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.     

Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay

We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamidoamine (G4-PAMAM) dendrimers carrying (i) (η⁵-cyclopentadienyl) iron dicarbonyl succinimidato complexes as infrared (IR) probes, (ii) nitroaniline entities as nuclear magnetic resonance (NMR) probes, (iii) acetamide groups for surface neutralization, and (iv) hydrazide-terminated spacer arms for the reaction with aldehyde. To preserve a high binding affinity, the conjugation was performed on the carbohydrate moieties located on the Fc fragment. The resulting conjugates were characterized by Fourier transform-IR, ultraviolet (UV), and high-mass matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. On the basis of relative concentration ratios of IR probes and antibody, an average labeling of 30 IR probes per antibody was reached (i.e., more than twice the value obtained with our previous strategy that generated no spacer arm). Immunoassays revealed that the antibody-dendrimer conjugates retained 55.1% of immunoreactivity on average with respect to underivatized antibody. Finally, the conjugates were used to quantify their antigen by solid-phase carbonyl metallo immunoassay (CMIA). Results showed a significant enhancement of the IR signal, demonstrating the efficiency of the new conjugation strategy and the potential of the new antibody-dendrimer conjugates as universal immunoanalytical reagents.     

Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode

We have developed an affinity biosensor system based on avidin-biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 x 10(-12) mol/cm(2) on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.     

Synthesis of a folate functionalized PEGylated poly(propylene imine) dendrimer as prospective targeted drug delivery system

Based on fourth generation diaminobutane poly(propylene imine) dendrimer, a novel targeted drug nanocarrier was prepared, bearing protective PEG chains and a folate targeting ligand. As a control a PEGylated derivative without folate was also synthesized. The encapsulation and release properties of these PEGylated derivatives were investigated employing etoposide, an anticancer hydrophobic drug. Enhanced solubility of etoposide was achieved inside the dendrimeric scaffold which was subsequently released in a controlled manner. These properties coupled with specificity towards the folate receptor and the low toxicity render folate functionalized PEGylated poly(propylene imine) dendrimer promising candidate for targeted drug delivery.     

Solid-phase synthesis of a library of functionalized aminodiol scaffolds.

A combinatorial library motif has been developed based on orthogonally protected aminodiol scaffolds. Amine functionality was derivatized by commercially available electrophiles including carboxylic acids, sulfonyl chlorides, isocyanates, and aldehydes. A hydroxyl moiety was converted to a carbamate linkage, allowing a variety of amines to be incorporated. The scaffold was anchored to TentaGel at the second hydroxyl via a succinyl linker, which was hydrolyzed by mild aqueous basic conditions. The method was used to make a library of about 17,000 different members in mixtures of 5 per sample.     

Efficient synthesis of C-11 functionalized steroids

An efficient synthesis from readily available materials of numerous steroids functionalized at C-11 position, interesting from a biological point of view, is described using our general approach. The characteristic (1)H and (13)C NMR spectroscopic features of the synthesized steroids are reported.     

Solid‐phase synthesis of a library of functionalized aminodiol scaffolds

A combinatorial library motif has been developed based on orthogonally protected aminodiol scaffolds. Amine functionality was derivatized by commercially available electrophiles including carboxylic acids, sulfonyl chlorides, isocyanates, and aldehydes. A hydroxyl moiety was converted to a carbamate linkage, allowing a variety of amines to be incorporated. The scaffold was anchored to TentaGel at the second hydroxyl via a succinyl linker, which was hydrolyzed by mild aqueous basic conditions. The method was used to make a library of about 17,000 different members in mixtures of 5 per sample. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:143–154, 1998/1999.     

One-pot synthesis of highly functionalized morphans from C-glycosides

Highly functionalized morphan derivatives were synthesized from nitroalkene 2′-(oxoalkyl)-C-glycosides by a tandem reaction that created three (two C–N and one C–C) new bonds and four stereogenic centers in a one-pot procedure under very mild conditions without the use of expensive reagents. The transformation was achieved from a β-elimination/Michael addition cascade, followed by Michael addition of the amine and intramolecular enamination. In the presence of sodium cyanoborohydride the iminium intermediate was reduced in situ to afford the desired morphans.     

First synthesis of a fully active spin-labeled peptide hormone

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. The ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac0-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for alpha-MSH.     

Synthesis of ferrocenyl-bearing dendrimers with a resorcinarene core

A series of ferrocenyl ended dendrons containing π-conjugated systems were obtained using Wittig and Heck reactions. The dendrons were attached to eight functionalized resorcinarenes via Williamson reaction obtaining high molecular weight dendrimers. No electronic communication between metal centers was observed by cyclic voltammetry. All the dendrimers were characterized by ¹H, ¹³C NMR, FTIR, UV-Vis, MALDI-TOF, elemental analyses, and electrochemical studies.     

Efficient synthesis of functionalized oligodeoxyribonucleotides with base-labile groups using a new silyl linker

In this study, we developed new 3′-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These reagents could increase the efficiency of introduction of 3′-terminal deoxyribonucleoside components into polymer supports to a level of 17–29 μmol/g. The efficiency was higher than that of previous T-loading reagents because reagents 1 contain a 4-aminobutyryl residue as a spacer. Moreover, we could synthesize not only unmodified DNA oligomers but also a base-labile modified DNA oligomer using resins 9a–d in the activated phosphite method without base protection.     

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.     

Chemical synthesis of a P(k)-antigenic globotriose analogue with a functionalized aglycon

6-Aminohexyl alpha-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside (2), a globotriose analogue with a functionalized aglycon, was synthesized, using alpha-D-galactopyranosyluronic acid-(1-->4)-D-galactopyranosyluronic acid [di-GalA (3)] as the starting material, which is commercially available or can be readily prepared from pectin.     

The synthesis of functionalized 13,14-seco-steroids via Grob fragmentation

A synthetic methodology for the synthesis of 13,14-seco-steroids with substituents at C-14 and C-17 is described. The approach involves Grob fragmentation of 14beta-hydroxy-17beta-tosylates, hydroboration-oxidation of the intermediate delta13(17)-olefin, and hydride reduction of the 14-ketone. An unambiguous structural assignment of (13R,14S,17S)-14,17-diacetoxy-3-methoxy-7alpha-methyl-13,14-secoestra-1,3,5(10)-triene was determined by X-ray analysis.     

Hyperbolic Modeling of Starburst Dendrimers

Starburst dendrimers are highly branched oligomers. A rigid dendritic hydrocarbon, C₁₁₃₄H₁₁₄₆, has recently been synthesized. It consists of 94 phenylacetylene units displayed in a self-similar two-dimensional skeleton isomorphous to the three-coordinated Bethe lattice. The three-dimensional representation of phenylacetylene dendrimer shows a globular architecture with large voids and niches in its interior, characteristic of hyperbolic surfaces. This work investigates the geometrical scaling behavior of this starburst dendrimer using the symmetry properties of a Bethe lattice embedded in the hyperbolic plane. The results for C₁₁₃₄H₁₁₄₆ provide its density profile and an upper bound for its macromolecular size. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of the functionalized congener approach to dendrimer-based signaling agents acting through A2A adenosine receptors

As a continued effort to develop multivalent ligands to enhance the pharmacological effects of monomeric drugs, DITC-APEC, a chemically reactive nucleoside A(2A) adenosine receptor (AR) agonist, was employed to derivatize the surface of third-generation (G3) polyamidoamine (PAMAM) dendrimers. The resulting conjugates carried multiple copies of the agonist attached through a thiourea linkage and differed in the number of attachments and in the presence of a fluorophore or additional surface modification. Computer modeling studies suggested that these DITC-APEC-loaded dendrimers extended the overall diameter of the previously reported PAMAM-CGS21680 dendrimer derivatives (Kim et al., Bioconjugate Chem 2008 19:406-411) by ca. 20 A, potentially increasing the conformational flexibility of the appended ligands to achieve optimal geometry for efficient binding at A(2A) ARs. Increased affinity and selectivity in binding in comparison to the CGS21680 conjugate were envisioned, due to the presence of an extended linker, i.e., a dithioureylenephenyl functionality. In vitro radioligand competition experiments showed effective binding of these PAMAM-DITC-APEC dendrimer conjugates at the human A(2A) and A(3) ARs with submicromolar K (i) values and selectivity in comparison to the human A(1) AR. Furthermore, these nucleoside-loaded dendrimers exhibited an A(2A) AR-mediated inhibitory effect on ADP-induced aggregation of human platelets. The present study demonstrates the potential of applying the functionalized congener concept to engineer dendrimer-based multivalent ligands for G protein-coupled receptors.     

The synthesis of amino-acid functionalized beta-carbolines as topoisomerase II inhibitors

The synthesis and biological activity of amino acid functionalized beta-carboline derivatives, which are structurally related to azatoxin and the tryprostatins, are reported. These compounds were assayed for their growth inhibition properties in H520 and PC3 cell lines and were examined for their abilities to inhibit topoisomerase II-mediated DNA relaxation.     

Enzymatic esterifications of functionalized phenols for the synthesis of lipophilic antioxidants

Of seven lipases used to esterify functionalized phenols and fatty acids to synthesize lipophilic antioxidants, that from Candida antarctica lipase B (CAL-B) had the highest catalytic activity. Esterification yields of benzoic acid derivatives catalyzed by CAL-B are below 2% after 7 days. In contrast, relatively high yields were obstained for the esterification of (poly)phenols bearing primary hydroxy groups and aliphatic acids (85% yield within 15 hours). Crude products were purified and used as lipophilic antioxidants in sunflower oil.     

First synthesis of a pentadeuterated 3'-hydroxystanozolol--an internal standard in doping analysis

The first synthesis of 16,16,20,20,20-pentadeuterio-3'-hydroxystanozolol (8) in 26% yield over nine steps is described using moderately priced starting materials and economic amounts of reagents. Compound 8 can be used as an internal standard in screening procedures for anabolic steroids as well as for the quantification of stanozolol metabolites via mass spectrometric techniques, such as LC-MS or gas chromatography-mass spectrometry (GC-MS).     

The synthesis and biological evaluation of a range of novel functionalized benzopyrans as potential potassium channel activators

A range of novel benzopyrans have been synthesised and biologically evaluated for K(ATP) channel activity employing cromakalim 1 as a benchmark K(ATP) channel opener. Although the compounds that were evaluated demonstrated a reduced ability to relax phenylephrine stimulated rat thoracic tissue, we provide evidence that benzopyrans 7a-h may be operating via an alternative mechanism than ATP-sensitive K(+) channel activity.