Junfang Zhao

Are the Radical Centers in Peptide Radical Cations Mobile? The Generation, Tautomerism, and Dissociation of Isomeric α-Carbon-Centered Triglycine Radical Cations in the Gas Phase

The mobility of the radical center in three isomeric triglycine radical cations[G(*)GG](+), [GG(*)G](+), and [GGG(*)](+) has been investigated theoretically via density functional theory (DFT) and experimentally via tandem mass spectrometry. These radical cations were generated by collision-induced dissociations (CIDs) of Cu(II)-containing ternary complexes that contain the tripeptides YGG, GYG, and GGY, respectively (G and Y are the glycine and tyrosine residues, respectively). Dissociative electron transfer within the complexes led to observation of [Y(*)GG](+), [GY(*)G](+), and [GGY(*)](+); CID resulted in cleavage of the tyrosine side chain as p-quinomethide, yielding [G(*)GG](+), [GG(*)G](+), and [GGG(*)](+), respectively. Interconversions between these isomeric triglycine radical cations have relatively high barriers (> or = 44.7 kcal/mol), in support of the thesis that isomerically pure [G(*)GG](+), [GG(*)G](+), and [GGG(*)](+) can be experimentally produced. This is to be contrasted with barriers < 17 kcal/mol that were encountered in the tautomerism of protonated triglycine [Rodriquez C. F. et al. J. Am. Chem. Soc. 2001, 123, 3006-3012]. The CID spectra of [G(*)GG](+), [GG(*)G](+), and [GGG(*)](+) were substantially different, providing experimental proof that initially these ions have distinct structures. DFT calculations showed that direct dissociations are competitive with interconversions followed by dissociation.

Conformation Switching in Gas-Phase Complexes of Histidine with Alkaline Earth Ions

Infrared multiple photon dissociation spectroscopy of gas-phase doubly charged alkaline earth complexes of histidine reveals a transition from dominance of the zwitterion (salt bridge, SB) conformation with Ba2+ to substantial presence of the canonical (charge-solvated, CS) conformation with Ca2+. This result is a clear illustration of the importance of metal-ion size in governing the delicate balance between these two modes of complexation of gas-phase amino acids. The two conformational motifs are clearly distinguished by characteristic spectral features, confirmed by density functional theory simulated IR spectra of the low-energy conformers. As a further illustration of histidine complexation possibilities, the spectrum of the Na+His complex shows purely CS character and emphasizes the greater tendency toward SB character induced by the higher charge in the alkaline earth complexes. Calculation of the complete series of alkaline earth/histidine complexes confirms the increasing stability of the SB conformations relative to CS with increasing metal ion size, as well as showing that among SB conformations the most highly chelated conformation (SB3) is favored for small metals, whereas the most extended conformation (SB1) is favored for large metals. A decomposition of the binding thermochemistry shows that these thermochemical trends versus metal-ion size are due to differences in electrostatic binding energies, with relatively little contribution from the deformation and rearrangement energy costs of distorting the ligand framework.

Structure of the Observable Histidine Radical Cation in the Gas Phase: A Captodative α-Radical Ion†

Protein-based radicals play crucial roles in some of the greatest biosynthetic challenges in nature, including photosynthesis and substrate oxidation. Radical centers have been located on aromatic and sulfur-containing amino acid residues, as well as glycine residues. Invariably these charged or neutral radical species are generated through involvement of an adjacent metal cofactor. The positions of charge and spin in the radical cations are paramount for reactivity modulation and proton-coupled electron transfer, but obtaining structural details is difficult even for the simplest models. 2] Experiments in vacuo permit the investigation of intrinsic properties of radical cations in the absence of a reactivity-modulating environment. Radical cations of amino acids and peptides have been produced in vacuo by one-electron transfer in collision-induced dissociations (CIDs) of a ternary complex system comprising copper(II), an auxiliary ligand, and the amino acid or peptide. Such ternary complexes are efficiently generated by electrospray ionization, and probed downstream by using mass spectrometry (MS). Under appropriate conditions, CID of the complex yields the radical cation of the amino acid or peptide that can be isolated and trapped for spectroscopic interrogation. Herein, we report the first infrared multiple photon dissociation (IRMPD) spectroscopic experiments on a prototypical amino acid radical cation, HisC, and its ternary complex ion. In a recent article, Ke et al. showed that, by judicious choice of the auxiliary ligand, HisC of different stabilities are formed through CID of the ternary complex ion. In particular, the use of 2,2’:6’,2’’-terpyridine (tpy) as the ligand leads primarily to a HisC that is stable on the MS timescale and can be isolated and fragmented at a subsequent MS stage; by contrast, employing acetone as the ligand results in a metastable HisC and only its fragment ions are observed. Furthermore, the former, relatively stable HisC fragments by losing a water molecule to give [b1-H]C + and then CO to give [a1-H]C , whereas the latter, metastable HisC dissociates spontaneously by losing first CO2 to give the 4-ethaniminoimidazole radical cation, which then loses methanimine to give the 4-methyleneimidazole radical cation. Density functional theory (DFT) calculations at the (unrestricted) UB3LYP/6-311 + + G(d,p) level of theory predicted five low-energy HisC structures. Scheme 1 shows these structures with additional, new information on the barriers against their interconversions (see Figures S2 and S3 in the Supporting Information for details). Ke et al. postulated that the stable and metastable HisC are His5 (the structure at the global minimum) and His2, respectively. His5 is a captodative aradical ion that differs from the canonical His1 structure in having the a-CH hydrogen migrated to the imino nitrogen of the imidazole ring; His2 is best described as a 4-ethaniminoimidazole radical cation solvated by CO2. His2–His5 are all unconventional structures, and experimental verification of the HisC structure is highly desirable for confirmation of the key roles played by spin and charge delocalization in HisC stabilization. Figure 1 compares the experimental IRMPD spectrum collected for HisC with the DFT-predicted IR spectra of His1–His5. It is apparent that only one predicted IR spectrum, that of His5, resembles the measured IRMPD spectrum. In particular, His5 is the only isomer predicted to exhibit two bands, 1596 and 1653 cm , which are assigned as NH2 scissoring and C=O stretching, respectively, that match the 1606 and 1666 cm 1 bands in the IRMPD spectrum. The lack of a strong band at around 1780–1790 cm 1 in the IRMPD spectrum rules out the presence of a significant fraction of His3 and His4. Similarly, His1 can be ruled out by the presence of the doublet, 1606 and 1666 cm , and the absence of spectroscopic details in the region of 1077– 1320 cm . His2 can be eliminated by the absence of peaks at around 810–820 cm 1 and by the low endothermicity against loss of the solvating CO2 (5 kcalmol ). We interpret the excellent match between the experimental IRMPD spectrum and the predicted IR spectrum of His5 to indicate that His5 is the only abundant species present. This degree of selectivity is feasible as His5 is positioned at the bottom of a deep well on the potential-energy surface of HisC. The barriers against His5 converting into the other His isomers and dissociating into [b1-H]C + are high (Scheme 1), [*] Dr. J. Zhao, Dr. C.-K. Siu, Y. Ke, Dr. U. H. Verkerk, Prof. A. C. Hopkinson, Prof. K. W. M. Siu Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street Toronto, ON M3J 1P3 (Canada) E-mail: kwmsiu@yorku.ca

Intramolecular hydrogen atom migration along the backbone of cationic and neutral radical tripeptides and subsequent radical-induced dissociations.

Dissociation of peptide radical ions involves competition between charge-induced and radical-induced reactions that can be preceded by isomerization. The isomeric radical cations of the peptide methyl ester [G˙GR-OMe](+) and [GG˙R-OMe](+) provide very similar collision-induced dissociation (CID) spectra, suggesting that isomerization occurs prior to fragmentation. They undergo characteristic radical-induced bond cleavage of the peptide N-terminal amide bond resulting in the y2(+) ion, and of the arginine side-chains Cα-Cβ bond giving protonated allylguanidine {[CH2[double bond, length as m-dash]CHCH2NHC(NH2)2](+), m/z 100}. The absence of a y2(+) fragment ion in the CID of the radical cationic tripeptide [ACH3G˙R](+) and of an m/z 100 ion in the spectrum of [G˙ACH3R](+) (where ACH3 is an α-aminoisobutyric acid residue, which cannot form an α-carbon-centered radical through hydrogen atom transfer) establishes the importance of hydrogen atom migration along the peptide backbone prior to specific radical-induced fragmentations. Herein we use density functional theory (DFT) at the B3LYP/6-31++G(d,p) level to evaluate the barriers for interconversion between the α-carbon-centered radicals and for dissociation. The radical cations [G˙GR](+) and [GG˙R](+) have their radicals located on the α-carbon atoms of the peptide backbone and their charge densities largely sequestered on the guanidine groups of the side-chain of arginine residues. This is in contrast to the isomeric radical cations of [GGG]˙(+), in which the charge resides necessarily on the peptide backbone. The lower charge densities on the backbones of [G˙GR](+) and [GG˙R](+) result in greater structural flexibility, decreasing the barrier for interconversion between these α-carbon-centered radicals to 36.2 kcal mol(-1) (cf. 44.7 kcal mol(-1) for [GGG]˙(+)). The total absence of charge, assessed by examining intramolecular hydrogen atom transfers among the three α-carbon centers of the isomeric neutral α-carbon-centered triglycine radicals [GGG-H]˙, leads to an additional but slight reduction in enthalpy, to approximately 34 kcal mol(-1).

Kinetics for tautomerizations and dissociations of triglycine radical cations

Fragmentations of tautomers of the α-centered radical triglycine radical cation, [GGG•]+, [GG•G]+, and [G•GG]+, are charge-driven, giving b-type ions; these are processes that are facilitated by a mobile proton, as in the fragmentation of protonated triglycine (Rodriquez, C. F. et al. J. Am. Chem. Soc. 2001, 123, 3006–3012). By contrast, radical centers are less mobile. Two mechanisms have been examined theoretically utilizing density functional theory and Rice-Ramsperger-Kassel-Marcus modeling: (1) a direct hydrogen-atom migration between two α-carbons, and (2) a two-step proton migration involving canonical [GGG]•+ as an intermediate. Predictions employing the latter mechanism are in good agreement with results of recent CID experiments (Chu, I. K. et al. J. Am. Chem. Soc. 2008, 130, 7862–7872).

Structure of the [M+H-H2O](+) Ion from Tetraglycine: A Revisit by Means of Density Functional Theory and Isotope Labeling

Collision-induced dissociations of protonated (18)O-labeled tetraglycines labeled separately at either the first or the second amide bond established that water loss from the backbone occurs from the N-terminal residue. Density functional theory at B3LYP/6-311++G(d,p) predicted that the low-energy [G(4) + H - H(2)O](+) product ion is an N(1)-protonated 3,5-dihydro-4H-imidazol-4-one. The ion at the lowest energy, III, is 24.8 kcal mol(-1) lower than the protonated oxazole structure, II, proposed by Bythell et al. (J. Phys. Chem A2010, 114, 5076-5082). In addition, structure III has a predicted IR spectrum that provides a better match with the published experimental IRMPD spectrum than that of structure II.

Infrared Multiple-Photon Dissociation Spectroscopy of Tripositive Ions: Lanthanum-Tryptophan Complexes

Collision-induced charge disproportionation limits the stability of triply charged metal ion complexes and has thus far prevented successful acquisition of their gas-phase IR spectra. This has curtailed our understanding of the structures of triply charged metal complexes in the gas phase and in biological environments. Herein we report the first gas-phase IR spectra of triply charged La(III) complexes with a derivative of tryptophan (N-acetyl tryptophan methyl ester), and an unusual dissociation product, a lanthanum amidate. These spectra are compared with those predicted using density functional theory. The best structures are those of the lowest energies that differ by details in the π-interaction between La(3+) and the indole rings. Other binding sites on the tryptophan derivative are the carbonyl oxygens. In the lanthanum amidate, La(3+) replaces an H(+) in the amide bond of the tryptophan derivative.

Dissociations of Complexes Between Monovalent Metal Ions and Aromatic Amino Acid or Histidine

AbstractThe fragmentations of [AA + M]+ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag•). Caption for Graphical AbstractCID products of Li+, Na+, and Ag+ complexes of Phe, Tyr, Trp, and His are reported and mechanisms by which they are formed are proposed.

Fragmentation of Peptide Radical Cations Containing a Tyrosine or Tryptophan Residue: Structural Features That Favor Formation of [x(n–1) + H]•+ and [z(n–1) + H]•+ Ions

Peptide radical cations A(n)Y(•+) (where n = 3, 4, or 5) and A5W(•+) have been generated by collision-induced dissociation (CID) of [Cu(II)(tpy)(peptide)](•2+) complexes. Apart from the charge-driven fragmentation at the N-Cα bond of the hetero residue producing either [c + 2H](+) or [z - H](•+) ions and radical-driven fragmentation at the Cα-C bond to give a(+) ions, unusual product ions [x + H](•+) and [z + H](•+) are abundant in the CID spectra of the peptides with the hetero residue in the second or third position of the chain. The formation of these ions requires that both the charge and radical be located on the peptide backbone. Energy-resolved spectra established that the [z + H](•+) ion can be produced either directly from the peptide radical cation or via the fragment ion [x + H](•+). Additionally, backbone dissociation by loss of the C-terminal amino acid giving [b(n-1) - H](•+) increases in abundance with the length of the peptides. Mechanisms by which peptide radical cations dissociate have been modeled using density functional theory (B3LYP/6-31++G** level) on tetrapeptides AYAG(•+), AAYG(•+), and AWAG(•+).

Fragmentation Chemistry of [Met-Gly]•+, [Gly-Met]•+, and [Met-Met]•+ Radical Cations

AbstractRadical cations [Met-Gly]•+, [Gly-Met]•+, and [Met-Met]•+ have been generated through collision-induced dissociation (CID) of [CuII(CH3CN)2(peptide)]•2+ complexes. Their fragmentation patterns and dissociation mechanisms have been studied both experimentally and theoretically using density functional theory at the UB3LYP/6-311++G(d,p) level. The captodative structure, in which the radical is located at the α-carbon of the N-terminal residue and the proton is on the amide oxygen, is the lowest energy structure on each potential energy surface. The canonical structure, with the charge and spin both located on the sulfur, and the distonic ion with the proton on the terminal amino group, and the radical on the α-carbon of the C-terminal residue have similar energies. Interconversion between the canonical structures and the captodative isomers is facile and occurs prior to fragmentation. However, isomerization to produce the distonic structure is energetically less favorable and cannot compete with dissociation except in the case of [Gly-Met]•+. Charge-driven dissociations result in formation of [bn – H]•+ and a1 ions. Radical-driven dissociation leads to the loss of the side chain of methionine as CH3-S-CH = CH2 producing α-glycyl radicals from both [Gly-Met]•+ and [Met-Met]•+. For [Met-Met]•+, loss of the side chain occurs at the C-terminal as shown by both labeling experiments and computations. The product, the distonic ion of [Met-Gly]•+, NH3+CH(CH2CH2SCH3)CONHCH•COOH dissociates by loss of CH3S•. The isomeric distonic ion NH3+CH2CONHC•(CH2CH2SCH3)COOH is accessible directly from the canonical [Gly-Met]•+ ion. A fragmentation pathway that characterizes this ion (and the distonic ion of [Met-Met]•+) is homolytic fission of the Cβ–Cγ bond to lose CH3SCH2•.