Experiment # 2: Piperine Synthesis and Isolation of a Natural

Product
INTRODUCTION
Piperine is the naturally occurring alkaloid that gives the spice black pepper its
characteristic biting taste. The stereoselective synthesis of this natural product, in good
yield from inexpensive and readily available starting materials, exemplifies the powerful
synthetic utility afforded by the Wittig reaction and its variations such as the HornerWadsworth-Emmons reaction. The important impact of the Wittig reaction on modern
synthetic organic chemistry was recognized by the 1979 Nobel Prize that was awarded
in part to Georg Wittig for his discovery and subsequent development of this reaction.
O
O

N
O

Piperine
The original Wittig reaction refers to the reaction of phosphonium ylides with aldehydes
or ketones to form alkenes:

O
+
R

R''
R'

P(C6H5)3
R'''

P(C6H5)3

R'''
R'

R''

R
R'

P(C6H5)3
+

R'''
R''

As shown, this reaction mechanism, which has been studied extensively, proceeds
through a four membered oxaphosphetane ring intermediate. This intermediate breaks
down to yield the corresponding alkene and phosphine oxide. Formation of the stable
P=O bond of the phosphine oxide is a major driving force for the reaction. Although the
original Wittig has proven to be a highly useful reaction in organic synthesis, there are

limitations with this reaction that have been largely overcome by the use of
phosphonate ester carbanions (see below). Phosphonate esters can be easily made by
reaction of an alkyl halide with a trialkylphosphite:
OCH2CH3
R

P(OCH2CH3)3

OCH2CH3

CH3CH2Br

The carbanion is then generated by reaction of a base with the parent phosphonate
ester:
OCH2CH3
R

OCH2CH3

Base

OCH2CH3

OCH2CH3

The phosphonate ester carbanions are significantly more nucleophilic than the
phosphonium ylides used in the classical Wittig reaction and therefore react readily
with a wider variety of aldehydes and ketones. Also, the phosphate side product
produced from these reactions is highly water soluble and generally easy to separate
from the alkene product in contrast with the phosphine oxide byproduct of the Wittig.
In order to undergo the Wittig type reaction with alkenes, the substituent on the carbon of the phosphonate ester must be capable of stabilizing the carbanion by
resonance. In the synthesis carried out here, the stabilizing group is methyl 2butenoate:

OEt

O
P
O

OEt

O
P

OEt
O

OEt

Reaction of these stabilized phosphonate ester carbanions with aldehydes and ketones
to form the corresponding alkenes constitutes the variant of the Wittig reaction known
as the Horner-Wadsworth-Emmons reaction shown at the top of the next page.

O
+
R

OEt
R'

(EtO)2PO2

P(OEt)2

OEt
R

R'

R'
R

A remarkable aspect of this reaction is its generally high degree of stereoselectivity for
the trans alkene when both cis and trans possibilities exist. The preference for the trans
product is understandable based on steric effects in the cyclic intermediate. The trans
alkene results from the less sterically strained four membered ring in which the largest
groups are on opposite sides of the ring:

(EtO)2PO2

P(OEt)2

+
R

R'

R'
R

Such stereoselectivity is very important in natural product syntheses where the

biologically important properties of a compound are usually specific to one
stereoisomer. In the case of piperine, the unique property of its desirable peppery taste
is specific to the (E,E) stereoisomer.
In this series of lab experiments, piperine will be obtained from two different sources,
one synthetic and one natural. The synthetic route will be a three-step synthesis utilizing
the Horner-Wadsworth-Emmons reaction. The crude synthetic product should be
sufficiently pure to complete its purification by recrystallization. The natural isolation
route will use an extraction procedure to isolate piperine from ground black pepper. The
crude piperine from pepper will be purified using flash column chromatography. You will
then compare the piperine from the two different sources using spectroscopic
characterization techniques (i.e., 1H NMR, 13C NMR).

SYNTHETIC SCHEME
The following scheme summarizes the reactions that will be used to synthesize
piperine:

Br
O

OCH2CH3

O
O

P(OCH2CH3)3

Methyl 4-bromobutenoate

OCH2CH3

Methyl 4-(diethoxyphosphinyl)-2-butenoate

O
NaOCH3

O
O

O
NaOCH3
O

N
H

Piperine

Methyl Piperate

In the first reaction, the phosphonate ester is prepared from methyl 4-bromo-2butenoate and triethylphosphite. The product, methyl 4-(diethoxyphosphinyl)-2butenoate, is reacted in the second step with sodium methoxide to generate the
phosphonate carbanion in the presence of piperonal. The phosphonate carbanion
undergoes a Wittig type reaction with the piperonal to form the trans alkene, methyl
piperate ((E,E)-5-(3,4-methylenedioxyphenyl)-2,4-pentadienoate). Finally, reaction of
methyl piperate with piperidine in the presence of sodium methoxide in refluxing
methanol solution gives piperine.

EXPERIMENTAL
Synthesis of Methyl 4-(diethoxyphosphinyl)-2-butenoate
Caution: Because of the toxicity of ethyl bromide and the stench of
triethylphosphite, this experiment should be carried out in a fume hood.
Methyl 4-bromo-2-butenoate (3.5 mL, 5.3 g, 30 mmol) (see Note 1) is placed in a 25 mL
round bottom flask equipped with a magnetic stirrer. To the neck of the flask is attached
a Claisen head adapter fitted with a thermometer adaptor allowing the thermometer to
extend down into the liquid in the flask (see Figure 1 below). The side arm of the
Claisen adapter is connected to a simple distillation apparatus. Triethylphosphite (5.0
mL, 4.8 g, 30 mmol) (see Note 2) is added to the stirred methyl 4-bromo-2-butenoate.
The stirred mixture is gently heated. After a brief induction period, an exothermic
reaction takes place and ethyl bromide (bp 37-40 oC) distills from the reaction mixture.
The mixture is heated to 120-130 oC and maintained in this temperature range for
20 min or longer if necessary until ethyl bromide ceases to distill over. The crude
product is characterized by IR and 1H NMR and saved as starting material for step two.

H2O out
H2O in

open

ice bath

heat applied using

heating mantle

Figure 1: Apparatus for Synthesis of Methyl 4-(diethoxyphosphinyl)-2-butenoate

Notes:
1. Methyl 4-bromo-2-butenoate is usually contaminated to some extent with 2(5H)furanone, a side product of its synthesis and purification. The boiling points of the
two compounds are nearly the same, so it is not possible to separate them by
distillation, but they are easily distinguishable by 1H NMR. The starting material
used in this experiment, purchased from Sigma-Aldrich, may contain as much as
15% of the 2(5H)-furanone which does not interfere with the reaction but might
be present in the product as a significant impurity.
2. Triethyl phosphate has an extremely disagreeable odor. It should be handled at
all time in a fume hood.
Synthesis of Methyl Piperate
Sodium metal (0.5 g, 22 mmol) cut into small pieces is placed in a dry 50 mL round
bottom flask equipped with a water-cooled reflux condenser and a magnetic stirrer
placed in a fume hood. Absolute methanol (25 mL) is rapidly added to the flask through
the condenser. Vigorous bubbling commences as hydrogen gas is evolved. (Caution:
make sure there are no open flames or ignition sources in the area.) After all of the
sodium metal has been consumed, the solution of sodium methoxide is allowed to cool
to room temperature.
Methyl 4-(diethoxyphosphinyl)-2-butenoate (5.0 g, 21 mmol), piperonal (3.2 g, 22 mmol)
and 50 mL of dimethoxyethane are placed in a 250 mL three-neck round bottom flask
equipped with a magnetic stirrer, a thermometer adapter, a drying tube, and a dropping
funnel. The sodium methoxide solution is added to the dropping funnel. The reaction
flask is cooled in an ice bath and the sodium methoxide solution is added slowly drop
wise to the mixture with continuous rapid stirring. After addition of the sodium
methoxide solution is complete, the ice bath is removed and the mixture is allowed to
warm to room temperature. The mixture is stirred at room temperature for 2 h, and then
stored in a refrigerator until the next lab period.
The reaction mixture is poured into 200 mL of cold water and the resulting mixture is
stirred for 45 min. The solid that forms is isolated by vacuum filtration and washed with
50 mL of cold water. The crude solid is recrystallized from ethyl acetate to afford methyl
piperate (methyl (E,E)-5-(3,4-methylenedioxyphenyl)2,4-pentadienoate) as light yellow
crystals, mp 146-148 oC. The recrystallized product is characterized by IR and 1H NMR.

Synthesis of Piperine
Sodium metal (0.2 g, 9 mmol) cut into small pieces, is placed in a clean, dry, 250 mL
round bottom flask fitted with a water-cooled reflux condenser. Absolute methanol (75
mL) is rapidly added to the flask through the condenser. Vigorous bubbling commences
as hydrogen gas is evolved. (Caution: make sure there are no open flames or
ignition sources in the area.) After all the sodium has been consumed, methyl
piperate (3.5 g, 15 mmol) and freshly distilled piperidine (17 mL, 170 mmol) are added
to the sodium methoxide solution. The solution is refluxed for 40 h and then allowed to
stand at room temperature until the next lab period. The resulting red-brown solution is
poured into 300 mL of cold water and stirred for 1 h. The precipitate that forms is
collected by suction filtration to afford piperine as a beige powder. The solid is
recrystallized from ethyl acetate/hexane to give fine, yellow-beige needles (mp123124oC). The recrystallized piperine is characterized by IR, 1H NMR, and 13C NMR.
O
O

N
O

Piperine

Piperine Isolation and Purification of the Natural Product

The chemistry of natural products is an integral part of organic chemistry. Around the
turn of this century, chemists endeavored to discover the active agent in black pepper.
In this project, you will isolate and purify this agent, piperine, from black pepper, study
its structure with 1D NMR experiments, and compare this structure to what you
synthesized in the laboratory.
Isolation of Piperine

Place 15 g of ground black pepper in a 100 mL round-bottom flask, add 30 mL of

CH2Cl2 and at least five boiling chips. Attach a water-jacketed condenser and heat at
reflux for 0.5 hr. IN YOUR FUME HOOD, using your large Buchner funnel, suction filter
the mixture, rinse the pepper solids with CH2Cl2 (up to 15 mL), and then concentrate the
resulting filtrate by use of a rotary evaporator. Remove the bulk of solvent.
To remove excess CH2Cl2, cool the residue in an ice bath, add 5-10 mL of cold diethyl
ether to the flask and triturate the residue until the ether is mixed in well. Precipitation
may begin at this point. Remove the ether using the rotary evaporator. If the remaining
residue still appears oily, repeat the cooling/trituration/evaporation process. When the
residue appears to have solidified, dissolve as much of it as possible in 10-15 mL of
95% ethanol.
To 10 mL of a 10% solution of potassium hydroxide in 95% ethanol, contained in a 125
mL Erlenmeyer flask, add the ethanolic pepper extract. Be sure to wash any
undissolved solid into the KOH flask with a small amount of 95% ethanol.
Warm the resulting solution (stean bath) and slowly add water with a Pasteur pipette
(trickle the water down the sides of the flask). The mixture should become cloudy and a
yellow precipitate should begin to form. Continue adding water in this fashion until you
have added a total of 100 mL. Cover the flask with Parafilm, label it, and allow the
mixture to stand until the next lab period.

This laboratory experiment is modified with permission from the Pomona College Departmental of
Chemistry 2003 Organic Chemistry Laboratory Manual.

Purification of Piperine
Collect the solid by suction filtration using the small Buchner funnel and two pieces of
filter paper (this helps avoid clogging of the filter paper). Wash the solid with two 5 mL
portions of water. If you observe solid in the filtrate, collect it also. To facilitate drying,
wash the solid with one 5 mL portion of cold diethyl ether. Allow the solid to dry under
vacuum (use the vacuum manifold on the vacuum cart and have a TA assist you with
this manipulation) before determining its mass. Determine and report the crude yield of
this powder as a weight percentage of the black pepper you started with.
After you have determined the crude yield, weigh 350 mg of the piperine into a small
vial. Please note: if you dont have 350 mg of piperine, contact your TA.
Dissolve/suspend the powder in 25 mL of CH2Cl2 and use TLC to determine the purity
of this crude material. Be sure to use the standard 3 spot procedure (lane 1 = crude
compound, lane 3 = authentic piperine, lane 2 = co-spot of lanes 1 and 3). Use a 40%
ethyl acetate/60% CH2Cl2 mixture as the elution solvent and UV irradiation to visualize
the developed plate. Draw the plate in your notebook and record the Rf values of
any significant spots.
Prepare your silica gel column using the following procedure. Weigh approximately 32 g
of silica gel into a 250 mL beaker and suspend the silica gel in CH2Cl2 (70 mL).
Dislodge any air bubbles by stirring the slurry with a clean spatula. Clamp the column to
the distillation rack in your hood. The outflow of the column should be directed to a 125
mL Erlenmeyer flask. Add a small quantity of sand (a powder funnel is handy here) so
that a 0.5 cm layer rests on top of the glass frit. Add a small quantity of CH2Cl2 so that
flow is established and arrest the flow with the stopcock when there is a one-inch layer
of CH2Cl2 covering the sand layer. Swirl the silica gel slurry (some settling may have
occurred) and slowly pour it down the inner surface of the column so that it disperses
and settles evenly when it reaches the one-inch layer of CH2Cl2 at the bottom of the
column. After most of the silica gel slurry has been transferred to the column, open the
stopcock and allow the excess CH2Cl2 to drain through the column. As this is
happening, use a Pasteur pipette and some of the excess CH2Cl2 to rinse down any
silica gel that might be adhering to the upper inside surface of the column. After the
CH2Cl2 has drained, the column should have a level upper surface. Add dry sand such
that a 0.5 cm layer rests on top of the silica gel. A few mL of CH2Cl2 can be used to
wash down any sand that might be adhering to the inner surfaces of the column. A well
poured column should be evenly packed and free of any visible air bubbles.
Use a Pasteur pipette to add the crude pepper extract (350 mg in 2-5 mL CH2Cl2, as
described above) to the top of the column bed. Add the solution in such a manner such
that it enters the column in an even manner. This can be accomplished with 1-2 smooth
pipette discharges using expanding concentric circles above the top of the column bed.
If any of the solution accidentally gets caught on the inner glass surface, dont worry too
much it will get washed down onto the column with subsequent rinses.

Allow the solution to enter the column and use 2 mL of fresh CH2Cl2 to rinse the inner
surface of the column and sand layer (some misers will use this volume of solvent to
rinse the residual vial contents onto the column this is a useful maneuver when
dealing with a precious compound). Repeat this rinse twice, always allowing the excess
solvent to drain from the bottom before adding new solvent to the top of the column.
The goal is to load the mixture onto the column in a non-polar solvent, in as
concentrated a solution as possible.
To the top of the column, carefully add the first elution solvent (100 mL of 40% v/v ethyl
acetate in CH2Cl2) and force it through the packed silica by applying a stream of
compressed air to the top of the column. Use care when adding solvent to the column:
you dont want to disturb the top of the column packing! When it appears that solvent
front (i.e., the blue fraction) is about to emerge from the column (the compounds are
yellow in color), begin to cut fractions with 13x100 mm test tubes (fill each ca. 3/4 full).
When the 40% ethyl acetate/60% CH2Cl2 eluent is running low, make a 100 mL batch of
60% ethyl acetate/40% CH2Cl2 and continue the column until this eluent has been used.
Use TLC to assess the chemical identity of each fraction. This is conveniently done by
spotting 4-5 fractions on one 1 inch-wide TLC plate. In order to pack this many spots
on one plate, you must use care with your spotting technique (use tight, non-diffuse
spots on a plate pre-marked with pencil spots to indicate the origin of each lane). Be
sure to draw these TLC plates in your notebook.
Using a pre-weighed round-bottom flask, consolidate the piperine fractions and remove
the solvents with a rotary evaporator. If the compound does not immediately form a
solid, try triturating the flask contents with a few mL of ether or hexane and then remove
the excess solvent again with the rotary evaporator. Collect the solid and determine its
mass and melting point. Calculate and report the yield as a weight percentage of
piperine contained in the crude material used for chromatography.
NMR Characterization of Piperine
Using the compound purified by column chromatography, prepare a sample for NMR
analysis by dissolving 10-15 mg in CDCl3. Obtain a 1H and 13C NMR spectra on the
sample. Compare these spectra to those you collected for synthetic piperine. Are they
the same? Include these spectra and your analyses in your lab report.

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