In the Laboratory edited by
The Microscale Laboratory
R. David Crouch Dickinson College Carlisle, PA 17013-2896
Creatine Synthesis: An Undergraduate Organic Chemistry Laboratory Experiment
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Andri L. Smith* and Paula Tan† Department of Chemistry, Quinnipiac University, Hamden, CT 06518; *
[email protected] Many students in introductory chemistry courses take chemistry as a prerequisite for another major. A way to help these students appreciate the relevance of chemistry in their lives is to link chemistry to themes such as sports and fitness. This strategy has been discussed in a recent article in this Journal (1). In this spirit, we have designed an undergraduate organic chemistry laboratory experiment in which students synthesize creatine monohydrate, which is widely used by athletes both to increase muscle mass and to improve performance in sports requiring short bursts of energy. Produced in the liver, kidneys, and pancreas from three amino acids—glycine, arginine, and methionine—creatine can also be obtained from dietary supplements and from foods such as meats and fish. Creatine is found primarily in skeletal muscle and is fundamental to the energy metabolism of muscle (2, 3). Creatine provides the energy necessary for vigorous muscle contraction and has been shown to enhance performance in high-intensity exercise (4, 5). In skeletal muscle creatine exists in equilibrium with phosphocreatine; the reversible
conversion of creatine into phosphocreatine is catalyzed by creatine kinase and involves the transfer of the γ-phosphate group of adenosine triphosphate (ATP) onto the guanidino group of creatine (Scheme I). The reverse reaction therefore provides an alternate pathway—besides glycolysis—for the regeneration of ATP (5), which is the “energy currency” in the body (6). Creatine appears to increase anaerobic capacity, aerobic recovery, and protein synthesis (2). For this reason, creatine has been used therapeutically for the treatment of gyrate atrophy (7, 8) (a metabolic disease of the eye leading to progressive loss of vision) (9) and muscular dystrophy (10). Creatine has also shown promise in animal studies for the treatment of traumatic brain injury (11) and amyotrophic lateral sclerosis (or Lou Gehrig’s disease) (12).1 Furthermore, although there are conflicting studies on the benefit of creatine to athletic performance, creatine supplements (most commonly available as creatine monohydrate) are in demand by athletes (5, 14). At present, creatine is not regulated by the Food and Drug Administration, nor is it banned by the National Collegiate Athletic Association or the International Olympic Committee (15–19). British sprinters and hurdlers have credited their
†
Current address: Ambulatory Services, Acadia Hospital, Bangor, ME 04401.
NH2 N O O
C
H2 C N
O
NH2 C
+
NH2
O
P
O O
P
N
O O
P
N
O
N
O O
CH3
O
O
creatine
OH OH adenosine triphosphate
creatine kinase
NH2 N O O
C
H2 C N CH3
NH2 C
O
O N
P
H
O
phosphocreatine
O
+
O
P
O
P
N
O O
O
N
O N
+
H
O OH OH adenosine diphosphate
Scheme I. Creatine and phosphocreatine exist in equilibrium in skeletal muscle and are interconverted through the action of creatine kinase. Conversion of phosphocreatine into creatine produces ATP, thereby providing energy required for high-intensity exercise.
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athletic success in the early 1990s with creatine supplementation (5). Today creatine is widely used among athletes at all levels. Baseball players Sammy Sosa and Mark McGwire and Olympic sprinters Donovan Bailey and Michael Johnson have all admitted to using the supplement (20, 21). Creatine use is also popular at the high-school level. A University of Wisconsin Sports Medicine survey conducted during the 1999–2000 school year revealed that 16.7% of high school student–athletes (25.3% of male athletes and 3.9% of female athletes) in Wisconsin (out of 4,011 who completed the survey) acknowledged using creatine and that 30.1% of the football players surveyed (1,388 total) admitted to using the supplement (21). College athletes use creatine extensively, as well. A recent survey revealed that 28% of athletes (out of 806 eligible varsity and junior varsity athletes, with a 93% response rate) in an NCAA Division I athletic program reported using creatine (22). Furthermore, in 1998 coaches of all eight teams participating in the College World Series admitted that their players all had easy access to the supplement (23). Overview
Experimental Procedure To a solution of sarcosine (N-methylglycine, 232 mg, 2.60 mmol) in distilled water (0.5 mL) was added sodium chloride (152 mg, 2.60 mmol) at room temperature. To a separate flask containing a solution of cyanamide (206 mg, 4.90 mmol) in distilled water (0.13 mL) was added concentrated ammonium hydroxide (1 drop, ∼0.7 mmol) at room temperature. The cyanamide mixture was added to the sarcosine mixture, and the new reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was removed from stirring and allowed to sit at room temperature for 1 week. After 1 week, the crude white creatine product was isolated by vacuum filtration (25). Creatine was recrystallized from boiling water to afford 261 mg of pure product (67% yield; white, needlelike crystals). Purity was determined by both thin-layer chromatography (Avicel TLC plate; 3:1 n-propanol兾water solvent system; ferricyanide nitroprusside dye) (26) and melting point (sealed capillary tube) (25, 27). In addition, the product was analyzed by low-field nuclear magnetic resonance spectroscopy. Hazards
Creatine monohydrate can be synthesized in the laboratory from N-methylglycine (sarcosine) and cyanamide (Scheme II). In fact, this is a common method for the commercial preparation of creatine (monohydrate) supplements (14). This is a useful laboratory experiment in an introductory organic chemistry course when discussing the chemistry of nitriles (whose reactivity is similar to that of certain carbonyl compounds) and amines. Addition of the sarcosine nucleophile to the cyanamide electrophile leads to the formation of the corresponding imine ion, which then undergoes two proton transfers to produce creatine (Scheme III). We based our synthetic procedure on a method developed at Oxford by Greenaway and Whatley (24). In this procedure, ammonium hydroxide and sodium chloride were added to an aqueous mixture of sarcosine and cyanamide. The presence of base increased the reaction rate, and the presence of salt increased the final creatine yield. We confirmed that the product was creatine monohydrate by CHN analysis.
Students should wear goggles and gloves and work in a fume hood when performing this experiment. For all the reagents used in this experiment, students should avoid contact with eyes, skin, and clothing, and avoid ingestion and inhalation. In particular, cyanamide is extremely irritating and
H2N
N + H
C
O H2 C C
N
OH
CH3 cyanamide
sarcosine ⴚ
ⴚ
OH
H2N
N C
H ⴙ
N
O H2 C C
OH
CH3
ⴚ
H2N
N C
N
O H2 C C
OH + H
OH
CH3
NH H2N
C
N
O H2 C C
OH
CH3 creatine
Scheme II. Creatine is synthesized from N-methylglycine (sarcosine) and cyanamide in the presence of salt and aqueous ammonia.
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Scheme III. The nucleophilic amino group of sarcosine forms a bond with the electrophilic carbon atom of the nitrile group of cyanamide to produce the imine ion. Subsequent deprotonation and protonation steps yield the creatine product.
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caustic when ingested, inhaled, or absorbed through the skin. Also, ammonium hydroxide causes severe skin irritation and skin burns, damage to the digestive and gastrointestinal tract, and irritation of the upper respiratory tract. Finally, students should use caution when dipping developed TLC plates into ferricyanide nitroprusside (FCNP) dye, as potassium ferricyanide, and sodium nitroprusside may cause irritation of the respiratory tract, digestive tract, gastrointestinal tract, and skin. Results and Discussion Our first experience using this experiment in an introductory organic chemistry course was a success. Due to the constraints of the existing schedule of laboratory experiments, this synthesis was carried out over a period of four weeks; however, it is possible to condense the experiment to a threeweek period.2 The reagents were combined in the first week (approximately 2 hours required for setup and stir time) and allowed to stir for one week. Crude creatine was isolated in the second week and recrystallized from boiling water (1 hour). The recrystallization was allowed to occur at room temperature over the course of the subsequent week. In our experience, recrystallization was necessary to remove excess cyanamide starting material. In the third week, pure creatine was isolated and analyzed by TLC (1 hour). According to their TLC results, all students were able to obtain pure creatine. As the purified creatine from the third week was generally wet, it was allowed to dry for one week, and final yields were determined in the fourth week. The average yield of pure creatine in this lab section of 16 students was 41%. Similar results were obtained when this experiment was carried out during a subsequent semester with more students (63 students in 6 lab sections): the average yield of pure creatine product was 45%. Conclusions We were pleased with our first attempt to incorporate a synthesis of creatine into the introductory organic chemistry laboratory curriculum. Students were able not only to appreciate the mechanism as it related to the carbonyl chemistry they had been studying in the lecture portion of the course but also to use synthetic, purification, and analytical techniques they had learned earlier in the year to make a substance that was familiar to them. One student, a hockey player, commented that this was the first time all year that his teammates were interested in what he was studying in organic chemistry. Acknowledgments The authors would like to thank Brian Geddes for helping to optimize the synthetic procedure and Dasan Thamattoor for verifying the presence of creatine hydrate by CHN analysis.
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Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Notes 1. To date, however, creatine has shown no benefit with respect to survival or disease progression in human trials involving A.L.S. patients (13). 2. Approximately 2 hours required for setup and stirring in the first week; 2–3 hours for isolation, recrystallization, and TLC analysis in the second week; and 2 hours for determination of final mass, melting point, and NMR in the third week
Literature Cited 1. Giffin, G. A.; Boone, S. R.; Cole, R. S.; McKay, S. E.; Kopitzke, R. J. Chem. Educ. 2002, 79, 813–817. 2. Clark, J. F. J. Athletic Training 1997, 32, 45–51. 3. Chiang, R. Athletic Supplements: Creatine. Ergogenic Aids. http://www.geocities.com/HotSprings/Spa/9971/creatine.html (accessed Jul 2006). 4. Balsom, P. D.; Soderlund, K.; Ekblom, B. Sports Med. 1994, 18, 268–280. 5. Wyss, M.; Kaddurah–Daouk, R. Physiol. Rev. 2000, 80, 1107– 1213. 6. Guyton, A. C.; Hall, J. E. Textbook of Medical Physiology, 10th ed.; W. B. Saunders Co.: Philadelphia, 2000. 7. Sipila, I.; Simell, O.; Vannas, A. N. Engl. J. Med. 1981, 304, 867–870. 8. Vannas–Sulonen, K.; Sipila, I.; Vannas, A.; Simell, O.; Rapola, J. Ophthalmology 1985, 92, 1719–1727. 9. Gyrate Atrophy of the Choroid and Retina. National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/ books/bv.fcgi?call=bv.View..ShowSection&rid=gnd.section.128 (accessed Jul 2006). 10. Rubin, M. Neurol. Alert 2000, 18, 87. 11. Sullivan, P. G.; Geiger, J. D.; Mattson, M. P.; Scheff, S. W. Ann. Neurol. 2000, 48, 723–729. 12. Zhang, W.; Narayanan, M.; Friedlander, R. M. Ann. Neurol. 2003, 53, 267–270. 13. Groeneveld, G. J.; Veldink, J. H.; van der Tweel, I.; Kalmijn, S.; Beijer, C.; de Visser, M.; Wokke, J. H. J.; Franssen, H.; van den Berg, L. H. Ann. Neurol. 2003, 53, 437–445. 14. Williams, M. H.; Kreider, R. B.; Branch, J. D. Creatine: The Power Supplement; Human Kinetics: Champaign, IL, 1999. 15. Creatine Report. Vitality Research Institute Metabolite Report. http://www.primev.com/Creatine.htm (accessed Jul 2006). 16. Juhn, M. S. The Physician and Sportsmedicine 1999, 27, 47– 52. 17. Meiggs, R. Committee Continues To Monitor Creatine Use in Sports. The NCAA News. http://www.ncaa.org/wps/portal/ newsdetail?WCM_GLOBAL_CONTEXT=/wps/wcm/connect/ NCAA/NCAA+News/NCAA+News+Online/Association-wide/ Committee+continues+to+monitor+creatine+use+in+sports+-+4-12-
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18.
19.
20.
21.
04&TITLE=Committee+continues+to+monitor+creatine+use+in+sports++4-12-04 (accessed Aug 2006). NCAA Banned-Drug Classes 2006-2007. National Collegiate Athletic Association. http://www1.ncaa.org/membership/ ed_outreach/health-safety/drug_testing/banned_drug_classes.pdf (accessed Aug 2006). The 2006 Prohibited List, International Standard. The World Anti-Doping Code. World Anti-Doping Agency. http:// www.wada-ama.org/rtecontent/document/2006_LIST.pdf (accessed Aug 2006). St. Philip, E. “Legal Steroid” Enhances Performance. Discovery Channel Canada. http://www.exn.ca/templates/printstory.asp? PageName=Discovery&story_id=1996112102 (accessed Jul 2006). McGuine, T. A.; Sullivan, J. C.; Bernhardt, D. A. Wisc. Med.
22. 23. 24. 25. 26. 27.
J. 2002, 101, 25–30. http://www.wisconsinmedicalsociety.org/ uploads/wmj/101-2-McGuine.pdf (accessed Aug 2006). LaBotz, M.; Smith, B. W. Clin. J. Sport Med. 1999, 9, 167–169. Olson, E. Creatine Use Common for CWS Teams. The Omaha World Herald, May 29, 1998, Sports, p 39. Greenaway, W.; Whatley, F. R. J. Labelled Compounds and Radiopharmaceuticals 1978, 14, 611–615. Williamson, K. L. Macroscale and Microscale Organic Experiments, 4th ed.; Houghton Mifflin Company: Boston, 2003. Cho, Y.–B.; Stedman, R. J.; Paik, W. K. J. Korean Chem. Soc. 1985, 29, 419–425. The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 11th ed.; Budavari, S., Eds.; Merck & Co. Inc.: Rahway, NJ, 1989.
The structures of a number of the molecules discussed in this article are available in fully manipulable Jmol and Chime format as JCE Featured Molecules in JCE Online (see page 1657).
Featured Molecules
http://www.JCE.DivCHED.org/JCEWWW/Features/MonthlyMolecules
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