Isotope labeling and theoretical study of the formation of a3* ions from protonated tetraglycine

Extensive 13C, 15N, and 2H labeling of tetraglycine was used to investigate the b3+ --> a3* reaction during low-energy collision-induced dissociation (CID) in a quadrupole ion-trap mass spectrometer. The patterns observed with respect to the retention or elimination of the isotope labels demonstrate that the reaction pathway involves elimination of CO and NH3. The ammonia molecule includes 2 H atoms from amide or amino positions, and one from an alpha-carbon position. The loss of NH3 does not involve elimination of the N-terminal amino group but, instead, the N atom of the presumed oxazolone ring in the b3+ ion. The CO molecule eliminated is the carbonyl group of the same oxazolone ring, and the alpha-carbon H atom is transferred from the amino acid adjacent to the oxazolone ring. Quantum chemical calculations indicate a multistep reaction cascade involving CO loss on the b3 --> a3 pathway and loss of NH=CH2 from the a3 ion to form b2. In the postreaction complex of b2 and NH=CH2, the latter can be attacked by the N-terminal amino group of the former. The product of this attack, an isomerized a3 ion, can eliminate NH3 from its N-terminus to form a3*. Calculations suggest that the ammonia and a3* species can form various ion-molecule complexes, and NH3 can initiate relay-type mobilization of the oxazolone H atoms from alpha-carbon positions to form a new oxazolone isomer. This multiple-step reaction scheme clearly explains the isotope labeling results, including unexpected scrambling of H atoms from alpha-carbon positions.     

Fluorescent Orthopalladated Complexes of 4-Aryliden-5(4H)-oxazolones from the Kaede Protein: Synthesis and Characterization

The goal of the work reported here was to amplify the fluorescent properties of 4-aryliden-5(4H)-oxazolones by suppression of the hula-twist non-radiative deactivation pathway. This aim was achieved by simultaneous bonding of a Pd center to the N atom of the heterocycle and the ortho carbon of the arylidene ring. Two different 4-((Z)-arylidene)-2-((E)-styryl)-5(4H)-oxazolones, the structures of which are closely related to the chromophore of the Kaede protein and substituted at the 2- and 4-positions of the arylidene ring (1a OMe; 1b F), were used as starting materials. Oxazolones 1a and 1b were reacted with Pd(OAc)₂ to give the corresponding dinuclear orthometalated palladium derivates 2a and 2b by regioselective C–H activation of the ortho-position of the arylidene ring. Reaction of 2a (2b) with LiCl promoted the metathesis of the bridging carboxylate by chloride ligands to afford dinuclear 3a (3b). Mononuclear complexes containing the orthopalladated oxazolone and a variety of ancillary ligands (acetylacetonate (4a, 4b), hydroxyquinolinate (5a), aminoquinoline (6a), bipyridine (7a), phenanthroline (8a)) were prepared from 3a or 3b through metathesis of anionic ligands or substitution of neutral weakly bonded ligands. All species were fully characterized and the X-ray determination of the molecular structure of 7a was carried out. This structure has strongly distorted ligands due to intramolecular interactions. Fluorescence measurements showed an increase in the quantum yield (QY) by up to one order of magnitude on comparing the free oxazolone (QY < 1%) with the palladated oxazolone (QY = 12% for 6a). This fact shows that the coordination of the oxazolone to the palladium efficiently suppresses the hula-twist deactivation pathway.     

Oxazolone versus macrocycle structures for leu-enkephalin b<Subscript>2</Subscript>–b<Subscript>4</Subscript>: Insights from infrared multiple-photon dissociation spectroscopy and gas-phase hydrogen/deuterium exchange

The collision-induced dissociation (CID) products b₂-b₄ from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium exchange (HDX). Infrared spectroscopy reveals that b₂ exclusively adopts oxazolone structures, protonated at the N-terminus and at the oxazolone ring N, based on the presence and absence of diagnostic infrared vibrations. This is correlated with the presence of a single HDX rate. For the larger b₃ and b₄, the IR-MPD measurements display diagnostic bands compatible with a mixture of oxazolone and macrocycle structures. This result is supported by HDX experiments, which show a bimodal distribution in the HDX spectra and two distinct rates in the HDX kinetic fitting. The kinetic fitting of the HDX data is employed to derive the relative abundances of macrocycle and oxazolone structures for b₃ and b₄, using a procedure recently implemented by our group for a series of oligoglycine b fragments (Chen et al. J. Am. Chem. Soc. 2009, 131(51), 18272–18282. doi: 10.1021/ja9030837). In analogy to that study, the results suggest that the relative abundance of the macrocycle structure increases as a function of b fragment size, going from 0% for b₂ to ∼6% for b₃, and culminating in 31% for b₄. Nonetheless, there are also surprising differences between both studies, both in the exchange kinetics and the propensity in forming macrocycle structures. This indicates that the chemistry of “head-to-tail” cyclization depends on subtle differences in the sequence as well as the size of the b fragment.     

Infrared spectroscopy and theoretical studies on gas-phase protonated leu-enkephalin and its fragments: direct experimental evidence for the mobile proton

The gas-phase structures of the protonated pentapeptide Leu-enkephalin and its main collision-induced dissociation (CID) product ions, b4 and a4, are investigated by means of infrared multiple-photon dissociation (IR-MPD) spectroscopy and detailed molecular mechanics and density functional theory (DFT) calculations. Our combined experimental and theoretical approach allows accurate structural probing of the site of protonation and the rearrangement reactions that have taken place in CID. It is shown that the singly protonated Leu-enkephalin precursor is protonated on the N-terminus. The b4 fragment ion forms two types of structures: linear isomers with a C-terminal oxazolone ring, as well as cyclic peptide structures. For the former structure, two sites of proton attachment are observed, on the N-terminus and on the oxazolone ring nitrogen, as shown in a previous communication (Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2005, 127, 17154-17155). Upon leaving the ions for longer radiative cooling delays in the ion cyclotron resonance (ICR) cell prior to IR spectroscopic investigation, one observes a gradual decrease in the relative population of oxazolone-protonated b4 and a corresponding increase in N-terminal-protonated b4. This experimentally demonstrates that the mobile proton is transferred between two sites in a gas-phase peptide ion and allows one to rationalize how the proton moves around the molecule in the dissociation process. The a4 fragment, which is predominantly formed via b4, is also confirmed to adopt two types of structures: linear imine-type structures, and cyclic structures; the former isomers are exclusively protonated on the N-terminus in sharp contrast to b4, where a mixture of protonation sites was found. The presence of cyclic b4 and a4 fragment ions is the first direct experimental proof that fully cyclic structures are formed in CID. These results suggest that their presence is significant, thus lending strong support to the recently discovered peptide fragmentation pathways (Harrison, A. G.; Young, A. B.; Bleiholder, B.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006, 128, 10364-10365) that result in scrambling of the amino acid sequence upon CID.     

Elimination of water from the carboxyl group of GlyGlyH+

The elimination of water from the carboxyl group of protonated diglycine has been investigated by density functional theory calculations. The resulting structure is identical to the b(2) ion formed in the mass spectrometric fragmentation of protonated peptides (therefore named "b2" in this study). The most stable geometry of the fragment ion ("b2") is an O-protonated diketopiperazine. However, its formation is kinetically disfavored as it requires a free energy of 58.2 kcal/mol. The experimentally observed N-protonated oxazolone is 3.0 kcal/mol less stable. The lowest energy pathway for the formation of the "b2" ion requires a free energy of 37.5 kcal/mol and involves the proton transfer from the amide oxygen of protonated diglycine to the hydroxyl oxygen. Fragmentation initiated by proton transfer from the terminal nitrogen has also a comparable free energy of activation (39.4 kcal/mol). Proton transfer initiating the fragmentation, from the highly basic terminal nitrogen or amide oxygen to the less basic hydroxyl oxygen is feasible at energies reached in usual mass spectrometric experiments. Amide N-protonated diglycine structures are precursors of mainly y(1) ions rather than "b2" ions. In the lowest energy fragmentation channels, proton transfer to the hydroxylic oxygen, bond breaking and formation of an oxazolone ring occur concertedly but asynchronously. Proton transfer to hydroxyl oxygen and cleavage of the corresponding C-O bond take place at the early stages of the fragmentation step, while ring closure to form an oxazolone geometry occurs at the later stages of the transition. The experimentally observed low kinetic energy release is expected to be due to the existence of a strongly hydrogen bonded protonated oxazolone-water complex in the exit channel. Whereas the threshold energy for "b2" ion formation (37.1 kcal/mol) is lower than for the y(1) ion (38.4 kcal/mol), the former requires a tight transition state with an activation entropy, DeltaS++ = -1.2 cal/mol.K and the latter has a loose transition state with DeltaS++ = +8.8 cal/mol.K. This leads to y(1) being the major fragment ion over a wide energy range.     

Scrambling of sequence information in collision-induced dissociation of peptides

Collision-induced dissociation (CID) of protonated YAGFL-NH2 leads to nondirect sequence fragment ions that cannot directly be derived from the primary peptide structure. Experimental and theoretical evidence indicate that primary fragmentation of the intact peptide leads to the linear YAGFLoxa b5 ion with a C-terminal oxazolone ring that is attacked by the N-terminal amino group to induce formation of a cyclic peptide b5 isomer. The latter can undergo various proton transfer reactions and opens up to form something other than the YAGFLoxa linear b5 isomer, leading to scrambling of sequence information in the CID of protonated YAGFL-NH2.     

An oxazol‐5(4H)‐one from benzyloxy­carbonyl‐(Aib)4‐OH

Benzyl N‐[8‐(4,4‐dimethyl‐5‐oxo‐4,5‐dihydrooxazol‐2‐yl)‐2,5,5,8‐tetra­methyl‐3,6‐dioxo‐4,7‐diazanon‐2‐yl]­carbamate, C₂₄H₃₄N₄O₆, an oxazol‐5(4H)‐one from N‐α‐benzyloxycarbonyl‐(Aib)₄‐OH (Aib = α‐amino­isobutyryl) represents the longest peptide oxazolone so far characterized by X‐ray diffraction. The overall geometry of the oxazolone ring compares well with literature data. The Aib(1) and Aib(2) residues are folded into a type III β‐bend, while the conformation adopted by Aib(3), preceding the oxazolone moiety, is semi‐extended. The disposition of the oxazolone ring relative to the preceding residue is stabilized by C—­H?N and C—H?O intramolecular interactions.     

Multi-stage collisionally-activated decomposition in an ion trap for identification of sequences, structures and bn --> bn-1 fragmentation pathways of protonated cyclic peptides

Cyclic penta-, hexa- and heptapeptides have been designed, synthesized and their fragmentations induced by multistage tandem mass spectrometry have been studied. Under low-energy collisionally activated decomposition (CAD), the protonated cyclic peptides mainly dissociate via ring opening pathways and the corresponding bn --> bn-1 pathways to form several sets of b ions as oxazolone rings (and b1 ions as aziridinone rings). Through repeated observation of these b ions in multistep CAD experiments, accurate sequencing and head-to-tail ring structure of cyclic peptides can be determined. The mistaken assignments of these b ions can be avoided by this sequencing method. Semiempirical molecular orbital calculations have been utilized to provide insight into the proposed dissociation mechanism. In addition, for cyclic peptides that include an Asn residue, the nitrogen of the Asn side chain is observed to be preferentially protonated, which can induce a unique ring-opening pathway with a loss of ammonia that competes with the conventional ring opening pathway.     

Dissociation of the peptide bond in protonated peptides

The dissociation of the amide (peptide) bond in protonated peptides, [M + H](+), is discussed in terms of the structures and energetics of the resulting N-terminal b(n) and C-terminal y(n) sequence ions. The combined data provide strong evidence that dissociation proceeds with no reverse barriers through interconverting proton-bound complexes between the segments emerging upon cleavage of the protonated peptide bond. These complexes contain the C-terminal part as a smaller linear peptide (amino acid if one residue) and the N-terminal part either as an oxazolone or a cyclic peptide (cyclic amide if one residue). Owing to the higher thermodynamic stability but substantially lower gas-phase basicity of cyclic peptides vs isomeric oxazolones, the N-terminus is cleaved as a protonated oxazolone when ionic (b(n) series) but as a cyclic peptide when neutral (accompanying the C-terminal y(n) series). It is demonstrated that free energy correlations can be used to derive thermochemical data about sequence ions. In this context, the dependence of the logarithm of the abundance ratio log[y(1)/b(2)], from protonated GGX (G, glycine; X, varying amino acid) on the gas-phase basicity of X is used to obtain a first experimental estimate of the gas-phase basicity of the simplest b-type oxazolone, viz. 2-aminomethyl-5-oxazolone (b(2) ion with two glycyl residues).     

Structure and fragmentation of b2 ions in peptide mass spectra

In a number of cases the b2 ion observed in peptide mass spectra fragments directly to the a1 ion. The present study examines the scope of this reaction and provides evidence as to the structure(s) of the b2 ions undergoing fragmentation to the a1 ion. The b2 ion H-Ala-Gly+ fragments, in part, to the a1 ion, whereas the isomeric b2 ion H-Gly-Ala+ does not fragment to the a1 ion. Ab initio calculations of ion energies show that this different behavior can be rationalized in terms of protonated oxazolone structures for the b2 ions provided one assumes a reverse activation energy of approximately 1 eV for the reaction b2-->a2; such a reverse activation energy is consistent with experimental kinetic energy release measurements. Experimentally, the H-Aib-Ala+ b2 ion, which must have a protonated oxazolone structure, fragments extensively to the a1 ion. We conclude that the proposal by Eckart et al. (J. Am. Soc. Mass Spectrom. 1998, 9, 1002) that the b2 ions which undergo fragmentation to a1 ions have an immonium ion structure is not necessary to rationalize the results, but that the fragmentation does occur from a protonated oxazolone structure. It is shown that the b2-->a1 reaction occurs extensively when the C-terminus residue in the b2 ion is Gly and with less facility when the C-terminus residue is Ala. When the C-terminus residue is Val or larger, the b2-->a1 reaction cannot compete with the b2-->a2 fragmentation reaction. Some preliminary results on the fragmentation of a2 ions are reported.     

Disfavoring Macrocycle b Fragments by Constraining Torsional Freedom: The “Twisted” Case of QWFGLM b<Subscript>6</Subscript>

While recent studies have shown that for some peptides, such as oligoglycines and Leu-enkephalin, mid-sized b fragment ions exist as a mixture of oxazolone and macrocycle structures, other primary structure motifs, such as QWFGLM, are shown to exclusively give rise to macrocycle structures. The aim of this study was to determine if certain amino acid residues are capable of suppressing macrocycle formation in the corresponding b fragment. The residues proline and 4-aminomethylbenzoic acid (4AMBz) were chosen because of their intrinsic rigidity, in the expectation that limited torsional flexibility may impede “head-to-tail” macrocycle formation. The presence of oxazolone versus macrocycle b₆ fragment structures was validated by infrared multiple photon dissociation (IRMPD) spectroscopy, using the free electron laser FELIX. It is confirmed that proline disfavors macrocycle formation in the cases of QPWFGLM b₇ and in QPFGLM b₆. The 4AMBz substitution experiments show that merely QWFG(4AMBz)M b₆, with 4AMBz in the fifth position, exhibits a weak oxazolone band. This effect is likely ascribed to a stabilization of the oxazolone structure, due to an extended oxazolone ring-phenyl π-electron system, not due to the rigidity of the 4AMBz residue. These results show that some primary structures have an intrinsic propensity to form macrocycle structures, which is difficult to disrupt, even using residues with limited torsional flexibility.     

Defying entropy: forming large head-to-tail macrocycles in the gas phase

Spectral fingerprints: Collision-induced dissociation (CID) of protonated peptides in the gas phase results in linear fragment ions with a five-membered oxazolone ring on their C-terminal side. Infrared spectroscopy confirms that smaller fragments adopt oxazolone structures. Conversely, in mid-sized and larger fragments an isomerization to "head-to-tail" macrocycles is observed (see picture).     

Excited state aromatization assisted push-pull abilities of two unsaturated oxazolone derivatives

Molecular structures of two unsaturated oxazolone derivatives involving furan rings, which are decorated with p-tolyl (FurM) and 4-nitro phenyl (FurN), were investigated by X-ray crystallography and quantum chemical calculations. Their ground and excited states were examined by DFT and TD-DFT computations with the aid of topological electron density studies, NBO and Charge Decomposition Analysis. Both compounds have push-pull (D–π–A) framework using oxazolone ring as π-linker. Depending on the transitions from the ground to excited states, intramolecular charge transfer (ICT) in both compounds results in aromatization of oxazolones. Push-pull ability of FurN has more pronounced than that of FurM. The use of furan instead of almost fully aromatic benzenoid ring reduces HOMO?LUMO band gap due to relatively low aromaticity level of furan. Introduction of nitro substituent leads to a further reduction in HOMO?LUMO gap. In addition, electronic redistribution in the excited state results in aromatization of oxazolone moieties without elongation of carbonyl bonds.     

Reaction of 4‐benzylidene‐2‐methyl‐5‐oxazolone with amines,Part 2: Influence of substituents in para‐position in the phenyl ring and a substituent on amine nitrogen atom on the reaction kinetics

An influence of a structure of the amine (benzylamine, N‐methyl‐benzylamine, N‐isopropyl‐benzylamine, N‐methyl‐butylamine, N‐ethyl‐butylamine, sec‐butylamine, and tert‐butylamine) on a rate constant of the ring‐opening reaction of 4‐benzylidene‐2‐methyl‐5‐oxazolone (Ox) was studied. The good correlation between logarithm of the rate constants and Charton's steric substituent constant ν as well as good correlation with a form of the simple branching equation indicate that there is a steric effect because of substitution at C1 carbon atom of nucleophile which decreases the reaction rate. Additionally, an influence of a structure of the benzylidene moiety of Ox on a rate of the oxazolone ring‐opening reaction was studied. The substituents (? OH, ? OCH₃, ? N(CH₃)₂, ? Cl, ? NO₂) in para‐position of the phenyl ring of Ox substantially modified the rate of the reaction with benzylamine in acetonitrile. The rate of the Ox ring‐opening reaction decreased with increase of the electron‐donating properties of the substituent. A good correlation between the rate constants of the reaction of 4‐(4′‐substituted‐benzylidene)‐2‐methyl‐5‐oxazolones with benzylamine and the electron density at the reaction center (carbon C5 of the oxazolone ring), calculated using ab initio method, and the Hammett substituent constants, and CR equation were established. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 148–155, 2002; DOI 10.1002/kin.10039     

Synthesis of 1-(oxiran-2-ylmethyl)-1<Emphasis Type="Italic">H</Emphasis>-indole-3-carbaldehyde and its reaction with active methylene compounds

Treatment of indole-3-carbaldehyde with epichlorohydrin gave 1-(oxiran-2-ylmethyl)-1H-indole-3-carb-aldehyde, reaction of which with 1,3-dimethylbarbituric acid or malononitrile gave crotonic condensation products with retention of the oxirane ring. The structure of the reaction products with aroylglycines depends on the conditions. In acetic anhydride a simultaneous formation of an oxazolone ring and bisacylation of the oxirane fragment occurs; the use of ethyl chloroformate in the presence of triethylamine allows to carry out only the heterocyclization process. When treated with the cyclic amines (N-methylpiperazine or morpholine) opening of the oxazolone ring in the products occurs with formation of the corresponding amides.     

Structural influences on preferential oxazolone versus diketopiperazine b(2+) ion formation for histidine analogue-containing peptides

Studies of peptide fragment ion structures are important to aid in the accurate kinetic modeling and prediction of peptide fragmentation pathways for a given sequence. Peptide b(2)(+) ion structures have been of recent interest. While previously studied b(2)(+) ions that contain only aliphatic or simple aromatic residues are oxazolone structures, the HA b(2)(+) ion consists of both oxazolone and diketopiperazine structures. The structures of a series of histidine-analogue-containing Xxx-Ala b(2)(+) ions were studied by using action infrared multiphoton dissociation (IRMPD) spectroscopy, fragment ion hydrogen-deuterium exchange (HDX), and density functional theory (DFT) calculations to systematically probe the influence of different side chain structural elements on the resulting b(2)(+) ion structures formed. The b(2)(+) ions studied include His-Ala (HA), methylated histidine analogues, including π-methyl-HA and τ-methyl-HA, pyridylalanine (pa) analogues, including 2-(pa)A, 3-(pa)A, and 4-(pa)A, and linear analogues, including diaminobutanoic acid-Ala (DabA) and Lys-Ala (KA). The location and accessibility of the histidine π-nitrogen, or an amino nitrogen on an aliphatic side chain, were seen to be essential for diketopiperazine formation in addition to the more typical oxazolone structure formation, while blocking or removal of the τ-nitrogen did not change the b(2)(+) ion structures formed. Linear histidine analogues, DabA and KA, formed only diketopiperazine structures, suggesting that a steric interaction in the HisAla case may interfere with the complete trans-cis isomerization of the first amide bond that is necessary for diketopiperazine formation.     

A Simple and Convenient Synthesis of 4‐Ylidene‐5(4H)oxazolone Derivatives: Oxazolone Ring Transformation Leading to Other Heterocyclic Structures

Simple, effective, and high yield synthetic procedure for the synthesis of 4‐ylidene‐5(4H)‐oxazolones 2a–m from arylidene‐malononitriles under solvent‐free conditions is described. The scope of this reaction was investigated, and it was found that the presence of anhydrous sodium acetate gave the corresponding oxazolones in excellent yields. The newly generated oxazolone derivative 2m underwent ring transformation into pyrroles, imidazoles, pyridazine, and triazines.     

1,3-Dipolar cycloaddition reactions of 2-phenyl-4-arylidene-5(4H)-oxazolones with nitrile oxides

Stable nitrile oxides are added stereospecifically and regioselectively to the carbon carbon double bond of 2-phenyl-4-arylidene-5(4H)-oxazolones resulting spiro-derivatives 3, 5 . The spectral properties of the reaction products are discussed. The cycloadducts give several substituted isoxazolines via an opening of the oxazolone ring with nucleophilic reagents.     

Characterization of the product ions from the collision-induced dissociation of argentinated peptides

Tandem mass spectrometry performed on a pool of 18 oligopeptides shows that the product ion spectra of argentinated peptides, the [bn + OH + Ag]+ ions and the [yn - H + Ag]+ ions bearing identical sequences are virtually identical. These observations suggest strongly that these ions have identical structures in the gas phase. The structures of argentinated glycine, glycylglycine, and glycylglycylglycine were calculated using density functional theory (DFT) at the B3LYP/DZVP level of theory; they were independently confirmed using HF/LANL2DZ. For argentinated glycylglycylglycine, the most stable structure is one in which Ag+ is tetracoordinate and attached to the amino nitrogen and the three carbonyl oxygen atoms. Mechanisms are proposed for the fragmentation of this structure to the [b2 + OH + Ag]+ and the [Y2 - H + Ag]+ ions that are consistent with all experimental observations and known calculated structures and energetics. The structures of the [b2 - H + Ag]+ and the [a2 - H + Ag]+ ions of glycylglycylglycine were also calculated using DFT. These results confirm earlier suggestions that the [b2 - H + Ag]+ ion is an argentinated oxazolone and the [a2 - H + Ag]+ an argentinated immonium ion.     

Reactivity of Unsaturated 5(4H)-Oxazolones with Hg(II) Acetate: Synthesis of Methyl N-Benzoylamino-3-arylacrylates

An efficient and high-yield procedure to prepare methyl N-benzoylamino-3-arylacrylates from unsaturated (Z)-2-aryl-4-arylidene-5-(4H)-oxazolones and Hg(OAc)₂ in methanol is described herein. The observed reactivity of mercury(II) acetate here is different than its usual metallating behavior and it cleaves the unsaturated oxazolone ring without change of stereochemistry.