Formation of the N(2)-acetyl-2,6-diaminopurine oligonucleotide impurity caused by acetyl capping.

The acetyl 'capping' reaction routinely employed during phosphorothioate oligonucleotide synthesis has been implicated in the formation of an impurity species with a mass 41 amu greater than the expected oligonucleotide molecule. The impurity has been found to arise by conversion of a protected guanine nucleobase to N(2)-acetyl-2,6-diaminopurine. A two-part mechanism is proposed consisting of transamidation of the protecting group on guanine and substitution of guanine's O(6) atom.

Structure-activity relationships among antifungal nylon-3 polymers: identification of materials active against drug-resistant strains of Candida albicans.

Fungal infections are a major challenge to human health that is heightened by pathogen resistance to current therapeutic agents. Previously, we were inspired by host-defense peptides to develop nylon-3 polymers (poly-ß-peptides) that are toxic toward the fungal pathogen Candida albicans but exert little effect on mammalian cells. Based on subsequent analysis of structure-activity relationships among antifungal nylon-3 polymers, we have now identified readily prepared cationic homopolymers active against strains of C. albicans that are resistant to the antifungal drugs fluconazole and amphotericin B. These nylon-3 polymers are nonhemolytic. In addition, we have identified cationic-hydrophobic copolymers that are highly active against a second fungal pathogen, Cryptococcus neoformans, and moderately active against a third pathogen, Aspergillus fumigatus.

C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

Nylon-3 polymers contain ß-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of ß-lactams, and use of an N-acyl-ß-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-ß-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new ß-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new ß-lactam should become attached at this position. If the terminating ß-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.

Biophysical mimicry of lung surfactant protein B by random nylon-3 copolymers.

Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequence-random copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of beta-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoid-based mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

Structure-activity relationships among random nylon-3 copolymers that mimic antibacterial host-defense peptides.

Host-defense peptides are natural antibiotics produced by multicellular organisms to ward off bacterial infection. Since the discovery of these molecules in the 1980s, a great deal of effort has been devoted to elucidating their mechanisms of action and to developing analogues with improved properties for possible therapeutic use. The vast majority of this effort has focused on materials composed of a single type of molecule, most commonly a peptide with a specific sequence of alpha-amino acid residues. We have recently shown that sequence-random nylon-3 copolymers can mimic favorable properties of host-defense peptides, and here we document structure-activity relationships in this polymer family. Although the polymers are heterogeneous in terms of subunit order and stereochemistry, these materials display structure-activity relationships comparable to those that have been documented among host-defense peptides and analogous synthetic peptides. Previously such relationships have been interpreted in terms of a specific and regular folding pattern (usually an alpha-helix), but our findings show that these correlations between covalent structure and biological activity do not require the adoption of a specific or regular conformation. In some cases our observations suggest alternative interpretations of results obtained with discrete peptides.

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides.

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.

Catechol: A minimal scaffold for non-peptide peptidomimetics of the i + 1 and i + 2 positions of the beta-turn of somatostatin.

The design, synthesis, and evaluation of a series of catechol-based non-peptide peptidomimetics of the peptide hormone somatostatin have been achieved. These ligands comprise the simplest known non-peptide mimetics of the i + 1 and i + 2 positions of the somatostatin beta-turn. Incorporation of an additional side chain to include the i position of the beta-turn induces a selective 9-fold affinity enhancement at the sst2 receptor.

Samarium-promoted coupling of 1,10-phenanthroline with carbonyl compounds for synthesis of new ligands.

1,10-Phenanthroline reacts with aldehydes and ketones in the presence of samarium diiodide to produce 2-(1-hydroxyalkyl)-1,10-phenanthrolines. The hydroxyalkyl substituent can be functionalized in numerous ways or removed to permit further ligand variation. The carbonyl coupling reaction can also be repeated to provide 2,9-disubstituted phenanthrolines. Taken together, these operations provide ready access to a large number of phenanthroline derivatives to serve as ligand libraries for catalyst exploration.