Carbodiimide chromatography

Liquid chromatography/mass spectrometry Lower limit of detection Limit of detection Limit of quantitation Florseshoe crab hemocyanin Liquid scintillation counting Matrix-assisted laser desorption/ ionization mass spectrometry m -Maleimidobenzoy 1-A -Hydroxysuccinimide 1 -Cyclohexyl-3-(2-Morptiolino-ethyl)carbodiimide rnetlio-/ -Toluenesulfonate (same as CDI)... 

Figure 9 A synthetic mixture of water-soluble carboxylic acids separated by anion-exchange chromatography. Column 0.3 cm x 300 cm Diaoion CA 08, 16-20 p (Mitsubishi Kasei Kogyo). Eluant 200 mM HC1. Detection reaction with Fe3-benzohy-droxamic acid-dicyclohexy carbodiimide-hydroxylamine perchlorate-triethyl amine with absorbance at 536 nm. Analytes (1) aspartate, (2) gluconate, (3) glucuronate, (4) pyroglutamate, (5) lactate, (6) acetate, (7) tartrate, (8) malate, (9) citrate, (10) succinate, (11) isocitrate, (12) w-butyrate, (13) a-ketoglutarate. (Reprinted with permission from Kasai, Y., Tanimura, T., and Tamura, Z., Anal. Chem., 49, 655, 1977. 1977 Analytical Chemistry).

CaM was purified from porcine brain. The purity of proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. CaM and PDE were cross-linked with l-ethyl-3(3-dimethylamino-propyl) carbodiimide (EDC) or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in a buffer solution of 0.1 M HEPES (pH 7.1) in the presence of 1 mM CaCl2. After a buffer solution containing 2 mM EGTA added into the reaction solution, the CaM-PDE hybrid was separated from other ingredients by gel chromatography on a Sepharose CL-6B solumn. 

The aroyl chloride (10 mmol) in PhMe (10 ml) is added dropwise to the carbodiimide (10 mmol), NaN (975 mg, 15 mmol) and TBA-Cl (0.28 g, l mmol) in PhMe (40 ml) at room temperature under N2. After ca. 3-4 h, the mixture is heated to 95 °C for 30 min. The cooled mixture is filtered and evaporated under reduced pressure to yield the tetra-zole, which is purified by chromatography from silica [e.g. 64% from PhN=C=NPh and PhCOCl 55% from TolN=C=NTol and PhCOCl 70% from PhN=C=NPh and TolCOCI],... 

Two equivalents of the tertiary amine base are required, and a significant improvement in the diastereoselectivity was observed with TMEDA over DIPEA. Purification and further enrichment of the desired RRR isomer to >98% ee was achieved by crystallization. Oxidative removal of the chiral auxiliary followed by carbodiimide mediated amide formation provides (3-keto carboxamide 14 in good yield. Activation of the benzylic hydroxyl via PPha/DEAD, acylation, or phosphorylation, effects 2-azetidinone ring-closure with inversion of stereochemistry at the C4 position. Unfortunately, final purification could not be effected by crystallization and the side products and or residual reagents could only be removed by careful chromatography on silica. 

The step 1 product was diluted with 1500 ml toluene to a final concentration of 0.013 mol/L. The mixture was then treated with 1,3-dimethyl-3-phospholene oxide and heated to 90°C while the cyclization reaction was monitored by Fouier transform infrared (FTIR). When the characteristic—NCO peak was eliminated in the macrocyclic carbodiimide (2134 cm-1), the reaction was stopped, concentrated, dried, and the crude product isolated. The crude product was purified by liquid column chromatography on silica gel using either ethyl acetate or ethyl acetate/ -hexane, 9 1, respectively, and the product isolated. 

A reactor charged with 4-ethynylbenzoic acid (2.01 mmol) dissolved in dimethylacetamide was treated with 1-hydroxybenzotriazole (2.01 mmol) and /V.V -dicyclohexyl-carbodiimide (2.07 mmol) and stirred for 2 hours at ambient temperature. The mixture was then treated with (S)-(+)-decylalaninate (2.13 mmol) and stirred at ambient temperature for 3 hours and then at 120°C for a further 3 hours. The reaction was filtered, concentrated, and the residue purified by a silica gel chromatography using chloroform and ethyl acetate. After recrystallization from hexane 0.33 g of product was isolated as a crystalline solid. 

The solvent is removed at the rotary evaporator, and the resulting residue is purified by chromatography. It can be advisable to filter the precipitate of N, iV -dicyclohexylurea8—formed when DCC is used—before removing the solvent. In order to avoid interferences from unreacting carbodii-mide, it can be advisable to transform it in the corresponding urea by careful addition of oxalic acid either solid or in a solution in methanol—to the stirred reaction mixture. Addition of oxalic acid produces a copious evolution of gas that signals the duration of the hydrolysis of the carbodiimide. 

The above obtained [(4S)-4-Methyl-5-oxo-2-(tribromomethyl)-l,3-dioxolan-4-yl]acetic acid (102.5 mg 0.250 mmol) and 2-mercaptopyridine- N-oxide (34.4 mg 0.280 mmol) were suspended in CBrCI3 (1.5ml). The reaction mixture was heated to reflux and a solution of dicyclohexyl carbodiimide (DCC) (103 mg 0.500 mmol) in CBrCI3 (1.0 ml) was added slowly over the course of 30 min. The reaction mixture was stirred for an additional hour. The product was purified by silica gel chromatography (CH2CI2/hexanes (1 2)) and was obtained as white needles from the same solvents. Yield 72 mg (65%) mp 110-113°C. 

Nitroindole-2-carboxylic acid (0.86 g), l-[3-(N-isopropyl)amino-2-pyridinyl]piperazine (0.43 g), l-(ethyl)-3-(dimethylaminopropyl)carbodiimide (0.45 g) and THF (4 ml), were stirred at 20-25°C for 3 hr then the reaction mixture was dissolved in chloroform (50 ml) and extracted with saturated aqueous sodium bicarbonate, saline, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by flash column chromatography (200 g silica) eluting with ethyl acetate/hexane (50/50), the appropriate fractions were pooled and concentrated to give l-[5-nitroindolyl-2-carbonyl]-4-[3-(l-methylethylamino)-2-pyridinyl]piperazine, mp 153°-154°C. 

The reactions of isoselenocyanates with carbodiimides in refluxing hexane afford l,3-selenazetidine-2,4-diimines 33 by a [2+2] cycloaddition in moderate to good yields (Scheme 12 and Table 4) <2005HCA766>. These compounds can be purified by silica gel chromatography and recrystallization. All products 33 are stable and can be stored at room temperature. 

The separation of this dicyclohexylurea urea is relatively laborious, that is, it has to be conducted by chromatography or crystallization. After the activation of carboxylic acid with carbodiimide B and subsequent acylation of a heteroatom nucleophile, one certainly obtains a urea as a side product, too. It has the structure C and thus is an amine, the separation of which can, however, be accomplished easily by extraction with aqueous hydrochloric acid. 

Urea A is the starting material for preparing the carbodiimide C, which activates carboxylic acids according to the same mechanism and for the same reason as DCC, with which you are already familiar (Figures 6.15 and 6.26). If the carbodiimide C from Figure 7.5 were not so much more expensive than DCC, everybody would use the former instead of the latter for carboxylic acid activation. There is a practical reason for this. When a heteroatom nucleophile is acylated with the DCC adduct of a carboxylic acid, besides the desired carboxylic acid derivative one obtains dicyclohexyl urea (formula B in Figure 7.5). This (stoichiometric) by-product must be separated from the acylation product, which is relatively laborious when realized by chromatography or by crystallization. When a carboxylic add has been activated with the carbodiimide C and the subsequent acylation of a heteroatom nucleophile has been effected, one also obtains a urea as a stoichiometric by-product. It has the structure D and is therefore... 

Purify the crude reaction mixture by column chromatography on silica gel, eluting with 50% diethyl ether in hexane to give the carbodiimide (223 mg, 64%) as a pale yellow solid. 

The product from Step 2 (220 mg) was mixed with (2R)-2-((lR)-3,3-difluorocyclopentyl)-2-hydroxy-2-phenylacetic acid (256 mg) dissolved in 8 ml chloroform, 1-hydroxybenzotriazole (203 mg) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (201 mg) added, and the mixture stirred 3 hours at ambient temperature. Thereafter, the mixture was diluted with EtOAc, washed with 1 M NaOH, brine, dried, and concentrated. The material was purified by chromatography on silica gel using hexane/EtOAc, 2 1, and 314 mg product isolated. 

Carbodiimides have chiral structures similar to allenes, i.e., they can exist in optically active forms. Schloegl and Mechtler were the first to report a partial optical separation of N,N"-diferrocenylcarbodiimide into enantiomers by chromatography on acetylated cellulose, but other authors doubt the validity of these results. According to theoretical calculations a separation of carbodiimide enantiomers is not possible. N,N -diferrocenylcarbodiimide was also obtained in optically active form by kinetic resolution in the reaction with (-)-S-6,6"-dinitrodiphenic acid. Cervinka and coworkers isolated both enantiomers of (R,S)-N,N"-bis(o -phenylethyl)carbodiimide, and they found that they undergo racemization at room temperature. A recent study on the racemization mechanism of macrocyclic carbodiimides indicates that the open chain as well as the large ring carbodiimides racemize by nitrogen inversion or tra 5-rotation, while medium size cyclic carbodiimides racemize by cw-rotation. ... 

Af-(D-Biotinyl)-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl)-L-threonine te/t-Butyl Ester 23 [34], A mixture of D-biotin (150 mg, 0.6 mmol), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC 580 mg, 3 mmol), and 1-hydroxy-benzotriazole (HOBt 540 mg, 4 mmol) in dimethylformamide (DMF 2 mL) is stirred under exclusion of moisture at 22 C. After 45 min, the biotin is dissolved, and a solution of freshly prepared glycosyl threonine ester 22 (0.3 mmol, preceding procedure) in dichloro-methane (2 mL) is added at 0°C. After stirring for 16 h at room temperature, the solvent is evaporated in vacuo, the remainder dissolved in dichloromethane (50 mL), extracted with ice-cold 0.2 N HCl (3 x 25 mL), water (25 mL), and saturated NaHCOj solution (2 x 25 mL), dried with MgSO, and concentrated in vacuo. Purification by flash chromatography on silica gel (20 g) in dichloromethane-ethanol (25 1) yields 23 200 mg (93%) [a] 96.5° (c 1, CHClj) Rf 0.29 (toluene-acetone 4 1). 

Carboxyl group reduction of the CPS and LPS samples was performed as previously described.55 Briefly, LPS (10 mg) was dissolved in distilled water (10 ml) and following the addition of l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonare (113 mg), the stirred mixture was maintained at pH 4.7 by titration with 0.1 moll-1 HC1 for 3 h. Following completion of the reaction a 2 moll-1 solution of sodium borohydride (12.5 ml) was added slowly and the reaction mixture was maintained at pH 7 by titration with 4moll-1 HC1. The reaction was allowed to proceed for 2 h at 22 °C, and the solution was dialyzed and lyophilized. The product was purified by gel permeation chromatography on Sephadex G-100 and lyophilized (yield 6 mg). 

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