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The hydrolysis of uridylyl(3′,5′)uridine promoted by Mg2+, Zn2+ and Zn2+[12]aneN3([12]aneN3= 1,5,9-triazacyclododecane) has been studied in imidazole, HEPES and triethanolamine buffers and the origin of marked electrolyte effects observed with Zn2+[12]ane N3 has been examined. The results obtained suggest that the basic buffer constituent does not serve as an external general base, but the catalytic activity of the metal ion species is influenced by the coordination to the buffer base and other Lewis bases present in the solution. The data lend additional support to a bifunctional mechanism, which consist of coordination of the metal ion to the anionic phosphodiester and intracomplex general base (or nucleophilic) catalysis by its hydroxo ligand.

Alanine and phenylalanine have been labelled in the 3-position, and 2-aminoadipic acid in the 6-position, with the short-lived positron-emitting radionuclide C-11 (t(1/2) = 20.3 min). (R)- and (S)-2-tert-butyl-1-tert-butyloxycarbonyl-3-methyl-4-imidazoli

Trivalent glycoconjugates, each bearing a biotin side arm in addition to three different sugar ligands, have been synthesized on a solid support. The conjugates were assembled on an orthogonally protected pentaerythrityl tetramine core that was anchored to the support through a backbone amide linker. Peptide coupling chemistry was applied to elongate three of the branches with β-alanine and a fully acylated glycosylacetic acid. The fourth branch was levulinoylated and oximated with aminooxy-derivatized D-biotin, followed by acidolytic release into solution. The acyl protecting groups were removed by methoxide-catalyzed transesterification in methanol.

Fully protected pA2'p5'A2'p5'A trimers 1a and 1b have been prepared as prodrug candidates for a short 2'-5' oligoadenylate, 2-5A, and its 3'-O-Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH 7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial removal of the 3'-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2',5'→3',5' phosphate migration and release of adenosine as side reactions.

Replacement of one of the phosphorus-bound oxygen atoms with sulfur has extensively been used for elucidation of mechanistic details of the cleavage of RNA phosphodiester bonds by ribozymes. Since sulfur atom is larger, less electronegative, and more readily polarizable than oxygen, this substitution affects in many ways metal ion binding and the ease of formation and breakdown of the phosphorane intermediate/transition state obtained by the attack of the entering hydroxyl group on the phosphorus atom. The factors that may be altered by thio substitution include the geometry of the phosphorane intermediate, relative apicophilicities of the ligands, the leaving group property, hydrogen bonding, solvation, and the affinity to metal ions. Experimental studies and theoretical calculations on various model systems have been undertaken to obtain a solid chemical basis for the mechanistic interpretations based on thio effects in ribozyme catalysis. The results of such studies are surveyed in this chapter.

Kinetics of the acid-catalysed hydrolysis of O5′,8-cycloadenosine, S5′, 8-cyclo-5′-thioadenosine and N5′, 8-cyclo-5′-amino-5′-deoxyadenosine have been studied. The S- and N-bridged cyclonucleosides are hydrolysed exclusively by the rupture of the N-glycosidic bond, while the O-bridged compound undergoes concurrent cleavage of the N-glycosidic and 5′,8-cyclo linkages, the proportion of the former reaction being markedly increased with increasing temperature. The conformations of the same cyclonucleosides have been elucidated by molecular modelling (SYBYL) and 1H NMR spectroscopy. The effect of the sugar ring puckering on the hydrolytic stability is discussed.

Syntheses of 5 non-nucleosidic solid supports (1–5) that enable preparation of oligonucleotides bearing a carboxy,-amino,-carboxamido,- or mercaptoalkyl spacer arm at their 3′-terminus are described. They all contain an ester bond of moderate susceptibility toward nucleophiles. Upon the completion of oligonucleotide chain assembly, this bond may be cleaved by a variety of nucleophiles. These release the oligonucleotide from the support and simultaneously introduce the desired functionality. Differences in the reactivity between the supports prepared are discussed.

The pH–rate profiles for the hydrolysis of the 3′,5′-phosphodiester bonds of poly(U) and their isomerization to the 2′,5′-bonds have been determined by following the progress of the reactions by enzymatic digestion with RNAase A and subsequent RP HPLC. The kinetics observed are compared with those of the hydrolysis and interconversion of uridylyl(3′,5′)uridine and uridylyl(2′,5′)uridine.

Anomeric 1-(3-deoxy-D-erythro-2-hexulofuranosyl)thymines and 9-(3-deoxy-D-erythro-2-hexulofuranosyl)adenines were prepared by tin(IV) chloride catalyzed N-glycosylation of trimethylsilylated thymine and N 6 -benzoyladenine with methyl 3-deoxy-D-erythro-2-hexulofuranoside triacetate or tribenzoate, respectively. These O-glycosides used as starting materials were obtained by deoxygenation of 1,2:4,5-di-O-isopropylidene-β-D-fructopyranose and subsequent acid-catalyzed methanolysis of the resulting 3-deoxy derivative. The anomeric configuration of the nucleosides prepared was assigned by a combination of X-ray crystallography and 2D 1 H NMR spectroscopy. The conformation and hydrolytic stability of these new nucleoside analogous are discussed

First-order rate constants for the hydrolysis of polyuridylic acid in the absence and presence of various metal ions and their chelates have been determined under neutral and slightly acidic conditions. The hydrolysis has been shown to proceed by cleavage of non-terminal bonds rather than by stepwise release of monomeric nucleotides. Only the hydrolysis of the 3′,5′-phosphodiester bonds is accelerated by metal ions, not their isomerization to 2′,5′-bonds. The catalytically active species has been shown to be the monohydroxo form of the metal aquo ion. The rate accelerations are parallel, but up to 20 times greater than those obtained with uridylyl (2′,5′)uridine. The mechanism of the metal-ion action is discussed.

Synthesis of two non-nucleosidic phosphoramidite building blocks, 1a, b, that enable attachment of various tether groups to oligonucleotides at their 5′-terminus (or 1′-OH of 3′-deoxypsiconucleoside units) is described. Introduction of these linkers during the oligonucleotide assembly on a solid support, and their subsequent derivatization upon deprotection, afforded amino-, carboxy-, and sulfanyl-alkyl-tethered oligonucleotides.

The potentiality of lanthanide chelates as photoluminescent markers and cleaving agents of nucleic acids is discussed, the main emphasis being on the chelates derived from aromatic nitrogen bases.

The rate constants for the acid-catalysed hydrolysis of 1-(1 -alkoxyethyl)uracils and 1-alkoxymethyluracils have been determined. With both series of compounds, the hydrolysis rate is rather insensitive to the polar nature of the alkoxy group, in striking contrast with the hydrolysis of the corresponding analogues of adenine and cytosine nucleosides, which react via rate-limiting formation of an oxocarbenium ion intermediate. Furthermore, it has been shown that the 3′,5′-cyclic monophosphates of thymidine and uridine undergo the hydrolysis of the N-glycosidic bond 760 and 260 times as fast as their parent nucleosides, while the cyclic monophosphates of 2′-deoxyadenosine and adenosine are depurinated much more slowly than the corresponding nucleosides. On this basis it is suggested that 4-oxopyrimidine nucleosides are hydrolysed by opening of the sugar ring. To obtain further evidence for this exceptional mechanism, comparative kinetic measurements with some seco- and 3′-C-alkyl nucleosides of uracil and adenine have been carried out.

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4'-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2'-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2'-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn(2+). Introduction of Zn(2+) moderately stabilized the duplex with U-bulged targets.

RNA molecules participate in many fundamental cellular processes either as a carrier of genetic information or as a catalyst, and hence, RNA has received increasing interest both as a chemotherapeutic agent and as a target of chemotherapy. In addition the dual nature of RNA has led to the RNA-world concept, i.e. an assumption that the evolution at an early stage of life was based on RNA-like oligomers that were responsible for the storage and transfer of information and as catalysts maintained primitive metabolism. Accordingly, the kinetics and mechanisms of the cleavage of RNA phosphodiester bonds have received interest and it is hoped they will shed light on the mechanisms of enzyme action and on the development of artificial enzymes. The major mechanistic findings concerning the cleavage by small molecules and ions and their significance for the development of efficient and biologically applicable artificial catalysts for RNA hydrolysis are surveyed in the present perspective.

Systematic variation of individual amino acid residues within the catalytic core by means of protein engineering has turned out to be a powerful tool for the mechanistic studies of ribonucleases. The results of such studies are, however, open to alternative interpretations, since the replacement of even a single residue may affect chain folding. This, in turn, alters the geometry, non-covalent interactions and mutual orientation of the catalytically active residues to such an extent that identification of the real origin of the observed influence on rate remains uncertain. Unambiguous structure–reactivity correlations based on studies with structurally simplified chemical models may help to distinguish between alternative mechanisms. The present review is aimed at summarizing the results of such model studies. Accordingly, cleavage of RNA phosphodiester bonds by solvent-derived species, general acids and bases, metal ions, and multifunctional small molecular entities is surveyed.

Oligodeoxyribonucleotides tethered with an amino-, carboxy,- amidocarbonyl-, or mercaptoalkyl spacer arm at 3′-terminus were obtained by assembling the chains on a modified aminoalkyl-CPG ( 1 ) and releasing them with appropriate reagents.

Two different building blocks derived from 3′deoxy-β-D-psicothymidine [1-(3-deoxy-β-D- erythro -2-hexulofuranosyl)thymidine] were prepared and used in the synthesis of modified oligonucleotides.

A new method to prepare oligonucleotides bearing a carboxy- or aminoalkyl spacer arm at their 3′-terminus is described.

The effects of several metal ions and metal ion complexes on the hydrolysis and interconversion of uridylyl(2′,5′)uridine and its 3′,5′-isomer were studied as a function of pH and metal ion concentration. The hydrolysis was shown to be markedly accelerated by Zn2+, Cd2+ and trivalent lanthanide ions and by tri- and tetraaza complexes of Zn2+. In contrast, none of these species appreciably promotes the interconversion of the 2′,5′- and 3′,5′-isomers, in spite of the fact that this reaction proceeds through the same pentacoordinated intermediate as the hydrolysis. Lanthanide ions also promote the hydrolytic dephosphorylation of uridine 2′- and 3′-monophosphates, but have a barely noticeable effect on their interconversion. The mechanisms of the metal ion-promoted reactions are discussed.

Diastereometric esters of O-arylated (R)-lactic acids have been studied by 1 H NMR spectroscopy with respect to their potential application as reagents for chiral analysis. The substitution pattern of the acid moiety of the ester was found to have a decisive influence on the magnitude of the resolution of the signals from the diastereoisomers

Bovine serum amine oxidase, porcine kidney diamine oxidase and human placental and pregnancy serum diamine oxidases have been purified by affinity chromatography and ion exchange chromatography. The purified enzymes were subjected to peptide mapping studies with trypsin, S. aureus V8 protease and pepsin. These studies revealed similarities between the enzymes and partial sequences from the bovine serum amine oxidase were obtained. The sequences obtained showed no homology to known sequences. Immunological studies using monoclonal antibodies to the purified enzymes revealed cross reactivity between the four enzymes. These results support the view that the Cu-dependent amine oxidases constitute a closely related group (E.C. 1.4.3.6).

2,6,10,14,18,22-Hexaazaspiro[11.11]tricosane 1 has been prepared in seven steps from pentaerythritol. The key steps include two successive cyclizations by displacement of two tosyloxy groups from the appropriate pentaerythritol derivatives ( 4 ; 6 ) with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), each reaction being followed by sodium borohydride reduction. Hydrolysis of the spiro bis(hexahydro-1 H ,4 H ,7 H -3a,6a,9a-triazaphenalene) formed 1 .

Oligodeoxyribonucleotide glycoconjugates bearing two trivalent glycoclusters have been synthesized by two alternative methods based on solid-supported oximation of aminooxy functionalized oligonucleotides with glycoclusters constructed by click chemistry. In more detail, the trivalent glycoclusters (5 and 6) bearing three sugar pendants were first assembled by treating a 4-[tri-O-propargylpentaerythrityloxy]benzaldehyde scaffold with methyl 6-azido-6-deoxyglycopyranoside under the click reaction conditions. Two phosphoramidite reagents containing a phthaloyl protected aminooxy function, viz., 2-cyanoethyl N,N-diisopropylphosphoramidites derived from 3-[3,5-bis(phthalimidoxymethyl)phenoxy]propanol (12) and 5-(4,4'-dimethoxytrityl)-1,2-dideoxy-1-C-(2-phthalimidoxyethyl)-beta-d-erythro-pentofuranose (16), were synthesized and incorporated as branching units in appropriate places of the oligonucleotide chains. On using 12, the phthaloyl protections of the branching unit were removed and two identical glycoclusters were attached via oxime linkage to the 5'-terminus of the support-bound oligonucleotide chain. With branching unit 16, the phosphoramidite coupling and the oximation were carried out alternately, allowing introduction of two dissimilar trivalent glycoclusters close to the 3'-end of the oligonucleotide chain. The products (20, 26) were released and deprotected by ammonolysis and purified by HPLC chromatography.

First-order rate constants for the acid-catalysed cleavage of adenine base from 2′-deoxyadenosine, its 3′ - and 5′- monophosphates, various dinucleoside monophosphates, trinucleoside diphosphates and hexameric 2′-deoxyadenylic acid were measured by the method of initial velocity. The results obtained were compared with the effect that hetroassociation with caffeine has on the pre-equilibrium protonation and rate-limiting heterolysis of 2′ -deoxyadenosine and its isosteric analogue, 1-(2-deoxy-β-D-erythro-pentofuranosyl) benzimidazole. The kinetics of the depurination of poly (dA-T), poly(dA) and a mixture of poly(dA) and poly(T) were determined, and the rate variations observed are discussed on the basis of the known structures of these polymers.

The effect of a wide variety of metal ious and metal ion complexes on the hydrolysis of uridine 2′,3′-cyclic monophosphate was studied over the pH range 4.5–8.0. The greatest rate accelerations observed were 107–108-fold. The kinetic data obtained are interpreted in terms of a mechanism involving arapid monodentate binding of the metal ion to the monoanionic phosphodiester and a subsequent intracomplex participation of the metal-bound hydroxo ligand, either as a nucleophile attacking the tetracoordinated phosphorus or as a base facilitating an intermolecular attack of a water molecule. No effect on the product distribution between uridine 2′- and 3′-monophosphates was observed.

Sugar ring conformations of a number of 2′ - and 3′ -substituted 2′,3′-dideoxyribonucleosides were determined in 2H2O by 1H NMR spectroscopy. First-order rate constants for the cleavage of their N-glycosidic bond in aqueous acid were measured. The dependence of sugar ring conformation and hydrolytic stability on the polar nature of the 2′/3′-substitutent is discussed.

2',3'-Cyclic monophosphates of 2'- and 3'-C-methyluridines have been prepared and shown to hydrolyze to a mixture of the correaponding 2'- and 3'-monophosphates. The predominant product isomer is the one having the tertiary hydroxyl group phosphorylated, but on longer treatment a phosphate migration from the tertiary to secondary hydroxyl function takes place. Hydrolytic dephosphorylation competes with the phosphate migration, the tertiary hydroxyl group being dephosphorylated 1 order of magnitude faster than the secondary one