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1 MLECULAR RECGNITIN F BILGICALLY RELEVANT TARGETS - FRM ANIN BINDING MTIFS T APPLICATIN IN CELL BILGY Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - vorgelegt von Diplom-Chemiker Hannes Yacu Kuchelmeister geboren in Illertissen erstellt am Institut für rganische Chemie an der Universität Duisburg-Essen 2011
3 Die vorliegende Arbeit wurde von März 2008 bis ktober 2011 in den Instituten für rganische Chemie der Universitäten Würzburg und Duisburg-Essen unter der Anleitung von Herrn Prof. Dr. Carsten Schmuck angefertigt. Ich erkläre hiermit des Eides statt, dass ich die vorliegende Arbeit selbst verfasst und mich dabei keiner anderen als der von mir bezeichneten Quellen und Hilfen bedient habe. Ich erkläre hiermit, dass ich an keiner anderen Stelle ein Prüfungsverfahren beantragt beziehungsweise die Dissertation in dieser oder anderer Form bereits anderweitig als Prüfungsarbeit verwendet oder einer anderen Fakultät als Dissertation vorgelegt habe. Essen, September 2011 Hannes Y. Kuchelmeister Disputation: 27. ktober 2011 Gutachter: Prüfungsvorsitzender: Prof. Dr. Carsten Schmuck Prof. Dr. Shirley Knauer Prof. Dr. Torsten C. Schmidt
5 DANKSAGUNG An erster Stelle möchte ich natürlich meinem Doktorvater, Prof. Dr. Carsten Schmuck, für die Möglichkeit danken, auf dem interessanten Gebiet der bioorganischen Chemie zu forschen. Durch seine hilfreiche fachliche Unterstützung, eine stets offene Tür und seine umgängliche, freundliche Art hat er wesentlich zum Gelingen dieser Arbeit beigetragen. Besonders bedanken möchte ich mich für die große Gestaltungsfreiheit bei der Bearbeitung meines Themas und dem stets für neue Ideen offenen hr. Für die finanzielle Unterstützung meiner Promotion möchte ich mich bei der Studienstiftung des deutschen Volkes bedanken. Der analytischen Abteilung der Universität Duisburg-Essen gilt mein Dank: Dr. Torsten Schaller und Heinz Bandmann für die Durchführung von NMR Messungen und Werner Karow für die Aufnahme der Massenspektren. Bei Wilhelm Sicking möchte ich mich für das DNA-Modelling bedanken. Ursula Nüchter gilt mein Dank für das Messen zwischen den Jahren. Allen Auszubildenden, Bachelor- und Vertiefungsstudenten möchte ich für ihren Einsatz und Beitrag zu dieser Doktorarbeit danken. Besonderer Dank gilt meinen Kooperationspartnern ohne deren Einsatz und Arbeit diese Dissertation nur die Hälfte wert wäre: Magdalena Gellner, Stephan Niebling und Prof. Dr. Sebastian Schlücker möchte ich für die angenehme und unkomplizierte Zusammenarbeit danken. Bei Dr. Ivo Crnolatac, Dr. Marijana Radić Stojković, Dr. Lidija-Marija Tumir und Dr. Ivo Piantanida möchte ich mich für die fachliche Hilfe in allen praktischen und theoretischen Belangen der Welt der Nukleinsäuren, die tolle Zeit in Zagreb, das schlechte Fußballspiel und das gute Essen bedanken. Aljona Gutschmidt, Sarah Tillmann und Prof. Dr. Shirley Knauer sei gedankt für die Zuverlässigkeit und Sparsamkeit, das schnelle Zellen-Zählen, die freundliche und unkomplizierte Art und die kurzfristige und unglaublich schnelle Hilfe im Juli. An den gesamten AK Schmuck und insbesondere an meine Würzburger und Essener Laborkollegen Dr. Volker Bickert, Dr. Nicholas Walden, Ute Michels, Pia Mereu und Tassilo Fenske geht mein Dank für die positive Atmosphäre und die gute Zusammenarbeit. Für das Korrekturlesen und die kritische Durchsicht des Manuskripts möchte ich Barbara Geibel, Sebastian Langolf, Dr. Ivo Piantanida und Waltraud Kuchelmeister danken. Ein herzliches Dankeschön geht an meine Eltern, Guido und Waltraud, die mich in meinem eingeschlagenen Lebensweg immer ermutigten und an meine Schwestern, Lena und Sophie, für die Ablenkung und die gute Zeit an stern als letztes Durchatmen vor dem Endspurt. Der größte Dank geht an Chris, die mich in all den Jahren begleitet hat und auch in den schwierigen Momenten meiner Arbeit immer für mich da war. Danke für all deine Mühen, dein Verständnis und die selbstlose Unterstützung.
7 Der Mensch hat dreierlei Wege klug zu handeln: durch Nachdenken ist der edelste, durch Nachahmen der einfachste, durch Erfahrung der bitterste. Konfuzius
9 TABLE F CNTENTS 1. INTRDUCTIN MLECULAR RECGNITIN F ANINS GENE THERAPY BACKGRUND INFRMATIN CARBXYLATE- AND LIGPEPTIDE RECGNITIN The GCP-xoanion Binding Site Recognition of Short Peptides with Linear Receptors Di- and Multivalent Receptors for Peptide Recognition NUCLETIDE RECGNITIN IN WATER Polyamine-Based Receptors Guanidinium-Based Receptors Calixarene- and Cyclodextrin-Based Receptors Peptide-Based Receptors NUCLEIC ACIDS DNA/RNA Structure CD-Spectroscopy of Polynucleotides Thermal Melting Experiments Isothermal Titration Calorimetry Specific DNA/RNA Recognition TRANSFECTIN Gene Therapy Viral Vectors Physical Vectors Chemical Vectors PRJECT AND BJECTIVES RESULTS AND DISCUSSIN RAMAN SPECTRSCPY AND MLECULAR RECGNITIN Resonance Raman Spectroscopy Surface Enhanced Raman Spectroscopy NN-SYMMETRIC TWEEZER RECEPTRS FR PEPTIDE RECGNITIN Synthesis of the rthogonally Protected Template Microwave-Assisted Solid Phase Synthesis of Two Non-Symmetric Tweezer Receptors Preliminary Binding Studies with N-Ac-Lys-D-Ala-D-Ala-H SYMMETRIC TWEEZER RECEPTR FR NUCLETIDE RECGNITIN Synthesis of the Template Synthesis of the Symmetric Tweezer Receptor Binding Studies TW- AND THREE-ARMED LIGANDS FR DNA/RNA-RECGNITIN Synthesis of the Two-Armed Ligand with an Inverse Amino Acid Sequence Synthesis of the Three-Armed Ligand Nucleic Acid Binding Studies Transfection Experiments CMBINATRIAL DNA-LIGAND LIBRARY Synthesis of the Library n-bead Screening with p(dadt) Synthesis of Additional Divalent Ligands Binding Studies in Solution
10 4.5.5 Identification of DNA-Ligands for Transfection Experiments AMPHIPHILIC GENE CARRIERS FR CELL TRANSFECTIN Combined Solution Phase and Solid Phase Peptide Synthesis Critical Micelle Concentration Transfection Experiments SUMMARY UTLK EXPERIMENTAL SECTIN GENERAL EXPERIMENTAL AND ANALYTICAL METHDS GENERAL PRCEDURES FR SPPS SYNTHESIS F SMALL LIGPEPTIDE SUBSTRATES DERIVATIZATIN F AU-NANPARTICLES GENERAL PRCEDURES FR MICRWAVE ASSISTED SPPS PREPARATIN F NN-SYMMETRIC TWEEZER RECEPTRS SYNTHESIS F SYMMETRIC TWEEZERS PREPARATIN F A TRIVALENT PLYNUCLETIDE LIGAND SPPS F THE DNA-BINDER LIBRARY SYNTHESIS F AMPHIPHILIC GENE CARRIERS BILGICAL TESTS APPENDICES A: ZUSAMMENFASSUNG UND AUSBLICK B: LIST F ABBREVIATINS C: SUPPLEMENTARY EXPERIMENTAL DATA C.1 HPLC Data C.2 UV/Vis and Fluorescence Titrations C.3 Thermal Melting Experiments C.4 EB Displacement Assays C.5 ITC Experiments C.6 DAPI Displacement Assays C.7 n-bead Screening of the Library D: CURRICULUM VITAE E: LIST F PUBLICATINS F: BIBLIGRAPHY
11 1. INTRDUCTIN 1 1. INTRDUCTIN 1.1 MLECULAR RECGNITIN F ANINS The recognition of anion plays a crucial role in a manifold of essential biological processes like enzyme-substrate interactions, 1 protein folding, 2 or DNA regulation. 3 The overwhelming majority of enzyme substrates for instance is of anionic nature such as carboxylates in the citric acid cycle or nucleotides for DNA polymerization (see Figure 1). 4 Hence, the importance of anion recognition processes in biological systems can hardly be overestimated. To gain a more detailed insight into these complex processes, which are up to this date not entirely understood in all their complexity, the development of synthetic model systems is worthwhile. Figure 1. X-ray crystal structure of a ternary complex between a DNA polymerase (green), a negatively charged double-stranded oligonucleotide helix (grey) and the anionic substrate thymidine triphosphate (red) (PDB access code: 3QEP 5 ). In order to create artificial model systems, which are similarly selective and efficient as their natural archetypes, a detailled comprehension of the interactions between host and guest on the molecular level is indispensable. Such molecular recognition events are based on the combination of various attractive non-covalent interactions between receptor and substrate like electrostatic, dipole and dispersion interactions, π-stacking, and hydrogen bonding together with entropic contributions like for example the liberation of solvent molecules. 6
12 MLECULAR RECGNITIN F BILGICALLY RELEVANT TARGETS While the selective recognition of cations by artificial receptor molecules was readily achieved by synthetic chemists, for example by binding alkaline metal ions with the crown ethers developed by Pederson as early as 1967, 7 the molecular recognition of anions has proven to be a somewhat more demanding task. Due to additional electron-electron repulsion, anions are larger in size than their corresponding neutral species and even larger in comparison to respective cations. Therefore, anion receptor systems need to be comparably larger to allow for efficient interaction with their target atom or molecule, thus increasing their complexity. Furthermore, even simple inorganic anions occur in a multitude of geometries like spherical halides, tetrahedral phosphates, planar nitrates, linear azides or in even more complex forms (e.g. oligophosphates). The substrate s geometry has to be matched by the artificial host system for efficient and selective binding. Furthermore, anions feature a higher free energy of solution in water. 8 Consequently anion receptors have to compete more strongly with the surrounding aqueous medium for binding to their substrates and thereby replacing the tightly bound water molecules. Finally, organic and also some inorganic anions often only exist within a rather small ph window which necessitates the receptors to be in an appropriate protonation state at this ph range to allow for efficient molecular recognition. In natural systems the amino acid arginine is very often involved in binding events between anionic substrates and proteins. 9 The energetic stabilization of carboxylates in the microenvironment of a protein by the guanidinium moiety in an arginine side chain for example outmatches the stabilization by the ammonium group of lysine by large because guanidinium is able to form a very stable bidentate hydrogen bonded salt bridge to the carboxylate. 10 Furthermore, the basicity of the guanidine group ensures protonation over a broad range of ph values due to the delocalization of the positive charge over three nitrogen atoms, thus guaranteeing for charge-charge interactions and the capability to act as efficient hydrogen bond donor. 11 These valuable features are the reason why chemists have regularly and successfully designed guanidinium-based artificial host systems for anion recognition. 12 Likewise in our working group, a guanidiniocarbonyl pyrrole (GCP) motif was developed for efficient binding of carboxylates, which can be equipped with a linear peptidic side chain for the formation of additional interactions with the substrate. 13 Within the framework of the work presented in this thesis, novel receptors containing the GCP binding motif in one to three peptidic side chains will be developed for the recognition of biologically relevant targets like oligopeptides, nucleotides, or nucleic acids. In order to selectively and efficiently bind to these polar substrates without the help of metal ions in aqueous medium at physiological ph these new host molecules need to overcome the competing influence of the surrounding water molecules by means of multiple non-covalent interactions. From these model systems one may then deduce general information on the binding events and proceed by applying the so obtained knowledge. ne potential application might for instance lie within the field of gene therapy. 1.2 GENE THERAPY Gene therapy can be defined as the treatment of human diseases by the transfer of genetic material into specific cells of the patients. 14 Advances in biotechnology paired with the decoding of the human genome lead to the identification of disease-causing genes. 15 These findings set the road for treating inherited or acquired genetic defects by introducing the
13 1. INTRDUCTIN 3 missing genetic information, replacing corrupted ones, or by deleting deleterious genes. This way the limitations of small molecule therapies can be overcome and yet non-druggable diseases are rendered curable. Therefore, the genetic information has to be delivered into the desired cell and implemented into the cell s own genetic code. Different from the majority of classical small molecule therapeutics, 16 nucleic acids are not able to enter the cell on their own. They are repelled from the negatively charged cell surface due to the negative charge of their ribophosphate backbone. Furthermore, they are rapidly degraded in the human body by DNAses or RNases. To surmount these obstacles transport systems, so called vectors, are necessary. Nowadays the most frequently utilized gene carriers are recombinant viruses. 17 The harmful parts of the virus RNA or DNA, e.g. their ability to replicate, are removed and replaced with the desired genetic information. Viruses are extremely efficient in delivering their genetic material into human cells, because this is the sole cause of their existence and ensures their survival. They were able to perfect this mechanism over millions of years. However, the gene transfer with designer viral vectors suffers from several severe drawbacks. First, the immune system recognizes the viral vectors as pathogens and reacts accordingly. This reaction is getting more severe with every treatment. Further, recombinant viruses are able to retrieve the removed harmful information by recombination with wild-type viruses and may thus become pathogenic again and spread. Another inconvenience is that the length of the therapeutic gene is limited to the size of the viral vector s own genome. Acceptance in public is not very good. Finally, the scale-up for industrial production on large scale is very difficult to achieve for these biological systems. Due to these inconveniences alternatives are necessary. Chemical vectors are the most promising ones because they offer the potential to overcome most of the difficulties associated with viral gene carriers. 18 These non-viral vectors are positively charged molecules which are able to condense the DNA into small, positively charged particles which are then able to cross the cell membrane. This way, the nucleic acid is also shielded from degrading enzymes and its half life in human serum is increased. Chemical vectors are not pathogenic and do not trigger an immune response. They can easily be produced in high quantities at low cost. However, there are still challenges which need to be addressed for efficient and safe delivery of genetic information by means of chemical vectors to enable application in gene therapy. Chemical vectors are not yet as efficient as viral vectors and due to their high positive charge they are often cytotoxic. Furthermore, they are not selective for a given cell type. Therefore, the development of novel chemical vectors with improved properties is necessary in order to overcome one of the major limitations of gene therapy the transport of genetic material into the cell. Within this work novel polynucleotide-ligands containing the GCP oxoanion binding motif combined with peptidic side chains will be developed and tested for their ability to shuttle genetic information into human cells. The results of these experiments will improve our knowledge about the interactions between small molecules with nucleic acids and help to progress the highly active and still developing field of non-viral vectors for the delivery of genetic material.
14 MLECULAR RECGNITIN F BILGICALLY RELEVANT TARGETS 2. BACKGRUND INFRMATIN 2.1 CARBXYLATE- AND LIGPEPTIDE RECGNITIN The GCP-xoanion Binding Site The binding motif 1 depicted in Figure 1 nukleotid enzyme dna ternary komplex Figure 2 was introduced by Schmuck in It consists of a relatively rigid, planar guanidiniocarbonyl pyrrole moiety, which allows efficient binding of planar oxoanions, e.g. carboxylates (2), in aqueous medium. In addition to the salt bridge between the guanidinio group and the carboxylate the complex is stabilized by several well-defined hydrogen bonds. The guanidinium group is connected to the pyrrole core by an acetyl group, thus decreasing the pk A value of free guanidine from 13.5 to approximately As a consequence the potency to act as a hydrogen donor increases dramatically. Furthermore, the intramolecular hydrogen bond between the carboxyl and guanidinium group preorganizes the binding motif in the correct conformation for oxoanion binding. Finally, by varying R the receptor can be fine tuned for a given substrate. 1 2 Figure 2. Guanidiniocarbonyl pyrrole receptor 1 for the recognition of carboxylates (2). This way binding constants between K 10 3 and 10 4 M -1 could be obtained in 10 % DMS/water for N-Ac-Ala-H. 21 This is a very good value when considering that water is an extremely competitive medium with regard to the recognition of polar substrates. Thus, by combining several weak, non-covalent interactions it was possible to devise artificial host systems for effective and selective binding of substrates in aqueous medium.
15 2. BACKGRUND INFRMATIN Recognition of Short Peptides with Linear Receptors Following Lehn s description the term receptor will be used throughout this work as a chemical host which binds to a given guest. 22 According to this definition, peptide receptors are synthetic organic molecules which selectively bind to a peptide sequence via multiple non-covalent interactions. The molecular recognition of short peptide sequences is a promising goal due to their importance in biochemical and medicinal processes. 23 Artificial model systems may not only contribute to gain a more substantial understanding of the yet not fully understood molecular recognition process itself, they may also serve as new starting points for drug development 24 or as sensors for diagnostics. 25 A prerequisite for potential application is always a strong and selective complexation of the target peptide sequence. A well studied example for a short peptide sequence of biological importance is the amino acid sequence D-Ala-D-Ala-H (3) and its depsipeptide analogue D-Ala-D-Lac-H (4), which are both shown in Figure 3. The first one is crucial for the mode of action of the antibiotic Vancomycin (5) against Gram positive bacteria: Vancomycin binds to this sequence with high affinity (K = M -1 ) thereby sterically blocking a transpeptidase enzyme from crosslinking the peptide side chains of peptidoglycan strands, which act as precursors during bacterial cell wall biosynthesis. This leads to a decrease of mechanical cell wall stability and ultimately to lysis upon osmotic pressure changes. The depsipeptide 4 can be found in Vancomycin resistant bacteria: By exchanging an amide for an ester bond the complex stability is reduced by a factor of 1,000. This dramatic decrease is due to the loss of one hydrogen bond which is instead replaced by an electrostatic repulsion between the oxygen lone pairs of the ester and the corresponding carbonyl group in the antibiotic Figure 3. Comparison of interactions between Vancomycin (5) with normal (3) and mutated (4) bacterial cell wall-forming peptide sequence. An attractive hydrogen bond is replaced for a repulsive electrostatic interaction thus decreasing the affinity of the antibiotic by a factor of 1,000. With the help of the combinatorial library 6 shown in Figure 4 comprising 39,304 members (34 3 proteinogenic and non-natural amino acids) Ellman was able to identify receptors which bind to the depsipeptide sequence N,N -Ac 2 -L-Lys-D-Ala-D-Lac-H (7) in aqueous solution. 27 The design of Ellman s receptor mimics that of Vancomycin: The right part corresponds to a simplified carboxylate binding pocket, which should be able to bind to both D-Ala-H and
16 MLECULAR RECGNITIN F BILGICALLY RELEVANT TARGETS D-Lac-H via three hydrogen bonds. The left side of the receptor on the other hand consists of a variable tripeptide, which is free to rotate and should therefore be able to avoid electrostatic repulsion between the antibiotic s carbonyl group and the lactate oxygen. And indeed, an on-bead screening with fluorescent labeled substrate revealed binding constants five times as high as for Vancomycin. The best receptors mainly contained the amino acid sequence L-Tpi-L-His (29 %) and L-N-Me-Phe-L-Disc (20 %) in position AA 1 and AA 2 (L- Disc 8 and L-Tpi 9 are shown in Figure 4). For AA 3 no clear tendency could be observed. To determine the binding constants and to validate the results from the on-bead screening, the best performing sequences were synthesized and tested in solution for their binding affinity towards N,N -Ac 2 -L-Lys-D-Ala-D-Lac-H (7) with the help of microcalorimetry. The overall best receptor 10 with the sequence L-N-Me-Phe-L-Disc-L-Tyr has a binding constant of M -1 in water. For the recognition of peptides in aqueous solution this is a good result. However, the substrate is fully acetylated and therefore rather non-polar. This facilitates binding based on van-der-waals interactions, but also is significantly different from the actual biological relevant substance AA 1-3 = L/D: Arg, Asn, His, Leu, N-Me-Leu, Lys, Phe, N-Me-Phe, Pro, Thr, Trp, Tyr, Tic L: Aib, Chg, Atc, Dapa, Disc, Isn, Tpi, N-Me-Gly = 39,304 receptors Figure 4. Schematic representation of receptor library 6 for the substrate N,N -Ac 2 -L-Lys-D-Ala-D-Lac-H (7) (left) and receptor of highest affinity 10 with the amino acid sequence L-N-Me-Phe-L-Disc-L-Tyr (right). As depicted in Figure 5 Schmuck was able to identify receptors for the dansyl-labeled polar, anionic peptide sequence N-Ac-D-Glu-L-Lys-D-Ala-D-Ala-H (EKAA, 12) with binding constants of up to K 10 4 M -1 in buffered water at ph 6 by means of screening a combinatorial library with 512 peptide-based artificial receptors (11) containing GCP as headgroup. 13 The best artificial receptor corresponds to the sequence R 1 = R 2 = lysine and R 3 = phenylalanine side chains (13). The C-terminus of the peptidic substrate 12 is bound by the GCP unit this is the main driving force for the complex formation. Additionally, the attached linear tripeptide chain interacts with the substrate by forming a hydrogen bond mediated β-sheet with the peptidic substrate. An additional salt bridge is formed between glutamate and the lysine ammonium group. In order to verify the substrate selectivity the library was also screened against the inverse substrate sequence N-Ac-D-Ala-D-Ala-L-Lys-D- Glu-H (AAKE), which resulted in binding constants that were lower by approximately one order of magnitude. 28 Thus, despite the fact that the complex stability is mainly based on undirected, long-range electrostatic interactions the binding constants are strongly dependent
17 2. BACKGRUND INFRMATIN 7 on the amino acid sequence of the substrate. For binding of a polar substrate in water the so achieved binding constants are very good AA 1-3 = Lys, Tyr, Ser, Glu, Phe, Val, Leu, Trp = 512 receptors Figure 5. General scheme of the tripeptide receptor library 11 and the dansyl-labeled substrate EKAA (12) (left) and calculated electrostatic surface potential (right), which shows the charge complementarity between receptor 13 (GCP-KKF) and tetrapeptide 14 (EKAA). The molecular modeling images are reprinted with permission from Schmuck, C.; Heil, M. Chem. Eur. J. 2006, 12, Copyright 2006 John Wiley and Sons. 28 In another set of experiments the best receptor for the EKAA sequence (CBS-KKF) was tested against a rather small combinatorial tetrapeptide library with 320 members. 29 With only three amino acids present in the library (Ala, Lys and Glu) all substrates were closely related, only differing in the absolute configuration of one amino acid (D/L-Ala). Binding constants were determined via fluorescence in buffered water at ph 6.1 and ranged from K < 50 M -1 to M -1. More importantly, a sequence-dependent stereoselectivity of the receptor could be observed as depicted in Figure 6 when the D/L-Ala amino acid of the substrate was fixed by strong electrostatic interactions between host and guest at both sides. Figure 6. Screening of a tetrapeptide library revealed that stereoselectivity is observed at positions where receptor and substrate are locked by strong electrostatic interactions at both sides of the stereogenic center (right). When this is not the case no stereoselectivity can be witnessed (left). These experiments clearly demonstrate the potential of small but focused libraries, which allow determining the binding properties of all members. A large and random library would not have elucidated the subtle differences of the binding properties. By characterizing all possible interactions it was possible to gain a more detailed insight into the binding event as would have been the case for a large and random library, in which only a few selected hit structures are isolated and characterized. f course, due to the limitations of small libraries with regard to their structural and functional diversity, the library has to be carefully designed to give the correct answer for the addressed problem. 30
18 MLECULAR RECGNITIN F BILGICALLY RELEVANT TARGETS Di- and Multivalent Receptors for Peptide Recognition Unlike linear receptors (vide supra) so called tweezer receptors contain two arms which are bridged via a template. Depending on the linker structure this kind of molecules are able to adopt a preorganized, yet flexible cavity where the substrate is held like between molecular tweezers. 31 When correctly designed the second side chain is able to form additional attractive interactions to the substrate, thus increasing the complex stability and the substrate selectivity. The second arm may either be identical to the first one in symmetric tweezer receptors or completely different in non-symmetric tweezers. This scheme may of course be further expanded to receptors with three or more arms. In general the aim of such a multivalent approach is to strengthen the complex stability by means of simultaneous interactions between multiple complementary functionalities between host and guest. ptimally the binding affinity is then higher than the sum of the corresponding monovalent interactions. This approach is also widely made use of in nature, be it bacterium-cell- 32, antibody-antigeninteractions 33 or the interplay of transcription factors with multiple sites of DNA. 34 In the following, this chapter will be limited to di- and trivalent receptors which also corresponds to the overall focus of the dissertation. Pioneering in the early 90s Still designed a library comprising 10,000 (10 4 ) peptidosteroidal receptors for enkephalin-like peptides (15, Figure 7). 35 The two arms are linked via a steroidal cheno-12-deoxycholic acid template and differ in their amino acid sequence. The nonsymmetric substitution was achieved by making use of the different chemical reactivity of the two hydroxyl groups at position C3 and C7. With the help of a two-colors-two-substratesassay receptors could be identified which were able to selectively bind to one of the two differently labeled substrates blue-dye-linker-l-tyr-gly-gly-l-phe-l-leu-h and red-dyelinker-l-tyr-d-ala-gly-l-phe-l-leu-h in chloroform, which only differ by one amino acid. Thus, by selecting beads which are stained by merely one color only those receptors which are selective for one of the two substrates could be isolated. When utilizing a flexible instead of the rigid steroidal backbone a weakening of binding strength by a factor of five and the complete loss of selectivity could be observed. In order to molecularly recognize the pentapeptidic substrates used in this work highly flexible receptors seem to be disadvantageous, probably due to a loss of preorganization. C3 Gly AA 1 AA 2 NHAc C7 Gly AA 3 AA 4 NHAc 15 HN AA 1-4 = Ala, Val, Leu, Phe, Pro, Ser, Thr, Lys, Asp, Glu = 10,000 receptors Figure 7. The peptidosteroidal tweezer receptor library 15 with two differently substituted side chains was simultaneously screened for two substrates labeled with either a blue or a red dye. The image of the screening (right) is reprinted with permission from Boyce, R. Li, G. Nestler, H. P. Suenaga, T.; Still, W. C. J. Am. Chem. Soc. 1994, 116, Copyright 1994 American Chemical Society. 35
19 2. BACKGRUND INFRMATIN 9 Wennemers synthesized five closely related, yet distinct tweezer receptors 19 which were labeled with a red dye as depicted in Figure 9. The two identical arms were linked via a rigid diamino diketopiperazine template. 36 A screening for substrate selectivity in chloroform was carried out with the help of a substrate library comprising 29 3 = 24,389 tripeptides (18). Binding constants of up to 10 3 M -1 (chloroform) and high selectivity were observed: nly one out of 5,000 sequences is bound, e.g. the receptor with the amino acid sequence L-Tyr-L- Asn(Trt)-L-Phe exclusively selects peptides containing D-His followed by two hydrophobic D- amino acids. In order to verify whether a simplified receptor design is still able to achieve effective binding, corresponding host systems with only two amino acids per side chain and one-armed analogues were screened with the same substrate library. However, as a result of the truncation the host system completely lost its binding affinity. 18 AA 1-3 = Gly, L/D: Ala, Val, Leu, Phe, Pro, Ser, Thr, Asp, Glu, Asn, Gln, His, Lys, Arg = 24, Figure 9. Schematic design of tweezer receptors 19 containing two identical tripeptidic arms labeled with a red dye (left) and alternative templates (right). Under the conditions used for the experiments 19 seems to be the minimum structure which is necessary for the recognition of the tripeptides under investigation. In order to evaluate the role of the template on the binding properties similar receptors with the same side chains but different diamine templates were synthesized as well. 37 A screening against the same substrate library revealed that with only a few exceptions these analogues do not bind to the substrates at all. The U-shaped geometry of the original trans-trans-diketopiperazine template in 19 highly preorganizes the two arms in a fashion which is suitable for tripeptide binding. n the other hand side-chains which are connected via the cis-cis-diastereomer 24 adopt an almost linear geometry. Hence, a defined angle between template and side chains as well as a distance of approximately 8 Å in between (as is the case for the original diketopiperazine scaffold) seem to be superior to all other templates that were tested in this work. Accordingly, the template plays a decisive role concerning the affinity between receptor and substrate and has to be carefully chosen. However, with the help of these host systems only rather moderate affinities could be achieved. Although the selectivity is quite good, it has to be noted that the binding studies were only carried out in organic solvent and would probably suffer in more demanding aqueous surroundings. Further studies concerning the role of the template were conducted by Liskamp. 38 As shown in Figure 10 tweezer receptors 25 consisting of two identical peptidosulfonamide side chains which were bridged by several templates 26-31, which differ in both their flexibility and the distance between the amine groups. A dye-label enables screening of a tripeptide library containing 29 3 = 24,389 members (18). The overall highest binding affinity in chloroform was
20 MLECULAR RECGNITIN F BILGICALLY RELEVANT TARGETS found for the scaffold 26 with a binding constant for the peptide D-Ala-L-Asp-D-Ser of M -1. Since no binding affinity could be observed for a one-armed analogue it was concluded that the second arm is necessary for efficient tripeptide binding. The second best receptors with binding constants of up to M -1 were those with template 27, which is more rigid and offers less space between the two arms. Third ranked are the two scaffolds 28 and 29. The distance between side chains seems to become too large for stable complexation of the peptidic substrates. When implementing 30 as linker, the corresponding receptor showed high selectivity (> 95 %) for the amino acid sequence Glu-His-X. However, the binding affinity was rather poor this was again probably due to the large distance between the two arms. No affinity at all could be observed for 31. The cause for this effect can probably be found in the repulsion between the free electron pairs of the sulfonamide oxygen groups, which would be in too close proximity in the depicted chair-chair-conformation of the template. Instead, it adopts a boat-chair-conformation which results in the loss of tweezer structure. A common feature of all examined host systems in this work is the high guest selectivity: the amino acid AA 1 almost always is an acid; AA 2 is a polar amino acid like asparagine or histidine. At position AA 3, which is bound directly to the resin, no tendency could be observed. 18 AA 1-3 = Gly, L/D: Ala, Val, Leu, Phe, Pro, Ser, Thr, Asp, Glu, Asn, Gln, His, Lys, Arg = 24, Figure 10. Schematic representation of tweezer receptors 25 and templates The binding studies of library 25 were again conducted in organic solvents. For further studies in aqueous solution two more combinatorial tweezer libraries (32, Figure 11) consisting of 1,728 members each were synthesized based on the best scaffold As before the two arms were identical but instead of sulfonamides they now consisted of tripeptides to facilitate the combinatorial approach. ne of the two libraries contained the template without change, for the preparation of the other one the template was derivatized with a hydrocarbon chain as illustrated in Figure 11 to reduce its flexibility. Both libraries were then tested for their binding affinity towards dansyl-labeled biologically relevant substrates Gly-D-Ala- D-Ala-H (33) and Gly-D-Ala-D-Lac-H (34) in aqueous phosphate buffer. Both substrates were linked to the fluorescent dansyl group via a glycine spacer. Characterization of hit structures revealed a highly consistent distribution of amino acids at position 3. For the dipeptide 33 lysine was reported with 40 % and proline and alanine each with 20 %. The results from the screening for the corresponding depsipeptide 34 show that position 3 was mainly occupied by proline (50 %), followed by lysine (25 %) and alanine (20 %). The conformity was less well pronounced for positions 1 and 2. However, in both cases mainly aromatic or aliphatic amino acids were present, which might be due to an interaction of the non-polar amino acid side chains with the dansyl label. The glycine linker is probably too
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text:
Hey, evil empire, regime changing provocateurs of treason....
Posted: Wed Sep 13, 2017 8:49 am
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Kaing Monika, deputy secretary-general of the Garment Manufacturers Association of in Cambodia (GMAC), said the industry body was working feverishly with the government to draft and submit a petition ahead of the October 17 deadline.
"[The petition] is still a work in progress, but together with the Ministry of Commerce we will have it submitted in October," he said. "If the US passes the law it would open up a whole new market."
According to Monika, footwear accounts for just $700 million of Cambodia's $6.5 billion garment and footwear industry, with the vast majority of locally produced shoes, boots and slippers shipped to the European and Japanese markets. Purchases by US buyers amount to less than 10 percent of Cambodia's footwear exports.
While this suggests enormous potential to increase exports to the US market, which imported $123 billion worth of footwear last year, local producers would need to adjust production to benefit from the bill's proposed duty-free exemptions.

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For the Berlin Art Week, residents open their studio and present their current work.
Of high voltage noise, alien worlds and the love life of octopuses. Electroacoustics in films (1929-1969)
Aufladetechnische Konferenz - music - video - dance - film - photo - lecture - bbq - performance - surprise - hangout
Art and Spatial Resistance in East Asia & Mullae Artist Village: Dancing on the gentrification


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Peer reviewed
ERIC Number: EJ403988
Record Type: CIJE
Publication Date: 1989
Pages: N/A
Abstractor: N/A
Reference Count: 0
Neuropsychological Profiles of Children with Learning Disabilities and Children with Documented Brain Damage.
Arffa, Sharon; And Others
Journal of Learning Disabilities, v22 n10 p635-40 Dec 1989
This study sought to identify clusters or subtypes of children with learning disabilities (LD, n=24) and brain damage (BD, n=36) using a neuropsychological battery. Five clusters were determined, but no one cluster was composed exclusively of LD or BD subjects. Significant qualitative and quantitative differences between clusters were found. (Author/PB)
Publication Type: Journal Articles; Reports - Research
Education Level: N/A
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A


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Gravestone Markers
Gravestone Markers
425 NW K St.
Richmond, Indiana 47374
Other Consumer Products & Services » Funeral Service And Crematories
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The Place Of Quality Headstone Markers. You Can Shop Or Design Online At Grassmarkers.com. Providing Headstone Monuments Or Cemetery Products For Funerals.
Gravestone Markers in Richmond is a company that specializes in Funeral Service And Crematories. Our records show it was established in Indiana.
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