Parent library

Each of the scaffolds reported in Scheme 24 can be used for the production of a stereo-isomeric sublibrary based on the appropriate peptide sequence. For example, with the sequence A-B-C-D-E and scaffold (1) two types of stereoisomeric sublibraries can be prepared. One type includes the sublibraries A and B of Scheme 26 in which within a given sequence the configuration of each residue is successively inverted thus, retaining the identical connectivity as in the parent linear peptide. In the second type 265 of sublibraries C and D (Scheme 26) the direction of the amide bond is inverted and hence the connectivity is not maintained. In most members of these sublibraries the overall conformation of the scaffold is maintained and therefore these components constitute stereoisomeric sublibraries of the parent library. Conversely, by introducing amide bond surrogates such as reduced amide bonds1465 or thioamide bonds 260,466 the conformation of the scaffolds are changed and their conformational flexibility enhanced. 

All those design alternatives require a virtual library as a fundamental basis, which refers to an electronic description of all possible compounds, which might be synthesized by a short sequence of specific transformations with particular reagents and one or multiple scaffolds. Appropriate fast and relevant searching techniques are available today, which can be used for selection and design of actual synthesis subsets in accordance with the level of biological knowledge [27,28] from such a parent library. 

Each parent library is selected for a distinct feature, then the aptamers selected are fused... 

Figure 3.1 Overview of DNA library creation strategies. Random mutagenesis introduces mutations at positions throughout the gene sequence. Semi-rational design randomizes only the specific position(s) of interest. Gene shuffling brings existing sequence diversity from different parental DNA sequences together to form a chimeric library...

A modified version of this protocol, called THIO-ITCHY, includes the random incorporation of a-phosphothioate nucleotide analogs into the parent genes [27,28]. Exonuclease III activity is inhibited at sites of analog incorporation, which relieves the efforts of producing incremental truncation aliquots. In combination with epPCR, the diversity of the fusion libraries created can be further expanded. 

Correlation with the EGA-mass spectra allows for the assignment to the initial loss of diethyl sulfide, as supported by the library fit and from the assignment of the fragment and parent ions (m/z = 90) (see Fig. 6.8a). After approximately 15 min, mass-spec EGA shows the absence of peaks assignable to Et2S... 

To be able to use MS/MS spectra library searching for general unknown screening, it is necessary to use an automatic process, called data dependent acquisition or information-dependent acquisition, to select the parent ions of interest, totally unexpected by definition, and to dissociate them and monitor their fragments. 

From the FIA—MS overview spectrum, speculation that there can be more than just one structurally defined molecule type behind an observable signal i.e. the presence of isobaric compounds, cannot be excluded whenever one signal defined by the m/z-ratio is examined in FIA-MS spectra. Consequently, the information obtained by FIA-MS is quite limited whenever we deal with complex mixtures of environmental pollutants rather than the analysis of pure products or formulations with a known range of ingredients. LC separation is inevitable when mixtures of isomeric compounds should be identified with MS-MS. Therefore, in FIA-MS-MS special attention has to be paid to avoid the generation of mixed product ion spectra from isomeric parent compounds. This would block identification by library search and may lead to misinterpretations of product ion spectra because of the fragmentation behaviour observed. 

FIA-MS-MS in parent or neutral loss mode on triple quad instruments can also be applied to screen mixtures of unknown compounds quite rapidly, so that compound classes can be recognised. Yet despite the information about molecular weight and the structural information by product ions, MS-data systems in their commercial form up to the mid-1990s provided no structural information for identification purposes in the form of libraries comparable with the NIST-library of El spectra in GC-MS analysis. It can be hoped that troubles arising out of the lack of computer-searchable library data for identification will be overcome with the gradual increase in trap applications in MS-MS mode. The situation in identification is set to change [36]. 

Figure 4, Selected daughter spectra of parent mass 105 from reference library.

The fact that, in both selection experiments, new solutions regarding the structure of the functional molecules have been adopted demonstrates that the best sequence for binding is not necessarily the best sequence for performing catalysis. It seems likely that many of the sequence solutions could also have been selected from completely randomized pools. This notion is confirmed by the aforementioned study by Hager and Szostak [82], in which the mutated ATP-aptamer motif was also included in the starting library but where the resulting ribozyme had no relationship to the parent ATP-binding motif. 

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