The adsorption of protein molecules at a crystal/solution interface observed by an improved TIRFM

We have improved the ordinary total internal reflection fluorescence microscopy (TIRFM). Two improvements have been achieved, one is the interface between opaque material and solution can be observed, another is the interface far away (usually several ten micro meters) the objective lens can be observed. By this improved TIRFM, the adsorption of protein molecules at a crystal/solution interface had been successfully observed. We have obtained the results of relationship between the amount of adsorbed protein molecules on bunched steps and the height of bunched steps of a protein crystal.

Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

Terahertz time domain spectroscopy of amino acids and sugars

A time-domain spectrometer for use in the terahertz (THz) spectral range was designed and constructed. Due to there being few existing methods of generating and detecting THz radiation, the spectrometer is expected to have vast applications to solid, liquid, and gas phase samples. In particular, knowledge of complex organic chemistry and chemical abundances in the interstellar medium (ISM) can be obtained when compared to astronomical data. The THz spectral region is of particular interest due to reduced line density when compared to the millimeter wave spectrum, the existence of high resolution observatories, and potentially strong transitions resulting from the lowest-lying vibrational modes of large molecules.

The heart of the THz time-domain spectrometer (THz-TDS) is the ultrafast laser. Due to the femtosecond duration of ultrafast laser pulses and an energy-time uncertainty relationship, the pulses typically have a several-THz bandwidth. By various means of optical rectification, the optical pulse carrier envelope shape, i.e. intensity-time profile, can be transferred to the phase of the resulting THz pulse. As a consequence, optical pump-THz probe spectroscopy is readily achieved, as was demonstrated in studies of dye-sensitized TiO₂, as discussed in chapter 4. Detection of the terahertz radiation is commonly based on electro-optic sampling and provides full phase information. This allows for accurate determination of both the real and imaginary index of refraction, the so-called optical constants, without additional analysis. A suite of amino acids and sugars, all of which have been found in meteorites, were studied in crystalline form embedded in a polyethylene matrix. As the temperature was varied between 10 and 310 K, various strong vibrational modes were found to shift in spectral intensity and frequency. Such modes can be attributed to intramolecular, intermolecular, or phonon modes, or to some combination of the three.

Reactions of small molecules facilitated by metal-acceptor interactions

The role of metal-acceptor interactions arising from M–BR3 and M–PR3 bonding is discussed with respect to reactions between first-row transition metals and N2, H2, and CO. Thermally robust, S = 1/2 (TPB)Co(H2) and (TPB)Co(N2) complexes (TPB = tris(2- (diisopropylphosphino)phenyl)borane) are described and the energetics of N2 and H2 binding are measured. The H2 and N2 ligands are bound more weakly in the (TPB)Co complexes than in related (SiP3)M(L) complexes (SiP3 = tris(2- (diisopropylphosphino)phenyl)silyl). Comparisons within and between these two ligand platforms allow for the factors that affect N2 (and H2) binding and activation to be delineated. The characterization and reactivity of (DPB)Fe complexes (DPB = bis(2- (diisopropylphosphino)phenyl)phenylborane) in the context of N2 functionalization and E–H bond addition (E = H, C, N, Si) are described. This platform allows for the one-pot transformation of free N2 to an Fe hydrazido(-) complex via an Fe aminoimide intermediate. The principles learned from the N2 chemistry using (DPB)Fe are applied to CO reduction on the same system. The preparation of (DPB)Fe(CO)2 is described as well as its reductive functionalization to generate an unprecedented Fe dicarbyne. The bonding in this highly covalent complex is discussed in detail. Initial studies of the reactivity of the Fe dicarbyne reveal that a CO-derived olefin is released upon hydrogenation. Alternative approaches to uncovering unusual reactivity using metal- acceptor interactions are described in Chapters 5 and 6, including initial studies on a new π-accepting tridentate diphosphinosulfinyl ligand and strategies for designing ligands that undergo site-selective metallation to generate heterobimetallic complexes.

Palladium-catalyzed asymmetric allylic alkylation: insights, application toward cyclopentanoid and cycloheptanoid molecules, and the total synthesis of several daucane sesquiterpenes

The asymmetric construction of quaternary stereocenters is a topic of great interest in the organic chemistry community given their prevalence in natural products and biologically active molecules. Over the last decade, the Stoltz group has pursued the synthesis of this challenging motif via a palladium-catalyzed allylic alkylation using chiral phosphinooxazoline (PHOX) ligands. Recent results indicate that the alkylation of lactams and imides consistently proceeds with enantioselectivities substantially higher than any other substrate class previously examined in this system. This observation prompted exploration of the characteristics that distinguish these molecules as superior alkylation substrates, resulting in newfound insights and marked improvements in the allylic alkylation of carbocyclic compounds.

General routes to cyclopentanoid and cycloheptanoid core structures have been developed that incorporate the palladium-catalyzed allylic alkylation as a key transformation. The unique reactivity of α-quaternary vinylogous esters upon addition of hydride or organometallic reagents enables divergent access to γ-quaternary acylcyclopentenes or cycloheptenones through respective ring contraction or carbonyl transposition pathways. Derivatization of the resulting molecules provides a series of mono-, bi-, and tricyclic systems that can serve as valuable intermediates for the total synthesis of complex natural products.

The allylic alkylation and ring contraction methodology has been employed to prepare variably functionalized bicyclo[5.3.0]decane molecules and enables the enantioselective total syntheses of daucene, daucenal, epoxydaucenal B, and 14-p-anisoyloxydauc-4,8-diene. This route overcomes the challenge of accessing β-substituted acylcyclopentenes by employing a siloxyenone to effect the Grignard addition and ring opening in a single step. Subsequent ring-closing metathesis and aldol reactions form the hydroazulene core of these targets. Derivatization of a key enone intermediate allows access to either the daucane sesquiterpene or sphenobolane diterpene carbon skeletons, as well as other oxygenated scaffolds.

Laser intensity dependence of high-order harmonic generation from aligned CO2 molecules

We investigate experimentally the high-order harmonic generation from aligned CO2 molecules and demonstrate that the modulation inversion of the harmonic yield with respect to molecular alignment can be altered dramatically by fine-tuning the intensity of the driving laser pulse for harmonic generation. The results can be modeled by employing the strong field approximation including a ground state depletion factor. The laser intensity is thus proved to be a parameter that can control the high-harmonic emission from aligned molecules.

Ionization suppression of diatomic molecules in strong laser fields

The ionization rate of molecules in intense laser fields may be much lower than that of atoms with similar binding energy. This phenomenon is termed the ionization suppression of molecules and is caused by the molecular inner structure. In this paper, we perform a comprehensive study of the ionization suppression of homonuclear diatomic molecules in intense laser fields of linear and circular polarizations. We find that for linear polarization the total ionization rate and the ionization suppression depend greatly on the molecular alignment, and that for circular polarization the ionization suppression of molecules in the antibonding (bonding) shells disappears (appears) for laser intensities around 10(15) W/cm(2). We also find that the molecular photoelectron energy spectra are greatly changed by the interference effect, even though the total ionization rate of molecules remains almost the same as that of their companion atoms.

Dynamics of rotationally resolved multiphoton ionization processes in molecules

This dissertation presents the results of studies of several rotationally- resolved resonance enhanced multiphoton ionization (REMPI) processes in some simple molecular systems. The objective of these studies is to quantitatively identify the underlying dynamics of this highly state-specific process which utilizes the narrow bandwidth radiation of a laser to ionize a molecule by first preparing an excited state via multiphoton absorption and subsequently ionizing that state before it can decay. Coupled with high-resolution photoelectron spectroscopy, REMPI is clearly an important probe of molecular excited states and their photoioniza tion dynamics.

A key feature of our studies is that they are carried out using accurate Hartree-Fock orbitals to describe the photoelectron orbitals of the molecular ions. The use of such photoelectron orbitals is important in rotationally-resolved studies where the angular momentum coupling in the photoelectron orbital plays a significant role in the photoionization dynamics. In these studies the Hartree-Fock molecular molecular photoelectron orbitals are obtained by numerical solution of a Lippmann-Schwinger integral equation.

Studies reported here include investigations of (i) ionic rotational branching ratios and their energy dependence for REMPI via the A^2Σ^+(3sσ) and D^2Σ^+(3pσ)states of NO, (ii) the influence of angular momentum constraints on branching ratios at low photoelectron energies for REMPI via low-J levels of the resonant intermediate state, (iii) the strong dependence of photoelectron angular distributions on final ionic rotational state and on the alignment in REMPI of the A^2Σ^+ state of NO, (iv) vibrational state dependence of ionic rotational branching ratios arising from rapid orbital evolution in resonant states (E'^2Σ^+(3pσ) of CH), (v) the influence of rovibronic interactions on the rotational branching ratios seen in REMPI via the D^2Σ^+(3pσ) state of NO, and (vi) effects of laser intensity on the photoionization dynamics of REMPI.

Quantum interference in laser-induced nonsequential double ionization in diatomic molecules: Role of alignment and orbital symmetry

We address the influence of the orbital symmetry and the molecular alignment with respect to the laser-field polarization on laser-induced nonsequential double ionization of diatomic molecules, in the length and velocity gauges. We work within the strong-field approximation and assume that the second electron is dislodged by electron-impact ionization, and also consider the classical limit of this model. We show that the electron-momentum distributions exhibit interference maxima and minima due to electron emission at spatially separated centers. The interference patterns survive integration over the transverse momenta for a small range of alignment angles, and are sharpest for parallel-aligned molecules. Due to the contributions of the transverse-momentum components, these patterns become less defined as the alignment angle increases, until they disappear for perpendicular alignment. This behavior influences the shapes and the peaks of the electron-momentum distributions.

Photoelectron angular distributions of molecules in bichromatic laser fields of circular polarization

Photoelectron angular distributions (PADs) from above-threshold ionization of O-2 and N-2 molecules irradiated by a bichromatic laser field of circular polarization are Studied. The bichromatic laser field is specially modulated such that it can be used to mimic a sequence of one-cycle laser pulses. The PADs are greatly affected by the molecular alignment, the symmetry of the initial electronic distribution, and the carrier-envelope phase of the laser pulses. Generally, the PADs do not show any symmetry, and become symmetric about an axis only when the symmetric axis of laser field coincides with the symmetric axis of molecules. This study shows that the few-cycle laser pulses call be used to steer the photoelectrons and perform the selective ionization of molecules. (C) 2008 Elsevier B.V. All rights reserved.

Surface-enhanced resonance Raman scattering spectroscopy of single R6G molecules

Surface-enhanced resonance Raman scattering (SERRS) of Rhodamine 6G (R6G) adsorbed on colloidal silver clusters has been studied. Based on the great enhancement of the Raman signal and the quench of the fluorescence, the SERRS spectra of R6G were recorded for the samples of dye colloidal solution with different concentrations. Spectral inhomogeneity behaviours from single molecules in the dried sample films were observed with complementary evidences, such as spectral polarization, spectral diffusion, intensity fluctuation of vibrational lines and even "breathing" of the molecules. Sequential spectra observed from a liquid sample with an average of 0.3 dye molecules in the probed volume exhibited the expected Poisson distribution for actually measuring 0, 1 or 2 molecules. Difference between the SERRS spectra of R6G excited by linearly and circularly polarized light were experimentally measured.

Coherent population accumulations of multiphoton transitions induced by an ultrashort pulse train in polar molecules

Coherent population accumulations of multiphoton transitions induced by an ultrashort pulse train in a two-level polar molecule are investigated theoretically by solving the density-matrix equations without invoking any of the standard approximations. It is shown due to the effects of permanent dipole moments, that the population accumulation of multiphoton transitions can be obtained in the polar molecule. Moreover, the population accumulations depend crucially on the relative phase between two sequential pulses, and the period in which the maximum population accumulation occurs is 2 pi/N in N-photon transitions.

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: An atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha 7 helix. (c) 2007 Elsevier Inc. All rights reserved.

Label-free detection of single biological molecules using microtoroid optical resonators

Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the `reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized.