Hermanson G. Bioconjugate Techniques, Second Edition

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230 3. Zero-Length Crosslinkers

mechanisms have been used successfully in peptide synthesis (Paul and Anderson, 1960, 1962).

In addition, activation of a styrene/4-vinylbenzoic acid copolymer with CDI was used to immo-

bilize the enzyme lysozyme through its available amino groups to the carboxyl groups on the

matrix (Bartling et al ., 1973).

CDI functions as a zero-length crosslinker if the activated species is a carboxylic acid, because

the attack of another nucleophile liberates the imidazole leaving group. However, if CDI is used

to activate a hydroxyl functional group, the reaction proceeds quite differently. The active inter-

mediate formed by the reaction of CDI with an OH group is an imidazolyl carbamate ( Figure

3.13 ). Attack by an amine releases the imidazole, but not the carbonyl. Thus, a hydroxyl-

containing molecule may be coupled to an amine-containing molecule with the result of a

one-carbon spacer, and forming a stable urethane ( N-alkyl carbamate) linkage. This coupling

rocedure has been applied to the activation of hydroxyl-containing chromatography supports

for the immobilization of amine-containing afﬁ nity ligands (Bethell et al., 1979; Hearn et al .,

1979, 1983) and also to the activation of polyethylene glycol for the modiﬁ cation of amine-

containing macromolecules (Beauchamp et al., 1983). In addition, Chapter 14 describes the use

of CDI for the activation of particles to immobilize proteins or other afﬁ nity ligands.

CDI-activated hydroxyls also may undergo a side reaction to form active carbonates. This

occurs when an imidazolyl carbamate reacts with another hydroxyl group before the second

hydroxyl has had a chance to get activated with CDI. Particularly with adjacent hydroxyls on

the same molecule, this can be a problem if a deﬁ ned reactive species is desired. Any carbonates

formed, however, are still reactive toward amines to create carbamate linkages.

Formation of the activated species, whether with a carboxylate or a hydroxyl, must take

place in nonaqueous environments due to the rapid breakdown of CDI by hydrolysis. Even in

solvents containing small amounts of water, CDI quickly hydrolyzes to CO

and imidazole. It

is best to use solvents with less than 0.1 percent water to prevent extensive CDI breakdown.

Figure 3.13 CDI reacts with hydroxyl groups to form an active imidazole carbamate intermediate. In the pres-

ence of amine-containing compounds, a carbamate linkage is created with loss of imidazole.

Characteristic bubble formation is an indication of reagent hydrolysis, although CO

also is

released upon reaction with a carboxylic acid. Activation of carboxylates or hydroxyls may be

done in dry organic solvents such as acetone, dioxane, DMSO, THF, or DMF. If an excess of

CDI is used during the activation step, it should be removed before adding the active interme-

diate to an amine-containing molecule for conjugation. Alternatively, equal molar quantities of

CDI and the molecule to be activated may be mixed to form the active species. After about an

hour of activation, add an equivalent molar quantity of the amine-containing target molecule

to be conjugated.

Aqueous reaction conditions that result in the best conjugation yields using CDI usually

reﬂ ect the relative p K

of the nucleophilic amine being coupled. Proteins are best coupled to

CDI-activated supports or molecules in an environment at least one pH unit above their pI

values. Frequently the greatest coupling yields occur in alkaline buffers within the range of

pH 8.0–10.0. In aqueous solutions, CDI-activated carboxylates or hydroxyls will hydrolyze

and slowly lose activity. N-Acylimidazoles hydrolyze by loss of imidazole and regenerate the

original carboxylate. The imidazole carbamate active species hydrolyzes by loss of CO

and

imidazole, regenerating in this case, the original hydroxyl group. CDI-activated carboxylic

acids hydrolyze faster in aqueous solutions than CDI-activated hydroxyls; however, both expe-

rience increasing hydrolysis with increasing pH.

Conjugation reactions using CDI also may be done in organic solutions. This is a distinct

advantage if the reactants are not very soluble in aqueous environments. In addition, organic

coupling will not result in concomitant loss of activity due to hydrolysis as water-based reac-

tions, thus nonaqueous reactions usually will provide greater yields.

A protocol for the use of CDI in the activation of poly(ethylene glycol) is discussed in

Chapter 25, Section 1.4, while CDI activation procedures for particles are described in Chapter 14.

4. Schiff Base Formation and Reductive Amination

Aldehydes and ketones can react with primary and secondary amines to form Schiff bases.

A Schiff base is a relatively labile bond that is readily reversed by hydrolysis in aqueous solution.

The formation of Schiff bases is enhanced at alkaline pH values, but they are still not completely

stable unless reduced to secondary or tertiary amine linkages ( Figure 3.14 ). A number of reduc-

ing agents can be used to convert speciﬁ cally the Schiff base into an alkylamine linkage. Once

reduced, the bonds are highly stable. The use of reductive amination to conjugate an aldehyde-

containing molecule to an amine-containing molecule results in a zero-length crosslink where

no additional spacer atoms are introduced between the molecules.

Reductive amination (or alkylation) may be used to conjugate an aldehyde- or ketone-

containing molecule with an amine-containing molecule. The reduction reaction is best facili-

tated by the use of a reducing agent such as sodium cyanoborohydride, because the speciﬁ city

of this reagent is toward the Schiff base structure and will not affect the original aldehyde

groups. By contrast, sodium borohydride also is used in this reaction, but its strong reducing

power rapidly converts any aldehydes not yet reacted into non-reactive hydroxyls, effectively

eliminating them from further participation in the conjugation process. Borohydride also may

affect the activity of some sensitive proteins, whereas cyanoborohydride is gentler, successfully

4. Schiff Base Formation and Reductive Amination 231

232 3. Zero-Length Crosslinkers

preserving the activity of even some labile monoclonal a ntibodies. Cyanoborohydride has been

shown to be at least 5 times milder than borohydride in reductive amination processes with

antibodies (Peng et al., 1987). Other reducing agents that have been explored for reductive

amination include various amine boranes and ascorbic acid (Cabacungan et al., 1982; Hornsey

et al ., 1986).

Immobilization by reductive amination of amine-containing biological molecules onto alde-

hyde-containing solid supports has been used for quite sometime (Sanderson and Wilson, 1971).

The reaction proceeds with excellent efﬁ ciency (Domen et al., 1990). The optimum pH for the

reaction is alkaline, although good yield can be realized from pH 7 to 10. At the high end of this

range (pH 9–10), the formation of the Schiff bases is more efﬁ cient, and the yield of conjugation

or immobilization reactions can be dramatically increased (Hornsey et al., 1986).

The introduction of aldehyde functional groups into proteins and other molecules can be

accomplished by a number of methods (Chapter 1, Section 4.4). Glycoproteins may be oxidized

at their carbohydrate residues using sodium periodate or a speciﬁ c sugar oxidase. Amine groups

may be modiﬁ ed to produce a formyl group by reacting with NHS-aldehydes or p-nitrophenyl

diazopyruvate. The following generalized protocol assumes that the requisite groups are present

on the two molecules to be conjugated.

Protocol

1. Dissolve the amine-containing protein to be conjugated at a concentration of 1–10 mg/ml

in a buffer having a pH between 7 and 10. Higher pH reactions will result in greater yield

of conjugate formation. Suitable buffers include 0.1 M sodium phosphate, 0.15 M NaCl,

pH 7.2; 0.1 M sodium borate, pH 9.5; or 0.05 M sodium carbonate, 0.1 M sodium cit-

rate, pH 9.5. Avoid amine-containing buffers like Tris.

2. Add a quantity of the aldehyde-containing molecule to the solution in step 1 to obtain

the desired molar ratio for conjugation. For instance, if the amine-containing protein is

Figure 3.14 Carbonyl groups can react with amine nucleophiles to form reversible Schiff base intermediates. In

the presence of a suitable reductant, such as sodium cyanoborohydride, the Schiff base is stabilized to a second-

ary amine bond.

an antibody and the aldehyde-containing protein is an enzyme such as horseradish per-

oxidase (HRP), a typical molar ratio for the reaction might be 2–4 moles of HRP per

mole of antibody.

3. Add 10 l of 5 M sodium cyanoborohydride in 1 N NaOH (Aldrich) per ml of the con-

jugation solution volume. Caution: Highly toxic compound. Use a fume hood and be

careful to avoid skin contact with this reagent.

4. React for 2 hours at room temperature.

5. To block unreacted aldehyde sites, add 20 l of 3 M ethanolamine (pH adjusted to

desired value with HCl) per ml of the conjugation solution volume. React for 15 minutes

at room temperature.

6. Purify the conjugate by dialysis or gel ﬁ ltration using a buffer suitable for the nature of

the proteins being crosslinked.

4. Schiff Base Formation and Reductive Amination 233

The ﬁ rst crosslinking reagents used for modiﬁ cation and conjugation of macromolecules

consisted of bireactive compounds containing the same functionality at both ends (Hartman

and Wold, 1966). Most of these homobifunctional reagents were symmetrical in design with

a carbon chain spacer connecting the two identical reactive ends ( Figure 4.1 ). Like molecular

rope, these reagents could tie one protein to another by covalently reacting with the same com-

mon groups on both molecules. Thus, the lysine -amines or N-terminal amines of one protein

could be crosslinked to the same functionalities on a second protein simply by mixing the two

together in the presence of the homobifunctional reagent.

The ability to so easily link two proteins or other molecules having different binding spec-

iﬁ cities or catalytic activities opened the potential for creating a new universe of unique and

powerful reagent systems for use in assay and targeting applications. The variety and reac-

tivity of homobifunctional reagents multiplied dramatically throughout the 1970s and 1980s.

Today, there are dozens of commercially available crosslinkers possessing almost every length

and reactivity desired.

The main disadvantage, however, of using simple homobifunctional reagents is the potential

for creating a broad range of poorly deﬁ ned conjugates (Avrameas, 1969). When crosslinking

two proteins, for example, the reagent may react initially with either one of the proteins, form-

ing an active intermediate. This activated protein may form crosslinks with the second protein

or react another molecule of the same type. It also may react intramolecularly with other functional

Homobifunctional Crosslinkers

Figure 4.1 The general design of a homobifunctional crosslinking agent. The two reactive groups are identi-

cal and typically are located at the ends of an organic spacer arm. The length of the spacer may be designed to

accommodate the optimal distance between two molecules to be conjugated.

234

groups on part of its own polypeptide chain. In addition, other crosslinking molecules may

continue to react with these intermediates to form various mixed oligomers, including severely

polymerized products that may even precipitate (see Chapter 1, Section 1.2).

The problem of poorly deﬁ ned conjugation products is exacerbated in single-step reaction

procedures using homobifunctional reagents. Single-step procedures involve the addition of

all reagents at the same time to the reaction mixture. This technique provides the least control

over the crosslinking process and invariably leads to a multitude of products, only a small per-

centage of which represent the desired conjugate. Excessive conjugation may cause the forma-

tion of insoluble complexes that consist of very high-molecular-weight polymers. For example,

one-step glutaraldehyde conjugation of antibodies and enzymes (Chapter 20, Section 1.2)

often results in signiﬁ cant oligomers and precipitated conjugates. To overcome this short-

coming, two-step reaction procedures have been developed using homobifunctional reagents.

Controlled, two-step conjugation protocols somewhat alleviate the polymerization problem

with homobifunctional reagents, but can never totally avoid it.

In two-step protocols, one of the proteins to be conjugated is reacted with the homobifunc-

tional reagent and excess crosslinker and by-products are removed. In the second stage, the

activated protein is mixed with the other protein or molecule to be conjugated, and the ﬁ nal

conjugation process occurs ( Figure 4.2 ).

One potential problem of such two-step procedures is hydrolysis of the activated intermedi-

ate before addition of the second molecule to be conjugated. For instance, N-hydroxysuccinimide

(NHS) ester homobifunctionals hydrolyze rapidly and may degrade before the second stage of

the crosslinking is initiated. In addition, the use of homobifunctional reagents in two-step pro-

tocols still produces many of the problems associated with single-step procedures, because the

ﬁ rst protein can crosslink and polymerize with itself long before the second protein is added.

Since the ﬁ rst protein to be activated has target functional groups on every molecule that can

couple with both reactive groups of the crosslinker, both ends of the reagent potentially can

react. This inherent capacity to polymerize uncontrollably unfortunately is characteristic of all

homobifunctional reagents, even in multi-step protocols.

Although their shortcomings in this regard are clearly recognized, homobifunctional rea-

gents continue to be popular choices for all kinds of conjugation applications. The fact is,

in many crosslinking functions, they work well enough to form effective conjugates. Even

glutaraldehyde-mediated antibody–enzyme conjugates still are commonly utilized in everything

from research to diagnostics.

The particular crosslinkers discussed in this section are the types most often referred to in

the literature or are commercially available. Many other forms of homobifunctional reagents

containing almost every conceivable chain length and reactivity can be found mentioned in the

scientiﬁ c literature.

1. Homobifunctional NHS Esters

Carboxylate groups activated with NHS esters are highly reactive toward amine nucle-

ophiles. In the mid-1970s, NHS esters were introduced as reactive ends of homobifunctional

crosslinkers (Bragg and Hou, 1975; Lomant and Fairbanks, 1976). Their excellent reactivity at

physiological pH quickly established NHS esters as viable alternatives to the imidoesters pre-

dominating at the time (Section 2, this chapter).

1. Homobifunctional NHS Esters 235

Figure 4.2 Homobifunctional crosslinkers may be used in a two-step process to conjugate two proteins or

other molecules. In the ﬁ rst step, one of the two proteins is reacted with the crosslinker in excess to create an

active intermediate. After removal of remaining crosslinker, a second protein is added to effect the ﬁ nal con-

jugate. Two-step reaction schemes somewhat limit the degree of polymerization obtained when using homo-

bifunctional reagents, but can ’t entirely prevent it.

Unfortunately, many NHS ester-containing crosslinkers are insoluble in aqueous buffers.

Most protocols involve dissolving the compound at a relatively high concentration in an

organic solvent and aliquoting the required quantity into the reaction medium. Prior dissolu-

tion helps to maintain at least some solubility in the buffered crosslinking environment. Most

of the time, however, the addition of an organic-solubilized crosslinker into a buffered solution

results in a microprecipitate that slowly goes into solution during the course of reaction.

In the early 1980s, Staros prepared a derivative of NHS that aids in the water solubility of

NHS ester crosslinkers (Staros, 1982). N-hydroxysulfosuccinimide (sulfo-NHS) esters possess a

negatively charged sulfonate group on carbon number 2 or 3 of the succinimide ring ( Figure 4.3 ).

Crosslinking reagents-containing sulfo-NHS esters have half-lives of hydrolysis on the order of

hours, sometimes even better than their NHS ester analogs (Anjaneyulu and Staros, 1987). The

sulfo-NHS ester often lends enough charge and polarity to a crosslinker to provide water solubil-

ity and thus eliminate the need for organic solvent dissolution. In addition, sulfo-NHS crosslinkers

may be used for surface only modiﬁ cation of membranes and cells, since they are more hydrophilic

and will not penetrate the lipid environment of the membrane (Staros, 1982, 1988; Staros et al.,

1987). By contrast, many of the more hydrophobic NHS ester crosslinkers can be used to traverse

the cell membrane and modify intracellular components.

NHS or sulfo-NHS ester-containing homobifunctional crosslinkers react with nucleophiles

to release the NHS or sulfo-NHS leaving group and form an acylated product. The reaction of

such esters with a sulfhydryl or hydroxyl group is possible, but doesn ’t yield stable conjugates,

forming thioesters and ester linkages which may hydrolyze in aqueous environments. Histidine

side chain nitrogens of the imidazolyl ring also may be acylated with an NHS ester reagent, but

they too hydrolyze rapidly (Cuatrecasas and Parikh, 1972). Reaction with primary and second-

ary amines, however, creates stable amide and imide linkages, respectively, that don ’t readily

Figure 4.3 In aqueous solution, a sulfo-NHS ester can either couple to an amine group to form an amide bond

or react with water to hydrolyze back to a carboxylate. Both processes release the sulfo-NHS leaving group.

1. Homobifunctional NHS Esters 237

238 4. Homobifunctional Crosslinkers

break down. In protein molecules, NHS ester crosslinking reagents primarily react with the  -

amines at the N-terminals and the abundant  -amines of lysine side chains.

NHS ester crosslinking reactions in aqueous solutions consist of the potential for hydrolysis

as well as the desired amide bond formation. Crosslinkers-containing NHS esters have fairly

good stability in aqueous solutions, despite their susceptibility to attack and breakdown by

water. Studies done on the NHS ester-containing homobifunctional reagent, dithio bis (succini

midylpropionate) (DSP), indicate that the activated carboxylates have half-lives on the order

of hours at physiological pH. However, hydrolysis and amine reactivity both increase with

increasing pH. At 0 °C at pH 7.0, the half-life of the crosslinking reagent DSP is 4–5 hours

(Lomant and Fairbanks, 1976). At pH 8.0 at 25 °C it falls to about 1 hour (Staros, 1988), and

at pH 8.6 and 4 °C the half-life is only 10 minutes (Cuatrecasas and Parikh, 1972).

The rate of hydrolysis may be monitored by measuring the increase in absorptivity at

260 nm as the NHS leaving group is cleaved. The molar extinction coefﬁ cient of the NHS

group in solution is 8.2  10

1

in Tris buffer at pH 9.0 (Carlsson et al., 1978), but

somewhat decreases to 7.5  10

1

 1

in potassium phosphate buffer at pH 6.5 (Partis

et al., 1983). Unfortunately, the sensitivity of this absorptivity usually does not allow for meas-

uring the rate of reaction in an actual crosslinking procedure.

To maximize the modiﬁ cation of amines and minimize the effects of hydrolysis, main-

tain a high concentration of protein or other target molecule. By adjusting the molar ratio of

crosslinker to target molecule(s), the level of modiﬁ cation and conjugation may be controlled

to create an optimal product.

The reaction buffer chosen for the conjugation reaction should be free of extraneous

amines. Avoid Tris or glycine buffers. Also, avoid imidazole buffers, since the nitrogens of the

imidazole ring may react with the active ester and then quickly hydrolyze. The effect is that

imidazole only acts to catalyze the hydrolysis process. The pH of the reaction should be in

the range of 7–9 to promote the unprotonated state of primary amines, which is the nucle-

ophilic species that most effectively attacks the activated carbonyl group. Dissolve NHS ester

crosslinkers that are insoluble in water in an organic solvent such as DMF or DMSO prior to

addition to the reaction medium. Sulfo-NHS crosslinkers may be added directly to the reaction

mixture or pre-dissolved in buffer at a higher concentration before adding an aliquot to the

reaction. Aqueous stock solutions should be used immediately to prevent extensive hydrolysis

of the active esters.

NHS ester crosslinking reagents also may be used in organic solvent-based reactions without

the competing hydrolysis problem provided the target molecules are soluble and stable in such

environments. In this case, both molecules to be conjugated must be soluble in the solvent.

DMF, DMSO, acetone, and dioxane are examples of solvents that can be utilized as the reac-

tion medium. Refer to any published solubility data on the crosslinking reagent of choice to see

which solvents are most appropriate.

1.1. DSP and DTSSP

Lomant ’s reagent [dithiobis(succinimidylpropionate), DSP] is a homobifunctional NHS ester

crosslinking agent containing an 8-atom spacer of 12 Å in length (Lomant and Fairbanks,

1976) (Thermo Fisher). It is symmetrically constructed around a central disulﬁ de group that is

cleavable after conjugation with typical disulﬁ de reducing agents (Chapter 1, Section 4.1).

DSP is water-insoluble and must be pre-dissolved in an organic solvent before addition to a

conjugation reaction. Concentrated stock solutions may be prepared in DMF or DMSO and an

aliquot added to a buffered reaction medium. The NHS ester reaction occurs most efﬁ ciently at

pH 7–9, with hydrolysis of the active species accelerating the greater the pH. The crosslinking

buffers should be free of amine-containing components other than the target molecules to be

conjugated. Avoid Tris or glycine buffers. A reaction buffer consisting of 0.1 M sodium phos-

phate, 0.15 M NaCl, pH 7.2–7.5 works well for most applications involving the crosslinking

of two puriﬁ ed proteins. The relatively high concentration of sodium phosphate is used to pre-

vent pH drift downward during the course of the reaction. For in vitro crosslinking of cellular

components such as membrane proteins, a more dilute PBS buffer-containing isotonic saline is

more appropriate. Since DSP is a hydrophobic reagent, it is able to penetrate the cell membrane

and conjugate membrane components. For this reason, it has become quite popular for use in

investigating the interactions of membrane proteins.

DSP reacts with -amine groups on the side chains of lysine residues or the -amine at the

N-terminal of proteins to form amide linkages. Amine-containing macromolecules may be

reversibly crosslinked with this reagent and later cleaved with dithiothreitol (DTT) or 2-mer-

captoethanol ( Figure 4.4 ). For reductive cleavage of conjugated molecules, add 10–50 mM

DTT, and incubate at 37 °C for 30 minutes. Alternatively, the conjugate may be reduced prior

to electrophoresis using SDS sample buffer with 5 percent 2-mercaptoethanol at elevated

temperatures.

Lomant’s reagent is one of the most popular of all crosslinking agents, especially for the

investigation of protein interactions. Hordern et al. (1979) used it to investigate the spatial rela-

tionships in the capsid polypeptides of the mengo virion. It has been used to study the interac-

tions of proteins involved with active transport (dePont et al., 1980; Joshi and Burrows, 1990),

identifying crosslinks involving cytochrome P-450 (Baskin and Yang, 1982), characterization

of cell surface receptors for colony-stimulating factor (Park et al., 1986), the determination of

various membrane antigens by crosslinking to speciﬁ c monoclonal antibodies (Hamada and

Tsuro, 1987), studying prothrombin self-association (Tarvers et al., 1982), investigating

1. Homobifunctional NHS Esters 239