Design and synthesis of 2-amino-pyrazolopyridines as Polo-like kinase 1 inhibitors.

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.

Chemoselective arylamidine cyclizations: mild formation of 2-arylimidazole-4-carboxylic acids.

A versatile, one-pot synthesis of 2-arylimidazole-4-carboxylic acids from arylamidines and methyl-2-chloroacetoacetate is described. The transformation is chemoselective, and reaction conditions are mild. Moreover, the flexibility of the strategy offers rapid access to two important classes of biaryl compounds, both 2-arylimidazoles and 2-arylpyrimidines, depending simply upon solvent and base selection. [reaction: see text]

Synthesis of dityrosine cross-linked peptide dimers using the Miyaura-Suzuki reaction.

[reaction: see text] Since peroxidase-catalyzed dityrosine formation is inefficient for peptides, we have developed alternative conditions for intermolecular dityrosine formation using the Miyaura-Suzuki reaction. A one-pot reaction is effective for cross-linking short peptides, but longer peptides inhibit the Suzuki step, mandating a traditional two-step procedure using potassium acetate for the Miyaura reaction and potassium carbonate for the Suzuki coupling. These palladium-based methods are complementary to the well-established peroxidase-catalyzed oxidative phenolic coupling of full-length proteins.

Indolocarbazole glycosides in inactive conformations.

Indolocarbazole glycosides related to rebeccamycin represent a promising category of antitumor agents targeting DNA and topoisomerase I. These drugs prefer to adopt a closed conformation with an intramolecular hydrogen bond between the indole NH group and the pyranose oxygen atom. Three pairs of indolocarbazole monoglycosides bearing an NH or an N-methyl indole moiety were synthesized and their biological properties investigated at the molecular and cellular level. Replacing the indole NH proton with a methyl group reduces DNA interaction and abolishes activity against DNA topoisomerase I. Surface plasmon resonance studies performed with a pair of water-soluble indolocarbazole glycosides and two hairpin oligonucleotides containing an [AT]4 or a [CG]4 sequence indicate that both the NH and the N-methyl derivative maintain a relatively high affinity for DNA (Keq = 2 - 6 x 10(5) M(-1)) but the incorporation of the methyl group restricts access to the DNA. The number of ligand binding sites (n) on the oligonucleotides is about twice as high for the NH compound compared to its N-methyl analogue. Modeling and 1H NMR studies demonstrate that addition of the N-methyl group drives a radical change in conformation in which the orientation of the aglycone relative to the beta-glucoside is reversed. The loss of the closed conformation by the N-methyl derivatives perturbs thir ability to access DNA binding sites and prevents the drug from inhibiting topoisomerase I. As a consequence, the NH compounds exhibit potent cytotoxicity against CEM leukemia cells with an IC50 value in the 1 microM range, whereas the N-methyl analogues are 10 to 100 times less cytotoxic. These studies offer circumstantial evidence supporting the importance of the closed conformation in the interaction of indolocarbazole glycosides with their molecular targets, DNA and topoisomerase I.

Dityrosine cross-linked Abeta peptides: fibrillar beta-structure in Abeta(1-40) is conducive to formation of dityrosine cross-links but a dityrosine cross-link in Abeta(8-14) does not induce beta-structure.

Recent reports by Galeazzi and co-workers demonstrated the susceptibility of Abeta(1-42) to undergo dityrosine formation via peroxidase-catalyzed tyrosine cross-linking. We have formed dityrosine cross-links in Abeta(1-40) using these enzymatic conditions as well as a copper-H(2)O(2) method. The efficiency of dityrosine cross-link formation is strongly influenced by the aggregation state of Abeta; more dityrosine is formed when copper-H(2)O(2) or horseradish peroxidase-catalyzed oxidation is applied to fibrillar Abeta vs soluble Abeta. Once formed, dityrosine cross-links are susceptible to further oxidative processes and it appears that cross-links formed in soluble Abeta react through these pathways more readily than those formed in fibrillar Abeta. Because preorganization of fibrils affects the efficiency of dityrosine formation, we examined the effect of dityrosine formation upon local peptide conformation by assessing the solution structure of a small dityrosine dimer derived from Abeta(8-14). Two-dimensional (1)H NMR studies of the short dityrosine dimer offer no evidence of structure. Thus, the fibrillar structure of Abeta enhances formation of dityrosine cross-links, but dityrosine cross-links do not seem to enhance local secondary structure.

A biaryl peptide crosslink in a MetJ peptide model confers cooperative, nonspecific binding to DNA that ablates both repressor binding and in vitro transcription.

The MetJ repressor is the archetypal example of the beta-ribbon-helix-helix DNA binding motif. A model of the MetJ beta-ribbon (residues 22-28) was prepared by forming a dityrosine crosslinked dimer from the heptapeptide KKYTVSI. Using SPR, the peptide dimer 2 was shown to bind to dsDNA under physiologically relevant conditions, whereas the monomeric peptide did not. The peptide dimer appeared to inhibit binding of the MetJ repressor to natural met operators. Based on the stoichiometry of binding, the binding of peptide dimer 2 seems both highly co-operative and to lack sequence specificity. Peptide binding also appears to prevent transcription in vitro.

Synthesis and study of a gramicidin B mutant possessing a ditryptophan crosslink.

Recent studies of peptide dimers linked by Trp-Trp (ditryptophan) crosslinks suggest that the crosslinks can reinforce antiparallel beta-structure. Depending on environment, gramicidins A, B and C form either helical ion channels with parallel beta-structure or non-functional pores with antiparallel beta-structure. In the channel conformation of the gramicidins Trp9 and Trp15 are close in space, but in the pore conformation Trp9 and Trp15 are far apart. We hypothesized that a ditryptophan crosslink between Trp9 and Trp15 could pre-organize gramicidin in an active conformation. To test the potential for preorganization, an intramolecular ditryptophan crosslink was formed between Trp9 and Trp15 in a W13F mutant of gramicidin B. Photooxidative conditions were shown to generate ditryptophan crosslinks in low yields. While not preparatively useful, photooxidative tryptophan crosslinking may have implications for protein aging processes like cataract formation. The ditryptophan crosslink in the gramicidin B mutant substantially lowered the antibiotic activity of the gramicidin B mutant, unlike the ditryptophan crosslink in the antibiotic X-indolicidin. The biaryl chromophore generated diagnostic Cotton effects in the CD spectrum that revealed the absolute stereochemistry of the biaryl chromophore, but the biaryl chromophore obscured diagnostic features below 220 nm. However, changes in peptide conformation were reflected in changes in the biaryl region of the CD spectrum above 240 nm.