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PubMed1988

Effects of reductive alkylation on peptide retention times.

Reductive alkylation of primary amino groups is used to introduce nuclear magnetic resonance or radioactive probes into proteins. Because of electrostatic and conformational effects, reductive alkylation is not always complete. We describe herein a rapid high-performance liquid chromatographic method for separating and quantitating mixtures of native and reductively alkylated peptides. Small synthetic peptides were chosen to illustrate the effects of methylation and isopropylation of primary amino groups on chromatographic retention times. Mixtures of unmodified and reductively methylated or isopropylated peptides (Gly-Leu-Tyr, Gly-Gly-Lys-Arg, Arg-Lys-Asp-Val-Tyr and Pro-Gly-Lys-Ala-Arg) could be separated. Chromatography was on a 5-micron, 25 cm x 0.4 I.D., C18 reversed-phase column with 0.1% trifluoroacetic acid in a 10 to 80% linear gradient of acetonitrile in water, a system appropriate for protein digests. The relative concentrations of native, and singly and doubly alkylated peptide were determined as well as the effective retention coefficients for dimethyl and isopropyl groups. The method shows promise for the peptide mapping of partially alkylated proteins.

Alkylation↗
PubMed1997

New tester strains of Salmonella typhimurium lacking O6-methylguanine DNA methyltransferases and highly sensitive to mutagenic alkylating agents.

Salmonella typhimurium YG7104 and YG7108 are derivatives of the Ames tester strain TA1535, and have chromosomal deletions of the ogtST gene or both the ogtST and adaST genes, respectively. The ogtST and adaST genes encode O6-methylguanine DNA methyltransferases that are involved in the repair of DNA damage caused by alkylating agents. The sensitivities of these strains to 15 mutagens with different structures were tested and compared with those of the parent strain TA1535. Deletion of ogtST or ogtST plus adaST substantially increased the sensitivity of strain TA1535 to the mutagenicity of alkylating agents, such as N-ethyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate or dimethylnitrosamine (DMN). Preincubation of the chemical with S9 mix and bacteria for 20 min at 37 degrees C before pouring them together on agar plates was not necessary to detect the mutagenicity of DMN when strain YG7104 or YG7108 was used as a tester strain. Introduction of plasmid pKM101 did not enhance but rather decreased the sensitivity of YG7104 and YG7108 to alkylating agents. Since the new strains are highly sensitive only to alkylating agents, they will be useful to detect the mutagenicity with high efficiency and to study the mechanism of mutagenesis induced by environmental alkylating agents.

Alkylating Agents↗
PubMed1999

Methionine reduces spontaneous and alkylation-induced mutagenesis in Saccharomyces cerevisiae cells deficient in O6-methylguanine-DNA methyltransferase.

The exposure of DNA to reactive intracellular metabolites is thought to be a major cause of spontaneous mutagenesis. DNA alkylation is implicated in the above process by the fact that bacterial and yeast cells lacking DNA alkylation-specific repair genes exhibit elevated spontaneous mutation rates. The origin of the intracellular alkylating molecules is not clear; however, S-adenosylmethionine (SAM) has been proposed as one source because it has a reactive methyl group known to methylate proteins and DNA. We supplemented yeast cultures with excess methionine and examined the effects of increased endogenous SAM concentration on spontaneous and alkylation-induced mutagenesis in the absence of various DNA repair pathways. Our results show that either the excess methionine, or the increased SAM produced as a result of this treatment, is able to protect yeast cells from mutagenesis, and that this effect is alkylation-damage-specific. The protective effect was observed only in the mgt1 mutant deficient in the O(6)-methylguanine-DNA repair methyltransferase, but not in the wild type or other DNA repair-deficient strains, indicating that the protection is specific for O-methyl lesions. Thus, our results may lend support to the recently reported chemopreventive effect of SAM in rodents and further suggest that the observed tumor prevention by SAM may be, in part, due to its suppression of spontaneous mutagenesis in mammals. Given that a strong correlation has been established between O(6)-methylguanine and carcinogenicity, this study may offer a novel approach to preventing carcinogenesis.

Alkylation↗
PubMed1999

Amino-alkyl-cyclohexanes are novel uncompetitive NMDA receptor antagonists with strong voltage-dependency and fast blocking kinetics: in vitro and in vivo characterization.

The present study characterized the in vitro NMDA receptor antagonistic properties of novel amino-alkyl-cyclohexane derivatives and compared these effects with their ability to block excitotoxicity in vitro and MES-induced convulsions in vivo. The 36 amino-alkyl-cyclohexanes tested displaced [3H]-(+)-MK-801 binding to rat cortical membranes with K(i)s between 1.5 and 143 microM. Current responses of cultured hippocampal neurones to NMDA were antagonized by the same compounds with a wide range of potencies (IC50s of 1.3-245 microM, at -70 mV) in a use- and strongly voltage-dependent manner (delta 0.55-0.87). The offset kinetics of NMDA receptor blockade was correlated with equilibrium affinity (Corr Coeff. 0.87 P < 0.0001). As an example, MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane HCl) had similar blocking kinetics to those previously reported for memantine (K(on) 10.67 +/- 0.09 x 10(4) M(-1) s(-1), K(off) 0.199 +/- 0.02 s(-1), K(d) = K(off)/K(on) = 1.87 microM c.f. IC50 of 1.29 microM). Most amino-alkyl-cyclohexanes were protective against glutamate toxicity in cultured cortical neurones (e.g. MRZ 2/579 IC50 2.16 +/- 0.03 microM). Potencies in the three in vitro assays showed a relatively strong cross correlation (all corr. coeffs. > 0.72, P < 0.0001). MRZ 2/579 was also effective in protecting hippocampal slices against 7 min. hypoxia/hypoglycaemia-induced reduction of fEPSP amplitude in CA1 with an EC50 of 7.01 +/- 0.24 microM. MRZ 2/579 showed no selectivity between NMDA receptor subtypes expressed in Xenopus oocytes but was somewhat more potent than in patch clamp experiments-IC50s of 0.49 +/- 0.11, 0.56 +/- 0.01 microM, 0.42 +/- 0.04 and 0.49 +/- 0.06 microM on NR1a/2A /2B, /2C and 2/D, respectively. In contrast, memantine and amantadine were both 3-fold more potent at NR1a/2C and NR1a/2D than NR1a/2A receptors. All Merz amino-alkyl-cyclohexane derivatives inhibited MES-induced convulsions in mice with ED50s ranging from 3.6 to 130 mg/kg i.p. The in vivo and in vitro potencies correlated indicating similar access of most compounds to the CNS. MRZ 2/579 administered at 10 mg/kg resulted in peak plasma concentrations of 5.3 and 1.4 microM following i.v. and p.o. administration respectively, which then declined with a half life of around 170-210 min. Analysis of A.U.C. concentrations indicates a p.o./i.v. bioavailability ratio for MRZ 2/579 of 60%. MRZ 2/579 injected i.p. at a dose of 5 mg/kg resulted in peak brain extracellular fluid (ECF) concentrations of 0.78 microM (brain microdialysates). Of the compounds tested MRZ 2/579, 2/615, 2/632, 2/633, 2/639 and 2/640 had affinities, kinetics and voltage-dependency most similar to those of memantine and had good therapeutic indices against MES-induced convulsions. We predict that these amino-alkyl-cyclohexanes, which all had methyl substitutions at R1, R2, and R5, at least one methyl or ethyl at R3 or R4 and a charged amino-containing substitution at R6, could be useful therapeutics in a wide range of CNS disorders proposed to involve disturbances of glutamatergic transmission.

Alkylation↗
PubMed2002

Successful treatment of serpiginous choroiditis with alkylating agents.

OBJECTIVE: To describe the management and long-term outcomes of patients with serpiginous choroiditis treated with alkylating agents. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Nine patients with active, vision-threatening serpiginous choroiditis who had progressive inflammation while on steroids and/or immunosuppressive agents other than alkylating agents treated at three tertiary care uveitis referral centers. METHODS: Patients received systemic immunosuppression with an alkylating agent, either chlorambucil or cyclophosphamide. Prednisone also was given initially and was tapered and discontinued. MAIN OUTCOME MEASURES: Visual acuity, clinical disease activity, duration of treatment, duration of drug-free disease remission, and side effects of alkylating agent therapy. RESULTS: No patients had recurrences while on therapy. No further visual loss was encountered after starting the therapy. Six of the patients regained vision. All but two patients achieved prolonged drug-free remissions, ranging in duration between 15 and 96 months (median, 78 months). Side effects included transient bone marrow suppression, nausea, and fatigue. Secondary malignancy was encountered in one patient, whose carcinoma of the urinary bladder was treated successfully. CONCLUSIONS: Adequate immunosuppression with alkylating agents may favorably alter the long-term prognosis of patients with serpiginous choroiditis.

Adult↗
PubMed2000

Ligand association with the rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y (NPY) receptors shows large subtype-related differences in sensitivity to chaotropic and alkylating agents.

The binding to rabbit kidney or hypothalamic particulates of the subtype-selective neuropeptide Y (NPY) receptor ligands [125I](Leu31,Pro34)hPYY (as Y1 site label at 2 nM human pancreatic polypeptide (hPP)), [125I]-hPYY(3-36) (Y2 label), and [125I]-hPP (Y5 label) displayed great differences in sensitivity to alkylators and chaotropic agents. Sensitivity to a nonionic chaotrope, urea, was much higher for the Y1 binding than for the Y5-like binding or the Y2 binding. The non-selective alkylator N-ethylmaleimide (NEM) and several alkylators selective for aminergic receptors were much more efficacious against the Y1 relative to the Y2 binding. Similar differences could be confirmed with the attachment of Y1 and Y2-selective tracers to CHO cells expressing the cloned guinea-pig Y1 or Y2 receptors. The Y5-like binding was quite insensitive to NEM, but sensitive to chloroethylclonidine (CEC) and prazobind, which were less potent at the Y1, and especially at the Y2 site. The unrestricted-access alkylator 2-aminoethyl methanethiosulfonate inhibited the binding to all subtypes, while the restricted-access agent 2-(trimethylammonium)ethylmethanethiosulfonate poorly inhibited the Y5-like binding, or the guanine nucleotide-insensitive Y2 binding. These results are compatible with an active conformation of the Y5-like site dependent on maintenance of a shared hydrophobic cavity. The Y2 sites resistant to guanosine polyphosphates and restricted-access alkylators were detected mainly in particulates slowly solubilized by cholate at 0-5 degrees C; these sites could be clustered.

Alkylating Agents↗
PubMed2000

Sequence specific alkylation of DNA by hairpin pyrrole-imidazole polyamide conjugates.

BACKGROUND: Pyrrole-imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification. RESULTS: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the gamma turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide-CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation. CONCLUSIONS: The ability of polyamide-CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms.

Alkylation↗
PubMed1998

Mutagenicity of isomeric alkanediazotates, precursors for ultimate alkylating species of carcinogenic N-nitroso compounds.

Alkanediazohydroxides are common key intermediates in carcinogenesis and mutagenesis of N-nitroso compounds, which are widely found in human environment. Mutagenicity of (E)- and (Z)-potassium alkanediazotates, as precursors of corresponding alkanediazohydroxides were evaluated to investigate the effect of geometric isomerism and also the effect of alkyl groups on their biological activity. Mutagenicity of N-nitroso-N-alkylureas which spontaneously produce alkanediazohydroxides after non-enzymatic hydrolysis were also tested in comparison to that of the corresponding diazotates and other activated chemical species of N-nitrosamines. When the mutagenicity was assayed in three microbial strains, Salmonella typhimurium TA1535, and Escherichia coli WP2 and WP2 uvrA, the order of mutagenic potency of the compounds with the same alkyl group was as follows; (E)-diazotates > (Z)-diazotates > nitrosoureas. The effect of alkyl groups on the mutagenic potency was different in Salmonella strain and in E. coli strains, and this result could be explained by the efficiency of O6-alkylguanine-DNA alkyltransferase. In each bacterial strain, this effect of alkyl groups was similar in mutagenicity induced by (E)- and (Z)-diazotates, N-nitroso-N-alkylureas and other activated N-nitrosodialkylamines such as alpha-hydroxy nitrosamines. The geometrical isomerism affected the mutagenicity of (E)- and (Z)-potassium alkanediazotates, and the result suggested that alkanediazohydroxides react through diazonium ions in a cage rather than through free alkyldiazonium ions which have no geometrical isomerism. Our results confirmed that (E)-potassium alkanediazotates, (Z)-potassium alkanediazotates and N-nitroso-N-alkylureas all decomposed through diazohydroxides, and that alkanediazohydroxides are the active alkylating species of N-nitroso compounds, and also that the geometrical isomerism is important for carcinogenic N-nitroso compounds to show their biological activity.

Alkylating Agents↗
PubMed2006

Synthesis and biological properties of sequence-specific DNA-alkylating pyrrole-imidazole polyamides.

In recent years, many diseases including cancer and hereditary and viral diseases have been understood at the DNA sequence level. Direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. N-Methylpyrrole (Py) and N-methylimidazole (Im) polyamides are a new type of small compound that precisely bind to the minor groove of the DNA duplex in a sequence-specific fashion and recruit alkylating agents to the target sequence. We designed and synthesized a series of sequence-specific alkylating Py-Im polyamide conjugates that selectively alkylate predetermined DNA sequences. We have demonstrated that sequence-specific alkylating agents possess gene-silencing activities and a promising potency against human cancer cell lines as well as against xenografts of human cancer cell lines. In this Account, we focus on recent progress in alkylating Py-Im polyamides with regard to sequence specificity and biological activities and the future direction of rational molecular design of genetic switches in the postgenome era is described.

Alkylation↗
PubMedDate not supplied

N-alkylated chitosan as a potential nonviral vector for gene transfection.

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Groove- and sequence-selective alkylation of DNA by sulfonate esters tethered to lexitropsins.

A series of sulfonate esters that are attached to a noncationic minor-groove-binding N-methylpyrrole dipeptide (Lex) related to netrospin have been synthesized. The compounds prepared differ in two respects: (1) the length [(CH2)2 vs (CH2)8] of the tether between the DNA affinity binding portion of the molecule and the sulfonate ester and (2) whether a methyl group [MeOSO2(CH2)n-Lex] or the dipeptide including the aliphatic tether [MeSO2O(CH2)n-Lex] is covalently transferred to the DNA. The DNA-cleavage patterns of these bimolecular alkylating compounds have been mapped in 32P-end-labeled restriction fragments using neutral thermal hydrolysis and alkali treatment to expose single-strand breaks at bases with thermally labile modifications. In contrast to the alkylation of DNA by simple alkyl alkanesulfonate esters, that predominantly yield major-groove alkylation at N7-guanine, the modification of DNA by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex occurs primarily at N3-adenine residues associated with previously footprinted Lex DNA affinity binding regions. The ratio for the formation of N3-methyladenine (minor groove) to N7-methylguanine (major groove) in calf thymus DNA is 1:7 for dimethyl sulfate, while only the former adenine product is observed with MeSO2O(CH2)n-Lex indicating the change in groove specificity. DNA cleavage by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex is efficiently inhibited by the coaddition of distamycin; however, only the DNA damage generated by the latter is blocked by NaCl. As expected, increasing the length of the (CH2)n tether from n = 2 to n = 8 moves the alkylation site by 1-2 base pairs further from the affinity binding domain. Finally, a comparison of the methylation patterns of MeOSO2(CH2)n-Lex as a function of tether length provides an insight into Lex sequence and orientational preferences.

Alkanesulfonates↗
PubMed1992

Recognition of specific DNA sequences by mitomycin C for alkylation.

Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3' to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C greater than T greater than A).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1991

In vitro transcription analysis of DNA alkylation by nitrogen mustard.

A synchronized in vitro transcription assay has been used to probe the sequence specificity of alkylation of DNA by nitrogen mustard. Transcriptional blockages were detected with use of a 497-base-pair PvuII/SalI restriction fragment of a modified pBR322 vector when initiation of transcription was commenced after the DNA had been alkylated but not if the initiated transcription complex was subjected to alkylation before the elongation phase. The intensity of transcriptional blockages increased with alkylation time and was maximal after 1.5 h at a mustard concentration of 200 microM. There was also evidence of alkylation of the promoter region with increasing mustard concentration. The transcriptional blockage pattern changed at some sites as elongation time was increased and three types of blockages were observed-partial transcription (one or two nucleotides) past an initial blockage site, delayed but normal transcription past some sites, and complete termination at most sites. Eight of the nine blockage sites detected were at G or GG sequences on the template strand, with an apparent specificity for 5'-CTGT sequences of the template strand. Seven of the nine sites were capable of inter- or intrastrand cross-links, including three possible G-G interstrand cross-links spanning an intervening base-pair. In the 103-bp segment probed by this procedure, transcriptional blockages were detected (with one exception) only at sites corresponding to G on the template strand where inter- or intrastrand cross-linking was possible but not for similar sequences on the non-template strand.

Alkylation↗
PubMed1985

Ubiquitin-dependent proteolysis of native and alkylated bovine serum albumin: effects of protein structure and ATP concentration on selectivity.

The susceptibility of bovine serum albumin to degradation by the ubiquitin-dependent system of proteolysis depends on the severity of the iodination conditions [Wilkinson, K.D., & Audhya, T.K. (1981) J. Biol. Chem. 256, 9235-9241]. To evaluate if other modifications of the protein changed its susceptibility to degradation, chemically modified derivatives of bovine serum albumin have been synthesized, characterized, and tested as substrates for the ubiquitin-dependent system. Serum albumin was reduced or reduced and alkylated with iodoacetic acid or iodoacetamide. Only the alkylated derivatives exhibit saturation kinetics. Both alkylated proteins competitively inhibit the degradation of the other. These substrates are useful for assay of the intact proteolysis system in crude extracts and in assays for other substrates using competitive alternate substrate inhibition. The physical properties of these proteins suggest that charge, denaturation, or aggregation is not correlated with the degradation rate of these proteins by this system. However, the selectivity of the ubiquitin-dependent proteolysis depends strongly on the ATP concentration. At saturating substrate concentrations, both alkylated substrates are degraded equally. At low ATP concentrations, there is a 2.4-fold difference in the degradation rates of the alkylated proteins. The results presented here indicate that the ubiquitin-dependent protein degradation system is selective and responsive to ATP concentrations and that not all abnormal proteins are equally preferred substrates. Thus, the system may be more selective than previously thought.

Adenosine Triphosphate↗
PubMed1975

The formation of tritiated O-alkyl lipid from acyldihydroxyacetone phosphate in the presence of tritiated water.

Previous studies from this laboratory on the mechanism of O-alkyl bond formation using a microsomal system from Tetrahymena pyriformis have shown that O-alkyl lipid synthesized from dihydroxyacetone phosphate has exchanged one hydrogen stereospecifically from the 1-sn position of the glycerol moiety. Indirect evidence suggested that acyldihydroxyacetone phosphate, an intermediate in )-alkyl lipid synthesis, is probably not the locus of the exchange. In the present study in was shown that stable acyldihydroxyacetone phosphate incubated in the presence of tritiated water and Tetrahymena microsomes does not become tritiated. When hexadecanol is added to the system O-alkyl lipid is produced which has incorporated one atom of hydrogen for each mole of hexadecanol at all time periods examined. Experiments in Ehrlich ascites tumor cells have shown that the hydrogen exchange also occurs in a mammalian system. The results indicate that the mechanism of O-alkyl lipid ether bond formation involves a hydrogen exchange and that this exchange occurs after the formation of acyldihydroxyacetone phosphate.

Acetone↗
PubMed2001

Amino acid nitrosation products as alkylating agents.

Nitrosation reactions of alpha-, beta-, and gamma-amino acids whose reaction products can act as alkylating agents of DNA were investigated. To approach in vivo conditions for the two-step mechanism (nitrosation and alkylation), nitrosation reactions were carried out in aqueous acid conditions (mimicking the conditions of the stomach lumen) while the alkylating potential of the nitrosation products was investigated at neutral pH, as in the stomach lining cells into which such products can diffuse. These conclusions were drawn: (i) The alkylating species resulting from the nitrosation of amino acids with an -NH(2) group are the corresponding lactones; (ii) the sequence of alkylating power is: alpha-lactones > beta-lactones > gamma-lactones, coming respectively from the nitrosation of alpha-, beta-, and gamma-amino acids; and (iii) the results obtained may be useful in predicting the mutagenic effectiveness of the nitrosation products of amino acids.

Alkylating Agents↗
PubMed2002

Ambident ethyl N-nitrosocarbamate anion: experimental and computational studies of alkylation and thermal stability.

Alkylation of N-nitrosourethane tetrabutylammonium salt (2-Bu(4)N) with four electrophiles (MeI, EtI, i-PrI, and PhCH(2)Br) was studied by (1)H NMR in CD(2)Cl(2) and CD(3)CN solutions. The ratio of the three regioisomers N-alkyl-N-nitrosourethane 3, azoxy 4, and O-alkyldiazotate 5 was practically independent of solvent but dependent on the nature of the electrophile. The anion 2 and O-alkyl derivative 5 are thermally unstable and decompose to ethyl carbonates 9 and 10, respectively, with a first-order rate constant (2-Bu(4)N: k = 18.5 +/- 0.1 x 10(-5) s(-1); 5b (R = Et): k = 1.77 +/- 0.02 x 10(-5) s(-1); 5d (R = PhCH(2)): k = 4.78 +/- 0.08 x 10(-5) s(-1) at 35 degrees C in CD(2)Cl(2)). Further kinetic measurements gave activation parameters for the decomposition of 2 (E(a) = 24.2 +/- 0.3 kcal/mol and ln A = 30.9 +/- 0.1). Gas-phase calculations at the MP2(fc)/6-31+G(d)//MP2(fc)/6-31G(d) level showed that the alkylation of 2 involves the lone electron pairs of the N-N-O atoms, and the calculated activation energies correspond well to the observed ratio of regioisomers 3-5. The theoretical analysis of the decomposition processes supports a concerted mechanism with a four-center transition state in the first step for all four compounds. The calculated activation energy order (2 < 5 < 3 < 4) is consistent with the observed order of stability. Decomposition of 2 and 5 is a unimolecular process, giving carbonates 9 and 10 in a single step. In contrast, rearrangement of 3 and 4 leads to alkyl diazonium ions. A detailed theoretical analysis indicates that the rate-determining step for thermal decomposition of 2 is the loss of molecular nitrogen, while in 5 it is the trans-cis isomerization process. The nonconcerted process involving homolytic cleavage of the O-N bond in 5 was found to be significantly less favorable.

Alkylation↗
PubMed2003

Inhibition of transcription at a coding sequence by alkylating polyamide.

Transcription from DNA sequence-specifically alkylated by a hairpin polyamide (ImPyPy-gamma-ImPyLDu86, 1) was investigated. High-resolution polyacrylamide gel electrophoresis demonstrated that conjugate 1 alkylated a 993-bp DNA fragment, in accordance with the Py-Im recognition rule, predominantly at the one match site on the GFP-encoding strand and at four sites (I'-IV') on the noncoding strand. Alkylation of DNA inhibited the formation of full-length mRNA and caused the transcription of truncated mRNA. Polyacrylamide gel electrophoresis demonstrated that the length of the truncated mRNA was consistent with the alkylated site on the coding strand. Complete inhibition of full-length mRNA synthesis was observed in the presence of 50 nM 1. In clear contrast, the hydrolyzed derivative of 1, designated 2, produced no truncated mRNA, nor did it significantly retard transcription: >80% transcription of full-length mRNA was observed at 50 nM. These results clearly indicate that inhibition of transcription can be achieved with alkylating Py-Im polyamide even in the coding regions of genes.

Alkylation↗