New materials for biological applications prepared by olefin metathesis reactions

With the advent of well-defined ruthenium olefin metathesis catalysts that are highly active and stable to a variety of functional groups, the synthesis of complex organic molecules and polymers is now possible; this is reviewed in Chapter 1. The majority of the rest of this thesis describes the application of these catalysts towards the synthesis of novel polymers that may be useful in biological applications and investigations into their efficacy. A method was developed to produce polyethers by metathesis, and this is described in Chapters 2 and 3. An unsaturated 12-crown-4 analog was made by template- directed ring-closing metathesis (RCM) and utilized as a monomer for the synthesis of unsaturated polyethers by ring-opening metathesis polymerization (ROMP). The yields were high and a range of molecular weights was accessible. In a similar manner, substituted polyethers with various backbones were synthesized: polymers with benzo groups along the backbone and various concentrations of amino acids were prepared. The results from in vitro toxicity tests of the unsubstituted polyethers are considered. The conditions necessary to synthesize polynorbornenes with pendent bioactive peptides were explored as illustrated in Chapter 4. First, the polymerization of various norbornenyl monomers substituted with glycine, alanine or penta(ethylene glycol) is described. Then, the syntheses of polymers substituted with peptides GRGD and SRN, components of a cell binding domain of fibronectin, using newly developed ruthenium initiators are discussed. In Chapter 5, the syntheses of homopolymers and a copolymer containing GRGDS and PHSRN, the more active forms of the peptides, are described. The ability of the polymers to inhibit human dermal fibroblast cell adhesion to fibronectin was assayed using an in vitro competitive inhibition assay, and the results are discussed. It was discovered that the copoymer substituted with both GRGDS and PHSR peptides was more active than both the GRGDS-containing homopolymer and the GRGDS free peptide. Historically, one of the drawbacks to using metathesis is the removal of the residual ruthenium at the completion of the reaction. Chapter 6 describes a method where the water soluble tris(hydroxymethyl)phosphine is utilized to facilitate the removal of residual ruthenium from RCM reaction products.</p

A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors--such as heat, mild and harsh acidic conditions, storage and proteolytic degradation--unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general

Angiogenic Biomarkers for Risk Stratification in Women with Suspected Preeclampsia

This poster presents the results of a single-center prospective cohort study of 315 pregnant women who presented to George Washington University Hospital Labor and Delivery service with a clinical suspicion of preeclampsia between February 2007 and November 2011. Informed consent was obtained. Baseline demographic information and medical history was collected on each patient including maternal age, ethnicity, body mass index, tobacco and other substance use, gestational age, medical problems and obstetric history. Serum samples were obtained from each enrolled subject within 24 hours of admission, and sFlt1, PlGF and sEng ELISA assays were performed in duplicate by a blinded laboratory technician at the University of Massachusetts

Trehalose Glycopolymer Enhances Both Solution Stability and Pharmacokinetics of a Therapeutic Protein

Biocompatible polymers such as poly(ethylene glycol) (PEG) have been successfully conjugated to therapeutic proteins to enhance their pharmacokinetics. However, many of these polymers, including PEG, only improve the in vivo lifetimes and do not protect proteins against inactivation during storage and transportation. Herein, we report a polymer with trehalose side chains (PolyProtek) that is capable of improving both the external stability and the in vivo plasma half-life of a therapeutic protein. Insulin was employed as a model biologic, and high performance liquid chromatography and dynamic light scattering confirmed that addition of trehalose glycopolymer as an excipient or covalent conjugation prevented thermal or agitation-induced aggregation of insulin. The insulin-trehalose glycopolymer conjugate also showed significantly prolonged plasma circulation time in mice, similar to the analogous insulin-PEG conjugate. The insulin-trehalose glycopolymer conjugate was active as tested by insulin tolerance tests in mice and retained bioactivity even after exposure to high temperatures. The trehalose glycopolymer was shown to be non-toxic to mice up to at least 1.6 mg/kg dosage. These results together suggest that the trehalose glycopolymer should be further explored as an alternative to PEG for long circulating protein therapeutics

Human Vault Nanoparticle Targeted Delivery of Antiretroviral Drugs to Inhibit Human Immunodeficiency Virus Type 1 Infection.

"Vaults" are ubiquitously expressed endogenous ribonucleoprotein nanoparticles with potential utility for targeted drug delivery. Here, we show that recombinant human vault nanoparticles are readily engulfed by certain key human peripheral blood mononuclear cells (PBMC), predominately dendritic cells, monocytes/macrophages, and activated T cells. As these cell types are the primary targets for human immunodeficiency virus type 1 (HIV-1) infection, we examined the utility of recombinant human vaults for targeted delivery of antiretroviral drugs. We chemically modified three different antiretroviral drugs, zidovudine, tenofovir, and elvitegravir, for direct conjugation to vaults. Tested in infection assays, drug-conjugated vaults inhibited HIV-1 infection of PBMC with equivalent activity to free drugs, indicating vault delivery and drug release in the cytoplasm of HIV-1-susceptible cells. The ability to deliver functional drugs via vault nanoparticle conjugates suggests their potential utility for targeted drug delivery against HIV-1

Synthesis of Norbornenyl Polymers with Bioactive Oligopeptides by Ring-Opening Metathesis Polymerization

Synthetic norbornenyl polymers with pendent cell adhesive sequences glycine-arginine-glycine-aspartic acid (GRGD) and serine-arginine-asparagine (SRN) were synthesized by ring-opening metathesis polymerization (ROMP) using newly developed ruthenium initiators. Initially, simpler polymers with pendent glycine, alanine, or penta(ethylene glycol) (EO_5) units attached directly or through ethyl and propyl spacers to various norbornenyl backbones were synthesized using Ru CHPh(Cl)_2(PCy_3)_2 (1) as the initiator. The molecular weights, PDI's, polymerization times, yields, and glass transition temperatures were compared for these polymers. As a result of this comparison, poly(5-norbornene-2-carboxyl) was chosen as the backbone for the more complex oligopeptide containing polymers, and norbornene monomers with pendent EO_5 (21), GRGD (24), and SRN (25) units were made. Monomers 21 and 24 were copolymerized to form a poly(norbornene) containing 9.2 mol % GRGD (26a) using 1 as the initiator. However, incorporating larger amounts of GRGD resulted in extremely low yields of polymers that exhibited bimodal molecular weight distributions. Homopolymers and copolymers with larger amounts of GRGD and SRN were synthesized in good yields (32−92%) with monomodal molecular weight distributions using the newly developed, more active, 2,3-dihydroimidazolylidene initiators, Ru CHPh(Cl)_2(PCy_3)(DHIMes) (2) and Ru CH−CH C(CH_3)_2(Cl)_2(PCp_3)(DHIMes) (3). In this way, EO_5 containing copolymers with 49 mol % GRGD (26b), 53 mol % SRN (27b), or 32 mol % GRGD and 21 mol % SRN (28a) were synthesized, as well as copolymer 28b with 53 mol % GRGD and 47 mol % SRN. To alter the presentation of the GRGD, an EO_5 containing copolymer with a propyl spacer between the GRGD and the backbone (30) was also synthesized

Inhibition of Cell Adhesion to Fibronectin by Oligopeptide-Substituted Polynorbornenes

Polynorbornenes substituted with two different peptide sequences from the RGD-containing integrin cell-binding domain of fibronectin are potent inhibitors of human foreskin fibroblast cell adhesion to fibronectin-coated surfaces. Ring-opening metathesis polymerization (ROMP) using Ru═CHPh(Cl)_2(PCy_3)(DHIMes) (1) as an initiator produced polymers substituted with GRGDS and PHSRN peptide sequences. The inhibitory activity was quantified for these polymers and compared to the free peptides and GRGES-containing controls. A homopolymer substituted with GRGDS peptides was significantly more active than the free GRGDS peptide (IC_(50) of 0.18 ± 0.03 and 1.33 ± 0.20 mM respectively), and the copolymer containing both GRGDS and PHSRN is the most potent inhibitor (IC_(50) of 0.04 ± 0.01 mM). These results demonstrate that significant enhancements of observed biological activity can be obtained from polymeric materials containing more than one type of multivalent ligand and that ROMP is a useful method to synthesize such well-defined copolymers

Development of a Grp94 inhibitor

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja303477g.Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein