Substrate Screening

As Lonza had complete freedom to modify its own process for (d)-(+)-biotin, and selective introduction of substituents at N-l (through choice of starting material) and N-3 (through functionalization of 2) was simple, the effect of substituents on the hydrogenation was briefly studied. The results for a selection of the N-l- and N-3-substituted analogues investigated in Fig. 6 are summarized in Tab. 2 and Tab. 3, respectively. 

The very high enantioselectivity of the hydrogenation of the 1-benzyl intermediate, even with the conventional MOD-DIOP ligand was probably the most impressive result. Development of the process with the N-l benzyl substituent would have made the later intermediates of the biotin process identical to those in the existent commercial process, which could have expanded the market opportunities 

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The printability of the LEP ink is determined by a number of factors, including the viscosity of the ink, the solvent evaporation rate, and the interactions between the ink and the screen, emulsion, and substrate. Screen-printable solutions normally require considerably... 

The influences of the ligand-to-metal ratio, reaction temperature and syngas pressure on the enantioselectivity and regioselectivity were also studied. A multi-substrate screening approach has recently been used by Dow Chemical Company to identify the best catalyst for the hydroformylation of vinyl acetate. Here, the chiral phosphite Kelliphite, 5 (Fig. 1) gave enantioselectivity up 88% ee and excellent regioselectivity for the branched isomer [24,25]. 

A substrate screening was also carried out and reported [67]. For both penta- and tetracoordinate complexes the reaction rate and selectivity increased on going from methyl to t-butyl ketone, thus following the steric hindrance and electrophilicity of the substrate. In the comparison between PPEI and APPEI, the former were markedly more selective in both the neutral (Table 4.7) and cationic complexes. An increase in the iodide Ir ratio also had an improving effect on the asymmetric induction for 77a, reaching a maximum 84% ee at [ "] [Ir] = 3. 

For our initial studies we chose to evaluate the hydrogenation of two unsaturated carbonyl model prochiral substrates with rhodium complexes of chiral ferrocene diphosphine and tetraphosphine ligands using a standard set of conditions. The substrates screened were methyl a-acetamido cinnamate (MAC) and dimethyl iticonate (DIMI). The substrates, catalysts, conditions, and experimental results are shown in Table 1. 

Table 2.2.7.2 Coupled enzyme-substrate screening for olefinic ketones 32 and 33 with relative activities of recLBADH, HLADH, TBADH, and CPCR.

Starting from the findings of the racemic cross-benzoin condensation [66], and assuming that aldehydes not accepted as donor substrates might still be suitable acceptor substrates, and vice versa, a mixed enzyme-substrate screening was performed in order to identify a biocatalytic system for the asymmetric cross-carboligation of aromatic aldehydes. For this purpose the reactions of 2-chloro-(40a), 2-methoxy- (40b) and 2-methylbenzaldehyde (40c), respectively, were studied with different enzymes in combination with benzaldehyde (Scheme 2.2.7.23) [67]. The three ortho-substituted benzaldehyde derivatives 40a-40c were... 

In the event of a first substrate screening, aryl iodides and bromides yielded mainly the reduced compound B with n-C6H13MgBr in presence of catalytic amounts of Fe(acac)3 in THF-NMP at 0°C (Table 5.4, entries 1 and 2). In contrast, the corresponding aryl chloride furnished the desired product in quantitative yield (entry 3) the triflate and even the tosylate, which turned out to be a difficult substrate in nickel- and palladium-catalyzed reactions [24], were converted in high yields (entries 4 and 5). 

Fig. 17. Nucleotidyltransferase substrate screening assay (NUSSA). A Nucleotide sugar-synthesizing nucleotidyltransferase (EC 2.7.7), B PPrdependent phosphofructokinase (EC 2.7.2.90), C Aldolase (EC 4.1.2.13), D Triose-phosphate isomerase (EC 5.3.1.1), E Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) [336]...

Satyanarayana, T. and Kagan, H.B. (2005) The multi-substrate screening of asymmetric catalysts. Adv. Synth. Catal., 347, 737. 

Active hits were found for every type of substrate screened, including those for which other known microbial epoxide hydrolases were ineffective. For example, hydrolysis of m-stilbenc oxide was not successful with several microbial EHs tested previously.4243 By contrast, several of our new enzymes actively hydrolyzed this substrate and exhibited excellent enantioselectivities (>99% ee). It is important to note that these enzymes were found to be capable of selectively hydrolyzing a wide range of mc.vo-cpoxidcs, including cyclic and acyclic alkyl- and aryl-substituted substrates. 

Table 3. Initial Substrate Screening for Asymmetric Oxidation Using the Kagan Method...

MS-based substrate specificity assays that are applied to NRPS domains are mainly aimed to characterize native and alternative biosynthetic substrates. Table 1 presents biosynthetic substrate pools for substrate screening of catalytic NRPS domains and tailoring enzymes. Biosynthetic substrate pools are specified for the two purposes of assaying NRPS substrate specificity as mentioned above (a) substrate identification and (b) determination of substrate tolerance. The biosynthetic substrate pools for each specific domain and for each experimental purpose are explained as follows. 

Most of the latest publications on NRPS substrate specificity are focused on A domain specificity because their substrate screening is straightforward in terms of biosynthetic substrate form (free amino acids/fatty acids/aryl acids) and T domain substrates (one T domain). Four studies focus on substrate specificity of NRPS loading modules of microcystin biosynthesis,97 mycosubtilin biosynthesis,51 daptomycin biosynthesis,108 and leinamycin biosynthesis.108 The A domains of microcystin, mycosubtilin, and daptomycin biosynthesis initiation showed fatty acid specificity. The initial domain from leinamycin biosynthesis has D-amino acid specificity. Another paper presents the elucidation of aryl acid-specific AsbC adenylation enzyme from petrobactin biosynthesis.104... 

Figure 14 In vivo and in vitro substrate screening of loading module of microcystin biosynthesis, (a) Microcystin and Adda, (b) Loading protein McyG of microcystin synthetase, (c) In vitro and in vivo substrate screening assays of McyG AT. (d) Characterized substrates of McyG AT, ,Vo and McyG AT, vitm by ESI-FTMS (observed and calculated mass shifts from holo McyG AT active site).

Figure 17 The loading module of leinamycin biosynthesis, (a) Loading module components (LnmQ and LnmP) of leinamycin synthetase and leinamycin structure, (b) Substrate screening assays of LnmQ. D-Alanine and glycine loading was detected by ESI-MS (observed and calculated mass shifts of holo LnmP).

Figure 29 MS-based in vitro reconstitution of orphan NRPS gene clusters. Substrates and chemical modifications of unknown NRP natural products can be dissected by FT-ICR-MS methods such as substrate screening and PEA. Based on these biosynthetic informations a structure related to the actual natural product can be drawn.

Some common ways of fabricating the MEA for microscale fuel cells include hot pressing the membrane onto preformed electrodes or hot pressing a complete MEA onto a substrate, screen printing a membrane onto a substrate and depositing the electrodes on either side, and using spin deposition to deposit the membrane followed by electrode deposition. 

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