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4 Cross-Linking Reagents Commonly Used for Immobilization of Biomolecules

4 Cross-Linking Reagents Commonly Used for Immobilization of Biomolecules

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416



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



Different carbodiimides, including water-soluble compounds described in Chapter 8, have been

used for coupling amino groups of proteins to carboxyl group–containing solid matrices.40 Among the

chloroformates, ethyl chloroformate, p-nitrophenyl chloroformate, 2,4,5-trichlorophenyl chloroformate, and N-hydroxysuccinimide chloroformate have been used.57,59 During the reaction, the carboxyl

group first reacts with the cross-linker to form an activated species, which is attacked by the amino

group nucleophile. Some reaction schemes for these reagents are shown in Chapter 8, Figure 8.5.

Other reagents that can be considered zero-length cross-linkers are sulfonyl chlorides and

2-fluoro-N-methylpyridinium tosylate.60 The most commonly used sulfonyl chlorides are tresyl

chloride (2,2,2-trifluoroethanesulfonyl chloride),61 although p-toluene sulfonyl chloride (tosyl chloride)

and colored sulfonyl chloride, 3,5-dinitro-4-dimethylaminobenzenesulfony chloride (diabsyl

chloride), have also been used.57,62 These reagents activate the primary hydroxyl groups into good

leaving groups as shown in Figures 14.9A for tresyl chloride. The activated function is then

displaced by a biological nucleophile, chiefly the amino side chain of lysine.

It should be pointed out that not only amino groups of proteins serve as nucleophiles; free sulfhydryl, if present, can potentially be coupled. The linking between a carboxyl group and an amino

group forms an amide bond, whereas a thioester bond is formed when a sulfhydryl group is involved.

Similarly, coupling primary alcohols using sulfonyl chloride and 2-fluoro-N-methylpyridinium salt

affords a secondary amine with amino group and a thioether bond with free thiol, as shown in

Figure 14.9.

Various different solid matrices have been activated by these zero-length cross-linkers, and

many enzymes and proteins have been immobilized this way. Carboxyl groups containing supports

such as carboxymethyl cellulose, acrylamide and acrylic acid copolymer, carboxymethyl Sephadex,

BioGel, carboxymethyl agarose, and polyacrylic acid have been used. Polyhydroxylic matrices that

have been activated by zero-length cross-linkers are agarose, glycerylpropyl-silica, cellulose, and

hydroxyethyl methacrylate.

Thiol-containing matrices such as thiopropyl-sepharose can be activated by 2,2′-dipyridyldisulfide through the thiol-disulfide interchange reaction.63,64 The protein is bonded via its free thiol

through a second thiol-disulfide interchange with the liberation of 2-thiopyridine according to

Figure 14.10. This method generates a disulfide bond between the protein and the solid support,

which is stable under nonreducing conditions.64 The enzyme can be released by low–molecularweight thiol compounds. For proteins that do not contain a free thiol, it can be thiolated using various reagents such as N-acetylhomocysteine thiolactone as described in Chapter 2.

(A)



OH



O

Cl S

O



CF3



O

O S

O



CF3



H2N



P



HS



P



(B)



F

OH



H2N



P



HS



P



H

N



P



S



P



H

N



P



S



P



N+

CH3

O



N+

CH3



FIGURE 14.9  Coupling proteins to hydroxyl matrix with (A) tresyl chloride and (B) 2-fluoro-1-methylpyridinium salt.



417



Application of Chemical Conjugation to Solid-State Chemistry



S



N

S



N



P



HS



N



S



SH



S

P



S



S



FIGURE 14.10  Coupling of proteins to thiol-containing matrices by thiol-disulfide interchange.



14.4.2  Use of Mono- and Homobifunctional Cross-Linkers

Almost any of the mono- and homobifunctional cross-linkers listed in Chapters 5 and 8 can be used

one way or another to immobilize proteins. Depending on the matrix functional groups, different

cross-linkers will have to be used. The following reactions demonstrate the use of these reagents.

14.4.2.1  Glutaraldehyde

Glutaraldehyde is the most·prevalent homobifunctional reagent used for the immobilization of biomolecules. The reaction is dependent on pH, temperature, and ionic strength.65 As presented in

Chapter 5, the chemistry of cross-linking by glutaraldehyde is complex. There is no consensus on

the main reactive species that participates in the cross-linking process because monomeric and

polymeric forms are in equilibrium. Although the amino group is assumed to be the primary function to react, other functional groups, such as imidazole, thiol, and hydroxyl, have also been implicated.66 Thus, glutaraldehyde has been used to couple proteins and other biomolecules to cellulosic

materials,67 silica gel,68 polyacrylhydrazide,69 nylon,70 polyethyleneimine-treated magnetite,71 and

carbon nanotube fiber72 as well as in the immobilization of glucose oxidase (GOD) for enzyme

glucose electrode use and cross-linking chitosan/polyethylene glycol beads for drug delivery.73 Both

one-step and two-step cross-linking procedures have been used. For the two-step procedure, the

matrix is first activated with glutaraldehyde, washed, and then coupled with the protein.

14.4.2.2  Chloroformates and Carbonyldiimidazole

A reaction similar to cyanogen bromide activation of vicinal hydroxyl groups is the formation

of the cyclic carbonate derivative with 1,1′-carbonyldiimidazole, ethyl chloroformate, or other

alkyl or aryl chloroformates (which are monofunctional agents, see Chapter 8) in anhydrous

organic solvents. The activated cyclic carbonate will react with nucleophiles in biomolecules at

pH 7–8 to form substituted carbonate bonds as shown in Figure 14.11.74 When an amino group

from a protein serves as the attacking nucleophile, a N-substituted carbamate bond is formed.

A thiol carbonate bond results when thiol is involved. A carbonyl moiety from the cross-linker

O

O



O



Cl



O R



O R



O

N



N



N



O



OH



OH

OH



O



H2N



OH

O

N



O



N



N



P



O



OH



P



H2N



O

O



N

H



P



OH

O

O



N

H



P



FIGURE 14.11  Immobilization of proteins on matrices containing vicinal diols with either chloroformates

or 1,1′-carbonyldiimidazole.



418



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation

Cl

N

Cl



OH



Cl



N

N



N



Cl

O



Cl

H2N



N

N



P



N



Cl



O



N

N



P

N

H



FIGURE 14.12  Immobilization of proteins with cyanuric chloride.



is incorporated into the bond. The carbamate and thiol carbonate bonds are not very stable and

may be hydrolyzed under extreme pHs.

14.4.2.3  Heterocyclic Halides

s-Triazines (see Chapter 5), such as cyanuric chloride and some of its dichloro derivatives, 2-amino

4,6-dichloro-s-triazine, 2-carboxy-methylamino-4,6-dichloro-s-triazine, and 2-carboxymethoxy4,6-dichloro-s-triazine, have been used to couple enzymes to hydroxyl and amino group containing

matrices such as cellulose, agarose, and polyvinylalcohol through the activation of the hydroxyl

group as shown in Figure 14.12.75,76 The coupling process is an alkylation reaction involving the

primary amino groups of proteins with the activated carbon of the s-triazine molecule.

14.4.2.4  Bisoxiranes

Introduction of epoxides onto matrix surfaces can be achieved by bisoxiranes, for example,

1,4-butanediol diglycidyl ether (1,4-bis(2,3-epoxypropoxy)butane). The reaction occurs readily at

alkaline pH to yield derivatives containing a long-chain hydrophilic function with a reactive epoxide. This method is suitable for hydroxyl-containing supports, which form ether linkages through

their hydroxyl groups.77 Other matrices containing the amino and thiol groups can also be modified. de Souza et al.78 immobilized ω-aminohexyl diamine onto bisoxirane-activated agarose gel for

purification of IgG by negative chromatography. Immobilization occurs when nucleophilic amino

group reacts with the epoxide as shown in Figure 14.13A. The bisoxiranes have also been used to

couple enzymes to agarose solid supports.64 Epoxides can be directly linked to hydroxyl group–

containing matrices like silica by direct silianization with silane compounds containing an epoxide

such as 3-glycidoxypropyltrimethoxysilane (which would be a heterofunctional agent).79 Like the

bisoxiranes, the epoxy function of the organomodified silica reacts with the amino function to

immobilize the protein as shown in Figure 14.13B. Due to its high reactivity, other nucleophiles

like hydroxyl or thiol moieties can also react with the epoxy group; hence, the selectivity to exclusively link specific moieties is low.



(A)



O

OH



O



OH



O

O



O

O

O Si



(B)



O



O



OH



O



O



O



H2N



P

O



OH



OH H

N



O



O



P



O

O

O

O Si



O



O



H2N



P



O

O

Si

O



O



OH H

N



P



FIGURE 14.13  Immobilization of proteins with (A) bisoxirane: 1,4-butanediol diglycidyl ether; (B)

3-glycidoxypropyltrimethoxysilane.



419



Application of Chemical Conjugation to Solid-State Chemistry



14.4.2.5  Divinylsulfone

Divinylsulfone has been used to modify hydroxyl-containing matrices to vinylsulfone similar to its

use with soluble proteins as discussed in Chapter 5.64,77 The vinyl group is very reactive and reacts

rapidly with nucleophiles of proteins, such as amines, alcohols, and phenols. However, the final

linkage is unstable. The product with a hydroxyl function is unstable above pH 9 and that with an

amino group is unstable above pH 8.77 However, Rekuc´ et al.80 showed that linkage to the amino

anchor of derivatized cellulose gave increased stability of the immobilized laccase compared to

other anchor groups.

14.4.2.6  Quinones

Proteins can be immobilized onto solid supports such as agarose by quinones, for example, p-benzoquinone.64 The reaction is similar to protein cross-linking as discussed in Chapter 5. The matrix

is first activated with the quinone. Proteins are then bound through their nucleophilic groups with

high yields. The coupling reaction can occur in a broad range from pH 3 to 10. Undesirable side

reactions may occur rendering a dirty color to the matrix. The reagent has also been used for the

activation of other matrices including silica and polyacrylamide gels.81,82

14.4.2.7  Transition Metal Ions

Transition metal ions that form stable hexaqua complexes with water molecules can be used

to activate certain polysaccharide matrices such as cellulose that contain vicinal diol groups.83

These diols are amenable to chelation by transition metal ions. In addition, amino and carboxyl

groups of solid matrices as in nylon can also be involved in the coordination chemistry.84 In

this process, the support, which may consist of materials as diverse as borosilicate, soda glass,

filter paper, cellulose derivatives, and nylon 66, is first reacted with a transition metal salt, such

as Ti(IV)Cl4, Co(II)Cl2, Cu(II)Cl2, Fe(II)Cl2, Mn(II)Cl2, Sn(IV)Cl4, Sn(II)Cl2, Cr(III)Cl3, Zr(IV)Cl4,

V(III)Cl3, or Fe(III)Cl3 for 24 h. After washing to remove unreacted metal salts, the immobilized metal ions further coordinate proteins through the carboxyl groups of C-terminus and

acidic amino acids, the hydroxyl groups of tyrosyl, seryl, and threonyl residues, the free sulfhydryl groups of cysteines, or amino groups of the N-terminus and ε-amino groups of lysyl residues as shown in Figure 14.14.85,86 Several enzymes, such as glucose dehydrogenase, α-amylase,

glucoamylase, GOD, chymotrypsin, and urease, have been bonded to various solid supports by

this method.83–86 Relatively high retention and operational stability are demonstrated for some

of these enzymes.

14.4.2.8  Other Homobifunctional Cross-Linkers

There are several other homobifunctional cross-linking reagents that have been used for immobilization of proteins such as toluene 2,4-diisocyanate and hexamethylene diisocyanate.87,88 A twostep reaction procedure is usually followed. First, the solid surface is activated by the bifunctional

reagent. The second step involves the reaction of the protein with the other end of the bifunctional

cross-linker. The advantage of this two-step procedure is that it permits removal of the unreacted

bifunctional reagent from the solid matrix before addition of the protein, thus preventing crosslinking of the protein in solution.



OH

OH

OH



Me(H2O)64+



O

O



O



OH2



Me

O



P



NH2



OH2



FIGURE 14.14  Immobilization of proteins with transition metal ions.



O

O



O



H

O



Me

O



P

N

H2



420



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



Bifunctional N-hydroxysuccinimide esters have been used to immobilize proteins to amino

group-containing solid matrices. Ethylene glycol bis(succinimidyl succinate) (EGS) and

dithiobis(succinimidyl propionate) (DSP) have been used to immobilize trypsin to hexamethylenediamine-sepharose CL-4B.89 The immobilized protein can be released by treatment with

hydroxylamine or thiol compounds, respectively, since these compounds contain cleavable bonds.

A  bis-sulfonated derivative of DSP, 3,3′-dithiobis(sulfosuccinimidyl propionate), which is more

water soluble, has also been used.



14.4.3  Use of Heterobifunctional Cross-Linkers

Heterobifunctional cross-linkers have been increasingly used to immobilize biomolecules to solid

carriers. A diverse array of reagents of different functionalities has been employed for this purpose.

The following paragraphs discuss some representative examples.

14.4.3.1  Monohalogenacetyl Halide

Jagendorf et al.90 used bromoacetyl bromide to immobilize serum globulins including antibodies to

cellulose. The hydroxyl group of cellulose was first activated to form bromoacetyl cellulose, which

alkylated a nucleophile from the protein, such as an amino group, to form conjugates as shown in

Figure 14.15. Sato et al.91 used the same approach to immobilize aminoacylase and found that, of the

three halides, iodide conferred the best reactivity and stability to the enzyme.

14.4.3.2  Epichlorohydrin

Epichlorohydrin activates matrices containing nucleophiles such as amino or hydroxyl groups to

an epoxide derivative as shown in Figure 14.16. This epoxide derivative reacts with nucleophilic

groups of proteins in the order of thiol > amino > hydroxyl, although aromatic hydroxyl, guanidino, and imidazole groups also react. Bayramoglu et al.92 used epichlorohydrin to both crosslink magnetic chitosan beads and to couple laccase under alkaline condition to the solid support.

In the first step, the heterobifunctional reagent reacted with the amino and hydroxyl groups of

chitosan to cross-link the polysaccharide chains. In the second step, laccase enzyme was coupled

to the epoxide-activated chitosan. Chymotrypsin was also coupled to epichlorohydrin-activated

chitosan, either in pure form or mixed with another biopolymer such as alginate, gelatin, or

carrageenan. The immobilized enzyme was evaluated and compared to other immobilization

methods.93 Other enzymes that have been immobilized include lipase on fibrous membranes94

and insoluble yeast beta-glucan,95 peroxidase on chitosan,96 catalase on starch-based polymers,97

acetylcholinesterase, and choline oxidase co-immobilized on poly(2-hydroxyethyl methacrylate)

O

Br



Br



O



OH



H2N



O



P



Br



O



H

N



O



P



FIGURE 14.15  Immobilization of proteins with monohalogenacetyl halide, such as bromoacetyl bromide.



Cl

OH



O

O



O



H2N



P



P

O

OH



FIGURE 14.16  Immobilization of proteins with epichlorohydrin.



N

H



421



Application of Chemical Conjugation to Solid-State Chemistry



membranes,98 α-amylase on poly(2-hydroxyethyl methacrylate) microspheres,99 and GOD on

poly(2-hydroxyethyl methacrylate) membranes.100

14.4.3.3  Amino- and Thiol-Group–Directed Reagents

Several heterobifunctional reagents of this type have been used to immobilize biomolecules.

Compounds containing a succinimidyl group (amino group directed) and a maleimide group (thiol

directed) are popular choices. The general reaction mechanism involves first reacting the crosslinker with the solid support. The activated matrices then react with the biomolecule to complete

the immobilization process. For example, N-sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) was used to immobilize cysteine-terminated peptides on gold surface.

SSMCC first reacted with an amine-modified gold surface to form an amide linkage. The solidbound maleimide function was then linked to a sulfhydryl group of cysteine-terminated peptides

through thioether bonds as shown in Figure 14.17.101 Schlapak et al.102 used another succinimidyl

maleimido cross-linker, N-succinimidyl 4-(p-maleimidophenyl)butyrate, to immobilize thiol-modified DNA oligonucleotides to the terminal amino groups of the diamine-modified poly(ethylene

glycol) (PEG) layer grafted onto silanized glass slides. The reaction of this type of cross-linkers

can also occur with thiol groups on the solid support. For instance, Charles and Kusterbeck103 used

N-succinimidyl 4-maleimidobutyrate (GMBS) to immobilize antibodies onto a microcapillary surface. GMBS first activated the surface by reacting with thiol-terminated silane. The antibodies were

then immobilized by reacting with the succinimidyl group. Bhatia et al.104 have used mercaptomethyldimethylethyoxysilane or mercaptopropyltrimethyloxysilane to convert the hydroxyl groups of

silica to thiol groups. N-γ-Maleimidobutyryloxy succinimide ester and N-succinimidyl 4-(p-maleidophenyl)butyrate, as well as other heterobifunctional reagents, N-succinimidyl-(4-iodoacetyl)

amino benzoate and N-succinimidyl-3-(2-pyridyldiothio)propionate (SPDP), were used to conjugate

immunoglobulins to the support.

As implied above, another group of commonly used heterobifunctional cross-linkers contain succinimidyl and pyridyldithio groups. The reagent, SPDP, whose N-hydroxysuccinimidyl ester reacts

with amino groups and whose 2-pyridyldisulfide moiety reacts with free thiols, has been used to

link calmodulin to thiol-sepharose 4B.105 The immobilized protein can be cleaved with dithiothreitol.

An analog with a longer flexible spacer arm, N-succinimidyl-6-[3′-(2-pyridyldithio) propionamido]

hexanoate (LC-SPDP), was used to immobilize 5′ end thiolated DNA to mica.106 The thiolated DNA

prepared by polymerase chain reaction reacted with the cross-linker to replace its 2-pyridyl disulphide group via sulfhydryl exchange. The modified DNA was deposited onto amino-silanized mica

where the NHS-ester moiety of the cross-linker reacted with the primary amino group on the surface.

Another analog, sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP),

which is more water soluble, was used to immobilize antibodies and acetylcholine esterase onto a

gold electrode surface in the piezoelectric quartz crystal.107,108 The proteins were thiolated by reacting

O



O

N



N O

–O S

3



NH2



O



O O



O

H

N



HS



N



P



O



O

O

H

N



S

N



P



O



O



FIGURE 14.17  Immobilization of proteins with succinimidyl and maleimido group containing heterobifunctional reagents, such as SSMCC.



422



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



with sulfo-LC-SPDP followed by dithiothreitol reduction. The free thiol released on the proteins then

formed a strong bond with gold on the electrode surface to immobilize the proteins.

Among other heterobifunctional cross-linkers, p-maleimidophenyl isocyanate (PMPI) was used

to immobilize thiol-modified oligonucleotides onto Si surface coated with high-density PEG molecules.109 The isocyanate moiety of PMPI first reacted with the –OH group of the surface to form a

stable carbamate linkage. To complete the immobilization process, the thiol-modified oligonucleotides were then added to react with the maleimido group.

Photosensitive heterobifunctional reagents have also been used to immobilize biomolecules.

Karakeỗili et al.110 used sulfosuccinimidyl-6-(4′-azido-2′-nitrophenyl-amino)hexanoate (sulfoSANPAH) to immobilize epidermal growth factor (EGF) to chitosan membrane. EGF was first

modified with the cross-linker though the reaction of an amino group with the sulfosuccinimidyl

moiety. The immobilization to chitosan was achieved by UV irradiation. Yan et al.111 synthesized a

series of NHS perfluorophenyl azides (PFPAs) for immobilization of proteins to polymer surfaces.

For example, N-succinimidyl-4-azido-2,3,5,6-tetrafluorobenzoate and N-succinimidyl-5-(4-azido2,3,5,6-tetrafluorobenzamido)pentanoate were covalently bonded to polystyrene beads by photolysis

at 254 nm. Horseradish peroxidase was immobilized on incubation with the NHS PFPAs-modified

surface by nucleophilic replacement of the NHS group.



14.5  I MMOBILIZATION BY CROSS-LINKING THROUGH

CARBOHYDRATE CHAINS

The immobilization of proteins to solid matrices via carbohydrate moieties was demonstrated by

Royer.112 The sugar moieties of the glycoprotein were first oxidized by sodium periodate to aldehydes, which form Schiff bases with either ethylenediamine or glycyltyrosine. Sodium borohydride

was then used to stabilize the bonds. The derivatized glycoprotein was then immobilized to NHS

ester-activated agarose or to diazotized arylamine supports as shown in Figure 14.18. Using this

procedure, glucoamylase, peroxidase, GOD, and carboxypeptidase Y have been immobilized to

solid supports.

Vicinal cis-hydroxyl groups of solid carriers such as agarose and many other polysaccharides

are also susceptible to oxidation by periodate to yield aldehydes that can be used to insolubilize

proteins by reductive amination.113 Schiff bases are formed between the protein amino groups and

the oxidized polysaccharide matrix. Subsequent reduction with sodium borohydride or pyridine

borane stabilizes the bonds. The aldehydes can be further converted to N-alkyl amines by reacting

with alkyl diamines or to hydrazides by reacting with dihydrazides such as adipic dihydrazide. The

hydrazide-modified matrix is preferred over that of alkyl amine because the amino group is positively charged at neutral pH (pKa = 10), whereas the hydrazide moiety is uncharged at pHs above

2.5 (pKa = 2.45). These derivatives can then be cross-linked to biomolecules by different methods

as mentioned above. The technique of reductive amination of Schiff bases formed between an aldehyde and an amine has been applied to the study of carbohydrate–protein and carbohydrate–carbohydrate interactions by surface plasmon resonance imaging and the construction of saccharide

microarrays.114,115



14.6  E XAMPLES OF APPLICATIONS OF SOLID-PHASE

IMMOBILIZATION CHEMISTRY

14.6.1  Affinity Chromatography

Affinity chromatography is one of the most powerful techniques in the purification and isolation

of biomolecules. The principle depends on the specific affinity between the target biomolecules

and the entity immobilized on a solid support. For example, the affinity between enzymes and

enzyme inhibitors, receptors and their ligands, and the specific binding of protein A and protein G



OH

Glycoprotein



OH

O



HO



OH



O

HN



IO4–



O

O



O



Glycoprotein



O



H

N



OH

O

N

H



OH



O



OH

NH2



H2N(CH2)2N

NaBH4



OH

Glycoprotein



O



Glycoprotein



N



Gly-Tyr

NaBH4

Glycoprotein

HOOC



N2+



OH

O



H

N

O



N

H



OH



Glycoprotein



HOOC



OH

O



H

N



N

H



OH



Application of Chemical Conjugation to Solid-State Chemistry



O



O



O



OH



N



N



OH



FIGURE 14.18  Immobilization of glycoproteins through oxidation and reductive amination of the carbohydrate moiety.



423



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4 Cross-Linking Reagents Commonly Used for Immobilization of Biomolecules

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