Protonated intermediates are postulated to be involved in the rate determining step of many sugar reactions. This paper presents a study of protonated sugar species, isolated in the gas phase, using a combination of infrared multiple photon dissociation (IRMPD) spectroscopy, classical ab initio molecular dynamics (AIMD) and quantum mechanical vibrational self-consistent field (VSCF) calculations. It provides a likely identification of the reactive intermediate oxocarbenium ion structure in a d-galactosyl system as well as the saccharide pyrolysis product anhydrogalactose (that suggests oxocarbenium ion stabilization), along with the spectrum of the protonated parent species: methyl d-galactopyranoside-H+. Its vibrational fingerprint indicates intramolecular proton sharing. Classical AIMD simulations for galactosyl oxocarbenium ions, conducted in the temperature range ∼300-350 K (using B3LYP potentials on-the-fly) reveal efficient transitions on the picosecond timescale. Multiple conformers are likely to exist under the experimental conditions and along with static VSCF calculations, they have facilitated the identification of the individual structural motifs of the galactosyl oxocarbenium ion and protonated anhydrogalactose ion conformers that contribute to the observed experimental spectra. These results demonstrate the power of experimental IRMPD spectroscopy combined with dynamics simulations and with computational spectroscopy at the anharmonic level to unravel conformer structures of protonated saccharides, and to provide information on their lifetimes.

Spectroscopic investigations of biomolecular structure and interactions in the gas phase, free from environmental ‘disturbance’, have become a growth industry over the last decade. The great majority however, have taken their ‘bio-pedigrees’ as a given rather than an issue to be addressed. This review attempts to move towards aspects of their biological, or perhaps more accurately, biochemical relevance, by reviewing some recent and current investigations of peptide and carbohydrate systems in the gas phase, chosen not just because they are ‘do-able’ but because of the biochemical questions they address. Hopefully, this will help to foster better communication between the molecular physics, biochemistry and molecular biology communities.

Protonated neurotransmitters have been produced in the gas phase via a novel photochemical scheme: complexes of the species of interest, 1-phenylethylamine, 2-amino-1-phenylethanol and the diastereo-isomers, ephedrine and pseudoephedrine, with a suitable proton donor, phenol (or indole), are produced in a supersonic expansion and ionized by resonant two photon ionization of the donor. Efficient proton transfer generates the protonated neurotransmitters, complexed to a phenoxy radical. Absorption of infrared radiation, and subsequent evaporation of the phenoxy tag, coupled with time of flight mass spectrometry, provides vibrational spectra of the protonated (and also hydrated) complexes for comparison with the results of quantum chemical computation. Comparison with the conformational structures of the neutral neurotransmitters (established previously) reveals the effect of protonation on their structure. The photochemical proton transfer strategy allows spectra to be recorded from individual laser shots and their quality compares favourably with that obtained using electro-spray or matrix assisted laser desorption ion sources.

The conformation and structure of phenyl-alpha-l-fucopyranoside (alpha-PhFuc), phenyl-beta-L-fucopyranoside (beta-PhFuc) and their singly hydrated complexes (alpha,beta-PhFuc.H(2)O) isolated in a molecular beam, have been investigated by means of resonant two photon ionization (R2PI) spectroscopy and ultraviolet and infrared ion-dip spectroscopy. Conformational and structural assignments have been based on comparisons between their experimental and computed near IR spectra, calculated using density functional theory (DFT) and their relative energies, determined from ab initio (MP2) calculations. The near IR spectra of "free" and hydrated alpha- and beta-PhFuc, and many other mono- and di-saccharides, provide extremely sensitive probes of hydrogen-bonded interactions which can be finely tuned by small (or large) changes in the molecular conformation. They provide characteristic "signatures" which reflect anomeric, or axial vs. equatorial differences, both revealed through comparisons between alpha/beta-PhFuc and alpha/beta-PhXyl; or similarities, revealed through comparisons between fucose (6-deoxy galactose) and galactose; or binding motifs, for example, "insertion" vs. "addition" structures in hydrated complexes. At the monosaccharide level (the first step in the carbohydrate hierarchy), these trends appear to be general. In contrast to the monohydrates of galactose (beta-PhGal) and glucose (beta-PhGlc), the conformations of alpha- and beta-PhFuc are unaffected by the binding of a single water molecule though changes in the R2PI spectra of multiply hydrated alpha-PhFucW(n) however, may reflect a conformational transformation when n> or = 3.

The intrinsic conformer specific vibrational spectra of two important subunits of the core pentasaccharide of N-linked glycans, the alpha(1,3) and alpha(1,6) dimannosides, have been recorded in the gas phase. Coupling these measurements with a computational exploration of their conformational landscapes has enabled their conformational assignment and has identified characteristic vibrational signatures associated with particular conformational families-including those that do or do not display inter-ring hydrogen bonding across the glycosidic linkage. In addition, the IR spectra of the monosaccharide moieties provide benchmarks, through which the conformational assignments can be refined. This introduces a general concept of modularity and secondary structure in oligosaccharides--essential for the success of similar studies on larger oligosaccharides in the future.

A computational (ab initio and molecular dynamics) and experimental exploration of the relative importance of molecular conformation and explicit solvent effects on the electronic circular dichroism (ECD) of chiral molecules, is presented. The exploration includes an assessment of the validity of angular correlation (sector) rules linking ECD to molecular conformation. It is based upon studies of 1-(R) phenylethanol (including its Raman optical activity spectrum), the corresponding 'benchmark' base, 1-(R)-phenylethylamine and its protonated cation; their hydrated clusters in the gas phase; and their non-polar and aqueous solutions. Emphasis is placed on the influence of specific, hydrogen bonded interactions with the aqueous solvent. The theoretical validity of the (otherwise empirical) sector rule in the neutral molecules and in their specifically hydrated clusters has been established—but with a reversal of the 'historical' sign convention. Protonation of the amine leads to a breakdown of the conventional sector rule but the change in its ECD intensity can still be related to the side chain dihedral angular dependence of its rotatory strength, computed ab initio for its explicitly hydrated clusters. Comparisons between ECD spectra measured in aqueous and in hydrocarbon solutions and the results of molecular dynamics calculations for aqueous solutions at 300 K, identify solvent induced structural change as the principal determinant of their relative ECD spectral intensities. Further links connecting the structures and conformations of chiral molecules and their explicitly solvated clusters in the gas phase, to their structures and conformational populations in solution can be expected through measurement, ab initio computation and analysis of their vibrational, ROA spectra.

The functional importance of carbohydrates in biological processes, particularly those involving specific molecular recognition, is immense. Characterizing the three-dimensional structures of carbohydrates and glycoconjugates and their interactions with other molecules, particularly the ubiquitous solvent, water, are key starting points on the road towards the understanding of these processes. The review introduces a new strategy, combining electronic and vibrational spectroscopy of mass-selected carbohydrate molecules and their hydrated ( and also protonated) complexes, conducted under molecular beam conditions, with ab initio computation. Its early successes have revealed a uniquely powerful means of characterizing carbohydrate conformations and hydrated structures, the hydrogen-bonded networks they support ( or which support them) and the specificity of their interactions with other molecules. The new information, obtained in the gas phase, complements that provided by more 'traditional' condensed phase methods such as NMR, X-ray diffraction, molecular mechanics and molecular dynamics calculations. The review concludes with a vision of the challenges and opportunities offered by applications of molecular beam spectroscopy and their relevance in a biological context.

The conformation of phenyl-substituted monosaccharides (mannose, galactose, and glucose) and their singly hydrated complexes has been investigated in the gas phase by means of a combination of mass selected, conformer specific ultraviolet and infrared double resonance hole burning spectroscopy experiments, and ab initio quantum chemistry calculations. In each case, the water molecule inserts into the carbohydrate at a position where it can replace a weak intramolecular interaction by two stronger intermolecular hydrogen bonds. The insertion can produce significant changes in the conformational preferences of the carbohydrates, and there is a clear preference for structures where cooperative effects enhance the stability of the monosaccharide conformers to which the water molecule chooses to bind. The conclusions drawn from the study of monosaccharide-water complexes are extended to the disaccharide lactose and discussed in the light of the underlying mechanisms that may be involved in the binding of carbohydrate assemblies to proteins and the involvement, or not, of key structural water molecules.

The gas phase structures of phenyl alpha- and beta-d-xylopyranoside (alpha- and beta-pXyl) and their mono-hydrates have been investigated using a combination of resonant two-photon ionization (R2PI), ultra-violet hole-burning and resonant infrared ion dip spectroscopy, coupled with density functional theory (DFT) and ab initio computation. The hole-burning experiments indicate the population of a single conformer only, in each of the two anomers. Their experimental and calculated infrared spectra are both consistent with a conformational assignment corresponding to the computed global minimum configuration. All three OH groups are oriented towards the oxygen atom (O1) on the anomeric carbon atom to form an all trans(ttt) counter-clockwise chain of hydrogen bonds. The mono-hydrates, alpha- and beta-pXyl(H(2)O) each populate two distinct structures in the molecular beam environment, with the water molecule inserted between OH4 and OH3 or between OH3 and OH2 in alpha-pXyl(H2O), and between OH2 and O1 in either of two alternative orientations, in beta-pXyl(H2O). In all of the mono-hydrated xyloside complexes, the water molecule inserts into the weakest link of the sugar molecules' hydrogen-bonded chain of hydroxy groups, creating a single extended chain, strengthened by co-operativity. The all-trans configuration of the xylose moiety is retained and the mono-hydrate structures correspond to those calculated to lie at the lowest relative energies.

The infrared spectra of hydrated complexes of tryptophan have been recorded in the gas phase over the range 160–800 cm−1 using double resonance IR-UV ion dip spectroscopy. Despite the problems arising from severe UV spectral overlap of unresolved resonances, the IR measurements, combined with new DFT and ab initio calculations have allowed a re-assessment of the spectral assignments proposed in an earlier combined near-infrared/quantum computational investigation [L. C. Snoek, R. T. Kroemer and J. P. Simons, Phys. Chem. Chem. Phys. 2002, 4, 2130]. It has reinforced the conclusion that hydration leads to conformational restructuring and also served to focus attention on the information that can be provided by spectroscopic measurements obtained in the mid and far IR.

The deoxynojirimycin (DNJ) family of imino sugars are glucose analogues with an NH group replacing the oxygen atom in the pyranose ring. They are powerful inhibitors of glycosidases and ceramide glucosyltransferases. The conformation of N-benzyl-DNJ, isolated in the gas phase, is studied using a combination of resonant two-photon ionization (R2PI), UV/UV hole-burn, and IR ion-dip spectroscopies in conjunction with electronic structure theory calculations. Three distinct conformers, one major and two minor, are present and all three are assigned to structures in which the exocyclic hydroxymethyl group is axial to the piperidine ring (gauche- to the ring nitrogen). This contrasts with the preferred equatorial (gauche+) orientation observed for simple glucosides and may well contribute to the stronger binding of some enzymes to DNJ-based inhibitors compared to their natural glucoside substrates.

The sensitivity of the electronic circular dichroism (ECD) of a chiral molecule to structural and environmental changes has been investigated using 1-(R)phenylethanol (1-PE) as the benchmark solute and cyclohexane and water as the trial solvents. Rotatory strengths associated with the pi --> pi* (L-1(b)) electronic transition have been calculated ab initio for: (a) isolated 1-PE, as a function of the dihedral angles within its chiral side chain and between the side chain and the aromatic ring: these confirm the validity of the empirical "sector rule" but with the signs reversed; (b) the singly and doubly hydrated clusters of 1-PE, isolated in the gas phase; (c) 1-PE and its singly and doubly hydrated clusters embedded in a polarizable dielectric continuum; and (d) 1-PE in an aqueous solution interacting with the local hydration shell and with the polarizable dielectric continuum, using averaged solute structures computed from the fluctuating solute and solvent configurations generated via molecular dynamics simulations.The ab initio calculations (conducted with GAUSSIAN 98) employed configuration interaction singles (CIS) or time-dependent density functional theory (TD-DFT) procedures using basis sets of double- and triple-zeta quality. The results have been related to experimental observations and discussed in terms of changes in the molecular geometry and solvent-induced perturbations.

The conformational preferences of two model anti-viral pharmaceuticals, phenylurea and 1,3-diphenylurea, isolated in the gas phase, have been explored using a combination of ultra-violet and infra-red ion-dip spectroscopy and quantum chemical computation. Two conformers have been assigned for each species associated with cis and trans configurations of the amide groups and stabilised by weak intramolecular hydrogen bonding between the amide group and the aromatic ring, NH --> pi (cis) or CH --> O=C (trails). In 1,3-diphenyl urea, the folded (global minimum) trans-cis and the extended trans trans conformers were both populated but the highly folded cis-cis structure, located at considerably higher energy, could not be detected.

The beta(1-->4) glycosidic linkage found in lactose is a prevalent structural motif in many carbohydrates and glycoconjugates. Using UV and IR ion-dip spectroscopies to probe benzyl lactoside isolated in the gas phase, we find that the disaccharide unit adopts only a single, rigid structure. Its fully resolved infrared ion-dip spectrum is in excellent agreement with that of the global minimum structure computed ab initio. This has glycosidic torsion angles of phi(H) (H1-C1-O-C4') approximately 180 degrees and psi(H) (C1-O-C4'-H4') approximately 0 degrees which correspond to a rotation of approximately 150 degrees about the glycosidic bond compared to the accepted solution-phase conformation. We discuss the biological implications of this discovery and the generality of the strategies employed in making it.

Partially resolved ultraviolet rotational band contours associated with the S1 ← S0 origin bands of the six most populated conformers of jet-cooled phenylalanine have been recorded via resonant two-photon ionization. The strong dependence of their transition moment orientation on the conformation of the alanyl side chain has facilitated their structural assignment through simulations based upon ab initio computation. The S1 lifetimes of all six conformers, measured through pump−probe delayed ionization, reveal an efficient nonradiative decay pathway in the most stable conformer, which is stabilized through a chain of intramolecular hydrogen bonds linking the side chain to the benzene ring.

The molecular conformation and structure of 2-phenoxy ethylamine and its hydrated complexes and of the generic beta-blocker molecule, 3-phenoxy propanolamine have been investigated and assigned using a combination of mass-selected, resonant two-photon ionisation (R2PI), coupled with infrared ion-dip spectroscopy and ab initio calculations. The results are compared with recent investigations of 2-phenoxy ethanol, 2-phenyl ethanolamine, the ephedra and noradrenaline. Attention is focused particularly on the degree of hydrogen bonding and rigidity or flexibility in the side chain and on the organisation and structural influence of bound water molecules.

The structural investigation of a hydrated monosaccharide, phenyl beta-D-glucopyranoside (PhebetaGlc), in the gas phase is presented. It is based upon ab initio computation coupled with the analysis of the resonant 2-photon ionization and IR ion-dip spectra of the singly hydrated clusters stabilized in a free jet expansion and follows an earlier investigation of the structure and conformations of the unhydrated sugar.(1) Despite the potentially large set of possible binding sites and conformations, only two singly hydrated complexes are formed in the free jet expansion. Tentative structural assignments are made on the basis of comparisons with those already established for related systems; comparisons between the observed O-H vibrational frequencies and those computed for structures optimized at the B3LYP/6-31+G(d) level of theory, and in light of the relative energies of these structures, calculated at the single-point MP2/6-311+G(d,p) level are made. A discrepancy between the latest revision of the Gaussian 98 package (revision A.11) and earlier revisions, which can lead to different computed structures when optimizing noncovalently bound molecular complexes, is discussed in an appendix.

Singly and multiply hydrated clusters of the noradrenaline analogue, 2-amino-I-phenyl-ethanol (APE) have been studied using a combination of resonant two-photon ionization time of flight spectroscopy (R2PI-TOF), infrared ion-dip spectroscopy and ab initio quantum chemical calculation. Singly hydrated clusters populate two distinct structures: the water molecule either hydrogen-bonds to the ethanol group in the extended AG conformer (leaving the intramolecular OH → N hydrogen bond intact) or inserts into the intramolecular hydrogen bond in the (distorted) ethanolamine side chain (promoting a weak NH → O bond). The observed doubly and triply hydrated clusters both display insertion structures only, with the water molecules arrayed as linear chains, hydrogen bonded to the functional groups of the side-chain and again promoting a weak NH → O bond along the distorted ethanolamine side-chain. The infrared spectrum of the 1:4 cluster of APE, which is very similar to that of the corresponding cluster of ephedrine, includes new features in the ‘window region’ (3500–3700 cm−1), indicating the onset of a three-dimensional assembly. Comparisons with ab initio computed spectra favour a structure that incorporates a cyclic water tetramer linked to the two functional groups on the ethanolamine side-chain.

The conformational structures of noradrenaline, isolated in the gas phase, have been explored through a combination of electronic structure computation (at the B3LYP/6-31 + G*, MP2/6-31 + G*, MP2/aug-cc-pVDZ and CIS/6-31 + G* levels of theory) and mass selected ultraviolet and infrared ion dip spectroscopy (following laser ablation of the neurotransmitter into a pulsed supersonic argon expansion). Despite the many possible low-lying conformational possibilities predicted by theory, almost the entire population of jet-cooled noradrenaline adopts the global minimum structure, associated with an extended, AG1a, ethanolamine side chain conformation. Intramolecular hydrogen bonds are formed between the neighbouring hydroxyl groups on the catechol ring and between the hydroxyl and amino groups on the side chain.

The conformational and molecular structures of singly hydrated noradrenaline complexes have been explored through a combination of electronic structure computation (at the B3LYP/ 6- 31+ G*, MP2/ 6- 31+ G* and MP2/ aug-cc-pVDZ levels of theory) and mass-selected ultraviolet and infrared ion-dip spectroscopy following laser ablation of the neurotransmitter into a freely expanding moist argon jet. Under these conditions, almost all the hydrated complexes are located in the global minimum energy configuration, associated with an extended, AG1a, ethanolamine side-chain conformation; the water molecule, which is located slightly above the plane of the catechol ring, is bound primarily as a proton acceptor to the m-OH substituent, and only weakly, as a proton donor, to the hydroxyl group on the side chain.