Spectroscopic evidence for an oxazolone structure of the b2 fragment ion from protonated tri-alanine

Infrared multiple photon dissociation (IRMPD) spectroscopy is used to identify the structure of the b ₂⁺ ion generated from protonated tri-alanine by collision induced dissociation (CID). The IRMPD spectrum of b ₂⁺ differs markedly from that of protonated cyclo-alanine-alanine, demonstrating that the product is not a diketopiperazine. Instead, comparison of the IRMPD spectrum of b ₂⁺ to spectra predicted by density functional theory provides compelling evidence for an oxazolone structure protonated at the oxazolone N-atom.     

Diagnostic NH and OH Vibrations for Oxazolone and Diketopiperazine Structures: b<Subscript>2</Subscript> from Protonated Triglycine

We present infrared multiple photon dissociation (IRMPD) spectra in the hydrogen stretching region of the simplest b fragment, b₂ from protonated triglycine, contrasted to that of protonated cyclo(Gly-Gly). Both spectra confirm the presence of intense, diagnostic vibrations linked to the site of proton attachment. Protonated cyclo(Gly-Gly) serves as a reference spectrum for the diketopiperazine structure, showing a diagnostic O-H⁺ stretch of the protonated carbonyl group at 3585 cm^–1. Conversely, b₂ from protonated triglycine exhibits a strong band at 3345 cm^–1, associated with the N-H stretching mode of the protonated oxazolone ring structure. Other weaker N-H stretches can also be discerned, such as the amino NH₂ and amide NH bands. These results demonstrate the usefulness of the hydrogen stretching region, and hence benchtop optical parametric oscillator/amplifier (OPO/A) set-ups, in making structural assignments of product ions in collision-induced dissociation (CID) of peptides.     

Proton affinities of the N- and C-terminal segments arising upon the dissociation of the amide bond in protonated peptides

Dissociation of the amide bonds in a protonated peptide leads to N-terminal sequence fragments with cyclic structures and C-terminal sequence fragments with linear structures. The ionic fragments containing the N-terminus (b _n ) have been shown to be protonated oxazolones, whereas those containing the C-terminus (y _n ) are protonated linear peptides. The coproduced neutral fragments are cyclic peptides from the N-terminus and linear peptides from the C-terminus. A likely determinant of these structural choices is the proton affinity (PA) of the described peptide segments. This study determines the PA values of such segments (Pep), i.e., cyclic and linear dipeptides and a relevant oxazolone, based on the dissociations of proton-bound dimers [Pep + B _i ]H⁺ in which B _i is a reference base of known PA value (Cooks kinetic method). The dissociations are assessed at different internal energies to thereby obtain both proton affinities as well as entropies of protonation. For species with comparable amino acid composition, the proton affinity (and gas phase basicity) follows the order cyclic peptide ≪ oxazolone ≈ linear peptide. This ranking is consistent with dissociation of the protonated peptide via interconverting proton-bound complexes involving N-terminal oxazolone (O) or cyclopeptide (C) segments and C-terminal linear peptide segments (L), viz. O ⋯ H⁺ ⋯ L ⇄ C ⋯ H⁺ ⋯ L. N-terminal sequence ions (b _n ) are formed with oxazolone structures which can efficiently compete for the proton with the linear segments. On the other hand, N-terminal neutral fragments detach as cyclic peptides, with H⁺ now being retained by the more basic linear segment from the C-terminus to yield y _n .     

Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: Formation of b 2 2+ ions

When ionized by electrospray from acidic solutions, the tripeptides Pro-His-Xaa (Xaa=Gly, Ala, Leu) form abundant doubly-protonated ions, [M+2H]²⁺. Collision-induced dissociation (CID) of these doubly-protonated species results, in part, in formation of b ₂ ²⁺ ions, which fragment further by loss of CO to form a ₂ ²⁺ ions; the latter fragment by loss of CO to form the Pro and His iminium [immonium is commonly used in peptide MS work] ions. Although larger doubly-charged b ions are known, this represents the first detailed study of b ₂ ²⁺ ions in CID of small doubly protonated peptides. The most abundant CID products of the studied doubly-protonated peptides arise mainly in charge separation involving two primary fragmentation channels, formation of the b ₂ /y ₁ pair and formation of the a ₁ /y ₂ pair. Combined molecular dynamics and density functional theory calculations are used to gain insight into the structures and fragmentation pathways of doubly-protonated Pro-His-Gly including the energetics of potential protonation sites, backbone cleavages, post-cleavage charge-separation reactions and the isomeric structures of b ₂ ²⁺ ions. Three possible structures are considered for the b ₂ ²⁺ ions: the oxazolone, diketopiperazine, and fused ring isomers. The last is formed by cleavage of the His-Gly amide bond on a pathway that is initiated by nucleophilic attack of one of the His side-chain imidazole nitrogens. Our calculations indicate the b ₂ ²⁺ ion population is dominated by the oxazolone and/or fused ring isomers.     

Differentiation of Positional Isomers of Hybrid Peptides Containing Repeats of β-Nucleoside Derived Amino Acid (β-Nda-) and L-Amino Acids by Positive and Negative Ion Electrospray Ionization Tandem Mass Spectrometry (ESI-MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript>)

A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS ⁿ ). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CH₃OH, NH₃, and C₂H₄O₂, whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y_n⁺ and b_n⁺ ions in MS ⁿ spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y_n⁺ and b_n⁺ ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y_n⁺ and b_n⁺ ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b_n⁺ ions are formed, presumably with stable oxazolone structures when the C-terminus of the b_n⁺ ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b_n⁺ ions.     

Structures of a n * Ions Derived from Protonated Pentaglycine and Pentaalanine: Results from IRMPD Spectroscopy and DFT Calculations

Infrared multiple-photon dissociation (IRMPD) spectroscopy and DFT calculations have been used to probe the most stable structures of a ₃ ^* and a ₄ ^* ions derived from both protonated pentaglycine (denoted G₅) and pentaalanine (A₅). The a ₃ ^* and a ₄ ^* ions derived from protonated A₅ feature a CHR=N-CHR’- group at the N-terminus and an oxazolone ring at the C-terminus, as proposed previously [J. Am. Soc. Mass Spectrom. 19, 1788–1798 (2008)]. The isomeric a ₄ ^* ion derived from A₅ with a 3,5-dihydro-4H-imidazol-4-one ring structure was calculated to have a slightly better energy than the oxazolone, but the barrier to its formation is higher and there was no evidence of this ion in the IRMPD spectrum. By contrast, the a ₄ ^* and [a ₄ – H₂O]⁺ (denoted a ₄ ⁰ ) ions from G₅ gave strikingly similar IRMPD spectra and both have the 3,5-dihydro-4H-imidazol-4-one ring structure similar to that recently reported for the [GGGG + H – H₂O]⁺ ion [Int. J. Mass Spectrom. 316–318, 268–272 (2012)]. In the absence of a solvent molecule, the pathway to the oxazolone is calculated to be lower than those to thermodynamically more stable products, the a ₄ ⁰ and the a ₄ ^* with the 3,5-dihydro-4H-imidazol-4-one ring structure. Incorporation of one water molecule is sufficient to reduce the barrier to formation of the a ₄ ⁰ of G₅ to below that for formation of the oxazolone. On the equivalent potential energy surface for protonated A₅ the barrier to formation of the a ₄ ⁰ ion is 12.3 kcal mol^–1 higher than that for oxazolone formation and the a ₄ ⁰ ion is not observed experimentally.

Effect of method of ion preparation on low-energy collision-induced dissociation mass spectra

The collision-induced dissociation (CID) mass spectra of protonated cocaine and protonated heroin have been measured using a triple quadrupole mass spectrometer at 50 eV ion/neutral collision energy for protonated molecules prepared by different protonating agents. The CID mass spectra of protonated cocaine using H⁺(H₂O)_n, H⁺(NH₃)_n and H⁺((CH₃)₂NH)_n as protonating agents are essentially identical and it is concluded that, regardless of the initial site of protonation, the fragmentation reactions occurring on collisional activation are identical. By contrast, protonated heorin prepared with H⁺(H₂O)_n and H⁺(NH₃)_n as protonating agents show substantial differences. That formed by reaction of H⁺(H₂O)_n shows a much more abundant peak corresponding to loss of CH₃CO₂H. From a comparison with model compounds, and from a consideration of the three-dimensional structure of heroin, it is concluded that with H⁺(H₂O)_n as protonating agent significant protonation occurs at the acetate group attached to the alicyclic ring, leading to acetic acid loss on collisional activation, but that reaction of H⁺(NH₃)_n leads to protonation at the nitrogen function. The proton attached to nitrogen cannot interact with the acetate group and, consequently, the probability of loss of acetic acid on collislional activation is greatly reduced.     

Spontaneous fragmentations of protonated benzaldimines mediated by intermediate ion-neutral complexes

4-Methoxymethylbenzaldimmonium ions (a) and the corresponding N-methylated ions (b) and N,N-dimethylated ions (c) were easily generated in the ion source by electron impact-induced dissociation from 1-(4-methoxymethylphenyl)ethylamine and its N-methylated derivatives. The spontaneous fragmentations of metastable ions a-c and of specifically deuterated derivatives in the second field-free region of a VG ZAB-2F mass spectrometer were studied by mass-analysed ion kinetic energy Spectrometry. The formation of an amino-p-quinodimethane radical cation by loss of the methoxy group is observed for all ions. In the case of a and b carrying at least one proton at the immonium group, competing fragmentations are the loss of CH₂O and CH₃OH, respectively, and the formation of ions CH₃OCH₂ ⁺, m/z 45, and C₇H₇ ⁺, m/z 91. Deuterium-labelling experiments indicated the migration of a proton from the protonated imino group of a and b to the aromatic ring followed by the loss of methanol from the methoxymethyl side-chain or protolysis of the bond to either side-chain to form ion-neutral complexes, in close analogy with the reactions of the corresponding protonated benzaldehydes. The intermediate ion-neutral complexes dissociate eventually by internal ion-neutral reactions resulting in the loss of CH₂ O and the formation of C₇H₇ ⁺, respectively.     

Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

Methylcyclopropane ion formation by elimination of water from n-butanol and 2-methylpropanol ions

C₄H₈⁺˙ ions generated from ionized n-butanol (a) and from ionized 2-methylpropanol (b) were characterized by reaction of the daughter ions with ammonia in a Fourier transform mass spectrometer. Hydrogen transfers occur in a in the ratio five-membered ring:six-membered ring = 1:3.2. However, both hydrogen-transfers are followed by the formation of ionized methylcyclopropane. Ionized methylcyclopropane is also produced on elimination of water from b.     

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.     

IRMPD Spectra of Protonated Hydroxybenzaldehydes: Evidence of Torsional Barriers in Carboxonium Ions

Protonation at the formyl oxygen atom of benzaldehydes leading to the formation of carboxonium ions yields two distinct isomers, depending on the relative orientation of the proton either cis or trans with respect to the hydrogen atom on the adjacent carbon. In this context, the IR multiple photon dissociation (IRMPD) spectra of protonated ortho, meta, and para-hydroxybenzaldehydes ( OH−BZH⁺ ), delivered into the gas phase by electrospray ionization of hydro-alcoholic solutions, are reported in the 3200–3700 cm⁻¹ spectral range. This range is characteristic of O−H stretching modes and thus able to differentiate cis and trans carboxonium isomers. Comparison between IRMPD spectra and DFT calculations at the B3LYP/6-311++G(2df2p) level suggests that for both p- OH−BZH⁺ and m- OH−BZH⁺ only cis conformers are present in the ion population analyzed. For o- OH−BZH⁺ , IRMPD spectroscopy points to a mixture comprising one trans and more than one cis conformers. The energy barrier for cis–trans isomerization calculated for each OH−BZH⁺ isomer is a measure of the degree of π-electron delocalization. Furthermore, IRMPD spectra of p- OH−BZH⁺ , m- OH−BZH⁺ and protonated phenol (this last used as reference) were recorded also in the fingerprint range. Both the observed C−O and O−H stretching vibrations appear to be a measure of π-electron delocalization in the ions.     

The structure and fragmentation of B n (n≥3) ions in peptide spectra

The unimolecular and low energy collision-induced fragmentation reactions of the MH⁺ ions of N-acetyl-tri-alanine, N-acetyl-tri-alanine methyl ester, N-acetyl-tetra-alanine, tetra-alanine, penta-alanine, hexa-glycine, and Leu-enkephalin have been studied with a particular emphasis on the formation and fragmentation of B _n (n=3,4,5) ions. In addition, the metastable ion fragmentation reactions of protonated tetra-glycine, penta-glycine, and Leu-enkephalin amide have been studied. B _n ions are prominent stable species in all spectra. The B _n ions fragment, in part, by elimination of CO to form A _n ions; this reaction occurs on the metastable ion time scale with a substantial release of kinetic energy (T _1/2=0. 3–0. 5 eV) that indicates that a stable configuration of the B _n ion fragments by way of a reacting configuration that is higher in energy than the fragmentation products, A _n + CO. Ab initio calculations strongly suggest that the stable configuration of the B₃ and B₄ ions is a protonated oxazolone formed by interaction of the developing charge with the next-nearest carbonyl group as HX is lost from the protonated species H-(Yyy) _n -X · H⁺. The higher B _n ions also fragment, in part, to form the next-lower B ion, presumably in its stable protonated oxazolone form. This reaction is rationalized in terms of the three-dimensional structure of the B _n ions and it is proposed that the neutral eliminated is an α-lactam.     

Lamellar aggregation and hydrogen‐bonding motifs in 3‐(amino­carbon­yl)­pyridinium perchlorate and hydrogen oxalate

In the title compounds, C₆H₇N₂O⁺·ClO₄⁻, (I), and C₆H₇N₂O⁺·C₂HO₄⁻, (II), the carboxamide plane is twisted from the plane of the protonated pyridine ring. Lamellar or sheet‐like structural features are observed through N—H⋯O and O—H⋯O hydrogen‐bonded motifs of cations and anions in (I) and (II), respectively. These sheets are aggregated through C(4) and C(5) chain motifs in (I) and (II), respectively. R₁²(4) ring motifs in (I) and R₁²(5) motifs in (II) are formed via pyridine–anion bifurcated N—H⋯O inter­actions. In (II), carboxamide groups form N—H⋯O dimers around the inversion centres of the unit cell, with R₂²(8) ring motifs. A 2₁ screw‐related helical or ribbon‐like structure along the b axis is formed in (II) through carboxamide and pyridinium N—H⋯O hydrogen bonds with the oxalate anions.     

Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala,Ser, Leu,Val, Phe,and Trp)

The product ion spectra of proline-containing peptides are commonly dominated by y _n ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y ₂ ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y ₂ /b ₃ abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y ₂ /b ₃ abundance ratio decreases.     

Protonenkatalysierte gasphasendehydratisierung von 1,4-diketonen : Ein massenspektrometrisches analogon zur furansynthese in kondensierter phase

It is shown that the gas phase proton catalyzed dehydration of both 2,5-hexanedione (1a: R CH₃) and 1,4-diphenyl-1,4-butanedione (1b: R Ph) yields protonated 2,5-disubstituted furans. the addition of H₃O⁺ to furans followed by ring opening and formation of protonated diketones has not been achieved. Reaction of 1a,b with NH₄⁺/NH₃ does not result in the formation of pyrrole. The main reaction corresponds to the formation of cluster ions, i.e. proton bounded dimers and trimers of diketone and ammonia.     

Diagnosing the Protonation Site of <Emphasis Type="BoldItalic">b</Emphasis><Subscript><Emphasis Type="BoldItalic">2</Emphasis></Subscript> Peptide Fragment Ions using IRMPD in the X–H (X = O,N, and C) Stretching Region

Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b ₂ ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800–3800 cm^–1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO₂ laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm^–1, corresponding to the asymmetric N–H stretch of the (nonprotonated) N-terminal NH₂ group, is a distinct vibrational signature of the ring-protonated oxazolone structure.     

Spectroscopic Evidence for an Oxazolone Structure in Anionic <Emphasis Type="BoldItalic">b</Emphasis>-Type Peptide Fragments

Infrared spectra of anionic b-type fragments generated by collision induced dissociation (CID) from deprotonated peptides are reported. Spectra of the b₂ fragments of deprotonated AlaAlaAla and AlaTyrAla have been recorded over the 800–1800 cm^–1 spectral range by multiple-photon dissociation (MPD) spectroscopy using an FTICR mass spectrometer in combination with the free electron laser FELIX. Structural characterization of the b-type fragments is accomplished by comparison with density functional theory calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric structures. Although diketopiperazine structures represent the energetically lowest isomers, the IR spectra suggest an oxazolone structure for the b₂ fragments of both peptides. Deprotonation is shown to occur on the oxazolone α-carbon, which leads to a conjugated structure in which the negative charge is practically delocalized over the entire oxazolone ring, providing enhanced gas-phase stability.     

Effects of Aromatic Fluorine Substitution on Protonated Neurotransmitters: The Case of 2‐Phenylethylamine

Fluorination of pharmaceutical compounds is a common tool to modulate their physiochemical properties. We determine the effects of site‐specific aromatic fluorine substitution on the geometric, energetic, vibrational, and electronic properties of the protonated neurotransmitter 2‐phenylethylamine (xF‐H⁺PEA, x=ortho, meta, para) by infrared multiphoton photodissociation (IRMPD) in the fingerprint range (600–1750 cm^?1) and quantum chemical calculations at the B3LYP‐D3/aug‐cc‐pVTZ level. The IRMPD spectra of all ions are assigned to their folded gauche conformers stabilized by intramolecular NH⁺???π hydrogen bonds (H‐bonds) between the protonated amino group and the aromatic ring. H→F substitution reduces the symmetry and allows for additional NH⁺???F interactions in oF‐H⁺PEA, leading to three distinct gauche conformers. In comparison to oF‐H⁺PEA, the fluorination effects on the energy landscape (energy ordering and isomerization barriers) in pF‐H⁺PEA and mF‐H⁺PEA with one and two gauche conformers are less pronounced. The strengths of the intramolecular NH⁺???F and NH⁺???π bonds are analyzed by the noncovalent interaction (NCI) method.     

Apparent inhibition by arginine of macrocyclic <Emphasis Type="Italic">b</Emphasis> ion formation from singly charged protonated peptides

There is now strong evidence for the existence of macrocyclic isomers of b_n⁺ ions, the formation and subsequent opening of which can lead to loss of sequence information from protonated peptides in multiple-stage tandem mass spectrometry experiments. In this study, the fragmentation patterns of protonated YARFLG and permuted isomers of the model peptide were investigated by collision-induced dissociation. Of interest was the potential influence of the arginine residue, and its position in the peptide sequence, on formation of the presumed macrocyclic b₅ ion isomer and potential loss of sequence information. We find that regardless of the sequence position (either internal or at the N- or C-terminus), only direct sequence ions or ions directly related to fragmentation of the arginine side chain are observed.