Hermanson G. Bioconjugate Techniques, Second Edition

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500 10. Bifunctional Chelating Agents and Radioimmunoconjugates

DTPA also can be used to modify amine-containing polymers, such as poly- L -lysine,

to create a chelating polymer possessing multiple metal-binding sites (Trubetskoy et al .,

1993). Subsequent polymer modiﬁ cation of antibodies provides much higher radioactivity

per molecule than if DTPA is directly coupled to the protein. Such chelating polymers can

introduce into proteins as much as 100 DTPA residues able to complex radiolabels per each

55,000 Dalton poly- L-lysine chain attached (Torchilin et al., 1993). Directed coupling to the

antibody through only the N-terminal of the poly- L-lysine chain limits the modiﬁ cation to a

single point along the polymer, thus avoiding crosslinking or multi-site attachment that can

affect antibody activity (Slinkin et al., 1991). This approach can dramatically increase the radi-

oactivity level at tumor sites, thus increasing cytotoxicity or enhancing imaging capability.

While DTPA has been used extensively as a BCA to prepare radiopharmaceutical reagents,

newer metal chelators such as those discussed below may show greater promise for in vivo

applications.

2. DOTA, NOTA, and TETA

DOTA is 1,4,7,10-tetraazacyclododecane- N , N,N,N-tetraacetic acid, a chelating ring struc-

ture containing 4 acetic acid carboxylate groups off the 4 nitrogens of its 12-atom cyclic

structure. C- or N-functionalized derivatives of this basic structure produce a BCA capable of

modifying proteins and binding radioactive metal ions in strong coordination complexes of up

to eight dative bonds (Cox et al., 1990; Renn and Meares, 1992). Perhaps the simplest method

of DOTA functionalization is through modiﬁ cation of one of its carboxylates to contain a short

spacer terminating in a reactive group capable of being coupled to proteins (Li and Meares,

1993). However, modiﬁ cation of carbons on its cyclic backbone also is possible (Brechbiel

et al., 1993). Complexes of metal ions with DOTA have been studied in detail (Sherry et al .,

1989; Aime et al ., 1992).

A similar BCA to DOTA is NOTA, which is 1,4,7-triazacyclononane- N,N,N-triacetic acid.

NOTA contains a smaller ring structure in comparison to DOTA and only has 3 chelating car-

boxylate groups and 3 nitrogens, creating a maximal coordination potential for six dative bonds

with metal ions. Synthesis of functional derivatives can be done through C- or N-modiﬁ cations,

creating reactive groups able to couple to proteins and other molecules (Cox et al., 1990). Cox

et al. (1990) have prepared an (S)-lysine derivative of a benzamide-protected, C-substituted

NOTA. Since coupling antibodies with NOTA through an amide linkage leaves only 2 free car-

boxylic acids, potentially making the reagent pentadentate for chelating purposes, metal com-

plexes formed with this reagent may have lower stability than those containing greater numbers

of chelating groups.

A third compound in the same category as DOTA and NOTA is TETA, which is 1,4,8,

11-tetraazacyclotetradecane-N , N,N , N-tetraacetic acid. TETA contains a larger ring structure

than the other two BCAs and has four chelating carboxylate groups and 4 nitrogens. Similar

to the other two chelators, C- and N-functionalized derivatives can be prepared with TETA

(Brechbiel et al., 1993). A p-bromoacetamidobenzyl-TETA derivative could be used to label

antibodies through sulfhydryl groups and securely bind radioactive copper for probing biologi-

cal systems in vivo (Moi et al ., 1985).

3 . D T T A

DTTA is N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N,N,N-tetraacetic acid. This

BCA contains four carboxylate groups and 3 nitrogens that can hold metals tightly in a coor-

dination complex of seven dative bonds. The compound is especially good at chelating lan-

thanide-series elements, such as europium, samarium, terbium, and dysprosium. Unlike the

previous BCAs which are used to prepare radiopharmaceutical reagents, this one is used pri-

marily for complexing metals to form ﬂ uorescent probes for time-resolved ﬂ uoroimmunoassays

3. DTTA 501

502 10. Bifunctional Chelating Agents and Radioimmunoconjugates

(Hemmila, 1988) (see Chapter 9, Section 9). The most commonly used lanthanides for this

purpose are europium (Eu

3

), terbium (Tb

3

), and samarium (Sm

3

). Proteins modiﬁ ed with

DTTA and complexed with lanthanide metal ions form the basis for unique ﬂ uorescent probes

possessing long lived signals upon excitation.

The isothiocyanate group of DTTA reacts with primary amines in proteins and other mol-

ecules to form stable thiourea bonds ( Figure 10.2 ) (Mukkala et al., 1989). The reagent is water-

soluble and can be reacted under relatively mild conditions (in 0.1 M sodium carbonate, pH 9.0).

The isothiocyanate group is reasonably stable in aqueous solution for short periods, but will

degrade. Best results will be obtained if fresh DTTA is used. The reaction involves attack of the

nucleophile on the central, electrophilic carbon of the isothiocyanate group. The resulting elec-

tron shift creates a thiourea linkage between the chelating compound and the protein with no

leaving group. Modiﬁ cation of antibodies can be done without loss of signiﬁ cant antigen-binding

activity, even when up to 10–15 DTTA chelates are substituted per immunoglobulin molecule

(Stahlberg et al., 1993). Diagnostic assays using DTTA–Eu

3

chelates are commercially available

employing the DELFIA ® (Wallac Oy, Turku, Finland) time-resolved ﬂ uoroimmunoassay system.

4 . D F A

DFA or deferoxamine is N-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]-

hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide, a naturally occurring product

Figure 10.2 The isothiocyanate group of DTTA can react with amine-containing molecules to form isothiourea

bonds.

isolated from Streptomyces pilosus. Its native activity is forming iron complexes, but it is also

very proﬁ cient at forming coordination chelates with other metals, particularly galium-68 (Motta-

Hennessy et al., 1985). Radiopharmaceutical agents for imaging can be produced by modiﬁ cation

of targeting molecules with DFA complexes containing radioactive metals. The amine group of DFA

can be utilized for direct labeling of antibodies and other proteins through coupling with available

carboxylates using carbodiimide-mediated conjugation with EDC (Chapter 3, Section 1.1). The

use of amine-reactive, homobifunctional crosslinkers (Chapter 4) also can be employed to modify

proteins with DFA at their amino groups.

DFA–polymer conjugates can be made containing multiple chelating groups along the length

of the polymer. The polymer backbone utilized for this synthesis can be either activated dextran

(Torchilin et al., 1989) or succinylated poly- L-lysine (Slinkin et al., 1990; Torchilin et al., 1993).

These DFA–polymer constructs can be attached to antibody molecules through additional func-

tional groups on the polymer. Chelating polymers can provide much higher signals than direct

attachment of DFA to antibodies, since each coupled polymer derivative can possess dozens of

chelated radioactive metals. In addition, high substitution levels of DFA directly coupled to anti-

bodies can signiﬁ cantly affect activity by denaturation or blocking of the antigen-binding sites.

5. Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups

Proteins containing sulfhydryl residues can be labeled with a radioactive element by direct com-

plexation to the  SH group through a dative bond (Chapter 2, Section 2.8), avoiding entirely

the use of a BCA. Particularly, reduced sulfhydryls in antibody molecules can be coupled with

99m

Tc to yield thiol-metal derivatives (Rhodes, 1991; Thakur and DeFulvio, 1991). However,

cleavage of disulﬁ de linkages within the antibody can lead to activity losses and fragmenta-

tion (Pimm et al., 1991). The required sulfhydryl groups can be introduced into antibodies

without disulﬁ de reduction through the use of a thiolating reagent that modiﬁ es amine residues

within the antibody (Joiris et al., 1991). Thiolating agents such as 2-iminothiolane or SATA

5. Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups 503

504 10. Bifunctional Chelating Agents and Radioimmunoconjugates

(N -Succinimidyl- S-acetylthioacetate) provide efﬁ cient ways of introducing multiple sulfhydryl

groups for this type of radiopharmaceutical preparation (Chapter 1, Section 4.1).

Site-directed thiolation at carbohydrate residues within the Fc region of antibody molecules

may prove to be the best choice for  SH group introduction while maintain antigen-binding

activity. Ranadive et al. (1993) have used the heterobifunctional crosslinking agent PDPH

(3-(2-pyridyldithio)propionyl hydrazide) (Chapter 5, Section 2.3) to react speciﬁ cally with

oxidized polysaccharide components of monoclonals. The polysaccharide chains are treated

ﬁ rst with sodium periodate (Chapter 1, Section 4.4) to generate reactive aldehyde residues.

PDPH then is coupled to these aldehydes via its hydrazide end to create stable hydrazone link-

ages. The other end of the crosslinker, containing a pyridyl disulﬁ de group, is reduced with

DTT under mild conditions (25 mM DTT, pH 4.5, 30 minutes) to produce the free sulfhy-

dryl groups. Since the thiolation occurs only at carbohydrate locations within the antibody, the

modiﬁ cation has a better chance of being away from the antigen-binding sites, thus preserv-

ing immunoglobulin activity. Subsequent treatment with sodium pertechnatate yields the

99m

derivative on the sulfhydryl groups ( Figure 10.3 ).

Figure 10.3 Antibody molecules oxidized with sodium periodate to create aldehyde groups on their polysac-

charide chains can be modiﬁ ed with PDPH to produce thiols after reduction of the pyridyl disulﬁ de. Direct

labeling of the sulfhydryls with

Tc produces a radioactive complex.

6. FeBABE

FeBABE (pronounced “ iron-babe ” ) is Fe(III) (S)-1-( p-bromoacetamido-benzyl)ethylene diamine

tetraacetic acid, a BCA designed to a hydroxyl radical probe to footprint interacting domains

between proteins. The bromoacetyl group reacts with thiols to form a thioether bond. The

EDTA chelating group coordinates Fe

3

to create a redox active complex that is able to form

reactive oxygen species in aqueous solution. Iron-EDTA chelates are effective at generating

hydroxyl radicals (

) that can react with peptide bonds to oxidize and cut them non-

selectively. The reaction is initiated with the addition of peroxide and ascorbate`, which catalyzes

the peptide cleavage reaction. Thus, one protein labeled with FeBABE can be allowed to interact

with a protein binding partner and the cleavage reaction initiated, which results in cuts in the

peptide structure immediately surrounding the region where FeBABE is attached. The pattern of

peptide cutting can be used to determine the area on both interacting proteins that constitutes

the binding region, because all other peptide regions will be affected by the oxidation reaction.

Chapter 28, Section 4 provides additional information about the FeBABE chelator and its

use in studying protein interactions.

6. FeBABE 505

The highly speciﬁ c interaction of (strept)avidin with the small vitamin biotin can be a useful

tool in designing assay, detection, and targeting systems for biological analytes (see Chapter 23).

The extraordinary afﬁ nity of (strept)avidin ’s interaction with biotin allows biotin-containing

molecules in complex mixtures to be discretely bound with (strept)avidin conjugates. If the

(strept)avidin–biotin complex contains detection components, then the targeted analytes can

be located or quantiﬁ ed. This assay concept is made possible through the ability of biotin to

be covalently attached to other targeting molecules, such as antibodies. In this sense, biotin

derivatives may be prepared which contain reactive portions able to couple with particular

functional groups in proteins and other molecules. Biotin modiﬁ cation of secondary molecules,

called biotinylation, results in covalent derivatives containing one or more bicyclic biotin rings

extending from the parent structure. These biotinylation sites are still capable of binding avidin

or streptavidin with the speciﬁ city and nearly the same avidity of free biotin in solution. Since

the biotin components are relatively small, macromolecules can be modiﬁ ed with these reagents

without signiﬁ cantly affecting their physical or chemical properties (Della-Penna et al ., 1986).

The basic design of a biotin labeling compound is illustrated in Figure 11.1 . Common to all

such modiﬁ cation reagents is the presence of the bicyclic biotin ring at one end of the struc-

ture and a reactive group at the other end that can be used to couple with other molecules.

Biotinylation reagents also possess various cross-bridges or spacer groups built off the valeric

acid side chain of the molecule. Since the binding sites for biotin on avidin and streptavidin are

pockets buried about 9 Å beneath the surface of the proteins, spacers can affect the accessibil-

ity of biotinylated compounds for efﬁ ciently binding avidin or streptavidin conjugates (Green

et al., 1971). In some applications, the use of a long spacer arm in the biotinylation reagent will

result in the greatest potential assay sensitivity. The rate of binding of an avidin or streptavidin

probe to a biotinylated molecule also is affected by the length of spacer in the biotinylation

reagent. When longer spacers are utilized to make biotinylated macromolecules, it potentially

can result in a 5-fold greater rate of streptavidin interaction (Bonnard et al ., 1984).

Another variable to consider in choosing biotinylation reagents is the use of a biotin analog

such as iminobiotin that has a moderated afﬁ nity constant in its binding of avidin or strepta-

vidin (Section 3.1, this chapter). Analogs may be useful if release of the (strept)avidin–biotin

bond is important for isolating a targeted analyte. Using native biotin, the interaction with avi-

din is so strong that up to 6–8 M guanidine at pH 1.5 is required to break the bond, possibly

Biotinylation Reagents

506

causing extensive denaturation of any other complexed molecules. By contrast, iminobioti-

nylated molecules can be released simply by adjusting the pH down to 4.

The following sections discuss some of the more common biotinylation reagents available

for modiﬁ cation of proteins and other biomolecules. Each biotin derivative contains a reactive

portion (or can be made to contain a reactive group) that is speciﬁ c for coupling to a particular

functional group on another molecule. Careful choice of the correct biotinylation reagent can

result in directed modiﬁ cation away from active centers or binding sites, and thus preserve the

activity of the modiﬁ ed molecule.

Additional biotinylation reagents are discussed in Chapter 18, Section 3, which describes

newer hydrophilic biotin compounds containing PEG spacers. These reagents offer signiﬁ cant

advantages over the more traditional aliphatic compounds, because the pronounced water-

solubility of the PEG cross-bridge can prevent aggregation of biotinylated molecules. With

some longer chain biotin compounds that contain hydrophobic hydrocarbon spacers, proteins

can precipitate or lose activity over time due to the insolubility of the biotin modiﬁ cations.

Biotinylated antibodies are particularly susceptible to aggregation and loss of antigen binding

ability if they are modiﬁ ed using hydrophobic biotin compounds. The PEG-based biotin reagents

show better solubility and longer stability than their corresponding aliphatic biotinylation com-

pounds of equivalent size.

1. Amine-Reactive Biotinylation Agents

Amine-reactive biotinylation reagents contain reactive groups off biotin ’s valeric acid side chain

that are able to form covalent bonds with primary amines in proteins and other molecules. Two

basic types are commonly available: N-hydroxysuccinimide (NHS) esters and carboxylates.

NHS esters spontaneously react with amines to form amide linkages (Chapter 2, Section 1.4),

Figure 11.1 The basic design of a biotinylation reagent includes the bicyclic rings and valeric acid side chain of

D-biotin at one end and a reactive group to couple with target groups at the other end. Spacer groups may be

included in the design to extend the biotin group away from modiﬁ ed molecules, thus ensuring better interac-

tion capability with avidin or streptavidin probes.

1. Amine-Reactive Biotinylation Agents 507

508 11. Biotinylation Reagents

whereas carboxylate-containing biotin compounds can be coupled to amines via a carbodiimide-

mediated reaction using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (Chapter 3,

Section 1.1).

1.1. D -Biotin and Biocytin

D-Biotin (hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid) is a naturally occur-

ring growth factor present in small amounts within every cell. It is a key component in

numerous processes involving carboxylation reactions, wherein it functions as a cofactor and

transporter of CO

(coenzyme R). Biotin is mainly found covalently attached to lysine  -amine

groups of proteins via its valeric acid side chain. The compound was originally discovered

through symptoms of deﬁ ciency caused by eating too many raw egg-whites. Biotin (or vitamin H)

was found to be complexed and inactivated by the egg-white protein avidin (Boas, 1927; du

Vigneaud, 1940). Treatment with additional vitamin H alleviated the symptoms.

The interaction of biotin with the proteins avidin and streptavidin is among the strongest

noncovalent afﬁ nities known ( Ka  10

 1

). The binding occurs between the bicyclic ring of

biotin and a pocket within each of the 4 subunits of the proteins. The valeric acid portion is not

directly involved with the interaction with avidin (Green, 1975; Wilchek and Bayer, 1988), but

elimination of its carboxylate group or changing the amide linkage in biotinylation compounds

can affect its afﬁ nity for (strept)avidin. Amide derivatives of biotin ’s valeric acid group, how-

ever, can be made without interfering with its high afﬁ nity interactions. This characteristic allows

modiﬁ cation of the valeric acid side chain without affecting the binding potential toward avidin

or streptavidin.

D-Biotin is thus the basic building block for constructing biotinylation reagents. The mol-

ecule may be attached directly to a protein via its valeric acid side chain or derivatized at this

carboxylate with other organic components to create spacer arms and various reactive groups.

Reaction of biotin with primary amine groups on proteins can be done using the water-soluble

carbodiimide, EDC (Chapter 3, Section 1.1). EDC activates the carboxylate to create a highly

reactive, intermediate ester. This ester then can couple to amines to form stable amide bond

derivatives ( Figure 11.2 ). Biotinylated molecules thus formed retain the ability to bind avidin

or streptavidin with high afﬁ nity.

The only potential deﬁ ciency in using D-biotin to modify directly a protein is the relatively

short spacer arm afforded by the indigenous valeric acid group. Some applications may require

longer spacers to maintain good binding potential toward avidin or streptavidin.

Biocytin is  - N -biotinyl-

L-lysine, a derivative of D-biotin containing a lysine group coupled

at its -amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of

biotin that is typically found in serum and urine, and probably represents breakdown products

of recycling biotinylated proteins. The enzyme biotinidase speciﬁ cally cleaves the lysine residue

and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987).

Biocytin has been used extensively as a labeling reagent for intracellular components within

neurons (Horikawa and Armstrong, 1988; King et al., 1989; Izzo, 1991; Granata and Kitai, 1992).

It is particularly good for anterograde tracing studies in the central nervous system, since it can

be easily injected into neurons using micropipettes. Subsequent visualization of biocytin loca-

tions may be done using an (strept)avidin–enzyme conjugate (Chapter 23, Section 3).

Figure 11.2 D-Biotin can be directly coupled to amine-containing molecules using the water-soluble carbodiim-

ide EDC to form an amide bond linkage.

1. Amine-Reactive Biotinylation Agents 509