Cyclohexane-l-carboxylate

The angularly condensed pyrimido[l,6- ]quinazoline 223 was prepared from [3+3] atom fragments by reacting 2,4,6-triaminopyrimidine and ethyl 2-oxo-cyclohexane-l-carboxylate under acidic conditions. The reaction also gave rise to the isomeric linear N-bridgehead tricycle, 224. The expected C-annelated product 225 was obtained under thermal conditions (Scheme 36) <1999JOC634>. 

Fig. 12 Illustration of the chemistry employed for preparation of a modified Si(lll) surface. Firstly, a layer of alkenyl acid is bound to the surface by covalent attachment. Secondly, a layer of polylysine is electrostatically bound to the carboxylic activated surface and finally the DNA is bound to the surface in a reaction mediated by sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate [27]...

Therefore, surface modification strategies for the formation of direct silicon-carbon bonds require, first, a special pre-treatment of the silicon surface to prevent oxidation and, second, an activation of the silicon surface for subsequent reaction with organic moieties. This has been achieved by treatment of the silicon surface with hydrofluoric acid to generate a hydrogen-terminated Si(lll) surface, which can further react with unsaturated co-functionahzed alkenes in the presence of UV irradiation or by thermal activation [27,44,45]. Using this method, carboxylic acid modified silicon substrates have been successfully generated and coupled to thiol modified ONDs via a polylysine/sulfosuccinimidyl 4-(M-maleimidomethyl)-cyclohexane-l-carboxylate couphng (Fig. 12). 

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) is a heterobifunctional cross-linker with significant utility in cross-linking proteins, particularly in the preparation of antibody-enzyme and hapten-carrier conjugates (Hashida and Ishikawa, 1985 Dewey et al., 1987). The NHS ester end of the reagent can react with primary amine groups on proteins to form stable amide bonds. The maleimide end of SMCC is specific for coupling to sulfhydryls when the reaction pH is in the range of 6.5-7.5 (Smyth et al., 1964) (Fig. 158). 

Preparation of intermediate 4-(4-chlorophenyl)cyclohexane-l-carboxylic acid was needed at first. It was made as follows acetyl chloride (30 g), aluminium chloride (60 g) in carbon disulfide (120 ml) were stirred at -50°C. Cyclohexen (30 g) previously cooled to -50°C was added dropwise during 10 minutes and the mixture was stirred for 60 minutes at -50°C. The solvent was decanted and 300 ml chlorobenzene was added, the so-obtained solution heated at 40°C for 3 hours with stirring, poured onto a mixture of ice and concentrated hydrochloric acid and the organic layer washed with 2 M HCI, 2 M NaOH and water, dried over anhydrous Na2S04. The product was distilled in vacuo, the fraction boiling at 140°-154°C (0.1 mm Hg) collected, diluted with an equal volume of petroleum ether, cooled to -6°C and a stream of nitrogen gas bubbled through. 

Heterobifunctional cross-linkers are employed to couple two different functional groups between the monolayer and the molecule for the subsequent immobilization. The wide range of these cross-linkers and their specific reactions are presented in Figure 14.7. One example in which the use of heterobifunctional cross-linkers was applied for immobilization of biomolecules was the work of Com and coworkers where the sulfosuccinimidyl 4-(fV-maleimidomethyl)cyclohexane-l-carboxylate (SSMCC) linker was applied to link thiol-modified DNA molecules with an amino-functionalized surface.54... 

Figure 11. Fixation of sweet and bitter compounds in rectangular coordinates— 1-amino-cyclohexane-l-carboxylic acid (sweet/bitter) p(—)p(—)a(- -x, y, z)...

A-succinimidyl-4-(iV-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC) and S-succinimidyl-S-acetyl-thioacetate (SATA) are marketed by various companies. 

By using a multistep procedure, DNA molecules have been covalently attached to SWNTs. First, the purified SWNTs were oxidized to form carboxylic acid groups at the ends and sidewalls, followed by reaction with thionyl chloride and ethylenediamine to produce amine-terminated sites. The amines were then reacted with the heterobifunctional cross-linker succinimi-dyl 4-(iV-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), leaving the surface terminated with maleimide groups. Finally, thiol-terminated DNA reacted with these groups to produce DNA-modified SWNTs [161]. AAHien DNA is covalently attached to SWNTs, a better stability, accessibility and selectivity are expected during competitive hybridization. 

Figure 13. (a) Formation of mixed SAM on Au electrode, (b) immobilization of Fc-D, (c) immobilization of thiolated capture probe with bifunctional linker (succinimidyl 4-(/V-maleimidomethyl)cyclohexane-l-carboxylate (SMCC)), (d) hybridization with target, (e) hybridization with biotinylated detection probe, (f) association with avidin-alkaline phosphatase, (g) description of the process of the electrocatalytic reaction of />-aminophenol (p-AP) via electronic mediation of ferrocenyl dendrimer (Adapted from Ref. [160])... 

IJ.2.3.2.1.2. 4-(N-Maleimidomethyl)-cyclohexane-l-carboxylic acid N-hydroxysuccinimide ester (CHM-NHS) a stable maleimide crosslinker. Ishikawa et al. (1978) synthesized alternative maleimide compounds of which CHM-NHS was particularly stable (decomposition rate less than 4% in 2 h at 30°C at pH 7.0, compared to 53% for OPDM and MBS. This cross-linker has been adopted successfully for the conjugation of GOase, APase, and POase (Yoshitake et... 

Fig. 11.13. Conjugation of enzyme (1) to thiolated antibody (2) by 4- /V-maleimi-domethyl)-cyclohexane-l-carboxylic acid A-hydroxysuccinimide ester.

Conjugation of enzymes to Fab or IgG using the A -hydroxysuccinimide ester of 4-(A -maleimidomethyl)cyclohexane-l-carboxylic acid (CHM-NHS)... 

The method developed by Bignami et al. is based on the generation of monse and rabbit monoclonal and polyclonal antibodies against conjugates of keyhole limpet hemocyanin and chemically dehned palytoxin haptens. Palytoxin haptens were prodnced by derivatization of the primary amino gronp with snlfosnccinimidyl 4-(A-maleimidomethyl)cyclohexane-l-carboxylate or snccinimidyl... 

Methyl l-bromomethyl-2-oxo-cyclopentanoate (235 mg, 1 mmol), 349 mg silylated cyclohexadiene (1.3 mmol), and 50 mg AIBN (0.3 mmol) were dissolved in 5 mL hexane under argon. The reaction mixture was refluxed for 7 h. Removal of the solvent in vacuo and purification by flash chromatography (Et20/pentane, 3 8) afforded 76 mg methyl 3-oxo-cyclohexane-l-carboxylate as a colorless oil, in a yield of 49%. 

Indium powder (2.08 mmol) was added to a refluxing solution of 0.4 mmol a-iodomethyl cyclic j8-keto ester in a mixed solvent of 2 mL tert-amyl alcohol and 1 mL water under an argon atmosphere. The mixture was refluxed for 2 h under stirring. After the reaction, the mixture was Altered through Celite, then the solvent was removed, and the residue was purified by siUca gel colunm chromatography to afford 59% methyl 3-oxo-cyclohexane-l-carboxylate as a colorless oil. 

Centricon 30 filtration units (Amicon) or Filtron Microsep 30 (Filtron) PDIO gel filtration medium (Pharmacia) 5(6)-carboxyfluoroscein-N-hydroxysuccinimide ester Boehringer cat. 10055089 stock solution, 10 mg/ml in DMSO Sulfo SMCC [sulfosuccinimidyl 4-(/V-maleimidomethyl)cyclohexane-l-carboxylate]. Pierce Chemical Company (cat. 22322), 20 mg/ml stock solution in 50 mM borate buffer, pH 7.6, freshly prepared... 

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