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Quoc Tran
Chemistry 213
Kristen Beiswenger
Final Formal Report #1
April 8, 2015
The Synthesis of a Coumarin Laser Dye via Knoevenagel Condensation
Introduction
If applied correctly, colors can serve to add emotion, visual aesthetics, and even life to a piece of
art. In the field of science, staining with dyes and colors proves to be of strong use in biology, the study
of life. Under the microscope, one can examine a cell and visually observe its image. One way to easily
observe the life of a cell is to use fluorescent probes to dye its biological systems. Fluorescent probes are
tools for cell biologists to create real-time imaging of live cells.1 Through this tool, cells do not
experience stress and damage. Therefore, fluorescent dyes provide a safe medium to preserve the life of
a cell. From visualizing enzyme activation to tracing diffusion across a membrane, advances in
fluorescent probing has allowed biologists to study complex cellular events.1
Depending on the solvent, Coumarin dyes suspended in a medium have the emission properties
of blue to green.2 It has the potential to be used as a laser dye for organic dye lasers in which its range of
emission can be controlled. For example, the tunable organic laser manipulates the emission of
Coumarin laser dye. This tuning ability of the laser can probe potential health risks to biological
systems.3 Coumarin dyes have been used as probes to detect harmful levels of thiophenols that can
causes muscle weakness, comas, paralysis, and even death.4
In this experiment, an analogue of Coumarin dye named 110 Coumarin laser dye was
synthesized using a Knoevenagel condensation (Figure 1). Since this particular reaction involves a
condensation pathway, carbon-carbon bonds were formed in synthesizing the product.5 More
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specifically, the Knoevenagel condensation involves a reaction between an aldehyde, ketone, or
compound with an active methylene group with an organic base to form an unsaturated compound.6
21°C, 30 min
1
2
3
Figure 1. Synthesis of Coumarin Laser Dye 3 from 4-(diethylamino)salicyaldehyde 1 and
ethyl acetoacetate 2.
In producing the Coumarin 110 3 analogue, the mechanism (Scheme 1) begins with the conversion
of ethyl acetoacetate 2 to an enolate ion by the catalytic base of piperdine. The enolate ion performs a
nucleophilic attack to the electrophilic carbonyl group of 4-(diethylamino)salicyaldehyde 1. The
resulting alkoxide ion intermediate protonates the conjugate acid and the base of piperdine is reformed.
The base then performs E1CB elimination on the hydrogen in the tetrahedral carbon and a hydroxyl
group is removed. The remaining hydroxyl group attached to the benzene ring performs another
nucleophilic attack on the ester carbonyl group to form a six membered ring. The enolate ion’s electrons
then move down to remove an ethoxide group. The ethoxide group then deprotonates the hydrogen on
the previous hydroxyl group to form ethanol and Coumarin 110 3.
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Scheme 1. Mechanism of Knoevenagel Condensation of ethyl acetoacetate and of 4-(diethylamino)salicyaldehyde.
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The purpose of this lab is to synthesize and purify product 3 through a condensation pathway. By
using the reagents of piperdine and ethanol, a Knoevenagel condensation converts the starting materials
of ethyl acetoacetate and of 4-(diethylamino)salicyaldehyde to the unsaturated compound. The products
of this reaction can then characterized by melting points with literature values and NMR
instrumentation.
Experimental
Coumarin 110. 4-(Diethylamino)salicyaldehyde (0.400 g, 2.07 mmol), ethyl acetoacetate (0.530 mL,
2.80 mmol), and piperdine (3 drops) were added to a flask and stirred for approximately 30 minutes at
room temperature. Upon completion of the reaction, the reaction mixture was refluxed for 20 minutes
and then cooled. The crystals were isolated using vacuum filtration and recrystallization was performed
using ethanol (20 mL) as a solvent. The flask was then cooled to room temperature and vacuum filtration
of mixture was performed again to afford a deep-yellow powder (430 mg, 80%). 1H NMR (400 MHz,
CDCl3): δ (ppm) 8.44 (s, 1H), 7.39 (d, 1H), 6.61(d, 1H), 6.46 (s, 1H), 3.49 (s, 4H), 2.68 (s, 3 H), 1.23 (s,
6H); 13C NMR (400 MHz, CDCl3): δ (ppm) 195.7, 160.9, 157.8, 153.1, 147.9, 131.9, 116.0, 109.9, 108.1,
96.5, 53.4, 45.2, 30.9, 30.6, 12.4., UV/Vis: δ (nm) 434.
Results and Discussion
The synthesis of Coumarin laser dye 3 experienced a Knoevenagel reaction with the organic base
of piperdine. The condensation of 2 ethyl acetoacetate and 1 4-(diethylamino)salicyaldehyde afforded
an unsaturated compound through the interactions with enolate ions and intramolecular cyclization.
To synthesize the product 3, small amounts of starting materials were needed because this
reaction can be very vigorous with the given catalyst of piperdine even running at room temperature.
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This amine base activates the carbonyl group by deprotonating the alpha hydrogen and initiates the
reaction between the starting materials. Throughout the course of the mechanism, the formation and
reformation of piperdine allowed functional groups such as the hydroxyl group to become nucleophiles
in order to attack carbonyls. Finally, it resulted in the movement of electrons to remove of leaving
groups of ethoxide. Since a dye was needed to be formed at the end of the synthesis, observing a color
change from a colorless solution to a dark red mixture helped indicate the progress of the reaction.
Assuming that most of the starting materials of 1 and 2 were converted to solid product 3,
recrystallization was an effective technique to purify the target solid with small amount of impurities.
Additionally, absolute ethanol was used as the solvent to partially dissolve the solid product of 3. Due
to its ability to have hydrogen bonding, ethanol is slightly higher in polarity than the target product 3
which does not exhibit hydrogen bonding. As a result of similar polarities, the product 3 was sparingly
soluble at room temperature and completely soluble upon heating in ethanol. Furthermore, since starting
material 1 exhibits hydrogen bonding, any small amounts of impurities remained in solution while solid
product 3 recrystallized upon cooling.
The identity of the product was characterized using 1H NMR spectrum (Figure 1, Supplemental
Information). The 400 MHz NMR provided key pieces of information that supported the synthesis
Coumarin 110 in this experiment. The first peak at approximately 1.25 ppm represents the hydrogens on
the two methyl groups at the end of the diethylamine functional group. This peak has an integration
value of six which represents the six total numbers of hydrogens on the two methyl groups. Its triplet
splitting pattern is caused by hydrogens adjacent to the methyl groups on the ethyl branch. These
hydrogens are found at about 3.475 ppm and have an integration value of four. The hydrogens
experience a splitting pattern of a doublet as well since they are adjacent to the hydrogens on the methyl
groups as previously noted. Furthermore, a methyl group found at 2.6804 ppm has an integration value
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of three and it exhibits a splitting pattern of a singlet because there are no adjacent hydrogens present.
Moving towards Coumarin 110’s aromatic structure, the overall lack of symmetry in structure causes
three distinct hydrogens to be found from the benzene ring. The two hydrogens on the adjacent carbons
located at one side of the ring cause a doublet splitting pattern for one another at 6.6173 and 7.3897
ppm. On the other side of the benzene ring, the lone hydrogen is found at 6.4691 ppm with a singlet
splitting pattern as there are no adjacent carbons with hydrogens attached. Its location right in between
electronegative atoms of nitrogen and oxygen limits the withdrawal of hydrogens for downfield shifts.
Finally on the second ring, the lone hydrogen is located far down field at 8.4357 ppm and has an
integration value of one. The starting material 1 contained very similar types of hydrogens as product 3
so only a few key types of hydrogen were indicative of the synthesis of product 3. For instance, the
peaks at 1.25 ppm were not helpful because both the starting material 1 and product 3 contained those
hydrogens on the two methyl groups at the end of the diethylamine functional group. On the other hand,
the presence of the hydrogen on the second ring of product 3 suggested a successful synthesis of
Coumarin laser dye. On the spectra, the lone hydrogen is located far down field at 8.4357 ppm and has
an integration value of one. It is located adjacent to a carbonyl group that withdraws electrons to cause
its extreme downfield shift.
The second spectral characterization using 13C NMR (Figure 2, Supplemental Information)
worked in conjunction with the 1H NMR in identifying the product as Coumarin laser dye. The peaks at
195.74 ppm and 160.91 ppm reveal the carbonyl groups in the structure. The ester carbons are found at
45.16 ppm and 53.47 ppm. The remainder of the carbons fall in between 10 to 30 ppm.
The UV/Vis analysis (Figure 3, Supplemental Information) provides both an exceptional
qualitative and quantitative source of evidence in identifying the product as Coumarin laser dye.
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UV/Vis analysis of the product exhibits the largest emission peak at 434 nm suspended in ethanol which
corresponds to the blue region of spectra.
Finally, the comparison of the product’s melting points to literature values supports the
characterization of the product as the Coumarin 110 analogue. Upon recording the melting point range
of 152.0-154.3 °C for the synthesized product. The starting materials 1 and 2 have respective melting
points of -43 °C and 60-62 °C. If the product contained substantial or trace amounts of impurities from
the starting material, the melting point range would have been observed to be lower. Comparing this
recorded melting point to the literature value of 151-153 °C, the close proximity between the melting
points asserts the fact that Coumarin laser dye was synthesized and purified.
In synthesizing Coumarin laser dye in this experiment, an 80% yield was achieved. This yield
shows a relatively efficient conversion of starting materials to the target product. Any loss of product
could have been from the purification technique of recrystallization. While no crude amount of product
was recorded before purification, too much of ethanol could have been used in the recrystallization
process and caused the loss of product by dissolving some of the crystals. From NMR instrumentation
to UV/Vis analysis, evidence from these sources of data asserts the successful synthesis of Coumarin
laser dye from a Knoevenagel condensation reaction. Possible sources of error exist in excessive
washing of the crystals with ethanol as a solvent. Additionally, adding too much hot ethanol to the
crude product during the recrystallization product could have forced the Coumarin laser dye to remain in
solution. One may want to avoid drastic changes in temperature upon cooling and reforming of crystals
as well because it could lead to inconsistent crystal formation. Of course, another source of error may
lie in the crystals sticking to glassware. Improvements for these possible sources of error include using
an appropriate amount of the correct solvent during recrystallization, avoiding excessive rinsing with the
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cold solvent after vacuum filtration, allowing for more time for the crystals to form, and checking if the
product was stuck to any glassware.
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References
(1) Zhang, J.; Campbell, R.; Ting, A.; Tsien, R. Creating New Fluorescent Probes for Cell Biology.
Nat Rev Mol Cell Biol. 2002, 3, 906-918.
(2) Jones, G.; Rahman, M. Fluorescence Properties of Coumarin Laser Dyes in Aqueous Polymer
Media. J. Phy. Chem. 1994, 98, 13028-13037.
(3) Duarte, F. Organic Dye Laser. Opt. & Photo. News. 2003, 14, 20-25.
(4) Li, J.; Zhang, C.; Yang, S.; Yang, W.; Yang, G. Anal. Chem. 2014, 86, 3037-3042.
(5) McMurry, J. Carbonyl Condensation Reactions. Org. Chem.; Brooks/Cole: Belmont, California,
2012; pp 904-905.
(6) Jones, G. The Knoevenagel Condensation. Org. Reac.; John Wiley & Sons, 2011, 15, 2, 204–
599.