Kasper K. Sørensen

A High-throughput O-Glycopeptide Discovery Platform for Seromic Profiling

Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introduction of a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products was achieved on an amine-reactive hydrogel-coated microarray glass surface, allowing high-throughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept, we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine-induced disease-free breast cancer patients and in patients with confirmed disease at time of diagnosis.

Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary N-Alkyl and N,N-Dialkyl Chitosan Derivatives

A highly efficient method for chemical modification of chitosan biopolymers by reductive amination to yield N,N-dialkyl chitosan derivatives was developed. The use of 3,6-O-di-tert-butyldimethylsilylchitosan as a precursor enabled the first 100% disubstitution of the amino groups with long alkyl chains. The corresponding mono N-alkyl derivatives were also synthesized, and all the alkyl compounds were then quaternized using an optimized procedure. These well-defined derivatives were studied for antibacterial activity against Gram positive S. aureus, E. faecalis, and Gram negative E. coli, P. aeruginosa, which could be correlated to the length of the alkyl chain, but the order was dependent on the bacterial strain. Toxicity against human red blood cells and human epithelial Caco-2 cells was found to be proportional to the length of the alkyl chain. The most active chitosan derivatives were found to be more selective for killing bacteria than the quaternary ammonium disinfectants cetylpyridinium chloride and benzalkonium chloride, as well as the antimicrobial peptides melittin and LL-37.

Direct chemoselective synthesis of glyconanoparticles from unprotected reducing glycans and glycopeptide aldehydes.

Chemoselective oxime coupling was used for facile conjugation of unprotected, reducing glycans and glycopeptide aldehydes with core?shell gold nanoparticles carrying reactive aminooxy groups on the organic shell.

Semi‐automated microwave‐assisted SPPS: Optimization of protocols and synthesis of difficult sequences

Precise microwave heating has emerged as a valuable addition to solid‐phase peptide synthesis. New methods and protocols are required to utilize this potential and make it a reliable technique. Here, we describe a new semi‐automated instrument for solid‐phase peptide synthesis with microwave heating and report protocols for its reliable use. The instrument features a flow‐through reaction vessel that is placed in the microwave instrument throughout the synthesis. Bubbling with N2 gas during the microwave irradiation proved important for temperature control. Washing and Fmoc removal steps were automated, while activated Fmoc‐amino acids were added manually. Several linkers and resins were evaluated and protocols for synthesis of difficult sequences were developed. These included on‐resin reductive amination of BAL handles under microwave heating. We believe that the instrument, as well as the protocols for its use, will be useful tools for peptide chemists.

An Intermolecular Binding Mechanism Involving Multiple Lysm Domains Mediates Carbohydrate Recognition by an Endopeptidase.

The crystal and solution structures of the T. thermophilus NlpC/P60 d,l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan.

Regioselective fluorescent labeling of N,N,N-trimethyl chitosan via oxime formation.

Fluorescent labeling of chitosan and its derivatives is widely used for in vitro visualization and is accomplished by random introduction of the fluorophore to the polymer backbone, conceivably altering the bioactivity of the polymer. Here, we report for the first time the regioselective conjugation of a fluorophore to the reducing end of a fully N,N,N-trimethylated chitosan (TMC) by oxime formation. End-labeled conjugation of 5-(2-((aminooxyacetyl)amino)ethylamino)naphthalene-1-sulfonic acid (EDANS-O-NH(2)) fluorophore to TMC to form TMC-oxime-EDANS (f-TMC) was confirmed by (1)H NMR and fluorescence spectroscopy. Average molecular weight calculations of f-TMC with (1)H NMR and fluorescence spectroscopy gave similar results or ∼7.7kDa. f-TMC in human bronchial epithelial cells was both cell membrane bound as well as intracellularly localized. This demonstrates the proof-of-concept for selective oxime formation at the reducing end of a chitosan derivative, which can be used for tracking chitosan in gene and drug delivery purposes and gives rise to further modifications with other functional groups.

Automated 'X-Y' robot for peptide synthesis with microwave heating: application to difficult peptide sequences and protein domains.

Precise microwave heating has emerged as a valuable method to aid solid‐phase peptide synthesis (SPPS). New methods and reliable protocols, as well as their embodiment in automated instruments, are required to fully use this potential. Here we describe a new automated robotic instrument for SPPS with microwave heating, report protocols for its reliable use and report the application to the synthesis of long sequences, including the β‐amyloid 1‐42 peptide. The instrument is built around a valve‐free robot originally developed for parallel peptide synthesis, where the robotic arm transports reagents instead of pumping reagents via valves. This is the first example of an ‘X‐Y’ robotic microwave‐assisted synthesizer developed for the assembly of long peptides. Although the instrument maintains its capability for parallel synthesis at room temperature, in this paper, we focus on sequential peptide synthesis with microwave heating. With this valve‐free instrument and the protocols developed for its use, fast and efficient syntheses of long and difficult peptide sequences were achieved. Copyright

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

Lipochitin Oligosaccharides Immobilized through Oximes in Glycan Microarrays Bind LysM Proteins

Glycan microarrays have emerged as novel tools to study carbohydrate–protein interactions. Here we describe the preparation of a covalent microarray with lipochitin oligosaccharides and its use in studying proteins containing LysM domains. The glycan microarray was assembled from glycoconjugates that were synthesized by using recently developed bifunctional chemoselective aminooxy reagents without the need for transient carbohydrate protecting groups. We describe for the first time the preparation of a covalent microarray with lipochitin oligosaccharides and its use for studying proteins containing LysM domains. Lipochitin oligosaccharides (also referred to as Nod factors) were isolated from bacterial strains or chemoenzymatically synthesized. The glycan microarray also included peptidoglycan‐related compounds, as well as chitin oligosaccharides of different lengths. In total, 30 ligands were treated with the aminooxy linker molecule. The identity of the glycoconjugates was verified by mass spectrometry, and they were then immobilized on the array. The presence of the glycoconjugates on the array surface was confirmed by use of lectins and human sera (IgG binding). The functionality of our array was tested with a bacterial LysM domain‐containing protein, autolysin p60, which is known to act on the bacterial cell wall peptidoglycan. P60 showed specific binding to Nod factors and to chitin oligosaccharides. Increasing affinity was observed with increasing chitin oligomer length.

3- Instead of 4-helix formation in a de novo designed protein in solution revealed by small-angle X-ray scattering.

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so‐called carboproteins. Here, we describe the preparation and structural studies of three α‐helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4‐α‐helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small‐angle X‐ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1‐helix folding topology in solution, while the free peptide formed a 3‐helix bundle. This finding is consistent with the calculated α‐helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4‐helix carboprotein monomers with the 2×2‐helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4‐helix (3+1) carboproteins based on D‐altropyranoside and D‐galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance‐geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability.