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2 Group-Selective Heterobifunctional Reagents for Protein Cross-Linking

2 Group-Selective Heterobifunctional Reagents for Protein Cross-Linking

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192



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 6.1

Representative Group Selective Heterobifunctional Cross-Linkers

I. Amino- and Sulfhydryl-Group–Directed Bifunctional Reagents

A. Succinimide and thiopyridine. General structure

O



O



N O



C



X



S



S



N



O



Example: N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP)

O



O



N O



C



S



(CH2)2



S

N



O

B. Succinimide and maleimide. General structure

O



O



N O



C



O

X



N



O



O



Example: N-Succinimidylmaleimidoacetate (AMAS)

O



O



N O



C



O

CH2



N

O



O

C. Succinimide and alkylhalide. General structure

O



O



N O



X



C



(I, Br, OR Cl)



CH2



O

Example: N-Succinimidylbromoacetate

O



O



N O



C



CH2



Br



O

D. Nitrophenol and maleimide. General structure

O



O

O



O2N



C



X



N

O



Example: p-Nitrophenyl-6-maleimidocaproate

O



O

O2N



O



C



(CH2)5



N

O



193



Heterobifunctional Cross-Linkers



TABLE 6.1 (Continued)

Representative Group Selective Heterobifunctional Cross-Linkers

E. Nitrophenol and alkylhalide. General structure

O

O



O2N



C



(I or Br)



CH2



X



Example: p-Nitrophenylbromoacetate

O

O2N



O



CH2Br



C



F. Acetimidate and alkylhalide. General structure

NH2Cl

CH3CH2



C



O



X



(I, Br, or Cl)



CH2



Example: Ethyl iodoacetimidate HCl

NH2Cl

O



CH3CH2



CH2



C



Br



G. Benzoyl derivative and maleimide. General structure

O



O

X



N



C



O

Example: 2-Hydroxy-4-(N-maleimido)benzoylazide (HMB)

O



O

C



N3



N

O



HO



II. Carboxyl- and Either Sulfhydryl- or Amino-Group–Directed Bifunctional Reagents

A. Diazoacetyl and thiopyridine. General structure

O

N2CH



C



X



S



S

N



Example: Pyridyl-2-2′-dithiobenzyldiazoacetate (PDD)

O

N2CH



C



O



CH2

S



S

N

(continued)



194



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 6.1 (Continued)

Representative Group Selective Heterobifunctional Cross-Linkers

B. Diazoacetyl and nitrophenol. General structure

O

C



N2CH



X



O



NO2



Example: p-Nitrophenyl diazoacetate

O

C



N2CH



O



NO2



III. Carbonyl- and Amino-Group–Directed Bifunctional Reagents

A. Hydrazide and succinimide. General structure

H

H2N



O



O

C



X



N



O



N

O



Example: N-Succinimidyl-4-hydrazidoterephthalate



H2N



H



O



O



N



C



C



O

O



N

O



IV. Carbonyl- and Sulfhydryl-Group–Directed Bifunctional Reagents

A. Aminooxy and thiopyridine. General structure

O 2N

O



H2N



X



S



S



N



Example: 1-Aminooxy-4-[(3-nitro-2-pyridyl)dithio]butane

O 2N

H2N



O



(CH2)4



S



S

N



B. Hydrazide and maleimide: General structure

O



O



NH2-NH



C



X



N

O



Example: N-(β-maleimidopropionic acid) hydrazide

O



O

NH2-NH



C



CH2-CH2



N

O



195



Heterobifunctional Cross-Linkers



TABLE 6.2

Representative Anchoring Agents of Photoactivatable

Heterobifunctional Cross-Linkers

I. Amino Group Anchoring Agents

A. Succinimides. General structure

O



O

C



O



Photosensitive moiety



O

Example: N-Succinimidyl-4-azidobenzolyglycinate

O



O



O



N O



CH2 N

H



C



N3



C



O

B. Imidates. General structure

NH2Cl

H3C O



Photosensitive moiety



C



Example: Methyl-[3-(4-azidophenyl)dithio]propionimidate HCl (MADP)

NH2Cl

H3C O



(CH2)2



C



S



N3



S



C. Phenylhalides. General structure

(I or F)



N3

X



Example: 4-Fluoro-3-nitrophenylazide (FNA)

F



N3



O2N

D. Phenylisocyanates. General structure

S= C=N



(Photosensitive group, e.g., N3)



Example: Benzophenone-4-isothiocyanate

O

S=C=N



C



E. Nitrophenols. General structure

O

N2O



O



C



X



CH = N2



Example: p-Nitrophenyl-3-diazopyruvate

N2O



O



O



O



C



C



CH=N2

(continued)



196



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 6.2 (Continued)

Representative Anchoring Agents of Photoactivatable

Heterobifunctional Cross-Linkers

II. Sulfhydryl Group—Anchored Agents

A. Disulfides. General structure

Photosensitive moiety—S—S— Photosensitive moiety

Example: 4,4′-Dithiobisphenylazide

N3



S



S



S



X



N3



B. Sulfenyls. General structure

(Leaving group)



N3



Example: 2-Nitro-4-azidophenylsulfenyl chloride

Cl



S



N3



C. Seleno derivatives. General structure

O

R



Se



C



Photosensitive moiety



Example: 3-(4-Azido-2-nitrobenzoylseleno)propionic acid

O



O

Se



HO-C-(CH2)2



C



N3



O2N

D. Maleimides. General structure

O

N



X



(Photosensitive group, e.g., N3)



O

Example: 4-Azidophenylmaleimide (APM)

O

N



N3



O

E. Akylhalides. General structure

(Br or I) CH2



N3



X



Example: 4-(Bromoaminoethyl)-3-nitrophenylazide

Br



CH2



CH2



N3



NH

O2N



197



Heterobifunctional Cross-Linkers



TABLE 6.2 (Continued)

Representative Anchoring Agents of Photoactivatable

Heterobifunctional Cross-Linkers

III. Guanidinal Group Anchoring Agent

A. Dicarbonyls. General structure

R



O



O



C



C



Photosensitive moiety



Example: 4-Azidophenylglyoxal



H



O



O



C



C



N3



IV. Carboxyl or Carboxamide Groups Anchoring Agent

A. Alkylamines. General structure

H2N–CH2—Photosensitive moiety

Example: N-[β-(β′-Aminoethyldithioethyl)]-4-azido-2-nitroaniline

H2N CH2CH2



S



S



N3



NH



CH2CH2



O2N



composed of the hydrosuccinimidyl (amino group selective) and maleimide (sulfhydryl group selective) moieties (XVI through LII in Appendix D), followed by compounds with hydrosuccinimidyl

and haloacetyl (sulfhydryl group selective) groups (LIII through LXIII). The rest of the compounds

have various combinations of nitrophenol, maleimide, haloacetyl, imidate, haloacyl, azidoacyl,

epoxide, disulfhydryl, and other groups. Since the maleimido group attached to aromatic rings is

labile at neutral pH, the most stable compound is probably N-succinimidyl-4-(N-maleimidomethyl)

cyclohexane-1-carboxylate (SMCC) and its sulfonated analog, sulfo-SMCC (XXXIII and XXXIV

in Appendix D).35–45 Compounds with nitrovinyl groups such as 2,4-dinitrophenyl-p-(β-nitrovinyl)

benzoate (LXX in Appendix D) react with the thiol through Michael addition to the double bond more

readily than N-maleimido derivatives under acidic conditions.20 In this particular cross-linker, the

dinitrophenyl ester at the other end of the compound also reacts with amines at a much faster rate than

N-hydroxysuccinimide esters under basic conditions. The cross-linking reaction is shown in Figure

6.1. Cross-linking of sulfhydryl and amino groups is also achievable with epichlorohydrin (ECH,

compound XCIX in Appendix D).32 Although its main application is in the immobilization of proteins

and carbohydrates, it has been shown to cross-link DNA,46 which will be further discussed below.

These amino- and sulfhydryl-group–directed heterobifunctional cross-linkers are not absolutely

specific; many of them can react with different amino acid side chains. Ethyl haloacetimidates

O

Protein-NH2 + O2N



O



C



CH



CH



NO2 + HS-Protein



NO2



Protein-NH



O



CH2NO2



C



CH



S-Protein



+



O2N



O–

NO2



FIGURE 6.1  Cross-linking reaction of 2,4-dinitrophenyl-p-(β-nitrovinyl)benzoate.



198



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



(LXXXIX through XCI in Appendix D), for example, have a broad reactivity. Although its imidate moiety reacts quite specifically with lysine residues through the amidination reaction, the

haloaetamido group can react with any nucleophile including histidine as shown by Diopoh and

Olomucki in cross-linking studies with RNAse.23,24 Since the thiol is a strong nucleophile, many of

the reagents such as compounds LXXXI and LXXXII in Appendix D may act as homobifunctional

reagents cross-linking two sulfhydryl groups if the thiol group is in excess. On the other hand, in

its absence, other nucleophiles will be cross-linked. In general, however, these reagents are used

to cross-link proteins with known amino acid side chains in a sequence of reaction steps. Proteins

that are known to contain free thiols will react first with the compound. After modification, the free

end of the bifunctional reagent reacts with another protein with desired amino acid side chains. For

example, to use 4-chloroactylphenylmaleimide (LXXXI in Appendix D) as a heterobifunctional

cross-linker, the maleimido end is first reacted with a protein containing a thiol group. The alkyl

halide end is then allowed to react with an amino group in another protein.26,29

There are several compounds in this category that are cleavable (e.g., I through XIV, L through LII,

LXII, LXIII, LXVI, LXVII, LXXXIII, LXXXV, LXXXVII, LXXXVIII, and XCV through XCVIII

in Appendix D). Obviously, compounds that result in the formation of disulfide bonds, for example,

N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (compound I in Appendix D), are cleavable

in the presence of a free thiol.2,21 The sulfone-containing compounds such as N-succinimidyl-3(2-bromo-3-oxobutane-1-sulfonyl)propionate (LXII in Appendix D) are cleavable by dithionite.

Compounds that lead to maleylation of amino groups such as N-[4-[(2,5-dioxo-3-furyl)methylsulfanyl]phenyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (LXXXIII in Appendix D) are cleavable under

mildly acidic conditions (see Figure 4.17).31

Among all these heterobifunctional compounds, few are particularly worth noting. 2-Hydroxy4-(N-maleimido)benzoylazide (HMB, compound LXXVII in Appendix D) is photosensitive but

undergoes nucleophilic substitution in the dark. This compound contains a phenolic ring and is

therefore iodionatable by various iodinating agents (see Chapter 4), providing the possibility of

introducing radioisotope 125I for various applications.25,26

There is a group of compounds referred to as equilibrium transfer alkylating cross-linkers (ETAC)

described by Mitra and Lawton and others.33,34 These compounds (CI through CXV in Appendix D)

contain a good leaving group and an electron-withdrawing group in resonance with a double bond. In

the first described compounds, 2-(p-nitrophenyl)allyl-4-nitro-3-carboxyphenylsulfide and 2-(p-nitrophenyl)allyl-4-trimethylammonium iodide (CI and CII in Appendix D), p-nitrophenyl served as an

electron-withdrawing group and 5-thio-2-nitrobenzoate and ammonium iodide, respectively, served as

leaving groups. As shown in Figure 6.2, protein nucleophilic residues undergo Michael addition at the

double bond, eliminating the leaving group to generate a new resonant double bond. A second Michael

addition is then possible with another nucleophile on the same or different protein, resulting in a crosslinkage. Alternatively, during the second Michael reaction process, the first added nucleophile may

be eliminated reforming a double bond. A nucleophile may then undergo similar Michael addition

and the process continues. The reagent can be transferred from the initial site of protein attachment to

other groups until the most thermodynamically stable bond is formed. With the protein side chains of

lysine, tyrosine, glutamic, and aspartic acids, the reagent will undergo Michael addition and Michael

elimination indefinitely, until a thermodynamically stable bond is formed. A more stable bond is

formed when thiol addition is encountered to form the thioether bridge. Since the publication of the

first ETAC, several compounds have been synthesized (CIII through CXV in Appendix D).34 Not only

can these compounds be used to cross-link different groups heterobifunctionally or homobifunctionally, they have also been used to introduce various reporter groups to reduced immunoglobulins.47

The amino- and sulfhydryl-directed heterobifunctional reagents have been extensively used to crosslink various proteins. SPDP is probably the most popular of all these reagents. It has been used to

conjugate antifibrin antibody to urokinase and tissue plasminogen activator,48,49 in the preparation of

bispecific antibodies and immunotoxins,50,51 and in the study of stomatal and fenestral diaphragm and

bacterial cell surface layer.52,53 Several SPDP analogs such as MSPDP, LC-SPDP, and SMPT have been



199



Heterobifunctional Cross-Linkers

:Nu1

w



w



Nu1



Nu1



Nu1



w



w



Nu2



X

:Nu2



Nu2



Nu1

:Nu3



w



Nu2



w



Nu3



Nu3



Nu3



w



w



Nu2



Nu2



Nu3



w



Nu2



Nu3

w



Nu4



:Nu4



Nu4



Nu3

w

Nu4



etc.



FIGURE 6.2  Mechanism of reaction of equilibrium transfer alkylating cross-linkers (ETAC). W designates

an electron withdrawing group, X a good leaving group, and Nu a nucleophile. (Adapted from Libertore, F. A.

et al., Bioconjug. Chem., 1, 36, 1990.)



synthesized and made more soluble by adding a sulfonyl group to the succinimide ring (II through

VII in Appendix D). N-Hydroxysuccinimidyl-3-methyl-3-(acetylthio)butanoate (SAMBA) (XV in

Appendix D) is a special SPDP analog.54 To cross-link proteins, it first reacts with an amino group. The

modified protein is then treated with hydroxylamine to remove the acetyl protecting group to expose

a free thiol, which then reacts with an activated protein disulfide to complete the cross-linking. The

reaction procedure is similar for iminothiolane (CXVI in Appendix D), which reacts with an amino

group and exposes a free thiol as discussed in Chapter 2. It has been used to immobilize avidin onto

gelatin nanoparticales.55 Goff and Carroll56 have synthesized a series of iminothiolane analogs (CXVII

through CXXII in Appendix D) and studied their effect on preparation of immunoconjugates.54 They

found that 5-methyl-2-iminothiolane (M2IT) (CXVII in Appendix D) has optimal properties for the

preparation of disulfide cross-linked immunoconjugates with enhanced disulfide bond stability.

The mercurial compounds, 3-(acetoxymercurio)-5-nitrosalicylaldehyde and 3-(chloromercurio)5-nitrosalicylaldehyde (CXXIII and CXXIV in Appendix D), are truly sulfhydryl specific. They

have been used to study the interactions between cobratoxin and acetylcholine receptor through

cross-linking of the reduced sulfhydryl group of AcChR and lysine 23 of cobratoxin.57 Many other

compounds have also been frequently used in protein structure and interaction,58 in the preparation

of enzyme–immunoglobulin conjugates,59–62 and the stabilization of microtubules.63



6.2.2  Cross-Linkers Directed toward Carboxyl and Either Sulfhydryl

or Amino Groups

The carboxyl group–selective agents used in these heterobifunctional compounds are shown in Table

6.1.II. As discussed in Chapter 5, compounds containing a diazoacetyl group are photosensitive. In the

dark, however, they are reactive toward the carboxyl group at acidic pHs (see Chapter 3).64 Therefore,



200



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



in addition to being photoaffinity labels, these cross-linking reagents (Appendix D.B) are potential

cross-linkers for carboxyl groups.65–68 The reactive group at the opposite end of the molecule determines

whether the cross-linker will react with sulfhydryl or amino group. For example, if the reagent contains a

disulfide bond such as p-(2′-pyridyldithio)benzyldiazoacetate (PPD, Appendix D.B.CXXVII), it will be

thiol specific because the disulfide bond can undergo thiol–disulfide interchange with sulfhydryl groups

on the protein. Compounds containing an alkylating or acylating group will react with any nucleophile.

For instance, in the presence of sulfhydryl groups, the compound, 1-diazoacetyl-1-bromo-2-phenylethane (Appendix D.B.CXXVIII), will be thiol directing and has been shown to react with a cysteine at

the active site of pepsin.66 In the absence of sulfhydryl groups, amino groups and other nucleophiles will

react. However, for acylating groups such as that in p-nitrophenyl diazoacetate (Appendix D.B.CXXIX),

more stable products will form with amino groups. These compounds have been used to photolabel the

active site of chymotrypsin67 and cross-link calmodulin and adenylate cyclase.68



6.2.3  Carbonyl- and Amino- or Sulfhydryl-Group–Directed Cross-Linkers

As discussed in Chapter 3, carbonyl groups form Schiff bases with amino groups. Thus, the heterobifunctional cross-linking reagents that have been used in literature contains an amino group,

either free or as a hydrazide (see Table 6.1.III and IV). Only one carbonyl and amino cross-linker

has been studied, N-succinimidyl-4-hydrazidoterephthalate (SHTH, Appendix D.E.CXLVIII). It has

been used to cross-link the oxidized carbohydrate moiety in Fc region of polyclonal antibodies and

lysine residues on the microtubule surface in a kinesin-based transport system.69

A few compounds are designed to cross-link carbonyl and sulfhydryl groups. Maleimido hydrazides (Appendix D.D.CXLIV through CXLVII) contain a maleimide and a hydrazide moiety. The

other reagents (Appendix D.D.CXLII and CXLIII) contain an alkoxylamino group with a disulfide bond. Through thiol–disulfide interchange, protein sulfhydryls will form new disulfide bonds

with these compounds.70 The alkoxylamino moiety of these compounds reacts readily with ketones

and aldehydes to produce stable alkoxime as shown in Figure 6.3. Thus, dialdehydes derived from

the carbohydrate moiety of glycoproteins on periodate oxidation will react with the alkoxylamino

group or hydrazide. These compounds have also been used to cross-link adriamycin and thiolated

antibody and the preparation of immunoconjugates.70,71



6.2.4  Miscellaneous Heterobifunctional Cross-Linkers with Undefined Specificity

In addition to the above categories of heterobifunctional cross-linkers, there are other heterobifunctional reagents that cross-link various nucleophiles. The Cyssor reagent, 2-methyl-N1-benzenesulfonylN4-bromoacetylquinonediimide (Appendix D.F.CXLIX), has been found to cross-link antibodies at

pH 8.0.72 This molecule is essentially an alkylating agent. Nucleophiles attack both the quinone ring

and the bromoacetyl group as shown in Figure 6.4. Although the nucleophiles in this particular reaction have not been identified, it is speculated that carboxyl groups of aspartate and glutamate and the

indolyl ring of tryptophan may serve as nucleophiles in addition to sulfhydryls and amino groups.

Compounds that contain an aldehyde group such as N-hydroxysuccinimidyl-p-formylbenzoate (HFB)

and methyl-4-(6-formyl-3-azidophenoxy)butyrimidate (FAPOB) (Appendix D.F.CL and CLI) will form



Protein-C-H + H2N-O-CH2-X-CH2-S-S

Protein-SH



O2N



O2N



O



Protein-C



Protein-C

N



N-O-CH2-X-CH2-S-S



H



N-O-CH2-X-CH2-S-S



N



Protein



H



FIGURE 6.3  Cross-linking carbonyl and sulfhydryl groups with aminooxypyridyldithio derivatives.



201



Heterobifunctional Cross-Linkers

H3C



O

S



O

N



N



O

CH2



C



Br



S



O



H3C



:Nu-Protein2



O

NH



NH



C



CH2



Br



O

Protein-Nu



Protein1-Nu:

O

S



H3C



O



NH



NH



C



CH2



Nu-Protein2



O

Protein1-Nu



FIGURE 6.4  Speculated mechanism of cross-linking reaction with 2-methyl-N1-benzenesulfonyl-N4-bromoacetylquinonediimide.



Schiff base with amino groups.72–76 The succinimidyl of HFB and the imidoester of FAPOB are also reactive toward the ammo group, making these two compounds function like a homobifunctional reagent.

FAPOB, for example, cross-links lysine 51 and lysine 29 of the ribosomal protein L7/L12.74,75 FAPOB,

containing an phenylazido group, is also photosensitive and may be used as a photochemical agent. In

acrolein (Appendix D.F.CLIII), the aldehyde group may form a Schiff base with an amino group. Its

double bond is subject to Michael addition with different nucleophiles and has been shown to cross-link

collagen.77 Ishii et al.78 used model peptides and mass spectrometric techniques to show that crosslinking took place between amino groups and the side chain of histidine in the peptide. Compounds that

contain either trimethoxysilyl or triethoxysilyl moiety such as 3-glycidyloxypropyltrimethoxy silane and

N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide (TPICC) (compounds

CXXXIV and CXXXVI in Appendix D) are capable of reacting with hydroxyl groups of silica on glass

surfaces. The other end of the molecules containing reactive epoxide or isothiocyanates can react with

various nucleophiles, thus immobilizing the molecules as shown in Figure 6.5. These compounds have

been used for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides, and proteins on the glass surface.79,80

Affinity labeling is a specially designed agent that will bind specifically to a desired location of a biomolecule, usually the active site. An example of an affinity bifunctional cross-linker is

EtO

CH2-N=C=S



EtO Si (CH2)3 NH-CO

EtO

Glass



Molecule-Nu:

OH



EtO

Glass



O



S



EtO

Si (CH2)3 NH-CO



CH2-N=C=S EtO



Si



(CH2)3 NH-CO



CH2-NH-C Nu-Molecule



EtO



EtO

Molecule-Nu:



O



OH

S



EtO

Glass



Glass



Si (CH2)3 NH-CO



CH2-NH-C



Nu-Molecule



EtO



FIGURE 6.5  Reaction mechanism of immobilization of macromolecules on to glass surface using N-(3triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide. (Adapted from Misra, A. et al.,

Bioorg. Med. Chem. Lett., 18, 5217, 2008.)



202



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



1-(4-methoxyphenyl)-3-acetamido-4-methoxyazetidin-2-one (Appendix D.F.CLVI), which is an analog of

β-lactam.81 It inhibits the class A β-lactamase from Bacillus cereus 569/H as an active site-directed inhibitor and cross-links ser 70 and lys 234 of the enzyme. However, the mechanism of reaction is unknown.



6.3  P

 ROTEIN-PHOTOSENSITIVE HETEROBIFUNCTIONAL

CROSS-LINKING REAGENTS

Photosensitive heterobifunctional cross-linkers represent by for the largest portion of the heterobifunctional reagents. For a comprehensive list of these compounds, please see Appendix E. Because these

functionalities are inert until they are photolyzed, these reagents are first linked to the protein in the dark

through a group-directed agent as shown in Table 6.2. The labeled protein is then irradiated to activate

the photosensitive group, which reacts indiscriminatively with its environment as discussed in Chapter 4.

The photosensitive cross-linkers are generally classified according to the active species they produce,

for example, the nitrenes and carbenes.82 These reactive species are generated from various groups. As

discussed in Section 4.5, the most widely used photoreactive groups are azides, diazo moiety, benzophenone, diazonium salts, and diazirines. Only a few carbene-generating diazo reagents are used

for cross-linking,65,67,68,82–87 probably because of the ability of carbenes to undergo a variety of reactions including the very efficient reaction with water.82 In addition, the parent diazoacetyl compounds

are generally unstable, particularly at low pH and are reactive toward nucleophiles including carboxyl

groups.64 However, 3-phenyl-3-(trifluoromethyl) diazirine (TPD) is gaining popularity and has been

incorporated into photoaffinity labels and cross-linkers.88,89 The reasons are the unexpected stability of

the TPD three-membered ring and its ability to be photoreactivated with light over 350 nm to generate

carbenes, which can rapidly form cross-links to biomolecules with short photoirradiation times.

Azido derivatives constitute the majority of the photoactivatable cross-linking agents (compounds

I through XXXVIII, L through LXI, LXXI through XCIV, CXVI, CXVII, and CXXI through

CXXXIX in Appendix E). Three types of azides have been synthesized: the aryl, alkyl, and acyl

azides. Alkylazides, however, are not used in cross-linking for several reasons. First, they have absorption maxima in the UV region in which irradiation may damage proteins, nucleic acids, and other

components. Second, the alkylnitrene intermediates readily undergo rearrangement to form inactive

imines. Last, alkylazides are reactive and may undergo nucleophilic displacement reactions. For the

same reasons, acylazides are generally used as acylating agents rather than photoaffinity labels.90 Only

arylazides have been extensively used in photoactivatable cross-linkers.90–138 Aryl azides have a low

activation energy and can be photolyzed in the long UV region.21,82,139 The presence of electron-withdrawing substituents such as nitro and hydroxyl groups further increases the wavelength of absorption

into the 300 nm region.64 Arylnitrenes have a half-life on the order of 10 −2–10 −4 s,140,141 and, therefore,

the cross-linking reaction is expected to be terminated within a short time. Arylazides are susceptible

to reduction to amino groups. They are not stable in the presence of thiols. The half-life of arylazides

is 5–15 min in 10 mM dithiothreitol (pH 8.0) and over 24 h in 50 mM mercaptoethanol (pH 8.0).142

The benzophenone derivatives (LXII through LXV, CXI, CXII, and CXL through CXLIV in

Appendix E) constitute yet another class of photoaffinity labels.143–145 These compounds, as shown in

Chapter 4, can form covalent adducts on irradiation with nearby amino acid residues leading to crosslinking. Unlike the azides, which are irreversibly photolyzed in most cases, the excited triplet state of

benzophenones may be resistant to reaction with water and may revert back to the starting material if no

photoreaction takes place. Since benzophenones can be reexcited, their cross-linking efficiency can, in

principle, reach 100%.143,144 These compounds have been used to study virus-induced proteins,146 ribosomal proteins,147 troponin and tropomyosin,148 actin,149 thin filament proteins,150 and chymotrypsin.151

Another class of photosensitive reagents is nitrophenyl ether (Appendix E.B.CXIV and CXV).152

These compounds react quantitatively with amines at slightly alkaline conditions (pH 8) on irradiation with 366 nm light. The reaction involves the transfer of nitrophenyl group from the alcohol to the

amine as shown in Figure 6.6 for the cross-linking reaction of N-(maleimidomethyl)-2-(O-methoxyp-nitrophenoxy)carboamidopropane. With this compound, the 2-methoxy-4-nitrophenyl ether is



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2 Group-Selective Heterobifunctional Reagents for Protein Cross-Linking

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