Laura Hartmann

Precision Polymers: Monodisperse, Monomer‐Sequence‐Defined Segments to Target Future Demands of Polymers in Medicine

The established technology platforms of solid-phase-supported oligopeptide and oligonucleotide synthesis can be expanded to access fully synthetic macromolecules, preserving both the monodisperse character and the defined monomer sequence. Precision polymers are sequentially assembled from a library of functional building blocks, enabling one to program interaction capabilities or generate functions by sequence-specific positioning of functionalities. Examples are provided, showing that these monodisperse macromolecules can be conjugated to oligonucleotides, oligopeptides, or poly(ethylene glycol)s. The resulting model systems can contribute to the understanding of complex biomedical-related processes. Due to the absence of chemical and molecular-weight distributions in these multifunctional segments, exact correlation of the monomer sequence and (bio)properties is attainable. This is demonstrated by the design of carrier systems that exhibit fine-tuned interactions with plasmid DNA, actively controlling important steps in DNA delivery and transfection, such as polyplex formation, DNA compression, and release of the cargo.

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.

Sequence-defined glycopolymer segments presenting mannose: synthesis and lectin binding affinity.

We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands.

Carbohydrate-Lectin Recognition of Sequence-Defined Heteromultivalent Glycooligomers

Multivalency as a key principle in nature has been successfully adopted for the design and synthesis of artificial glycoligands by attaching multiple copies of monosaccharides to a synthetic scaffold. Besides their potential in various applied areas, e.g. as antiviral drugs, for the vaccine development and as novel biosensors, such glycomimetics also allow for a deeper understanding of the fundamental aspects of multivalent binding of both artificial and natural ligands. However, most glycomimetics so far neglect the purposeful arranged heterogeneity of their natural counterparts, thus limiting more detailed insights into the design and synthesis of novel glycomimetics. Therefore, this work presents the synthesis of monodisperse glycooligomers carrying different sugar ligands at well-defined positions along the backbone using for the first time sequential click chemistry and stepwise assembly of functional building blocks on solid support. This approach allows for straightforward access to sequence-defined, multivalent glycooligomers with full control over number, spacing, position, and type of sugar ligand. We demonstrate the synthesis of a set of heteromultivalent oligomers presenting mannose, galactose, and glucose residues. All heteromultivalent structures show surprisingly high affinities toward Concanavalin A lectin receptor in comparison to their homomultivalent analogues presenting the same number of binding ligands. Detailed studies of the ligand/receptor interaction using STD-NMR and 2fFCS indeed indicate a change in binding mechanism for trivalent glycooligomers presenting mannose or combinations of mannose and galactose residues. We find that galactose residues do not participate in the binding to the receptor, but they promote steric shielding of the heteromultivalent glycoligands and thus result in an overall increase in affinity. Furthermore, the introduction of nonbinding ligands seems to suppress receptor clustering of multivalent ligands. Overall these results support the importance of heteromultivalency specifically for the design of novel glycoligands and help to promote a fundamental understanding of multivalent binding modes.

Magnetic Porous Sugar-Functionalized PEG Microgels for Efficient Isolation and Removal of Bacteria from Solution

Here, we present a new microparticle system for the selective detection and magnetic removal of bacteria from contaminated solutions. The novelty of this system lies in the combination of a biocompatible scaffold reducing unspecific interactions with high capacity for bacteria binding. We apply highly porous poly(ethylene glycol) (PEG) microparticles and functionalize them, introducing both sugar ligands for specific bacteria targeting and cationic moieties for electrostatic loading of superparamagnetic iron oxide nanoparticles. The resulting magnetic, porous, sugar-functionalized (MaPoS) PEG microgels are able to selectively bind and discriminate between different strains of bacteria Escherichia coli . Furthermore, they allow for a highly efficient removal of bacteria from solution as their increased surface area can bind three times more bacteria than nonporous particles. All in all, MaPoS particles represent a novel generation of magnetic beads introducing for the first time a porous, biocompatible and easy to functionalize scaffold and show great potential for various biotechnological applications.

Metal-Mediated Molecular Self-Healing in Histidine-Rich Mussel Peptides

Mussels withstand high-energy wave impacts in rocky seashore habitats by fastening tightly to surfaces with tough and self-healing proteinaceous fibers called byssal threads. Thread mechanical behavior is believed to arise from reversibly breakable metal coordination cross-links embedded in histidine-rich protein domains (HRDs) in the principle load-bearing proteins comprising the fibrous thread core. In order to investigate HRD behavior at the molecular level, we have synthesized a histidine-rich peptide derived from mussel proteins (His5-bys) and studied its reversible adhesive self-interaction in the presence and absence of metal ions using PEG-based soft-colloidal probes (SCPs). Adhesion energies of greater than 0.3 mJ/m(2) were measured in the presence of metal ions, and the stiffness of the modified SCPs exhibited a 3-fold increase, whereas no adhesion was observed in the absence of metals. Raman spectroscopy confirmed the presence of metal-coordination via histidine residues by the peptide-supporting the role of His-metal complexes in the mechanical behavior of the byssus.

Solid-Phase Synthesis of Asymmetrically Branched Sequence-Defined Poly/Oligo(amidoamines)

We present for the first time the synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines) (PAAs) using solid-phase synthesis with the capability of introducing diversity at the side chains. We introduce two new versatile (diethylenetriamine) building blocks for solid-phase synthesis bearing Fmoc/Boc and Fmoc/Alloc protecting groups expanding recently used Fmoc/Boc protecting group strategy for linear PAAs to an Fmoc/Alloc/Boc strategy. This allows for orthogonal on-resin cleavage of Fmoc and Alloc protecting groups during solid-phase synthesis of PAAs with backbones differing in chain length and sequence. With these structures we then demonstrate the potential for generating asymmetrical branching by automated multiple on-resin cleavage of Alloc protecting groups as well as the introduction of side chains varying in length and number. Such systems have high potential as nonviral vectors for gene delivery and will allow for more detailed studies on the correlation between the degree of branching of PAAs and their resulting biological properties.

Synthesis of Porous PEG Microgels Using CaCO3 Microspheres as Hard Templates

Porous poly(ethylene glycol) (PEG) microgels of both 17.6 and 8.3 μm in diameter are synthesized via hard templating with calcium carbonate (CaCO(3)) microparticles. The synthesis is performed in three steps: loading of PEG macromonomers into CaCO(3) microparticles, crosslinking via photopolymerization, and removal of the CaCO(3) template under acidic conditions. The resulting porous PEG microgels are inverse replicates of their templates as indicated by light microscopy, cryo-scanning electron microscopy (cryo-SEM), and permeability studies. Thus this process allows for the straightforward and highly reproducible synthesis of porous hydrogel particles of two different diameters and porosities that show great potential as carriers for drugs or nanomaterials.

Electrochemical displacement sensor based on ferrocene boronic acid tracer and immobilized glycan for saccharide binding proteins and E. coli

Pathogens such as viruses and bacteria use their envelope proteins and their adhesin lectins to recognize the glycan residues presented on the cell surface of the target tissues. This principle of recognition is used in a new electrochemical displacement sensor for the protein concanavalin A (ConA). A gold electrode was first modified with a self-assembled monolayer of a thiolated mannose/OEG conjugate and a ferrocene boroxol derivative was pre-assembled as reporter molecule onto the mannose surface. The novel tracer molecule based on a 2-hydroxymethyl phenyl boronic acid derivative binds even at neutral pH to the saccharides which could expand the application towards biological samples (i.e., urine and feces). Upon the binding of ConA, the tracer was displaced and washed away from the sensor surface leading to a decrease in the electrochemical signal. Using square wave voltammetry (SWV), the concentration of ConA in the sample solution could be determined in the dynamic concentration range established from 38nmolL(-1) to 5.76µmolL(-1) with a reproducible detection limit of 1µgmL(-1) (38nmolL(-1)) based on the signal-to-noise ratio (S/N=3) with fast response of 15min. The new reporter molecule showed a reduced non-specific displacement by BSA and ribonuclease A. The sensor was also successfully transferred to the first proof of principle for the detection of Escherichia coli exhibiting a detection limit of approximately 6×10(2)cells/mL. Specificity of the displacement by target protein ConA and E. coli was demonstrated since the control proteins (i.e., BSA and RNaseA) and the control E. coli strain, which lack of type 1 fimbriae, were ineffective.

Mechanical carbohydrate sensors based on soft hydrogel particles.

Elastic sensors: A simple method is presented for the measurement of specific biomolecular interactions with soft colloidal hydrogel particles (SCPs) as sensors. Carbohydrate/lectin interactions (see picture; green: carbohydrate molecules) were studied by optical detection of the mechanical deformation of the particles on a lectin surface. The affinity of various carbohydrate inhibitors could also be readily determined.