Final Exam
Name :
Ekki Widya Lestary
Student Number :
RSA1C110001
1. Explain the triterpenoid biosynthetic pathway, identify
important factors that determine the quantities produced many triterpenoids.
Answer :
Triterpenoids including
steroids are a highly diverse group of natural products widely distributed in
plants (Vincken et al., 2007). Plants often accumulate these
compounds in their glycosylated form – saponin. 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. In triterpenoid biosynthesis the cyclization of
2,3-oxidosqualene catalyzed by oxidosqualene cyclase (OSC; Abe et al., 1993; Figure 1). In general, animals
and fungi have only one OSC, lanosterol synthase (LAS), for sterol
biosynthesis. However, higher plants have several OSCs not only for sterol
biosynthesis, such as cycloartenol synthase (CAS) and LAS (Ohyama et al., 2009), but also for triterpenoid
biosynthesis.
The important factor of triterpenoid
biosynthesis is the enzymatic conversion of oxidosqualene to cyclic triterpenes
represents which is the key step in the biosynthesis of more than 20,000
triterpenoids. These compounds are widely distributed in nature among plants,
animals, fungi, and some bacteria.
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.
Answer :
NMR spectra
Nuclear Magnetic
Resonance spectroscopy, hereafter simply designated by NMR,
is one the most powerful research techniques used to investigate the structure
and some properties of molecules. One of the main applications of NMR in flavonoid
research is the structural elucidation of novel compounds, for which nothing is
known; although NMR traditionally requires large amounts of sample, which is
not easy to obtain when analysing novel compounds, the technical developments
in the last decade, both in NMR instrumentation, pulse programs and in
computing power, have allowed the complete assignment of all proton and carbon
signals using amounts in the order of 1 mg.
NMR
spectroscopy is an extremely powerful analytical technique for the determination
of flavonoid structures, but it is limited by poor sensitivity, slow
throughput, and difficulties in analysis of mixtures. Some recent publications
reporting flavonoid coupling constants include: NMR studies on flavones after
the incorporation of 13C at the carbonyl group, which allowed the measurement
of two- and three-bond carbon–carbon coupling constants, ranging from 1.4 to3.5
Hz, and the measurement of two-, three-, and four-bond carbon–hydrogen coupling
constants, which ranged from 0.3 to 3.8 Hz; complete assignment of the 1H
and 13CNMR spectra of several flavones and their proton–proton and
carbon–proton coupling constants, including the extreme seven-bond long-range
coupling between H-7 and H-3 in 6-hydroxyflavone (0.52 Hz) and flavone (0.27
Hz). Typical one-bond 1H–13C coupling constants of mono
saccharides in anthocyanins have been observed within magnitudes of 125 and 175
Hz.
IR spectra
Light and matter can
interact. The examination of this interaction is termed spectroscopy. The
interactions are characterized by the energy of the radiation and its effects
on materials. IR radiation supplies sufficient energy to produce translational,
rotational, and vibrational motion in molecules. The measurement of the characteristic
IR energies (photons) that correspond to these transitions results in a
spectrum. Based on its atomic structure, each molecule produces a unique and
characteristic IR spectrum. When a material is
irradiated with infrared radiation, absorbed IR radiation usually excites
molecules into a higher vibrational state. The wavelength of light absorbed by
a particular molecule is a function of the energy difference between the
at-rest and excited vibrational states. The wavelengths that are absorbed by
the sample are characteristic of its molecular structure.
To identify the material being analyzed, the
unknown IR absorption spectrum is compared with standard spectra in computer
databases or with a spectrum obtained from a known material. Spectrum matches
identify the polymer or other constituent(s) in the sample. Absorption bands in
the range of 4000 - 1500 wavenumbers are typically due to functional groups
(e.g., -OH, C=O, N-H, CH3, etc.). The region from 1500 - 400 wavenumbers is
referred to as the fingerprint region. Absorption bands in this region are
generally due to intramolecular phenomena and are highly specific to each
material. The specificity of these bands allows computerized data searches
within reference libraries to identify a material. Quantitative
concentration of a compound can be determined from the area under the curve in
characteristic regions of the IR spectrum. Concentration calibration is
obtained by establishing a standard curve from spectra for known
concentrations.
IR spectra for Catechin
IR spectra [υmax (KBr)]
showed band at 2600-3400 (broad), 1620, 1520, 1470, 1380, 1280, 1240, 1150,
1120, 1080, 1020, 820 cm-1.
NMR
spectra for Catechin
Carbon atoms showed
peaks at 22.7 (C-4), 62.3 (C-3), 80.9 (C-2), 93.9 (C-6), 95.1 (C-8), 114.5 (C-2
َ), 115.1 (C-5َ ), 18.4 (C-6َ ) and other aromatic carbons showed peaks at δ
of 99.1, 130.6, 144.6, 144.8, 155.3, 156.1 and 156.4.
IR
spectra of quercetin
The description and
interpretation of the spectra ignored the wavenumber range of > 2000 cm-1
with regard to the occurrence of a very broad peak of vibrations (-OH), which
overlaps other bands. The vibrations stretch –O-H bonds that occur due to
moisture which is difficult to be removed from the investigated compounds.
Peaks of < 2000 cm-1, mainly valence bands of the carbonyl group,
were considered as diagnostic bands.
NMR
spectra of quercetin
3. In
the isolation of alkaloids, in the early stages of acid or base required
conditions. Explain the reason of the use of those reagents, and give examples
of at least three kinds of alkaloids.
Answer
:
The general methods of isolation of
alkaloids largely depend upon several vital factors, for instance: (a) the
alkaline nature of most alkaloids, (b) the ability and ease of formation of
alkaloidal salts with acids, and (c) the relative solubilities of the resulting
alkaloidal salts either in polar organic solvents e.g., ethanol, chloroform,
isopropanol etc., or in aqueous medium.
To isolate alkaloids from plants, the
dried and powdered plant material is extracted with pet ether (or hexane,
colemans etc.) first. This removes fats, oils, terpenes, waxes etc. This
extract is discarded.The material is now subjected to an alcohol extraction, eg
with methanol or ethanol. The extract is evaporated to leave the crude
alkaloids mixture.
This extract is partitioned between an
diluted aq. tartaric acid solution and ethyl acetate. Other acids like citric
acid (acid reagen) can be used, and
other solvents may substitute here. The ethyl acetate layer contains neutral
and weakly basic alkaloids. Evaporate the solvent to isolate them.
The aq. layer is neutralised with NH3
or Na2CO3 (basic
reagen) and again extracted with ethyl acetate (acid reagen). The organic layer now contains basic alkaloids, while
the aq. layer contains quarternary ammonium ions.
Based on the explanation above there
will be formed two layers after the extract is partitioned, that is ethyl
acetate layer and aqueous layer. The using of acid reagen here is to change the
alkaline nature of ethyl acetate layer formed after partition of the extract
into neutral or weakly basic alkaloids. While the using of basic reagen is to
neutralize the aqueous layer formed. So at the end the organic layer (ethyl
acetate layer) now contains basic alkaloids, while the aq. layer contains
quarternary ammonium ions.
ISOLATION OF THEOBROMINE FROM COCOA
POWDER
Prepare the mixture of cocoa powder (10 g), MgO (3g) in
water (20 mL) and methanol (10 mL) in a round bottom flask (250 mL). The
mixture is stirred with a glass rod and heated in heating mantle to dryness. It
takes approximately 1 hour. To the dry substance received add 170 mL of
methylene chloride and heat under reflux for 30 min. Next, filter the contents
on a Büchner funnel. Dry the solution
over MgSO4. Crush the solid substance and once more put it into a
round bottom flask, add 170 mL of methylene chloride. Heat the mixture under
reflux for additional 30 min and once more filter on the Büchner funnel.
Dry the extract over MgSO4, then
filtrate through the funnel with cotton plug to remove MgSO4.
Transfer the combined fractions into a clean and dry round-bottom flask (100
mL) and concentrate the solution to 10 mL of. Move the solution to a beaker
(100 mL) wash carefully with chloroform and transfer also to the beaker. Add 45
mL of ether and leave to crystallization to obtain micro-crystals then wash
them on a Büchner funnel 5 times with 10 mL of ether. Yield ca. 0.15 g
theobromine, mp. 35oC.
ISOLATION OF PIPERINE FROM BLACK PEPPER
Place powdered black pepper (20 g) in the thimble of a Soxhlet
apparatus and extract with chloroform for 2 h to obtain the piperine solution.
At the end of this operation, the extract obtained is colorless. All of the
solvent is removed in vacuo and a brown oil remains.
The extract contains all lipophilic constituents of low
polarity. In the concentrated extract, triglycerides present are cleaved by
saponification with aqueous ethanolic KOH solution, whereas crude piperine
crystallizes on standing in the cold.
Add 20 mL of a 10% KOH solution in 50% aqueous ethanol. Stir
the mixture for 10 min and filter on the Büchner funnel. Allow to stand the
filtrate overnight in a refrigerator at 4 °C. Filter the obtain crystals of
crude piperine on the Büchner funnel and wash with 2 mL of cold water to remove
the adhering base. Air-dry the crystals and recrystallize from
cyclohexane/toluene (4:1, v/v). Use 10 mL of this solvent for each 200 mg of
crude piperine (recovery ca. 60%). Piperine crystallizes on standing in a beaker
as shiny, pale yellow crystals Filter the crystals and wash them with a few mL
of cyclohexane, mp 130-131 °C
Yield: 200-500 mg depending on the
pepper.
ISOLATION OF EPHEDRINE
FROM MA HUANG POWDER
1 kilo of powdered Ma Huang was extracted with cold benzene in the
presence of dilute Na2CO3solution, and the benzene
extract was shaken up with a sufficient quantity of dilute HCl to remove the
basic substances. The acid solution was made alkaline with solid K2CO3 and
the liberated base was then extracted with chloroform. The chloroform solution,
when dried over anhydrous Na2SO4 and distilled, gave
2.6 g of crude base.
Preparation of Ephedrine HCl by Fractional Crystallization
The crude base obtained as above was taken up with about twice its
weight of alcohol and neutralized with concentrated HCl diluted with twice its
volume of alcohol. Nearly pure ephedrine hydrochloride crystallized out on
standing. After filtering it was washed with a mixture of alcohol and ether,
and then with pure ether, and dried. A further quantity of ephedrine
hydrochloride may be got by concentrating the mother liquors and washings. The
final mother liquor was kept for the isolation of pseudoephedrine. Ephedrine
hydrochloride crystallized out from alcohol in prismatic needles, mp 216°C, [α]D22 -32.5°.
The salts prepared by fractional crystallization show no change in the melting
point when recrystallized seven times. In many of our experiments the salts
were recrystallized twelve times.
4. Describe
the correlation between biosynthesis, methods of isolation and structural
determination of compounds of natural ingredients. Give an example.
Answer :
Biosynthesis explained about the formation of a compound
from several substrates that are also accompanied by the enzymatic reaction. By
understanding the biosynthesis will be known the chemical or enzymatic
reactions that occur to form a natural product compound which is then become
not possible to make a synthesis of the compound. Isolation of a sample is
essentially an attempt to capture the natural compound material that we want to
separate it from other compounds. By doing isolation, structure elucidation of
a wanted compound can be determined through a variety of spectroscopy such as
IR, NMR, mass spectroscopy, and others.
Example
:
Biosynthesis
of morphine
Summary of the overall pathway starting from L-tyrosine with a focus on the late demethylation
events. T6ODM first acts on thebaine, removing the methyl group at the 6
position (shown in red) to generate codeine, followed by demethylation at the 3
position (blue) to complete the pathway to morphine. An alternate route (not
shown) involving first removal of the 3-methyl of thebaine leads through
oripavine. Both routes require the action of codeinone reductase (after T6ODM
as diagrammed). (b) Demethylation via
oxidation. In the case of the α-ketoglutarate–dependent,
non-heme iron O-demethylases, the enzyme family binds iron with a conserved HXDXnH motif and,
through reaction with reducing equivalents and α-ketoglutarate,
generates a high-energy iron(IV)-oxo species
capable of hydroxylating the methyl ether. The intermediate hemiacetal
spontaneously decomposes, releasing the carbon as formaldehyde.
Isolation of morphine from opium by lime method
Opium is dissolved in three times its weight of
hot water and filtered. The residue is re-extracted and the two filtrates are
combined. The total filtrate volume is reduced to half and then poured into a
solution of boiling calcium hydroxide. Additional precipitants form and they
are captured by filtration. The capture residues are re-extracted with three
parts of water, and again filtered to obtain a clean filtrate. The filtrates
are combined, and the residues are removed from the process. The total combined
filtrates are concentrated to a weight approximately twice that of the original
opium, and the resulting solution is again filtered, with any captured residues
being removed from the process. Then the solution is brought to a boil, and
ammonium chloride is added; upon cooling, the solution is filtered to collect
the precipitated morphine base. The morphine is then dissolved in a minimum
volume of warm hydrochloric acid. As the solution cools, morphine hydrochloride
precipitates, whereupon it is isolated by filtration.
In the field of clandestine opium extraction,
there is a great variance in the effort expended to produce a pure morphine,
and several different purification methods are known to be employed. However,
in general, it can be said that a product of high purity is often produced by
those laboratories that produce morphine as the hydrochloride salt and/or where
the morphine is the end product. In some cases, the product is near pharmaceutical
quality. Conversely, in general, those laboratories that process from opium ti
heroin and produce the intermediate morphine as the free base, make a product
that is often substantially less pure.
Structure elucidation of morphine
Morphine
has characteristic peaks at around 3200 cm-1(O-H stretching), 1471 cm-1
(O-H bend), 1442 cm-1 (C-C stretch) 1241 cm-1, 1117 cm-1,
1086 cm-1, 941 cm-1, 800 cm-1 and 757 cm-1.
