Isolation of 6-hydroxy-L-tryptophan from the fruiting body of Lyophyllum decastes for use as a tyrosinase inhibitor

ABSTRACT

Tyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastes inhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the N^α-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver–Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC₅₀ values of 6-hydroxy-L-tryptophan was 0.23 mM.     

Nω-Hydroxyamino-α-amino acids as a new class of very strong inhibitors of arginases

 The effects of various compounds bearing an N-OH group such as N-hydroxy-guanidines, amidoximes, and hydroxylamines, on bovine and rat liver arginases was studied. Some of these compounds with an l-α-amino acid function at an appropriate distance from the N-OH group acted as strong competitive liver arginase inhibitors, displaying Ki values between 4 and 150 μM. Two compounds, N ^ε-hydroxy-l-lysine and N ^ω-hydroxy-d,l-indospicine, which exhibited Ki values of 4 and 20 μM (at pH 7.4), were the most potent inhibitors of arginase described to date. The distance between the α-amino acid and N-OH functions appeared to be crucial for potent inhibition of arginase, as N ^δ-hydroxy-l-ornithine, which has one -CH₂ group less than N ^ε-hydroxy-l-lysine, exhibited a 37-fold higher Ki value than N ^ε-hydroxy-l-lysine. Based on these results, a model for the interaction of N ^ω-hydroxyamino-l-α-amino acids with the arginase active site is proposed. This model involves the binding of the N-OH group of the inhibitors to the arginase Mn(II) center and suggests that N ^ε-hydroxy-l-lysine is a good transition state analog of arginase.     

New Amides from Buckwheat Seeds (Fagopyrum esculentum Moench)

Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentum Moench. One of them called BN-II is composed of salicylamine and allo-4-hydroxy-l-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and l-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2′-hydroxybenzyl)-allo-4-hydroxy-l-glutamine and N⁵-(4′-hydroxybenzyl)-l-glutamine respectively from their chemical and spectrometry properties.     

Structure of acinetoferrin,a new citrate-based dihydroxamate siderophore fromAcinetobacter haemolyticus

From low-iron cultures of Acinetobacter haemolyticus ATCC 17906, a new hydroxamate siderophore was purified by XAD-7 adsorption followed by preparative thin layer chromatography. The siderophore, named acinetoferrin, released citric acid, 1,3-diaminopropane and (E)-2-octenoic acid upon hydrolysis with HCl, reductive hydrolysis with HI and oxidation with periodate, respectively. Structure elucidation by a combination of NMR spectroscopy and positive fast atom bombardment mass spectrometry revealed that acinetoferrin is a derivative of citric acid, both of its terminal carboxyl groups being symmetrically amide-linked with the 1-amino-3-(N-hydroxy-N-2-octenylamino)propane residues. The (E)-2-octenoic acid is novel as a component of the siderophores.     

Thermodynamics of site-specific small molecular ion interactions with DNA duplex: a molecular dynamics study

The stability and dynamics of a double-stranded DNA (dsDNA) is affected by the preferential occupancy of small monovalent molecular ions. Small metal and molecular ions such as sodium and alkyl ammonium have crucial biological functions in human body, affect the thermodynamic stability of the duplex DNA and exhibit preferential binding. Here, using atomistic molecular dynamics simulations, we investigate the preferential binding of metal ion such as Na⁺ and molecular ions such as tetramethyl ammonium (TMA⁺) and 2-hydroxy-N,N,N-trimethylethanaminium (CHO⁺) to double-stranded DNA. The thermodynamic driving force for a particular molecular ion-DNA interaction is determined by decomposing the free energy of binding into its entropic and enthalpic contributions. Our simulations show that each of these molecular ions preferentially binds to the minor groove of the DNA and the extent of binding is highest for CHO⁺. The ion binding processes are found to be entropically favourable. In addition, the contribution of hydrophobic effects towards the entropic stabilisation (in case of TMA⁺) and the effect of hydrogen bonding contributing to enthalpic stabilisation (in case of CHO⁺) have also been investigated.     

Synthesis of Karahanaenone Derivatives and Their Inhibition Properties toward Tyrosinase and Superoxide Scavenging Activity†

Fourteen amides condensing with aminophenols, anisidines, or aniline were synthesized from karahanaenone 1 as the starting material. The tyrosinase inhibitory activity and superoxide scavenging activity of these derivatives were examined in order to develop whitening agents for cosmetics. Of the compounds, N-p-hydroxyphenyl-1,3,3,6-trimethyl-5-cyclohepten-2-on-1-carboxamide 9, 2-hydroxy-N-o-hydroxyphenyl-3,3,6-trimethyl-5-cyclohepten-1-carboxamide 13, and 2-hydroxy-N-p-hydroxyphenyl-3,3,6-trimethyl-5-cyclohepten-1-carboxamide 15 showed strong tyrosinase inhibitory activity. 13 and 5 possessed a hydroxy group in the karahana skeleton and on the aromatic ring, respectively. These inhibitory rates were higher than that of arbutin that is used for commercial cosmetics (77.4%, 73.6%, and 72.3% against 63.0% for arbutin). Furthermore, 13 indicated 51.0% for superoxide scavenging activity.     

Structural versatility of peptides from Cα,α-dialkylated glycines. I. A conformational energy computation and x-ray diffraction study of homo-peptides from Cα,α-diethylglycine

Conformational energy computations on a derivative and a homo-dipeptide of C^α,α-diethylglycine were performed. In both cases the N- and C-terminal groups are blocked as acetamido and methylamido moieties, respectively. It was found that the C^α,α-diethylglycine residues are conformationally restricted and that the minimum energy conformation corresponds to the fully extended C₅ structure when the N? C^α? C′ bond angle is smaller than 108° (as experimentally observed). The results of the theoretical analysis are in agreement with the crystal-state structural propensity of the complete series of N-trifluoroacetylated homo-peptides of this C^α,α-dialkylated residue from monomer to pentamer, determined by x-ray diffraction and also described in this work. Interestingly, for the first time, a crystallographically planar peptide backbone was observed (in the protected tripeptide). A comparison with peptides of C^α,α-dimethylglycine, C^α-methyl, C^α-ethylglycine, and C^α,α-di-n-propylglycine indicates that the fully extended conformation becomes more stable than the helical structures when both amino acid side-chain C^β atoms are substituted.     

Nucleosides and Nucleotides. 192. Toward the Total Synthesis of Cyclic ADP-Carbocyclic-Ribose. Formation of the Intramolecular Pyrophosphate Linkage by a Conformation-Restriction Strategy in a Syn-form Using a Halogen Substitution at the 8-Position of the Adenine Ring

Abstract

The synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was investigated. Construction of the 18-membered backbone structure was successfully achieved by condensation of the two phosphate groups of 19, possibly due to restriction of the conformation of the substrate in a syn-form using an 8-chloro substituent at the adenine moiety. SN2 reactions between an optically active carbocyclic unit 8, which was constructed by a previously developed method, and 8-bromo-N ⁶-trichloroacetyl-2′,3′-O-isopropylideneadenosine 9c gave N-1-carbocyclic derivative, which was deprotected to give 5′,5′-diol derivatives 18. When 18 was treated with POCl₃ in PO(OEt)₃, the bromo group at the 8-position was replaced to give N-1-carbocyclic-8-chloroadenosine 5′,5′-diphosphate derivative 19 in 43% yield. Treatment of 19 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride gave the desired intramolecular condensation product 20 in 10% yield. This is the first chemical construction of the 18-membered backbone structure containing an intramolecular pyrophosphate linkage of a cADPR-related compound with an adenine base.     

Divers Stereoisomers of N-Acetylhydroxyprolinol as Sugar Substitute in Oligonucleotides

Abstract

A detailed comparison of the hybridisation characteristics of oligonucleotides with 4-hydroxy-N-acetylprolinol or 3-hydroxy-N-acetylprolinol as sugar substitute, reveals dramatic differences. The 4-HO oligonucleotides are able to form stable complexes which are in general duplexes when natural nucleic acids are used as the complement, and the system has a strong preference for isochiral interaction. For the 3-HO oligonucleotides on the other hand complexes are generally weak, triple stranded, and often isochiral and heterochiral hybrids have a similar stability.     

Basidiochrome – A Novel Siderophore of the Orchidaceous Mycorrhizal Fungi Ceratobasidium and Rhizoctonia spp.

A novel trishydroxamate siderophore, named basidiochrome, was isolated as the principal siderophore from low-iron culture filtrates of Ceratobasidium and Rhizoctonia species which are known as mycorrhizal fungi associated with orchid roots. Ion-exchange chromatography and preparative HPLC yielded a pure compound which contained two components according to GC–MS analysis: l-N⁵-hydroxy-ornithine and 3-methyl-2-cis-pentenedioic acid (3-methyl-cis-glutaconic acid). FTICR-ESI-MS of both the iron-free and ferric form indicated an elemental composition of C₃₃H₄₇N₆O₁₆Fe (MW = 839) for the ferric form of basidiochrome. The connectivity was further elucidated by 2D-NMR techniques (HSQC, HMBC, COSY, NOESY) indicating that basidiochrome is a novel linear tripeptide consisting of three l-N⁵-hydroxy-ornithines each linked to 3-methyl-2-cis-pentenedioic acid residues.     

Synthesis of sequential oligopeptides and polypeptide having the sequence of L-alanyl-L-leucylglycine

A series of sequential oligopeptides having simple nonpolar side chains, Nps-(L -Ala-L -Leu-Gly)_n- OEt has been prepared by a stepwise fragment-condensation method using Nps-L -alanyl-L -leucylglycine N-hydroxysuccinimide ester, which was prepared by the Nps-N-carboxy α-amino-acid-anhydride method. The success of the synthesis of the peptide having a high-molecular weight, such as octadecapeptide, results from the highest solubility of the tripeptide unit, L -alanyl-L -leucylglycine. The sequential polypeptide having the same tripeptide sequence was also prepared by polycondensation of the tripeptide N-hydroxy-succinimide ester.     

A new non-natural arginine-like amino acid derivative with a sulfamoyl group in the side-chain

Sulfamoylation of the l-ornithine methyl ester side-chain generates a non-natural arginine isostere which can be coupled with N-Fmoc-l-proline to synthesize analogues which maintain the structural characteristics of the biologically important Pro-Arg dipeptide sequence. As a probe of its biological importance, the sulfamoylated amino acid derivative was also incorporated as P1 residue in tripeptide structures matching the C-terminal subsequence of fibrinogen. The reported results demonstrate that the functionalization of l-ornithine side-chain with a neutral sulfamoyl group can generate an arginine bioisostere which can be used for the synthesis of prototypes of a new class of human thrombin inhibitors.     

Mutational spectrum of N-hydroxy-N-acetyl-4-aminobiphenyl at exon 3 of the HPRT gene

4-Aminobiphenyl is a human bladder carcinogen present in many environmental sources, including cigarette smoke. It can be metabolized in two steps to the mutagen N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP). In this study the mutational spectrum of N-OH-AABP-exposed human lymphoblastoid cells (TK6) was determined using HPRT     

Synthesis of a Highly Substituted N-Linked Immobilized NAD Derivative Using a Rapid Solid-Phase Modular Approach: Suitability for Use with the Kinetic Locking-on Tactic for Bioaffinity Purification of NAD-Dependent Dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD⁺-dependent dehydrogenases. Specifically, the synthesis of highly substituted N⁶-linked immobilized NAD⁺ derivatives is described using a rapid solid-phase modular approach. Other modifications of the N⁶-linked immobilized NAD⁺ derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 Å) in an attempt to minimize nonbiospecific interactions. Analysis of the N⁶-linked NAD⁺ derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S⁶-linked immobilized NAD⁺ derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. -phenylalanine dehydrogenase ( -PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and -phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD⁺-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S⁶-linked immobilized cofactor derivatives than for the new N⁶-linked derivatives. In contrast, the NAD⁺-dependent phenylalanine dehydrogenase showed no affinity for the S⁶-linked immobilized NAD⁺ derivative, but was locked-on strongly to the N⁶-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N⁶-linked immobilized NADP⁺ derivative in the presence of -phenylalanine. This differential locking-on of NAD⁺-dependent dehydrogenases to N⁶-linked and S⁶-linked immobilized NAD⁺ derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD⁺ immobilized via N⁶-linkage as compared to thiol-linkage.     

Synthesis and bioactivity of chemotactic tetrapeptides: fMLF-OMe analogues incorporating spacer aminoacids at the lateral positions

A small library of N-For and N-Boc tetrapeptidic analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe), obtained by incorporating three different spacer aminoacids (Gly, βAla and Pro) between the native residues of Met and Leu (N-For- and N-Boc-Met-Xaa-Leu-Phe-OMe; Xaa² series) and Leu and Phe (N-For- and N-Boc-Met-Leu-Xaa-Phe-OMe; Xaa³ series), have been synthesized and examined for their biological activity as agonists and antagonists. Chemotaxis, lysozyme release and superoxide anion production have been measured. All the N-For analogues maintain good to moderate chemotactic activity with the βAla³ 15 model reaching the maximum value. All the N-Boc tetrapeptides are efficient chemotactic antagonists. Conversely, with the exception of the moderate antagonistic activity exhibited by the N-Boc Xaa² models against lysozyme release, all the other N-Boc analogues do not show significant activity against both superoxide anion and lysozyme release.     

Synthesis and Anti-HIV Activity of 5-Fluorocytallene: N-Dimethylaminomethylene as a Facilitating Group in Acetylene → Allene Isomerization

Abstract

The synthesis and biological activity of 5-fluorocytallene (3a) is described. 5-Fluorocytosine (4) was alkylated with 1-benzoyloxy-4-bromo-2-butyne (5) to give N¹-(4-benzoyloxy-2-butyn-1-yl)-5-fluorocytosine (6). Debenzoylation led to N¹-(4-hydroxy-2-butyn-1-yl)-5-fluorocytosine (7a). The latter compound was transformed to the N⁴-dimethylaminomethylene derivative 8 which was isomerized in situ to the corresponding allene 9. Deprotection afforded 5-fluorocytallene (3a). Compound 3a suppressed the infectivity and replication of both laboratory and primary HIV-1 strains in vitro at nontoxic concentrations.     

Synthesis,conformation, and activity of HCO-Met-ΔZ Leu-Phe-Ome,an active analogue of chemotactic N-formyltripeptides

In order to induce a β-turn conformation into the chemotactic linear tripeptide N-formyl-L -methionyl-L -leucyl-L -phenylalanine (fMLP), the new analogue N-formyl-L -methionyl-Δ^Zleucyl-L -phenylalanine methyl ester [ Δ^ZLeu]²f MLP-OMe ( 1 ) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-α,β-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1 . The x-ray analysis shows that 1 adopts in the crystal a type II β-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. ¹H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl₃ solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by f MLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity. © 1993 John Wiley & Sons, Inc.     

An Improved Synthesis of N6-(6-Aminohexyl)FAD

Abstract

We report an improved synthesis of N ⁶-(6-aminohexyl)FAD (1) using an efficient one-pot conversion of inosine to the N-trifluoroacetyl protected N ⁶-(6-aminohexyl)adenosine 3. The 5′-O-phosphorylated AMP derivative 4, activated as the imidazolide, was coupled with commercial sodium riboflavin phosphate by using 18-crown-6 in DMF.     

Deacetylation ofN-acetylthienamycin to thienamycin by a cell-free extract ofStreptomyces cattleya,the thienamycin producer

Summary A cell-free extract from the thienamycin producer,Streptomyces cattleya, has been found to deacetylate the co-product,N-acetylthienamycin. The pH optimum of the reaction is 7.5. Due to the lability ofN-acetylthienamycin, we used thed andl forms of the synthetic substrateN-chloroacetylvaline. We found that the enzyme is anl-deacetylase, has a molecular weight of 58 000, is stable up to 40°C, acts optimally at 45°C, is stable at pH 5–8, is not activated by divalent metal ions and is inhibited by Hg⁺⁺, Cu⁺⁺ andp-chloromercuribenzoate. This is the first report of an extract from a carbapenem producer which carries out the deacetylation ofN-acetylthienamycin, suggesting that the acetylated derivative is a precursor of thienamycin.Abbreviations THM thienamycin - N-AcTHM N-acetylthienamycin - CFE cell-free extract - N-Cl-Ac-l-Val N-chloroacetyl-l-valine - N-Cl-Ac-d-Val N-chloroacetyl-d-valine     

DISTRIBUTION OF FOLIC ACID COENZYMES AND FOLATE DEPENDENT ENZYMES IN MOUSE BRAIN1

—Folic acid coenzymes were found to be distributed equally between post-nuclear particulate and soluble fractions from whole Swiss mouse brain. Mitochondria isolated from the particulate fraction contained essentially only the N⁵-methyl derivative of folate, virtually all of which was in a polyglutamate form. Isolated synaptosomes contained significantly more folate than did mitochondria, with the greater proportion being non-N⁵-methyl derivatives. Osmotic lysis of synaptosomes released only a small portion of the folate; approximately 80 per cent remained with the particulate components of the synaptosome. The enzymes serine transhydroxymethylase and N⁵, N¹⁰-methylenetetrahydrofolate dehydrogenase were found in both the soluble and particulate fractions while formiminoglutamic acid:tetrahydrofolate formiminotransferase activity could not be detected. These findings may be of importance with respect to the synaptic functions of folate coenzymes, including the methylation of biogenic amines.