Hermanson G. Bioconjugate Techniques, Second Edition

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1.1. Isothiocyanates

Isothiocyanates can be formed by the reaction of an aromatic amine with thiophosgene (Rifai and

Wong, 1986). The group reacts with nucleophiles such as amines, sulfhydryls, and the phenolate

ion of tyrosine side chains (Podhradsky et al., 1979). The only stable product of these reactions,

however, is with primary amine groups. Therefore, isothiocyanate compounds are almost entirely

selective for modifying -amino groups in lysine side chains and N-terminal -amines in proteins

or primary amines in other molecules (Jobbagy and Kiraly, 1966). The reaction involves attack of

the nucleophile on the central, electrophilic carbon of the isothiocyanate group (Reaction 1). The

resulting electron shift and proton loss creates a thiourea linkage between the isothiocyanate-

containing compound and the amine with no leaving group involved.

(Reaction 1)

Isothiocyanate compounds react best at alkaline pH values where the target amine groups are

mainly unprotonated. Many reactions are done in 0.1 M sodium carbonate buffer at pH 9.0.

Reaction times vary from 4 to 24 hours at 4°C. Rana and Meares (1990) found that by reacting

isothiocyanate-containing chelates at pH 7 they could selectively modify a monoclonal anti-

body only at its N-terminal -amines while leaving lysine amines unmodiﬁ ed. This is an excel-

lent method for selectively modifying only a single site on a protein or peptide molecule. Since

the isothiocyanate group is relatively unstable in aqueous conditions, reagents containing this

function should be stored desiccated at refrigerator or freezer temperatures.

1.2. Isocyanates

Isocyanates are similar to the isothiocyanates discussed above, except an oxygen atom

replaces the sulfur. An isocyanate can be formed from the reaction of an aromatic amine with

phosgene (Rifai and Wong, 1986). The group also can be created from acyl azides by treat-

ment at 80°C in the presence of an alcohol (Section 4.7, this chapter). Under these conditions,

the acyl azide rearranges to form an isocyanate. Isocyanates can react with amine-containing

molecules to form stable isourea linkages (Reaction 2). The reactivity of isocyanates is greater

than that of isothiocyanates, but for the same reason their stability can be a problem. Many

commercial suppliers of bioconjugate reagents have found isocyanate compounds too unstable

to offer them for sale, since moisture rapidly decomposes them, releasing CO

and leaving an

aromatic amine.

(Reaction 2)

170 2. The Chemistry of Reactive Groups

Isocyanate-containing reagents also can be used to crosslink or label hydroxyl-containing

molecules. Recently, a heterobifunctional compound containing a isocyanate group on one end

and a maleimide group on the other end was reported (Annunziato et al., 1993). PMPI, or

p-maleimidophenyl isocyanate, can be used to conjugate hydroxyl-containing compounds such

as polysaccharides with sulfhydryl-containing molecules (available from Thermo Fisher).

1.3. Acyl Azides

Acyl azides are activated carboxylate groups that can react with primary amines to form

amide bonds. The azide function is a good leaving group similar to the N -hydroxysuccinimide

group of NHS ester compounds. An acyl azide can be formed by treatment of a hydrazide with

sodium nitrite at 0°C (Lowe and Dean, 1974). A coupling reaction with an amine group occurs

by attack of the nucleophile at the electron-deﬁ cient carbonyl group (Reaction 3). Optimum

conditions for the reaction are a pH range of 8.5–10 in buffers which contain no competing

amines or other nucleophiles.

(Reaction 3)

The major competing reaction in acyl azide coupling is hydrolysis. The higher the pH, the

faster the reactivity, both with regard to amine conjugation and hydrolysis. Crosslinkers or

modiﬁ cation reagents containing this compound must be kept dry to preserve activity. Reactions

are complete in 2–4 hours at room temperature.

1.4. NHS Esters

An N-hydroxysuccinimide (NHS) ester is perhaps the most common activation chemistry for

creating reactive acylating agents. NHS esters were ﬁ rst introduced as reactive ends of homobi-

functional crosslinkers (Bragg and Hou, 1975; Lomant and Fairbanks, 1976). Today, the great

majority of amine-reactive crosslinking or modiﬁ cation reagents commercially available utilize

NHS esters. An NHS ester may be formed by the reaction of a carboxylate with NHS in the

presence of a carbodiimide. To prepare stable NHS ester derivatives, the activation reaction

must be done in non-aqueous conditions using water-insoluble carbodiimides or condensing

agents, such as DCC (Chapter 3, Section 1.4).

NHS or sulfo-NHS ester-containing reagents react with nucleophiles with release of the

NHS or sulfo-NHS leaving group to form an acylated product (Reaction 4). The reaction

of such esters with a sulfhydryl or hydroxyl group does not yield stable conjugates, form-

ing thioesters or ester linkages, respectively. Both of these bonds potentially can hydrolyze in

aqueous environments or exchange with neighboring amines to form amide bonds. Histidine

1. Amine Reactions 171

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

they hydrolyze very rapidly in aqueous environments (Cuatrecasas and Parikh, 1972). Thus,

the presence of imidazole in reaction buffers only serves to increase the hydrolysis rate of the

active ester. Reaction with primary and secondary amines, however, creates stable amide and

imide linkages, respectively, that don ’t readily break down. Thus, in protein molecules, NHS

ester crosslinking reagents couple principally with the -amines at the N-terminals and the

 -amines of lysine side chains.

(Reaction 4)

NHS esters also may be formed in situ to react immediately with target molecules in aque-

ous reaction media. Using the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) a

carboxylate-containing molecule can be transformed into an active ester by reaction in the

presence of NHS or sulfo-NHS ( N-hydroxysulfosuccinimide) (Chapter 3, Section 1.2). Sulfo-

NHS esters are hydrophilic active groups that couple rapidly with amines on target molecules

with the same speciﬁ city and reactivity as NHS esters (Staros, 1982). Unlike NHS esters that

are relatively water insoluble and must be ﬁ rst dissolved in organic solvent before being added

to aqueous solutions, sulfo-NHS esters are relatively water soluble, longer-lived, and hydrolyze

more slowly in water. In the presence of amine nucleophiles that can attack at the electron-

deﬁ cient carbonyl of the active ester, the sulfo-NHS group rapidly leaves, creating a stable amide

linkage with the amine compound. Sulfhydryl and hydroxyl groups also will react with such

active esters, but the products of such reactions, thioesters and esters, are unstable in aqueous

environments or in the presence of amine nucleophiles.

NHS esters have a half-life on the order of hours under physiological pH conditions.

However, hydrolysis and amine reactivity both increase with increasing pH. At 0°C at

pH 7.0, the half-life is typically 4–5 hours (Lomant and Fairbanks, 1976). At pH 8.0 at

25°C it falls to 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 extinc-

tion 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

/cm

1

in potassium phos-

phate buffer at pH 6.5 (Partis et al., 1983). Unfortunately, the relatively low sensitivity of this

absorptivity measurement does not allow for determining the rate of reaction in an actual

crosslinking procedure.

To maximize the modiﬁ cation of amines and minimize the effects of hydrolysis, maintain a

high concentration of protein or other target molecule in the reaction medium. 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. Water-insoluble crosslinkers containing NHS esters may

be reacted in organic solvents, eliminating the hydrolysis problem, provided the target molecule

is soluble and stable in such environments. For non-aqueous reactions, an organic base (proton

acceptor) typically is added, such as triethylamine or 4-(dimethylamine)pyridine (DMAP).

172 2. The Chemistry of Reactive Groups

1.5. Sulfonyl Chlorides

Sulfonyl chlorides are reactive sulfonic acid derivatives similar in properties and reactivity to

acid chlorides of carboxylates. The sulfonic acid group, however, is a highly hindered mol-

ecule, containing a tetrahedral conﬁ guration of substituents. The attack of a nucleophile on a

sulfonyl chloride involves temporary formation of a pentavalent intermediate which is highly

crowded and unstable. Unlike the capability of using other condensing agents such as carbodi-

imides when preparing amide linkages between carboxylate groups and amines, sulfonic acids

are too hindered to allow such bulky active intermediates to be formed. Thus, the main activa-

tion chemistry employed with sulfonates is to create the sulfonyl chloride derivative. Reaction

of a sulfonyl chloride compound with a primary amine-containing molecule proceeds with loss

of the chlorine atom and formation of a sulfonamide linkage (Reaction 5).

(Reaction 5)

Sulfonic acids are frequent constituents of ﬂ uorescent probes (Chapter 9, Section 1). The

sulfonyl chloride derivative allows simple conjugation of these molecules with proteins and

other amine-containing compounds. The derivative is prepared by reaction of the sulfonate

with thionyl chloride or phosphorus pentachloride in non-aqueous conditions. The reaction

of a sulfonyl chloride with an amine proceeds under alkaline pH conditions (typically done at

pH 9–10). It may also be done in organic solvent for the modiﬁ cation of water-insoluble com-

pounds. Hydrolysis is the major competing reaction in aqueous environments, although the

overall rate of sulfonyl chloride reactivity and hydrolysis is less than that of the corresponding

acid chlorides of carboxylates. However, sulfonyl chloride-containing reagents should be stored

under nitrogen or in a desiccator to prevent breakdown by moisture.

1.6. Aldehydes and Glyoxals

Carbonyl groups such as aldehydes, ketones, and glyoxals can react with amines to form

Schiff base intermediates which are in equilibrium with their free forms. The interaction is

pH dependent, being more efﬁ cient at low pH and especially efﬁ cient at high pH conditions.

Certain compounds, particularly some reducing sugars, may undergo an Amadori rearrange-

ment after Schiff base formation to a stable ketoamine structure (Chapter 1, Section 2.1). This

occurs in vivo as glucose modiﬁ es amine-containing components in the blood to form glycated

derivatives. Such modiﬁ cation is thought to be related to aging and is a signal for protein and

cellular regeneration.

The rather labile Schiff base interaction can be chemically stabilized by reduction. The addi-

tion of sodium borohydride or sodium cyanoborohydride to a reaction medium containing an

aldehyde compound and an amine-containing molecule will result in reduction of the Schiff

1. Amine Reactions 173

base intermediate and covalent bond formation, creating a secondary amine linkage between

the two molecules (Reaction 6).

(Reaction 6)

Although both borohydride and cyanoborohydride have been used for reductive amination

purposes, borohydride will reduce the reactive aldehyde groups to hydroxyls at the same time

it converts any Schiff bases present to secondary amines. Cyanoborohydride, by contrast, is

a milder reducing agent. It has been shown to be at least 5 times milder than borohydride

in reductive amination processes with antibodies (Peng et al., 1987). While cyanoborohydride

does not reduce aldehydes, it is very effective at Schiff base reduction. Thus, higher yields of

conjugate formation can be realized using cyanoborohydride instead of borohydride. Other

reducing agents also have been explored for reductive amination processes, including various

amine boranes and ascorbic acid (Cabacungan et al., 1982; Hornsey et al., 1986). See Chapter 3,

Section 4 for a protocol for reductive amination coupling.

1.7. Epoxides and Oxiranes

An epoxide or oxirane group will react with nucleophiles in a ring-opening process. The

reaction can take place with primary amines, sulfhydryls, or hydroxyl groups to create

secondary amine, thioether, or ether bonds, respectively. During the coupling process, ring

opening forms a -hydroxy group on the epoxy compound (Reaction 7). The reaction of the

epoxide functionalities with hydroxyls requires high pH conditions, usually in the range of pH

11–12. Amine nucleophiles react at more moderate alkaline pH values, typically needing buffer

environments of at least pH 9.0. Sulfhydryl groups are the most highly reactive nucleophiles

with epoxides, requiring a buffered system closer to the physiological pH range of 7.5–8.5 for

efﬁ cient coupling.

(Reaction 7)

The principal side reaction to epoxide coupling is hydrolysis. Particularly at acid pH values,

the epoxide ring can hydrolyze to form adjacent hydroxyls. This diol can be oxidized with perio-

date to create a terminal aldehyde residue with loss of one molecule of formaldehyde (Chapter 1,

Section 4.4). The aldehyde then can be used in reductive amination reactions. The reaction of an

epoxide group with an ammonium ion generates a terminal primary amine group that also can

be used for further derivatization.

174 2. The Chemistry of Reactive Groups

1.8. Carbonates

Carbonates are diester derivatives of carbonic acid formed from its condensation with hydroxyl

compounds. These groups may be created from the reaction of a bifunctional carbonic acid

compound like phosgene or carbonyldiimidazole (CDI) (Chapter 3, Section 3) with two alco-

hols. Carbonates can rapidly react with nucleophiles to form carbamate linkages, which are

extremely stable bonds (Reaction 8). A commonly used bifunctional carbonate compound,

disuccinimidyl carbonate, can be used to activate hydroxyl-containing molecules to form

amine-reactive succinimidyl carbonate intermediates (Section 4.3, this chapter). This carbonate

activation procedure can be used with great success in coupling polyethylene glycol (PEG) to

proteins and other amine-containing molecules (Chapter 25, Section 1.2).

(Reaction 8)

Nucleophiles, such as the primary amino groups of proteins, can react with the succinimidyl

carbonate functional groups to give stable carbamate (aliphatic urethane) bonds. The linkage

is identical to that obtained through CDI activation of hydroxyl groups with subsequent cou-

pling of amines (Chapter 3, Section 3 and Chapter 25, Section 1.4). However, the reactivity

of the succinimidyl carbonate is much greater than that of the imidazole carbamate formed as

the active species in CDI activation. A succinimidyl carbonate group may hydrolyze in aque-

ous solution to release NHS and CO

, essentially regenerating the underivatized hydroxyl.

Carbonates formed from esteriﬁ cation of two alcohol groups similarly hydrolyze to release

plus the original hydroxyl compounds.

The coupling reaction of a carbonate functional group with an amine is best done in slightly

alkaline pH (7–9) and in the absence of any competing amine or sulfhydryl components.

1.9. Arylating Agents

Aryl halide compounds such as ﬂ uorobenzene derivatives can be used to form covalent bonds

with amine-containing molecules like proteins. The reactivity of aryl halides, however, is not

totally speciﬁ c for amines. Other nucleophiles such as thiol, imidazolyl, and phenolate groups

of amino acid side chains also can react (Zahn and Meinhoffer, 1958). Conjugates formed with

sulfhydryl groups are reversible by cleaving with an excess of thiol (Shaltiel, 1967).

Fluorobenzene-type compounds have been used as functional groups in homobifunctional

crosslinking agents (Chapter 4, Section 4). Their reaction with amines involves nucleophilic

displacement of the ﬂ uorine atom with the amine derivative, creating a substituted aryl amine

bond (Reaction 9). Detection reagents incorporating reactive aryl chemistry include 2,4-

dinitroﬂ uorobenzene and trinitrobenzenesulfonate (Eisen et al., 1953). These compounds form

1. Amine Reactions 175

colored complexes with target amine groups. The relative rate of reactivity for aryl compounds

is: F  Cl  Br  Sulfonate.

(Reaction 9)

1.10. Imidoesters

The Imidoester (or imidate) functional group is one of the most speciﬁ c acylating agents avail-

able for modifying primary amines. Unlike most other coupling chemistries, imidoesters pos-

sess minimal cross-reactivity toward other nucleophilic groups in proteins. The -amines and

-amines of proteins may be targeted and crosslinked by reacting with homobifunctional imi-

doesters at a pH of 7–10 (optimal pH 8–9). The product of this reaction, an imidoamide (or

amidine) (Reaction 10), is protonated and thus carries a positive charge at physiological pH

(Kiehm and Ji, 1977; Liu et al. , 1977; Ji, 1979; Wilbur, 1992).

(Reaction 10)

The amidine bond formed is quite stable at acid pH; however, it is susceptible to hydrolysis

and cleavage at high pH. A typical reaction condition for using imidate crosslinkers is a buffer

system consisting of 0.2 M triethanolamine in 0.1 M sodium borate, pH 8.2. After conjugating

two proteins with a bifunctional imidoester crosslinker, excess imidoester functional groups

may be blocked with ethanolamine.

1.11. Carbodiimides

Carbodiimides are zero-length crosslinking agents used to mediate the formation of an amide

or phosphoramidate linkage between a carboxylate group and an amine or a phosphate and an

amine, respectively (Hoare and Koshland, 1966; Chu et al., 1986; Ghosh et al., 1990). They

are called zero-length reagents because in forming these bonds no additional chemical structure

is introduced between the conjugating molecules.

N-substituted carbodiimides can react with carboxylic acids to form highly reactive,

o-acylisourea derivatives that are extremely short-lived (Reaction 11). This active species then

can react with a nucleophile such as a primary amine to form an amide bond (Reaction 12)

176 2. The Chemistry of Reactive Groups

(Williams and Ibrahim, 1981). Other nucleophiles also are reactive. Sulfhydryl groups may

attack the active species and form thioester linkages, although these are not as stable as the

bond formed with an amine.

(Reaction 11)

(Reaction 12)

Hydrazide-containing compounds also can be coupled to carboxylate groups using a

carbodiimide-mediated reaction. Using bifunctional hydrazide reagents, carboxylates can be

modiﬁ ed to possess terminal hydrazide groups able to conjugate with other carbonyl compounds

(Chapter 4, Section 8).

In addition, oxygen atoms may act as the attacking nucleophile, such as those in water mol-

ecules. In aqueous solution, hydrolysis by water is the major competing reaction, both inac-

tivating EDC itself and cleaving off the activated ester intermediate, forming an isourea, and

regenerating the carboxylate group (Gilles et al. , 1990).

Nakajima and Ikada (1995) investigated the reactions of EDC amide bond formation in

aqueous solution using a hydrogel of poly(acrylic acid) to contribute the carboxylate groups

and ethylenediamine or benzylamine as the amine functional groups. Their results indicate

that carboxylate activation occurs most effectively at pH 3.5–4.5, while amide bond formation

occurs with highest yield at pH 4–6. However, the maximal rate of hydrolysis of EDC occurs

at acidic pH values with increasing stability of the carbodiimide in solution at or above pH 6.5.

When working with proteins and peptides, experience indicates that EDC-mediated amide bond

formation effectively occurs between pH 4.5 and 7.5. Buffer systems using MES or phosphate

may be used to stabilize the pH during the course of the reaction. For additional information

on speciﬁ c carbodiimides used in bioconjugate chemistry, see Chapter 3, Section 1.

1. Amine Reactions 177

Molecules containing phosphate groups, such as the 5  phosphate of oligonucleotides, also

may be conjugated to amine-containing molecules by using a carbodiimide-mediated reaction

(Chapter 27, Section 2.1). The carbodiimide activates the phosphate to an intermediate phos-

phate ester similar to its reaction with carboxylates (Chapter 3, Section 1). In the presence of

an amine, the ester reacts to form a stable phosphoramidate bond (Reaction 13).

(Reaction 13)

1.12. Anhydrides

Acid anhydrides, as their name implies, are formed from the dehydration reaction of two car-

boxylic acid groups. Anhydrides are highly reactive toward nucleophiles and are able to acylate

a number of the important functional groups of proteins and other macromolecules. Upon

nucleophilic attack, the anhydride yields one carboxylic acid for every acylated product. If the

anhydride was formed from monocarboxylic acids, such as acetic anhydride, then the acylation

occurs with release of one carboxylate group. However, for dicarboxylic acid anhydrides, such

as succinic anhydride, upon reaction with a nucleophile the ring structure of the anhydride

opens, forming the acylated product modiﬁ ed to contain a newly formed carboxylate group

(Reaction 14). Thus, anhydride reagents may be used to both block functional groups and to

convert an existing functional group into a carboxylic acid.

(Reaction 14)

Protein functional groups able to react with anhydrides include the -amines at the

N-terminals, the -amine of lysine side chains, cysteine sulfhydryl groups, the phenolate ion of

tyrosine residues, and the imidazolyl ring of histidines. However, acylation of cysteine, tyro-

sine, and histidine side chains forms unstable complexes that are easily reversible to regenerate

the original group. Only amine functional groups of proteins are stable to acylation with anhy-

dride reagents, forming amide bonds (Fraenkel-Conrat, 1959; Smyth, 1967).

Another potential site of reactivity for anhydrides in protein molecules is modiﬁ cation of

any attached carbohydrate chains. In addition to amino group modiﬁ cation in the polypeptide

chain, glycoproteins may be modiﬁ ed at their polysaccharide hydroxyl groups to form esteriﬁ ed

178 2. The Chemistry of Reactive Groups

derivatives. Esteriﬁ cation of carbohydrates by acetic anhydride, especially cellulose, is a major

industrial application for this compound. In aqueous solutions, however, esteriﬁ cation will be a

minor product, since the oxygen of water is about as strong a nucleophile as the hydroxyls of

sugar residues.

The major side reaction to the desired acylation product is hydrolysis of the anhydride. In

aqueous solutions, anhydrides may breakdown by the addition of one molecule of water to

yield two unreactive carboxylate groups. The presence of an excess of the anhydride in the

reaction medium usually is used to minimize the effects of competing hydrolysis.

Since both hydrolysis and acylation result in the release of carboxylic acid functionalities,

the medium becomes acidic during the course of the reaction. This requires either the presence

of a strongly buffered environment to maintain the pH or periodic monitoring and adjustment

of the pH with base as the reaction progresses.

1.13. Fluorophenyl Esters

Another type of carboxylic acid derivative that reacts with amines consists of the ester of a

ﬂ uorophenol compound, which creates a group capable of forming amide bonds with proteins

and other molecules. Several types of ﬂ uorophenyl esters have been used as reactive groups:

a pentaﬂ uorophenyl (PFP) ester, a tetraﬂ uorophenyl (TFP) ester, and a sulfo-tetraﬂ uorophenyl

(STP) ester. All of these derivatives have similar reactivity with amines, but the uncharged ones

are hydrophobic and the sulfonated one is negatively charged in aqueous solution, thus provid-

ing water solubility to active ester compounds (Gee et al ., 1999) ( Figure 2.1).

Fluorophenyl esters react with amine-containing molecules at slightly alkaline pH values to

give the same amide bond linkages as NHS esters (Reaction 15). However, in most cases, the

ﬂ uorophenyl ester compound will display better stability toward hydrolysis in aqueous solution.

It has been reported that a TFP ester has over twice the half-life in basic pH buffers (pH 8)

than a corresponding NHS ester on the same compound (Molecular Probes).

Figure 2.1 Three types of ﬂ uorophenyl esters have been used for coupling to amine-containing molecules. The

PFP and TFP esters are relatively hydrophobic and typically have better stability toward hydrolysis in aqueous

solution than NHS esters. The STP ester is water-soluble due to the negatively charged sulfonate group, and it

provides better solubility to associated crosslinkers or bioconjugation reagents similar to that of a sulfo-NHS

ester group.

1. Amine Reactions 179