Synthesis and characterization of 1‐substituted 5‐alkylphenazine derivatives carrying functional groups

Abstract

The following 1-substituted derivatives of Smethylphenazine and 5-ethylphenazine were synthesized: 1-(3-carboxypropyloxy)-5-methylphenazine (lB), l-(3-carboxypropyloxy)-5-ethylphenazine (2B), 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine (2C) and l-[N-(2-aminoethyl)carbamoylpropyloxy]-5-~thylphenazine (2D); their spectra, stability and reactivity as electron mediators were investigated, together with those of 5-methylphenazine (1A) and 5-ethylphenazine (2A). The 1-substituted derivatives are all insensitive to light and the derivatives of 5-ethylphenazine are more stable than those of 5-methylphenazine under neutral and alkaline conditions; 2 8 is the most stable of all the derivatives. The spectral properties of the decomposed compounds showed that photodecomposition of 1A and 2A is associated with hydroxylation at position 1, alkali decomposition of 1A and 1 B with elimination of the 5-methyl group and alkali decomposition of 2A, 2B, and 2D with a ring-opening reaction. The second-order rate constant kl for the reaction of the phenazine derivatives with NADH was measured under steady-state conditions. The k l values vary depending on the substituents at positions 1 and 5 :the values for lA, lB, 2A, 2B, 2C and 2D are 1.83 m M -' s-', 3.33 mM-' s-', 0.75 mM-' s-', 1.42 mM-' s-', 1.68 mM-' s -' and 2.03 mM-' s -I , respectively. The rate constants, k, and k3, for the reactions of the reduced form of 2B with oxygen and with 3-(4,5'-dimethyl~hiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.21 mM-' sC1 and k3 = 91 mM-' s-'. These phenazine derivatives have potential applications in the biochemical field. (Fig. 1, 1A) is a unique electron mediator as it can accept electrons from NAD(P)H and is widely used in various redox reactions. However, its application is still limited, mainly because of its instability to light and to alkaline solution. 5-Ethylphenazine (Fig. 1, 2A) has been recommended as a more stable electron mediator in alkaline solutions [l, 21 and light-insensitive derivatives have also been prepared and used in the assay of dehydrogenases [3 -61, but the mechanisms of these instabilities are still unclear. 5-MethylphenazineWe have studied co-enzyme-recycling systems [7 -91 and now we intend to use phenazine derivatives as NAD(P)+ regenerators. For this purpose, it is necessary to prepare stable phenazine derivatives with a functional group suitable for binding them to other molecules, such as water-soluble polymers, solid supports and enzymes. These derivatives are also useful for investigating the mechanisms of the electron-transfer reactions from NAD(P)H. Here, we describe the preparation of new phenazine derivatives carrying a carboxyl group (Fig. I , 1 B and 2B) and an amino group (Fig. 1, 2D) and also their stability and electron-transfer activity. The kinetic constants obtained in this work are necessary for understanding the coupled-reaction system of NAD(P)+ regeneration.

Preparation and kinetic properties of 5‐ethylphenazine‐glucose‐dehydrogenase‐NAD+ conjugate, a semisynthetic glucose oxidase

“…In fact, there is another cycle in which the oxidized forms are EP t -GlcDH-NADH and EPH-GlcDH-NAD + and the reduced form is EPH-GlcDH-NADH. The ratio of the fluxes of these two cycles changes depending on the ratio of the rate constants for the reduction of the NAD' moiety and for the oxidation of the reduced form of the ethylphenazine moiety, as shown in our previous paper on EP+-PEG-NAD' [2].…”

“…The concentrations of glucose dehydrogenase, NAD +, NADH, PEG-NAD + , PEG-NADH, Sethylphenazine, and EP' -PEG were measured spectrophotometrically using the following molar absorption coefficients : the subunit of glucose dehydrogenase, 33 000 M ' cm-' at 280 nm ( M , 28 100) [7]; NAD+, ISOOOM-' cm-' at 260nm; PEG-NAD', 21 500 M-' cm-' at 266 nm [2]; NADH and PEG-NADH, 6300 M-' cm-' at 340 nm; Sethylphenazine, 26300 M-' cm-' at 386nm [8]; and EP+-PEG, 17000M-' cm-' at 386 nm [2]. The concentrations of the ethylphenazine moiety, the enzyme moiety (as a subunit) and the NAD' moiety in EP+-GlcDH-NAD' were calculated as described by MHnsson et al [9] from the absorption spectrum of the conjugate using the following molar absorption coefficients : for the ethylphenazine moiety, 17000 M-' cm-' at 386 nm, 43000M-' cm-' at 280nm, and 28000M-' cm-' at 266 nm; for the enzyme moiety, zero at 386 nm, 33000 M-' cm-' at 280 nm, and 23000 M-' cm-' at 266 nm; for the NAD+ moiety, zero at 386 nm, 12000 M-' cm-' at 280 nm, and 21 500 M-' cm-' at 266 nm.…”

“…In fact, there is another cycle in which the oxidized forms are EP t -GlcDH-NADH and EPH-GlcDH-NAD + and the reduced form is EPH-GlcDH-NADH. The ratio of the fluxes of these two cycles changes depending on the ratio of the rate constants for the reduction of the NAD' moiety and for the oxidation of the reduced form of the ethylphenazine moiety, as shown in our previous paper on EP+-PEG-NAD' [2].The preparation of EP+-GlcDH-NAD' is not homogeneous in the reactivities of the NAD' and the ethylphenazine moieties (see Appendix), but the experimental results shown in Figs 3 and 4 fit the simple Michaelis-Menten equation. This means that each EP+-GlcDH-NAD ' molecule has similar V,,, and K, values.…”

“…It should be noted that the reactivity of the ethylphenazine moiety toward the NADH moiety cannot be affected by the nature of the electron acceptor, but the number of the actively recycling ethylphenazine moieties could be affected by some physical interactions. In our previous work, we found that the rate constant of the reduced form of the ethylphenazine moiety toward MTT is decreased to about one third by covalently linking the moiety to poly(ethy1ene glycol), but that the rate constant toward oxygen is not affected by the linking [2]. There may be some common mechanism between these phenomena, because the polymer chain of poly(ethy1ene glycol) is attached to the ethylphenazine moiety in both cases, and the dehydrogenase molecule is further attached in the case of EP'-GlcDH-NAD'.…”

“…This means that each EP+-GlcDH-NAD ' molecule has similar V,,, and K, values. To confirm this homogeneity, we assumed that the preparation of EP+-GlcDH-NAD' is composed of four groups with the same V,,, value but with different K, values (Km,l, Km,2, Km,3 and Km,4), and obtained these values by fitting the following equation to the data points for the MTT system by the Marquardt method [ll] with a standard deviation of 0.021 ; the values are: V,,, = 0.65 pM/ min, K,,l = K,,2 = 0.115mM, Km,3 = 0.116mM, and Km,4 = 0.024 mM.(1) The resulting curve for Eqn (1) is shown in Fig. 3A as a broken line.…”