Thomas Reiner

Towards Quantitative Catalytic Lignin Depolymerization

The products of base-catalyzed liquid-phase hydrolysis of lignin depend markedly on the operating conditions. By varying temperature, pressure, catalyst concentration, and residence time, the yield of monomers and oligomers from depolymerized lignin can be adjusted. It is shown that monomers of phenolic derivatives are the only primary products of base-catalyzed hydrolysis and that oligomers form as secondary products. Oligomerization and polymerization of these highly reactive products, however, limit the amount of obtainable product oil containing low-molecular-weight phenolic products. Therefore, inhibition of concurrent oligomerization and polymerization reactions during hydrothermal lignin depolymerization is important to enhance product yields. Applying boric acid as a capping agent to suppress addition and condensation reactions of initially formed products is presented as a successful approach in this direction. Combination of base-catalyzed lignin hydrolysis with addition of boric acid protecting agent shifts the product distribution to lower molecular weight compounds and increases product yields beyond 85%.

A Pretargeted PET Imaging Strategy Based on Bioorthogonal Diels–Alder Click Chemistry

The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels–Alder click reaction between tetrazine and transcyclooctene. Methods: A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and 64Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with 64Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen–expressing, SW1222 colorectal cancer xenografts. Results: The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the 64Cu-NOTA–labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9–10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with 64Cu-NOTA–labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either 64Cu or 89Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the 64Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with 89Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively). Conclusion: The high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.

Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo

Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.

Ubiquitous Detection of Gram-Positive Bacteria with Bioorthogonal Magnetofluorescent Nanoparticles

The ability to rapidly diagnose gram-positive pathogenic bacteria would have far reaching biomedical and technological applications. Here we describe the bioorthogonal modification of small molecule antibiotics (vancomycin and daptomycin), which bind to the cell wall of gram-positive bacteria. The bound antibiotics conjugates can be reacted orthogonally with tetrazine-modified nanoparticles, via an almost instantaneous cycloaddition, which subsequently renders the bacteria detectable by optical or magnetic sensing. We show that this approach is specific, selective, fast and biocompatible. Furthermore, it can be adapted to the detection of intracellular pathogens. Importantly, this strategy enables detection of entire classes of bacteria, a feat that is difficult to achieve using current antibody approaches. Compared to covalent nanoparticle conjugates, our bioorthogonal method demonstrated 1-2 orders of magnitude greater sensitivity. This bioorthogonal labeling method could ultimately be applied to a variety of other small molecules with specificity for infectious pathogens, enabling their detection and diagnosis.

Accurate measurement of pancreatic islet β-cell mass using a second-generation fluorescent exendin-4 analog

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K12 position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4×12-VT750) had a high binding affinity (∼3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4×12-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.

An efficient protocol for the palladium-catalysed Suzuki–Miyaura cross-coupling

The palladacyclic catalyst precursor received by ortho-palladation of ([1,1′-biphenyl]-2-yloxy)diisopropyl-phosphine represents a highly active system for Suzuki–Miyaura cross-coupling reactions when used in neat water. An efficient, broadly applicable and sustainable aqueous protocol was developed using 2.5 eq. of Na2CO3 as base, allowing the reaction to be performed under air and at ambient temperature with Pd loadings of 0.04 mol%. Coupling products are obtained in high yields and excellent purity by simple filtration with no organic solvents needed throughout the whole reaction. A broad variety of functional groups are tolerated and a large number of substrates can be applied with this protocol. The crystal structure of the palladacyclic catalyst precursor is presented as well as investigations targeting the nature of catalyst activation and the active catalytic species.

Synthesis and In Vivo Imaging of a 18F‐Labeled PARP1 Inhibitor Using a Chemically Orthogonal Scavenger‐Assisted High‐Performance Method

Pedal to the metal: Using inverse Diels-Alder catalyst free TCO/Tz cycloadditions, we were able to quickly and selectively generate an 18F-labeled AZD2281-derivative from multiple different scaffolds (A–E). Excess cold material was removed within minutes using a TCO scavenger resin. This protocol allows the parallel synthesis of a library of potential PET imaging agents in a short time, increasing the efficiency of lead compound detection. The novel PET probe was successfully tested in biological assays and its potency and targeted accumulation was confirmed in vivo.

High‐Yielding, Two‐Step 18F Labeling Strategy for 18F‐PARP1 Inhibitors

Positron emission tomography (PET) of labeled metabolites, drugs, proteins and nanomaterials[1-3] is rapidly emerging as a powerful imaging tool to detect and stage disease, to study human biology, to investigate pharmacokinetics and pharmacodynamics of new drugs, or to measure treatment efficacy in clinical trials.[4-8] 18F is one of the most commonly used isotopes for clinical imaging given its half-life, ease of production, wide availability, and compatibility with microfluidics syntheses.[9] Despite extensive use and well established procedures of labeling some small molecules, facile 18F platform-type universally adaptable labeling strategies are still largely missing. This is especially true for rapid labeling of small molecules that emerge from high throughput screens or for optimizing hybrid and modular imaging agents. Bioorthogonal chemistries represent one avenue to develop such generic labeling platforms.

Bioorthogonal Imaging of Aurora Kinase A in Live Cells

Small molecule imaging: Aurora kinase A (AKA) was imaged in live cells using an in cellulo bioorthogonal two-step reaction with a small molecule and a fluorescent reporter. The small molecule was localized to spindle poles and microtubules during metaphase, consistent with the localization of both endogenous and GFP-/RFP-tagged AKA. Using this approach, changes in AKA distribution during mitosis were also observed.

Bioorthogonal Probes for Polo-like Kinase 1 Imaging and Quantification†

A nuclear protein target, polo-like kinase 1 (PLK1) was imaged using a biocompatible bioorthogonal ligation between a specific drug and a fluorescent dye in live cells. Colocalization of the dye and the protein target was confirmed by antibody staining and by expressing a GFP construct of PLK1. The two-step PLK1 imaging procedure was used to quantify PLK1 expression levels in cancer cell lines of various tissue origins.