Transcript organic - vGloop

NATURAL PRODUCTS
NATURAL PRODUCTS
• Compounds that occur naturally in plants and animals
• Ubiquitous compounds are usually not classified as
natural products.
Ubiquitous = occurs everywhere in nature, universal
excludes amino acids, nucleic acids, all compounds
found in major metabolic pathways.
• Natural products are unique to only one species or family
of organisms.
Natural Product - how did the term arise?
1769-85 Scheele showed that tartaric acid in grapes, citric
acid in lemons, malic acid in apples, gallic acid in galls,
lactic acid in milk, uric acid in urine.
1772-7 Lavoisier burned sugar, ethanol and acetic acid in
oxygen and found only CO2 and H2O, thus the burned
chemicals must have been made of carbon and hydrogen
only. Quantification showed that they must also contain
oxygen. Later studies by others found that some natural
substances when burned also gave off nitrogen hence
must also contain nitrogen.
1807 Berzelius introduced the terms organic and inorganic , to
refer to chemicals made by living organisms and found in minerals
respectively
1828 Wöhler showed that he could make urea hence the “vital
force” of living organisms was not needed to make organic
chemicals.
1833 Persoz and Payen first noted enzyme activity. The concept of
vitalism now transferred to enzymes in whole cells. However in 1897
Büchner showed enzyme activity in cell-free solutions so vitalism
suffered a final blow.
19th C. The blossoming of the study of organic molecules gradually
split into the study of man-made organic chemicals (the organic
chemicals and organic chemistry of today) and organic chemicals
made by organisms (=Natural Products and Natural Product
Chemistry of today).
1891 Kössel, a German physiological chemist, proposed that the
metabolism of organisms could be divided into two type. Primary
metabolism was the basic biochemistry common to all cells.
Secondary metabolism was the type of biochemistry found only in
some species. Thus to physiological chemists, later to be called
biochemists, Secondary Metabolites are what chemists call Natural
Products.
20th C Physiological chemistry split off from Chemistry Departments
and became Biochemistry ... but that split normally left Natural
Products being studied in Chemistry Departments and now ignored by
biochemists. None of the major introductory biochemistry texts in the
library give the term Natural Product in their indices
Synthetic chemicals are made by the use of chemically reactive
reagents. The chemicals tend to fairly crude in bring about changes to
structures – addition, subtraction, substitution and rearrangements
Natural Products are made by enzymes which can be much more
selective – they can target their action on parts of the molecule by
bringing the active site of the enzyme into close proximity with one
part of the molecule to be changed.
A primary metabolite is a kind of metabolite that is directly involved
in normal growth, development, and reproduction. It usually
performs a physiological function in the organism (i.e. an intrinsic
function). A primary metabolite is typically present in many organism
or cell. It is also referred to as a central metabolite, which has an
even more restricted meaning (present in any autonomously
growing cell or organism).
Conversely, a secondary metabolite is not directly involved in those
processes, but usually has an important ecological function (i.e. a
relational function). A secondary metabolite is typically present in a
taxonomically restricted set of organisms or cells (Plants, Fungi,
Bacteria...).
Secondary metabolites are organic compounds that are not directly
involved in the normal growth, development, or reproduction of an
organism. Unlike primary metabolites, absence of secondary
metabolites does not result in immediate death, but rather in long-term
impairment of the organism's survivability, fecundity ( ‫)الخصوبة‬, or
aesthetics (‫(جماليات‬, or perhaps in no significant change at all.
Secondary metabolites are often restricted to a narrow set of species
within a phylogenetic (‫ )النشوء والتطور‬group. Secondary metabolites often
play an important role in plant defense against herbivory and other
interspecies defenss. Humans use secondary metabolites as
medicines, flavorings, and recreational drugs ( ‫) العقاقير المنشطة‬.
1803-Derosne extracted a mixture of narcotine and morphine
1806-Serturner recognised the alkaline nature of the principle content of
opium
However the structures of these complex molecules were made known only in
the 2nd half of the 20th century
NMR & X-ray- the tedious task has become much easier.
Many drugs were discovered from natural products and their complex
structures have intrigued many chemists and due to this, synthesis of them
were of great challenge.
1952-morphine was synthesised.
1950’s –reserpine was synthesized.
PRELIMINARY BIOLOGICAL SCREENING OF SAMPLES
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Plant collection
Identify the botanical name
Prepare a herbarium sample
Dry and grind sample
Extract the samples with various solvents
Send extracts for biological screening
Natural products are expected to play an important role as one of the
major sources of new drugs in the years to come because of
(i) their incomparable structural diversity,
(ii) the relatively small dimensions of many of them (<2000 Da, 1
kilogram is equal to 6.022 E+26 dalton), and
(iii) their ‘‘drug-like’’ properties, i.e. their ability to be absorbed and
metabolized.
Isolation of natural products from higher plants, marine organisms and
microorganisms is therefore still urgently needed, calling for state-of-theart methodologies for separation and isolation procedures.
Taking into consideration that a plant may contain thousands of
constituents, the separation and isolation process can be long and
tedious.
Isolation of natural products generally combines various separation
techniques, which depend on the solubility, volatility and stability of the
compounds to be separated.
Several sample preparation, pre-purification and
clean-up steps are used prior to isolation and/or
analysis of natural products.
Initial extraction with low-polarity solvents yields
the more lipophilic components, while ethanolic
solvents obtain a larger spectrum of non-polar
and polar material. If a more polar solvent is
used for the first extraction step subsequent
solvent partition allows a finer division into
different polarity fractions.
Extraction methods are therefore used as a pre-purification step to
selectively remove interfering components and/or to isolate the active
compounds. Other pre-purification methods are filtration,
precipitation, removal of chlorophyll, waxes and tannins, solid-phase
extraction (SPE) using pre-packed cartridges with a variety of packing
material, both normal- and reversed-phase silica gel, or short columns
with other suitable packing material such as alumina, Celite, Amberlite
resins and Sephadex LH-20. Pre-packed cartridges for SPE operate on
the principle of liquid–solid extraction and may be used in one of two
modes: a) the interfering matrix elements of a sample are retained on
the cartridge while the components of interest are eluted; b) the
components of interest are retained while the interfering matrix
elements are eluted. In the latter case, a concentration effect can be
achieved. The required compounds are then eluted from the cartridge
by changing the solvent.
Extraction techniques used for separation and isolation
The first step in the analysis and isolation of natural
products is extraction to separate the compounds from
the cellular matrix.
Extraction and recovery of a solute from a solid matrix
may be regarded as a five-stage process: (i) desorption
of the compound from the active sites of the matrix; (ii)
diffusion into the matrix itself; (iii) solubilisation of the
analyte in the extractant; (iv) diffusion of the
compound in the extractant; (v) collection of the
extracted solutes. Ideally, an extraction process should
be exhaustive with respect to the constituents to be
analysed or isolated, rapid, simple, inexpensive, and –
at least for routine analysis – amenable for automation.
Conventional methods for the extraction of natural products include
Soxhlet extraction, maceration, percolation, turbo-extraction and
sonication. These traditional methods present major drawbacks,
including long extraction times, labour-intensive procedures, large
amounts of organic solvents, unsatisfactory extraction efficiency, and
potential degradation of labile compounds.
Example of pre-packed cartridges
Soxhlet extractor
Normally a solid material containing some of the desired compound is placed inside a
thimble made from thick filter paper, which is loaded into the main chamber of the
Soxhlet extractor. The extraction solvent to be used is taken into a distillation flask and
the Soxhlet extractor is now placed onto this flask. The Soxhlet is then equipped with a
condenser.
The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods
into the chamber housing the thimble of solid. The condenser ensures that any solvent
vapour cools, and drips back down into the chamber housing the solid material.
The chamber containing the solid material is slowly filled with warm solvent. Some of the
desired compound will then dissolve in the warm solvent. When the Soxhlet chamber is
almost full, the chamber is automatically emptied by a siphon side arm, with the solvent
running back down to the distillation flask. The thimble ensures that the rapid motion
of the solvent does not transport any solid material to the still pot. This cycle may be
allowed to repeat many times, over hours or days.
During each cycle, a portion of the non-volatile compound dissolves in the solvent. After
many cycles the desired compound is concentrated in the distillation flask. The
advantage of this system is that instead of many portions of warm solvent being passed
through the sample, just one batch of solvent is recycled.
After extraction the solvent is removed, typically by means of a rotary evaporator, yielding
the extracted compound. The non-soluble portion of the extracted solid remains in the
thimble, and is usually discarded.
A schematic representation of a Soxhlet extractor
1: Stirrer bar 2: Still pot (the still pot should not be overfilled
and the volume of solvent in the still pot should be 3 to 4
times the volume of the soxhlet chamber) 3: Distillation path
4: Thimble 5: Solid 6: Siphon top 7: Siphon exit 8: Expansion
adapter 9: Condenser 10: Cooling water in 11: Cooling water
out
Maceration Processes (Steady – State Extraction)
Maceration :
This simple widely used procedure involves leaving the pulverized plant to
soak in a suitable solvent in a closed container .simple maceration is
performed at room temperature by mixing the ground drug with the solvent
(drug solvent ratio : 1:5 or 1:10) and leaving the mixture for several days
with occasional shaking or stirring. The extract is then repeated from the
plant particles by straining . The process is repeated for once or twice with
fresh solvent .Finally the last residue of extract is pressed out of the plant
particles using a mechanical press or a centrifuge.kinetic maceration
differe from simple one by continous stirring.
-The method is suitable for both initial and bulk extraction.
-The main disadvantage of maceration is that the process can be quite
time-consuming, taking from a few hours up to several weeks
Percolation :
the powdered plant material is soaked initially in a solvent
in a percolator . Additional solvent is then poured on top of the
plant material and allowed to percolate slowly (dropwise) out of
the bottom of the percolator. Additional filtration of the extract
is not required because there is a filter at the outlet of the
percolator.
-Percolation is adequate for both initial and large-scale
extraction.
-The main disadvantages are :
1-fine powders and materials such as resins and plants that
swell excessively (e.g., those containing mucilages) can clog
the percolator. 2-if the material is not distributed
homogenously in the container, the solvent may not reach all
areas and the extraction will be incomplete.
Simple percolator
Turbo Distillation Extraction:
Turbo distillation is suitable for hard-to-extract or coarse plant
material, such as bark, roots, and seeds. In this process, the
plants soak in water and steam is circulated through this plant
and water mixture. Throughout the entire process, the same
water is continually recycled through the plant material. This
method allows faster extraction of essential oils from hard-toextract plant materials
In recent years new extraction techniques with significant advantages
over conventional methods have been developed for extracting
analytes from solid matrices, e.g. reduction in organic solvent
consumption and in sample degradation, reduction of extraction and
clean-up times or even elimination of additional sample clean-up and
concentration steps, improvement in extraction efficiency, selectivity,
and/or kinetics, ease of automation. These recent extraction
techniques include supercritical fluid extraction (SFE),
pressurised liquid extraction (PLE), microwave-assisted
extraction (MAE), solid-phase microextraction (SPME), ultrasoundassisted extraction (UAE), superheated liquid extraction,
and extraction with supercritical or subcritical water. Most of
these methods have similar pros and cons with regard to solvent
volume, extraction time and extraction efficiency.
Supercritical fluid extraction (SFE)
Supercritical fluid extraction (SFE) represents an interesting alternative
technique to conventional solid–liquid extraction (e.g. Soxhlet
extraction) with lower solvent consumption and lower working
temperature. It is a form of liquid extraction where the usual liquid
solvent phase has been replaced by a supercritical fluid—a substance
that is above its critical point. Amongst a wide variety of supercritical
fluids, carbon dioxide is essentially the only convenient supercritical
extraction solvent used because of its comparatively low critical
temperature (31.1C) and pressure (73.8 bar/7.38 MPa).
An organic solvent (also called modifier) may be added to the
supercritical fluid to enhance its solvating properties. In case of CO2,
volatile polar solvents such as ethanol, methanol or acetonitrile are
preferred. By using CO2 as the supercritical fluid, extractions can be
performed under mild conditions, thus reducing both the risks of
thermal degradation and the poor collection efficiencies of volatile
analytes. CO2 is most effective for dissolving organic compounds,
particularly molecules displaying some degree of lipophilicity, such
as esters, ethers and lactones. The modifier component may be
introduced into the fluid either using a separate pump and suitable
mixing device or may be added to the sample matrix in the extraction
cell prior to pressuring with CO2. Frequently, an off-line valve is
incorporated between the pump and the extraction vessel and
between the vessel and the restrictor. In this set-up static or dynamic
extraction or a combination of the two may be performed. The
restrictor maintains the pressure within the extraction vessel by flow
control.
The use of SFE both at the analytical and processing scales is quite
widespread in the food industry for the extraction of fats and oils from
seeds, foodstuffs, and other materials. The technique has also been
applied to the extraction of active compounds from medicinal
plants, such as steroids, terpenes, alkaloids, various oxygen containing
heterocyclic compounds, as well as aromatic and phenolic compounds.
Pressurised liquid extraction (PLE)
PLE is a solid–liquid extraction process using organic
solvents at an elevated temperature (usually between 50 and 200 C)
and applying higher pressure (between 10–15 MPa) to extract
samples in an extraction cell. Extractions are carried out under
pressure in order to maintain the solvent in its liquid state, even at
temperatures above boiling point. Moreover, pressure allows the
extraction cell to be filled more quickly, and helps to force the
solvent into the matrix pores. Thus, the efficiency of the extraction
process is improved. Extraction at elevated temperatures
increases solubility, diffusion rate, and mass transfer, coupled
with the ability of the solvent to disrupt the analyte– matrix
interactions
PLE thus allows fast extraction owing to increased solubility, better
desorption and enhanced diffusion, and the extraction is generally
completed within a few minutes. PLE was developed especially for
laboratories with increased sample throughput. Comparison with
conventional extraction methods has demonstrated faster extraction,
higher extraction efficiency, and lower solvent consumption, along
with comparable recoveries in most cases. No evidence was seen for
thermal degradation during the extraction of temperature-sensitive
compounds
Scheme for PLE System
There are two ways to perform PLE. The first is the static mode in
which the extraction cell is filled with a solvent, followed by
heating to generate pressure in the cell. After a period of time (5–
10 min is usually sufficient), the system is rinsed with fresh
solvent to ensure that all of the extract reaches the collection
vials, and is purged with gas to avoid any losses or ‘memory’
effects. In the second method, the dynamic mode, fresh solvent is
continuously percolated through the cartridge under pressure at
a constant flow rate for a fixed period of time. The extraction cell
is placed in a thermostatted oven. In both cases, under conditions
of elevated pressure and temperature, the mass transfer
rates are accelerated. The typical volume collected depends on
the cell size. Volumes between 10 and 100 ml may be required,
and hence repeated evaporation steps are needed to concentrate
the final extracts. Both commercially available and laboratoryassembled PLE systems are used.
Microwave-assisted extraction (MAE)
Microwave-assisted extraction (MAE) is a recent
technology for extracting soluble products into a
fluid from a wide range of materials using
microwave energy. It provides a technique which
allows one to extract compounds more selectively
and more rapidly (usually in less than 30 min) with
similar or better recovery than traditional extraction
processes. Microwaves directly heat the solvent or
solvent mixture, thus accelerating the speed of
heating. Besides the advantage of high extraction
speed, MAE also enables a significant reduction in
the consumption of organic solvent.
The application of microwave energy to the samples may be
performed using two technologies: either closed vessels under controlled
pressure and temperature, or open vessels at atmospheric
pressure. The two technologies are commonly named pressurized MAE
(PMAE), with a multi-mode cavity, or focused MAE (FMAE) using the
waveguide as a single-mode cavity, respectively. Both systems are shown
in Fig. 6. Whereas in open vessels the temperature is limited by the boiling
point of the solvent at atmospheric pressure, in closed vessels the solvent
can be heated above its boiling point at atmospheric pressure by simply
applying suitable pressure, thus enhancing both extraction speed and
efficiency. However, after extraction with closed vessels, one needs to
wait for the temperature to decrease before opening the vessel, thereby
increasing the overall extraction time (by approximately 20 min). Open
systems use focused microwaves, resulting in homogenous and very
efficient heating of the sample. In closed systems using diffuse
microwaves, the electric field in the cavity is non-homogenous, and
therefore the vessels are placed on a turntable.
Scheme of the two microwave systems, using diffused or focused
microwaves.
MOST COMMON CHROMATOGRAPHIC TECHNIQUES
• Column chromatography
• Thin layer chromatography
• MPLC ( medium pressure liquid chromatography)
• HPLC (High pressure liquid chromatography)
STATIONARY PHASE
• Silica-nonpolar
• Bonded silica-C18, C-8, CN etc
• Polyacrylamide
• Polysaccharides; sephadex
SOME CLASSES OF NATURAL PRODUCTS
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Alkaloids
Terpenes
Polyketides
Steroids
ALKALOIDS
WHAT IS AN ALKALOID ?
• Naturally-occurring compounds that contain nitrogen
• Many have heterocyclic rings as a part of their structure
• They are found mostly in plants and also in certain animals
• Many are physiologically active (often spectacularly)
• Many are used by native peoples for religious or medicinal
purposes.
• Many are basic (“alkaline”, due to an unshared pair on N)
• Those nitrogen compounds that are found in all organisms
(i.e., amino acids, nucleic acids, etc.) are not considered
alkaloids.
• Alkaloids are “secondary metabolites”, they are not
involved in primary metabolism.
Classification Schemes for Alkaloids
• The heterocyclic ring systems found as a part of the
compound’s structure
• The plant or plant family where they originate
Also alkaloids are classified as:
1- True alkaloids ( Characterized by heterocyclic ring with a
nitrogen atom, and are derived from amino acids).
2- Proto alkaloids ( characterized by absence of the
heterocyclic ring but also derived from amino acids)
3- Pseudo alkaloids ( characterized by a heterocyclic ring
with a nitrogen atom, but are not derived from amino
acids. ( Steroidal alkaloids)
In general the alkaloids are classified according to the chemical
structure in two broad divisions:1- Non-heterocyclic or typical alkaloids or biological amines.
2- Heterocyclic or typical alkaloids , divided into 14 groups
according to their structure, as :
1) Pyrrol or pyrrolidine 2) pyrrolizidine 3) Pyridine and
piperidine 4) Tropane 5) Quinoline 6) Isoquinoline
7) Aporphine 8) Norlupinane 9) Indole 10) Indolizidine
11) Imidazole 12) purine 13) steroid 14) Terpenoid
HETEROCYCLIC RING SYSTEMS
N
N
N
H
H
H
pyrrole
piperidine
pyrrolidine
N
N
pyridine
N
N
H
quinoline
isoquinoline
indole
N
H
dihydroindole
HETEROCYCLIC RING SYSTEMS
H
N
N
N
tropane
pyrrolizidine
quinolizidine
N
N
N
C C N
N
N
H
benzylisoquinoline
purine
-phenylethylamine
(cont)
Alkaloids with Exocyclic Nitrogen
(Protoalkaloids)
(Non-Heterocyclic Alkaloids)
Phenyl alkylamine alkaloids
*This group of alkaloids have the nitrogen atom located in an amino
group and is not a member of a heterocyclic ring.
i.e. alkaloids characterized by the absence of heterocyclic ring in their
molecules.
*Many are simple derivatives of -Phenylethylamine and as such, are
derived from the common amino acids Phenyl alanine or Tyrosine.
*They are Sympathomimetic drugs (as they rise the blood pressure).
*They includes the alkaloids of:
1) Ephedra alkaloids
2) Khat alkaloids
3) Capsicum alkaloids
4) Colchicum alkaloids
Prefixes
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Nor-" designates N-demethylation or Ndemethoxylation, e.g. norpseudoephedrine and
nornicotine.
"Apo-" designates dehydration e.g. apomorphine.
"Iso-, pseudo-, neo-, and epi-" indicate different types
of isomers.
• Suffixes:
"-dine" designates isomerism as quinidine and
cinchonidine.
"-ine" indicates, in case of ergot alkaloids, a lower
pharmacological activity e.g. ergotaminine is less
potent than ergotamine.
"
Ephedrine
Recently banned
from organic
weight-loss
supplements!
found in
OH CH3
CH CH N
ephedra spp
CH3
Extract of ephedra was used in
the early american west as a
cure for asthma (Mormon Tea).
H
ephedrine
Sympathomimetic Amine
Chinese medicine “Ma Huang”
for hay fever.
“Organic” appetite suppressant.
compare
CH3
OH
CH CH2 N
HO
HO
R= H
R
CH2 CH N
H
Norepinephrine
R = CH3 Epinephrine (adrenalin)
neurotransmitter / hormone
R
H
R=H
amphetamine
R = CH3 methamphetamine
“speed” or crystal “meth”
UTAH
Ephedra
Ephedrine biosynthesis
NH2
N
N
N
CH2
N
OH HO
S CH2 CH2 CH COOH
CH3
NH2
O
S-adenosylmethionine
SAM
S-Adenosylmethionine is the
principal methylating agent in
the secondary metabolism of
plants.