Modified Bleomycin Disaccharides Exhibiting Improved Tumor Cell Targeting

The bleomycins (BLMs) are a family of antitumor antibiotics used clinically for anticancer chemotherapy. Their antitumor selectivity derives at least in part from their ability to target tumor cells, a property that resides in the carbohydrate moiety of the antitumor agent. In earlier studies, we have demonstrated that the tumor cell selectivity resides in the mannose carbamoyl moiety of the BLM saccharide and that both the BLM disaccharide and monosaccharide containing the carbamoyl moiety were capable of the delivery/uptake of a conjugated cyanine dye into cultured cancer cell lines. Presently, the nature of the participation of the carbamoyl moiety has been explored further to provide compounds of utility for defining the nature of the mechanism of tumor cell recognition and uptake by BLM saccharides and in the hope that more efficient compounds could be identified. A library of seven disaccharide–Cy5** dye conjugates was prepared that are structural analogues of the BLM disaccharide. These differed from the natural BLM disaccharide in the position, orientation, and substitution of the carbamoyl group. Studies of these compounds in four matched sets of tumor and normal cell lines revealed a few that were both tumor cell selective and internalized 2–4-fold more efficiently than the natural BLM disaccharide

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro

Plasmids containing 23S rRNA randomized at positions 2057–2063 and 2502–2507 were introduced into <i>Escherichia coli</i>, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puro­mycin, a natural product which acts as an analogue of the 3′-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puro­­mycin derivative in this study contained the dipeptide <i>p</i>-methoxy­phenyl­alanyl­glycine, implying the ability of the modified ribosomes in clones sensitive to this puro­mycin analogue to recognize dipeptides. Several clones inhibited by the puro­mycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (<b>2</b>) and Phe-Gly (<b>3</b>)), as well as a thiolated dipeptide analogue (<b>4</b>) and a fluorescent oxazole (<b>5</b>) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide <b>4</b> and a 7-methoxy­coumarin fluoro­phore was found to undergo fluorescence quenching. Introduction of the oxazole fluoro­phore <b>5</b> into dihydro­folate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues

The Dynamic Character of the <i>BCL2</i> Promoter i‑Motif Provides a Mechanism for Modulation of Gene Expression by Compounds That Bind Selectively to the Alternative DNA Hairpin Structure

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The <i>BCL2</i> promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the <i>BCL2</i> gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers <i>BCL2</i> levels. The <i>BCL2</i> levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression

Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA–Protein Interaction

Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of <i>Escherichia coli</i> dihydrofolate reductase (<i>ec</i>DHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (<b>A</b>) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)­ethylglycine (HCO, <b>D</b>) at position 17. Further, 6-CNTrp (<b>A</b>) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving <b>A</b> as a fluorescence donor and benzo­[<i>g</i>]­quinazoline-2,4-(1<i>H</i>,3<i>H</i>)-dione 2′-deoxyriboside (T_f) or 4-aminobenzo­[<i>g</i>]­quinazoline-2-one 2′-deoxyriboside (C_f) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with T_f), or the domain-specific association between hnRNP LL and the <i>BCL2</i> i-motif DNA (labeled with C_f). Thus, the non-natural amino acid could be used as a FRET partner for studying protein–nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (<b>A</b>) as a fluorescence donor for the study of protein conformational events