Search

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetyl-protected natural aromatic α-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotected ones. The N-acetylated TFMD phenylalanine derivative afforded slightly higher H/D exchange than unprotected derivatives. An effective post-deuteration method for cross-linkable α-amino acid derivatives will be useful for the analysis of biological functions of bioactive peptides and proteins by mass spectrometry.

The Friedel-Crafts reaction with 3-(3-methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine and optically active N-TFA-Asp(Cl)-OMe in triflic acid afforded homophenylalanine derivatives without any loss of the optical purity.

As a result of recent developments in molecular biology, the investigation of biofunctional machinery at ligand-accepting interfaces has become one of the challenging and important subjects in the post-genome field. The technique of photoaffinity labeling has become increasingly appreciated as a powerful methodology for this purpose. This microreviewfocuses on the synthesis of 3-phenyl-3-(trifluoromethyl)diazirine, one of the most promising photophores, and its application to biomolecules.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Photoaffinity labeling enables the direct probing of a target protein through a covalent bond between a ligand and its binding protein, and even a complex formed by weak interactions can be isolated by the method. The photochemical fishing approach accelerates the throughput, isolating crosslinked complexes and analyzing the structure of the ligand binding site within the protein. We used carbene-generating phenyldiazirine for this approach because practical examinations had shown that the phenyldiazirine functioned as the powerful barb on the hook. Improving the synthetic pathways of the photoprobes and using chemoselective-integrated photoreactive units makes possible the easy and rapid preparation of carbene-generating photoreactive probes including the derivatives in peptides, proteins, DNAs, and carbohydrates. This review also shows several recent impacts of photoaffinity labeling, including the in vivopreparation of photoreactive proteins in living cells.

In a model system of a water-soluble hemin, tetrakis(4-methylpyridyl)porphinatoiron(III) pentachloride (FeTMPyPCl5), with 3-methyl-2-benzothiazolinone hydrazone (MBTH), superoxide anion radical (O2•-), hydrogen peroxide, and then hydroxyl radical were generated in a phosphate buffer solution (pH 6.0–7.5) under aerobic conditions. The hemin was degraded in this system. The reactive oxygen species were deduced from inhibition of the degradation by adding SOD and catalase, and one of them was trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a DMPO–hydroxyl radical adduct, which was detected by ESR. The mechanism for activating dioxygen and the degradation of hemin was examined, and it was proposed to be the initial one-electron reduction of hemin by MBTH forming a dioxygen complex and liberating superoxide as one of the routes during the reduction–autoxidation cycle in aqueous media.

Sophoragrin, a mannose/glucose-specific lectin in Sophora japonica (Japanese pagoda tree) bark, was the first lectin found to show self-aggregation that is dependent on the sugar concentration accompanying the interconversion between solubility and insolubility [Ueno, Ogawa, Matsumoto and Seno (1991) J. Biol. Chem. 266, 3146–3153]. The interconversion is regulated by the concentrations of Ca2+ and specific sugars: mannose, glucose or sucrose. The specific glycotopes for sophoragrin were found in the sophoragrin subunit and an endogenous galactose-specific lectin, B-SJA-I (bark S. japonica agglutinin I), and the lectin subunit that binds to the glycotope was identified by photoaffinity glycan probes. Remarkably, the insoluble polymer of sophoragrin is dissociated by interaction with B-SJA-I into various soluble complexes. Based on these results, self-aggregation of sophoragrin was shown to be a unique homopolymerization due to the sugar-specific interaction. An immunostaining study indicated that sophoragrin localizes mainly in vacuoles of parenchymal cells coincidently with B-SJA-I. These results indicate that sophoragrin can sequester endogenous glycoprotein ligands via sugar-specific interactions, thus providing new insights into the occurrence and significance of the intravacuolar interaction shown by a legume lectin.

The GLUT5 transporter catalyses the specific uptake of d-fructose and can accept this hexose in its furanose and pyranose ring forms. The transporter does not accept fructose epimers and has very limited tolerance of bulky groups substituted at the 2-, 3-, 4- and 5-OH positions [Tatibouët, Yang, Morin and Holman (2000) Bioorg. Med. Chem. 8, 1825—1833]. To further explore whether bulky groups can be tolerated at the primary OH positions, a d-fructose analogue with an allylamine group substitution to replace the 1-OH group was synthesized and was found to be quite well tolerated (Ki = 27.1mM). However, this analogue occurs in multiple ring forms. By contrast, 2,5-anhydro-d-mannitol is a symmetrical molecule that occurs only in a furanose ring form in which C-1 and C-6 are equivalent. We have therefore synthesized new 2,5-anhydro-d-mannitol analogues (substituted at the equivalent of the 6-OH of d-fructose) and from studies in Chinese hamster ovary cells expressing GLUT5 cells report that (i) the allylamine derivative of 2,5-anhydro-d-mannitol is well tolerated (Ki = 2.66mM); (ii) introduction of a di-nitrophenyl-substituted secondary amine group enhances affinity (Ki = 0.56mM); (iii) introduction of amide-linked biotinylated photolabel moieties is possible without loss of affinity relative to 2,5-anhydro-d-mannitol but a small secondary amine spacer between the biotinylated photolabelling moiety and the fructofuranose ring increases affinity (fructose photolabel 2; Ki = 1.16mM); (iv) introduction of a hydrophilic tartarate spacer between biotin and the diazirine photoreactive groups can be accomplished without reduction in affinity and (v) photoactivation of biotinylated fructose photolabels leads to specific biotin tagging of GLUT5. These data suggest that substitution of a secondary amine group (-NH) to replace the C-6 (or C-1) -OH of 2,5-anhydro-d-mannitol results in compounds of high affinity; the affinity is enhanced over 10-fold compared with d-fructose.

During a chemical study of Indonesian medicinal plants, we examined the constituents of fruits of Helicteres isora L. (Sterculiaceae), one of the famous Jamu medicines. From a H2O extract of the fruits, we isolated six new neolignans, the helicterins A – F (1 – 6), and elucidated their structures by spectral analyses. Helicterins A – F (1 – 6) are dimeric (7.5',8.2')-neolignans with a bicyclo[2.2.2]octene C-framework, and showed mild inhibitory activity against reverse transcriptase from avian myeloblastosis virus.

During a chemical study of Indonesian medicinal plants, we examined the constituents of fruits of Helicteres isora L. (Sterculiaceae), one of the famous Jamu medicines. From a water extract of the fruits, we isolated three new neolignans, helisterculins A (1) and B (2) and helisorin (3), and elucidated their structures by spectral analyses. Helisterculins A (1) and B (2) are (7.5',8.2')-neolignans with a bicyclo[2.2.2]octene C-framework, while helisorin (3) is a (6.4',7.5',8.2')-neolignan with a very rare 4,4a,9,9a-tetrahydro-3,9-methano-3H-fluorene C-framework. The natural product with the latter C-framework has no literature precedent. The neolignans 1 – 3 showed weak inhibitory activity against reverse transcriptase from avian myeloblastosis virus.

Three photoreactive derivatives of μ-conotoxin G IIIA have been prepared as photoaffinity labeling reagents for muscle-type sodium channels. The reagents competitively inhibited the binding of saxitoxin to the eel sodium channel with Kivalues of 11–18 nM. The introduced chromogenic phenyldiazirine group on the toxin was photolyzed efficiently, and spectroscopic properties of the reagents demonstrated that irradiation and detection can be performed in a spectral region where the absorptions due to most of biological macromolecules are negligible.

Abstract(4R)-Musclide-A2 and (2R,5R)- and (2S,5R)-musclide-B were synthesized from L-leucine and their enantiomers from D-leucine. Comparisons of spectral and [α]Ddata of synthetic and natural musclides indicated that the absolute configuration at C-4 of natural musclide-A2 and that at C-5 of natural musclide-B should be 4S. Thus, the structures of natural musclides-A2 and -B are revised to (4S)-1 -hydroxy-6-methylhept-4-yl hydrogen sulfate and (2R,5S)-2-hydroxy-7-methyloct-5-yl hydrogen sulfate, respectively.

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxianawere obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor'sα, β, γ, orδsubunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.

New cell-impermeant bis-mannose photolabels have been developed with biotinyl groups attached to 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) by either a polyethoxy spacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, Ki values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359±10 and 273±28 μM for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respectively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced cross-linking of the biotinylated photolabels to rat adipocytes, the biotinylated glucose transporter isoform 4 (GLUT4) could be detected by non-radioactive and radioactive methods that utilized the interaction with streptavidin. Biotinylated GLUT4 from 1-2 μg of adipose cell membranes, precipitated onto magnetic streptavidin beads, could be sensitively and quantitatively detected using an electrochemiluminescent assay method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent signal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 could be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotting. Data obtained using the non-radioactive methods compared favourably with those using tritiated versions of the biotinylated probes. Insulin treatment of adipocytes increased the levels of signals from surface biotinylated GLUT4 by ~ 10-fold or ~ 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.

Three photoreactive derivatives of μ‐conotoxin G IIIA have been prepared as photoaffinity labeling reagents for muscle‐type sodium channels. The reagents competitively inhibited the binding of saxitoxin to the eel sodium channel with Kivalues of 11–18 nM. The introduced chromogenic phenyldiazirine group on the toxin was photolyzed efficiently, and spectroscopic properties of the reagents demonstrated that irradiation and detection can be performed in a spectral region where the absorptions due to most of biological macromolecules are negligible.

(4R)-Musclide-A2 and (2R,5R)- and (2S,5R)-musclide-B were synthesized from L-leucine and their enantiomers from D-leucine. Comparisons of spectral and [α]D data of synthetic and natural musclides indicated that the absolute configuration at C-4 of natural musclide-A2 and that at C-5 of natural musclide-B should be 4S. Thus, the structures of natural musclides-A2 and -B are revised to (4S)-1 -hydroxy-6-methylhept-4-yl hydrogen sulfate and (2R,5S)-2-hydroxy-7-methyloct-5-yl hydrogen sulfate, respectively.

A molecular docking study has been performed on the interaction of β1,4-galactosyltransferase with an acceptor site photoprobe. This is based on an acceptor site peptide fragment which was recently identified by the use of a photoprobe. The present model strongly suggests that the carboxylate group of Asp318 could be involved in the activation of the acceptor sugar 4-OH for the efficient galactosyltransfer. The result also exemplified that the combination of photoaffinity labeling with crystallography is a powerful method for the detailed structural analysis of ligand–protein complex.

The photolytic decomposition of trifunctional carbene generating photoaffinity probes in methanolic solution was studied, a cleavage reaction with butylamine in water, the conjugation with a ligand (moenomycin), and experiments that demonstrate that the fully armed probes interact with penicillin-binding protein 1b.

Deuterium incorporation into diazirinyl acetophenone derivative (I) proceeds at the α‐position of the carbonyl group without decomposition of the diazirinyl ring.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.