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.

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.

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.

Covalently surface-attached coatings and free-standing membranes of some 20 µm in thickness were prepared by photocopolymerization of a bitelechelic methacrylate-terminated poly-(dimethylsiloxane) (PDMS) and silyl-protected 2-hydroxyethyl acrylate. After cleaving the silyl groups, the resulting amphiphilic conetworks exhibit two nanoseparated phases in bulk as well as on their surface as shown by atomic force microscopy (AFM). Thereby, the surface structure is controlled by the chemical nature of the surface covering material. Comparing the swelling behavior of 1D swelling of the coatings with the 3D swelling of the membranes revealed perfect interconnectivity of the hydrophilic phase in all compositions prior to and after water treatment. The hydrophobic PDMS phase, on the other hand, is not interconnected in dry state at compositions with less than 80 wt % PDMS. Diffusion experiments indicate that the PDMS phase reorganizes upon swelling and becomes continuous at PDMS contents of about 50 wt % and more.

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.