Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

Periodate Oxidation of Glycols and Carbohydrates

Carbohydrates and other biological molecules that contain polysaccharides, such as glycopro-

teins, can be speciﬁ cally modiﬁ ed at their sugar residues to produce reactive formyl function-

alities. With proteins, this method often allows modiﬁ cation to occur only at speciﬁ c locals,

usually away from critical active centers or binding sites.

Periodate oxidation is perhaps the simplest route to transforming the relatively unreactive

hydroxyls of sugar residues into amine-reactive aldehydes. Periodate cleaves carbon–carbon

bonds that possess adjacent hydroxyls, oxidizing the OH groups to form highly reactive

aldehydes (Bobbit, 1956; Rothfus and Smith, 1963). Terminal cis-glycols result in the loss of

one carbon atom as formaldehyde and the creation of an aldehyde group on the former No. 2

carbon atom. Varying the concentration of sodium periodate during the oxidation reaction

gives some speciﬁ city with regard to what sugar residues are modiﬁ ed. Sodium periodate at a

concentration of 1 mM at near 0 °C speciﬁ cally cleaves only at the adjacent hydroxyls between

carbon atoms 7, 8, and 9 of sialic acid residues (Van Lenten and Ashwell, 1971; Wilchek and

Bayer, 1987). The product is the formation of one aldehyde group on the No. 7 carbon and

liberation of two molecules of formaldehyde ( Figure 1.102 ).

Since sialic acid is a frequent terminal sugar constituent of the polysaccharide trees on glyco-

proteins, this method selectively forms reactive aldehydes on the most accessible parts for sub-

sequent modiﬁ cations. The carbohydrate polymer of a protein provides a long spacer arm that

can be used to conjugate another large macromolecule, such as a second protein, with little

steric problems.

Oxidation of polysaccharides using 10 mM or greater concentrations of sodium periodate

at room temperature results in the cleavage of adjacent hydroxyl-containing carbon–carbon

bonds on other sugars besides just sialic acid residues (Lotan et al., 1975). High concentrations

130 1. Functional Targets

Figure 1.102

The reaction of sodium periodate with sugar residues can produce aldehydes for conjugation

reactions.

of periodate result in sugar ring opening and the creation of many aldehydes on each polysac-

charide tree.

Using these methods, carbohydrate-containing proteins may be altered to contain alde-

hydes for conjugation with other proteins or for detection using hydrazide-containing probes

(Chapter 9). The aldehydes thus formed then can be coupled to other amine-containing mol-

ecules by Schiff base formation and reductive amination (Chapter 2, Section 5.3 and Chapter 3,

Section 4). For instance, the enzyme HRP can be activated with periodate for conjugation with

antibodies (Nakane and Kawaoi, 1974). Alternatively, such reactive formyl groups may be con-

jugated to hydrazide-containing molecules to form hydrazone bonds (Chapter 4, Section 8; and

Chapter 26, Section 2.1). Cell-surface polysaccharides may be probed with hydrazide-containing

reagents for sialic acid groups or total glycoconjugates. Glycoproteins or glycopeptides in solu-

tion also may be tagged in this manner. Gangliosides and other glycolipids may be modiﬁ ed

with hydrazide reagents as well (Spiegel et al., 1982).

Protocol

1. The glycoprotein or diol-containing molecule is dissolved in deionized water or a buffer

at physiological pH. Sodium phosphate buffer (0.01–0.1 M), pH 7.0, is an appropriate

choice. When oxidizing cell-surface glycoconjugates, use a buffer suitable for cellular

stability requirements. Avoid amine-containing buffers such as Tris and glycine, because

they may interact with the aldehyde groups as they are formed. For glycoproteins in solu-

tion, a concentration range of 1–10 mg/ml will produce acceptable results in this proce-

dure. For sialic acid modiﬁ cation, place the sample in ice to cool to near 0 ° C.

2. Dissolve sodium periodate (MW 213.91) in water at a concentration of 10 mg/ml

(0.046 M). Protect from light. To obtain approximately a 1 mM concentration of sodium

periodate in the reaction solution (suitable for oxidizing only sialic acid residues), add

21.8 l of this stock solution to each ml of the glycoprotein solution to be oxidized.

Maintain the solution on ice. For general oxidation of carbohydrates other than just

sialic acid, add 218 l of the stock solution to obtain an approximate ﬁ nal concentration

of 10 mM periodate in the reaction. Use room temperature conditions for general carbo-

hydrate oxidation. Wrap the vial containing the reaction solution with aluminum foil to

protect from light. The use of an amber vial also is suitable for this purpose.

3. React for 15–30 minutes at room temperature.

4. Quench the reaction by the addition of 0.1 ml of glycerol per ml of reaction solution.

Alternatively, the reaction may be stopped by immediate gel ﬁ ltration on a desalting column.

If a dextran-based resin is used for the chromatography, the support itself will react with

sodium periodate to quench excess reagent. Alternatively, N-acetylmethionine may be added

to quench the reaction, because the thioether of the methionine side chain will react with

periodate to form sulfoxide or sulfone products (Geoghegan and Stroh, 1992). In addition,

sodium sulﬁ te (Na

) was used by Stolowitz et al. (2001) to quench the periodate oxida-

tion of HRP in solution. To quench the reaction with cellular samples, wash the cells with

buffer to remove remaining traces of periodate.

Oxidase Modiﬁ cation of Sugar Residues

Another method of forming aldehyde groups on carbohydrates and glycoproteins involves the use

of speciﬁ c sugar oxidases. These enzymes only affect the monosaccharide they are speciﬁ c toward,

4. Creating Speciﬁ c Functionalities 131

leaving other sugar residues within polysaccharides unaffected. Probably the most often used oxi-

dase for this purpose is galactose oxidase, which can form C-6 aldehydes on terminal D-galactose

or N-acetyl-D-galactose residues (Avigad et al., 1962) ( Figure 1.103 ). When galactose residues are

penultimate to sialic acid residues, another enzyme, neuraminidase, must be used to remove the

sialic acid sugars and expose galactose as the terminal residue (Wilchek and Bayer, 1987). The

speciﬁ city of using glycosidases to create aldehyde residues on carbohydrates may be the meth-

od’s greatest advantage. However, the use of a simple chemical reagent such as sodium periodate

still may be the easiest way to create aldehydes on carbohydrates (Section 4.4, this chapter).

The following protocol was used by Wilchek and Bayer (1987) to label cell-surface galactose

residues.

Protocol

1. Prepare a 5 percent cell suspension in an appropriate buffer. Avoid amine-containing

buffers, as these will interact with aldehydes.

2. Add 0.05 units of Vibrio cholerae neuraminidase and 5 units of galactose oxidase per ml

of cell suspension.

3. Incubate for 60 minutes at 37 °C.

4. Wash cells with PBS to remove excess enzymes.

Modiﬁ cation of Amines with NHS-Aldehydes (SFB and SFPA)

Succinimidyl p-formylbenzoate (SFB) and succinimidyl p-formylphenoxyacetate (SFPA) are

amine-reactive reagents that contain terminal aldehyde residues. Their NHS ester ends react

with primary amines in proteins and other molecules at pH 7–9 to yield amide linkages (see

Chapter 2, Section 1.4 and Chapter 17, Section 2) ( Figure 1.104 ). The resulting formyl deriva-

tives may be utilized to couple to other amine or hydrazinecontaining molecules (Kraehenbuhl

et al., 1974; Galardy et al., 1978). In particular, SFB can be used to produce aldehyde groups

on alkaline phosphatase for conjugation with 5 -hydrazide modiﬁ ed DNA for use in hybridi-

zation assays (Chapter 27, Section 2.4) (Ghosh et al., 1989). SFB and SFPA are insoluble in

water, but may be pre-dissolved in DMF or acetonitrile before adding a small quantity to an

aqueous reaction mixture. Both reagents contain aromatic phenyl rings and have absorptivity

at wavelengths less than 300 nm. Their structures may contribute a signiﬁ cant degree of

132 1. Functional Targets

Figure 1.103 Galactose oxidase may be used to transform speciﬁ cally the C-6 hydroxyl group of galactose into

an aldehyde.

hydrophobicity to macromolecules being modiﬁ ed, especially if high-density couplings are

achieved. For this reason, modiﬁ ed proteins and other soluble molecules may have a tendency

to precipitate if modiﬁ cation is done too heavily. The optimal amount of modiﬁ cation may

have to be adjusted to maintain solubility in each application.

Protocol

1. Dissolve a macromolecule containing amine groups at a concentration of 1–10 mg/ml

in a buffer having a pH of 7.0–9.0 (i.e., 0.1 M sodium phosphate, pH 7.5). Higher pH

conditions will increase the hydrolysis rate of the NHS ester. Avoid amine containing or

nucleophilic buffers such as Tris, glycine, or imidazole (see Chapter 2, Section 1.4).

2. Dissolve SFB (Thermo Fisher, Solulink) or SFPA (Molecular Probes) in DMF. The con-

centration should be such that a small aliquot can be added to the reaction medium to

obtain at least a 10-fold molar excess of modifying reagent over the amount of amines

4. Creating Speciﬁ c Functionalities 133

Figure 1.104 SFB reacts with primary amines to form amide bond derivatives containing aldehyde groups.

to be modiﬁ ed. Add no more than 100 l of the modiﬁ er/DMF solution to each ml of the

macromolecule solution prepared in (1).

3. React for 2 hours at room temperature.

4. Purify the modiﬁ ed macromolecule from excess reagent and reaction by-products by dial-

ysis or gel ﬁ ltration.

Modiﬁ cation of Amines with Glutaraldehyde

Amino groups on proteins may be reacted with the bis-aldehyde compound glutaraldehyde to

form activated derivatives able to crosslink with other proteins. The reaction mechanism for

this modiﬁ cation proceeds by one of several possible routes. In the ﬁ rst option, one of the alde-

hyde ends can form a Schiff base linkage with -amines or -amines on proteins to leave the

other aldehyde terminal free to conjugate with another molecule. Alternatively, a glutaralde-

hyde polymer may undergo vinyl addition to create stable secondary amine bonds, leaving the

aldehydes exposed for subsequent reductive amination reactions. Finally, a cyclized form of

glutaraldehyde also may react with the -amines of two neighboring lysine side chains to form

a quaternary pyridinium crosslink ( Figure 1.105 ).

Schiff base interactions between aldehydes and amines typically are not stable enough to

form irreversible linkages. These bonds may be reduced with sodium cyanoborohydride or

a number of other suitable reductants (Chapter 2, Section 5) to form permanent secondary

amine bonds. However, proteins crosslinked by glutaraldehyde without reduction nevertheless

show stabilities unexplainable by simple Schiff base formation. The stability of such unreduced

glutaraldehyde conjugates has been postulated to be due to the vinyl addition mechanism,

which doesn ’t depend on the creation of Schiff bases.

Glutaraldehyde modiﬁ cation readily proceeds at alkaline pH. The higher the pH, the more

efﬁ cient is Schiff base formation. Using a reductant like sodium cyanoborohydride that does

not affect the aldehyde groups, while efﬁ ciently transforming Schiff bases into a secondary

amines, provides the best possible yields. In many cases, the degree of glutaraldehyde-induced

crosslinks is so severe that conjugate precipitation occurs. This is especially well documented in

antibody–enzyme conjugation schemes employing this reagent (Chapter 20, Section 1.2).

Glutaraldehyde also can be used to create aldehydes on amine-containing polymers. The

use of this reagent in derivatizing chromatography supports and other soluble polymers is well

known (Hermanson et al ., 1992).

The following protocol may be used as the ﬁ rst stage of a two-step glutaraldehyde conju-

gation reaction. In this initial reaction, glutaraldehyde modiﬁ cation converts available protein

amines into reactive formyl groups. The subsequent addition of a second protein or another

amine-containing molecule causes the activated protein to crosslink with the amines and forms

a conjugate. Glutaraldehyde also may be used in single-step conjugation procedures where

the aldehyde-modiﬁ ed protein is not isolated before addition of a second protein. In single-

step conjugation both proteins to be crosslinked are together in solution and glutaraldehyde is

added to effect crosslinking (Chapter 20, Section 1.2).

Protocol

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

centration of 1–10 mg/ml in a buffer having a pH from 7 to 10. The higher the pH, the

134 1. Functional Targets

more efﬁ ciently Schiff base formation will occur. Phosphate, borate, and carbonate buff-

ers at 0.01–0.1 M are acceptable. Avoid amine-containing buffers like Tris and glycine,

since they will react with glutaraldehyde.

2. Add a quantity of glutaraldehyde equal to a 10-fold molar excess over the amount of

amines to be modiﬁ ed. A typical concentration of glutaraldehyde in the reaction mixture

is 1.25 percent. In some cases, trial experiments will have to be done to check for solubil-

ity of the resultant modiﬁ ed protein. Scale back the quantity of glutaraldehyde added if

precipitation occurs.

3. React for at least 2 hours at 4 ° C.

4. Quickly isolate the modiﬁ ed protein by gel ﬁ ltration using a desalting resin.

4. Creating Speciﬁ c Functionalities 135

Figure 1.105 Glutaraldehyde can undergo complex reactions with amine groups, resulting in aldehyde-containing

derivatives that can be used in conjugation reactions.

In some cases, the modiﬁ ed protein may be stored for long periods before conjugation with

another amine-containing molecule by immediate freezing and lyophilization. If stability is a

problem, however, the modiﬁ ed protein should be conjugated immediately.

Periodate Oxidation of N-Terminal Serine or Threonine Residues

Sodium periodate can be used to form aldehydes on unmodiﬁ ed N-terminal serine or threo-

nine residues in proteins and peptides (Geoghegan and Stroh, 1992). Periodate cleaves carbon–

carbon bonds that possess on both of them primary or secondary hydroxyls or amines (i.e.,

diols or 2-amino alcohol groups). If a primary hydroxyl is present, such as in the case of

N-terminal Serine residues, then the reaction liberates formaldehyde and forms an aldehyde

group (an -N-glyoxylyl) at the end of the peptide ( Figure 1.106 ). This reaction can be used to

direct bioconjugation to a site-speciﬁ c point on biomolecules, provided that there are no other

periodate-oxidizable groups within the protein structure. A synthetic peptide designed to have

an N-terminal serine or threonine residue can provide a site of coupling at the end of the chain.

This strategy is a viable alternative to the incorporation of a cysteine group for bioconjugation

at the end of a peptide.

If other oxidizable groups are present in a protein, such as carbohydrates or sensitive amino

acids side chains (Section 1.1, this chapter), then this method should be avoided, because modi-

ﬁ cation can occur at sites other than just the N-terminal. This method has been used successfully

to conjugate tags with small peptides, to attach ﬂ uorescent probes to enzyme substrates, to effect

the conjugation of lactamase to a Fab  antibody fragment, for the coupling of antibodies to lipo-

somes, and to couple a PEG polymer to the amino terminus of proteins (Geoghegan et al., 1993;

Gaertner et al., 1994; Mikolajczyk et al., 1994; Gaertner and Offord, 1996; and Koning et al.,

1999, respectively).

When using sodium periodate to oxidize an N-terminal serine or threonine residue on a large

molecule like a protein, excess oxidant can be removed simply by dialysis or size exclusion chro-

matography. However, when using periodate to oxidize a low-molecular-weight peptide, it can

become problematic to remove excess reactant by size separation methods alone. For this reason,

the addition of a reducing agent may be used to scavenge any remaining periodate, so long as

the reductant chosen doesn ’t also reduce the aldehydes that have been formed. Geoghegan and

Stroh (1992) used N-acetylmethionine for this purpose, because the thioether of the methionine

side chain readily can react with periodate to form sulfoxide or sulfone products, but it won ’t

affect the aldehyde groups formed at the end of the peptide chains or interfere with subsequent

coupling reactions. A less expensive reagent, sodium sulﬁ te (Na

) was used by Stolowitz

et al. (2001) to quench the periodate oxidation of HRP in solution, and ultimately this may

prove to be the best choice for stopping the reaction.

Conjugation of molecules to periodate-oxidized N-terminal serine or threonine residues in

peptides is best done using hydrazine or hydrazide reagents to avoid potential cross-reactions

with lysine amino groups in the peptide structure. The aldehyde group preferentially reacts

with the hydrazino group even in the presence of other amines to form a hydrazone bond

(Figure 1.107 ). After the reaction, the hydrazone may be reduced with sodium cyanoborohy-

dride to stabilize the linkage.

The following protocol is based on the methods of Geoghegan and Stroh (1992) and

Stolowitz et al . (2001).

136 1. Functional Targets

Protocol

1. Dissolve the peptide containing an N-terminal serine or threonine group at a concentra-

tion of at least 2 mg/ml in 0.04 M sodium phosphate, pH 7.0. Higher concentrations of

peptides or proteins may be used without modiﬁ cation to the rest of the protocol, because

the amount of periodate used in the reaction is in sufﬁ cient molar excess, even when

low-molecular-weight peptides are being oxidized. Peptides that are initially insoluble

4. Creating Speciﬁ c Functionalities 137

Figure 1.106 An N-terminal serine or threonine residue can be oxidized with sodium periodate to produce an

aldehyde group. The reaction can be quenched with sodium sulﬁ te to eliminate excess periodate.

at physiological pH ﬁ rst may be dissolved at higher concentration in 0.01 percent TFA

before adding a small aliquot to the buffer. Adjust the pH back to neutral, if needed.

2. With mixing, add sodium periodate to a ﬁ nal concentration of 2.5 mM. Periodate should

be pre-dissolved as a stock solution at a higher concentration in buffer or water, and then

138 1. Functional Targets

Figure 1.107 The N-terminal aldehyde group on a peptide formed from periodate oxidation of serine or threo-

nine residues can be conjugated with a hydrazide-containing molecule to produce a hydrazone bond.

4. Creating Speciﬁ c Functionalities 139

a small aliquot added to the peptide solution to start the reaction. Pre-dissolving the perio-

date will facilitate immediate dissolution in the ﬁ nal reaction medium without the creation

of regions of high concentration, as would occur if solid sodium periodate is added directly

to the peptide solution. Protect all solutions containing periodate from exposure to light.

3. React for 3 minutes at room temperature. Longer reactions increase the likelihood of

oxidative damage to other amino acids within the peptide structure.

4. Quench the oxidation by the addition to the peptide solution of at least a 4-fold molar

excess of N-acetylmethionine or sodium sulﬁ te over the concentration of periodate in the

reaction mixture. Pre-dissolve the quencher in buffer at a higher concentration prior to

adding an aliquot of it to the reaction solution. React for 10 minutes.

5. The oxidized peptide may be reacted with an amine- or hydrazide-containing molecule by

reductive amination to conjugate with the newly formed aldehyde at the N-terminal (for

protocols see the following section this chapter; Chapter 2, Sections 5.1–5.3; and Chapter 17,

Section 2). For conjugation with amine-containing molecules, the peptide must not have

any other competing amines (e.g., lysine residues) present or else ring formation or pep-

tide-to-peptide coupling may occur. If lysines are present within the peptide, then the use

of a hydrazide (or hydrazine) conjugation process will eliminate interference from lysine

amines.

4.5. Introduction of Hydrazine or Hydrazide Functionalities

Hydrazide-containing reagents can be used for probing or conjugation of carbonyl-containing

compounds, including macromolecules possessing aldehydes and ketones. Fluorescent or enzy-

matic probes containing hydrazide functionalities can be used to assay or label carbohydrates,

glycoproteins, the polysaccharide portion of cell surfaces, gangliosides, and glycoconjugates on

blots (Lotan et al ., 1975; Hurwitz et al., 1980; Spiegel et al., 1982; Gershoni et al., 1985; Wilchek

and Bayer, 1987). Multivalent forms of hydrazide reagents created by modifying enzymes, ferri-

tin, and polymers such as dextran and polypeptides with bis-hydrazides can be used to target

formyl groups with high avidity and sensitivity (Roffman et al., 1980; Kaplan et al., 1983).

The creation of hydrazide probes often is based on the derivatization of a detectable molecule

with a bis-hydrazide compound. Although hydrazine itself (in the form of hydrazine hydrate)

can be used in a methanolic solution to modify activated carboxylate molecules forming

hydrazides, the availability of the bifunctional hydrazides provides a built-in spacer to accom-

modate greater steric accessibility.

The following protocols make use of the compounds adipic acid dihydrazide and carbohy-

drazide to derivatize molecules containing aldehydes, carboxylates, and alkylphosphates. The

protocols are applicable for the modiﬁ cation of proteins, including enzymes, soluble polymers

such as dextrans and poly-amino acids, and insoluble polymers used as micro-carriers or chro-

matographic supports.

The addition of hydrazide groups into macromolecules containing aldehydes, carboxylates,

or alkylphosphates has the effect of increasing the pI or net charge. In the case of carboxylates

or alkylphosphates, blocking these groups with hydrazide compounds eliminates the negative

charge contribution of the original functionality and adds a potential positive charge contribu-

tion due to the terminal hydrazide. The consequence of raising the pI of a macromolecule can

have dramatic effects on the molecule ’s conformation and activity or on its relative nonspeciﬁ city