John F. Trant

Structurally diverse disaccharide analogs of antifreeze glycoproteins and their ability to inhibit ice recrystallization

The β-d-galactosyl-(1,3)-α-N-acetyl-d-galactosamine disaccharide is present in antifreeze glycoproteins (AFGPs). Analogs of this disaccharide including the β-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl galactosamine and the β-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The results from this study demonstrate that the β-linked-(1,4) disaccharide exhibits more potent IRI activity than the native β-linked-(1,3) disaccharide. The C2 N-acetyl group of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice recrystallization inhibitors.

Developing highly active small molecule ice recrystallization inhibitors based upon C-linked antifreeze glycoprotein analogues

Ice recrystallization during cryopreservation results in a significant amount of cellular damage making compounds that exhibit ice recrystallization inhibition (IRI) activity desirable as a novel class of cryoprotectants. Herein, we report a systematic structure–function study on a previously identified IRI active C-linked antifreeze glycoprotein (C-AFGP) analogue (1). These studies indicate that while C-AFGPs containing three tripeptide repeats exhibit weak IRI activity 5.5 μM, a minimum number of four tripeptide repeats is required for potent IRI activity at this concentration. In addition, the galactosyl–ornithine building block 5 exhibited only moderate activity at 22 mM, but IRI activity was significantly increased upon addition of two glycine units to the C-terminal end of the amino acid bearing the C-linked galactopyranose residue. Finally, we report that conjugation of long alkyl chains (octyl, nonyl and decyl) to the C-linked galactosyl moiety of 1 can furnish IRI active small molecules. The “ideal” hydrocarbon chain length was 10 carbons for potent activity in this series of compounds. Longer hydrocarbon chain lengths dramatically decreased solubilities. The results of this study emphasize the importance of hydrophobic moieties for IRI activity and while consistent with previously reported small molecule carbohydrate-based and lysine-based ice recrystallization inhibitors, is the first example where a large IRI active glycoconjugate has been successfully truncated to small molecule IRI active components.

Synthetic glycopolypeptides: synthesis and self-assembly of poly(γ-benzyl-L-glutamate)-glycosylated dendron hybrids

Nano-assemblies prepared from glycosylated macromolecules are promising systems for modulating or mimicking the interactions between natural carbohydrates and their receptors. In the current work, polyester dendrons bearing focal point alkynes and peripheral C-linked α-galactose moieties were synthesized and coupled to helical poly(γ-benzyl-L-glutamate) (PBLG) to afford synthetic linear-dendritic glycopolypeptides. Both the dendrimer generation and the length of the PBLG were varied to provide a small library of amphiphiles with hydrophilic mass fractions ranging from 0.07 to 0.54. The self-assembly of the copolymers in water using a solvent exchange method was optimized and studied in detail. While the linear-dendritic copolymers composed of lower generation dendrons tended to aggregate, a copolymer composed of a 4th generation galactose-functionalized dendron and PBLG with a degree of polymerization of 28 formed micellar nano-assemblies whose size could be tuned by varying the self-assembly process. Overall, this study provides new insights into the effects of polymer architecture on self-assembly properties, while at the same time introducing a new platform for the preparation of bioactive nanoparticles.

Diels–Alder Click-Cross-Linked Hydrogels with Increased Reactivity Enable 3D Cell Encapsulation

Engineered hydrogels have been extensively used to direct cell function in 3D cell culture models, which are more representative of the native cellular microenvironment than conventional 2D cell culture. Previously, hyaluronan-furan and bis-maleimide polyethylene glycol hydrogels were synthesized via Diels-Alder chemistry at acidic pH, which did not allow encapsulation of viable cells. In order to enable gelation at physiological pH, the reaction kinetics were accelerated by replacing the hyaluronan-furan with the more electron-rich hyaluronan-methylfuran. These new click-cross-linked hydrogels gel faster and at physiological pH, enabling encapsulation of viable cells, as demonstrated with 3D culture of 5 different cancer cell lines. The methylfuran accelerates Diels-Alder cycloaddition yet also increases the retro Diels-Alder reaction. Using computational analysis, we gain insight into the mechanism of the increased Diels-Alder reactivity and uncover that transition state geometry and an unexpected hydrogen-bonding interaction are important contributors to the observed rate enhancement. This cross-linking strategy serves as a platform for bioconjugation and hydrogel synthesis for use in 3D cell culture and tissue engineering.

Synthesis, self-assembly, and immunological activity of α-galactose-functionalized dendron-lipid amphiphiles

Nanoassemblies presenting multivalent displays of biologically active carbohydrates are of significant interest for a wide array of biomedical applications ranging from drug delivery to immunotherapy. In this study, glycodendron-lipid hybrids were developed as a new and tunable class of dendritic amphiphiles. A modular synthesis was used to prepare dendron-lipid hybrids comprising distearylglycerol and 0 through 4th generation polyester dendrons with peripheral protected amines. Following deprotection of the amines, an isothiocyanate derivative of C-linked α-galactose (α-Gal) was conjugated to the dendron peripheries, affording amphiphiles with 1 to 16 α-Gal moieties. Self-assembly in water through a solvent exchange process resulted in vesicles for the 0 through 2nd generation systems and micelles for the 3rd and 4th generation systems. The critical aggregation concentrations decreased with increasing dendron generation, suggesting that the effects of increasing molar mass dominated over the effects of increasing the hydrophilic weight fraction. The binding of the assemblies to Griffonia simplicifolia Lectin I (GSL 1), a protein with specificity for α-Gal was studied by quantifying the binding of fluorescently labeled assemblies to GSL 1-coated beads. It was found that binding was enhanced for amphiphiles containing higher generation dendrons. Despite their substantial structural differences with the natural ligands for the CD1d receptor, the glycodendron-lipid hybrids were capable of stimulating invariant natural killer T (iNKT) cells, a class of innate-like T cells that recognize lipid and glycolipid antigens presented by CD1d and that are implicated in a wide range of diseases and conditions including but not limited to infectious diseases, diabetes and cancer.

Covalent Polyisobutylene–Paclitaxel Conjugates for Controlled Release from Potential Vascular Stent Coatings

The development of covalent polyisobutylene (PIB)-paclitaxel (PTX) conjugates as a potential approach to controlling drug release from vascular stent coatings is described. PIB-PTX materials containing ∼24 and ∼48 wt % PTX, conjugated via ester linkages, were prepared. The PTX release profiles were compared with those of physical mixtures of PTX with carboxylic acid-functionalized PIB and with the triblock copolymer polystyrene-b-PIB-b-polystyrene (SIBS). Covalent conjugation led to significantly slower drug release. Atomic force microscopy imaging of coatings of the materials suggested that the physical mixtures exhibited multiple domains corresponding to phase separation, whereas the materials in which PTX was covalently conjugated appeared homogeneous. Coatings of the conjugated materials on stainless steel surfaces suffered less surface erosion than the physically mixed materials, remained intact, and adhered well to the surface throughout the thirty-five day study. Tensile testing and rheological studies suggested that the incorporation of PTX into the polymer introduces similar physical changes to the PIB as the incorporation of a glassy polystyrene block does in SIBS. Cytotoxicity assays showed that the coatings did not release toxic levels of PTX or other species into a cell culture medium over a 24 h period, yet the levels of PTX in the materials were sufficient to prevent C2C12 cells from adhering to and proliferating on them. Overall, these results indicate that covalent PIB-PTX conjugates have promise as coatings for vascular stents.

Preparation and Characterization of a Small Library of Thermally-Labile End-Caps for Variable-Temperature Triggering of Self-Immolative Polymers

The reaction between furans and maleimides has increasingly become a method of interest as its reversibility makes it a useful tool for applications ranging from self-healing materials, to self-immolative polymers, to hydrogels for cell culture and for the preparation of bone repair. However, most of these applications have relied on simple monosubstituted furans and simple maleimides and have not extensively evaluated the potential thermal variability inherent in the process that is achievable through simple substrate modification. A small library of cycloadducts suitable for the above applications was prepared, and the temperature dependence of the retro-Diels-Alder processes was determined through in situ 1H NMR analyses complemented by computational calculations. The practical range of the reported systems ranges from 40 to >110 °C. The cycloreversion reactions are more complex than would be expected based on simple trends expected based on frontier molecular orbital analyses of the materials.

Endo-/exo- and halogen-bonded complexes of conformationally rigid C-ethyl-2-bromoresorcinarene and aromatic N-oxides

The host–guest complexes of conformationally rigid C-ethyl-2-bromoresorcinarene with aromatic N-oxides were studied using single crystal X-ray crystallography. Unlike that of the conformationally more flexible C-ethyl-2-methylresorcinarene, the C-ethyl-2-bromoresorcinarene cavity forms endo-complexes only with the small pyridine-N-oxides, such as pyridine N-oxide, 2-methyl-, 3-methyl- and 4-methylpyrdine N-oxide, and quinoline N-oxide. The larger 2,4,6-trimethylpyridine, 4-phenylpyridine and isoquinoline N-oxide, and 4,4-bipyridine N,N′-dioxide and 1,3-bis(4-pyridyl)propane N,N′-dioxide do not fit into the host cavity. Instead endo-acetone complexes are formed. Remarkably, differing from the anti–gauche endo-complex with C-ethyl-2-methylresorcinarene, the flexible 1,3-bis(4-pyridyl)propane N,N′-dioxide guest forms an anti–anti exo-complex with C-ethyl-2-bromoresorcinarene. The endo- and exo-complexes of C-ethyl-2-bromoresorcinarene and studied N-oxides manifest C–O⋯Br, C–H⋯π and C–Br⋯π interactions.

Halogen-bonded solvates of tetrahaloethynyl cavitands

The formation and structures of halogen-bonded solvates of three different tetrahaloethynyl cavitands with acetone, chloroform, acetonitrile, DMF and DMSO were prepared and investigated. The inclusion and host–guest behaviour of the resorcinarene cavitands was found to be highly dependent on the flexibility of the ethylene-bridging unit.

Host-guest complexes of C-propyl-2-bromoresorcinarene with aromatic N-oxides*

Abstract The host-guest complexes of C-propyl-2-bromoresorcinarene with pyridine N-oxide, 3-methylpyridine N-oxide, quinoline N-oxide and isoquinoline N-oxide are studied using single crystal X-ray crystallography and 1H NMR spectroscopy. The C-propyl-2-bromoresorcinarene forms endo-complexes with the aromatic N-oxides in the solid-state when crystallised from either methanol or acetone. In solution, the endo-complexes were observed only in methanol-d4. In DMSO the solvent itself is a good guest, and crystallisation provides only solvate endo-complexes. The C-propyl-2-bromoresorcinarene shows remarkable flexibility when crystallised from either methanol or acetone, and packs into one-dimensional self-included chains. Of special note, crystallising C-propyl-2-bromoresorcinarene with 3-methylpyridine N-oxide from acetone results in a 2:2 dimeric capsular assembly organised through both C−H···πhost and N−O···(H−O)host interactions.