Sugumaran M, Semensi V, Kalyanaraman B, Bruce J M, Land E J
Department of Biology, University of Massachusetts, Boston 02125.
J Biol Chem. 1992 May 25;267(15):10355-61.
1,2-Dehydro-N-acetyldopamine (dehydro-NADA) is an important catecholamine derivative involved in the cross-linking of insect cuticular components during sclerotization. Since sclerotization is a vital process for the survival of insects, and is closely related to melanogenesis, it is of interest to unravel the chemical mechanisms participating in this process. The present paper reports on the mechanism by which dehydro-NADA is oxidatively activated to form reactive intermediate(s) as revealed by pulse radiolysis, electron spin resonance spectroscopy, high performance liquid chromatography, and ultraviolet-visible spectroscopic analysis. Pulse radiolytic one-electron oxidation of dehydro-NADA by N3. (k = 5.3 x 10(9) M-1 s-1) or Br2.- (k = 7.5 x 10(8) M-1 s-1) at pH6 resulted in the rapid generation of the corresponding semiquinone radical, lambda max 400 nm, epsilon = 20,700 M-1 cm-1. This semiquinone decayed to form a second transient intermediate, lambda max 485 nm, epsilon = 8000 M-1 cm-1, via a second order disproportionation process, k = 6.2 x 10(8) M-1 s-1. At pH 6 in the presence of azide, the first order decay of this second intermediate occurred over milliseconds; the rate decreases at higher pH. At pH 6 in the presence of bromide, the intermediate decayed much more slowly over seconds, k = 0.15 s-1. Under such conditions, the dependence of the first order decay constant upon parent dehydro-NADA concentration led to a second order rate constant of 8.5 x 10(2) M-1 s-1 for reaction of the intermediate with the parent, probably to form benzodioxan "dimers." (The term dimer is used for convenience; the products are strictly bisdehydrodimers of dehydro-NADA (see "Discussion" and Fig. 11)) Rate constants of 5.9 x 10(5), 4.5 x 10(5), 2.8 x 10(4) and 3.5 x 10(4) M-1 s-1 were also obtained for decay of the second intermediate in the presence of cysteine, cysteamine, o-phenylenediamine, and p-aminophenol, respectively. By comparison with the UV-visible spectroscopic properties of the two-electron oxidized species derived from dehydro-NADA and from 1,2-dehydro-N-acetyldopa methyl ester, it is concluded that the transient intermediate exhibiting absorbance at 485 nm is the quinone methide tautomer of the o-quinone of dehydro-NADA. Sclerotization of insect cuticle is discussed in the light of these findings.
1,2 - 脱氢 - N - 乙酰多巴胺（脱氢 - NADA）是一种重要的儿茶酚胺衍生物，参与昆虫硬化过程中表皮成分的交联。由于硬化是昆虫生存的关键过程，且与黑色素生成密切相关，因此揭示参与此过程的化学机制具有重要意义。本文报道了通过脉冲辐解、电子自旋共振光谱、高效液相色谱和紫外 - 可见光谱分析所揭示的脱氢 - NADA被氧化活化形成反应性中间体的机制。在pH6条件下，N3·（k = 5.3×10⁹ M⁻¹ s⁻¹）或Br2·⁻（k = 7.5×10⁸ M⁻¹ s⁻¹）对脱氢 - NADA进行脉冲辐解单电子氧化，导致迅速生成相应的半醌自由基，λmax 400 nm，ε = 20,700 M⁻¹ cm⁻¹。该半醌通过二级歧化过程（k = 6.2×10⁸ M⁻¹ s⁻¹）衰变形成第二个瞬态中间体，λmax 485 nm，ε = 8000 M⁻¹ cm⁻¹。在pH6且存在叠氮化物的情况下，该第二个中间体的一级衰变发生在毫秒级；在较高pH下速率降低。在pH6且存在溴化物的情况下，中间体在数秒内衰变要慢得多，k = 0.15 s⁻¹。在这种条件下，一级衰变常数对母体脱氢 - NADA浓度的依赖性导致中间体与母体反应的二级速率常数为8.5×10² M⁻¹ s⁻¹，可能形成苯并二恶烷“二聚体”。（术语二聚体是为方便起见使用；产物严格来说是脱氢 - NADA的双脱氢二聚体（见“讨论”和图11））在分别存在半胱氨酸、半胱胺、邻苯二胺和对氨基苯酚的情况下，第二个中间体衰变的速率常数分别为5.9×10⁵、4.5×10⁵、2.8×10⁴和3.5×10⁴ M⁻¹ s⁻¹。通过与源自脱氢 - NADA和1,2 - 脱氢 - N - 乙酰多巴甲酯的双电子氧化物种的紫外 - 可见光谱性质进行比较，得出在485 nm处有吸收的瞬态中间体是脱氢 - NADA邻醌的醌甲基互变异构体。根据这些发现对昆虫表皮的硬化进行了讨论。
