Hydantoin Products Research Articles

NEIL3 promoter G-quadruplex with oxidatively modified bases shows magnesium-dependent folding that stalls polymerase bypass

Oxidative stress unleashes reactive species capable of oxidizing 2′-deoxyguanosine (G) nucleotides in G-rich sequences of the genome, such as the potential G-quadruplex forming sequencing (PQS) in the NEIL3 gene promoter. Oxidative modification of G yields 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) that can be further oxidized to hydantoin products. Herein, OG was synthesized into the NEIL3 PQS that was allowed to fold to a G-quadruplex (G4) in K+ ion solutions with varying amounts of Mg2+ in the physiological range. The Mg2+ dependency in the oxidatively modified NEIL3 G4 to stall a replicative DNA polymerase was evaluated. The polymerase was found to stall at the G4 or OG, as well as continue to full-length extension with dependency on the location of the modification and the concentration of Mg2+. To provide some clarity on these findings, OG or the hydantoins were synthesized in model NEIL3 G4 folding sequences at the positions of the polymerase study. The model G4 sequences were allowed to fold in K+ ion solutions with varying levels of Mg2+ to identify how the presence of the divalent metal impacted G4 folding depending on the location of the modification. The presence of Mg2+ either caused the transition of the NEIL3 G4 folds from an antiparallel to parallel orientation of the strands or had no impact. Structural models are proposed to understand the findings using the literature as a guide. The biological significance of the results is discussed.

Synthesis and Characterization of Amino Acid-Derived Hydantoins

The Urech hydantoin synthesis is the chemical reaction of amino acids with potassium cyanate and hydrochloric acid, which allows access to hydantoins and its derivatives. Most of them are biologically active with varieties of structural diversity that make them synthetically attractive. A series of hydantoins were synthesized from various α-amino acids in a reaction with hydrochloric acid and ethanol as the first step, and with potassium cyanate as the second step for the formation of ureido derivatives. Lastly, the cyclization of ureido derivatives yielded the desired hydantoin products. All hydantoins were obtained in a good yield and fully characterized by NMR and IR spectroscopy. This strategy provides a variety of biologically significant hydantoin derivatives.

8-Oxo-7,8-dihydro-2'-deoxyguanosine and abasic site tandem lesions are oxidation prone yielding hydantoin products that strongly destabilize duplex DNA.

In DNA, 2'-deoxyguanosine (dG) is susceptible to oxidative modification by reactive oxygen species (ROS) yielding many products, one of which is 8-oxo-7,8-dihydro-2'-deoxyguanosine (dOG). Interestingly, dOG is stable but much more labile toward oxidation than dG, furnishing 5-guanidinohydantoin-2'-deoxyribose (dGh) that is favored in the duplex context or spiroiminodihydantoin-2'-deoxyribose (dSp) that is favored in the oxidation of single-stranded contexts. Previously, exposure of DNA to ionizing radiation found ∼50% of the dOG exists as a tandem lesion with an adjacent formamide site. The present work explored oxidation of dOG in a tandem lesion with a THF abasic site analog (F) that models the formamide on either the 5' or 3' side. When dOG was in a tandem lesion, both dGh and dSp were observed as oxidation products. The 5' versus 3' side in which F resided influenced the stereochemistry of the dSp formed. Further, tandem lesions with dOG were found to be up to two orders of magnitude more reactive to oxidation than dOG in an intact duplex. When dOG is in a tandem lesion it is up to fivefold more prone to formation of spermine cross-links during oxidation compared to dOG in an intact duplex. Lastly, dOG, dGh, and each dSp diastereomer were synthesized as part of a tandem lesion in a duplex DNA to establish that dOG tandem lesions decrease the thermal stability by 12-13 °C, while dGh or either dSp diastereomer in a tandem lesion decrease the stability by >20 °C. The biological consequences of these results are discussed.

Pseudoephedrine-Directed Asymmetric α-Arylation of α-Amino Acid Derivatives.

Available α-amino acids undergo arylation at their α position in an enantioselective manner on treatment with base of N'-aryl urea derivatives ligated to pseudoephedrine as a chiral auxiliary. In situ silylation and enolization induces diastereoselective migration of the N'-aryl group to the α position of the amino acid, followed by ring closure to a hydantoin with concomitant explulsion of the recyclable auxiliary. The hydrolysis of the hydantoin products provides derivatives of quaternary amino acids. The arylation avoids the use of heavy-metal additives, and is successful with a range of amino acids and with aryl rings of varying electronic character.

Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA.

The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.

Structural Context Effects in the Oxidation of 8-Oxo-7,8-dihydro-2′-deoxyguanosine to Hydantoin Products: Electrostatics, Base Stacking, and Base Pairing

8-Oxo-7,8-dihydroguanine (OG) is the most common base damage found in cells, where it resides in many structural contexts, including the nucleotide pool, single-stranded DNA at transcription forks and replication bubbles, and duplex DNA base-paired with either adenine (A) or cytosine (C). OG is prone to further oxidation to the highly mutagenic hydantoin products spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in a sharply pH-dependent fashion within nucleosides. In the present work, studies were conducted to determine how the structural context affects OG oxidation to the hydantoins. These studies revealed a trend in which the Sp yield was greatest in unencumbered contexts, such as nucleosides, while the Gh yield increased in oligodeoxynucleotide (ODN) contexts or at reduced pH. Oxidation of oligomers containing hydrogen-bond modulators (2,6-diaminopurine, N(4)-ethylcytidine) or alteration of the reaction conditions (pH, temperature, and salt) identify base stacking, electrostatics, and base pairing as the drivers of the key intermediate 5-hydroxy-8-oxo-7,8-dihydroguanine (5-HO-OG) partitioning along the two hydantoin pathways, allowing us to propose a mechanism for the observed base-pairing effects. Moreover, these structural effects cause an increase in the effective pK(a) of 5-HO-OG, following an increasing trend from 5.7 in nucleosides to 7.7 in a duplex bearing an OG·C base pair, which supports the context-dependent product yields. The high yield of Gh in ODNs underscores the importance of further study on this lesion. The structural context of OG also determined its relative reactivity toward oxidation, for which the OG·A base pair is ~2.5-fold more reactive than an OG·C base pair, and with the weak one-electron oxidant ferricyanide, the OG nucleoside reactivity is >6000-fold greater than that of OG·C in a duplex, leading to the conclusion that OG in the nucleoside pool should act as a protective agent for OG in the genome.

Domino synthesis of 1,3,5-trisubstituted hydantoins: a DFT study

The mechanism of the reaction between carbodiimides and activated α,β-unsaturated carboxylic acids yielding fully substituted hydantoins and variable amounts of N-acyl urea by-products was studied using density functional theory calculations. Two alternative pathways featuring N-acyl ureas and imino-oxazolidinones as intermediates for the formation of the hydantoin product were taken into account. The results obtained using two different computational models indicate that the overall barriers are similar for the two pathways considered. In all cases, inclusion of a second molecule of carboxylic acid was required to mediate tautomerizations and rearrangement steps. The calculations successfully reproduce the experimentally observed regioselectivity with respect to both N-acyl urea and hydantoin products.

An iminophosphorane-based approach for the synthesis of spiropyrrolidine–imidazole derivatives

A method based on the reaction of an E-phosphazide, an intermediate in the Staüdinger reaction between triphenylphosphine and an azide, with heterocumulenes allows the one-pot, two-component synthesis of a number of pyrrole–imidazole derivatives. The procedure, which involves sequential treatment of the appropriate α-azido ester with triphenylphosphine and isocyanate leads to the hydantoin product after aqueous work-up. The cyclization conditions can also be adapted for the synthesis of thiohydantoins by using isothiocyanates. These hybrids pyrrole–thiohydantoins undergo a novel oxidative spirocyclization by action of DDQ to give a tricyclic derivative (pyrrole–pyrrolidine–imidazole), which displays an interesting cytotoxic activity.

Solid-phase synthesis of α-substituted proline hydantoins and analogs

The manual solid-phase preparation of racemic α-substituted bicyclic proline hydantoins and analogs, which can potentially contain up to four sites of structural diversity (ring size and substitution on the ring or at the ring fusion), is described. Key steps involved alkylation of aldimines of resin-bound amino acids with α,ω-dihaloalkanes and intramolecular displacement of the halide to generate α-substituted prolines and homologs. After formation of resin-bound ureas by reaction of these sterically-hindered secondary amines with isocyanates, base-catalyzed cyclization/cleavage yielded the desired hydantoin products.

Oxidation of 5-hydroxy-2'-deoxyuridine into isodialuric acid, dialuric acid, and hydantoin products.

Oxidation products of cytosine, including 5-hydroxycytosine and 5-hydroxyuracil, are highly susceptible to subsequent oxidation. Here, the oxidation products of 5-hydroxy-2'-deoxyuridine have been studied by NMR and MS analyses. The initial products were diastereomers of isodialuric acid nucleoside. These products subsequently decomposed into corresponding dialuric acid derivatives at neutral pH. The position of the carbonyl and hydroxyl groups, at C5 and C6 for isodialuric acid and at C6 and C5 for dialuric acid derivatives, respectively, was determined by 1H- and 13C NMR analyses. In addition, these analyses revealed that the carbonyl groups of both isodaluric and dialuric acid derivatives exist in their fully hydrated form in aqueous solution. Finally, the dialuric acid derivatives were observed to undergo subsequent decomposition into the corresponding 5-hydroxyhydantoin derivatives. Studies of a trinucleotide containing 5-hydroxyuracil suggest that the reactions described herein for the monomer can be extrapolated to DNA.

Structure and potential mutagenicity of new hydantoin products from guanosine and 8-oxo-7,8-dihydroguanine oxidation by transition metals.

In vitro work in this laboratory has identified new DNA lesions resulting from further oxidation of a common biomarker of oxidative damage, 8-oxo-7,8-dihydroguanine (OG). The major product of oxidation of OG in a nucleoside, nucleotide, or single-stranded oligodeoxynucleotide using metal ions that act as one-electron oxidants is the new nucleoside derivative spiroiminodihydantoin (Sp). In duplex DNA an equilibrating mixture of two isomeric products, guanidinohydantoin (Gh) and iminoallantoin (Ia), is produced. These products are also formed by the overall four-electron oxidation of guanosine by photochemical processes involving O(2). DNA template strands containing either Sp or Gh/Ia generally acted as a block to DNA synthesis with the Klenow exo(-) fragment of pol I. However, when nucleotide insertion did occur opposite the lesions, only 2'-deoxyadenosine 5-triphosphate and 2'-deoxyguanine 5-triphosphate were used for primer extension. The Escherichia coli DNA repair enzyme Fpg was able to remove the Sp and Gh/Ia lesions from duplex DNA substrates, although the efficiency was depended on the base opposite the lesion.

Characterization of hydantoin products from one-electron oxidation of 8-oxo-7,8-dihydroguanosine in a nucleoside model.

Use of one-electron oxidants such as Na(2)IrCl(6) to oxidize 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) residues in oligodeoxynucleotides was previously shown to lead to predominant formation of a base lesion of mass M - 10 compared to starting material [Duarte et al. (1999) Nucleic Acids Res. 27, 596-502]. To thoroughly characterize the structure of this lesion, the oxidation of the nucleoside 9-N-(2',3',5'-tri-O-acetyl-beta-D-erythro-pentanosyl)-8-oxo-7,8-dihydroguanine with one-electron oxidants at pH 2-4 was used as a model for duplex DNA oxidation of OG residues. (1)H NMR and H,H COSY NMR studies in CD(3)OD along with LC-ESI-MS/MS fragmentation analysis are consistent with the assignment of the M - 10 species as a mixture of two pH-dependent equilibrating isomers, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) nucleoside, both present as mixtures of epimers at the C5 position of the hydantoin ring, i.e., four total isomers are formed. The Gh/Ia mixture is formed from hydration and decarboxylation of the initially formed intermediate 5-hydroxy-8-oxo-7,8-dihydroguanosine, a species that is also produced by four-electron oxidation (e.g., singlet oxygen) of guanosine. The product mixture can be further oxidized to a species designated Ia(ox), a hydrolytically unstable material at pH 7 that has been characterized by ESI-MS and (1)H NMR. Competition studies with 8-oxo-7,8-dihydroadenosine placed the redox potential of Gh/Ia at about 1.0 V vs NHE. These studies provide important information concerning the structures of lesions obtained when OG, a "hot spot" for oxidative damage, serves as a "hole trap" in long-range electron-transfer studies.

Radiation chemistry of d(ApCpGpT).

The radiation chemistry of the DNA tetranucleoside triphosphate d(ApCpGpT) was investigated. Irradiations were carried out on aqueous solutions saturated with oxygen (with and without added Cu++), nitrogen or nitrous oxide. When oxygen was present, principal products were formed by hydroxylation at the 8-position of guanine and by degradation of thymine leaving a formamido remnant. Products were also formed containing both of the aforementioned lesions at adjacent deoxyguanosine and pyrimidine nucleosides. Other products resulted from rearrangement of the thymine ring generating two diastereoisomers of the 5-methyl-5-hydroxyhydantoin modification of d(ApCpGpT). Rearrangement of the cytosine ring occurred generating imidazolidine products and a hydantoin product. The product profiles are similar when either an N2O or N2 gaseous environment is maintained. However, in the latter case the dihydrothymine modifications of d(ApCpGpT) are markedly enhanced. Other products include an 8,5' cyclized product formed from the 2'-deoxyadenosine nucleoside and the 8-hydroxyguanine modification. 6-Hydroxy-5,6-dihydrothymine and 5,6-dihydroxy-5,6-dihydrothymine modifications of the thymidine nucleoside were also observed. A strand break product formed in oxygenated solution is also produced in nitrous oxide saturated solutions. Scission of the deoxyadenosine terminus was also observed. The effect of several of these lesions on d(ApCpGpT) as substrate for nuclease P1, bovine spleen phosphodiesterase and snake venom phosphodiesterase was studied.

Aqueous stability and solubility of CI-988, a novel "dipeptoid" cholecystokinin-B receptor antagonist.

The aqueous solubility and solution stability of the N-methylglucamine and sodium salts of CI-988 (CI-988 NMG and CI-988 Na) were evaluated to aid in the development of a parenteral formulation for preclinical and clinical testing. CI-988 ([R-(R*,R*)]-4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2- [[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)- carbonyl]amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) is a selective "dipeptoid" cholecystokinin-B receptor antagonist. The shape of the pH-solubility profile, generated at 30 degrees C, is consistent with the ionization of the terminal carboxyl group (pKa of 4.34). The pH-rate profile is independent of the salt form and is well described by two reaction pathways: spontaneous or water-catalyzed degradation of the nonionized form and specific base-catalyzed catalyzed degradation of the ionized form. The primary mechanism of degradation from the former pathway is consistent with intramolecular, carboxyl-assisted, amide-bond cleavage, whereas the primary mechanism of degradation from the latter pathway appears to be intramolecular cyclization to a hydantoin product with expulsion of 2-adamantanol. The pH dependencies of the solubility and stability show that a simple aqueous buffered solution of CI-988 has a predicted t90 of 2.1 years and a solubility of 0.94 mg/ml at pH 6.5, the theoretical pH of maximum stability, and 30 degrees C.

Preparative High-Performance Liquid Chromatography and Preparative Thin-Layer Chromatography Isolation of Tocainide Carbamoyl-Glucuronide: Structural Characterization by Gas Chromatography-Mass Spectrometry and Fast Atom Bombardment-Mass Spectrometry

Tocainide carbamoyl-O-β-D-glucuronide, a major urinary metabolite of the antiarrhythmic drug tocainide [2-amino-N-(2′, 6′-xylyl)propanoxylidide], was isolated by preparative-TLC and preparative-HPLC. The isolated glucuronide was hydrolyzed in sodium hydroxide (pH > 12) to 3-(2′, 6′-xylyl)-5-methylhydantoin. This hydantoin product was also identified when tocainide was reacted with urea in urine. Structural characterization of the isolated tocainide glucuronide was carried out using GC-MS of the permethylated derivative. The molecular ion of the permethylated glucuronide was not observed, but ion fragments at m/z 232(244), 277(288), and 334(349) were found to correspond to the postulated novel carbamoyl ester structure of the permethylated (perdeuteromethylated) glucuronide. Structural evidence for the underivatized tocainide glucuronide was obtained using fast atom bombardment-MS. The [M+H]+ ion at m/z413 was observed. Characteristic sodium ion adducts [M+Na]+ and [M−H+2Na]+ were also observed at m/z435 and 457, respectively.

1 Final Report on the Safety Assessment of DMDM Hydantoin

DMDM Hydantoin is a preservative, which is used in cosmetic products at concentrations up to 1%. This ingredient is a formaldehyde donor containing up to 2% of the free aldehyde in equilibrium with the hydantoin. When 14C-DMDM Hydantoin was applied to the middorsal area of Sprague-Dawley rats, more than 98% of the recovered radioactivity was confined to the dose site. The LD50 dermal and oral toxicity of DMDM Hydantoin was greater than 2 g/kg. No significant toxic effects were noted in a subchronic oral toxicity study. In skin irritation studies using product formulations, results ranged from non-irritating to moderate skin irritation. At most, transient minimal irritation was noted in albino rabbits treated with DMDM Hydantoin formulations. The mutagenicity of DMDM Hydantoin formulations varies in accordance with the test system. The ingredient was not mutagenic in one Salmonella/microsome assays but was in another. Both positive and negative mutagenic activities were reported when DMDM Hydantoin was tested in the L5178y TK± mouse lymphoma assay. The ingredient was mutagenic in the chromosome aberrations and unscheduled DNA synthesis assays but was not mutagenic in unscheduled DNA synthesis and DNA strand breaks/crosslinks assays. A comparison of Ames test results from studies of DMDM Hydantoin product and formaldehyde indicates a similar number of reversants per formaldehyde equivalent. In clinical studies, skin irritation ranged from none to observations of intense erythema and edema when various formulations containing DMDM Hydantoin were applied. DMDM Hydantoin formulations did not induce sensitization in some clinical studies. DMDM Hydantoin formulations were neither phototoxic nor photoallergenic. Use of DMDM Hydantoin at its current concentration of use in cosmetic products would not expose the consumer to levels of formaldehyde above the limit previously considered as acceptable in cosmetic products. Based on the available data included in this report, it is concluded that DMDM Hydantoin is safe as a cosmetic ingredient in the present practices of use.