EDC/sulfo-NHS reaction

A number of BODIPY derivatives that contain reactive groups able to couple with amine-containing molecules are commonly available. The derivatives either contain a carboxy-late group, which can be reacted with an amine in the presence of a carbodiimide to create an amide bond, or an NHS ester derivative of the carboxylate, which can react directly with amines to form amide linkages. The three discussed in this section are representative of this amine-reactive BODIPY family. The two NHS ester derivatives react under alkaline conditions with primary amines in molecular targets to form stable, highly fluorescent derivatives. The carboxylate derivative can be coupled to an amine using the EDC/sulfo-NHS reaction discussed in Chapter 3, Section 1.2. 

BODIPY 530/550 C3 is insoluble in aqueous solution, but it may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction as discussed in Chapter 3, Section 1.2 (Figure 9.29). However, modification of proteins with this fluorophore probably won t yield satisfactory results, since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry. For labeling molecules which contain only one amine group, such as DNA probes modified at the 5 end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C3 will give intensely fluorescent derivatives. 

Figure 9.61 QDs containing carboxylate groups can be coupled to amine-containing proteins or other molecules using the EDC/sulfo-NHS reaction to form amide bond linkages. The intermediate sulfo-NHS ester is negatively charged and will help maintain particle stability due to like charge repulsion between particles.

QD nanoparticles containing carboxylate groups also may be reacted in a two-step EDC/ sulfo-NHS reaction to couple proteins and other molecules containing both amines and car-boxylates. This type of reaction is designed to remove excess EDC activating agent before addition of protein, so protein polymerization cannot occur. 

The following protocol can be used to biotinylate carboxylate-containing molecules in aqueous solution using the EDC/sulfo-NHS reaction. 

Figure 18.22 Biotin-PEG -amine can be used to add a biotin label to carboxylate-containing molecules using the EDC/(sulfo)NHS reaction, which forms a stable amide linkage.

Another method of NHS ester mediated hapten—carrier conjugation is to create reactive sulfo-NHS esters directly on the carboxylates of the carrier protein using the EDC/sulfo-NHS reaction described in Chapter 3, Section 1.2. A carbodiimide reaction in the presence of sulfo-NHS activates the carboxylate groups on the carrier protein to form amine-reactive sulfo-NHS esters. The activation reaction is done at pH... 

In addition to the potential side reactions of EDC as mentioned previously (Section 1.1, this chapter), the additional efficiency obtained by the use of a sulfo-NHS intermediate in the process may cause other problems. In some cases, the conjugation actually may be too efficient to result in a soluble or active complex. Particularly when coupling some peptides to carrier proteins, the use of EDC/sulfo-NHS often causes severe precipitation of the conjugate. Scaling back the amount of EDC/sulfo-NHS added to the reaction may be done to solve this problem. However, eliminating the addition of sulfo-NHS altogether may have to be done in some instances to preserve the solubility of the final product. 

To each ml of QD solution, add 50 pi of the EDC/sulfo-NHS stock solution. Maintain the pH at 7.0 by the addition of base, if necessary. Small volume reactions may be controlled using a pH stat. 

Add EDC (Pierce) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.05—0.1 M EDC concentration in the reaction usually works well. Also, add sulfo-NHS (Pierce) to the reaction to bring its final concentration to 5 mM. To make it easier to add the correct quantity of EDC or sulfo-NHS, higher concentration stock solutions may be prepared if they are dissolved and used rapidly. Mix to dissolve. If this ratio of EDC/sulfo-NHS to peptide or protein results in precipitation, scale back the amount of addition until a soluble conjugate is obtained. 

Fig. 4 Reaction of EDC/sulfo-NHS activated heparin with amine-end-functionahsed star-PEG to form biohybrid gels. Gel materials are additionally modified with adhesion ligands (integrin binding RGD peptides) and loaded with soluble signalling molecules (growth factors, e.g. FGF-2). The covalent cross-links (dashed lines) could he replaced by use of enzymatically cleavable crosslinks (e.g. matrix metalloprotease sensitive peptide sequences) to allow for remodelling of the matrix by invading cells...

There are some side reactions that may occur when using EDC with proteins. In addition to reacting with carboxylates, EDC itself can form a stable complex with exposed sulfhydryl groups (Carraway and Triplett, 1970). Tyrosine residues can react with EDC, most likely through the phe-nolate ionized form of its side chain (Carraway and Koshland, 1968). The imidazolyl group of histidine may react with sulfo-NHS esters, resulting in an active carbonyl imidazole group which subsequently hydrolyzes (Cuatrecasas and Parikh, 1972). Finally, EDC may promote unwanted polymerization due to the usual abundance of both amines and carboxylates on protein molecules. 

Figure 3.2 The efficiency of an EDC-mediated reaction may be increased through the formation of a sulfo-NHS ester intermediate. The sulfo-NHS ester is more effective at reacting with amine-containing molecules. Thus, higher yields of amide bond formation may be realized using this two-step process as opposed to using a single-step EDC reaction.

Add to the solution in step 1 a quantity of EDC and sulfo-NHS (Thermo Fisher) to obtain a concentration of 2 mM EDC and 5 mM sulfo-NHS. To aid in aliquoting the correct amount of these reagents, they may be quickly dissolved in the reaction buffer at a higher concentration, and then a volume immediately pipetted into the protein solution to obtain the proper molar quantities. 

CMC should be able to participate in the two-step reaction using a sulfo-NHS ester intermediate similar to EDC, however there are no reports in the literature to this effect. Protocols for the use of this reagent in biological crosslinking applications should be essentially the same as those given previously for EDC, except substituting a molar equivalent quantity of CMC. See Sections 1.1 and 1.2 in this chapter for additional information concerning carbodiimide reactions. 

Carbodiimide coupling to carboxylate-containing QDs usually involves the use of EDC in a single-step or two-step process to form an amide bond. If a one-step reaction is done, the QD is activated with EDC in the presence of an amine-containing molecule, such as a protein. Many protocols use this method, but it can result in protein polymerization in addition to coupling, because proteins contain both carboxylates and amines. A two-step protocol results in better control of the reaction (Figure 9.61). In the first step, EDC is used in the presence of sulfo-NHS to activate the carboxylates on the particles to intermediate sulfo-NHS esters. After a quick separation step to remove excess reactants, the activated QDs are added to the protein solution to be coupled. This then results in amide bond formation without polymerization of the protein in solution. See Chapter 3, Section 1 and Chapter 14, Section 1 for additional information on this process. 

Add 100 mg of EDC and 100 mg of sulfo-NHS. Mix to dissolve. To facilitate faster dissolution, EDC and sulfo-NHS may be dissolved immediately before use as a concentrated stock solution in reaction buffer and then an aliquot of this solution added to the particle suspension to obtain the correct final concentration. 

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