Incorporating Carbohydrates into Laboratory Curriculadepa.fquim.unam.mx/amyd/archivero/ARTICULO-CARBOHIDRATOS_3… · Incorporating Carbohydrates into Laboratory Curricula Jennifer

Incorporating Carbohydrates into Laboratory CurriculaJennifer Koviach-Cote† 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

ABSTRACT: As an important but underappreciated field, carbohydrate chemistry is acritical topic for undergraduate students to learn. With applications to nutrition, foodscience, and diabetes, carbohydrates provide real-world relevance to students. Thisreview summarizes the literature of undergraduate laboratory experiments which usecarbohydrates since the year 2000. Experiments explore important chemical conceptssuch as synthesis, kinetics, analysis, and computational chemistry. Experiments designedfor general chemistry, organic chemistry, biochemistry, and analytical chemistry at avariety of skill levels are presented.

CONTENTS

1. Introduction 79862. General Chemistry 7987

2.1. Analysis of Sugar Content in Food 79872.2. Kinetics of Mutarotation 79872.3. Molecular Modeling and Computational

Chemistry 79883. Organic Chemistry 7990

3.1. Selective Transformations 79903.2. Esterification 79903.3. Glycosidation 79913.4. Synthesis 7992

4. Biochemistry 79964.1. Glucose Oxidase 79964.2. Blood Glucometer Experiments 79974.3. Glycosidase Studies and Michaelis−Menten

Kinetics Measurement 79985. Analytical Chemistry 80016. Conclusions and Looking Forward 8002Author Information 8002

Corresponding Author 8002ORCID 8002Notes 8002Biographies 8002

References 8002

1. INTRODUCTIONAlthough carbohydrate chemistry has been a subject of studysince Fischer’s work in the 19th century, carbohydrates havereceived much less attention than other biomolecules such asproteins and nucleic acids. However, recent advances inglycobiology and developments in synthesis have allowed formuch expanded study of carbohydrates by scientists in fieldsincluding chemistry, biochemistry, immunology, and molecularbiology. As scientists begin to realize the importance ofcarbohydrates in biological systems, it becomes increasinglyimportant to expose students to this long-established but stillemerging field. A challenge with incorporating carbohydrates

into various levels of education has perhaps been under-standing even the basics of the complexity within carbohydratestructure and function. Possible adopters may feel that thereare numerous complex background concepts that must beunderstood before any work with carbohydrates can beaccomplished. However, it is increasingly important thatcarbohydrates are brought into undergraduate and earliereducation to bring more exposure and understanding to thefield.In this review, we summarize the literature describing

carbohydrates in the undergraduate curriculum, specifically inthe undergraduate laboratory. As far as we can tell, this has notbeen the subject of a review to date, so we have limited thispaper to experiments published since 2000, based upon adesire to incorporate modern experiments that include themost advanced instrumentation. Not only are carbohydrates animportant class of compounds to understand; they also provideexcellent teaching opportunities in the gamut of undergraduate(or high school) chemistry courses. Many students are alreadyfamiliar with carbohydrates in the context of nutrition, foodscience, and diabetes, which provides relevance to the realworld. Carbohydrates are nontoxic, generally inexpensive, lowwaste, and often easy to purify, and with many years of study,experiments involving carbohydrates are reliable. Manycarbohydrate experiments may be performed in a 3 h timeblock required of the teaching laboratory, but are also stablefor extended periods that can span multiweek experiments. Inaddition, analysis of carbohydrates, glucose in particular, maybe easily performed using inexpensive commercially availableblood glucose monitors. Since carbohydrates are importantbiomolecules across the curriculum, experiments have beendeveloped for general chemistry, organic chemistry, biochem-istry, and analytical chemistry at a variety of skill levels. Inaddition, the versatility of carbohydrates allows investigations

Special Issue: Carbohydrate Chemistry

Received: December 20, 2017Published: August 16, 2018

Review

pubs.acs.org/CRCite This: Chem. Rev. 2018, 118, 7986−8004

© 2018 American Chemical Society 7986 DOI: 10.1021/acs.chemrev.7b00757Chem. Rev. 2018, 118, 7986−8004

Dow

nloa

ded

via

UN

IV N

AC

ION

AL

AU

TO

NO

MA

ME

XIC

O o

n O

ctob

er 2

4, 2

018

at 2

1:06

:23

(UT

C).

Se

e ht

tps:

//pub

s.ac

s.or

g/sh

arin

ggui

delin

es f

or o

ptio

ns o

n ho

w to

legi

timat

ely

shar

e pu

blis

hed

artic

les.

pubs.acs.org/CR

http://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.chemrev.7b00757

http://dx.doi.org/10.1021/acs.chemrev.7b00757

of selective chemical transformations, kinetics, computationalchemistry, and quantitative analysis, among many others.While this review covers specifically the literature of

carbohydrate-based laboratory experiments, carbohydrates arealso used as teaching tools in nonlaboratory settings at thesecondary-school and college levels. The blue bottledemonstration is a classic example, updated recently byCampbell. In this demonstration, a blue dye is reduced to acolorless species, as various sugars are air-oxidized.1,2 Manyother chemical and biochemical concepts involving carbohy-drates have been described in the context of blood glucosetesting,3 food science and nutrition,4−7 and wine and beerproduction.8−12 Other educators describe pedagogical meth-ods to help students better learn concepts related tocarbohydrate chemistry. Pedagogical techniques includeresearch-based courses,13 active learning,14 writing informa-tional pamphlets for nonscientists,15 using the book Napoleon’sButtons,16 mnemonics for learning Fischer projections,17 analgorithm to convert aldoses into straight-chain conforma-tions,18 card games,19 computer-based three-dimensional (3D)simulations,20 and specific homework or educational exercisesfor students.21−31 Risley has also addressed the issue ofincorrect structures of the ABO blood group in numerousorganic and biochemistry texts.32 In addition, Milenkovic hasdeveloped an assessment tool to determine misconceptions ofcarbohydrates among upper level undergraduates.33

2. GENERAL CHEMISTRYCarbohydrates make ideal substrates for general chemistryexperiments. They are inexpensive and nontoxic, and they havea variety of interesting physical and chemical propertiessuitable for study at the first-year undergraduate level. Inaddition, there are many commercially available methods forthe analysis of carbohydrates in blood and food samples, whichcan be used for chemical analysis in the laboratory. Finally,carbohydrates are critical for the understanding of food scienceand have important biological functions, which providestudents context for the real-world application of chemicalexperiments.2.1. Analysis of Sugar Content in Food

Experiments which involve food and nutrition providerelevance to the real world and increase student interest. Inaddition, these topics provide the opportunity to discuss

differences in structure and physical nature of differentcarbohydrate forms, namely starch and smaller free sugars, aswell as their roles in nutrition. Quantitative analysis experi-ments also provide students with a variety of laboratorytechniques.Deal et al. have developed an introductory level experiment

designed for nutrition students, also appropriate for first-yearor organic students, in which students performed quantitativecarbohydrate 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 percentageof 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 componentsof the banana using glass homogenizers and refrigeratedcentrifuges.34 A later modification showed that homogeniza-tion could be performed as effectively using a mortar andpestle, and tabletop microcentrifuges.35 Pedagogically, thisexperiment ties together students’ familiarity with food scienceand chemical analysis.Since it has been shown previously that the insoluble portion

of banana is predominantly starch, students could determinestarch content directly by weighing the centrifuged and driedpellet. A 3,5-dinitrosalicyclic acid−potassium sodium tartrate(DNS) assay was then used as a quantitative test for reducingsugars in the soluble portion. Reaction of DNS with reducingsugars results in reddish-brown product which can bemeasured spectrophotometrically and compared to a standardcurve. In a subsequent paper, the authors show that thecolorimetric DNS assay could be replaced with commerciallyavailable glucose test strips with no loss in reproducibility.Finally, students investigated several methods of storage,including refrigeration, commercial banana hangers, a brownpaper bag, and the lab drawer. After storing for 1 week,students repeated the quantitative analysis. As expected,refrigeration slows ripening, while the other three storagemethods have no effect.

2.2. Kinetics of Mutarotation

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

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

Chemical Reviews Review

DOI: 10.1021/acs.chemrev.7b00757Chem. Rev. 2018, 118, 7986−8004

7987


relatively slow, it pedagogically represents a simple method forundergraduates to perform kinetics experiments.

A convenient method to study mutarotation is to useglucose biosensors which employ enzymes that selectivelyoxidize the β-anomer to gluconolactone (Scheme 2). Bloodglucose meters and glucose test strips typically use glucosedehydrogenase (GDH) combined with a coenzyme such aspyrroloquinoline quinone (PQQ) to provide easy colorimetricanalysis 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, thesemethods are not easily amenable to the undergraduatelaboratory. Instead, Volpe and Perles have found a methodin which the blood glucose concentration recorded byinexpensive commercial blood glucose meters may becorrelated to optical rotation values.36 This way, undergraduategeneral chemistry students can study the kinetics ofmutarotation without the need for a polarimeter. As describedabove, blood glucose meters use the GDH−PQQ enzymesystem to determine the concentration of β-D-glucopyranose,through its selective enzymatic conversion to D-glucolactone.The authors predetermined that the blood glucose concen-tration provided by the blood glucose meter (Cbgm) is linearlyrelated to the optical rotation, α, of the sample through eq 1. Inaddition, the concentrations of α- and β-anomers may becalculated through eqs 2 and 3.

α = ° − ° C0.767 (0.0054 L/g)( )bgm (1)

α= − − °°

×βC0.8064

9.371000 g/L

ikjjj

y{zzz

(2)

= −α βC Ctotal concentration (3)

To obtain kinetics data, aqueous D-glucose solutions wereprepared and placed into a 20 °C water bath. Aliquots werethen removed every 20 min for 3−4 h and glucoseconcentration was determined using the blood glucosemeter. Commercially available D-glucose contains approx-imately 90% of the α-anomer, but after 3−4 h, it was 38% α-anomer and 62% β-anomer, in agreement with previousmeasurements. Although students did not measure kineticsdirectly, this method was fast and reliable, and consistent withprevious results. Typical results are shown in Figure 2.Teruel and Jenkins have also developed an experiment for

undergraduate general chemistry students in which students

measure the kinetics of D-glucose mutarotation.37 In thisexperiment, students first prepared glucose electrodes whichwere coated with GOx and polymerized with a poly-o-phenylenediamine film. They then selectively crystallized β-D-glucopyranose from a mixture of anomers, taking advantage ofthe difference in solubility of the two anomers at highertemperatures. The purified sugar was dissolved in an EDTAbuffer, and the concentration of the α-anomer was determinedby measurement of the current used by the glucose biosensor.Since the biosensor allows for real-time measurements, a plotof current vs time provides kinetics information. Typicalstudent data is presented in Figure 3.2.3. Molecular Modeling and Computational Chemistry

Molecular modeling has become a staple of the undergraduateteaching lab, especially as computing power has become fasterand more accessible. Pedagogically, computational chemistryprovides students with the ability to visualize molecules andtheir interactions with each other in ways that are difficult intwo dimensions. In addition, undergraduates can quickly andeasily generate large amounts of data, which allows them tolearn methods of data analysis and gives them the opportunityto recognize trends within similar systems.Tribe and co-workers have developed a computational

chemistry laboratory for first-year students taking chemistry asfood science, human nutrition, and pharmacy majors as well asfirst-year general chemistry students.38 The pedagogical goal of

Scheme 1. Mutarotation of D-Glucopyranose

Scheme 2. Glucopyranose Oxidation Reactions in Commercially Available Glucose Biosensors

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-D-glucose and β-D-glucose calculated by eqs 2 and 3. Reprinted from ref36. Copyright 2008 American Chemical Society.



7988


the experiment was for students to use computationalmodeling to calculate the energy released upon combustionof food items. The calculated energy was then compared to thenutrition information on each food. As a model for theingredients listed in the food’s composition, carbohydrateswere represented completely by glucose, fats by stearin, andamino acids by a single amino acid or dipeptide. Degradationproducts of glucose and stearin were represented as CO2 andH2O, while protein degradation products were assumed to beurea, CO2, and H2O (Figure 4).To perform the calculations, students built the biomolecules

in a modeling software program such as Hyperchem.6 orSpartan and optimized the geometry using molecular

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

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 isnormalized 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 AmericanChemical Society.

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



7989


values and reference data range only from 3 to 22%. Theauthors note that the computational experiment can becombined with more traditional thermochemistry experimentsto compare computational and experimental results.38

Hayes took a different approach to the use of molecularmodeling in an experiment designed specifically for first-yearundergraduate students. In this experiment, students visualizedthe molecular basis of target−drug interactions, to betterunderstand medicinal chemistry (Figure 5). After an initialexploration of the Protein Data Bank (PDB) database andvisualization of DNA, students performed a structuralinvestigation 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 theanalysis, students visualized different levels of protein structure,determined the structural components of the GlcClU ligand,isolated the PLP cofactor, analyzed the enzyme−ligandinteractions, and proposed structural modifications to theinhibitor to improve activity and its pharmacokinetic profile.39

This activity is designed to give first-year students experiencein modeling as well as understanding the “real-world”implications of the topics discussed in their first-year classes.

3. ORGANIC CHEMISTRY

The organic chemistry of carbohydrates has been well-studiedsince the 19th century. As such the chemistry is wellunderstood and relatively easy to perform. In addition,carbohydrates provide excellent examples of selective reac-tivity: acetals vs ethers, primary vs secondary alcohols,thermodynamic vs kinetic products, etc. With a variety offunctional groups in one sugar, they are highly suitable formultistep synthesis, and many of the products can be purifiedthrough crystallization, a benefit to the teaching laboratory.Finally, most sugars provide well-resolved NMR spectra,suitable for one- and two-dimensional (1D and 2D) character-ization. An analysis of the 1H spectra may provide examples ofdiastereotopic relationships, and provide ideal methods ofcorrelating dihedral angles to coupling constants.

3.1. Selective Transformations

Sugars are particularly well-suited for the demonstration ofselective transformations, since they contain similar functionalgroups which differ in their steric environments. In thefollowing experiments, students perform selective reactions,then must use spectroscopic methods to determine theproduct, and finally justify selectivity on the basis of structureand reactivity.Norris et al. have developed a two-step synthesis of a stable

xylose based azide suitable for an upper level organic synthesislaboratory (Scheme 3).40 In this experiment, students firstselectively converted monoacetone xylose 1 into primarytosylate 2. Following recrystallization, the tosylate underwentSN2 displacement with NaN3 to form 3. Students probed thesteric discrimination of a primary hydroxyl group over asecondary hydroxyl group, and confirmed the productstructure using IR spectroscopy through the azide stretch. Inaddition, both 1D and 2D NMR spectroscopies were used todetermine coupling constants.Demchenko et al. introduced sophomore-level organic

students to a common protecting group in carbohydratechemistry by selectively converting α-D-methylglucopyranoside(4) into its 4,6-O-benzylidene derivative 5, a commonintermediate in the synthesis of many carbohydrate buildingblocks (Scheme 4).41 As an extension of this experiment,students in an advanced organic laboratory confirmed theregioselectivity of benzylidene formation by further formingthe diacetate 6 followed by comparison of the chemical shiftsfor H2 and H3 in 5 compared to 6. This experiment providedthe opportunity to discuss regioselective preferences forformation of a six-membered acetal ring instead of a five-membered ring, the formation of a new chiral center on thebenzylidene, and the preference for the phenyl group to adoptan equatorial position.3.2. Esterification

The esterification of sugars is straightforward, and typicallyprovides crystalline products with well-resolved 1H NMRspectra. Pedagogically, acetylation of reducing sugars allows fora discussion of kinetic vs thermodynamic products in the formof axial and equatorial anomeric acetates and furanose vs

Scheme 3. Selective Tosylation and Azide Formation

Scheme 4. Selective Benzylidenation and Acetylation



7990


pyranose ring forms. The anomeric effect may also beintroduced, in order to explain increased stability of axialsubstituents at the anomeric center. Acetylation experimentsalso allow for analysis of more complex 1D NMR spectra thanstudents have typically seen to date and the introduction of 2DNMR. The protons of peracetylated sugars are shifteddownfield and across a larger chemical shift range comparedto the unprotected sugars, which allows for simplified peakassignment. In addition, students may be introduced tocomplex NMR splitting patterns and coupling constantanalysis, through which they may determine dihedral anglesand 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 beisolated and recrystallized within a single lab period. The ratioof anomers was determined through examination of thecoupling constant of the anomeric hydrogen.42

Sorensen also described a peracetylation experiment inwhich third-year advanced organic students were providedwith either α-methyl glucopyranoside (4) or α-methylgalactopyranoside (9) (Scheme 6).43 Peracetylation with

pyridine and acetic anhydride provided the tetraacetatederivative 10 or 11 respectively, which was then separatedchromatographically from unreacted starting material. Analysisof the 1D 1H and 2D-COSY NMR spectra allowed students toassign all peaks and determine the coupling constants.

Through this analysis, students determined the identity oftheir 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, changingonly the temperature of the reaction. The reaction at roomtemperature selectively provided the kinetic product, α-anomer, 8, while heating at reflux selectively provided the β-anomer, 12, as the thermodynamic product. Through thisexperiment, students observed how differences in temperatureaffected the product outcome, which they then explainedthrough an analysis of relative reaction rates and productstabilities. Both products were purified by crystallization, andcharacterized according to their coupling constants in 1HNMR.Rhoad described a similar peracetylation experiment, but in

this case, students were provided with either D-glucose (7) orD-galactose (13), for peracetylation with sodium acetate andacetic anhydride to afford pentaacetate 12 or 14, respectively(Scheme 8).45 Following purification, students obtained 1D 1Hand 13C NMR spectra as well as 2D-COSY and inverseHETCOR spectra. Through interpretation of these spectra,students assigned each of the proton peaks. Analysis of thecoupling constants then allowed them to determine whichsugar they began with, as well as the anomeric configurationsof the product. In addition, the β-selectivity of this reactionallowed for a discussion of C-2 protecting group participation.

3.3. Glycosidation

Glycosidation is one of the oldest and most importantreactions for carbohydrate chemistry. Simple traditionalmethods of Fischer glycosidation are easily accessible tostudents, while methods using more sophisticated glycosyldonors provide students with experience in modern organicsynthesis. Pedagogically, the product ratio of anomers providesa discussion of mechanism, neighboring group participation,and thermodynamic control.In an experiment developed by Hovinen et al., under-

graduate organic chemistry students perform a Fischerglycosidation of D-fructose (15) with methanol46 (Scheme9.) The choice of D-fructose was important for several

Scheme 5. Acetylation of Glucose with Iodine

Scheme 6. Acetylation of Methyl-α-D-Glucose and Galactose

Scheme 7. Kinetic and Thermodynamic Peracetylation of D-Glucose



7991


pedagogical reasons. First, many students are familiar withfructose from everyday life. Second, in theory, four productscould be formed. However, the two furanosides (16 and 17)are the kinetic products, while the pyranosides (18 and 19) arethermodynamic products. This facilitated a discussion ofactivation energies and product stability, and students couldselectively form the furanosides by careful monitoring with 1Dor 2D thin layer chromatography (TLC). Two-dimensionalTLC was particularly useful in this experiment, as the firsteluent separated the furanosides from the pyranosides, whilethe second eluent separated the α- and β-anomers. Since theproduct was formed as a 1:1 mixture of anomers, students usedion-exchange chromatography to separate the products, whichwere then characterized by optical rotation.Bendinskas et al. also described a Fischer glycosidation

reaction with methanol, this time with ribose (20) as thesubstrate (Scheme 10), for upper division undergraduatestudents.47 As above, the methyl furanoside products (21 and22) are the kinetic products, while the pyranosides (23 and24) are the thermodynamic products. In this experiment,students performed the glycosidation for either 30 or 60 min atroom temperature or 65 °C. Students then determined theratio of the four products by integration of the 1H NMRspectrum with D2O as solvent, at 30 °C. Under theseconditions, all four anomeric hydrogens were clearlydistinguished from each other and the solvent. The class

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

the differences in composition at each temperature, studentsevaluated the mechanism for formation of each glycoside, anddiscussed activation energies for each reaction intermediate.3.4. Synthesis

Multistep synthesis provides a variety of pedagogicaladvantages. It provides students with a variety of synthesistechniques and characterization methods, and it emphasizesthe need for planning and experiment design.Callam and Lowary have developed a two-step synthesis of

compound 27 for honors organic chemistry lab students, asshown in Scheme 11.48 The relevance of this experiment isreinforced, since polymers of D-arabinofuranose are prevalentin the cell walls of mycobacteria. In the first week, students

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

Table 1. Representative Data for the Relative Compositionof Methyl Glycosides of D-Ribose in Refluxing Methanol

reaction products (%)

time(min)

methylα-furanoside

methylβ-furanoside

methylα-pyranoside

methylβ-pyranoside

30 21.4 51.5 4.82 12.660 20.0 47.7 6.90 18.1



7992


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 Datawith Molecular Modeling Data



7993


performed a Fischer glycosidation to form the methyl glycosidefrom arabinose using acetyl chloride and methanol. The resultis an approximate 1:1 mixture of α- and β-methylfuranosides,26. The results of this reaction allowed for a discussion ofmutarotation and the pyranose and furanose forms of pentosesugars. In addition, kinetic and thermodynamic control werediscussed, since the furanose form of arabinose is thekinetically formed product. In the second week of theexperiment, students treated the methyl glycoside withpyridine and benzoyl chloride to form the tribenzoate 27. Atthis point, the anomers could be separated throughrecrystallization, since the α-anomer is a solid, while the β-anomer is an oil. Following isolation of the product, studentsused 1D and 2DNMR spectroscopies to assign 1H and 13Cpeaks.Jackson et al. used carbohydrate chemistry to demonstrate

stereochemical inversion in an SN2 reaction.49 In thisexperiment, advanced undergraduate or beginning master’sstudents converted commercially available bromide 28 into thecorresponding azide 29 (Scheme 12). Students obtained a 1HNMR spectrum of both the starting bromide and the productazide. Since the spectra of both compounds were particularlywell resolved, students could determine the stereochemistry ofthe anomeric position through coupling constant analysis.Using this method, students demonstrated inversion ofconfiguration at the anomeric center, which indicated thatthe reaction proceeds through an SN2 rather than an SN1mechanism.Norris and Fluxe have developed a two-step synthesis for

their advanced organic chemistry laboratory sequence.50 In thissequence, students first prepared the tosylate 31 fromdiacetone-D-glucose 30 using standard reaction conditions(Scheme 13). Compound 31 was then purified by recrystal-lization and treated with KOt-Bu, a bulky base. An E2elimination resulted in formation of alkene 32, which waspurified by flash column chromatography. This experimenthighlights several pedagogical concepts: the use of tosylates asleaving groups, the use of a bulky base to favor E2 over SN2products, and the need for an antiperiplanar hydrogen forelimination to occur. In this case, there are two β-protons, butH4 is antiperiplanar, while H2 is synperiplanar. Therefore, onlythe C3−C4 alkene was formed, in lieu of the C2−C3 alkene.Sales and Silveira developed an experiment for an upper-

division organic chemistry laboratory course in which studentstreated D-ribonolactone 33 with either acetone or benzalde-hyde and 12 M HCl to form an acetal (Scheme 14).51 Due tothe differences in reaction conditions, the acetonide wasformed as kinetic lactone 34, while the benzylidene was formedas the thermodynamic six-membered ring lactone 35, alsoknown as the Zinner lactone. In an extension of theexperiment, students also acylated the remaining alcoholgroup with an aromatic acyl chloride. While five- and six-membered lactones can typically be distinguished from oneanother using 13C NMR, the spectra of compounds 36 and 37is misleading, and cannot be used to definitively determine ringsize. Instead, students used a 2D-NOESY NMR spectrum tocharacterize their products. In compounds 34 and 36, H3 andH4 are trans, and do not exhibit a cross-peak. In contrast, H3and H4 are cis in the six-membered rings, and do show amedium-intensity cross-peak for compounds 35 and 37.Finally, students used ChmBio3D software to perform MM2energy minimization, and then uploaded their minimizedstructure to JANOCCHIO to compare their experimental

coupling constant and NOE data to theoretical values. Usingthis method, students performed conformational analysis ofeach molecule in solution, determined the dihedral angles, andconfirmed the structure of each lactone.Simeonov and Afonso have used carbohydrate chemistry to

illustrate the ideas of biorefinery batch and flow processchemistry as well as materials recycling to second-yearpharmaceutical science students.52 In this experiment, studentsused either batch or flow process techniques to prepare 5-hydroxymethylfufural (HMF, 38) from fructose (15) (Scheme15). The relevance of this experiment is important, as HMF

can be used as a precursor to building blocks for polymerproduction or biofuels. In the batch experiment, studentsconverted fructose into HMF using tetraethylammoniumbromide (TEAB) and Amberlyst 15. Following completionof the reaction, the TEAB and Amberlyst resin were recovered,and could be reused several times. To perform the flow processexperiment, a standard glass chromatography column wasconnected to a glass reactor, which was placed in a boilingwater bath. A round-bottom flask was connected to the reactorto collect the product. TEAB was mixed with 5% aqueousH2SO4, and combined with fructose. This mixture was addedto the glass column, and the flow was controlled by slightpositive air pressure until the reaction in the glass column wascompletely discharged. Each student assessed the purity oftheir product using HPLC, and determined the E-factor fortheir process. Students then compared the results of the twoprocesses in terms of E-factor and purity.Penverne and Ferrieres have introduced a four-step synthesis

to their fourth-year organic chemistry students (Scheme 16).53

The pedagogical goals for this experiment were to introducethe reactivity of the anomeric center and to discuss theimportance of anchimeric assistance through neighboringgroup participation. The target molecule has been used inbiological studies as an inhibitor and as a fluorogenic substrate,giving students motivation for its synthesis. However, thetarget molecule cannot be prepared using traditional Fischerglycosidation, so a multistep synthesis involving protectinggroup manipulation was required. The synthetic sequence

Scheme 15. Flow Synthesis of Compound 38 and ApparatusUsed (Reprinted from ref 52. Copyright 2013 AmericanChemical Society.)



7994


began with selective removal of the anomeric acetate from 39to afford compound 40, followed by formation of trichlor-oacetimidate 41, which was purified by flash chromatography.Students then characterized 41 by 1H NMR, and determinedthat a mixture of α- and β-anomers was formed. Glycosylationbetween donor 41 and acceptor 42 afforded 43, which waspurified by crystallization. Compound 43 was formed as asingle anomer, and could be characterized using 2D-COSY andHMQC experiments. Students then employed the theory ofanchimeric assistance to explain the stereoselectivity. Finally,the tetraacetate 43 was deacetylated to afford the fullydeprotected glycoside 44, which was purified by crystallization.The entire sequence could also be performed using glucosepentaacetate as the initial substrate, but the intermediateglucoside was not readily purified by crystallization, soadditional 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 sequenceintroduces the ideas of increased anomeric reactivity, kinetic vsthermodynamic control, and stereoselectivity of glycosylationreactions. In the first step, one member of the pair performed a

kinetic acetylation using sodium acetate and acetic anhydrideto form compound 12 while the other used zinc chloride andacetic anhydride to give the thermodynamic product, 8. Bothproducts were purified through recrystallization, and theanomeric acetate was selectively deprotected to give 45. Theproduct could be purified by crystallization or chromatographyif additional experience with chromatographic separation wasdesired. Students then converted alcohol 45 into thetrichloroacetimidate donor 46, which was purified by flashchromatography. Finally, the donor 46 underwent glycosyla-tion with one of three acceptors. As the glycosylation requiredanhydrous conditions, students gained experience withhandling 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 NMRtechniques. As each step involved transformation at theanomeric center, students made use of the Karplus relationshipto identify the anomeric configuration of each product, andused concepts such as kinetic and thermodynamic reactionconditions, mutarotation, the anomeric effect, and anchimericassistance to explain the selectivity of each reaction.

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

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



7995


Pontrello developed a multistep synthetic sequence for thefirst-semester organic laboratory, to prepare a small diverselibrary of carbohydrate-based HIV inhibitor mimics. One ofthe main pedagogical goals of this four-reaction, six-weekexperiment was to expose students to the primary literature. Assuch, students were assigned background literature describingthe molecular mechanisms of HIV infection, with attention tosmall molecule inhibition of binding between the HIV Tatprotein and TAR-RNA. In addition, students searched theProtein Data Bank (PDB) for the crystal structure of HIVTAR-RNA with a bound peptide mimic. Finally, students werenot provided with the procedure for each experiment, butinstead were given references from the primary literature toperform each step of the synthesis as described in Scheme18.55

To provide the library of compounds, every studentperformed reaction 1 using commercially available D-glucal(48) as starting material, and the products were pooled. Asubset of students (20−50%) continued the synthetic sequencefrom compound 49, while the remainder incorporated diversityinto the library by following a parallel set of reactions, butbeginning with pyran 53. Additional diversity was incorporatedinto the library in reaction 3, in which alcohols of varying chainlength were used in a glycosylation reaction. Finally, studentsused one of two Grubbs’s catalysts to carry out the metathesisin reaction 4. All reactions could be monitored by TLC andcharacterized easily by IR or 13C NMR spectroscopy. Throughthis synthetic sequence, students were exposed to a small-scaleversion of the methods used by pharmaceutical companies tosearch for novel bioactive compounds.

4. BIOCHEMISTRY

Methods for teaching undergraduates about carbohydrates ingeneral chemistry and organic chemistry laboratories havealready been discussed. Biochemistry is often a third- or fourth-year undergraduate course, very much building upon principlesdeveloped in earlier chemistry and biology classes. Incorporat-ing more carbohydrates into the earlier collegiate curriculummay decrease the barrier to having carbohydrates included in

later curricula, such as biochemistry, where understanding thebulk of carbohydrate functions resides. Furthermore, bio-chemistry lectures may not be accompanied by a laboratorysection, in part because biochemistry requires a dedicated setof reagents and equipment. Though some current carbohy-drate research relies on expensive instrumentation andtechniques, laboratory experiments have been developed forseveral areas of biochemistry that are fairly accessible to a rangeof institutions with respect to required chemicals andequipment, with appropriate intellectual challenges forstudents.4.1. Glucose Oxidase

As mentioned earlier, a common method for determining theconcentration of glucose in a solution is using the glucoseoxidase (GOx) enzyme. Under standard conditions, GOxconverts β-D-glucose and one molecule of oxygen into oneequivalent each of β-D-gluconolactone and hydrogen peroxide,as shown in Scheme 19a.56 As the gluconolactone structure can

be challenging to observe through regular spectroscopicmethods, the initial hydrogen peroxide product is more oftenused in a subsequent reaction. This second reaction gives areadout that is therefore proportional to the amount of glucosethat was oxidized. So long as it is kinetically faster than theGOx reaction, several measurements can be taken from thissecond reaction: the initial concentration of glucose inunknown samples (proportional to the output of the secondreaction) and kinetics data for these enzyme combinations.Data collection can be done on several types of instruments in

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

Scheme 19. General Scheme Using Glucose OxidaseCoupled with Horseradish Peroxidase To Measure GlucoseConcentrations



7996


the laboratory, including commonplace spectrophotometers,though whether these will work depends on the secondcoupled reaction’s readout being in the UV or visible spectrumor some other sort of electrochemical readout.Gooding et al. coupled the GOx activity with horseradish

peroxidase’s (HRP) ability to oxidize ferrocyanide toferricyanide in an aqueous solution. This oxidation cantherefore provide a colorimetric response in proportion tothe amount of glucose found in solutions, namely in sportsdrinks (Scheme 19b).57 For this experiment, the students weretasked with finding the proper dilutions for the sports drinks.There have been other experiments developed along thesesame lines. For example, Bare and colleagues used GOx/HRPto oxidize a ruthenium-based dye via to produce luminescencemeasurable on a home-built fluorimeter. This inexpensiveinstrument gave the students results nearly identical to thoseobtained on a far costlier research-grade fluorimeter, evidencethat carbohydrate-related experiments are available to depart-ments with limited instrument availability. From this data,students determined kinetics information based on the activityof glucose oxidase.58 Similarly, Vasilarou described anotherGOx/HRP experiment based on the amount of glucose in fruitdrinks and carbonated beverages using a colorimetric reactionon a UV−vis spectrometer.59

The amount of endogenous oxygen available will affect thekinetics of the GOx reaction; therefore, the GOx can be thesecond in a chain of biochemical reactions rather than the first.Choi and Wong studied, via a datalogger, several changes in asolution using an immobilized GOx enzyme on an eggmembrane along with an oxygen electrode. This system givesstudents a view of a coupled biosensor and can be modified towork with any biological reaction that releases or usesoxygen.60 More recently, Hobbs et al. coupled the GOxenzyme with a carbon nanotube electrode that measures thecurrent produced by the breakdown of hydrogen peroxide intooxygen, protons and electrons, the setup for which is shown inFigure 6A. Using data produced from this electrode (Figure6B), students can build a calibration curve and measure theconcentration of glucose in various sports or soft drinks.61

Similarly, Blanco-Lo pez et al. used ferrocene as thecolorimetric reductant and a carbon paste electrode to measurecurrent flow during the GOx reaction.62

4.2. Blood Glucometer Experiments

As mentioned, most personal blood glucose monitors takeadvantage of the GOx/HRP pairing to give a fast, reliable, andrelatively inexpensive blood glucose concentration readout fordiabetic patients. The HRP induces a colorimetric responsewhich is read by the instrument to give the relative bloodglucose concentration. Over the past several years, the costs ofthese monitors and the requisite test strips have significantlydecreased. Given the improvement in glucometer technology,readings of blood glucose can now happen nearly instanta-neously, and in addition to the convenience and the potentiallylife-saving aspect for diabetes patients, these improvementshave made laboratory experiments using blood glucometerspossible for undergraduate institutions. Lazarim and colleagueswanted students to gain a better physical understanding of theglycemic index of foods and the impact on the glycemic load inthe body. Students ingested sugar via drinking juice or eatingfood, and then every 30 min over the course of 2 h, monitoredthe changes in their blood sugar levels.63 This study hadstudents comprehend the link between what food items andquantity thereof they ingested and what happened with theirblood sugar. Another study in this area involves postunder-graduate medical students studying the blood glucose andtriglyceride content in diabetic and nondiabetic rats.64

Hardee et al. have published a procedure for finding theMichaelis−Menten kinetic parameters for the enzyme-catalyzed mutarotation of glucose using a commercial bloodglucose monitoring system. This procedure can be adapted foruse in several college-level courses depending on the type ofdata desired. The experimenters could show that a bloodglucometer was able to give readings similar to a polarimeter.65

Heinzerling et al. used the same method to observe thehydrolysis of sucrose and lactose by the invertase enzymefound in baker’s yeast (Saccharomyces cerevisiae) by monitoringglucose production. Students were asked to make a standardcurve containing a series of dilutions, from which they coulddiscern the Michaelis constant and later other kineticparameters through conversion to Lineweaver−Burke plots.66

In addition to helping students understand Michaelis−Mentenparameters, this exercise also gave students hands-on under-standing of the impacts of temperature and pH on enzymeactivity.Recently, Amor-Gutierrez et al. used a student-built

printable carbon electrode to fabricate a glucose biosensor in

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



7997


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

traditional glucose-sensing manner (by coupling GOx toHRP). The laboratory required students to understand theconcepts and fabrication of screen-printed carbon electrodesand electrochemistry coupled with the biochemical concept ofenzymatic analysis.67 In building and using these disposableelectrodes, students learned about the concepts of cyclicvoltammetry as well as its applicability in biological reactions.Furthermore, the students were also responsible for optimizingenzyme solutions, with this association leading to greaterunderstanding of biochemical concepts in an analytical-chemistry-type course. Lastly, at the end of the experiment,students discussed accuracy, precision, and the time and costaspects of these reactions.4.3. Glycosidase Studies and Michaelis−Menten KineticsMeasurement

Enzymes are used to break down larger carbohydrate chainsinto smaller ones for the uses of metabolism and cleaning,among others. These enzymes are present to aid in digestion inhumans, as the amylase enzyme in saliva helps to break downstarch prior to food’s arrival in the stomach. Humans do notpossess the enzyme α-galactosidase, however. This enzymepromotes breakdown of other long polysaccharides such asthose found in peas, and as such humans’ inability to digestthese polysaccharides leads to flatulence. Hardee and co-workers tasked students with using the pharmaceutical Beano,a glycosidase, to look at the basics of enzyme kinetics for healthscience majors. The students measured the production ofglucose from a solution of pea polysaccharides using a bloodglucometer.68 Mulimani and Dhananjay introduced students to

the concept of enzyme immobilization using the same enzymeand a different substrate.69

Yet others have used amylase to quantify the rate of activityand the effects of the reaction parameters on the activity ofenzymes, helping teach the concepts of Michaelis−Mentenkinetics. Cochran and co-workers had undergraduate biologystudents study reaction kinetics via a colorimetric analysis withtwo solutions: KI/I2 and Benedict’s solution. Working with acommercial flatbed scanner, the students used the proportion-ally decreasing concentrations of starch and increasing glucoseconcentrations in samples of starch exposed to α-amylase tomake a standard curve. Using this curve, the students couldthen determine the concentrations of glucose in unknownsamples.70 Valls and colleagues used amylase isolated fromhuman saliva and compared it to industrial detergents,71 whileMunegumi used same-sourced α-amylase to look at thesimilarities between saliva and detergent functions.72 Thesearticles each help students understand the impact of thereaction parameters: Munegumi and co-workers specificallyfocused on changing the temperature of the solution whileValls and co-workers changed several conditions, ranging fromthe pH to adding oxidizing agents to the mixture. Whencomparing saliva enzymes to the detergents, Munegumi andcolleagues noted that, among other conclusions, the salivaenzymes denatured at higher temperatures whereas thedetergent-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 mainadvantages to this method is the ability to reuse catalyst andreduce overall chemical consumption and waste.73 So far flowchemistry been slow to find purchase outside of industrialapplications, perhaps because of the types of reactors requiredthat may have limited use in university settings. Taipa andcolleagues have begun to bridge this gap by having advancedlaboratory students work on three types of industrial-like flowsystems: continuous stirred tank reactors (CSTR), plug flowreactors (PFR), and fluidized bed reactors (FBR) (Figure 9).74

The researchers measured the rate constants of animmobilized invertase enzyme in comparison to the freeenzyme. Here, the enzyme is immobilized in an alginatesuspension and placed in one of the flow systems, with theexpectation that the so-bound enzyme will be less efficient thana free version of the same enzyme. Groups of students worked

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

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


with each type of flow reactor and compared their results,requiring them to work together, to understand the ideasbehind flow synthesis and accurately complete their dataanalysis.74 Though flow chemistry is a promising area of futurecarbohydrate research, it is still not as common to find inuniversities as other instrumentation. In the future, flowchemistry experiments may grow in popularity when the partsand mechanics of them are less expensive and can beimplemented in undergraduate education.Carbohydrate conformation and structural details, though

somewhat covered in organic chemistry, are rarely covered indepth before the first semester of biochemistry. NMR is alsoone of the most powerful and versatile techniques available tochemists and biochemists alike using time-course and orquantitative experiments as mentioned earlier. Analyzing the J-

values from coupling constants or integration of peaks canrepresent a large amount of possible data toward structuralconcepts or can report kinetics data over time. NMRinstruments are also a nearly ubiquitous element in theuniversity setting, allowing most institutions to incorporatethese 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 theenzyme’s starting material, its products or both. For example,NMR can monitor the release of viral particles in a modelsystem using a commercial neuraminidase and a glycoprotein.In an influenza infection the virus finds a healthy cell bybinding to neuraminic acids on the cell’s surface. After theinitial infection, the virus will then cleave the neuraminic acidfrom the rest of the glycan structure, thereby allowing newly

Figure 9. (A−C) Types of flow reactors used in the flow-chemistry laboratory (CSTR, PFR and FBR, respectively). (D) Schematic representationof 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 GrowthCorresponding to N-Acetylneuraminic Acid in Solution (Reprinted from ref 75. Copyright 2011 American Chemical Society.)

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



7999


made viral particles to leave and infect other cells (Scheme20A). Barb et al. had students use NMR to monitor the rate ofrelease of neuraminic acid 58 from a commercial glycoprotein,generically shown in Scheme 20A.75 Rather than using the fullvirus, the students prepared samples of a commercialneuraminidase with a glycoprotein (either α1-glycoprotein orfetuin) and watched the neuraminidase progress via time-course NMR experiments over 2 h. Students could observe thegrowth of a peak corresponding to the N-acetate of freeneuraminic acid as well as a shifting signal that corresponds toH3 (Scheme 20B). Though early undergraduates may stop atthis point, students later in their undergraduate studies mayadd statistical analysis of the data to find the half-life of thereaction. Additionally, Periyannan and colleagues introducedstudents to studying β-glucosidase activity in an undergraduatebiochemistry laboratory using the breakdown of cellobiose toglucose by tracking the coupling constants, peak widths, andintegrations of the corresponding anomeric proton peaks.76

Both Kehlbeck and Her utilized NMR spectroscopy to studythe hydrolysis of sucrose by invertase.77,78 These groups usedtime-course experiments to observe the decreasing concen-tration of sucrose and the growing concentrations of glucoseand fructose by measuring initial rates (Kehlbeck et al.) orusing qNMR (Her et al.). Using these data, students

investigated the Michaelis−Menten kinetics of the invertaseenzyme.Krishnan and co-workers described a quantitative NMR

experiment for determining Michaelis−Menten kinetic param-eters using statistical analysis aimed at undergraduate studentswith a physical chemistry or biochemistry focus. This exercisehelps students to better understand Michaelis−Mentenkinetics, statistics, mathematical interpretation, and NMRoptimization by learning to quantify peaks based on relativeareas (Figure 10).78

Guerra has recently published a Michaelis−Menten kineticsstudy on a multienzyme complex that looks at the breakdownof larger carbohydrate polymers into smaller polymers or intomonomeric units. The entire Viscozyme L complex of enzymesis less expensive to purchase than the corresponding singleenzyme components, each of which breaks down differingtypes of carbohydrate polymers. With the complex moremarketable to educators, students look at the breakdown ofseveral different carbohydrate polymers in a mixture.Furthermore, this experiment tasked the students with fittingtheir data to several equations, helping the students come upwith the strengths and weaknesses of each type of plot.79

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.



8000


5. ANALYTICAL CHEMISTRY

Before more complex spectrometry or spectroscopy techniqueswere invented, qualitative analysis used to make up nearly theentirety of the chemical process, with Fischer’s determinationof glucose and the isomers of glucose being among the mostwell-known process among organic chemists. Many of thesetests have fallen by the wayside when they are replaced withmore instrumentation that could provide far more data or asprocedures have gone greener. Qualitative testing is oftenuseful for determining the presence or absence of a desiredanalyte in a solution, and as such, qualitative testing is stillincorporated into chemistry curricula at various levels.80

Recently, Dickman and colleagues described two new testsfor distinguishing a sample of a pure carbohydrate fromanother carbohydrate. Using seven qualitative analyses in aspecific order, students can identify if their given unknown is amonosaccharide or disaccharide and later what the identity ofthese are based on positive or negative results to the chemicaltests (Figure 11).81 This type of qualitative experiment can bedone at the high school or college level and does not requiremuch by way of instrumentation or complicated chemicaltechniques.Determining the reducing sugar concentration in a solution

has in the past been carried out using hazardous conditions orwith expensive electrodes; either way, the reaction of reducingsugars with copper is a nonstoichiometric process, leading to achallenging analysis with respect to the copper used. Morescoand Sanson described a potentiometric determination of theconcentration of reducing sugars in solution using aninexpensive copper electrode at room temperature. Studentsform a standard curve of known solutions of glucose bymeasuring the voltage versus the mass of glucose in the sampleto determine the reducing power of glucose. This method canbe repeated with other reducing sugars, or it can be used tofind the amount of reducing sugars in products such as honeyor sugary drinks.82

Despite structural similarities that make carbohydrates achallenge to separate on large scale, these biomolecules areoften easily separable via HPLC. However, given carbohy-drates’ lack of chromophores, they are often hard to detect vianormal means, and through oxidative or reductive processes,they tend to poison the corresponding electrodes. Jensenpublished a method of detecting carbohydrates using pulsedamperometric detection (PAD), which removes most electrode

poisoning possibilities. In this experiment, students generated astandard curve of sugar concentrations and, using HPLCtraces, could determine the amounts of glucose, fructose, andsucrose in commercial fruit juices.83

Farris et al. studied the charge density of large biomolecularpolymers using conductometric titration. This set of experi-ments is designed for upper-level students and can be furthermodified 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 chargedensity of biomolecules by measuring the ionic conductivity(χ) and relative pH during the addition of a strong acid orbase.84 As expected for the anionic polysaccharide pectin, therewere three zones corresponding to the changes in ionicconductivity and pH as the volume of sodium hydroxide wasincreased.Wei et al. tried to provide a research-like experience for

undergraduates in an upper-level analytical chemistry courseusing isothermal titration calorimetry (ITC). Experimentsusing ITC allow upper-level students to better understand thecore concepts of enzymatic inhibition in biochemical reactions;however, in most undergraduate biochemistry laboratories,binding between ligands and receptors is intentionallymediocre. By not having particularly strong or weakinteractions, binding can be directly monitored via titration.In this case, students are asked to work with a competitivesystem where a strong binding ligand is titrated into an existingmixture of the receptor and a weak-binding ligand. Studentscan then study the decrease in the binding affinity as thestronger ligand has to compete with the weak ligand.Furthermore, students can then visualize the enzyme shapeusing PyMol. Visualization of the shape/reactivity relationshipthrough 3D representations helps students visualize things on amolecular scale. Students were divided into groups of three tofour students who worked with one titration of lysozyme andan N-acetylglucosamine oligosaccharide, one of which is thestrong-binding NAG3, on the ITC. Students then imported thestructures into PyMol and were responsible for answeringseveral exercise questions based on their visualization data.These questions included asking the number of α helices and βsheets, and had the students perform a literature search todetermine the catalytic amino acids of the enzyme, amongother questions that they could use commands in PyMol to

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.



8001


obtain answers for. Through this laboratory experiment, thestudents could better understand the concepts of molecularrecognition by using the modeling program PyMol to visualizethe changing active site of lysozyme and pool class data tobetter understand the reaction parameters of a competitivelybinding enzyme.85

6. CONCLUSIONS AND LOOKING FORWARDFrom a perusal of the chemistry education literature,carbohydrates comprise an increasingly important part of anundergraduate’s education. In the past several years, numerousmethods have been developed that help students learnconcepts related to carbohydrates through both laboratoryand lecture materials. There are now experiments using manymajor types of instrumentation, from NMR to ITC, that areapplicable for many levels of students depending on thecontent desired for that group. Going forward, however, it isimperative that the field of carbohydrates continues to developeducational and pedagogical methods for all levels of students,with a focus on educating earlier college students. Whenfaculty and students are less cautious around carbohydrates,there will be an increased understanding of carbohydrates bythe time the students enter undergraduate and graduateprograms, which will later translate to greater numbers ofresearch scientists studying (or, at minimum, understanding)carbohydrates. To continue moving the field along as it is or togrow the field, more methods of teaching carbohydrates toyounger undergraduates through lecture and laboratorymaterials are necessary. Laboratory experiments wouldpreferably be within one of two general areas: first, utilizereadily available reagents; second, determine inexpensive andfacile ways of obtaining, interpreting, and displaying data.

AUTHOR INFORMATIONCorresponding Author

*E-mail: [email protected]

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

The authors declare no competing financial interest.

Biographies

Jennifer Koviach-Cote obtained her Ph.D. from the University ofMinnesota in 1999, where she worked on natural product synthesiswith Craig Forsyth. She was then a postdoctoral researcher at theUniversity of Colorado, Boulder, where she developed methods forcarbohydrate synthesis with Randall Halcomb. She is currently anassociate professor in the Department of Chemistry and Biochemistryat Bates College, where she studies natural product and carbohydratesynthesis.

Alyssa L. Pirinelli is an assistant professor of chemistry at theUniversity of Minnesota, Morris (UMM). She received her B.S. inchemistry at St. Lawrence University in 2010 and her Ph.D. in organicchemistry in 2017 from the research group of Nicola L. B. Pohl atIndiana University, Bloomington. Her present research focus at UMMincludes the synthesis and study of aromatic glycosides and resins.

REFERENCES(1) Staiger, F. A.; Peterson, J. P.; Campbell, D. J. Variations on the“Blue-Bottle” Demonstration Using Food Items That Contain FD&CBlue #1. J. Chem. Educ. 2015, 92, 1684−1686.

(2) Anderson, L.; Wittkopp, S. M.; Painter, C. J.; Liegel, J. J.;Schreiner, R.; Bell, J. A.; Shakhashiri, B. Z. What Is Happening Whenthe Blue Bottle Bleaches: an Investigation of the Methylene Blue-Catalyzed Air Oxidation of Glucose. J. Chem. Educ. 2012, 89, 1425−1431.(3) Kerber, R. C. The A1c Blood Test: an Illustration of PrinciplesFrom General and Organic Chemistry. J. Chem. Educ. 2007, 84,1541−1545.(4) Miles, D. T.; Bachman, J. K. Science of Food and Cooking: aNon-Science Majors Course. J. Chem. Educ. 2009, 86, 311−315.(5) Bell, P. Design of a Food Chemistry-Themed Course forNonscience Majors. J. Chem. Educ. 2014, 91, 1631−1636.(6) Pogozelski, W.; Arpaia, N.; Priore, S. The Metabolic Effects ofLow-Carbohydrate Diets and Incorporation Into a BiochemistryCourse. Biochem. Mol. Biol. Educ. 2005, 33, 91−100.(7) Sherman, M. B.; Evans, T. A. PopcornWhat’s in the Bag? J.Chem. Educ. 2006, 83, 416A.(8) Luck, L. A.; Blondo, R. M. The Grapes of Class: TeachingChemistry Concepts at a Winery. J. Chem. Educ. 2012, 89, 1264−1266.(9) McDermott, M. L. Lowering Barriers to Undergraduate ResearchThrough Collaboration with Local Craft Breweries. J. Chem. Educ.2016, 93, 1543−1548.(10) Pelter, M. W.; McQuade, J. Brewing Science in the ChemistryLaboratory: 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 theLaboratory: Grain Amylases and Yeast’s Sweet Tooth. J. Chem.Educ. 2010, 87, 1244−1247.(13) McReynolds, K. D. Glycobiology, How to Sugar-Coat anUndergraduate Advanced Biochemistry Laboratory. Biochem. Mol.Biol. Educ. 2006, 34, 369−377.(14) Smith, A. L.; Purcell, R. J.; Vaughan, J. M. Guided InquiryActivities for Learning About the Macro- and Micronutrients inIntroductory Nutrition Courses. Biochem. Mol. Biol. Educ. 2015, 43,449−459.(15) Harrison, M. A.; Dunbar, D.; Lopatto, D. Using Pamphlets toTeach Biochemistry: a Service-Learning Project. J. Chem. Educ. 2013,90, 210−214.(16) Bucholtz, K. M. Spicing Things Up by Adding Color andRelieving 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 inLearning Structures, Names, and Interconversion of FischerProjection Formulas and Pyranose Chair Forms. J. Chem. Educ.2015, 92, 395−398.(18) Dias, D. A. A. Simple Algorithm to Convert Complex OrganicMolecules Into Their Straight-Chain Conformations. J. Chem. Educ.2009, 86, 194.(19) Costa, M. J. Carbohydeck: a Card Game to Teach theStereochemistry of Carbohydrates. J. Chem. Educ. 2007, 84, 977−978.(20) Gunersel, A. B.; Fleming, S. A. Qualitative Assessment of a 3DSimulation Program: Faculty, Students, and Bio-Organic ReactionAnimations. 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 Home-work Exercise: Deriving Potential Mechanisms for Glucose RingOpening in Mutarotation. J. Chem. Educ. 2014, 91, 2146−2147.(22) Farmer, S. C.; Schuman, M. K. A Simple Card Game to TeachSynthesis in Organic Chemistry Courses. J. Chem. Educ. 2016, 93,695−698.(23) Sims, P. A. Big-Picture” Worksheets to Help Students Learnand Understand the Pentose Phosphate Pathway and the CalvinCycle. J. Chem. Educ. 2014, 91, 541−545.(24) Nassiff, P.; Czerwinski, W. A. Using Paperclips to ExplainEmpirical Formulas to Students. J. Chem. Educ. 2014, 91, 1934−1938.



8002

mailto:[email protected]

http://orcid.org/0000-0002-1230-3581


(25) Rostejnska, M.; Klímova, H. Biochemistry Games: AZ-Quizand Jeopardy! J. Chem. Educ. 2011, 88, 432−433.(26) Chen, H.; Ni, J.-H. Teaching Arrangements of CarbohydrateMetabolism in Biochemistry Curriculum in Peking University HealthScience Center. Biochem. Mol. Biol. Educ. 2013, 41, 139−144.(27) Figueira, A. C. M.; Rocha, J. B. T. A Proposal for TeachingUndergraduate Chemistry Students Carbohydrate Biochemistry byProblem-Based Learning Activities. Biochem. Mol. Biol. Educ. 2014, 42,81−87.(28) Kalogiannis, S.; Pagkalos, I.; Koufoudakis, P.; Dashi, I.;Pontikeri, K.; Christodoulou, C. Integrated Interactive Chart as aTool for Teaching Metabolic Pathways. Biochem. Mol. Biol. Educ.2014, 42, 501−506.(29) Meany, J. E. Lactate Dehydrogenase Catalysis: Roles of Keto,Hydrated, and Enol Pyruvate. J. Chem. Educ. 2007, 84, 1520.(30) Park, B.; Holman, R. W.; Slade, T.; Murdock, M.; Rodnick, K.J.; Swislocki, A. L. M. A Biochemistry Question-Guided Derivation ofa Potential Mechanism for HbA1c Formation in Diabetes MellitusLeading to a Data-Driven Clinical Diagnosis. J. Chem. Educ. 2016, 93,795−797.(31) Zhu, L. Teaching Glycoproteins with a Classical Paper:Knowledge and Methods in the Course of an Exciting Discovery.Biochem. Mol. Biol. Educ. 2008, 36, 336−340.(32) Risley, J. M. Structures for the ABO(H) Blood Group: WhichTextbook Is Correct? J. Chem. Educ. 2007, 84, 1546−1547.(33) Milenkovic, D. D.; Hrin, T. N.; Segedinac, M. D.; Horvat, S.Development of a Three-Tier Test as a Valid Diagnostic Tool forIdentification of Misconceptions Related to Carbohydrates. J. Chem.Educ. 2016, 93, 1514−1520.(34) Deal, S. T.; Farmer, C. E.; Cerpovicz, P. F. CarbohydrateAnalysis: Can We Control the Ripening of Bananas? J. Chem. Educ.2002, 79, 479−480.(35) Davis-McGibony, C. M.; Bennett, R. R.; Bossart II, A. D.; Deal,S. T. An Alternative Procedure for Carbohydrate Analysis of Bananas:Cheaper and Easier. J. Chem. Educ. 2006, 83, 1543.(36) Perles, C. E.; Volpe, P. A Simple Laboratory Experiment toDetermine the Kinetics of Mutarotation of D-Glucose Using a BloodGlucose Meter. J. Chem. Educ. 2008, 85, 686−688.(37) Reyes-de-Corcuera, J. I.; Teruel, M. A.; Jenkins, D. M.Crystallization of β-D-Glucose and Analysis with a Simple GlucoseBiosensor. J. Chem. Educ. 2009, 86, 959−961.(38) Barbiric, D.; Tribe, L.; Soriano, R. Computational ChemistryLaboratory: Calculating the Energy Content of Food Applied to aReal-Life Problem. J. Chem. Educ. 2015, 92, 881−885.(39) Hayes, J. M. An Integrated Visualization and Basic MolecularModeling Laboratory for First-Year Undergraduate MedicinalChemistry. J. Chem. Educ. 2014, 91, 919−923.(40) Norris, P.; Freeze, S.; Gabriel, C. J. Synthesis of a PartiallyProtected Azidodeoxy Sugar. A Project Suitable for the AdvancedUndergraduate Organic Chemistry Laboratory. J. Chem. Educ. 2001,78, 75−76.(41) Demchenko, A. V.; Pornsuriyasak, P.; De Meo, C. AcetalProtecting Groups in the Organic Laboratory: Synthesis of Methyl-4,6-O-Benzylidene-α-D-Glucopyranoside. J. Chem. Educ. 2006, 83,782−784.(42) Schatz, P. F. An Improved Preparation of α-D-(+)-Glucopyr-anose Pentaacetate. J. Chem. Educ. 2001, 78, 1378−1378.(43) Sorensen, J. L.; Witherell, R.; Browne, L. M. Use of 1H NMR inAssigning Carbohydrate Configuration in the Organic Laboratory. J.Chem. Educ. 2006, 83, 785−797.(44) Pandita, S.; Goyal, S.; Passey, S. A Convenient MicroscaleSynthesis of α -and β-D-(+)-Glucopyranose Pentaacetate. Chem.Educ. 2007, 12, 402−408.(45) Rhoad, J. S. Determination of Relative Stereochemistry of anUnknown Carbohydrate: Application of Two-Dimensional NMR andthe Karplus Relationship. Chem. Educ. 2011, 16, 304−306.(46) Nurminen, E.; Poijarvi, P.; Koskua, K.; Hovinen, J. Synthesis ofAnomeric Methyl Fructofuranosides and Their Separation on an Ion-Exchange Resin. J. Chem. Educ. 2007, 84, 1480−1483.

(47) Simon, E.; Cook, K.; Pritchard, M. R.; Stripe, W.; Bruch, M.;Bendinskas, K. Glycosidation of Methanol with Ribose: anInterdisciplinary Undergraduate Laboratory Experiment. J. Chem.Educ. 2010, 87, 739−741.(48) Callam, C. S.; Lowary, T. L. Synthesis of Methyl 2,3,5-Tri-O-Benzoyl- α -D-Arabinofuranoside in the Organic Laboratory. J. Chem.Educ. 2001, 78, 73−74.(49) Adesoye, O. G.; Mills, I. N.; Temelkoff, D. P.; Jackson, J. A.;Norris, P. Synthesis of a D-Glucopyranosyl Azide: SpectroscopicEvidence for Stereochemical Inversion in the S N2 Reaction. J. Chem.Educ. 2012, 89, 943−945.(50) Norris, P.; Fluxe, A. Preparation of a D-Glucose-DerivedAlkene. An E2 Reaction for the Undergraduate Organic ChemistryLaboratory. J. Chem. Educ. 2001, 78, 1676−1678.(51) Sales, E. S.; Silveira, G. P. Synthesis and 1H NMRSpectroscopic Elucidation of Five- and Six-Membered D-Ribonolac-tone Derivatives. J. Chem. Educ. 2015, 92, 1932−1937.(52) Simeonov, S. P.; Afonso, C. A. M. Batch and Flow Synthesis of5-Hydroxymethylfurfural (HMF) From Fructose as a BioplatformIntermediate: an Experiment for the Organic or Analytical Laboratory.J. Chem. Educ. 2013, 90, 1373−1375.(53) Penverne, C.; Ferrieres, V. Synthesis of 4-Methylumbellifer-7-Yl-α-D-Mannopyranoside: an Introduction to Modern GlycosylationReactions. J. Chem. Educ. 2002, 79, 1353−1354.(54) Dangerfield, E. M.; Stocker, B. L.; Batchelor, R.; Northcote, P.T.; Harvey, J. E. Stereochemical Control in Carbohydrate Chemistry.J. Chem. Educ. 2008, 85, 689−691.(55) Pontrello, J. K. Bringing Research Into a First Semester OrganicChemistry Laboratory with the Multistep Synthesis of Carbohydrate-Based HIV Inhibitor Mimics. Biochem. Mol. Biol. Educ. 2015, 43,417−427.(56) Johnson, K. A.; Kroa, B. A.; Yourey, T. Factors AffectingReaction Kinetics of Glucose Oxidase. J. Chem. Educ. 2002, 79, 74−76.(57) Gooding, J. J.; Yang, W.; Situmorang, M. J. Chem. Educ. 2001,78, 788−790.(58) Cuber, M.; Demas, J. N.; Bare, W. D.; Pham, C. V. AnImproved Method for Studying the Enzyme-Catalyzed Oxidation ofGlucose Using Luminescent Probes. J. Chem. Educ. 2007, 84, 1511−1514.(59) Vasilarou, A.-M. G.; Georgiou, C. A. Enzymatic Spectrophoto-metric Reaction Rate Determination of Glucose in Fruit Drinks andCarbonated Beverages. an Analytical Chemistry Laboratory Experi-ment for Food Science-Oriented Students. J. Chem. Educ. 2000, 77,1327−1329.(60) Choi, M. M. F.; Wong, P. S. Application of Datalogger inBiosensing: a Glucose Biosensor. J. Chem. Educ. 2002, 79, 982−984.(61) Hobbs, J. M.; Patel, N. N.; Kim, D. W.; Rugutt, J. K.;Wanekaya, A. K. Glucose Determination in Beverages Using CarbonNanotube Modified Biosensor: an Experiment for the UndergraduateLaboratory. J. Chem. Educ. 2013, 90, 1222−1226.(62) Blanco-Lopez, M. C.; Lobo-Castanon, M. J.; Miranda-Ordieres,A. J. Homemade Bienzymatic-Amperometric Biosensor for BeveragesAnalysis. J. Chem. Educ. 2007, 84, 677−680.(63) Lazarim, F. L.; Stancanelli, M.; Brenzikofer, R.; Vaz de Macedo,D. Understanding the Glycemic Index and Glycemic Load and TheirPractical Applications. Biochem. Mol. Biol. Educ. 2009, 37, 296−300.(64) Jiao, L.; Xiujuan, S.; Juan, W.; Song, J.; Lei, X.; Guotong, X.;Lixia, L. Comprehensive Experiment-Clinical Biochemistry: Determi-nation of Blood Glucose and Triglycerides in Normal and DiabeticRats. Biochem. Mol. Biol. Educ. 2015, 43, 47−51.(65) Hardee, J. R.; Delgado, B.; Jones, W. Kinetic Parameters for theNoncatalyzed and Enzyme-Catalyzed Mutarotation of Glucose Usinga Blood Glucometer. J. Chem. Educ. 2011, 88, 798−800.(66) Heinzerling, P.; Schrader, F.; Schanze, S. Measurement ofEnzyme Kinetics by Use of a Blood Glucometer: Hydrolysis ofSucrose and Lactose. J. Chem. Educ. 2012, 89, 1582−1586.



8003


(67) Amor-Gutierrez, O.; Rama, E. C.; Fernandez-Abedul, M. T.;Costa-García, A. Bioelectroanalysis in a Drop: Construction of aGlucose Biosensor. J. Chem. Educ. 2017, 94, 806−812.(68) Hardee, J. R.; Montgomery, T. M.; Jones, W. H. Chemistry andFlatulence: an Introductory Enzyme Experiment. J. Chem. Educ. 2000,77, 498−500.(69) Mulimani, V. H.; Dhananjay, K. Immobilized α-Galactosidasein the Biochemistry Laboratory. J. Chem. Educ. 2007, 84, 1974−1975.(70) Cochran, B.; Lunday, D.; Miskevich, F. Kinetic Analysis ofAmylase Using Quantitative Benedict’s and Iodine Starch Reagents. J.Chem. Educ. 2008, 85, 401−403.(71) Valls, C.; Rojas, C.; Pujadas, G.; Garcia-Vallve, S.; Mulero, M.Characterization of the Activity and Stability of Amylase From Salivaand Detergent: Laboratory Practicals for Studying the Activity andStability of Amylase From Saliva and Various Commercial Detergents.Biochem. Mol. Biol. Educ. 2012, 40, 254−265.(72) Munegumi, T.; Inutsuka, M.; Hayafuji, Y. Investigating theHydrolysis of Starch Using Α-Amylase Contained in DishwashingDetergent and Human Saliva. J. Chem. Educ. 2016, 93, 1401−1405.(73) Plutschack, M. B.; Pieber, B.; Gilmore, K.; Seeberger, P. H. TheHitchhiker’s Guide to Flow Chemistry. Chem. Rev. 2017, 117,11796−11893.(74) Taipa, M. A.; Azevedo, A. M.; Grilo, A. L.; Couto, P. T.;Ferreira, F. A. G.; Fortuna, A. R. M.; Pinto, I. F.; Santos, R. M.;Santos, S. B. Student Collaboration in a Series of IntegratedExperiments to Study Enzyme Reactor Modeling with ImmobilizedCell-Based Invertase. J. Chem. Educ. 2015, 92, 1238−1243.(75) Barb, A. W.; Glushka, J. N.; Prestegard, J. H. Kinetics ofNeuraminidase Action on Glycoproteins by One- and Two-Dimen-sional NMR. J. Chem. Educ. 2011, 88, 95−97.(76) Periyannan, G. R.; Lawrence, B. A.; Egan, A. E. 1H NMRSpectroscopy-Based Configurational Analysis of Mono- and Dis-accharides and Detection of β-Glucosidase Activity: an Under-graduate Biochemistry Laboratory. J. Chem. Educ. 2015, 92, 1244−1249.(77) Kehlbeck, J. D.; Slack, C. C.; Turnbull, M. T.; Kohler, S. J.Exploring the Hydrolysis of Sucrose by Invertase Using NuclearMagnetic Resonance Spectroscopy: a Flexible Package of KineticExperiments. J. Chem. Educ. 2014, 91, 734−738.(78) Her, C.; Alonzo, A. P.; Vang, J. Y.; Torres, E.; Krishnan, V. V.Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy andDetermination of the Michaelis−Menten Constant Using theLambert-W Function. J. Chem. Educ. 2015, 92, 1943−1948.(79) Guerra, N. P. Enzyme Kinetics Experiment with theMultienzyme Complex Viscozyme L and Two Substrates for theAccurate Determination of Michaelian Parameters. J. Chem. Educ.2017, 94, 795−799.(80) Ruiz-Chica, A. J.; Urdiales, J. L.; Medina, M. A.; Sanchez-Jimenez, F. One Century After Fischer: Better Tools for Teaching theStereochemistry of Carbohydrates. Biochem. Educ. 1999, 27, 7−8.(81) Herdman, C.; Diop, L.; Dickman, M. Carbohydrate AnalysisExperiment Involving Mono- and Disaccharides with a Twist ofGlycobiology: Two New Tests for Distinguishing Pentoses andGlycosidic Bonds. J. Chem. Educ. 2013, 90, 115−117.(82) Seoane, G.; Moresco, H.; Sanson, P. Simple PotentiometricDetermination of Reducing Sugars. J. Chem. Educ. 2008, 85, 1091−1093.(83) Jensen, M. B. Integrating HPLC and Electrochemistry: aLabVIEW-Based Pulsed Amperometric Detection System. J. Chem.Educ. 2002, 79, 345−348.(84) Farris, S.; Mora, L.; Capretti, G.; Piergiovanni, L. ChargeDensity Quantification of Polyelectrolyte Polysaccharides by Con-ductometric Titration: an Analytical Chemistry Experiment. J. Chem.Educ. 2012, 89, 121−124.(85) Wei, C.-C.; Jensen, D.; Boyle, T.; O’Brien, L. C.; De Meo, C.;Shabestary, N.; Eder, D. J. Isothermal Titration Calorimetry andMacromolecular Visualization for the Interaction of Lysozyme and ItsInhibitors. J. Chem. Educ. 2015, 92, 1552−1556.



8004


Documents

Incorporating Carbohydrates into Laboratory Curriculadepa.fquim.unam.mx/amyd/archivero/ARTICULO-CARBOHIDRATOS_3… · Incorporating Carbohydrates into Laboratory Curricula Jennifer