Enzymatic synthesis of l‐tryptophan and 5prime‐hydroxy‐l‐tryptophan labeled with deuterium and tritium at the alpha‐carbon position

The enzymatic synthesis of l‐tryptophan and its derivative 5′‐hydroxy‐l‐tryptophan labeled with deuterium and tritium at the α‐carbon position is reported. The mixture containing S‐methyl‐l‐cysteine, indole or 5‐hydroxyindole dissolved in deuteriated or tritiated water has been converted to [2‐²H]‐l‐tryptophan, [2‐³H]‐l‐tryptophan, 5′‐hydroxy‐[2‐²H]‐l‐tryptophan, and 5′‐hydroxy‐[2‐³H]‐l‐tryptophan, respectively, in a one‐pot reaction using the enzyme tryptophanase. The same reaction carried out in heavy water with THO added yielded either doubly labeled [2‐²H/³H]‐l‐tryptophan or 5′‐hydroxy‐[2‐²H/³H]‐l‐tryptophan. Copyright © 2003 John Wiley & Sons, Ltd.     

Simple single‐step single‐enzyme synthesis of [14C]‐GSH

The tri‐peptide [¹⁴C]‐glutathione ([¹⁴C]‐GSH) was synthesized in a single step by GSH synthetase catalyzed reaction of L ‐γ‐glutamyl‐L ‐cysteine and [¹⁴C]‐glycine. Preparative reverse phase HPLC afforded [¹⁴C]‐GSH in 30% yield and 98% purity. Preparation of GSH synthetase from E. coli via recombinant DNA and the interconversion of [¹⁴C]‐GSH to the disulfide [¹⁴C]‐GSSG for storage are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Syntheses of halogen derivatives of L‐tryptophan,L‐tyrosine and L‐phenylalanine labeled with hydrogen isotopes

Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L ‐tryptophan, i.e. 5′‐bromo‐[2‐³H]‐, 5′‐bromo‐[2‐²H/³H]‐, 5′‐fluoro‐[2‐³H]‐5′‐fluoro‐[2‐²H/³H]‐, 6′‐fluoro‐[2‐³H]‐, 6′‐fluoro‐[2‐²H/³H]‐L ‐tryptophan, as well as, L ‐tyrosine, i.e. 3′‐fluoro‐[2‐³H]‐, 3′‐fluoro‐[2‐²H/³H]‐, 3′‐chloro‐[2‐³H]‐, and 3′‐chloro‐[2‐²H/³H]‐L ‐tyrosine, and also L ‐phenylalanine, i.e. 2′‐fluoro‐[(3S)‐³H]‐, 2′‐fluoro‐[(3S)‐²H/³H]‐, 2′‐chloro‐[(3S)‐³H]‐, 2′‐chloro‐[(3S)‐²H/³H]‐, 4′‐chloro‐[(3S)‐³H]‐, and 4′‐chloro‐[(3S)‐²H/³H]‐L ‐phenylalanine were synthesized using enzymatic methods. Isotopomers of L ‐tryptophan were synthesized by coupling of halogenated indoles with S‐methyl‐L ‐cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L ‐tyrosine were obtained by the enzymatically supported exchange between halogenated L ‐tyrosine and isotopic water. Labeled halogenated isotopologues of L ‐Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D₂O) with addition of HTO were used.     

Thirteen week toxicity study of dietary l‐tryptophan in rats with a recovery period of 5 weeks

Although l ‐tryptophan is nutritionally important and widely used in medical applications, toxicity data for its oral administration are limited. The purpose of this study was to evaluate the potential toxicity of an experimental diet containing added l ‐tryptophan at doses of 0 (basal diet), 1.25%, 2.5% and 5.0% when administered to Sprague–Dawley rats for 13 weeks. There were no toxicological changes in clinical signs, ophthalmology, urinalysis, hematology, necropsy, organ weight and histopathology between control rats and those fed additional l ‐tryptophan. Body weight gain and food consumption significantly decreased throughout the administration period in males in the 2.5% group and in both sexes in the 5.0% group. At the end of the dosing period, decreases in water intake in males in the 5.0% group and in serum glucose in females in the 5.0% group were observed. The changes described above were considered toxicologically significant; however, they were not observed after a 5 week recovery period, suggesting reversibility. Consequently, the no‐observed‐adverse‐effect level of l ‐tryptophan in the present study was 1.25% for males and 2.5% for females (mean intake of l ‐tryptophan: 779 mg kg^–1 body weight day^–1 [males] and 1765 mg kg^–1 body weight day^–1 [females]). As the basal diet used in this study contained 0.27% of proteinaceous l ‐tryptophan, the no‐observed‐adverse‐effect level of overall l ‐tryptophan was 1.52% for males and 2.77% for females (mean intake of overall l ‐tryptophan: 948 mg kg^–1 body weight day^–1 (males) and 1956 mg kg^–1 body weight day^–1 (females)). We conclude that l ‐tryptophan has a low toxicity profile in terms of human use.     

Design and synthesis of enantiopure 18F‐labelled [18F]trifluoromethyltryptophan from 2‐halotryptophan derivatives via copper(I)‐mediated [18F]trifluoromethylation and evaluation of its in vitro characterization for the serotonergic system imaging

We synthesized [¹⁸F]trifluoromethyl‐l ‐tryptophan ([¹⁸F]CF₃‐l ‐Trp) using Cu(I)‐mediated [¹⁸F]trifluoromethylation to image serotonergic system. Radiochemical yield was 6 ± 1.5% (n = 9), and radiochemical purity was over 99%. The molar activity was 0.44 to 0.76 GBq/μmol. [¹⁸F]CF₃‐l ‐Trp was stable for up to 6 hours in mouse and human sera at 37°C. Protein‐binding was 0.26 ± 0.03% and 0.34 ± 0.02% in human and mouse serum at 60 minutes, respectively. In conclusion, enantiopure [¹⁸F]CF₃‐l ‐Trp was synthesized as a feasible imaging agent for the serotonergic system.     

An asymmetric synthesis of l‐[3‐13C]alanine

l ‐[3‐¹³C]Alanine was synthesized from [¹³C]methyl iodide by using Dellaria's oxazinone, prepared from phenyl[2‐¹³C]bromoacetate and (S)‐2‐phenylglycinol, as a chiral glycine equivalent. Alkylation of the oxazinone with [¹³C]methyl iodide was achieved with high diastereoselectivity. Hydrolysis and removal of the chiral auxiliary of the alkylated oxazinone gave l ‐[3‐¹³C]alanine. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of (2‐mercaptoacetyl)‐L‐[2‐14C]tryptophan as a selective metallo‐β‐lactamase inhibitor via [2‐14C]indole based on chiral pool strategy

Metallo‐beta‐lactamase enzymes make bacteria resistant to a broad range of commonly used beta‐lactam antibiotics. Several thiol derivatives of L‐amino acids have been shown their inhibitory effects against the metallo‐β‐lactamase IMP‐1. In this study, (2‐mercaptoacetyl)‐L‐tryptophan as a new inhibitor of metallo‐β‐lactamases labeled with carbon‐14 in the 2‐position of the indole ring was prepared from [2‐¹⁴C]indole as a key synthetic intermediate based on chiral pool strategy. The overall synthesis was performed in 10 steps with the overall radiochemical yield 3.6% on the basis of the barium [¹⁴C]carbonate as a starting material.     

Asymmetric synthesis of L‐[4‐13C]glutamic acid and glutamine

L ‐[4‐^l3C]Glutamic acid ( 1 ) and L ‐[4‐¹³C]glutamine ( 2 ) were synthesized from sodium [2‐¹³C]acetate ( 5 ) and Dellaria's oxazinone 3 as a chiral glycine equivalent. Sodium [2‐¹³C]acetate ( 5 ) was converted to [2‐¹³C]acrylate 4 . Diastereoselective Michael addition of the enolate of 3 to the acrylate 4 proceeded with high diastereoselectivity to give the adduct 12 . Reductive cleavage of the C–S bond, ethanolysis, hydrogenolysis and hydrolysis gave L ‐[4‐¹³C]glutamic acid ( 1 ). L ‐[4‐¹³C]Glutamine ( 2 ) was synthesized from 1 in 4 steps. Copyright © 2006 John Wiley & Sons, Ltd.     

Partial [αMe]Aun scan of [l‐Leu11‐OMe]‐trichogin GA IV,a membrane active synthetic precursor of the natural lipopeptaibol

Abstract: We synthesized using solution‐phase methods three analogs of [l ‐Leu¹¹‐OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N‐terminal n‐octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic C^α,α‐disubstituted glycine l ‐(αMe)Aun, respectively [partial (αMe)Aun scan]. FT‐IR absorption and CD analyses unequivocally show that the main three‐dimensional structural features of [l ‐Leu¹¹‐OMe] trichogin GA IV are preserved in the analogs. Also, [l ‐Leu¹¹‐OMe] trichogin GA IV and the three N^α‐acetylated l ‐(αMe)Aun analogs exhibit strictly comparable membrane‐modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the N^α‐blocking group to the side‐chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l ‐Leu¹¹‐OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.     

A simple convenient synthesis of l‐[4‐13C]glutamine

l ‐[4‐¹³C]Glutamine was synthesized from sodium [2‐¹³C]acetate in 12 steps and 18% overall yield. A Wittig reaction of (R)‐benzyl 4‐formyl‐2,2‐dimethyloxazolidine‐3‐carboxylate and ethyl 2‐(triphenylphosphoranylidene)[2‐¹³C]acetate prepared from d ‐serine and sodium [2‐¹³C]acetate, respectively, gave (4S)‐4‐(2‐ethoxycarbonyl[2‐¹³C]vinyl)‐2,2‐dimethyloxazolidine‐3‐carboxylic acid α,β‐isopropylidene group, oxidation of the resulting hydroxyl group to a carboxyl group and transamidation of the ester moiety gave l ‐N‐Cbz‐[4‐¹³C]glutamine (Cbz = benzyloxycarbonyl). Finally, removal of the Cbz group gave l ‐[4‐¹³C]glutamine. l ‐[4‐¹³C]Glutamine can be prepared in fewer steps and higher yield by this method compared with previously reported methods.     

A rapid microwave assisted synthesis of [U‐14C]isosorbide and dimethyl[U‐14C]isosorbide from D‐[U‐14C]glucose

[U‐¹⁴C]Isosorbide and [U‐¹⁴C]dimethyl isosorbide with a specific activity of 462 MBq/mmol was prepared from D ‐[U‐¹⁴C]glucose, in an overall yield of 79%, under microwave heating conditions. Copyright © 2006 John Wiley & Sons, Ltd.     

[125I], [127I]‐and [14C]‐Labelling of the GLP‐1‐(7‐37) derivative NN2211

Arg³⁴Lys²⁶(N^ε‐(γ‐L ‐glutamyl(N^α‐palmitoyl)))‐GLP‐1(7‐37) (NN2211) is currently in development as a diabetes type 2 drug. The fatty acid attached to the GLP‐1(7‐37) ensures a long and controlled duration of action. The synthesis of [¹²⁵I]NN2211, [¹²⁷I]NN2211 and [¹⁴C]NN2211 used for preclinical ADME studies are described. NN2211 was iodinated using the lactoperoxidase/hydrogen peroxide method, and [¹⁴C]NN2211 was synthesized in 4 steps by two routes both starting from an α‐protected [U‐¹⁴C]glutamic acid. Copyright © 2003 John Wiley & Sons, Ltd.     

A short synthesis of d‐[1‐14C]‐serine of high enantiomeric purity

Herein, we report a short, three‐step synthesis of d ‐[1‐¹⁴C]‐serine (4) in high enantiomeric purity. Starting from [¹⁴C]‐KCN and 2‐(benzyloxy)acetaldehyde, Strecker reaction using (R)‐1‐phenylethylamine as the chiral auxiliary gave two diastereomeric aminonitriles 1 and 2 in the ratio of 4:3, which were conveniently separated and purified chromatographically. Following hydrolysis and subsequent hydrogenolysis, the purified major diastereomer 1, was smoothly converted to d ‐[1‐¹⁴C]‐serine (4) in an enantiomeric excess of >99%, thus circumventing time intensive chiral HPLC enantiomeric resolution.     

Synthesis of [14C]‐labelled repinotan hydrochloride and its major metabolite M‐6

For studies of pharmacokinetics and drug metabolism of the new 5‐HT_1A agonist repinotan, the ¹⁴C‐labelled version was synthesized. Starting from [U‐¹⁴C]phenol, a 10‐step synthesis led to 457 mg (1.58 GBq) of [U‐¹⁴C]repinotan hydrochloride, labelled uniformly in the aromatic ring of the chromane moiety. For a study in man, a mono‐carbon‐14 labelled substance was required. Therefore a 7‐step synthesis was performed starting from [carbonyl‐¹⁴C]2‐hydroxy‐acetophenone. The yield was 106 mg (0.396 GBq) of [4‐chromane‐¹⁴C]repinotan hydrochloride. The carbon‐14 labelled major metabolite, hydroxylated in the 6‐position of the chromane moiety, was synthesised as reference compound. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of [13C]‐isotopomers of indole and tryptophan for use in the analysis of indole‐3‐acetic acid biosynthesis

The direct conversion of indole to indole‐3‐acetic acid without tryptophan as an intermediate has previously been shown to occur in vivo, as well as in vitro, with seedlings of plants. In order to facilitate the purification of the enzymes that carry out the enzymatic synthesis of indole‐3‐acetic acid from labeled indole, it was necessary to develop an assay that had both high sensitivity and analytical precision. To obtain the required analytical resolution and to allow definitive product identification, [¹³C₆]indole was synthesized for use in GC‐MS assays of the enzymatic conversion. Plants have been shown to be able to synthesize indole‐3‐acetic acid either directly from indole as well as by degradation of tryptophan. Thus, in order to allow the biochemical discrimination between these processes, the synthesized [¹³C₆]indole was used as a starting material for a novel enzymatic synthesis of [¹³C]isotopomers of L ‐tryptophan labeled at specific positions. Together, these isotope labeled indolic compounds offer powerful new approaches to understanding and differentiating routes of indole‐3‐acetic acid biosynthesis in vitro and in vivo. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of [7‐14C]bergapten

Bergapten (1) is a furocoumarin natural product and currently employed to treat skin disorders. Since past attempts to radiolabel 1 with ¹⁴C were limited to only its 5‐methoxy group, a synthesis of the required ring [7‐¹⁴C]1 is now described. The literature reported precursor 4‐methoxy‐6‐hydroxybenzofuran‐5‐carboxaldehyde (3) was Wittig reacted with stabilized [carbonyl‐¹⁴C]methoxycarbonylmethylenetriphenylphosphorane (4) to obtain [7‐¹⁴C]1 in 47% radiochemical yield, with the desired product being characterized by thin‐layer chromatography, HPLC, m.p. and proton NMR.     

Synthesis of [4‐14C]‐pelargonidin chloride and [4‐14C]‐delphinidin chloride

The synthesis of [4‐¹⁴C]‐pelargonidin chloride and [4‐¹⁴C]‐delphinidin chloride via [formyl‐¹⁴C]‐2‐(benzoyloxy)‐4,6‐dihydroxybenzaldehyde, ω,4‐diacetoxyacetophenone and ω,3,4,5‐tetraacetoxyacetophenone is described. The first step comprised labelling of the carbonyl group of 2‐(benzoyloxy)‐4,6‐dihydroxybenzaldehyde, verifying that the coupling with ω,4‐diacetoxyacetophenone or ω,3,4,5‐tetraacetoxyacetophenone under hydrogen chloride atmosphere resulted in the formation of [4‐¹⁴C] labelled anthocyanidins. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of a carbon‐14 labeled 1‐(indole‐6‐carbonyl‐D‐phenylglycinyl)‐4‐(1‐methylpiperidin‐4‐YL)piperazine‐[carbonyl‐14C], LY517717‐[14C], a factor Xa inhibitor

Human Factor Xa is a trypsin‐like serine protease, which serves a critical role in blood coagulation events. LY517717 is currently under clinical investigation as a Factor Xa inhibitor. To support the ADME studies, LY517717 was synthesized using D ‐phenylglycine with a carbon‐14 labeled carboxyl moiety. This key component, D ‐phenylglycine‐[carboxyl‐¹⁴C], was synthesized by a Strecker synthesis on benzaldehyde with potassium [¹⁴C]cyanide, followed by a resolution of DL ‐phenyl‐glycine methyl ester‐[carbonyl‐¹⁴C] with (+)‐tartaric acid in the presence of benzaldehyde. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of [14C]‐ and [35S]‐labelled PI‐88 for pharmacokinetic and tissue distribution studies

PI‐88, uniformly labelled with [¹⁴C] was prepared by incorporating D ‐[¹⁴C]glucose into the fermentation of Pichia (Hansenula) holstii NRRL Y‐2448 under controlled conditions to produce [¹⁴C]‐labelled extracellular phosphomannan. Subsequent acid catalyzed hydrolysis gave the [¹⁴C]‐labelled oligosaccharide phosphate fraction which was sulfonated with excess sulfur trioxide pyridine complex to give [¹⁴C]PI‐88. [³⁵S]‐labelled PI‐88 was similarly prepared by the sulfonation of unlabelled oligosaccharide phosphate fraction with [³⁵S] sulfur trioxide pyridine complex. Copyright © 2002 John Wiley & Sons, Ltd.     

Syntheses of [14C]‐labeled 2‐(3‐chlorophenyloxy)‐3‐[3‐(3‐hydroxy) pyridin‐4‐yl propoxy]pyridine,a phosphodiesterase 4 inhibitor and its metabolites

We describe here the synthesis of [¹⁴C]‐2‐(3‐chlorophenyloxy)‐3‐[3‐(3‐hydroxy)pyridin‐4‐yl propoxy]pyridine (1), a phosphodiesterase 4 inhibitor. [¹⁴C]‐Labeled 1 was prepared in three steps from [¹⁴C]‐2‐bromopyridin‐3‐ol in an overall yield of 32%. Preparation of [¹⁴C]‐labeled 2 and 3, two metabolites of 1, is also described. Copyright © 2014 John Wiley & Sons, Ltd.