Heyns Rearrangement Research Articles

Structure of the adduct of 16α-hydroxyestrone with a primary amine: evidence for the heyns rearrangement of steroidal D-ring α-hydroxyimines

16α-Hydroxyestrone, a product of estrogen 16α-hydroxylotion in humans that is suspected to be implicated in cell transformation, has been found to form stable adducts with nuclear components. The stable covalent adduct formed from 16α-hydroxyestrone with 2-methoxyethylamine via the Heyns rearrangement of the α-hydroxyimine was identified as 3-hydroxy-17β-(2-methoxyethylamino)estra1, 3,5(10)-trien-16-one. Since the same product was obtained from 16β-hydroxyestrone with the amine, the α-hydroxyenamine is the most likely intermediate of the Heyns rearrangement. The adduct was fairly stable at 37 C in phosphate buffer (pH 7.4)/methanol (1:1 v/v), while the adduct formed from 16-oxoestradiol was disrupted reversely and completely within 6 hours. The evidence suggests that N-(3hydroxy-16-oxoestra-1,3,5 (10)-trien-17β-yI)amine is the partial structure of the stable adducts formed from D-ring α-ketol estrogens with proteins.

Preparation of Nε-(3-hydroxy-16-oxoestra-1,3,5(10)-trien-17β-YL)- l-lysine, 16α-hydroxyestrone-lysing adduct, via heyns rearrangement

Preparation of a stable covalents adduct from 16α-hydroxyestrone with the ε-amino group of lysine via two step reactions involving Schiff base formation followed by Heyns rearrangement is described.

The Maillard reaction of DNA with D-fructose 6-phosphate.

The Maillard reaction of DNA with ketoses was investigated. Several days of incubation of d-fructose 6-phosphate with deoxyribonucleotides or with polymer DNA in an aqueous buffer resulted in the formation of chromophores and fluorophores. Aminoguanidine and sodium cvanoborohydride inhibited the formation of fluorophores. Transition metal ions such as Cu2+, Fe3+, Fe2+, or Mn2 + promoted the formation of chromophores and fluorophores. Metal-chelating agents such as DETAPAC, citrate, and Desferal inhibited the formation of fluorophores. Superoxide dismutase and catalase also inhibited the formation of fluorophores. The transition metal ion-catalyzed autoxidation of d-fructose 6-phosphate or of the Heyns rearrangement products were to be partially involved in the glycation of DNA and subsequent formation of chromophores and of fluorophores.

Browning reactions of Heyns rearrangement products

AbstractThe browning rate of Heyns rearrangement products at 110°C under free access of oxygen is slower that that of Amadori rearrangement products. The maximum browning rate was observed at pH = 8.0. The browning was accelerated by Fe(III) and Cu(II) ions. The presence of Fe(II) ions, propyl gallate, and rutin caused lag periods but had no pronounced effect during the subsequent browning. Hydrogen peroxide bleached the melanoidin pigments but did not destroy the browning precursors. Sulphites inhibited the browning reaction. Fluorescence spectra differed from those of the respective Amadori products.

Structure of lysine adducts with 16α-hydroxyestrone and cortisol

Recent studies indicate that steroids containing a vicinal hydroxyketone moiety can react with proteins both in vitro and in vivo to form covalent addition products. This reaction is non-enzymatic and occurs via the Heyns rearrangement of an initial Schiff base adduct between the steroid carbonyl and the ϵ-amino group of lysine residues. The present study describes the synthesis, isolation, and structural analysis of model adducts prepared by the incubation of 16α-hydroxyestrone or cortisol with NaCNBH 3 and lysine derivatives blocked in the N α-position. The product formed from the reaction of 16α-hydroxyestrone and lysine was found to have the structure predicted for a reduced Schiff base between these molecules. A stable, cortisol-lysine adduct was similarly synthesized and isolated. This conjugate was found not to be the expected reduced Schiff base but rather a C-20 cyano amine. This compound most likely was formed by the nucleophilic addition of cyanide during the course of the incubation. The observation that the cortisol-lysine Schiff base is not reducible with NaCNBH 3 accounts for the observation that the incorporation rate of glucocorticoids into proteins is not increased by the presence of NaCNBH 3.

Characterization of antisera to the addition product formed by the nonenzymatic reaction of 16α-hydroxyestrone with albumin

16α-Hydroxyestrone (16αOHE) has been shown previously to react nonenzymatically with proteins via a Heyns rearrangement of a Schiff base intermediate. Albumin modified by the addition of 16αOHE is immunogenic, despite a relatively low molar substitution. High-litre antisera can be elicited which have a very high affinity toward the estrogen hapten. Cross-reactivity analysis reveals a high specificity for the phenolic A-ring and a lack of specificity to chemical substituents in the D-ring, the site of covalent linkage. The antisera reacts equally well with 16αOHE-peptides as with 16αOHE-lysine, suggesting the utility of this antisera in analyzing either enzymatically or chemically hydrolyzed proteins for the presence of 16αOHE adducts. Immunochemical analysis of proteins modified by 16αOHE may provide insight into the pathogenesis of systemic lupus erythematosus, an autoimmune disease in which elevated levels of 16αOHE are known to occur.

Formation of covalent adducts between cortisol and 16 alpha-hydroxyestrone and protein: possible role in the pathogenesis of cortisol toxicity and systemic lupus erythematosus.

The incubation of albumin with cortisol or 16 alpha-hydroxyestrone results in the formation of covalent steroid-protein adducts. The rate of adduct formation increases in the presence of sodium cyanoborohydride (NaCNBH3), indicating that the reaction proceeds nonenzymatically through a Schiff base intermediate. Under nonreducing conditions, a stable adduct forms with cortisol and 16 alpha-hydroxyestrone but not with estrone, which lacks a hydroxyl group adjacent to the reactive carbonyl. It is hypothesized that a Heyns rearrangement involving the adjacent hydroxyl group traps the Schiff base and produces a stable ketoamine adduct. The binding of 16 alpha-hydroxyestrone and cortisol to albumin is significantly inhibited by acetylsalicylic acid, which has been shown to acetylate an epsilon-amino group of a lysine residue in albumin. High-pressure liquid chromatography analysis of an acid hydrolysate of 16 alpha-hydroxyestrone-albumin shows that a product containing 16 alpha-hydroxyestrone coelutes with a standard prepared by reacting 16 alpha-hydroxyestrone with the epsilon-amino group of lysine. We propose that the formation of covalent steroid-protein adducts is a generalized phenomenon which may contribute to the pathological effects produced by elevated levels of certain endogenous steroids.