David Sweedler

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2′-O-Me nucleotides, 1-2 other 2′-modified nucleotides at positions U4 and U7, and a 3′-3′-linked nucleotide “cap.” Eight 2′-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2′-NH substitutions at U4 and U7, or 2′-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only 0.1 min. The addition of a 3′-3′-linked nucleotide “cap” (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers

The pharmacokinetics and tolerability of a chemically stabilized synthetic ribozyme (ANGIOZYME™) targeting the Flt‐1 VEGF receptor mRNA were evaluated in healthy volunteers. In a placebo‐controlled, single‐dose escalation study, ribozyme was administered as a 4‐hour IV infusion of 10 or 30 mg/m2 or as a SC bolus of 20 mg/m2. Peak ribozyme plasma concentrations of 1.5 and 3.8 μg/mL were observed after the 10 and 30 mg/m2 IV infusions, respectively. When normalized to dose, AUC values as well as peak concentrations increased proportionally as the dose was increased from 10 to 30 mg/m2. Peak concentrations of 0.9 μg/mL were observed approximately 3.25 hours after a 20 mg/m2 SC bolus of ribozyme. The dose‐normalized AUCs obtained after SC dosing were compared to the mean dose‐normalized AUC after IV dosing to estimate an absolute SC bioavailability (f) of approximately 69%. An average elimination half‐life of 28 to 40 minutes was observed after IV administration, which increased to 209 minutes after SC administration. Only 4 of 12 reported adverse events were possibly related to administration of ribozyme (headache and somnolence). Thus, ribozyme administration was well tolerated after a single 4‐hour IV infusion of up to 30 mg/m2 or a single SC bolus of 20 mg/m2.

Improved Synthetic Approaches Toward 2′-O-Methyl-Adenosine and Guanosine and Their N-Acyl Derivatives

Abstract We developed several improved approaches toward 2′-O-methyl adenosine and guanosine and their N-acyl derivatives. (a) Transglycosylation of N4-acetyl-5′, 3′-di-O-acetyl-2′-O-methyl cytidine with N6-Bz-adenine provided N6-benzoyl-5′3′-di-O-acetyl-2′-O-methyl adenosine in 50% yield. (b) Regioselective methylation of 2-amino-6-chloro purine riboside with MeI/NaH followed by hydrolysis provided 2′-O-Me-guanosine in high yield. The same 2′-O-Me-precursor was transformed into 2′-O-Me-adenosine in 58% yield. (c) Very efficient transformation of 2,6-diamino-purine riboside into N2-isobutyryl (isopropylphenoxyacetyl) 2′-O-Me-guanosine through methylation of 5′,3′-O-TIPDSi derivative followed by selective N2-acylation, deamination and desylilation provided target compounds in 70% combined yield. (d) Mg2+ and Ag+ directed methylation of N1-Bzl-guanosine proceeded in >80% yield with ratio of 2′-O-Me/3′-O-Me=9:1. The same methylation of adenosine with Ag+ and Sr2+ acetylacetonates provided 2′-O-Me-adenosine in 75–80% yield.

[3] Base-modified phosphoramidite analogs of pyrimidine ribonucleosides for RNA structure-activity studies

Publisher Summary This chapter describes the design, synthesis, and utilization of base-modified analogs of natural ribonucleosides originally developed for the structure–activity studies of the hammerhead ribozyme. These compounds are also applied to study RNA–ligand interactions in the hairpin ribozyme, group II intron, VP vaccinia virus, and the development of a “subtraction mutagenesis” approach with basic modifications in the hammerhead system. The most frequently used method for the incorporation of modified nucleosides in synthetic RNAs is via solid-phase oligonucleotide synthesis based on phosphoramidite chemistry. This method utilizes the repetitive coupling of appropriately protected monomeric nucleotides to the growing oligonucleotide chain in the 3´ to 5 direction. After assembly of the chain is completed, removal of the phosphate, base, and 2´-OH protecting group results in target RNA with or without incorporated modified monomeric units. To incorporate a specifically modified nucleotide at a desired position, it is necessary to prepare a monomeric building block protected at the phosphate, base, and 2´-OH with protecting groups that are compatible with the general methodology of incorporation of regular unmodified monomers. Thus, synthesis of the target monomer includes the standard procedure for introduction of 5´-O-dimethoxytrityl group, 2´-O-tert-butyldimethylsilyl group and introduction of 3´-diisopropylaminocyanoethylphosphoramidite, and protection of the base.

SYNTHESIS OF MODIFIED NUCLEOSIDE 5′-TRIPHOSPHATES FOR IN VITRO SELECTION OF CATALYTIC NUCLEIC ACIDS [1]

2′-Modified pyrimidine nucleoside 5′-triphosphates comprising amino, imidazole and carboxylate functionality attached to the 5-position of the base were synthesized. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.