Download Procedure for making nitrotyrosine

Green Electrophilic Aromatic Substitution: Nitration of Tyrosine
prepared by T. Michelle Jones-Wilson, 2003
modified by Angela King, 2005, 2006, 2012
Objectives:
To synthesize 3-nitrotyrosine via electrophilic aromatic substitution (EAS)
To gain understanding of green chemistry and the need for green chemical processes
Prelaboratory
1. Complete your notebook with all relevant sections as described on the course web page.
2. Prepare your detailed procedure and a table in which to record your data.
3. Read sections in your organic text about electrophilic aromatic substitution.
4. Please review the following before coming to lab:
http://college.wfu.edu/chemistry/course-materials/classroom-materials: 223L’s Filtration section.
Background and Introduction
Green Chemistry
Remarkable technological advances in the field of chemistry continue to improve the human living condition in
many respects. Unfortunately, the chemical waste generated by scientists as they achieve such progress has had
harmful consequences for human beings and the environment. The burning of the Cuyahoga River in the early
1970s exemplifies this problem. The magnitude of the issue, though, has become especially apparent in the last
two decades. According to the Toxics Release Inventory (TRI) of the United States Environmental Protection
Agency, 2.26 billion pounds of more than 300 hazardous chemicals were released into the environment in 1994
alone (Anastas and Warner, 1998). Of the top ten industrial sectors responsible for the production of hazardous
waste according to the TRI, the chemical industry maintains a substantial lead as the head contributor. In fact, a
greater quantity of hazardous waste is released annually by the single chemical industry than the other nine
sectors tracked by the TRI combined. The United States Environmental Protection Agency has established strict
guidelines for determining the hazard of a substance.
The endangered state of the environment has necessitated significant governmental action. Prior to the
investigation of issues such as chronic toxicity, carcinogenicity, and bioaccumulation, the solution was thought to
be dilution of hazardous wastes. As experts discovered the fallacy of this theory, laws serving to limit the
quantities of such wastes being released were affected. These command and control policies were only partially
successful, though, for they failed to take into account synergistic effects. Hazardous chemicals, although only
present in limited concentrations, often possessed the capability of reacting with unregulated substances also
present in solution. The resulting products were potentially toxic as well. Subsequent environmental regulations
were then primarily concentrated on the treatment of toxic waste prior to or immediately following their release.
The focus of environmental action began to change in 1990 with the passing of the Pollution Prevention Act. In
this policy, the United States designated waste prevention as their central ethic when addressing environmental
predicaments (U.S. Congress, 1990). Preventing the formation of waste at the source would thus eliminate the
need for the remediation activities that had been previously employed. This novel preventative approach led to
the onset of the green chemistry initiative.
General Overview of Green Chemistry
Green chemistry is a form of pollution prevention governed by twelve primary principles (Anastas and Warner,
1998). It is most simply defined as the use of chemistry techniques and methodologies that reduce or eliminate
the use or generation of feedstocks, products, by-products, solvents, reagents, etc. that are hazardous to human
health or the environment (Anastas and Williamson, 1996). Green chemistry is described as a fundamental form
of preventative action. It seeks to solve the problem of pollution by attacking the relevant molecular science at the
onset rather than attempting to find solutions after the fact. The basic principle governing the green chemistry
concept arises from the fact that the risk caused by toxic chemicals is a function of both exposure and hazard
(Anastas and Warner, 1998). In the past, environmental initiatives have been aimed at reducing risk via
decreasing exposure. Hazard, however, has been held relatively constant. Green chemistry involves an
alternative approach. If fundamental changes can be made to a hazardous chemical process that succeed in
transforming it into one that is environmentally benign, the hazard factor goes to zero and the risk is therefore
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The Economics of Green Chemistry
From an economic standpoint, the green chemistry approach has many advantages. The costs associated with
regulatory compliance including factors such as waste treatment, disposal costs, waste control, and the necessity
for personal protective gear amount to 100 to 150 billion dollars per year for industry in the United States alone
(Anastas and Williamson, 1998). Environmental compliance budgets of individual chemical companies often rival
those funds allotted for research and development. In addition, cleanup of existing hazardous waste sites incur a
substantial expense. On the other hand, implementation of green procedures could potentially succeed in
lowering feedstock costs, increasing conversion rates, decreasing reaction times, improving selectivity, enhancing
separations, and lowering the energy required for reactions. As a result, industry could experience significant
financial gain if green methods were to be successfully adopted.
The State of Green Chemistry in Industry and Academia
Prominent agencies in the United States have recognized the need to explore green methods as possible
solutions to the environmental problems created by industry. Under the EPA/NSF Technologies for a Sustainable
Environment Program, the Environmental Protection Agency and the National Science Foundation have focused
on providing support for research into the development of environmentally harmless industrial processes. A great
deal of success has already been achieved in industrial applications of green processes. In addition, Anastas has
outlined key catalytic procedures with potential industrial applications that illustrate each of the twelve green
chemistry guidelines (Anastas et al., 2000). The list of industrially useful green methods is long and continually
growing.
Although the major portion of chemical waste produced in the United States comes from industry, significant
amounts of hazardous materials are released into the environment by academic laboratories. Antiquated
procedures employing traditional and hazardous reagents are common, especially in undergraduate laboratories.
While the adoption of microscale techniques has substantially improved the environmental position of academic
laboratories, a considerable problem still remains (Singh et al., 1999). Disposal costs often comprise a large
portion of a department’s budget (that's your tuition dollars!). In addition to the financial drain placed on the
chemistry department, the use and production of hazardous waste pose a constant danger for the student. Little
progress, however, has been made in making the academic laboratory environmentally sound.
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. 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.
All aromatic compounds 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. However, for aromatic compounds, substitution is
the favored reaction. This can be explained by following the course of the reaction illustrated above. 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
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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, 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 NO2 . The aromatic
ring on tyrosine can then react with the nitronium ion via electrophilic aromatic substitution to produce 3nitrotyrosine. 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 3nitrotyrosine. The deactivating effect of the nitro substituent restricts any additional nitration from occurring.
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. As always: wear your safety goggles! Mixtures of concentrated sulfuric and nitric acids
violently oxidize acetone. All glassware containing the acids should be thoroughly cleaned and rinsed.
Any paper used to wipe up nitric acid spills should be thoroughly rinsed to avoid fire hazard.
Laboratory Exercise
Reaction—Synthesis of 3-nitrotyrosine
1. Weigh 2.5 g L-tyrosine in a 100-mL 2-neck round-bottomed flask.
2. Add a stir bar and fit with a reflux condenser. Fit open neck with lightly greased stoppers. Correctly attach
the reflux condenser to the water supply. Water enters the bottom and exits the top!
3. Add 12 mL of deionized water to create a suspension. Stir and turn on the water supply (trickle only!).
4. Cool a second 50-ml round-bottomed-flask in an ice water bath. Equip with a stir bar. See the Hazards
section for additional cautions. Carefully, add 2 mL of concentrated H2SO4 and 2.5 mL of
concentrated HNO3 using disposable glass pipettes. 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 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, stir the reaction for 15 minutes in the ice water bath. In the meantime
prepare a 40 °C water bath for the next step. 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
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. Allow your product to dry in your drawer until next week. Obtain its mass, determine your percent yield.
Propose elements of the reaction or technique that could affect your yield.
Post-laboratory Questions
1. Show the reaction and mechanism for the electrophilic nitration of methyl benzoate (PhCO2CH3).
2. Explain the difference between the nitration in question 1 and the reaction done for this lab.
3. The tyrosine used in this experiment is designated as L-tyrosine. What does the L designation mean?
4. Below are mass spectra of nitrotyrosine (left) and a reaction product (right). Does this data support the
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References
(1) Anastas, P.T.; Barlett, L.B.; Kirchhoff, M.M.; Williamson, T.C. Catalysis Today 2000, 55, 12.
(2) Anastas, P.T.; Warner, J.C. Green Chemistry: Theory and Practice; Oxford University Press: New York, 1998.
(3) Anastas, P.T.; Williamson, T.C.; Green Chemistry: Designing Chemistry for the Environment; American Chemical
Society: Washington, DC, 1996.
(4) Anastas, P.T.; Williamson, T.C. Green Chemistry: Frontiers in Benign Chemical Syntheses and Processes; Oxford
University Press: New York, 1998.
(5) Bible, K.C.; Boerner, S.A.; Kaufmann, S.H. Analytical Biochemistry 1999, 267, 217–221.
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