Piotr Guga

P-Chirality-Dependent Immune Activation by Phosphorothioate CpG Oligodeoxynucleotides

Many of the biologic activities of phosphorothioate oligodeoxynucleotides (PS-oligos) are affected by the sense of chirality of the phosphorus atoms of the internucleotide linkages. Some of the activities are increased by the Rp stereoisomer, and others are increased by the Sp stereoisomer. In previous studies, we showed that PS-oligos containing unmethylated CpG dinucleotides in particular sequence contexts can stimulate B cells and other immune cells. These CpG PS-oligos trigger mitogenactivated protein kinase (MAPK) signaling pathways, causing the induction of B cell proliferation and cytokine and immunoglobulin secretion. We investigated whether the immune stimulation by CpG PS-oligos depends on the sense of their P-chirality. CpG PS-oligos synthesized with internucleotide phosphorothioates of Rp configuration at P-atom showed much stronger MAPK activation and induction of I kappa B degradation after 40 minutes of stimulation compared with PS-oligos synthesized with Sp linkages. In order to determine if the enhanced stimulatory effects of the Rp stereoisomer may result from differential cellular uptake, we examined the rates at which fluorescently labeled Rp or Sp CpG PS-oligos were taken up by B cells, but these were found to be identical to each other and to stereorandom PS-oligos. The stronger stimulatory effect of the R stereoisomer did not last for 48 hours, and (3)H-thymidine incorporation assays at this point showed that only the S stereoisomer was active--to approximately the same level as induced by PS-oligos with stereorandom phosphorothioate linkages. This loss of activity of the R stereoisomer most likely resulted from rapid degradation of the oligonucleotides rather than from reduced interaction with the CpG receptor because PS-oligos in which only the CpG dinucleotide was stereodefined were most stimulatory when the CpG was Rp but not when the CpG was Sp. These studies demonstrate that the sense of Pchirality of PS-oligos plays a major role in determining the biologic activities of CpG motifs. Rp-chirality at the CpG is preferred for best stimulation at early time points, but Sp-chirality of the PS-oligo appears to improve stability and may provide more durable effects in prolonged tissue culture systems.

Synthesis and absolute configuration of P-chiral O-isopropyl oligonucleotide triesters

Abstract New O -isopropylphosphomorpholidite reagents provided the title compounds as mixtures of P-chiral diastereomers, which were separated by HPLC for enzymatic digestion studies and assignment of configuration at phosphorus by chemical correlation with known phosphorothioates.

Recent Advances in Stereocontrolled Synthesis of P-Chiral Analogues of Biophosphates

Oligonucleotides and their analogues have recently found broad application in biochemistry and molecular biology as new tools for studying interactions of nucleic acids with other biomolecules, and as potential candidates for therapeutics. In this chapter the synthetic methods leading to P-chiral oligonucleotide analogues are reviewed, including synthesis of isotopomeric phosphates and various nucleotide and oligonucleotide conjugates. In this respect special emphasis has been put on the application of oxathiaphospholane methodology, recently developed in the authors’ laboratory, for the stereocontrolled synthesis of P-chiral biophosphate analogues.

Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages

A method for solid‐phase synthesis of stereodefined PS‐oligos via an oxathiaphospholane approach using pure P‐diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5′‐O‐DMTr‐N‐protected deoxyribonucleosides with 2‐chloro‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O‐labeled mercaptoalcohol, the corresponding 18O‐labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS‐oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s into their P‐diastereomers, and for manual solid‐phase synthesis of PS‐oligos. Oxidation of 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s with selenium dioxide yields their 2‐oxo‐analogs, which are suitable either for elongation of stereodefined PS‐oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O‐labeled PO‐oligos of predetermined P‐chirality.

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site‐specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′‐phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg‐308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti‐hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site‐specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns.

Stereoretentive conversion of dialkyl phosphorothioates into [18O]-phosphates

Abstract Dicyclohexylammonium salts of cis - and trans -2-hydroxy-2-thiono-4-methyl-1,3,2-dioxaphosphorinane are quantitatively converted by means of styrene [ 18 O]-oxide into the corresponding 2-[ 18 O]-oxo-derivatives with retention of configuration at the phosphorus atom.

An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective

Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been employed to trace the topological features of DNA within high-order DNA interactions established by protein machines. The directed evolution of altered specificity YRs, combined with their spatially and temporally regulated expression, heralds their emergence as vital tools in genome engineering projects with wide-ranging biotechnological and medical applications.

Inhibition of human immunodeficiency virus type 1 replication by P‐stereodefined oligo(nucleoside phosphorothioate)s in a long‐term infection model

Oligo(nucleoside phosphorothioate)s (S‐ODNs), if prepared by conventional methods, consist of a mixture of diastereomers by virtue of the asymmetry of the phosphorus atom involved in the internucleotide linkages. This may affect the stability of the complexes formed between S‐ODNs and complementary oligoribonucleotides, which is commonly accepted as the most important factor in determining the efficacy of an antisense approach. Using HIV‐1‐infected MOLT‐4 cells via a long‐term culture approach, we studied the influence of the P‐chirality sense of stereodefined 28mer oligo(nucleoside phosphorothioate)s, [All‐Rp]‐S‐ODN‐gag‐28‐AUG and [All‐Sp]‐S‐ODN‐gag‐28‐AUG, complementary to the sequence starting at the AUG initiation codon of the gag mRNA of HIV‐1, upon the anti‐HIV‐1 activity. The [All‐Sp]‐S‐ODN‐gag‐28‐AUG at a low concentration of 0.5 μM can completely suppress HIV‐1gag p24 antigen expression in HIV‐1‐infected MOLT‐4 clone 8 cells for 32 days. Cells treated with [All‐Rp]‐S‐ODN‐gag‐28‐AUG (0.5 μM) showed a high level of the antigen expression at day 16. Furthermore, satisfactory suppression could not be achieved from a random [Mix]‐S‐ODN‐gag‐28‐AUG, consisting of a diastereomeric mixture of the oligonucleotides. Our results suggest that chemotherapy based upon the use of stereodefined antisense [All‐Sp] S‐ODN may be a more effective method for reducing the viral burden in HIV‐1‐infected individuals.

Stereospecific conversion of P-chiral nucleoside phosphorothioates into [18O]phosphates

Abstract P-chiral phosphorothioate analogs of thymidine and adenosine nucleotides are transformed in high yield with retention of configuration by [ 18 O] chloral and [ 18 O] styrene oxide into corresponding nucleoside [ 18 O]phosphates.

Reactions of Cre with methylphosphonate DNA: similarities and contrasts with Flp and vaccinia topoisomerase.

Background Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis. Methodology/Principal Findings We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step. Conclusions/Significance Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.