Hermanson G. Bioconjugate Techniques, Second Edition

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Annunziato et al. (1993) have reported on the synthesis and use of a novel heterobifunc-

tional crosslinking reagent containing a hydroxyl-reactive isocyanate group on one end and a

sulfhydryl-reactive maleimide group on the other end. The compound can be useful in labeling

hydroxylic molecules for subsequent conjugation with thiol-containing molecules.

5. Aldehyde and Ketone Reactions

Aldehyde and ketone groups are important reactive sites in molecules for many bioconjugate

strategies. Although some pharmacological agents contain ketones, these groups usually are

not present in proteins and other biological macromolecules. Even when a molecule does not

contain these functionalities, however, they may be created through a number of processes

(Chapter 1, Section 4.4). The following sections discuss the major reactions that can be done

with aldehydes and ketones to modify or crosslink molecules containing them.

5.1. Hydrazine Derivatives

Derivatives of hydrazine, especially the hydrazide compounds formed from carboxylate groups,

can react speciﬁ cally with aldehyde or ketone functional groups in target molecules. Reaction

with either group creates a hydrazone linkage (Reaction 44)—a type of Schiff base. This bond

is relatively stable if it is formed with a ketone, but somewhat labile if the reaction is with an

aldehyde group. However, the reaction rate of hydrazine derivatives with aldehydes typically

is faster than the rate with ketones. Hydrazone formation with aldehydes, however, results in

much more stable bonds than the easily reversible Schiff base interaction of an amine with an

aldehyde. To further stabilize the bond between a hydrazide and an aldehyde, the hydrazone

may be reacted with sodium cyanoborohydride to reduce the double bond and form a secure

covalent linkage.

(Reaction 44)

5.2. Schiff Base Formation

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

a dehydration reaction yielding an imine (Reaction 45). However, Schiff base formation is a

relatively labile, reversible interaction that is readily cleaved in aqueous solution by hydrolysis.

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

enough to use for crosslinking applications unless they are reduced by reductive amination (see

below).

200 2. The Chemistry of Reactive Groups

(Reaction 45)

The reaction of dicarbonyl compounds, such as glyoxal or phenylglyoxal, with a guanidinyl

group, such as that of an arginine residue, proceeds to yield a more stable linkage due to the

formation of a cyclic derivative (Reaction 46).

(Reaction 46)

5.3. Reductive Amination

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

ing molecule with an amine-containing molecule. Schiff base formation between aldehydes and

amines occurs readily in aqueous solutions, especially at elevated pH. This type of linkage,

however, is not stable unless reduced to secondary or tertiary amine bonds. A number of reduc-

ing agents can be used to convert speciﬁ cally the Schiff base interaction into an alkylamine

linkage (Reaction 47). Once reduced, the bonds are highly stable and will not readily hydrolyze

in aqueous environments. The use of reductive amination to conjugate an aldehyde containing

molecule to an amine containing molecule results in a zero-length crosslinking procedure

where

no additional spacer atoms are introduced between the molecules (Chapter 3, Section 4). Reaction

of ammonia or a diamine compound with an aldehyde by reductive amination is a

method of cre-

ating a primary amine functional group (Chapter 1, Section 4.3).

(Reaction 47)

5.4. Mannich Condensation

Aldehydes may participate in a condensation reaction with an amine compound and a sub-

stance containing a sufﬁ ciently-active hydrogen, yielding an alkylated derivative that effectively

5. Aldehyde and Ketone Reactions 201

crosslinks the two molecules through the carbonyl group of the aldehyde. Strictly speaking,

the Mannich reaction consists of the condensation of formaldehyde (or sometimes another

aldehyde) with ammonia, in the form of its salt, and another compound containing an active

hydrogen. Instead of using ammonia, however, this reaction can be done with primary or sec-

ondary amines, or even with amides. An example is illustrated in the condensation of phenol,

formaldehyde, and a primary amine salt (Reaction 48).

(Reaction 48)

The Mannich reaction provides an often-superior alternative to diazonium conjugation

(Section 6.1, this chapter), because of the disadvantages inherent in the instability of both

the diazonium group and the resultant diazo linkage. By contrast, conjugations done through

Mannich condensations result in stable covalent bonds.

The crosslinking scheme using this method can make use of the native - and N-terminal

amines on proteins as the source of primary amine for the condensation reaction. Added to

the conjugation reaction is formaldehyde and the desired molecule to be coupled containing an

appropriately active hydrogen. The Mannich reaction should not be used for molecules con-

taining both an amine and a reactive hydrogen, since polymerization may occur. It is especially

useful for preparing hapten–carrier conjugates when the hapten contains no other available

functionalities suitable for crosslinking, but does contain an active hydrogen (Chapter 19,

Section 6.2).

6. Active Hydrogen Reactions

Many compounds contain reactive (or replaceable) hydrogens that are able to participate in

conjugation procedures using certain chemical reactions. These hydrogens typically are associ-

ated with aromatic systems wherein an electron donating group activates positions on the ring

toward substitution reactions. At such carbons, the hydrogen is easily displaced by an attack-

ing electrophilic group able to form a new covalent linkage. Several common modiﬁ cation

reactions are used in bioconjugate chemistry to label or crosslink molecules at active hydrogen

sites. The following three sections discuss these chemical reactions.

6.1. Diazonium Derivatives

Diazonium groups react with active hydrogen sites on aromatic rings to give covalent diazo

bonds. Generation of a diazonium functional group usually is done from an aromatic amine

202 2. The Chemistry of Reactive Groups

by reaction with sodium nitrite under acidic conditions at 0°C (Chapter 1, Section 4.3 and

Chapter 19, Section 6.1). The highly reactive and unstable diazonium is reacted immediately

with an active hydrogen-containing compound at pH 8–10. In general, at pH 8.0 the diazo-

nium group will react principally with histidinyl residues, attacking the electron-rich nitrogens

of the imidazole ring. At higher pH, the phenolic side chain of tyrosine groups can be modiﬁ ed

(Reaction 49). The reaction proceeds by electrophilic attack of the diazonium group toward

the electron rich points on the target molecules. Phenolic compounds are modiﬁ ed at positions

ortho and para to the aromatic hydroxyl group. For tyrosine side chains, only the ortho modi-

ﬁ cation is available.

(Reaction 49)

Crosslinking using diazonium compounds usually creates deeply colored products charac-

teristic of the diazo bonds. Occasionally, the conjugated molecules may turn dark brown or

even black. The diazo linkages are reversible by addition of 0.1 M sodium dithionite in 0.2 M

sodium borate, pH 9.0. Upon cleavage, the color of the complex is lost.

6.2. Mannich Condensation

The Mannich reaction consists of the condensation of an active hydrogen-containing com-

pound with an amine-containing compound in the presence of formaldehyde. See Section 5.4

(this chapter) for addition details.

6.3. Iodination Reactions

Radioiodination involves the substitution of radioactive iodine atoms for reactive hydrogen

sites in target molecules. The process usually involves the action of a strong oxidizing agent

to transform iodide ions into a highly reactive electrophilic iodine compound (typically I

or a

mixed halogen species such as ICl). Formation of this electrophilic species leads to the poten-

tial for rapid iodination of aromatic compounds containing strong activating groups, such as

aryl compounds. Particularly, aromatic constituents that have electron donating groups can

sufﬁ ciently activate the carbons on the ring to undergo electrophilic substitution reactions.

Therefore, phenols, aniline derivatives, or alkyl anilines that contain OH, NH

, or NHR con-

stituents, respectively, are very susceptible to being iodinated. In proteins, this translates into

tyrosine side chain phenolic groups and histidine side chain imidazole groups (Reaction 50).

See Chapter 12 for further details on iodination reactions.

6. Active Hydrogen Reactions 203

(Reaction 50)

7. Photo-Chemical Reactions

Photoreactive groups can be induced to couple with target molecules by exposure to UV light.

Until they are photolyzed, photosensitive functional groups are relatively non-reactive in typi-

cal thermochemical processes. For this reason, reagents designed with a photoreactive group

can be used in highly controlled reactions. The labeling reaction can be induced by a UV ﬂ ash

at predetermined points in an experimental protocol. For instance, covalent bond formation

can be initiated after binding of photo-labeled ligands to receptors or after some other bio-

chemical process has taken place. In this regard, photoreactive chemistry has become an impor-

tant device for numerous bioconjugate applications. The following sections describe the major

photosensitive groups that can be used in the design of modiﬁ cation or crosslinking reagents.

(Chapter 4, Section 5; Chapter 5, Sections 3–7; Chapter 6; and Chapter 11, Section 4 describe

the reagents that utilize these functional groups.)

7.1. Aryl Azides and Halogenated Aryl Azides

The most popular type photosensitive functional group is the aryl azide derivative. Upon pho-

tolysis, phenyl azide groups form short-lived nitrenes that react rapidly with the surrounding

chemical environment (Gilchrist and Rees, 1969). Nitrenes can insert nonspeciﬁ cally into chem-

ical bonds of target molecules, including undergoing addition reactions with double bonds and

insertion reactions into active hydrogen bonds at C H and N H sites. Abundant evidence,

however, indicates that the photolyzed intermediates of aryl azides principally undergo ring

expansion to create nucleophile-reactive dehydroazepines. Instead of inserting non-selectively

at active carbon–hydrogen bonds, dehydroazepines have a tendency to react preferentially with

nucleophiles, especially amines (Reaction 51).

(Reaction 51)

204 2. The Chemistry of Reactive Groups

However, some investigators have shown that aryl azides that possess a perﬂ uorinated ring

structure or are substituted completely with halogen atoms are quite efﬁ cient at forming the

desired nitrene intermediate (Keana and Cai, 1990; Cai et al., 1993; Schnapp and Platz, 1993;

Soundararajan et al., 1993; Schnapp et al., 1993; Yan et al., 1994). The ring substitution pre-

vents ring expansion after nitrene formation, thus allowing the reactive intermediate to survive

long enough to react with target molecules. Halogenated phenyl azides undergo the insertion

reactions that were typically attributed to unsubstituted aryl azides in the past (Reaction 52).

(Reaction 52)

7.2. Benzophenones

A photoreactive group consisting of a benzophenone residue photolyzes upon exposure to UV

light to give a highly reactive triplet-state ketone intermediate (Walling and Gibian, 1965).

Similar to the reactive nitrene of photolyzed phenyl azides, the energized electron of an activated

benzophenone can insert in hydrogen–carbon bonds and other active groups to give covalent

linkages with target molecules (Reaction 53). Unlike phenyl azides, however, the decomposition

or decay of the photoactivated species does not yield an inactive compound. Instead, benzophe-

nones that have become deactivated without forming a covalent bond can be once again pho-

tolyzed to an active state. As a result of this multiple-activation characteristic, a benzophenone

reagent has more than one chance to form a covalent bond with its intended target. Thus it typi-

cally gives much higher yields of photo-crosslinking than comparable phenyl azide crosslinkers.

(Reaction 53)

The use of a benzophenone photoactivatable group in the design of bioconjugate reagents

is rare. Two sulfhydryl-reactive ones incorporating a maleimide group and a iodoacetyl group

opposite the benzophenone are described in Chapter 5, Sections 4.3 and 4.4. A newer benzophe-

none crosslinker containing a water-soluble PEG spacer is described in Chapter 18, Section 3.6.

7.3. Anthraquinones

Anthraquinone groups are highly photoreactive by exposure to long UV light ranging from 340

to 360 nm. Unlike photoreactive groups that form intermediate nitrene or carbine precursors

7. Photo-Chemical Reactions 205

after photoactivation, anthraquinones react by a radical generation process, which is much

more efﬁ cient in coupling to C H substrates. Photoactivation results in a highly reactive,

excited species that involves the formation of a triplet n,*-state, which becomes a powerful

electron acceptor. If an organic substrate is present containing a reactive C H bond, then the

excited anthraquinone is able to cause rapid proton abstraction, resulting in the formation of

a reduced, phenoxy radical intermediate with a second radical formed on the hydrogen donat-

ing substrate. This radical pair then can react to link covalently the anthraquinone group to the

substrate, forming an ether linkage and effectively immobilizing the reagent to the substrate

(Brennan and Beutel, 1969; Koch et al., 2000) ( Figure 2.6 ).

206 2. The Chemistry of Reactive Groups

Figure 2.6 Anthraquinone derivatives can photoreactively couple to substrates by means of a free radical gen-

eration process. The reactive intermediate also can be regenerated back to the initial anthraquinone by proton

abstraction and oxidation, resulting in the possibility of again being photolyzed and successfully coupled to the

substrate.

Modiﬁ cation or crosslinking reagents containing an anthraquinone group can be made from

the 2-carboxylic acid derivative (Kumar et al., 2004) (Exiqon). A spacer arm can be added to

the carboxylate to terminate in a reactive group for bioconjugation or an afﬁ nity group (e.g.,

biotin) for interaction with other molecules. Anthraquinone photoreactive linking reagents can

be used to modify any surfaces or particles containing C H groups, including polymer-based

microplates, slides, and particles. Inorganic surfaces can be modiﬁ ed with an organosilane com-

pound (Chapter 13) and then further reacted using an anthraquinone compound, however the

beneﬁ t of this strategy may be negated by choice of the proper reactive group on the silane.

Reactions with surfaces usually are done with anthraquinone concentrations in aqueous buffers

ranging from 100 ng/ml to about 2  g/ml. Protect all photoreactive reagents and solutions from

light until ready to photoactivate them.

7.4. Certain Diazo Compounds

Certain diazo compounds can be photolyzed with UV light to generate highly reactive carbenes

(Reaction 54). Similar to nitrenes, carbenes can insert into active C H or N H bonds or

add to double bonds, forming covalent linkages with target molecules (Gilchrist and Rees,

1969). Few diazo photoreactive reagents have been synthesized, probably due to their ten-

dency to react with water molecules after photoactivation, thus severely decreasing coupling

yields with intended molecules. One heterobifunctional crosslinker, PNP-DTP, containing an

amine-reactive end and a photosensitive diazotriﬂ uoropropionate group is available (Chapter 5,

Section 3.12).

(Reaction 54)

Diazopyruvates are another class of photoreactive diazo compounds that have a unique cou-

pling mechanism (Chapter 5, Section 3.11). The diazo functional group can by photolyzed by

exposure to irradiation at 300 nm, forming a highly reactive carbene, which can undergo a

Wolff rearrangement to produce a ketene amide intermediate. In the presence a nucleophilic

species on a target molecule, the ketene can undergo an acylation reaction to form a stable

malonic acid derivative. The photolyzed product thus can couple to hydrazide or amine con-

taining targets to form covalent linkages (Reaction 55).

(Reaction 55)

7. Photo-Chemical Reactions 207

208 2. The Chemistry of Reactive Groups

7.5. Diazirine Derivatives

Diazirine compounds are similar in their photoreactivity to diazo groups, forming highly

reactive carbene intermediates upon exposure to UV light of about 360 nm (Reaction 56).

Diazirines consist of a three-member ring system containing two nitrogen atoms connected

through a double bond. First developed by Smith and Knowles (1973), the photosensi-

tive diazirine is perhaps second in popularity to phenyl azides in the design of photoreactive

crosslinking agents.

(Reaction 56)

Some diazirines, particularly the 3-triﬂ uoromethyl-3-aryldiazirines, can rearrange upon

photolysis to a linear diazo derivative, similar in structure to the photosensitive end of the

crosslinker PNP-DTP (Chapter 5, Section 3.12). These isomerized products themselves can be

photolyzed to the reactive carbene.

Carbene generation from photolysis of diazirine compounds leads to efﬁ cient insertion into

CH or N H bonds and also causes addition reactions with points of unsaturation within

target molecules. Diazirine-containing photoafﬁ nity probes have been used to study numerous

ligand–receptor interactions (Bergmann et al., 1994). Heterobifunctional crosslinkers contain-

ing a diazirine photosensitive group also have been used to attach macromolecules to surfaces

such as polystyrene and glass (Collioud et al., 1993). In addition, diazirine-containing photore-

active amino acid analogs, photo-leucine and photo-methionine, have been developed to study

protein interactions within cells (Suchanek et al ., 2005).

7.6. Psoralen Compounds

Psoralen, or derivatives of 9-methoxy-7 H -furo[3,2- g]chromen-7-one tricyclic ring structures,

are used as photoreactive groups in crosslinkers, biotinylation compounds, and nucleic acid

probes. Psoralens have been used for many years as photochemotherapy agents for treatment

of psoriasis and vitiligo (Smith and Barker, 2006). Psoralens react when exposed to UV light

ranging from 320 to 400 nm to form an excited triplet state intermediate that can insert in cer-

tain double bond structures, especially at the 5,6-double bond of thymine bases.

Psoralens can react by two different routes upon photoactivation (Parsons, 1980; Pathak,

1984). The ﬁ rst route is through the well-known photoreaction mechanism that principally

involves intercalation within double-stranded DNA or RNA with the formation of adducts

with adjacent thymine bases. The furan-side and pyrone-side rings in psoralen both can form

cycloaddition products with the 5,6-double bond of thymine to create a crosslink between two

DNA strands (Reaction 57) or to a lesser extent, within double-strand regions of RNA.

(Reaction 57)

Psoralen also can undergo reactions with oxygen to produce reactive oxygen species, includ-

ing the formation of singlet oxygen (

), superoxide anion (





), and hydroxyl radicals

(



). The production of reactive oxygen species by psoralen derivatives can damage biological

molecules and structures. See Chapter 1, Section 1.1 for additional information on the oxida-

tion of amino acids.

Psoralen-biotin compounds have been used to label double-stranded DNA for detection using

(strept)avidin reagents (Henriksen et al., 1991; Wygrecka et al., 2007). The compound psoralen-

PEG

-biotin is commercially available for this purpose (Thermo Fisher). Crosslinking agents also

can be built using a photoreactive psoralen ring system at one end. The reagent succinimidyl-

[4-(psoralen-8-yloxy)]butyrate (SPB) contains an NHS ester to covalently link a psoralen group

to proteins or other amine-containing molecules (Thermo Fisher, Molecular Biosciences). Oser

et al. (1988) created a lanthanide chelate with a psoralen group to label DNA with a time-

resolved ﬂ uorescent probe. Psoralen derivatives also can be coupled to polymeric surfaces by a

7. Photo-Chemical Reactions 209