Cyclopropanone Hydrate Research Articles

The photodissociation of solvated cyclopropanone and its hydrate explored via non-adiabatic molecular dynamics using ΔSCF.

The decay of cyclopropanone is a typical example of a photodecomposition process. Ethylene and carbon monoxide are formed following the excitation to the first singlet excited state through a symmetrical or asymmetrical pathway. The results obtained with non-adiabatic molecular dynamics (NAMD) using the delta self-consistent field (ΔSCF) method correspond well to previous experimental and multireference theoretical studies carried out in the gas phase. Moreover, this efficient methodology allows NAMD simulations of cyclopropanone in aqueous solution to be performed, which reveal analogue deactivation mechanisms, but a shorter lifetime and reduced photodissociation as compared to the gas-phase. The excited state dynamics of cyclopropanone hydrate, an enzyme inhibitor, in an aqueous environment are reported as well. Cyclopropanone hydrate strongly interacts with the surrounding solvent via the formation of hydrogen bonds. Excitation to the first singlet excited state shows an asymmetric pathway with cyclopropanone hydrate and propionic acid as the main photoproducts.

Cytochrome P450-Catalyzed Oxidation of N-Benzyl-N-cyclopropylamine Generates Both Cyclopropanone Hydrate and 3-Hydroxypropionaldehyde via Hydrogen Abstraction, Not Single Electron Transfer

The suicide substrate activity of N-benzyl-N-cyclopropylamine (1) and N-benzyl-N-(1'-methylcyclopropyl)amine (2) toward cytochrome P450 and other enzymes has been explained by a mechanism involving single electron transfer (SET) oxidation, followed by ring-opening of the aminium radical cation (protonated aminyl radical) and reaction with the P450 active site. Although the SET oxidation of N-cyclopropyl-N-methylaniline (3) by horseradish peroxidase leads exclusively to ring-opened (non-cyclopropyl) products, P450 oxidation of 3 leads to formation of cyclopropanone hydrate and no ring-opened products, and 3 does not inactivate P450. To help reconcile these discrepant behaviors we have determined the complete metabolic fate of 1 with P450 in vitro. 3-Hydroxypropionaldehyde (3HP), the presumptive "signature metabolite" for SET oxidation of a cyclopropylamine, was observed for the first time in 57% yield, along with cyclopropanone hydrate (34%), cyclopropylamine (9%), benzaldehyde (6%), benzyl alcohol (12%), and benzaldoxime (19%). Unexpectedly, N-benzyl-N-cyclopropyl-N-methylamine (4) was found not to inactivate P450 and not to give rise to 3HP as a metabolite without first undergoing oxidative N-demethylation to 1. These and other observations argue against a role for SET mechanisms in the P450 oxidation of cyclopropylamines. We suggest that a conventional hydrogen abstraction/hydroxyl recombination mechanism (or its equivalent as a one-step "insertion" mechanism) at C-H bonds in 1-4 leads to nonrearranged carbinolamine intermediates and thereby to "ordinary" N-dealkylation products including cyclopropanone hydrate. Alternatively, hydrogen abstraction at the N-H bond of secondary cyclopropylamines 1 gives a neutral aminyl radical which could undergo rapid ring-opening leading either to enzyme inactivation or 3HP formation.

Studies on the cytochrome P450 catalyzed oxidation of 13C labeled 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine by 13C NMR

A recent study from Hanzlik’s laboratory ( J. Am. Chem. Soc. 2002, 124, 8268) has provided compelling evidence of a hydrogen atom transfer pathway for the cytochrome P450-catalyzed oxidative N-decyclopropylation of N-cyclopropyl- N-methylaniline. In the present paper, we report an analogous pathway for the oxidative decyclopropylation of a 13C-labeled 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridinyl substrate. Three 13C-enriched metabolites were characterized: (1) a diastereomeric pair of N-cyclopropyl- N-oxides; (2) the N-cyclopropylpyridinium species; and (3) cyclopropanone hydrate. These results extend the hydrogen atom transfer pathway to include aliphatic tertiary amine substrates. Consideration of all of the available evidence, however, leads us to conclude that the cytochrome P450-catalyzed α-carbon oxidations of cyclopropylamines may proceed via both the single electron and hydrogen atom transfer pathways.

Formation of cyclopropanone during cytochrome P450-catalyzed N-dealkylation of a cyclopropylamine.

The role of single electron transfer (SET) in P450-catalyzed N-dealkylation reactions has been studied using the probe substrates N-cyclopropyl-N-methylaniline (2a) and N-(1'-methylcyclopropyl)-N-methylaniline (2b). In earlier work, we showed that SET oxidation of 2a by horseadish peroxidase leads exclusively to products arising via fragmentation of the cyclopropane ring [Shaffer, C. L.; Morton, M. D.; Hanzlik, R. P. J. Am. Chem. Soc. 2001, 123, 8502-8508]. In the present study, we found that liver microsomes from phenobarbital pretreated rats (which contain CYP2B1 as the predominant isozyme) oxidize [1'-(13)C, 1'-(14)C]-2a efficiently (80% consumption in 90 min). Disappearance of 2a follows first-order kinetics throughout, indicating a lack of P450 inactivation by 2a. HPLC examination of incubation mixtures revealed three UV-absorbing metabolites: N-methylaniline (4), N-cyclopropylaniline (6a), and a metabolite (M1) tentatively identified as p-hydroxy-2a, in a 2:5:2 mole ratio, respectively. 2,4-Dinitrophenylhydrazine trapping indicated formation of formaldehyde equimolar with 6a; 3-hydroxypropionaldehyde and acrolein were not detected. Examination of incubations of 2a by (13)C NMR revealed four (13)C-enriched signals, three of which were identified by comparison to authentic standards as N-cyclopropylaniline (6a, 33.6 ppm), cyclopropanone hydrate (11, 79.2 ppm), and propionic acid (12, 179.9 ppm); the fourth signal (42.2 ppm) was tentatively determined to be p-hydroxy-2a. Incubation of 2a with purified reconstituted CYP2B1 also afforded 4, 6a, and M1 in a 2:5:2 mole ratio (by HPLC), indicating that all metabolites are formed at a single active site. Incubation of 2b with PB microsomes resulted in p-hydroxylation and N-demethylation only; no loss or ring-opening of the cyclopropyl group occurred. These results effectively rule out the participation of a SET mechanism in the P450-catalyzed N-dealkylation of cyclopropylamines 2a and 2b, and argue strongly for the N-dealkylation of 2a via a carbinolamine intermediate formed by a conventional C-hydroxylation mechanism.

N-dealkylation of an N-cyclopropylamine by horseradish peroxidase. Fate of the cyclopropyl group.

Cyclopropylamines inactivate cytochrome P450 enzymes which catalyze their oxidative N-dealkylation. A key intermediate in both processes is postulated to be a highly reactive aminium cation radical formed by single electron transfer (SET) oxidation of the nitrogen center, but direct evidence for this has remained elusive. To address this deficiency and identify the fate of the cyclopropyl group lost upon N-dealkylation, we have investigated the oxidation of N-cyclopropyl-N-methylaniline (3) by horseradish peroxidase, a well-known SET enzyme. For comparison, similar studies were carried out in parallel with N-isopropyl-N-methylaniline (9) and N,N-dimethylaniline (8). Under standard peroxidatic conditions (HRP, H(2)O(2), air), HRP oxidizes 8 completely to N-methylaniline (4) plus formaldehyde within 15-30 min, whereas 9 is oxidized more slowly (<10% in 60 min) to produce only N-isopropylaniline (10) and formaldehyde (acetone and 4 are not formed). In contrast to results with 9, oxidation of 3 is complete in <60 min and affords 4 (20% yield) plus traces of aniline. By using [1'-(14)C]-3, [1'-(13)C]-3, and [2',3'-(13)C]-3 as substrates, radiochemical and NMR analyses of incubation mixtures revealed that the complete oxidation of 3 by HRP yields 4 (0.2 mol), beta-hydroxypropionic acid (17, 0.2 mol), and N-methylquinolinium (16, 0.8 mol). In buffer purged with pure O(2), the complete oxidation of 3 yields 4 (0.7 mol), 17 (0.7 mol), and 16 (0.3 mol), while under anaerobic conditions, 16 is formed quantitatively from 3. These results indicate that the aminium ion formed by SET oxidation of 3 undergoes cyclopropyl ring fragmentation exclusively to generate a distonic cation radical (14+*) which then partitions between unimolecular cyclization (leading, after further oxidation, to 16) and bimolecular reaction with dissolved oxygen (leading to 4 and 17 in a 1:1 ratio). Neither beta-hydroxypropionaldehyde, acrolein, nor cyclopropanone hydrate are formed as SET metabolites of 3. The synthetic and analytical methods developed in the course of these studies should facilitate the application of cyclopropylamine-containing probes to reactions catalyzed by cytochrome P450 enzymes.

Rearrangement of alpha-amino cyclopropanone hydrate: a novel route to labeled amino acids.

Rearrangement of alpha-amino cyclopropanone hydrate: a novel route to labeled amino acids.

Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach.

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.

Oxidation of Cycloalkylamines by Cytochrome P-450: Mechanism-based Inactivation, Adduct Formation, Ring Expansion, and Nitrone Formation

Mechanism-based destruction of cytochrome P-450 (P-450) and P-450 heme is observed during the oxidation of N-cyclopropyl and N-cyclobutyl benzylamines. The slower inactivation by the cyclobutylamines relative to cyclopropylamines is consistent with known relative rates of ring opening of cycloalkyl-substituted aminium radicals. Evidence was found that porphyrin meso adducts of the type reported for horseradish peroxidase and cyclopropanone hydrate (Wiseman, J. S., Nichols, J. S., and Kolpak, M. X. (1982) J. Biol. Chem. 257, 6328-6332) were not formed. Radiolabels from cyclopropylamine substrates were covalently attached to protein but essentially only from the cyclopropyl portion and not the benzylic portion. Neither label appeared to be bound to extractable heme; however, during oxidations with cyclopropylamines, labeled P-450 heme became covalently attached to protein. Oxidation of 1-phenylcyclobutylamine by P-450 yielded 2-phenyl-1-pyrroline and 2-phenylpyrrolidine, and the ring expansion is interpreted as evidence for the existence of aminium radicals based on precedents with monoamine oxidase (Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138). In addition, purified P-450PB-B oxidized N-(1-phenylcyclobutyl)-benzylamine to N-(1-phenyl)cyclobutyl phenyl nitrone, identified using spectral techniques. This transformation involves two sequential oxidations with either a hydroxylamine or benzylidene intermediate. While P-450 oxidized the amine to both compounds, only the hydroxylamine was rapidly oxidized to give the nitrone. The ring expansion and nitrone products are interpreted in the context of aminium radical intermediates involved in the mechanism of P-450-catalyzed amine oxidation.

The Cyclopropyl Group in Studies of Enzyme Mechanism and Inhibition

AbstractThe cyclopropyl group—particularly in compounds such as cyclopropylmethanol, phenylcyclopropylamine, and cyclopropanone hydrate—has proved to be very useful in the study of enzyme reactions. Whether the cyclopropane ring is opened to form a radical or remains intact can provide much information about reactions occurring at the active site.

Inactivation of quinoprotein alcohol dehydrogenases with cyclopropane-derived suicide substrates. .

Quinoprotein alcohol dehydrogenases can be inactivated by cyclopropanol, cyclopropanone hydrate, and, depending on whether they can oxidize secondary alcohols, also by cyclopropanone ethyl hemiketal. Only enzyme molecules containing the oxidized coenzyme (PQQ), but not those with the coenzyme in the semiquinone form (PQQH), become inactivated with these compounds. The inactivation process proceeds without proton production or electron acceptor consumption and free radical is not observed in the inactivated enzyme. It could be demonstrated that a stoichiometric relationship exists between enzyme inactivation, PQQ converted, PQQ adduct formed, and cyclopropanol added. Thus the dimeric and monomeric enzyme become fully inactivated with two and one molecule of cyclopropanol, respectively, indicating that the dimeric enzyme contains two independently acting catalytic sites. Inactivation of the enzyme by cyclopropanol and cyclopropanone hydrate produces chromatographically different PQQ adducts. Since cyclopropanemethanol, cyclobutanol and cyclohexanol are not suicide substrates, the inactivation presumably proceeds via a ring opening such as proposed for the metal-ion-catalysed degradation of cyclopropane derivatives. The results are in accordance with our view on the reaction mechanism of these enzymes but not with that of others [Mincey et al. (1981) Biochemistry 20, 7502-7509]. The reasons why their model has to be refuted are discussed.

Mechanism of inhibition of horseradish peroxidase by cyclopropanone hydrate.

Cyclopropanone hydrate irreversibly inactivates horseradish peroxidase in a time-dependent manner in the presence of oxidizing agent, hydrogen peroxide. The inhibition reaction is a second order reaction of cyclopropanone hydrate with compound I, the 2 electron-oxidized form of peroxidase, and results in covalent modification of the heme cofactor. A new propionic acid side chain is substituted for one of the methine protons of the heme. A mechanism for inhibition is proposed to involve oxidative ring opening of cyclopropanone hydrate to give the primary free radical of propionic acid, which subsequently alkylates the heme. An isoporphyrin pi cation intermediate is predicted by this mechanism, and this intermediate has been detected spectroscopically.

Irreversible inactivation of the flavoenzyme alcohol oxidase by cyclopropanone

Incubation of the flavoenzyme alcohol oxidase from Candida boidinii with cyclopropanone hydrate in phosphate buffer at pH 7.5 and 20°C leads to a time dependent inactivation of the enzyme which cannot be reversed by removal of the cyclopropanone hydrate. The rate of inactivation is reduced in the presence of substrate, product, or glutathione, and the inactivation is prevented completely by reduction of the FAD coenzyme prior to addition of the inactivator. Inactivation of the enzyme is accompanied by a change in the absorption spectrum of the enzyme which indicates loss of the oxidized flavin chromophore. Denaturation of the inactivated enzyme under a variety of conditions does not cause reoxidation of the flavin which therefore must be covalently modified by cyclopropanone hydrate. A mechanism for the inactivation is proposed which involves radical intermediates and which suggests a similarity in reaction mechanism between alcohol oxidase and flavoprotein amine oxidases.

Kinetics of the reaction of cyclopropanone hydrate with yeast aldehyde dehydrogenase: a model for enzyme-substrate interaction

Kinetics of the reaction of cyclopropanone hydrate with yeast aldehyde dehydrogenase: a model for enzyme-substrate interaction

Mechanism of inhibition of aldehyde dehydrogenase by cyclopropanone hydrate and the mushroom toxin coprine

Mechanism of inhibition of aldehyde dehydrogenase by cyclopropanone hydrate and the mushroom toxin coprine

The chemistry of cyclopropanones. Part VI: Addition of 1,1‐dihydroxycyclopropane

AbstractOn adding a slight excess of water to cyclopropanone, 1,1‐dihydroxycyclopropane (cyclopropanone hydrate) can be isolated in a high yield.The hydrate adds to cyclopropanone giving a mixture of α,ω‐dihydroxypoly‐(oxycyclopropylidenes), which are isolated as the acetates.

Oxidation of cyclopropanone hydrate and hemiacetal by metal ions: (Preliminary communication)

AbstractThe isomerisation of cyclopropanone hemiacetal to propionic ester can be suppressed by metal ions such as Ag+ and Cu2+. These mild oxidising agents can lead to adipic ester (up to 80%) through dimerisation of a radical CH2CH2COOR (III) detectable by ESR. This radical is not formed via propionic ester, but by isomerisation of a cyclic radical II, the primary oxidation product of the hemiacetal. With ferric chloride the radical can pick up chlorine to form β‐chloropropionic ester.The reactions are equally facile with cyclopropanone hydrate leading to adipic acid and β‐chloropropionic acid.