PTT 202 ORGANIC CHEMISTRY FOR BIOTECHNOLOGY CARBOHYDRATES PREPARED

PTT 202: ORGANIC CHEMISTRY FOR BIOTECHNOLOGY CARBOHYDRATES PREPARED BY: NOR HELYA IMAN KAMALUDIN helya@unimap. edu. my

Introduction of Carbohydrates are aldehydes or ketones of higher polyhydric alcohols or components that yield these derivatives on hydrolysis. Difficulties are encounted in the qualitative and quantitative analysis of carbohydrates mixtures because of the structural and chemical similarity of many compounds. Analytical specificity may be improved by the preliminary separation of the components of the mixture using a chromatographic technique prior to quantitation.

Chemical methods of carbohydrate analysis Reaction with aromatic amines Reduction methods Reactions of the carbonyl group Reactions with strong acids and a phenol

Reduction methods Reduction of cupric ions (Cu 2+) to cuprous ions (Cu+) • In alkaline solution, it will form yellow cuprous hydroxide, which in turn converted by the heat of the reaction to insoluble red cuprous oxide (Cu 2 O). • Production of a yellow or orange-red precipitate indicates the presence of a reducing carbohydrate. • Different carbohydrates will result in the formation of different amounts of cuprous oxide. • The methods of measuring cuprous oxide frequents used involves the reduction of either phosphomolybdic acid or arsenomolybdic acid to lower oxides of molybdenum by cuprous oxide. • The intensity of the colored complex produced is related to the concentration of the reducing substances. Reduction of ferricyanide ions to ferrocyanide ions • The color will change from yellow solution to colorless solution by reducing carbohydrates when heated in alkaline solution. Disadvantages • Lack of specificity • Result in positive error as non-carbohydrate reducing substances present in the sample.

Reaction with aromatic amines Various aromatic amines will condense with aldoses and ketoses in glacial acetic acid to form coloured products • Maxima absorbance are often characteristic of an individual carbohydrate or group. The examples of aromatic amines that have been used • p-aminobenzoic acid, diphenylamine and p-aminophenol. These amines has ability to react preferentially with a particular carbohydrate or class of carbohydrate. • e. g. p-aminophenol shows some specificity for ketoses compared with aldoses and is useful for measuring fructose. These reagents have proved particularly useful for the visualization and identification of carbohydrates after separation of mixtures by paper or thinlayer chromatography.

Reactions with strong acids and a phenol Formation of furfural and hydroxymethylfurfural • Pentoses and hexoses are dehydrated to form furfural and hydroxymethylfurfural derivatives respectively when heated with a strong acid (Figure 1). • The aldehyde groups will condense with a phenolic compound to form a colored product. • The reaction forms the basis of some of the oldest qualitative tests for the detection of carbohydrates, e. g. the Molisch test using concentrated sulphuric acid and α-naphthol. Advantage • Give some degree of specificity through careful choice of both the reaction conditions and the phenolic compound used which may be possible to produce a color that is characteristic of a particular carbohydrate or related group. Disadvantage • Non-carbohydrate substances may be present in a biological sample that will decompose on heating and will react in a similar manner to a carbohydrate and may give a false positive reaction for carbohydrates.

Figure 1: Formation of furfural and 5 hydroxymethylfurfural

Enzymic methods of carbohydrate analysis Assay of glucose using glucose dehydrogenase Assay of glucose using glucose oxidase Assay of glucose using hexokinase Enzymic methods

Assay of glucose using glucose oxidase Glucose oxidase enzyme catalyses the oxidation of β-D-glucose by atmospheric oxygen to produce D-gluconolactone, which is converted to gluconic acid with the production of hydrogen peroxide. The oxidation of α-D-glucose occurs at less than 1% the rate of oxidation the β anomer • • Because of these two forms exist in solution in equilibrium, mutarotation of the α to the β form must be allowed to reach equilibrium in the sample and standards for consistent results. The inclusion of aldose-1 -epimerase (glucomutarotase) in the glucose oxidase reagent will permit rapid restoration of the α-β equilibrium, effectively enabling the reaction to go to completion.

Assay of glucose using glucose oxidase Measurement of hydrogen peroxide formed Quantitation of glucose using glucose oxidase during the reaction Measurement of oxygen consumed Spectrophotometric methods Electrochemical method

Assay of glucose using glucose oxidase Measurement of the hydrogen peroxide formed Spectrophotometric methods • H 202 formed was measured by monitoring the change in color of a chromogenic oxygen acceptor in the presence of the enzyme peroxidase. • Such chromogens are colorless in their reduced form but exhibit characteristic colors when oxidized. • Disadvantages: less specific, errors may be introduced in the second stage of assay reaction by interference of some substances in the reaction. Electrochemical methods • The instruments used for electrochemical measurement of the H 202 produced is glucose analysers especially in an immobilized form.

Assay of glucose using glucose oxidase Measurement of the oxygen consumed The initial oxidation of glucose can be monitored and this is most easily achieved by measuring the amount of oxygen consumed during the reaction. An electrochemical method using a polographic Clark oxygen electrode has been used and the first oxygen electrode to be described for the measurement of glucose in contained soluble glucose oxidase held between cuprophane membrane. Recent modifications have been resulted in the incorporation of a variety of forms of immobilized glucose oxidase into the electrode.

Assay of glucose using glucose dehydrogenase Glucose can be measured using bacterial glucose dehydrogenase • Catalyses the dehydrogenation of β-D-glucose to Dgluconolactone • The hydrogen being transferred to NAD+ or NADP+. • The method involves measuring the increase in absorbance at 340 nm caused by the production of NADH using either a kinetic or fixed time assay technique. • A mutarotase should be included in the assay to accelerate the conversion of a α to the β form.

Assay of glucose using hexokinase Measuring of glucose using hexokinase enzyme • Catalyses the phosphorylation of glucose to produce glucose-6 phosphate with adenosine triphosphate (ATP) as the phosphate donor and magnesium ions as an activator. • The rate of formation of glucose-6 -phosphate can be linked to the reduction of NADP by the enzyme glucose-6 -phosphate dehydrogenase. • The indicator reaction can be monitored spectrophotometrically at 340 nm as below:

Assay of glucose using hexokinase Characteristic of hexokinase enzyme • Not specific for glucose only • Capable of converting some other hexoses to their corresponding 6 -phosphate derivatives. • Additionally, the specificity of the enzyme may vary slightly depending on its source. • For example, yeast hexokinase will catalyse the phosphorylation of a number of hexoses as well as glucose including D-mannose, D-fructose, D-glucosamine. • This provides an assay system for mannose or fructose if the initial reaction is linked to a suitable second reaction (Figure 1)

Mannose, fructose and glucose may all be assayed independently using hexokinase and the appropriate additional enzymes. All the reactions can be monitored in absorbance at 340 nm as NADP+ is reduced to NADPH. Figure 1: Assay systems for mannose, fructose and glucose using hexokinase.

Separation and identification of carbohydrate mixtures Gas-liquid chromatography High performance liquid chromatography Paper and thinlayer chromatography Techniques of separation and identification

Paper and thin-layer chromatography Both ascending and descending paper chromatography techniques have been used, when thin-layer supports are employed As the number of carbohydrates present in the sample is often small, careful choice of solvent will generally make it unnecessary to perform the two-dimensional separations. Thus, the separation of carbohydrate mixtures depends on the solvent systems and locating reagents.

Paper and thin-layer chromatography Solvent systems • Several monophasic solvent systems are useful for the separation of carbohydrate mixtures as listed in Table 1. • The smallest solute molecules, the fastest of mobility. • Thus, pentoses have higher RF values than hexoses, followed by disaccharides and oligosaccharides. Locating reagents • A variety of reagents can be used for visualization of the separated components. • Useful to run duplicate chromatograms and use a different stain on each one to assist in identification of unknown spots. • The actual reagent composition may be modified either in terms of concentration of components or by substitution of one chemical for another similar one. • Such variations in reagent composition may be advantageous in promoting the production of characteristic colors for different carbohydrates.

Table 1: Some monophasic solvents for thin-layer chromatography of carbohydrates

Gas-liquid chromatography This technique is necessary to identify or to quantitate one or more carbohydrates especially when they are present in small amounts. GLC can be carried out using volatile derivatives of carbohydrates such as O-trimethylsilyl (TMS) derivatives which probably used most frequently due to simple preparation and have been successfully applied to wide variety of compounds. The choice of stationary phase will depend upon the nature of the carbohydrates to be separated. For example; OV-17 stationary phase useful for isometric separations while OV-1 useful for wider range of carbohydrates.

Figure 2: Separation of equimolar concentrations of methyglycosides by GLC. The analysis was performed on an OV-1 stationary phase using a temperature gradient from 120 to 220˚C

High performance liquid chromatography Function • For separation and quantitation of carbohydrate mixtures. • This method does not necessitate the formation of a volatile derivative as in GLC • Techniques used: Partition and Ion-exchange Techniques. Partition chromatography • Is usually performed in the reverse phase mode using a chemically bonded stationary phase and acetonitrile (80: 20) in 0. 1 mol/l acetic acid as the mobile phase. Ion-exchange chromatography • Anion and cation exchange resins can be used. • Carbohydrates form anionic complexes in alkaline borate buffers and quaternary ammonium anion-exchange resins in hdroxyl form can be used for their separation. • Sulphonated cationic exchange resins with metal counter-ions are also useful for carbohydrate analysis.

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