Electron ionization mass spectral fragmentation of cholestane-3beta,4alpha,5alpha-triol and cholestane-3beta,5alpha,6alpha/beta-triol bis- and tris-trimethylsilyl derivatives

The electron ionization (EI) mass spectral fragmentation of the bis- and tris-trimethylsilyl derivatives of cholestane-3beta,4alpha,5alpha-triol, cholestane-3beta,5alpha,6beta-triol and cholestane-3beta,5alpha,6alpha-triol was investigated. The EI mass spectrum of the 3beta,4alpha-bis-trimethylsilyl derivative of cholestane-3beta,4alpha,5alpha-triol exhibits interesting fragment ions at m/z 142 and 332 resulting from the initial loss of TMSOH between the carbons 2 and 3 and subsequent retro-Diels-Alder (RDA) cleavage of the ring A. Trimethylsilyl transfer between the 4alpha- and the 5alpha-hydroxy groups acts significantly before RDA cleavage affording an ion at m/z 404. Complete silylation of cholestane-3beta,4alpha,5alpha-triol strongly stabilizes the molecule, affording an abundant molecular ion at m/z 636 and decreasing the abundance of the RDA cleavage. Loss of water (from the non-silylated 5alpha-hydroxy group) plays a very important role during the decomposition of the molecular ion of 3beta,6alpha/beta-bis-trimethylsilyl derivatives of cholestane-3beta,5alpha,6alpha/beta-triols. These derivatives appear to be very useful in assigning the configuration of the carbon 6. This assignment is based on the abundance of the fragment ions at m/z 321, 367 and 403, which are more prominent in the EI mass spectrum of the beta-isomer. In contrast, EI mass spectra of the tris-trimethylsilyl derivatives of cholestane-3beta,5alpha,6beta-triol and cholestane-3beta,5alpha,6alpha-triol differ only slightly and appear to be poorly informative.     

Hydrolysis of 2',3'-O-methyleneadenosin-5'-yl bis-5'-O-methyluridin-3'-yl phosphate: the 2'-hydroxy group stabilizes the phosphorane intermediate, not the departing 3'-oxyanion, by hydrogen bonding

Hydrolytic reactions of 2',3'-O-methyleneadenosin-5'-yl bis-5'-O-methyluridin-3'-yl phosphate (1a) have been followed by RP HPLC over a wide pH range to elucidate the role of the 2'-OH group as an intermolecular hydrogen bond donor facilitating the cleavage of 1a. At pH < 2, where the decomposition of 1 is first-order in hydronium-ion concentration, the P-O5' and P-O3' bonds are cleaved equally rapidly. Over a relatively wide range from pH 2 to 4, the hydrolysis is pH-independent and the P-O5' bond is cleaved 1.6 times as rapidly as the P-O3' bond. At pH 6, the reaction becomes first-order in hydroxide-ion concentration and cleavage of the P-O3' bond starts to predominate, accounting for 89% of the overall hydrolysis in 10 mmol L(-)(1) aqueous sodium hydroxide. Under alkaline conditions, the 2'-OH group facilitates the cleavage of 1 by a factor of 27 compared to the 2'-OMe counterpart, the influence on the P-O3' and P-O5' bond cleavage being equal. Accordingly, the 2'-hydroxy group stabilizes the phosphorane intermediate, not the departing 3'-oxyanion, by hydrogen bonding.     

Characterization of C-glycosyl quinochalcones in Carthamus tinctorius L. by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

C-Glycosyl quinochalcones are unique components in Carthamus tinctorius L. The reported C-glycosyl quinochalcones have the same quinochalcone skeleton with a hydroxyl group at the 5'-position and a glucose linked to this position with a carbon-carbon bond. In this study, the standard hydroxysafflor yellow A and water-extracted fraction of Carthamus tinctorius L. were analyzed by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOFMS) in both positive and negative ion modes. The fragmentation pathways of C-glycosyl quinochalcones were interpreted and validated by accurate mass measurement. Their fragmentation showed a special cleavage at the C-C bond except for the typical internal cleavage at the sugar moiety of other C-glycosyl flavonoids. In positive ion mode, cleavage of the 5'-glucose produced an [M+H-162](+) ion by a neutral loss, while cleavage of the 5'-glucose in negative ion mode led to an [M-H-163](-.) ion by radical cleavage. The cleavage from the carbonyl group produced fragment ions containing an A or a B ring. The fragment ions containing an A ring were common product ions of seven compounds in both ion modes, and fragment ions containing the B ring were used to judge the different substituent groups at the 3'-position. The fragmentation patterns of seven structurally related C-glycosyl quinochalcones were analyzed systematically and the formation of the fragment ions in two modes is explained in detail in this report. UPLC/Q-TOFMS is an effective tool for characterizing a complex sample, which gives higher resolution separation and generates accurate mass measurement of the product ions.     

Isolation of an oxomanganese(V) porphyrin intermediate in the reaction of a manganese(III) porphyrin complex and H2O2 in aqueous solution

The reaction of [Mn(TF(4)TMAP)](CF(3)SO(3))(5) (TF(4)TMAP=meso-tetrakis(2,3,5,6-tetrafluoro-N,N,N-trimethyl-4-aniliniumyl)porphinato dianion) with H(2)O(2) (2 equiv) at pH 10.5 and 0 degrees C yielded an oxomanganese(V) porphyrin complex 1 in aqueous solution, whereas an oxomanganese(IV) porphyrin complex 2 was generated in the reactions of tert-alkyl hydroperoxides such as tert-butyl hydroperoxide and 2-methyl-1-phenyl-2-propyl hydroperoxide. Complex 1 was capable of epoxidizing olefins and exchanging its oxygen with H(2) (18)O, whereas 2 did not epoxidize olefins. From the reactions of [Mn(TF(4)TMAP)](5+) with various oxidants in the pH range 3-11, the O-O bond cleavage of hydroperoxides was found to be sensitive to the hydroperoxide substituent and the pH of the reaction solution. Whereas the O-O bond of hydroperoxides containing an electron-donating tert-alkyl group is cleaved homolytically, an electron-withdrawing substituent such as an acyl group in m-chloroperoxybenzoic acid (m-CPBA) facilitates O-O bond heterolysis. The mechanism of the O-O bond cleavage of H(2)O(2) depends on the pH of the reaction solution: O-O bond homolysis prevails at low pH and O-O bond heterolysis becomes a predominant pathway at high pH. The effect of pH on (18)O incorporation from H(2) (18)O into oxygenated products was examined over a wide pH range, by carrying out the epoxidation of carbamazepine (CBZ) with [Mn(TF(4)TMAP)](5+) and KHSO(5) in buffered H(2) (18)O solutions. A high proportion of (18)O was incorporated into the CBZ-10,11-oxide product at all pH values but this proportion was not affected significantly by the pH of the reaction solution.     

Hydrolytic reactions of diribonucleoside 3',5'-(3'-N-phosphoramidates): kinetics and mechanisms for the P-O and P-N bond cleavage of 3'-amino-3'-deoxyuridylyl-3',5'-uridine

Hydrolytic reactions of 3'-amino-3'-deoxyuridylyl-3',5'-uridine (2a), an analogue of uridylyl-3',5'-uridine having the 3'-bridging oxygen replaced with nitrogen, have been followed by RP HPLC over a wide pH range. The only reaction taking place under alkaline conditions (pH > 9) is hydroxide ion-catalyzed hydrolysis (first-order in [OH(-)]) to a mixture of 3'-amino-3'-deoxyuridine 3'-phosphoramidate (7) and uridine (4). The reaction proceeds without detectable accumulation of any intermediates. At pH 6-8, a pH-independent formation of 3'-amino-3'-deoxyuridine 2'-phosphate (3) competes with the base-catalyzed cleavage. Both 3 and in particular 7 are, however, rather rapidly dephosphorylated under these conditions to 3'-amino-3'-deoxyuridine (5). In all likelihood, both 3 and 7 are formed by an intramolecular nucleophilic attack of the 2'-hydroxy function on the phosphorus atom, giving a phosphorane-like intermediate or transition state. Under moderately acidic conditions (pH 2-6), the predominant reaction is acid-catalyzed cleavage of the P-N3' bond (first-order in [H(+)]) that yields an equimolar mixture of 5 and uridine 5'-phosphate (6). The reaction is proposed to proceed without intramolecular participation of the neighboring 2'-hydroxyl group. Under more acidic conditions (pH < 2), hydrolysis to 3 and 4 starts to compete with the cleavage of the P-N bond, and this reaction is even the fastest one at pH < 1. Formation of 2'-O,3'-N-cyclic phosphoramidate as an intermediate appears probable, although its appearance cannot be experimentally verified. The rate constants for various partial reactions have been determined. The reaction mechanisms and the effect that replacing the 3'-oxygen with nitrogen has on the behavior of the phosphorane intermediate are discussed.     

Mass spectrometry of 4-amino-4′-nitroazobenzene compounds

The mass spectra of 32 substituted 4-amino-4′-nitroazobenzene compounds have been recorded and the most intense peaks have been used to characterize these spectra. It was found that the spectra of 4-amino-4′-nitroazobenzene compounds are characterized by peaks due to: (1) molecular ions, (2) fragment ions formed by cleavage of one of the carbon-nitrogen bonds adjacent to the azo linkage with the positive charge remaining with the amine fragment, (3) ions formed by cleavage alpha to the amine nitrogen with the charge remaining with the amine substituent, (4) ions formed by cleavage beta to the amine nitrogen with the loss of the amine substituent fragment, (5) secondary ions formed by cleavage beta to the amine nitrogen with the loss of the amine substituent fragment from the primary amine fragment (2), and (6) ions formed by loss of NO from the molecular ion. This work shows that 4-amino-4′-nitroazobenzene compounds exhibit fragmentation which is dependent in a consistent manner on the types of substituents. This work provides a basis for a systematic approach to the identification of 4-amino-4′-nitroazobenzene compounds.     

Tethered dinuclear europium(III) macrocyclic catalysts for the cleavage of RNA

Dinuclear europium(III) complexes of the macrocycles 1,3-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-m-xylene (1), 1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene (2), and mononuclear europium(III) complexes of macrocycles 1-methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (3), 1-[3'-(N,N-diethylaminomethyl)benzyl]-4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (4), and 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (5) were prepared. Studies using direct excitation ((7)F0 --> (5)D0) europium(III) luminescence spectroscopy show that each Eu(III) center in the mononuclear and dinuclear complexes has two water ligands at pH 7.0, I = 0.10 M (NaNO3) and that there are no water ligand ionizations over the pH range of 7-9. All complexes promote cleavage of the RNA analogue 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at 25 degrees C (I = 0.10 M (NaNO3), 20 mM buffer). Second-order rate constants for the cleavage of HpPNP by the catalysts increase linearly with pH in the pH range of 7-9. The second-order rate constant for HpPNP cleavage by the dinuclear Eu(III) complex (Eu2(1)) at pH 7 is 200 and 23-fold higher than that of Eu(5) and Eu(3), respectively, but only 7-fold higher than the mononuclear complex with an aryl pendent group, Eu(4). This shows that the macrocycle substituent modulates the efficiency of the Eu(III) catalysts. Eu2(1) promotes cleavage of a dinucleoside, uridylyl-3',5'-uridine (UpU) with a second-order rate constant at pH 7.6 (0.021 M(-1) s(-1)) that is 46-fold higher than that of the mononuclear Eu(5) complex. Methyl phosphate binding to the Eu(III) complexes is energetically most favorable for the best catalysts, and this supports an important role for the catalyst in stabilization of the developing negative charge on the phosphorane transition state. Despite the formation of a bridging phosphate ester between the two Eu(III) centers in Eu2(1) as shown by luminescence spectroscopy, the two metal ion centers are only weakly cooperative in cleavage of RNA and RNA analogues.     

Chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids

The mechanism of chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids is studied by kinetic measurements. The Schiff bases are shown to be fairly stable in neutral media. In acid media, the Schiff bases are hydrolyzed into the initial components. In alkaline media, cleavage of α-hydrogen from the amino acid fragment and structural rearrangement into the quinoid form followed by hydrolysis of the latter with elimination of pyridoxamine and keto acid take place. The rate constants of the chemical transformations of the Schiff bases are found to depend on the pH of the medium. It is shown for the first time that the phosphate group in the pyridoxal 5′-phosphate fragment catalyzes the α-hydrogen cleavage and strongly accelerates alkaline decomposition of the Schiff bases.     

Intramolecular Participation of Amino Groups in the Cleavage and Isomerization of Ribonucleoside 3′‐Phosphodiesters: The Role in Stabilization of the Phosphorane Intermediate

A dinucleoside‐3′,5′‐phosphodiester model, 5′‐amino‐4′‐aminomethyl‐5′‐deoxyuridylyl‐3′,5′‐thymidine, incorporating two aminomethyl functions in the 4′‐position of the 3′‐linked nucleoside has been prepared and its hydrolytic reactions studied over a wide pH range. The amino functions were found to accelerate the cleavage and isomerization of the phosphodiester linkage in both protonated and neutral form. When present in protonated form, the cleavage of the 3′,5′‐phosphodiester linkage and its isomerization to a 2′,5′‐linkage are pH‐independent and 50–80 times as fast as the corresponding reactions of uridylyl‐3′,5′‐uridine (3′,5′‐UpU). The cleavage of the resulting 2′,5′‐isomer is also accelerated, albeit less than with the 3′,5′‐isomer, whereas isomerization back to the 3′,5′‐diester is not enhanced. When the amino groups are deprotonated, the cleavage reactions of both isomers are again pH‐independent and up to 1000‐fold faster than the pH‐independent cleavage of UpU. Interestingly, the 2′‐ to 3′‐isomerization is now much faster than its reverse reaction. The mechanisms of these reactions are discussed. The rate accelerations are largely accounted for by electrostatic and hydrogen‐bonding interactions of the protonated amino groups with the phosphorane intermediate.     

Selective Cleavage of the HIV-1 TAR-RNA with a Peptide–Cyclen Conjugate

Covalent linkage of the arginine-rich fragment of the Tat protein to 1,4,7,10-tetraazacyclododecane (cyclen) results in the selective cleavage of the TAR-RNA of HIV-1 (see picture; the biotin at the 5′ end acts as a label for the subsequent analysis of the cleavage fragments). The cleavage occurs at room temperature and is diminished when Eu^III ions are present—at a concentration of about 1/10 of the concentration of the peptide–cyclen conjugate. The pH dependence indicates that two ammonium ions are responsible for the cleavage reaction. The white arrows in the schematic diagram mark the cleavage sites in RNase T1, and the black arrows the sites in the peptide–cyclen conjugate.     

Phosphodiester cleavage of guanylyl-(3',3')-(2'-amino-2'-deoxyuridine): rate acceleration by the 2'-amino function

Hydrolytic reactions of the structural analogue of guanylyl-(3',3')-uridine, guanylyl-(3',3')-(2'-amino-2'-deoxyuridine), having one of the 2'-hydroxyl groups replaced with an amino function, have been followed by RP HPLC in the pH range 0-13 at 90 degrees C. The results are compared to those obtained earlier with guanylyl-(3',3')-uridine, guanylyl-(3',3')-(2',5'-di-O-methyluridine), and uridylyl-(3',5')-uridine. Under basic conditions (pH > 8), the hydroxide ion-catalyzed cleavage of the P-O3' bond (first-order in [OH(-)]) yields a mixture of 2'-amino-2'-deoxyuridine and guanosine 2',3'-cyclic phosphate which is hydrolyzed to guanosine 2'- and 3'-phosphates. Under these conditions, guanylyl-(3',3')-(2'-amino-2'-deoxyuridine) is 10 times less reactive than guanylyl-(3',3')-uridine. Under acidic and neutral conditions (pH 3-8), where the pH-rate profile for the cleavage consists of two pH-independent regions (from pH 3 to pH 4 and from 6 to 8), guanylyl-(3',3')-(2'-amino-2'-deoxyuridine) is considerably reactive. For example, in the latter pH range, guanylyl-(3',3')-(2'-amino-2'-deoxyuridine) is more than 2 orders of magnitude more labile than guanylyl-(3',3')-(2',5'-di-O-methyluridine), while in the former pH range the reactivity difference is 1 order of magnitude. Under very acidic conditions (pH < 3), the isomerization giving guanylyl-(2',3')-(2'-amino-2'-deoxyuridine) and depurination yielding guanine (both first-order in [H(+)]) compete with the cleavage. The Zn(2+)-promoted cleavage ([Zn(2+)] = 5 mmol L(-)(1)) is 15 times faster than the uncatalyzed reaction at pH 5.6. The mechanisms of the reactions of guanylyl-(3',3')-(2'-amino-2'-deoxyuridine) are discussed, particularly focusing on the possible stabilization of phosphorane intermediate and/or transition state via an intramolecular hydrogen bonding by the 2'-amino group.     

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine)

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine) have been followed by RP HPLC over a wide pH range at 363.2 K in order to elucidate the role of the 2'-hydroxyl group as a hydrogen-bond donor upon departure of the 3'-uridine moiety. Under neutral and basic conditions, guanosyl-(3',3')-uridine undergoes hydroxide ion-catalyzed cleavage (first order in [OH(-)]) of the P-O3' bonds, giving uridine and guanosine 2',3'-cyclic monophosphates, which are subsequently hydrolyzed to a mixture of 2'- and 3'-monophosphates. This bond rupture is 23 times as fast as the corresponding cleavage of the P-O3' bond of guanosyl-(3',3')-(2',5'-di-O-methyluridine) to yield 2',5'-O-dimethyluridine and guanosine 2',3'-cyclic phosphate. Under acidic conditions, where the reactivity differences are smaller, depurination and isomerization compete with the cleavage. The effect of Zn(2+) on the cleavage of the P-O3' bonds of guanosyl-(3',3')-uridine is modest: about 6-fold acceleration was observed at [Zn(2+)] = 5 mmol L(-)(1) and pH 5.6. With guanosyl-(3',3')-(2',5'-di-O-methyluridine) the rate-acceleration effect is greater: a 37-fold acceleration was observed. The mechanisms of the partial reactions, in particular the effects of the 2'-hydroxyl group on the departure of the 3'-linked nucleoside, are discussed.     

Photoreactivity and photopolymerization of silicon-bridged [1]ferrocenophanes in the presence of terpyridine initiators: unprecedented cleavage of both iron-cyclopentadienyl bonds in the presence of chlorosilanes

The photopolymerisation of sila[1]ferrocenophane [Fe(eta-C5H4)2SiMe2] (3) with 4,4',4'-tri-tert-butyl-2,2':6',2'-terpyridine (tBu3terpy) as initiator has been explored. High-molecular-weight polyferrocenylsilane (PFS) [{Fe(eta-C5H4)2SiMe2}n] (5) was formed in high yield when a stoichiometric amount of tBu3terpy was used at 5 degrees C. Photopolymerisation of ferrocenophane 3 at higher temperatures gave PFS 5 in lower yield and with a reduced molecular weight as a result of a slower propagation rate. Remarkably, when Me3SiCl was added as a capping agent before photopolymerisation, subsequent photolysis of the reaction mixture resulted in the unprecedented cleavage of both iron-Cp bonds in ferrocenophane 3: iron(II) complex [Fe(tBu3terpy)2Cl2] (7Cl) was formed and the silane fragment (C5H4SiMe3)2SiMe2 (8) was released. The iron-Cp bond cleavage reaction also proceeded in ambient light, although longer reaction times were required. In addition, the unexpected cleavage chemistry in the presence of Me3SiCl was found to be applicable to other photoactive ferrocenes such as benzoylferrocene. For benzoylferrocene and ferrocenophane 3, the presence of metal-to-ligand charge transfer (MLCT) character in their low-energy transitions in the visible region probably facilitates photolytic iron-Cp bond cleavage, but this reactivity is suppressed when the strength of the iron-Cp bond is increased by the presence of electron-donating substituents on the cyclopentadienyl rings.     

Hydrolytic reactions of 3'-N-phosphoramidate and 3'-N-thiophosphoramidate analogs of thymidylyl-3',5'-thymidine

The diastereomeric thiophosphoramidate analogs [(R(P))- and (S(P))-3[prime or minute],5[prime or minute]-Tnp(s)T] and the phosphoramidate analog [3[prime or minute],5[prime or minute]-TnpT] of thymidylyl-3[prime or minute],5[prime or minute]-thymidine were prepared and their hydrolytic reactions over the pH-range 1-8 at 363.2 K were followed by RP HPLC. At pH < 6, an acid-catalyzed P-N3[prime or minute] bond cleavage (first-order in [H(+)]) takes place with both 3[prime or minute],5[prime or minute]-Tnp(s)T and 3[prime or minute],5[prime or minute]-TnpT, the former being about 12 fold more stable than the latter. At pH > 4, Tnp(s)T undergoes two competing pH-independent reactions, desulfurization (yielding TnpT) and depyrimidination (cleavage of the N-glycosidic bond) the rates of which are of the same order of magnitude. Also with 3[prime or minute],5[prime or minute]-TnpT the pH-independent depyrimidination competes with P-N3[prime or minute] cleavage at pH > 5.     

Substrate specificity of an active dinuclear Zn(II) catalyst for cleavage of RNA analogues and a dinucleoside

The cleavage of the diribonucleoside UpU (uridylyl-3'-5'-uridine) to form uridine and uridine (2',3')-cyclic phosphate catalyzed by the dinuclear Zn(II) complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (Zn(2)(1)(H(2)O)) has been studied at pH 7-10 and 25 degrees C. The kinetic data are consistent with the accumulation of a complex between catalyst and substrate and were analyzed to give values of k(c) (s(-)(1)), K(d) (M), and k(c)/K(d) (M(-)(1) s(-)(1)) for the Zn(2)(1)(H(2)O)-catalyzed reaction. The pH rate profile of values for log k(c)/K(d) for Zn(2)(1)(H(2)O)-catalyzed cleavage of UpU shows the same downward break centered at pH 7.8 as was observed in studies of catalysis of cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) and uridine-3'-4-nitrophenyl phosphate (UpPNP). At low pH, where the rate acceleration for the catalyzed reaction is largest, the stabilizing interaction between Zn(2)(1)(H(2)O) and the bound transition states is 9.3, 7.2, and 9.6 kcal/mol for the catalyzed reactions of UpU, UpPNP, and HpPNP, respectively. The larger transition-state stabilization for Zn(2)(1)(H(2)O)-catalyzed cleavage of UpU (9.3 kcal/mol) compared with UpPNP (7.2 kcal/mol) provides evidence that the transition state for the former reaction is stabilized by interactions between the catalyst and the C-5'-oxyanion of the basic alkoxy leaving group.     

Identification of the products of oxidation of quercetin by air oxygen at ambient temperature

Oxidation of quercetin by air oxygen takes place in water and aqueous ethanol solutions under mild conditions, namely in moderately-basic media (pH approximately 8-10) at ambient temperature and in the absence of any radical initiators, without enzymatic catalysis or irradiation of the reaction media by light. The principal reaction products are typical of other oxidative degradation processes of quercetin, namely 3,4-dihydroxy-benzoic (proto-catechuic) and 2,4,6-trihydroxybenzoic (phloroglucinic) acids, as well as the decarboxylation product of the latter--1,3,5-trihydroxybenzene (phloroglucinol). In accordance with the literature data, this process involves the cleavage of the gamma-pyrone fragment (ring C) of the quercetin molecule by oxygen, with primary formation of 4,6-dihydroxy-2-(3,4-dihydroxybenzoyloxy)benzoic acid (depside). However under such mild conditions the accepted mechanism of this reaction (oxidative decarbonylation with formation of carbon monoxide, CO) should be reconsidered as preferably an oxidative decarboxylation with formation of carbon dioxide, CO2. Direct head-space analysis of the gaseous components formed during quercetin oxidation in aqueous solution at ambient temperature indicates that the ratio of carbon dioxide/carbon monoxide in the gas phase after acidification of the reaction media is ca. 96:4%. Oxidation under these mild conditions is typical for other flavonols having OH groups at C3 (e.g., kaempferol), but it is completely suppressed if this hydroxyl group is substituted by a glycoside fragment (as in rutin), or a methyl substituent. An alternative oxidation mechanism involving the direct cleavage of the C2-C3 bond in the diketo-tautomer of quercetin is proposed.     

Mass spectrometry of heterocyclic compounds—II: Electron-impact induced fragmentation of 3,5-diphenyl-1,2,4-oxadiazole

The electron-impact induced fragmentation of 3,5-diphenyl-1,2,4-oxadiazole has been investigated by labelling experiments, defocused metastable ion detections and high resolution mass measurements. The main fragmentation process suggests heterocyclic cleavage at the 1 to 5 and 3 to 4 bonds confirming our previous interpretation. The structure of the major fragment ion [C₇H₅NO]⁺· has been interpreted as being represented by the isomeric benzonitrile oxide and phenylisocyanate structures, the latter isomerising irreversibly from the former. The benzonitrile oxide structure is consistent with [C₇H₅NO]⁺· formation by cleavage of the 1 to 5 and 3 to 4 bonds.     

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.     

The amyloidogenicity of gelsolin is controlled by proteolysis and pH.

BACKGROUND: Normally, gelsolin functions in plasma as part of the actin-scavenging system to assemble and disassemble actin filaments. The Asp 187-->Asn (D187N) Asp 187-->Tyr (D187Y) gelsolin mutations facilitate two proteolytic cuts in the parent protein generating a 71-residue fragment that forms amyloid fibrils in humans, putatively causing Finnish type familial amyloidosis (FAF). We investigated the role of the D187N mutation in amyloidogenicity using biophysical studies in vitro. RESULTS: Both the recombinant wild-type and D187N FAF-associated gelsolin fragments adopt an ensemble of largely unfolded structures that do not self-associate into amyloid at pH 7. 5. Incubation of either fragment at low pHs (6.0-4.0) leads to the formation of well-defined fibrils within 72 hours, however. CONCLUSIONS: The D187N mutation has been suggested to destabilize the structure of the gelsolin parent protein (specifically domain 2), facilitating two proteolytic cleavage events. Our studies demonstrate that generating the largely unstructured peptide is not sufficient alone for amyloid formation in vitro (on a time scale of months). A drop in pH or an analogous environmental change appears necessary to convert the unstructured fragment into amyloid fibrils, probably through an associative mechanism. The wild-type gelsolin fragment will make amyloid fibrils from pH 6 to 4 in vitro, but neither the wild-type fragment nor fibrils have been observed in vivo. It is possible that domain 2 of wild-type gelsolin is stable in the context of the whole protein and not susceptible to the proteolytic degradation that affords the 71-residue FAF-associated peptide.     

Hydrolysis of 2',3'-O-methyleneadenos-5'-yl bis(2',5'-di-O-methylurid-3'-yl) phosphate, a sugar O-alkylated trinucleoside 3',3',5'-monophosphate: implications for the mechanism of large ribozymes

Hydrolytic reactions of 2',3'-O-methyleneadenos-5'-yl bis(2',5'-di-O-methylurid-3'-yl) phosphate (1), a sugar O-alkylated trinucleoside 3',3',5'-monophosphate, have been followed by RP HPLC over a wide pH range. Under neutral and mildly acidic conditions, the only reaction observed was a pH-independent cleavage of the O-C5' bond of the 5'-linked nucleoside. Under more alkaline conditions nucleophilic attack by hydroxide ion starts to compete. The reaction is first order in [OH(-)] and becomes predominant at pH 10. Each of the 3'-linked nucleosides is displaced 2.9 times as readily as the 5'-linked one. To determine the beta(lg) value for the hydroxide ion catalyzed hydrolysis of 1, two diesters (2a,b) having 2',3'-O-methyleneadenosine (7) and 2',5'-di-O-methyluridine (4) as leaving groups were hydrolyzed under alkaline conditions. Since the beta(lg) value for this reaction is known, DeltapK(a) between 4 and 7 could be calculated. The beta(lg) for the hydrolysis of 1 was estimated to be -0.5 with use of this information. The mechanisms of the partial reactions and the role of leaving group properties in ribozyme reactions of large ribozymes are discussed.