Reporter ligand

EPR investigations are necessarily carried out in frozen solution at low temperature. Room temperature binding of thiols to FeMoco has been monitored by F NMR spectroscopy using /J-CF3C6H4S as the reporter ligand. These experiments revealed that the binding of thio-late is characterized by a dynamic equilibrium between the FeMoco and thiolate (159) and that cyanide and methyl isocyanide can bind to isolated FeMoco complexed with thiol (160). 

Scheme 7 Recently reported ligands for iron-catalyzed asymmetric epoxidations...

Fig. 5.10 Principle of competitive ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). The extent of complex forming is monitored by the addition of a bioactive reporter ligand (R) resulting in the formation of protein-reporter complex (PR). The concentration free R is directly dependent on the concentration and affinity of L R is monitored by ESI-MS at its corresponding m/z trace.

Figure 5.11 illustrates the basic performance of the on-line MS assay. For comparison, a homogenous fluorescence assay has been set up in parallel. For this purpose, the carrier flow was split after the second microcoU reactor, with 90% of the total flow being directed to a fluorescence detector (Fig. 5.11a) and 10% to the MS (Fig. 5.11b). The affinity interaction between streptavidin and biotin was chosen to study the characteristics of an on-line MS biochemical assay. Fluorescein-biotin was used as reporter ligand for both fluorescence and MS in the SIM mode (m/z 390) detection. In the fluorescence mode, the homogeneous biochemical assay is based on the quenching of the fluorescein-biotin fluorescence upon binding to streptavidin.

Fig. s.n On-line continuous-flow monitoring of biochemical interaction with (a) fluorescence and (b) MS SIM (m/z 390) detection. Fluorescein-biotin (96 nM), streptavidin (32 nM), 20-pL loop injections of 1000 nM biotin (n = 3). MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Point 1 Carrier pump, protein and reporter ligand pumps... 

Because the interaction between biotin and streptavidin is strong (fCa = 0.6 X 10 L mol ) with a relatively fast association rate (k+i = 2.4 x 10 L mol s ) and slow dissociation rate (k i = 0.4 x 10 s ), the reaction times are fast, i.e. 10-20 s. Furthermore, the addition of reporter ligand is performed only after the analyte protein reaction has taken place in coil I, avoiding a displacement reaction that would substantially increase the overall reaction time. 

The biochemical MS assay performance was studied for various biotin derivatives, such as biotin [m/z 245), N-biotinyl-6-aminocaproic acid hydrazide (m/z 372), biotin-hydrazide (m/z 259), N-biotinyl-L-lysine (m/z 373) and biotin-N-succinimi-dylester m/z 342). These five different bioactive compounds were consecutively injected into the biochemical MS assay. Figure 5.12 shows triplicate injections in the biochemical MS-based system of the different active compounds. Each compound binds to streptavidin, hence the MS responses of peaks of the reporter ligand (fluorescein-biotin, m/z 390) are similar. The use of SIM allows specific components to be selected and monitored, e.g. protonated molecule of the biotin derivatives. In this case, no peaks were observed for biotin-N-succinimidylester (m/z 342), because under the applied conditions fragmentation occurred to m/z 245. In combination with full-scan MS measurements, the molecular mass of active compounds can be determined simultaneously to the biochemical measurement. 

The assay principle shown in Fig. 5.10 has the potential of multiplexing, i.e. performing several assays in parallel, by pumping mixtures of receptors, i.e. streptavidin and anti-digoxigenin and reporter ligands, i.e. fluorescein-biotin and digoxin [29]. Clearly this approach will only be feasible for those assays... 

The first example of a metal complex of the dithiaporphyrin (400) has been reported. Ligand (400) reacts with [Ru(cod)Cl2] to give tra 5-[RuCl2(400)] which has been characterized in solution using NMR spectroscopy and electrochemical methods, and in the solid state by X-ray diffraction. The latter reveals that the two thiophene rings in coordinated ligand (400) are tilted out of the mean porphyrin plane. 

Early NOE studies on cationic Pd(ii) allyl complexes used reporter ligands [97, 98], i. e. simple bidentate nitrogen ligands, such as bipy or phenanthroline, whose... 

The bidentate oxazoline ligands 85 and 86 (and derivatives thereof) are excellent reporter ligands, and several studies have used NOEs to determine the nature of their chiral pockets [61, 113, 114, 126]. NOESY studies on the cations [Ir(l,5-COD)(86)]+ and several cationic tri-nudear Ir(iii)(hydrido) compounds [110], e. g. [Ir3(p3-H)(H)5(86)3] +, 87, in connection with their hydrogenation activity, allowed their 3-D solution structures to be determined. In addition to the ortho P-phenyl protons, the protons of the oxazoline alkyl group are helpful in assigning the 3-D structure of both the catalyst precursors and the inactive tri-nudear dusters. Specifically, for one of these tri-nudear Ir(iii) complexes, 87 [110], with terminal hydride ligands at d -17.84 and d -21.32 (and a triply bridging hydride at 5 -7.07), the P-phenyl and oxazoline reporters define their relative positions, as shown in Scheme 1.5. 

Binding Experiments. Some of these chromophoric glycosides proved also to be valuable in cases where no hydrolysis by the cellulases occurred. This was shown in the case where the 4-methylumbelliferyl glucoside and cellobioside were not hydrolyzed by the CBH II from Trichoderma (see above) but could be used as reporter ligands in a series of binding experiments. Typically the fluorescence of the cellobioside was quenched in the presence of CBH II and was restored by the addition of excess amounts of non-chromophoric ligands, i.e., cellobiose (Fig. 3). Thus association constants... 

Sorba, G. Tertuik, W. Ganellin, C. R. Synthesis and authentication of iodoazidophenpyr-amine, a photoaffmity reporter ligand previously used for histamine Hj-receptor labelling. Arch. Pharm. (Weinheim) 1995, 328, 677-680. 

In this work we have paid attention to the most frequently reported ligand systems from within the enormous number of ligands used in modern coordination chemistry, as reported in the many pieces of literature dedicated to this topic. In the material that follows we have separated inorganic and organic compounds. In general, the data summarized in monographs [1,9] and our previous reviews, [10-17a] forms the basis for the presentation of this work. It must be said that not only neutral molecules are examined throughout, but also anions. Considerable attention is paid to the reviewed literature of the last decade. Due to the synthetic direction of this book, classic theories on the structure of coordination compounds [17b] are not examined here. 

From 1995 to the present, several advances in asymmetric hydroformylations have been reported. Ligands that have been used with reasonable success with benchmark substrates, such as styrene and vinyl acetate (Schemes 12.70 and 12.71), are shown in Figure 12.8. 

In 1995 Schwarz et al.159 described novel heterocyclic structures able to adsorb selectively immunoglobulins. The structures contained sulfur and nitrogen. Reported ligands were 2-mercapto-pyridine,2-mercapto-pyrimidine, and mercapto-thiazoline. These structures were chemically immobilized on silica and agarose beads using epoxy-activated matrices. Binding capacities for silica based material were about 25 mg of IgG per milliliter of resin, while for agarose beads it was about 18 mg/mL. 

An alternative approach is competition-based screening. " A known medium-affinity binder (reporter ligand) is added to the solution of the target. If other ligands... 

Figure 7 Depiction of competition-based screening. Low- to medium-affinity binders are displaced from their binding site by higher affinity binders. In this experiment, only the signals of these low- to medium-affinity binders (reporter ligands) are monitored...

---