The synthesis of prostaglandin analogs containing hydroxycyclohexenyl rings.

The synthesis of prostaglandin analogs incorporating the 3-hydroxycyclohexenyl moiety in place of the natural C13-C20 side-chain has been accomplished via copper-assisted conjugate addition of the cycloalkenyllithium 2 to the cyclopentenous intermediates 4, 7 and 10.     

Modified oligonucleotides containing 1-beta-D-galactopyranosylthymine: synthesis and substrate properties

A convenient method of regioselective introduction of 1-beta-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymic reaction of formation and hydrolysis of internucleotide bonds. The English version of the paper.     

The synthesis of prostaglandin analogs containing hydroxycyclohexenyl rings

The synthesis of prostaglandin analogs incorporating the 3-hydroxycyclohexenyl moiety in place of the natural C₁₃–C₂₀ sidechain has been accomplished via copper-assisted conjugate addition of the cycloalkenyllithium to the cyclopentenone intermediates , and .     

Influence of substrate and product diffusion on the heterogeneous kinetics of enzymic reversible reactions

When a reversible reaction is catalyzed by a surface bound enzyme, the diffusion of both substrate and product can considerably modify the kinetic properties of the reaction. According to this theoretical analysis, the enzyme activity is decreased due to the presence of substrate and product concentration gradients in the enzyme microenvironment, and the relative kinetic importance of the two diffusion steps mainly depend on the value of a dimensionless criterion inversely proportional to the equilibrium constant. Moreover diffusional effects increase with increasing bound enzyme activity, but decrease with increasing substrate and product concentration. Analytical expressions are established for the limiting values of substrate and product concentrations in the enzyme microenvironment, as well as for the increase in half-maximal-activity substrate concentration in the presence of substrate and product diffusional limitations.     

Oligonucleotides containing disaccharide nucleosides: synthesis, physicochemical, and substrate properties

The efficient synthesis of oligonucleotides containing 2'-O-beta-D-ribofuranosyl (and beta-D-ribopyranosyl)nucleosides, 2'-O-alpha-D-arabinofuranosyl (and alpha-L-arabinofuranosyl)nucleosides. 2'-O-beta-D-erythrofuranosylnucleosides, and 2'-O-(5'-amino-5-deoxy-beta-D-ribofuranosyl)nucleosides have been developed.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.     

The synthesis and biological activity of prostaglandin analogs containing spirocyclic rings

The synthesis of prostaglandin analogs ( - ) incorporating the spiro[3.3]hept-2-yl moeity in place of the natural C₁₆–C₂₀ side-chain has been accomplished via reaction of the mixed cuprate with cyclopentenone intermediates , , and . The biological activities of these new analogs are discussed.     

The synthesis and properties of four spin-labeled analogs of adenosylcobalamin

Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.     

The synthesis and properties of oligodeoxyribonucleotides containing N6-methoxyadenine.

Oligodeoxyribonucleotides containing N6-methoxyadenine (M) have been synthesized. The order of stability of duplexes consisting of synthesized oligodeoxyribonucleotides, 5'd(CCTGGTAXCAGGTCC)3'-5'd(GGACCTGNTACCAGG)3' (X = M, A, G. N = A, G, T, C), was M: A (Tm = 52 degrees C) greater than M: T (50 degrees C) greater than M: G (48 degrees C) greater than M: C (46 degrees C) observed by thermal denaturation in a buffer of 0.01 M Na cacodylate, and 0.1 M NaCl at pH 7.0. The Tms are within a range of 6 degrees of difference, which is smaller than those of Tms of the duplexes containing A:N pairs (11 degrees) and G:N pairs (11 degrees). DNA replication study on a template-primer system, 5'd(32p-CAGCTTTCGC)3' 3'd(GTCGAAAGCGMAGTCG)5', showed that TTP and dCTP were incorporated into DNA strands at a site opposite to M by Klenow DNA polymerase, but dATP and dGTP were not.     

The 5'-O-phosphonomethyl analog of ATP. Studies of substrate properties in ligase-dependent reactions

The 5-O-phosphonomethyl analog of ATP (ATPc) and its interaction with different enzymes, catalysing synthesis of C-O-, C-S-, C-N- and C-C-bond were investigated. It was shown ATPc to have substrate properties and to be involved in all studied ligase-dependent reactions. Kinetic parameters of the interaction of alternative substrate - ATPc with all studied enzymes were established and the comparison of ATPc with other phosphonate analogs of ATP was performed.     

The synthesis of prostaglandin analogs containing the (hydroxycyclooctylidene)methyl ring system

The synthesis of prostaglandin analogs incorporating the (2-hydroxycyclooctylidene)methyl moiety in place of the natural C₁₃–C₂₀ sidechain has been accomplished via copper-assisted conjugate addition of the (cyclooctylidene)methyllithium to the cyclopentenone intermediates and .     

The synthesis of oligonucleotides containing a primary amino group at the 5''-terminus.

Oligonucleotides containing a primary amino group at their 5'-termini have been prepared and further derivatised with amino specific probes. The sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed with N-monomethoxytrityl-0-methoxydiisopropylaminophosphinyl 3-aminopropan(1)ol. After cleavage from the resin, removal of the phosphate and base protecting groups and purification gives a monomethoxytrityl-NH(CH2)3PO4-oligomer. The monomethoxytrityl group can be removed with acetic acid to give the desired amino containing oligomer. The amino group can be further derivatised with amino specific probes yielding fluorescent or biotinylated oligonucleotide products.