Fall Organic Chemistry Experiment #5
Project: Natural Products Isolation and Characterization
Suggested Reading:
"The Student's Lab Companion: Laboratory Techniques for Organic Chemistry”, by John W.
Lehman
OP-15 pages 70-81 (if you have not already read)
OP-17 pages 86-93
OP-18 pages 93-104
OP-19 pages 104-111
OP-20 pages 111-115
OP-35 pages 214-218
Try these links as well:
http://pubs.acs.org/cen/coverstory/8141/8141pharmaceuticals2.html (and links at the bottom of
the page)
http://www.sciam.com/article.cfm?articleID=000DE779-9600-1C759B81809EC588EF21&sc=I100322
Introduction:
For centuries, natural products (primarily from plants) have been used for food, shelter,
medicine, clothing, hunting, and religious practices. Indeed, many of our "modern" products
(both synthetic and natural) have distinct connections with historical predecessors. Think about
it. We utilize extraction to obtain sugar from sugar cane, perfumes from flowers, flavorings and
spices from seeds/leaves, and alkaloids from leaves or bark. In particular, we rely very strongly
on natural animal and plant resources to provide us with medicinal agents. For example, Taxol
(paclitaxel) was discovered in the bark of the Pacific Yew tree some 20 years ago and found to be
a potent and effective anticancer agent. But, this is really nothing "new". Alchemists, witch
doctors, medicine men, shaman, pharmacognosists, pharmacists, and physicians have routinely
used botanically based compounds for the treatment of disease for centuries. In fact, nearly 50%
of all prescriptions written today by physicians contain at least one drug that has been derived
from a natural source. In addition, many of the synthetic drugs that are on the market are simply
molecular modifications of a natural product. Today's pharmaceutical companies rely heavily
upon the exploration of botanicals for providing "new leads". Shown below (Table 1) are some
traditional examples of natural product leads that have worked their way into common
pharmaceutical use.
Table 1
Name of Compound
d-tubocuraine
quinine
pilocarpine
amphotericin
cephalosporin
reserpine
cocaine
morphine
mitomycin
mescaline
Botanical Source
Chondodendron tomentosum
Cinchona
Pilocarpus jaborandi
Streptomyces nodosus
Cephalosporium
Rauwolfia serpentina
Erythroxylon coca
Papaver somniferum
Streptomyces caespitosus
Lophophora williamsii
Use
skeletal muscle relaxant
anti-malarial
glaucoma
anti-fungal
antibiotic
tranquilizer
local anesthetic
analgesic
anti-cancer
hallucinogen
The exploration of plant extracts for effective therapeutic molecules is certainly ongoing.
In fact, the industrial drug discovery process has historically relied heavily upon the application
of chemical methods as a means of screening plant/animal species for new drugs. It is the job of
the chemist to utilize certain techniques to isolate compounds that may eventually lead to the
discovery of the discrete components of a molecule (pharmacophore) that are necessary for a
molecule to elicit a biological action. In turn, the pharmacophore can then be refined into a new
molecule that can ultimately be synthesized in the laboratory. The goal is to synthesize new
molecules (that may, in some cases, look radically different from the original compound) that
retain the intrinsic activity of the natural compound or even enhance its potency and efficacy.
As an example, we may consider the history of the compound shown below
(apomorphine). Upon isolation from a random screening of plants, apomorphine was originally
found to be active at dopamine receptors in the brain. However, scientists soon realized that it
was not a very potent or selective compound. It required the patient to take large quantities (low
potency).
OH
HO
N
CH3
apomorphine
In addition, patients suffered from numerous side effects (low selectivity) because the compound
can interact with other (non-dopamine) receptors. In order to alleviate some of these problems,
chemists discovered that they could slightly alter the structure of the natural compound by
incorporating a propyl substituent in place of the N-methyl group to generate N-propyl
apomorphine. Although the new molecule did not look radically different from apomorphine, the
result was a molecule that had enhanced potency and selectivity for dopamine receptors.
Therefore, a new therapeutic agent for the treatment of dopamine related disease states (e.g.
Parkinson's disease) was developed starting from the simple isolation of a natural dopaminergic
from a plant.
There are numerous examples of compounds that are produced by plants which can
biochemically interact with the human body. Take, for instance, caffeine. Many consider
caffeine to be the most widely used (possibly, abused) drug in America. After all, it is present in
many of the products that we use on a daily basis. It is found in tea, coffee, cola, chocolate,
weight-loss drugs, pain-relievers, and sleep inhibitors. It has been documented that caffeine is a
stimulant of the central nervous, cardiac, and respiratory systems. In addition, caffeine can be
psychologically addictive. Chemists classify caffeine as an alkaloid. Other common alkaloids
include nicotine, morphine, and cocaine. More examples of common everyday products and their
primary natural constituent(s) are shown below (Table 2). The structures of the major
constituents can be found in the Merck Index (Wilmot Library or Smyth 303) along with relevant
physical properties. Some popular texts that have been written on the subject of medicinal
chemistry or natural products chemistry can be found in the Wilmot Library as well. Try "An
Honest Herbal" by Varro Tyler for a start. You can also consult with Dr. Beverly Brown of the
Nazareth College Biology Department as she is an expert in ethnobotany.
Table 2
Plant Source
tomato
carrots
spinach leaves
caraway seeds
cloves
cinnamon
spearmint
black pepper
allspice
nutmeg
vanilla
lemons
oranges
lemongrass
Major Constituent
lycopene
b-carotene
chlorophyll
(-)-carvone
eugenol
cinnamaldehyde
(-)-carvone
piperine
eugenol
trimyristin
vanillin
citral
limonene
citral
Mechanism of Action:
The basic theory regarding the mechanism of action centers upon the idea that these
constituents are volatile (can readily travel through the air). Eventually some of the volatile
molecules will arrive at a receptor site somewhere in the organism. These sites may be the
olfactory receptors in your nose or the taste receptors in your mouth. The molecule will become
specifically bound to the receptor usually causing some sort of secondary biochemical cascade to
occur. One of the more common biochemical pathways involves the coupling of an enzyme to a
biological receptor. For example, the enzyme adenylate cyclase is coupled to certain dopamine
receptors in the brain. Once a stimulus reaches the receptor, the enzyme is either positively or
negatively influenced and a corresponding increase or decrease of an important biological second
messenger (cAMP) results. The relative increase or decrease of cAMP will result in a defined
course of action for the organism. The organism will be programmed to follow a set series of
biochemical reactions that will eventually result in an observable response (for example, an
increase in pupillary size).
Another example occurs when you peel an orange releasing the volatile constituents
(limonene among others). These molecules travel to your olfactory receptors and eventually you
realize that odor of oranges. In some cases (banana), it is a few volatile constituents that elicit a
response. In others (coffee), it may be hundreds of components. In addition, 3-dimensional
structure (or stereochemistry) can play a significant role. For example, carvone can exist as the
(+) or the (-) enantiomer. It is the (+) enantiomer that gives rise to the essence of spearmint and
the (-) that gives rise to the essence of caraway seeds. Suffice it to say that biological receptors
are quite selective in what they can bind to. If you are interested in learning more about the
mechanism of action of drugs, consider signing up for CHM 447 Medicinal Chemistry next fall
Isolating Natural Products from their Sources:
How is that we can isolate, purify, and identify the major organic components from plant
material? Well, there are actually quite a few general methods that have been traditionally used
to derive natural products from plant material including extraction, chromatography and steam
distillation. These methods generally serve as the initial screening methodology for isolating
bioactive constituents from a particular plant species. Chemists screen the plant material hoping
to find leads for the development of new drugs, pesticides, herbicides, nutrition supplements, etc.
We have already introduced the concepts of extraction (Exp. #4). Hopefully, you have read about
distillation and chromatography in the suggested reading from Lehman. Therefore, you should be
familiar with the basic theory behind these processes. If not, here’s some basic information.
Chromatography:
Chromatography is an efficient method for the separation of compounds distributed
between two phases. These two phases are the stationary phase and the mobile phase. Do not
confuse these phases with the two phases in extraction. Separation occurs because the
compounds have different affinities (attraction) for the stationary phase and the mobile phase.
Let us consider the following analogy.
Imagine that you and an alien are standing on the top of a mountain. The alien is
covered from head to toe in super glue. You do not have the super glue on you.
Now the mountain you are standing on is on Mars and is also made of a very
sticky Martian material. Both of you decide that you want to get down off of the
mountain and dive into a shallow olive oil slick running over the sticky surface of
the mountain. Who will get there faster? If we think about the chemistry, we
might say that each of you has a different affinity for the sticky material
(stationary phase) on the mountain. In this case, the alien who is covered in glue
will "stick" better to the sticky Martian stationary phase and has the higher
affinity compared to you. On the other hand, you (without glue) will have a
lower affinity for the sticky material (and, by default, a higher affinity for the
olive oil mobile phase). Therefore, you should be able to slide down the
mountain much faster.
The basic gist here is that depending upon the individual physical characteristics, molecules may
have a relatively high or low affinity for either phase. Certain molecules move along readily with
the mobile phase (like you in the analogy above), while others are held up by their affinity for the
stationary phase (like the alien). All types of chromatography (column, gas, liquid, etc.) involve
this same sort of separation scheme.
Thin-Layer Chromatography:
In thin layer chromatography (TLC), a plastic or glass plate is coated with silica on one
side. The silica functions as the stationary phase. The mobile phase is the solvent that moves up
the plate by capillary action. The basic process involves applying a small drop of solution at one
end of the plate (origin), immersing the same end of the plate in a developing solvent, letting the
solvent run just about to the top of the plate, and visualizing the resulting chromatogram.
In the thin layer chromatography experiment you are doing today, some molecules will
adhere to the plate (higher affinity for the stationary phase) and will not move very far up the
plate. Others will move along more readily with the solvent (higher affinity for the mobile phase)
and may move as far up the plate as the solvent front. In general, polar compounds, such as
alcohols, amines, and carboxylic acids, stick tightly to the stationary phase and do not move far
from the origin. As a result, a very polar developing solvent (mobile phase) is required to move
polar compounds further up the plate. Nonpolar compounds have modest affinity for the plate
and will move quickly up the plate in nonpolar solvents.
Designing the polarity of the developing solvent is a critical step in any TLC experiment.
The solvent polarity plays a direct role in the efficiency of the separation of components (spots).
The general rule of thumb is for the mobile phase to be polar enough to move the components up
the plate away from the origin and nonpolar enough to trail behind the solvent front. Keep in
mind that the lower the affinity of a compound for the silica and the higher the solubility in the
developing solvent, the closer the compound will move to the solvent front. The silica is a
constant in terms of polarity, but the mobile phase is not. We can adjust the polarity of the
mobile phase. We can create the ideal solvent system by using a single solvent or by using a
solvent mixture. Experience is the best teacher in this regard. It is useful to try a number of
different single solvent systems along with some mixtures to achieve the desired separation. You
may also consider the CRC Handbook of Chromatography which lists compounds and their
separation efficiency in various solvent systems. Table 3 lists some common laboratory solvents
in order of increasing polarity.
Table 3
Cyclohexane
Least Polar
Petroleum Ether
|
Benzene
Toluene
Dichloromethane
Chloroform
Diethyl Ether
Ethyl Acetate
Ethanol
|
|
|
|
|
|
|
Acetone
Acetic acid
Methanol
Water
|
|
|
Most Polar
Once you have designed a reasonable developing system, it is time to run the chromatogram and
interpret the results. The results of a TLC experiment are measured by a quantity called the Rf
value. This value corresponds to the relative distance a particular compound has moved up the
plate. The Rf value is the ratio of the distance a compound moves from the origin to the distance
the solvent front moves (Figure 1).
Figure 1
The Rf value is specific to the solvent conditions of the chromatogram. Normally, these
conditions are difficult to reproduce. Therefore, if you wish to identify a particular compound,
and if an authentic sample is available, the unknown is chromatographed on the same plate with
the authentic sample by co-spotting the unknown and authentic compound on top of one another.
Once the run has been completed the spots are visualized. There are a variety of
visualization methods that can be used. The most common method is to use a plate impregnated
with a fluorescent material that allows spots to be visualized under UV light. Compounds with
conjugated bond systems (e.g. benzene) will show up nicely because they absorb UV light.
Another method is to place the plate in a chamber containing iodine crystals. The spots will turn
dark brown due to a weak electronic interaction of the iodine with the compound on the plate. A
final method involves dipping the plate in a solution of phosphomolybdic acid. Molybdenum
(+6) will oxidize most organic compounds to yield a permanently colored spot.
Thin layer chromatography is a simple, rapid method for analyzing mixtures of
compounds. As a result, TLC is routinely used in the organic chemistry laboratory for a variety
of functions. The two most common applications include using TLC to test the fractions
collected from a (column) chromatography experiment and using TLC to monitor the course of a
chemical reaction. We will see examples of these two applications throughout the year.
Column Chromatography:
The same principles apply to column chromatography as they do to TLC. The difference
lies in the amounts of material and the characteristics of the stationary and mobile phases.
Typically, there are three types of column-based chromatography employed to separate mixtures
of organic compounds: (a) gravity or flash column chromatography, (b) high-performance liquid
chromatography (or LC), and (c) gas-chromatography (or GC). You can read more about gas
chromatography in OP-34 (pages 205-214) and LC in OP-35 (pages 214-218).
Steam Distillation:
Steam distillation is simply a variation of simple distillation (see OP-27 pages 148-161).
In a steam distillation, a water insoluble organic compound codistills as a two-phase mixture with
water. The organic phase can be separated from the water, dried, and recovered to yield a
purified oil of the natural product. The importance of this application has to do with the fact that
we can isolate organic compounds by distilling below their normal boiling point. In general,
steam distillation is used for the isolation of organic compounds from a purified material (usually
an essential oil). Essential oils are "pure", natural oils that have been extracted from plant
material. They are much more potent (up to 100 times) than the dried herb form. Essential oils
have a long tradition of therapeutic use. Some of the more common oils and their primary
therapeutic effects are listed in Table 4. The use of steam distillation allows the volatile
components of essential oils to be isolated without the decomposition to tar that is likely to occur
at elevated temperatures of a normal distillation. In many cases, a steam distillation is employed
when either simple or fractional distillation cannot isolate a desired component of an essential oil.
Table 4
Essential Oil
citronella
geranium
basil
cardamom
eucalyptus
oregano
rosemary
Effect(s)
insect repellant
anti-inflammation, diuretic, anti-depressant
anti-spasmodic, anti-depressant, fever reduction
sexual stimulant
asthma, sinus problems, bronchitis, acne
antiseptic, anti-bacterial, antiviral
stimulates memory, stimulant, relieves PMS, aching muscles
This experiment:
This experiment will occur over the next two weeks (possibly spilling over into a third
week). Our objective is to isolate and characterize a flavonoid metabolite from a natural product.
Flavonoids consist of a C6-C3-C6 tricyclic array as shown in Figure 2 below. Notice that both C6
rings are benzenes and that C3 portion is tied into a 6-membered oxygen-containing ring (called a
chromane). Variations in structure lead to the different sub-groupings of flavonoids (see Figure
2).
Other variations can be seen at http://www.friedli.com/herbs/phytochem/flavonoids.html.
Figure 2
C6
O
Basic Flavonoid Structure
C3
C6
C1
C2
OR
OR
RO
RO
O
O
Flavanone
OR
OH
OR
O
Flavanol
O
OR
RO
O
Flavone
OR
O
R = H or alkyl (typically, methyl) if the aglycone
R = sugar (usually a disaccharide containing glucose or rhammanose)
Flavonoids occur in nearly every part of the plant – root, heartwood, sapwood, bark, leaf, fruit
and flower. In order to isolate a particular flavonoid, we must have a pre-determined strategy.
The routine procedure involves the following steps:
(a) Drying and maceration of the plant material OR acquisition of the essential oil.
(b) Pre-extraction with hot petroleum ether or hexanes to remove sterols/fats, carotenoids,
chlorophylls, etc.
(c) Our choice of a preliminary isolation strategy is a critical one. The standard methods are
(a) steam distillation of the essential oil, (b) sequential solvent extraction (usually performed with
solvents of varying degrees of polarity), (c) extraction with refluxing (hot) solvent – also, called a
soxhlet extraction (usually, acetone, methanol, isopropanol, or water for flavonoid glycosides and
polar aglycones – less polar aglycones can be extracted with diethyl ether or ethyl acetate), (d)
column chromatography -- here, the selection of mobile phase (solvent) and stationary phase
(solid, usually it’s cellulose, but you may also consider using silica gel or even Sephadex) are
important variables in determining success, or (e) recrystallization (solvent selection is critical
here).
NOTE: Selection of an appropriate preliminary separation strategy is complicated by
the fact that flavonoids exist in the plant as both glycoside (coupled to a sugar
molecule, see above) and/or aglycone (with out the coupled sugar molecule). Of
course, the strategy that we employ to isolate the flavonoid must either (a) isolate
both the aglycone and the glycoside or (b) just isolate the aglycone. More than
likely the isolation will achieve the former of the two (a). Achieving the isolation of
the aglycone (our ultimate objective) is complicated as we must be able to cleave the
sugar moiety through chemical means. Usually, the sugar is cleaved under acidic
conditions with heating (reflux).
(d) Once we have the aglycone in hand, we can then perform the required analyses to
determine its purity. Purity is most effectively ascertained by thin-layer chromatography (TLC)
and by HPLC. In the case of TLC, we will use silica gel coated plates. Your objective will be to
determine an optimal solvent system for separating spots (components of the mixture). You will
also need to determine how you are going to visualize the spots. For HPLC, we will be using a
standard C-18 reverse-phase column with diode-array detection (useful for detecting aromatics,
which comprise flavonoids, so no problem here). You will have to select an appropriate solvent
system (mobile phase) for elution of the components in the mixture.
(e) Structural elucidation (determining the identity of your extract) can be most effectively
assessed by using a battery of spectroscopic techniques. You will be able to choose among 1HNMR, 13C-NMR, 2-D NMR, 13C-DEPT, LCMS, uv-vis, and IR spectroscopy.
Procedure
In this week's experiment, you will have to design an experiment to extract and
characterize a substance isolated from a natural product.
First of all you need to choose a material from which you will isolate a flavonoid. Here are some
possibilities:
1.
2.
3.
4.
Hesperitin from oranges – you can use peels or juice
Apigenin from chamomile, parsley, alfalfa, or sage
Naringenin from grapefruit – peels or juice
If you are game, you may find a plant source that contains one of the flavonoids listed above
and extract from it. I’d consult with Dr. Brown if you choose this route.
You will have to also choose the method of isolation (drying, maceration, extraction, column
chromatography, steam distillation, etc.) and design a procedure to achieve your goal. There are
a number of ways to tackle this assignment. However, the best bet is to (a) follow the guidelines
above and (b) go to the literature for example procedures. We have access here at Nazareth to
ACS publications. Here’s how to access them:
1. Go to: http://www.naz.edu/dept/library/resources/database/subject.cfm
2. Find chemistry and click on it
3. Click on “ACS Web Editions”
4. Here you can search ACS journals – I’d suggest using keywords that will lead you to
flavonoid research – e.g. “flavonoid” or “hesperidin”, etc.
You may also search “Google” as well. Be advised that you may have to use a combination of
strategies to get your flavonoid isolated from the plant material.
Next, you’ll need to determine how you will convert the glycoside to the aglycone.
Finally, you’ll need to have some idea of how you are going to assess purity and do the structural
elucidation. Of course, all you really need to do here is to select appropriate solvent conditions
for TLC and/or HPLC. Again, this information is readily available in the literature.
I will also post on “Andromeda” in the “Organic Chemistry” folder some useful pdf files that
should be helpful in designing an experiment.
YOU MUST GAIN MY APPROVAL OF YOUR PROCEDURE BEFORE STARTING IN
LAB NEXT WEEK!
As this experiment will serve as our semester project, you will be required to write this up as your
formal report.