent free radicals that enter the body. One is to prevent cancer and many more benefits.

CHOLESTEROL

I. Structure

fig. 1 cholesterol structure

Cholesterol is a fat-sterol-containing metabolites (English: waxy steroid) which is found in cell membranes and circulated in the blood plasma. Is a kind of lipid molecules that are fat or like it. Cholesterol is a special type of lipid called steroids. Steroids are lipids that have special chemical structure. This structure consists of four rings of carbon atoms.

Steroids include steroid hormones such as cortisol, estrogen, and testosterone. In fact, all steroid hormones are made from chemical changes in the basic structure of cholesterol. At about making a molecule of molecular conversion easier, scientists call it synthetic.

Hypercholesterolemia means that the levels of cholesterol in the blood is too high.

Cholesterol can be made ​​synthetically. Cholesterol synthetic currently implemented in widescreen technology (billboards) as an alternative to the LCD.

II. Properties of cholesterol

Properties
Molecular formula	C27H46O
Molar mass	386.65 g/mol
Appearance	white crystalline powder[2]
Density	1.052 g/cm3
Melting point	148–150 °C[2]
Boiling point	360 °C (decomposes)
Solubility inwater	0.095 mg/L (30 °C)
Solubility	soluble in acetone, benzene,chloroform, ethanol, ether, hexane,isopropyl myristate, methanol
Hazards
Flash point	209.3 ±12.4°C [1

III. Biosynthesis of Cholesterol

Slightly less than half of the cholesterol in the body derives from biosynthesis de novo. Biosynthesis in the liver accounts for approximately 10%, and in the intestines approximately 15%, of the amount produced each day. Cholesterol synthesis occurs in the cytoplasm and microsomes (ER) from the two-carbon acetate group of acetyl-CoA.

The acetyl-CoA utilized for cholesterol biosynthesis is derived from an oxidation reaction (e.g., fatty acids or pyruvate) in the mitochondria and is transported to the cytoplasm by the same process as that described for fatty acid synthesis (see the Figure below). Acetyl-CoA can also be synthesized from cytosolic acetate derived from cytoplasmicoxidation of ethanol which is initiated by cytoplasmic alcohol dehydrogenase (ADH3). All the reduction reactions of cholesterol biosynthesis use NADPH as a cofactor. The isoprenoid intermediates of cholesterol biosynthesis can be diverted to other synthesis reactions, such as those for dolichol (used in the synthesis of N-linked glycoproteins, coenzyme Q (of the oxidative phosphorylation pathway) or the side chain of heme-a. Additionally, these intermediates are used in the lipid modification of some proteins.

Pathway for the movement of acetyl-CoA units from within the mitochondrion to the cytoplasm for use in lipid and cholesterol biosynthesis. Note that the cytoplasmic malic enzyme catalyzed reaction generates NADPH which can be used for reductive biosynthetic reactions such as those of fatty acid and cholesterol synthesis.

The process of cholesterol synthesis has five major steps:

1. Acetyl-CoAs are converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)

2. HMG-CoA is converted to mevalonate

3. Mevalonate is converted to the isoprene based molecule, isopentenyl pyrophosphate (IPP), with the concomitant loss of CO₂

4. IPP is converted to squalene

5. Squalene is converted to cholesterol.

athway of cholesterol biosynthesis. Synthesis begins with the transport of acetyl-CoA from the mitochondrion to the cytosol. The rate limiting step occurs at the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reducatase, HMGR catalyzed step. The phosphorylation reactions are required to solubilize the isoprenoid intermediates in the pathway. Intermediates in the pathway are used for the synthesis of prenylated proteins, dolichol, coenzyme Q and the side chain of heme a. The abbreviation "PP" (e.g. isopentenyl-PP) stands for pyrophosphate. Place mouse over intermediate names to see structure.

Acetyl-CoA units are converted to mevalonate by a series of reactions that begins with the formation of HMG-CoA. Unlike the HMG-CoA formed during ketone body synthesis in the mitochondria, this form is synthesized in the cytoplasm. However, the pathway and the necessary enzymes are similar to those in the mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase reaction, forming acetoacetyl-CoA. The cytoplasmic thiolase enzyme involved in cholesterol biosynthesis is acetoacetyl-CoA thiolase encoded by the ACAT2 gene. Although the bulk of acetoacetyl-CoA is derived via this process, it is possible for some acetoacetate, generated duringketogenesis, to diffuse out of the mitochondria and be converted to acetoacetyl-CoA in the cytosol via the action of acetoacetyl-CoA synthetase (AACS). Acetoacetyl-CoA and a third mole of acetyl-CoA are converted to HMG-CoA by the action of HMG-CoA synthase.

HMG-CoA is converted to mevalonate by HMG-CoA reductase, HMGR (this enzyme is bound in the endoplasmic reticulum, ER). HMGR absolutely requires NADPH as a cofactor and two moles of NADPH are consumed during the conversion of HMG-CoA to mevalonate. The reaction catalyzed by HMGR is the rate limiting step of cholesterol biosynthesis, and this enzyme is subject to complex regulatory controls as discussed below.

Mevalonate is then activated by two successive phosphorylations (catalyzed by mevalonate kinase, and phosphomevalonate kinase), yielding 5-pyrophosphomevalonate. In humans, mevalonate kinase resides in the cytosol indicating that not all the reactions of cholesterol synthesis are catalyzed by membrane-associated enzymes as originally described. After phosphorylation, an ATP-dependent decarboxylation yields isopentenyl pyrophosphate, IPP, an activated isoprenoid molecule. Isopentenyl pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate, DMPP. One molecule of IPP condenses with one molecule of DMPP to generate geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to yield farnesyl pyrophosphate, FPP. Finally, the NADPH-requiring enzyme, squalene synthase catalyzes the head-to-tail condensation of two molecules of FPP, yielding squalene. Like HMGR, squalene synthase is tightly associated with the ER. Squalene undergoes a two step cyclization to yield lanosterol. The first reaction is catalyzed by squalene monooxygenase. This enzyme uses NADPH as a cofactor to introduce molecular oxygen as an epoxide at the 2,3 position of squalene. Through a series of 19 additional reactions, lanosterol is converted to cholesterol.

The terminal reaction in cholesterol biosynthesis is catalyzed by the enzyme 7-dehydrocholesterol reductase encoded by the DHCR7 gene. Functional DHCR7 protein is a 55.5 kDa NADPH-requiring integral membrane protein localized to the microsomal membrane. Deficiency in DHCR7 (due to gene mutations) results in the disorder calledSmith-Lemli-Opitz syndrome, SLOS. SLOS is characterized by increased levels of 7-dehydrocholesterol and reduced levels (15% to 27% of normal) of cholesterol resulting in multiple developmental malformations and behavioral problems.

we can also see of cholesterol biosynthesis with this page :
http://www.cholesterol-and-health.com/Synthesis-Of-Cholesterol.html

IV. Identification Structure

The 13C NMR spectrum below is for an unknown compound with 38 carbons. Several carbons in the spectrum are overlapping with each other, thus, making the interpretation difficult.

To facilitate the process of elucidating the unknown, the 13C NMR spectrum for the unknown (drawn in black) is compared to the 13C NMR spectrum for a pure sample of cholesterol (drawn in red). The signals that line up are part of the same cholesterol scaffold; in all ~25 signals match up. The remaining 13 signals belong to the unknown part that requires further work.

The structures for the unknown and cholesterol are shown below