Dominik K. Kölmel

Luminescent cell-penetrating pentadecanuclear lanthanide clusters.

A novel pentadecanuclear lanthanide hydroxy cluster [{Ln15(μ3-OH)20(PepCO2)10(DBM)10Cl}Cl4] (Ln = Eu (1), Tb (2)) featuring the first example with peptoids as supporting ligands was prepared and fully characterized. The solid-state structures of 1 and 2 were established via single-crystal X-ray crystallography. ESI-MS experiments revealed the retention of the cluster core in solution. Although OH groups are present, 1 showed intense red fluorescence with 11(1)% absolute quantum yield, whereas the emission intensity and the quantum yield of 2 were significantly weaker. In vitro investigations on 1 and 2 with HeLa tumor cells revealed an accumulation of the clusters in the endosomal-lyosomal system, as confirmed by confocal microscopy in the TRLLM mode. The cytotoxicity of 1 and 2 toward the HeLa cells is moderate.

Azides – Diazonium Ions – Triazenes: Versatile Nitrogen-rich Functional Groups

For more than 100 years, nitrogen-rich compounds such as azides, diazonium ions, and triazenes have proved to be extremely valuable. Because these functional groups can be easily introduced into various substrates, they are frequently used nowadays. More importantly, they can be converted into a great number of other functional groups. The scope of this article is thus to summarize possible synthetic routes for the formation of these functional groups as well as to highlight some of the most prominent applications of these exciting moieties in chemical biology and combinatorial chemistry. Many of the most famous name reactions such as the Staudinger reduction, Staudinger ligation, Sandmeyer reaction, Wallach reaction, Mitsunobu reaction, Huisgen reaction, Balz–Schiemann reaction, Meerwein arylation, Pschorr reaction or Gomberg–Bachmann reaction are covered.

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chemistry, biomaterials and hydrogels, dynamic combinatorial chemistry, organic synthesis, and chemical biology. Here we outline chemical developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomolecules are described. The basic chemical properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reactions mechanism and how reactant structure controls rates and equilibria in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified especially fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addition, the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A number of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomolecules and biomaterials.

Peptoid‐Ligated Pentadecanuclear Yttrium and Dysprosium Hydroxy Clusters

A new family of pentadecanuclear coordination cluster compounds (from now on simply referred to as clusters) [{Ln15 (OH)20 (PepCO2 )10 (DBM)10 Cl}Cl4 ] (PepCO2 =2-[{3-(((tert-butoxycarbonyl)amino)methyl)benzyl}amino]acetate, DBM=dibenzoylmethanide) with Ln=Y and Dy was obtained by using the cell-penetrating peptoid (CPPo) monomer PepCO2 H and dibenzoylmethane (DBMH) as supporting ligands. The combination of an inorganic cluster core with an organic cell-penetrating peptoid in the coordination sphere resulted in a core component {Ln15 (μ3 -OH)20 Cl}(24+) (Ln=Y, Dy), which consists of five vertex-sharing heterocubane {Ln4 (μ3 -OH)4 }(8+) units that assemble to give a pentagonal cyclic structure with one Cl atom located in the middle of the pentagon. The solid-state structures of both clusters were established by single-crystal X-ray crystallography. MS (ESI) experiments suggest that the cluster core is robust and maintained in solution. Pulsed gradient spin echo (PGSE) NMR diffusion measurements were carried out on the diamagnetic yttrium compound and confirmed the stability of the cluster in its dicationic form [{Y15 (μ3 -OH)20 (PepCO2 )10 (DBM)10 Cl}Cl2 ](2+) . The investigation of both static (dc) and dynamic (ac) magnetic properties in the dysprosium cluster revealed a slow relaxation of magnetization, indicative of single-molecule magnet (SMM) behavior below 8 K. Furthermore, the χT product as a function of temperature for the dysprosium cluster gave evidence that this is a ferromagnetically coupled compound below 11 K.

Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials

The advent of antibody–drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation. Selectively targeting native amino acids for late-stage protein modification is a significant challenge, but now it has been shown that photoredox catalysis can be used to specifically target protein C-termini toward decarboxylative-alkylation with Michael acceptors. This technology harnesses innate differences in side-chain oxidation potentials to select between the various functional groups typical among proteins in order to form a single modified product.

Novel pyridinium dyes that enable investigations of peptoids at the single-molecule level.

Single-molecule microscopy is a powerful tool for investigating various uptake mechanisms of cell-penetrating biomolecules. A particularly interesting class of potential transporter molecules are peptoids. Fluorescence labels for such experiments need to comply with several physical, chemical, and biological requirements. Herein, we report the synthesis and photophysical investigation of new fluorescent pyridinium derived dyes. These fluorescent labels have advantageous structural variations and spacer units in order to avoid undesirable interactions with the labeled molecule and are able to easily functionalize biomolecules. In our case, cell-penetrating peptoids are successfully labeled on solid supports, and in ensemble measurements the photophysical properties of the dyes and the fluorescently labeled peptoids are investigated. Both fluorophores and peptoids are imaged at the single-molecule level in thin polymer gels. With respect to bleaching times and fluorescence lifetimes the dye molecules and the peptoids show only slightly perturbed optical behaviors. These investigations indicate that the new fluorophores fulfill well single-molecule microscopy and solid-phase synthesis requirements.

Cell-penetrating peptoids: introduction of novel cationic side chains.

During the last decade peptoid-based molecular transporters have been broadly applied. They are highly valued for their easy synthesis and their superior stability against enzymatic degradation. The special structure of peptoids generally allows introducing a variety of different side chains. Yet, the cationic side chains of cell-penetrating peptoids displayed solely lysine- or arginine-like structures. Thus this report is intended to extend the spectrum of cationic peptoid side chains. Herein, we present novel functional groups, like polyamines, aza-crown ethers, or triphenylphosphonium ions that are introduced into peptoids for the first time. In addition, the obtained peptoids were tested for their cell-penetrating properties.

Photophysical properties of fluorescently-labeled peptoids

Fluorescently-labeled biomolecules are often utilized in biochemical or cellular experiments without further detailed spectroscopical characterization. This report is intended to narrow this gap and therefore presents the photophysical investigation of a library of 17 fluorescently-labeled molecules, namely peptoid transporters. First, one peptoid structure is labeled with seven different fluorophores and the spectroscopical properties are examined. Absorption and fluorescence maxima are almost identical for free dyes and conjugated dyes, suggesting free choice of a spectrally suitable fluorophore for different applications. Otherwise, extinction coefficients and quantum yields, and therefore the brightness of all seven dyes are strongly influenced. For the fluorophores, e.g. rhodamine B, the extent of this influence depends on the peptoid itself. This is shown by comparing different structures in the second part of this report. Especially the side chain functionalities influence the brightness. And finally, peptoids having two identical fluorescent labels are presented, which show decreased quantum yields. Possible reasons for the observed photophysical properties are discussed.

Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans.

Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines)] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.

Novel Prodrug of Doxorubicin Modified by Stearoylspermine Encapsulated into PEG-Chitosan-Stabilized Liposomes

Here, we report a new modification of doxorubicin based on an amphiphilic stearoylspermine anchor, enabling loading into liposomal membranes. Doxorubicin is coupled with stearoylspermine through an acid-labile hydrazone linker to ensure the release of the drug in the acidic interstitium of tumors. Using ATR-FTIR spectroscopy (Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy), the mechanism of interaction of doxorubicin with the anionic liposomal membrane was studied: incorporation of stearoyl chains leads to an increase in local microfluidity, and the amino groups of spermine interact with the phosphate groups of lipids. To stabilize liposomes against aggregation, we applied the copolymer PEG-chitosan as a coating: complex formation leads to charge neutralization, and the liposomes grow in size. According to MTT tests and confocal microscopy for cell lines A459 and Caco-2, PEG-chitosan-coated liposomes are as effective as neutral liposomes but are much more stable.