Hermanson G. Bioconjugate Techniques, Second Edition

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370 7. Dendrimers and Dendrons

used. For modiﬁ cation of amine-dendrimers that are not soluble in the recommended

organic solvents, the glycan or carbohydrate initially may be solubilized in DMSO and

then an aliquot added to the aqueous reaction buffer.

2. Add to the glycan solution the amine-dendrimer to be labeled. Dendrimer concentrations

of at least 10 mg/ml will help to increase the reaction kinetics, although the reaction pro-

ceeds slowly, mainly due to the limited quantity of open-form reducing sugar available for

coupling at any given time.

3. In a fume hood, add to the reaction mixture a quantity of reducing agent (e.g., sodium

cyanoborohydride or borane dimethylamine (BDA)) to give a ﬁ nal concentration of

1.0 M. The high concentration of reductant will aid in accelerating the reaction to com-

pletion. Cyanoborohydride is extremely toxic and should not be handled outside of a

fume hood. Use proper protective clothing when handling such compounds.

4. When using nonaqueous reaction conditions, incubate for 1–2 hours at 60–80°C. For

reactions in an aqueous environment, the reaction may be done at room temperature

or 37°C. In this case, the reaction time should be extended to at least 24 hours. Longer

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.

reaction times are not unusual when modifying carbohydrates by reductive amination at

ambient temperature.

5. Purify the modiﬁ ed dendrimer from reactants and reaction by-products by dialysis, gel

ﬁ ltration, or ion-exchange chromatography.

Synthesis of other reactive groups on sugars for conjugation with amine-dendrimers also has

been done with success. For example, André et al. (1999) used the p-isothiocyanato derivative

of p-aminophenyl--

D-lactoside to couple with the amine groups on a G-3 PAMAM dendrimer,

forming isothiourea linkages between the sugar and dendrimer. Davis et al. (2005) used glycosyl

derivatives containing the thiol-reactive group methanethiosulfonate to couple with dendrons

modiﬁ ed with thioacetic acid to contain terminal thiol groups. This reactive functionality was

again used to conjugate to thiol-containing proteins to create glyco-dendrimer-protein reagents.

Woller and Cloninger (2001) used another isothiocyanate derivative of mannose to couple with

amine-dendrimers of several different generations. Finally, Kensinger et al. (2004a) synthesized

a thiopropionic acid derivative of galactose and coupled it to amine-dendrimers using the amide

bond forming agent HATU (Aldrich) in acetonitrile. The galactose-functionalized dendrimers

were used to study their binding to HIV-1 gp120 protein (Kensinger et al., 2004b).

Conjugation of Carboxylate Organic Molecules to Amine-Dendrimers

Amine-dendrimer molecules of various generations have been used as carriers for small organic

molecules, such as chemotherapeutic agents or small ligand, cell-targeting compounds, such as

folic acid (Quintana et al., 2002; Shukla et al., 2003; Islam et al., 2005). The presence of folic

acid on the dendrimer surface allows the drug complex to bind with over-expressed folate recep-

tors on certain tumor cells (Ross et al., 1994). Adding a chemotherapeutic drug to the folate–

dendrimer conjugate creates a toxic payload deliverable directly to the cancer cells in vivo .

Majoros et al. (2005) synthesized such dendrimer conjugates using the carbodiimide EDC to

activate the carboxylate group of folic acid or methotrexate for reaction with the amines on the

PAMAM dendrimer surface. A G-5 amine-dendrimer ﬁ rst was partially blocked by acetylation

according to the previously described procedure. The result was about 82 of the available amines

were capped, leaving about 28 amines still free for coupling with other molecules. In some

derivatives, a ﬂ uorescent probe (FITC) also was conjugated to the partially blocked dendrimers

to provide a detectable tag for following binding to cells. Then both folic acid and methotrex-

ate were conjugated to the labeled dendrimer in separate reactions to yield the ﬁ nal conjugate.

Careful tuning of the molar ratios used during each reaction resulted in a modiﬁ cation level per

dendrimer of 4 FITC labels, 4 folic acids, and 5 methotrexate groups. In some cases, glycidol

also was used to block about 14 of the amine groups and form hydrophilic hydroxylated regions

on the dendrimer surface to promote water solubility. Figure 7.16 illustrates the ﬁ nal conjugate

composition with a G-5 dendrimer. These conjugates were extensively studied by HPLC to deter-

mine properties and modiﬁ cation levels (Islam et al., 2005).

Hong et al. (2007) investigated the binding afﬁ nity of such folate–dendrimer derivatives

toward the folate-binding protein (FBP), typically over expressed on cancer cells. Using SPR

analysis, they were able to measure the binding constants of a series of folate–dendrimer con-

jugates containing different levels of folate modiﬁ cation (from 2 to 14 folates–dendrimer). The

results indicate that the combined avidity of multiple folates on a single dendrimer enhanced the

binding to FBP by up to 5 orders of magnitude (2,500- to 170,000-fold enhancement). This is

2. Conjugation to Dendrimers 371

372 7. Dendrimers and Dendrons

the ﬁ rst deﬁ nitive study on the beneﬁ ts of multivalent dendrimer drugs as compared to the same

drug interactions individually in solution. Using this approach to drug targeting, Kukowska-

Latallo et al. (2005) found that a dendrimer conjugate improved the therapeutic response toward

tumor cells in animal models of human epithelial cancer.

Similar dendrimer conjugates for therapeutic application also have been designed for boron

neutron capture therapy in the treatment of cancer (Barth et al., 1994; Newkome et al., 1994;

Qualmann et al., 1996). In one design, a G-3 PAMAM dendrimer was modiﬁ ed with folate

and 12–15 polyhedral decaborate clusters (Alam et al., 1989) along with an average of 1 to

1.5 PEG

2000

units to make the therapeutic conjugate (Shukla et al., 2003). The folate-targeting

component was not attached to the surface amines, but to the end of an additional amino-

PEG

800

unit. The PEG groups also facilitated increased in vivo half-life and prevented immedi-

ate uptake of the complex into the liver.

Most of these conjugation reactions involve amide bond formation between a small, organic

molecule containing a carboxylate and the amines on the dendrimer surface. There are two

potential reaction strategies for creating such conjugates: aqueous or nonaqueous reactions.

For the coupling of folic acid to PAMAM dendrimers, Majoros et al. (2005) used an organic

solvent environment according to the following protocol.

Protocol

1. Dissolve 33 mg of folic acid in a mixture of 24 ml DMF and 8 ml DMSO at room

temperature.

2. Add a 14-fold molar excess of EDC (200 mg) with mixing.

3. React for 1 hour at room temperature. The product of this reaction forms an amine-

reactive ester on folate for coupling to the dendrimer.

Figure 7.16 The creation of a tumor-targeting dendrimer conjugate can take advantage of the multivalent

character of the dendrimer polymer. This ﬁ gure illustrates the attachment of ﬁ ve different groups to an amine-

dendrimer to produce a chemotherapeutic construct.

4. Dissolve an amine-containing dendrimer in water at a concentration of at least 4.5 mg/

ml. Majoros et al. (2005) used 403 mg of a partially acetylated G-5 dendrimer that con-

tained 82 blocked amines and 28 available amines for coupling, dissolved in 90 ml of

water.

5. Slowly add with mixing, the activated folic acid from step 3 to the amine-dendrimer solu-

tion to give a ﬁ nal molar ratio of folate-to-dendrimer of 5.5:1.

6. React for 1 hour at room temperature with mixing.

7. Purify the modiﬁ ed dendrimer from excess reactants and reaction byproducts by dialysis

against water or buffer.

The same type of modiﬁ cation with carboxylate molecules can be done in aqueous solution

using EDC. If the ligand to be coupled only has a single carboxylate with no amines or other

nucleophiles present, then the dendrimer and ligand may be dissolved at a similar molar ratio

in aqueous buffer and EDC added to facilitate the coupling reaction.

Amine-containing dendrimers also have been used to quantify phosphorylated peptides

for phosphoproteome analysis by mass spec (Tao et al., 2005). In this novel application, pro-

teins undergoing analysis are reduced and alkylated with iodoacetamide to block sulfhydryls,

then trypsin digested. The peptides are then desalted and lyophilized. The dried peptides are

methylated in methanolic HCl to eliminate interference by carboxylate groups and then the sol-

vent is removed in vacuo. The mixture of blocked peptides next is reacted with a G-5 PAMAM

dendrimer using 50 mM EDC and 100 mM imidazole in 0.2 M MES buffer, pH 6.0 (see Chapter

27, Section 2.1). The reaction activates the phosphoryl groups on the phosphorylated peptides,

which in turn react with the amines on the dendrimers to create phosphoramidate linkages

(Figure 7.17 ). The conjugated phosphopeptides can be separated from non-phosphorylated

peptides by simple ﬁ ltration and washing. Finally, the isolated phosphopeptides can be cleaved

from the dendrimer using 10 percent TFA treatment and analyzed by mass spec.

Epoxy Activation of Amine-Dendrimers

Epoxy groups can be formed on the surface of amine-containing dendrimers by reaction with

either bis-epoxide compounds or epibromohydrin (or epichlorohydrin). Modiﬁ cation with

these compounds forms terminal epoxide groups that can be used for subsequent conjugation

with amine-, thiol-, or hydroxyl-containing ligands. Singh (1998) described a simple procedure

using epibromohydrin to form an epoxy-activated PAMAM dendrimer for conjugation with

thiol-modiﬁ ed alkaline phosphatase enzymes ( Figure 7.18 ).

The following protocol is based on the method of Singh (1998). Other amine-containing den-

drimers besides the PAMAM type, such as the PrioStar dendrimers from Dendritic Nanotechnologies

may be used as well.

Protocol

1. Dissolve a dendrimer in 50 percent methanol/water at a concentration of at least

10 mg/ml. Singh (1998) used about 165 mg of a G-5 PAMAM dendrimer dissolved in

1 ml of the alcohol/water mixture. Use a fume hood for all operations.

2. Add 200 mg of sodium carbonate per ml of the dendrimer solution prepared in step 1

and a quantity of epibromohydrin equal to a 285-fold molar excess over the amount of

2. Conjugation to Dendrimers 373

374 7. Dendrimers and Dendrons

dendrimer present. The large excess of reagent assures that the amines get fully blocked

during the reaction and will prevent any amines from crosslinking by reaction with the

epoxy ends of the modiﬁ ed dendrimer.

3. React for 4 hours with mixing in a fume hood.

4. Dilute the reaction mixture 10-fold with 50 percent methanol/water and purify the

epoxy-activated dendrimer by repeated ultraﬁ ltration using a membrane with a molecular

Figure 7.17 Phosphopeptides can be separated for mass spec analysis using an amine-dendrimer. A control

and test sample ﬁ rst are both methylated to block their carboxylate groups and then mixed together in equal

amounts. An amine-containing dendrimer then is used to capture the phosphorylated peptides by conjugation

with the phospho groups using EDC and imidazole to form phosphoramidate bonds. After separation of the

phosphopeptide-dendrimer conjugates using size exclusion chromatography, the phosphopeptides are cleaved

off using TFA and analyzed by mass spec.

weight cutoff able to retain the size of dendrimer being activated. Repeat the dilution and

concentration steps until the ﬁ ltrate is neutral in pH.

5. The epoxy-activated dendrimer may be conjugated to thiol-containing proteins by reac-

tion in 50 mM sodium phosphate, pH 7.2. The reaction can be done at 4°C or at room

temperature for 8–16 hours to form thioether linkages.

6. Purify the conjugate by gel ﬁ ltration to separate protein-dendrimer complexes from

excess protein or dendrimer.

Figure 7.18 Amine-containing dendrimers can be activated with epibromohydrin to result in the formation of

reactive epoxy groups on the dendrimer surface. This reactive intermediate then can be used to conjugate with

thiol-containing proteins, such as thiolated alkaline phosphatase. The reaction results in the formation of a

thioether bond.

2. Conjugation to Dendrimers 375

376 7. Dendrimers and Dendrons

Biotinylation of Amine-Dendrimers

Amine-containing dendrimers can be modiﬁ ed to contain one or more biotin groups for inter-

action with avidin or streptavidin reagents. The polyvalent nature of a dendrimer permits the

formation of a biotin multimeric structure for potential enhancement of avidin–biotin assays.

Since a biotin-dendrimer scaffold allows many biotin-binding proteins to dock simultaneously

and form larger complexes, these conjugates can recruit more detection molecules to bind at

the site of an analyte. This can have direct effect on the sensitivity of immunoassays, such as

ﬂ uorescence detection, enzyme linked immunoadsorbent assays (ELISAs), and western blotting

procedures.

This type of biotinylated dendrimer-based signal ampliﬁ cation technique has been done to

increase the detection of genomic DNA in suspension arrays (Borucki et al., 2005), wherein a

DNA dendrimer was biotinylated multiple times and also modiﬁ ed to contain a targeting oligo

sequence. The DNA dendrimer, which in that case was a construct consisting of partially hybrid-

ized oligonucleotide sequences branching out from a central core, was modiﬁ ed to contain as

many as 850–900 biotin molecules on its surface along with at least one targeting oligonucleotide

sequence. After genomic DNA was allowed to bind to a capture oligo on a ﬂ uorescence micro-

particle, the biotinylated dendrimer was added and it bound to the genomic DNA via hybridiza-

tion with the targeting oligo. Finally, streptavidin- phycoerythrin detection conjugate was added

and it then was bound to the biotin groups on the dendrimer ( Figure 7.19 ). The resultant ﬂ uores-

cent signal was ampliﬁ ed beyond that possible using a simple biotinylated oligo directly.

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA micro-

array detection by 100-fold over that obtainable using traditional avidin–biotin reagent sys-

tems (Stears, 2000; Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is

able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or ﬂ uo-

rescent probes for assay detection. In addition, if the biotinylated dendrimer and the strepta-

vidin detection agent is added at the same time, then at the site of a captured analyte, the

biotin–dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or

streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of mul-

tivalent biotin–dendrimers can become universal enhancers of DNA hybridization assays or

immunoassay procedures.

Another example of an immunoassay enhancement using biotinylated dendrimers involves

a novel detection technique called carbonylmetallo-immunoassay (CMIA). This technology

involves the use of an NHS-4-pentynoate-(dicobalt hexacarbonyl) transition metal chelate label-

ing reagent or ( n

-cyclopentadienyl)iron dicarbonyl ( n

-N-maleimidato) group, which can be

detected using Fourier transform infrared spectroscopy (Salmain et al., 1991, 1992; Varenne

et al., 1992, 1995; Philomin et al., 1994; Vessières et al., 1999). These compounds can be cou-

pled to amine groups on PAMAM dendrimers to yield amide linkages. Salmain et al. (2002)

developed a dendrimer-based signal enhancement method to increase the sensitivity of the IR

detection chelate. A G-4 PAMAM dendrimer containing 64 primary amine groups was labeled

with NHS-biotin and the iron chelate to yield a complex containing 45 chelate groups and

4 biotins ( Figure 7.20 ). The ratios in the ﬁ nal dendrimer conjugate were of course dependent on

the molar ratios in the initial reaction mixture and could be controlled through careful planning

of the conjugation process.

Biotinylated dendrimers also have been used to develop targeting conjugates for thera-

peutic use in targeting cancer cells. Wilbur et al. (1998) studied several different PAMAM

generations in the coupling of a water-soluble biotin analog with unique biotinidase insensitivity.

The biotin-dendrimer conjugates were compared with biotin trimers or biotin tetramers in their

ability to recruit radionuclide-labeled streptavidin complexes after binding to a streptavidin–

antibody conjugate already bound to antigen on a tumor cell surface. It was found that the

Figure 7.19 Biotinylated dendrimers can be used to enhance signals in assay procedures using the

(strept)avidin–biotin interaction. In this example, a ﬂ uorescent microsphere containing capture oligos is able to

interact with target DNA while a biotinylated dendrimer also containing detection oligo sequences binds to the

genomic DNA target. The interaction of the dendrimer is detected using streptavidin–phycoerythrin conjugates,

which are intensely ﬂ uorescent.

2. Conjugation to Dendrimers 377

378 7. Dendrimers and Dendrons

biotinylated dendrimer increased the number of radionuclide-streptavidin binding events by a

factor of 4.

The efﬁ ciency of biotinylation also was determined relative to the size of the amine-

dendrimer (Wilbur et al., 1998). The reactions were done in DMF with triethylamine added

as a proton acceptor, so competing hydrolysis of the NHS ester on the biotin compound was

not an issue. In reacting each generation of dendrimer with a large excess of biotinylation

agent, it was found that G-0, G-1, G-2, and G-3 PAMAM dendrimers all could be completely

biotinylated by modiﬁ cation of all of the pendent amine groups. Only when reaching the size of

a G-4 dendrimer was the biotinylation yield less than the number of total amine groups present

(51 of 64 possible amines modiﬁ ed). This indicates that, unlike with protein labeling, the reac-

tivity and accessibility of available amines on dendrimers remains high even when working

with mid-sized molecules.

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.

Mamede et al. (2003) developed a biotinylated G-4 PAMAM dendrimer containing mul-

tiple diethylenetriamine pentaacetic acid (DTPA) chelating groups for use in radiotherapy of

intraperitoneally disseminated tumors. The G-4 dendrimer was reacted with sulfo-NHS-LC-

biotin at a molar ratio that resulted in approximately 2.5 biotin molecules per dendrimer.

This conjugate then was conjugated with 2-( p -isothiocyanatobenzyl)-6-methyldiethylenetri-

aminepentaacetic acid to give about 52 DTPA chelating groups per dendrimer. Finally, the com-

plex was loaded with radioactive

111

In and also incubated with avidin to result in 2–3 avidin

molecules per dendrimer binding to the biotin groups on the surface. The positive charge of

the avidin protein molecules facilitated entry into the tumor cells, while the radioactive cargo

killed the cells.

The biotinylation of amine-dendrimers may be accomplished using either an organic reac-

tion environment or an aqueous medium. For modiﬁ cation 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

-biotin, which closely compares to the

biotinidase insensitive compound used in the published procedure.

Protocol

1. In a fume hood, dissolve an amine-containing dendrimer in DMF at a concentration of

at least 10 mg/ml with mixing. For the modiﬁ cation of a G-3 dendrimer, Wilbur et al .

(1998) used approximately 240 mg of dendrimer dissolved in 10 ml of DMF. Other con-

centrations will work well in this procedure.

2. Add 15.7  l of triethylamine (0.12 mmol) to the dendrimer solution with mixing.

3. Dissolve a quantity of NHS-PEG

-biotin (MW 588.67) in the dendrimer solution with

mixing to bring the ﬁ nal reagent-to-dendrimer ratio in the reaction medium to at least

1.25 times greater than the molar amount of amines to be modiﬁ ed. To saturate com-

pletely a G-3 dendrimer initially containing 32 primary amine groups, add at least 8 mg

of biotinylation reagent for each mg of dendrimer being modiﬁ ed. Using this molar ratio

of biotin-to-dendrimer in the reaction will result in a high-modiﬁ cation yield of all the

amine groups on the dendrimer, especially for G-1 through G-3 size dendrimers. If a

lower modiﬁ cation level is desired, then scale back the amount of biotinylation reagent

added accordingly. Note: The most important consideration is optimizing the molar ratio

of biotinylation reagent to dendrimer to obtain the desired modiﬁ cation level in the ﬁ nal

conjugate. This entirely depends on the intended application of the biotinylated den-

drimer, and a series of reactions containing different biotinylation ratios may have to be

done to determine the best ratio to use.

4. React for a minimum of 1 hour at room temperature with mixing. Longer reaction times

may be used, particularly if a maximal modiﬁ cation level of biotin is desired.

5. Purify the biotinylated dendrimer by diluting it with an equal volume of water and then

using dialysis, ultraﬁ ltration, or size exclusion chromatography.

Another method that can be used to biotinylate an amine-dendrimer is to do a similar reac-

tion in aqueous buffer conditions. The following protocol is based on the methods of Tomalia

et al . (1998) and Mamede et al . (2003).

2. Conjugation to Dendrimers 379