In the Laboratory edited by
Green Chemistry
Mary M. Kirchhoff ACS Green Chemistry Institute Washington, DC 20036
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A Green Starting Material for Electrophilic Aromatic Substitution for the Undergraduate Organic Laboratory T. Michelle Jones-Wilson* Department of Chemistry, East Stroudsburg University of Pennsylvania, East Stroudsburg, PA 18301; *
[email protected] Elizabeth A. Burtch 3463 West 151 Street, Cleveland, OH 44111
Green chemistry is a form of pollution prevention. It is most simply defined as “the use of chemistry techniques and methodologies that reduce or eliminate the use or generation of feedstocks, products, byproducts, solvents, reagents, and so forth that are hazardous to human health or the environment” (1). Green chemistry 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. In the past, environmental initiatives have been aimed at reducing risk via decreasing exposure (2). The hazard factor, 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 eliminated. Green chemistry is based on this premise. Applications in Academia Although the major portion of chemical waste produced in the United States comes from industry, significant quantities 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. Disposal costs often comprise a large portion of a department’s budget. In addition to the financial drain placed on the chemistry department, the production of hazardous wastes pose a constant danger for the student. Microscale experiments have been widely employed and combined with green methodologies in some cases in the academic laboratory (3). Little progress, however, has been made in transforming the academic laboratory into a space that is environmentally sound and even less progress has been made in educating our future chemists in green methodologies.
commonly prepared by EAS. Common EAS reactions are employed to halogenate, nitrate, sulfonate, alkylate, and acylate aromatic compounds. Unfortunately, many common aromatic compounds are toxic. Most undergraduate organic laboratory texts include EAS experiments. However, the traditional experiments often employ nongreen or hazardous starting materials. In this experiment, designed for the second-semester undergraduate organic chemistry laboratory, the principles of green chemistry can be discussed and illustrated in conjunction with the presentation of electrophilic aromatic substitution. Nitration of Tyrosine Amino acids are green starting materials as they are nontoxic in nature. They contain a variety of functional groups useful for the illustration of common organic reactions. In addition, reactions of amino acids can often be carried out in water, a green solvent. An alternative to the traditional methods employed to demonstrate EAS makes use of the aromatic moiety on the amino acid tyrosine. Characterization of 3-nitrotyrosine has been reported (4). A method of utilizing peroxynitrite, a known initiator of aromatic nitration, was also explored (5). One technique reported for nitrating tyrosine is potentially adaptable for use in a green undergraduate organic chemistry laboratory (6). The complete reaction is presented in Scheme I. Nitronium, a strong electrophile, is generated in situ from the reaction of the sulfuric and nitric acids. The aromatic ring on tyrosine can then react with the nitronium ion via electrophilic aromatic substitution to produce 3-nitrotyrosine. The hydroxyl group is a strong elec-
⫹
O
H C
H3N
C
CH2
⫹
OH
H 3N HNO3, H2SO4
H C
O C
OH
CH2
H2O
Electrophilic Aromatic Substitution Electrophilic aromatic substitution (EAS) is arguably the most significant reaction type experienced by aromatic compounds and is fundamental to the study of organic chemistry. Thousands of substituted aromatic compounds are 616
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NO2 OH
Scheme I. Nitration of tyrosine.
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In the Laboratory
tron donating group and serves as an activating o and p director. Therefore, substitution occurs at the ortho position to produce 3-nitrotyrosine. The deactivating effect of the nitro substituent restricts any additional nitration from occurring (4). Use of a substituted aromatic ring allows for discussion of activating and deactivating substituents and o, p, or m directing effects.
acetone. All glassware containing the acids should be thoroughly cleaned and rinsed. Alternatively, the instructor can prepare the mixture immediately before the laboratory period. Any paper used to wipe up nitric acid spills should be thoroughly rinsed to avoid fire hazard.
Chemicals and Equipment
The final product can be characterized by melting point, UV–vis spectroscopy, or NMR. Melting point provides a quick characterization, (theoretical melting point is 234 ⬚C; ref 6 ) but impure products may decompose before melting. 3-Nitrotyrosine has an absorbance maximum due to the aromatic ring at 358 nm (4), while the conjugated ring in L-tyrosine produces an absorbance maximum at approximately 275 nm. As a result, UV–vis spectroscopy is a suitable method for product characterization. 1 H NMR spectroscopy can also employed to characterize the nitrated product in D2O at field strength of 90 MHz or greater. The key peaks in the spectrum are located in the aromatic proton region (6.5–8.5 ppm). Tyrosine shows two aromatic doublets peaks with equal peak height (n = 2); 3-nitrotyrosine shows three aromatic protons, 2 doublets, and a singlet shifted downfield (n = 1). The average overall yield was 43.4% and average percent recovery was 56.7%.
L-tyrosine, nitric, sulfuric acids, and ethyl acetate can be obtained from a variety of chemical suppliers. Melting points were obtained using a Mel-Temp apparatus. UV–vis spectra were obtained using a PerkinElmer Lambda II spectrometer at concentrations of approximately 10᎑4 M. 1H NMR were obtained on a Varian 90 MHz spectrometer in D2O relative to TSP.
Experimental Procedure
Synthesis of 3-Nitrotyrosine Five grams of L-tyrosine are weighed into a 100-mL 3neck round-bottomed flask fitted with a reflux condenser and suspended in 20 mL of deionized water (alternatively a Claisen adapter and a single-necked flask could be used). In a separate reaction vessel, 3.6 mL of concentrated H2SO4 and 4.7 mL of concentrated HNO3 should be carefully mixed while cooling in an ice water bath. (To minimize the hazards associated with handling strong acids, this step can be accomplished by students using bottles fitted with acid resistant pumps or by the instructor immediately prior to class). The cooled acid mixture is added dropwise under water reflux and with gentle stirring to the tyrosine suspension. Cooling in an ice-water bath should be maintained throughout the addition. The reaction should be cooled sufficiently and the rate of addition kept slow enough to prevent reflux. After the addition is completed, the reaction should be left in the ice-water bath with stirring for 15 minutes. The reaction vessel is then removed from the ice-water bath and allowed to warm to room temperature followed by heating to 40 ⬚C in a water bath. The reaction should be maintained for 30 minutes at approximately 40 ⬚C. Product Isolation and Purification Following the reaction period the solution is cooled in an ice-water bath until crystallization occurs. The solution is then filtered using a Büchner funnel and the crude product collected. Recrystallization is performed to purify the product. The crude product is rinsed with a 10-mL aliquot of ethyl acetate and then recrystallized in approximately 50 mL of deionized water. If crystallization does not occur the ethyl acetate rinse can be repeated. Hazards Students work with a mixture of concentrated sulfuric and nitric acids. Nitric and sulfuric acids are corrosive and the additions are highly exothermic. Burns are a potential problem if the acids are not handled judiciously. Appropriate eye protection and gloves should be worn. Mixtures of concentrated sulfuric and nitric acids violently oxidize www.JCE.DivCHED.org
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Results and Discussion
Conclusion The nitration of tyrosine fits well into a green laboratory curriculum. Tyrosine is a naturally occurring amino acid and a green reagent. Although concentrated sulfuric and nitric acids are hazardous chemicals, only small quantities are necessary for the reaction to occur successfully. If handled properly, these reagents pose little or no danger for students. In addition, the aqueous acidic waste produced from the procedure can be neutralized with an appropriate base and disposed of down the drain as harmless aqueous salts. The complete scheme generates no toxic byproducts other than the 3-nitrotyrosine itself. WSupplemental
Material
Instructions for the students and notes for the instructor, including the answers to the prelab questions and comments about the postlab questions, are available in this issue of JCE Online. Literature Cited 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. Singh, M. M.; Szafran, Z.; Pike, R. M. J. Chem. Educ. 1999, 76, 1684. 4. Bible, K. C.; Boerner, S. A.; Kaufmann, S. H. Anal. Biochem. 1999, 267, 217–221. 5. Oury, T. D.; Tatro, L.; Ghio, A. J.; Piantadosi, C. A. Free Radical Research 1995, 23, 537–547. 6. Waser, E.; Lewandowski, M. Helvetica Chimica Acta 1921, 4, 657–666.
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