Enzymatic synthesis of tritium‐labelled isotopomers of L‐DOPA

The synthesis of two isotopomers of L ‐DOPA labelled selectively with tritium is reported. In the intermediate step [3S‐³H]‐, and [3′,5′‐³H₂]‐L ‐tyrosine, have been obtained using a combination of chemical and enzymatic methods. The labelled isotopomers of L ‐tyrosine, L ‐Tyr, have been converted into, [3S‐³H]‐, and [5′‐³H]‐L ‐DOPA using the enzyme mushroom tyrosinase (monophenol oxidase, EC 1.14.18.1) from Neurospora crassa. Copyright © 2005 John Wiley & Sons, Ltd.     

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.     

Isotopically labelled tropane alkaloids. The synthesis of (RS)‐[3′, 3′‐2H2]‐ and (RS)‐[1′‐13C, 3′, 3′‐2H2]‐ hyoscyamines for metabolism studies in plants

A synthetic route to isotopically labelled forms of the tropane alkaloid hyoscyamine, including (RS)‐[3′, 3′,‐²H₂]‐ ( 2a ) and (RS)‐[1′‐¹³C, 3′, 3′,‐²H₂]‐ ( 2b ) hyoscyamines, involving the reaction between phenylacetyl tropine and formaldehyde is described. The isotopically labelled products enable the metabolism of hyoscyamine to be studied in plants such as Datura stramonium. Copyright © 2002 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 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.     

Radiosynthesis of 1′‐[18F]fluoroethyl‐β‐D‐lactose ([18F]‐FEL) for early detection of pancreatic carcinomas with PET

Introduction: The hepatocellular carcinoma–intestine–pancreas and pancreatitis‐associated proteins, also known as lactose‐binding protein, is upregulated in peritumoral pancreatic tissue. Previously, we reported ethyl‐ β ‐D ‐galactopyranosyl‐(1,4′)‐2′‐deoxy‐2′‐[¹⁸F]fluoro‐ β ‐D ‐glucopyranoside (Et‐[¹⁸F]‐FDL), a radiofluorinated lactose analog for positron emission tomography (PET) of small pancreatic carcinomas in mice. However, synthesis of the precursor for Et‐[¹⁸F]‐FDL involves 11 steps, which is quite lengthy, and produces overall low yields. Here, we report on synthesis and radiolabeling of another analog of lactose, the 1′‐[¹⁸F]fluoroethyl‐ β ‐D ‐lactose for PET imaging of pancreatic carcinomas. Methods: Two precursor compounds, 1′‐bromoethyl‐2′,3′,6′,2,3,4,6‐hepta‐O‐acetyl‐ β ‐D ‐lactose 4, and 1′‐p‐toluenesulfonylethyl‐2′,3′,6′,2,3,4,6‐hepta‐O‐acetyl‐ β ‐D ‐lactose 5, were synthesized in two and three steps, respectively; then, cold fluorination and radiofluorination of these precursors were performed. The reaction mixture was passed through a silica gel Sep‐pack cartridge, eluted with EtOAc, and the 1′‐[¹⁸F]fluoroethyl‐2′,3′,6′,2,3,4,6‐hepta‐O‐acetyl‐ β ‐D ‐lactose ([¹⁸F]‐6) purified by HPLC. After hydrolysis of the protecting groups, the 1′‐[¹⁸F]fluoroethyl‐ β ‐D ‐lactose [¹⁸F]‐7 was neutralized, diluted with saline, filtered through a sterile Millipore filter, and analyzed by radio‐TLC. Results: The average decay‐corrected radiochemical yield was 9% (n = 7) with>99% radiochemical purity and specific activity of 55.5 GBq/ µ mol. Conclusion : A new analog of lactose, 1′‐[¹⁸F]fluoroethyl‐ β ‐D ‐lactose, has been synthesized in good yields, with high purity and high specific activity suitable for PET imaging of early pancreatic carcinomas. Copyright © 2011 John Wiley & Sons, Ltd.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

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.     

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.     

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.     

Synthesis of tritium‐labeled (R,R)‐4‐methoxyfenoterol

The preparation of 2′,4′,6′‐[³H₃]‐(R,R)‐4‐methoxyfenoterol, a tritium‐labeled derivative of (R,R)‐4‐methoxyfenoterol was demonstrated on a 15 mCi scale providing material with a specific activity of 57 Ci/mmol. 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.     

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.     

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.     

Synthesis and 11C‐labelling of (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl) benzene for PET imaging of amyloid deposits†

Carboxylic acid derivatives of the amyloid‐binding dye Congo red do not enter the brain well and are thus unable to serve as in vivo amyloid‐imaging agents. A neutral amyloid probe, (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl)benzene ( 3 ), devoid of any carboxylate groups has been designed and synthesized via a 12‐step reaction sequence with a total yield of 30%. The unsymmetric compound 3 has also been labelled with C‐11 via [¹¹C]methyl iodide ([¹¹C]CH₃I) methylation of a symmetric 4,4′‐dimesyl protected precursor followed by deprotection. Preliminary evaluation indicated that compound 3 selectively stained plaques and neurofibrillary tangles in post‐mortem AD brain, and exhibited good binding affinity (K_i=38±8 nM) for Aβ(1–40) fibrils in vitro. In vivo pharmacokinetic studies indicated that [¹¹C] 3 exhibited higher brain uptake than its carboxylic acid analogs and good clearance from normal control mouse brain. [¹¹C] 3 also exhibited specific in vivo binding to pancreatic amyloid deposits in the NOR‐beta transgenic mouse model. These results justify further investigation of 3 and similar derivatives as surrogate markers for in vivo quantitation of amyloid deposits. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of (E)‐2,3′,4,5′‐tetramethoxy[2‐11C]stilbene

In this paper, we describe the radiosynthesis of the compound (E)‐2,3′,4,5′‐tetramethoxy[2‐¹¹C]stilbene, a potential, universal tumour positron emission tomography imaging agent. The production of (E)‐2,3′,4,5′‐tetramethoxy[2‐¹¹C]stilbene was carried out via ¹¹C‐methylation of (E)‐2‐(hydroxy)‐3′,4,5′‐trimethoxystilbene by using [¹¹C]methyl trifluoromethanesulfonate ([¹¹C]methyl triflate). (E)‐2,3′,4,5′‐tetramethoxy[2‐¹¹C]stilbene was obtained with a radiochemical purity greater than 95% in a 20 ± 2% decay‐corrected radiochemical yield, based upon [¹¹C]carbon dioxide. Synthesis, purification and formulation were completed on an average of 30 min following the end of bombardment (EOB). The specific radioactivity obtained was 1.9 ± 0.6 GBq/µmol at EOB. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of 3′‐deoxy‐3′‐[18F]fluoro‐1‐β‐D‐xylofuranosyluracil ([18F]‐FMXU) for PET

The synthesis of a pyrimidine analog, 3′‐deoxy‐3′‐[¹⁸F]‐fluoro‐1‐β‐D ‐xylofuranosyluracil ([¹⁸F]‐FMXU) is reported. 5‐Methyluridine 1 was converted to its di‐methoxytrityl derivatives 2 and 3 as a mixture. After separation the 2′,5′‐di‐methoxytrityluridine 2 was converted to its 3′‐triflate 4 followed by derivatization to the respective N³‐t‐Boc product 5 . The triflate 5 was reacted with tetrabutylammonium[¹⁸F]fluoride to produce 6 , which by acid hydrolysis yielded compound 7 . The crude preparation was purified by HPLC to obtain the desired product [¹⁸F]‐FMXU. The radiochemical yields were 25–40% decay corrected (d. c.) with an average of 33% in four runs. Radiochemical purity was >99% and specific activity was >74 GBq/µmol at the end of synthesis (EOS). The synthesis time was 67–75 min from the end of bombardment (EOB). Copyright © 2005 John Wiley & Sons, Ltd.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.