**9.6. Oxidation of N6-carboxymethyl-NADH to N6-carboxymethyl-NAD+ (compound 3 in Figure 19)**

The reaction mixture containing N6-carboxymethyl-NADH was treated with 17.5 mL 3M Tris buffer (pH7.0) and the pH was adjusted to 7.5 using 5M HCl aqueous solution. 3.5 mL acetaldehyde (62.6mmol) was added, immediately followed by 10.5 mg yeast alcohol dehydrogenase (s*accharomyces cerevisiae*) (~300U/mg) before allowing agitating at ambient temperature to deoxidize the nicotinamide moiety. After 18 hours, the reaction mixture (c.a. 485 mL) was concentrated *in vacuo* (30°C/10-15bar) to approximately 1/3 volume and poured into 1800 mL pre-cooled (-5°C) mixture of acetone/IMS (1:1). The fine slurry was left to age for 18 hours at 3°C. The resulting precipitate was collected by centrifugation and washed on a glass sinter with 40 mL IMS then 120 mL dry diethyl ether before air-drying under dry nitrogen for 10 minutes. Further drying overnight in a desiccator over fused CaCl2 afforded 3.99g crude N6-carboxymethyl-NAD+ as a tan coloured hygroscopic solid.

1.0 g of the above-prepared crude N6-carboxymethyl-NAD+ was taken up in 20 mL water and passed through a Sephadex G10 gel filtration column (2x10cm, 20 mL). All eluted fractions containing UV active material were combined (60 mL total volume) and added to a column of Dowex 1-X2 ion exchange resin (Cl- , 4x50cm, 200 mL) which had been preequilibrated with water. A linear gradient of 0-50 mM LiCl (buffered to pH 3.0), at 10 mL per minute over 65 minutes, was applied using "*Presearch Combiflash Companion"* chromatography equipment. The fractions eluted between 25-35 mM were combined (c.a. 100 mL), neutralized to pH 7.0 with 5M LiOH and evaporated to approximately 1/3 volume and poured into 300 mL pre-cooled (-5°C) mixture of acetone/IMS (1:1). The fine slurry was left to age for 18 hours at 3°C. The resulting precipitate was collected by centrifugation and washed on a glass sinter with 30 mL IMS then 50 mL dry diethyl ether before air-drying under dry nitrogen for 10 minutes. Further drying overnight in a desiccator over fused CaCl2 afforded 0.307g purified N6-carboxymethyl-NAD+ as a cream coloured hygroscopic solid.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 349

which require any exogenous coenzyme to function. This makes the manufacture of the sensor relatively straight forward compared to those that require a modified electrode, an enzyme and in addition exogenous coenzyme added to the enzyme ink. In addition, switching to a NAD-dehydrogenase system may be incompatible with the manufacturing equipment currently used by manufacturers and thus prevent the adoption of this

[1] Evans JMM, Newton RW, Ruta DA, MacDonald TM, Stevenson RJ, Morris AD (1999) Frequency of blood glucose monitoring in relation to glycemic control: observational

[2] Benjamin EM (2002) Self-Monitoring of Blood Glucose: The Basics. Clinical Diabetes

[3] Franciosi M, Pellegrini F, De Bernardis G, Belfiglio M, Nicolucci A (2001) The impact of blood glucose self-monitoring on metabolic control and quality of life in type 2 diabetic

[4] Diabetes Education Study Group of the European Association for the Study of Diabetes (2003) Type 2 diabetes patient education basics. Blood glucose monitoring: a must in

[5] Peel E (2004) Blood glucose self-monitoring in non-insulin treated Type 2 diabetes: a qualitative study of patients' perspectives. British Journal of General Practice. 54:183-8

[7] White HB (1982) Evolution of coenzymes and the origin of pyridine nucleotides, in: Everse J, Anderson B, You K-S, editors. The Pyridine Nucleotide Cofactors, Academic

[8] Rodkey FL (1955) Oxidation-reduction potentials of the diphosphopyridine nucleotide

[9] Rodkey FL (1959) The effect of temperature on the oxidation-reduction potential of the

[10] Blaedal WJ, Jenkins RA (1975) Study of the electrochemical oxidation of reduced

[11] Nassef HM, Radi A-E, O'Sullivan CK (2006) Electrocatalytic sensing of NADH on a glassy carbon electrode modified with electrografted o-aminophenol film

[12] Compton RG, Hancock G (1999) Comprehensive Chemical Kinetics, Vol 37, Application

[6] Updike SJ, Hicks GP (1967) The Enzyme Electrode. Nature. 214:986 - 988

diphosphopyridine nucleotide system. J. Biol. Chem. 234:188 - 90

nicotinamide adenine dinucleotide. Anal. Chem. 47:1337–1343

Electrochemistry Communications. 8:1719–1725

of Kinetic modelling (eds), Elsevier, pp.49 -85

technology.

**Author details** 

**11. References** 

January. 20:45-47

diabetes management.

Marco Cardosi and Zuifang Liu

*LifeScan Scotland Limited, a Johnson & Johnson Company, UK* 

study with diabetes database. BMJ 319:83–86,

patients. Diabetes Care. 24:1870–1877

Press, New York, 1982, pp. 1 -17

system, J. Biol. Chem., 213:777–786

**Figure 19.** Synthesis of N6-carboxymethyl-NAD+

The synthesized N6-carboxymethyl-NAD+ is an important intermediate for NAD immobilization at electrodes for continuous monitoring biosensors. An extension of this work could involve synthesis of various polymeric NADs with tailor-made chemical properties to meet biosensor requirements for continuous monitoring of different analytes.
