Communication Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
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Synthesizing a Berberine Derivative and Evaluating Antimicrobial Activity To Reinforce with Students the Potential Significance of Small Chemical Structure Changes for Biological Systems Catarina A. B. Rodrigues,† Iris Neto,‡ Patricia Rijo,†,‡ and Carlos A. M. Afonso*,† †
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal ‡ CBIOS - Research Centre for Biosciences & Health Technologies, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal S Supporting Information *
ABSTRACT: The convenient synthesis of dihydroberberine by the reduction of berberine is described as an experiment for an upper-division undergraduate organic chemistry laboratory course. Students obtained up to 74% yield of the desired pure product without the use of chromatographic techniques. The antimicrobial activities of both compounds against Staphylococcus aureus and Candida albicans were then assessed and compared in an upper-division undergraduate microbiology laboratory course. The students verified that berberine shows higher antimicrobial activity than its derivative dihydroberberine, demonstrating that small changes in a chemical structure can result in great biological differences.
KEYWORDS: Upper-Division Undergraduate, Organic Chemistry, Biochemistry, Interdisciplinary/Multidisciplinary, Hands-On Learning/Manipulatives, Applications of Chemistry, Medicinal Chemistry, Natural Products, Oxidation/Reduction
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INTRODUCTION
are characterized by the addition of hydrogen to an unsaturated group (e.g., carbonyl groups or C−C double bonds) or addition of hydrogen followed by fission of a bond between two atoms. On the other hand, oxidations11 are defined by the addition of oxygen to the substrate, removal of hydrogen, or removal of one electron. An important and simple method of reduction of functional groups, such as carbonyls and imines, is the use of NaBH4.12−14 This versatile reagent is also able to reduce the quinolizinium functionality. The synthesis of a berberine derivative by reduction of the quinolizinium group15 and an evaluation and comparison of its antimicrobial activity with that of berberine (Scheme 1) is described. This experiment is divided into two dependent laboratory sessions: the first one introduces students to a common reaction performed in any organic chemistry lab using a substrate with pharmaceutical value; in the second part of this work, students test the antimicrobial activity of the previously synthesized product and compare it with that of berberine (the starting material). This work shows students the importance of
Quinolizinium is a functional group with broad application in medicinal chemistry. This cationic group is widely applied in the design of new probes and sensors1,2 and new drugs with multitarget purposes. Of special interest are applications in anticancer studies, DNA intercalation, and microbiology.3,4 The natural product berberine is an example of a compound in which this functionality can be found. This isoquinoline alkaloid is isolated from plants of the Berberidaceae, Ranunculaceae, and Papaveraceae families and has been widely used in traditional Chinese medicine because of its antimicrobial and antiprotozoal activities.5−8 Berberine and derivatives have gained much attention in recent years because of the multiple pharmacological effects, including anticancer, antiviral, and antibacterial activities, also shown by other quinoliziniumcontaining molecules.5−9 Berberine can be functionalized at different positions on its skeleton (Figure 1): the typical functionalizations of the berberine core are nucleophilic addition to C-8, derivatization at C-9, and alkylation at C-13 through enamine activation.6−8 In a teaching lab, it is very important to show students the importance of organic chemistry and how it can be used as a tool to synthesize new molecules for applications in different fields. As such, reduction and oxidation reactions are of crucial importance in this subject. In organic chemistry, reductions10 © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: June 27, 2017 Revised: January 6, 2018
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DOI: 10.1021/acs.jchemed.7b00458 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 1. Typical functionalizations of berberine.
read and interpret, and it has a good correlation to the reference Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards or NCCLS).16−18 In this assay, small wells are made in the agar of a plate inoculated to form a microbial lawn (even, confluent bacterial growth), and the chemical agents to be tested are placed in these wells. The plates are incubated (24 to 48 h at 37 °C) to allow the growth of the microorganism and time for the chemicals to diffuse into the agar. As a chemical diffuses into the agar, it becomes less concentrated.16 If an organism is susceptible to a chemical, a clear zone of inhibition will appear around the well where the growth has been inhibited. The size of this zone of inhibition (in mm) depends on the sensitivity of the organism to the specific chemical and the chemical’s ability to diffuse through the agar.16
Scheme 1. Synthesis of Dihydroberberine (DB) from Berberine (B)
organic chemistry and how small structural transformations can produce considerable differences in biological activity.
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EXPERIMENTAL PROCEDURES
Chemistry
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Upper-division undergraduate students may work either individually or in groups of two students during a 3 h laboratory session. The students then compare and discuss their results as a class at the end of the experiment. Students synthesize the target compound, dihydroberberine (DB), by reduction of the iminium cation of the natural compound berberine (B) using conditions adapted from the literature: a solution of sodium borohydride (1.43 mmol) in aqueous sodium hydroxide (1 mL, 5% w/v) is added to a mixture of berberine chloride (1.34 mmol) and potassium carbonate (4.03 mmol) in methanol (18 mL). The resulting suspension is stirred at room temperature for 15 min (see the Supporting Information for a detailed description). The orange mixture becomes green, and the desired product is collected by vacuum filtration after workup by washing with water and then ethanol/water (30% w/v). If needed, the product is purified by recrystallization from ethanol. Students calculate the yield of the reaction and ascertain the purity of the product by thinlayer chromatography (TLC) and by determining the melting point.
HAZARDS Gloves and safety goggles should be used during the experiment. All manipulations should be carried out in a fume hood. Berberine chloride, potassium carbonate, and sodium borohydride are hazardous, can cause eye and skin irritation, and are harmful if swallowed or inhaled. Sodium hydroxide is corrosive and should be handled with care. Methanol and ethanol are flammable and volatile organic solvents. Dichloromethane and dimethyl sulfoxide (DMSO) can cause eye and skin irritation and are harmful if swallowed or inhaled. Waste should be disposed in the appropriate waste containers. The toxicological properties of dihydroberberine have not been investigated. Students should use appropriate procedures and handle it with care, avoiding inhalation or skin contact. Vancomycin is hazardous on eye contact (irritant) and may cause an allergic skin reaction and damage to organs upon prolonged or repeated exposure. Nystatin may be a potential irritant to the eyes, respiratory system, and skin. This product may also be harmful if ingested. Staphylococcus aureus Rosenbach (ATCC 25923) is a bacterium that is classified as biosafety level 2 (BSL-2),
Microbiology
The antimicrobial activities of berberine and its derivative dihydroberberine were tested using the well diffusion test. This test is simple, easy to reproduce, inexpensive, and easy both to B
DOI: 10.1021/acs.jchemed.7b00458 J. Chem. Educ. XXXX, XXX, XXX−XXX
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the occurrence of incomplete removal of the solvent in some samples. The antimicrobial activities of B and DB were analyzed by another group of upper-division students in a pharmaceutical science course (five-year course of study). The students compared the biological activities with those of positive controls against the bacteria Staphylococcus aureus and the fungus Candida albicans using the well diffusion test. The results are summarized in Table 2. The derivative dihydroberberine was found to have moderate activity against S. aureus and C. albicans compared with the corresponding positive controls. However, smaller inhibition zones formed around the wells compared with those for berberine. These results were reproducible among the samples, showing that a small structural difference can greatly influence the antimicrobial activity. The expected student learning outcomes for this experiment are (1) experience with standard laboratory techniques such as TLC, melting point determination, and filtration; (2) introduction to reduction/oxidation reactions; (3) experience with NMR spectroscopy as a structural analysis technique; (4) experience with standard microbiological laboratory techniques; and (5) understanding of the influence of small structural modifications on biological activity. These outcomes were all successfully addressed in the postlab quizzes.
presents a moderate risk of infection, and should be handled under BSL-2 guidelines from the Centers for Disease Control.19 Candida albicans (Robin) Berkhout (ATCC 10231) is a yeast that is classified as BSL-1 and requires a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing.19
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RESULTS AND DISCUSSION The synthesis of dihydroberberine was reproduced by 11 upper-division students in a pharmaceutical science course (five-year course of study), by five students individually, and by three teams of two students during a 3 h laboratory session. The students obtained the desired product in yields ranging from ∼41 to 74%. The purity of the product was assessed by TLC analysis and determination of the melting range during the laboratory class and later by the instructors by 1H NMR analysis and determination of the melting range. The results are summarized in Table 1. Table 1. Results from Student Experiments Using Previously Optimized Conditions Student Groupa
Mass of Product (g)b
Yield (%)
Melting Range (°C)c
1 2 3 4 5 6 7 8
0.233 0.297 0.244 0.322 0.334 0.270 0.184 0.322
51 66 54 71 74 60 41 71
152 146−150 152 158d 155−160d 142−156d 145−162d 149−160
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SUMMARY The reduction of berberine to dihydroberberine for use in upper-division undergraduate teaching laboratories is described. Students learn different topics, including the borohydride reduction mechanism of a quinolizinium cation, improvement of preparative organic chemistry, and structural elucidation skills. The antimicrobial activities of the product dihydroberberine against S. aureus and C. albicans were determined in the microbiology teaching lab. Students compared their results for dihydroberberine with those for berberine and the corresponding positive controls vancomycin and nystatin. In this part of the experiment students develop skills in standard microbiological lab techniques.
a One or two students per group. bDihydroberberine starting from berberine (∼0.5 g). cThe literature melting range is 155−161 °C.20 d These data reflect another laboratory session with several groups.
The melting temperature ranges determined by the students had a difference of more than 13 °C in some cases compared with the literature value of 155−161 °C.20 The students concluded that these differences are justified by contaminations with solvent (resulting from insufficient product drying time) or with starting material (resulting from difficulties in the workup). The TLC analyses performed by the students (Figure S5 in the Supporting Information) did not show any visible impurities. Analysis by 1H NMR spectroscopy showed that no impurities were present in the students’ products, which were identical to the dihydroberberine product prepared by the instructors (Figure S2). In addition, after the student samples were left in the open air for a long time, the observed melting range was comparable to the reported value (see the Supporting Information). This combined information supports
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00458. Student handout containing an overview of the chemical and biological importance of berberine and the reduction reaction, detailed experimental procedures, and pre- and postlab questionnaires; instructor notes (list of chemicals, equipment, hazards and safety information, instrumentation, and answers to the hints for student
Table 2. Antimicrobial Activity Results for the Derivative Dihydroberberine (DB) Obtained by Student Groups (G1−G7) Compared with Those for Berberine (B) and Positive Controls Using the Well Diffusion Test Diameter of Inhibition Zone (mm) by Student Group Microorganism
a
B
G1
G2
G3
G4
G5
G6
G7
Diameter of Inhibition Zone (mm) (Positive Control)b
S. aureus C. albicans
20 20
15 16
15 15
16 15
16 16
15 16
16 16
16 15
23 (Vancomycin) 17 (Nystatin)
a Relative to B, the observed error is ≤1 mm between assays with a standard deviation of 0.53. bThe inhibition zone result obtained for the negative control, DMSO, was 5 mm.
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DOI: 10.1021/acs.jchemed.7b00458 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Undergraduate Organic Chemistry Laboratory. J. Chem. Educ. 2008, 85 (11), 1535−1537. (14) Touchette, K. M. Reductive Amination: A Remarkable Experiment for the Organic Laboratory. J. Chem. Educ. 2006, 83 (6), 929−930. (15) Liu, H.; Wang, L.; Li, Y.; Liu, J.; An, M.; Zhu, S.; Cao, Y.; Jiang, Z.; Zhao, M.; Cai, Z.; Dai, L.; Ni, T.; Liu, W.; Chen, S.; Wei, C.; Zang, C.; Tian, S.; Yang, J.; Wu, C.; Zhang, D.; Liu, H.; Jiang, Y. Structural Optimization of Berberine as a Synergist to Restore Antifungal Activity of Fluconazole against Drug-Resistant Candida albicans. ChemMedChem 2014, 9 (1), 207−216. (16) Brown, A. Benson’s Microbiological Applications: Laboratory Manual in General Microbiology, 12th ed.; McGraw-Hill Education: New York, 2012. (17) Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement; CLSI document M100-S25; Clinical and Laboratory Standards Institute: Wayne, PA, 2015. (18) Magaldi, S.; Mata-Essayag, S.; Hartung de Capriles, C.; Perez, C.; Colella, M. T.; Olaizola, C.; Ontiveros, Y. Well diffusion for antifungal susceptibility testing. Int. J. Infect. Dis. 2004, 8 (1), 39−45. (19) U.S. Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th ed.; U.S. Government Printing Office: Washington, DC, 2009. (20) Vennerstrom, J. L.; Klayman, D. L. Protoberberine alkaloids as antimalarials. J. Med. Chem. 1988, 31 (6), 1084−1087.
discussion); and copies of the characterization data collected from student samples (PDF, DOC)
AUTHOR INFORMATION
Corresponding Author
*E-mail: carlosafonso@ff.ulisboa.pt. ORCID
Patricia Rijo: 0000-0001-7992-8343 Carlos A. M. Afonso: 0000-0002-7284-5948 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the first-year students of the Integrated Master Course on Pharmaceutical Science from the Faculty of Pharmacy, University of Lisbon, Portugal, as well as Fundaçaõ para a Ciência e a Tecnologia (FCT) (ref SFRH/ BPD/100677/2014, UID/DTP/04138/2013, COMPETE Programme (SAICTPAC/0019/2015)), REDE/1518/REM/2005 for providing access to the Mass Spectrometry Facility, and the European Research Area Network, ERANet LAC (ref ELAC2014/BEE-0341) for financial support.
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REFERENCES
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DOI: 10.1021/acs.jchemed.7b00458 J. Chem. Educ. XXXX, XXX, XXX−XXX
