**9.5. Synthesis of N6-carboxymethyl-NADH (compound 2 in Figure 19)**

346 Dehydrogenases

an enzyme(s), the ferrocene polymeric mediator and a polymeric NAD in an electrode with the 3 dimentional nano-porous structure (see Figure 10). A polymeric NAD is normally synthesized by two routes. One is coupling NAD or NAD analogue with a polymer bearing functional groups. [66, 70-73] The other route involves synthesis of a NAD monomer and then its copolymerisation with another co-monomer(s). One of the challenges for NAD immobilization is to keep NAD coenzymic activity while achieving effective retention at electrode. Yamazaki *et al* synthesized three NAD monomers (N6-[N-(6-methacrylamidohexyl)carbamoylmethyl]-, N6[N-[2-[N-(2-

methacrylamidopropyl) carbamoylmethyl] carbamoylmethyl]-NAD) and then copolymerized them with various co-monomers (acrylamide, N-(2-hydroxyethyl)-, N-ethyl-, N,N-diethyl-, and N,N-dimethylacrylamide, acrylic acid, and 6-methacrylamidohexylammonium) by free radical polymerisation to form a series of polymeric NADs. [74] Their studies revealed that hydrophilicity and length of the spacers linking NAD moieties and the polymer backbone had the most important effects on coenzymic activity of the polymeric NADs. This suggests that keeping mobility of the NAD moieties covalently attached to a polymer chain is critical to NAD coenzymic activity. Chemically modification to native NAD is required to tailor chemical properties of the spacers. N6-amino group on the adenine ring of NAD (see Figure 18) is normally selected as the site for this purpose. [75, 76] Lindberg *et al* reported alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement to give N6-carboxymethyl-NAD+.

However, the reaction between NAD+ and iodoacetic acid took 10 days in the dark at room temperature. We successfully synthesized N6-carboxymethyl-NAD+ by a modified method with

**9.4. Synthesis of N1-carboxymethyl-NAD+ (compound 1 in Figure 19)** 

1.0g NAD+ (1.51mmol) was dissolved in 3.5mL 0.1M pH 7.0 sodium phosphate buffer in a 5ml Biotage microwave reaction tube. Then, 1.5 g (8.06mmol, 5.34eq) iodoacetic acid was added and pH was adjusted to 7.0 by using 5.0M NaOH aqueous solution. The reaction vessel was sealed and the mixture was heated to 50°C for 10 minutes by using microwave irradiation. After that, the pink solution (c.a. 5.0 mL) was acidified to pH3.0 using 5M HCl aqueous solution before being poured into 25 mL pre-cooled (-5°C) mixture of acetone/IMS (1:1). The resulting precipitate was filtered, washed first with 5.0 mL IMS, then 15 mL dry diethyl ether before air drying under dry nitrogen for 10 minutes. Further drying overnight in a desiccator over fused CaCl2 gave 1.62 g crude N1-carboxymethyl-NAD+ as a pink amorphous solid.

**OH <sup>H</sup> <sup>H</sup>**

**N**

**N**

**OH <sup>H</sup> <sup>H</sup> O N**

**N**

**O P O P <sup>O</sup> HO**

**O**

**O**

**OH**

**H2N**


[75]

methacrylamidoethyl)carbamoyl]ethyl] carbamoylmethyl]-, and N6

a dramatic reduction in the reaction time.

**N+**

**NH2 O**

> **OH H H**

**OH <sup>H</sup> <sup>H</sup> O**

**Figure 18.** Chemical structure of NAD+

9.1g (c.a. 10.57mmol) above prepared crude N1-carboxymethyl-NAD+ was dissolved in 1.3% w/v NaHCO3 in 450 mL aqueous solution and the solution was deoxygenated by sparging with nitrogen for 10 minutes. 3.5 g (20.1mmol) sodium dithionite was added in one portion and the mixture was stirred at ambient temperature to affect reduction of the nicotinamide moiety. After 1.0 hour, the solution colour changed from pink to yellow. The solution was then sparged with air for 10 minutes to destroy any excess dithionite and the pH was brought to 11.0 by using 5M NaOH aqueous solution. The mixture was heated at 70°C for 90 minutes, to promote Dimroth rearrangement to N6-carboxymethyl-NADH, before cooling to 25°C. Thin-layer chromatography (silica gel, isobutryic acid/water/32% NH4OH (aq), 66/33/1.5 by volume) showed no evidence for the presence of N1 carboxymethyl-NADH.
