The screening of some microorganisms for their ability to N-dealkylate drug molecules

Summary Selected microorganisms were screened for their ability to N-dealkylate drug molecules. The compounds studied enabled the investigation of N-alkyl groups in different chemical environments, including alkylaminoalkyl chains, saturated cyclic structures and in amide functions. Transformation products were extracted from the transformation mixtures, derivatised and analysed by gas liquid chromatography (GLC). For the purposes of screening, important transformation products were identified by comparison of their GLC retention data with similar derivatives of authentic standards. N-demethylation was effected by all test strains of the fungus Cunninghamella, except C. elegans, and also by three of the Streptomyces species tested. The Cunninghamella demonstrated the widest spectrum of transformation activity, N-demethylating the alkylaminoalkyl side chains of amitriptyline and chlorpromazine, the N-methylpiperidine function of codeine, and the cyclic amide function of diazepam. The N-demethylation of the latter substrate, which is only slightly water soluble, occurred in surprisingly high yield. There was no evidence that bulky groups such as N-dimethylallyl were celaved and selectivity for N-demethylation rather than O-demethylation was demonstrated when both N-and O-methyl functions were present in the same molecule. Comparisons are made with the mammalian metabolic pathways of the drug compounds studied.     

Biotransformation of Flurbiprofen by Cunninghamella Species

The biotransformation of the fluorinated anti-inflammatory drug flurbiprofen was investigated in Cunninghamella spp. Mono- and dihydroxylated metabolites were detected using gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy, and the major metabolite 4′-hydroxyflurbiprofen was isolated by preparative high-pressure liquid chromatography (HPLC). Cunninghamella elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a phase II (conjugated) metabolite, which was identified as the sulfated drug via deconjugation experiments.One of the objectives of the recent European Union legislation governing the testing and evaluation of chemicals, REACH (Regulation, Evaluation, Authorisation and Restriction of Chemicals), is to further reduce the need for animals in the testing process. Some microorganisms, such as the zygomycete fungus Cunninghamella and actinomycetes bacteria, have been shown to metabolize xenobiotic compounds in a fashion analogous to that of mammals (3, 5, 11, 17). It was suggested over 3 decades ago that microorganisms had potential as models of mammalian metabolism (16), although there are concerns about their predictive value (8). Nevertheless, certain microorganisms can be applied to the generation of useful quantities of drug metabolic intermediates (13), which is more desirable than isolation of these compounds from dosed animals, and avoids the concerns often associated with chemical synthesis, such as the use of toxic reagents and harsh reaction conditions.Owing to the desirable physicochemical properties of the fluorine atom (small Van der Waals radius, electronegativity, and strength of the carbon-fluorine bond), approximately 25% of drugs either currently on the market or in the pipeline are fluorinated (12). One such example is flurbiprofen [(RS)-2-(2-fluoro-4-biphenyl)propionic acid], which is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of inflammation caused by arthritis. In humans it is transformed to the phase I (oxidative) metabolites 4′-hydroxyflurbiprofen, 3′,4′-dihydroxyflurbiprofen, and 3′-hydroxy,4′-methoxyflurbiprofen; glucuronide and sulfate conjugates (phase II metabolites) have also been detected (9, 15). In equine urine additional hydroxylated and methoxylated metabolites were detected (20). Tracy et al. (18) demonstrated that only one cytochrome P450 isoform (2C9) is involved in the oxidation of flurbiprofen, which makes the drug a potentially useful in vivo probe for this particular isoform. Despite the prevalence of fluorinated drugs, only a few investigations have been undertaken to determine the microbial biotransformation of these compounds (7, 21). Here we describe the biotransformation of flurbiprofen by Cunninghamella species and the determination of the metabolites by nuclear magnetic resonance (NMR) spectroscopy (¹H and ¹⁹F), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC).Three species of Cunninghamella were selected for the biotransformation experiments: C. elegans (strains DSM 1908, DSM 8217, and DSM 63299), C. echinulata DSM 1905, and C. blakesleeana DSM 1906. The fungi were grown on Sabouraud dextrose agar plates (Sigma) for 5 days at 26°C before being homogenized in 100 ml of sterile saline solution. The homogenate (10%, vol/vol) was used to inoculate 50 ml of fresh Sabouraud dextrose broth in 250-ml Erlenmeyer flasks, which were incubated at 28°C with shaking at 150 rpm. Following previously established procedures (2), 5 mg of flurbiprofen (Sigma) dissolved in dimethyl formamide (20 μl) was added to the cultures after 72 h, and the incubation was continued up to a further 120 h. Control experiments were conducted in the absence of either flurbiprofen or fungus. The cultures (supernatant and cells) were sonicated on ice (Sonicator U200S control; IKA Labortechnik) for 5 min at 50% amplitude, with intervals of 30 s after each minute to prevent overheating. The sonicates were centrifuged, the supernatant was extracted with 50 ml of ethyl acetate, and the extracts were evaporated to dryness.     

Synthesis,Anticonvulsant Activity and Metabolism of 4‐chlor‐3‐methylphenoxyethylamine Derivatives of Trans‐2‐aminocyclohexan‐1‐ol

In this study, we report the synthesis, spectral characterization, antiepileptic activity and biotransformation of three new, chiral, N‐aminoalkyl derivatives of trans – 2 aminocyclohexan‐1‐ol: 1 (R enantiomer), 2 (S enantiomer) and 3 (racemate). Antiepileptic activity of the titled compounds was studied using MES and scMet. Moreover, in this study, the biotransformation of 1 , 2 and 3 in microbial model (Cunninghamella), liver microsomal assay as well as in silico studies (MetaSite) was evaluated. Studies have indicated that 1 , 2 and 3 have good antiepileptic activity in vivo, comparable to valproate. Biotransformation assays showed that the most probable metabolite (indicated in every tested assays) was M1 . The microbial model as well as in silico study showed no difference in biotransformation between tested enantiomers. However, in a rat liver microsomal study compound 1 and 2 (R and S enantiomer) had different main metabolite – M2 for 1 and M1 for 2 . MS/MS fragmentation allowed us to predict the structures of obtained metabolites, which were in agreement with 1°alcohol ( M1 ) and carboxylic acid ( M2 ). Our research has shown that microbial model, microsomal assay, and computational methods can be included as useful and reliable tools in early ADME‐Tox assays in the process of developing new drug candidates. Chirality 27:163–169, 2015. © 2014 Wiley Periodicals, Inc.     

Initial oxidative and subsequent conjugative metabolites produced during the metabolism of phenanthrene by fungi

Three filamentous fungi were examined for the ability to biotransform phenanthrene to oxidative (phase I) and conjugative (phase II) metabolites. Phenanthrene metabolites were purified by high-performance liquid chromatography (HPLC) and identified by UV/visible absorption, mass, and¹H NMR spectra.Aspergillus niger ATCC 6275,Syncephalastrum racemosum UT-70, andCunninghamella elegans ATCC 9245 initially transformed [9-¹⁴C]phenanthrene to produce metabolites at the 9,10-, 1,2-, and 3,4- positions. Subsequently, sulfate conjugates of phase I metabolites were formed byA. niger, S. racemosum, andC. elegans. Minor glucuronide conjugates of 9-phenanthrol and phenanthrenetrans-9,10-dihydrodiol were formed byS. racemosum andA. niger, respectively. In addition,C. elegans produced the glucose conjugates 1-phenanthryl -d-glucopyranoside and 2-hydroxy-1-phenanthryl -d-glucopyranoside, a novel metabolite. [9-¹⁴C]Phenanthrene metabolites were not detected in organic extracts from biotransformation experiments with the yeasts,Candida lipolytica 37-1,Candida tropicalis ATCC 32113, andCandida maltosa R-42.     

Microbial metabolism of the \-adrenoreceptor antagonist bisoprolol

Eighteen filamentous fungi and six actinomycetes species were screened for their ability to metabolize bisoprolol, a \-blocking drug. All strains of Cunninghamella tested accumulated metabolite M4 = EMD 46193 ([4-(2-hydroxy-3-isopropylaminopropoxy)benzyloxy]ethanol). Among the strains investigated only Gliocladium deliquescens excreted the corresponding carbonic acid M1 = EMD 44025 into the culture medium. Biotransformation of bisoprolol by fungi occurred only during growth in complex medium or with resting cells after cultivation in complex medium. The screened Actinomycetes showed no biotransformation of the drug. Correspondence to: H. Schwartz     

Structural elucidation of metabolites of tanshinone I and its analogue dihydrotanshinone I in rats by HPLC–ESI-MSn

Tanshinone I and its analogue dihydrotanshinone I are the major active components isolated from Salvia miltiorrhiza Bunge and Salvia Przewalskii Maxim. These compounds have been found to possess significant antibacterial, anti-dermatophytic, antioxidant, anti-inflammatory and anticancer activities. Fifteen phase I metabolites and two phase II metabolites of tanshinone I and dihydrotanshinone I in rat bile were elucidated and identified by a sensitive HPLC–ESI-MSⁿ method. The molecular structures of the metabolites are presented on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The results indicate that the phase I metabolites are biotransformed through four main pathways: dehydrogenation, hydroxylation, furan ring cleavage and oxidation metabolism. Phase II metabolites were mainly identified as the sulfated conjugates which showed a characteristic neutral loss of 80 Da. The biotransformed pathways of tanshinone I and dihydrotanshinone I were proposed on the basis of the investigation.     

Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58–86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.     

Effect of colchicine on the nitrogen metabolism and fate of phosphates absorbed during the formation of fungal felts of cunninghamella sp.

Summary During formation of fungal mats of Cunninghamella sp., colchicine seemed to have no effect on the mycelial dry weight but increased the phosphorus and nitrate uptake only when present in 20 p.p.m. All treatments increased the organic phosphorus content and peptide fraction of the tissue medium systems. These phenomena were furthered by raising the concentration of the drug. Colchicine showed its inhibitory effects on protein synthesis only when administered in the high concentration. The mechanism of nitrate utilisation in presence of colchicine was also discussed.     

Comparison of 2,4,6-Trinitrotoluene Degradation by Seven Strains of White Rot Fungi

The degradation of 2,4,6-trinitrotoluene (TNT) by seven strains of white rot fungi was examined in two different media containing 50 mg L⁻¹ of TNT. When TNT was added into a nutrient-rich YMG medium at the beginning of the incubation, four of the fungal strains completely removed TNT during several days of incubation and showed higher removal rates than those of Phanerochaete chrysosporium. TNT added into YMG medium after a 5-day preincubation period completely disappeared within 12 hours, and the removal rates were higher than those in N-limited minimal medium. Isomers of hydroxylamino-dinitrotoluene were identified as the first detectable metabolites of TNT. These were transformed to amino-dinitrotoluenes, which also disappeared during further incubation from cultures of Irpex lacteus. During the initial phase of TNT degradation by I. lacteus, dinitrotoluenes were also detected after the transient formation of a hydride-Meisenheimer complex, indicating that I. lacteus used two different pathways of TNT degradation simultaneously. Received: 29 March 2000 / Accepted: 23 May 2000     

Synthesis of potential buprenorphine intermediates by selective microbial <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">O</Emphasis>-demethylation

A group of filamentous fungi from the genus Cunninghamella have been identified as potential biocatalysts in the generation of potential buprenorphine intermediates. A simple batch fermentation procedure results in the formation of dealkylated alkaloid compounds that are readily available for analysis and further chemical modification.     

Inhibitory effect of extracts from Brazilian medicinal plants on the adhesion of <Emphasis Type="Italic">Candida albicans</Emphasis> to buccal epithelial cells

Plants are known to produce a plethora of secondary metabolites which are recognized as a useful source of new drugs or drug leads. Extracts and fractions of Schinus terebinthifolius Raddi (Anacardiaceae), Piper regnellii C.D.C. (Piperaceae), Rumex acetosa L. (Polygonaceae), and Punica granatum L. (Punicaceae) were assessed for their antifungal activity against eight clinical isolates of C. albicans. They were also evaluated for their effect on the adhesion of these C. albicans isolates to buccal epithelial cells (BECs). The ethyl acetate fraction from the leaves of S. terebinthifolius showed promising activity, inhibiting the growth of three C. albicans isolates at 7.8 μg ml⁻¹ and significantly inhibiting their adhesion to BEC at 15 μg ml⁻¹ . In addition, this fraction did not show cytotoxic activity against murine macrophages. The results show the potential of the plant extracts studied as a source of new antifungal compounds. Further studies are necessary for isolation and characterization of the active compounds of these plants.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.     

Development of an <Emphasis Type="Italic">in vitro</Emphasis> assay for the investigation of metabolism-induced drug hepatotoxicity

In a number of adverse drug reactions leading to hepatotoxicity drug metabolism is thought to be involved by generation of reactive metabolites from nontoxic drugs. In this study, an in vitro assay was developed for measurement of the impact of metabolic activation of compound on the cytotoxicity toward a human hepatic cell line. HepG2 cells were treated for 6 h with compound in the presence or absence of rat liver S9-mix, and the viability was measured using the MTT test. The cytotoxicity of cyclophosphamide was substantially increased by S9-mix in the presence of NADPH. Three NADPH sources were tested: NADPH (1 mmol/L) or NADPH regenerating system with either NADP⁺/glucose 6-phosphate (G6P) or NADP⁺/isocitrate. All three NADPH sources increased the cytotoxicity of cyclophosphamide to a similar extent. Eight test compounds known to cause hepatotoxicity were tested. For these, only the cytotoxicity of diclofenac was increased by S9 enzymes when an NADPH regenerating system was used. The increased toxicity was NADPH dependent. Reactive drug metabolites of diclofenac, formed by NADPH-dependent metabolism, were identified by LC-MS. Furthermore, an increase in toxicity, not related to enzymatic activity but to G6P, was observed for diclofenac and minocycline. Tacrine and amodiaquine displayed decreased toxicity with S9-mix, and carbamazepine, phenytoin, bromfenac and troglitazone were nontoxic at all tested concentrations, with or without S9-mix. The results show that this method, with measurement of the cytotoxicity of a compound in the presence of an extracellular metabolizing system, may be useful in the study of cytotoxicity of drug metabolites.     

Application of Two‐Dimensional NMR Spectroscopy to Metabotyping Laboratory Escherichia coli Strains

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one‐dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two‐dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed ¹H,¹³C‐HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1‐Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D‐NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains.     

Transformation of Verapamil by Cunninghamella blakesleeana

A filamentous fungus, Cunninghamella blakesleeana AS 3.153, was used as a microbial model of mammalian metabolism to transform verapamil, a calcium channel antagonist. The metabolites of verapamil were separated and assayed by the liquid chromatography-ion trap mass spectrometry method. After 96 h of incubation, nearly 93% of the original drug was metabolized to 23 metabolites. Five major metabolites were isolated by semipreparative high-performance liquid chromatography and were identified by proton nuclear magnetic resonance and electrospray mass spectrometry. Other metabolites were characterized according to their chromatographic behavior and mass spectral data. The major metabolic pathways of verapamil transformation by the fungus were N dealkylation, O demethylation, and sulfate conjugation. The phase I metabolites of verapamil (introduction of a functional group) by C. blakesleeana paralleled those in mammals; therefore, C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of verapamil.     

Non‐invasive imaging of allogeneic transplanted skin graft by 131I‐anti‐TLR5 mAb

Although ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) uptake can be used for the non‐invasive detection and monitoring of allograft rejection by activated leucocytes, this non‐specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a ¹³¹I‐radiolabelled anti‐Toll‐like receptor 5 (TLR5) mAb for non‐invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non‐specific radiotracer ¹⁸F‐FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo‐treated group or allo‐rejection group), were injected with ¹³¹I‐anti‐TLR5 mAb, ¹⁸F‐FDG, or mouse isotype ¹³¹I‐IgG, respectively. Whole‐body phosphor‐autoradiography and ex vivo biodistribution studies were obtained. Whole‐body phosphor‐autoradiography showed ¹³¹I‐anti‐TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The ¹³¹I‐anti‐TLR5 mAb had significantly higher graft uptake and target‐to‐non‐target ratio in the allo‐treated group, as determined by semi‐quantification of phosphor‐autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high ¹⁸F‐FDG uptake was not observed in the allo‐treated group. The highest allograft‐skin‐to‐native‐skin ratio (A:N) of ¹³¹I‐anti‐TLR5 mAb uptake was significantly higher than the ratio for ¹⁸F‐FDG (7.68 versus 1.16, respectively). ¹³¹I‐anti‐TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of ¹³¹I‐IgG was comparable in both groups. We conclude that radiolabelled anti‐TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.     

p38 MAPK as a signal transduction component of heavy metals stress in <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Living organisms are subject to stress, and among these stressors, heavy metals exposure triggers accumulation of sulfur metabolites. Among these metabolites, glutathione and phytochelatins are found in several organisms, such as Euglena gracilis. Pre-exposing E. gracilis to low concentrations of Hg²⁺ generates a population with resistance to even 0.2 mM Cd²⁺, and this resistance relies partly on phytochelatins. p38 MAPK is stimulated by stress and is involved in apoptotic as well as survival mechanisms. In this study, we explored its participation in heavy metal-induced stress and its possible role in sulfur metabolite accumulation. We found that about 51% of the E. gracilis pretreated with Hg²⁺ becomes resistant to Cd²⁺ and proliferates despite the presence of this metal. The accumulation of the sulfur metabolites γ-glu-cys, glutathione and phytochelatin 2 displayed cyclic patterns that were disturbed by a challenge with Cd²⁺. We observed a p38 MAPK-like activity that was stimulated by acute or chronic heavy metal exposure, and its inhibition by SB203580 slightly diminished the accumulation of sulfur compounds. p38 MAPK inhibition also affected basal levels of glutathione in either pretreated or control cells. Thus, it appears that p38 MAPK mediates redox stress component of the signal pathway induced by heavy metals.     

Microbial production of testosterone and testololactone in the culture of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Two metabolites were obtained by microbial transformation of androstendione in the culture of Aspergillus terreus PTCC 5283, a fungus isolated from soil. Their structures were established as testosterone and testololactone on the basis of the spectral data including ¹H NMR, ¹³C NMR, FTIR, MS and physical constants such as melting point and optical rotation. Aspergillus terreusproduced both metabolites after 3 days incubation at 27 °C. The bioconversion reactions observed were 17-carbonyl reduction and biological Baeyer–Villiger oxidation.     

Secondary metabolites during ontogenetic phase of reproductive structures in Hypericum maculatum

The distribution patterns of flavonoids hyperoside, isoquercitrin, quercitrin, quercetin, I3,II8-biapigenin and naphtodianthrones hypericin and pseudohypericin were studied in reproductive structures during ontogenetic phase of flowering in Hypericum maculatum Crantz. Considerable differences in the content of these secondary metabolites, in the particular flower parts were found. The content of all the metabolites studied is stable during the whole period of flowering in green flower parts (sepals). In petals, stamens and pistils their content undergoes considerable change associated with the biological functions of particular metabolites. The most conspicuous changes during ontogenetic phase of flowering were the decrease of hyperoside and isoquercitrin content in petals (average content in buds 1.589 mg g⁻¹ dry weight, average content in overblown flowers 0.891 mg g⁻¹ dry weight), the decrease of the I3,II8-biapigenin content in stamens (in buds 1.189 mg g⁻¹ dry weight, in overblown flowers 0.319 mg g⁻¹ dry weight), and the increase of hypericin and pseudohypericin content in both petals (total average content of hypericins in the buds 0.547 mg g⁻¹ dry weight; in overblown flowers 0.792 mg g⁻¹ dry weight) and stamens (in buds 0.189 mg g⁻¹ dry weight; in overblown flowers 0.431 mg g⁻¹ dry weight). Hypericins are absent in the pistil. The flavonoids hyperoside and isoquercitrin, the content of which decreased during ontogenetic phase of flowering, reach the highest contents in the pistil.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.