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5 Phenolate and Imidazolyl Group–Directed Cross-Linking Reagents

5 Phenolate and Imidazolyl Group–Directed Cross-Linking Reagents

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103



Homobifunctional Cross-Linking Reagents



H



O



O



O



O



C



C



C



C



H



p-Phenylenediglyoxal



H



O



O



O



O



C



C



C



C



H



4,4΄-Biphenyldiglyoxal



FIGURE 5.13  Chemical structures of homobifunctional arginine reagents.



i­ ntramolecular movements of the myosin subfragment-1 molecule.403 Both PDG and BDG have been

used to demonstrate the feasibility of elucidation of biomolecular structures by employing peptide

mapping with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.404



5.7  METHIONINE RESIDUE CROSS-LINKING AGENT

The sulfur atoms of methionine residues in proteins sometimes compete with other nucleophiles

in cross-linking reactions as shown in various instances discussed above. No methionine-specific

cross-linkers have been published. Although not specifically designed for cross-linking methionine residues, cis-diaminedichloroplatinum(II) (Cisplatin, cis-DDP) as shown in Figure 5.14, has

been found to cross-link α2-macroglobulin with methionine residues at or near its receptor recognition site.405–407 The cross-linking can be reversed by diethyldithiocarbamate, which is a potent

platinum chelator.407 Trans-diaminedichloroplatinum(II) (trans-DDP, Figure 5.14) also reacts with

α2-macroglobulin, but is less efficient at inhibition. Both complexes have been shown to react with

albumin and other plasma α-1 proteinase inhibitors, however, cysteines may be involved.408,409 In

addition, they cross-link DNAs as will be discussed more fully below.



5.8  CARBOHYDRATE MOIETY–SPECIFIC REAGENTS

As discussed in Section 3.3.1.4, the monosaccharide unit of glycoproteins can be oxidized by sodium

periodate to yield dialdehydes. These dialdehydes will form Schiff bases with amines, which can

be stabilized by reducing agents in the process of reductive amination. Thus, amines can be used as

cross-linkers after oxidation of the carbohydrate moiety of glycoproteins. Adipic acid dihydrazide

(Figure 5.15) has been used to cross-link glycoproteins, such as acid phosphatase and invertase.410

Other diamines of different chain lengths may also be used in this process. The efficiency of reductive amination can be increased by including 500 mM sodium sulfate in the reaction media.411

Cl



NH3



Pt(II)

Cl



NH3



NH3



Cl



Pt(II)



Cl



NH3



cis-DDP



Trans-DDP



FIGURE 5.14  Chemical structure of cis-diaminedichloroplatinum (cis-DDP) and trans-diaminedichloroplatinum (trans-DDP).

O



O



H2N-NH-C-(CH2)4-C-NH-NH2



FIGURE 5.15  Chemical structure of adipic acid dihydrazide (AAD).



104



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 5.4

Photoactivatable Homobifunctional Cross-Linking Reagents

A. 4,4′-Diazidobiphenyl (DABP):



N3



N3

N3



B. 1,5-Diazidonaphthalene (NAD):

N3

NO2



OH



C. N,N′-Bis(p-azido-o-nitrophenyl)-1,3-diamino-2-propanol:



N3



NH-CH2-CH-CH2-NH



D. 4,4′-Dithiobisphenylazide (DTPA, DTBPA):



N3



S



S



O2N

N3

N3



5.9  NONDISCRIMINATORY PHOTOACTIVATABLE CROSS-LINKERS

Among the photoactivatable moieties, diazoalkanes and diazirines have not been incorporated

into homobifunctional cross-linkers, although they are found in heterobifunctional reagents, which

will be discussed in the next chapter. The more commonly used group is the azido function. Alkyl

azides, however, have not been favorably used.10,412 Acylazides as well as sulfonyl and phosphoryl azides are generally not used as photoactivatable reagents because of their nucleophilic reactivity in the dark.35 The most common photoactivatable homobifunctional reagents are derived

from aryl azides as shown in Table 5.4.413,414 These aryl azides are photolyzed at wavelengths of

300–400 nm so that the biological component, be it protein or nucleic acid, would not be damaged by photoirradiation. They are used mainly to label erythrocyte membrane proteins.413,415

4,4′-Dithiobisphenylazide (DTBPA) containing a disulfide bond was synthesized by Mikkelsen

and Wallach413 as a cleavable reagent in the presence of free thiols. It has been used to identify

erythrocyte membrane proteins. It should be noted that the reagent may be used as a heterobifunctional reagent by reaction first with a sulfhydryl group through the disulfide–thiol interchange

reaction and followed by photoactivation.



5.10  NONCOVALENT HOMOBIFUNCTIONAL CROSS-LINKING REAGENTS

While chemical cross-linking has the connotation that the reagents react to form covalent bonds,

there are species that physically associate with proteins or other entities to form such tight complexes that binding is essentially irreversible. The avidin–biotin complex is a well-known example

of such a system. Some immunoglobulins can also bind to antigens with dissociation constants in

the order of femtomolar. Lectins are another class of proteins that have specific binding targets.

These molecules may be important under certain circumstances and are hence useful for some

applications.

The avidin–biotin and the lectin systems will be discussed in the context of multifunctional

reagents in Chapter 7. Avidin and streptavidin are proteins with four high affinity binding sites

for biotin.415 They will cross-link four biotin moieties bound to biological molecules. The lectins

are complex sugar binding proteins with multivalent binding sites for specific carbohydrates.416

These proteins will cross-link multiple glycoproteins. In Chapter 7, these systems will be further

explored in detail.



105



Homobifunctional Cross-Linking Reagents

OH

H2O3As



N=N



N=N



AsO3H2



CH2

NH2-CH-COOH



FIGURE 5.16  Structure of a bifunctional antigen: l-tyrosine-bis(p-azobenzenearsenate) (Bis-RAT).



For the immunological systems, some antibodies can be used as homobifunctional cross-linkers

while others have been designed to be heterobifunctional.417 The latter will be discussed in the

next chapter. The basic unit (monomer) of immunoglobulins contains two Fab (antigen-binding)

fragments and one Fc (crystallizable) fragment. These molecules are therefore bifunctional in that

they can bind specifically two antigens, thus cross-linking them. With monoclonal antibodies, the

dissociation constant of antibody–antigen complex can be as low as 10 −15 M, making the complex

essentially irreversible. There are five classes of immunoglobulins, designated as IgG, IgA, IgM,

IgD, and IgE. All the antibodies exist as monomers except IgM, which is pentameric. In adult animals, the majority of serum immunoglobulins are in the IgG class. IgG is thus the most common

monospecific reagent. Treatment of IgG with pepsin yields a F(ab)12 fragment, which retains the two

antigen binding sites. F(ab)12 can be purified and used as a cross-linking reagent.

Homobifunctional antibodies are used in various immunoassays, including agglutination precipitin assays, and more recently in proteomic tissue profiling by antibody-based microarray.418,419 They

are also used to construct immunotoxins.7 However, their use as cross-linking agents for chemical

studies is limited.

Similarly, compounds that contain two antigenic determinants will cross-link antibody molecules. Simple molecules such as l-tyrosine-bis(p-azobenzenearsenate) (Bis-RAT, see Figure 5.16)

have been shown to cross-link anti-RAT antibodies antibodies.420 Similar cross-linking of the immunoglobulins is achieved by bis-azobenzenearsenate separated by various spacers. Other examples of

multiepitopic molecules are hapten-conjugated antigens such as dinitrophenyl labeled bovine serum

albumin.421 In addition to proteins, polysaccharides have also been used as carriers. The antigen–

antibody complexes have been used in various studies.



5.11  NUCLEIC ACID CROSS-LINKING REAGENTS

From the discussions in earlier sections, it is obvious that there are reagents that not only cross-link

proteins but also react with nucleic acids. Reagents like bisimidates, dicarbonyl compounds, mustards, aziridine benzoquinones, diepoxides, and platinum complexes, can also cross-link protein

and nucleic acids, and DNAs. However, there are series of compounds that have been reported to

mainly cross-link nucleic acids. These DNA cross-linking reagents, listed in Table 5.5, are generally

antitumor agents and will be described below.422



5.11.1  Metal Compounds

In addition to cis- and trans-DDP mentioned in Section 5.7, there are di-, tri-, and poly-nuclear

platinum complexes that will react with DNA (Table 5.5.I.A).423–425 The active form of the reagent

is believed to be the aquatic species, formed by the displacement of the chlorides by water. For

cis-DDP this would be cis-[Pt(NH3)2(H2O)2]2+.426 These compounds are thought to exert their antitumor activities through DNA adducts formation with the N7-nitrogen of purines, mainly guanine.

The main products of cis-DDP and DNA reactions are intrastrand cross-links between guanine

residues in 5′-GG-3′, 5′-GNG-3′, and between adenine and guanine in 5′-AG-3′ sequences.427



106



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 5.5

Nucleic Acid Cross-Linking Reagents

I. Metal complexes

A. Platinum complexes

1. Dinuclear platinum complexes



Pt



Pt

CH2



NH2



NH3



2+



Cl



NH3



NH3



Cl



n



NH3



NH2



n = 2,4,6

2. Trinuclear platinum complex

Cl



NH3



NH2-(CH2)6-NH2



NH3

Pt



Pt

NH3



4+



Pt

NH3



NH2-(CH2)6-NH2



NH3

Cl



NH3



B. Phenyl selenides:

1. 2,6-Bis(phenylselenylmethyl)phenol

OH

Se



Se

2. 2,5-Bis(phenylselenylmethyl)benzene-1,4-diol

OH



Se

Se

OH

3. 3,3′-Bis(phenylselenylmethyl)-4,4′-dihydroxy-1,1′-biphenyl

OH

Se



Se

OH

C. Aluminum(III) complex

1. (R,R)-N,N′-Bis[5-methyl-3-(4-methylpiperazinyl)-salicylidene]-1,2-diphenylethanediamine aluminum(III)

chloride ([SalenAlIII]Cl)

+



N

H 3C



H3C



N



N

Al



O

N



CH3



O

N



N



CH3



107



Homobifunctional Cross-Linking Reagents



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

II. Azinomycin derivatives

A. Azinomycin bisepoxides with methylene spacer

O



O

H3C



CH3

-CO-O-CH-CO-NH



H3C



CH2



n



NH-CO-CH-O-CO-



O-CH3



CH3

O-CH3



1. n = 2: 1,2-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]ethane

2. n = 3: 1,3-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]propane

3. n = 4: 1,4-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]butane

4. n = 5: 1,5-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]pentane

5. n = 6: 1,6-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]hexane

B. 2,2′-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]diethylmethylamine

O



O



CH3



CH3

-CO-O-CH-CO-NH



H3C



H3C



(CH2)2-N-(CH2)2



NH-CO-CH-O-CO-



CH3

O-CH3



O-CH3

C. Azinomycin bisepoxides with aromatic spacer

O



O

CH3



H3C



H3C



-CO-O-CH-CO-NH



NH-CO-CH-O-CO-



O-CH3



CH3

O-CH3



1. p-Aryl: 1,4-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]benzene

2. m-Aryl: 1,3-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]benzene

3. o-Aryl: 1,2-Bis[(2S,3S)-3,4-epoxy-2-(3-methoxy-5-methyl-1-naphthoyloxy)-3-methylbutanamido]benzene

D. 1,4-Bis[(2S,3S)-3,4-epoxy-2-(1-naphthoyloxy)-3-methylbutanamido]benzene

O



O

CH3



H3C

NH-CO-CH-O-CO-



-CO-O-CH-CO-NH

III. Bis-pyrrolobenzodiazepine (PBD) derivatives

A. C7/C7′-Linked PBD dimers



N



N



N



X



N

O



O



1. X=S-(CH2)3-S: 1,3-Bis[7-sulfo-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]benzodiazepin-5-one]propane

(continued)



108



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

2. X = O-(CH2)2-N-(CH2)2-O: 2,2′-Bis[7-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]benzodiazepin-5one]ethylmethylamine

CH3

3. X = NH-(CH2)3-N-(CH2)3-HN: 2,2′-Bis[7-amino-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]benzodiazepin-5-one]ethylmethylamine

CH3

B. C8/C8′-Alkanediyldioxy-linked PBD dimers

N



O



N



CH2



n



O-CH3

O



N



O



N



CH3-O

O



n = 3,4,5,6



1. n = 3: 1,1′-[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]

benzodiazepin-5-one] (DSB-120)

2. n = 4: 1,1′-[(Butane-1,4-diyl)dioxy]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]

benzodiazepin-5-one]

3. n = 5: 1,1′-[(Pentane-1,5-diyl)dioxy]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]

benzodiazepin-5-one]

4. n = 6: 1,1′-[(Hexane-1,6-diyl)dioxy]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[1,2-c][1,4]

benzodiazepin-5-one]

C. C8/C8′-Alkanediyldioxy-linked C2-difluorinated PBD dimers

N



O



N



F



CH2



O-CH3



F



O



n



N



O



N



CH3-O

O



n = 3,4,5



F

F



1. n = 3: 1,1′-[Propane-1,3-diyl)dioxy]-bis[(11aS)-7-methoxy-2,2-difluoro-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]

[1,4]benzodiazepin-5-one]

2. n = 4: 1,1′-[Butane-1,4-diyl)dioxy]-bis[(11aS)-7-methoxy-2,2-difluoro-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]

[1,4]benzodiazepin-5-one]

3. n = 5: 1,1′-[Pentane-1,5-diyl)dioxy]-bis[(11aS)-7-methoxy-2,2-difluoro-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]

[1,4]benzodiazepin-5-one]

D. C8/C8′-Alkanediyldioxy-linked C2-exo/endo unsaturated PBD dimers

1. C2-Exo unsaturated PBD dimer: 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] (SJG-136)

N

N



O



(CH2)3



O-CH3



O



N

N



CH3-O



O



O



2. C2-Exo-difluoromethylene PBD dimers

N

N



F

F



O



CH2



O-CH3

O



n



O



N

N



CH3-O



n = 3,4,5



O



F

F



109



Homobifunctional Cross-Linking Reagents



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

a. n = 3: 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-fluoromethylidene-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepin-5-one]

b. n = 4: 1,1′-[[(Butane-1,4-diyl)dioxy]bis[(11aS)-7-methoxy-2-fluoromethylidene-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepin-5-one]

c. n = 5: 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-7-methoxy-2-fluoromethylidene-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepin-5-one]

3. C2-C3/C2′-C3′-endo unsaturated PBD dimer

N

O



O



N



CH3-O-C-CH2-CH2



(CH2)3



O-CH3



N



O



O



N



CH3-O



O



CH2-CH2-C-O-CH3



O



E. C8/C8′-Polyheterocyclic-linked PBD dimers

1. Tripyrrole-containing PBD dimer (AT-235)

O

N



H

N



O-(CH2)3-C



H

N



C



CH3



O



O-CH3



N

O



N



N-(CH2)3-O



N



CH3-O



3



O

2. Polypyrrole-polyamide-containing PBD dimers

H



N



O-CH3



N



N-C



O-(CH2)3-C-N-(CH2)2-N-C-CH2

H



N-C-(CH2)3-O



H



H



O



CH3



O



O



O



N



N

N



CH3-O

O



n



n = 1,2,3



O

3. Polyimidazole-polyamide-containing PBD dimers



H

O

N

N



H



H



O-(CH2)3-C-N-(CH2)2-N-C-CH2

O-CH3



H



N-C



O



N

CH3



O



O



N-C-(CH2)3-O



N



N



CH3-O



N

O



n



n = 1,2,3



O

F. C8/C8′-Linked-1,2,3-triazole-containing PBD dimers

N

N



N

O-(CH2)n

O-CH3



O



n = 3,4,5,6,8



N



N

CH2



O



N

N



CH3-O

O



(continued)



110



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

G. C8/C8′-piperazine-linked PBD dimers

N

N



O-(CH2)n



N



N



O-CH3



(CH2)n-O



N

N



CH3-O



O



O



1. n = 2: 1,1′-[[1,4-Di(ethane-1,2-diyl)hexahydropiperazine]dioxy]-bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]

2. n = 3: 1,1′-[[1,4-Di(propane-1,3-diyl)hexahydropiperazine]dioxy]-bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]

3. n = 4: 1,1′-[[1,4-Di(butane-1,4-diyl)hexahydropiperazine]dioxy]-bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]

4. n = 5: 1,1′-[[1,4-Di(pentane-1,5-diyl)hexahydropiperazine]dioxy]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]

H. C8/C8′-Piperazine-linked C2-monoflourinated PBD dimers

N

F



O-(CH2)n



N



N



N



O-CH3



F



N



CH3-O



O



H



N



(CH2)n-O



H



O



1. n = 3: 1,1′-{[1,4-Di(propane-1,3-diyl)hexahydropiperazine]dioxy}-bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

2. n = 4: 1,1′-{[1,4-Di(butane-1,4-diyl)hexahydropiperazine]dioxy}-bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

3. n = 5: 1,1′-{[1,4-Di(pentane-1,5-diyl)hexahydropiperazine]dioxy}-bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

I. C8/C8′-piperazine-linked C2-difluorinated PBD dimers

N

F



O-(CH2)n



N



N



N



O-CH3



F



N



CH3-O



O



F



N



(CH2)n-O



O



F



1. n = 3: 1,1′-{[1,4-Di(propane-1,3-diyl)hexahydropiperazine]dioxy}-bis[(11aS)-2,2-di-fluoro-7-methoxy1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

2. n = 4: 1,1′-{[1,4-Di(butane-1,4-diyl)hexahydropiperazine]dioxy}-bis[(11aS)-2,2-di-fluoro-7-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

3. n = 5: 1,1′-{[1,4-Di(pentane-1,5-diyl)hexahydropiperazine]dioxy}-bis[(11aS)-2,2-di-fluoro-7-methoxy1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]

J. C8/C8′-Piperazine-linked C2-exo-difluoromethylene PBD dimer: 1,1′-{[1,4-Di(propane-1,3-diyl)

hexahydropiperazine]dioxy}-bis[(11aS)-2-difluoromethylidine-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]

[1,4]benzodiazepine-5-one]

N

N



F

F



O-(CH2)3

O-CH3



O



N



N



(CH2)3-O



N

N



CH3-O

O



F

F



111



Homobifunctional Cross-Linking Reagents



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

K. C8/C8′-anthraquinone-linked PBD dimers

N



(CH2)n-O



O



O



CH3-O

O

N



(CH2)n-O



O



O



N



N



CH3-O

O



1. n = 3: 1,4-Bis-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one]

propyloxy}anthracene-9,10-dione

2. n = 4: 1,4-Bis-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one]

butyloxy}anthracene-9,10-dione

3. n = 5: 1,4-Bis-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one]

pentyloxy}anthracene-9,10-dione

L. C2/C2′-Linked PBD dimers

O



N



O

CH2



CH-C-N



N



n



H



O



N



N-C-CH



N



H



O



n = 5–7



M. A-C8/C-C2 Amide-linked PBD dimers

N



CH3-O



OH

C-N



N



CH3-O



N



O

N



X

X = H or O-CH3



O



N. A-C8/C-C2 Alkoxyamido-linked PBD dimers

BnO



N

O

N



CH3-O

O



N



N-C-(CH2)n-O

H



N



CH3-O



n = 1,2,3



O



O. C8/C2-Linked-1,2,3-triazole-containing PBD dimers

R1



N

N



R2

O



N



i. R1 = R2 = H

ii. R1 = R2 = OCH3

iii. R1 = OBn, R2 = OCH3



N



N



O



N

N



CH3-O

O



(continued)



112



Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

IV. Cyclopropylpyrroloindole (CPI)-based dimers

A. Bis-Duocarmycin SA enantiomer analogs

N

N

MeO2C



C

O



N

H



O

i. (+)(+)-DSA2

iii. (–)(+)-DSA2



N

H



boc



O



ii. (+)(–)-DSA2

iv. (–)(–)-DSA2



B. Bis(N-methylimidazole-N-methylpyrrole-CPI)

O



H

HOOC



N-C



N

H3C



C



N



N



O



O



N



NH



C-(CH2)n-C-



N



n = 3,4,5



CH3



CH3



O



O



2



C. Seco-CPI-based dimers

1. Bizelesin, 1,3-bis[2-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-3H-pyrrolo[3,2-e]indole-6carbonyl]-1H-indol-5-yl]urea

H



Cl



Cl



N-C-N



H3C



N



C

O



N

H



H



O

N

H



N

H



CH3



N



C

O



N

H



OH



OH



2. U78779

HO

Cl



N-C



H3C

N

N

H



C



OH

O



Cl



C-N

N

H



N

H



O



C



CH3



N



O



N

H

OH



OH

3. Bis(3-methoxycarbonyl-2-trifluoromethyl-CPI) (MCTFCPI)

CO2Me



Cl



Cl



CO2Me



F3 C



CF3

N



N

H



C

HO



O

a. X = O



X

N

H

b. X = CH2



c. X = C = C



N



C

N

H



O



d. X = NH-CO-NH



NH

OH



113



Homobifunctional Cross-Linking Reagents



TABLE 5.5 (Continued)

Nucleic Acid Cross-Linking Reagents

4. Methoxycarbonyl/methyl-CPI dimer linked by phenylenediacryloyl group

Cl



Cl



R2

R1



N

H



CH=CH



CH=CH



N C

O



R2



C N

O



OH



a. R1=CF3, R2=CO2Me

d. R1=CO2Me, R2=H



N

H



R1



OH

c. R1 = H, R2 = CO2Me



b. R1=Me, R2=CO2Me

e. R1=H, R2=Me



5. Phenyl-CPI dimer linked by phenylenediacryloyl group

Cl



Cl

N



N

H



CH=CH



CH=CH



C

O



C N

O



OH



OH



N

H



V. Seco-CBI (cyclopropanebenz[e]indoline)-based dimers

A. C7-C7 Seco-CBI dimers

1. With methylene spacer arm

Cl

boc



Cl



N



OH

N-C

OH



CH2



HO

a. n = 3 b. n = 4



n



boc



C-N

OH

d. n = 6



c. n = 5



2. With pyrrole polyamide spacer arm

Cl

boc



N



Cl



O H



O H



N-C-(CH2)2-C N



OH



HO



N



C N-CH2-C-N

OH



CH3

a. n = 1



boc



OH



n



b. n = 2



c. n = 3



3. With imidazole polyamide spacer arm

Cl

boc



N



Cl



O H

OH



N-C-(CH2)2-C N

HO



N

N



O H

C



CH3

a. n = 1



b. n = 2



OH



boc



N-CH2-C-N-CH2-C-NOH



OH



n



c. n = 3

(continued)



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