Incorporating Carbohydrates into Laboratory Curricula - Chemical

Review Cite This: Chem. Rev. 2018, 118, 7986−8004

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Incorporating Carbohydrates into Laboratory Curricula Jennifer Koviach-Côté† and Alyssa L. Pirinelli*,‡ †

Department of Chemistry and Biochemistry, Bates College, Lewiston, Maine 04240, United States Department of Chemistry, University of Minnesota, Morris, Morris, Minnesota 56267, United States

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‡

ABSTRACT: As an important but underappreciated field, carbohydrate chemistry is a critical topic for undergraduate students to learn. With applications to nutrition, food science, and diabetes, carbohydrates provide real-world relevance to students. This review summarizes the literature of undergraduate laboratory experiments which use carbohydrates since the year 2000. Experiments explore important chemical concepts such as synthesis, kinetics, analysis, and computational chemistry. Experiments designed for general chemistry, organic chemistry, biochemistry, and analytical chemistry at a variety of skill levels are presented.

CONTENTS 1. Introduction 2. General Chemistry 2.1. Analysis of Sugar Content in Food 2.2. Kinetics of Mutarotation 2.3. Molecular Modeling and Computational Chemistry 3. Organic Chemistry 3.1. Selective Transformations 3.2. Esterification 3.3. Glycosidation 3.4. Synthesis 4. Biochemistry 4.1. Glucose Oxidase 4.2. Blood Glucometer Experiments 4.3. Glycosidase Studies and Michaelis−Menten Kinetics Measurement 5. Analytical Chemistry 6. Conclusions and Looking Forward Author Information Corresponding Author ORCID Notes Biographies References

into various levels of education has perhaps been understanding even the basics of the complexity within carbohydrate structure and function. Possible adopters may feel that there are numerous complex background concepts that must be understood before any work with carbohydrates can be accomplished. However, it is increasingly important that carbohydrates are brought into undergraduate and earlier education to bring more exposure and understanding to the field. In this review, we summarize the literature describing carbohydrates in the undergraduate curriculum, specifically in the undergraduate laboratory. As far as we can tell, this has not been the subject of a review to date, so we have limited this paper to experiments published since 2000, based upon a desire to incorporate modern experiments that include the most advanced instrumentation. Not only are carbohydrates an important class of compounds to understand; they also provide excellent teaching opportunities in the gamut of undergraduate (or high school) chemistry courses. Many students are already familiar with carbohydrates in the context of nutrition, food science, and diabetes, which provides relevance to the real world. Carbohydrates are nontoxic, generally inexpensive, low waste, and often easy to purify, and with many years of study, experiments involving carbohydrates are reliable. Many carbohydrate experiments may be performed in a 3 h time block required of the teaching laboratory, but are also stable for extended periods that can span multiweek experiments. In addition, analysis of carbohydrates, glucose in particular, may be easily performed using inexpensive commercially available blood glucose monitors. Since carbohydrates are important biomolecules across the curriculum, experiments have been developed for general chemistry, organic chemistry, biochemistry, and analytical chemistry at a variety of skill levels. In addition, the versatility of carbohydrates allows investigations

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1. INTRODUCTION Although carbohydrate chemistry has been a subject of study since Fischer’s work in the 19th century, carbohydrates have received much less attention than other biomolecules such as proteins and nucleic acids. However, recent advances in glycobiology and developments in synthesis have allowed for much expanded study of carbohydrates by scientists in fields including chemistry, biochemistry, immunology, and molecular biology. As scientists begin to realize the importance of carbohydrates in biological systems, it becomes increasingly important to expose students to this long-established but still emerging field. A challenge with incorporating carbohydrates © 2018 American Chemical Society

Special Issue: Carbohydrate Chemistry Received: December 20, 2017 Published: August 16, 2018 7986

DOI: 10.1021/acs.chemrev.7b00757 Chem. Rev. 2018, 118, 7986−8004

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Figure 1. Sample student results in determining starch and reducing sugar content in bananas.

differences in structure and physical nature of different carbohydrate forms, namely starch and smaller free sugars, as well as their roles in nutrition. Quantitative analysis experiments also provide students with a variety of laboratory techniques. Deal et al. have developed an introductory level experiment designed for nutrition students, also appropriate for first-year or organic students, in which students performed quantitative carbohydrate analysis of bananas in varying stages of ripening (Figure 1). Each pair of students was assigned either a green, yellow, or overripe banana, and then calculated the percentage of starch and free sugar content in relation to the overall mass. In the first iteration of the experiment, published in 2002, students first separated the soluble and insoluble components of the banana using glass homogenizers and refrigerated centrifuges.34 A later modification showed that homogenization could be performed as effectively using a mortar and pestle, and tabletop microcentrifuges.35 Pedagogically, this experiment ties together students’ familiarity with food science and chemical analysis. Since it has been shown previously that the insoluble portion of banana is predominantly starch, students could determine starch content directly by weighing the centrifuged and dried pellet. A 3,5-dinitrosalicyclic acid−potassium sodium tartrate (DNS) assay was then used as a quantitative test for reducing sugars in the soluble portion. Reaction of DNS with reducing sugars results in reddish-brown product which can be measured spectrophotometrically and compared to a standard curve. In a subsequent paper, the authors show that the colorimetric DNS assay could be replaced with commercially available glucose test strips with no loss in reproducibility. Finally, students investigated several methods of storage, including refrigeration, commercial banana hangers, a brown paper bag, and the lab drawer. After storing for 1 week, students repeated the quantitative analysis. As expected, refrigeration slows ripening, while the other three storage methods have no effect.

of selective chemical transformations, kinetics, computational chemistry, and quantitative analysis, among many others. While this review covers specifically the literature of carbohydrate-based laboratory experiments, carbohydrates are also used as teaching tools in nonlaboratory settings at the secondary-school and college levels. The blue bottle demonstration is a classic example, updated recently by Campbell. In this demonstration, a blue dye is reduced to a colorless species, as various sugars are air-oxidized.1,2 Many other chemical and biochemical concepts involving carbohydrates have been described in the context of blood glucose testing,3 food science and nutrition,4−7 and wine and beer production.8−12 Other educators describe pedagogical methods to help students better learn concepts related to carbohydrate chemistry. Pedagogical techniques include research-based courses,13 active learning,14 writing informational pamphlets for nonscientists,15 using the book Napoleon’s Buttons,16 mnemonics for learning Fischer projections,17 an algorithm to convert aldoses into straight-chain conformations,18 card games,19 computer-based three-dimensional (3D) simulations,20 and specific homework or educational exercises for students.21−31 Risley has also addressed the issue of incorrect structures of the ABO blood group in numerous organic and biochemistry texts.32 In addition, Milenković has developed an assessment tool to determine misconceptions of carbohydrates among upper level undergraduates.33

2. GENERAL CHEMISTRY Carbohydrates make ideal substrates for general chemistry experiments. They are inexpensive and nontoxic, and they have a variety of interesting physical and chemical properties suitable for study at the first-year undergraduate level. In addition, there are many commercially available methods for the analysis of carbohydrates in blood and food samples, which can be used for chemical analysis in the laboratory. Finally, carbohydrates are critical for the understanding of food science and have important biological functions, which provide students context for the real-world application of chemical experiments.

2.2. Kinetics of Mutarotation

Most commercial D-glucose consists predominantly of the αanomer, due to its reduced solubility over the β-anomer, which results in its selective crystallization. However, in solution, the α-anomer equilibrates to a mixture of the two anomers through the process of mutarotation (Scheme 1). Since this process is

2.1. Analysis of Sugar Content in Food

Experiments which involve food and nutrition provide relevance to the real world and increase student interest. In addition, these topics provide the opportunity to discuss 7987


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relatively slow, it pedagogically represents a simple method for undergraduates to perform kinetics experiments. Scheme 1. Mutarotation of D-Glucopyranose

A convenient method to study mutarotation is to use glucose biosensors which employ enzymes that selectively oxidize the β-anomer to gluconolactone (Scheme 2). Blood glucose meters and glucose test strips typically use glucose dehydrogenase (GDH) combined with a coenzyme such as pyrroloquinoline quinone (PQQ) to provide easy colorimetric analysis of blood glucose concentrations. Glucose oxidase (GOx) is another convenient enzyme used in sugar monitors, in that it may be used in electrochemical sensors. Traditional methods for the measurement of glucose mutarotation use polarimetric analysis. However, these methods are not easily amenable to the undergraduate laboratory. Instead, Volpe and Perles have found a method in which the blood glucose concentration recorded by inexpensive commercial blood glucose meters may be correlated to optical rotation values.36 This way, undergraduate general chemistry students can study the kinetics of mutarotation without the need for a polarimeter. As described above, blood glucose meters use the GDH−PQQ enzyme system to determine the concentration of β-D-glucopyranose, through its selective enzymatic conversion to D-glucolactone. The authors predetermined that the blood glucose concentration provided by the blood glucose meter (Cbgm) is linearly related to the optical rotation, α, of the sample through eq 1. In addition, the concentrations of α- and β-anomers may be calculated through eqs 2 and 3. α = 0.767° − (0.0054° L/g)(C bgm)

i α − 0.8064° yz zz × 1000 g/L Cβ = −jjj 9.37° k {

(1)

Cα = total concentration − Cβ

(3)

Figure 2. Results obtained by one pair of students in a 4 h laboratory: (A) optical rotation estimated by eq 1 and (B) concentrations of α-Dglucose and β-D-glucose calculated by eqs 2 and 3. Reprinted from ref 36. Copyright 2008 American Chemical Society.

measure the kinetics of D-glucose mutarotation.37 In this experiment, students first prepared glucose electrodes which were coated with GOx and polymerized with a poly-ophenylenediamine film. They then selectively crystallized β-Dglucopyranose from a mixture of anomers, taking advantage of the difference in solubility of the two anomers at higher temperatures. The purified sugar was dissolved in an EDTA buffer, and the concentration of the α-anomer was determined by measurement of the current used by the glucose biosensor. Since the biosensor allows for real-time measurements, a plot of current vs time provides kinetics information. Typical student data is presented in Figure 3.

(2)

2.3. Molecular Modeling and Computational Chemistry

Molecular modeling has become a staple of the undergraduate teaching lab, especially as computing power has become faster and more accessible. Pedagogically, computational chemistry provides students with the ability to visualize molecules and their interactions with each other in ways that are difficult in two dimensions. In addition, undergraduates can quickly and easily generate large amounts of data, which allows them to learn methods of data analysis and gives them the opportunity to recognize trends within similar systems. Tribe and co-workers have developed a computational chemistry laboratory for first-year students taking chemistry as food science, human nutrition, and pharmacy majors as well as first-year general chemistry students.38 The pedagogical goal of

To obtain kinetics data, aqueous D-glucose solutions were prepared and placed into a 20 °C water bath. Aliquots were then removed every 20 min for 3−4 h and glucose concentration was determined using the blood glucose meter. Commercially available D-glucose contains approximately 90% of the α-anomer, but after 3−4 h, it was 38% αanomer and 62% β-anomer, in agreement with previous measurements. Although students did not measure kinetics directly, this method was fast and reliable, and consistent with previous results. Typical results are shown in Figure 2. Teruel and Jenkins have also developed an experiment for undergraduate general chemistry students in which students

Scheme 2. Glucopyranose Oxidation Reactions in Commercially Available Glucose Biosensors

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Figure 3. Relaxation of α-D-glucose (○) and β-D-glucose (●) to equilibrium following dissolution in 5 mM EDTA buffer of pH 7.4. Current scale is normalized to the equilibrium current observed at time i∞. Reprinted from ref 37. Copyright 2009 American Chemical Society.

Figure 4. Computational analysis energy of various foods and comparison with food labels. Reprinted from ref 38. Copyright 2015 American Chemical Society.

Figure 5. Molecular modeling of glycogen phosphate and an inhibitor. Reprinted from ref 39. Copyright 2014 American Chemical Society.

mechanics and semiempirical methods. Students then recorded the heat of formation for each of the compounds, as well as the combustion products. Using a balanced equation of the combustion reaction, students used Hess’s law to determine the molar heat of combustion for that molecule as well as the kilocalories per gram released. Each group of students then used the ingredient list or nutritional information on a food item to determine the energy released for every 100 g or per serving as described on the food label. The computational results were then compared to the nutritional information on the food item. Although models were used for actual food components and calculations were performed in the gas phase, errors of energy calculations compared to the conventional

the experiment was for students to use computational modeling to calculate the energy released upon combustion of food items. The calculated energy was then compared to the nutrition information on each food. As a model for the ingredients listed in the food’s composition, carbohydrates were represented completely by glucose, fats by stearin, and amino acids by a single amino acid or dipeptide. Degradation products of glucose and stearin were represented as CO2 and H2O, while protein degradation products were assumed to be urea, CO2, and H2O (Figure 4). To perform the calculations, students built the biomolecules in a modeling software program such as Hyperchem.6 or Spartan and optimized the geometry using molecular 7989


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Scheme 3. Selective Tosylation and Azide Formation

Scheme 4. Selective Benzylidenation and Acetylation

3.1. Selective Transformations

values and reference data range only from 3 to 22%. The authors note that the computational experiment can be combined with more traditional thermochemistry experiments to compare computational and experimental results.38 Hayes took a different approach to the use of molecular modeling in an experiment designed specifically for first-year undergraduate students. In this experiment, students visualized the molecular basis of target−drug interactions, to better understand medicinal chemistry (Figure 5). After an initial exploration of the Protein Data Bank (PDB) database and visualization of DNA, students performed a structural investigation of the enzyme glycogen phosphorylase (GP) and an inhibitor, 1-(β-D-glucopyranosyl)-5-Cl-uracil (GlcClU) using structures from PDB (code 3T3E). As part of the analysis, students visualized different levels of protein structure, determined the structural components of the GlcClU ligand, isolated the PLP cofactor, analyzed the enzyme−ligand interactions, and proposed structural modifications to the inhibitor to improve activity and its pharmacokinetic profile.39 This activity is designed to give first-year students experience in modeling as well as understanding the “real-world” implications of the topics discussed in their first-year classes.

Sugars are particularly well-suited for the demonstration of selective transformations, since they contain similar functional groups which differ in their steric environments. In the following experiments, students perform selective reactions, then must use spectroscopic methods to determine the product, and finally justify selectivity on the basis of structure and reactivity. Norris et al. have developed a two-step synthesis of a stable xylose based azide suitable for an upper level organic synthesis laboratory (Scheme 3).40 In this experiment, students first selectively converted monoacetone xylose 1 into primary tosylate 2. Following recrystallization, the tosylate underwent SN2 displacement with NaN3 to form 3. Students probed the steric discrimination of a primary hydroxyl group over a secondary hydroxyl group, and confirmed the product structure using IR spectroscopy through the azide stretch. In addition, both 1D and 2D NMR spectroscopies were used to determine coupling constants. Demchenko et al. introduced sophomore-level organic students to a common protecting group in carbohydrate chemistry by selectively converting α-D-methylglucopyranoside (4) into its 4,6-O-benzylidene derivative 5, a common intermediate in the synthesis of many carbohydrate building blocks (Scheme 4).41 As an extension of this experiment, students in an advanced organic laboratory confirmed the regioselectivity of benzylidene formation by further forming the diacetate 6 followed by comparison of the chemical shifts for H2 and H3 in 5 compared to 6. This experiment provided the opportunity to discuss regioselective preferences for formation of a six-membered acetal ring instead of a fivemembered ring, the formation of a new chiral center on the benzylidene, and the preference for the phenyl group to adopt an equatorial position.

3. ORGANIC CHEMISTRY The organic chemistry of carbohydrates has been well-studied since the 19th century. As such the chemistry is well understood and relatively easy to perform. In addition, carbohydrates provide excellent examples of selective reactivity: acetals vs ethers, primary vs secondary alcohols, thermodynamic vs kinetic products, etc. With a variety of functional groups in one sugar, they are highly suitable for multistep synthesis, and many of the products can be purified through crystallization, a benefit to the teaching laboratory. Finally, most sugars provide well-resolved NMR spectra, suitable for one- and two-dimensional (1D and 2D) characterization. An analysis of the 1H spectra may provide examples of diastereotopic relationships, and provide ideal methods of correlating dihedral angles to coupling constants.

3.2. Esterification

The esterification of sugars is straightforward, and typically provides crystalline products with well-resolved 1H NMR spectra. Pedagogically, acetylation of reducing sugars allows for a discussion of kinetic vs thermodynamic products in the form of axial and equatorial anomeric acetates and furanose vs 7990


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Scheme 5. Acetylation of Glucose with Iodine

Through this analysis, students determined the identity of their initial unknown compound. Pandita et al. also employed pyridine and acetic anhydride to peracetylate glucose (Scheme 7).44 In this experiment, students performed two experiments simultaneously, changing only the temperature of the reaction. The reaction at room temperature selectively provided the kinetic product, αanomer, 8, while heating at reflux selectively provided the βanomer, 12, as the thermodynamic product. Through this experiment, students observed how differences in temperature affected the product outcome, which they then explained through an analysis of relative reaction rates and product stabilities. Both products were purified by crystallization, and characterized according to their coupling constants in 1H NMR. Rhoad described a similar peracetylation experiment, but in this case, students were provided with either D-glucose (7) or D-galactose (13), for peracetylation with sodium acetate and acetic anhydride to afford pentaacetate 12 or 14, respectively (Scheme 8).45 Following purification, students obtained 1D 1H and 13C NMR spectra as well as 2D-COSY and inverse HETCOR spectra. Through interpretation of these spectra, students assigned each of the proton peaks. Analysis of the coupling constants then allowed them to determine which sugar they began with, as well as the anomeric configurations of the product. In addition, the β-selectivity of this reaction allowed for a discussion of C-2 protecting group participation.

pyranose ring forms. The anomeric effect may also be introduced, in order to explain increased stability of axial substituents at the anomeric center. Acetylation experiments also allow for analysis of more complex 1D NMR spectra than students have typically seen to date and the introduction of 2D NMR. The protons of peracetylated sugars are shifted downfield and across a larger chemical shift range compared to the unprotected sugars, which allows for simplified peak assignment. In addition, students may be introduced to complex NMR splitting patterns and coupling constant analysis, through which they may determine dihedral angles and thus relative stereochemistry of coupled protons. Schatz found glycosylation of glucose (7) with iodine easily formed α-8 as shown in Scheme 5. The pentaacetate could be isolated and recrystallized within a single lab period. The ratio of anomers was determined through examination of the coupling constant of the anomeric hydrogen.42 Sorensen also described a peracetylation experiment in which third-year advanced organic students were provided with either α-methyl glucopyranoside (4) or α-methyl galactopyranoside (9) (Scheme 6).43 Peracetylation with Scheme 6. Acetylation of Methyl-α-D-Glucose and Galactose

3.3. Glycosidation

Glycosidation is one of the oldest and most important reactions for carbohydrate chemistry. Simple traditional methods of Fischer glycosidation are easily accessible to students, while methods using more sophisticated glycosyl donors provide students with experience in modern organic synthesis. Pedagogically, the product ratio of anomers provides a discussion of mechanism, neighboring group participation, and thermodynamic control. In an experiment developed by Hovinen et al., undergraduate organic chemistry students perform a Fischer glycosidation of D-fructose (15) with methanol46 (Scheme 9.) The choice of D-fructose was important for several

pyridine and acetic anhydride provided the tetraacetate derivative 10 or 11 respectively, which was then separated chromatographically from unreacted starting material. Analysis of the 1D 1H and 2D-COSY NMR spectra allowed students to assign all peaks and determine the coupling constants.

Scheme 7. Kinetic and Thermodynamic Peracetylation of D-Glucose

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Scheme 8. Peracetylation of D-Glucose and D-Galactose

Scheme 9. Kinetic and Thermodynamic Formation of Methyl-D-fructosides

Scheme 10. Fischer Glycosidation of D-Ribose

data were then pooled, and the percent composition of each product was plotted vs time, as shown in Table 1. To explain

pedagogical reasons. First, many students are familiar with fructose from everyday life. Second, in theory, four products could be formed. However, the two furanosides (16 and 17) are the kinetic products, while the pyranosides (18 and 19) are thermodynamic products. This facilitated a discussion of activation energies and product stability, and students could selectively form the furanosides by careful monitoring with 1D or 2D thin layer chromatography (TLC). Two-dimensional TLC was particularly useful in this experiment, as the first eluent separated the furanosides from the pyranosides, while the second eluent separated the α- and β-anomers. Since the product was formed as a 1:1 mixture of anomers, students used ion-exchange chromatography to separate the products, which were then characterized by optical rotation. Bendinskas et al. also described a Fischer glycosidation reaction with methanol, this time with ribose (20) as the substrate (Scheme 10), for upper division undergraduate students.47 As above, the methyl furanoside products (21 and 22) are the kinetic products, while the pyranosides (23 and 24) are the thermodynamic products. In this experiment, students performed the glycosidation for either 30 or 60 min at room temperature or 65 °C. Students then determined the ratio of the four products by integration of the 1H NMR spectrum with D2O as solvent, at 30 °C. Under these conditions, all four anomeric hydrogens were clearly distinguished from each other and the solvent. The class

Table 1. Representative Data for the Relative Composition of Methyl Glycosides of D-Ribose in Refluxing Methanol reaction products (%) time (min)

methyl α-furanoside

methyl β-furanoside

methyl α-pyranoside

methyl β-pyranoside

30 60

21.4 20.0

51.5 47.7

4.82 6.90

12.6 18.1

the differences in composition at each temperature, students evaluated the mechanism for formation of each glycoside, and discussed activation energies for each reaction intermediate. 3.4. Synthesis

Multistep synthesis provides a variety of pedagogical advantages. It provides students with a variety of synthesis techniques and characterization methods, and it emphasizes the need for planning and experiment design. Callam and Lowary have developed a two-step synthesis of compound 27 for honors organic chemistry lab students, as shown in Scheme 11.48 The relevance of this experiment is reinforced, since polymers of D-arabinofuranose are prevalent in the cell walls of mycobacteria. In the first week, students 7992


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Scheme 11. Two-Step Synthesis of an Arabinofuranoside

Scheme 12. Synthesis of β-D-Gucopyranosyl Azide through SN2 Inversion

Scheme 13. Two-Step Tosylation/Elimination Sequence from Diacetone-D-glucose

Scheme 14. Formation of Kinetic and Thermodynamic Ribonolactone Acetals Followed by Comparison of Experimental Data with Molecular Modeling Data

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coupling constant and NOE data to theoretical values. Using this method, students performed conformational analysis of each molecule in solution, determined the dihedral angles, and confirmed the structure of each lactone. Simeonov and Afonso have used carbohydrate chemistry to illustrate the ideas of biorefinery batch and flow process chemistry as well as materials recycling to second-year pharmaceutical science students.52 In this experiment, students used either batch or flow process techniques to prepare 5hydroxymethylfufural (HMF, 38) from fructose (15) (Scheme 15). The relevance of this experiment is important, as HMF

performed a Fischer glycosidation to form the methyl glycoside from arabinose using acetyl chloride and methanol. The result is an approximate 1:1 mixture of α- and β-methylfuranosides, 26. The results of this reaction allowed for a discussion of mutarotation and the pyranose and furanose forms of pentose sugars. In addition, kinetic and thermodynamic control were discussed, since the furanose form of arabinose is the kinetically formed product. In the second week of the experiment, students treated the methyl glycoside with pyridine and benzoyl chloride to form the tribenzoate 27. At this point, the anomers could be separated through recrystallization, since the α-anomer is a solid, while the βanomer is an oil. Following isolation of the product, students used 1D and 2DNMR spectroscopies to assign 1H and 13C peaks. Jackson et al. used carbohydrate chemistry to demonstrate stereochemical inversion in an SN2 reaction.49 In this experiment, advanced undergraduate or beginning master’s students converted commercially available bromide 28 into the corresponding azide 29 (Scheme 12). Students obtained a 1H NMR spectrum of both the starting bromide and the product azide. Since the spectra of both compounds were particularly well resolved, students could determine the stereochemistry of the anomeric position through coupling constant analysis. Using this method, students demonstrated inversion of configuration at the anomeric center, which indicated that the reaction proceeds through an SN2 rather than an SN1 mechanism. Norris and Fluxe have developed a two-step synthesis for their advanced organic chemistry laboratory sequence.50 In this sequence, students first prepared the tosylate 31 from diacetone-D-glucose 30 using standard reaction conditions (Scheme 13). Compound 31 was then purified by recrystallization and treated with KOt-Bu, a bulky base. An E2 elimination resulted in formation of alkene 32, which was purified by flash column chromatography. This experiment highlights several pedagogical concepts: the use of tosylates as leaving groups, the use of a bulky base to favor E2 over SN2 products, and the need for an antiperiplanar hydrogen for elimination to occur. In this case, there are two β-protons, but H4 is antiperiplanar, while H2 is synperiplanar. Therefore, only the C3−C4 alkene was formed, in lieu of the C2−C3 alkene. Sales and Silveira developed an experiment for an upperdivision organic chemistry laboratory course in which students treated D-ribonolactone 33 with either acetone or benzaldehyde and 12 M HCl to form an acetal (Scheme 14).51 Due to the differences in reaction conditions, the acetonide was formed as kinetic lactone 34, while the benzylidene was formed as the thermodynamic six-membered ring lactone 35, also known as the Zinner lactone. In an extension of the experiment, students also acylated the remaining alcohol group with an aromatic acyl chloride. While five- and sixmembered lactones can typically be distinguished from one another using 13C NMR, the spectra of compounds 36 and 37 is misleading, and cannot be used to definitively determine ring size. Instead, students used a 2D-NOESY NMR spectrum to characterize their products. In compounds 34 and 36, H3 and H4 are trans, and do not exhibit a cross-peak. In contrast, H3 and H4 are cis in the six-membered rings, and do show a medium-intensity cross-peak for compounds 35 and 37. Finally, students used ChmBio3D software to perform MM2 energy minimization, and then uploaded their minimized structure to JANOCCHIO to compare their experimental

Scheme 15. Flow Synthesis of Compound 38 and Apparatus Used (Reprinted from ref 52. Copyright 2013 American Chemical Society.)

can be used as a precursor to building blocks for polymer production or biofuels. In the batch experiment, students converted fructose into HMF using tetraethylammonium bromide (TEAB) and Amberlyst 15. Following completion of the reaction, the TEAB and Amberlyst resin were recovered, and could be reused several times. To perform the flow process experiment, a standard glass chromatography column was connected to a glass reactor, which was placed in a boiling water bath. A round-bottom flask was connected to the reactor to collect the product. TEAB was mixed with 5% aqueous H2SO4, and combined with fructose. This mixture was added to the glass column, and the flow was controlled by slight positive air pressure until the reaction in the glass column was completely discharged. Each student assessed the purity of their product using HPLC, and determined the E-factor for their process. Students then compared the results of the two processes in terms of E-factor and purity. Penverne and Ferrières have introduced a four-step synthesis to their fourth-year organic chemistry students (Scheme 16).53 The pedagogical goals for this experiment were to introduce the reactivity of the anomeric center and to discuss the importance of anchimeric assistance through neighboring group participation. The target molecule has been used in biological studies as an inhibitor and as a fluorogenic substrate, giving students motivation for its synthesis. However, the target molecule cannot be prepared using traditional Fischer glycosidation, so a multistep synthesis involving protecting group manipulation was required. The synthetic sequence 7994


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Scheme 16. Four-Step Synthesis of Pharmaceutically Relevant Fluorogenic α-Arylmannoside

Scheme 17. Four-Step Synthesis of Three β-D-Glucopyranosides

kinetic acetylation using sodium acetate and acetic anhydride to form compound 12 while the other used zinc chloride and acetic anhydride to give the thermodynamic product, 8. Both products were purified through recrystallization, and the anomeric acetate was selectively deprotected to give 45. The product could be purified by crystallization or chromatography if additional experience with chromatographic separation was desired. Students then converted alcohol 45 into the trichloroacetimidate donor 46, which was purified by flash chromatography. Finally, the donor 46 underwent glycosylation with one of three acceptors. As the glycosylation required anhydrous conditions, students gained experience with handling solvents and working in an inert atmosphere. In this experiment, students fully characterized their products at every stage using a variety of 1D and 2D NMR techniques. As each step involved transformation at the anomeric center, students made use of the Karplus relationship to identify the anomeric configuration of each product, and used concepts such as kinetic and thermodynamic reaction conditions, mutarotation, the anomeric effect, and anchimeric assistance to explain the selectivity of each reaction.

began with selective removal of the anomeric acetate from 39 to afford compound 40, followed by formation of trichloroacetimidate 41, which was purified by flash chromatography. Students then characterized 41 by 1H NMR, and determined that a mixture of α- and β-anomers was formed. Glycosylation between donor 41 and acceptor 42 afforded 43, which was purified by crystallization. Compound 43 was formed as a single anomer, and could be characterized using 2D-COSY and HMQC experiments. Students then employed the theory of anchimeric assistance to explain the stereoselectivity. Finally, the tetraacetate 43 was deacetylated to afford the fully deprotected glycoside 44, which was purified by crystallization. The entire sequence could also be performed using glucose pentaacetate as the initial substrate, but the intermediate glucoside was not readily purified by crystallization, so additional chromatographic separation was necessary. Stocker et al. have developed a similar four-step synthetic sequence for their final-year organic chemistry students, working in pairs (Scheme 17).54 Pedagogically, this sequence introduces the ideas of increased anomeric reactivity, kinetic vs thermodynamic control, and stereoselectivity of glycosylation reactions. In the first step, one member of the pair performed a 7995


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Scheme 18. Multistep Synthesis of Small Library of HIV Inhibitor Mimics

later curricula, such as biochemistry, where understanding the bulk of carbohydrate functions resides. Furthermore, biochemistry lectures may not be accompanied by a laboratory section, in part because biochemistry requires a dedicated set of reagents and equipment. Though some current carbohydrate research relies on expensive instrumentation and techniques, laboratory experiments have been developed for several areas of biochemistry that are fairly accessible to a range of institutions with respect to required chemicals and equipment, with appropriate intellectual challenges for students.

Pontrello developed a multistep synthetic sequence for the first-semester organic laboratory, to prepare a small diverse library of carbohydrate-based HIV inhibitor mimics. One of the main pedagogical goals of this four-reaction, six-week experiment was to expose students to the primary literature. As such, students were assigned background literature describing the molecular mechanisms of HIV infection, with attention to small molecule inhibition of binding between the HIV Tat protein and TAR-RNA. In addition, students searched the Protein Data Bank (PDB) for the crystal structure of HIV TAR-RNA with a bound peptide mimic. Finally, students were not provided with the procedure for each experiment, but instead were given references from the primary literature to perform each step of the synthesis as described in Scheme 18.55 To provide the library of compounds, every student performed reaction 1 using commercially available D-glucal (48) as starting material, and the products were pooled. A subset of students (20−50%) continued the synthetic sequence from compound 49, while the remainder incorporated diversity into the library by following a parallel set of reactions, but beginning with pyran 53. Additional diversity was incorporated into the library in reaction 3, in which alcohols of varying chain length were used in a glycosylation reaction. Finally, students used one of two Grubbs’s catalysts to carry out the metathesis in reaction 4. All reactions could be monitored by TLC and characterized easily by IR or 13C NMR spectroscopy. Through this synthetic sequence, students were exposed to a small-scale version of the methods used by pharmaceutical companies to search for novel bioactive compounds.

4.1. Glucose Oxidase

As mentioned earlier, a common method for determining the concentration of glucose in a solution is using the glucose oxidase (GOx) enzyme. Under standard conditions, GOx converts β-D-glucose and one molecule of oxygen into one equivalent each of β-D-gluconolactone and hydrogen peroxide, as shown in Scheme 19a.56 As the gluconolactone structure can Scheme 19. General Scheme Using Glucose Oxidase Coupled with Horseradish Peroxidase To Measure Glucose Concentrations

be challenging to observe through regular spectroscopic methods, the initial hydrogen peroxide product is more often used in a subsequent reaction. This second reaction gives a readout that is therefore proportional to the amount of glucose that was oxidized. So long as it is kinetically faster than the GOx reaction, several measurements can be taken from this second reaction: the initial concentration of glucose in unknown samples (proportional to the output of the second reaction) and kinetics data for these enzyme combinations. Data collection can be done on several types of instruments in

4. BIOCHEMISTRY Methods for teaching undergraduates about carbohydrates in general chemistry and organic chemistry laboratories have already been discussed. Biochemistry is often a third- or fourthyear undergraduate course, very much building upon principles developed in earlier chemistry and biology classes. Incorporating more carbohydrates into the earlier collegiate curriculum may decrease the barrier to having carbohydrates included in 7996


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Figure 6. (A) Three-electrode electrochemical setup. (B) Student-generated amperogram showing increase in current vs time upon addition of glucose. Glucose was added to the solution at points a−d. Reprinted from ref 61. Copyright 2013 American Chemical Society.

4.2. Blood Glucometer Experiments

the laboratory, including commonplace spectrophotometers, though whether these will work depends on the second coupled reaction’s readout being in the UV or visible spectrum or some other sort of electrochemical readout. Gooding et al. coupled the GOx activity with horseradish peroxidase’s (HRP) ability to oxidize ferrocyanide to ferricyanide in an aqueous solution. This oxidation can therefore provide a colorimetric response in proportion to the amount of glucose found in solutions, namely in sports drinks (Scheme 19b).57 For this experiment, the students were tasked with finding the proper dilutions for the sports drinks. There have been other experiments developed along these same lines. For example, Bare and colleagues used GOx/HRP to oxidize a ruthenium-based dye via to produce luminescence measurable on a home-built fluorimeter. This inexpensive instrument gave the students results nearly identical to those obtained on a far costlier research-grade fluorimeter, evidence that carbohydrate-related experiments are available to departments with limited instrument availability. From this data, students determined kinetics information based on the activity of glucose oxidase.58 Similarly, Vasilarou described another GOx/HRP experiment based on the amount of glucose in fruit drinks and carbonated beverages using a colorimetric reaction on a UV−vis spectrometer.59 The amount of endogenous oxygen available will affect the kinetics of the GOx reaction; therefore, the GOx can be the second in a chain of biochemical reactions rather than the first. Choi and Wong studied, via a datalogger, several changes in a solution using an immobilized GOx enzyme on an egg membrane along with an oxygen electrode. This system gives students a view of a coupled biosensor and can be modified to work with any biological reaction that releases or uses oxygen.60 More recently, Hobbs et al. coupled the GOx enzyme with a carbon nanotube electrode that measures the current produced by the breakdown of hydrogen peroxide into oxygen, protons and electrons, the setup for which is shown in Figure 6A. Using data produced from this electrode (Figure 6B), students can build a calibration curve and measure the concentration of glucose in various sports or soft drinks.61 Similarly, Blanco-Lóp ez et al. used ferrocene as the colorimetric reductant and a carbon paste electrode to measure current flow during the GOx reaction.62

As mentioned, most personal blood glucose monitors take advantage of the GOx/HRP pairing to give a fast, reliable, and relatively inexpensive blood glucose concentration readout for diabetic patients. The HRP induces a colorimetric response which is read by the instrument to give the relative blood glucose concentration. Over the past several years, the costs of these monitors and the requisite test strips have significantly decreased. Given the improvement in glucometer technology, readings of blood glucose can now happen nearly instantaneously, and in addition to the convenience and the potentially life-saving aspect for diabetes patients, these improvements have made laboratory experiments using blood glucometers possible for undergraduate institutions. Lazarim and colleagues wanted students to gain a better physical understanding of the glycemic index of foods and the impact on the glycemic load in the body. Students ingested sugar via drinking juice or eating food, and then every 30 min over the course of 2 h, monitored the changes in their blood sugar levels.63 This study had students comprehend the link between what food items and quantity thereof they ingested and what happened with their blood sugar. Another study in this area involves postundergraduate medical students studying the blood glucose and triglyceride content in diabetic and nondiabetic rats.64 Hardee et al. have published a procedure for finding the Michaelis−Menten kinetic parameters for the enzymecatalyzed mutarotation of glucose using a commercial blood glucose monitoring system. This procedure can be adapted for use in several college-level courses depending on the type of data desired. The experimenters could show that a blood glucometer was able to give readings similar to a polarimeter.65 Heinzerling et al. used the same method to observe the hydrolysis of sucrose and lactose by the invertase enzyme found in baker’s yeast (Saccharomyces cerevisiae) by monitoring glucose production. Students were asked to make a standard curve containing a series of dilutions, from which they could discern the Michaelis constant and later other kinetic parameters through conversion to Lineweaver−Burke plots.66 In addition to helping students understand Michaelis−Menten parameters, this exercise also gave students hands-on understanding of the impacts of temperature and pH on enzyme activity. Recently, Amor-Gutiérrez et al. used a student-built printable carbon electrode to fabricate a glucose biosensor in 7997


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the concept of enzyme immobilization using the same enzyme and a different substrate.69 Yet others have used amylase to quantify the rate of activity and the effects of the reaction parameters on the activity of enzymes, helping teach the concepts of Michaelis−Menten kinetics. Cochran and co-workers had undergraduate biology students study reaction kinetics via a colorimetric analysis with two solutions: KI/I2 and Benedict’s solution. Working with a commercial flatbed scanner, the students used the proportionally decreasing concentrations of starch and increasing glucose concentrations in samples of starch exposed to α-amylase to make a standard curve. Using this curve, the students could then determine the concentrations of glucose in unknown samples.70 Valls and colleagues used amylase isolated from human saliva and compared it to industrial detergents,71 while Munegumi used same-sourced α-amylase to look at the similarities between saliva and detergent functions.72 These articles each help students understand the impact of the reaction parameters: Munegumi and co-workers specifically focused on changing the temperature of the solution while Valls and co-workers changed several conditions, ranging from the pH to adding oxidizing agents to the mixture. When comparing saliva enzymes to the detergents, Munegumi and colleagues noted that, among other conclusions, the saliva enzymes denatured at higher temperatures whereas the detergent-based enzymes did not (Figure 8). Flow chemistry and reactions using flow techniques have grown in popularity over the past few years; one of the main advantages to this method is the ability to reuse catalyst and reduce overall chemical consumption and waste.73 So far flow chemistry been slow to find purchase outside of industrial applications, perhaps because of the types of reactors required that may have limited use in university settings. Taipa and colleagues have begun to bridge this gap by having advanced laboratory students work on three types of industrial-like flow systems: continuous stirred tank reactors (CSTR), plug flow reactors (PFR), and fluidized bed reactors (FBR) (Figure 9).74 The researchers measured the rate constants of an immobilized invertase enzyme in comparison to the free enzyme. Here, the enzyme is immobilized in an alginate suspension and placed in one of the flow systems, with the expectation that the so-bound enzyme will be less efficient than a free version of the same enzyme. Groups of students worked

an advanced analytical chemistry class (Figure 7). This electrode measured the reduction of ferrocyanide in a

Figure 7. Setup of the screen-printed carbon electrode experiment. Reprinted from ref 67. Copyright 2017 American Chemical Society.

traditional glucose-sensing manner (by coupling GOx to HRP). The laboratory required students to understand the concepts and fabrication of screen-printed carbon electrodes and electrochemistry coupled with the biochemical concept of enzymatic analysis.67 In building and using these disposable electrodes, students learned about the concepts of cyclic voltammetry as well as its applicability in biological reactions. Furthermore, the students were also responsible for optimizing enzyme solutions, with this association leading to greater understanding of biochemical concepts in an analyticalchemistry-type course. Lastly, at the end of the experiment, students discussed accuracy, precision, and the time and cost aspects of these reactions. 4.3. Glycosidase Studies and Michaelis−Menten Kinetics Measurement

Enzymes are used to break down larger carbohydrate chains into smaller ones for the uses of metabolism and cleaning, among others. These enzymes are present to aid in digestion in humans, as the amylase enzyme in saliva helps to break down starch prior to food’s arrival in the stomach. Humans do not possess the enzyme α-galactosidase, however. This enzyme promotes breakdown of other long polysaccharides such as those found in peas, and as such humans’ inability to digest these polysaccharides leads to flatulence. Hardee and coworkers tasked students with using the pharmaceutical Beano, a glycosidase, to look at the basics of enzyme kinetics for health science majors. The students measured the production of glucose from a solution of pea polysaccharides using a blood glucometer.68 Mulimani and Dhananjay introduced students to

Figure 8. Figure showing enzyme reactivity of amylase on starch is dependent on temperature. The detergents used: C, Charmy Crysta Clear Gel; J, Joy; K, Kyukyutto at 17% concentration. −, no reaction; ±, weak reaction; +, strong reaction; + +, very strong reaction. Reprinted from ref 72. Copyright 2016 American Chemical Society. 7998


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Figure 9. (A−C) Types of flow reactors used in the flow-chemistry laboratory (CSTR, PFR and FBR, respectively). (D) Schematic representation of a CSTR. (E) Schematic representation of a PFR and/or a FBR. Reprinted from ref 74. Copyright 2015 American Chemical Society.

Scheme 20. (A) Action of a Neuraminidase on a Terminal Neuraminic Acid;a (B) Time-Course NMR of Peak Growth Corresponding to N-Acetylneuraminic Acid in Solution (Reprinted from ref 75. Copyright 2011 American Chemical Society.)

a

In vivo, R is the cell, whereas in this experiment, R is the remainder of a glycoprotein.

with each type of flow reactor and compared their results, requiring them to work together, to understand the ideas behind flow synthesis and accurately complete their data analysis.74 Though flow chemistry is a promising area of future carbohydrate research, it is still not as common to find in universities as other instrumentation. In the future, flow chemistry experiments may grow in popularity when the parts and mechanics of them are less expensive and can be implemented in undergraduate education. Carbohydrate conformation and structural details, though somewhat covered in organic chemistry, are rarely covered in depth before the first semester of biochemistry. NMR is also one of the most powerful and versatile techniques available to chemists and biochemists alike using time-course and or quantitative experiments as mentioned earlier. Analyzing the J-

values from coupling constants or integration of peaks can represent a large amount of possible data toward structural concepts or can report kinetics data over time. NMR instruments are also a nearly ubiquitous element in the university setting, allowing most institutions to incorporate these types of experiments into laboratory curricula. NMR is limited in scale and time frame; however, it can be used to monitor the activity of enzymes by analyzing either the enzyme’s starting material, its products or both. For example, NMR can monitor the release of viral particles in a model system using a commercial neuraminidase and a glycoprotein. In an influenza infection the virus finds a healthy cell by binding to neuraminic acids on the cell’s surface. After the initial infection, the virus will then cleave the neuraminic acid from the rest of the glycan structure, thereby allowing newly 7999


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Figure 10. Real-time 1H NMR showing the decreasing signal from sucrose (S) and the increase in concentration of the α-anomer of glucose (Pα). The later equilibrium with mutarotation to the β-anomer is also shown. Reprinted from ref 78. Copyright 2015 American Chemical Society.

Figure 11. Flowchart depicting possible order of testing using seven wet tests to determine the identity of an unknown. Reprinted from ref 81. Copyright 2013 American Chemical Society.

made viral particles to leave and infect other cells (Scheme 20A). Barb et al. had students use NMR to monitor the rate of release of neuraminic acid 58 from a commercial glycoprotein, generically shown in Scheme 20A.75 Rather than using the full virus, the students prepared samples of a commercial neuraminidase with a glycoprotein (either α1-glycoprotein or fetuin) and watched the neuraminidase progress via timecourse NMR experiments over 2 h. Students could observe the growth of a peak corresponding to the N-acetate of free neuraminic acid as well as a shifting signal that corresponds to H3 (Scheme 20B). Though early undergraduates may stop at this point, students later in their undergraduate studies may add statistical analysis of the data to find the half-life of the reaction. Additionally, Periyannan and colleagues introduced students to studying β-glucosidase activity in an undergraduate biochemistry laboratory using the breakdown of cellobiose to glucose by tracking the coupling constants, peak widths, and integrations of the corresponding anomeric proton peaks.76 Both Kehlbeck and Her utilized NMR spectroscopy to study the hydrolysis of sucrose by invertase.77,78 These groups used time-course experiments to observe the decreasing concentration of sucrose and the growing concentrations of glucose and fructose by measuring initial rates (Kehlbeck et al.) or using qNMR (Her et al.). Using these data, students

investigated the Michaelis−Menten kinetics of the invertase enzyme. Krishnan and co-workers described a quantitative NMR experiment for determining Michaelis−Menten kinetic parameters using statistical analysis aimed at undergraduate students with a physical chemistry or biochemistry focus. This exercise helps students to better understand Michaelis−Menten kinetics, statistics, mathematical interpretation, and NMR optimization by learning to quantify peaks based on relative areas (Figure 10).78 Guerra has recently published a Michaelis−Menten kinetics study on a multienzyme complex that looks at the breakdown of larger carbohydrate polymers into smaller polymers or into monomeric units. The entire Viscozyme L complex of enzymes is less expensive to purchase than the corresponding single enzyme components, each of which breaks down differing types of carbohydrate polymers. With the complex more marketable to educators, students look at the breakdown of several different carbohydrate polymers in a mixture. Furthermore, this experiment tasked the students with fitting their data to several equations, helping the students come up with the strengths and weaknesses of each type of plot.79 8000


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Figure 12. (A) Setup for a conductometric titration. (B) Conductometric and potentiometric titration curves for pectin. Reprinted from ref 84. Copyright 2012 American Chemical Society.

5. ANALYTICAL CHEMISTRY Before more complex spectrometry or spectroscopy techniques were invented, qualitative analysis used to make up nearly the entirety of the chemical process, with Fischer’s determination of glucose and the isomers of glucose being among the most well-known process among organic chemists. Many of these tests have fallen by the wayside when they are replaced with more instrumentation that could provide far more data or as procedures have gone greener. Qualitative testing is often useful for determining the presence or absence of a desired analyte in a solution, and as such, qualitative testing is still incorporated into chemistry curricula at various levels.80 Recently, Dickman and colleagues described two new tests for distinguishing a sample of a pure carbohydrate from another carbohydrate. Using seven qualitative analyses in a specific order, students can identify if their given unknown is a monosaccharide or disaccharide and later what the identity of these are based on positive or negative results to the chemical tests (Figure 11).81 This type of qualitative experiment can be done at the high school or college level and does not require much by way of instrumentation or complicated chemical techniques. Determining the reducing sugar concentration in a solution has in the past been carried out using hazardous conditions or with expensive electrodes; either way, the reaction of reducing sugars with copper is a nonstoichiometric process, leading to a challenging analysis with respect to the copper used. Moresco and Sansón described a potentiometric determination of the concentration of reducing sugars in solution using an inexpensive copper electrode at room temperature. Students form a standard curve of known solutions of glucose by measuring the voltage versus the mass of glucose in the sample to determine the reducing power of glucose. This method can be repeated with other reducing sugars, or it can be used to find the amount of reducing sugars in products such as honey or sugary drinks.82 Despite structural similarities that make carbohydrates a challenge to separate on large scale, these biomolecules are often easily separable via HPLC. However, given carbohydrates’ lack of chromophores, they are often hard to detect via normal means, and through oxidative or reductive processes, they tend to poison the corresponding electrodes. Jensen published a method of detecting carbohydrates using pulsed amperometric detection (PAD), which removes most electrode

poisoning possibilities. In this experiment, students generated a standard curve of sugar concentrations and, using HPLC traces, could determine the amounts of glucose, fructose, and sucrose in commercial fruit juices.83 Farris et al. studied the charge density of large biomolecular polymers using conductometric titration. This set of experiments is designed for upper-level students and can be further modified for earlier audiences. Using pectin and chitosan, which are anionic and cationic polysaccharides respectively, they looked at the overall charge density change upon titration (Figure 12). This method successfully observed the charge density of biomolecules by measuring the ionic conductivity (χ) and relative pH during the addition of a strong acid or base.84 As expected for the anionic polysaccharide pectin, there were three zones corresponding to the changes in ionic conductivity and pH as the volume of sodium hydroxide was increased. Wei et al. tried to provide a research-like experience for undergraduates in an upper-level analytical chemistry course using isothermal titration calorimetry (ITC). Experiments using ITC allow upper-level students to better understand the core concepts of enzymatic inhibition in biochemical reactions; however, in most undergraduate biochemistry laboratories, binding between ligands and receptors is intentionally mediocre. By not having particularly strong or weak interactions, binding can be directly monitored via titration. In this case, students are asked to work with a competitive system where a strong binding ligand is titrated into an existing mixture of the receptor and a weak-binding ligand. Students can then study the decrease in the binding affinity as the stronger ligand has to compete with the weak ligand. Furthermore, students can then visualize the enzyme shape using PyMol. Visualization of the shape/reactivity relationship through 3D representations helps students visualize things on a molecular scale. Students were divided into groups of three to four students who worked with one titration of lysozyme and an N-acetylglucosamine oligosaccharide, one of which is the strong-binding NAG3, on the ITC. Students then imported the structures into PyMol and were responsible for answering several exercise questions based on their visualization data. These questions included asking the number of α helices and β sheets, and had the students perform a literature search to determine the catalytic amino acids of the enzyme, among other questions that they could use commands in PyMol to 8001


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(2) Anderson, L.; Wittkopp, S. M.; Painter, C. J.; Liegel, J. J.; Schreiner, R.; Bell, J. A.; Shakhashiri, B. Z. What Is Happening When the Blue Bottle Bleaches: an Investigation of the Methylene BlueCatalyzed Air Oxidation of Glucose. J. Chem. Educ. 2012, 89, 1425− 1431. (3) Kerber, R. C. The A1c Blood Test: an Illustration of Principles From General and Organic Chemistry. J. Chem. Educ. 2007, 84, 1541−1545. (4) Miles, D. T.; Bachman, J. K. Science of Food and Cooking: a Non-Science Majors Course. J. Chem. Educ. 2009, 86, 311−315. (5) Bell, P. Design of a Food Chemistry-Themed Course for Nonscience Majors. J. Chem. Educ. 2014, 91, 1631−1636. (6) Pogozelski, W.; Arpaia, N.; Priore, S. The Metabolic Effects of Low-Carbohydrate Diets and Incorporation Into a Biochemistry Course. Biochem. Mol. Biol. Educ. 2005, 33, 91−100. (7) Sherman, M. B.; Evans, T. A. PopcornWhat’s in the Bag? J. Chem. Educ. 2006, 83, 416A. (8) Luck, L. A.; Blondo, R. M. The Grapes of Class: Teaching Chemistry Concepts at a Winery. J. Chem. Educ. 2012, 89, 1264− 1266. (9) McDermott, M. L. Lowering Barriers to Undergraduate Research Through Collaboration with Local Craft Breweries. J. Chem. Educ. 2016, 93, 1543−1548. (10) Pelter, M. W.; McQuade, J. Brewing Science in the Chemistry Laboratory: a “Mashing” Investigation of Starch and Carbohydrates. J. Chem. Educ. 2005, 82, 1811−1812. (11) Korolija, J. N.; Plavsic, J. V.; Marinkovic, D.; Mandic, L. M. Beer as a Teaching Aid in the Classroom and Laboratory. J. Chem. Educ. 2012, 89, 605−609. (12) Gillespie, B.; Deutschman, W. A. Brewing Beer in the Laboratory: Grain Amylases and Yeast’s Sweet Tooth. J. Chem. Educ. 2010, 87, 1244−1247. (13) McReynolds, K. D. Glycobiology, How to Sugar-Coat an Undergraduate Advanced Biochemistry Laboratory. Biochem. Mol. Biol. Educ. 2006, 34, 369−377. (14) Smith, A. L.; Purcell, R. J.; Vaughan, J. M. Guided Inquiry Activities for Learning About the Macro- and Micronutrients in Introductory Nutrition Courses. Biochem. Mol. Biol. Educ. 2015, 43, 449−459. (15) Harrison, M. A.; Dunbar, D.; Lopatto, D. Using Pamphlets to Teach Biochemistry: a Service-Learning Project. J. Chem. Educ. 2013, 90, 210−214. (16) Bucholtz, K. M. Spicing Things Up by Adding Color and Relieving Pain: the Use of Napoleon’s Buttons in Organic Chemistry. J. Chem. Educ. 2011, 88, 158−161. (17) Zheng, S. Mnemonics for the Aldohexoses That Aid in Learning Structures, Names, and Interconversion of Fischer Projection Formulas and Pyranose Chair Forms. J. Chem. Educ. 2015, 92, 395−398. (18) Dias, D. A. A. Simple Algorithm to Convert Complex Organic Molecules Into Their Straight-Chain Conformations. J. Chem. Educ. 2009, 86, 194. (19) Costa, M. J. Carbohydeck: a Card Game to Teach the Stereochemistry of Carbohydrates. J. Chem. Educ. 2007, 84, 977−978. (20) Günersel, A. B.; Fleming, S. A. Qualitative Assessment of a 3D Simulation Program: Faculty, Students, and Bio-Organic Reaction Animations. J. Chem. Educ. 2013, 90, 988−994. (21) Murdock, M.; Holman, R. W.; Slade, T.; Clark, S. L. D.; Rodnick, K. J. An Introductory Organic Chemistry Review Homework Exercise: Deriving Potential Mechanisms for Glucose Ring Opening in Mutarotation. J. Chem. Educ. 2014, 91, 2146−2147. (22) Farmer, S. C.; Schuman, M. K. A Simple Card Game to Teach Synthesis in Organic Chemistry Courses. J. Chem. Educ. 2016, 93, 695−698. (23) Sims, P. A. Big-Picture” Worksheets to Help Students Learn and Understand the Pentose Phosphate Pathway and the Calvin Cycle. J. Chem. Educ. 2014, 91, 541−545. (24) Nassiff, P.; Czerwinski, W. A. Using Paperclips to Explain Empirical Formulas to Students. J. Chem. Educ. 2014, 91, 1934−1938.

obtain answers for. Through this laboratory experiment, the students could better understand the concepts of molecular recognition by using the modeling program PyMol to visualize the changing active site of lysozyme and pool class data to better understand the reaction parameters of a competitively binding enzyme.85

6. CONCLUSIONS AND LOOKING FORWARD From a perusal of the chemistry education literature, carbohydrates comprise an increasingly important part of an undergraduate’s education. In the past several years, numerous methods have been developed that help students learn concepts related to carbohydrates through both laboratory and lecture materials. There are now experiments using many major types of instrumentation, from NMR to ITC, that are applicable for many levels of students depending on the content desired for that group. Going forward, however, it is imperative that the field of carbohydrates continues to develop educational and pedagogical methods for all levels of students, with a focus on educating earlier college students. When faculty and students are less cautious around carbohydrates, there will be an increased understanding of carbohydrates by the time the students enter undergraduate and graduate programs, which will later translate to greater numbers of research scientists studying (or, at minimum, understanding) carbohydrates. To continue moving the field along as it is or to grow the field, more methods of teaching carbohydrates to younger undergraduates through lecture and laboratory materials are necessary. Laboratory experiments would preferably be within one of two general areas: first, utilize readily available reagents; second, determine inexpensive and facile ways of obtaining, interpreting, and displaying data. AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Alyssa L. Pirinelli: 0000-0002-1230-3581 Notes

The authors declare no competing financial interest. Biographies Jennifer Koviach-Côté obtained her Ph.D. from the University of Minnesota in 1999, where she worked on natural product synthesis with Craig Forsyth. She was then a postdoctoral researcher at the University of Colorado, Boulder, where she developed methods for carbohydrate synthesis with Randall Halcomb. She is currently an associate professor in the Department of Chemistry and Biochemistry at Bates College, where she studies natural product and carbohydrate synthesis. Alyssa L. Pirinelli is an assistant professor of chemistry at the University of Minnesota, Morris (UMM). She received her B.S. in chemistry at St. Lawrence University in 2010 and her Ph.D. in organic chemistry in 2017 from the research group of Nicola L. B. Pohl at Indiana University, Bloomington. Her present research focus at UMM includes the synthesis and study of aromatic glycosides and resins.

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Incorporating Carbohydrates into Laboratory Curricula - Chemical

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