Identification of the Enzymes Mediating the Maturation of the Seryl‐tRNA Synthetase Inhibitor SB‐217452 during the Biosynthesis of Albomycins

Albomycin δ₂ is a sulfur‐containing sideromycin natural product that shows potent antibacterial activity against clinically important pathogens. The l ‐serine‐thioheptose dipeptide partial structure, known as SB‐217452, has been found to be the active seryl‐tRNA synthetase inhibitor component of albomycin δ₂. Herein, it is demonstrated that AbmF catalyzes condensation between the 6′‐amino‐4′‐thionucleoside with the d ‐ribo configuration and seryl‐adenylate supplied by the serine adenylation activity of AbmK. Formation of the dipeptide is followed by C3′‐epimerization to produce SB‐217452 with the d ‐xylo configuration, which is catalyzed by the radical S‐adenosyl‐l ‐methionine enzyme AbmJ. Gene deletion suggests that AbmC is involved in peptide assembly linking SB‐217452 with the siderophore moiety. This study establishes how the albomycin biosynthetic machinery generates its antimicrobial component SB‐217452.     

An efficient synthetic method for Z-fluoroalkene dipeptide isosteres: Application to the synthesis of the dipeptide isostere of Sta-Ala

Reaction of γ,γ-difluoro-α,β-enoates having a δ-hydroxyl group with trialkylaluminum (R₃Al) was found to be promoted by CuI·2LiCl and to proceed in SN2′ manner giving rise to the α-alkylated (Z)-γ-fluoro-β,γ-enoates, while reductive defluorination of γ,γ-difluoro-α,β-enoates with Me₂CuLi followed by reaction with alkyl halides provided the corresponding (Z)-α-alkylated products in high yields. The latter reaction was applied to the preparation of the dipeptide (Z)-fluoroalkene isostere of Sta-Ala, which is the central dipeptide unit in Pepstatin, a natural inhibitor of aspartate proteases.     

Larval Tenebrio molitor (Coleoptera: Tenebrionidae) Fat Body Extracts Catalyze Firefly D-Luciferin- and ATP-Dependent Chemiluminescence: A Luciferase-like Enzyme*

Ultraweak light emission was detected upon injection of firefly luciferin into live Tenebrio larvae. A chemilumi-nescent enzymatic activity dependent on molecular oxygen, D-luciferin and MgATP was then isolated from larval fat body extracts by precipitation with 70% ammonium sulfate. D-Luciferin and ATP can be replaced by luciferyl-adenylate. Pyrophosphate is a main product from the chemiluminescent reaction. The in vitro chemiluminescence intensity was not affected by peroxidase inhibitors such as N₃^?_- (0.5 mM) and CN^? (1 mM), attesting to its nonperoxidatic nature but was strongly inhibited by AMP (1 mM), luciferin 6′-ethyl ether (1 mM) and sodium pyrophosphate (2 mM), well-known firefly lucifer-ase inhibitors. Some physical-chemical properties of this enzymatic activity were similar to those of firefly lucif-erase (K_MATP = 195 μM; K_0.5 luciferin - 0.8 mM; optimum pH 8.5; δ_max= 610 nm at pH 8.5; firefly lucifer-ase: δ_max= 565 nm at pH 8.0 and 619 mm at pH 6.0), but the chemiluminescence was not affected by addition of polyclonal antibodies raised against Photinus pyralis luciferase. These data suggest that this chemiluminescence results from a ligase with luciferase activity.     

Die absolute Konfiguration der 3,5-Diaminohexansäure aus der β-Lysin-Mutase-Reaktion

Absolute configuration of the 3,5-diaminohexanoic acid produced in the β-lysine mutase reaction The (3S, 5S)-configuration of the 3,5-diaminohexanoic acid 3 produced by the coenzyme-B₁₂-dependent β-lysine mutase from Clostridium sticklandii has been determined by two different methods: by comparison of the ¹H-NMR.-spectrum of its δ-lactam with that of synthetic (±)-cis-and (±)-trans-4-amino-6-methyl-piperidones ( 1 and 2 ) and by chemical correlation with (+)-(6S)-6-methyl-piperidone-2 ( 9 ).     

On the Purported Structure of Cyclodeca[1,2,3-de: 6,7,8-d′e′]dinaphthalene

The reaction of the bis(sulfonium salt) 7 in a solution of Na₂CO₃ in H₂O/EtOH yielded three main products 8 – 10 . The spectroscopic data of 8 were identical to those which led Mithcell and Sondheimer to assign them to cyclodeca[1,2,3-de: 6,7,8-d′e′]dinaphthalene ( 3 ). Our investigations show, however, that the correct structural assignment leads to the structure of 7,7a-dihydrodibenzo[de,mn]naphthacene ( 8 ).     

Phosphoramidites of Chiral (Rp)-and (Sp)-Configurated d(T[P-18O]-A): Synthesis,Configurational Assignment,and Use as Dimer Blocks in Oligonucleotide Synthesis

The N,N-diisopropylphosphoramidites 10a and 10b of appropriately protected chiral diastereoisomers of d(T[P-¹⁸O]-A) ( 8a and 8b , resp.), chiral by virtue of the isotope ¹⁸O at the P-atom, have been synthesized. The ¹⁸O-isotope was incorporated by oxidation of the phosphite triester 3 with H₂[¹⁸O]/I₂. Separation of the diastereoisomers was accomplished by flash chromatography of the O-3′-deprotected phosphate triesters 5a/b . The absolute configuration at the chiral P-atom was deduced from the methylation products of the fully deprotected diastereoisomers 8a and 8b . Phosphinylation of 5a and 5b yielded the configurationally pure phosphoramidites 10a and 10b , respectively, which were then employed in solid-phase synthesis to yield the self-complementary oligomers d(G-A-G-T-(R_p)-[P-¹⁸O]-A-C-T-C) ( 13 ) and d(G-A-G-T-(S_P)-[P-¹⁸O]-A-C-T-C) ( 14 ), respectively.     

Nucleotides. Part XLV. Synthesis of new (2′–5′)adenylate trimers,containing 5′-amino-5′-deoxyadenosine residues at the 5′-end of the oligoadenylate chain,and of its analogues,carrying a 9-[(2-hydroxyethoxy)methyl]adenine residue at the 2′-terminus

The 5′-amino-5′-deoxy-2′,3′-O-isopropylideneadenosine ( 4 ) was obtained in pure form from 2′,3′-O-isopropylideneadenosine ( 1 ), without isolation of intermediates 2 and 3 . The 2-(4-nitrophenyl)ethoxycarbonyl group was used for protection of the NH₂ functions of 4 (→7) . The selective introduction of the palmitoyl (= hexadecanoyl) group into the 5′-N-position of 4 was achieved by its treatment with palmitoyl chloride in MeCN in the presence of Et₃N (→ 5 ). The 3′-O-silyl derivatives 11 and 14 were isolated by column chromatography after treatment of the 2′,3′-O-deprotected compounds 8 and 9 , respectively, with (tert-butyl)dimethylsilyl chloride and 1H-imidazole in pyridine. The corresponding phosphoramidites 16 and 17 were synthesized from nucleosides 11 and 14 , respectively, and (cyanoethoxy)bis(diisopropylamino)phosphane in CH₂Cl₂. The trimeric (2′–5′)-linked adenylates 25 and 26 having the 5′-amino-5′-deoxyadenosine and 5′-deoxy-5′-(palmitoylamino)adenosine residue, respectively, at the 5′-end were prepared by the phosphoramidite method. Similarly, the corresponding 5′-amino derivatives 27 and 28 carrying the 9-[(2-hydroxyethoxy)methyl]adenine residue at the 2′-terminus, were obtained. The newly synthesized compounds were characterized by physical means. The synthesized trimers 25–28 were 3-, 15-, 25-, and 34-fold, respectively, more stable towards phosphodiesterase from Crotalus durissus than the trimer (2′–5′)ApApA.     

Synthesis of porphyrinyl-nucleosides

Several porphyrinyl-nucleosides were prepared in the reaction of the OH group of one, two or four meso-p-hydroxyphenyl substituents of porphyrin with 5′-O-tosylates of 2′,3′-O-isopropylidene-adenosine or -uridine, or 5′-O-tosylthymidine; the remaining porphyrin meso-substituents were p-tolyl, p-hydroxyphenyl or 4-pyridyl. The following porphyrinyl-nucleosides were obtained with 8–17% yield: meso-di(p-tolyl)di(p-phenylene-5′-O-2′,3′-O-isopropylidene-adenosine) (or -uridine)porphyrins 1,2 , the respective meso-tetranucleosideporphyrins 3,4 -meso-mono(p-phenylene-5′-O-thymidine)porphyrins 5–7 , meso-di(p-tolyl)di(p-phenylene-5′-O-thymidine)porphyrins 8,9 and the meso-di(p-hydroxyphenyl)di(p-phenylene-5′-O-thymidine)porphyrins 10. Other compounds prepared belonged to the series: meso(4-pyridyl)_4?n(p-phenylene-5′-O-2′,3′-O-isopropylideneuridine)_nporphyrin, n = 1, 2 or 4, 11–13. N-Methylation gave the water soluble iodide salts: (N-methyl-4-pyridinium)4_4?n(p-phenylene-5′-O-2′,3′-isopropylideneuridine)_nporphyrins, n = 1, 2 or 4, 14–16. The ms fab showed in most cases stepwise detachment of the CH₂(5′)-nucleoside fragments. The porphyrins meso disubstituted by thymidine represent a convenient substrate for the build-up of both nucleoside units into the oligo/polynucleotide chains.     

2′-Deoxy-β-D-ribofuranosides of N6-Methylated 7-Deazaadenine and 8-Aza-7-deazaadenine: Solid-phase synthesis of oligodeoxyribonucleotides and properties of self-complementary duplexes

The syntheses of 7-deaza-N⁶-methyladenine N⁹-(2′-deoxy-β-D -ribofuranoside) ( 2 ) as well as of 8-aza-7-deaza-N⁶-methyladenine N⁸? and N⁹?(2′-deoxyribofuranosides) ( 3 and 4 , resp.) are described. A 4,4′-dimeth-oxylritylation followed by phosphitylation yielded the methyl phosphoramidites 12–14 . They were employed together with the phosphoramidite of 2′-deoxy-N⁶v-methyladenosine ( 15 ) in automated solid-phase oligonucleotide synthesis. Alternating or palindromic oligonucleotides derived from d(A-T)₆ or d(A-T-G-C-A-G-A*-T-C-T-G-C-A) but containing one methylated pyrrolo[2,3-d]pyrimidine or pyrazolo[3,4-d]pyrimidine moiety in place of a N⁶-methylaminopurine (A*) were synthesized. Melting experiments showed that duplex destabilization induced by a N⁶-Me group of 2′-deoxy-N⁶-methyladenosine is reversed by incorporation of 8-aza-7-deaza-2′-deoxy-N⁶-meihyladenosine, whereas 7-deaza-2′-deoxy-N⁶-methyladenostne decreased the T_m value further. Regiospecific phosphodiester hydrolysis of d(A-T-G-C-A-G-m⁶A-T-C-T-G1-C-A) by the endodeoxyribonuclease Dpn I, yielding d(A-T-G-C-A-G-m⁶A) and d(pT-C-T-G-C-A), was prevented when the residue c⁷m⁶A_d ( 2 ), c⁷m⁶z⁸A_d ( 3 ), or c⁷m⁶z⁸A_d′ ( 4 ) replaced m⁶A_d ( 1 ) indicating that N(7) of N⁶-methyladenine is a proton-acceptor site for the endodeoxyribonuclease.     

13C nuclear magnetic resonance spectroscopy of selected adenine nucleosides: Structural correlation and conformation about the glycosidic bond

Structural correlations have been carried out from ¹³C chemical shifts (δ) and by analysis of ¹J(CH) coupling constants, and the conformation about the glycosidic bond has been studied by means of the ³J(CH) vicinal coupling constants between C-8 and H-1′ of some adenine nucleosides such as adenosine (Ado), N(7)-β-D-ribofuranosyladenine (N(7)-Ado), N(9)- and N(7)-β-D-xylofuranosyladenine (N(9)-xylAde and N(7)-xylAde), N(9)-(3-chloro-3-deoxy-β-D-xylofuranosyl)adenine (3′-Cl-xylAde) and N(9)-(2-chloro-2-deoxy-β-D-arabinofuranosyl)adenine (2′-Cl-araAde). The analysis of the influence on δ¹³C of the nature and configuration of the substituent in the carbohydrate fragment of the molecule has revealed two types of effects, namely, 1,2-cis and 1,2-trans. This approach, as well as the ³J(CH) values and the analysis of the C-3′-endo?C-2′-endo equilibrium of the carbohydrate fragment of nucleosides, and circular dichroism (CD) data, provides important information on the conformation about the glycosidic bond. The magnitudes of ³J(C-4, H) are indicative of the position of attachment of the carbohydrate fragment to the heterocyclic base.     

Highly Stereoselective Photochemical Oxidative Addition Reactions. Preliminary Communication

Cyclometallated complexes of the type cis-bis(2-phenylpyridine)platinum(II) (C₂₂H₁₆N₂Pt) and cis-bis(2-(2′-thienyl)pyridine)platinum(II) (C₁₈H₁₂N₂S₂Pt) undergo thermal or photochemical oxidative addition (TOA or POA) reactions with a number of substrates. TOA (with CH₃I, CH₃CH₂I etc.) yield mixtures of several isomers which rearrange slowly (within ca. one week at room temperature) to one of the possible cis-isomers. CH₂Cl₂, CHCl₃, or (E)? ClCH?CHCl, e.g., do not react thermally. POA yield directly complexes of Pt(IV) with the halide and a σ-bonded C-atom in cis-position. The configuration, as assigned by extensive use of ¹H-NMR data, can be characterized for the two chelating ligands C …? N and C′ …? N′ by C,C′-cis; N,N′-cis and C(chelate), Cl-trans.     

Ruthenium(II) Complexes with Three Different Diimine Ligands

The synthesis of tri-heteroleptic complex of Ru(II) with diimine ligands is describe. Ten compounds [Ru(R₂bpy) (biq) (L)][PF₆]₂ (R = H, CH₃); L = 2,2′-bipyridine (bpy), 4,4′-dimethyl-2,2′-bipyridine (Me₂bpy), 2,2′-bipyrimidine (bpm), 2,2′-biisoquinoline (biiq), 1,10-phenanthroline (phen), dipyrido[3,2-c:2′,3′-e]pyridazine (taphen), 2,2′-biquinoline (biq), 6,7-dihydrodipyrido[2,3-b:3,2-j][1,10]-phenanthroline (dinapy), 2-(2[pyridyl)quinoline (pq), 1-(2-pyrimidyl)pyrazole] (pzpm), 2,2′-biimidazole (H₂biim) are characterized by elemental analysis, electronic and ¹H-NMR spectroscopy. The relative photosustitution rates of biq in MeCN are given at three temperatures.     

Selective acylation of ribonucleotides and ribonucleosides with acylimidazoles

Selective acylation of ribonucleotides and ribonucleosides can be achieved by using N-acylimidazole on a preparative scale with good yields (50–80%). For uridine 3′-phosphate (Up): in the presence of MDCAI, the 2′-O-acyl-derivative is the main product, while in the presence of an excess of TEAH, the 5′-O-acyl-derivative is the main product. For ribonucleosides (U_R or A_R or ψ_R): in the presence of MDCAI, the acylations take place preferably at 2′-OH or 3′-OH of ribonucleosides and only 3′-O-acyl-derivatives can be isolated by crystallization; in the presence of an excess of TEAH. 5′-O-acyl-derivative is obtained as the main product. Arabinonucleoside and deoxyribonucleoside are only slowly acylated to form 5′-O-acyl-derivatives as the main products by acylimidazole in the presence of MDCAI. Possible mechanisms of these acylations have been discussed.     

Syntheses,crystal structures,and properties of optically active Lu(III) and Yb(III) complexes of N,N′-bis(2-hydroxybenzyl)-N,N′-bis(2-pyridylmethyl)-R-1,2-propanediamine with chloride or thiocyanate

The reaction of N,N′-bis(2-hydroxybenzyl)-N,N′-bis(2-pyridylmethyl)-R-1,2-propanediamine (R-bbppnH₂) with LnCl₃?6H₂O (Ln: Lu, Yb) stereoselectively gave an optically active complex, [LnCl(R-bbppn)], which crystallizes in the acentric space group of P2₁2₁2₁. The central lutetium(III) is coordinated by two oxygens from two phenolates, four nitrogens from two pyridines, and one bidentate propanediamine of R-bbppn^2?, one chloride to form a seven-coordinate distorted pentagonal bipyramidal geometry. Although two optical isomers, ΔΛΔ and ΛΔΛ, are possible for such a structure, the absolute configuration of the complex is stereoselectively unified to ΔΛΔ. The coordinated chloride of the complex is readily replaced by other ligands, and hence the reaction with thiocyanate results in formation of another seven-coordinate complex, [Ln(NCS)(R-bbppn)].     

Synthesis of 5-Methyluridine and Its 5′-Mercapto-, 2-Amino-, and 4′,5′-Unsaturated Analogues

The stereospecific cis-hydroxylation of 1-(2,3-dideoxy-β-D -glyceropent-2-enofuranosyl)thymine (1) into 1-β-D -ribofuranosylthymine (2) by osmium tetroxide is described. Treatment of 2′,3′-O, O-isopropylidene-5-methyl-2,5′-anhydrouridine (8) with hydrogen sulfide or methanolic ammonia afforded 5′-deoxy-2′,3′-O, O-isopropylidene-5′-mercapto-5-methyluridine (9) and 2′,3′-O, O-isopropylidene-5-methyl-isocytidine (10) , respectively. The action of ethanolic potassium hydroxide on 5′-deoxy-5′-iodo-2′,3′-O, O-isopropylidene-5-methyluridine (7) gave rise to the corresponding 1-(5-deoxy-β-D -erythropent-4-enofuranosyl)5-methyluracil (13) and 2-O-ethyl-5-methyluridine (14) . The hydrogenation of 2 and its 2′,3′-O, O-isopropylidene derivative 4 over 5% Rh/Al₂O₃ as catalyst generated diastereoisomers of the corresponding 5-methyl-5,6-dihydrouridine ( 17 and 18 ).     

1-(2′-Deoxy-β-D-xylofuranosyl)thymine Building Blocks for Solid-Phase Synthesis and Properties of Oligo(2′-Deoxyxylonucleotides)

1-(2′-Deoxy-β-D -threo-pentofuranosyl)thymine (= 1-(2′-deoxy-β-D -xylofuranosyl)thymine; xT_d; 2 ) was converted into its phosphonate 3b as well as its 2-cyanoethyl phosphoramidite 3c . Both compounds were used for solid-phase synthesis of d[(xT)₁₂-T] ( 5 ), representing the first DNA fragment build up from 3′–5′-linked 2′-deoxy--β-D -xylonucleosides. Moreover, xT_d was introduced into the innermost part of the self-complementary dodecamer d(G-T-A-G-A-A-xT-xT-C-T-A-C)₂ (9). The CD spectrum of d[(xT)₁₂–T] ( 5 ) exhibits reversed Cotton effects compared to d(T₁₂) ( 6 ; see Fig. 1), implying a left-handed single strand. With d(A₁₂) ( 7 ) it could be hybridized to form a propably Left-handed double strand d(A₁₂) · d[(xT)₁₂–T] ( 7 · 5 ) which was confirmed by melting experiments in combination with temperature-dependent CD spectroscopy. While 5 was hydrolyzed by snake-venom phosphodiesterase, it was resistant towards calf-spleen phosphodiesterase. The modified, self-complementary duplex 9 was hydrolyzed completely by snake-venom phosphodiesterase, at a twelvefold slower rate compared to unmodified 8 ; calf-spleen phosphodiesterase hydrolyzed 9 only partially.     

Synthesis of [D-alanine, 4'-azido-3',5'-ditritio-L-phenylalanine, norvaline4[alpha-melanotropin as a 'photoaffinity probe' for hormone-receptor interactions (author's transl)]

Synthesis of [D -alanine¹, 4′-azido-3′, 5′-ditritio-L -phenylalanine², norvaline⁴]α-melanotropin as a ‘photoaffinity probe’ for hormone-receptor interactions. The synthesis of an α-MSH derivative containing 4′-azido-3′,5′-ditritio-L -phenylalanine is described: Ac · D -Ala-Pap(³H₂)-Ser-Nva-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val · NH₂. This hormone analogue is being used for specific photoaffinity labelling of receptor molecules. The synthesis was performed in a way to minimize the number of radioactive steps and to introduce the radio-active and the photoaffinity label exclusively into position 2. The dipeptide N(α)-acetyl-D -alanyl- (4′-amino-3′,5′-diiodo)-L -phenylalanine was tritriated and transformed into the azido compound, N(α)-acetyl-D -alanyl-(4′-azido-3′,5′-ditritio)-L -phenylalanine which was then condensed with H · Ser-Nva-Glu(OtBu)-His-Phe-Arg-Trp-Gly-Lys(BOC)-Pro-Val · NH₂ to the tridecapeptide. The α-MSH analog displayed a specific activity of 11 Ci/mmol, and a biological activity of about 4 · 10⁹ U/mmol (10% of α-MSH).     

Applicability of the Mosher MPTA-Ester Methodology to Monosaccharides

Caryophyllose 1 is a novel twelve carbon 4-C-branched monosaccharide and a component of the polysaccharide chains found in the lypopolysaccharide fraction from Pseudormnas caryophylli bacterium.¹ Its absolute stereochemistry was elucidated² by applying the exciton chiral coupling method to two fragments obtained by NaIO₄, oxidation of the polysaccharide chain. The absolute configuration of a chiral secondary alcohol can be defined by Mosher's method.³ It analyzes the signs of the differences between the chemical shifts of the protons vicinal to the chiral Center in the (S)- and (R)-α-methoxy-α-trifluoromethylphenylacetate (MPTA) esters obtained from the compound. However, the Mosher ester methodology failed to give completely reliable results when applied to the bisisopropylidene derivative 2 of caryophyllose. In fact, whereas Δδ_H(δ_R-δ_S) was positive for 3′-H and negative for 5′-H, indicating R configuration for the 4′ chiral centre, it was positive for 1-H but of opposite sign for the two protons at C-3 (negative for 3_eq and positive for 3_ax), failing to indicate the configuration at C-2. This result, however, was in line with Mosher's warning³ about circumspection in applying his correlation to molecules which “contain additional chiral centres, possess heteroatoms, or show unusual conformational restraints”. It also prompted us to an investigation of applicability of Mosher arguments to sugar MPTA esters, in view of our interest in carbohydrate structure elucidation.     

Synthese von diastereo- und enantio-selektiv deuterierten β,ε-, β,β-, β,γ- und γ,γ-Carotinen

Synthesis of Diastereo- and Enantioselectively Deuterated β,ε-, β,β-, β,γ- and γ,γ-Carotenes We describe the synthesis of (1′R, 6′S)-[16′, 16′, 16′-²H₃]-β, εcarotene, (1R, 1′R)-[16, 16, 16, 16′, 16′, 16′-²H₆]-β, β-carotene, (1′R, 6′S)-[16′, 16′, 16′-²H₃]-γ, γ-carotene and (1R, 1′R, 6S, 6′S)-[16, 16, 16, 16′, 16′, 16′-²H₆]-γ, γ-carotene by a multistep degradation of (4R, 5S, 10S)-[18, 18, 18-²H₃]-didehydroabietane to optically active deuterated β-, ε- and γ-C₁₁-endgroups and subsequent building up according to schemes \documentclass{article}\pagestyle{empty}\begin{document}${\rm C}_{11} \to {\rm C}_{14}^{C_{\mathop {26}\limits_ \to }} \to {\rm C}_{40} $\end{document} and C₁₁ → C₁₄; C₁₄+C₁₂+C₁₄→C₄₀. NMR.- and chiroptical data allow the identification of the geminal methyl groups in all these compounds. The optical activity of all-(E)-[²H₆]-β,β-carotene, which is solely due to the isotopically different substituent not directly attached to the chiral centres, is demonstrated by a significant CD.-effect at low temperature. Therefore, if an enzymatic cyclization of [17, 17, 17, 17′, 17′, 17′-²H₆]lycopine can be achieved, the steric course of the cyclization step would be derivable from NMR.- and CD.-spectra with very small samples of the isolated cyclic carotenes. A general scheme for the possible course of the cyclization steps is presented.