Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

340 6. Trifunctional Crosslinkers

Protocol

1. Dissolve 5 mg of STI in 0.5 ml 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2.

2. In a fume hood, dissolve 1.12 mg of sulfo-SBED in 25 l of DMSO. Prepare fresh and

protect from light.

3. Add 11  l of the sulfo-SBED solution to the STI solution. Mix well.

4. React for 30 minutes at room temperature or for 2 hours at 4°C.

5. If some precipitation occurs, clarify the solution by centrifugation using a microfuge.

Remove excess reactant by gel ﬁ ltration using a desalting resin.

6. Mix the puriﬁ ed sulfo-SBED-modiﬁ ed STI with 5 mg of trypsin dissolved in 0.1 M

sodium phosphate, 0.15 M NaCl, pH 7.2.

7. Incubate at room temperature 3.5 minutes to allow the speciﬁ c binding of the two mol-

ecules to occur.

8. Photolyze the solution with long UV light (about 365 nm) at a distance of about 5 cm for 15

minutes. This process may be done with the solution on ice to prevent heating of the sample.

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester

side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink for-

mation with a second interacting protein. The biotin portion provides puriﬁ cation or labeling capability using

avidin or streptavidin reagents. The disulﬁ de bond on the NHS ester arm provides cleavability using disulﬁ de

reductants, which effectively transfers the biotin label to an unknown interacting protein.

Isolation of complexed molecules may be done by afﬁ nity chromatography using a column of

immobilized avidin or immobilized streptavidin. Cleavage of the disulﬁ de bond of the crosslinker

may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the

use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1.

3. MTS-ATF-Biotin and MTS-ATF-LC-Biotin

MTS-ATF-biotin and MTS-ATF-LC-biotin are trifunctional crosslinkers similar in design

to sulfo-SBED discussed previously, but in addition to the biotin handle, they contain a

3. MTS-ATF-Biotin and MTS-ATF-LC-Biotin 341

342 6. Trifunctional Crosslinkers

thiol-reactive group and an enhanced photoreactive, perﬂ uorinated phenyl azide group.

The two reagents differ only in the length of the cross-bridge in the photoreactive arm, with

the LC version containing an extended aminocaproyl spacer. Relative to the spacing possible

between the reactive groups on these compounds, the LC version therefore provides nearly

twice the maximal molecular distance over its shorter analog (21.8 Å versus 11.1 Å). Thus,

interacting proteins may be captured either through use of a long or short crosslink, depend-

ing on the optimal distances between the proteins—or at least to the nearest thiol on the bait

protein.

Both MTS-ATF-biotin and MTS-ATF-LC-biotin contain a methanethiolsulfonate group

(MTS) on one arm, which is able to couple with thiols. This reaction proceeds with loss of the

methyl sulfonate leaving group (sulﬁ nic acid) and forms a disulﬁ de linkage ( Figure 6.3 ). Unlike

a pyridyl disulﬁ de group, however, which also reacts with thiols to form disulﬁ de linkages, the

MTS group is unstable to hydrolysis in aqueous solution, especially if other strong nucleophiles

are present. Therefore, most MTS compounds dissolved or brought into PBS buffer at physi-

ological pH will hydrolyze with a half-life on the order of 10–15 minutes. However, they also

have very rapid reactivity with thiols (Stauffer and Karlin, 1994; Holmgren et al., 1996; Liu

et al., 1996). The reaction of an MTS group with a thiol on a bait protein can take place with

high yields in just a matter of minutes. Both of these trifunctional label transfer compounds are

hydrophobic, so their MTS reactivity in the aqueous phase may be somewhat slower than cor-

responding hydrophilic MTS reagents.

For use in studying protein interactions, these compounds ﬁ rst are reacted with a thiol on

a puriﬁ ed bait protein to form a disulﬁ de bond. Since the reagents are water-insoluble, they

must be dissolved in an organic solvent such as DMF or DMSO and then an aliquot added to

the bait protein in an aqueous buffer to initiate the reaction. Once modiﬁ ed, the biotinylated

bait protein then is incubated with a sample containing potentially interactive prey proteins.

After an incubation period, initiating the photoreaction by exposure to UV irradiation captures

the interacting proteins. Any interaction complexes thus formed can be isolated or detected

using the biotin handle. In addition, the disulﬁ de bond formed with the bait protein during the

crosslinking reaction can be reduced to cleave the conjugates and transfer the biotin label to

the unknown interacting prey proteins. This is a powerful way of labeling unknown interacting

proteins for subsequent analysis.

Although MTS-ATF-biotin and MTS-ATF-LC-biotin are available commercially (Thermo

Fisher and Toronto Research), they are relatively new and don ’t have the publications or appli-

cations backing up their use as sulfo-SBED. A protocol for the use of these compounds in the

study of protein interactions can be found in Chapter 28, Section 3.2.

4. Hydroxymethyl Phosphine Derivatives

Although phosphine compounds often are used as disulﬁ de reducing agents (Chapter 1, Section

4.1), there are classes of organo-phosphine reagents containing three hydroxymethyl groups

that can act as trifunctional bioconjugation agents for coupling or crosslinking purposes. Tris

(hydroxymethyl)phosphine (THP) and -[tris(hydroxymethyl)phosphino] propionic acid (THPP;

Thermo Fisher) are small trifunctional compounds that spontaneously react with nucleophiles,

such as amines, to form covalent linkages (Henderson et al., 1994; Katti, 1996; Katti et al.,

Figure 6.3 Mts-Atf-Biotin can be used to label bait proteins at available thiol groups using the MTS

group, which forms a disulﬁ de linkage after reaction. The modiﬁ ed protein then is allowed to interact with

a protein sample and photoactivated with UV light to cause a covalent crosslink with any interacting

proteins. Cleavage of the disulﬁ de bond effectively transfers the biotin label to the unknown interacting

protein.

4. Hydroxymethyl Phosphine Derivatives 343

344 6. Trifunctional Crosslinkers

1999). Nucleophiles react with the hydroxymethyl arms by attack on the electron-deﬁ cient car-

bon atom with loss of water to form secondary or tertiary amine bonds ( Figure 6.4 ).

Both THP and THPP are stable in aqueous solution, as the only potential product

of hydrolysis is the reformation of the hydroxymethyl groups. It is unusual for an amine-

reactive functional group to have long-term stability in water or buffer, which makes these

reagents uniquely suitable for creating reactive surfaces or reactive molecules for subsequent

Figure 6.4 THPP reacts with amine-containing molecules to form secondary or tertiary amine bonds.

conjugation with proteins or other amine-containing compounds. Hydroxylic chromatographic

supports also have been activated with hydroxymethyl phosphine derivatives for immobiliza-

tion of enzymes (Petach et al. , 1994).

Hydroxymethyl phosphines are susceptible to oxidation to form the phosphine oxide.

Therefore, avoid excess oxygen, oxidizing agents, or azide compounds, which react with phos-

phines in the Staudinger reaction (Chapter 17, Section 5). In addition, metallic surfaces can be

modiﬁ ed via the phosphine group to result in hydroxymethyl group substitutions.

4. Hydroxymethyl Phosphine Derivatives 345

1. Dendrimer Construction

The science of nanotechnology has employed many different constructs having low nanometer

dimensions, including inorganic scaffolds, biological macromolecules, and various forms of

polymers and particles. One of the most deﬁ ned nanoparticle constructs is a unique polymeric

assemblage called a dendrimer. First described in 1983 by Tomalia and Dewald in the applica-

tion to U.S. patent 4,507,466 and again in 1985 by Tomalia et al. and Newkome et al., den-

drimers are monodisperse, globular macromolecules grown by successive synthetic steps from

a central core molecule. Each step, called a generation, adds a distinct layer to the previous one

so that a dendrimer grows out from the core like the branches of a tree. In fact, the name den-

drimer comes from the Greek word for tree, or “dendron”. The result is a polymeric molecule

having a fractal dimensional quality with properties and shape that is determined by the types

of monomers used to grow the branches.

Each step in dendrimer synthesis occurs independent of the other steps; therefore, a dendrimer

can take on the characteristics deﬁ ned by the chemical properties of the monomers used to con-

struct it. Dendrimers thus can have almost limitless properties depending on the methods and

materials used for their synthesis. Characteristics can include hydrophilic or hydrophobic regions,

the presence of functional groups or reactive groups, metal chelating properties, core/shell dissim-

ilarity, electrical conductivity, hemispherical divergence, biospeciﬁ c afﬁ nity, photoactivity, or the

dendrimers can be selectively cleavable at particular points within their structure.

Dendrimers have been used for many diverse applications within the biological, chemical, poly-

mer, and nanotechnology ﬁ elds. Some major applications include their use as multivalent biocon-

jugation scaffolds, for enhancement of signals in assays, to solubilize hydrophobic molecules in

aqueous environments by internal entrapment, to functionalize surfaces and particles for conjuga-

tion, as transfection agents for cells, to create targeted therapeutic constructs for the treatment of

disease, as carriers of afﬁ nity ligands, and as additives for other polymer mixtures. For excellent

reviews of dendrimer technology, see Fréchet and Tomalia, 2002, as well as Boas et al., 2006.

The synthesis and structure of a dendrimer can be illustrated by the well-known poly

(amidoamine) type (called PAMAM), which describes the monomers making up the complete pol-

ymer. The synthesis starts from a core diamine (or ammonia) molecule. The diamine can be of var-

ious lengths and spacer arm properties and even contain cleavable components. Typically, the core

Dendrimers and Dendrons

346

is a short diamine, such as ethylenediamine. The ﬁ rst reaction that is done to form a dendrimer is

reacting the core with methyl acrylate to form the Michael addition product, a tetra-methyl ester

branched molecule. Next ethylenediamine is again added in large excess to the tetra-methyl ester

intermediate, which undergoes amidation to form the tetra-amidoethylamine generation-0 (G-0)

product containing 4 pendent amines. Another round of methyl acrylate addition followed by

ethylenediamine yields the 8-amine generation 1 (G-1) PAMAM dendrimer ( Figure 7.1 ).

Similar successive additions of methyl acrylate and ethylenediamine result in progressively

higher generation dendrimers, which branch out to larger diameters and contain greater num-

bers of amines on their surface. For dendrimers of the classic PAMAM type, each additional gen-

eration results in doubling the number of pendent amine groups decorating its surface, because

alkylation of each terminal amine can be done twice with two molecules of methyl acrylate. Since

each step in dendrimer synthesis adds greater branching of monomer units as they grow out from

the core, the design has become known as “ starburst ” dendrimers. Figure 7.2 illustrates graphi-

cally how the growth of dendrimers built from a bifunctional core results in ever more branching

as the generational size increases. The corresponding G-3 and G-4 PAMAM dendrimer chemical

structures are shown in Figures 7.3 and 7.4 . Although a two-dimensional depiction of this den-

drimer structure may look like the molecule has nearly perfect circular symmetry, its true three-

dimensional structure actually appears more asymmetrical for lower generation dendrimers and

like a complex globular protein ( Figure 7.5 ) for generations above G-4.

There are two main methods of synthesizing dendrimers: (1) the divergent method, which

involves building the core outward in successive steps or generations as described above for

PAMAM dendrimers and (2) the convergent method (Hawker and Fréchet, 1990; Hodge, 1993;

Grayson and Fréchet, 2001), which consists of building a single branched tree as it would grow

out from the core in the divergent method, but in this case, after synthesis of individual trees,

they are linked to the core structure as single units ( Figure 7.6 ). Thus, convergent dendrimers

are constructed from the outside in. One advantage of the convergent synthesis strategy is that

different dendritic starting materials (dendrons) can be built and combined to form a segment-

block dendrimer or a layer-block dendrimer, which consists of polymers of different types within

the same dendrimer structure. The only major disadvantage of using the convergent approach to

making dendrimers is that the result is limited to rather small dendritic molecules, because as the

size of the building blocks increases steric crowding prevents efﬁ cient reaction of all the dendrons

with the core.

A third method of constructing dendrimers is through self-assembly of engineered building

blocks. When the blocks are put together under the correct conditions in solution, they spon-

taneously assemble into dendritic structures. For instance, dendrimers have been assembled

through use of chelating components, which assemble into dendritic structures upon addition

of the appropriate metal ions (Denti et al., 1992; Balzani et al., 1996; Kawa and Fréchet, 1998).

Oligonucleotide dendrimers also have been formed by using intelligently designed sequences

that hybridize to other oligos in such a way that dendritic molecules spontaneously are created

in solution (Genisphere technology). In addition, dendrimers have been made from dendron

trees containing interior groups that can self-assemble through hydrogen bonding with groups

on neighboring trees, thus forming the complete dendrimer as the adjacent groups interact at

the core (Hudson et al., 1997; Percec et al., 1998).

Finally, dendrimers have been synthesized using solid phase peptide synthesis resins, wherein

the core is linked to the resin and the half-dendrimer (dendron) is built out from it in sequential

steps (Marsh et al., 1996; Swali et al., 1997; Wells et al., 1998). The advantage of this method

1. Dendrimer Construction 347

348 7. Dendrimers and Dendrons

Figure 7.1 The synthesis of a PAMAM-type dendrimer proceeds from a diamine core [e.g., ethylene diamine

(EDA)] by initial addition of the amines to the double bonds of methacrylate. Subsequent reaction of the methyl

ester groups with EDA produces a G-0 dendrimer with four pendent amine groups. Another round of methacr-

ylate and EDA additions results in a G-1 PAMAM dendrimer containing eight primary amines.

Relevant A Head 349

Figure 7.3 The chemical structure of a G-3 PAMAM dendrimer.

Figure 7.2 A graphical illustration of the growth of dendrimer structures from an initial bifunctional core to a

G-4 dendrimer containing 64 terminal groups on its outer surface.