Friedrich Hammerschmidt

Enzymes in organic chemistry, part 1: Enantioselective hydrolysis of α-(acyloxy)phosphonates by esterolytic enzymes

Abstract α-Hydroxyphosphonates (±)- 3 were prepared and transformed into esters (±)- 5 . Eight lipases as well as pig liver esterase were tested as catalysts for enantioselective hydrolyses of α-(acyloxy)phosphonates in a biphasic system. Two of them proved to be useful. The highest enantioselectivity was obtained with lipase F-AP 15 and α-(acetyloxy)phenylmethylphosphonates(±)- 5a and (±)- 5b as substrates. The (S)-enantiomers were exclusively hydrolyzed to give optically pure alcohols (S)-(−)- 3a and (S)-(−)- 3b . Lipases AP 6 and F-AP 15 were used to prepare phosphonates (S)-(−)- 3b , (S)-(+)- 3d and (S)-(−)- 3e on a preparative scale with an enantiomeric excess of 81%, 87%, and 89%, respectively. The absolute configurations ofthe α-hydroxyphosphonates were assigned by Horeaus method and 1 H-NMR spectroscopy of Moshers derivatives.

Determination of absolute configuration of α-hydroxyphosphonates by 31P NMR spectroscopy of corresponding mosher esters

Abstract ( R )-MTPA esters of α-hydroxyphosphonates 1a-h of known absolute configuration were prepared and their 31 P NMR spectra were recorded. The chemical shifts of the 31 P signals of the derivatized ( S )-alcohols are consistently downfield relative to the signals of the corresponding ( R )-alcohols. A variety of racemic and diastereomeric α-hydroxyphosphonates 1i-o and their ( R )-MTPA esters were prepared. The 31 P chemical shift differences are large enough to securely assign absolute configurations to α-hydroxyphosphonates.

Enzymes in organic chemistry, part 2: Lipase-catalysed hydrolysis of 1-acyloxy-2-arylethylphosphonates and synthesis of phosphonic acid analogues of L-phenylalanine and L-tyrosine☆

Abstract α-Hydroxyphosphonates (±)-3, prepared by base catalysed addition of phosphites 2 to aldehydes 1, were acylated to give esters (±)-4. Diethyl 1-acyloxy-2-arylethylphosphonates (±)-4a, 4b, and 4e were hydrolysed by lipase from Aspergillus niger in a biphasic system to afford (R)-α-hydroxyphosphonates of low enantiomeric purity. The corresponding diisopropyl phosphonates (±)-4c, 4f and 4g gave (S)-α-hydroxyphosphonates with an ee of up to 78%. The absolute configuration of the α-hydroxyphosphonates was assigned by 31P NMR spectroscopy of their (R)-MTPA-esters. (S)- 3 b and 3e were chemically transformed via their azides to phosphonic acid analogues of L-phenylalanine and L-tyrosine, respectively.

Direct liquid chromatographic enantioseparation of chiral α- and β-aminophosphonic acids employing quinine-derived chiral anion exchangers: determination of enantiomeric excess and verification of absolute configuration

Four types of quinine-derived chiral anion exchangers were used for the direct liquid chromatographic separation of enantiomers of chiral but N-protected α- and β-aminophosphonic acids. The respective and easily prepared 3,5-dinitrobenzyloxycarbonyl and dinitrophenyl derivatives were well separated, yielding α-values between 1.1 and 2.8 and resolution values in the range 2.0–9.0. Besides the chromatographic investigations, data on enantiomeric excesses (ees) and the absolute configurations of some enantiomerically enriched α- and β-aminophosphonic acids prepared by stereoselective synthesis were also determined. The results obtained by this method (ee and absolute configuration) correspond well with the results previously obtained by a 31P-NMR spectroscopic method.

Enzymes in organic chemistry. Part 9:Chemo-enzymatic synthesis of phosphonic acid analogues of l-valine, l-leucine, l-isoleucine, l-methionine and l-α-aminobutyric acid of high enantiomeric excess

Abstract Diisopropyl α-chloroacetoxyphosphonates derived from propanal, isobutanal, 3-methylmercaptopropanal, 3-methylbutanal and ( S )-2-methylbutanal were resolved by enzyme-catalysed hydrolysis. Lipases preferentially hydrolysed the ( S )-esters and protease Chirazyme ® P-2 the ( R )-esters. Replacing the isopropyl groups by t -butyl groups reduced the reaction rate; replacing them by a 2,2-dimethylpropane-1,3-diyl group increased the reaction rate. ( S )-α-Hydroxyphosphonates (ee 92–99%), obtained with the protease except one, were transformed into phosphonic acid analogues of l -valine, l -leucine, l -isoleucine, l -methionine and l -α-aminobutyric acid. Their enantiomeric purity was determined by HPLC on a chiral stationary phase after derivatisation at nitrogen.

Enzymes in organic chemistry. Part 10: Chemo-enzymatic synthesis of l-phosphaserine and l-phosphaisoserine and enantioseparation of amino-hydroxyethylphosphonic acids by non-aqueous capillary electrophoresis with quinine carbamate as chiral ion pair agent

Abstract Diisopropyl 2-azido-1-acetoxyethylphosphonate (±)- 7 was hydrolysed with high enantioselectivity by lipase SP 524 to give α-hydroxyphosphonate ( S )-(−)- 6 and ester ( R )-(−)- 7 , which was saponified to give ( R )-(+)- 6 . The two α-hydroxyphosphonates ( R )- and ( S )- 6 were transformed into l-phosphaisoserine and l -phosphaserine, respectively. Their enantiomeric excesses were determined to be 97% by HPLC on an chiral stationary phase. A mixture of all four stereoisomeric amino-hydroxyethylphosphonic acids can be separated by non-aqueous capillary electrophoresis with quinine carbamate as the chiral ion pair agent applying the partial filling technique.

Preparation and configurational stability of chiral chloro-[D1]methyllithiums of 98% enantiomeric excess.

Enantiopure stannyl-[D1]methanol was converted to chloro-[D1]methylstannane under complete inversion of configuration using Ph3P/N-chlorosuccinimide in THF. It was transmetalated to stereospecifically give chloro-[D1]methyllithium (ee up to 98%). Its microscopic configurational stability was tested by performing tin-lithium exchange in the presence of benzaldehyde as the electrophile under various conditions. The macroscopic configurational stability was addressed by using the same electrophile but by adding it 30 s after the addition of MeLi used for transmetalation. Chloro-[D1]methyllithium is chemically very labile, however completely configurationally stable on both time scales up to the temperature of rapid decomposition (-78 degrees C).

New Selective Inhibitors of Steroid 11β-Hydroxylation in the Adrenal Cortex. Synthesis and Structure–Activity Relationship of Potent Etomidate Analogues

Derivatives of etomidate were evaluated as inhibitors of adrenal steroid 11beta-hydroxylations. Stereoselective coupling by Mitsunobu produced chirally pure analogues to study the effect of configuration, modification of the ester, and substitution in the phenyl ring, with the aim to probe specific sites for introducing a radionuclide. Iodophenyl metomidate (IMTO) labeled with iodine-131 served as radioligand for structure-affinity relationship studies. We have characterized the kinetic parameters of specific (131)I-IMTO binding on rat adrenal membranes and used the displacement of (131)I-IMTO binding to evaluate functionalized MTO analogues. Our results indicated that (1) ( R)-configuration is essential for high affinity, (2) highest potency resides in the ethyl, 2-propyl, and 2-fluoroethyl esters, and (3) substitution of the phenyl ring is well tolerated. The clinically used inhibitors metyrapone and ketoconazole inhibited (131)I-IMTO binding with low affinity. Incubation of selected analogues with human adrenocortical NCI-h295 cells demonstrated a high correlation with the inhibitory effect on cortisol secretion.

Low‐molecular‐weight chiral cation exchangers: Novel chiral stationary phases and their application for enantioseparation of chiral bases by nonaqueous capillary electrochromatography

Cation exchange type chiral stationary phases (CSPs) based on 3,5‐dichlorobenzoyl amino acid and amino phosphonic acid derivatives as chiral selectors (SOs) and silica as chromatographic support were developed and applied to enantiomer separations of chiral bases by nonaqueous capillary electrochromatography (NA‐CEC). As a rationale for efficient CSP development we adopted the combined use of the “reciprocity principle of chiral recognition” and nonaqueous ion‐pair CE as screening assay. Thus, (S)‐atenolol was employed as chiral counter‐ion added to the BGE in CE and a series of N‐derivatized amino acids and amino phosphonic acids were screened to derive reciprocally information on their chiral recognition abilities for atenolol enantiomers. Two SO candidates, namely N‐(3,5‐dichlorobenzoyl)‐O‐allyl‐tyrosine and N‐(4‐allyloxy‐3,5‐dichlorobenzoyl)‐1‐amino‐3‐methylbutane phosphonic acid that have been identified as potential SOs in the CE screening were, after immobilization on thiol‐modified silica, evaluated in cation‐exchange NA‐CEC. The strong chiral cation exchanger with the free phosphonic acid group exhibited enhanced enantioselectivity compared to the weak chiral cation exchanger with the carboxylic acid group. A wide variety of chiral bases could be successfully resolved on the strong chiral cation exchanger with α‐values up to 2.2 and efficiencies up to 375 000 m–1 including β‐blockers and other amino alcohols, local anesthetics like etidocaine, antimalarial agents like mefloquine, Tröger’s base, phenothiazines like promethazine, and antihistaminics. The influence of several experimental parameters (electrolyte concentration, acid‐base ratio and acetonitrile‐methanol ratio) was evaluated *.

Aspects of 6-[18F]fluoro-L-DOPA preparation: precursor synthesis, preparative HPLC purification and determination of radiochemical purity

A modified method for the synthesis of the intermediate product N-Boc-3,4-di(Boc-O)-6-iodo-L-phenylalanine ethyl ester of the [18F]FDOPA precursor preparation was developed. With the application of bis-(trifluoroacetoxy)-iodobenzene for the iodination step with elemental iodine the yield of the intermediate can be increased from 12% to 50-60%. By replacing silica-gel-based RP HPLC column by a polymer-based column for semi-preparative purification of [18F]FDOPA from the reaction mixture the radiochemical purity of the final product can be increased up to >99%. For the determination of the radiochemical impurity [18F]fluoride a HPLC method using a column with polymer-based RP material was introduced.