Expanding the scope of poly(ethylene glycol)s for bioconjugation to squaric acid-mediated PEGylation and pH-sensitivity

Poly(ethylene glycol) (PEG) is used in a broad range of applications due to its unique combination of properties and is approved use in formulations for body-care products, edibles and medicine. This thesis aims at the synthesis and characterization of novel heterofunctional PEG structures and the establishment of diethyl squarate as a suitable linker for the covalent attachment to proteins. Chapter 1 is an introduction on the properties and applications of PEG as well as the fascinating chemistry of squaric acid derivatives. In Chapter 1.1, the synthesis and properties of PEG are described, and the versatile applications of PEG derivatives in everyday products are emphasized with a focus on PEG-based pharmaceuticals and nonionic surfactants. This chapter is written in German, as it was published in the German Journal Chemie in unserer Zeit. Chapter 1.2 deals with PEGs major drawbacks, its non-biodegradability, which impedes parenteral administration of PEG conjugates with polyethers exceeding the renal excretion limit, although these would improve blood circulation times and passive tumor targeting. This section gives a comprehensive overview of the cleavable groups that have been implemented in the polyether backbone to tackle this issue as well as the synthetic strategies employed to accomplish this task. Chapter 1.3 briefly summarizes the chemical properties of alkyl squarates and the advantages in protein conjugation chemistry that can be taken from its use as a coupling agent. In Chapter 2, the application of diethyl squarate as a coupling agent in the PEGylation of proteins is illustrated. Chapter 2.1 describes the straightforward synthesis and characterization of squaric acid ethyl ester amido PEGs with terminal hydroxyl functions or methoxy groups. The reactivity and selectivity of theses activated PEGs are explored in kinetic studies on the reactions with different lysine and other amino acid derivatives, followed by 1H NMR spectroscopy. Further, the efficient attachment of the novel PEGs to a model protein, i.e., bovine serum albumin (BSA), demonstrates the usefulness of the new linker for the PEGylation with heterofunctional PEGs. In Chapter 2.3 initial studies on the biocompatibility of polyether/BSA conjugates synthesized by the squaric acid mediated PEGylation are presented. No cytotoxic effects on human umbilical vein endothelial cells exposed to various concentrations of the conjugates were observed in a WST-1 assay. A cell adhesion molecule - enzyme immunosorbent assay did not reveal the expression of E-selectin or ICAM-1, cell adhesion molecules involved in inflammation processes. The focus of Chapter 3 lies on the syntheses of novel heterofunctional PEG structures which are suitable candidates for the squaric acid mediated PEGylation and exhibit superior features compared to established PEGs applied in bioconjugation. Chapter 3.1 describes the synthetic route to well-defined, linear heterobifunctional PEGs carrying a single acid-sensitive moiety either at the initiation site or at a tunable position in the polyether backbone. A universal concept for the implementation of acetal moieties into initiators for the anionic ring-opening polymerization (AROP) of epoxides is presented and proven to grant access to the degradable PEG structures aimed at. The hydrolysis of the heterofunctional PEG with the acetal moiety at the initiating site is followed by 1H NMR spectroscopy in deuterium oxide at different pH. In an exploratory study, the same polymer is attached to BSA via the squarate acid coupling and subsequently cleaved from the conjugate under acidic conditions. Furthermore, the concept for the generation of acetal-modified AROP initiators is demonstrated to be suitable for cholesterol, and the respective amphiphilic cholesteryl-PEG is cleaved at lowered pH. In Chapter 3.2, the straightforward synthesis of α-amino ω2-dihydroxyl star-shaped three-arm PEGs is described. To assure a symmetric length of the hydroxyl-terminated PEG arms, a novel AROP initiator is presented, who’s primary and secondary hydroxyl groups are separated by an acetal moiety. Upon polymerization of ethylene oxide for these functionalities and subsequent cleavage of the acid-labile unit no difference in the degree of polymerization is seen for both polyether fragments.

Tailored epoxide monomers as building units for multifunctional poly(ethylene glycol)

Poly(ethylenglykol) (PEG) ist eines der wichtigsten Polymere für pharmazeutische und biomedizinische Zwecke. Dies lässt sich vor allen Dingen auf seine ausgezeichnete Biokompatibilität, seine hohe chemische Stabilität sowie seine sehr gute Wasserlöslichkeit zurückführen. Neben seiner Anwendung in Produkten wie Lebensmitteln und Kosmetika ist PEG vor allem im pharmazeutischen Bereich unersetzlich geworden. Hier dient PEG als Grundlage für Salben, es kommt aber auch in der sogenannten „PEGylierung“ zum Einsatz. Unter PEGylierung versteht man die kovalente Verknüpfung von PEG mit Wirkstoffmolekülen, beispielsweise Proteinen oder niedermolekularen Medikamenten. In der akademischen Forschung sind aber auch PEGylierte Nanopartikel oder durch PEG stablisierte Liposomen für die Applikation im Bereich der Medizin von hohem Interesse. Trotz seiner breiten Verwendung hat PEG zwei entscheidende Nachteile: Zum einen benötigt man gerade im Hinblick auf PEGylierungen viele funktionelle Gruppe, jedoch trägt PEG maximal zwei Hydroxyl-Gruppen (die Endgruppen), die für kovalente Verknüpfungen genutzt werden können. Zum anderen ist PEG nicht in physiologischer Umgebung abbaubar und kann daher in vivo oberhalb eines Molekulargewichts von 40 000 g/mol nicht eingesetzt werden, da sonst eine Ausscheidung über die Niere nicht möglich ist und eine ungewollte Anreicherung im Körper stattfindet.rnDie durch die geringe Anzahl an Endgruppen limitierte Beladungsdichte kann durch das Design neuer Epoxid-Derivate und deren statistischen Einbau in das PEG Rückgrat deutlich verbessert werden. Im ersten Teil dieser Arbeit werden drei neuartige funktionelle Oxirane vorgestellt, die systematisch mit Ethylenoxid copolymerisiert wurden, was die selektive Einführung verschiedener funktioneller Gruppen am Polymerrückgrat ermöglicht. Im Vordergrund der Betrachtungen standen die Eigenschaften der neuartigen multifunktionellen (mf)-PEG Copolymere im Hinblick auf ihr thermisches Verhalten sowie die Verteilung der funktionellen Gruppen (Mikrostruktur) innerhalb des PEG-Rückgrats. Die gezielte Adressierbarkeit der funktionellen Gruppen konnte durch verschiedene Modellreaktionen bestätigt werden. Darüber hinaus konnte gezeigt werden, dass sich mit der vorgestellten Synthesestrategie komplexe Hybridmaterialien, beispielsweise metallhaltige Polyether, darstellen lassen. Mit Hinblick auf die biomedizinischen Anwendungen und die Konkurrenz zu etablierten PEG-Hompolymeren, standen die Wasserlöslichkeit und die Toxizität der synthetisierten Materialien im Zentrum weiterer Untersuchungen. Alle dargestellten Polymere zeigten einen Trübungspunkt in Wasser, der sich in Abhängigkeit der Zusammensetzung und Hydrophobizität der Comonomere über ein weites Temperaturspektrum variieren und somit systematisch einstellen ließ. Die Toxizität der statistischen mf-PEGs lag im Bereich von PEG, was die mf-PEGs interessant für biomedizinische Anwendung macht.rnIm zweiten Teil der Arbeit wurden Copolymerisationen verwendet, um über erstmals hergestellte Epoxid-Inimere sauer spaltbare Einheiten in das Polyetherrückgrat einzuführen. Die neuen, verzweigten Strukturen wurden auf die Zersetzung in physiologisch relevantem Milieu untersucht. Die erzielte pH-abhängige Spaltbarkeit, kann für potenzielle Anwendungen beispielsweise in der Krebstherapie, von Vorteil sein.rn

Protein-Protein- und Protein-Lipid-Wechselwirkungen beeinflussen die Oligomerisierung und Funktion des E. coli Aquaglyceroporins GlpF

Aquaporine sind hochselektive Transmembrankanäle, die in allen Lebensformen den Fluss von Wasser und kleinen, polaren Molekülen wie Glycerol über Lipidmembranen ermöglichen. Obwohl die Kanalpore für den Substratfluss im Monomer lokalisiert ist, liegen Aquaporine innerhalb biologischer Membranen als Homotetramere vor. Im Rahmen dieser Arbeit wurden proteinbezogene und lipidmembranassoziierte Einflüsse auf die Oligomerisierung und Funktion des bakteriellen Aquaglyceroporins GlpF sowohl in vitro als auch in vivo untersucht. rnDie erhöhte Stabilität der Aquaporinpore sowie Interaktion zwischen den GlpF-Monomeren sind Triebkräfte der Aquaporin-Tetramerisierung. Ferner erfordern die GlpF-Tetramerisierung und -Aktivität bei Abschirmung der Ladung anionischer Lipide und einer minimalen Membrandicke von 27 Å keine spezielle Lipidumgebung. Da anionische Lipide die GlpF-Funktion jedoch störten, kann die GlpF-Aktivität in vivo möglicherweise durch die selektive Anreicherung von anionischen Lipiden in der unmittelbaren Proteinumgebung reguliert werden. Ungünstige Lipid-GlpF-Interaktionen können jedoch in Lipidumgebungen mit hoher Ordnung in der Acylkettenregion entstehen, die zu einer Aggregation der GlpF-Tetramere und reduzierten Aktivität führen. rnFerner wurde die Auswirkung der nephrogenen Diabetes insipidus verursachenden Aquaporin 2-Punktmutation V71M auf die Oligomerisierung und Funktion des homologen, bakteriellen Aquaglyceroporins GlpF untersucht. Da weder die Oligomierisierung noch die Aktivität des homologen, bakteriellen Aquaglyceroporins eingeschränkt sind, beruht der Krankheitsmechanismus der Aquaporin 2-Mutante V71M vermutlich auf einem defekten Transportmechansimus im Menschen. rn

Sulfur-containing polymers for surface attachment

Different concepts for the synthesis of sulfur-containing polymers as well as their adsorption onto gold surfaces were studied. The present work is divided into three parts. The main part focuses on the synthesis of poly(1,2-alkylene sulfides) (“polysulfides”) with complex architectures on the basis of polyether-based macroinitiators by the anionic ring-opening polymerization of ethylene sulfide and propylene sulfide. This synthetic tool kit allowed the synthesis of star-shaped, brush-like, comb-like and pom-pom-like polysulfides, the latter two with an additional poly(ethylene glycol) chain. Additionally, the number of polysulfide arms as well as the monomer composition could be varied over a wide range to obtain copolymers with multiple thioether functionalities.rnThe second section deals with the synthesis of a novel lipoic acid-based initiator for ring-opening polymerizations for lactones and epoxides. A straightforward approach was selected to accomplish the ability to obtain tailored polymers with a common used disulfide-anchoring group, without the drawbacks of post-polymerization functionalization. rnIn the third part, a new class of block-copolymers consisting of polysulfides and polyesters were investigated. For the first time this approach enabled the use of hydroxyl-terminated poly(propylene sulfide) as macroinitiator for the synthesis of a second block.rnThe adsorption efficiency of those different polymer classes onto gold nanoparticles as well as gold rnsupports was studied via different methods.rn

Investigation of the Potential Energy Surface for the First Step in the Alkaline Hydrolysis of Methyl Acetate

The potential energy surface for the first step of the alkaline hydrolysis of methyl acetate was explored by a variety of methods. The conformational search routine within SPARTAN was used to determine the lowest energy am1 and pm3 structures for the anionic tetrahedral intermediate. Ab initio single point and geometry optimization calculations were performed to determine the lowest energy conformer, and the linear synchronous transition (lst) method was used to provide an initial structure for transition state optimization. Transition states were obtained at the am1, pm3, 3-21G, and 3-21 + G levels of theory. These transition states were compared with the anionic tetrahedral intermediates to examine the assumption that the intermediate is a good model for the transition state. In addition, the Cramer/Truhlar sm3 solvation model was used at the semiempirical level to compare gas phase and aqueous alkaline hydrolysis of methyl acetate.

A New Crystalline LiPON Electrolyte: Synthesis, Properties, and Electronic Structure

The new crystalline compound, Li2PO2N, was synthesized using high temperature solid state methods starting with a stoichiometric mixture of Li2O, P2O5, and P3N5. Its crystal structure was determined ab initio from powder X-ray diffraction. The compound crystallizes in the orthorhombic space group Cmc2(1) (# 36) with lattice constants a = 9.0692(4) angstrom, b = 53999(2) angstrom, and c = 4.6856(2) angstrom. The crystal structure of SD-Li2PO2N consists of parallel arrangements of anionic chains formed of corner sharing (PO2N2) tetrahedra. The chains are held together by Li+ cations. The structure of the synthesized material is similar to that predicted by Du and Holzwarth on the basis of first principles calculations (Phys. Rev. B 81,184106 (2010)). The compound is chemically and structurally stable in air up to 600 degrees C and in vacuum up to 1050 degrees C. The Arrhenius activation energy of SD-Li2PO2N in pressed pellet form was determined from electrochemical impedance spectroscopy measurements to be 0.6 eV, comparable to that of the glassy electrolyte LiPON developed at Oak Ridge National Laboratory. The minimum activation energies for Li ion vacancy and interstitial migrations are computed to be 0.4 eV and 0.8 eV, respectively. First principles calculations estimate the band gap of SD-Li2PO2N to be larger than 6 eV. (C) 2013 Elsevier B.V. All rights reserved.

Iodide interaction with natural pyrite

29I is one of the major dose-determining nuclides in the safety analysis of deep storage of radioactive waste. Iodine forms anionic species that hardly sorb on the surfaces of common host-rock minerals. Recently, interest has arisen on the role of pyrite, an accessory mineral capable of binding anionic selenium. Whereas the interaction of selenium with pyrite is well documented, corresponding results on iodine sorption are still scarce and controversial. Pyrite is present in argicilleous rocks which are being considered in many countries as potential host rocks for a radioactive waste repository. The uptake of iodide (I−) on natural pyrite was investigated under nearly anoxic conditions (O2 < 5 ppm) over a wide concentration range (10−11–10−3 M total I−) using 125I as the radioactive tracer. Weak but measurable sorption was observed; distribution coefficients (R d) were less than 0.002 m3 kg−1 and decreased with increasing total iodide concentration. Iodide sorption was connected to the presence of oxidized clusters on the pyrite surface, which were presumably formed by reaction with limited amounts of dissolved oxygen. The results obtained indicated that pyrite cannot be considered as an effective scavenger of 129I under the geochemical conditions prevailing in underground radioactive waste geologic storage.

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.

Surfactant mediated adsorption of negatively charged latex particles to a cellulose surface

The adsorption of anionic, carboxyl functionalized latex particles, recharged by a cationic surfactant acting as fabric softener/conditioner, to a cellulose surface was investigated with evanescent wave video microscopy. This technique allows to monitor the deposition and release of individual particles in real-time with an excellent selectivity and sensitivity. Since the recharged particles and the conditioner compete for the free surface, the initial deposition rate and final surface coverage are found to be strongly dependent on the ratio of particle and conditioner concentrations.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix−hinge−helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

High-resolution computer simulation of electrophoretic mobilization in isoelectric focusing

Cationic and anionic electrophoretic mobilization for focusing of hemoglobins (Hb's) in the presence of 100 carrier ampholytes covering a pI range of 6.00-7.98 was studied by computer simulation at a constant current density of 300 A/m(2). Electropherograms that would be produced by whole column imaging and by single detectors placed at different locations along the focusing column are presented. Upon mobilization, peak heights of the Hb zones decrease, but the zones retain a relatively sharp constant profile and are migrating at a constant velocity. A further peak decrease occurs during readjustment at the locations of the original buffer/column interfaces, indicating that detection sensitivity is the lowest at these locations. An anionic carrier ampholyte mobility smaller than that of its cationic species produces a cathodic drift which is smaller than the transport rate used for electrophoretic mobilization. Compared to the case with equal mobilities of carrier ampholyte species, a small increase (decrease) is predicted for the cationic (anionic) mobilization rate within the focusing column. Simulation data suggest that electrophoretic mobilization after focusing and focusing with concurrent electrophoretic mobilization are comparable isotachophoretic processes that occur when there is an uninterrupted flux of an ion through the focusing column. Cathodic drift caused by unequal mobilities of the species of carrier ampholytes, electrophoretic mobilization, and decomposition occurring at the pH gradient edges are related electrophoretic processes.

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.

Cooperative Effects of Electron Donors and Acceptors for the Stabilization of Elusive Metal Cluster Frameworks: Synthesis and Solid-State Structures of Pt-19(CO)(24)(mu(4)-AuPPh3)(3)](-) and Pt-19(CO)(24){mu(4)-Au-2(PPh3)(2)}(2)]

The anionic cluster Pt-19(CO)(22)](4-) (1), of pentagonal symmetry, reacts with CO and AuPPh3+ fragments. Upon increasing the Au:Pt-19, molar ratio, different species are sequentially formed, but only the last two members of the series could be characterized by X-ray diffraction, namely, Pt-19(CO)(24)(mu(4)-AuPPh3)(3)](-) (2) and Pt-19(CO)(24){mu(4)-Au-2(PPh3)(2)}(2)] (3).The metallic framework of the starting cluster is completely modified after the addition of CO and AuL+, and both products display the same platinum core of trigonal symmetry, with closely packed metal atoms. The three AuL+ units cap three different square faces in 2, whereas four AuL+ fragments are grouped in two independent bimetallic units in the neutral cluster 3. Electrochemical and spectroelectrochemical studies on 2 showed that its redox ability is comparable with that of the homometallic 1.