Structural mimicry of DNA is utilized in nature as a strategy to evade molecular defences mounted by host organisms. One such example is the protein Ocr – the first translation product to be expressed as the bacteriophage T7 infects E. coli. The structure of Ocr reveals an intricate and deliberate arrangement of negative charges that endows it with the ability to mimic ∼24 base pair stretches of B-DNA. This uncanny resemblance to DNA enables Ocr to compete in binding the type I restriction modification (R/M) system, and neutralizes the threat of hydrolytic cleavage of viral genomic material. Here, we report the de novo design and biophysical characterization of DNA mimicking peptides, and describe the inhibitory action of the designed helical bundles on a type I R/M enzyme, EcoR124I. This work validates the use of charge patterning as a design principle for creation of protein mimics of DNA, and serves as a starting point for development of therapeutic peptide inhibitors against human pathogens that employ molecular camouflage as part of their invasion stratagem.

Structural and quantitative changes in the expression of sialic acid residues on the surface of eukaryotic cells profoundly influence a broad range of biological processes including inflammation, antigen recognition, microbial attachment and tumour metastasis. Uptake and incorporation of sialic acid analogues in mammalian cells enable structure–function studies and perturbation of specific recognition events. Our group has recently shown that a trifluorobutyryl-modified sialic acid metabolite diminishes the adhesion of mammalian cells to E and P-Selectin, presumably by leading to the expression of fluorinated sLex epitopes on cell surfaces, and interfering with the sLex–selectin interactions that are well known in mediating tumour cell migration (J. Med. Chem.2010, 53, 4277). For studies directed towards understanding the molecular basis of this reduced adhesion, chemical synthesis of trifluorobutyrylated sialyl Lewis X (C4F3-sLex) was crucial. We have developed a highly efficient [2+2] approach for the assembly of C4F3-sLex on a preparative scale that contains versatile protective groups allowing the glycan to be surface immobilized or solubilized as needed for biophysical studies to investigate selectin interactions. This strategy can, in principle, be used for preparation of other N-modified sLex analogues.

The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d > 3 μm); (3) nanometer-scale domains (d = 100 nm to 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.

Herein we report the synthesis of N-acetyl neuraminic acid derivatives as 4-methylumbelliferyl glycosides and their use in fluorometrically quantifying human and bacterial sialidase activity and substrate specificities. We found that sialidases in the human promyelocytic leukemic cell line HL60 were able to cleave sialic acid substrates with fluorinated C-5 modifications, in some cases to a greater degree than the natural N-acetyl functionality. Human sialidases isoforms were also able to cleave unnatural substrates with bulky and hydrophobic C-5 modifications. In contrast, we found that a bacterial sialidase isolated from Clostridium perfringens to be less tolerant of sialic acid derivatization at this position, with virtually no cleavage of these glycosides observed. From our results, we conclude that human sialidase activity is a significant factor in sialic acid metabolic glycoengineering efforts utilizing unnatural sialic acid derivatives. Our fluorogenic probes have enabled further understanding of the activities and substrate specificities of human sialidases in a cellular context.

We describe herein the design, synthesis, and thermodynamic characterization of fluorinated β-hairpin constructs. Introduction of hexafluoroleucine (Hfl) did not perturb β-hairpin formation, as judged by 1H NMR structures of four peptides determined to <1 Å backbone RMSDs, allowing direct comparison of thermodynamic stabilities of fluorinated peptides to their hydrocarbon counterparts. Judicious fluorination of peptides often results in increased thermal and chemical stability of the resultant folded structures. However, we found that when cross-strand residue partners were varied, the side-chain interaction energies followed the order Leu-Leu > Hfl-Leu > Hfl-Hfl. All peptides were more structured in 90% MeOH than in aqueous buffers. The peptides with Hfl-Leu or Hfl-Hfl cross-strand partners showed increased interaction energies in this solvent compared to those in water, in contrast to the insignificant effect on Leu-Leu. Our results inform the binding and assembly of peptides containing Hfl in the context of β-sheet structures and may be useful in interpreting binding of fluorinated ligands and peptides to biological targets.

Aberrant glycosylation of lipid and protein molecules on cellular surfaces is responsible for many of the pathophysiological events in tumor progression and metastasis. Sialic acids in particular, are overexpressed on the glycocalyx of malignant tumor cells and sialic acid-mediated cell adhesion is required for metastasis. We report here that replacement of sialic acids on cell surfaces with fluorinated congeners dramatically decreases cell adhesion to E- and P-selectin-coated surfaces. Comparison of adhesion of fluorinated cells with those modified with nonfluorinated analogues suggests that both reduce binding of the modified sialosides to their cognate lectins to a similar extent on a per molecule basis. The overall reduction in cell adhesion results from greater cell surface presentation of the fluorinated congeners. This work suggests an avenue for inhibition of metastasis by administration of small molecules and concomitant noninvasive imaging of tumor cells by 19F MRI before they are visible by other means.

Fluorinated lipids get rapidly internalized into living cells and are also displayed on the cell surface. The uptake of lipids is energy dependent and is likely via the clathrin-mediated endocytic pathway. Fluorinated lipids are 3−5-fold more efficient in acting as molecular transporters of noncovalently bound proteins than their hydrocarbon counterparts. These materials could serve as efficient molecular transporters for molecules that function in the cytoplasm such as short interfering RNAs (siRNAs)

The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis, and characterization of fluorinated analogues of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogues displayed higher proteolytic stability with simultaneous retention of biological activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand−receptor interactions.

Metabolic oligosaccharide engineering has been employed to introduce fluorine-containing groups onto mammalian cell surfaces. Incubation of HeLa, Jurkat, and HL60 cells in culture with fluorinated sialic acid and mannosamine analogues resulted in cell-surface presentation of fluorinated glycans. Metabolic conversion of fluorinated precursors was detected and quantified by DMB-derivatization and HPLC ESI-MS analysis. Between 7% and 72% of total membrane-associated sialosides were fluorinated, depending on the precursor used and the cell type. Fluorination of mammalian cell surfaces provides a means for introducing a bioorthogonal surface for modulating noncovalent interactions such as those involved in cell adhesion.The surfaces of living cells have been fluorinated by incubation with N-fluoroacyl mannosamines or N-fluoroacyl neuraminic acids. Fluorinated cells showed reduced adhesion to extracellular matrix biomolecules.

We describe here the use of polyfluorinated trivalent iodonium salts as efficient and robust capping reagents during solid phase peptide synthesis using either t-Boc or Fmoc chemistry. Standard protocols established for solid phase peptide synthesis can be utilized without any change in solvent or reagent conditions. The capping reaction was carried out at sites where amino acid coupling steps failed. At the termination of the synthesis, the crude peptide mixture obtained from release of materials from the resin, is either simply centrifuged in aqueous solution to yield pure peptide, or purified by passage through fluorous silica gel in solvents with high water content. We envision that the chemistry and reagents described here will find wide use in peptide and protein chemistry, and also in combinatorial library synthesis where terminal amines are coupled to reaction partners.Peptides synthesized on the solid phase using the Fmoc protocol on commercial automated synthesizers or using manual synthesis were capped using fluorous trivalent iodonium salts at sites of incomplete couplings. Deletion products are removed by simple centrifugation of aqueous solutions.

Control of structure and function in membrane proteins remains a formidable challenge. We report here a new design paradigm for the self-assembly of protein components in the context of nonpolar environments of biological membranes. An incrementally staged assembly process relying on the unique properties of fluorinated amino acids was used to drive transmembrane helix–helix interactions. In the first step, hydrophobic peptides partitioned into micellar lipids. Subsequent phase separation of simultaneously hydrophobic and lipophobic fluorinated helical surfaces fueled spontaneous self-assembly of higher order oligomers. The creation of these ordered transmembrane protein ensembles is supported by gel electrophoresis, circular dichroism spectroscopy, equilibrium analytical ultracentrifugation, and fluorescence resonance energy transfer.

Lipid bilayers consisting of lipids with terminally perfluoroalkylated chains have remarkable properties. They exhibit increased stability and phase-separated nanoscale patterns in mixtures with nonfluorinated lipids. In order to understand the bilayer properties that are responsible for this behavior, we have analyzed the structure of solid-supported bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and of a DPPC analogue with 6 terminal perfluorinated methylene units (F6-DPPC). Polarized attenuated total reflection Fourier-transform infrared spectroscopy indicates that for F6-DPPC, the tilt of the lipid acyl chains to the bilayer normal is increased to 39° as compared to 21° for native DPPC, for both lipids in the gel phase. This substantial increase of the tilt angle is responsible for a decrease of the bilayer thickness from 5.4 nm for DPPC to 4.5 nm for F6-DPPC, as revealed by temperature-controlled imaging ellipsometry on microstructured lipid bilayers and solution atomic force microscopy. During the main phase transition from the gel to the fluid phase, both the relative bilayer thickness change and the relative area change are substantially smaller for F6-DPPC than for DPPC. In light of these structural and thermotropic data, we propose a model in which the higher acyl-chain tilt angle in F6-DPPC is the result of a conformational rearrangement to minimize unfavorable fluorocarbon−hydrocarbon interactions in the center of the bilayer due to chain staggering.