A Mn2(+)-dependent ribozyme.

An RNA hairpin identical in sequence with the one formed during autocyclization of the 414-nucleotide Tetrahymena intervening sequence undergoes strand scission at a specific site in the presence of Mn2+. In addition to representing one of the smallest and simplest ribozymes possible, strand scission occurs readily under physiological conditions, is unaffected by the presence of Mg2+, and displays salt, pH, and temperature optima of potential use in exploiting Mn2+ as a regulatory switch in intact cells. The chemistry of strand scission of the RNA hairpin is described, as is the Mn2(+)-dependent solvolysis of a 231-nucleotide RNA transcript containing this structural motif.

Cytokinin from soluble RNA of Escherichia coli: 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine.

We have isolated a compound responsible for the cytokinin activity of soluble RNA from Escherichia coli. The structure, indicated as 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine, C(16)H(23)N(5)0(4)S, on the basis of low-and high-reso!ution mass spectrometry, was established by unequivocal synthesis. The mass spectra, chromatographic behavior, and ultraviolet spectra of the compounds from natural and synthetic sources were identical.

Cytokinin of wheat germ transfer RNA: 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine.

A new modified nucleoside is responsible, in part, for the cytokinin activity of transfer RNA from wheat germ. The structure as judged by mass spec-trometry is 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuran-osylpurine. Unequivocal synthesis afforded material having ultraviolet, mass spectral, and chromatographic properties identical with those of the natural product.

Facile characterization of translation initiation via nonsense codon suppression.

A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Substrate specificity of bleomycin hydrolase.

Bleomycin (BLM) hydrolase is believed to protect both malignant and normal tissue from the toxicity of the antitumor drug BLM. Little is known about the substrate specificity of BLM hydrolase. Thus, we developed ion-paired reverse phase high speed liquid chromatography systems to assay for the metabolism of several BLM analogs. We found that BLM A2, BLM B2, tallysomycin S10b (TLM S10b), peplomycin (PEP), butylamino-3-propylamino-3-propylamine bleomycin (BAPP), deglyco bleomycin A2 (dgBLM A2) and bleomycinic acid were each metabolized by rabbit lung BLM hydrolase to a single metabolite. When compared to their corresponding parent compounds, these metabolites were 6- to 35-fold less potent in their ability to inhibit the proliferation of A-253 human head and neck squamous carcinoma cells in culture. Furthermore, we found that substitutions in various regions of the BLM molecule greatly affected the kinetic parameters of BLM hydrolase. For example, the Km with BLM B2 (0.056 +/- 0.005 mM) was 15-fold lower than that seen with BLM A2 (0.83 +/- 0.11 mM). In contrast, the Vmax was not affected markedly by these terminal amine substitutions but was influenced greatly by deletion of the carbohydrate groups of BLM. For example, a 4-fold higher Vmax was observed with dgBLM A2 compared to BLM A2. Thus, these results demonstrate that BLM hydrolase can recognize and metabolize a broad spectrum of BLM analogs regardless of their structural features. This enzymatic conversion resulted in the inactivation of the BLMs as demonstrated by a substantial decrease in their cytotoxicity. Furthermore, the terminal amine and carbohydrate regions, respectively, dictate the apparent affinity and the rate of metabolism of BLM hydrolase substrates.

Studies on the total synthesis of tallysomycin. Synthesis of the threonylbithiazole moiety containing a structurally unique glycosylcarbinolamide.

[structure: see text]. Tallysomycins are glycopeptide antibiotics that were first isolated from fermentation broths of Streptoalloteichus hindustanus. They are structurally related to the bleomycins but contain an additional talose sugar attached via a unique glycosylcarbinolamide linkage. Herein we report the synthesis of a key tallysomycin intermediate that incorporates the glycosylcarbinolamide moiety unique to the tallysomycins.

Analogues of bleomycin: synthesis of conformationally rigid methylvalerates.

[structure: see text]. Several conformationally rigid analogues of the methylvalerate subunit contained within the linker domain of the antitumor antibiotic bleomycin have been prepared. These compounds have been protected in a fashion suitable for the solid-phase synthesis of bleomycin. Bleomycin congeners containing these analogues should facilitate a more detailed understanding of the nature of the conformational bend that the methylvalerate moiety is thought to impart to the natural product.

Synthesis and DNA cleavage activity of a novel bleomycin A(5) glycoconjugate.

[structure in text] To explore the possibility of modifying bleomycin in a fashion that could alter its physiological distribution in a therapeutic setting, a new analogue of bleomycin has been prepared. This analogue is intended to target the asialoglycoprotein receptor on liver cells. Critically, despite the large C-substituent, the bleomycin conjugate was found to degrade DNA in the same fashion as bleomycin A(5) itself, and with only modestly decreased efficiency.

Solid-phase synthesis of bleomycin group antibiotics. Elaboration of deglycobleomycin A(5).

The solid-phase syntheses of two deglycobleomycin A(5) analogues were achieved using a commercially available polystyrene resin containing triphenylmethyl-linked spermidine. The final products were deblocked and released from the resin, analyzed, and purified by C(18) reversed phase HPLC and characterized by high-field (1)H NMR spectroscopy and mass spectrometry. The purified products relaxed supercoiled plasmid DNA in a concentration-dependent fashion and to the same extent as authentic material derived from natural BLM A(5).

Camptothecins as probes of the microenvironments of topoisomerase I--DNA complexes.

By uncoupling the cleavage and ligation reactions of DNA oligonucleotides mediated by topoisomerase I, it has been possible to demonstrate modification of DNA oligonucleotide structure by the enzyme. These modifications indicate an unusual flexibility inherent in the behavior of topoisomerase I and may reflect some of the cellular roles played by the enzyme. The ability of individual camptothecin analogues to inhibit these modification processes differentially provides insight into the relative nature of the microenvironments present. To the extent that these enzyme-mediated structural modifications do constitute models of cellular roles for the enzyme, the observed differential inhibition also provides a potential strategy for assessing the function and importance of such modifications.

Studies toward the site specific incorporation of sugars into proteins: synthesis of glycosylated aminoacyl-tRNAs.

A series of glycosylated serine derivatives was synthesized from peracetylated sugars and Fmoc-protected serine; these were chemically esterified with the tris-(tetrabutylammonium) salt of pdCpA. The fully protected and deprotected glycosylated aminoacyl pdCpAs were ligated enzymatically to an abbreviated tRNA (tRNA-C(OH)) to provide the title compounds that are key intermediates in the elaboration of glycoproteins using readthrough of a nonsense codon.

DNA strand scission by activated bleomycin group antibiotics.

The bleomycins (BLMs) are a structurally related group of antitumor antibiotics used clinically for the treatment of certain malignancies. The mechanism of action of the BLM is believed to involve DNA strand scission, a process that requires O2 and an appropriate metal ion; the therapeutically relevant metal is probably iron or copper. DNA strand scission by activated Fe X BLM involves oxygenation C-4' of deoxyribose and leads to two sets of products. One set results from scission of the C-3'--C-4' bond of deoxyribose, with concomitant cleavage of the DNA chain. The other set of products consists of free bases and an alkali-labile lesion, the latter of which leads to DNA chain cleavage on subsequent treatment with base. The structures of all of these degradation products have now been established by direct comparison with authentic synthetic samples. Also studied was the activation of BLM with (mono)oxygen surrogates such as iodosobenzene. The chemistry of the activated BLM so formed was remarkably similar to that of activated cytochrome P-450 and structurally related metalloporphyrins, which suggests a mechanistic analogy between the two. Remarkably, both Fe X BLM and Cu X BLM were also shown to be activated by NADPH cytochrome P-450 reductase in a transformation that was dependent on metal ion, O2 and NADPH.