Synthesis of tritium labeled isotopomers of L‐tyrosine

The synthesis of four selectively labeled isotopomers of L ‐tyrosine, (L ‐Tyr), using chemical and enzymatic methods is reported. Four tritium labeled isotopomers of L ‐phenylalanine (L ‐Phe) – [2‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐ and [3S‐³H]‐ have been synthesized using a combination of chemical and enzymatic methods. The labeled isotopomers of L ‐Phe have been converted into [2 ‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐, and [3S‐³H]‐L ‐Tyr by using the enzyme L ‐phenyl‐alanine 4′‐monooxygenase. Copyright © 2002 John Wiley & Sons, Ltd.     

Enzymatic synthesis of isotopomers of tyramine labeled with deuterium and tritium

The combined chemical and enzymatic methods of synthesis of five isotopomers of L ‐tyrosine, L ‐Tyr, and their derivatives, i.e. corresponding isotopomers of tyramine (TA), labeled with deuterium and tritium have been reported. Two‐step synthesis consists with introduction of deuterium or tritium label into intermediate L ‐Tyr using isotope exchange followed by enzymatic decarboxylation using enzyme tyrosine decarboxylase (EC 4.1.1.25). This way five isotopomers of L ‐tyrosine, i.e. [2‐²H]‐L‐, [2‐³H]‐L‐,[2‐²H/³H]‐L‐, [3′,5′‐²H₂]‐L‐,[3′,5′‐³H₂]‐L ‐Tyr, and six isotopomers of tyramine i.e. [1S‐²H]‐, [1S‐³H]‐, [1S‐²H/³H]‐, [3′,5′‐²H₂]‐, [3′,5′‐²H₂]‐, [2′,6′‐²H₂]‐TA were obtained. Copyright © 2007 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.     

Synthesis of isotopomers of dopamine labeled with deuterium or tritium

The synthesis of four isotopomers of dopamine labeled with deuterium or tritium is reported. The ring labeled [2′,5′,6′‐²H₃]‐, and [2′,5′,6′‐³H₃]‐dopamine were obtained using acid catalyzed isotopic exchange between dopamine and heavy or tritiated water respectively. Two selectively labeled isotopomers, i.e. [1R‐²H]‐, and [1R‐³H]‐dopamine were synthesized by enzymatic decarboxylation of L ‐DOPA using the enzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis. Copyright © 2006 John Wiley & Sons, Ltd.     

Enzymatic synthesis of tritium‐labelled isotopomers of histamine

Three tritium‐labelled isotopomers of histamine (HA) have been synthesized using combined chemical and enzymatic methods. In the first step the tritium‐labelled isotopomers of histidine have been obtained by catalysed exchange with tritiated water. These intermediates have been converted into [1S‐³H]‐HA, [1R‐³H]‐HA and [2′,4′,1S,‐³H₃]‐HA using the enzyme histidine decarboxylase (HDC, EC 4.1.1.22) from Lactobacillus 30a. Copyright © 2004 John Wiley & Sons, Ltd.     

Trifluoromethanesulfonic acid,an alternative solvent medium for the direct electrophilic fluorination of DOPA: new syntheses of 6‐[18F]fluoro‐L‐DOPA and 6‐[18F]fluoro‐D‐DOPA

Previous work from this laboratory has shown that the direct fluorination of 3, 4‐dihydroxy‐phenyl‐L ‐alanine (L ‐DOPA) in anhydrous HF (aHF) or BF₃/HF with F₂ is an efficient method for the synthesis of 6‐fluoro‐L ‐DOPA. Since then, ¹⁸F‐labeled 6‐fluoro‐L ‐DOPA ([¹⁸F]6‐fluoro‐L ‐DOPA) has been used to study presynaptic dopaminergic function in the human brain and to monitor gastrointestinal carcinoid tumors. This work demonstrates that the reactivity and selectivity of F₂ toward L ‐DOPA in CF₃SO₃H is comparable with that in aHF. This new synthetic procedure has led to the production of [¹⁸F]fluoro‐L ‐DOPA and [¹⁸F]fluoro‐D‐DOPA isomers in 17±2% radiochemical yields (decay corrected with respect to [¹⁸F]F₂). The 2‐ and 6‐FDOPA isomers were separated by HPLC and subsequently characterized by ¹⁹F NMR spectroscopy. The corresponding [¹⁸F]‐FDOPA enantiomers have been obtained in clinically useful quantities by a synthetic approach that avoids the use of aHF. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of tritium labelled [2′,6′]‐L‐tyrosine

The synthesis of a specifically ring labelled isotopomer of L ‐tyrosine, (L ‐Tyr), using a combination of chemical and enzymatic methods is reported. The tritium labelled [2′,6′]‐L ‐Tyr has been synthesized via catalytic exchange of phenol with tritiated water in the presence of K₂PtCl₄, reverse acid catalysed removal of tritium from the o‐ and p‐positions of phenol, and subsequent condensation of the resulting [3′,5‐³H₂]‐phenol with S‐methyl‐L ‐cysteine using the enzyme β‐tyrosinase from Citrobacter freundii. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of a technetium‐99m labelled L‐tyrosine derivative with the fac‐99mTc(I)(CO)3‐core using a simple kit‐procedure

The synthesis of a novel technetium‐99m labelled derivative of L ‐tyrosine as a potential tumour imaging agent for nuclear medicine diagnosis is reported. The synthesis involved the labelling precursor fac‐[^99mTc(OH₂)(CO)₃]⁺ which was synthesized using the commercially available Isolink^®‐labelling kit and the tyrosine derivative O‐(N,N‐bis(carboxymethyl)aminoethyl)‐L ‐tyrosine trifluoroacetate. The labelled compound O‐(^99mTc(I)‐tricarbonyl‐N,N‐bis(carboxymethyl)aminoethyl)‐L ‐tyrosine was obtained in a radiochemical yield of 70–80% within 60 min with a radiochemical purity greater than 98% without any HPLC purification step. Purification was achieved merely by solid phase extraction. Chemical as well as chiral purity was determined using gradient‐ and chiral HPLC. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of tritium‐labelled 3′‐azido‐3′‐deoxythymidine

New approaches to the synthesis of 3′‐azido‐3′‐deoxythymidine labelled with tritium in the heterocyclic base have been developed. With this aim, enzymatic transribosylation with [³H]thymine using the enzyme preparation from rat liver and a three‐step chemical synthesis with use of the tritium labelled precursor were studies. The enzyme preparation did not catalyse the transfer of the 3′‐azido‐3′‐deoxyribosyl fragment to the [³H]thymine residue. 5′‐O‐Benzoyl‐2,3′‐anhydrothymidine was taken as a precursor for the tritium labelling by the chemical methods. The resulting [³H]3′‐azido‐3′‐deoxythymidine was obtained with a specific radioactivity of 18.3 Ci/mmol, the tritium is located in the C‐6 position of the thymine residue. Copyright © 2003 John Wiley & Sons, Ltd.     

Enzymatic synthesis of N‐methylhistamine labeled with deuterium and tritium

The isotopomers of N^π‐methylhistamine (πMeHA) and N^τ‐methylhistamine (τMeHA) labeled with deuterium and tritium at the α‐carbon atom of the side chain were obtained using the enzyme histidine decarboxylase (HDC, EC 4.1.1.22) from Lactobacillus 30a. The deuterium labeled isotopomers [(αR)‐²H]‐πMeHA and [(αR)‐²H]‐τMeHA were synthesized by enzymatic decarboxylation of N^π‐methyl‐, and N^τ‐methyl‐L ‐histidines (respectively) in a fully deuteriated incubation medium. The same decarboxylation carried out in a tritiated medium resulted in tritiated [αR‐³H]‐πMeHA and [αR‐³H]‐τMeHA isotopomers of N‐methylhistamine. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and biodistribution studies of two novel radioiodinated areno‐annelated estra‐1,3,5(10),16‐tetraene‐3‐ols as promising estrogen receptor radioligands

Two novel radioiodinated areno‐annelated estra‐1,3,5(10),16‐tetraenes, [¹²⁵I]2‐iodo‐1′‐methoxybenzo[4′,3′:16,17]estra‐1,3,5(10),16‐tetraene‐3‐ol ( 2 ‐[ ¹²⁵ I ]‐ MEBE ) and [¹²⁵I]4‐iodo‐1′‐methoxybenzo[4′,3′:16,17]estra‐1,3,5(10),16‐tetraene‐3‐ol, ( 4 ‐[ ¹²⁵ I ]‐ MEBE ) were synthesized for evaluation as potential ligands for the estrogen receptor. Radioiodination of 1′‐methoxybenzo[4′,3′:16,17]estra‐1,3,5(10),16‐tetraene‐3‐ol at the A ring was accomplished by electrophilic aromatic substitution using [¹²⁵I] sodium iodide and chloramine‐T as oxidant. After purification by reverse phase HPLC, the two radioisomers ( 2 ‐[ ¹²⁵ I ]‐ MEBE and 4 ‐[ ¹²⁵ I ]‐ MEBE ) were obtained in a radiochemical yield of 42 and 48%, respectively, in a radiochemical purity of greater than 95% and a high specific activity. The effect of the site of radioiodination (C₂ vs C₄) on the biological behaviour of the molecules was evaluated through biodistribution studies in immature female Sprague‐Dawley rats. Both 2 ‐[ ¹²⁵ I ]‐ MEBE and 4 ‐[ ¹²⁵ I ]‐ MEBE are stable in vivo and are mainly excreted through the hepatobiliary pathway. Both localize in the uterus and ovaries via a receptor‐mediated process, where the 2 ‐[ ¹²⁵ I ]‐ MEBE isomer has the higher specific ER binding and uterus selectivity. The favourable in vitro/in vivo stability and biodistribution profiles suggest that these radioligands are good candidates for further exploration of their potential clinical application. Copyright © 2006 John Wiley & Sons, Ltd.     

A novel method for stereospecific fluorination at the 2′‐arabino‐position of pyrimidine nucleoside: synthesis of [18F]‐FMAU

Direct fluorination of a pyrimidine nucleoside at the 2′‐arabino‐position has been deemed to be extremely difficult, if not impossible. The conventional synthesis of 2′‐deoxy‐2′‐fluoro‐5‐methy‐1‐β‐D ‐arabinofuranosyluracil (FMAU) and its 5‐substituted analogs involves stereospecific fluorination of the 1,3,5‐tri‐O‐benzoyl‐α‐D ‐ribofuranose‐2‐sulfonate ester followed by bromination at the C₁‐postion, and then coupling with pyrimidine‐bis‐trimethylsilyl ether. Several radiolabeled nucleoside analogs, including [¹⁸F]FMAU, and other 5‐substituted analogs, were developed according to this methodology. However, routine production of these compounds using this multi‐step process is inconvenient and limits their clinical application. We developed a novel precursor and method for direct fluorination of preformed nucleoside analogs at the 2′‐arabino position, exemplified via radiosynthesis of [¹⁸F]FMAU. The 2′‐methylsulfonyl‐3′,5′‐O‐tetrahydropyranyl‐N³‐Boc‐5‐methyl‐1‐β‐D ‐ribofuranosiluracil was synthesized in multiple steps. Radiofluorination of this precursor with K¹⁸F/kryptofix produced 2′‐deoxy‐2′‐[¹⁸F]fluoro‐3′,5′‐O‐tetrahydropyranyl‐N³‐Boc‐5‐methyl‐1‐β‐D ‐arabinofuranosiluracil. Acid hydrolysis followed by high‐performance liquid chromatography purification produced the desired [¹⁸F]FMAU. The average radiochemical yield was 2.0% (decay corrected, n=6), from the end of bombardment. Radiochemical purity was >99%, and specific activity was >1800 mCi/µmol. Synthesis time was 95–100 min from the end of bombardment. This direct fluorination is a novel method for synthesis of [¹⁸F]FMAU, and the method should be suitable for production of other 5‐substituted pyrimidine analogs, including [¹⁸F]FEAU, [¹⁸F]FIAU, [¹⁸F]FFAU, [¹⁸F]FCAU, and [¹⁸F]FBAU. Copyright © 2010 John Wiley & Sons, Ltd.     

13C‐ and 14C‐labelling of N‐[1‐(4‐chlorophenyl)‐1H‐pyrrol‐2‐yl‐methyleneamino] guanidinium acetate

N‐[1‐(4‐chlorophenyl)‐1H‐pyrrol‐2‐yl‐¹³C₄‐methyleneamino]guanidinium acetate has been synthesized by a four‐step procedure. This involved reduction of the Weinreb amide N,N′‐dimethyl‐N,N′‐dimethyloxybutane‐1,4‐diamide‐1,2,3,4‐¹³C₄ by Dibal‐H to give the corresponding unstable dialdehyde which is reacted in situ with 4‐chloroaniline to form 1‐(4‐chlorophenyl)‐1H‐pyrrole‐¹³C₄. This pyrrole analogue underwent a Vilsmeyer acylation with POCl₃/DMF followed by final reaction with aminoguanidine bicarbonate to produce the desired labelled compound with 99% atom ¹³C. By using DMF [α‐¹⁴C] a radio‐labelled analogue was synthesized with a specific activity of 60 mCi/mmol. Copyright © 2005 John Wiley & Sons, Ltd.     

Enzymatic synthesis of phenylpyruvic acid labeled with deuterium,tritium, and carbon‐14

The synthesis of isotopomers of phenylpyruvic acid, PPA, selectively labeled with hydrogen isotopes in the 3‐position of the side‐chain is reported. Three deuterium or tritium labeled isotopomers of L‐phenylalanine, L‐Phe, i.e. [(3S)‐²H]‐L‐, [(3S)‐³H]‐L‐, and doubly labeled [(3S)‐²H/³H]‐L‐Phe were synthesized using the enzyme phenylalanine ammonia lyase (EC 4.3.1.5). In the second step these isotopomers of L‐Phe were converted into [(3S)‐²H], [(3S)‐³H]‐, and [(3S)‐²H/³H]‐isotopomers of PPA using the enzyme L‐phenylalanine dedydrogenase (EC 1.4.1.20). The isotopomer of PPA labeled with ¹⁴C in carboxylic group, [1‐¹⁴C]‐PPA, was obtained in a two‐step enzymatic reaction using [1‐¹⁴C]‐cinnamic acid as the starting substrate. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of selectively labeled histidine and its methylderivatives with deuterium,tritium, and carbon‐14

Isotopologues of l ‐histidine and its N‐methylderivatives labeled with deuterium and tritium at the 5‐position in the imidazole ring were obtained using the isotope exchange method. The deuterium‐labeled isotopologues [5‐²H]‐l ‐histidine, [5‐²H]‐N^τ‐methyl‐l ‐histidine, [5‐²H]‐N^π‐methyl‐l ‐histidine, and [2,5‐²H₂]‐l ‐histidine were synthesized by isotope exchange method carried out in a fully deuterated medium with. The same reaction conditions were applied to synthesize [5‐³H]‐N^τ‐methyl‐l ‐histidine, [5‐³H]‐N^π‐methyl‐l ‐histidine, and [5‐³H]‐l ‐histidine with specific activity of 2.0, 5.0, and 2.6 MBq/mmol, respectively. The N^π‐[methyl‐¹⁴C]‐histamine was obtained with specific activity of 0.23 MBq/mmol in a one‐step reaction by the direct methylation of histamine by [¹⁴C]iodomethane.     

Synthesis of isotopically labelled glycyl‐L‐prolyl‐L‐glutamic acid (Glypromate®) and derivatives

The related tripeptides glycyl‐L ‐prolyl‐L ‐glutamic acid (GPE) and glycyl‐L ‐2‐methylprolyl‐L ‐glutamic acid (G‐2‐MePE) were labelled with commercially available [1,2,3,4,5‐¹³C₅, 2‐¹⁵N₁]‐L ‐glutamic acid in 3 steps in excellent overall yield with high isotope incorporation. A related cyclic dipeptide was labelled with [2,2‐²H₂, 2‐¹⁵N₁]glycine giving a mixture of compounds resulting from deuterium scrambling. Copyright © 2006 John Wiley & Sons, Ltd.     

Radiosynthesis of O‐[11C]methyl‐L‐tyrosine and O‐[18F]Fluoromethyl‐L‐tyrosine as potential PET tracers for imaging amino acid transport

Two positron‐emitting analogues of tyrosine, O‐[¹¹C]methyl‐L ‐tyrosine and O‐[¹⁸F]fluoromethyl‐L ‐tyrosine were prepared as new tumor imaging agents. The alkylating agent, [¹¹C]methyl triflate or [¹⁸F]fluoromethyl triflate, was simply bubbled through a dimethylsulfoxide solution of L ‐tyrosine disodium salt at room temperature. After subsequent HPLC purification the labeled L ‐tyrosine analogues were obtained in decay‐corrected radiochemical yields of over 50%, based on their corresponding labeling agent, with radiochemical purities always higher than 98%. The quite straightforward preparation, together with the high radiochemical yields achieved, make both these syntheses suitable for routine production. Copyright © 2003 John Wiley & Sons, Ltd.     

Syntheses of (R,S)‐naproxen and its 6‐O‐desmethylated metabolite labelled with 2H

Naproxen, a well‐known non‐steroidal anti‐inflammatory drug, and its 6‐O‐desmethylated metabolite have been labelled with ²H. (R,S)‐Naproxen 7 labelled with ²H was obtained in five steps using the commercially available [²H₃]iodomethane 5 as the stable labelled reagent. The demethylation of 7 using 48% HBr in 1‐butyl‐3‐methylimidazolium tetrafluoborate gave the corresponding ²H‐labelled 6‐O‐desmethylated metabolite 8. Copyright © 2008 John Wiley & Sons, Ltd.     

3H‐labelled alkyl‐nucleotides, ‐nucleosides and ‐bases for the immunoanalytical quantification of DNA damage and repair

Analysis of the formation and repair of structurally modified DNA is of particular interest in the study of carcinogenesis, cancer therapy and aging. The quantification of specific DNA lesions by sensitive immunoanalytical methods requires radiotracers with high specific activity. We describe the synthesis of ³H‐labelled adenine‐, cytosine‐, guanine‐ and thymine‐alkyl derivatives by nucleophilic N‐ and O‐alkylation using alkyl halides and diazoalkanes: 3‐alkyl‐[8‐³H]adenine (Alkyl = Me, Et, n‐Bu); O⁶‐alkyl‐deoxy[1′,2′‐³H]guanosine (Alkyl = Me, Et, i‐Pro, n‐Bu); O⁶‐ethyl‐deoxyguanosine‐5′‐triphosphate ([2‐³H‐Ethyl]; [8‐³H]); O⁶‐alkyl‐9‐hydroxyhexyl‐[8‐³H] guanine (Alkyl=Me, Et); 7‐ethyl‐[8,5′‐³H]guanosine‐3′,5′‐cyclic‐phosphate; O²‐andO⁴‐alkyl‐[methyl, 1′,2′‐³H]thymidine (Alkyl=Me, Et); the conversion of ³H‐labelled thymidine to the corresponding 5‐methylcytidine; the synthesis of three different 8‐oxo‐guanine tracers; and the generation of thymidine glycol (5,6‐dihydroxy‐5,6‐dihydro‐[methyl‐³H]thymidine) from thymidine. All radiotracers were sucessfully employed in competitive radioimmunoassays for the quantification of defined DNA alkylation products in DNA repair analyses. Copyright © 2003 John Wiley & Sons, Ltd.     

The synthesis of isotopically labeled (+)‐2‐aminobicyclo[3.1.0]hexane‐2,6‐carboxylic acid and its 2‐oxa‐ and 2‐thia‐analogs

As part of a program aimed at the design of conformationally constrained analogs of glutamic acid, (+)‐2‐aminobicyclo[3.1.0]hexane‐2,6‐carboxylic acid ( 1 ), identified as a highly potent, selective, group II metabotropic glutamate receptor agonist has been synthesized and studied clinically. Heterocyclic analogs of 1 were subsequently synthesized in which the C‐2 methylene has been replaced by an oxygen atom ( 2 ) or a sulfur atom ( 3 ). C‐14 labeled isotopomers of 1 , 2 and 3 have been synthesized to facilitate pre‐clinical ADME studies. A tritium labeled isotopomer of 1 was also synthesized for use in in vitro experiments. A stable labeled isotopomer of rac‐1 was prepared for use as an internal standard for bioanalytical assays. The key step in each of these syntheses was the reaction of chiral ketone 4 , 5 or 6 with K¹⁴CN/(NH₄)₂CO₃ using the Bucherer–Berg protocol. In the preparation of the stable labeled isotopomer, rac‐4 ‐[¹³ C ₂] was prepared in two steps from ethyl bromoacetate‐[UL‐¹³C₂]; subsequent reaction of rac‐4 ‐[¹³ C ₂] with K¹³CN/¹⁵NH₄Cl/Na₂CO₃, followed by hydrolysis of the hydantoin yielded rac‐1 ‐[¹³ C ₃,¹⁵ N ]. Copyright © 2005 John Wiley & Sons, Ltd.