Hermanson G. Bioconjugate Techniques, Second Edition
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360 7. Dendrimers and Dendrons
to 24 repeating units. The PEG spacer chain increases the overall water solubility of modi-
fi ed molecules and decreases nonspecifi c binding potential of the fi nal conjugate. The following
protocol may be used with any of these discrete PEG crosslinkers with the appropriate adjust-
ments in the quantity of reagent added to take into account differences in molecular weight
due to the PEG length. The longer of these discrete NHS-PEG-maleimide crosslinkers will
provide the greatest degree of hydrophilicity after modifi cation of an amine-dendrimer sur-
face. Intermediate-length NHS-PEG-maleimides probably provide a suffi cient combination of
hydrophilicity while maintaining a smaller conjugate size. One caution should be noted when
using PEG-based reagents with amine-containing dendrimers. The polyether cross-bridge of
PEG compounds has the ability to hydrogen bond to the dendrimer surface, especially when
using dendrimers with a high density of amines. Reactions done at higher levels of PEG-con-
taining reagents may be done to overcome this tendency. Alternatively, the amine surface may
be partially blocked or derivatized with another molecule prior to using the PEG compound to
avoid hydrogen bond interactions.
Figure 7.10 An NHS-PEG-maleimide compound can be used to functionalize dendrimers to provide a hydrophilic
spacer terminating in thiol-reactive groups. Thiol-containing proteins then can be conjugated to this reactive inter-
mediate to form covalent thioether bonds.
Protocol
1. Dissolve 10 mg of an amine-containing dendrimer into 1 ml of 50 mM sodium phosphate,
0.15 M NaCl, pH 7.5 (coupling buffer) with mixing.
2. Dissolve NHS-PEG
6
-maleimide (MW 601.6) into DMSO at a concentration of 20 mM.
Short, PEG-type crosslinkers often exist as a thick oily mass, and preparing the solution
typically involves dissolving an entire vial of the compound into DMSO to determine accu-
rately the required concentration. Use only dry DMSO to avoid hydrolysis of the NHS ester.
3. Add 50 l of the NHS-PEG
6
-maleimide solution to the 1 ml dendrimer solution and mix
thoroughly to dissolve. This represents approximately a 14-fold molar excess of crosslinker
over the quantity of dendrimer present, if a G-3 PAMAM dendrimer is used with an
ethylenediamine core. The optimum molar ratio of crosslinker-to-dendrimer should be
determined experimentally for best performance of the resultant conjugate in its intended
application. If enough material is available, doing a series of experiments at different mole
ratios of crosslinker-to-dendrimer will help to optimize the resultant conjugate.
4. React for 1 hour at room temperature with mixing.
5. Purify the derivatized dendrimer from excess crosslinker and reaction by-products using
gel fi ltration (size exclusion chromatography) on a 10 ml desalting column or through
use of ultrafi ltration spin-tubes. If a dendrimer of at least G-3 size is being modifi ed, the
separation should be done on a support with an exclusion limit of no more than 2,500–
5,000 Daltons to avoid losing the derivatized molecule through the membrane or not
obtaining suffi cient separation during the chromatography.
6. Add 1–10 mg of a protein or antibody containing an available thiol group to the puri-
fi ed, modifi ed dendrimer from step 5. Alternatively, add the protein to be coupled to the
dendrimer suspension in an amount equal to 1–10 molar excess over the estimated
number of maleimide groups on the modifi ed dendrimer. The amount of maleimide func-
tionality may be determined using the protocol in Chapter 19, Section 5. Creating thiol
groups from disulfi des in proteins may be done according to the procedures in Chapter 1,
Section 4.1. Alternatively, the use of a thiolation reagent may be done to add thiols to the
protein surface for coupling to the maleimide groups. The optimal amount of protein to
be added to the dendrimer should be determined experimentally.
7. React the protein with activated dendrimer for 2 hours at room temperature with mix-
ing. At the completion of the reaction, cysteine may be added at 50 mM to block excess
maleimide-reactive sites, which are not coupled with protein.
8. Purify the conjugate and remove excess protein by gel fi ltration using a column with an
exclusion limit that is able to accommodate both the protein being conjugated and the
dendrimer-protein conjugate. If the conjugate elutes in the void volume, while the protein
is retained in the pores, this also will result in suffi cient separation to purify the conjugate.
Store the dendrimer conjugate frozen (especially if it ’s a PAMAM-type) or lyophilized.
The addition of a stabilizing excipient to the freeze-dried conjugate may be done to pro-
tect the coupled protein during lyophilization (i.e., sucrose).
Coupling Glycoproteins to Amine-Dendrimers by Reductive Amination
The amines on the surface of PAMAM-type and other amine-containing dendrimers may be
used to couple to aldehyde groups in other molecules, including those formed after periodate
2. Conjugation to Dendrimers 361
362 7. Dendrimers and Dendrons
oxidation of sugar groups in glycosylated proteins. Mild treatment of glycoproteins with sodium
meta periodate results in cleavage of diol carbon–carbon bonds with concomitant oxidation
of the hydroxyls to aldehyde groups. These aldehydes can be used to conjugate the proteins to
amine-dendrimers through Schiff base formation and reduction to secondary amine linkages
using sodium cyanoborohydride (Chapter 1, Section 4.4 and Chapter 2, Section 5) ( Figure 7.11 ).
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
Figure 7.11 Oxidation of glycoproteins with periodate, such as glycosylated antibodies, results in the forma-
tion of aldehyde groups that can be used for conjugation to dendrimers containing amine groups. Reductive
amination with sodium cyanoborohydride results in coupling via secondary (or tertiary) amine bonds.
to dendrimers may be used even after the dendrimer has been initially modifi ed with a
limited number of heterobifunctional crosslinking molecules. Thus, an amine-dendrimer fi rst
may be reacted with crosslinkers such as sulfo-NHS-LC-SPDP or NHS-PEG
6
-maleimide as
described previously, and the remaining amines on the dendrimer surface used to couple with
an oxidized glycoprotein. This would create that contained a covalently linked glycoprotein
with thiol-reactive groups available for further conjugation with another molecule containing a
sulfhydryl group. Singh (1998) used this technique to produce a dendrimer conjugate contain-
ing alkaline phosphatase for detection and a Fab fragment of an antibody directed against cre-
atine kinase MB isoenzyme. The phosphatase enzyme was coupled through its carbohydrates
by reductive amination and the Fab fragment was coupled through a thiol group.
Protocol
1. Dissolve an amine-containing dendrimer in 50 mM sodium phosphate, 0.15 M NaCl,
pH 7.5, at a concentration of at least 10 mg/ml. Note: The use of a buffer at pH 9–10
(i.e., 0.1 M sodium carbonate) for the initial Schiff base formation (step 2) will result in
higher effi ciency of conjugation. However, if a higher pH is used during this fi rst stage,
then the pH must be adjusted back down to more neutral conditions before the addition
of reductant (step 4), as the reducing agent is not effective in the higher pH environment.
2. Add a quantity of a periodate-oxidized glycoprotein to the dendrimer solution (made
according to Chapter 1, Section 4.4) to provide a molar ratio of protein-to-dendrimer of
1:1–10:1. Mix to dissolve. The optimal level of protein addition should be adjusted to
provide maximal performance and activity in the intended application. If another protein
also is to be attached to the dendrimer to make the fi nal complex, then limiting the den-
sity of the periodate-oxidized protein may be necessary to leave room on the surface for
additional protein coupling. In addition, some proteins with multiple glycosylation sites
may cause dendrimer oligomerization if the conjugation reaction is done with too low of
a protein-to-dendrimer ratio.
3. React for 2 hours at room temperature with mixing.
4. In a fume hood, add 10 l of 5 M sodium cyanoborohydride (Sigma) per ml of reaction
solution. Caution: cyanoborohydride is extremely toxic. All operations should be done
with care in a fume hood. Also, avoid any contact with the reagent, as the 5 M stock
solution is dissolved in 1 N NaOH. If a higher pH buffer was used for the Schiff base
formation, then adjust the solution to pH 7.5 before adding the cyanoborohydride.
5. React for 30 minutes at room temperature in a fume hood.
6. Block unreacted aldehydes by the addition of 50 l of 1 M ethanolamine, pH 7.5, per ml
of reaction solution. Block for 30 minutes at room temperature with mixing.
7. Purify the conjugate from unreacted protein or unreacted dendrimer using gel fi ltra-
tion chromatography with a matrix having an exclusion limit appropriate to accommo-
date the size of the molecules being separated (i.e., a HiPrep 16/60 column packed with
Sephacryl S-200 HR, GE Healthcare).
Blocking of Amines on PAMAM Dendrimers
The number of amine groups on dendrimers of medium to large size (G-3 and above) often is
many more than what is required to prepare a conjugate. Since amines can become protonated
2. Conjugation to Dendrimers 363
364 7. Dendrimers and Dendrons
and carry a positive charge even under physiological conditions, they may interact nonspecifi -
cally with biomolecules. Ionic interactions can be a signifi cant source of high backgrounds in
assays or create off-target effects if using a dendrimer conjugate in the development of in vivo
applications (cell-based or whole animal). In particular, nonspecifi c binding to the positively
charged amine groups on dendrimers, if left unblocked, can be cytotoxic to normal cells in vivo
especially if the conjugate is designed to carry chemotherapeutic components, which can be
used to kill cancer or diseased cells (Patri et al., 2002; Kukowska-Latallo et al., 2005). To over-
come the positive charge character of amine-dendrimers, excess amine groups can be covalently
blocked to eliminate the possibility of ionic interactions. This blocking process can occur before
activation of the dendrimer with a crosslinker or after modifi cation, providing that the blocking
agent used to couple with the remaining amines does not affect the activation chemistry.
Singh (1998) used succinylation with succinic anhydride to block excess amines and convert
them into negatively charged carboxylates, which often have lower interaction potential with
biomolecules than positive charges. Thomas et al. (2004) used acetic anhydride to acetylate
and block the amines of a G-4 PAMAM dendrimer before or after activation with sulfo-NHS-
LC-SPDP. Similarly, Patri et al. (2004) also used acetic anhydride to block amines on a G-5
PAMAM dendrimer prior to activation with sulfo-NHS-LC-SPDP, but the blocking step was
done to eliminate about 80 of the 128 amine groups before effecting the conjugation with an
antibody. In all these instances, the amine groups on the dendrimer are converted into amides,
which carry no charge at physiological pH.
Reactions with succinic anhydride or acetic anhydride to block dendrimer amines can be
done in aqueous or methanolic solution. If organic solvent is used for the reaction, then it is
typical to include triethylamine as a proton acceptor, which helps drive the reaction. Such reac-
tions, however, can ’t be done to dendrimer amines once a protein containing amines also has
been conjugated, as the protein too will get modifi ed.
Another method of blocking excess amines on dendrimers is to use small, hydrophilic block-
ers that contain hydroxyl or ether groups. Islam et al. (2005) used the short epoxy compound
glycidol to modify amine groups to reduce nonspecifi c interactions in a dendrimer conjugate.
The primary amine groups can be alkylated a maximum of 2 times with this reagent in metha-
nol, resulting in 4 hydroxyl groups for each amino functionality (Shi et al., 2007). The reaction
with glycidol, however, was shown to be not as effi cient in creating a homogeneous product
as acylation with either succinic anhydride or acetic anhydride (Shi et al., 2005). This likely
is due to steric issues coming into play in trying to alkylate each amine twice with glycidol as
opposed to a single acylation product with either anhydride compound. Glycidol modifi cation
of an amine-dendrimer is done according to the following protocol.
Protocol
1. Dissolve the amine-dendrimer to be modifi ed in methanol at a concentration of 10 mg/ml
(all operations are done in a fume hood).
2. Add glycidol to the dendrimer solution in a 4-fold molar excess over the number of
amines to be blocked (Shi et al., 2007). The number of amines may be calculated from
the generation number of the dendrimer and the degree of blocking desired. Obtaining
the optimal molar ratio for a particular conjugate application may have to be done by
experimentation using a series of different glycidol-to-dendrimer molar ratios.
3. The reaction is continued for 24 hours in the fume hood with mixing.
4. Dialyze the modifi ed dendrimer against PBS buffer and then water to remove excess
reactants.
Another option to limit the nonspecifi c binding character of amine-dendrimers is the
use of PEG compounds. Amine-reactive PEG derivatives can be used to covalently link to
excess amines on dendrimers and create a hydrophilic tether, which can dramatically limit non-
specifi c interactions with the exposed surface. For this purpose, PEG reagents containing an
activated carboxylate on one end and a methyl ether group (mPEG) on the other end work
well (Chapter 18, Section 4). NHS-mPEG derivatives that are spontaneously reactive to the
dendrimer surface amines will form amide bonds and eliminate the positive charge character
of the pendent amino groups. Such PEG compounds may be a superior option to either suc-
cinylation or acetylation of amines to eliminate charge, because the PEG component also adds
considerable hydrophilicity and low interaction potential to the resultant dendrimer derivative
(Figure 7.12 ).
Acylation reactions to block amine groups on PAMAM dendrimers with anhydride com-
pounds are done in a similar manner to glycidol modifi cation. The following protocol is based
on the method of Majoros et al ., 2005.
Protocol
1. Dissolve an amine-containing dendrimer in methanol at a concentration of 15 mg/ml with
mixing. All operations should be done in a well-ventilated fume hood. A G-5 dendrimer was
used in this reaction by Majoros et al. (2005), which contained 110 available amine groups.
Figure 7.12 Amine-containing dendrimers can be modifi ed using a number of common reactive modifi cation
agents. Excess amine groups can be blocked using acetic anhydride, glycidol, or an NHS-mPEG compound.
Amines also can be converted into carboxylates using succinic anhydride.
2. Conjugation to Dendrimers 365
366 7. Dendrimers and Dendrons
2. Add to the dendrimer solution a quantity of acetic anhydride that represents a molar ratio
of anhydride-to-amines of 0.72:1.0 (680 l of acetic anhydride was added for the G-5 den-
drimer). Using a molar quantity of anhydride that is less than the amount of amines present
on the dendrimer assures that only a portion of the amines will become blocked, so that
further modifi cation remains possible.
3. Add a quantity of triethylamine to the solution so that a 25 percent molar excess over
the amount of anhydride will result (1.25 ml for the G-5 dendrimer).
4. React for 2 hours at room temperature with mixing (in a fume hood).
5. Extensively dialyze the reaction mixture against water or buffer to remove excess reactants.
Preparation of Sugar-Dendrimer Derivatives
The multifunctional nature of an amine-dendrimer can be used to advantage to mimic multi-
dentate interactions of molecules with cell surfaces or virus particles. For instance, the binding
affi nity of carbohydrate binding proteins (lectins) for individual sugar molecules typically is
weak, on the order of 10
6
M
1
. In the native method used to increase the binding strength of
these interactions, lectins on cell surfaces usually engage in multipoint attachments with car-
bohydrates or glycans. The conjugation of sugars to the pendent amine groups on dendrimers
provides a scaffold for similar multi-site interactions with lectins, which effectively increases
the avidity of the resultant binding complex (Aoi et al., 1995; Bertozzi and Kiessling, 2001).
This design is structurally similar to the multiple tree branched structures of glycans on glyco-
proteins. The terminal sugar groups on such glycoconjugates are capable of interacting with
more than one binding site or more than one receptor on cell surfaces. This effectively turns a
single low affi nity interaction into a high affi nity binding event, which forms the basis for many
life processes, including cellular recognition, adhesion, transport, and cell signaling (Clarke
and Wilson, 1988; Sharon and Lis, 1989). For a review of glycobiology, see the entire issue of
Science: Carbohydrates and Glycobiology, Vol. 291, March 23, 2001, pages 2263–2502.
A series of different mannose-dendrimers was synthesized to investigate their interaction with
Concanavalin A (Con A) (Woller and Cloninger, 2002; Woller et al., 2003). It was discovered that
the sugars on the dendrimer surface were able to bind to the Con A binding sites just like free
methyl mannose in solution. However, as the size of the dendrimer increased and number of mul-
tivalent mannose residues became available for binding to multiple Con A interaction sites, the
affi nity of the interaction dramatically increased. For a G-3 mannose-dendrimer derivative, the
sugar complex was about 45 times more active than methyl mannose in solution. For larger sized
mannose-dendrimer complexes, the increase in activity of binding was up to 660-fold greater
than free methyl mannose. However, the interaction potential for the mannose-dendrimer deriva-
tives also was shown to be dependent on the degree of mannose loading. For large dendrimers,
steric crowding of sugar molecules on the dendrimer surface decreased its binding activity toward
Con A if the mannose loading was greater than about 50 percent of the amines modifi ed. Thus,
both dendrimer size and the level of modifi cation must be carefully considered when designing
sugar-dendrimer conjugates.
Such sugar-dendrimer complexes ( “sugar balls ”) have been used to inhibit the interactions of
viruses with cell surfaces. Many viruses bind to particular carbohydrate residues on cell surfaces,
which in turn facilitate their entry into cells and the resultant infection process. A virus particle
presents a multi-dentate surface consisting of many carbohydrate-binding proteins able to inter-
act with multiple cell-surface carbohydrates. The surface of a dendrimer that is modifi ed with
sugar molecules is able to mimic the glycan rich fi eld on the surface of a cell. In this way, sugar-
dendrimer complexes are able to interfere with virus binding to cellular targets. For instance,
Borges et al. (2005) used dendrimers modifi ed with a carbohydrate usually found on immune
cell-surface glycosphingolipids to function as an inhibitor of HIV-1 infection. The polyvalent
nature of the sugar-dendrimer derivative was able to bind effectively to the virus particles with
high avidity, thus preventing the virus from binding to the carbohydrates on the immune cell
surface (Borges and Schengrund, 2005). The sugar-dendrimer complex in the vaginal ointment
Vivagel, developed by Starpharma, is undergoing clinical trials as a preventative for HIV infection
(Halford, 2005). The dendrimer formulation also is said to be active against other STDs, includ-
ing chlamydia, herpes simplex virus, hepatitis B, and human papilloma virus.
Sugar-dendrimer complexes also may be used as affi nity ligands for purifi cation of carbohy-
drate or sugar-binding proteins. Szwergold et al. (2001) used a polyvalent fructose-dendrimer
derivative immobilized on a chromatography matrix to purify human fructosamine-3-kinase.
The ligand was coupled to the support through excess amine groups on the dendrimers, which
were not coupled with sugar molecules, and gel functioned with high affi nity toward binding
the enzyme out of complex samples.
The synthesis of sugar-dendrimer derivatives may be done using a number of reaction strate-
gies. Aoi et al. (1995) used the lactone derivatives of lactose and maltose to react with the ter-
minal amine groups on G2–G4 PAMAM dendrimers. The reaction of a lactone with an amine
involves nucleophilic attack on the lactone carbonyl with ring opening to form an amide bond
(Figure 7.13 ). The conjugation was done in organic solvent (DMSO), but the reaction also may
be done in aqueous buffers under alkaline conditions.
The preparation of sugar-dendrimer conjugates may encompass a variety of modifi cation lev-
els and sizes, depending on the generation of dendrimer used and the density of sugar molecules
modifying its surface. Interactions with lectins vary according to the optimal sugar-to-sugar dis-
tance within the fi nal complex. For instance, it is known that the 3 binding sites on the mam-
malian hepatic lectin for interaction with galactose and N-acetylgalactosamine are arranged on
the protein in a triangle with a separation of 1.5 nm, 2.2 nm, and 2.5 nm (Lee et al., 1984). Aoi
et al. (1995) found that the molecular distances between sugar molecules on a G-3 PAMAM
dendrimer prepared according to their protocol was between 1.3 nm and 2.9 nm. Thus, the
sugar ball dendrimer had an optimal inter-sugar spacing to interact with the binding sites on the
targeted lectin.
The following protocol represents the method of Aoi et al. (1995) for the coupling of lac-
tone sugars to amine-dendrimers in organic solvent.
Figure 7.13 Lactone sugar derivatives can be used to react with amine-containing dendrimers, which results in
coupling via amide bonds. The “sugar ball ” dendrimers then can be used to specifi cally bind to carbohydrate
binding proteins.
2. Conjugation to Dendrimers 367
368 7. Dendrimers and Dendrons
Protocol
1. In a fume hood, dissolve 270 mg of a G-4 dendrimer (PAMAM or other amine contain-
ing) in 2 ml of dry DMSO. Maintain a nitrogen blanket over the reaction to prevent oxi-
dation. The use of a 3-necked fl ask and a heating mantle is recommended to control
mixing and the proper temperature.
2. Add with mixing, a 300-fold molar excess of a sugar lactone derivative, such as o -b- D -
galactopyranosyl-(1 ; 4)- D -glucono-1,5-lactone (2.6 gm) dissolved in 3 ml of DMSO.
3. React at 40°C for 9 hours or overnight with stirring.
4. After cooling to room temperature, the sugar-dendrimer derivative may be precipitated
with a large volume of methanol. The precipitate may be purifi ed from reaction products
by dialysis against water or buffer using a membrane with a molecular weight cutoff of
3500 Daltons. The fi nal product may be stored frozen or lyophilized to a white powder.
The modifi cation of amine-dendrimers with lactose also can be done using mono
(lactosylamido) mono(succinimidyl)suberate (Thermo Fisher). The amine-reactive compound
contains a lactose group at the end of a suberate bridge and terminates at the other end in an
NHS ester (Vetter et al., 1995). The NHS ester spontaneously reacts with the amine groups on a
dendrimer at neutral or slightly basic pH values to form an amide bond ( Figure 7.14 ). The pres-
ence of the lactose disaccharide provides a hydrophilic modifying group that maintains the water
solubility of the resultant conjugate.
Figure 7.14 The reaction of an amine-containing dendrimer with mono(lactosylamido) mono(succinimidyl)sub
erate results in the attachment of a lactose unit via an amide bond.
Protocol
1. Dissolve mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to prepare a
concentrated stock solution. The compound is extremely soluble in DMF, and solutions
of 100 mg/ml may be prepared.
2. 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,
0.15 M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the den-
drimer in the reaction buffer should be at least 10 mg/ml. Other dendrimer concentrations
also will work, but highly dilute solutions will result in less effi cient modifi cation yields.
3. With mixing, add a quantity of the mono(lactosylamido) mono(succinimidyl)suberate in
dry DMF to the dendrimer solution to result in at least a 10- to 20-fold molar excess
of reagent over the quantity of amines to be modifi ed in the dendrimer. Depending on
the desired application for the lactosyl-modifi ed dendrimer, several different molar ratios
may have to be tried to optimize the resulting modifi cation level.
4. React for 30–60 minutes at room temperature with gentle mixing.
5. Purify the modifi ed dendrimer away from reactants and reaction by-products using dialy-
sis or size exclusion chromatography.
Another method for coupling carbohydrates or sugar derivatives to amine-dendrimers is to take
advantage of the reducing end of sugars to reductively aminate the amines on the dendrimer sur-
face. This reaction will form stable secondary amine linkages between carbon-1 of the sugars and
the dendrimer ( Figure 7.15 ). Saccharides with reducing ends have carbonyl groups that can be
coupled to an amine-dendrimer in the presence of a reducing agent. However, since most reducing
ends of sugars or glycans exist mainly in an acetal (or ketal) ring form, and only the open form
with the exposed aldehyde can participate in a reductive amination reaction, the rate of modifi ca-
tion by this process can be slow. The open form of a reducing sugar is available at any given time
in only a small percentage of the total saccharide present, usually far less than 1 percent. For this
reason, reactions done with primary amines at room temperature may take days to reach accepta-
ble coupling yields. Typically, such reactions are done at elevated temperatures and over the course
of at least several days, or sometimes weeks, to reach completion.
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 carbohy-
drates, and even complex glycans after release from a protein (see Chapter 1, Section 4.6).
Protocol
1. Dissolve a carbohydrate, saccharide, or glycan sample having a free reducing end in
0.1 M sodium acetate, pH 5.0. Alternative coupling conditions that can be used for the
modifi cation reaction include 30 percent glacial acetic acid in DMSO (v/v) or acetic acid/
pyridine (1:2, v/v), if the dendrimer is soluble in these solutions. The use of DMSO or
pyridine often facilitates solubilization of a greater range of carbohydrates or glycans
than aqueous buffers. The presence of acetic acid has been found to accelerate the reduc-
tive amination reaction when the organic solvent conditions are used (Bigge et al., 1995).
A well-ventilated fume hood should be used for all organic reactions. The concentra-
tion of the carbohydrate should be 5–100 M for glycans, but for the modifi cation of
dendrimers with other more abundant carbohydrates, higher concentrations should be
2. Conjugation to Dendrimers 369