Preparation of potential cell-permeant nucleoside-2',3'-cyclic phosphate precursors

Uridine-3'-phosphorothiolate triesters bearing lipophilic moieties were prepared via Michaelis-Arbuzov chemistry. Subsequent deprotection of the S-cholesteryl phosphorothiolate triester afforded the corresponding diester which underwent spontaneous Cyclization to cleanly afford uridine 2',3'-cyclic phosphate. This transesterification reaction could be expedited by treatment with iodine under mild, neutral conditions.     

Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation.

The cyclic pyrimidine nucleotide, cCMP, is an endogenous substance in mammalian cells but little is known on its functional role. We studied the effects of cCMP, its cell-permeant analogue, N4,2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate (Bt2cCMP), and of butyrate on superoxide (O2-) formation and cytosolic Ca2+ [( Ca2+]i) in human neutrophils. Bt2cCMP inhibited O2- formation and the rise in [Ca2+]i induced by a chemotactic peptide at submaximally effective concentrations. O2- formation induced by platelet-activating factor was potentiated by Bt2cCMP, whereas the cyclic nucleotide had no effect on the rise in [Ca2+]i induced by this agonist. Bt2cCMP enhanced O2- formation induced by tau-hexachlorocyclohexane at a submaximally effective concentration. O2- formation stimulated by complement C5a, concanavalin A, NaF, A 23187, phorbol myristate acetate and arachidonic acid was not affected by Bt2cCMP. cCMP was less effective than Bt2cCMP to inhibit fMet-Leu-Phe-induced O2- formation, and butyrate was without effect on any of the functional parameters studied. Our data show that a cell-permeant analogue of cCMP differentially inhibits and potentiates activation of human neutrophils.     

Nucleotide 2',3'-cyclic monophosphokinase from actinomycetes

Streptomyces nucleotide 3'-pyrophosphokinase does not only transfer the 5'-beta, gamma-pyrophosphoryl group of ATP, ATP 3'-pyrophosphate or dATP to a variety of nucleotides at the 3'-OH site, but also adds 2',3'-cyclic terminal monophosphate to some suitable nucleotides with the use of diadenosine 5',5'-polyphosphates (n = 3-5). Examples are pA greater than p, ppA greater than p, pG greater than p, CpG greater than p, etc.     

Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate.

2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.     

2',3'-cyclic nucleotide 2'-phosphodiesterase from Fusarium culmorum

We describe the properties of a 2',3'-cyclic nucleotide 2'-phosphodiesterase (EC 3.1.4.16), found in Fusarium culmorum, which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 3'-phosphates. In contrast with a similar enzyme found in bacteria, the Fusarium enzyme does not exhibit nucleotidase activity and does not show a requirement for metal ions, but is inhibited by micromolar concentrations of Cu++ and Zn++, and is very stable to heat. This cyclic phosphodiesterase hydrolyzes the four major nucleoside 2',3'-cyclic monophosphates and has greater affinity for purine (Kms for Ado-2',3'-P = 0.3 mM and for Guo-2',3'-P = 0.1 mM) than for pyrimidine nucleotides (Kms for Cyd-2',3'-P = 0.6 mM and for Urd-2',3'-P = 2 mM). The respective Vmax for Urd-2',3'-P; Cyd-2',3'-P; Ado-2',3'-P; and Guo-2',3' are 100:45:16:5. The efficacy of the phosphodiesterase to hydrolyze the four major 2',3' cyclic nucleotides (based on the relative values of Vmax/Km) is not significantly different. The Fusarium enzyme differs from a previously described 2',3' cyclic phosphodiesterase from Neurospora, in that it is inactive on 3',5'-nucleoside monophosphates and nucleoside 2' or 3' phosphates.     

Activity of tubercidin-, toyocomycin-, and sangivamycin-3',5'-cyclic phosphates and related compounds with some enzymes of adenosine-3',5'-cyclic phosphate metabolism

Rat liver nuclei contain at least two DNA polymerases that can be separated by extracting the nuclei with 5% Brij 58. The loosely-bound activity increases little or not at all after partial hepatectomy and is insensitive to cytosine arabinoside triphosphate (araCTP). The tightly-bound enzyme activity rises along with DNA replication and is inhibited by araCTP.     

Diastereomeric specificity of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

The diastereomers of adenosine and uridine 2',3'-cyclic phosphorothioates were tested as substrates for 2',3'-cyclic nucleotide 3'-phosphodiesterase from bovine brain. The enzyme cleaves the Sp (or exo) diastereomers efficiently, whereas the Rp (or endo) diastereomers are resistant to hydrolysis, even after long incubation. As the enzyme exhibits strong substrate inhibition the precise determination of kinetic parameters posed problems, particularly with phosphorothioates. The stereoselectivity of this enzyme is opposite to that of RNase T1 and RNase A and thus could be a useful complement in determination of the configuration of nucleoside 2',3'-cyclic phosphorothioates resulting from hydrolysis reactions of unknown stereochemical course.