Amine-Dendrimers

Modification of Amine-Dendrimers with Suifo-NHS-LC-SPDP 

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Synthetic pathways have been deliberately aimed at producing megamers. For example, poly(amido amine) dendrimers of different generations have been combined to give well-defined core-shell megamers in which a central, large core dendrimer is surrounded by a well-defined number of smaller dendrimers. 

Dye molecules can also be hosted into poly(propylene amine) dendrimers peripherally modified with OPV units [71]. In these systems, energy transfer from the OPV fluorescent units nm) to the enclosed dye molecules is... 

Dendritic hosts can be used in aqueous solution to encapsulate water-soluble fluorescent probes. Changes in the photophysical properties of these encapsulated probes are useful to understand the properties of the microenvironment created by the dendritic interior. For example, adamantyl-terminated poly(pro-pylene amine) dendrimers from the first to the fifth generation (36 represents the third generation) can be dissolved in water at pH<7 in the presence of -cyclodextrin because of encapsulation of the hydrophobic adamantyl residue inside the /1-cyclodextrin cavity and the presence of protonated tertiary amine units inside the dendrimer [72]. Under these experimental conditions, 8-anifi-... 

It has been demonstrated that dendrimers can be used also as fluorescent sensors for metal ions. Poly(propylene amine) dendrimers functionalized with dansyl units at the periphery like 34 can coordinate metal ions by the aliphatic amine units contained in the interior of the dendrimer [80]. The advantage of a dendrimer for this kind of application is related to the fact that a single analyte can interact with a great number of fluorescent units, which results in signal amplification. For example, when a Co ion enters dendrimer 34, the fluorescence of all the 32 dansyl units is quenched with a 32-fold increase in sensitivity with respect to a normal dansyl sensor. This concept is illustrated in Fig. 3. 

Vogtle et al. have prepared chiral poly(imine) dendrimers of various generations by condensation of non-racemic 5-formyl-4-hydroxy[2.2]paracyclophane moieties with poly(amine) dendrimers [71]. They have found that the optical activity of these dendrimers was nearly constant with increasing generation number. 

K. Aoi, K. Itoh, and M. Okada, Globular carbohydrate macromolecules sugar balls Synthesis of novel sugar-persubstituted poly(amido amine) dendrimers, Macromolecules, 28 (1995) 5391-5393. 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

Coupling Glycoproteins to Amine-Dendrimers by Reductive Animation... 

The following protocol involves the conjugation to an amine-dendrimer of a periodate-xidized glycoprotein, such as an antibody, which has been treated to produce aldehydes according to the protocols in Chapter 1, Section 4.4. This type of conjugation reaction... 

Dissolve the amine-dendrimer to be modified in methanol at a concentration of lOmg/ml (all operations are done in a fume hood). 

The following protocol represents the method of Aoi et al. (1995) for the coupling of lactone sugars to amine-dendrimers in organic solvent. 

Prepare the amine-dendrimer to be glycosylated in a buffer at a slightly basic pH (avoid amine-containing buffers, such as Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the den-drimer in the reaction buffer should be at least lOmg/ml. Other dendrimer concentrations also will work, but highly dilute solutions will result in less efficient modification yields. 

The following method for carbohydrate conjugation to dendrimers may be used to couple a variety of reducing sugars to amine-dendrimers, including saccharides, longer-chain carbohydrates, and even complex glycans after release from a protein (see Chapter 1, Section 4.6). 

Figure 7.15 The reducing end of a glycan or a carbohydrate can be used to conjugate to an amine-dendrimer by reductive amination, which results in the formation of a secondary amine linkage.

Figure 7.16 The creation of a tumor-targeting dendrimer conjugate can take advantage of the multivalent character of the dendrimer polymer. This figure illustrates the attachment of five different groups to an amine-dendrimer to produce a chemotherapeutic construct.

Slowly add with mixing, the activated folic acid from step 3 to the amine-dendrimer solution to give a final molar ratio of folate-to-dendrimer of 5.5 1. 

Figure 7.20 The multivalent surface of dendrimers can be used to couple biotin groups and labels for detection in immunoassays. One such conjugate was made by coupling NHS-biotin and a maleimido-iron chelate to an amine-dendrimer for use in an unique carbonyl metallo assay method.

The biotinylation of amine-dendrimers may be accomplished using either an organic reaction environment or an aqueous medium. For modification of PAMAM dendrimers with a biotinidase resistant biotin compound, Wilbur et al. (1998) performed the reaction in DMF with triethylamine as catalyst (proton acceptor). The following protocol illustrates this type of procedure using the biotinylation reagent NHS-PEG/pbiotin, which closely compares to the biotinidase insensitive compound used in the published procedure. 

Another method that can be used to biotinylate an amine-dendrimer is to do a similar reaction in aqueous buffer conditions. The following protocol is based on the methods of Tomalia et al. (1998) and Mamede et al. (2003). 

Figure 7.22 Fluorescent dyes such as an amine-reactive Cy5 derivative can be coupled to amine-dendrimers at relatively high substitution levels to create intensely fluorescent detection agents. If the dendrimer also is deriva-tized to contain an affinity group or a targeting group then specific fluorescent detection at high sensitivity can be realized.

Dissolve the amine-dendrimer to be modified in DMF or buffer (50mM sodium borate, pH 8.5) at a concentration of at least lOmg/ml. Avoid the use of amine-containing buffers for an aqueous reaction, such as Tris or imidazole, as these will react with the... 

Figure 7.23 Chelating groups such as the isothiocyanate derivative of DTPA can be used to create multivalent chelating complexes with amine-dendrimers. Such complexes are able to coordinate multiple metal ions for detection, imaging, or radioimmunotherapy purposes.

The following protocol for the modification of an amine-dendrimer with an SCN-Bzl-DTPA chelator is based on the literature references previously cited. Dendrimers of other generations will work well in this procedure provided that the molar ratios of reactants are adjusted for the size of dendrimer being used and the substitution level desired. 

Dissolve lOmg of a G-4 amine-dendrimer in 1ml of 0.1 M sodium carbonate buffer,... 

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