Xxn

Electrophilic selenium anchors as multifunctional linkers. When considering the use of a multifunctional linker, however, one must take into account the nature of the cleavage reagent and the cleavage step. Cleavage consisting of, for example, addition of a Grignard reagent to an ester with a huge excess of the organometallic component requires aqueous work-up and, therefore, potential loss of valuable material. Excess reagents should, therefore, be easy to remove (volatile, low,...

Biosynthesis of bAmino Acids

Peter Spiteller and Franz von Nussbaum 1.6.1 The biosynthetic pathways leading to genesis of -amino acids that are structurally related to proteinogenic a-amino acids can be divided roughly into two groups. Most -amino acids are derived from a-amino acids by a 2,3-shift of the a-amino group to the ft position. In contrast, -alanine and -aminoisobutyrate are generated by degradation reactions. These fundamentally different biosynthetic pathways are discussed in the following sections.

Conclusion A New Function of Myoglobin

Although hemoglobin and myoglobin are probably the most thoroughly studied proteins, it has recently been proposed that these proteins might have additional biological functions these remain a matter of continuing investigation. The original role attributed to myoglobin (Box 10) has recently been challenged. The discovery that a mutant mouse devoid of myoglobin is capable of apparently normal muscle function raised questions about the real significance of this protein 34 . Our research in this...

Ywm

Another possible means of conversion of functionalized arenes into the corresponding hydrocarbons is the reduction of diazonium compounds 49 . By appli cation of this method cinnamic esters were synthesized in a sequence starting from the iodoarene resin 135. Heck coupling with acrylates, using palladium catalysis, affords an immobilized cinnamate. This can be detached after a sequence of transformations, yielding to an allyl amine in a traceless fashion (Scheme 6.1.34).

Info

Groups enables additional anion complexation so that overall, 3 acts as a ditopic receptor in the presence of suitable anions with the ability to bind both components of an ion pair simultaneously. The aggregates formed are stabilized by strong electrostatic interactions between the oppositely charged ions, an effect reflected in the increase in cation complex stability with increasing stability of the anion complexes of 3 (Table 2.2.1) 15 . The corresponding dependence of cation complex...

Mirrorimage DNA Inhibiting Vasopressin in Cell Culture

The first bioactivity of a functional mirror image DNA oligonucleotide directed against the hormone peptide vasopressin was shown by Williams et al. in 1997 22 . An initial in-vitro selection experiment using a library containing 60 random positions that was brought into contact with pre-immobilized D-vasopressin revealed only one binding sequence after 13 cycles. The sequence was truncated to a functional 68-mer. In a second in-vitro selection experiment a new pool containing the original...

P

Enzyme was able to promote nucleotide insertion opposite F efficiently with remarkable selectivity. Steady-state analysis revealed that the formation of the artificial dA-F pair was only a factor of four lower than that of the natural pair 8 . In the next set of experiments Kool et al. turned the base pair around. They found that dFTP was still inserted highly efficiently by the polymerase, and that the selectivity was as high as for the natural substrates 9 . On the basis of these results Kool...

References

Cookson, Nature 1999, 402 (Suppl), B5-B11. 2 W. W. Busse, R. F. Lemanske Jr, N. Engl. J. Med. 2001, 344(5), 350-362 and references cited therein. 3 Nat. Biotechnol. 2000, 18 (Suppl.), IT10-11 (http www.biotech.nature. com). 4 D. H. Smith, D. C. Malone, K. A. Lawson, L. J. Okamoto, C. Battista, W. B. Saunders, Am. J. Respir. Crit. Care. Med. 1997, 156, 787-793. 5 P. J. Gergen, J. Allergy Clin. Immunol. 2001, 107(5), S445-S448. 6 Academic Press Dictionary of Science Technology (http...

O

However, NCA polymerization neither affords perfectly monodisperse materials nor enables precise control of the composition and a-amino acid sequence. This is, of course, in great contrast with natural proteins, and automatically limits the utility of this approach for the generation of biologically-inspired or biomimetic architectures. Nevertheless, because of its simplicity, NCA polymerization has been widely used for preparation of a range of polypeptide homopolymers, random copolymers,...

Single Label Interactions

Label Hydrogen Bond Dna

For achieving a specific detection most assays rely on use of labeled probe molecules. Only one label is necessary when the hybridization strongly affects the Fig. 4.2.2. Binding modes of typical stains used for homogenous detection of double stranded DNA. Binding to the minor groove or intercalation between base pairs can lead to fluorescence enhancement. Fig. 4.2.2. Binding modes of typical stains used for homogenous detection of double stranded DNA. Binding to the minor groove or...

Screening for Polymerases with Altered Substrate Tolerance

Over the past 15 years a group of chemists worked to develop a novel strategy for DNA sequencing that is based on fluorescence detection at the single molecule level 43-47 . The proposed ''single molecule sequencing'' requires the complete and faithful synthesis of DNA copies exclusively from labeled analogs of the four types of nucleobase, A, G, C, and T, and thus unusual polymerase activity. Most natural DNA polymerases are known to discriminate against bulky fluorescent nucleotide analogs...

Alternative Scenarios for Assaying Polymerase Activity

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays 56, 57 or fluorescence-based assays 58-61 . Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer 62 . Their...

Mdw

To design a traceless linker one must start from a heteroatom-carbon bond which is labile towards protogenolytic, hydrogenolytic, or hydridolytic cleavage. Because most heteroatom-carbon single bonds are less stable than carbon-carbon bonds, traceless linkers can be synthesized on the basis of nearly all heteroatoms. The enthalpies of C-X bonds are, however, only relevant for homolytic bond scission. Many linkers are cleaved heterolytically, and kinetic stability toward hetero-lytic bond...

Bgdg

Benzyl)acetamide, potassium tert-butoxide, dmf, 88 (c) l< 2C03, methanol, 85 (d) N-(+)-biotinyl-6-aminocaproic acid N-succinimidyl ester, triethylamine, DMF, 69 (e) acid N-succinimidyl ester, DMF, triethylamine, 87 (f) 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (mixture of isomers), triethylamine, DMF, 8 (BGFL), 2 (BGAF). Fig. 4.4.2. Labeling of W160hAGT fusion proteins in vitro and in vivo. (A) Analysis of the in vitro reaction ofW160hAGT (0.4 im) with BGBT (15 im). Aliquots were...

Lowvalent Glycoclusters for Directed Multivalency

Low-valent glycoclusters have been important for defining the structural features required for high-affinity binding to multivalent receptors. As discussed above, rigid miniclusters are particularly affine ligands - if the carbohydrates are properly oriented, enabling unstrained multidentate binding. Rigid miniclusters are, in principle, moreover, able to differentiate between various multivalent lectins with the same carbohydrate specificity but varying orientation of their binding sites. If,...

Proteindependent Charge Transfer in DNA

From spectroscopic and biochemical studies it has become clear that DNAmediated CT is extremely sensitive to the re-stacking of the intervening DNA bases and to disruption and perturbation of the DNA structure or conformation. This indicates that sensing of DNA damage could be accomplished, at least in part, on the basis of CT chemistry. In considering these possibilities, it is important to discover whether DNA-mediated CT does occur within the cell. Charge transfer in HeLa cell nuclei has...

Homo

Differences between oxidative HT and reductive ET. In both a photoexcited fluorophore is used to initiate the CT process. In oxidative HTan electron is removed from the HOMO of the DNA (A) into the HOMO of D. In reductive ETthe photoexcited electron is shifted from the LUMO of D into the LUMO of the DNA (A). In the former DNA is oxidized whereas in the latter case DNA is reduced. intramolecular ET yielding the corresponding pyrimidine radical anion and the pyrenyl radical cation...

M0c1o

Ty r j le-G I u - Ala- Leu - Ly s- Al a- GI lil C- y- k iif M 17,45, m - Jfi n 1 Jri f) 1,2,3 the self-organization of the block copolymers. The self-assembly of PEO-b-peptide block copolymers inspired by coiled-coil protein-folding motifs can be described as an equilibrium between unimeric block copolymer molecules with partially ordered peptide blocks and intermolecular coiled-coil dimers and tetramers (Figure 6.6.4). The folding and unfolding of the peptide blocks is sensitive to temperature...

Biochemical and Structural Studies

Valuable insights into how DNA polymerases process their substrates were obtained as a result of detailed kinetic studies of the enzymes. Benkovic and co-workers employed rapid quenching techniques to study the kinetics of transient intermediates in the reaction pathway of DNA polymerases 5 . Intensive studies revealed that E. coli DNA polymerase I follows an ordered sequential reaction pathway when promoting DNA synthesis. Important aspects of these results for DNA polymerase fidelity are...

Pna

Split-pool synthesis of PNA-encoded combinatorial libraries. R element of diversity present in library, B base of the peptidonucleic acid, x number of bases encoding a single element of diversity, n number of chemical diversity-introducing steps, PG protecting group. hybridization to an oligonucleotide microarray Scheme 6.2.2. Split-pool synthesis of PNA-encoded combinatorial libraries. R element of diversity present in library, B base of the peptidonucleic acid, x number of bases...

Chemistry

Because of their high selectivity against other serine proteases, for example thrombin, trypsin, or factor Xa, the development of bifunctional tryptase inhibitors has attracted much attention during recent years. Several classes of effective dibasic tryptase inhibitor have been reported, recently 13, 14, 23 . Herein, we describe syn- Fig. 3.2.1. Crystal structure of tetrameric j-tryptase, consisting of monomers A, B, C, and D, with a symbolized bifunctional inhibitor interacting with two active...

1

Mutation analysis of the tripartite ribozyme version (absolutely conserved nucleotides are in black and underlined). The activity profile is shown on a logarithmic scale. between RNA and substrate was omitted 10 . By employing UV-visible spectroscopy the progress of the reaction could be observed by following the decrease of the anthracene absorption at 365 nm. The catalytic action of this ribozyme on different anthracene and maleimide derivatives was explored and turned out to be...

Oh

Chorismate and isochorismate as branch points toward the synthesis of many different primary and secondary metabolites. is formed from chorismate in one catalytic step, are ideal starting materials for different functionalized aromatic cell products 3 . Thus, biosynthetically both chorismate and isochorismate are characterized by their diversity-oriented bias. Because of their high functionalization these molecules are also very interesting substances chemically and biochemically,...

B25

Solid-phase peptide synthesis (SPPS), first introduced by R. B. Merrifield 1 , involves a repeated sequence of a-amino acid coupling and deprotection reactions performed on an insoluble solid support 2 . The support enables the use of excess a-amino acids and reagents, which helps to drive each of the reactions to completion. By-products and excess reagents are easily removed by filtration. After attachment of the last a-amino acid the linker connecting the support and the peptide, and...

Xx

Synthesis of a benzodiazepine library using a silyl linker. aryl halide. Because the silyl arene can be cleaved in the unwanted direction to give silylated arenes, further improvement led to the development of a germanium linker 40 this gives rise to the formation of pure material. In addition, cleavage of these linkers can be accomplished by electrophiles, for example iodine, bromine, and chlorine, to yield halogenated residues thus converting this linker into a multiple cleavage...

Ho

The enzyme has a monomer weight of 30 kDa and a Km and Vmax for L-pan-tolactone of 7 mM and 30 U mg-1, respectively. X-ray fluorescence spectroscopy of crystals, and renaturation of urea EDTA-denatured Lph in the presence of Zn2+, Mn2+, Co2+, or Ni2+ indicated Lph to be a Zn2+-hydrolase. Kinetic resolution of rac-pantolactone proceeds similarly to the fungal process mentioned above except that L-pantolactone is hydrolyzed and D-pantolactone is left behind. Repeated batches with isolated Lph and...

R

Peptide synthesis, Acid anhydrides, Oxidation Fig. B.14.2. The first polymer reagents, either in the form of ion-exchange resins or covalently attached, were employed for a variety of simple transformations in the 1960s and 70s. solution of the respective salt is added to the resin in excess, and removed after equilibration by washing with non-ionic solvents. Leaching of the reactive ions is, however, a general problem of this type of support. In principle, the immobilized ions can be exchanged...

UVVisible Spectroscopy

In a complex with DNA the guest molecule is positioned in an environment which is different from that of the uncomplexed molecule in solution. The guest molecules, especially solvatochromic compounds such as organic dyes 23 , usually have different absorption properties in the complexed and uncomplexed forms. Thus, on addition of DNA to a solution of an intercalator or groove binder, a shift of the absorption maximum to longer wavelengths (bathochromic shift or red shift) and a decrease of the...

[20

Schematic representations of all seven isomers of the complexes 12(OCH3)2Ti2 2 . Catecholate units at the N-termini are indicated as gray bars, those at the C-termini as black bars. Fig. 1.3.6. Schematic representations of all seven isomers of the complexes 12(OCH3)2Ti2 2 . Catecholate units at the N-termini are indicated as gray bars, those at the C-termini as black bars. 1.3.2 Dinuclear Coordination Compounds from Amino Acid-bridged Dicatechol Ligands 37 internal catecholate...

Groove Binding

The DNA helix has two grooves of different size, the minor and major grooves, which can serve as binding sites for guest molecules. Whereas relatively large molecules such as proteins bind preferentially to the major Fig. B.9.1. Molecules such as organic dyes, polycyclic aromatic compounds, organometallic complexes, saccharides, peptides, and polyamides bind to nucleic acids. Two major binding modes are possible - intercalation (left) and groove binding (right). Fig. B.9.1. Molecules such as...

Application of Ribozymes

Because of their ability to cleave a suitable RNA target in trans, ribozymes have great potential as gene inhibitors. Concepts for using ribozymes in gene therapy were developed over the past decade and have been transformed into a number of clinical trials, although success is still limited. In general, ribozyme-based strategies focus on suppression of pathological gene products to treat diseases caused by viral infections or malignancies. The major aim is to block gene expression by...

N

Therefore be of interest to use the geometrical conformation at a metal ion to induce three dimensional structure ( conformation) in amino acids (Nature also uses metal ions in this context - Box 3). Examples have been reported of modified peptides in which metal bonding units are attached to different positions of the peptide and induce different structures. Starting from random coil peptides, examples have been reported in which the metal can induce either a-helical- (Figures 1.3.3A or B) or...

Ypx

Affinity chromatography with immobilized target molecule affinity chromatography with immobilized target molecule lection of active species from nucleic acid libraries by separation from unreactive molecules and enzymatic amplification of the enriched library. Because of these two key steps the method is called SELEX (systematic evolution of ligands by exponential enrichment). Two aims can thus be attempted. The first is the selection of molecules with specific binding properties, called...

The Peroxynitritemediated Oxidation of OxyMb and OxyHb

Despite the presence of a large concentration of carbon dioxide in the blood (ca. 1 mM), it has been reported that peroxynitrite can diffuse across the red-blood-cell membrane and react with oxyHb 24 . The anionic form (ONOO ) crosses the ery-throcyte membrane by using the anion channel band 3 whereas peroxynitrous acid crosses the lipid membranes by rapid passive diffusion 24 . We have recently shown that the peroxynitrite-mediated oxidation of oxyMb and oxyHb proceeds via intermediate...

Oh Oh

Activation of glass slides and the covalent attachment of alcohols. Scheme 6.2.1. Activation of glass slides and the covalent attachment of alcohols. Lam et al. have introduced two new ligation methods for glyoxylic acid-modified glass slides with which they could immobilize biotin or peptide ligands derivatized either via an amino-oxy group by oxime formation or a 1,2-aminothiol group by thiazolidine ring formation 19, 28 . The microrarray of immobilized ligands was analyzed in...

Preparation of Peptide Aldehyde Collections

Peptide aldehydes are important biochemical tools for specific and reversible inhibition of serine, cysteine, and aspartate proteases in-vitro and in-vivo. Whereas several methods for the preparation of peptide aldehydes have been described 26-28 , their reliable parallel synthesis remained a tedious endeavor. Peptide aldehydes suffer from configurational lability in the a-carbonyl position and from high chemical reactivity. A polymer-supported approach employing oxidizing IBX-resin could be a...

GSecretase Inhibitors

Several peptidic aldehydes have been reported to be inhibitors for either y-secretase or S-secretase or both. Common to both series are lipophilic di- and tripeptides with bulky N-terminal protection, e.g. Z-LLL-CHO (MG132), Z-YIL-CHO, and Boc-GVV-CHO. The general lack of specificity of these aldehydes and their simultaneous inhibition of serine and cysteine proteases makes interpretation of data rather cumbersome. Indirect mechanisms through general protease inhibition result in complex...

Mechanistic Aspects of Multivalent Interactions

Multivalent interactions are characterized by the simultaneous binding of several ligands on one biological entity (surface, macromolecule) with several receptors on another entity (Figure 2.7.1) 8 . This type of interaction has unique collective properties that are qualitatively different from those of the corresponding monovalent systems. Not only it is possible to regulate the strength of an interaction by the number of receptor-ligand contacts, multivalent interactions also have different...

Ocxdo

Step 1 (1st cycle) splitting of the beads step 1 (2nd cycle) splitting of the beads Fig. B.11.1. Basic concept of split synthesis. pounds. Because each bead reacts with only one set of reagents in each cycle, each bead carries only a single compound (''one-bead-one-compound'') in other words each compound is located on a different bead 1, 2 . Split-and-mix libraries have found numerous applications in the search for a substrate for a given receptor and vice versa. A typical affinity assay...

I

Secondary structure of the branched ribozyme HP-RJBR and structure of the branching nucleoside. The arrow denotes the cleavage site. The single segments within the ribozyme strand are marked. Fig. 5.2.11. Secondary structures of twin ribozymes. The arrows mark the cleavage sites. The substrates are fluorescein-labeled at both ends. Fig. 5.2.11. Secondary structures of twin ribozymes. The arrows mark the cleavage sites. The substrates are fluorescein-labeled at both ends. TW2....

Hybridization

Homogeneous DNA detection with DNA-intercalator dye conjugates. Fig. 4.2.5. Homogeneous DNA detection with DNA-intercalator dye conjugates. jugates 7 was up to 16 times higher than single-strand fluorescence. In one instance a hybridization-induced fluorescence decrease was observed. Similar fluorescence enhancements were determined on binding to single mismatched DNA as long as measurements were performed at temperatures below the melting temperature of the corresponding PNA-DNA...

Rotating couette

Reference axis hydrodynamic field vector Fig. 2.5.5. Schematic illustration of the orientation of a cylindrical Couette flow cell relative to the linearly polarized light. (a) the generation of an electric field (''electric LD'') which forces the molecules to arrange in this field depending on their intrinsic dipole moment and (b) the implementation of a hydrodynamic field in a rotating couette (''flow LD'', Figure 2.5.5), in which molecules can align along the flow field. The latter method is...

Flap I

BACE complexed to Glu-Val-Asn-C(Leu-Ala)-Ala-Glu-Phe (3, OM99-2) (1FKN). Fig. 3.5.1. BACE complexed to Glu-Val-Asn-C(Leu-Ala)-Ala-Glu-Phe (3, OM99-2) (1FKN). ment, although their normal regulatory function in healthy tissue is not fully understood (Ab or peptide models thereof are also dealt with in Chapters 2.3 and 2.4). Although several reviews on secretase inhibition have been published 10-12 , the rapid progress in the field demands continuous survey. And despite this...

Molecular Beacons

A growing number of fluorescence-based assays makes use of distance-dependent interactions between a fluorophore and a chromophore 1 . For example, once excited by absorption of light a fluorescent group can pass its energy by radiationless fluorescence resonance energy-transfer (FRET) to an acceptor group, assuming donor emission overlaps (at least partially) with acceptor absorption. The acceptor group dissipates the energy as heat, or emits light if the acceptor is fluorescent itself. As a...

LD Spectroscopy

Linear dichroism is defined as the differential absorption of linearly polarized light AI I is the absorbance of the sample when the light is polarized parallel to a reference axis, and A is the absorbance of light which is polarized perpendicular to this axis. The strength of the absorption depends on the orientation of the electric field vector of the light and the transition moment of the chromophore - parallel orientation results in maximum absorption whereas perpendicular orientation leads...

C C U G Gc Gc Au Au Gc Gc Gacc

The identical sequence partition CGGAAGGUCC (gray) of the GGAA- and the UCCG reference (center) allows the design of two competing secondary structures within a single sequence (below). equilibria of short RNA sequences. The NH protons that are involved in Watson-Crick pairing (''imino protons'') are easily detectable for RNA in submillimolar concentrations. In principle, each Watson-Crick base pair is assigned to a single signal. Because of base-pair fraying at the double helix...

RAS The Good The Bad and The Ugly

We chose H-RAS as the first target protein for several reasons. RAS proteins are, perhaps, the most intensively studied and best understood signal transduction proteins from the perspective of structure and function 8 . The H-RAS crystal structure has been determined to the level of 1.5 A, and the domains involved in guanine nucleotide binding and hydrolysis, effector interactions, interactions with regulating proteins, and processing have been extensively characterized. RAS proteins are...

Ssu

A topologically chiral rotaxane. Arrows indicate the directionality imposed by the sulfonamide group in the wheel. 6.5.5). Photochemical E Z isomerization produces a sterically strained state in which the methyl substituents are positioned in close proximity. The strain is released when the methyl groups and the naphthyl moieties switch their positions. This second step is followed by back-isomerization from the Z to the E form of the double bond and again steric hindrance...

Recognition of Proteins with Aminopyrazoles

Protein-folding diseases are characterized by the formation of insoluble protein plaques in the brains' nerve cells. The mechanism of these misfolding events is often unclear. In BSE CJD an infectious protein particle could be identified, leading to the famous prion hypothesis 22 . It is called prion protein scrapie (PrPSc). After refolding of the native protein newly formed -sheet domains aggregate with those in other PrPSc molecules and lead to precipitation of the protein from physiological...

BSecretase Inhibitors

Several inhibitors of b-secretase have been identified in cellular assays but, more often than not, the true nature of the inhibition mechanism was not reported. Broad-spectrum protease inhibitors, for example pepstatin 1, known aspartic protease inhibitors from renin, and HIV protease programs and cocktails thereof, had little inhibitory effect and gave misleading results. But the consequent utilization of the Swedish mutation and structure-activity relationships for an early Bristol-Myers...

RNA Folding

RNA plays a central role in the life of cells. It is, therefore, important to understand how RNA forms functional native structures endowed with properties such as catalysis, binding of small-molecular-weight ligands, or recognition of proteins. Termed the ''RNA folding problem'' 1 , the question is how the primary structure of RNA, a linear polynucleotide, encodes its functional three-dimensional (tertiary) structure. On the basis of early studies on tRNA and consistent in the first order with...

G

Assay for spectroscopic investiga- reactivity. The radical of Lys-Trp-Lys forms tion of how aromatic amino acids modulate oxidized peptide products in the presence of DNA-mediated HT. The peptide radicals gen- O2 whereas the radical Lys-Tyr-Lys forms cross- erated by DNA-mediated HT have different links with the DNA bases at the binding site.

Tms

Synthesis of (Sa)-10 and (all-Sa)-11. (a) t-BuLi, THF, -78 C, then ZnCl2, -78 C to rt, 2 h (b) Pd2dba3-CHCl3, t-Bu3P, THF, 83 (c) KF, MeOH, 97 (d) NaH, DMF, MOMCl, 80 (e) n-BuLi, Et2O, rt, then I2, Et2O, -78 C, 42 monoiodo and 40 diiodo compound Fig. 2.1.7. 'H NMR spectra (500.1 MHz, 11 0 ca. 10 mmol L-1 in THF-d8 CD3CN at 300 K) (a) (all-Sa)-11, (b) (all-Sa)-11 + 1 2 equiv. Cu(CH3CN)4 BF4, (c) (all-Sa)-11 + 1 2 equiv. Ag(CH3CN)2 BF4. Fig. 2.1.8. Positive ESI MS of ca. 5 x 104 mol...

Aspects of Chirality

The essential step during in-vitro selection is amplification of the rare species represented in the combinatorial oligonucleotide library that bind to the target molecule. Amplification of nucleic acids can be accomplished very easily by employing enzymes - DNA polymerases (Box 16) - in a process called PCR. On the other hand, the degradation of aptamers occurs as a result of abundant nucleases which attack specific sites within the naturally configured nucleic acids. Most enzymes are proteins...

D

Electron transfer in DNA hairpins capped with a stilbene diether derivative as the charge donor D. Fig. 4.6.10. Py-dU and Py-dC as nucleoside models for ET in DNA. When Py-dX (X U or C) is excited at 340 nm intramolecular ET occurs. Subsequently, protonation can occur, yielding the biradical Py.+-dX(H)

Polymersupported Acylanion Equivalents [30

Polymer-supported carbanion equivalents are the obvious supplement to polymer-supported access to reactive electrophiles either by oxidizing polymers or by release of carbenium ions. The combination of an oxidizing resin with a support carrying carbanion equivalents will be especially rewarding, enabling reaction sequences with C-C coupling steps and thus opening access to a wealth of potentially relevant products. Bestatin 6a (Figure 3.6.8) is the lead structure of a family of highly potent...

B26

Peptide ligation involves regiospecific coupling of unprotected peptide segments in aqueous solution. The concept of peptide ligation is outlined in Scheme B.26.1. It consists of two steps, a chemoselective capture reaction and an intramolecular acyl rearrangement 1 . Chemoselective ligation requires a suitable combination of electrophilic and nucleophilic groups

Screening Polymerase Libraries for Altered Activity

Obviously, an adaptation of Loeb's scheme to the selection of RNA polymerases (DNA-dependent transcriptases, or RNA-dependent replicases), reverse tran-scriptases, or DNA polymerases with altered substrate tolerance is difficult because these enzymes hardly enable the conferring of a growth advantage to host cells. In an exceptional approach to this problem, Andrew Ellington and colleagues focused Fig. 4.3.1. Schemes for genetic selection of DNA polymerase function based on complementation. (a)...

Omom Omom

Synthesis of (Sa)-10 and (all-Sa)-11. (a) t-BuLi, THF, -78 C, then ZnCl2, -78 C to rt, 2 h (b) Pd2dba3-CHCl3, t-Bu3P, THF, 83 (c) KF, MeOH, 97 (d) NaH, DMF, MOMCl, 80 (e) n-BuLi, Et2O, rt, then I2, Et2O, -78 C, 42 monoiodo and 40 diiodo compound (f) Pd(OAc)2, t-Bu2(2-biph)P, Zn, DMF, rt, 62 (g) KF, MeOH, rt, 79 (h) Et3N, Cul, Pd2dba3-CHCl3, dppf, 50 C, 77 (i) conc. HCl, THF, MeOH, 95 (j) Et3N, Cul, Pd2dba3-CHCl3, dppf, 50 C, 96 (k) conc. HCl, THF, MeOH, 97 .

RNA and DNA Spiegelmers Binding to GnRH

More biological data for mirror-image oligonucleotides were generated by evaluating the properties of GnRH (gonadotropin-releasing hormone) binding spiegelmers 23 . GnRH is a ten-residue peptide released from the hypothalamus which acts as a key hormone of mammalian reproduction. The hormone binds to a single class of high affinity G-protein-coupled receptors on the plasma membranes of pituitary gonadotrophs and regulates the secretion of LH (luteinizing hormone) and FSH (follicle stimulating...

Binding Modes

In general, two binding modes are possible between guest molecules and the host DNA (a) minor or major groove binding, and (b) intercalation (Figure 2.5.1). A third binding mode, external binding, is also known this results exclusively from attractive electrostatic interactions between a positively charged molecule and the negatively charged phosphate backbone of the DNA. In contrast, groove binding Fig. 2.5.1. Binding modes of small molecules with DNA and intercalation may be viewed as the...

Protein Structure Determination

Even with modern supercomputers it is impossible to predict the absolute thermodynamic minimum of an unknown folded protein chain. If the Fig. B.8.2. Double-stranded antiparallel 0-sheet with intermodular hydrogen bonds (dotted lines) 4 . structures of related proteins with similar sequences are known, homology modeling will produce very good estimates. In recent years conventional structure determination by X-ray crystallography has been complemented by the new NMR NOESY technique used for...

Serine Proteases

Clay Filter Kerosene Treatment Unit

The serine proteases are a class of proteolytic enzyme (they catalyze the hydrolysis of either ester or peptide bonds in proteins) that require an active site residue for covalent catalysis. The active site residue, the catalytic Ser-195, is particularly activated by hydrogen-bonding interactions with His-57 and Asp-102. Crystal structures show that Ser-195, His-57, and Asp-102 are close in space. Together these three residues, which are located in the substrate binding (S1) pocket, form the...

Exploring the Capabilities of Nucleic Acid Polymerases by Use of Directed Evolution 329

Susanne Brakmann and Marina Schlicke 4.3.2 Directed Evolution of Nucleic Acid Polymerases 330 4.3.3 Practical Approaches to the Directed Evolution of Polymerase Function Selection or Screening 331 4.3.3.1 Selection of Polymerases with Altered Activity and Fidelity 331 4.3.3.2 Screening Polymerase Libraries for Altered Activity 331 4.3.4 Genetic Selection of an Error-prone Variant of Bacteriophage T7 RNA Polymerase 333 4.3.5 Screening for Polymerases with Altered Substrate Tolerance 335 4.3.6...

Oxidations with Immobilized Periodinanes

Ibx Reagent

In recent years hypervalent iodine compounds have experienced extensive investigations yielding many results of practical synthetic importance. Supported iodi-nanes, i.e. iodoso or iodine(III) reagents, have been prepared by several groups, mainly as the bis-acetoxyiodoso derivatives 15-18 or as the respective dihalogeno compounds 19 . Iodoso reagents are employed in the oxidation of hydroquinones and phenols that have been exploited in the formation of spiroketals from a variety of tyrosines....

Introduction

''Alzheimer's disease (AD) is a devastating illness that robs humans of their ability to remember, to think and to understand all the things we cherish most about being human.'' (P.F. Chapman) Alzheimer's disease (AD) is an epidemic neurodegenerative disorder claiming millions of victims per year. The aging of the world population will be accompanied by an even higher toll. According to the World Health Organization the prevalence of Alzheimer's disease is 5.5 above 60 years of age and...

Analogs with Modified Sugar Moieties

Most functional studies have focused on nucleobase-recognition processes. Little is known about the impact of DNA polymerase interactions with the 2 '-deoxyribose moiety and their participation in processes which contribute to fidelity. Crystal structures of DNA polymerases together with enzyme mutation studies suggest that the sugar moiety of the incoming triphosphate is fully embedded in the nu-cleotide binding pocket and undergoes essential interactions with the enzyme 3, 6 . This issue has...

Genetic Selection of an Errorprone Variant of Bacteriophage T7 RNA Polymerase

Bacteriophage

Studies with several RNA viruses have demonstrated their exceptionally high replication error rates that confer genetic flexibility, while resulting in substantial decreases of infectivity 35, 36 . Enhanced mutagenesis might even result in virus extinction, as predicted from virus entry into an ''error catastrophe'' 37, 38 . We are interested in studying and utilizing the link between error-prone polymerases and virus viability and started our search for an error-prone variant with the RNA...

Hemoglobin and Myoglobin

Hemoglobin, the constituent of red blood cells responsible for transport of dioxygen, and myoglobin, present in skeletal muscles to store dioxygen, are probably the most thoroughly studied proteins. Hemoglobins were first found in blood because of their very high concentration 15 g per 100 mL in normal human blood . Until recently it seemed that the oxygen carrier function was so specialized that it appeared to be the only role of these Scheme B.10.1. The active site of hemoglobin and...

Spiegelmers Binding to Small Molecules

Spiegelmers

The first functional mirror-image oligonucleotides, spiegelmers, described in 1996 were generated to bind to small molecules, i.e. the amino acid arginine and the nucleoside adenosine 18, 19 . For in-vitro selection against adenosine, the target in the enantiomeric configuration was covalently coupled to a matrix. A standard characterized and tested for binding to the mirror-image form of the naturally occurring target. The sequences of identified binders are then synthesized in the...

Bi2

Taxus brevifolia T. ba. Taxus baccata C. v. Cortinarius violaceus B. b. Bacillus brevis Vm4 a Involvement of B12 is unlikely bAn 15 N label in tyrosine is lost in the product -tyrosine laceus. These enzymes are unlikely to use B12. These enzymes have, on the other hand, not yet been isolated nor investigated in detail, so their classification remains speculative. The tyrosine 2,3-aminomutase of Bacillus brevis Vm4 differs fundamentally from all the...

The NOmediated Reduction of FerrylMb and FerrylHb

It has been suggested that heme-mediated redox reactions contribute to the organ dysfunction and or tissue damage which occur in some pathological states characterized by release of Hb and Mb into the extracellular environment 29 . Autoxidation of oxyHb and oxyMb to their met-forms releases superoxide which dis-mutates to dioxygen and hydrogen peroxide, which in turn can cause tissue damage 30 . In addition, oxidation of these two proteins generates potentially cy-totoxic products such as the...