Multistep purification procedures

The following example illustrates how the impact of these kinetic processes can escalate rapidly in multistep purification procedures. Let us assume that the purification of a particular protein to a level of purity of 99.95% required a 10-step procedure. If the average yield of bioactivity-mass per step in this 10-step method was 60% (a relatively favorable situation), predominately due to conformation changes leading to denatured-unfolded product then the overall yield of the desired native protein would be only 0.6%... 

FIGURE 30 Plot of the product yield versus the number of steps in a multistep purification procedure. These plots correspond to the situations that apply when the repetitive yield is 95, 90, 80, 60 and 40%, respectively. 

However, wider prachcal applicahons of aldolases require cheap and ready access to DHAP. Expensive or toxic reagents, multistep purification procedures, and functional group protechon complicate the chemical strategies. A promising alternative would combine a short and inexpensive DHAP synthesis followed by an in situ aldolization reaction catalyzed by aldolase. By this approach, DHAP was recently obtained by DHA phosphorylation catalyzed by DHA kinase with ATP... 

PEG precipitation works well for IgM, but is less efficient for IgG, and salt precipitation methods are usually recommended for the latter. PEG precipitation may be preferred in multistep purifications that use ion-exchange columns, because the ionic strength is not altered and therefore does not require dialysis before ion-exchange chromatography. Furthermore, it is a very mild procedure that usually results in little denaturation of antibody. This procedure is applicable to both polyclonal antisera and most MAb-containmg fluids. 

The library synthetic steps were carefully adjusted to avoid complex purification procedures, but even the simple washing/drying/extraction procedures made this library synthesis rather laborious and time requiring. The authors, in fact, synthesised only 600 discrete compounds of the 3078 which were planned in the library design. This either calls for simpler and effective work-up procedures for multistep solution libraries, which are very difficult to identify, or new automated techniques to overcome the purification/work-up bottleneck (see some of the following paragraphs). 

Recently, the multistep combined procedures became fairly popular in the separations of complicated biological samples, especially in their purification, preseparation and preconcentration. 

Another example of a multistep synthesis using only PSQ purification procedures was described by Hodges etal. In the first step, 1,3-diketone 47 was reacted with an excess of hydrazine 48 in the presence of morpholinomethyl-polystyrene (49). Removal of excess 48 by quenching with polymer-bound isocyanate 50 afforded pyrazole 51 with a purity of 97% (HPLC) but in quite moderate yield (48%). In the second step, 51 was converted into a mixed anhydride (by treatment with isobutyl chloroformate in the presence of 49) and subsequently transformed in situ into 5 3 by reaction with an amine. Addition of the polymer-bound reagents 50 and 52 removed excesses of chloroformate and amine by filtration and 53 was isolated in 75% yield with a purity of 97% (HPLC). 

Multistep, one-pot sequential reactions are considered to be an ideal synthetic methodology because they do not require isolation and purification procedures for intermediates between reaction steps. They also reduce the necessary time and reagents. Multiple active components are necessary to promote several reactions however, active components are often opposing and mutually destructive when contacting with other components such as Hquid add and base reagents. Such difficulties could be setded by the concept of site isolation [127]. Sol-gel entrapment [128] and polymeric reagents [129-131] have been utilized to achieve the site... 

Besides this fundamental reason one has to realize that the result of even a most accurate multistep synthesis on polymer can be questioned by the chemical reaction necessary to release the final product from its support. Parts of the target sequence can be destroyed and desired protecting groups on side functions may be cleaved. These facts furthermore multiply the possibility for contaminations. Therefore, the overall success of any Merrifield peptide synthesis is inherently related also to the quality of isolation and purification procedures (see for example [168]). 

Salt concentration or pH highly influenced the partition coefficients (Fig. 14.7) and both effects could be exploited to improve the separation. Using a multistep extraction procedure (i.e. collecting the phase of interest and mixing it, at each step, with the desired amount of fresh opposite phase) isomerase was obtained in five operations with a 50% yield and a purification ratio of 170. 

The preparation of fluorinated alcohols was carried out in multistep routes according to the reported procedures.1012 The synthesis of acrylic and methacrylic esters as shown in Table 11.1 was carried out in a fluorocarbon solvent such as Freon 113 by the reaction of the respective fluorinated alcohol with acryloyl chloride or methacryloyl chloride and an amine acid acceptor such as triethyla-mine with examples shown in Scheme 1. Other attempts to esterify the fluoroalcohols directly with acrylic acid or acrylic anhydride were not successful.11 Product purification by distillation was not feasible because of the temperature required, but purification by percolation of fluorocarbon solutions through neutral alumina resulted in products of good purity identified by TLC, FTIR, and H-, 13C-, and 19F- FTNMRs. 

Cosford et al. [60] have used a commercial Syiris Africa system [14] to S3mthe-size a library of 1,2,4-oxadiazoles (Scheme 27). By a multistep procedure using connected microreactors in a single sequence, the procedure was performed in an efficient way the intermediate product purification was avoided and the reaction time was considerably reduced from several days to 30 min. 

Whether an enzyme is obtained commercially or prepared in a multistep procedure, an experimental method must be developed to detect and quantify the specific enzyme activity. During isolation and purification of an enzyme, the assay is necessary to determine the amount and purity of the enzyme and to evaluate its kinetic properties. An assay is also essential for a further study of the mechanism of the catalyzed reaction. 

Different procedures are used for analysis of simple and complex pathways. Analysis of single-step pathways often begins with the isolation and characterization of the enzyme involved. During enzyme isolation each purification step is monitored by a specific assay that measures the conversion of substrate to product. Multistep pathway analysis ideally begins with complementation analysis, a genetic technique that entails the isolation of mutants with genetic blocks in each step of the pathways. Once the numbers of enzymes and intermediates are established, each enzyme can be isolated with the help of a specific assay. 

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