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Hypothesis: The application of ferritin containers as a promising drug delivery vehicle is limited by their low bioavailability in blood circulation due to unfavorable environments, such as degradation by protease. The integration of ferritin containers into the polymeric network of microgels through electrostatic interactions is expected to be able to protect ferritin against degradation by protease. Furthermore, a stimuli-responsive microgel system can be designed by employing an acid-degradable crosslinker during the microgel synthesis. This should enable ferritin release in an acidic environment, which will be useful for future drug delivery applications.Experiments: Nanoparticle/fluorophores-loaded ferritin was integrated into microgels during precipitation polymerization. The integration was monitored by transmission electron microscopy (TEM)2 and fluorescence microscopy, respectively. After studying ferritin release in acidic solutions, we investigated the stability of ferritin inside microgels against degradation by chymotrypsin.Findings: About 80% of the applied ferritin containers were integrated into microgels and around 85% and 50% of them could be released in buffer pH 2.5 and 4.0, respectively. Total degradation of the microgels was not achieved due to the self-crosslinking of N-isopropylacrylamide (NIPAM). Finally, we prove that microgels could protect ferritin against degradation by chymotrypsin at 37 degrees C. (C) 2021 Elsevier Inc. All rights reserved.

This minireview provides an overview on the current knowledge of protein–protein interactions, common characterisation methods to characterise them, and their role in protein complex formation with some examples. A deep understanding of protein–protein interactions and their molecular interactions is important for a number of applications, including drug design. Protein–protein interactions and their discovery are thus an interesting avenue for understanding how protein complexes, which make up the majority of proteins, work.

The assembly of molecular building blocks into highly ordered structures is crucial, both in nature and for the development of novel functional materials. In nature, noncovalent interactions, such as hydrogen bonds or hydrophobic interactions, enable the reversible assembly of biopolymers, such as DNA or proteins. Inspired by these design principles, scientists have created biohybrid materials that employ natural building blocks and their assembly properties. Thus, structures and materials are attainable that cannot be made through other synthetic procedures. Herein, we review current concepts and highlight recent advances.

A large-scale screen to target SARS-CoV-2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, Mpro, is essential to yield functional viral proteins, making it a key drug target. Günther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to Mpro. High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site. Science, this issue p. 642, A repurposed drug-library screen reveals two allosteric drug binding sites of the SARS-CoV-2 main protease., The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

Protein containers are suitable building blocks for bioinorganic materials. Here, we show that high concentrations of magnesium ions induce the formation of a unitary protein scaffold, whereas low magnesium concentration leads to a binary protein scaffold. The molecular interactions in the protein scaffold were characterized with X-ray crystallography to high resolution. We show that the unitary framework can be applied for the assembly of inorganic nanoparticles such as metal oxides into highly ordered bioinorganic structures. Our work emphasizes the structural tunability of protein-container-based materials, important for adjusting emerging properties of such materials.

Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding a ffi nity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. We developed an extracorporeal, whole- blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. Atime- dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding a ffi nity to the adsorber particles. Further, the particle showed a su ffi cient hemocompatibility without significant e ff ects on complement component 5a, thrombin- antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. In conclusion, the bifunctional adsorber particle with cross- linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse e ff ects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the e ffi ciency of conventional dialysis techniques.

Nanoscale 11(13), 6497-6497 (2019). doi:10.1039/C9NR90066G, Published by RSC Publ., Cambridge

Biomolecules can be combined with inorganic compounds to unite biological features with the chemical and physical properties of abiotic materials. In particular, protein containers, with their inherent ability to encapsulate cargo molecules, are perfect platforms for the generation of multifunctional assemblies. However, encapsulation of foreign cargo is immensely challenging due to the lack of specific interactions between cargo and container. Here, we demonstrate that the highly specific cargo-loading mechanism of the bacterial nanocompartment encapsulin can be employed for encapsulation of artificial cargo like inorganic nanoparticles. For this purpose, container-filling gold nanoparticles were decorated with a small number of encapsulin cargo-loading peptides. By lock-and-key interaction between the peptides and the peptide-binding pockets on the inner container surface, the nanoparticles are encapsulated into encapsulin with extremely high efficiency. Most notably, peptide binding is independent from external factors such as ionic strength. Cargo-loading peptides may serve as generally applicable tool for efficient and specific encapsulation of cargo molecules into a proteinaceous compartment.

Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution.

The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition.

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.

Glucokinase (GCK) is responsible for maintaining glucose homeostasis in the human body. Dysfunction or misregulation of GCK causes hyperinsulinemia, hypertriglyceridemia, and type 2 diabetes. In the liver, GCK is regulated by interaction with the glucokinase regulatory protein (GKRP), a 68 kDa polypeptide that functions as a competitive inhibitor of glucose binding to GCK. Formation of the mammalian GCK-GKRP complex is stimulated by fructose 6-phosphate and antagonized by fructose 1-phosphate. Here we report the crystal structure of the mammalian GCK-GKRP complex in the presence of fructose 6-phosphate at a resolution of 3.50 Å. The interaction interface, which totals 2060 Å(2) of buried surface area, is characterized by a small number of polar contacts and substantial hydrophobic interactions. The structure of the complex reveals the molecular basis of disease states associated with impaired regulation of GCK by GKRP. It also offers insight into the modulation of complex stability by sugar phosphates. The atomic description of the mammalian GCK-GKRP complex provides a framework for the development of novel diabetes therapeutic agents that disrupt this critical macromolecular regulatory unit.

Biomolecules can act as functional templates for the organization of inorganic particles. Here we use two protein containers, engineered with opposite surface charge, as building blocks for the construction of a new type of biohybrid material. Binary structures with crystalline order were obtained, adopting a tetragonal lattice. Moreover, the cavity of the engineered protein containers can be filled with inorganic nanoparticles. The controlled assembly of these protein–nanoparticle composites yields highly ordered binary nanoparticle superlattices as free-standing crystals, with up to a few hundred micrometers in size. Because the structure and lattice parameters of the protein–nanoparticle crystals are independent of their nanoparticle cargo, the binary protein material may serve as a generally applicable matrix for the assembly of a variety of nanoparticles types.

Monomeric three-coordinate (amidinato)tin chloride [PhC-(NtBu) 2 SnCl] (1) was prepared by the reaction of N,N'-di-tert-butylcarbodiimide, phenyllithium and SnCl 2 . The metathesis reaction of 1 with AgSO 3 CF 3 and (Me 3 Si) 2 NLi afforded the formation of PhC(NtBu) 2 SnOTf (Tf = CF 3 SO 2 ) (2) and PhC(NtBu) 2 SnN(SiMe 3 ) 2 (3). The reductive dehalogenation of 1 with L-selectride resulted in the formation of the four-coordinate homoleptic tin compound Ph 2 C 2 (NtBu) 4 Sn (4). Compounds 1, 2, 3, and 4 were characterized by single-crystal structural analysis. Furthermore, 1 was treated with Fe 2 (CO) 9 to afford the Lewis acid-base adduct 5.

The enantioselective organocatalytic synthesis of arylglycines has been developed employing 1 mol% of an enantiopure N-triflyl phosphoramide Bronsted acid as organocatalyst. Various differently substituted phenylglycine derivatives can be synthesized in good to excellent yields and enantiomeric excesses based on a Friedel―Crafts alkylation of electron-rich arenes with a glyoxylate imine. A novel protocol for the deprotection of the N-tert-butylsulfonyl (Bus) group has also been developed.

5-Amino-2,4,6-tribromoisophthalic acid is used as a phasing tool for protein structure determination by MAD phasing. It is the second representative of a novel class of compounds for heavy-atom derivatization that combine heavy atoms with amino and carboxyl groups for binding to proteins., Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

The acid catalyzed rearrangement of two cyclohexanols of spiroannelated four-membered rings has been studied. In accordance with molecular mechanics calculations, far-reaching reorganizations with formation of unsaturated hexacyclic systems, including a fully cycloalkylated cyclohexene with a bispropellane partial structure, were observed. Attempts to convert this bispropellane to a trispropellane failed.

Obtaining phase information for the solution of macromolecular structures is still one of the bottlenecks in X-ray crystallography. 5-Amino-2,4,6-triiodoisophthalic acid (I3C), in which three covalently bound iodines form an equilateral triangle, was incorporated into proteins in order to obtain phases by single-wavelength anomalous dispersion (SAD). An improved binding capability compared with simple heavy-metal ions, ready availability, improved recognition of potential heavy-atom sites and low toxicity make I3C particularly suitable for experimental phasing.

The reaction of the diaminonaphthalene dilithium salt(THF)2Li2[(NSiMe3)2C10H6] with BX3 (X = Cl, Br) in hexane provides the monomeric compounds BX[(NSiMe3)2C10H6] [3 (X = Cl), 4 (X = Br)] with a tricoordinate boron atom. Attempts to reduce 3 and 4 with lithium, sodium, potassium or sodium/potassium alloy with the intention to isolate a boranide resembling a carbene were unsuccessful. However, the diaminonaphthalene dilithium salt (THF)2Li2[(NSi-iPr3)2C10H6] (2) reacts with SnCl2 in THF to provide the carbene analog 5. Compounds 2, 3 and 5 were characterized by single-crystal X-ray structure analysis. The structures of 3 and 5 show the naphthalene backbone to be nearly planar, while the heterocycles in 3 and 5 containing boron and tin atoms, respectively, have torsion angles (3: 30.06°; 5: 37.27°) with respect to the naphthalene plane. The boron atom has a trigonal-planar environment, and the stannylene tin–nitrogen bonds form a very acute N–Sn–N angle of 92.2°.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Reaction of an excess of hydrated LnCl3 (Ln = La, Gd, Ho, Yb, Y) with sodium 5-(2-pyridyl)tetrazolate (Napytz) in water led to the formation of either cationic disubstituted complexes (La, Gd, Ho) or neutral μ-hydroxo-bridged dimers (Y, Yb). Interestingly, there is a lack of propensity for these disubstituted complexes to redistribute forming trisubstituted ones, which is a common behaviour in lanthanoid chemistry. Also, compared to their corresponding lanthanoid carboxylates, all these products are discrete molecular entities forming an extensive hydrogen-bonded and/or π stacking network rather than hybrid polymeric frameworks.

Photodynamic therapy (PDT) has received increased attention as a treatment modality for malignant tumors as well as non-oncologic diseases such as age-related macular degeneration (AMD). An alternative to excite the photosensitizer by the common one-photon absorption is the method of two-photon excitation (TPE). This two-photon photodynamic therapy has the potential of improving the therapeutic outcome due to a highly localized photodynamic effect. The present study investigated the two-photon excited PDT performing in vitro experiments where C6 rat glioma cells were irradiated with a pulsed and focused fs Ti:sapphire laser emitting light at 800 nm. The irradiance distribution of the laser beam was carefully analyzed before the experiment and the applied irradiance was known for each position within the irradiated cell layer. Cells were divided into four groups and one group was incubated with 5-ALA and irradiated 4–5 h later. The survival of this group was tested after irradiation by means of ethidium bromide and acridine orange staining and compared to a control group, which was irradiated under the same conditions, but not incubated with 5-ALA before. Both groups showed necrotic areas depending on the applied irradiance, the value of which at the margin of the necrotic area could be deduced from its size. 5-ALA incubated cells became necrotic after irradiation with a mean irradiance above 6.1 × 10 10 W/cm 2 , while non-incubated cells remained viable. Cells of both groups became necrotic when treated with an irradiance above 10.9 × 10 10 W/cm 2 . The observed affected area of the cell layers was between 0.13 mm 2 and 1.10 mm 2 . Since the irradiation of non-incubated cells below the mean power density of 10.9 × 10 10 W/cm 2 induced no necrosis, apparently no thermal damage was induced in the cells and necrosis of the 5-ALA incubated cells can be ascribed to the photodynamic effect induced by two-photon excitation. The successful photodynamic treatment of a large area of a monolayer cell culture induced by two-photon excitation offers new perspectives for photodynamic treatment modalities.

Background and Objective Limited knowledge of the light and temperature distribution within the target volume in combination with non-selective accumulation of the applied photosensitizers (PS) has hampered the clinical relevance of interstitial photodynamic therapy (iPDT) for treatment of malignant glioma patients. The current pilot study focused on the development and the clinical implementation of an accurate and reproducible irradiation scheme for iPDT using 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) as a selectively working PS. Study Design/Materials and Methods Monte Carlo simulations of fluence rate and heat transport simulations were performed using the optical properties of normal brain tissue infiltrated by tumor cells (absorption coefficient µa = 0.2 cm−1, reduced scattering coefficient: µ′s = 20 cm−1). A modified 3-D treatment-planning software was used to calculate both, the treatment-volume and the exact position of the light diffusers within the lesion. The feasibility and the risk of iPDT were tested in 10 patients with small and circumscribed recurrent malignant gliomas. Results The optimum distance between the implanted light diffusers was determined to be 9 mm with regard to both fluence rate and temperature distribution. For this distance a temperature increase above 42°C was not expected to occur. Up to six cylindrical light diffusers were stereotactically implanted to achieve a complete irradiation of the tumor volume, which was possible in every single patient (mean tumor volume: 5.9 cm3). The total applied light fluence was between 4,320 J and 11,520 J. Side effects of iPDT were not observed. Median survival was 15 months. Conclusion 5-ALA iPDT in combination with a 3-D treatment-planning (which was based on optical and thermal simulations) is a safe and feasible treatment modality. The clinical impact of these findings deserves further prospective evaluation. Lasers Surg. Med. 39:386–393, 2007. © 2007 Wiley-Liss, Inc.

BACKGROUND Oral application of 20 mg/kg body weight of 5-aminolevulinic acid (ALA) leads to a highly specific accumulation of fluorescent Protoporphyrin IX (PPIX) in malignant glioma tissue. In the past few years, we have participated in several clinical studies designed to investigate fluorescence guided resection (FGR) and photodynamic therapy (PDT). METHODS PPIX selectivity and PPIX bleaching during PDT were assessed with spectroscopic measurements. FGR was performed in 18 clinics in Germany (ALA-Glioma Study Group, participants see end of paper) in a phase III trial comprising an ALA group and a white-light group. PDT was performed with microlens fibers or cylindrical diffusers postsurgically to the resection bed. Additionally, a protocol for the interstitial stereotactic placement of cylindrical diffusers was established and applied on patients with recurrent, inoperable glioblastoma. RESULTS Compared to normal cortex, mean PPIX fluorescence in vital tumor was found more than 100-fold increased. During PDT, the PPIX fluorescence bleached to 8%, 16%, and 1% of the initial intensity for the 100, 150, and 200 J/cm2 groups (median values). FGR: Contrast-enhancing tumor was completely resected in 65% of patients in the ALA group compared to 36% in the white-light group (p < 0.0001). Progression-free survival was superior in the ALA group compared to white-light patients with cumulative 6 months progression-free survival rates of 41% and 21% (p = 0.0003), respectively. Interstitial PDT can be performed with multiple radial diffusers approximately 10 mm apart, 200 mW/cm, and an irradiation time of one hour.

Designing nanoscaled hierarchical structures with increasing levels of complexity is challenging. Here we show that electrostatic interactions between two complementarily supercharged protein nanocages can be effectively utilized to create nested Matryoshka-type structures. Cage-within-cage complexes containing spatially ordered iron oxide nanoparticles spontaneously self-assemble upon mixing positively supercharged ferritin compartments with AaLS-13, a larger shell-forming protein with a negatively supercharged lumen. Exploiting engineered Coulombic interactions and protein dynamics in this way opens up new avenues for creating hierarchically organized supramolecular assemblies for application as delivery vehicles, reaction chambers, and artificial organelles.

The title compound, C(8)H(4)Br(3)NO(4), shows an extensive hydrogen-bond network. In the crystal structure, molecules are linked into chains by COO-H...O bonds, and pairs of chains are connected by additional COO-H...O bonds. This chain bundle shows stacking interactions and weak N-H...O hydrogen bonds with adjacent chain bundles. The three Br atoms present in the molecule form an equilateral triangle. This can be easily identified in the heavy-atom substructure when this compound is used as a heavy-atom derivative for experimental phasing of macromolecules. The title compound crystallizes as a nonmerohedral twin.

The enantioselective organocatalytic synthesis of the arylglycines (III) is reported to proceed with good to excellent yields (60-89%) and enantioselectivities (68-96%).

New photochromic switches based on helical alkenes can quickly and efficiently be accessed by Pd-catalyzed domino reactions using a modular approach; this allows a wide variability in product formation with the advantages of a convergent synthetic route. The alkenes have been synthesized in excellent enantioselectivity and their switching properties assessed by stimulation with nanosecond laser pulses at two different wavelengths in over 1000 switching cycles.

The magic triangle 5-amino-2,4,6-triiodoisophthalic acid (I3C) and the MAD triangle 5-amino-2,4,6-tribromoisophthalic acid (B3C) are two representatives of a novel class of compounds that combine heavy atoms for experimental phasing with functional groups for protein interactions. These compounds are readily available and provide easy access to experimental phasing. The preparation of stock solutions and the incorporation of the compounds into protein crystals are discussed. As an example of incorporation via cocrystallization, the incorporation of B3C into bovine trypsin, resulting in a single site with high occupancy, is described.

A summary of clinical trials employing photodynamic diagnosis (PDD) and photodynamic therapy (PDT) for the diagnosis and treatment of brain malignancies is presented. Intra-cavity PDT has been performed within the surgical cavity following FGR, employing oral administration of 5-aminolevulinic acid (5-ALA), either targeting fluorescing tissue regions that were not removed during FGR due to safety reasons (referred to as focal PDT, n=20) or illuminating the entire resection cavity (referred to as integral PDT, n=9). Both approaches proved technically feasible and safe. Spectroscopic measurements performed pre-, during and post-PDT revealed Protoporphyrin IX (PpIX)-photobleaching of more than 95% after the delivery of 200 J/cm2. This light dose did not induce any side effects. Furthermore, interstitial PDT (iPDT) has been employed within one feasibility trial (n=10) and one Phase I/II trial (n=15). Here, three to six cylindrical light diffusors (20-30 mm length, 200 mW/cm, 720 J/cm) were positioned within the target tissue under stereotactic guidance. Pre-treatment planning was performed with the intent to target the entire tumour volume with a sufficient light dose while also minimising the risk of any light-induced temperature increase. For the feasibility trial patients with small, recurrent gliomas were included, resulting in a median survival of 15 months as well as some unexpected longterm survivals (up to 5 years). The Phase I/II trial employed the same clinical procedures. Here, the 12-month survival was 35% and the median progression-free survival was 6 months. In summary, stereotactic iPDT in combination with treatment-planning could be shown to be a safe and feasible treatment modality. These trials are presently being extended to also include on-line monitoring of PpIX fluorescence and photobleaching kinetics. Preliminary data has revealed dramatically different PpIX levels and photobleaching kinetics. Such data could possibly be employed for realtime treatment monitoring and as an early prognostic marker for the PDT response.

The stable beta-diketiminate tin(II) complexes LSnX [L = HC(CMeNAr)2, Ar = 2,6-iPr2C6H3] with terminal methyl, amide, fluoride, and iodide (X = Me, N(SiMe3)2, F, I) are described. LSnMe (2) is synthesized by salt metathesis reaction of LSnCl (1) with MeLi and can be isolated in the form of yellow crystals in 88% yield. Compound LSnN(SiMe3)2 (3) was obtained by treatment of LH with 2 equiv of KN(SiMe3)2 in THF followed by adding 1 equiv of SnCl2. Reaction of 2 and 3 respectively with Me3SnF in toluene provided the tin(II)fluoride LSnF (4) with a terminal fluorine as colorless crystals in 85% yield. 4 is highly soluble in common organic solvents. The reaction of LLi(OEt2) with 1 equiv of SnI2 in diethyl ether afforded the LSnI (5). Compounds 2, 3, 4, and 5 were characterized by microanalysis, multinuclear NMR spectroscopy, and X-ray structural analysis. Single crystal X-ray structural analyses indicate that all the compounds (2, 3, 4, 5) are monomeric and the tin center resides in a trigonal-pyramidal environment.

The title compound, C8H4I3NO4·H2O, shows an extensive hydrogen-bond network; in the crystal structure, molecules are linked by O—H...O, N—H...O and O—H...N hydrogen bonds involving all possible donors and also the water molecule.

Background: Oral application of 20 mg/kg body weight of 5-aminolevulinic acid (ALA) results in a highly specific accumulation of fluorescent Protoporphyrin IX (PpIX) in malignant glioma tissue. In a clinical phase II study, Photodynamic Therapy (PDT) to postsurgical tumor-remnants was monitored by fluorescence spectroscopy and photobleaching of PpIX was detected. Methods: Fluorescence spectra were measured from the tumor remnant pre and at intervals during PDT with 100, 150 and 200 J/cm 2 and from adjacent normal cortex. The spectra obtained were fitted with spectra from pure PpIX, autofluorescence and photoproducts, the fit-parameters quantifying the contributions of these components to the measured spectrum. Results: The PpIX-fluorescence bleached to 8%, 16% and 1% of the initial intensity for the 100, 150 and 200 J/cm 2 -groups (median values). Photoproduct formation was minimal. PpIX-fluorescence of normal cortex was 6% of tumor remnant fluorescence (median, range: 0% to 49%).

With upcoming interest of performing Photodynamic Therapy (PDT) interstitially in organs like the prostate, pancreas, liver or brain it becomes desirable to measure the photosensitizer concentration interstitially inside and outside the tumor volume. Knowledge about the sensitizer concentrations in tumor and normal tissue is a precondition for a proper dosimetry. With Monte Carlo simulations and liquid phantom experiments the reliability of quantitative fluorescence measurements of fluorochromes in turbid media by a single-fiber device has been investigated. For a single optical fiber that both delivers excitation light and gathers fluorescence, the signal obtained is rather insensitive to variations of the concentrations of scatterers or absorbers within a certain range. A detailed analysis shows that the most reliable results are obtained with fiber core radii r of 0.1 10) results in detectable but small differences in the detected signal. Compared to other geometries of detecting fluorescence in turbid media, single fiber detection promises to be sufficiently quantitative for most applications. Other aspects relevant for measurements in live tissue, like sensitivity to local inhomogeneities and artifacts remain to be investigated.

Oral application of 20 mg/kg bw of 5-aminolevulinic acid results in a highly specific accumulation of fluorescent and phototoxic Protoporphyrin IX in malignant glioma tissue. Surgical removal with fluorescence guidance is studied in a phase III clinical trial, adjuvant Photodynamic Therapy (PDT) to the surgical cavity is in phase II and for interstitial PDT of recurrent gliomas, a phase I/II study has started. Fluorescence guided resections have been shown to be safe and effective in augmenting neurosurgical removal of malignant gliomas in 52 consecutive patients. Intra-operative fluorescence spectroscopy showed statistically significant higher sensitizer accumulation in vital brain tumor versus the infiltration zone and in the infiltration zone versus adjacent normal brain, which contained very little PPIX. This is promisingly exploited for PDT - both to the surgical cavity by surface irradiation and for stereotactically guided interstitial irradiation.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several different mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, for over 30 years the paradigm has been that this initial radical is always channeled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction. Such a cysteine residue has been present in the structure of each of the many RNRs determined to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), both alone and in complex with allosteric effector dATP and substrate CTP. Remarkably, tmNrdD lacks a cysteine for radical transfer to the substrate, and is the first structurally or biochemically characterized RNR to do so. However in many other respects tmNrdD appears to be a normal anaerobic RNR, including gene structure, expression levels, metal cofactor and binding of allosteric effectors and substrates in the expected conformations. Furthermore, it is possible to generate a glycyl radical as expected. We present evidence that the structure of tmNrdD is representative for the new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria, all lacking the canonical cysteine residue. The wide distribution provides further evidence that the subclass IIIh is a functional RNR. Taken together, the results imply that an alternative initiation route for the RNR reduction reaction must exist that do not require channeling through a cysteine side chain.

Objective The visualization of cancer-specific enzymatic activities holds potential as a useful tool for early cancer detection and identification of aggressive tumors. DNaseX, which is typically found in early lesions, and TKTL1, which identifies aggressive carcinomas with a high metastatic potential, have been proposed as such cancer-specific enzymes. The enzymatic activity of DNaseX can be detected via molecular beacons i.e. the reversed fluorescence quenching produced when DNase cleavage of the DNA-hairpin occurs, containing fluorochrome and quencher. Furthermore, an increase in rhodamine B hydrazide (RBH) fluorescence can be used to detect the accumulation of putatively specific intermediates of TKTL1-related metabolism. However, for quantitative analysis of fluorophore concentration it is important to disentangle the effects of light absorption and scattering of the sample being investigated. Material and methods A fiber-based system was developed for the quantitative detection of small concentrations of fluorescent dyes in scattering and absorbing media. Both the raw fluorescence and the intrinsic fluorescence signals were employed for fluorophore quantification. The intrinsic fluorescence was evaluated via a theoretical model for light distribution based on the diffusion approximation, and the measured reflectance and fluorescence spectra. The ability of the algorithm to assess the intrinsic fluorescence spectra was evaluated for different fluorophores, wavelength ranges and background optical properties. Results The resulting intrinsic fluorescence spectra show less dependence on the optical properties of the sample than on the raw fluorescence signals. The ability to separate the effects of light absorption caused by the surrounding material was particularly good. Furthermore, the intensity of the intrinsic fluorescence correlated with the fluorophore concentration. Fluorophore concentrations and concentration variations could be detected in tens of picomolar. Conclusion The fluorescence diagnostic system and evaluation algorithm showed good detection efficiency and quantification of the fluorescent probes investigated. The lower detection limit was in the range expected for fluorophore concentration within clinically obtained samples. This configuration thus presents a useful method for quantifying fluorophore concentration in clinically obtained tissue samples, which are related to the enzymatic activity of TKTL1 and DNaseX.

Reaction of LaCl(3) and dibenzoylmethane (Ph(2)acacH) in methanol with an excess of triethylamine resulted in the unexpected formation of a dodecanuclear hydroxo-bridged La cluster templated by CO(3)(2-) and phenylglyoxylate (Phgly), as determined by X-ray crystallography.