The study and characterization of nucleosides by mass spectrometry—II: Comparisons between the mass spectra of 3′,5′-substituted,isomeric pairs of 2,2′-anhydrouridines

The mass spectra of 2,2′-anhydrouridines substituted in the 3′ and 5′ positions were studied. When the substituents were acetyl, pivaloyl, trifluoroacetyl or trimethylsilyl, it was found that specific fragmentations occured which could identify the position of the substituent. The specific fragmentations often resolve earlier ambiguities in the interpretation of mass spectra of 2,2′-anhydropyrimidine derivatives. Mono-acetyl and mono-pivaloyl 2,2′-anhydrouridines are not easily distinguished because of thermal reactions in the sample probe. They may be readily distinguished, however, by acetylation (with acetic anhydride-d₆ for the mono-acetyl isomers), trifluoroacetylation or trimethylsilylation, followed by mass spectral analysis of the reaction product.     

Mass spectrometry of trialkylsilyl and acyl derivatives of 2′-deoxynucleosides

The electron impact mass spectra of monosilyl and mixed acyl-silyl derivatives of 2′-deoxynucleosides are described in detail. (Silyl = tert-butyldimethylsilyl, cyclo-tetramethylene-isopropylsilyl, or cyclo-tetramethylene-tert-butylsilyl; acyl = acetyl or trifluoroacetyl.) The interpretation of the fragmentation pathways was aided by metastable ion decomposition studies, precise mass and deuterium labelling measurements. Mass spectrally, the acyl substituents are mostly ‘passive’ and have (with possibly one exception) little fragmentation directing capability. In contrast, the silyl groups have powerful fragmentation directing properties. Elimination of the bulky alkyl radical R^˙ (tert-butyl or isopropyl) from the molecular ion produces the siliconium ion, [M–R]⁺, which is the precursor for most of the other prominent ions in the spectra. These arise from ‘siliconium ion rearrangements’ resulting from the interaction of the positively charged siliconium ion centre with the electron dense regions (i.e. oxygens) in the molecule, to form cyclic silyloxonium ions which subsequently decompose. Since the interacting oxygen and silicon must be sterically accessible, the fragment ion types and their abundances are very dependent upon structure. Consequently, [M–R]⁺ ions formed from 3′- or 5′-O-silyl groups give rise to different sets of daughter ions which, for the most part, are not found, or have very low abundances, in the mass spectra of underivatized or trimethylsilylated nucleosides. Detailed information on sugar and base moieties and isomeric substitution is readily obtained.     

The mass spectra of the trimethylsilyl esters of acetyl,schiff base and isothiocyanate derivatives of some aminoalkylphosphonic acids

Trimethylsilyl esters of acetyl, Schiff base and isothiocyanate derivatives of a series of aminoalkylphosphonic acids were prepared for the purpose of characterizing these phosphorus compounds by combined gas chromatography and mass spectrometry. The mass spectra of these derivatives were investigated by means of high resolution mass measurments and deuterium labeling. Ions characteristic of the presence of the trimethylsilylphosnate group were observed at m/e 121, 195, 211 and 225 to 227 in the spectra of all the derivatives. Several ions produced by interaction between the trimethylsilyl group and the derivatized amino function were present, particularly in the spectra of the acetate derivatives ([M — 56]⁺, [M — R]⁺ and [M — 153]⁺, where R is the side chain attached to C-1), and the isothiocynate derivatives (m/e 268, 253, 241, 190 and 116).     

Mass spectra of sterically crowded trialkylsilyl derivatives of nucleosides

The electron impact mass spectra of tert-butyldimethylsilyl-, cyclo-tetramethylene-tert-butylsilyl and cyclo-tetramethylene-isopropylsilyl- ether derivatives of ribo- and 2′-deoxyribonucleosides are described in detail. The interpretation of fragmentation pathways of full and mixed derivatives was aided by metastable ion decomposition studies, precise mass and deuterium labelling measurements, and spectra of mixed derivatives containing the ‘passive’ (in these spectra) trimethylsilyl group. The sterically crowded silyl groups have a powerful fragmentation directing effect. Elimination of a bulky radical, R˙ (tert-butyl or isopropyl), from the molecular ion produces the siliconium ion [M? R]⁺, which is the precursor for most of the other prominent ions in the spectra. These arise from ‘siliconium ion rearrangements’ resulting from the interaction of the positively charged siliconium ion center with electron dense regions (i.e. oxygens) in the molecule, to form cyclic silyloxonium ions which subsequently decompose. Since the interacting oxygen and silicon must be sterically accessible, the fragment ion types and their abundances are very dependent upon structure. Consequently, [M? R]⁺ ions formed from 2′, 3′ or 5′-O-silyl groups give rise to different sets of daughter ions which, for the most part, are not found, or have very low abundances, in the mass spectra of underivatized or trimethylsilylated nucleosides. Detailed information on sugar and base moieties and isomeric substitution is readily obtained.     

Silylgruppenwanderung und CO2-Eliminierung beim massenspektrometrischen Zerfall von N-Trifluoracetyl-α-aminosäretrimethylsilylestern

The molecular ions of N-trifluoroacetyl α-amino acid trimethylsilyl esters exhibit a characteristic elimination of CO₂, in contrast to other amino acid derivatives and apparently caused by migration of the ester trimethylsilyl group to the oxygen atom of the N-trifluoroacetyl function. Fragmentation of the [M – CO₂]⁺˙ ions gives rise to a series of intense peaks, especially for the aliphatic amino acid derivatives. In the case of the isomers leucine and isoleucine, different base peaks are formed for the 20 eV spectra. Amino acids which can easily split off a group in their β-position possibly fragment by synchronous elimination of CO₂ and this group. With serine, threonine and cysteine a concurrent ester silyl migration to the oxygen of the β-function is observed, accompanied by the expulsion of CO₂.     

Mass spectral hydrogen rearrangements of 1,1′-bicycloalkyldiols under electron impact

1,1′-Bicycloalkyl-2,2′-diols (mixtures of stereoisomers) and 1,1′-diols on electron impact give rise to strong peaks equivalent to [M-1]⁺ions, corresponding to cycloalkenone fragments and obviously involving hydrogen atom migrations. Deuterium labelling and substitution techniques reveal the operation of different rearrangement mechanisms for the two series of isomeric compounds. Thus, in the 1,1′-bicycloalkyl-2,2′-diols a ring hydrogen atom is involved in the rearrangement, whereas in the 1,1′-bicycloalkyl-1,1′-diols (pinacols) the hydroxylic hydrogen atoms migrate.     

The mass spectral fragmentation of 2,2′-bis-trimethylsilylbenzhydrylethers. An indication for transannular hydrogen/deuterium rearrangements and transannular elimination reactions

The mass spectra of the methyl-, trideuteromethyl-, ethyl- and pentadeuteroethylethers of 2,2′-bis-trimethylsilylbenzhydrol are reported. The most significant ions arise from the [M – CH₃]⁺ ion, formed by loss of a methyl radical from one of the trimethylsilyl groups. After ring formation by interaction of the siliconium ion centre with an aromatic nucleus, the ion loses (CH₃)₃Si? OR (R = CH₃, C₂H₅, CD₃ and C₂D₅), giving ion m/e 223. The fragment (CH₃)₃Si? OCH₃ is also eliminated in the four ethers investigated from the ion [M – R]⁺. Attack of the siliconium ion. Indications are found for a transannular hydrogen/deuterium rearrangement and a transannular elimination reaction. The intensity of some peaks in the spectra are discussed in relation to group R.     

The mass spectra of the trimethylsilyl derivatives of some alkyl and aminoalkyl phosphonates

The mass spectra of the trimethylsilyl derivatives of a series of alkyl and aminoalkyl phosphonates were investigated with the aid of deuterium labeling and high resolution mass measurements. The spectra contained several prominent ions characteristic of the phosphonate system and a number of ions produced by inter and intra molecular migration and interaction of trimethylsilyl groups.     

Fragmentation of trimethylsilyl derivatives of 2-alkoxyphenols: A further violation of the ‘even-electron rule’

The mass spectra of trimethylsilyl (TMS) ethers of 2-methoxyphenols show abundant [M–30]⁺˙ ions originating from consecutive loss of two methyl radicals. This is illustrated by comparison of the accurate mass-measured and linked-scan spectra of the TMS derivatives of 2-methoxyphenol (guaiacol), 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid methyl ester (ferulic acid methyl ester) with those of the TMS derivatives of phenol, 4-hydroxybenzaldehyde, 3-(4-hydroxyphenyl)-2-propenoic acid methyl ester (p-coumaric acid methyl ester), 3-methoxyphenol and 4-methoxyphenol. This distinctive ortho effect is valuable in the identification of isomeric phenolic compounds. In the spectra of the TMS derivatives of 2-ethoxyphenol and 2-propoxyphenol the sequential loss of two radicals is less pronounced, because elimination of the side-chain and a methyl group with rearrangement and hydrogen migration is competitive.     

Negative-ion mass spectrometry of substituted adenine bases and adenosine nucleosides

The negative-ion chemical ionization (ammonia, 5 Pa source pressure) mass spectra of a series of substituted adenine bases, adenosine nucleosides, and the trimethylsilyl derivatives of the nucleosides are described. Selected ions from these spectra were subject to collisionally activated dissociation with mass-analysed ion kinetic energy (CAD/MIKE) analysis of the products and the spectra assessed for information content. In addition to observing strong peaks due to quasimolecular ions and heterocyclic-base ions, it proved possible to differentiate between 2'-, 3'- and 5'-deoxy and between 2'- and 3'-O-methyl isomers. The negative-ion chemical ionization spectra of four methyladenines are essentially identical, but could be clearly distinguished from each other by CAD/MIKE analysis.     

Fast atom bombardment mass spectra of telluronium salts

The fast atom bombardment (FAB) mass spectra of telluronium salts were studied. The spectra exhibit the intact cation (C⁺) and cluster ions ([M + C]⁺). The principal fragment ions in the FAB mass spectra of telluronium salts are [RTe]⁺, [R₂Te]⁺˙, [R₂Te − H]⁺, [RTeR′]⁺˙, and [RTeR′ + H]⁺. When the anion was [BPh₄]⁻, interesting cluster ions such as [M + C − BPh₃]⁺ appeared.     

Structural determination of sildenafil and its analogues in dietary supplements by fast‐atom bombardment collision‐induced dissociation tandem mass spectrometry

Sildenafil and its analogues, which are used as illegal additives in several dietary supplements, were isolated by liquid‐liquid extraction and column chromatography and analyzed by fast‐atom bombardment mass spectrometry (FAB‐MS). Structures of sildenafil and its derivatives were elucidated by FAB‐tandem mass spectrometry (MS/MS) with exact mass measurement in the positive‐ion mode. To find structurally diagnostic ions for the sildenafil analogues, authentic sildenafil was preferentially analyzed by high‐energy collision‐induced dissociation (CID)‐MS/MS. The CID‐MS/MS spectra of [M+H]⁺ precursor ions resulted in the formation of numerous characteristic ions via a series of dissociative processes. The product ions formed by CID provided important information on the modification of the piperazine ring, the phenylsulfonyl group and the pyrazolopyrimidine moiety of sildenafil. By interpreting their MS/MS spectra, the chemical structures of sildenafil analogues isolated from dietary supplements could be elucidated and fragmentation patterns were proposed. To clearly identify the sidenafil derivatives in dietary supplements, some of the derivatives such as acetildenafil, homosildenafil and hydroxyhomosildenafil which are not commercially available were synthesized and compared with their MS/MS spectra. In addition, high‐resolution mass measurements were conducted to obtain the elemental compositions of sildenafil and its analogues. Copyright © 2009 John Wiley & Sons, Ltd.     

Tandem mass spectrometric analysis of fixed-charge derivatized oligosaccharides

Various derivatives of a set of three isomeric (linear and branched) pentasaccharides (LNF-1, LNF-2 and LNF-3) were prepared and investigated by high-performance tandem mass spectrometry in order to compare their collision-induced dissociation (CID) behaviour. The fast atom bombardment tandem mass spectra of [M + H]⁺ ions of peracetylated derivatives mainly display fragments containing the non-reducing terminus (B-type ions) and allow a straightforward assignment of sugar sequence and branching together with the identification of some interglycosidic linkages. Permethylated derivatives, which better accommodate the mass range of tandem mass spectrometers in the case of larger oligosaccharides, yield similar results, i.e. predominance of B ions, but the necessary information has to be retrieved from incomplete B_i and Y_i ion series. By contrast, CID of permethyl or peracetyl derivatives carrying a preformed charge due to prior reductive amination of the oligosaccharides with trimethyl(p-aminophenyl)ammonium chloride yields exclusively fragment ions comprising the reducing end. In this case, the four distinct series of fragments observed involve charge-remote fragmentation processes. As a consequence, the spectral patterns are not significantly affected by the nature of the sugar O-substituents additionally introduced (i.e. methyl or acetyl).     

Cyclic ions in the mass spectra of trimethylsilyl derivatives of substituted o-dihydroxybenzenes

The mass spectra of trimethylsilyl (TMS) ethers/methyl esters of phenolic acids containing o-dihydroxybenzene groups have base peaks at [M?119]⁺ instead of the usual [M?15]⁺ and [M?31]⁺ that are characteristic of TMS/methyl esters of monohydroxyphenolic acids. These ions, formed by the loss of 31+88 u from the parent ion, possess a cyclic moiety as proven by substitution of deuterium atoms for hydrogen atoms in the TMS groups of the methyl esters of 3,4,5-trihydroxybenzoic (gallic), 3,4-dihydroxybenzoic (protocatechuic) and β-(3,4-dihydroxyphenyl)propenoic (caffeic) acids. Although these cyclic ions are the base peaks in TMS-derivatized o-dihydroxyphenolic acid esters, similar ions represent intense peaks but not necessarily the base peak in other derivatized compounds such as 1,2-dihydroxybenzene, 1,2-dihydroxy-3-methyl- and 1,2-dihydroxy-4-methyl-benzenes and flavan-3-ols that possess o-dihydroxybenzene groups. Compounds possession m- or p-dihydroxybenzene groups do not form these cyclic ions; therefore, this procedure for derivatization and interpretation of mass spectra is valuable for the identification of compounds containing o-dihydroxybenzene groups in complex mixtures of isomeric compounds.     

Identification of butyryl derivatives of cyclic nucleotides by positive ion fast atom bombardment mass spectrometry and mass-analysed ion kinetic energy spectrometry

The syntheses of the dibutyryl derivatives of the 3′,5′-cyclic monophosphates of adenosine, guanosine and cytidine are described. The fast atom bombardment mass spectra of these compounds are discussed, together with the mass-analysed ion kinetic energy spectra of their protonated molecular ions and of diagnostic fragments. A protocol for the identification of the derivatives is reported which includes criteria for confirming retention of the cyclic phosphodiester moiety, substitution of both heterocyclic base and ribose ring, and butyrylation of the 2′-O-position. The origins of significant fragments in the spectra are discussed.     

Characterization of blocked nucleosides by mass spectrometry. Benzyl derivatives

The mass spectra of eleven model monobenzylated nucleosides were studied using low and high resolution mass spectrometry. Structural assignments to the major ions were made and several decomposition mechanisms proposed, with the goal of establishing the uses and limitations of mass spectrometry for the characterization of benzylaled nucleosides. Mass spectra generally permit determination of the extent and site of benzylation, with particular regard to base vs. sugar substitution, 0–2′ vs. 0–3′ or 0–5′, and in some cases 0–5′ vs. other isomers.     

Fast atom bombardment mass spectra of iodonium salts

Positive-ion fast atom bombardment mass spectra and linked-scan tandem mass spectra were measured for aromatic iodonium salts. The mass spectra usually contain the intact cation of the iodonium salt as the base peak, fragment ions of lower abundance resulting from simple cleavages or rearrangements, and a characteristic loss of atomic iodine. High-level semi-empirical calculations suggest that an obtuse ring? I⁺? ring angle facilitates loss of atomic iodine through concomitant ring? to? ring bond formation.     

Mass spectrometry of hydroxy dicarboxylic acids as trimethylsilyl derivatives. Rearrangement fragmentations

Gas chromatography—mass spectrometry offers a convenient method for the separation and identification of hydroxy dicarboxylic acids as open-chain trimethylsilyl (TMS) derivatives. Mass spectra were studied of aldaric (tartronic, tartaric, pentaric and hexaric) acids and deoxyaldaric (malic, 2-deoxypentaric, 2-deoxyhexaric, 3-deoxypentaric and 3-deoxyhexaric) acids. The different structural types can be readily identified from their characteristic spectra. The most prominent fragmentations involving the rupture of one bond are the loss of a siliconlinked methyl group and the formation of α-cleavage ions by carbon chain cleavage. The further decay is characterized by a number of significant rearrangements specific of TMS derivatives. Several of these can be classified as involving migration of a TMS group to an oxygen atom or migration of an ester OTMS group to a silicon atom. Concomitant loss of a stable molecule often provides a driving force. Prominent odd-electron ions are formed by a McLafferty-type rearrangement of a TMS group. The decomposition of several even-electron ions can be regarded as analogous to that rearrangement.     

Daughter ion mass spectra of 11-pentafluorobenzyl ester derivatives of 11-dehydiothromboxane B2 and B3

Collisionally activated decomposition (CAD) mass spectra of the [M – C₆F₅CH₂?]^? ions of 1-methyl cster-11-pentafluorobenzyl ester-9,12,15-tris(trimethylsilyl) and 9,12,15-tris(ethyldimethylsilyl) ether derivatives of 11-dehydrothromboxane B₂ are presented and discussed. The spectra are interpreted with the aid of those of a corresponding 3,3,4,4-tetradeutero compound and of an analogous derivative of 11-dehydrothromboxane B₃. Proposed fragmentation pathways are based on internal consistency of data from all four compounds. The migration of a trimethylsilyl or an ethyldimethylsilyl group is the salient feature of all the CAD spectra.     

The mass spectra of cinnolines and their N-oxides

The mass spectra of cinnoline and various alkyl derivatives, and their 1- and 2-mono-oxides and 1,2-dioxides and a quaternary salt have been investigated. The spectra are interpreted in the light of experiments with deutero and ¹⁵N labelled derivatives whose syntheses are described. The major fragmentation path for cinnoline is loss of nitrogen molecule while that for its homologues is loss of nitrogen plus a hydrogen atom in some instances. Various initial fragmentations occur with the N-oxides, and deoxygenation appears to be the dominant process; in both 4-methylcinnoline 1-oxide and 1,2-dioxide an important path is formation of a 3-methyl-anthranil cation by loss of HCN and HCNO respectively.