Hermanson G. Bioconjugate Techniques, Second Edition

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(Reaction 15)

Fluorophenyl ester compounds can be coupled to amines at a pH range of 7–9, with 0.1 M

sodium bicarbonate, pH 8, a suggested reaction medium.

1.14. Hydroxymethyl Phosphine Derivatives

Phosphine compounds often are thought of only in terms having reductant properties, espe-

cially in biological applications. However, there are classes of phosphine derivatives with

hydroxy-methyl group substitutions that also can act as bioconjugation agents for coupling or

crosslinking purposes. Tris(hydroxymethyl)phosphine (THP) and  -[tris(hydroxymethyl)phosp

hino] propionic acid (THPP; Thermo Fisher) are small trifunctional compounds that sponta-

neously react with nucleophiles, such as amines, to form covalent linkages (Henderson et al .,

(Reaction 16)

180 2. The Chemistry of Reactive Groups

1994; Katti, 1996; Katti et al., 1999). Nucleophiles react with the hydroxymethyl arms by

attack on the electron-deﬁ cient carbon atom with loss of water to form secondary or tertiary

amine bonds (Reaction 16).

Both THP and THPP are stable in aqueous solution, as the only potential product of

hydrolysis is the reformation of the hydroxymethyl groups. It is unusual for an amine-reactive

functional group to have long-term stability in water or buffer, which makes these reagents

uniquely suitable for creating reactive surfaces or reactive molecules for subsequent conjuga-

tion with proteins or other amine-containing compounds. Hydroxylic chromatographic sup-

ports also have been activated with hydroxymethyl phosphine derivatives for immobilization

of enzymes (Petach et al ., 1994).

Hydroxymethyl phosphines are susceptible to oxidation to form the phosphine oxide deriva-

tive. Therefore, avoid excess oxygen, oxidizing agents, or azide compounds, which react with

phosphines in the Staudinger reaction (Chapter 17, Section 5). In addition, metallic surfaces

can be modiﬁ ed via the phosphine group to result in hydroxymethyl group substitutions.

1.15. Guanidination of Amines

The addition of a guanidino group to amine-containing molecules can be done using the com-

pound o-methylisourea (as the hemisulfate salt). Guanidination has been used to increase the ion-

ization potential of lysine-containing peptides for greater sensitivity in mass spec analysis (Brancia

et al., 2000). The process also has been used to add stable isotope labels (

N) to tryptic peptides

(Cristea et al., 2004). Upon trypsin digestion, protein samples are cleaved at arginine and lysine

residues to yield peptide fragments containing these amino acids at their C-terminal. The guani-

dine group of arginine is known to aid in the ionization of peptides for MS analysis. The reaction

of o-methylisourea hemisulfate with lysine -amino groups produces homoarginine (Reaction

17), which ionizes far better than lysine and aids in the detection of these peptides (Warwood

et al., 2006). The addition of a guanidine group to a lysine residue adds 42.02 Daltons to the

resultant modiﬁ ed peptide, which needs to be taken into account for mass spec purposes.

(Reaction 17)

Protocols for guanidination reactions typically use basic conditions to deprotonate all the

lysine -amino groups, which is necessary to achieve efﬁ cient yields. The amount of N-terminal

1. Amine Reactions 181

-amine labeling is minimal, but may occur to some extent, especially for peptides containing

an N-terminal glycine residue (Beardsley and Reilly, 2002). The initial protocols for guanidina-

tion usually use reaction times of several hours, but optimization has resulted in decreasing this

to 5–10 minutes at elevated temperature. The following protocol is based on the method of

Beardsley and Reilly (2002).

Protocol

1. Dissolve 50 mg of o -methylisourea hemisulfate in 51  l of water.

2. Prepare 5 l of a peptide solution to undergo guanidination at a concentration of 1 pmol/  l

and add 5.5 l of 7 N ammonium hydroxide.

3. Add 1.5 l of the o-methylisourea hemisulfate solution to the peptide solution with

mixing.

4. React for 5–10 minutes at 65°C in an oven.

5. Stop the reaction with the addition of 15  l of 10 percent TFA (v/v).

2 . Thiol Reactions

Reactive groups able to couple with sulfhydryl-containing molecules are perhaps the second

most common functional groups present on crosslinking or modiﬁ cation reagents. Especially in

the design of heterobifunctional crosslinkers, sulfhydryl-reactive groups frequently are present

on one of the two ends. The other end of such crosslinkers is often an amine-reactive group

that is coupled to a target molecule before the sulfhydryl-reactive end, due to the labile nature

of amine acylation chemistries. The primary coupling reactions for modiﬁ cation of sulfhydryls

proceed by one of two routes: alkylation or disulﬁ de interchange. Many of the reactive groups

that undergo these reactions are stable enough in aqueous environments to allow a two-step

conjugation strategy to be used (Chapter 5, Section 1). Once initiated, most of these reactions

are rapid and occur in high yield to give stable thioether or disulﬁ de bonds.

2.1. Haloacetyl and Alkyl Halide Derivatives

Three forms of activated halogen derivatives can be used to create sulfhydryl-reactive com-

pounds: haloacetyl (see Chapter 1, Section 5.2), benzyl halides that react through a resonance

activation process with the neighboring benzene ring, and alkyl halides that possess the halo-

gen  to a nitrogen or sulfur atom, as in N- and S-mustards. In each of these compounds, the

halogen group is easily displaced by an attacking nucleophilic substance to form an alkylated

derivative with loss of HX (where X is the halogen and the hydrogen comes from the nucle-

ophile). Haloacetyl compounds and benzyl halides typically are iodine or bromine derivatives,

whereas the halo-mustards mainly employ chlorine and bromine forms (see Chapter 4, Section

10 for examples of homobifunctional reagents that employ reactive halogen groups).

Although the primary utility of active halogen compounds is to modify sulfhydryl groups in

proteins or other molecules, the reaction is not totally speciﬁ c. Iodoacetyl (and bromoacetyl)

derivatives can react with a number of functional groups within proteins: the sulfhydryl group

of cysteine, both imidazolyl side chain nitrogens of histidine, the thioether of methionine, and

182 2. The Chemistry of Reactive Groups

the primary -amine group of lysine residues and N-terminal -amines (Gurd, 1967). The

relative rate of reaction with each of these residues is generally dependent on the degree of

ionization and thus the pH at which the modiﬁ cation is done. The exception to this rule is

methioninyl thioethers which react rapidly at nearly all pH values above 1.7 (Vithayathil and

Richards, 1960). The only reaction resulting in one deﬁ nitive product is that of the alkyla-

tion of cysteine sulfhydryls, giving the carboxymethylcysteinyl derivative (Cole et al., 1958)

(Reaction 18). Histidine groups may be modiﬁ ed at either nitrogen atom of its imidazolyl side

chain, thus producing the possibility of either mono-substituted or di-substituted products

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

dues, the products of the reaction are either the secondary amine, monocarboxymethyllysine,

or the tertiary amine derivative, dicarboxymethyllysine. Methionine thioether groups give the

most complicated products, some of which rearrange or decompose unpredictably. The only

stable carboxy derivative of methionine is where the terminal methyl group is lost to form car-

boxymethylhomocysteine, the same product as the reaction of iodoacetate with homocysteine.

(Reaction 18)

The relative reactivity of -haloacetates toward protein functional groups is sulfhydryl 

imidazolyl  thioether  amine. Among halo derivatives the relative reactivity is I  Br 

Cl  F, with ﬂ uorine being almost unreactive. The -haloacetamides have the same trend of

relative reactivities, but will create a terminal amide group not a terminal carboxylate.

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

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

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

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

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

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

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

2.2. Maleimides

Maleic acid imides (maleimides) are derivatives of the reaction of maleic anhydride and ammo-

nia or an amine derivative. This functional group is a popular constituent of many heterobi-

functional crosslinking agents (Chapter 5). The double bond of maleimides may undergo an

alkylation reaction with sulfhydryl groups to form stable thioether bonds. Maleimide reactions

are speciﬁ c for thiols in the pH range of 6.5–7.5 (Smyth et al., 1964; Gorin et al., 1966; Heitz

et al., 1968; Partis et al., 1983). At pH 7.0, the reaction of the maleimide with sulfhydryls

proceeds at a rate 1000 times greater than its reaction with amines. At higher pH values, some

cross-reactivity with amino groups takes place (Brewer and Riehm, 1967). One of the carbons

adjacent to the maleimide double bond undergoes nucleophilic attack by the thiolate anion

to generate the addition product (Reaction 19). When sufﬁ cient quantities of SH groups

2. Thiol Reactions 183

are being alkylated, the reaction may be followed spectrophotometrically by the decrease in

absorbance at 300 nm as the double bond reacts and disappears.

(Reaction 19)

The maleimide group also may undergo hydrolysis to an open maleamic acid form that is

unreactive toward sulfhydryls (Chapter 19, Section 5). Hydrolysis may occur after sulfhydryl

coupling to the maleimide, as well. This ring-opening reaction typically happens faster the

higher the pH becomes. Hydrolysis is also dependent on the type of chemical group next to the

maleimide function. For instance, the cyclohexane ring of SMCC (Chapter 5, Section 1.3) pro-

vides increased stability to maleimide hydrolysis probably due to its steric effects and its lack

of aromatic character. However, the adjacent phenyl ring of MBS allows much greater rates of

hydrolysis to occur at the maleimide ring (Chapter 5, Section 1.4).

2.3. Aziridines

An Aziridine reactive group is a small ring system composed of one nitrogen and two carbon

atoms. The highly hindered nature of this heterocyclic ring gives it strong reactivity toward

nucleophiles. Sulfhydryls will react with aziridine-containing reagents in a ring-opening proc-

ess, forming thioether bonds (Reaction 20). The simplest aziridine compound, ethylenimine,

can be used to transform available sulfhydryl groups into amines (Chapter 1, Section 4.3).

(Reaction 20)

The reaction of an aziridine with a thiol is highly speciﬁ c at slightly alkaline pH values. In

aqueous solution, the major side reaction is hydrolysis.

Substituted aziridines have been used to form homobifunctional and trifunctional crosslink-

ing agents, although their use has been limited (Ross, 1953; Alexander, 1954). The func-

tional group has found use, however, in the design of the ﬂ uorescent probe dansyl aziridine

(5-dimethylaminonaphthalene-2-sulfonyl aziridine) (Johnson et al., 1978; Grossman et al., 1981).

2.4. Acryloyl Derivatives

Reactive double bonds are capable of undergoing addition reactions with sulfhydryl groups.

A popular example of this type of functional group is the maleimide group (Section 2.2, this

184 2. The Chemistry of Reactive Groups

chapter). However, derivatives of acrylic acid also are able to participate in this reaction,

although the rate of sulfhydryl addition is somewhat slower than that of maleimides. The reac-

tion of an acryloyl compound with a sulfhydryl group occurs with the creation of a stable

thioether bond (Reaction 21).

(Reaction 21)

Although acryloyl crosslinking agents have not been common, the reactive group has found

use in the design of the sulfhydryl-reactive ﬂ uorescent probe, 6-acryloyl-2-dimethylaminonaph-

thalene (acrylodan; Molecular Probes) (Epps et al ., 1992; Yem et al ., 1992).

2.5. Arylating Agents

Arylating agents are reactive aromatic compounds containing a constituent on the ring that

can undergo nucleophilic substitution. The most common arylating agents are derivatives of

benzene which possess either halogen or sulfonate groups on the ring. The presence of elec-

tron withdrawing constituents, such as nitro groups, increases the reactivity of the replace-

able group. Although aryl halides are commonly used to modify amine-containing molecules to

form aryl amine derivatives, they also react quite readily with sulfhydryl groups.

Fluorobenzene-type compounds have been used as functional groups in homobifunctional

crosslinking agents (Chapter 4, Section 4). Their reaction with nucleophiles involves bimolecu-

lar nucleophilic substitution, causing the replacement of the ﬂ uorine atom with the sulfhydryl

derivative, and creating a substituted aryl bond (Reaction 22). Conjugates formed with sulfhy-

dryl groups are reversible by cleaving with an excess of thiol (such as DTT) (Shaltiel, 1967).

Detection reagents incorporating reactive aryl chemistry include 2,4-dinitroﬂ uorobenzene and

trinitrobenzenesulfonate (Eisen et al., 1953). The relative rate of reactivity for aryl compounds

is: F  Cl  Br  Sulfonate.

(Reaction 22)

2.6. Thiol-Disulﬁ de Exchange Reagents

Compounds containing a disulﬁ de group are able to participate in disulﬁ de exchange reactions

with another thiol. The disulﬁ de exchange (also called interchange) process involves attack of the

thiol at the disulﬁ de, breaking the SS bond, with subsequent formation of a new mixed

disulﬁ de comprising a portion of the original disulﬁ de compound (Reaction 23). The reduction

2. Thiol Reactions 185

of disulﬁ de groups to sulfhydryls in proteins using thiol-containing reductants proceeds through

the intermediate formation of a mixed disulﬁ de (Chapter 1, Section 4.1). If the thiol is present in

excess, the mixed disulﬁ de can go on to form a symmetrical disulﬁ de consisting entirely of the

thiol reducing agent—thus completely reducing the original disulﬁ de to free sulfhydryls. If the

thiol reductant is not present in large enough excess, the mixed disulﬁ de product is the end result.

(Reaction 23)

Crosslinking or modiﬁ cation reactions using disulﬁ de exchange processes form disulﬁ de linkages

with sulfhydryl-containing molecules. These bonds are reversible using disulﬁ de reducing agents.

Thus, conjugates may be created and latter released for analysis by incubation with DTT or other

disulﬁ de reductants (e.g., TCEP). The disulﬁ de bond within these crosslinks also permits important

reactions to occur in vivo, such as the release of the toxin component of immunotoxin conjugates,

allowing the cytotoxic portion to penetrate target cells and cause cell death (Chapter 21).

Disulﬁ de exchange reactions occur over a broad range of conditions—from acid to basic

pH—and in a wide variety of buffer constituents. Most crosslinking reactions involving

disulﬁ de exchange are done under physiological conditions or those most appropriate to main-

tain stability of the protein or other molecule being modiﬁ ed.

Pyridyl Disulﬁ des

A pyridyl dithiol is perhaps the most popular type of thiol-disulﬁ de exchange functional group

used in the construction of crosslinkers or modiﬁ cation reagents. Pyridyl disulﬁ des can be cre-

ated from available primary amines on molecules through the reaction of 2-iminothiolane in

tandem with 4,4 -dipyridyl disulﬁ de (King et al., 1978). For instance, the simultaneous reac-

tion between a protein, 2-iminothiolane, and 4,4 -dipyridyl disulﬁ de yields a modiﬁ cation con-

taining reactive pyridyl disulﬁ de groups in a single step (Chapter 1, Section 4.1).

A pyridyl disulﬁ de will readily undergo an interchange reaction with a free sulfhydryl to

yield a single mixed disulﬁ de product. This is due to the fact that the pyridyl disulﬁ de contains

a leaving group that is easily transformed into a non-reactive compound not capable of par-

ticipating in further mixed disulﬁ de formation. Thus, the thiol-disulﬁ de exchange reaction can

be controlled to occur with only one-half of the original disulﬁ de compound. For instance, a

reagent system containing a pyridyl disulﬁ de group, such as SPDP (Chapter 5, Section 1.1), is

able to react with sulfhydryl groups by releasing the electron-stabilized compound, pyridine-2-

thione (Reaction 24). Since the leaving group does not possess a free thiol, it can not disulﬁ de

exchange with another molecule of the attacking sulfhydryl compound. Thus, only one end of

the reagent has potential for becoming attached to the sulfhydryl-containing molecule.

(Reaction 24)

186 2. The Chemistry of Reactive Groups

Pyridyl dithiol containing crosslinking and modiﬁ cation reagents are highly efﬁ cient in form-

ing disulﬁ de bonds with sulfhydryl-containing molecules. In addition, the pyridine-2-thione

leaving group has unique spectral properties that allow the measurement of sulfhydryl cou-

pling by monitoring the increase in absorbance at 343 nm (   8.08  10

1

). Once a

disulﬁ de linkage is formed, it may be cleaved using standard disulﬁ de reducing agents (Chapter 1,

Section 4.1).

TNB-Thiol

Sulfhydryl groups activated with the leaving group 5-thio-2-nitrobenzoic acid can be used to

couple free thiols by disulﬁ de interchange similar to pyridyl disulﬁ des, as discussed previously.

A TNB-thiol-activated species may be created by reaction of a sulfhydryl group with Ellman ’s

reagent, 5,5  -dithio- bis(2-nitrobenzoic acid) or DTNB, a compound useful for the quantita-

tive determination of sulfhydryls in solution (Ellman, 1958, 1959) (Chapter 1, Section 4.1).

The disulﬁ de of Ellman ’s reagent readily undergoes disulﬁ de exchange with a free sulfhydryl to

form a mixed disulﬁ de with concomitant release of one molecule of the chromogenic substance

5-sulﬁ do-2-nitrobenzoate, also called 5-thio-2-nitrobenzoic acid (TNB). The TNB-thiol group

can again undergo interchange with a sulfhydryl-containing target molecule to yield a disulﬁ de

crosslink. Upon coupling with a sulfhydryl compound, the TNB group is released (Reaction 25).

The intense yellow color produced by the TNB anion can be measured by its absorbance at

412 nm (   1.36  10

1

at pH 8.0). Since each sulfhydryl which is coupled gener-

ates one molecule of TNB per molecule of Ellman ’s reagent, the possibility for quantifying the

reaction exists.

(Reaction 25)

Disulﬁ de exchange with a TNB-thiol group occurs efﬁ ciently at physiological to slightly

alkaline pH conditions. Avoid the presence of disulﬁ de reducing agents, as these will cleave the

TNB group and prevent speciﬁ c coupling.

Disulﬁ de Reductants

Disulﬁ de reduction by the use of disulﬁ de interchange can be done using thiol-containing com-

pounds such as TCEP, DTT, 2-mercaptoethanol, or 2-mercaptoethylamine (Chapter 1, Section

4.1). The formation of free sulfhydryls from a disulﬁ de group occurs in two stages. First, one

molecule of the reducing agent undergoes disulﬁ de exchange, cleaving the disulﬁ de and forming a

new, mixed disulﬁ de. In the next stage, a second molecule of the thiol cleaves the mixed disulﬁ de,

releasing a free sulfhydryl and forming a molecule of oxidized reducing agent (Reaction 26).

2. Thiol Reactions 187

(Reaction 26)

Disulﬁ de reduction occurs over a broad pH range and in a variety of buffer environments. The

reaction can be done in denaturants, chaotropic agents, detergents, and in high salt conditions.

2.7. Vinylsulfone Derivatives

A vinylsulfone group can be used to conjugate with nucleophiles, especially thiol groups, in

aqueous solution and under mild conditions (Masri and Friedman, 1988). In addition to thiols,

they can react with amines and hydroxyls under higher pH conditions. As opposed to a male-

imide group, the vinylsulfone is not as strong an electrophile, but efﬁ ciently couples with thiols

at slightly alkaline pH values to give stable -thiosulfonyl linkages (Reaction 27). The product

of the reaction of a thiol with a vinylsulfone gives a single stereoisomer structure, unlike conju-

gation with maleimides, which produces two potential stereoisomers. In addition, crosslinkers

and modiﬁ cation reagents containing a vinylsulfone can be used to activate surfaces or mol-

ecules to contain thiol-reactive groups. These vinylsulfone groups are stable in aqueous solu-

tion for extended periods, as they are not subject to hydrolysis at neutral pH. Thus, they retain

excellent coupling potential for thiol-containing proteins or other molecules even when used in

aqueous buffer conditions.

(Reaction 27)

Vinylsulfone activated chromatography supports long have been used for coupling afﬁ nity

ligands that contain thiols or other nucleophiles (Porath, 1974). The reactive group also has

been used to activate PEG polymers for modiﬁ cation of thiol-containing molecules (Morpurgo

et al., 1996). There now are homobifunctional and heterobifunctional crosslinking agents com-

mercially available that use the vinylsulfone reactive group (Thermo Fisher and Molecular

Biosciences).

2.8. Metal-Thiol Dative Bonds

Thiol-containing molecules can interact with metal ions and metal surfaces to form dative

bonds. Dative bonds also are known as coordinate covalent bonds. They differ from normal

188 2. The Chemistry of Reactive Groups

covalent linkages, because they are formed by two electrons coming from a single atom, instead

of two atoms each sharing one electron. In a coordinate bond formed with a thiol, the unshared

pair of electrons on the sulfur atom is able to form a dative bond with a metal atom. In this

sense, even disulﬁ des are able to datively link to a metal surface without prior reduction to thi-

ols (Reaction 28).

(Reaction 28)

Other atoms containing a lone pair of electrons also are effective at forming coordinate

bonds. Oxygen- and nitrogen-containing organic molecules often are used to chelate metal

ions, such as in various lanthanide chelates (Chapter 9, Section 9), bifunctional metal-chelating

compounds (Chapter 10), and FeBABE (Chapter 28, Section 4.1). In addition, amino acid side

chains and prosthetic groups in proteins frequently form bioinorganic motifs by coordinating a

metal ion as part of an active center (Degtyarenko, 2000). Thiol organic compounds, however,

are used routinely to coat metallic surfaces or particles to form biocompatible layers or create

functional groups for further conjugation of biomolecules.

Thiol-ligand modiﬁ cation in particular has been used extensively to create water solu-

ble quantum dots (Sapsford et al., 2006) and gold nanoparticles having reduced nonspeciﬁ c

binding character to the metallic surface and to form surface groups for coupling proteins and

other afﬁ nity molecules (Chapter 9, Section 10 and Chapter 24). For instance, thiol-containing

aliphatic/PEG linkers have been used to form self-assembled monolayers (SAMs) on planar

gold surfaces and particles (Prime and Whitesides, 1991).

A monodentate thiol-metal bond is not as strong as a true covalent linkage. These bonds are

subject to displacement by other molecules containing thiols or other atoms with a lone pair

of electrons. The thiol also may oxidize off the surface if exposed to oxygen in air or aqueous

solutions. Instead of monodentate compounds, the use of multidentate molecules can dramati-

cally increase the stability of a coordination bond. A bidentate DOPA ligand, for instance, was

found to have far greater bonding adhesion to a surface than monodentate ligands and nearly

as much as a covalent bond (Lee et al ., 2006).

To increase the strength of thiol dative linkages, the application of multivalent

thiols has been used to increase the overall strength of a single molecule tether bound to a

metal. Bidentate thiol ligands that have been designed for metal surface modiﬁ cation include

lipoic acid (thioctic acid) derivatives (Cheng and Brajter-Toth, 1992; Willey et al., 2004;

Hahn et al., 2007), dithiobis(succinimidyl)propionate (DSP) modiﬁ cations (Grubor et al .,

2004) (Chapter 4, Section 1.1), and a dithiol linker built from a central phenyl ring, which

contains a PEG spacer for hydrophilicity on the SAM surface (Spangler et al., 2004) ( Figures

2.2 and 2.3 ).

2. Thiol Reactions 189