Synthesis of amino-analogs of bacteriochlorophyll-d and their self-aggregation in an aqueous micelle solution.

Zinc methyl 3-aminomethyl- and 3-(1-aminoethyl)-pyropheophorbides-a were prepared by modifying naturally occurring chlorophyll-a. The synthetic amino-analogs of bacteriochlorophyll-d self-aggregated in an aqueous micelle solution to give large oligomers with red-shifted and broadened electronic absorption bands. The spectra of these self-aggregates were similar to those of bacteriochlorophyll self-aggregates in the main light-harvesting antennas of green photosynthetic bacteria. The 3(1)-amino groups were alternative to the 3(1)-hydroxy groups in natural bacteriochlorophylls-c/d/e/f.

Synthesis of chlorophyll-amino acid conjugates as models for modification of proteins with chromo/fluorophores.

A chlorophyll-a derivative bonded directly with epoxide at the peripheral position of the chlorin π-system was reacted with N-urethane and C-ester protected amino acids bearing an alcoholic or phenolic hydroxy group as well as a carboxy group at the residue to give chlorophyll-amino acid conjugates. The carboxy residues of N,C-protected aspartic and glutamic acids were esterified with the epoxide in high yields. The synthetic conjugates in dichloromethane had absorption bands throughout the visible region including intense red-side Qy and blue-side Soret bands. By their excitation at the visible bands, strong and efficient fluorescence emission was observed up to the near-infrared region. The chromo/fluorophores are promising for preparation of functional peptides and modification of proteins.

Peptidomimetic modification improves cell permeation of bivalent farnesyltransferase inhibitors.

Bivalent enzyme inhibitors, in which a surface binding module is linked to an active site binding module through a spacer, are a robust approach for site-selectively delivering a minimally-sized agent to a protein surface to regulate its functions, such as protein-protein interactions (PPIs). Previous research revealed that these agents effectively disrupt the interaction between farnesyltransferase (FTase) and the C-terminal region of K-Ras4B protein. However, the whole cell activity of these peptide-based agents is limited due to their low membrane permeability. In this study, we tested a peptidomimetic modification of these bivalent agents using a previously developed inhibitor, FTI-249, and evaluated their cell permeability and biological activity in cells. Confocal cell imaging using fluorescently-labeled agents showed that the peptidomimetic 3-BODIPY penetrated cells, while the peptide-based 1-BODIPY did not. Cell-based evaluation demonstrated that peptidomimetic 3 at a concentration of 100µM inhibited HDJ-2 processing in cells, indicating that this peptidomimetic modification improves cell permeability, thus leading to enhanced whole cell activity of the bivalent compounds.

Modification of 3-substituents in (bacterio)chlorophyll derivatives to prepare 3-ethylated, methylated, and unsubstituted (nickel) pyropheophorbides and their optical properties.

Methyl mesopyropheophorbide-a possessing an ethyl group at the 3-position, its 3(1)-demethyl analogue (3-methyl homologue), and its 3(1)-deethyl analogue (3-unsubstituted chlorin) were prepared by modifying naturally occurring (bacterio)chlorophylls bearing 3-vinyl, formyl, acetyl, and 1-hydroxyethyl groups. These synthetic 3-(un)substituted chlorophyll derivatives and their nickel complexes are probable intermediates during degradation of (bacterio)chlorophylls to chemically stable porphyrinoids. The optical properties (visible absorption, circular dichroism, and fluorescence emission) of the catabolic candidates in a solution were measured, and the substitution effect was investigated.

Bivalent inhibitors for disrupting protein surface-substrate interactions and for dual inhibition of protein prenyltransferases.

Low-molecular-weight compounds that disrupt protein−protein interactions (PPIs) have tremendous potential applications as clinical agents and as chemical probes for investigating intracellular PPI networks. However, disrupting PPIs is extremely difficult due to the large, flat interfaces of many proteins, which often lack structurally defined cavities to which drug-like molecules could bind in a thermodynamically favorable manner. Here, we describe a series of bivalent compounds that anchor to the enzyme active site to deliver a minimally sized surface-binding module to the targeted surface involved in transient PPI with a substrate. These compounds are capable of significantly inhibiting enzymatic reactions involving protein surface−substrate interaction in the single-digit nanomole range. Inhibitors of farnesyltransferase (FTase), which possesses a negatively charged local area on its α-subunit, were designed by attaching a module derived from a branched monoamine-containing gallate to a conventional active-site-directed CVIM tetrapeptide using an alkyl spacer. A significant improvement in inhibitory activity (>200-fold) against farnesylation of the K-Ras4B peptide was observed when the gallate module was attached to the CVIM tetrapeptide. Furthermore, the bivalent compounds had submicromolar inhibitory activity against geranylgeranylation of the K-Ras4B peptide catalyzed by GGTase I, which has an α-subunit identical to that of FTase. The anchoring strategy we describe would be useful for designing a new class of PPI inhibitors as well as dual enzyme inhibitors targeting common surface structures.

Module assembly for protein-surface recognition: geranylgeranyltransferase I bivalent inhibitors for simultaneous targeting of interior and exterior protein surfaces.

Synthetic chemical probes designed to simultaneously targeting multiple sites of protein surfaces are of interest owing to their potential application as site specific modulators of protein-protein interactions. A new approach toward bivalent inhibitors of mammalian type I geranylgeranyltransferase (GGTase I) based on module assembly for simultaneous recognition of both interior and exterior protein surfaces is reported. The inhibitors synthesized in this study consist of two modules linked by an alkyl spacer; one is the tetrapeptide CVIL module for binding to the interior protein surface (active pocket) and the other is a 3,4,5-alkoxy substituted benzoyl motif that contains three aminoalkyl groups designed to bind to the negatively charged protein exterior surface near the active site. The compounds were screened by two distinct enzyme inhibition assays based on fluorescence spectroscopy and incorporation of a [(3)H]-labeled prenyl group onto a protein substrate. The bivalent inhibitors block GGTase I enzymatic activity with K(i) values in the submicromolar range and are approximately one order of magnitude and more than 150 times more effective than the tetrapeptide CVIL and the methyl benzoate derivatives, respectively. The bivalent compounds 6 and 8 were shown to be competitive inhibitors, suggesting that the CVIL module anchors the whole molecule to the GGTase I active site and delivers the other module to the targeting protein surface. Thus, our module-assembly approach resulted in simultaneous multiple-site recognition, and as a consequence, synergetic inhibition of GGTase I activity, thereby providing a new approach in designing protein-surface-directed inhibitors for targeting protein-protein interactions.