David Vander Velde

The effect of β-turn structure on the passive diffusion of peptides across Caco-2 cell monolayers

AbstractPurpose. To investigate the relationships between the β-turn structure of a peptide and its passive diffusion across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. Methods. Linear hydrophilic peptides (Ac-TyrProXaaZaaVal-NH2; Xaa = Gly, Ile and Zaa = Asp, Asn) and hydrophobic (Ac-YaaPro-XaaIleVal-NH2; Yaa = Tyr, Phe and Xaa = Gly, Ile: and Ac-PhePro-XaaIle-NH2; Xaa = Gly, He) peptides were synthesized and their effective permeability coefficients (Peff) were determined across Caco-2 cell monolayers. The lipophilicities of the peptides were estimated by measuring their partition coefficients (Po/w) between 1-octanol and HBSS. Two-dimensional NMR (2D-NMR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the solution structures of these model peptides. Results. Using 2D-NMR spectroscopy and CD spectroscopy, the hydrophilic Gly-containing peptides (Ac-TyrProGlyZaaVal-NH2; Zaa = Asp, Asn) were shown to exhibit a higher degree of β-turn structure in solution than the Ile-containing peptides (Ac-TyrProIleZaaVal-NH2; Zaa = Asp, Asn). CD spectroscopy was used to show that the Gly-containing hydrophobic peptides (Ac-YaaProGlyIleVal-NH2; Yaa = Tyr, Phe: and Ac-PheProGlyIle-NH2) exhibited a higher degree of β-turn structure in solution than the Ile-containing hydrophobic peptides. The Peff values of all four hydrophilic peptides across unperturbed Caco-2 cell monolayers were very low and no statistically significant differences were observed between the Gly- and Ile-containing penta-peptides within either the Asp or Asn series. The Peff values for the hydrophobic Gly-containing peptides were significantly greater than the Peff values determined for their Ile-containing counterparts. The Gly-containing penta- and tetrapeptides in the Phe series, which exhibited high permeation, were shown to be metabolically unstable. In contrast, the Gly- and Ile-containing pentapeptides in the Tyr series and the Ile-containing penta- and tetrapeptides in the Phe series, which exhibited low permeation, were metabolically stable. Conclusions. Hydrophobic peptides that exhibit significant β-turn structure in solution are more lipophilic as measured by log Po/w and more readily permeate Caco-2 cell monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. The ability of these peptides to permeate Caco-2 cell monolayers via the transcellular route also exposed them to metabolism, presumably by cytosolic endopeptidases. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties fo the peptides so as to alter their paracellular flux through unperturbed Caco-2 cell monolayers.

Effect of restricted conformational flexibility on the permeation of model hexapeptides across Caco-2 cell monolayers

AbstractPurpose. To determine how restricted conformational flexibility of hexapeptides influences their cellular permeation characteristics. Methods. Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X-Ala]; X = Asp, Asn, Lys) hexapeptides were synthesized, and their transport characteristics were assessed using the Caco-2 cell culture model. The lipophilicities of the hexapeptides were determined using an immobilized artificial membrane. Diffusion coefficients used to calculate molecular radii were determined by NMR. Two-dimensional NMR spectroscopy, circular dichroism, and molecular dynamic simulations were used to elucidate the most favorable solution structure of the cyclic Asp-containing peptide. Results. The cyclic hexapeptides used in this study were 2−3 times more able to permeate (e.g., Papp = 9.3 ± 0.3 × 10−8 cm/sec, X = Asp) the Caco-2 cell monolayer than were their linear analogs (e.g., Papp = 3.2 ± 0.3 × 10−8 cm/sec, X = Asp). In contrast to the linear hexapeptides, the flux of the cyclic hexapeptides was independent of charge. The cyclic hexapeptides were shown to be more lipophilic than the linear hexapeptides as determined by their retention times on an immobilized phospholipid column. Determination of molecular radii by two different techniques suggests little or no difference in size between the linear and cyclic hexapeptides. Spectroscopic data indicate that the Asp-containing linear hexapeptide exists in a dynamic equilibrium between random coil and β-turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two β-turns. Conclusions. Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two β-turns, explain its greater lipophilicity.

Comprehensive Study of Sansalvamide A Derivatives and their Structure–Activity Relationships against Drug-Resistant Colon Cancer Cell Lines

We report an extensive structure-activity relationship (SAR) of 62 compounds active against two drug-resistant colon cancer cell lines. Our comprehensive evaluation of two generations of compounds utilizes SAR, NMR, and molecular modeling to evaluate the key 3D features of potent compounds. Of the seven most potent compounds reported here, five are second-generation, emphasizing our ability to incorporate potent features found in the first generation and utilize their structures to design potency into the second generation. These analogs share no structural homology to current colon cancer drugs, are cytotoxic at levels on par with existing drugs treating other cancers, and demonstrate selectivity for drug-resistant colon cancer cell lines over noncancerous cell lines. Thus, we have established sansalvamide A as an excellent lead for treating multiple drug-resistant colon cancers.

A tandem prins/schmidt reaction approach to marine alkaloids: Formal and total syntheses of lepadiformines A and C

The tricyclic core of the cylindricine or lepadiformine families of alkaloid natural products was assembled via a Prins addition/intramolecular Schmidt rearrangement under Lewis acid conditions. Both single-pot and two-stage variations of this process were examined, with particular attention to the stereochemical outcome of the processes. This technology has been applied to a formal total synthesis of lepadiformine A and a total synthesis of lepadiformine C.

Characterization of the complexation of fluoroquinolone antimicrobials with metal ions by nuclear magnetic resonance spectroscopy

The complexation of the fluoroquinolone antimicrobials is important because it has been implicated in reduced oral bioavailability and reduced antimicrobial activity when the drugs are co-administered with antacids or multi-vitamin preparations containing iron. The complexation of two model compounds, lomefloxacin and norflaxacin was studied using NMR. With aluminum ions, exchange between free and bound drug molecules was slow on the NMR time-scale. Two complexes, proposed to have stoichiometries of 2:1 and 3:1 (drug:metal) based on peak widths and variable temperature studies, were observed. The crystal structure of lomefloxacin, which shows intermolecular self association previously reported to be crucial to the drugs mode of action, is also reported. Because the metal ion complexes could not be crystallized, the crystal structure of uncomplexed lomefloxacin together with the NMR data on the aluminum complexes were used in the molecular modelling of the lomefloxacin-aluminum complexes.

Relative stereochemistry and solution conformation of the novel paclitaxel-like antimitotic agent epothilone A

Abstract A nearly complete set of 1 H 1 H coupling constants and NOEs were determined for the novel paclitaxel-like antimitotic agent epothilone A from phase sensitive DFQ COSY and NOESY experiments in 75% DMSO- d 6 /D 2 O. This data was employed as distance constraints for molecular modeling studies. Molecular dynamic simulations were carried out on a complete series of diastereoisomers, and the results show only two relative stereochemistries to be in agreement with the NOE data.

The Effect of Conformation on Membrane Permeability of an Acyloxyalkoxy-linked Cyclic Prodrug of a Model Hexapeptide

AbstractPurpose. To determine the different conformations of the acyloxyalkoxy-linked cyclic prodrug 1 of the model hexapeptide 2 in solution and to investigate the relationship between these solution conformations and the cellular permeability characteristics of this prodrug. Methods. Two-dimensional Homonuclear Hartmann-Hahn spectroscopy, Rotating-Frame Overhouser effect spectroscopy, circular dichroism and molecular dynamics simulations were used to find the solution conformers of cyclic prodrug 1. Results. Our spectroscopic findings suggest that cyclic prodrug 1 exhibits a major and a minor conformer in solution. The major conformer appears to have a well-defined secondary structure, which involves a β-turn and 4 → 1 intramolecular hydrogen bond, creating a compact structure with a reduced average hydrodynamic radius compared to the model hexapeptide 2. Conclusions. The increased ability of cyclic prodrug 1 to permeate membranes compared to the model hexapeptide 2 could be due to reduction in the average hydrodynamic radius of the molecule facilitating paracellular flux and/or the reduction in the hydrogen bonding potential facilitating transcellular flux.

Galactose-Imines in the staudinger reaction

Abstract Staudinger reaction of imines 1 and 2 derived from galactose with a variety of acid chlorides resulted in the formation of two β-lactams with cis configuration, typically in a 60:40 ratio and in good yields. The β-lactams were separated and the absolute stereochemistry of the diastereoisomers was determined by single crystal X-ray analysis and correlation of their 1 H-NMR data.

Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier.

In keeping with the advance of biotechnology, cell culture becomes an important tool for investigating the transport and the metabolism phenomena. A cell line of human origin, the BeWo choriocarcinoma cell line, was used for the study of the transport and metabolism of opioid peptides across the in vitro model of the placental barrier. Opioid peptides, both naturally occurring and their synthetic analogs, are of interest to be developed as potent analgesics and were included in this study. The apparent permeability coefficients (Pe)s of the peptides containing 4-11 amino acid or analog residues were in the range of 0.23-14.6 x 10(-5) cm/s. The (Pe)s of these peptides were comparable to those of sucrose or dextrans, hydrophilic markers. The (Pe)s of low molecular weight (MW) peptides was not dependent on their MW or molecular size, whereas an inversely linear correlation between (Pe)s and molecular size was observed with the larger peptides. Molecular sieving of the BeWo monolayer restricted the transport of the peptides with MW> or =1033 Da or molecular size > or =6.6 A. Membrane partitioning ability and charge of the peptides were also investigated and found to be the minor factors regulating the extent of peptide permeation. Contrasting to the transport of Tyr-[D-pen-Gly-Phe-D-Pen] (DPDPE) peptide analog across the blood-brain barrier, the transport of DPDPE across the BeWo monolayers were not indicated to be via carrier-mediated transport. The major transport pathway of the opioid peptides across the BeWo monolayers was found to be via paracellular route. In metabolism studies, aminopeptidase was found to be a major enzyme type responsible for the degradation of naturally occurring peptides but not for the synthetic analogs. The finding obtained from the present study reveals the applicability of the BeWo cell line as an in vitro model for investigating placental transport and metabolism of opioid peptides.

Comparative effects of (SBE)7m‐β‐CD and HP‐β‐CD on the stability of two anti‐neoplastic agents, melphalan and carmustine

Abstract The purpose of this study was to evaluate and compare the potential use of two parenterally safe β‐cyclodextrins derivatives, (SBE) 7m ‐β‐CD and HP‐β‐CD, as solubilizers and stabilizers for melphalan and carmustine, two very unstable antineoplastic agents. Phase solubility and chemical stability of the compounds in the presence of the cyclodextrins were studied. UV, fluorescence, and several NMR techniques were used to probe the potential causes for the differences observed. The phase solubility method was found to provide only qualitative data on the binding of melphalan to the cyclodextrins since rapid degradation and the presence of products of degradation complicated the interpretation of the results. Qualitatively, however, the solubilizing potential was similar for the two cyclodextrins. The chemical stability studies indicate that both of the drugs had similar binding constants for both cyclodextrins; however, the intrinsic reactivities in the complexes were significantly lower with (SBE) 7m ‐β‐CD than for HP‐β‐CD. The main cause for this distinct difference appeared to correlate with differences in the site of binding and the polarity of the binding site.