Fall Experiment #4
Using Thin-Layer Chromatography to monitor and Column Chromatography to purify
organic reactions
(Multi-step Synthesis of 5-alkoxy-4-methoxy-2-nitro-trans-cinnamic acids)
Introduction
This multi-step organic synthesis project consists of two interconnected pieces. In Part A
of the project, our research team is interested in developing a multistep synthesis of a key
synthetic intermediate. This synthetic intermediate can then be employed in Part B as a critical
synthon for the creation a small molecule combinatorial library analogous to the example target
structure shown below. The merit of this project involves the creation of a novel 1,4benzothiazine heterocycle (1, below) that can be adorned with amino acid units creating a
potential drug-like molecule or useful biological probe.
O
O
OH
OH
X
X
NH2
NO2
O
O
OH
OH
S
X
N
H
S
X
NH
R
N
NH
R
1
The starting point for synthesis of molecules like 1, is the nitrocinnamic acid structure shown in
red. As a primary research objective, we would like to employ diversified nitrocinnamic acids
that can be varied depending upon the substitution pattern in the aromatic ring (represented by the
X group). This objective can be accomplished using commercially available 2-nitrocinnamic
acids or through the synthesis of novel compounds like the one shown below.
O
OH
6
RO
5
1
4
H3CO
2
3
NO2
Herein, we have an excellent opportunity to provide diversity of structure through variation of the
substituents in the aromatic ring (here, crudely represented by the R group). Our goal is to create
molecules with enough molecular diversity to allow full investigation of the effects of
substitution in the aromatic ring above. As part of the investigation we can ask what effect does
substitution of the aromatic ring have on the binding of the target structures to other molecules of
biological significance (for example proteins, nucleic acids, etc)? Some such example structures
are shown below.
Position #5
We are particularly interested in structures like the one appearing in the oval. The synthesis of
structures like this can be accomplished using the strategy outlined in the following scheme. The
idea is to substitute various R groups in place of the isoamyl group (also called an isopentyl
group) that is present on the oxygen at position #5 of the ring.
Procedure
Step 1 (already completed for you)
The first conversion involves the protection of the aldehyde as the ethylene glycol acetal. This is
important as we do not want this particular position to be influenced by the chemistry that we will
be employing in subsequent steps. Often, organic chemists introduce protecting groups and then
remove them when they are no longer needed or to reveal functionality later on that can be
manipulated.
6-Nitroveratraldehyde (47.4 mmol) is mixed with 120 mL of toluene in a 250 mL round-bottom
flask. Ethylene glycol (1 equivalent) and a catalytic amount of p-toluenesulfonic acid (0.03
equivalent) are added to the mixture, which is heated at reflux for 3 hours and the water is
removed by Dean-Stark apparatus. Upon completion, the solvent is removed via rotary
evaporation to yield a yellow solid. The product is dissolved in the minimum amount of hot
EtOAc and the product is left to crystallize. The solid is isolated by vacuum filtration and is
weighed. Finally, a 30 mg sample is set aside for mp, IR, 1H-NMR and 13C- NMR analyses.
Step 2 (already completed for you)
The next reaction involves the selective deprotection under basic conditions of the metamethoxy ether (position #5, but not at position #4 – see numbering on the structure at the bottom
of page 1).
You will set up a 48 hr reflux reaction for the selective deprotection of the aromatic methoxy
group. The product from the previous step (16.11 mmol) is dissolved in 15 mL of 1,4-dioxane
and 75 mL of 20% KOH(aq) is added to the mixture. The resulting solution is heated under reflux
for 48 hrs. The resulting solution is cooled in an ice-bath and neutralized to slightly acidic (pH =
6.5) at which point the crude product precipitates from solution. BE CARFUL NOT TO OVERACIDIFY!!!!!!!!!!!!!!!!!!!!! It will ruin your reaction!!!!!!!!!!!!! The crude product is isolated by
vacuum filtration, washed with water and recrystallized from EtOAc. The recrystallized product
is isolated by vacuum filtration and dried on a vacuum pump overnight. The mass of product is
determined and a 30 mg sample is set aside for mp, IR, 1H-NMR and 13C- NMR analyses.
Step 3 (this is the work you will actually perform in lab – START HERE)
From here, the free phenolic group (phenol = an alcohol group attached to aromatic ring – can
you find this structure?) can be reacted with a primary or sterically-accessible secondary alcohol
under Mitsunobu conditions (alcohol, PPh3, and DEAD or DIAD).
1st week: To 2-neck 100 mL RBF, add the starting phenol (750 mg) and 1.3 equiv. of PPh3. Place
a septum over the side outlet and a gas inlet adapter over the top outlet and introduce a strong
stream of N2 gas to flush the flask. Under an N2 atmosphere, add 10 mL of dry THF by syringe
followed by your unknown alcohol (1.5 eq.), by syringe and place the entire mixture in an ice
bath for 15 minutes. At 0 ºC, add dropwise via syringe 1.6 eq. of DIAD
(diisopropylazodicarboxylate) that has been dissolved in 2 mL of dry THF in a small glass vial.
After addition, the reaction is stirred at 0 ºC for 15 minutes followed by room temperature for 1
hour. An aliquot is removed from the reaction mixture and the progress of the reaction is checked
by TLC (See the videos on the MIT Virtual Lab Manual site – Videos #3 and #4) in 3:1
hexane:EtOAc spotting against PPh3 and the starting phenol. The spots can easily be visualized
by viewing under UV light (the conjugated aromatic (benzene) ring absorbs UV light). If the
reaction is incomplete an additional 0.3 eq. of PPh3 is added and the reaction is continued for
another hour. The cycle of TLC monitoring and addition of PPh3 is continued until the reaction
has gone to completion. Upon completion, the solvent is removed in vacuo. The residue is
redissolved in CH2Cl2 and then a small amount of alumina (neutral) is added to the flask. The
contents are swirled and the solid is removed by filtration through a plugged filtration adapter.
The solvent is again removed in vacuo. The product is purified by column chromatography as
described below.
2nd week: Our crude reaction mixture can be efficiently purified using standard column
chromatography. (See the video on the MIT Virtual Lab Manual site – Video #10) The theory
underlying the separation of components is the SAME as that for TLC. In this technique, we will
pack a glass column with silica gel (the same material that is on the rough surface of your TLC
plate) that will serve as the stationary phase. In order to pack the column, we must saturate
completely with our mobile phase before we develop it. During the development part of the
experiment, we will collect fractions of the solution that elutes from the column. These fractions
can be analyzed using the mobile phase/TLC system we developed above. We combine like
fractions containing our spot of interest and remove the solvent using rotary evaporation. The
remaining residue in the round-bottom flask should be our PURE product.
Here's the column chromatography procedure in a little more detail:
We begin by introducing a small (1-2 cm) layer of washed sea sand, followed by 10-15 cm of
silica gel (70-200 mesh) -- see the figure below. We then select a relatively nonpolar mobile
phase to pack the column (90% hexane/10% EtOAc should work well). The solvent is carefully
introduced into the column and then is forced through with the help of an air or compressed gas
line. Additional solvent is introduced so that the column does not dry out. Once the column is
saturated (we determine by visual inspection), the solvent level is lowered to just even with the
top of the silica gel. At this point, we prepare a concentrated solution of our crude material. This
solution is CAREFULLY pipetted onto the top of the column being careful to not disrupt the
surface. The solvent level is returned to even with the top of the solid material and capped with a
3 cm layer of washed sea sand. Our mobile phase of choice is then reintroduced and fractions are
collected (usually in medium size test tubes). Be careful to never let the column dry out and to
drain solvent to the top of the column when changing to a different mobile phase. Fractions
are TLC'd and combined as appropriate for concentration.
Step-by-step summary of the procedure:
(1) clean the column and dry before packing.
(2) mount the column using the clamps (provided)
(3) fill the bottom of the column with approximately 1/2 inch of sea sand
(4) fill the next 10 inches of the column with 60 x 200 micron silica gel
(5) introduce the initial mobile phase (9:1 hexane:EtOAc) and pack with nitrogen gas
(6) once you've generated a homogeneous mixture, introduce your sample by dissolving in the
minimum amount of dichloromethane
(7) drain off solvent until the liquid level matches the top of the solid silica gel column
(8) cap your column with 1/2 inch of sea sand
(9) fill the column with the initial mobile phase
From here you will run solvent through until the level of yellow bands is approximately 1 inch
above the frit. At this point, begin collecting fractions in 15 x 125 mm test tubes. Periodically,
check your fractions by TLC using 3:1 hexane:EtOAc as the mobile phase in the chamber. If it
does not appear that your sample has come off the column by fraction #10, consult with your
instructor to see if you might switch mobile phases (to something more polar). Once you've run
all of your desired sample off of the column, TLC fractions to determine which ones can be
combined. Combine like fractions in a large RBF and remove the solvent on the rotary
evaporator, followed by further drying under a stream of N2 gas or exposure to high vacuum to a
constant mass. Be sure to preweigh this flask! Be sure to record the mass of product recovered.
***Additional information on TLC and column chromatography can be found in Lehman
Operation #21 (pages 120-132) and Operation #22 (pages 133-140).
The DRY acetal-protected product isolated from column chromatography is saved for
confirmation of the structure by 1H- and 13C NMR, IR, and mp analyses (subsequent weeks).
Steps 4 and 5 (will not be completed)
The acetal protecting group is then removed (deprotection) under acidic conditions (step 4) and
then immediately reacted via Knoevenagel condensation (step 5) to generate the desired
nitrocinnamic acid target molecule. The chemistry for this series of steps has been derived in
large part from J. Chem. Soc. Perkin I, 1978, 440-446. Once complete, the nitrocinnamic acid
can be incorporated as a reagent in the construction of a much larger molecule that is adorned
with amino acids units creating a potential drug-like molecule or useful biological probe.