Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.

A mechanism for posttranslational modifications of proteins by yeast protein farnesyltransferase.

Protein farnesyltransferase catalyzes the alkylation of cysteine in C-terminal CaaX sequences of a variety of proteins, including Ras, nuclear lamins, large G proteins, and phosphodiesterases, by farnesyl diphosphate (FPP). These modifications enhance the ability of the proteins to associate with membranes and are essential for their respective functions. The enzyme-catalyzed reaction was studied by using a series of substrate analogs for FPP to distinguish between electrophilic and nucleophilic mechanisms for prenyl transfer. FPP analogs containing hydrogen, fluoromethyl, and trifluoromethyl substituents in place of the methyl at carbon 3 were evaluated as alternative substrates for alkylation of the sulfhydryl moiety in the peptide dansyl-GCVIA. The analogs were alternative substrates for the prenylation reaction and were competitive inhibitors against FPP. A comparison of kcat for FPP and the analogs with ksolv, the rate constants for solvolysis of related p-methoxybenzenesulfonate derivatives, indicated that protein prenylation occurred by an electrophilic mechanism.

CC-1065 and the duocarmycins: unraveling the keys to a new class of naturally derived DNA alkylating agents.

Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined.

Estudos visando à síntese de sesquiterpenos bacanos

Nesta tese, efetuamos estudos visando à síntese de sesquiterpenos bacanos, cuja etapa chave consistiu na construção do sistema cis-hidrindânico, através de reação de contração de anel de cis-octalinas e 2-octalonas mediada por trinitrato de tálio (TTN). Apenas as cis-octalinas como, por exemplo, o cis-4a-metil-l,2,3,4,4a,5,8,8a-octahidronaftaleno e o cis-4a, 7-dimetil-l,2,3,4,4a,5,8,8a-octa-hidronaftaleno, foram passíveis de reação de contração de anel em rendimentos satisfatórios; já a cis-5,10-dimetil-l(9)-octal-2-ona levou ao produto de contração em baixo rendimento. Tentamos utilizar a reação de cis-4a-metil-l,2,3,4,4a,5,8,8a-octa-hidronaftaleno com TTN na síntese da nor-baquenolida-A, porém não conseguimos completar a síntese desta, pois não foi possível efetuar a última etapa sintética, nas várias abordagens testadas. Grandes esforços também foram empregados na preparação diastereosseletiva da cis-5,10-dimetil-l(9)-octal-2-ona através de três abordagens diferentes que foram investigadas, sendo duas delas com êxito. Contudo, o baixo rendimento (38%) da etapa de contração de anel da cis-5,10-dimetil-l(9)-octal-2-ona não permitiu a continuação da rota sintética proposta para a baquenolida-A. Também realizamos a resolução cinética de três diferentes cis-octalóis que foram preparados através da reação de Diels-Alder seguida de redução diastereosseletiva - com a lipase Novozym 435, e os produtos resolvidos foram obtidos em excelentes rendimentos isolados (≥ 40% para cada enantiômero) e excelentes excessos enantioméricos (≥ 98%).

Domino 1,3-Dipolar Cycloadditions of N-Alkyl-α-Amino Esters with Paraformaldehyde: A Direct Access to α-Hydroxymethyl α-Amino Acids

N-Alkyl-α-amino esters undergo a domino reaction, based on the iminium cation generation, with paraformaldehyde, followed by a 1,3-dipolar cycloaddition of the stabilized azometh­ine ylide with another equivalent of formaldehyde. The resulting products are oxazolidines, which can be transformed after hydrolysis into α-hydroxymethyl α-amino acid or its derivatives. The diastereoselective 1,3-dipolar cycloaddition was performed using sarcosine (–)-menthyl or (–)-8-phenylmenthyl esters affording the cyclic product with moderate enantiomeric ratio.

Asymmetric 1,3-Dipolar Cycloadditons of Stabilized Azomethine Ylides with Nitroalkenes

This review highlights the biological importance of many polysubstituted nitro-prolines and -pyrrolidines. Their preparation using asymmetric 1,3-dipolar cycloadditions of azomethine ylides with nitroalkenes using diastereoselective and enantioselective strategies is described remarking the scope and main features of each one.

Ablative Pyrolysis of Beetle-killed Trees

Thesis (Ph.D.)--University of Washington, 2016-06

Immobilization of aluminum chloride on MCM-41 as a new catalyst system for liquid-phase isopropylation of naphthalene

A great deal of effort has been made at searching for alternative catalysts to replace conventional Lewis acid catalyst aluminum trichloride (AlCl3). In this paper, immobilization of AlCl3 on mesoporous MCM-41 silica with and without modification was carried out. The catalytic properties of the immobilized catalyst systems for liquid-phase isopropylation of naphthalene were studied and compared with those of H/MCM-41 and H/mordenite. The structures of the surface-immobilized aluminum chloride catalysts were studied and identified by using solid-state magic angle spinning nuclear magnetic resonance (MAS NMR), Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), nitrogen adsorption, and X-ray diffraction (XRD) techniques. The catalytic activity of the immobilized catalysts was found to be similar to that of acidic mordenite zeolite. A significant enhancement in the selectivity of 2,6-diisopropylnaphthalene (2,6-DIPN) was observed over the immobilized aluminum chloride catalysts. Immobilization of aluminum chloride on mesoporous silica coupled with surface silylation is a promising way of developing alternative catalyst system for liquid-phase Friedel-Crafts alkylation reactions. (C) 2002 Elsevier Science B.V. All rights reserved.

Calibration of the channel that determines the omega-hydroxylation regiospecificity of cytochrome P4504A1 - Catalytic oxidation of 12-halododecanoic acids

The fatty acid omega-hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in (H2O)-O-18 show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O-2. The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an omega-1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the omega-aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[H-2](2)-12-chlorododecanoic acid causes a 2-3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl >> I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 angstrom but smaller than 4.30 angstrom.

Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands

A novel synthetic approach towards N1-alkylated 3-propyl-1,4-benzodiazepines was developed in five synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as a key intermediate. The structure-activity relationship optimization of this 3-prophyl-1,4-benzodiazepine template was carried out on the N1-position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK2) receptor. The N-allyl-3-propyl-benzodiazepine 6d displayed an affinity towards the CCK2 (CCK-B) receptor of 170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-1,4-benzodiazepine 6d was subsequently determined in in-vivo psychotropic assays. This novel ligand had ED50 values of 4.7 and 5.2 mg kg-1 in the black and white box test and the x-maze, respectively, and no significant sedation/muscle relaxation was observed.

Synthesis and evaluation of asperlicin analogues as non-peptidal cholecystokinin-antagonists

The SAR of Asperlicin analogues is reported, leading to bioactive 1,4-benzodiazepine-2-ones, which were prepared in a 3 step reaction sequence. The Asperlicin substructure was built up using Tryptophan and readily available 2-amino-acetophenones. This template, containing a 1,4-benzodiazepin-2-one moiety with a 3-indolmethyl side chain, was transformed into mono- and di-substituted 3-indol-3 '-yl-methyl-1,4-benzodi-azepine-2-ones by selective alkylation and acylation reactions. The SAR optimization of the 1,4-benzodiazepine scaffold has included variations at the 5-, 7-, 8-position, at the N1, N-indole nitrogen and the configuration of the C3-position. The most active Asperlicin analogue, having an IC50 of 1.6 microM on the CCKA receptor subtype, was obtained from Tryptophan in only 3 steps in an overall yield of 48%.

Organic reactions in ionic liquids: ionic liquid-accelerated facile synthesis of 3-alkyl-2,4-thiazolidinediones

The room temperature ionic liquid [bmim]PF6 is a new green solvent for the N-alkylation of 2,4-thiazolidinones. Significant rate enhancement and improved yields have been observed.

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.