Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.     

Design, synthesis, and evaluation of a mechanism-based inhibitor for gelatinase A

[structure: see text] Matrix metalloproteinases (MMPs), of which 26 are known, have been implicated in a number of pathological conditions, including tumor metastasis. We have previously described the first mechanism-based inhibitor for MMPs (J. Am. Chem. Soc. 2000, 122, 6799-6800), which in chemistry mediated by the active site zinc ion selectively and covalently inhibits MMP-2, -3, and -9. Computational analyses indicated that this selectivity in inhibition of MMPs could be improved by design of new variants of the inhibitor class. We report herein the syntheses of methyl 2-(4-{4-[(2-thiiranylpropyl)sulfonyl]phenoxy}phenyl)acetate (3) and 2-(4-{4-[(2-thiiranylpropyl)sulfonyl]phenoxy}phenyl)acetic acid (4), and show that compound 3 serves as a mechanism-based inhibitor exclusively for MMP-2. This molecule should prove useful in delineating the functions of MMP-2 in biological systems.     

Structural Basis for Carbapenemase Activity of the OXA-23 β-Lactamase from Acinetobacter baumannii

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Coating of titanium implant materials with thin polymeric films for binding the signaling protein BMP2

A fast and simple approach for immobilization using copolymers as interlayers is reported. The synthesized copolymers form stable self-assembled layers on implant materials like, e.g., titanium in a simple coating/drying/washing sequence and have functional groups which can bind proteins from an aqueous solution. The copolymer films have been characterized via ellipsometry and contact angle measurements and were tested for biocompatibility. An immunoassay was used to determine the amount of BMP2 and demonstrated an approximately 10-fold increase as compared to previously used self-assembled monolayers. A BMP2-responsive cell line with luciferase detection was used to determine the biological activity of the bound signaling protein.     

Enantiospecific formal total syntheses of (-)-salicylihalamides A and B from D-glucose and L-rhamnose

[reaction: see text] Two formal chiral pool syntheses of the (-)-salicylihalamides A and B were achieved from commercially available 1,2,5,6-diacetone-d-glucose and l-rhamnose.     

Synthesis and structure of novel 1lambda4,2,6-thiadiazines

S,S-Disubstituted sulfodiimines 9 are known to be versatile building blocks for the synthesis of various sulfur-nitrogen-containing heterocycles. Addition-condensation reactions of 9 with three different activated carbonylsubstrates 14-16 lead to several new 1lambda(6),2,6-thiadiazine-3-ones 17-20. S-Debenzylation via a novel synthetic approach utilizing different electrophiles such as TMSCl, Alk(3)O(+)BF(4)(-), Tos(2)O, and Mes(2)O gives rise to a variety of so far hardly known 1lambda(4),2,6-thiadiazine-3-ones 25 and 26. Structure elucidation reveals NH/CH tautomers in solution as well as a tetrahedral asymmetric sulfur attached to the conjugated planar N/C skeleton. The structural features of the 1lambda(4),2,6-thiadiazines 25 and 26 as well as their reactivity toward certain electrophiles will be discussed in detail. Alkylation with Alk(3)O(+)BF(4)(-) preferably results in formation of the 3-alkoxy-1lambda(4)-2,6-thiadiazines 29. In the presence of acid or base, also the N-2-alkyl isomers 30 are formed. Mesylation of 26 with methanesulfonic acid anhydride followed by aminolysis furnishes a variety of the desired heterocycles 5 in moderate overall yield.     

Formal total syntheses of the (-)-salicylihalamides A and B from D-glucose and L-rhamnose

[reaction: see text] Two formal total syntheses of the (-)-salicylihalamides, based on chiral pool approaches, are reported. D-glucose and L-rhamnose were used to prepare advanced intermediates 23 and 54, which can be converted in three or four steps, respectively, to the target compounds. The synthesis of 23 from a known D-glucose-derivative was accomplished in 12 steps and 17% overall yield, and the synthesis of 54 from a known L-rhamnose-derivative was done in nine steps and 6% overall yield. A key step in the synthesis was a ring-closing metathesis reaction to prepare the macrocyclic ring system. It was demonstrated that the phenolic protecting group was critical for inducing the preferential formation of the desired E isomer. It was further shown that the protecting group at the C13 hydroxyl group had no significant influence on the E:Z ratio during the ring-closing metathesis reaction.     

What Kind of Image is Drawn by a MALDI-Experiment of Grafted Nanoparticles? - How to Distinguish between Chemically and Physically Adsorbed Parts

Summary: The modern MALDI-TOF mass spectrometry is a powerful technique for the fast and accurate determination of a number of polymer characteristics like end group analysis. It can also be used to analyze the grafted shell around silane modified nanoparticles. This shell contains physically and chemically adsorbed parts. It is a question of great interest what kind of adsorbent can be visualized by using MALDI-TOF mass spectrometry, while it is not possible to ionize the whole AEROSIL® particle. Therefore we chose the system MPTMS/AEROSIL®200 to address this issue. We grafted AEROSIL®200 with methacryloxy(propyl)-trimethoxysilane (MPTMS) under acid conditions. The grafted nanoparticles were treated via Soxhlet extraction. The resulting fractions (the washed nanoparticles and the Soxhlet extract) were systematically analyzed by using MALDI-TOF MS as well as the grafted nanoparticles themselves. As a crosscheck MPTMS was homopolymerized and analyzed. Our results show that for the system under investigation there is strong evidence that only the physically adsorbed part can be analyzed via MALDI-TOF MS.     

Assessing the RAFT Equilibrium Constant via Model Systems: An EPR Study

Reversible addition‐fragmentation chain transfer (RAFT) equilibrium constants, K_eq, for the model system cyano‐iso‐propyl dithiobenzoate (CPDB) – cyano‐iso‐propyl radical (CIP) have been deduced via electron paramagnetic reso1494nce (EPR) spectroscopy. The CIP species is produced by thermal decomposition of azobis‐iso‐butyronitrile (AIBN). In solution of toluene at 70 °C, K_eq has been determined to be (9 ± 1) L · mol⁻¹. Measurement of K_eq = k_ad/k_β between 60 and 100 °C yields ΔE_a = (–28 ± 4) kJ · mol⁻¹ as the difference in the activation energies of k_ad and k_β. The data measured on the model system are indicative of fast fragmentation of the intermediate radical produced by addition of CIP to CPDB.