Hiram College, Chem. 320: Spring 2010
Student Notes: Green Nitration of L-Tyrosine
“An Electrophilic Aromatic Substitution (EAS)”
(ref: Jones-Wilson, T.M.; Burtch, E.A. JCE., 2005, 84, 616-617)
In this experiment you will:
Successfully perform an electrophilic aromatic substitution reaction on an organic substrate and
characterize the product.
Techniques: refluxing, suction filtration, and recrystallization
Characterization: melting point, 1H NMR Spectroscopy, and UV-Vis Spectroscopy
Chemicals Used: L-Tyrosine, concentrated nitric acid, concentrated sulfuric acid, ethyl acetate, and D2O
(NMR solvent). (All of these should be in table of reagents including the overall product)
Pre-Lab Assignments
Notebook
After table of reagents include: overall reaction (draw the structures)
convert all amounts to moles
label the limiting reagent
calculate the theoretical yield
Questions (on a separate sheet of paper, turn in at the beginning of lab)
1. Show the reaction for the electrophilic nitration of tyrosine
2. Provide a list of at least 5 strong ortho, para directing substituents and 5 strong meta
directing substituents in EAS reactions. What type of substituents are on the starting
material?
3. Describe the expected results from UV-Vis analysis and 1H NMR (i.e. absorbance
wavelength and important chemical shifts).
4. Using 0.05 g of product (3-nitrotyrosine), show the calculation for a 1 x 10-4M
solution in water. You will be given two 20 ml volumetric flasks, you will need to
carry out 2 dilutions.
Waste Disposal: Aqueous waste can be disposed of in the sink with water. Ethyl acetate and all solid
wastes should be collected in the proper waste containers in the hood.
Electrophilic Aromatic Substitution
Electrophilic aromatic substitution is the most significant reaction type experienced by aromatic
compounds and is fundamental to the study of organic chemistry. Thousands of substituted aromatic
compounds can be prepared by means of this reaction. In electrophilic aromatic substitution, the
nucleophilic aromatic ring reacts with a strong electrophile and addition occurs. A hydrogen atom is
then eliminated and aromaticity is restored to the substituted ring.
Hiram College, Chem. 320: Spring 2010
All aromatic compounds, including benzene and substituted benzene rings, are capable of undergoing
electrophilic aromatic substitution. Conjugated systems contain a substantial amount of electron
density, thus allowing them to act as efficient nucleophiles. The driving force behind the electrophilic
aromatic substitutive process is the extreme stability of the aromatic ring. In general, alkenes react with
strong electrophiles by means of addition. In the first step, the nucleophilic aromatic ring attacks a
strong electrophile. This process is identical to that of addition and is the rate-limiting step for the
aromatic substitution. A nonaromatic carbocation intermediate is produced.
This intermediate is doubly allylic and can be resonance stabilized. Nevertheless, this first step is
endothermic and requires significant activation energy due to the loss of aromaticity experienced by the
ring. As a result, addition to the carbocation intermediate does not occur. Instead, elimination of
hydrogen occurs and a neutral, aromatic compound is produced. The stabilization energy of the
aromatic ring is restored and the net reaction is exothermic. Substituents already on the aromatic ring
affect both the reactivity of the substrate as well as the orientation of the reaction. Functional groups
that are capable of donating electron density generally serve to activate the ring and are ortho/para
directors.
Electron withdrawing substituents are most often deactivators and direct further substitution onto the
meta position. Reactions of activated aromatic rings proceed more quickly than those of deactivated
substrates because electron donating groups are capable of stabilizing the carbocation intermediate
formed. This lowers the activation energy of the addition step. Deactivating groups, however,
destabilize this intermediate and so the rate of formation is decreased. The position of substitution can
also be explained by considering resonance and inductive effects. Ortho and para substitution occurs
because the carbocation intermediate is most stabilized in these configurations when electron donating
groups are present. On the other hand, with the presence of electron withdrawing groups, the
carbocation is most stabilized when meta addition occurs. Common electrophilic aromatic substitution
reactions are employed to halogenate, nitrate, sulfonate, alkylate, and acylate aromatic compounds.
Electrophilic aromatic substitution reactions differ drastically from nucleophilic substitution reactions in
that no leaving group is expelled. As a result, the primary environmental consideration for electrophilic
substitution reactions involves the nature of the aromatic substance. Unfortunately, though, many
common aromatic compounds are toxic.
Nitration of Tyrosine
Amino acids are green reagents because they are nontoxic. They contain a variety of functional groups
useful for the illustration of common organic reactions. An alternative to the traditional starting
materials employed to demonstrate electrophilic aromatic substitution makes use of the aromatic
moiety on the amino acid tyrosine. Concentrated sulfuric acid and nitric acid can be used to generate a
nitronium electrophile. The aromatic ring on tyrosine can then react with the nitronium ion via
electrophilic aromatic substitution to produce 3-nitrotyrosine. Although both the hydroxyl group and
the alkyl substituent serve to activate the aromatic ring, the hydroxyl is a stronger electron donating
group. Therefore, substitution occurs at the ortho position to produce 3-nitrotyrosine. The deactivating
effect of the nitro substituent restricts any additional nitration from occurring.
Hiram College, Chem. 320: Spring 2010
Procedure
Reaction Synthesis of 3-nitrotyrosine
1. Weigh 5.0 g L-tyrosine in a 100-mL round-bottomed flask attached to a Claisen adapter.
2. Add a stir bar and fit with a reflux condenser. Fit the open neck of the claisen adapter with a lightly
greased stopper. Correctly attach the reflux condenser to the water supply (in the bottom and out the
top!)
3. Add 20 mL of DI water to create a suspension. Stir and turn on the water supply.
4. Cool a second 50-ml round-bottomed-flask in an ice water bath. See the Hazards section for
additional cautions. Add 3.6 mL of concentrated H2SO4 and 4.7 mL of concentrated HNO3 carefully via
the flask neck using a disposable glass pipette, swirling the flask occasionally. The addition should be
dropwise and at a rate to minimize the effects of the highly exothermic reaction.
5. When the acid mixture has cooled move the ice water bath to the 100 mL flask. See the Hazards
section for additional cautions. Add the acid mixture dropwise to the tyrosine suspension under water
reflux and with gentle stirring. Cooling in an ice water bath should be maintained throughout the
addition. Make sure your condenser is operational throughout the reaction.
6. After the addition is completed, leave the reaction in the ice water bath with stirring for 15 minutes (if
a stir plate is not available gently swirl the flask in the ice water bath). In the meantime prepare a 40°C
water bath for the next step.
7. Remove the ice water bath and allow the reaction to warm to room temperature while stirring. Place
the reaction mixture in the 40°C water bath for 30 minutes. Continue stirring.
Product Isolation and Purification
1. Transfer the reaction solution quickly to an Erlenmeyer flask. Cool the Erlenmeyer flask in an ice water
bath until crystallization occurs.
2. Filter using a Buchner funnel and collect the crude product.
3. Rinse the product cake with a 10-mL aliquot of ethyl acetate.
4. Recrystallize in the minimal volume of deionized water.
5. If crystallization does not occur see your instructor. Check with your instructor about which methods
you will be using to characterize your product.
6. Obtain the melting point of your dry product.
7. Prepare a 10-4 M solution of your product in deionized water and obtain a UV spectrum. Scan from
240nm-400nm.
8. Prepare a 10% solution of your product in D2O and obtain an 1H NMR.
Hiram College, Chem. 320: Spring 2010
Hazards
In part of this experiment you will handle concentrated acids. ACIDS BURN! Take care when handling
the sulfuric and nitric acids. Wear gloves and safety goggles and make sure you carefully follow the
procedure. Additions of acids are HIGHLY exothermic. Add acids SLOWLY and COOL and stir as
directed. If you spill any of the acid on yourself immediately notify your instructor and rinse with
large quantities of water.
Reactivity Hazard
Mixtures of concentrated sulfuric and nitric acids violently oxidize acetone. All glassware
containing the acids should be thoroughly cleaned and rinsed with water before drying with
acetone. Any paper used to wipe up nitric acid spills should be thoroughly rinsed to avoid fire
hazard. As always: wear your safety goggles!
LAB REPORTS (20 points) -You must have in order:
1) Title (in bold and centered)
2) Objectives-1 pt
3) Procedure -a few sentences, reference where the entire procedure can be found -1 pt
4) Results -must include the overall reaction, equation for percent yield, one sample calculation for
percent yield, a table with all obtained percent yields (if only one product was obtained no table is
needed for percent yield), a table for melting points with both experimental and literature values and
reference where it was found, a table with important chemical shifts from 1H NMR-labeling each shift
with it’s characteristic bond, and a table including the wavelength max from UV-Vis spectroscopy
including both the experimental and literature values- 6 pt
5) Discussion-discuss the results from the percent yields, melting points, NMR, and UV-Vis. Compare
and contrast your results to what is known in the literature and reference where you obtained that
information. -9 pt
6) Conclusion- be brief, restate results, include error, and provide enhancements for future
experiments-3 pt