Isolation tags

This chapter is divided in five sections. The first one highlights the potential of immobilizing a natural product core structure on solid phase to facilitate its diversification into a library of analogues. The next three sections cover the de novo synthesis of natural products using solid phase synthesis, immobilized reagents or isolation tags respectively. Finally, the last section reviews six landmark libraries based on natural products or natural product motifs. 

Synthesis of Natural Products Using Isolation Tags 627... 

An alternative method to overcome the limitations of solid phase synthesis is the use of isolation tags. For example, long polyethylene glycol (PEG) chains are... 

Scheme 21.13 Synthesis of prostaglandin using isolation tags (Janda et a .). NCPS noncross-linked polystyrene.

Scheme 21.15 Synthesis of radicicol A and analogues using fluorous isolation tags (Winssinger et ai.).

The isolated tagged peptides are separated and analyzed by microcapillary tandem MS which provides both identification of peptides by fragmentation in MS-mode and relative quantitation of labeled pairs by comparing signal intensities in MS mode. 

Adequate precautions shall be taken to prevent electrical equipment that has been made dead from becoming live while work is carried out on or near the equipment. This will include means of locking off isolators, tagging equipment, permits to work and removing fuses. 

PSA prostate specific antigen RCT randomized controlled trial ROS reactive oxygen species SF soy foods SI soy isoflavones SP soy protein SPl soy protein isolate TAG triacylglycerol TNF-a tumor necrosis factor a TVP texturized vegetable protein VCAM-1 vascular cell adhesion molecule-1... 

Are machines shut down in an orderly fashion before energy isolating devices are locked out or tagged so as to avoid any hazards to employees as a result of equipment deenergization [OSHA Reference. 147(d)(2)]... 

Isolation The disablement and tagging-out of appropriate interfacing components prior to initiating maintenance on another component. 

Ensure equipment has been electrically isolated and suitably locked out and tagged. 

Before carrying out any inspections or maintenance procedures on belt drives, make sure they are not in operation. Equipment operators should electrically isolate the drive system and lock-out and tag the machine that is to undergo maintenance. Once this precaution has been taken, the belts and pulleys can be removed from the machine for inspection, cleaning, and repair. 

Products Isolated Assayed for Tagged Atom Content... 

In the carbon-14 expts, HMX/RDX product was isolated qualitatively, separated Into its components, and each component assayed for carbon-14 beta radioactivity using a liquid scintillation counting technique (Ref 11). DPT-l4C was isolated as an intermediate product from the reaction mixt and similarly radioassayed. For the nitrogen-15 tagged AN expts, HMX and RDX were assayed mass spectrometrically for i5N/i4N ratios from which atom %1SN contents were calcd. In die course of these expts, each tagged species was added initially and also at subsequent stages of the reaction process. The important observations and results are summarized as ... 

All fires should be extinguished, fuel lines isolated, valves closed, and lock out/tag out programs implemented to prevent accidents through the sudden release of some form of energy. 

A third method consists of measuring the time taken for a tagged panicle (e.g. radioactive or magnetic) to travel between two points 75 . The method gives results applicable only to an isolated particle which may not be representative of the bulk of the particles. These techniques can readily be used in experimental equipment but are not practicable for industrial plant. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Figure 3.3. Structure of the ICAT reagent. The reagent contains a biotin affinity tag that is used to isolate ICAT-labeled peptides. The reagent also contains a linker that exists in a heavy (where X= deuterium) or light form (X= hydrogen) and a reactive group with specificity towards the thiol groups of cysteine residues. Figure adapted from Gygi et al. (1999).

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

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