Hermanson G. Bioconjugate Techniques, Second Edition

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The following protocol describes the modiﬁ cation of a protein with mono(lactosylamido)

mono(succinimidyl)suberate. The reagent is available from Thermo Fisher. The use of this rea-

gent to couple to amine-containing surfaces, such as polystyrene beads, also has been done

using similar reaction conditions (Vetter et al ., 1995).

Protocol

1. Dissolve mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to prepare a

concentrated solution from which an aliquot may be taken and added to a ﬁ nal aqueous

reaction medium. The compound is extremely soluble in DMF, and solutions of 100 mg/

ml may be prepared. The use of dry solvent is essential to prevent hydrolysis of the NHS

ester. However, make only enough of this stock solution so that a small amount added to

the protein reaction will provide the appropriate molar excess desired for the modiﬁ ca-

tion reaction.

2. Prepare the protein to be modiﬁ ed in a non-amine-containing buffer at a slightly basic

pH (i.e., avoid Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15 M NaCl, pH

7.2 works well for NHS ester reactions. The concentration of the protein in the reaction

buffer may vary from g/ml to mg/ml, but highly dilute solutions will result in less efﬁ -

cient modiﬁ cation yields. A protein concentration from 1 to 10 mg/ml works well in this

reaction.

3. With mixing, add a quantity of the mono(lactosylamido) mono(succinimidyl)suberate

in dry DMF to the protein solution to result in a 10–20 fold molar excess of reagent

over the amount of protein present. Depending on the desired application for the lactosyl-

modiﬁ ed protein, several different molar ratios of reactant-to-protein may have to be

tried to optimize the resulting modiﬁ cation level.

4. React for 30–60 minutes at room temperature with gentle mixing.

5. Purify the modiﬁ ed protein away from reactants and reaction by-products using dialysis

or size exclusion chromatography.

Modiﬁ cation of Amine or Hydrazide Molecules by Carbohydrates and Glycans

The modiﬁ cation of proteins, surfaces, or other molecules with reducing sugars through a

reductive amination process still is perhaps the most common method for glycosyl addition.

A saccharide or glycan molecule containing a reducing end typically has a masked carbonyl

group (i.e., an aldehyde or ketone) that can be reacted with an amine or hydrazide in the pres-

ence of a reducing agent (such as sodium cyanoborohydride or borane compounds) to form a

secondary amine or a reduced hydrazone bond ( Figure 1.114 ). However, since most reducing

ends of sugars or glycans exist mainly in an acetal (or ketal) ring form, while only the open

form with the exposed aldehyde can participate in a reductive amination reaction, the rate of

modiﬁ cation by this process can be slow and the yield low.

To help overcome the predominance of the cyclic acetal form, which inhibits the desired

amination reaction, functional groups other than amines (having greater reactivity) have been

used, along with elevated temperatures and high concentrations of reducing agent, to form

more efﬁ ciently the initial Schiff base and thus drive the coupling reaction to completion. In

particular, hydrazide or hydrazine groups have been used successfully to modify reducing

150 1. Functional Targets

4. Creating Speciﬁ c Functionalities 151

glycans and saccharides with or without a reducing agent present (Rothenberg et al., 1993;

Toomre and Varki, 1994; Leteux et al., 1998; Srikrishna et al., 2001) ( Figure 1.115 ). Whereas

reactions done with primary amines at room temperature may take days to reach acceptable

coupling yields, the modiﬁ cation of hydrazino groups with the reducing end of a sugar can be

done in hours at 60–80 °C. Also, the modiﬁ cation of glycans with hydrazines can be done with-

out the addition of cyanoborohydride, because the resultant hydrazone linkage is more stable

compared to the Schiff base formed between an aldehyde and an amine. Although when cou-

pling hydrazino compounds to the reducing end of a glycan, the addition of a reductant often

Figure 1.114 The reaction of a reducing sugar with an amine-containing compound in the presence of sodium

cyanoborohydride results in ring opening with the formation of a secondary amine derivative.

Figure 1.115 The reaction of a reducing sugar with a hydrazide-containing molecule can proceed by one of

two routes depending on whether a reducing agent is present. If the reaction is done in the presence of sodium

cyanoborohydride, then ring opening will occur followed by the formation of a reduced hydrazone linkage.

If no reducing agent is present, then the reaction gives a glycosylhydrazide derivative with retention of the ring

structure of the sugar group.

is done to drive the reaction to completion and further stabilize the hydrazone, reduction of the

terminal saccharide can cause changes in the binding potential of some lectins that recognize

the core sugar structure (Leteux et al ., 1998).

The following protocol may be used to conjugate the available reducing end of a saccha-

ride or glycan with an amine, hydrazide, or hydrazine group. These functional groups may be

present on a variety of molecules, such as biotin groups for labeling or molecules having ﬂ uores-

cent properties, which allows detection of the glycan derivative. For speciﬁ c modiﬁ cation details,

see the methods of Rothenberg et al. (1993); Toomre and Varki (1994); Leteux et al. (1998); and

Srikrishna et al. (2001). The following method is based on the optimized conditions as deter-

mined by Bigge et al. (1995). The use of organic solvents should be done in a fume hood.

Protocol

1. Dissolve a carbohydrate, saccharide, or glycan sample having a free reducing end in

0.1 M sodium acetate, pH 5.0 ( Note: Glycans may be released from glycoconjugates by

hydrazinolysis using pure hydrazine or by endoglycosidase treatment with PNGase F).

Alternative coupling conditions that can be used for the modiﬁ cation reaction include

30 percent glacial acetic acid in DMSO (v/v) or acetic acid/pyridine (1:2, v/v). The use

of DMSO or pyridine often facilitates solubilization of a greater range of carbohydrates

or glycans than aqueous buffers. The presence of acetic acid has been found to acceler-

ate the reductive amination reaction when the organic solvent conditions are used (Bigge

et al., 1995). The concentration of the carbohydrate should be 5–100 M for glycans.

For modiﬁ cation of other more abundant carbohydrates, higher concentrations can be

used if required. For glycan modiﬁ cation of biomolecules that are not compatible with

organic solvents, such as proteins, the glycan initially may be solubilized in DMSO and

then an aliquot added to the aqueous reaction buffer.

2. Add to the glycan solution the molecule to be labeled containing an available amine,

hydrazine, or hydrazide group. For small molecule derivatization, the ﬁ nal concentration

of the nucleophile in the glycan solution should be about 0.3 M to result in maximal efﬁ -

ciency of labeling. For protein modiﬁ cation, an aqueous reaction buffer should be used,

and the protein should be as concentrated as possible.

3. Add to the reaction mixture a quantity of reducing agent (e.g., sodium cyanoborohydride

or borane dimethylamine (BDA)) to give a ﬁ nal concentration of 1.0 M.

4. When using nonaqueous reaction conditions, incubate for 1–2 hours at 60–80 °C. For

reactions in an aqueous environment with temperature-sensitive molecules, the reaction

may be done at room temperature or 37 °C. In this case, the reaction time should be

extended to at least 24 hours. Longer reaction times are not unusual when modifying

carbohydrates by reductive amination at ambient temperature. For instance, the coupling

of heparin through its reducing end to a solid phase containing a hydrazide group takes

up to 72 hours at room temperature to obtain maximal yield.

5. Purify the modiﬁ ed glycan from reactants and reaction by-products by dialysis, gel ﬁ ltra-

tion, or ion-exchange chromatography, depending on the size and type of the molecule

being modiﬁ ed by the carbohydrate. For instance, Rothenberg et al. (1993), fractionated

glycans modiﬁ ed with a biotin–diaminopyridine derivative from excess biotin compound

by size exclusion chromatography on a 1.5  48 cm Toyopearl HW40S column equili-

brated with 50 percent acetonitrile/10 mM sodium acetate.

152 1. Functional Targets

Labeling Glycans with Fluorescent 2-Aminopyridine, 2-Amino Benzamide, or Anthranilic Acid

The ability to label glycans released from glycoproteins and other glycoconjugates is

important for tracking complex carbohydrates through puriﬁ cation or analysis, such as

HPLC, electrophoresis, and mass spec. Glycan molecules typically don ’t have spectrally detect-

able groups and therefore beneﬁ t by being labeled with a detectable component for easy assay

and detection. Small, amine-containing ﬂ uorescent compounds have been found to be par-

ticularly useful in this regard. The compounds 2-aminopyridine, benzamide, and anthranilic

acid (2-aminobenzoic acid) have been used to label the reducing end of glycans and other

carbohydrates.

Glycosylated proteins and other glycoconjugates may be treated to release the modify-

ing carbohydrate component, making available the reducing end for conjugation. N -linked

glycans may be released from glycoconjugates by PNGase F, PNGase A or released by anhy-

drous hydrazine and regenerated to reducing oligosaccharides, thus yielding free reducing ends

for bioconjugation. In addition, the enzymes Endo H and Endo F release N-linked glycans

by cleaving the GlcNAc( 1–4)GlcNAc core, thereby resulting in one less GlcNAc moiety at

the reducing end. o-Linked glycans may be released using o-glycanase treatment, or by mild

hydrazinolysis followed by regeneration to the reducing oligosaccharides. o-Linked glycans

also may be released by non-reductive -elimination. However, one should avoid strong reduc-

tive glycan releasing methods using sodium borohydride, as these reduce the C1 aldehyde on

the core sugar group and make it unreactive for further derivatization.

Bigge et al. (1995) describe a method to label released glycans by reductive amination with

the small ﬂ uorescent compounds 2-amino benzamide and anthranilic acid (2-aminobenzoic

acid). The result is a secondary amine bond with the C1 carbon atom of the reducing end of

the glycan ( Figure 1.116 ). The derivatives provide stable ﬂ uorescent modiﬁ cations on glycans,

which can be used for detection during electrophoresis, separation, and puriﬁ cation techniques

(such as HPLC). The 2-amino benzamide label was found to work best for chromatographic

separations, enzymatic modiﬁ cations, and mass spec analysis, while the anthranilic acid deriva-

tive was more suitable for electrophoretic separations, because its negative charge produced

sharper bands.

Rothenberg et al. (1993) describe the synthesis of a very useful ﬂ uorescent glycan labeling

agent that contains a biotin group. Diaminopyridine was reacted with sulfo-NHS-biotin to

form 2-amino-(6-amidobiotinyl)pyridine (BAP). This reagent contains the ﬂ uorescent diami-

nopyridine group for detection and a biotin handle for puriﬁ cation or immobilization using its

strong interaction with streptavidin. BAP can be used to label the reducing ends of glycans and

other carbohydrates by reductive amination to form a secondary amine linkage with the ami-

nopyridine group.

The ﬂ uorescent 2-aminopyridine group also may be used without a biotin handle for

modiﬁ cation of glycans for detection, similar to the use of anthranilic acid and 2-amino

benzamide. In this case, the tag just functions as a ﬂ uorescent label, which can be used

to track glycans during puriﬁ cation or analysis. Modiﬁ cation of glycans with the bifunc-

tional 2,6-diaminopyridine group using reductive amination produces a ﬂ uorescently labeled

carbohydrate with an available amine for further coupling to surfaces or other biomolecules.

Care should be taken, however, when using amine-reactive reagents to modify such amino

glycans, because many electrophilic compounds cross-react with hydroxyls on the sugar

molecules.

4. Creating Speciﬁ c Functionalities 153

A similar ﬂ uorescent biotin label to the BAP reagent was created by Leteux et al. (1998) for

labeling glycans without reduction of the reducing end of the monosaccharide. Biotinyl- L -3-

(2-naphthyl)-alanine hydrazide (BNAH) contains a strongly UV absorbing naphthalene group,

which has ﬂ uorescent characteristics, and a hydrazide group for conjugation to the reducing

end of glycans. This compound was found to label effectively glycans at the reducing end,

while retaining the unreduced nature of the carbohydrate, thus preserving critical protein inter-

actions, which may recognize this region of the sugar.

The reductive amination protocol for coupling these types of small tags to glycans can be

found in the previous section.

154 1. Functional Targets

Figure 1.116

Released glycans can be labeled with small ﬂ uorescent compounds containing amines for sub-

sequent detection upon chromatographic separation. In the presence of sodium cyanoborohydride these com-

pounds react at the reducing end of glycans to form secondary amine derivatives with characteristic spectral

properties.

Synthesis of Glycosylamines for Conjugating Glycans

Reducing sugars and carbohydrates or glycans containing an unmodiﬁ ed reducing end may be

derivatized in a simple reaction to provide an amine group on C1 for further conjugation. The

anomeric hydroxyl group at the reducing end of such sugars can be converted into an amino

group by reaction in an aqueous, saturated solution of ammonium carbonate. Kochetkov ami-

nation, as it is called (Likhosherstov et al., 1986), can be used to modify a wide variety of

reducing carbohydrates and glycans, including neutral and charged monosaccharides, disaccha-

rides, and oligosaccharides (Kallin et al., 1989; Urge et al., 1991, 1992; Manger et al., 1992;

Cohen-Anisfeld and Langburg, 1993).

Modiﬁ cation using this protocol yields saccharides that are highly stereochemically pure,

with typical reactions giving  95 percent glycosylamines of the -anomer structure ( Figure

1.117 ). The resulting primary amine group can be further coupled with amine-reactive bio-

conjugation reagents or used for carbohydrate immobilization onto surfaces or insoluble sup-

ports. The major side reaction of amination is the dimeric diglycosylamine derivative, which

has two sugar groups attached to a single secondary amine in the middle. This by-product can

be present in up to 10 percent of the resulting mass of product formed by the reaction.

The following protocol for synthesis of glycosylamines from reducing sugars is based on the

method of Likhosherstov et al . (1986).

Protocol

1. Prepare a solution of the saccharide to be aminated at a concentration of up to 1 percent

(w/v) in an aqueous solution of saturated ammonium carbonate. Even higher concentra-

tions of saccharides may be used if the carbohydrate being modiﬁ ed is abundant and

inexpensive.

2. React with stirring at room temperature for up to 5 days. Over the course of the reac-

tion, add to the solution solid ammonium carbonate at a rate of about 40 mg per mg of

saccharide to assure continued saturation.

3. After the reaction, freeze the solution and lyophilize to remove excess ammonium carbonate.

Complete removal of volatile salt can be accomplished by re-dissolving the solid in warm

methanol. After the completion of CO

evolution, dry the saccharide by evaporation under

vacuum. Removal of ammonium carbonate is essential, as the ammonium ion will interfere

with any subsequent conjugations attempted with the glycosylamine derivative.

4. Creating Speciﬁ c Functionalities 155

Figure 1.117 Glucosylamine derivatives can be prepared at the reducing end of glycans or other reducing car-

bohydrates by reaction with ammonium carbonate. The resultant amine derivative can be used to conjugate the

carbohydrate with other proteins or molecules without disturbing the cyclic character of the reducing end.

5. Blocking Speciﬁ c Functional Groups

It is often necessary to block speciﬁ c groups on macromolecules to prevent them from partici-

pating in modiﬁ cation or conjugation reactions. In most blocking procedures, a chemical group

is covalently coupled to an undesired functional group on the macromolecule to mask or elimi-

nate its reactivity. In this sense, the modiﬁ cation is done with a compound that is relatively inert

in whatever application the macromolecule is intended for use. The blocking agent is usually

a small organic compound containing a functional group able to couple with the group to be

masked. The blocking molecule may contain another functional group of its own, converting

the blocked group into a chemical function of another type, but this conversion is all right, pro-

viding the newly created function does not interfere in subsequent reactions or applications.

In some cases, a blocking procedure is done to direct a conjugation reaction to discrete sites

in a macromolecule. In other instances, blocking a group on one of two macromolecules can

prevent self-polymerization and promote the desired intermolecular conjugation. For instance,

HRP can be blocked with an amine-speciﬁ c coupling reagent prior to periodate oxidation to

prevent the reactivity of its two amino groups during subsequent conjugation with an antibody

molecule (Chapter 20, Section 1.3).

In other uses of blocking reagents, proteins dissociated into subunits by the use of denaturants

and disulﬁ de reductants may be prevented from re-association or oxidation of their sulfhydryls by

blocking the SH groups with the appropriate reagent. Alternatively, sulfhydryls may be blocked

on a protein prior to activation with a heterobifunctional crosslinking agent that contains both

amine-reactive and sulfhydryl-reactive ends. The amine-reactive end will couple to the amines of

the protein without reaction of the sulfhydryl-reactive end. This can prevent oligomer formation

during the activation process and thus ensure that the sulfhydryl-reactive function is available for

conjugation with the desired molecule.

Controlled functional studies of a protein ’s active center also may be done by blocking speciﬁ c

groups and observing its effect on activity. Often, this blocking procedure is performed through

the use of a reversible blocking agent to subsequently regenerate activity; therefore demonstrat-

ing the affect was directed at functionalities present in the active site (Perham and Jones, 1967).

Blocking also may be done to quench further modiﬁ cation or conjugation through a targeted

functional group. After a conjugation reaction, excess functional groups may be masked from

nonspeciﬁ cally reacting with other molecules. For instance, periodate-oxidized glycoproteins

may still contain aldehyde groups after conjugation with another protein by reductive amination.

Blocking the aldehydes with a small amine-containing molecule prevents unwanted reactions

from occurring when the conjugate is used in an assay or targeting operation. This also is true of

excess sulfhydryl groups that may undergo disulﬁ de interchange with other sulfhydryl molecules

subsequent to a conjugation reaction. Blocking these groups with the appropriate reagent pre-

vents this type of side reaction from occurring.

Blocking of amine groups on proteins also has been used to create a sensitive reagent for

measuring protease activity (Hatakeyama, 1992). With nearly all the primary amines of casein

blocked, an amine detection reagent such as TNBS will react only minimally with the pro-

tein and form its typical orange derivative. As proteases cleave the protein, however, primary

-amines are created from cleavage of the -chain peptide bonds, and TNBS then can react

with them. The more protease activity present, the more color is formed.

The choice and application of a speciﬁ c blocking reagent can produce a modiﬁ ed macromol-

ecule with unique and useful properties. Many of the common blocking reagents are discussed

156 1. Functional Targets

in this section. Beyond the scope of this book, however, is a discussion of the numerous block-

ing agents used in peptide or nucleic acid synthesis to temporarily block speciﬁ c reactive groups

during growth of the polymer chain.

5.1. Blocking Amine Groups

The amine functionalities most commonly found in macromolecules are primary amines such

as those at the N-terminal of polypeptide chains ( -amines) and the side chain -amino groups

of lysine residues. Several acylation reagents can effectively block these primary amines, some

of which are reversible under the right conditions. It should be noted that the cyclic anhydrides

mentioned in this section react with amino groups to form amide bonds, opening the anhydride

ring and effectively transforming the amine function into a carboxylate. There are additional

compounds described in Section 4.2 (this chapter) that also create carboxylates from amines,

but in this section the two cyclic anhydrides discussed, maleic anhydride and citraconic anhy-

dride, both are reversible and designed more for temporary masking than permanent blocking.

For more stable blocking of amines, the compounds sulfo-NHS acetate and acetic anhydride

are the best choices.

Sulfo-NHS Acetate

Sulfo-NHS acetate is the N-hydroxysulfosuccinimide ester of acetic acid. The NHS ester end

provides high reactivity with the amino groups of proteins at a pH range of 7–9, acylating the

amines and forming nonreversible acetamide modiﬁ cations ( Figure 1.118 ). The sulfonate deriva-

tive of the NHS ester provides good water solubility to the reagent. Thus, the compound can be

added directly to an aqueous solution of the protein to be blocked, or a stock solution may be

prepared and a small aliquot added to the reaction medium. Stock solutions should be dissolved

rapidly and used immediately. In aqueous solutions, the main competing reaction is hydroly-

sis of the active ester to release nonreactive sulfo-NHS and acetic acid. The use of a 10- to

50-fold molar excess of sulfo-NHS acetate over the molar amount of groups to be blocked

should provide good yields of acylated amines. Reaction buffers should contain no extraneous

amines that could cross-react with the sulfo-NHS acetate. Avoid Tris, glycine, and imidazole

containing buffers. Phosphate, borate, or bicarbonate buffers work well at a concentration of

0.05–0.1 M. React for at least 1 hour at room temperature.

It should be noted that complete blocking of all amines on proteins with sulfo-NHS ace-

tate may cause precipitation or loss of native structure and function. The acetate modiﬁ cations

5. Blocking Speciﬁ c Functional Groups 157

Figure 1.118 Sulfo-NHS acetate may be used to block amine groups, forming permanent amide bond

derivatives.

are uncharged and relatively hydrophobic, which may decrease solubility of proteins or other

molecules.

Protocol

1. Dissolve the protein or other amine-containing macromolecule at a concentration of

1–10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5.

2. Add a 25-molar excess of sulfo-NHS acetate over the amount of amines present in the

sample. If the precise amount of amines is not known, adding an equal mass of reagent to

the mass of protein will provide a large excess of reactivity to completely block all amines.

3. React at room temperature for at least 1 hour.

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

Acetic Anhydride

Acetic anhydride is the only monocarboxylic acid anhydride that is important in modiﬁ cation

reactions. The acetylation of the amino groups of proteins can be made relatively speciﬁ c if the

reaction is done in saturated sodium acetate, since the o-acetyltyrosine derivative is unstable to

an excess of acetate ions (Fraenkel-Conrat, 1959). The tyrosine derivative rapidly hydrolyzes

in alkaline reaction conditions, even in the absence of added acetate buffer (Uraki et al., 1957;

Smyth, 1967). Treatment with hydroxylamine also cleaves any o-acetyltyrosine modiﬁ cations,

forming acetylhydroxamate, which can be followed by its purple complex with Fe



at 540 nm

(Balls and Wood, 1956).

At physiological pH values, acetylation of amine groups proceeds rapidly, requiring less than

an hour to go to completion ( Figure 1.119 ).

158 1. Functional Targets

Protocol

1. Dissolve the macromolecule to be modiﬁ ed at a concentration of 1–10 mg/ml in a buff-

ered solution having a pH between 6.5 and 7.5. Avoid amine-containing buffers such as

glycine and Tris. Sodium phosphate buffer at a concentration of 0.1 M works well. The

addition of an equal volume of a saturated solution of sodium acetate may be done to

prevent tyrosine derivatization.

2. Cool the solution on ice. With stirring, add an amount of acetic anhydride equal to the

mass of macromolecule to be modiﬁ ed. Alternatively, add a 10-fold molar excess of ace-

tic anhydride over the amount of amines present. The addition of the anhydride slowly

or in several aliquots over the course of 1 hour will assure good yield of acetylation.

3. React with stirring for at least 1 hour while cooling in an ice bath.

4. Purify the acetylated macromolecule by gel ﬁ ltration or dialysis.

Citraconic Anhydride

Citraconic anhydride (or 2-methylmaleic anhydride) is a derivative of maleic anhydride that is

reversible after acylation of amine groups. At alkaline pH values (pH 7–8) the reagent reacts

with amines to form amide linkages with an extending terminal carboxylate. However, at acid

pH (3–4), these bonds rapidly hydrolyze to release citraconic acid and free the amine (Dixon

and Perham, 1968; Habeeb and Atassi, 1970; Klapper and Klotz, 1972; Shetty and Kinsella,

1980). Thus, citraconic anhydride is useful in temporarily blocking amine groups while other

parts of a molecule are undergoing derivatization. Once the modiﬁ cation is complete, the

amines can be then unblocked to create the original structure. See Section 4.2 (this chapter) for

additional information and a protocol for modiﬁ cation of proteins with citraconic anhydride.

Maleic Anhydride

Maleic acid is a linear four carbon molecule with carboxylate groups on both ends and a dou-

ble bond between the central carbon atoms. The anhydride of maleic acid is a cyclic molecule

containing ﬁ ve atoms. Although the reactivity of maleic anhydride is similar to other cyclic

anhydrides, the products of maleylation are much more unstable toward hydrolysis, and the

site of unsaturation lends itself to additional side reactions. Acylation products of amino

groups with maleic anhydride are stable at neutral pH and above, but they readily hydrolyze

at acid pH values around pH 3.5 (Butler et al., 1967). Maleylation of sulfhydryls and the phe-

nolate of tyrosine are even more sensitive to hydrolysis. Thus, maleic anhydride is an excellent

reversible blocker of amino groups to temporarily mask them from reactivity while another

5. Blocking Speciﬁ c Functional Groups 159

Figure 1.119 Acetic anhydride reacts with amines to form amide bond derivatives.