Library first-generation

A given ligand system was first prepared on the solid support, and a small collection of catalysts (library 1) generated by adding 11 different metals and in one case no metal (Scheme 23).119 It turned out that the best ee-value (19%) was in fact obtained in the metal-free system. Based on this... 

Early work in the field has established the synthetic strategies and analytical tools for such class of libraries (for reviews see refs[111 112 456]). As listed in Table 13, the first generation of cyclic peptide libraries focused on biologically active sequences such as the cell adhesion RGD motif, the antileukemic heptapeptide stylostatin, or endothelin antagonists, but also on metal-binding sequence motifs and on the de novo discovery of bioactive cyclic peptides without sequence-biased motifs. Moreover, synthetic questions were addressed such as the sequence dependency of peptide cyclization reactions (see Table 13). 

Every time the library must be produced for selection, it should be prepared from the first-generation library by infection. 

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

Of the nine thermostabilized S41 variants identified in the first generation, three had substitutions at residue 211, and one contained a substitution at 212. Saturation mutagenesis was performed at both sites in order to explore a greater sequence diversity than that accessible through error-prone PCR (Miyazaki and Arnold, 1999). One hundred and five of the clones from the library that showed reasonable activity were assayed... 

Fig. 14. Distribution of activity and stability in a first generation random mutant library of S41. Ellipse shows one standard deviation of the values obtained for wild-type clones in this assay.

Three mutants with improved activity at 10°C were identified in the first generation library. Recombination of these resulted in variant (P3C9) whose k,M at 10°C is 6.6 times that of wild-type SSII and 4.5 times that of the naturally cold-adapted S41 (Fig. 17). The mutant SSII Km is 9.6 times that of wild type and 6.6 times S41 s (Table II). The activities of naturally psychrophilic enzymes are generally less temperature dependent than those of their mesophilic counterparts. The temperature dependence of enzyme activity is often expressed in terms of the increase in kcM brought about by raising the reaction temperature by 10°C ( (Tjo ) The (Tjo of P3C9 is —1.2, more than two times lower than wild-type SSII and comparable to the value of—1.3 determined for S41. 

Upon generating a library of only 1000 variants in the first generation, about twelve improved variants were identified, the best one resulting in an ee value of 31 % ( = 2.1) in the test reaction. The process was repeated in the second, third and fourth round of mutagenesis as outlined in Fig. 11.2 with formation of slightly larger libraries (2000 -... 

Fig. 17.6 Solution and solid phase synthesis from the racemic first-generation indoline scaffold to obtain a 100-membered library using an intramolecular Mitsunobu approach.

Two other isomeric libraries of self-assembling AB2 monodendrons based on 3,4- and 3,5-disubstituted benzyl ether internal repeat units containing four first generation monodendrons, i.e., 3,4,5-tris( -dodecan-l-... 

The MS/MS analysis of the molecular ion will also yield a structurally informative set of fragment ions. Again, if exact mass data can be obtained for these, a further set of elemental formulas may be obtained. Unfortunately, because there are only limited collections of library spectra generated by MS/MS (see Section 9.10.3.2), the experimenter will generally have to resort directly to a first-principles interpretation. 

---