Reactivity derivatives

Nitrogen forms two series of oxohalides — the nitrosyl halides XNO and the nitryl halides XNO2. There are also two halogen nitrates FONO2 (bp -46°) and CIONO2 (bp 22.3°), but these do not contain N-X bonds and can be considered as highly reactive derivatives of nitric acid, from which they can be prepared by direct halogen ation ... 

A special type of ammonio group is represented by 4-( 1 -pyridinium)-pyridine and other azinium analogs. Such products often result from self-quaternization of highly reactive derivatives. A -(4-Pyridyl)-and A -(3-nitro-4-pyridyl)-pyridinium chloride hydrochlorides (121) react with aniline, chloride ion, and water to give 4-substituted pyridines plus pyridine. l-(2-Quinolyl)- and l-(4-quinolyl)-pyridinium salts undergo 2- and 4-substitution, respectively, with amines, anilines, hydroxylamine, phenols, alkoxides, mercaptans, and chloride... 

No kinetic data or semi-quantitative comparisons among themselves or with other diazines are available. The most reactive derivative is expected to be the 4-substituted-l,6-naphthyridine (425), with 2-substituted-1,6- and l-substituted-2,7-naphthyridines (426) somewhat less reactive, all three positions being activated by two ring-nitrogens by resonance. Other positions also activated in this way... 

Rate coefficients and kinetic parameters for iododeboronation were determined for the benzene- and thiophene-boronic acids, and the results are given in Table 256. The relative reactivities derived from this work correlated well with those obtained in a number of other electrophilic substitutions572, which is perhaps surprising in view of the large variation in the entropies of activation. These differences were explained by Brown et al.132 in terms of the transition state for the phenyl compound occurring earlier along the reaction coordinate than for the... 

Normally, reactive derivatives of sulfonic acids serve to transfer electrophilic sulfonyl groups259. The most frequently applied compounds of this type are sulfonyl halides, though they show an ambiguous reaction behavior (cf. Section III.B). This ambiguity is additionally enhanced by the structure of sulfonyl halides and by the reaction conditions in the course of electrophilic sulfonyl transfers. On the one hand, sulfonyl halides can displace halides by an addition-elimination mechanism on the other hand, as a consequence of the possibility of the formation of a carbanion a to the sulfonyl halide function, sulfenes can arise after halide elimination and show electrophilic as well as dipolarophilic properties. 

Nylon 66 can be drawn from a dish containing two starting materials. The bottom layer is an aqueous solution of 1,6-hexanediamine, and the top layer is adipoyl chloride (a more reactive derivative of adipic acid). Nylon 66 forms at the interface between the two liquids. 

Instead of direct halogenation of ketones, reactions with more reactive derivatives such as silyl enol ethers and enamines have advantages in certain cases. 

However, not all of the vinyloxyphosphazene monomers will undergo radical polymerization) those with amino substituents are unreac-tive. The i C nmr data indicate that these species electronically resemble vinyl ethers (which do not undergo radical polymerization) whereas the reactive derivatives resemble vinyl acetate. These data demonstrate an excellent example of electronic effect transmission in cyclophosphazene systems. 

The high cost of these compounds, mainly those of the reactive derivatives pose restrictions, when a considerable quantity is needed. However, the less expensive commercially available rhodamines have been used either as such or as precursors in the preparation of other derivatives for various assays. 

Tetrahydroepoxides as models. Since the quantum chemical calculations apply most rigorously to the simple benzo-ring tetrahydroepoxides and since the calculations neglect influences of the hydroxyl groups in the diol epoxides, it is instructive first to examine the benzo-ring tetrahydroepoxides as simplified models for the reactive site in the diol epoxides. Most of the information about tetrahydroepoxide reactivity derives from studies of the kinetics of their hydrolysis reactions, in which cis- and trans-diols, as well as tetrahydroketones can be formed (Equation 5). 

Prolonged residence in the intestine or urinary bladder lumen could allow time for significant reaction with tissue components however, N-glucuronyloxy-AAF was only weakly carcinogenic at local subcutaneous sites of application (89). Enzymatic deacetylation to N-glucuronyloxy-AF has been detected in hepatic tissue but this activity in different species does not correlate with their relative susceptibility to AAF hepatocarcinogenesis (94). On the other hand, the alkaline pH-induced conversion to a reactive derivative may play an important role in urinary bladder carcinogenesis (87) by AAF and other arylamides in those species or individuals where normal urine pH is alkaline (e.g. normal rabbit urine pH is 8.5-9.0). 

Halogenation of pyrimidine bases may be done with bromine or iodine. Bromination occurs at the C-5 of cytosine, yielding a reactive derivative, which can be used to couple diamine spacer molecules by nucleophilic substitution (Figure 1.48) (Traincard et al., 1983 Sakamoto et al., 1987 Keller et al., 1988). Other pyrimidine derivatives also are reactive to bromine compounds... 

Figure 5.9 SIAX can be used to modify amine-containing molecules to produce sulfhydryl-reactive derivatives. Subsequent reaction with a thiol compound produces a thioether linkage.

Figure 7.9 Amine-containing dendrimers can be activated with SPDP to create thiol-reactive derivatives. Alternatively, the pyridyl dithiol group may be reduced to create free thiols on the dendrimer surface for subsequent conjugation.

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The result is reactivity directed toward amine-containing molecules, forming amide linkages with the AMCA fluorophore (Figure 9.23). Proteins labeled with AMCA show little-to-no effect on the isoelectric point of the molecule. 

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

Figure 21.10 Cystamine may be used to make immunotoxin conjugates by a disulfide interchange reaction. Modification of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol can be coupled to the cystamine-modified antibody to form disulfide crosslinks.

Figure 22.14 Intact liposomes containing PE components may be modified with SPDP to produce thiol-reactive derivatives.

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

The following sections describe the major activation and coupling methods used with dextran polymers. The reactive derivatives may be used to couple with proteins and other molecules containing the appropriate functional groups. 

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to create reactive derivatives that subsequently can be used to label antibodies or other targeting molecules at their amine groups. The aldehyde-HRP intermediate may be stored for extended periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting in precipitation and loss of activity. 

Reduction of the pyridyl disulfide end after SPDP modification releases the pyridine-2-thione leaving group and generates a terminal—SH group. This procedure allows sulfhydryl-reactive derivatives such as maleimide-activated enzymes (Chapter 26, Section 2.3) to be conjugated with DNA probes for use in hybridization assays (Malcolm and Nicolas, 1984). 

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