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3 Protein Immobilization by Matrix Activation

3 Protein Immobilization by Matrix Activation

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413



Application of Chemical Conjugation to Solid-State Chemistry



14.3.2  Activation of Carboxyl Groups

Matrix carboxyl groups can be activated with N-hydroxybenzotriazole in the presence of a watersoluble carbodiimide.38 Before coupling to proteins, the activated matrix is washed to remove excess

carbodiimide. The reactive ester reacts very rapidly with amino groups of the ligand to form stable

amide bonds as shown in Figure 14.3. Among the other nucleophiles, only sulfhydryl groups compete effectively with the amino group during the reaction.

p-Nitrophenol39 and NHS34,40,41 are also commonly used to form active esters with carboxylic

acids in the presence of a carbodiimide. These active ester derivatives are stable when stored in

dioxane. The coupling reaction is similar to that of N-hydroxybenzotriazole ester. Sam et al.41 used

EDC/NHS to activate porous silicon layers grafted with carboxyl-terminated alkyl chains.

Carboxylate functional groups can also be directly activated to the acyl chloride by reacting with

thionyl chloride. The very reactive acyl chloride reacts with proteins at low temperatures and is used

to immobilize alpha-amylase on poly(methyl methacrylate-acrylic acid) microspheres.42 Thionyl

chloride is also used to activate carboxyl groups of multiwalled carbon nanotubes for coupling

poly(l-lactic acid).43



14.3.3  Activation of Acyl Hydrazide

As mentioned above, reaction of a hydrazide-modified surface with glutaraldehyde generates aldehyde groups, which can be used to couple to proteins by forming Schiff bases with the proteins’

amino groups. Acyl hydrazides can also be converted to acyl azides with nitrous acid via the diazotization reaction.44 The azide group will be replaced by amino groups on the proteins to form amide

bonds as shown in Figure 14.4. Thus, by a series of reactions, surface hydroxyls can be activated to

acyl azides as illustrated in Figure 14.4. First, the hydroxyl is carboxymethylated with chloroacetic

acid, which is converted to an acyl hydrazide through an ester. The final activated acyl hydrazide

then reacts with the proteins. Since matrix amide groups can also be converted to acyl hydrazide, they

can be linked to proteins through this reaction sequence.



N

N



O



N

HO



O

OH



N C N



R



O

R



N



O



P



H2N



N

H



N N



P



FIGURE 14.3  Immobilization of proteins to carboxylate matrix with carbodiimide and N-hydroxybenzotriazol.



OH



Cl

O



OH



OH CH3OH/HCl



O



H

N

O



NH2



HNO2



CH3



NH2NH2



O



O



O



O



O



N3



O

O



H2N



P



H

N



O



FIGURE 14.4  Coupling of proteins to hydroxyl matrix via diazotization of acyl hydrazide.



O



P



414



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



14.3.4  Activation of Amines

14.3.4.1  Use of Nitrous Acid

Aromatic amino groups from poly-p-aminostyrene can be diazotized with nitrous acid to form the

corresponding diazonium derivative.34 The diazonium group will react with phenolic, imidazole,

and amino side chains of a protein to form a diazo bond (see Chapter 2). By this method, phenyl

groups on polystyrene can be coupled to proteins through nitration and amination.45 Reaction of the

benzene ring on polystyrene with fuming red nitric acid results in nitrostyrene. The nitro benzene

group is then converted to amino benzene by reduction with sodium dithionite and then diazotized

for coupling with proteins. This sequence of steps is shown in Figure 14.5. Hydroxyl-containing

carriers, such as cellulose, can also be coupled through the diazonium group after reaction with

p-nitrobenzylchloride.34,46

14.3.4.2  Use of Phosgene and Thiophosgene

Aromatic amines can also be activated at alkaline pH with phosgene or thiophosgene to yield the

corresponding isocyanate and isothiocyante, respectively, as shown in Figure 14.6.34,47 These active

derivatives react with free amino groups of proteins to form an amide or thioamide bond of the

substituted urea or thiourea (see Chapter 2). The isothiocyanate derivative can also be obtained from

aliphatic amines and acyl azides.

14.3.4.3  Use of Cyanogen Bromide

Like hydroxyl groups, amino group containing matrices can also be activated with cyanogen bromide to the corresponding cyanamides, which reacts with proteins amino groups to form guanidine

linkages as shown in Figure 14.7.48 It was claimed that amine matrices yielded more stable products

than the hydroxyl group–based materials.

HNO3/NO2



NO2



P

N



Na2S2O4



HNO2



NH2



OH



HO



N Cl



N

N



P



FIGURE 14.5  Coupling of proteins to aromatic amines via diazotization.



O



H2N

Cl



Cl



N C



P



O



Cl



Cl



N C



S



H

N



N

H



NH2

S



O



H2N



P



S

N

H



FIGURE 14.6  Coupling of proteins to aromatic amines through isocyanates and isothiocyanates.



P

H

N

P



415



Application of Chemical Conjugation to Solid-State Chemistry



NH2



N

H



HN



P



H2N



CNBr



H

N



N

H



N



P



FIGURE 14.7  Coupling of proteins to cyanogen bromide-activated amine matrices.



CN



C2H5OH/HCl



H2N



NH

O



P



C2H5



NH

N

H



P



FIGURE 14.8  Coupling proteins to polyacrylonitrile after activation to imidoester.



14.3.5  Activation of Polyacrylonitrile

Polyacrylonitrile can be activated with absolute ethanol and bubbling hydrogen chloride to an imidoester, which is readily attacked by amino groups of proteins at basic pH to yield amidine as shown

in Figure 14.8.49 There are several other methods to activate polyacrylonitrile. Jain et al.50 reduced

the pendant nitrile group of polyacrylonitrile with lithium aluminum hydride to an amine functionality, which was further activated by using glutaraldehyde for the covalent linking of immunoglobulins. The nitrile groups may also be transformed into the corresponding carboxyl group by strong

acids or bases.51,52 Alkaline hydrolysis introduces amide and carboxylic groups, which improve its

hydrophilicity.53 These group can be further chlorinated with thionyl chloride to form acyl chloride

derivatives, which react with amino and hydroxyl groups of proteins to form amide and ester bonds,

respectively.54 Furthermore, Battistel et al.55 have used an enzyme, nitrile hydratase, a member of

the class of nitrile-converting enzymes, to selectively convert the pendant nitrile into the corresponding amides.



14.4  C

 ROSS-LINKING REAGENTS COMMONLY

USED FOR IMMOBILIZATION OF BIOMOLECULES

Theoretically, any of the cross-linking reagents discussed earlier in this book can be used to couple

biomolecules to solid supports. The choice of reagents depends on the system to be studied. Suitable

cross-linkers target the functionalities on the matrix and the functional groups of the biological

molecules to be cross-linked. The use of these cross-linkers is, in a way, similar to the activation of

the matrix as narrated in the earlier sections. In almost all cases, the matrix is first reacted with the

cross-linker (an activation process), and the biological compound to be immobilized is then added

to complete the conjugation reaction. The following sections illustrate the use of various types of

cross-linking reagents to couple proteins to solid supports.



14.4.1  Use of Zero-Length Cross-Linking Reagents

The commonly known zero-length cross-linkers that have been used for immobilization of biomolecules include carbodiimides, Woodward’s reagent K,56 chloroformates,57 and carbonyldiimidazole.58 While all of these compounds condense carboxyl and amino groups to form amide bonds,

the latter two also activate hydroxyl groups. When hydroxyl groups are involved, a carbonyl moiety is incorporated. These compounds function as monobifunctional reagents as will be discussed

below (see also Chapter 8).



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.



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