Hermanson G. Bioconjugate Techniques, Second Edition

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in assay systems due to the presence of additional positive charge. For instance, the modiﬁ cation

of avidin with adipic acid dihydrazide by coupling through the protein ’s carboxylate groups sig-

niﬁ cantly increases the net charge of an already highly cationic molecule, and therefore increases

its overall cross-reactivity in avidin–biotin assays (Chapter 23, Section 5).

Modiﬁ cation of Aldehydes with Bis-Hydrazide Compounds

Aldehyde-containing macromolecules will react spontaneously with hydrazide compounds

to form hydrazone linkages. The hydrazone bond is a form of Schiff base that is more stable

than the Schiff base formed from the interaction of an aldehyde and an amine. The hydrazone,

however, may be reduced and further stabilized by the same reductants utilized for reductive

amination purposes (Chapter 3, Section 4.8). The addition of sodium cyanoborohydride to

a hydrazide–aldehyde reaction drives the equilibrium toward formation of a stable covalent

complex. Mallia (1992) found that adipic acid dihydrazide derivatization of periodate-oxidized

dextran (containing multiple formyl functionalities) proceeds with much greater yield when

sodium cyanoborohydride is present.

The reaction of an excess of adipic acid dihydrazide with aldehyde groups present on glyco-

proteins or other molecules will result in modiﬁ ed proteins containing alkylhydrazide groups

(Figure 1.108 ). Another bis-hydrazide compound, carbohydrazide, also may be employed

with similar results, except the spacer afforded through its use is considerably shorter. Target

aldehydes may be created on macromolecules according to the protocols described in Section 4.4,

this chapter. Thus, glycoproteins and other molecules containing polysaccharides may be peri-

odate oxidized to contain formyl groups and then modiﬁ ed with a bis-hydrazide compound

to create the hydrazide-activated reagent. Modiﬁ cation of proteins through glycan residues

obviates the blocking of negatively charged carboxylates and only adds a limited number of

hydrazides at discrete points on the molecule. The enzyme HRP is conveniently modiﬁ ed with

hydrazide functionalities using this approach (Chapter 26, Section 2.4).

Protocol

1. Dissolve a macromolecule (such as a protein) containing aldehyde functionalities at a

concentration of about 1–10 mg/ml in 100 mM sodium citrate, 150 mM NaCl, pH 6.0.

Alternatively, a buffered solution at a pH of about 7–8.5 also will work well in this

protocol. Phosphate, carbonate, borate, or similar buffers adjusted to this pH range work

well. Avoid amine-containing buffers (i.e., glycine or Tris) or other components contain-

ing strong nucleophiles, since these may react with the aldehydes. To modify a molecule

to contain aldehyde groups, see Section 4.4, this chapter.

2. Add a quantity of adipic acid dihydrazide or carbohydrazide (Aldrich) to the protein

solution to obtain at least a 10-fold molar excess over the amount of aldehyde function-

ality present. High molar ratios are necessary to avoid protein conjugation during the

reaction process. If the concentration of aldehydes is unknown, the addition of 32 mg

adipic acid dihydrazide per ml of the protein solution to be modiﬁ ed should work well.

3. React for 2 hours at room temperature. While hydrazone formation does not require the

addition of a reductant to create a linkage, including sodium cyanoborohydride in the

reaction considerably increases the yield and stability of bonds formed. If the presence

140 1. Functional Targets

of a reducing agent will not cause harm to the macromolecule being modiﬁ ed, the addi-

tion of 10 l of 5 M sodium cyanoborohydride (Sigma) per ml of reaction solution may

be done. Caution: Cyanoborohydride is extremely toxic. All operations should be done

with care in a fume hood. Also, avoid any contact with the reagent, as the 5 M solution

is prepared in 1 N NaOH.

4. Purify the modiﬁ ed protein by dialysis or gel ﬁ ltration using a desalting resin.

Hydrazide-activated proteins are stable to long-term storage at 4 °C in the presence of a pre-

servative (0.05 percent sodium azide) or in a frozen or lyophilized state.

4. Creating Speciﬁ c Functionalities 141

Figure 1.108 Glycoproteins that have been treated with sodium periodate to produce aldehyde groups can be

further modiﬁ ed with adipic acid dihydrazide to result in a hydrazide derivative.

142 1. Functional Targets

Modiﬁ cation of Carboxylates with Bis-Hydrazide Compounds

Carboxylic acids may be covalently modiﬁ ed with adipic acid dihydrazide or carbohydrazide

to yield stable imide bonds with extending terminal hydrazide groups. Hydrazide functionali-

ties don ’t spontaneously react with carboxylate groups the way they do with aldehyde groups

(Section 4.5, this chapter). In this case, the carboxylic acid ﬁ rst must be activated with another

compound that makes it reactive toward nucleophiles. In organic solutions, this may be accom-

plished by using a water-insoluble carbodiimide (Chapter 3, Section 1.4) or by creating an

intermediate active ester, such as an NHS ester (Chapter 2, Section 1.4).

In aqueous solutions, the easiest method for forming this type of bond is to use the water-

soluble carbodiimide EDC (Chapter 3, Section 1.1). For proteins and other water-soluble

macromolecules, EDC reacts with their available carboxylate groups to form an intermediate,

highly reactive, o-acylisourea. This active ester species may further react with nucleophiles such

as a hydrazide to yield a stable imide product ( Figure 1.109 ).

Most proteins contain an abundance of carboxylic acid groups from C-terminal function-

alities and aspartic and glutamic acid side chains. These groups are readily modiﬁ ed with bis -

hydrazide compounds to yield useful hydrazide-activated derivatives. Both carbohydrazide and

adipic acid dihydrazide have been employed in forming these modiﬁ cations using the carbodi-

imide reaction (Wilchek and Bayer, 1987).

Protocol

1. Dissolve 32 mg of adipic acid dihydrazide per ml of 0.1 M sodium phosphate, 0.15 M

NaCl, pH 7.2.

2. Dissolve 5 mg of the protein or other macromolecule to be modiﬁ ed per ml of the above

solution.

Figure 1.109 Carboxylate groups on proteins may be modiﬁ ed with adipic acid dihydrazide in the presence of

a carbodiimide to produce hydrazide derivatives.

3. Add 16 mg EDC and react at room temperature for 2–4 hours.

4. Purify the modiﬁ ed protein by dialysis or gel ﬁ ltration using a desalting resin.

Modiﬁ cation of Amines with SANH, SHNH, or SHTH

The introduction of hydrazine or hydrazide functional groups may be done using the bifunc-

tional crosslinkers that are a part of the hydrazine-aldehyde chemoselective ligation reagents,

which are described in Chapter 17, Section 2 (Wong, 1991; Hartmann et al., 2002; Zhong

et al., 2003; Kozlov et al., 2004). These are amine-reactive compounds that all have NHS esters

on one end, and which form amide bonds when coupled to primary amines, such as on the side

chain of lysine. SANH (succinimidyl 4-hydrazinonicotinate acetone hydrazone) and SHNH

(succinimidyl hydraziniumnicotinate hydrochloride) both are hydrazine derivatives of nicotinic

acid, while SHTH (succinimidyl 4-hydrazidoterephthalate hydrochloride) is a hydrazide deriv-

ative of terephthalate. Hydrazines and hydrazides react with carbonyl compounds, such as

aldehydes and ketones, to form hydrazone bonds. The hydrazone linkage formed between a

hydrazine and an aldehyde is much more stable than that formed between a hydrazide and an

aldehyde. Thus, hydrazine–aldehyde bonds typically don ’t require further reduction to stabi-

lize the linkage, as is often the case with hydrazide–aldehyde linkages. For both hydrazide and

hydrazine groups, however, the bond that is formed with ketones is typically weaker than the

hydrazone formed with aldehydes.

The reaction of SANH, SNHN, or SHTH with an amine-containing molecule proceeds at

physiological pH or at slightly basic pH conditions to form an amide bond ( Figure 1.110 ).

Reported reaction conditions used the following buffers to modify proteins, amine-modiﬁ ed

DNA, or amine-modiﬁ ed surfaces: (a) 0.1 M sodium borate buffer, pH 8.1–8.4 or (b) 0.1 M

sodium phosphate, 0.15 M NaCl, pH 7.2–7.4. Whereas NHS esters are reactive throughout

this range, the use of pH buffers closer to neutrality will provide optimal yields while reducing

the amount of competing hydrolysis as compared to higher pH conditions. Avoid the use of

amine or thiol components in the reaction medium, as these will react with the NHS ester (e.g.,

avoid Tris buffer, glycine, DTT, or imidazole).

The following protocol describes a general method for modifying amines with SANH,

SNHN, or SHTH and assaying for the incorporation of hydrazino groups in the ﬁ nal derivative.

4. Creating Speciﬁ c Functionalities 143

Protocol

A. Modiﬁ cation of amine groups on proteins with SANH, SHNH, or SHTH

1. Dissolve SANH, SHNH, or SHTH in DMF to prepare a stock solution at a concentra-

tion of 2.0–4.0 mg in 100–200 l. Use highly pure and dry solvent (H

O content  0.1

percent or treat with molecular sieves) to prevent hydrolysis of the NHS ester.

2. Dissolve the protein to be modiﬁ ed in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2–

7.4, at a concentration of at least 1–10 mg/ml.

3. With mixing, add an aliquot of the crosslinker solution to the protein solution such that

the desired molar excess is attained of reagent over the protein present in the reaction.

144 1. Functional Targets

Figure 1.110 The reaction of SANH with an amine-containing molecule results in an amide bond derivative

that terminates in a protected hydrazine group. Reaction with an aldehyde-containing molecule results in release

of the acetone-protecting group and formation of a stable hydrazone bond.

For most applications, molar ratios in the range of 5:1 to 20:1 will work best to generate

a number of hydrazine groups on the protein. Maintain the ﬁ nal percentage of DMF in

the reaction mixture at less than 10 percent to avoid precipitation of protein.

4. React at room temperature for at least 30–60 minutes or 2–3 hours at 4 ° C.

5. Purify the modiﬁ ed protein away from excess reagent and reaction by-products by gel

ﬁ ltration using a desalting column or dialysis.

6. Calculate the protein concentration in the ﬁ nal preparation using its absorbance at

280 nm or a colorimetric method, such as the Coomassie assay. ( Note: The presence of

hydrazine or hydrazide groups on the protein will interfere with the BCA assay for total

protein concentration.)

B. Measurement of hydrazine modiﬁ cation level

The number of hydrazine groups per protein molecule can be determined by reacting a small

portion of the hydrazine-modiﬁ ed protein with p-nitrobenzaldehyde, which forms a chromog-

enic product upon formation of the hydrazone derivative ( Figure 1.111 ).

1. Prepare a 0.5 mM p-nitrobenzaldehyde buffer by initially dissolving the compound at a

higher concentration in organic solvent (e.g., methanol, DMF, or DMSO) and then adding

the appropriate aliquot to 100 mM acetate, pH 5.0 to result in the ﬁ nal concentration.

2. Add an aliquot of the hydrazine-modiﬁ ed protein solution to the p -nitrobenzaldehyde

solution and incubate at 37 °C for 1 hour or at room temperature for 2 hours. To assure

accuracy, determine the linear response range of the test by adding a series of different

concentrations of the hydrazine-modiﬁ ed protein solution to the p -nitrobenzaldehyde

buffer. This is done by preparing a set of serial dilutions of the protein solution and

4. Creating Speciﬁ c Functionalities 145

Figure 1.111 An SANH-modiﬁ ed molecule can be detected and measured by reaction with p -nitrobenzalde-

hyde, which forms a chromogenic derivative with a characteristic absorbance at 350 nm.

adding an equal volume of each dilution to an aliquot of the p-nitrobenzaldehyde solu-

tion in separate tubes. Determine the absorbance of each solution at 380 nm versus a

blank prepared by the addition of an equal aliquot of buffer alone.

3. Calculate the substitution level of hydrazine groups into the protein by determin-

ing the hydrazine concentration using the molar extinction coefﬁ cient of the resultant

p-nitrobenzyl derivative (22,000 M

 1

) and dividing this value by the protein con-

centration (moles/liter). Any sample falling within the linear response range of the test

can be used for this calculation.

Modiﬁ cation of Alkylphosphates with Bis-Hydrazide Compounds

Alkylphosphate groups such as those present at the 5 -end of RNA and DNA molecules may

be speciﬁ cally modiﬁ ed with bis-hydrazide compounds. Mediated by the addition of the water-

soluble carbodiimide EDC and imidazole, adipic acid dihydrazide or carbohydrazide will

react with the phosphate group in a two-step process to form phosphoramidate bonds with

short linker arms containing terminal hydrazides ( Figure 1.112 ) (Ghosh et al., 1989). In the

ﬁ rst stage, EDC activates the phosphate group forming a short-lived, but highly reactive phos-

phodiester species, which in turn reacts with a molecule of imidazole to form a longer-lived,

active phosphorimidazolide. The second stage involves addition and attack of the hydrazide

nucleophile, releasing imidazole and forming the phosphoramidate bond. In a modiﬁ cation of

the two-stage reaction, Zanocco (1993) developed a single-pot reaction in which the alkylphos-

phate molecule is reacted in the presence of EDC, imidazole, and the bis-hydrazide compound.

The modiﬁ cation reaction proceeds rapidly at room temperature.

Protocol

1. Weigh out 1.25 mg of the carbodiimide EDC hydrochloride (Thermo Fisher); into a

microfuge tube.

2. Add to the tube 7.5 l of RNA or DNA containing a 5 -phosphate group. The concentra-

tion of the oligonucleotide should be 7.5–15 nmol or total of about 57–115.5 g. Also,

immediately add 5 l of 0.25 M adipic acid dihydrazide or carbohydrazide dissolved in

0.1 M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the

oligo and bis-hydrazide/imidazole solutions should occur quickly.

3. Mix by vortexing, then place the tube in a microcentrifuge and spin for 5 minutes at

maximal rpm.

146 1. Functional Targets

Figure 1.112 Phosphate groups can be modiﬁ ed with adipic acid dihydrazide in the presence of a carbodi-

imide to produce hydrazide derivatives. This is a common modiﬁ cation route for the 5 -phosphate group of

oligonucleotides.

4. Add an additional 20 l of 0.1 M imidazole, pH 6.0. Mix and react for at least 2 hours

at room temperature. The additional buffer prevents pH drift during the carbodiimide

reaction.

5. Purify the hydrazide-labeled oligo by gel ﬁ ltration on a desalting resin using 10 mM

sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The hydrazide-containing probe

now may be used to conjugate with a molecule containing an aldehyde-reactive group.

4.6. Introduction of Saccharide or Glycan Groups

The modiﬁ cation of proteins with sugar groups occurs in vivo through both enzymatic and non-

enzymatic processes. Approximately 1 percent of proteins encoded in the genomes of mammals

are enzymes that are involved with carbohydrate production or modiﬁ cation. Many of these

enzymes digest carbohydrate in foods to provide energy for cellular metabolism, but others

are involved with the controlled modiﬁ cation of proteins or other biomolecules to create com-

plex polysaccharide structures. This process results in carbohydrates, called glycans, covalently

attached to proteins at discrete locations on only certain amino acid residues within a polypep-

tide sequence (Section 2, this chapter). The presence of carbohydrate modiﬁ cations on proteins

has a pronounced effect on biological activity in vivo .

Non-enzymatic modiﬁ cation of proteins with saccharides also occurs in vivo through uncon-

trolled glycation of lysine amines with the reducing end of sugars, especially glucose. This reac-

tion results in the formation of an initial Schiff base with a subsequent rearrangement to form

a stable ketoamine derivative. The non-enzymatic glycation reaction has been studied exten-

sively as a result of it being a major factor in the development of the complications associated

with diabetes (for review, see Singh et al ., 2001).

In vitro modiﬁ cation of protein can be done synthetically to add speciﬁ c sugars or com-

plex carbohydrates to proteins for further bioconjugation or for subsequent study of the

glycan-derivative in vivo. Investigations of the effect of these synthetic carbohydrate–protein

conjugates (neoglycoproteins) on the immune response date back many decades with the dia-

zonium-mediated coupling of aminophenol glucosides to study type-II and type-III pneumonia

polysaccharides (Goebel et al., 1932). More recently, conjugation of carbohydrates to pro-

tein carriers has been done to illicit a speciﬁ c immune response to glyco-antigens of infectious

diseases or tumor cells (Toyokuni and Singhal, 1995; Koganty et al., 1996; Ragupathi et al .,

1997; Pozsgay, 1998; Mawas et al., 2002; Karsten et al., 2004). Synthetic peptide–glycan con-

jugates also have been prepared by conjugation of carbohydrates to peptide sequences that can

be presented by MHC (major histocompatibility complex) molecules to enhance the immune

response against the carbohydrate component (Kihlberg and Magnusson, 1996). For an excel-

lent review of glycoconjugation, see Davis (1999).

Sugar residues also can be used to modify a protein, molecule, or surface for subsequent use

in a bioconjugation procedure or to increase the hydrophilicity of the modiﬁ ed molecule. For

bioconjugation purposes, a sugar group can be added to facilitate the covalent conjugation of

another molecule. Since many saccharides contain diols that can be oxidized by periodate to cre-

ate aldehydes, certain sugars can be used after glycol oxidation to couple with amine-contain-

ing molecules by reductive amination. For instance, the amine group on the monosaccharide

glucosamine can be coupled to an amine-reactive surface or to a protein through its carboxy-

late groups using EDC (Chapter 3, Section 1.1). The glucosamine-modiﬁ ed surface or molecule

4. Creating Speciﬁ c Functionalities 147

subsequently can be oxidized by sodium periodate to create aldehydes (Section 2 and 4.4, this

chapter). This reactive intermediate ﬁ nally can be conjugated to another amine-containing mol-

ecule to create the ﬁ nal conjugate through reductive amination (Chapter 2, Section 5).

Periodate oxidation of carbohydrates should be avoided, however, if generating an immune

response against the saccharide component is desired or antibody recognition needs to be

retained against the carbohydrate. Woodward et al. (1985) demonstrated that periodate oxida-

tion of glycans effectively destroys antibody binding if the speciﬁ city of the antibody truly is

toward the carbohydrate. The assay of antibody binding to a carbohydrate with and without

periodate oxidation often is used to demonstrate antibody speciﬁ city. A speciﬁ c antibody will

fail to bind to periodate-oxidized carbohydrate, but will bind to the non-oxidized glycan.

The modiﬁ cation of molecules with saccharides also has the effect of increasing the

hydrophilicity of the resultant complex due to the presence of multiple hydroxyl groups. Native

glycan modiﬁ cation of proteins functions in much the same manner, because the carbohydrate

148 1. Functional Targets

Figure 1.113 The NHS ester–suberate derivative of lactose can be used to add lactose groups to amine-contain-

ing molecules. The reaction results in the formation of amide bonds containing terminal lactose groups.

“ tree ” becomes fully hydrated through the ability of the hydroxyls to hydrogen bond with the

surrounding water molecules. In addition, post-translational modiﬁ cation of proteins helps

polypeptides fold properly by assuring that certain regions are maintained near the solvent sur-

face. Chemical modiﬁ cation with saccharides thus can be done to increase the hydrophilicity

and solubility of proteins or other molecules in aqueous solution.

Some reports even indicate that the conjugation of proteins or peptides with carbohydrates

can increase dramatically their activity compared to that of the native state (Susaki et al .,

1998). Carbohydrates also can provide a protective effect on modiﬁ ed peptides toward pro-

teolytic digestion (Rudd et al., 1994) or mask recognition of a peptide by the immune system

(Harding et al., 1993). The creation of neoglycoproteins thus can affect the activity of peptides

and proteins, which are not normally glycosylated in vivo .

The following sections describe several examples of saccharide modiﬁ cation for the purpose

of bioconjugation, the study of glycan function, to prepare immunogens, or to increase the

water solubility of a modiﬁ ed molecule.

Modiﬁ cation of Amines with Mono(lactosylamido) mono(succinimidyl)suberate

The amine-reactive compound mono(lactosylamido) mono(succinimidyl)suberate contains a

lactose group at the end of a suberate bridge, which terminates at the other end in an NHS

ester. Vetter et al. (1995) prepared this reagent by reacting the corresponding glycosylamine

derivative with disuccinimidyl suberate (DSS). The starting lactosylamine derivative was pre-

pared via a 5-day reaction of the reducing end of lactose with aqueous ammonia solution in

sodium carbonate. Other similar 1-amino, 1-deoxy sugar derivatives may be synthesized in

a like manner for subsequent bioconjugation. The ﬁ nal product after reaction with DSS, the

mono-NHS ester derivative of lactose containing an 8-carbon suberate spacer, can be used to

modify proteins or other amine-containing molecules with a lactose group. The NHS ester

spontaneously reacts with an amine at neutral or slightly basic pH values to form an amide

bond ( Figure 1.113 ). The presence of the lactose disaccharide provides a hydrophilic modify-

ing group that increases the water solubility of the resultant conjugate. Other glycosylamine

derivatives may be prepared using a similar strategy to modify biomolecules or surfaces with

speciﬁ c saccharide compounds.

4. Creating Speciﬁ c Functionalities 149