Intramolecular Base Research Articles

Evolution of transcript structure and base composition of rDNA expansion segment D3 in ticks.

Four to thirty-two copies of the rDNA 28S gene expansion segment D3 and flanking H14 stem were sequenced in six species of ticks (Ixodes: Ixodidae: Acari). Sequence match among species varied from 66% to 97%. Sequence length averaged 130 bases in I. persulcatus across eight Eurasian sites and averaged 186 bases in five other species across 19 Eurasian and North American sites. The difference in length represents one or more deletions totalling about 60 bases that correspond to stems S3 or S4 of the folded transcript. The typical transcript conformation was observed as one possible low energy structure in the five species of longer D3. The structure entails a basal loop with four stem/loop structures, S1-S4 (moving 5' to 3') atop stem H14. A secondary structure lacking S4 but possessing all other putative standard features of the D3 transcript is possible with the shorter I. persulcatus sequences. Interspecific sequence differences occur at higher frequency in loops and bulges vs. complementary pairing regions of stems. Insertion/deletion events (indels) and base substitutions accounted equally for sequence differences. Indels are flanked by similar sequences, suggesting that they occur by slippage during replication. The D3 of Ixodes species is composed of a degenerate set of subrepeats. Thus, unequal exchange among subrepeats may have caused the reduction in length of the I. persulcatus D3. Compensatory base substitution and compensatory insertion/deletion events are indicated by the failure of mutations to affect secondary structure. Transversions accounted for 64% of sequence differences and were biased toward the gain of G and U and the loss of A and C. This bias could re-establish intramolecular base pairing when disrupted by insertions or deletions that shift one side of a stem relative to the other. The distribution of sequence differences, biased substitution, and conservation of transcript conformation in D3 suggest selective constraint.

Analysis of RNA by Northern Blot Hybridization

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. The Basic Protocol describes blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support protocol.

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra- and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stem-loops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.

Water-soluble cyclopalladated aryl oxime: a potent ‘green’ catalyst

Orthopalladated aryl oxime with a 15-crown-5 motif is prepared in a 72% yield by the exchange of cyclopalladated ligands from oxime of 4′-acetylbenzo-15-crown-5 and orthometalated N, N-dimethylbenzylamine, [Pd( o-C 6H 4CH 2NMe 2)Cl] 2. Its solubility in water is more than ten times higher than that of the related complex without the crown fragment and increases further in the presence of magnesium(II) salts. Potential applications of the newly synthesized palladacycle in catalysis is demonstrated by the example of biomimetic hydrolysis of 4-nitrophenyl 2,3-dihydroxybenzoate. It has been demonstrated that a 120-fold rate increase is achieved by realization of the intramolecular general base mechanism.

Mechanism of hydrogen peroxide dismutation by a dimanganese catalase mimic: dominant role of an intramolecular base on substrate binding affinity and rate acceleration.

Several modifications of the manganese coordination environment and oxidation states of a family of synthetic dimanganese complexes have been introduced in search of the structural features that promote high rates of hydrogen peroxide dismutation (catalase activity). The X-ray structure of reduced catalase (T thermophilus) reveals a dimanganese(II,II) site linked by three bridges: mu 13-glutamate-, mu-OH-, and mu-OH2. The roles of a bridging hydroxide vs mu-aqua and the carboxylate have been examined in the reduced Mn2(II,II) complexes, [(L1,2)Mn2(mu-O2CCH3)(mu-X)]2+ for X- = OH- (7A) or X = H2O (1-4), and their oxidized Mn2(III,III) analogues, [(L1,2)Mn2(mu-O)(O2CCH3)(OH)]+ (6) (L1 is N,N,N',N'-tetrakis(2-methylenebenzamidazolyl)-1,3-diaminopropan- 2-ol, and L2 is the tetrakis-N-ethylated analogue of L1, which has all amine protons replaced by ethyl groups). The steady-state catalase rate is first-order in concentration of both substrate and reduced catalyst and saturates at high peroxide concentrations in all cases, confirming peroxide/catalyst complex formation. No catalyst decomposition is seen after > 2000 turnovers. Catalysis proceeds via a ping-pong mechanism between the Mn2(II,II/III,III) redox states, involving complexes 6 and 7A/7A'. The Mn2(III,IV) oxidation state was not active in catalase activity. Replacement of the mu-aqua bridge by mu-hydroxide eliminates a kinetic lag phase in production of the O2 product, increases the affinity for substrate peroxide in the rate-limiting step as seen by a 5-fold. decrease in the Michaelis constant (KM), and accelerates the maximum rate (kcat) by 65-fold The kinetic and spectroscopic data are consistent with substrate deprotonation by the hydroxide bridge, yielding a hydroperoxyl bridge coordinated between the Mn ions (mu, eta 2 geometry, "end-on") as the basis for catalysis: mu-OH- + H2O2-->mu-O2H- + H2O. Binding of a second hydroxide ion to 7A causes a further increase in kcat by 4-fold with no further change in substrate affinity (KM). By contrast, free (noncoordinating) bases in solution have no effect on catalysis, thus establishing intramolecular sites for both functional hydroxide anions. Solution structural studies indicate that the presence of 2-5 equiv of hydroxide in solution leads to formation of a bishydroxide species, [(L1,2)Mn2(mu 13-O2CCH3)(OH)2], which in the presence of air or oxygen auto-oxidizes to yield complex 6, a Mn2(III,III)(mu-O) species. Complex 6 oxidizes H2O2 to O2 without a kinetic lag phase and is implicated as the active form of the oxidized catalyst. A maximum increase by 240-fold in catalytic efficiency (kcat/KM = 700 s-1 M-1) is observed with the bishydroxide species versus the aquo complex 1, or only 800-fold less efficient than the enzyme. Deprotonation of the amine groups of the chelate ligand L was shown not to be involved in the hydroxide effects because identical results were obtained using the catalyst with tetrakis(N-ethylated)-L. Uncoupling of the Mn(II) spins by protonation of the alkoxyl bridge (LH) was observed to lower the catalase activity. Comparisons to other dimanganese complexes reveals that the Mn2(II,II)/Mn2(III,III) redox potential is not the determining factor in the catalase rate of these complexes. Rather, rate acceleration correlates with the availability of an intramolecular hydroxide for substrate deprotonation and with binding of the substrate at the bridging site between Mn ions in the reductive O-O bond cleavage step that forms water and complex 6.

Kinetics and mechanism of the bleaching of a triarylmethane dye by hydrogen peroxide and water: evidence for intramolecular base catalysis

This work describes the ionisation of the triarylmethane dye Green S and the kinetics of its oxidation by hydrogen peroxide over a wide range of pH. Spectrophotometric titration yields pKa values for the equilibria D2− + H2O = D(OH)3− + H+, HD− = D2− + H+, and H2D = HD− + H+ of 11.72, 7.66, and 1.31 respectively. The forward rate constant for the first reaction is 1.3 × 10−4 s−1. The UV-visible spectrum of HD− indicates that the ortho-O− substituent is protonated. The rate constant for the reaction of D2− and HOO−, determined at high pH, is 0.31 dm3 mol−1 s−1. Rate constants for the reactions of D2− and HOO− associated with one, two and three protons, respectively, have been estimated from the pH dependence of the reaction. From the application of the transition-state pKa approach and consideration of the changes in the UV-visible spectra during the reactions, the rate constants have been assigned to the reactions of H2O2 and D2− (0.48 dm3 mol−1 s−1); H2O2 and HD− or H3O2+ and D2− (respective upper limits, 5.7 × 10−6 or 1.3 × 107 dm3 mol−1 s−1) and H3O2+ and HD− (5.1 × 102 dm3 mol−1 s−1). Comparisons between these rate constants and published rate constants for alkali and peroxide bleaching of similar triarylmethane dyes show the exceptionally high reactivity of D2− toward H2O and H2O2. This is attributed to intramolecular base catalysis by the ortho-O− substituent of D2− and is discussed in terms of the position of the catalytic site and the pKa of its conjugate acid. The relevance of this work to oxygen atom transfer catalysts is considered.

Role of the dimethylbenzimidazole tail in the reaction catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase.

The recent structures of cobalamin-dependent methionine synthase and methylmalonyl-CoA mutase have revealed a striking conformational change that accompanies cofactor binding to these proteins. Alkylcobalamins have octahedral geometry in solution at physiological pH, and the lower axial coordination position is occupied by the nucleotide, dimethylbenzimidazole ribose phosphate, that is attached to one of the pyrrole rings of the corrin macrocycle via an aminopropanol moiety. In contrast, in the active sites of these two B12-dependent enzymes, the nucleotide tail is held in an extended conformation in which the base is far removed from the cobalt in cobalamin. Instead, a histidine residue donated by the protein replaces the displaced intramolecular base. This unexpected mode of cofactor binding in a subgroup of B12-dependent enzymes has raised the question of what role the nucleotide loop plays in cofactor binding and catalysis. To address this question, we have synthesized and characterized two truncated cofactor analogues: adenosylcobinamide and adenosylcobinamide phosphate methyl ester, lacking the nucleotide and nucleoside moieties, respectively. Our studies reveal that the nucleotide tail has a modest effect on the strength of cofactor binding, contributing approximately 1 kcal/mol to binding. In contrast, the nucleotide has a profound influence on organizing the active site for catalysis, as evidenced by the retention of the base-off conformation in the truncated cofactor analogues bound to the mutase and by their inability to support catalysis. Characterization of the kinetics of adenosylcobalamin (AdoCbl) binding by stopped-flow fluorescence spectroscopy reveals a pH-sensitive step that titrates to a pKa of 7.32 +/- 0.19 that is significantly different from the pKa of 3.7 for dimethylbenzimidazole in free AdoCbl. In contrast, the truncated cofactors associate very rapidly with the enzyme at rates that are too fast to measure. Based on these observations, we propose a model in which the base-on to base-off conformational change is slow and is assisted by the enzyme, and is followed by a rapid docking of the cofactor in the active site.

Are intramolecularly stabilized compounds of aluminum suitable structural models of the S N2 transition state? Molecular structure of [( tBu) 2Al(μ-OC 6H 4-2-OMe)] 2

The synthesis and crystallographic characterization of [( t Bu) 2Al(μ-OC 6H 4-2-OMe)] 2, when combined with the previously reported methyl, ethyl and iso-butyl analogs, allows for the structural comparison of the homologous series [R 2Al(μ-OC 6H 4-2-OMe)] 2 with the ideal structural changes that occur during the S N2-like cleavage of an aluminum alkoxide dimer with an intramolecular Lewis base. Ab initio calculations on the model system, [H 2Al(μ-OCH 2CH 2OH)] 2, confirm that in the absence of steric effects, intramolecularly stabilized compounds of aluminum are suitable structural models of the S N2 transition state. However, real compounds are not good models since the bond distances are controlled by the steric bulk of the alkyl substituents on adjacent aluminum centers rather than the extent of coordination of the fifth ligand.

GEL MATRICES IN N-METHYLFORMAMIDE FOR SEPARATION OF DNA FRAGMENTS

Capillary gel electrophoresis is a powerful method for separation of DNA fragments. Usually, water based poly-(acrylamide) gels are employed. In order to release compressions due to intra-molecular base pairing within DNA fragments, it is often necessary to incorporate denaturing agents (e.g. urea, formamide) and/or to operate at elevated temperatures. In this work we have studied the performance of gels in non-aqueous solvents. Poly(dimethylacrylamide) gels in both formamide and N-methyl formamide were prepared. The formamide based gels showed a poor stability under normal capillary electrophoresis conditions,1 rapidly developing voids at moderate electric field strengths. However, N-methyl formamide based gels were more stable and could be employed for electrophoretic separations of oligonucleotides. Sieving properties similar to those of regular water-based poly(acrylamide) gels, were observed. Non-aqueous gels without any additives had a considerable denaturing power, in fact, comparable to water-based gels containing 7 M urea. Interesting influences on the separation selectivity when utilizing different concentrations of N-methyl formamide were observed, which are discussed in the paper.

The change in the electronic character upon cisplatin binding to guanine nucleotide is transmitted to drive the conformation of the local sugar-phosphate backbone—a quantitative study

The microstructure alteration as a result of cisplatin binding to N7 of guanines in DNA has been herein assessed through the multinuclear temperature-, pH*- and concentration-dependent NMR study of the effect of Pt2+ complexation to 2′-deoxyguanosine 3′,5′-bis(ethyl hydrogen phosphate) 1 and its ribo analogue 3 which mimic the central nucleotide moiety in a trinucleoside diphosphate in the complete absence of intramolecular base–base stacking interactions. The N S pseudorotational equilibrium shifts towards N-type conformers by 17 and 21 percentage points at 298 K, respectively, thereby showing that free energy of platination is transmitted to drive the sugar conformation. The increase in the population of N-type conformers was rationalized with the strengthening of the anomeric effect in both 2′-deoxy and ribo nucleotides upon the formation of the Pt–N7 bond which promotes nO4′ → σ*C1′-N9 orbital interactions due to the reduction of π-electron density in the imidazole part of guanine. The additional stabilization of N-type conformers in Pt2+ complexes of ribonucleotides is due to the tuning of the gauche effect of the [N9–C1′–C2′–O2′] fragment, which is absent in 2′–deoxy-ribo counterparts. The platination of N7 favours N1 deprotonation in 2 and 4 by ΔpKa of 0.7 and 0.9 units in comparison with parent nucleotides 1 and 3, respectively. The N S pseudorotational equilibrium in 1–4 showed classical sigmoidal dependence as a function of pH with pKa-values at the inflection points. The population of S-type conformers has increased upon N1 deprotonation in 1–4 because the anomeric effect weakened due to the increased π-electron density in the imidazole part of the guanine moiety. The formation of the Pt–N7 bond in bifunctional complexes 2 and 4 simultaneously causes a shift of the syn anti equilibrium towards anti by 43 and 63 percentage points, and the increase in the population of εt,N conformers by 20 and 32 percentage points at 278 K, respectively. Only a minor conformational redistribution along β, γ, β+1 and ε–1 torsion angles has been observed, which suggests their weak conformational cooperativity with the N S pseudorotational equilibrium as a result of platination to guanine. In comparison with nucleotide phosphodiesters, apurinic 3′,5′-bis(ethyl hydrogen phosphate) sugars 5 and 6 showed no interaction with Pt2+ and therefore no conformational changes.

Electrochemical Oxidation ofN-Acyldopamines and Regioselective Reactions of Their Quinones withN-Acetylcysteine and Thiourea

The metabolism of catechols often involves their oxidation to quinones and subsequent nucleophilic addition reactions with sulfur-containing compounds. Adducts formed during these reactions may play important roles in many biological systems. We have studied the electrochemical oxidation ofN-acetyldopamine (NADA) andN-β-alanyldopamine (NBAD) in the presence of two sulfur-centered nucleophiles,N-acetylcysteine (NACySH) and thiourea (TU), and have characterized the adducts and reaction pathways. NADA and NBAD react similarly, but their adducts with NACySH and TU were formed regioselectively. NACySH yields mainly 5-adducts and TU only 6-adducts. The NACySH adducts are oxidized more easily than the parentN-acyldopamine, and their oxidations are chemically reversible. However, the TU adducts are more difficult to oxidize, and their oxidation products undergo further chemical reactions. An intramolecular base catalysis mechanism for adduct formation with NACySH is proposed, which facilitates removal of the proton from the sulfhydryl group of NACySH and directs formation of the 5-adduct via a 1,6-Michael addition reaction. The absence of a proton on the thioureylene sulfur atom leads to formation of the 6-thioureylene adduct via a 1,4-Michael addition reaction of TU. This mechanism is consistent with the formation of other sulfur-centered adducts of catechols previously reported in the literature.

An intramolecularly stabilized arylboron dibromide

A new boron dibromide featuring intramolecular Lewis base coordination, ArBBr2 (4; Ar = 2-[(dimethylamino)methyl]phenyl) has been prepared via the salt-elimination reaction of [2-(Me2NCH2)C6H4]Li with BBr3. Single-crystal X-ray diffraction experiments reveal that 4 rystallizes in two polymorphs, both of which ave orthorhombic. The principal difference between the two polymorphs is the presence or absence of weak Br ... Br intermolecular contacts. The metrical parameters for the individual molecules of 4 are very similar for both polymorphs; the amine arm is coordinated strongly, and the boron atom adopts a somewhat distorted tetrahedral geometry. Compound 4 is surprisingly unreactive toward a variety of hydride transfer reagents. (C) 1998 John Wiley & Sons, Inc.

ABUNDANT PLASMID‐LIKE DNA IN VARIOUS MEMBERS OF THE ORDERS SIPHONOCLADALES AND CLADOPHORALES (CHLOROPHYTA)1

ABSTRACTIn all seven genera belonging to the order Siphonocladales and in two genera of the Cladophorales (Chlorophyta) that were examined, extracts of total DNA contained abundant low molecular weight (LMW) molecules. This DNA typically ranged in size from 1.5 to 3.0 kb among the different genera studied. The complexity of the populations of molecules found in different genera also varied with regard to sizes and numbers of bands evident in electrophoresis gels. Our data suggest that the LMW DNA is ubiquitous among these algae since it occurred in each of our six isolates of Ernodesmis verticillata (Kützing) Børgesen collected in both the Caribbean and the Gulf of California. The LMW DNA from Ernodesmis and Ventricaria ventricosa (J. Agardh) Olsen et J. West was denser than high molecular weight (HMW) DNA in CsCl/bisbenzimide gradients. Ultrastructurally, the LMW DNA molecules were linear, averaging 0.65 μm (Ernodesmis) and 1.0 μm (Ventricaria) in length. Approximately 2%–5% of the former possessed an apparent loop or lariat at one end, as visualized in the electron microscope after rotary shadowing. The LMW DNA molecules appeared to be predominantly double‐stranded DNA based upon staining with acridine orange. The 2.2‐kb DNA from Ernodesmis has features typical of plasmids in that it rapidly reannealed after heat denaturation, and it was selectively enriched in an alkaline‐lysis extraction procedure. In denaturing gels, this DNA migrated at >4.0 kb, indicating that the molecules may actually be single‐stranded DNA species with intramolecular base pairing, each containing a long inverted repeat folded back on itself in a hairpin conformation in the native state. Digestion with mung bean nuclease revealed that additional single‐stranded regions may occur in many of the LMW molecules, in addition to the hairpin loop. Although the endonuclease DNase I rapidly digested the LMW DNA, neither exonuclease III nor lambda exonuclease digested it. This suggests that the ends of these DNA molecules are protected. Collectively, the data indicate that members of the Siphonocladales and Cladophorales contain linear plasmid‐like DNA molecules that appear to have a novel combination of features.

The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein.

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

Synthesis of RNAse active site model systems using a steroid template

Abstract An RNAse active site model system is described. The rigid steroid backbone is used as the template on which guanidinium and imidazole moieties, necessary for the transesterification/cleavage, are assembled. By changing the stereochemistry at C11 of 2 , and varying the guanidinium side chains, active compounds 9 , 11 , 13 , 15 with different hydrolytic behavior are obtained. Comparison of the steroid compounds clearly demonstrates that changes in the geometry can influence the cleavage reaction of RNA analogs. Furthermore, an intramolecular base can enhance the cleavage rate. The pK a values of the most active bis(guanidinium) compound 9 has been determined and the pH dependence of the cleavage reaction is discussed.

Functional importance of RNA interactions in selection of translation initiation codons.

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA-small ribosomal subunit-initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis-acting mRNA elements and trans-acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.

Mechanism of esterification of 1,3-dimethylamino alcohols by N-acetylimidazole in acetonitrile and the influence of alkyl and geminal dialkyl substitution upon the rate

3-(Dimethylamino)propan-1-ol and seven derivatives with alkyl substituents at the 2-position have been prepared by conventional methods, and second-order rate constants for their esterification by N-acetylimidazole in acetonitrile have been measured under pseudo-first-order conditions both by 1H NMR spectroscopy at a single temperature (23 °C) and by a UV spectroscopic method over the temperature range 25–65 °C. Evidence is presented that the intermolecular esterifications proceed via an initial rate-determining intramolecular general base catalysed formation of a cyclic tetrahedral intermediate. Effective molarities compared with the third-order reactions of simpler alcohols with acetylimidazole catalysed by triethylamine are estimated to be 13–14 mol dm–3, but alkyl substitution at the 2-position of the amino alcohol has only a modest effect upon reaction rates. All reactions have substantial negative entropies of activation and only modest enthalpies of activation as expected for concerted bimolecular reactions with highly ordered transition structures. Three structurally related carbocyclic amino alcohols constitute a short isokinetic series with the isokinetic temperature very close to the experimental range. Along this series, decreasing enthalpies of activation are almost exactly balanced in their contributions to the overall free energy of activation near room temperature by increasingly negative entropies of activation.

Entropic strain and conformational preference in the hydrolysis of some N-alkyl-6-acetylaminotriazinediones

The rate–pH profiles for hydrolysis of the title compounds 1 show four distinct regions: kA, kB and kC for rate plateaux corresponding to cationic, neutral and anionic species, plus kD for attack of OH- on the anion. At the ends of the pH scale the reaction is much slower than for model amides of comparable pKlg, due to exceptional charge dispersal in reactant and leaving group. The plateau rates kB and kC are due to hydrolysis by water, not to some kinetically equivalent process, and are much faster than model calculations would predict. This is traced to intramolecular general base catalysis via solvent bridges, and leads to remarkable rate enhancements in aqueous alcohols. The considerable, and quite independent, variations in kB, kC and acid pKa with only alkyl substitution in the amide moiety points to a dominant effect of conformation which has been explored using a number of techniques, notably octanol–water partitioning, and appears best rationalised in terms of Taft’s ‘steric hindrance of motions’ or Tillett’s ‘entropic strain’. The overall picture for the effect of pH is of successively increasing C–O bond formation in the transition state along the sequence kA → kB → kC but with C–N bond breaking quite out of line and largely dependent on conformational factors.Given pKcat < 0, the presence of effective intramolecular general base catalysis in kB is unexpected. We explain this as being due to a unique feature of 1 whereby catalyst and leaving group are part of the same conjugated structure, leading to pKcat → pK lg as C–N bond-breaking proceeds. Further light on kB comes from the ring-N-methylated analogue 3d, which cannot form the intramolecular hydrogen bond found elsewhere and whose otherwise similar rate–pH profile shows an anomalous ‘apparent pKa’ that can be explained as a consequence of this.

REACTIONS OF CIS-(AQUA)(AMINE)BIS(ETHYLENEDIAMINE)COBALT(III) COMPLEXES WITH SULFUR(IV) IN AQUEOUS MEDIUM

The kinetics of the formation/acid-catalysed aquation (SO2 elimination), CoIII-OSO2+→CoIII-SO3+ isomerization, intramolecular reduction and base hydrolysis of the O-bonded sulfito complexes, cis-[Co(en)2(RNH2) (OSO2)]+ (en=ethylenediamine; R=H, Me, Et, PhCH2 and C6H11) have been investigated. The spontaneous and base-catalysed isomerization involve loss of monodentate amine ligands from the CoIII centre to give trans-[Co(en)2(SO3-S)2]– (in excess SO32–) or trans-[Co(en)2(OH)(SO3-S)] (under base hydrolysis conditions). This result is presumably associated with a cis-labilizing action of the O-bonded sulfite. Steric retardation is observed for the formation and acid-catalysed aquation of the O-sulfito complex, the effect being relatively larger for the latter reaction. Steric acceleration is observed in the isomerization and intramolecular reduction and base hydrolysis of the O-sulfito complexes.¶ The trans-S-disulfito complex is prone to fast H+-catalysed aquation {k=(1.52±0.06)×104 dm3 mol–1 s–1, ΔH‡=81 ± 2 kJ mol–1, ΔS‡= 108 ± 5 J K–1 mol–1 at 25°C, I = 1.0 mol dm−3} yielding trans-[Co(en)2 (SO3-S)(OH2)]+.

Analysis of RNA by Northern and Slot Blot Hybridization

AbstractSpecific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. Because they are single‐stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The describes blotting and hybridization of RNA fractionated in an agarose‐formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot‐blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a .