HOBt (anhydrous)

HOBt was proposed as an additive to DCC to reduce racemization. Today, majority of peptide drugs up to 13-15 amino acids are synthesized using solution approach, Boc chemistry and conventional DCC/ HOBt method

Studies on the metabolic clearance of ciclotropium to alpha-phenylciclopentylacetic acid using a new enantiospecific metabolite assay.[Pubmed:1492851]

Arzneimittelforschung. 1992 Nov;42(11):1354-8.

Ester hydrolysis represents an important biotransformation pathway for various parasympatholytic agents. Cleavage of the ciclotropium ester bond results in the formation of alpha-phenylciclopentylacetic acid (PCA). The relevance of this metabolic route for ciclotropium bromide (HIT-PCE, CAS 85166-20-7) including its stereochemical aspects was studied in a preliminary pharmacokinetic study. An enantiospecific assay for biological material was developed that is based on chiral derivatization of PCA with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) and the primary amine S-FLOPA, a chiral coupling component for carboxylic acids derived from S-flunoxaprofen, followed by HPLC resolution. R-(--)-Ibuprofen was used as internal standard. From plasma or urine PCA can be extracted into n-hexane/ethanol (9:1) at pH 4 under addition of sodium chloride. Derivatization with EDAC/FLOPA was performed under addition of 1-hydroxybenzotriazole in anhydrous dichloromethane that contained trace amounts of pyridine (ambient temperature; 2 h reaction time). The chromatographic separation was performed on a silica gel stationary phase (Zorbax Sil) using n-hexane-chloroform-ethanol (100:10:1, by vol.) as mobile phase (flow rate, 2 ml/min; fluorescence-detection, 305/355 nm; elution order of the derivatives, (-) before (+)). Limit of quantification was 1.0 ng/ml for plasma and 10 ng/ml for urine. In the pharmacokinetic study in two healthy volunteers who received a single i.v. dose of 10 mg ciclotropium race-mate the PCA concentrations in plasma were below the detection limit, but approx. 1.5% of the administered dose were excreted into urine as the respective glucuronides.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and catalytic property of surfactant-laccase complex in organic media.[Pubmed:10933832]

Biotechnol Prog. 2000 Jul-Aug;16(4):583-8.

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.

Synthesis and pharmacological properties of [1-L-penicillamine,4-L-leucine]oxytocin.[Pubmed:1159679]

J Med Chem. 1975 Oct;18(10):1020-2.

For the synthesis of [1-L-penicillamine,4-L-leucine]oxytocin (2), Z-Tyr(Bzl)-Ile-Leu-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 was treated with anhydrous HBr, and the resulting partially deprotected octapeptide was coupled with Z-penicillamine(Bzl) in a condensation reaction mediated by dicyclohexylcarbodiimide and 1-hydroxybenzotriazole. The protected nonapeptide Z-penicillamine(Bzl)-Tyr-Ile-Leu-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 was treated with Na in NH3 and the resulting disulfhydryl compound was subjected to oxidative cyclization in H2O-CH3OH with ICH2CH2I, Purification of 2 was effected by partition chromatography and gel filtration. The analog possesses antioxytocic and antiavian vasodepressor pA2 values of 6.77 and 7.21, respectively, and has no antipressor or anti-ADH activity. Its biological activity spectrum is qualitatively identical with that of [1-penicillamine]oxytocin. In contrast to the marked natriuretic-diuretic and anti-antidiuretic activity of [Leu4]oxytocin, 2 exhibits none of these effects on the rat kidney.

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids.[Pubmed:1446968]

Int J Pept Protein Res. 1992 Aug;40(2):114-8.

When L-proline or O-benzyl-trans-4-hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the C alpha of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.

Pharmacokinetic studies with the lipid-regulating agent beclobrate: enantiospecific assay for beclobric acid using a new fluorescent chiral coupling component (S-FLOPA).[Pubmed:2039683]

Chirality. 1991;3(1):35-42.

The major biotransformation pathway for the chiral lipid-regulating agent beclobrate is conversion to the corresponding carboxylic acid, which is then metabolized to the acyl glucuronide. An enantiospecific assay for biological material was developed that is based on chiral derivatization with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) and the primary amine S-FLOPA, a new chiral coupling component for carboxylic acids derived from the 2-arylpropionic acid S-flunoxaprofen. Conversion of beclobric acid to the acyl chloride prior to coupling with the amine is also feasible. From plasma or urine beclobric acid was extracted into n-hexane/ethanol (9:1) at pH 4 after addition of sodium chloride. Clofibric acid was used as internal standard. Derivatization with EDAC/FLOPA was performed under addition of 1-hydroxybenzotriazole in anhydrous dichloromethane containing trace amounts of pyridine (ambient temperature/2 h reaction time). The chromatographic separation was performed on a silica gel stationary phase (Zorbax Sil) using n-hexane-chloroform-ethanol (100:10:0.75, by vol) as mobile phase [flow rate, 2 ml/min; fluorescence detection, 305/355 nm; elution order of the derivatives, (-) before (+)]. Coefficients of variation were between 1.3 and 9.3% for both plasma and urine. Limit of quantification was 20-25 ng/ml for plasma based on a sample volume of 0.2 ml. Application of the assay in a pilot pharmacokinetic study showed significant differences between the kinetics of the two enantiomers. In plasma and urine, the concentrations of the dextrorotatory enantiomer exceeded those of the levorotatory enantiomer significantly.