Cleavage Of Intermediates Research Articles

Transport of proteins to the mitochondrial intermembrane space: the 'sorting' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.

The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain. This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al. (1987) EMBO J., 6, 2433-2439). In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments. The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane. In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes. We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane. Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism.

Self-cleavage of rna in the replication of viroids and virusoids

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

The products of hydrolysis of cyclic orthoesters as a function of pH and the theory of stereoelectronic control

The acid hydrolysis of cyclic orthoesters 1, 3–6 (R = Me), and 2 (R = Me and Et) as a function of pH was studied. The bicyclic orthoester 5 yields mainly the hydroxy-ester (less than 5% lactone), and this result is essentially independent of pH. For the other orthoesters, the relative percentage of products differs for each case and varies with pH. At pH ≤ 3, the percentage of lactone is always larger than at pH > 3. These results are explained on the basis of the stereoelectronic theory for the cleavage of tetrahedral intermediates.

Formation of collagen fibrils by enzymic cleavage of precursors of type I collagen in vitro.

Two systems were used to generate collagen fibrils in vitro by enzymic cleavage of intermediates in the conversion of procollagen to collagen. In one system fibrils were generated by using procollagen NH2-terminal proteinase to cleave pNcollagen, the intermediate which contains the NH2-terminal but not the COOH-terminal propeptides found in procollagen. When pNcollagen was incubated with procollagen NH2-terminal proteinase, the NH2-terminal propeptides were enzymically cleaved from the protein, and there was an increase in the turbidity of the solution over and above the turbidity observed with pNcollagen alone. Electron microscope examination of the samples demonstrated that the increase in turbidity was associated with the assembly of collagen fibrils. The fibrils had a mean diameter of 104 nm +/- 51.7 S.D. or about the same as fibrils formed from pNcollagen alone. However, the fibrils formed by enzymic cleavage of pNcollagen had a more distinct gap-overlap pattern and they appeared to be more tightly packed than fibrils of pNcollagen. Varying the concentration of enzyme varied both the rate of enzymic cleavage of the pNcollagen and the rate of fibril assembly, but there was no consistent effect on the diameter or morphology of the fibrils. In the second system, fibrils were generated with a recently described procedure (Miyahara, M., Njieha, F. K., and Prockop, D. J. (1982) J. Biol. Chem. 257, 8442-8448) in which procollagen COOH-terminal proteinase is used to cleave pCcollagen, the intermediate containing the COOH-terminal but not the NH2-terminal propeptides found in procollagen. When incubated with procollagen COOH-terminal proteinase, the COOH-terminal propeptides were cleaved and collagen fibrils assembled. The collagen fibrils were unusually thick with a mean diameter of 1184 nm +/- 291 S.D. The large diameters of the fibrils made it possible to demonstrate by scanning electron microscopy that each fibril was comprised of a bundle of subfibrils packed into a right-handed helix. The fibrils frequently had branch points which appeared to consist of subfibrils which separated from the main axis of the structure. Also, the surface of the fibrils was scalloped at 270- to 300-nm intervals, suggesting that some of the collagen molecules on the surface were in a 4D staggered array. The results suggested the hypothesis that the order in which the NH2-terminal and COOH-terminal propeptides are cleaved in the conversion of procollagen to collagen may provide a mechanism for controlling the diameter, or both the diameter and morphology, of collagen fibrils.

Oxidation of triaryl- and diarylmethanes by oxygen in the system KOH-dimethoxyethane-18-crown-6-ether and cleavage of triarylcarbinol and diaryl ketone intermediates

1. We have developed a method for oxidation by oxygen of carbanions obtained from di-and triarylmethanes in the system KOH-DME-18-crown-6-ether. The reaction products are diarylketones and triarylcarbinols. 2. Under the reaction conditions, diarylketones (XC6H4)2CO may be converted to triarylcarbinols (X=H, F, Br). Under the same conditions, (NO2C6H4)2 CO is cleaved with formation of p-nitrophenol and p-nitrobenzoic acid. 3. In the case of tris(p-nitrophenyl)carbinolate we have shown the exceptionally strong dependence of the stability of the carbinolate on the counterion: potassium carbinolate is cleaved in the presence of O2 with formation of p-NO2C6H4OH and p-NO2C6H4COOH, lithium carbinolate is stable.

Synthetic analogues and biosynthetic intermediates of bleomycin. Metal-binding, dioxygen interaction, and implication for the role of functional groups in bleomycin action mechanism.

In order to clarify the role of bleomycin functional groups in action mechanism, the metal-binding, dioxygen activation, and DNA cleavage of several synthetic analogues and biosynthetic intermediates of bleomycin have been investigated. The present results support that 1) the beta-aminoalaninepyrimidine-beta-hydroxyhistidine portion of the bleomycin molecule substantially participates in the Fe(II) and dioxygen interactions, 2) the transposition of the pyrimidine (or pyridine) and imidazole groups in the Fe(II)-coordination is essential for the effective binding and activation of molecular oxygen by the bleomycin ligands, and 3) the gulose-mannose moiety plays an important role as an environmental factor for the efficient dioxygen reduction and DNA cleavage, although the sugar portion does not contribute significantly to the nucleotide specificity in the DNA strand scission. Certain oligopeptides are able to mimic the metal-binding and dioxygen activation by bleomycin, but not induce the effective DNA cleavage. Probably, the bithiazole DNA interaction site of bleomycin delivers the iron/dioxygen chemistry to particularly the DNA (formula, see text) nucleotide sequences.

Hydrolytic behavior of two β-lactams and their corresponding imidate salts. New evidence for stereoelectronic control

The basic hydrolysis and the concurrent carbonyl–oxygen exchange of 18O-labelled N-phenyl-4-phenyl-2-azetidinone (5*) and N-2,6-dimethylphenyl-2-azetidinone (6*) have been studied. β-Lactam 5* was easily hydrolysed and showed no carbonyl–oxygen exchange with 0.1 N sodium hydroxide (dioxane–water, 9:1). Under the same conditions, β-lactam 6* gave no exchange and was found resistant to basic hydrolysis. Hydrolysis and exchange was observed when 6* was refluxed with 0.1 N and 1 N sodium hydroxide in water.The hydrolysis of the corresponding imidate salts 7 and 8 was also investigated. Under basic conditions, salt 7 gave an 8:2 mixture of ester amine 9 and β-lactam 5, while under acidic conditions the hydrochloride salt of ester amine 9 was the only product. Under basic conditions, the imidate salt 8 produced only the β-lactam 6, and under acidic conditions, a 3:7 mixture of the hydrochloride salt of 10 and β-lactam 6. Under stronger acidic conditions (≥3 N HCl), 8 gave only the starting β-lactam 6.The results of these hydrolysis reactions are easily explained on the basis of the stereoelectronic theory for the cleavage of tetrahedral intermediates, and by taking into account that the nitrogen of tetrahedral intermediates must be either protonated under acidic conditions, or hydrogen bonded with the solvent under basic conditions in order to observe the cleavage of the C—N bond.

ChemInform Abstract: HYDROLYSIS OF CYCLIC UNSYMMETRICAL ANTI IMIDATE SALTS. NEW EVIDENCE FOR STEREOELECTRONIC CONTROL

The synthesis and the hydrolytic behavior of the cyclic imidate salts 7, 8, and 9 is reported. Under acidic conditions, the salt 7 gives the ester ammonium salt 21 and the salt 9 gives a 1:1 mixture of the ester ammonium salt 22 and the amide alcohol 23 as the rotamer 23B only. Under basic conditions, the salt 7 (or 8) gives a 2:8 (or 1:9) mixture of the amide alcohol rotamers 25A and 25B (or 27A and 27B) under kinetically controlled conditions. The salt 9 gives, as the kinetic product, the amide alcohol rotamer 23B only. These results can be rationalized on the basis of the stereoelectronic theory for the cleavage of tetrahedral intermediates and by taking into account that the nitrogen in tetrahedral intermediates must be either protonated under acidic conditions, or hydrogen bonded with the solvent under basic conditions, in order to observe the cleavage of a C—N bond.

Oxidative cleavage of conformationally rigid vinyl orthoesters. Evidence for primary and secondary stereoelectronic control in the cleavage of tetrahedral intermediates

Permanganate oxidation of either axial vinyl orthoester 1 or equatorial vinyl orthoester 7 yields essentially hydroxy ester 5 only. These results can be interpreted on the basis of stereoelectronic effects which control the cleavage of tetrahedral intermediates 2 and 8.

Hydrolysis of cyclic unsymmetrical anti imidate salts. New evidence for stereoelectronic control

The synthesis and the hydrolytic behavior of the cyclic imidate salts 7, 8, and 9 is reported. Under acidic conditions, the salt 7 gives the ester ammonium salt 21 and the salt 9 gives a 1:1 mixture of the ester ammonium salt 22 and the amide alcohol 23 as the rotamer 23B only. Under basic conditions, the salt 7 (or 8) gives a 2:8 (or 1:9) mixture of the amide alcohol rotamers 25A and 25B (or 27A and 27B) under kinetically controlled conditions. The salt 9 gives, as the kinetic product, the amide alcohol rotamer 23B only. These results can be rationalized on the basis of the stereoelectronic theory for the cleavage of tetrahedral intermediates and by taking into account that the nitrogen in tetrahedral intermediates must be either protonated under acidic conditions, or hydrogen bonded with the solvent under basic conditions, in order to observe the cleavage of a C—N bond.

Oxygenation of 3-aryl-2-hydroxyacrylic acids. The question of linear fragmentation vs. cyclization and cleavage of intermediates

Oxygenation of 3-aryl-2-hydroxyacrylic acids. The question of linear fragmentation vs. cyclization and cleavage of intermediates

ChemInform Abstract: STEREOELECTRONIC PROPERTIES OF TETRAHEDRAL SPECIES DERIVED FROM CARBONYL GROUPS. AB INITIO STUDY OF AMINODIHYDROXYMETHANE, CH(OH)2NH2, A MODEL TETRAHEDRAL INTERMEDIATE IN AMIDE HYDROLYSIS

An ab initio theoretical study of all fifteen fully staggered conformations of aminodihydroxymethane, CH(OH)2NH2 has been performed. Optimization of the CO and CN bond lengths, population analyses and orbital localisation reveal the presence of marked conformation dependent stereoelectronic effects which influence bond lengths and overlap populations. These effects may be parametrized as a function of number and nature of antiperiplanar (app) oriented electronic lone pairs (1p) and polar bonds. In a YCX fragment an app orientation between a lone pair on Y and the CX bond increases the length and weakens the CX bond, shortens and strengthens the CY bond. Thus a CX (X O, N) bond of CH(OH)2NH2 is longest and weakest when: (i) it is app to two vicinal lp's; (ii) the X 1p's are not app to a vicinal polar bond; (iii) the conformation of the molecule has as many axially oriented lp's as possible. Results (i) and (ii) agree with a simple hyperconjugation picture involving interaction between an electronic 1p and an app oriented antibonding bond orbital σ* (CX). Bond properties, relative energies and effects on reactivity of the tetrahedral species are discussed, as well as their relations to experimental results on the cleavage of tetrahedral intermediates and to enzyme catalysis.

Stereoelectronic Properties of Tetrahedral Species Derived from Carbonyl Groups. Ab initio study of aminodihydroxymethane, CH(OH)2NH2, a model tetrahedral intermediate in amide hydrolysis

An ab initio theoretical study of all fifteen fully staggered conformations of aminodihydroxymethane, CH(OH)2NH2 has been performed. Optimization of the CO and CN bond lengths, population analyses and orbital localisation reveal the presence of marked conformation dependent stereoelectronic effects which influence bond lengths and overlap populations. These effects may be parametrized as a function of number and nature of antiperiplanar (app) oriented electronic lone pairs (1p) and polar bonds. In a YCX fragment an app orientation between a lone pair on Y and the CX bond increases the length and weakens the CX bond, shortens and strengthens the CY bond. Thus a CX (X  O, N) bond of CH(OH)2NH2 is longest and weakest when: (i) it is app to two vicinal lp's; (ii) the X 1p's are not app to a vicinal polar bond; (iii) the conformation of the molecule has as many axially oriented lp's as possible. Results (i) and (ii) agree with a simple hyperconjugation picture involving interaction between an electronic 1p and an app oriented antibonding bond orbital σ* (CX). Bond properties, relative energies and effects on reactivity of the tetrahedral species are discussed, as well as their relations to experimental results on the cleavage of tetrahedral intermediates and to enzyme catalysis.

α‐ジケトンモノチオケタール基の開裂反応を利用するアルカロイドの合成

Recent synthetic works on the alkaloids including emetine, tabersonine, quebrachamine, vincamine, eburnamine, velbanamine, 4 α-, and 4 β-dihydrocleavamine, through the cleavage of cyclic α-diketone monothioketal intermediates are reviewed. Formations and synthetic utilities of α-diketone monothioketal are also briefly reviewed.

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.

Torsional-strain considerations in enzymology: Some applications to proteases and ensuing mechanistic consequences

A stereoxplicit analysis of amide torsional strain reveals two distinct rehybridization modes, each of which correlates with a unique hydrolysis mechanism. These may be characterized as anti distortion of cis ( Z) substitutents (yielding trans addition of HOR) and syn distortion of trans ( E) substituents (correlating with cis addition). The latter mechanism is known to apply in the cases of the serine proteases, and supporting evidence for torsional activation is sought in crystallographic structures of trypsin with its inhibitors as well as from kinetic data. A contrast is made with the mechanism of carboxypeptidase A, which operates on the former principle. For this enzyme it is demonstrated that an unusual minor-protonation state is most probably the catalytically active species, a conclusion with some important ramifications. A general comparison of the torsional strain hypothesis is made with a recently propounded theory concerning stereoelectronic control of cleavage of hemiorthoamide tetrahedral intermediates. When these approaches to enzyme mechanism are used in tandem, a unique comprehension is achievable.

The reduction of acetals with cobalt carbonyl catalysts

When heated with a dicobalt octacarbonyl (Co2(CO)8) catalyst under synthesis gas, acetals of aromatic aldehydes (in contrast to aliphatic acetals) gave good yields of the products of reductive cleavage, but no products of hydroformylation. A mechanism is proposed to explain the products of cyclic acetals of benzaldehyde which yielded not only the expected hydroxy-ethers, but also considerable amounts of dibenzyl ethers. The room temperature reductive cleavage of aromatic acetals by pure HCo(CO)4 in organic solvents indicates that HCo(CO)4 might be the active species in the catalytic reductions. Since acetals of benzaldehyde para-substituted with electron-releasing groups cleaved faster than those substituted with electron-withdrawing groups, the mechanism probably involves a carbonium ion intermediate.The previously observed formation of dibenzyl ether during benzaldehyde reduction with cobalt carbonyl catalysts can now be explained via the reductive cleavage of hemiacetal or acetal intermediates.

ChemInform Abstract: SYNTHESIS AND BIOLOGICAL ACTIVITY OF LH‐RH ANALOGS MODIFIED AT THE CARBOXYL TERMINUS

Des(Pro9,Gly10)-LH-RH ethylamide, des(Pro9,Gly10)-LH-RH butylamide, desGly10-LH-RH 2-aminoethylamide, and desGly10-LH-RH hydrazide were synthesized by a solid-phase method involving cleavage of protected peptide intermediates from their resin support by reaction with ethylamine, butylamine, ethylenediamine, and hydrazine, respectively. In the assay utilizing steroid pretreated, ovariectomized rats, the peptides were found to have the following LH-releasing activities when compared with natural LH-RH: ethylamide, 0.2%; butylamide, 0,1%; 2-aminoethylamide, 2.4%; hydrazide, 12%. DesGly10-LH-RH hydrazide was used as a precursor in the synthesis of desGly10-LH-RH allylamide and desGly10-LH-RH propargylamide by conversion to the azide and reaction with allylamine and propargylamine, respectively. LH and FSH levels were measured over a 4-hr period after subcutaneous injection of these two peptides into immature male rats in order to detect any prolongation of activity. The allylamide analog was quite active, releasing 1.7 times more LH and 1.3 times more FSH than the same dose of LH-RH. The propargylamide analog was considerably less active, exhibiting 50% LH-releasing activity and 64% FSH-releasing activity. Neither peptide appeared to be longer acting than LH-RH.

The Hydrolysis of Cyclic Orthoesters. Stereoelectronic Control in the Cleavage of Hemiorthoester Tetrahedral Intermediates

The synthesis of several cyclic dialkoxy orthoesters is reported. Acid hydrolysis of these orthoesters under kinetically controlled conditions gives the corresponding hydroxy esters only. Conformationally rigid cyclic mixed orthoesters give a hydroxy ester by exclusive loss of the axial alkoxy group. A cyclic orthoester can have nine different gauche conformations. It is shown that they are hydrolyzed through only one gauche conformer. These results bring further experimental evidence that there is a stereoelectronic control in the cleavage of tetrahedral intermediates in the hydrolysis of esters.

Synthesis and biological activity of LH-RH analogs modified at the carboxyl terminus.

Des(Pro9,Gly10)-LH-RH ethylamide, des(Pro9,Gly10)-LH-RH butylamide, desGly10-LH-RH 2-aminoethylamide, and desGly10-LH-RH hydrazide were synthesized by a solid-phase method involving cleavage of protected peptide intermediates from their resin support by reaction with ethylamine, butylamine, ethylenediamine, and hydrazine, respectively. In the assay utilizing steroid pretreated, ovariectomized rats, the peptides were found to have the following LH-releasing activities when compared with natural LH-RH: ethylamide, 0.2%; butylamide, 0,1%; 2-aminoethylamide, 2.4%; hydrazide, 12%. DesGly10-LH-RH hydrazide was used as a precursor in the synthesis of desGly10-LH-RH allylamide and desGly10-LH-RH propargylamide by conversion to the azide and reaction with allylamine and propargylamine, respectively. LH and FSH levels were measured over a 4-hr period after subcutaneous injection of these two peptides into immature male rats in order to detect any prolongation of activity. The allylamide analog was quite active, releasing 1.7 times more LH and 1.3 times more FSH than the same dose of LH-RH. The propargylamide analog was considerably less active, exhibiting 50% LH-releasing activity and 64% FSH-releasing activity. Neither peptide appeared to be longer acting than LH-RH.