A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism.

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N(1)-acetylspermidine (N(1)AcSpd), N(8)-acetylspermidine (N(8)AcSpd), N(1)-acetylspermine, N(1),N(8)-diacetylspermidine, and N(1),N(12)-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N(1)AcSpd and N(8)AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with (13)C(2)-N(1)AcSpd and (13)C(2)-N(8)AcSpd which have the (13)C(2)-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N(1)-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N(1)-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-(15)N(3)]-N(1)-acetylspermine and [1,4,8-(15)N(3)]spermidine ((15)N(3)-Spd), respectively; for SMO, [1,4,8,12-(15)N(4)]spermine and (15)N(3)-Spd, respectively; and for SSAT, (15)N(3)-Spd and [1,4,8-(15)N(3)]-N(1)-acetylspermidine, respectively.

Efficient identification and quantification of peptides containing nitrotyrosine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after derivatization.

Protein nitration at tyrosine residues proceeds to form 3-nitrotyrosine; this product is today used as a biomarker of nitrative stress. We have developed an efficient method with which to identify nitrated peptides and to quantify protein nitration levels in different biological samples by a combination of the chemical derivatization of nitrotyrosine-containing peptides and mass spectrometry. Our strategy includes: 1) the protection of both N-terminal amines and epsilon-amines of lysine residues by acetylation with (13)C(0)/(13)C(4)- or D(0)/D(6)-acetic anhydride; 2) the reduction of nitrotyrosine to aminotyrosine with sodium hydrosulfite; and 3) the derivatization of aminotyrosine with 1-(6-methyl[D(0)/D(3)]nicotinoyloxy)succinimide. The utility of our method is demonstrated with nitrotyrosine-containing angiotensin II and bovine serum albumin as the model compounds.

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry.

A method for the quantification of acetylpolyamines, N(1),N(12)-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N(1),N(8)-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using (15)N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of alpha,omega-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N(1)-acetylspermidine and N(8)-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).

Syntheses of water-soluble [60]fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells.

Water-soluble [60]fullerene (C(60)) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C(60) derivatives together with some reported ones were tested. Among the compounds tested, C(60)/(gamma-CyD)(2), C(60)-bis(gamma-CyD) (5) containing C(60)-mono(gamma-CyD) (5'), and C(60)/PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.

Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on beta-elimination of the phosphate group from serine/threonine phosphorylation sites under alkaline conditions, followed by Michael addition reaction with N-(2-mercaptoethyl)-6-methylnicotinamide (MEMN). As a result of the derivatization reaction, the negatively charged phosphate group is substituted with the nicotinoyl moiety to improve the ionization efficiency of the derivatized peptide. The combination of d(3)-labeled MEMN (d(3)-MEMN) and MEMN (d(0)-MEMN) generates a 3 Da mass difference between d(3)-MEMN-labeled and d(0)-MEMN-labeled peptides, which is a useful signature for the identification of peptides containing serine/threonine phosphorylation sites in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum. Moreover, the mass difference is useful for the quantitative analysis of serine/threonine phosphorylation in proteins. In this paper, we describe the synthesis of d(0)/d(3)-labeled MEMN and an application of our approach to model peptides and proteins.

Preparation of C60-based active esters and coupling of C60 moiety to amines or alcohols.

We report the syntheses of C(60)-based active esters and the coupling of their C(60) moiety to various amines or alcohols. Methano[60]fullerene carboxylic acid was activated by esterification with N-hydroxysuccinimide (NHS) or pentafluorophenol (PFP) and the active esters were isolated. Reactions of the active esters with amines or alcohols proceeded easily to give a variety of compounds having the C(60) moiety.

Efficient identification and quantification of proteins using isotope-coded 1-(6-methylnicotinoyloxy)succinimides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a convenient and useful method for the identification and relative quantification of proteins using light and heavy reagents, 1-(6-methylnicotinoyloxy)succinimides (6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS, respectively). This method is based on the chemical derivatization of amino groups of tryptic peptides with these reagents, i.e., the basic moiety of the reagents thus incorporated into both the N-terminal amino group and the epsilon-amino group of the lysine residue would improve the ionization efficiency of tryptic peptides. An increase in protein sequence coverage is achieved by derivatization with these reagents or by combination of mass values before and after derivatization. Since a combination of 6-CH(3)-Nic-NHS and d(3)-labeled reagent (6-CD(3)-Nic-NHS) generates a 3 Da mass difference per reaction site, the d(3)-labeled reagent shifts the mass values of d(0)-labeled peptides according to the number of reactive amino groups in the peptides. In the case of tryptic peptides, the mass values of C-terminal arginine and lysine peptides are shifted by 3 and 6 Da, respectively. Further, the 3 Da mass difference between 6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS offers a means for the relative quantification of protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Electrospray ionization and time-of-flight mass spectrometric method for simultaneous determination of spermidine and spermine.

A sensitive method for the determination of polyamines in mammalian cells was described using electrospray ionization and time-of-flight mass spectrometer. This method was 50-fold more sensitive than the previous method using ionspray ionization and quadrupole mass spectrometer. The method employed the partial purification and derivatization of polyamines, but allowed a measurement of multiple samples which contained picomol amounts of polyamines. Time required for data acquisition of one sample was approximately 2 min. The method was successfully applied for the determination of reduced spermidine and spermine contents in cultured cells under the inhibition of aminopropyltransferases. In addition, a new proper internal standard was proposed for the tracer experiment using (15)N-labeled polyamines.

Balancing risks.

Regulatory policies designed to reduce the health risk of environmental and/or synthetic chemicals generally aim for zero or negligible levels. Foods, on the other hand, especially those with a long history in the human diet, have been treated as essentially safe, even though they too contain various chemicals including nutrients. The recent debate on the presence in food of acrylamide, a possible human carcinogen, is likely to shake up the traditional paradigm held by regulatory agencies on chemical health risks. The current stance on the safety of acrylamide in food seems to be an extension of the traditional approach to assessment of environmental and/or synthetic chemicals. However, even foods which have long been a part of the human diet contain components that do not necessarily meet the safety margins applied to environmental and/or synthetic chemicals. In the future, a greater understanding of the effect of these agents on biological systems as well as the development of analytical methods for testing will result in many questions being raised concerning chemicals in foods, such as acrylamide which is under scrutiny today. Regulatory policies currently employ various standards for controlling chemical risk. These standards are dependent upon the labeling of the chemical in question, e.g., whether carcinogenic or non-carcinogenic, synthetic or natural, or whether a food or industrial chemical. Regardless of labeling, all chemicals to which we are exposed should be evaluated on an equal footing. Then, according to the level of the identified health risk, regulations could or could not be applied based on local circumstances, e.g., public acceptance, voluntary risk vs. involuntary risk, etc. In order to create a standardized system for chemical risk assessment, the introduction of uniform measures is essential. Loss of life expectancy (LLE) is one possible measure to assess chemical health risk. When LLE has been used, animal toxicity data have indicated that an ad libitum diet intake has considerably more impact on health risk than the acrylamide concentration of the ingested food. Reassessing the health effects of chemicals with a system of uniform measures could reveal many risks that need to be preferentially addressed above and beyond keeping minor toxicants to zero or negligible levels. Recognition of such risks may result in changes that conflict with existing regulations. In any case, whether consciously or unconsciously, people have always been exposed to a certain degree of chemical risk in their daily life. Based on the premise that the public can accept some degree of chemical risk in balance with other risks in their lives, regulatory bodies should be able to take a flexible and effective approach. In order to efficiently and comprehensively maximize the protection of our health against potential harm from chemicals using limited public resources, it is now time for regulatory agencies to restructure their policy frameworks across categories for controlling chemical health risks.

Synthesis of oligodeoxynucleotides containing a single 6alpha- or 6beta-diastereoisomer of N2-(estradiol-6-yl)-2'-deoxyguanosine.

DNA damage induced by estrogens is associated with developing breast, ovary, and endometrial cancers. The quinone of 2-hydroxyestrogen (2-OHE), a major estrogen metabolite, produces 2-OHE-derived dG and dA adducts in DNA. N(2)-[Estradiol-6(alpha or beta)-yl]-2'-deoxyguanosine [dG-N(2)-6(alpha or beta)-E(2)] lacking a 2-OH moiety may also be formed through sulfonation of 6-hydroxyestrogen. To explore the biological properties of such estrogen-DNA adducts, oligodeoxynucleotides modified by estrogen-derived DNA adduct were prepared by chemical synthesis. Initially, 6alpha- and 6beta-aminoestradiol 17-acetate (6alpha- and 6beta-NH(2)-E(2) 17Ac) were prepared by reductive amination of 6-oxo-estradiol 3,17-diacetate. The DMT-phosphoramidite derivative of N(2)-(3,17-diacetoxyestradiol-6alpha-yl)-2'-deoxyguanosine and its 6beta-isomer were prepared by coupling 5'-O-(4,4'-dimethoxytrityl)-2-fluoro-O(6)-[2-(4-nitrophenyl)ethyl]-2'-deoxyinosine separately with 6alpha- and 6beta-forms of NH(2)-E(2) 17Ac, respectively, followed by selective acetylation of the steroidal 3-hydroxyl group. The desired oligodeoxynucleotide containing a single dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) was prepared efficiently by an automated DNA synthesizer. Synthesis of these site-specifically modified oligodeoxynucleotides will benefit further research into the biological properties and three-dimensional structure of 6alpha- and 6beta-diastereoisomers of estrogen-DNA adducts.

Design, synthesis, and biological activity of novel PPARgamma ligands based on rosiglitazone and 15d-PGJ2.

To develop novel PPARgamma ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which are designed based on the structures of rosiglitazone and 15d-PGJ2. Among these compounds, compound 9 was found to be as potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARgamma protein where the alkyl chain of 15d-PGJ2 is expected to be located.

1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1-amino-4-phenylpyridinium salt, the 1-amino analogues of neurotoxins, MPTP and MPP+, induce apoptosis in PC12 cells: detection of apoptotic cells by Comet assay and flow cytometric analysis.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to induce parkinsonism in humans when it is oxidized to the 1-methyl-4-phenylpyridinium salt (MPP+). We previously reported the syntheses of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine (APTP) and 1-amino-4-phenyl-pyridinium salt (APP+), the 1-amino analogues of the dopaminergic neurotoxins, MPTP and MPP+, respectively, and demonstrated that both APTP and APP+ are cytotoxic to PC12 cells. In this study, we found that both APTP and APP+ induce apoptotic cell death in PC12 cells. Apoptosis was determined by the Comet assay and flow cytometric analysis. Prior to using the Comet assay for detection of apoptotic PC12 cells, Comet images of apoptotic and necrotic cells were first distinguished by using several standards. Comet images were classified into four groups (A to D) according to their shapes. Class D consisted of the apoptotic cells and was easily distinguished. We also demonstrated that apoptotic and necrotic PC12 cells can be easily differentiated and quantified using the convenient Comet assay.

Product analyses of ozone mediated nitration of benzimidazole derivatives with nitrogen dioxide: formation of 1-nitrobenzimidazoles and conversion to benzotriazoles.

Several benzimidazole derivatives having electron-withdrawing or -donating substituent(s) at the benzene moiety were used as models of the imidazole moiety of purine bases and their nitration with nitrogen dioxide and ozone (so-called Kyodai nitration) were examined. Products were extracted from the reaction mixture with AcOEt and their structures were analyzed. 1-Nitrobenzimidazole derivatives and unexpected 1-nitrobenzotriazole derivatives were identified. Although the yields of 1-nitrobenzimidazole derivatives were quite low, these were all new compounds that could be obtained only by Kyodai nitration. It was speculated that benzotriazoles were formed via 1-nitrobenzimidazoles and subsequent nitration toward benzotriazoles resulted in the formation of 1-nitrobenzotriazoles.

Thiol-based SAHA analogues as potent histone deacetylase inhibitors.

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.

Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition, and binding mode study of SAHA-based non-hydroxamates.

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i). substrate (acetyl lysine) analogues (compounds 3-7), (ii). analogues bearing various functional groups expected to chelate zinc ion (compounds 8-15), and (iii). analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16-18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.

Preparation of deuterated methyl and dimethyl substituted nicotinoylating agents for derivatization of the N-terminal of protein.

Methyl groups of 6-methylnicotinic acid and 2,6-dimethylnicotinic acid were deuterated by an H-D exchange reaction under conditions of 1% NaOD/D(2)O on heating. With a condensation reaction between the D-labeled nicotinic acid derivative and N-hydroxysuccinimide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the nicotinoylating agents, 1-(6-methyl[D(3)]nicotinoyloxy)succinimide (2c) and 1-(2,6-dimethyl[D(6)]nicotinoyloxy)succinimide (2f) were prepared. Both D-labeled nicotinoylating agents and their unlabeled counterparts quantitatively modified the N-terminal of protein.

Formation of 2'-deoxy-2-nitroadenosines by reaction of 2'-deoxyadenosines with copper(II) nitrate/acetic anhydride.

Nitration of 9-substituted [ethyl, (Ac)2-2'-deoxyribosyl, (Ac)3-ribosyl] N6-acetyladenine derivatives with Cu(NO3)2.3H2O/Ac2O was examined. Nitration proceeded at the 2-position, although the yield was low. Removal of the acetyl groups gave 2'-deoxy-2-nitroadenosine derivatives.