Hermanson G. Bioconjugate Techniques, Second Edition

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Both mono-substituted derivatives and di-substituted products of the imidazole ring are possible

(Crestﬁ eld et al., 1963). With primary amine groups such as in the side chain of lysine residues,

the products of the reaction are the secondary amine (monocarboxymethyllysine) or the tertiary

amine derivative (dicarboxymethyllysine). Methionine thioether groups give the most compli-

cated products, some of which rearrange or decompose unpredictably. The only stable derivative

of methionine is where the terminal methyl group is lost to form carboxymethylhomocysteine,

the same product as the reaction of iodoacetate with homocysteine (Gundlach et al., 1959).

The relative reactivity of -haloacetates toward protein functionalities is sulfhydryl  imid

azolyl  thioether  amine. Among halo derivatives the relative reactivity is I  Br  Cl  F,

with ﬂ uorine being almost unreactive. The -haloacetamides have the same trend of relative

110 1. Functional Targets

Figure 1.87 Iodoacetate can modify a number of amino acid side chains in proteins, forming alkylated deriva-

tives containing a terminal carboxylate.

reactivities, but will obviously not create a carboxylate functional group. The acetamide deriv-

atives typically are used only as blocking agents.

Thus, iodoacetate has the highest reactivity toward sulfhydryl cysteine residues and may be

directed speciﬁ cally for SH modiﬁ cation. If iodoacetate is present in limiting quantities (rela-

tive to the number of sulfhydryl groups present) and at slightly alkaline pH, cysteine modiﬁ -

cation will be the exclusive reaction. The speciﬁ city of this modiﬁ cation has been used in the

design of heterobifunctional crosslinking reagents, where one end of the crosslinker contains an

iodoacetamide derivative and the other end contains a different functionality directed at another

chemical target (see SIAB; Chapter 5, Section 1.5).

Protocol

1. Dissolve the sulfhydryl-containing protein or macromolecule to be modiﬁ ed at a con-

centration of 1–10 mg/ml in 50 mM Tris, 0.15 M NaCl, 5 mM EDTA, pH 8.5. EDTA is

present to prevent metal-catalyzed oxidation of sulfhydryl groups. The presence of Tris,

an amine-containing buffer, should not affect the efﬁ ciency of sulfhydryl modiﬁ cation.

Not only do amines generally react slower than sulfhydryls, the amine in Tris buffer is of

particularly low reactivity. If Tris does pose a problem, however, use 0.1 M sodium phos-

phate, 0.15 M NaCl, 5 mM EDTA, pH 8.0.

2. Add iodoacetate to a concentration of 50 mM in the reaction solution. Alternatively, add

a quantity of iodoacetate representing a 10-fold molar excess relative to the number of

SH groups present. An estimation of the sulfhydryl content in the protein to be modi-

ﬁ ed can be accomplished by performing an Ellman ’s assay (Chapter 1, Section 4.1).

Readjust the pH if necessary. To aid in adding a small quantity of iodoacetic acid to

the reaction, a concentrated stock solution may be made in the reaction buffer, the pH

re-adjusted, and an aliquot added to the protein solution to give the desired concentration.

3. Mix and react for 2 hours at room temperature. To avoid the possibility of methionine

modiﬁ cation, limit the reaction to 30 minutes.

4. Purify the modiﬁ ed protein from excess iodoacetate by dialysis or gel ﬁ ltration.

5. An Ellman ’s assay comparing the unmodiﬁ ed protein to the iodoacetylated protein may

be done to assess the degree of modiﬁ cation.

Modiﬁ cation of Sulfhydryls with BMPA

BMPA is N--maleimidopropionic acid (or 3-maleimidopropionic acid), which contains a thiol-

reactive maleimide group at one end and a carboxylate group on the other end (Rich et al ., 1975;

Moroder, 1983, 1987). The compound is the acid precursor to the short, heterobifunctional

crosslinker 3-maleimidopropionic acid N-hydroxysuccinimide ester (BMPS).

4. Creating Speciﬁ c Functionalities 111

Like BMPS, BMPA is spontaneously reactive toward sulfhydryls through its maleimide, but

unlike BMPS it must be activated using a carbodiimide, such as EDC, to couple to amines or

hydrazides through its carboxylic acid end. In its use as a blocking or modiﬁ cation agent for

sulfhydryls, BMPA may be reacted with a thiol-containing molecule to form a stable thioether

bond. The blocking of thiols takes place in buffered aqueous conditions from slightly acidic to

moderately basic pH by addition to the double bond of the maleimide group. The ﬁ nal product,

which then contains the short propionic acid spacer, terminates in a negatively charged carboxy-

late. Thus, thiols can be modiﬁ ed and transformed into carboxylate-containing molecules using

this reagent.

BMPA also has been used as a crosslinking agent in a number of applications, including the

preparation of peptide–protein conjugates for immunogens Cruz et al ., to prepare immunomodu-

lating adducts (Gemeiner et al., 1992; Cruz et al., 2001), in the preparation of novel trifunctional

compounds containing the metal-chelating group lysine nitrilotriacetic acid (NTA) for conjuga-

tion with His-tagged proteins (Meredith et al., 2004), for the immobilization of proteins onto

surfaces (Jung and Wilson, 1996), to create thiol-reactive quantum dots for labeling biomolecules

(Evident Technologies, Inc., web site protocols, 2005), to form albumin–insulin conjugates for

slow-release drugs (Shechter et al., 2005), and to synthesize thiol-reactive luminescent chelates

for time-resolved ﬂ uorescence applications (Chen and Selvin, 1999).

Figure 1.88 shows the reactions of BMPA for the modiﬁ cation of thiols and a subsequent

reaction using an EDC-mediated amide bond formation for coupling to its carboxylate end.

The maleimide–thiol reaction proceeds at physiologic pH to form a stable thioether linkage with

sulfhydryl-containing molecules. The combination of EDC and sulfo-NHS (Chapter 3, Section 1.2)

also may be used to form an intermediate sulfo-NHS ester, which can enhance the yield of amide

bond formation. Cysteine groups in proteins and peptides may be permanently blocked using this

reagent, yielding a modiﬁ cation that terminates in the negatively charged carboxylate.

112 1. Functional Targets

Figure 1.88 The maleimide group of BMPA reacts with a thiol-containing molecule to result in a modiﬁ cation

having a terminal carboxylate group. Amine-containing molecules then can be conjugated to the carboxylate

using a carbodiimide reaction with EDC.

The use of BMPA to block a thiol and create a terminal carboxylate is illustrated in the fol-

lowing protocol. The protocol relates to the modiﬁ cation of proteins, but similar reaction condi-

tions can be used to modify other thiol-containing molecules or surfaces.

Protocol

1. Dissolve a thiol-containing protein in phosphate buffered saline (PBS), pH 6.5–7.5, at

a concentration 1–10 mg/ml. Disulﬁ des may be reduced to yield free thiols using DTT,

TCEP, or other reducing agents, but reductants containing sulfhydryls should be com-

pletely removed by dialysis or desalting prior to reaction with BMPA.

2. Dissolve BMPA in DMSO or DMF to prepare a stock solution at a higher concentra-

tion such that adding an aliquot of this solution to the protein solution will result in the

desired molar excess of the maleimide over the concentration of thiols present.

3. Add a quantity of BMPA to the protein solution to obtain at least a 5-fold molar excess

of maleimide reagent over the amount of thiol present in the protein. The ﬁ nal concen-

tration of organic solvent in the protein solution should not exceed 10 percent to prevent

protein precipitation. Mix thoroughly to dissolve.

4. React for 2 hours at room temperature.

5. Purify the modiﬁ ed protein from reactants and reaction by-products by dialysis or gel

ﬁ ltration.

Modiﬁ cation of Hydroxyls with Chloroacetic Acid

Chloroacetic acid can be used to transform a rather unreactive hydroxyl into a carboxylate

group that can be used in a variety of conjugation reactions. The reaction proceeds under basic

conditions, yielding a stable ether bond terminating in a carboxymethyl group ( Figure 1.89 )

(Plotz and Rifai, 1982; Brunswick et al., 1988). Side reactions will occur with other nucle-

ophiles, such as amines, if they are present in the molecule to be modiﬁ ed. The reagent is used

most often to modify pure polysaccharides or hydroxyl-containing polymers that contain no

other functionalities.

4. Creating Speciﬁ c Functionalities 113

Figure 1.89 Chloroacetic acid can be used to create a carboxylate group from a hydroxyl.

The following protocol illustrates the modiﬁ cation of a dextran polymer with chloroace-

tic acid.

Protocol

1. In a fume hood, prepare a solution consisting of 1 M chloroacetic acid in 3 M NaOH.

2. Immediately add dextran polymer to a ﬁ nal concentration of 40 mg/ml. Mix well to

dissolve.

3. React for 70 minutes at room temperature with stirring.

4. Stop the reaction by adding 4 mg/ml of solid NaH

and adjusting the pH to neutral

with 6 N HCl.

5. Remove excess reactants by dialysis.

4.3. Introduction of Primary Amine Groups

Primary amine groups on proteins consisting of N-terminal -amines and lysine side chain

-amines are typically present in abundant quantities for modiﬁ cation or conjugation reactions.

Occasionally, however, a protein or peptide will not contain sufﬁ cient amounts of available

amines to allow for an efﬁ cient degree of coupling to another molecule or protein. For instance,

HRP, a popular enzyme to employ in the preparation of antibody conjugates, only possesses

two free amines that can participate in conjugation protocols. Creating additional amines on

HRP allows for higher amounts of modiﬁ cation and thus produces more active conjugates.

Other non-protein molecules, such as nucleic acids and oligonucleotides, may not nor-

mally possess primary amines of sufﬁ cient nucleophilicity to react with common modiﬁ cation

reagents. The ability to add amine functionalities to these molecules is sometimes the only

route to successful conjugation. Creating amines at speciﬁ c sites within these molecules allows

for site-directed modiﬁ cation at known positions, thus better assuring active conjugates once

formed.

The following reagents and techniques can be used to directly transform carboxylates or

sulfhydryls into reactive amine functional groups. In addition, sugars, polysaccharides, or gly-

can containing macromolecules may be modiﬁ ed to contain amines after mild periodate activa-

tion to form aldehyde groups or through modiﬁ cation at the reducing end of a carbohydrate.

Modiﬁ cation of Carboxylates with Diamines

Carboxylic acids may be covalently modiﬁ ed with short compounds containing primary amines

at either end to form amide linkages. The result of such alterations is to block the carboxylates

and form terminal amino groups. Reacting the diamine in high molar excess assures that

only one end of the compound couples to each carboxylate and does not crosslink the mol-

ecules being modiﬁ ed. Amide bond formation may be accomplished by several methods includ-

ing carbodiimide-mediated coupling (Chapter 3, Section 1), active ester intermediates such as

N-hydroxysuccinimide esters (Chapter 2, Section 1.4), and the use of carbonylating compounds like

N,N -carbonyldiimidazole (Chapter 3, Section 3). A combination of the water-soluble carbodiimide

EDC and sulfo-NHS also is an efﬁ cient way of creating amide linkages (Chapter 3, Section 1.2).

114 1. Functional Targets

Diamines that can be used for aminoalkylation include ethylene diamine, 1,3-diaminopro-

pane, 3,3  -imino bispropylamine (also known as diaminodipropylamine), 1,6-diaminohexane,

and the Jeffamine-type compounds containing a hydrophilic chain consisting of polyethylene-

or polypropylene-oxide (formerly from Texaco Chemical Co., now Huntsman Corporation).

Ethylene diamine is perhaps the most popular choice for protein carboxylate modiﬁ cation. Its

short chain length assures minimal steric effects and virtually no hydrophobic interactions.

Diaminodipropylamine provides a longer spacer arm and has been used extensively as a bridg-

ing molecule for coupling carboxylate-containing ligands to insoluble supports (Hermanson

et al., 1992). The long hydrocarbon chain of 1,6-diaminohexane, however, may induce

hydrophobic effects and probably should be avoided. The longest diamine of the group is the

Jeffamine compound. Its chain is extremely hydrophilic and should function as an excellent

modiﬁ er of carboxylates when a longer spacer is desired.

In addition, there are other diamine spacers containing discrete PEG chains available with

one end blocked using either a t-BOC group or a CBZ-amido group (Quanta BioDesign). These

diamine spacers are extremely hydrophilic due to the presence of a PEG

or PEG

cross-bridge

units. Since one end of these compounds is masked with a reversible blocking group commonly

used in peptide synthesis, the free amine end can be conjugated to a carboxylic acid without

the possibility of crosslinking. The blocked end then can be removed with TFA (for t-BOC

groups) or by reduction using hydrogen in the presence of a Pd/C catalyst (for CBZ-amido

groups). This type of deblocking reaction only should be used with molecules that can tolerate

these conditions, such as organic molecules and short peptides. Complex proteins, however,

may be denatured or loose activity under those conditions.

4. Creating Speciﬁ c Functionalities 115

Diamine modiﬁ cation of proteins can have dramatic effects on the net charge of the molecule,

usually signiﬁ cantly raising the pI from the native state. The amide linkage eliminates the negative

potential of the carboxylate and the terminal amine adds the potential for a positive charge. Thus,

diamine modiﬁ cation may have the net effect of changing the overall charge by plus two for every

carboxylate residue coupled. Proteins heavily modiﬁ ed with diamines may exhibit vital changes in

activity due to the alteration of microenvironmental charge at each site of modiﬁ cation. In some

cases, native conformation may be changed and activity completely lost.

Raising the pI of macromolecules also can signiﬁ cantly alter the immune response toward

them upon in vivo administration. Cationized proteins (those modiﬁ ed with diamines to increase

their net charge or pI) are known to generate an increased immune response compared to their

native forms (Muckerheide et al., 1987a, b; Apple et al., 1988; Domen et al., 1987; Domen and

Hermanson, 1992). The use of cationized BSA as a carrier protein for hapten conjugation can

result in a dramatically higher antibody response toward a coupled hapten (Chapter 19).

The following protocol using the carbodiimide EDC is an efﬁ cient way of modifying protein

carboxylates with diamines to either increase the amount of amines present for further conju-

gation or to create a cationized protein having an increased net charge ( Figure 1.90 ). Note that

116 1. Functional Targets

Figure 1.90 Cationization of protein molecules can be done using ethylene diamine to modify carboxylate

groups using a carbodiimide reaction process.

glycoproteins containing sialic acid may be modiﬁ ed at this sugar ’s COOH group in addition

to coupling at C-terminal, aspartic acid, and glutamic acid functions on the polypeptide chain.

Other carboxylate-containing macromolecules may be modiﬁ ed using this procedure as well.

Protocol

1. Dissolve the protein to be modiﬁ ed at a concentration of 1–10 mg/ml in 0.1 M MES, pH 4.7

(coupling buffer). Other buffers may be used as long as they don ’t contain groups that can

participate in the carbodiimide reaction. Avoid carboxylate- or amine-containing buffers

such as citrate, acetate, glycine, or Tris. Higher pH conditions may be used up to about pH

7.5 (in sodium phosphate buffer) without severely affecting the yield of modiﬁ cation. The

protein in solid form also may be added directly to the diamine solution prepared in (2).

2. Dissolve the diamine chosen for modiﬁ cation at a concentration of 1 M made up in the

coupling buffer. If a free-base form of diamine is used, then the solution will become

highly alkaline upon dissolution. This operation also will generate heat—the solution

process being highly exothermic. The easiest way to dissolve such a diamine is to initially

add the correct amount to a beaker containing a quantity of crushed ice equal to the ﬁ nal

solution volume desired. The ice should be made from deionized water or the equivalent

to maintain purity. All operations should be done in a fume hood. Next, add an equivalent

weight of concentrated HCl and mix. As the mixing becomes complete, the ice will almost

totally melt and provide nearly the correct ﬁ nal solution volume. Finally, add an amount

of MES buffer salt to bring its concentration to 0.1 M, and adjust the solution pH to 4.7.

In some cases, the dihydrochloride form of the diamine is commercially available and can

be used to avoid such unpleasant pH adjustments. For instance, ethylene diamine dihy-

drochloride is available from Aldrich. It can be added to the 0.1 M MES buffer without a

signiﬁ cant change in pH.

3. Add the protein solution to an equal volume of diamine solution and mix. Alternatively,

the solid protein can be dissolved directly in the diamine solution (after pH adjustment)

at the indicated concentration.

4. Add EDC hydrochloride; Thermo Fisher to a ﬁ nal concentration of 2 mg/ml in the reac-

tion solution. To aid in the addition of a small amount of EDC, a higher concentration

stock solution may be prepared in water and an aliquot added to the reaction to give the

proper concentration. Since EDC is labile in aqueous solutions, the stock solution must

be made quickly and used immediately.

5. React for 1–2 hours at room temperature.

6. Purify the modiﬁ ed protein by extensive dialysis against 0.02 M sodium phosphate,

0.15 M NaCl, pH 7.4 (PBS) or another suitable buffer.

The changes that occur in the pI of a protein modiﬁ ed with diamines may be assessed by

isoelectric focusing or by general electrophoresis based upon relative migration due to charge.

A cationized protein will possess a higher pI value or migrate further toward the anode than its

native form. Using the above protocol typically alters the net charge of BSA from a native pI of

4.9 to the highly basic range of pI 9.5 to over pI 11.0.

Modiﬁ cation of carboxylate groups with diamines also may be done in organic solvent for

those molecules insoluble in aqueous buffers. Some peptides are quite soluble in solvents such

as DMF and DMSO, but relatively insoluble in water. Such molecules may be reacted in these

4. Creating Speciﬁ c Functionalities 117

solvents with the carbodiimide DCC (dicyclohexyl carbodiimide) using the same basic reactant

ratios as given above for EDC in aqueous solutions (Chapter 3, Section 1.4).

Modiﬁ cation of Sulfhydryls with N -(  -Iodoethyl)triﬂ uoroacetamide [Aminoethyl-8]

The conversion of sulfhydryl groups on cysteine residues or other molecules to amine-

containing groups may be accomplished by aminoethylation with N -( -iodoethyl) triﬂ uoro-

acetamide (Schwartz et al., 1980). The haloalkyl group speciﬁ cally reacts with sulfhydryls to

form the aminoalkyl derivative in one step. Under the conditions of the reaction, the triﬂ uor-

oacetate amine-protecting group spontaneously hydrolyzes to expose the free primary amine

without the need for a secondary deblocking step ( Figure 1.91 ). This reagent is commercially

available from Thermo Fisher Chemical under the name Aminoethyl-8 ™.

Aminoethyl-8 has an advantage over ethylenimine modiﬁ cation (see next section), due to the

potential polymerization of ethylenimine in aqueous solutions. Such polymers are highly cati-

onic and may nonspeciﬁ cally block the protein. The speciﬁ city of Aminoethyl-8 for sulfhydryls

makes it an optimum choice for modiﬁ cation.

For small molecules containing sulfhydryls or for low-molecular-weight peptides contain-

ing cysteine residues, modiﬁ cation may proceed without deforming agents. However, for intact

proteins containing both disulﬁ des and free sulfhydryls, a denaturant and a disulﬁ de reducing

118 1. Functional Targets

Figure 1.91 Aminoethyl-8 can be used to transform a sulfhydryl group into an amine. The intermediate spon-

taneously undergoes deblocking to release the primary amine group.

agent may be required to open buried or structurally inaccessible groups if complete modiﬁ ca-

tion is desired.

Protocol

1. Dissolve the protein to be aminoalkylated at a concentration of 1–10 mg/ml in 6 M gua-

nidine hydrochloride, 0.2 M N-ethylmorpholine acetate, pH 8.1. All water used in pre-

paring buffers should be deoxygenated by boiling followed by cooling and bubbling with

nitrogen. Small molecules that don ’t require denaturants to expose internal disulﬁ des or

sulfhydryls may be modiﬁ ed without using guanidine treatment.

2. Add DTT to obtain a 20-fold molar excess over the amount of disulﬁ des present.

3. React for 4 hours at room temperature, maintaining a blanket of nitrogen over the solution.

4. Adjust the pH to 8.6 with NaOH, and heat the solution to 50 ° C.

5. Add a quantity of Aminoethyl-8 in methanol to equal a 25-fold molar excess over the

amount of sulfhydryl present (including the amount of DTT added). The solution in

methanol should be made concentrated enough so only a small amount of methanol has

to be added to the reaction solution (i.e., no more than 10 percent of the ﬁ nal volume).

A second addition of modifying agent may be made after 1 hour to drive the reaction

more completely toward total SH aminoalkylation.

6. React for 3 hours at 50 ° C.

7. Purify the modiﬁ ed protein or other macromolecules by gel ﬁ ltration or dialysis.

Occasionally, complete modiﬁ cation with Aminoethyl-8 will cause precipitation of the

protein.

Modiﬁ cation of Sulfhydryls with Ethylenimine

The cyclic compound ethylenimine reacts with protein sulfhydryl groups causing ring open-

ing and forming the aminoalkyl derivative, S-(2-aminoethyl)cysteine (Raftery and Cole, 1963,

1966) ( Figure 1.92 ). Under physiological conditions ethylenimine is virtually speciﬁ c for sulf-

hydryls with no cross-reactivity toward other protein functionalities. At acid pH, a small

degree of reactivity occurs with methionine residues, forming S-(2-aminoethyl)methionine sul-

fonium ion (Schroeder et al., 1967). Since aminoethylated cysteine groups resemble the side-

chain structure of lysine residues, except for the replacement of one methylene group with

a thioether, these modiﬁ cations make them susceptible to tryptic hydrolysis, although at an

abbreviated rate (Plapp et al ., 1967; Wang and Carpenter, 1968).

4. Creating Speciﬁ c Functionalities 119

Figure 1.92 The small compound ethylenimine can react with sulfhydryls to form aminoethyl derivatives.