A proposal for a convenient notation for P-chiral nucleotide analogues. Part 2. Dinucleoside monophosphate analogues

A configuration of ligands around a phosphorus atom in P-chiral dinucleoside monophosphate analogues can be described using D_P/L_P stereochemical notation, which allows immediate correlation between the notation of configuration and the actual spatial arrangement of the phosphorus ligands. The area of applications of this new stereochemical nomenclature covers dinucleoside units bridged by virtually any type of tri-and tetra-coordinated phosphorus moieties, that is, phosphorothioates, phosphoramidates, phosphoramidites, boranephosphates, methanephosphonates, H-phosphonates, and many others.     

A proposal for a convenient notation for P-chiral nucleotide analogues. Part 3. Compounds with one nucleoside residue and nonnucleosidic derivatives

Recently, we have proposed a new DP/LP stereochemical notation for P-chiral dinucleoside monophosphate analogues that permits simple correlation between spatial arrangement of the substituents and the configuration at the phosphorus center. As an extension of this work, we present here applications of the DP/LP notation to derivatives containing only one nucleoside unit (e.g., alkyl nucleoside phosphodiesters, nucleoside phosphomonoesters, cyclic phosphate derivatives, nucleoside di-, and triphosphates) and to nonnucleosidic phosphorus compounds.     

A proposal for a convenient notation for P-chiral nucleotide analogues. Part 3. Compounds with one nucleoside residue and nonnucleosidic derivatives

Recently, we have proposed a new D_P/L_P stereochemical notation for P-chiral dinucleoside monophosphate analogues that permits simple correlation between spatial arrangement of the substituents and the configuration at the phosphorus center. As an extension of this work, we present here applications of the D_P/L_P notation to derivatives containing only one nucleoside unit (e.g., alkyl nucleoside phosphodiesters, nucleoside phosphomonoesters, cyclic phosphate derivatives, nucleoside di-, and triphosphates) and to nonnucleosidic phosphorus compounds.     

DNA-Triesters - The Synthesis and Absolute Configuration Assignments at P-Stereogenic Centres

Abstract

Decadeoxyribonucleotide GGGAATTCCC and nine diastereomeric pairs of its mono-O-ethyl ester analogues were synthesized via phosphoramidite approach using the combination of 5′-DMT-base protected (except T) nucleoside 3′-(2-cyanoethyl N,N-diisopropyl phosphoramidites) and 3′-(0-ethyl N,N-diisopropyl phosphoramidites). Under conditions of release from solid support and removal of base-protecting groups (25% NH₄OH, 25°C, 48 h) 2-cyanoethyl groups were removed while O-ethyl phosphate triester functions were practically intact. Isolation of products and separation of diastereomers were performed by means of RP-HPLC. Absolute configuration at P-stereogenic centres was established via degradation of decamers into corresponding dinucleoside O-ethyl phosphates and stereochemical correlation with dinucleoside phosphorothioates of known configuration at phosphorus. Decadeoxyribonucleotide mono-O-ethyl esters were used for mapping the contact points between DNA and Eco RI endonuclease - the restriction enzyme which recognizes canonical sequence. GAATTC and cleaves unmodified DNA strands giving G and p AATTC.     

The case for configurational stability of H-phosphonate diesters in the presence of diazabicyclo[5.4.0]undec-7-ene (DBU).

Configurational stability of dinucleoside H-phosphonates and the stereochemical course of their sulfurisation in the presence of diazabicyclo[5.4.0]undec-7-ene (DBU) were investigated using 31P NMR spectroscopy. It was found that under the reaction conditions and irrespective of the type of protecting groups present in the nucleoside moieties, the H-phosphonate diesters investigated did not undergo any detectable epimerisation at the phosphorus centre, and their sulfurisation with elemental sulfur in the presence of DBU, proceeded stereospecifically. Thus, we could not confirm reports from another laboratory on a stereoselective course of sulfurisation of H-phosphonate diesters and the corresponding acylphosphonates in the presence of DBU.     

Synthesis and properties of dithymidine phosphate analogues containing 3''-thiothymidine.

Dithymidine-3'-S-phosphorothioate (d(TspT)) has been prepared from a 5'-O-monomethoxytritylthymidine-3'-S-phosphorothioamidite (7) by activation with 5-(p-nitrophenyl)tetrazole in the presence of 3'-O-acetylthymidine. The resulting dinucleoside phosphorothioite is readily oxidised to the corresponding 3'-S-phosphorothioate using either tetrabutylammonium (TBA) periodate or TBA oxone and has been deprotected under standard conditions to yield d(TspT). This dithymidine phosphate analogue is comparatively resistant to hydrolysis by nuclease P1, but the P-S bond is readily cleaved by aqueous solutions of either iodine or silver nitrate. Dithymidine-3'-S-phosphorodithioate (d[Tsp(s)T]) was prepared in an analogous fashion using sulphur to oxidise the intermediate dinucleoside phosphorothioite. Absolute stereochemistry has been assigned to the diastereoisomers of d[Tsp(s)T] by comparing their physical and chemical properties to those of the dinucleoside phosphorothioates.     

Molecular mechanics studies of dinucleoside methylphosphonates: influence of methylphosphonate and its chirality on the phosphodiester conformation.

Molecular mechanics studies are performed on single stranded as well as base paired forms of dinucleoside methylphosphonates comprising different base sequences for both the S- and R-isomers of methylphosphonate (MP). S-MP produces noticeable distortions in the geometry, locally at the phosphate center, and this enables the stereochemical feasibility of compact g- g- phosphodiester. Besides, it tends to perturb the conformations around the P-O3' and glycosyl bonds, causing minor variations in stacking interactions. In single stranded dinucleosides, the gain in adjacent base stacking interaction energies seems to be sufficient to overcome the barrier to P-O3' bond rotation arising due to S-MP...sugar interaction, and this results in transition to a compact phosphodiester (BI-type) from an initial extended phosphodiester (BII-type) conformation. Such a thing seems rather difficult in base pair constrained duplexes. Dinucleosides with R-MP behave analogous to normal phosphate duplexes as the methyl group is away from the sugar. It is found that dinucleoside methylphosphonates are energetically less favoured than the corresponding dinucleoside phosphates mainly due to the depletion of contributions from electrostatic attractive interactions involving the base and sugar with the methylphosphonate consequent to the nonionic nature of the latter. Neither S-MP nor R-MP seem to significantly alter the stereochemistry of duplex structure.     

Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase.

Adenosine 5'-(thiophosphate) AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the "pro-R" and "pro-S" oxygens. When we converted the AMPS to the isomer A of adenosine 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-CoA synthetase is specific for the isomer B of ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.     

Molecular and crystal structure of Sp-thymidin-3'-yl 4-thiothymidin-5'-yl methylphosphonate.

The molecular structure of one diastereomer of the dinucleoside methylphosphonate Tp(Me)sT (1) has been determined by X-ray diffraction methods. The crystal asymmetric unit contains one molecule of 1 and one methanol in an orthorhombic unit cell of dimensions a = 13.241(4), b = 13.844(3), c = 14.944(7) A, space group P2(1)2(1)2(1). Both pyrimidine bases in 1 are oriented anti relative to the 2'-deoxyribose rings, and the sugar conformations are 2E and 2(3)T in the 4-thiothymidine and thymidine moieties, respectively. The deoxyribose-phosphonate backbone has an extended conformation with the bases completely unstacked and almost parallel. The absolute configuration at the phosphorus center in 1 is Sp.     

Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase.

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.     

Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.     

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli has been determined. Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of the elongation factor and ribosomes. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate ws analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus atom. It is therefore likely that the hydrolysis occurs in a single step by direct, in-line transfer of the phosphorus from GDP to a water oxygen, without a phosphoenzyme intermediate.     

Phosphoenolpyruvate synthetase and pyruvate, orthophosphate dikinase: stereochemical consequences at both the beta-phospho and gamma-phospho groups of ATP

[(R)-16O,17O,18O]Phosphoenolpyruvate and adenosine 5'-O-[(gamma S)-beta gamma-17O,gamma-17O,18O](3-thiotriphosphate) have been synthesized and used to determine the stereochemical course of the several displacements at phosphorus catalyzed by phosphoenolpyruvate synthetase and by pyruvate, orthophosphate dikinase, two enzymes that catalyze the formation of phosphoenolpyruvate from pyruvate and ATP. The catalytic mechanisms for each of these enzymes are believed to involve both phospho- and pyrophospho-enzyme intermediates. The stereochemical results are entirely in accord with these pathways: the beta-phospho group of ATP suffers overall retention of configuration that is presumably the consequence of two displacements with inversion, and the gamma-phospho group of ATP gamma S suffers inversion of configuration that is most probably the consequence of a single displacement at this center.     

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.     

P-chiral oligonucleotides. 2D Roesy NMR assignment of absolute configuration at phosphorus and conformational analysis of 5'-O-monomethoxytrityl-(2'-O-deoxyribonucleoside) 3'-O-[O-(4-nitrophenyl)]methanephosphonates

Fast and simple methodology for the assignment of the absolute configuration at the phosphorus atom in diastereomerically pure Rp and Sp 5'-O-monomethoxytrityl-2'-O-deoxynucleoside 3'-O-(O-4-nitrophenyl) methanephosphonate (3) was established. The method utilizes 2D ROESY NMR and can be used for the stereochemical analysis of other P-chiral mononucleotides. Configurational analysis shows that the major conformation of the sugar residue in 3 is of the S (South) type. This study will facilitate synthesis of stereoregular methylphosphonate oligonucleotide analogues via the transesterification method.     

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.     

Combined tachykinin receptor antagonist: synthesis and stereochemical structure-activity relationships of novel morpholine analogues

We report herein the synthesis and stereochemical structure-activity relationships of novel morpholine analogues 12 and 13 with regards to NK1, NK2 and NK3 tachykinin receptor binding affinity. An essential requirement for more potent binding affinities was controlled by absolute configuration. (S,R)-12 and (S,R)-13 exhibited high binding affinities for NK1, NK2 and NK3 receptors.