Activation and Coupling

The activation of a carboxylate group with CDI proceeds to give an intermediate imide with imidazole as the active leaving group. In the presence of a primary amine-containing compound, the nucleophile attacks the electron-deficient carbonyl, displacing the imidazole and forming a 

There are a few precautions that should be noted when doing a CDI activation and coupling experiment. First, CDI itself is extremely unstable to aqueous environments, much more so than the active imidazolyl carbamate that s formed after PEG activation. Therefore, the activation step must be done in a solvent that is free of water. If unacceptable amounts of water are present, CDI will be immediately broken down to C02 and imidazole. The evolution of bubbles upon addition of CDI to a PEG solution is the telltale sign of high water content. Only freshly obtained solvents analyzed to be extremely low in moisture or those dried over a molecular sieve should be used. A water content of less than 0.1 percent in the solvent is usually all right for a CDI activation procedure. 

A second precaution is to carry out the activation step in a fume hood away from sources of ignition. Most CDI activation protocols use flammable or toxic solvents and care should be taken in handling and disposing of them. 

To remove excess CDI and reaction by-products, Beauchamp et al. (1983) dialyzed against water at 4°C. However, the imidazole carbamate groups on mPEG formed during the activation process are subject to hydrolysis in aqueous environments. A better method may be to precipitate the activated mPEG with diethyl ether as in the protocol described previously for SC activation (Section 1.2, this chapter). 

Finally, dry the isolated product by lyophilization (if the water dialysis method is used) or by use of a rotary evaporator (if the ether precipitation method is used). 

There are a few precautions that should be noted when doing a CDI activation and 

Protein Modification with Activated Polyethylene Glycols 

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This section is designed to provide a general overview of activation and coupling chemistry. Some of the reagents discussed in this chapter are not themselves crosslinking or modification compounds, but may be used to form active intermediates with another functional group. These active intermediates subsequently can be coupled to a second molecule that possesses the correct chemical constituents, which allows bond formation to occur. 

If a NHS-PEG-maleimide compound is used for this type of activation and coupling, the intermediate maleimide-activated dendrimer should be quickly purified of excess crosslinker and reaction by-products and immediately used to couple ligand. This is due to the fact that the maleimide hydrolyzes in aqueous solution at a higher rate than an SMCC-type crosslinker, because of the extreme hydrophilicity of the PEG spacer arm compared to the cyclohexane spacer of SMCC. 

Note The amount of crosslinker solution to be transferred is dependent on the level of activation desired. Suitable activation levels can be obtained for the following proteins by adding the indicated quantities of the sulfo-SMCC solution. The degree of sulfo-SMCC modification often determines whether the carrier will maintain solubility after activation and coupling to a hapten. Multimeric KLH in particular, is sensitive to the amount of crosslinker addition. KLH usually retains solubility at about 0.1-0.2 times the mass of crosslinker added to BSA. This level of addition still results in excellent activation yields, since KLH is significantly larger than most of the other protein carriers. 

The following sections describe the major activation and coupling methods used with dextran polymers. The reactive derivatives may be used to couple with proteins and other molecules containing the appropriate functional groups. 

The two-step nature of SPDP crosslinking provides control over the conjugation process. Complexes of defined composition can be constructed by adjusting the ratio of enzyme to secondary molecule in the reaction as well as the amount of SPDP used in the initial activation. The use of SPDP in conjugation applications is extensively cited in the literature, perhaps making it one of the more popular crosslinkers available. It is commonly used to form immunoto-xins, antibody-enzyme conjugates, and enzyme-labeled DNA probes. A standard activation and coupling procedure can be found in Chapter 5, Section 1.1. 

When Wa = RC(=0), that is, acyl (Figure 1.11), Wa is not removable without destroying the peptide bond. When Wa = ROC(=0) with the appropriate R, the 0C(=0)-NH bond of the urethane is cleavable. When Wb = NHR, Wb is not removable without destroying the peptide bond. When Wb = OR, the 0=C-0R bond of the ester is cleavable. During activation and coupling, activated residue Xaa may undergo isomerization, and aminolyzing residue Xbb is not susceptible to isomerization. 

The phosphonium and carbenium salts are efficient reagents for activating and coupling A-alkoxycarbonylamino acids as well as peptide acids. However, the requirement for tertiary amine to effect the reaction has several implications. The base renders hydroxyl groups subject to acylation. Hence, the side chains of serine and threonine and any hydroxymethyl groups of a resin that have not been derivatized... 

Activation and Coupling Fmoc-TOAC-OH (lmmol) was dissolved in a DMF soln (2mL), 0.45M in both HBTU and HOBt. 139 To this soln was added 2M DIPEA in DMF (lmL). After 5 min, the activated mixture and NMP (1 mL) were added to the peptide-resin (0.25 mmol). The resulting mixture was shaken for at least 30 min. 

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