Kanosamine biosynthesis: a likely source of the aminoshikimate pathway's nitrogen atom

The biosynthetic source of the nitrogen atom incorporated into the aminoshikimate pathway has remained a question for some time. 3-Amino-3-deoxy-D-fructose 6-phosphate has previously been demonstrated to be a precursor to 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid via the inferred intermediacy of 1-deoxy-1-imino-D-erythrose 4-phosphate in Amycolatopsis mediterranei cell-free extract. This investigation examines the possibility that the natural product kanosamine might be a precursor to 3-amino-3-deoxy-D-fructose 6-phosphate. Kanosamine 6-phosphate was synthesized by a chemoenzymatic route and incubated in A. mediterranei cell-free lysate along with D-ribose 5-phosphate and phosphoenolpyruvate. Formation of 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid was observed. Subsequent incubation in A. mediterranei cell-free lysate of glutamine and NAD with UDP-glucose resulted in the formation of kanosamine. The bioconversion of UDP-glucose into kanosamine along with the bioconversion of kanosamine 6-phosphate into 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid suggests that kanosamine biosynthesis is the source of the aminoshikimate pathway's nitrogen atom.     

Characterization of the early stage aminoshikimate pathway in the formation of 3-amino-5-hydroxybenzoic acid: the RifN protein specifically converts kanosamine into kanosamine 6-phosphate

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), precursor of the ansamycin and mitomycin antibiotics, proceeds by the aminoshikimate pathway from 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP). Identification of RifN, product of one of three genes from the rifamycin biosynthetic gene cluster known to be essential for aminoDAHP formation, as a specific kanosamine (3-deoxy-3-amino-D-glucose) 6-kinase establishes kanosamine and its 6-phosphate as specific intermediates in AHBA formation. This suggests a hypothetical reaction sequence for aminoDAHP formation, and thus for the early steps of AHBA biosynthesis, starting from UDP-D-glucose and introducing the nitrogen by oxidation and transamination at C-3.     

Syntheses of 4-deoxy-d-fructose and enzymatic affinity study

Enantiomerically pure 4-deoxy-d-fructose has been prepared and characterised in a protected form, acidic hydrolysis of which led to an aqueous solution of 4-deoxy-d-fructose. Activities of this compound with enzymes of the glycolysis pathway involved in glucose metabolism make possible access to 4-deoxy-d-fructose-6-phosphate, 4-deoxy-d-glucose-6-phosphate and 4-deoxy-d-gluconate-6-phosphate.     

Creation of a shikimate pathway variant

The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.     

Biosynthesis of flavocoenzymes

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 2,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.     

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate aldolase to replace 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbial synthesis of shikimate pathway products provides an alternate strategy to circumvent the competition for phosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and the phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGal aldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, and multiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-fold improvement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the addition of pyruvate to d-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2 contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression of NR8.276-2 in the DAHP synthase and shikimate dehydrogenase-deficient E. coli strain NR7 under fed-batch fermentor-controlled cultivation conditions resulted in synthesis of 13 g/L 3-dehydroshikimic acid in 6.5% molar yield from glucose. Increased coexpression of the irreversible downstream enzyme 3-dehydroquinate synthase increased production of 3-dehydroshikimic acid to 19 g/L in 9.7% molar yield from glucose. Coamplification with transketolase, which increases d-erythrose 4-phosphate availability, afforded 16 g/L 3-dehydroshikimic acid in 8.5% molar yield.     

Reactions of 4-amino-1,3,5-dithiazine with ammonia and amines

It is shown that selective replacement of the sulfur atom in the 3 position by a nitrogen atom, which leads to 2,6-dimethyl-4-amino-3H-2,6-dihydro-1,3,5-thiadiazine, occurs when 2H,6H-2,6-dimethyl-4-amino-1,3,5-dithiazine is treated with ammonium hydroxide. Under the same conditions, amines cause profound destruction of 2H,6H-2,6-dimethyl-4-amino-1,3,5-dithiazine with the production of thiourea.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 652–655, May, 1982.     

The dioxanone approach to (2S,3R)-2-C-methylerythritol 4-phosphate and 2,4-cyclodiphosphate, and various MEP analogues

Efficient syntheses of the non-mevalonate pathway intermediates 2-C-methylerythritol 4-phosphate (MEP) and 2-C-methylerythritol 2,4-cyclodiphosphate (ME-2,4-cycloPP), as well as the parent tetrol 2-C-methylerythritol, in enantiopure form from (2S,4R)-cis-2-phenyl-4-tert-butyldimethylsilyloxy-1,3-dioxan-5-one are reported. The 2S configuration of the C-methyl group was installed by highly axial-face selective addition of CH3MgBr (20:1) to the chiral dioxanone carbonyl group. Primary selective mono-phosphorylation and 2,4-bis-phosphorylation, followed by desilation and hydrogenolysis to the free mono- and diphosphates, and, in the latter case, cyclization to form the eight-membered phosphoryl anhydride, afforded MEP and ME-2,4-cycloPP in good yields. The C2 epimeric analogues, 2-C-methylthreitol and its 4-phosphate, were accessed by LiAlH4 reduction of the cis,cis epoxide of (2S,4R)-4-tert-butyldimethylsilyloxymethyl-5-methylene-2-phenyl-1,3-dioxane, primary-selective phosphorylation, and cleavage of the silyl, benzylidene, and benzyl protecting groups. Regioselective cleavage of the acetal ring of 1,3-benzylidene 2-C-methylerythritol silyl ether by ozonolysis afforded a 1,2,3-triol 3-monobenzoate intermediate that was converted to the novel amino sugar, 1-amino-1-deoxy-2-C-methylerythritol.     

O-Acetyl Protection of 6-Aminoaldopyranosides and 1-Aminoalditols

Abstract

Methyl 6-amino-6-deoxy-α-D-glycopyranosides having the D-gluco, D-manno and D-galacto configurations (1a–3a), 2-aminoethanol (4a), 1-amino-1-deoxy-D-glucitol (5a), and 1-amino-1-deoxy-4-O-β-D-glucopyranosyl-D-glucitol (6a) were transformed into the corresponding per-O-acetyl amine hydrochlorides 1d–6d in excellent yields by using the 2,2-(diethoxycarbonyl)vinyl group for temporary amine protection. Deprotection of the peracetylated enamines 1c–6c was effected with chlorine in chloroform and no O→N acetyl migration occurred when short reaction times were used. Treatment of 1d–6d with thiophosgene resulted in the formation of peracetyl isothiocyanates (1e–6e).     

Halogenated 7-deazapurine nucleosides: stereoselective synthesis and conformation of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides

The stereoselective syntheses of 5-halogenated 7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine nucleosides 3b-d, 4a-c as well as 7-deaza-2'-deoxyisoguanosine are described. Nucleobase anion glycosylation of 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (5) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranosyl bromide (6) exclusively gave the beta-D-anomer, which was deblocked (--> 8), aminated at C4 (--> 3a) and selectively deaminated at C2 to yield 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl 7-deazaisoguanine (2). Condensation of the 5-halogenated 4-chloro-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidines 9a-c with 6 furnished the N7-nucleosides 10a-c together with N2,N7-bisglycosylated compounds 11a-c. The former was converted to the corresponding 2,4-diamino-compounds 3b-d, and the latter was deblocked by NaOMe/MeOH to yield the 4-methoxy-nucleosides 4a-c. Conformational analysis of the sugar moiety of the nucleosides 2 and 3a-d was performed on the basis of vicinal [1H,1H] coupling constants. The fluorine atom in the sugar moiety shifts the sugar conformation from S towards N by about 10%, while the halogen substituents in the base moiety increase the hydrophobicity and polarizability of the nucleobases.     

Nucleoside und Nucleotide. Teil 15. Synthese von Desoxyribonucleosid-monophosphaten und -triphosphaten mit 2(1H)-Pyrimidinon, 2(1H)-Pyridinon und 4-Amino-2(1H)-pyridinon als Basen

Nucleosides and Nucleotide. Part 15. Synthesis of Deoxyribonucleoside Monophosphates and Triphosphates with 2(1H)-Pyrimidinone, 2(1H)-Pyridinone and 4-Amino-2(1H)-pyridinone as the Bases The phosphorylation of the modified nucleosides 1-(2′-deoxy-β-D -ribofuranosyl)-2(1 H)-pyrimidinone (M_d, 4 ), 4-amino-1-(2′-deoxy-β-D -ribofuranosyl)-2(1 H)-pyridinone (Z_d, 6 ) and the synthesis of 1–2′-deoxy-β-D -ribofuranosyl-2(1 H)-pyrimidinone-5′-O-triphosphate (pppM_d, 1 ), 1-(2′-deoxy-β-D ribofuranosyl)-2(1 H)-pyridinone-5′-O-triphosphate (pppII_d, 2 ), and 4-amino-1-(2′-deoxy-βD -ribofuranosyl)-2(1 H)-pyridinone-5′-O-triphosphate (pppZ_d, 3 ) are described. The nucleoside-5′-monophosphates pM_d (5) and pZ_d (7) were obtained by selective phosphorylation of M_d (4) and Z_d (6) , respectively, using phosphorylchloride in triethyl phosphate or in acetonitril. The reaction of pM_d (5) pII _d (8) or pZ_d (7) with morpholine in the presence of DCC led to the phosphoric amides 9, 10 and 11 , respectively, which were converted with tributylammonium pyrophosphate in dried dimethylsulfoxide to the nucleoside-5′triphosphates 1, 2 and 3 , respectively.     

New glycolipid inhibitors of Myt1 kinase

A crude extract of a marine alga showed activity against the enzyme Myt1 kinase. Bioassay-directed fractionation led to the isolation of two bioactive glycoglycerolipids. Lipid 1 was identified as sn-1,2-dipalmityl-3-(N-palmityl-6-deoxy-6-amino-α-d-glucosyl) glycerol and lipid 2 as sn-1-palmityl-2-myristyl-3-(N-stearyl-6-deoxy-6-aminoglucosyl)glycerol. Compounds 1 and 2 had IC₅₀ values of 0.12 and 0.43 μg/mL, respectively, in the Myt1 kinase inhibitory bioassay, and were inactive against Akt and Chk1 kinases.     

Mechanisms for NH3 decomposition on Si(100)-(2 x 1) surface: a quantum chemical cluster model study

In this paper, we present a detailed mechanism for the complete decomposition of NH3 to NHx(a) (x = 0-2). Our calculations show that the initial decomposition of NH3 to NH2(a) and H(a) is facile, with a transition-state energy 7.4 kcal mol-1 below the vacuum level. Further decomposition to N(a) or recombination-desorption to NH3(g) is hindered by a large barrier of approximately 46 kcal mol-1. There are two plausible NH2 decomposition pathways: 1) NH2(a) insertion into the surface Si-Si dimer bond, and 2) NH2(a) insertion into the Si-Si backbond. We find that pathway (1) leads to the formation of a surface Si = N unit, similar to a terminal Si = Nt pair in silicon nitride, Si3N4, while pathway (2) leads to the formation of a near-planar, subsurface Si3N unit, in analogy to a central nitrogen atom (Nc) bounded to three silicon atoms in the Si3N4 environment. Based on these results, a plausible microscopic mechanism for the nitridation of the Si(100)-(2 x 1) surface by NH3 is proposed.     

Amadori ketoses with calcium hydroxide and the Kiliani reaction on 1-deoxy ketoses: two approaches to the synthesis of saccharinic acids

Saccharinic acids (2-C-methyl aldonic acids) may be formed by treatment of Amadori ketoses with calcium hydroxide or by the Kiliani reaction of 1-deoxy ketoses with cyanide. Thus (i) N,N-dibenzyl or N,N-dimethyl-1-amino-1-deoxy-d-fructose with aqueous calcium hydroxide afforded 2-C-methyl-d-ribono-1,4-lactone under green conditions and (ii) reaction of methyl magnesium bromide with 2,3-O-isopropylidene-d-erythronolactone gave 1-deoxy-3,4-O-isopropylidene-d-ribulose, which on subsequent treatment with aqueous sodium cyanide and hydrolysis, formed 2-C-methyl-d-arabinono-1,4-lactone. Such branched sugar lactones are likely to be of value as chirons containing branched carbon chains.     

Importance of specific hydrogen-bond donor-acceptor interactions for the key carbocycle-forming reaction catalyzed by 2-deoxy-scyllo-inosose synthase in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics

A crucial enzyme in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous D-glucose 6-phosphate (G-6-P) into the specific carbocycle 2-deoxy-scyllo-inosose. Among all the oxygenated carbons of the substrate, C-1, -4, -5, and -6 are directly involved in the chemical transformation. To get insight into the roles of C-2 and C-3 hydroxy groups, 2-deoxy-2-fluoro-, 3-deoxy-3-fluoro-, 2-amino-2-deoxy-, and 3-amino-3-deoxy-D-glucose 6-phosphates (2-F-G-6-P, 3-F-G-6-P, 2-NH(2)-G-6-P, and 3-NH(2)-G-6-P, respectively) were subjected to the DOIS reaction as probe, since a fluorine substituent generally acts as a hydrogen-bond acceptor, and an ammonium functionality derived physiologically from an amino group as a hydrogen-bond donor. Among those tested, 2-F-G-6-P and 3-NH(2)-G-6-P were used as substrates by DOIS and were converted into the corresponding deoxyfluoro- and aminodeoxy-scyllo-inososes, respectively. In contrast, 3-F-G-6-P and 2-NH(2)-G-6-P were inactive in the cyclization reaction. Clearly, DOIS recognizes the G-6-P substrate through specific hydrogen-bonding interactions, i.e., through a hydrogen-donating group for C-2 and an accepting group for C-3 of the substrate. Modeling of DOIS based on the structure of evolutionary-related dehydroquinate synthase is also described.     

(MN)nHm(M=Ga,In; n=1-4; m=1, 2)团簇的结构与稳定性

采用密度泛函理论(DFT)的B3LYP方法, 在6-31G**和Lanl2dz水平上分别对(MN)nHm(M=Ga, In; n=1-4; m=1, 2)进行了优化和振动频率计算. 得到了上述团簇的最稳定构型、H原子的结合能以及它们的能隙. 结果表明, (MN)nH(M=Ga, In; n=1-4)的基态构型均为双重态, (MN)nH2(M=Ga, In; n=1-4)的基态构型均为单重态; 当氢的个数为1时, 加在N原子上比加在M(M=Ga, In)原子上稳定, 如有N3单元, 那么加在N3单元两侧的构型是相同的, 且它是最稳定的; 当氢的个数为2时, 除n=1外, 分别加在两个N原子上的构型是最稳定的, 如有N3单元, 那么分别加在N3单元分离最远的两个N原子的构型是最稳定的. GaNH、(GaN)3H 和InNH的结合能和能隙都很大, 说明这些团簇都有很高的稳定性.     

1H, 13C and 15N NMR studies on adducts formation of rhodium(II) tetraacylates with some azoles in CDCl3 solution

Adduct formations of rhodium(II) tetraacetate and tetratrifluoroacetate with some 1H-imidazoles, oxazoles, thiazoles, 1H-pyrazoles and isoxazole have been investigated by the use of 1H, 13C, 15N NMR and electronic absorption spectroscopy (VIS) in the visible range. Azoles tend to form axial adducts containing rhodium(II) tetraacylates bonded via nitrogen atom. Bulky substituents close to the nitrogen atom prevent the Rh--N bond formation, and in several cases switch over the binding site to the oxygen or sulphur atoms. The (15)N adduct formation shift Deltadelta(15N) (Deltadelta = delta(adduct) - delta(ligand)) varied from ca - 40 to - 70 ppm for the nitrogen atom involved in complexation, and of a few parts per million only, from ca - 6 to 3 ppm, for the non-bonded nitrogen atom within the same molecule. The Deltadelta(1H) values do not exceed one ppm; Deltadelta(13C) ranges from - 1 to 6 ppm. Various complexation modes have been proved by electronic absorption spectroscopy in the visible region (VIS). For comparison purposes, some adducts of pyridine, thiophene and furan derivatives have been measured as well. The experimental findings were compared with calculated chemical shifts, obtained by means of DFT B3LYP method, using 6-311 + G(2d,p), 6-31(d)/LanL2DZ and 6-311G(d,p) basis set.     

基于主客体作用的多重刺激响应型超分子水凝胶的制备及性能

利用单-6-脱氧-6-氨基-β-环糊精(β-CDNH2)与双二茂铁亚甲基丁二胺季铵盐(FBI)之间的主-客体作用制备了一种性能特异的超分子水凝胶, 该水凝胶具有显著的热可逆性和pH刺激响应性, (NH4)2Ce(NO3)6的引入导致凝胶液化, 体系的颜色也由淡黄色转变为墨绿色. 扫描电镜结果表明, 在凝胶中胶凝剂β-CDNH2和FBI结合组成以带状纤维为特征的三维网络结构. 1H NMR, FTIR, XRD以及循环伏安等表征结果表明, FBI与β-CDNH2间的主客体包结作用和β-CDNH2分子间的氢键作用是凝胶形成并能稳定存在的主要原因.     

Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics

Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an L-methionine:uridine-5'-aldehyde aminotransferase; (ii) LipP, a 5'-amino-5'-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-D-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5'-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.     

Deamination of the Radical Cation of the Base Moiety of 2′‐Deoxycytidine: A Theoretical Study

Five pathways leading to the deamination of cytosine (to uracil) after formation of its deprotonated radical cation are investigated in the gas phase, at the UB3LYP/6‐311G(d,p) level of theory, and in bulk aqueous solvent. The most favorable pathway involves hydrogen‐atom transfer from a water molecule to the N3 nitrogen of the deprotonated radical cation, followed by addition of the resulting hydroxyl radical to the C4 carbon of the cytosine derivative. Following protonation of the amino group (N4), the C4? N4 bond is broken with elimination of the NH₃^?+ radical and formation of a protonated uracil. The rate‐determining step of this mechanism is hydrogen‐atom transfer from a water molecule to the cytosine derivative. The associated free energy barrier is 70.2 kJ mol^?1.