Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A

The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.     

The stereochemical course of the first step of pre-mRNA splicing.

We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

Stereochemistry of internucleotidic bond formation by tRNA nucleotidyltransferase from baker's yeast.

Isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) is a substrate for tRNA nucleotidyltransferase from baker's yeast, whereas isomer B is a competitive inhibitor. The tRNA resulting from this reaction has a phosphorothioate instead of a phosphate diester linkage at the last internucleotidic linkage between cytidine and adenosine. On limited digestion of this tRNA with RNase A, one can isolate cytidine 2',3'-cyclic phosphorothioate which can be deaminated to uridine 2',3'-cyclic phosphorothioate. It can be shown that this compound is the endo isomer and that, therefore, the phosphorothioate diester bond in the tRNA must have had the R configuration. This result indicates that no racemization during the condensation of ATP alpha S, isomer A, onto the tRNA had occurred. Whether inversion or retention of configuration had taken place awaits elucidation of the absolute configuration of isomer A of ATP alpha S.     

The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase

The stereochemical course of the reaction catalyzed by the soluble form of bovine lung guanylate cyclase has been investigated using [alpha-18O]guanosine 5'-triphosphate (Rp diastereomer) and guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) as substrates. The product from the 3-thiomorpholino-1',1'-dioxide sydnonimine-stimulated enzymatic cyclization of [alpha-18O] guanosine 5'-triphosphate was esterified with diazomethane. 31P NMR analysis of the triesters indicated that all of the 18O label was present in the axial position. Guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) was cyclized under stimulated and basal enzyme activities and, in both cases, the Rp diastereomer of guanosine 3',5'-cyclic phosphorothioate was formed. This was determined by direct comparison with material synthesized chemically from guanosine 5'-phosphorothioate. The results from these experiments show that the reaction catalyzed by guanylate cyclase proceeds with inversion of configuration at phosphorus and this indicates that the reaction proceeds by way of a single direct displacement reaction.     

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.     

Phospholipids chiral at phosphorus. Stereochemical mechanism of reactions catalyzed by phosphatidylinositide-specific phospholipase C from Bacillus cereus and guinea pig uterus

(Rp)- and (Sp)-1,2-dipalmitoyl-sn-glycero-3-thiophosphoinositol (DPPsI) were synthesized as a mixture and their configurations assigned on the basis of the stereospecific hydrolysis catalyzed by phospholipase A2 (PLA2) from bee venom. PLA2 is known to be stereospecific to the Rp isomer of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) and 1,2-dipalmitoyl-sn-glycero-3-thiophosphoethanolamine (DPPsE). Since the configurations of (Rp)- and (Sp)-DPPsI correspond to those of (Sp)- and (Rp)-DPPsC, respectively, due to a change in priority, the isomer specifically hydrolyzed by PLA2 was assigned to (Sp)-DPPsI. The DPPsI analogues were then used to probe the mechanism and to elucidate the steric course of the reaction catalyzed by phosphatidylinositide-specific phospholipase C (PI-PLC) from Bacillus cereus and for both isozyme I and isozyme II of PI-PLC from guinea pig uterus. It was found that the Rp isomer of DPPsI is the preferred substrate for all three PI-PLCs. Thus PI-PLC shows the same stereospecificity as phosphatidylcholine-specific PLC (PC-PLC), which prefers the Sp isomer of DPPsC. The ratio of the two products inositol 1,2-cyclic phosphorothioate (cIPs) and inositol phosphorothioate (IPs) was not significantly perturbed by the use of phosphorothioate analogue for all three PI-PLCs, which implies that IPs is not produced by enzyme-mediated ring opening of cIPs and supports a parallel pathway for the formation of both products. In order to elucidate the steric course of the cyclization reaction, exo and endo isomers of cIPs were synthesized and their absolute configurations at phosphorus were determined by nuclear magnetic resonance and other techniques. It was found that exo-cIPs is the product produced by all three PI-PLCs. Thus the steric course of the conversion DPPsI to cIPs catalyzed by all three PI-PLCs was inversion of configuration at phosphorus. These results taken together suggest that the reaction catalyzed by PI-PLC most likely proceeds via direct attack by the 2-OH group to generate the cyclic product, and parallelly by water to generate the noncyclic inositol phosphates, without involving a covalent enzyme-phosphoinositol intermediate.     

P alpha-chiral phosphorothioate analogues of bis(5''-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.     

Activity of acid deoxyribonuclease towards diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate). Stereochemistry of transnucleotidylation reaction

The (Rp)- and (Sp)-diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate) (1) were found to act as unusual substrates for acid deoxyribonuclease (DNase II). Instead of the expected thymidine 3'-phosphorothioate, the product resulting from the reaction of (Rp)-1 catalyzed by DNase II was identified as (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), while that from (Sp)-1 has been recognized as a 10:1 mixture of (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 5'-(4-nitrophenyl phosphorothioate) and (Rp, Sp)-thymidyl (3'-5')-thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), respectively. Both types of transnucleotidylations were found to occur with retention of configuration at phosphorus. Stereochemical results may be interpreted in terms of two step mechanisms involving the formation of the intermediate, covalent substrate enzyme complexes.     

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.     

Nucleotide stereochemistry in the formyltetrahydrofolate synthetase reaction

In a recent study, we have shown that N10-formyltetrahydrofolate synthetase prefers (Sp)-MgATP beta S over the Rp isomer in the forward reaction. In this report the stereochemistry of ATP beta S produced from prochiral ADP beta S in the reverse reaction was determined. The ATP beta S product was purified and tested as a substrate for hexokinase (preference for the Rp isomer), adenylate kinase (preference for the Sp isomer) and N10-formyltetrahydrofolate synthetase. A comparison of kinetic constants for the product and the authentic Sp and Rp isomers shows that the product is the Sp diastereomer. 31P NMR was also used to identify the product as (Sp)-ATP beta S.     

Mung bean (Phaseolus aureus) nuclease. A mechanistic investigation of the DNA-cleavage reaction using a dinucleoside phosphorothioate.

Mung-bean (Phaseolus aureus) nuclease has been found to cleave the Sp diastereoisomer of 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl)phosphorothioate, (Sp)-d[Ap(S)T], in 18O-labelled water with inversion of configuration at phosphorus to give (Sp)-thymidine 5'-[16O, 18O]phosphorothioate, the stereochemistry of which was deduced by methylation to (Rp,Sp)-thymidine 5'-S-methyl-O-methyl-[16O,18O]phosphorothioate and 31P-n.m.r. analysis. This result is consistent with a mechanism involving a direct 'in-line' attack of water on DNA for the nuclease-catalysed reaction without the involvement of a covalent nucleotidylated-enzyme intermediate.     

Enzymatic synthesis of the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate on a preparative scale

(Rp)-Adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) is a highly specific antagonist of the cAMP-dependent protein kinase from eukaryotic cells and is a very poor substrate for phosphodiesterases. It is therefore a useful tool for investigating the role of cAMP as a second messenger in a variety of biological systems. Taking advantage of stereospecific inversion of configuration around the alpha-phosphate during the adenylate cyclase reaction, we have developed a method for the preparative enzymatic synthesis of the Rp diastereomer of adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) from the Sp diastereomer of adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP alpha S). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes (Sp)-ATP alpha S approximately 40-fold more slowly than ATP, but binds (Sp)-ATP alpha S with about 10-fold higher affinity than ATP. The triethylammonium salt of the reaction product can be purified by elution from a gravity flow reversed-phase C18 column with a linear gradient of increasing concentrations of methanol. Yields of the pure (Rp)-cAMPS product of a synthesis with 2 mg of substrate are about 75%.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.     

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.     

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

The emergence of antisense and antigene oligonucleotides as potential sequenceselective inhibitors of gene expression is evidenced by the growing number of ongoing clinicals trials against a variety of diseases. First generation antisense therapeutics utilize a uniformly modified oligodeoxyribonucleotide phosphorothioate where one non-bridging oxygen atom is formally replaced by sulfur, because natural DNA is unstable towards extra- and intracellular enzymes. Phosphoramidite chemistry has been widely used for the synthesis of phosphorothioate oligonucleotides because of its potential for automation, high coupling efficiency, ease of site-specific thioate linkage incorporation, and ready scalability. The large scale solid-supported synthesis of phosphorothioates is presently carried out by initial formation of the internucleotidic phosphite linkage followed by sulfurization of the phosphite triester to phosphorothioate using the Beaucage reagent. The resulting O,O-linked phosphorothioate diester linkage in the oligonucleotide is a chiral functional group. For a typical 20-mer there are 524,288 (2¹⁹) possible diastereoisomers. Separation and individual quantification of this number of diastereomers is currently not feasible. In addition, the best reported methods for stereocontrolled synthesis of phosphorothioate oligomers are not presently useful for drug synthesis; that is, since net 100% enantiomeric excess is not achieved in the coupling step, the oligomeric product still consists of the same mixture of Sp and Rp diastereomers, except that the levels of all but one isomer are reduced to low individual levels. As a result, even a small change in the and Sp phosphorothioate diesters, due to racemization during coupling, indicating that the overall synthetic process is stereo reproducible and under inherent process control.