Reversible metal-dependent destabilization and stabilization of a stem-chelate-loop probe binding to an unmodified DNA target.

Herein, we report the discovery of a novel DNA probe with a stem-chelate-loop structure, wherein the stability of the probe-target duplex can be modulated lower or higher using a narrow concentration range of dilute transition metal ions (0.1-10 µM). Oligonucleotide probes containing two terpyridine (TPY) ligands separated by 15 bases of single-stranded DNA, with or without a flanking 5 base self-complementary DNA stem, were tested in thermal transition studies with linear target DNA and varying amounts of ZnCl(2). Without the stem, addition of Zn(2+) resulted only in reversible destabilization of the probe-target duplex, consistent with assembly (up to 1 equiv Zn(2+)) and disassembly (excess Zn(2+)) of the intramolecular Zn(2+)-(TPY)(2) chelate. Surprisingly, probes including both the intramolecular chelate and the stem gave a probe-target duplex that was reversibly destabilized and stabilized upon addition of Zn(2+) by ±5-7 °C, a phenomenon consistent with assembly and then disassembly of the entire stem-Zn(2+)-(TPY)(2) motif, including the DNA stem. Stem-chelate-loop probes containing dipicolylamine (DPA) ligands exhibited no metal-dependent stabilization or destabilization. The stem-Zn(2+)-(TPY)(2) motif is readily introduced with automated synthesis, and may have broad utility in applications where it is desirable to have both upward and downward, reversible metal-dependent control over probe-target stability involving an unmodified DNA target.

Intestinal transport of aminopterin enantiomers in dogs and humans with psoriasis is stereoselective: evidence for a mechanism involving the proton-coupled folate transporter.

N-[4-[[(2,4-diamino-6-pterdinyl)methyl]amino]benzoyl]-L/D-glutamic acid (L/D-AMT) is an investigational drug in phase 1 clinical development that consists of the L-and D-enantiomers of aminopterin (AMT). L/D-AMT is obtained from a novel process for making the L-enantiomer (L-AMT), a potent oral antiinflammatory agent. The purpose of these studies was to characterize oral uptake and safety in the dog and human of each enantiomer alone and in combination and provide in vitro evidence for a mechanism of intestinal absorption. This is the first report of L /D-AMT in humans. In dogs (n = 40) orally dosed with L-AMT or D-AMT absorption was stereoselective for the L-enantiomer (6- to 12-fold larger peak plasma concentration after oral administration and area under the plasma concentration-time curve at 0-4 h; p < 0.001). D-AMT was not toxic at the maximal dose tested (82.5 mg/kg), which was 100-fold larger than the maximal nonlethal L-AMT dose (0.8 mg/kg). Dogs (n = 10) and humans with psoriasis (n = 21) orally administered L-AMT and L /D-AMT at the same L-enantiomer dose resulted in stereoselective absorption (absent D-enantiomer in plasma), bioequivalent L-enantiomer pharmacokinetics, and equivalent safety. Thus, the D-enantiomer in L/D-AMT did not perturb L-enantiomer absorption or alter the safety of L-AMT. In vitro uptake by the human proton-coupled folate transporter (PCFT) demonstrated minimal transport of D-AMT compared with L-AMT, mirroring the in vivo findings. Enantiomer selectivity by PCFT was attributable almost entirely to decreased binding affinity rather than changes in transport rate. Collectively, our results demonstrate a strong in vitro-in vivo correlation implicating stereoselective transport by PCFT as the mechanism underlying stereoselective absorption observed in vivo.

Clusters of ligands on dendrimer surfaces.

The development of methodology that is designed to allow a significant increase in the patterning and in the functionalization of the dendrimer is the ultimate goal of the research described here. Glycoside clusters based on TRIS were formed using click chemistry and were attached to PAMAM dendrimers. A series of dendrimers bearing tris-mannoside and an ethoxyethanol group was synthesized, and the binding interactions of these dendrimers with Concanavalin A were evaluated using inhibition ELISAs. The results of the inhibition ELISAs suggest that tris-mannoside clusters can replace individual sugars on the dendrimer without loss of function. Since tris-mannoside clustering allows for a redistribution of the dendrimers' surface functionalities, from this chemistry one can envision patterned dendrimers that incorporate multiple groups to increase the function and utility of the dendrimer.

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain.

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0-2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K(d) = 0.6 microM) to the Src SH2 domain when compared with Ac-pYEEI (K(d) = 1.7 microM), an optimal Src SH2 domain ligand, and peptides 2-4 (K(d) = 2.9-52.7 microM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K(d) = 1.6 microM) upon addition of Ni(2+) (300 microM), possibly due to modest structural effect of Ni(2+) on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 microM) (K(d) = 0.79 microM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.

Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity.

We describe snap-to-it probes, a novel probe technology to enhance the hybridization specificity of natural and unnatural nucleic acid oligomers using a simple and readily introduced structural motif. Snap-to-it probes were prepared from peptide nucleic acid (PNA) oligomers by modifying each terminus with a coordinating ligand. The two coordinating ligands constrain the probe into a macrocyclic configuration through formation of an intramolecular chelate with a divalent transition metal ion. On hybridization with a DNA target, the intramolecular chelate in the snap-to-it probe dissociates, resulting in the probe 'snapping-to' and binding the target nucleic acid. Thermal transition analysis of snap-to-it probes with complementary and single-mismatch DNA targets revealed that the transition between free and target-bound probe conformations was a reversible equilibrium, and the intramolecular chelate provided a thermodynamic barrier to target binding that resulted in a significant increase in mismatch discrimination. A 4-6 degrees C increase in specificity (DeltaT(m)) was observed from snap-to-it probes bearing either terminal iminodiacetic acid ligands coordinated with Ni(2+), or terminal dihistidine and nitrilotriacetic acid ligands coordinated with Cu(2+). The difference in specificity of the PNA oligomer relative to DNA was more than doubled in snap-to-it probes. Snap-to-it probes labeled with a fluorophore-quencher pair exhibited target-dependent fluorescence enhancement upon binding with target DNA.

Cyanovirin-N binding to Manalpha1-2Man functionalized dendrimers.

Manalpha1-2Man functionalized G(3) and G(4)-PAMAM dendrimers have been synthesized and characterized by MALDI-TOF MS and NMR spectroscopy. Precipitation assays to assess the binding of the dimannose-functionalized dendrimers to Cyanovirin-N, a HIV-inactivating protein that blocks virus-to-cell fusion through high mannose mediated interactions, are presented.

Altering the strength of lectin binding interactions and controlling the amount of lectin clustering using mannose/hydroxyl-functionalized dendrimers.

Protein-carbohydrate interactions play a critical role in many biological recognition events. Multivalent therapeutic agents that utilize protein-carbohydrate interactions have proven difficult to design, primarily because the fundamental requirements of protein-carbohydrate interactions are not well understood. Here, we report a systematic study of the effect on lectin binding of varying the loading of mannose surface residues on generations three through six PAMAM dendrimers. The degree of mannose functionalization was controlled by stoichiometric addition, and dendrimers were characterized using NMR and MALDI-TOF MS. Hemagglutination assays and quantitative precipitation assays were performed to determine the relative activity of the dendrimers. Using the mannose/hydroxyl-functionalized dendrimers reported here, we could systematically control both the degree of lectin clustering and the overall activity of the lectin with the dendrimer.

Heterogeneously functionalized dendrimers.

Significant timely advances have been made in the synthesis of heterogeneously functionalized dendrimers. Convergent, divergent and other functionalization techniques have been used to create a variety of complex dendrimer motifs with more than one type of surface group. Synthetic advances, as well as pertinent applications of heterogeneously functionalized dendrimers, are reviewed.