Hydrogen Bonding Shuts Down Tunneling in Hydroxycarbenes: A Gas-Phase Study by Tandem-Mass Spectrometry, Infrared Ion Spectroscopy, and Theory.

Hydroxycarbenes can be generated and structurally characterized in the gas phase by collision-induced decarboxylation of α-keto carboxylic acids, followed by infrared ion spectroscopy. Using this approach, we have shown earlier that quantum-mechanical hydrogen tunneling (QMHT) accounts for the isomerization of a charge-tagged phenylhydroxycarbene to the corresponding aldehyde in the gas phase and above room temperature. Herein, we report the results of our current study on aliphatic trialkylammonio-tagged systems. Quite unexpectedly, the flexible 3-(trimethylammonio)propylhydroxycarbene turned out to be stable─no H-shift to either aldehyde or enol occurred. As supported by density functional theory calculations, this novel QMHT inhibition is due to intramolecular H-bonding of a mildly acidic α-ammonio C-H bonds to the hydroxyl carbene's C-atom (C:···H-C). To further support this hypothesis, (4-quinuclidinyl)hydroxycarbenes were synthesized, whose rigid structure prevents this intramolecular H-bonding. The latter hydroxycarbenes underwent "regular" QMHT to the aldehyde at rates comparable to, e.g., methylhydroxycarbene studied by Schreiner et al. While QMHT has been shown for a number of biological H-shift processes, its inhibition by H-bonding disclosed here may serve for the stabilization of highly reactive intermediates such as carbenes, even as a mechanism for biasing intrinsic selectivity patterns.

Breslow Intermediates (Amino Enols) and Their Keto Tautomers: First Gas-Phase Characterization by IR Ion Spectroscopy.

Breslow intermediates (BIs) are the crucial nucleophilic amino enol intermediates formed from electrophilic aldehydes in the course of N-heterocyclic carbene (NHC)-catalyzed umpolung reactions. Both in organocatalytic and enzymatic umpolung, the question whether the Breslow intermediate exists as the nucleophilic enol or in the form of its electrophilic keto tautomer is of utmost importance for its reactivity and function. Herein, the preparation of charge-tagged Breslow intermediates/keto tautomers derived from three different types of NHCs (imidazolidin-2-ylidenes, 1,2,4-triazolin-5-ylidenes, thiazolin-2-ylidenes) and aldehydes is reported. An ammonium charge tag is introduced through the aldehyde unit or the NHC. ESI-MS IR ion spectroscopy allowed the unambiguous conclusion that in the gas phase, the imidazolidin-2-ylidene-derived BI indeed exists as a diamino enol, while both 1,2,4-triazolin-5-ylidenes and thiazolin-2-ylidenes give the keto tautomer. This result coincides with the tautomeric states observed for the BIs in solution (NMR) and in the crystalline state (XRD), and is in line with our earlier calculations on the energetics of BI keto-enol equilibria.

Free radical-initiated peptide sequencing (FRIPS)-based cross-linkers for improved peptide and protein structure analysis.

Free radical-initiated peptide sequencing (FRIPS) has recently been introduced as an analytical strategy to create peptide radical ions in a predictable and effective way by collisional activation of specifically modified peptides ions. FRIPS is based on the unimolecular dissociation of open-shell ions and yields fragments that resemble those obtained by electron capture dissociation (ECD) or electron transfer dissociation (ETD). In this review article, we describe the fundamentals of FRIPS and highlight its fruitful combination with chemical cross-linking/mass spectrometry (MS) as a highly promising option to derive complementary structural information of peptides and proteins. FRIPS does not only yield exhaustive sequence information of cross-linked peptides, but also defines the exact cross-linking sites of the connected peptides. The development of more advanced FRIPS cross-linkers that extend the FRIPS-based cross-linking/MS approach to the study of large protein assemblies and protein interaction networks can be eagerly anticipated.

IRMPD Spectroscopic and Theoretical Structural Investigations of Zinc and Cadmium Dications Bound to Histidine Dimers.

Metallated gas-phase structures consisting of a deprotonated and an intact histidine (His) ligand, yielding M(His-H)(His)+, where M = Zn and Cd, were examined with infrared multiple photon dissociation (IRMPD) action spectroscopy utilizing light from a free-electron laser (FEL). In parallel, quantum chemical calculations identified several low-energy isomers for each complex. Experimental action spectra were compared to linear spectra calculated at the B3LYP level of theory using the 6-311+G(d,p) and def2-TZVP basis sets for the zinc and cadmium complexes, respectively. For both Zn and Cd species, the definitive assignment is complicated by conflicting relative energetics, which were calculated at B3LYP, B3LYP-GD3BJ, B3P86, and MP2(full) levels. Spectral comparison for both species indicates that the dominant conformation, [Nα,Nπ,CO-][CO2-](NπH+), has the deprotonated His chelating the metal at the amine nitrogen, π nitrogen of the imidazole ring, and the deprotonated carbonyl oxygen and that the intact His ligand adopts a salt-bridge bidentate binding motif, coordinating the metal with both carboxylate oxygens. There is also evidence for a conformation where the deprotonated His coordination is maintained, but the intact His ligand adopts a more canonical structure, coordinating with the metal atom at the amine nitrogen and π nitrogen, [Nα,Nπ,CO-][Nα,Nπ]gtgg. For both metallated species, B3LYP, B3P86, and B3LYP-GD3BJ levels of theory appear to describe the relative stability of the dominant zwitterionic species more accurately than the MP2(full) level.

Intermediates of N-Heterocyclic Carbene (NHC) Dimerization Probed in the Gas Phase by Ion Mobility Mass Spectrometry: C-H⋠⋠⋠:C Hydrogen Bonding Versus Covalent Dimer Formation.

N-Heterocyclic carbenes (NHCs, :C) can interact with azolium salts (C-H+ ) by either forming a hydrogen-bonded aggregate (CHC+ ) or a covalent C-C bond (CCH+ ). In this study, the intramolecular NHC-azolium salt interactions of aromatic imidazolin-2-ylidenes and saturated imidazolidin-2-ylidenes have been investigated in the gas phase by traveling wave ion mobility mass spectrometry (TW IMS) and DFT calculations. The TW IMS experiments provided evidence for the formation of these important intermediates in the gas phase, and they identified the predominant aggregation mode (hydrogen bond vs. covalent C-C) as a function of the nature of the interacting carbene-azolium pairs.

Hydrogen tunneling avoided: enol-formation from a charge-tagged phenyl pyruvic acid derivative evidenced by tandem-MS, IR ion spectroscopy and theory.

A charge-tagged phenyl pyruvic acid derivative was investigated by tandem-MS, infrared (IR) ion spectroscopy and theory. The tailor-made precursor ions efficiently lose CO2 in collision induced dissociation (CID) experiments, offering access to study the secondary decay reactions of the product ions. IR ion spectroscopy provides evidence for the formation of an enol acid precursor ion structure in the gas phase and indicates the presence of enol products formed after CO2 loss. Extensive DFT computations however, suggest intermediate generation of hydroxycarbene products, which in turn rearrange in a secondary process to the enol ions detected by IR ion spectroscopy. Quantum mechanical tunneling of the hydroxycarbene can be excluded since no evidence for aldehyde product ion formation could be found. This finding is in contrast to the behavior of methylhydroxycarbene, which cleanly penetrates the energy barrier to form exclusively acetaldehyde at cryogenic temperatures in an argon matrix via quantum mechanical hydrogen tunneling. The results presented here are attributed to the highly excited energy levels of the product ions formed by CID in combination with different barrier heights of the competing reaction channels, which allow exclusive access over one energy barrier leading to the formation of the enol tautomer ions observed.

Hydrogen Tunneling above Room Temperature Evidenced by Infrared Ion Spectroscopy.

While hydrogen tunneling at elevated temperatures has, for instance, often been postulated in biochemical processes, spectroscopic proof is thus far limited to cryogenic conditions, under which thermal reactivity is negligible. We report spectroscopic evidence for H-tunneling in the gas phase at temperatures around 320-350 K observed in the isomerization reaction of a hydroxycarbene into an aldehyde. The charge-tagged carbene was generated in situ in a tandem mass spectrometer by decarboxylation of oxo[4-(trimethylammonio)phenyl]acetic acid upon collision induced dissociation. All ion structures involved are characterized by infrared ion spectroscopy and quantum chemical calculations. The charge-tagged phenylhydroxycarbene undergoes a 1,2-H-shift to the corresponding aldehyde with an half-life of about 10 s, evidenced by isomer-selective two-color (IR-IR) spectroscopy. In contrast, the deuterated (OD) carbene analogue showed much reduced 1,2-D-shift reactivity with an estimated half-life of at least 200 s under the experimental conditions, and provides clear evidence for hydrogen atom tunneling in the H-isotopologue. This is the first spectroscopic confirmation of hydrogen atom tunneling governing 1,2-H-shift reactions at noncryogenic temperatures, which is of broad significance for a range of (bio)chemical processes, including enzymatic transformations and organocatalysis.

Ergothioneine and related histidine derivatives in the gas phase: tautomer structures determined by IRMPD spectroscopy and theory.

l-Ergothioneine (ET) is a sulfur-containing derivative of the amino acid histidine that offers unique antioxidant properties. The enzyme independent redox-chemistry of ET relies on the availability of the thiol tautomer to allow oxidative formation of disulfide bridges, i.e., the tautomeric equilibrium. To study the intrinsic properties of ET the tautomeric equilibrium is studied in the gas-phase by infrared multiphoton dissociation (IRMPD) spectroscopy. The IR ion spectra of isolated molecular ions of ET and of the biosynthetic precursors of ET, i.e., hercynine and Nε-methyl-hercynine are acquired. The analyte structures are independently investigated by density functional theory (DFT) and computed linear IR-spectra of tautomer ion structures are compared with the gas-phase spectra for identification. For the molecular ion of ET the simulated IR spectra of thione and thiol structures match the recorded IRMPD spectrum and that prevents an individual structure assignment. On the other hand, theory suggests that ET adopts a thione tautomer in MeOH solution which could be carried over from the condensed phase to gas phase and could be kinetically trapped after effective electrospray phase transfer and desolvation. Such a non-thermal behavior is also found for the molecular ions of protonated hercynine and Nε-methyl-hercynine. Contrary to that, the sodium complex ions of ET, hercynine and Nε-methyl-hercynine adopt the respective ground structures predicted by theory, which are reliably identified spectroscopically. For ET the thione tautomer is by far the most stable isomer in the sodium complex molecular ion.

An Integrated Mass Spectrometry Based Approach to Probe the Structure of the Full-Length Wild-Type Tetrameric p53 Tumor Suppressor.

We present an integrated approach for investigating the topology of proteins through native mass spectrometry (MS) and cross-linking/MS, which we applied to the full-length wild-type p53 tetramer. For the first time, the two techniques were combined in one workflow to obtain not only structural insight in the p53 tetramer, but also information on the cross-linking efficiency and the impact of cross-linker modification on the conformation of an intrinsically disordered protein (IDP). P53 cross-linking was monitored by native MS and as such, our strategy serves as a quality control for different cross-linking reagents. Our approach can be applied to the structural investigation of various protein systems, including IDPs and large protein assemblies, which are challenging to study by the conventional methods used for protein structure characterization.

Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS Cleavable Cross-Linker.

Cross-linking combined with mass spectrometry (MS) has evolved as an alternative strategy in structural biology for characterizing three-dimensional structures of protein assemblies and for mapping protein-protein interactions. Here, we describe an integrated workflow for an automated identification of cross-linked products that is based on the use of a tandem mass spectrometry (MS/MS) cleavable cross-linker (containing a 1,3-bis-(4-oxo-butyl)-urea group, BuUrBu) generating characteristic doublet patterns upon fragmentation. We evaluate different fragmentation methods available on an Orbitrap Fusion mass spectrometer for three proteins and an E. coli cell lysate. An updated version of the dedicated software tool MeroX was employed for a fully automated identification of cross-links. The strength of our cleavable cross-linker is that characteristic patterns of the cross-linker as well as backbone fragments of the connected peptides are already observed at the MS/MS level, eliminating the need for conducting MS(3) or sequential CID (collision-induced dissociation)- and ETD (electron transfer dissociation)-MS/MS experiments. This makes our strategy applicable to a broad range of mass spectrometers with MS/MS capabilities. For purified proteins and protein complexes, our workflow using CID-MS/MS acquisition performs with high confidence, scoring cross-links at 0.5% false discovery rate (FDR). The cross-links provide structural insights into the intrinsically disordered tetrameric tumor suppressor protein p53. As a time-consuming manual inspection of cross-linking data is not required, our workflow will pave the way for making the cross-linking/MS approach a routine technique for structural proteomics studies.

Ligand-modulated chemical and structural implications in four-, five-, and six-fold coordinated aluminum heteroaryl alkenolates.

Synthesis and characterization (gas phase, solution, and solid-state) of a series of four-, five- and six-fold coordinated heteroaryl-alkenolato aluminum complexes were performed to demonstrate the delicate interplay of structural and chemical influences of ligands in the design of new precursors for chemical vapor deposition. We are investigating the properties of heteroaryl alkenols as O^N chelating ligands [where O^N is 3,3,3-trifluoro(pyridin-2-yl)propen-2-ol (H-PyTFP), 3,3,3-trifluoro(1,3-benzimidazol-2-yl)propen-2-ol (H-BITFP), 3,3,3-trifluoro(dimethyl-1,3-oxazol-2-yl)propen-2-ol (H-DMOTFP), 3,3,3-trifluoro(1,3-benzoxazol-2-yl)propen-2-ol (H-BOTFP), 3,3,3-trifluoro(1,3-benzthiazol-2-yl)propen-2-ol (H-BTTFP), and 3,3,3-trifluoro(dimethyl-1,3-thiazol-2-yl)propen-2-ol (H-DMTTFP)] to prepare volatile and air-stable compounds. All three methyl groups in highly reactive AlMe3 could be replaced by H-PyTFP, H-BITFP, H-DMOTFP, and H-BOTFP yielding octahedral complexes of the type Al(O^N)3; under similar conditions H-BTTFP and H-DMTTFP produced heteroleptic MeAl(O^N)2 compounds with five-fold coordinated aluminum centers. Various attempts to obtain tris-alkenolato derivatives by choosing higher temperatures and prolonged reaction times were not successful. The reaction of H-PyTFP with [Al(O(t)Bu)3]2 produced the dimeric heteroleptic [Al(PyTFP)(O(t)Bu)2]2 complex with Al atoms present in both octahedral (Oh) and tetrahedral (Td) coordination in a single molecular unit. The introduction of the chelating ligand H-PyTFP in the dimeric framework of [Al(O(t)Bu)3]2 enhanced the stability against hydrolyses significantly. The tendency of Al(III) centers to preferably coordinate in Td or Oh environment was elucidated by hydrolysis studies of monomeric Al(PyTFP)3, Al(BOTFP)3, and MeAl(BTTFP)2 that produced hydroxo-bridged dimers to retain the octahedral environment for Al atoms. Surprisingly, hydrolysis of monomeric MeAl(DMTTFP)2 yielded an oxo-bridged dimer with two five-fold coordinated aluminum centers. The structural features of all new complexes were investigated in solution, vapor, and solid state by multinuclear NMR spectroscopy, EI-MS spectrometry, and single-crystal X-ray diffraction analyses, respectively.

Aryl-phenyl scrambling in intermediate organopalladium complexes: a gas-phase study of the Mizoroki-Heck reaction.

The intramolecular aryl-phenyl scrambling reaction within palladium-DPPP-aryl complex (DPPP=1,3-bis(diphenylphosphino)propane) ions was analyzed by state-of-the-art tandem MS, including gas-phase ion/molecule reactions. The Mizoroki-Heck cross-coupling reaction was performed in the gas phase, and the intrinsic reactivity of important intermediates could be examined. Moreover, linear free-energy correlations were applied, and a mechanism for the scrambling reaction proceeding via phosphonium cations was assumed.

A comprehensive gas chromatography electron ionization high resolution mass spectrometry study of a new steroidal selective androgen receptor modulator (SARM) compound S42.

The synthetic 20-keto-steroid S42 (1) demonstrated selective androgen receptor modulator (SARM) properties in preclinical studies and, consequently, received growing attention also in the context of sports drug testing programs. Fundamental understanding of the behavior of S42 (1) and of relevant derivatives in gas chromatography-electron ionization MS experiments at high resolution (GC-EI-HRMS) is indispensable to develop a reliable qualitative and quantitative doping control method for S42 (1) and its metabolites in body fluid matrices. We present important fundamental mechanistic data on the EI fragmentation behavior of S42 (1) and of silyl ether derivatives as well as of stable isotope-labelled reference material.

New linear lipopeptides produced by Pseudomonas cichorii SF1-54 are involved in virulence, swarming motility, and biofilm formation.

Pseudomonas cichorii is the causal agent of lettuce midrib rot, characterized by a dark-brown to green-black discoloration of the midrib. Formation of necrotic lesions by several plant-pathogenic pseudomonads is associated with production of phytotoxic lipopeptides, which contribute to virulence. Therefore, the ability of P. cichorii SF1-54 to produce lipopeptides was investigated. A cell-free culture filtrate of SF1-54 showed surfactant, antimicrobial, and phytotoxic activities which are typical for lipopeptides. High-performance liquid chromatography analysis of P. cichorii SF1-54 culture filtrate revealed the presence of seven compounds with lipopeptide characteristics. Two related lipopeptides, named cichofactin A and B, were studied in more detail: they are linear lipopeptides with a decanoic and dodecanoic lipid chain, respectively, connected to the N-terminus of an eight-amino-acid peptide moiety. Both cichofactins are new members of the syringafactin lipopeptide family. Furthermore, two nonribosomal peptide synthethase-encoding genes, cifA and cifB, were identified as responsible for cichofactin biosynthesis. A cifAB deletion mutant no longer produced cichofactins and was impaired in swarming motility but showed enhanced biofilm formation. Upon spray inoculation on lettuce, the cichofactin-deficient mutant caused significantly less rotten midribs than the wild type, indicating that cichofactins are involved in pathogenicity of P. cichorii SF1-54. Further analysis revealed that P. cichorii isolates vary greatly in swarming motility and cichofactin production.

Cobalt catalysis in the gas phase: experimental characterization of cobalt(I) complexes as intermediates in regioselective Diels-Alder reactions.

In situ-formed cobalt(I) complexes are proposed to act as efficient catalysts in regioselective Diels-Alder reactions of unactivated substrates such as 1,3-dienes and alkynes. We report the first experimental evidence for the in situ reduction of CoBr2(dppe) [dppe = 1,2-bis(diphenylphosphino)ethane] by Zn/ZnI2 to [Co(I)(dppe)](+) by means of electrospray MS(n) experiments. Additionally, the reactivities of Co(II) and Co(I) dppe complexes toward the Diels-Alder substrates isoprene and phenylacetylene were probed in gas-phase ion/molecule reactions (IMRs). Isoprene and phenylacetylene were introduced into the mass spectrometer via the buffer gas flow of a linear ion trap. The IMR experiments revealed a significantly higher substrate affinity of [Co(I)(dppe)](+) compared with [Co(II)Br(dppe)](+). Furthermore, the central intermediate of the solution-phase cobalt-catalyzed Diels-Alder reaction, [Co(I)(dppe)(isoprene)(phenylacetylene)](+), could be generated via IMR and examined in the gas phase. Collision activation of this complex ion delivered evidence for the gas-phase reaction of isoprene with phenylacetylene in the coordination sphere of the cobalt ion. The experimental findings are consistent with the results of quantum-chemical calculations on all of the observed Co(I) dppe complex ions. The results constitute strong analytical evidence for the formation and importance of different cobalt(I) species in regioselective Diels-Alder reactions of unactivated substrates and identify [Co(I)(dppe)](+) as the active Diels-Alder catalyst.

Examination of the coordination sphere of Al(III) in trifluoromethyl-heteroarylalkenolato complex ions by gas-phase IRMPD spectroscopy and computational modelling.

A series of aluminium complex ions with trifluoromethyl-heteroarylalkenolato (TMHA) ligands are studied by gas-phase infrared multiphoton-dissociation (IRMPD) spectroscopy and computational modelling. The selected series of aluminium TMHA complex ions are promising species for the initial study of intrinsic binding characteristics of Al(III) cations in the gas phase as corresponding molecular ions. They are readily available for examination by (+) and (-) electrospray ionization mass spectrometry (ESI-MS) by spraying of [Al(3+)⋅(L(-))(3)] solutions. The complex ions under investigation contain trivalent Al(3+) cations with two chelating anionic enolate ligands, [Al(3+)⋅(L(-))(2)](+), providing insights in the nature of the heteroatom-Al bonds. Additionally, the structure of a deprotonated benzimidazole ligand, L(-,) and an anionic complex ion of Al(III) with two doubly deprotonated benzimidazole ligands, [Al(3+)⋅(L(2-))(2)](-), are examined by (-)ESI-IRMPD spectroscopy. Experimental and computational results are highly consistent and allow a reliable identification of the ion structures. In all complex ions examined the planar TMHA ligands are oriented perpendicular to each other around the metal ion, leading to a tetrahedral coordination sphere in which aluminium interacts with the enolate oxygen and heteroaryl nitrogen atoms available in each of the bidentate ligands.

Unidirectional Double- and Triple-Hydrogen Rearrangement Reactions Probed by Infrared Ion Spectroscopy.

Unidirectional double-hydrogen (2H) and triple-hydrogen (3H) rearrangement reactions occur upon electron-ionization-induced fragmentation of trans-2-(4-N,N-dimethylaminobenzyl)-1-indanol (1), trans-2-(4-methoxybenzyl)-1-indanol (2), 4-(4-N,N-dimethylaminophenyl)-2-butanol (3), and related compounds, as reported some 35 years ago (Kuck, D.; Filges, U. Org. Mass Spectrom. 1988, 23, 643-653). These unusual intramolecular redox processes were found to dominate the mass spectra of long-lived, metastable ions. The present report provides independent evidence for the structures of the product ions formed by the 2H and 3H rearrangement in an ion trap instrument. The radical cations 1•+ and 3•+ as well as ionized 1-(4-N,N-dimethylaminophenyl)-5-(4-methoxyphenyl)-3-pentanol, 5•+, were generated by electrospray ionization from anhydrous acetonitrile solutions. The 2H and 3H fragment ions were obtained by collision-induced dissociation and characterized by IR ion spectroscopy and density functional theory calculations. Comparison of the experimental and calculated infrared ion spectra enabled the identification of the 2H rearrangement product ion, C9H14N+ (m/z 136), as an N,N-dimethyl-para-toluidinium ion bearing the extra proton ortho to the amino group, a tautomer which was calculated to be 31 kJ/mol less stable than the corresponding N-protonated form. The 3H rearrangement product ion, C8H13N•+ (m/z 123), formerly assumed to be a distonic ammonium ion bearing a cyclohexadienyl radical, was now identified as a conventional radical cation, ionized N,N-dimethyl-2,3-dihydro-para-toluidine. Thus, the 3H rearrangement represents an intramolecular transfer hydrogenation between a secondary alcohol and an ionized aromatic ring. Based on these structural assignments, more detailed mechanisms for the unidirectional 2H and 3H rearrangement reactions are proposed.

Evidence for the role of tetramethylethylenediamine in aqueous Negishi cross-coupling: synthesis of nonproteinogenic phenylalanine derivatives on water.

The structure of the alkylzinc-tetramethylethyl-enediamine (TMEDA) cluster cation 3 has been determined in the gas phase by a combination of tandem mass spectrometry, infrared multiphoton dissociation (IRMPD) spectroscopy, and DFT calculations. Both sets of experimental results establish the existence of a strongly stabilizing interaction of TMEDA with the zinc cation. High-level DFT calculations on the alkylzinc-TMEDA cluster cation 3 allowed the identification of two low energy conformers, each featuring a four-coordinate zinc atom with a bidentate TMEDA ligand, and internal coordination from the carbonyl group of the Boc group to zinc. The experimental IRMPD spectrum is reproduced with an appropriately weighted combination of the IR spectra of the two conformers identified by theory. DFT calculations on the structure of the alkylzinc halide 2 with coordinated TMEDA using the PCM model of water solvent suggest that TMEDA can promote ionization of the zinc-iodine bond in organozinc iodides under aqueous conditions, providing a credible explanation for the role of TMEDA in stabilizing the carbon-zinc bond. Reaction of the serine-derived iodide 1 with aryl iodides "on water", promoted by nano zinc in the presence of PdCl(2)(Amphos)(2) (5 mol %) and TMEDA, leads to the formation of protected phenylalanine derivatives 4 in reasonable yields. In the case of ortho-substituted aryl iodides and aryl iodides that are solids at room temperature, conducting the reaction at 65 °C gives improved results. In all cases, the product 5 of reductive dimerization of the iodide 1 is also isolated.

A universal matrix-assisted laser desorption/ionization cleavable cross-linker for protein structure analysis.

The concept of protein cross-linking in combination with mass spectrometry holds great promise to derive structural information on protein conformation and protein-protein interactions. We recently presented a dissociative amine-reactive cross-linker (NHS-BuUrBu-NHS) that is shown herein to be universally applicable to protein structure analysis under matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) conditions, based on the examples of the peptides substance P, luteinizing hormone releasing hormone (LHRH), and the 32-kDa ligand-binding domain of peroxisome proliferator-activated receptor alpha (PPARα). The characteristic fragment ion patterns and constant neutral losses of the cross-linker greatly simplify the identification of different cross-linked species from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Therefore, this cross-linker holds an enormous potential for deriving structural information of proteins and protein complexes in a highly automated fashion.

Gas-phase study of new organozinc reagents by IRMPD-spectroscopy, computational modelling and tandem-MS.

An extensive set of organozinc iodides, useful for Negishi-type cross-coupling reactions, are investigated as respective cations after formal loss of iodide in the gas phase. Firstly, two new alkylzinc compounds derived from Tyrosine (Tyr) and Tryptophan (Trp) are closely examined. Secondly, the influence of specific protecting groups on the subtle balance between intra- and intermolecular coordination of zinc in these reagents is probed through trifluoroacetyl (TFA)-derivatized alkylzinc compounds. Finally, the influence of the strongly coordinating bidentate ligand N,N,N',N'-tetramethylethylenediamine (TMEDA) on the structure of alkylzinc cations is further explored in order to better understand the stability of the respective complexes towards water. A combination of electrospray (ESI)-MS/MS, accurate ion mass measurements, infrared multiple-photon dissociation (IRMPD) spectroscopy and computational modelling allowed the full characterisation of all dimethylformamide (DMF)-solvated and TMEDA-coordinated alkylzinc cations in the gas phase. The calculations indicate that the zinc cation in gas-phase alkylzinc-DMF or TMEDA-complex ions preferentially adopts a tetrahedral coordination sphere with four ligands. Additionally, conformers with only three binding partners bound to zinc but with effectively combined hydrogen-bond interactions are also found. Collision induced dissociation (CID) patterns demonstrate that the zinc-DMF interaction in tetrahedral four-coordinate mono-DMF-zinc complex ions as well as the interaction between TMEDA and zinc in the corresponding complex ions is even stronger than typical covalent bonds. In most cases, all major features of the IRMPD spectra are consistent with only a single major isomer, allowing secured identification and assignment.