In the Laboratory
Benzoylation of Ergosterol through Nucleophilic Acyl Substitution and Subsequent Formation of Ergosterol Benzoate Endoperoxide by Reaction with Singlet Oxygen Generated by Photosensitization Mary C. Roslaniec* Science, Mathematics and Engineering, Modesto Junior College, Modesto, California 95350, United States *
[email protected] Elizabeth M. Sanford Hope College Department of Chemistry, Holland, Michigan 49422-9000, United States
An innovative and multifaceted two-part laboratory experience has been designed that coordinates well with lecture material while providing students the opportunity to develop pertinent laboratory skills. Nucleophilic acyl substitution (NAS) is a hallmark of carboxylic acid chemistry. Ergosterol (ERG), a sterol, is benzoylated through NAS forming ergosterol benzoate (ERGB) (1). In addition to strengthening the students' grasp of NAS, use of ERG as a substrate introduces students to the steroid ring system as well as the electrocyclic reaction and [1,7]-sigmatropic rearrangement that converts ERG to vitamin D2 (2). In the second step, singlet oxygen (1O2) acts as a unique and reactive dienophile in a reaction analogous to a Diels-Alder addition and adds to the diene of the B ring of ERGB resulting in ergosterol benzoate endoperoxide (ERGBO2) (Scheme 1) (3, 4). The 1O2 is generated through photosensitization using meso-tetraphenylporphyrin (TPP) (5) as a sensitizer. The sensitizer absorbs visible light promoting it from the ground state to an excited singlet state (sensitizer1). Sensitizer1 undergoes an intersystem cross (isc) from the singlet to the triplet state (sensitizer3). Sensitizer3 then transfers energy (et) to the ground-state triplet oxygen (3O2) boosting it to the excited state, 1O2 (Scheme 2) (6). The procedure also introduces the students to reactive oxygen species (ROS) and photochemical processes. Many students in organic chemistry classes are pursuing health-related careers and should be familiar with ROS and their role in biology and medicine as well as photochemical processes as they relate to the many phototherapies now available. Furthermore, a microscale version of the photosensitization is provided using a red, 650 nm photodiode laser pointer as a light source. The experiment is well suited for introductory organic chemistry as well as upper-level mechanistic chemistry; the narrative for the upper-level student delves into electronic states and energy transfer. It can be incorporated into the laboratory schedule once the students have an understanding of conjugated compounds, the Diels-Alder reaction, UV-vis and NMR spectroscopy, and carboxylic acid chemistry as well as previous experience with TLC and column chromatography. ERGB was chosen as the diene due to the well-known stability of its endoperoxide. Endoperoxides of aromatic compounds are less
_
stable and decompose upon heating releasing some of the oxygen in the singlet state (3, 7). Endoperoxides of smaller aliphatic compounds are also unstable, for example, ascaridole, the endoperoxide of terpinene, is prone to explosion when treated with heat or organic acids (8). Therefore, it is important not to vary the diene unless full consideration is given to the safety of the resulting endoperoxide. Experimental Overview The procedure was performed with a group of introductory organic chemistry students at a community college. Prelaboratory lectures were given for both the benzoylation and photooxidation covering the reaction schemes and mechanisms, the laboratory procedure, and safety hazards. The students were required to prepare a laboratory notebook in advance with customary information. All students successfully completed the benzoylation of ERG in approximately 60% yield (Scheme 1). In a fume hood, ERG was dissolved in freshly opened pyridine at room temperature. Benzoyl chloride was then added dropwise. (Students should be aware of the high reactivity of benzoyl chloride and instructed to add it slowly and allow any bubbling, HCl gas evolution, to dissipate between additions). The temperature of the mixture was slowly increased to approximately 60 °C and heated for 30 min. TLC samples were taken at strategic points noted in the procedure. The TLCs were run in dichloromethane and viewed with a fluorescent lamp. Although lower Rf values were obscured by pyridine, ERGB formation was quite obvious; ERGB is less polar, hence, has a higher Rf value. After 30 min, the reaction was cooled and the remaining benzoyl chloride was completely converted to benzoic acid by careful addition of water. The remaining benzoyl chloride was no longer evident by TLC. At room temperature, neither the benzoic acid nor the ERGB was soluble in pyridine. The combined crystals were collected by vacuum filtration. Cold water and cold acetone washes were performed to remove the pyridine and benzoic acid, respectively. The product was recrystallized from methanol. 1H NMR (CDCl3) and IR spectra were taken. The 1H NMR spectrum of ERGB indicated the presence of the benzoate group not
_
r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 2 February 2011 10.1021/ed100487a Published on Web 11/17/2010
_
Journal of Chemical Education
229
In the Laboratory
Scheme 1. Reaction Sequence for the Synthesis of ERGB and Its Subsequent Photooxidation
Scheme 2. Generation of 1O2 via Photosensitization
Hazards
present in ERG. The IR clearly showed the presence of a broad alcohol peak at 3375 cm-1 and a sharp carbonyl peak at 1760 cm-1 for ERG and ERGB, respectively. In a second session, ERGB was photooxidized to ERGBO2. All students were also successful in producing ERGBO2 in approximately 60% yield. In a fume hood with dim lighting, ERGB was dissolved in dichloromethane. Owing to the small amount of TPP required per student, it was dispensed from a stock solution. The total volume of the reaction solution was brought to 25.0 mL in a 50.0 mL graduated cylinder. The solution was cooled in an ice bath to prevent thermal reactions and aerated throughout the procedure. The reaction solution was irradiated for 15 min with a 150 W tungsten light bulb. Use of Pyrex glassware and a tungsten light source is adequate to prevent radiation of less than 300 nm wavelength from reaching the reaction solution. This is important because absorption of UV radiation by the diene chromophore of ERGB could cause ringopening, an undesired side reaction (2). A final TLC indicated absence of ERGB and presence of ERGBO2. After rotary evaporation to remove solvent, the product was purified by silica gel column chromatography. The initial mobile phase was dichloromethane. Once TPP and ERGB eluted from the column, the mobile phase was switched to (98 dichloromethane)/ (2 methanol) (v/v) to remove the more polar ERGBO2. The solvent was then removed by rotary evaporation. 1H NMR (CDCl3) and IR spectra were taken. In the transition from ERGB to ERGBO2, emphasis was placed on the sp2 hybridized C and vinylic H atoms of the B ring of the steroid, both of which are shifted downfield upon addition of the peroxide group. Because of the complexity of the steroid structure, in-depth NMR analysis of the alkyl region is not recommended for the undergraduate laboratory. Minor products are possible, in particular, 9,11-dehydroergosterol (9,11-dehydroERG) (3, 4). The microscale procedure, performed in an NMR tube, has advantages in addition to lower cost and less waste. First, the lifetime of 1O2 is significantly longer in deuterated and halogenated solvents (9-13). Although it would be prohibitive to carry out the macroscale procedure with CDCl3, it is reasonable on a microscale with irradiation and analysis performed completely in a single NMR tube. Second, the light source for this procedure is a red, 650 nm laser pointer. This negates any concern regarding UV radiation reaching the diene. 230
Journal of Chemical Education
_
Vol. 88 No. 2 February 2011
_
The procedures should be carried out in a fume hood. Safety goggles are required and a lab coat or apron is recommended. Dichloromethane, pyridine, benzoyl chloride, methanol, and meso-tetraphenylporphyrin are harmful and irritating if inhaled, ingested, or in contact with skin. Dichloromethane acts as a narcotic in high concentrations and there is limited evidence of a carcinogenic effect. Pyridine and methanol are flammable. Benzoyl chloride is a lachrymator and causes burns on skin contact. It is extremely reactive with water releasing hydrochloric acid and is corrosive. Methanol is extremely toxic. MSDSs for ERGB and ERGBO2 are not available; however, safety information on the substrate, ERG, a vitamin precursor found in foods (primarily yeasts), states that it is extremely toxic by ingestion. ERGB is light sensitive and should be stored in a dark, sealed container. All of the compounds used that are harmful or irritants should be handled in a manner consistent with the appropriate safety data (MSDS). Conclusion A multifaceted procedure is provided that correlates well with introductory organic chemistry or upper-level mechanistic chemistry. Lecture topics including carboxylic acid chemistry, the Diels-Alder reaction, photochemistry, and UV-vis and NMR spectroscopy are reinforced and the student is provided the opportunity to become more adept with laboratory procedures, in particular, TLC and column chromatography. The student will also gain exposure to topics that he or she will see in future courses such as ROS, steroid structures, and aspects of vitamin D chemistry. Acknowledgment We acknowledge Joseph Caddell for running successful trials of the procedures with his organic chemistry class at Modesto Junior College. Literature Cited 1. Dolle, R. E.; Kruse, L. I. J. Org. Chem. 1986, 51, 4047–4053. 2. Lowry, T. H.; Schueller Richardson, K. Mechanism and Theory in Organic Chemistry, 3rd ed.; Harper and Row, New York, 1987; p 1018. 3. Roslaniec, M C. Evaluation of Ergosterol, Salicylate Anion and 5,5Dimethyl-1-pyrroline-N-oxide as Singlet Oxygen Quenchers and Four Near-IR Absorbing Dyes as Potential PDT Agents, Ph.D. Dissertation, University of California at Los Angeles, Los Angeles, CA, 1993. 4. Ponce, M. A.; Ramirez, J. A.; Galagovsky, L. R.; Gros, E. G.; ErraBalsells, R. Photochem. Photobiol. Sci. 2002, 1 (10), 749–756. 5. Falvo, R.A. E.; Mink, L. M. J .Chem. Educ. 1999, 76, 237–239.
pubs.acs.org/jchemeduc
_
r 2010 American Chemical Society and Division of Chemical Education, Inc.
In the Laboratory
6. Cox, A.; Kemp, T. J. Introductory Photochemistry; McGraw-Hill: London, 1971; p 134. 7. Turro, N. J.; Chow, M.-F.; Rigaudy, J. J. Am. Chem. Soc. 1981, 103, 7218–7224. 8. Merck Index, 10th ed.; Merck and Co., Inc.: Rahway, NJ, 1983; entry 842, p 119. 9. Ogilby, P. R.; Foote, C. S. J. Am. Chem. Soc. 1983, 105, 3423–3430. 10. Rodgers, M. A. J. J. Am. Chem. Soc. 1983, 105, 6201–6205. 11. Okamoto, M.; Tanaka, F. J. Phys. Chem. 1993, 97, 177–180.
r 2010 American Chemical Society and Division of Chemical Education, Inc.
_
12. Krasnovsky, A. A., Jr. J. Am. Chem. Soc. 1993, 115, 6013– 6016. 13. Jenny, T. A.; Turro, N. J. Tetrahedron Lett. 1982, 23, 2923–2926.
Supporting Information Available Detailed student instructions and materials, instructor notes, TLC shifts, NMR data, a list of chemicals and their hazards. This material is available via the Internet at http://pubs.acs.org.
pubs.acs.org/jchemeduc
_
Vol. 88 No. 2 February 2011
_
Journal of Chemical Education
231
