Controlled modification of biomolecules by ultrashort laser pulses in polar liquids

Targeted chemical modification of peptides and proteins by laser pulses in a biologically relevant environment, i.e. aqueous solvent at room temperature, allows for accurate control of biological processes. However, the traditional laser methods of control of chemical reactions are applicable only to a small class of photosensitive biomolecules because of strong and ultrafast perturbations from biomolecule-solvent interactions. Here, we report excitation of harmonics of vibration modes of solvent molecules by femtosecond laser pulses to produce controlled chemical modifications of non-photosensitive peptides and proteins in polar liquids under room conditions. The principal modifications included lysine formylation and methionine sulfoxidation both of which occur with nearly 100% yield under atmospheric conditions. That modification occurred only if the laser irradiance exceeded certain threshold level. The threshold, type, and extent of the modifications were completely controlled by solvent composition, laser wavelength, and peak irradiance of ultrashort laser pulses. This approach is expected to assist in establishing rigorous control over a broad class of biological processes in cells and tissues at the molecular level.

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title: Coherent nonlinear spectroscopy: from femtosecond dynamics to control.
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title: Hydrogen bond dynamics in primary alcohols: a femtosecond infrared study.
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title: Measuring vibrational coherence lifetimes in liquid methanol using transient coherent Raman scattering
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title: Structure and dynamics of monohydroxy alcohols—Milestones towards their microscopic understanding, 100 years after Debye
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abstract: We study the ultrafast O-H stretch vibrational relaxation dynamics of protonated water clusters embedded in a matrix of deuterated acetonitrile, using polarization-resolved mid-IR femtosecond spectroscopy. The clusters are produced by mixing triflic (trifluoromethanesulfonic) acid and H2O in molar ratios of 1:1, 1:2, and 1:3, thus varying the degree of hydration of the proton. At all hydration levels the excited O-H stretch vibration of the hydrated proton shows an ultrafast vibrational relaxation with a time constant T1 < 100 fs, leading to an ultrafast local heating of the protonated water cluster. This excess thermal energy, initially highly localized to the region of the excited proton, first re-distributes over the aqueous cluster and then dissipates into the surrounding acetonitrile matrix. For clusters with a triflic acid to H2O ratio of 1:3 these processes occur with time constants of 320 ± 20 fs and 1.4 ± 0.1 ps, respectively. The cooling of the clusters reveals a long-living, underlying transient absorption change with high anisotropy. We argue that this feature stems from the vibrational predissociation of a small fraction of the proton hydration structures, directly following the ultrafast infrared excitation.

title: Ultrafast vibrational energy flow in water monomers in acetonitrile
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title: The growing landscape of lysine acetylation links metabolism and cell signalling
abstract: Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation and crotonylation. The intricate link between lysine acylation and cellular metabolism has been clarified by the occurrence of several such metabolite-sensitive acylations and their selective removal by sirtuin deacylases. These emerging findings point to new functions for different lysine acylations and deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes.

title: N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage
abstract: The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N6-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3′-formylphosphate residues arising from 5′-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N6-amino groups of lysine side chains. A liquid chromatography (LC)–tandem mass spectrometry (MS) method was developed to quantify the resulting N6-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04–0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose–response relationship for the formation of N6-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N6-formylation of the linker histone H1. The N6-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N6-acetylation recognized as an important determinant of gene expression in mammalian cells. The N6-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

title: A convenient method for the N-formylation of secondary amines and anilines using ammonium formate
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title: A facile method for the N-formylation of primary and secondary amines by liquid phase oxidation of methanol in the presence of hydrogen peroxide over basic copper hydroxyl salts
abstract: N-formylation reactions by catalytic oxidation of methanol in the presence of primary or secondary amines and hydrogen peroxide has been investigated using a liquid phase reaction system over basic copper hydroxyl salts. A series of basic copper hydroxyl salts was prepared by the conventional precipitation method using aqueous ammonia and sodium hydroxide as precipitating agents. PXRD, SEM, FT-IR, BET were employed for physical characterization of the prepared basic copper hydroxyl salts. The composition of the catalytic material obtained was found dependent on the nature of the anion associated with the copper salt precursor. The copper hydroxyl chloride catalyst has shown the best catalytic performance in terms of the reaction rate and product selectivity whereas for the copper oxide catalyst the reaction rate was extremely slow. It is interesting to observe that 4-piperidone protected with acid-sensitive functional groups such as N-acetyl piperazine and ethylene glycol can also be formylated from moderate to good yields by these catalysts.

title: New Route to N-Formylation of Primary Amines with Amino Acids as a Source of CO Using Polyaniline Catalyst
abstract: The treatment of primary amines with amino acids as a CO source in the presence of polyaniline emeraldine salts bring about N-formylation to give the corresponding formamides. This route to N-formylation methodology of primary amine represents the first reported example of a CO-transfer via radical mechanism by highly nanoporous polyaniline emeraldine salts catalyst.

title: Convenient N-Formylation of Amines in Dimethylformamide with Methyl Benzoate under Microwave Irradiation
abstract: -dimethylformamide (DMF) with methyl benzoate as a promoter under microwave irradiation. Although DMF as a formylating source has been employed for the formylation reaction with 2,3-dihydro-1,4-phthalazinedione as a promoter, it needed long reaction time of more than 40 hours and was limited to the formylation of primary amines.

title: A remarkably simple protocol for the N-formylation of amino acid esters and primary amines.
abstract: A simple, convenient, and wide scope protocol for the N-formylation of amino acid esters and primary amines has been developed utilizing only imidazole in warm DMF.

title: Nonlinear protein – nucleic acid crosslinking induced by femtosecond UV laser pulses in living cells
abstract: We investigate the interactions between proteins and DNA in solutions of living cells, triggered by UV, 200-fs laser pulses. DNA-protein crosslinking is obtained with two different techniques: conventional chemical or laser-induced. Our results evidence a nonlinear response of the cells to the laser irradiation in the investigated intensity range. The laser induced crosslink efficiency can reach a value approximately twice higher than that obtained with ordinary chemical methods. The experimental results are very well reproduced by a simple phenomenological model based on the interplay between two-photon absorption by DNA bases and cell damage induced by high intensity laser pulse.

title: Femtosecond quantum control of molecular bond formation
abstract: Ultrafast lasers are versatile tools used in many scientific areas, from welding to eye surgery. They are also used to coherently manipulate light–matter interactions such as chemical reactions, but so far control experiments have concentrated on cleavage or rearrangement of existing molecular bonds. Here we demonstrate the synthesis of several molecular species starting from small reactant molecules in laser-induced catalytic surface reactions, and even the increase of the relative reaction efficiency by feedback-optimized laser pulses. We show that the control mechanism is nontrivial and sensitive to the relative proportion of the reactants. The control experiments open up a pathway towards photocatalysis and are relevant for research in physics, chemistry, and biology where light-induced bond formation is important.

title: Ultraviolet laser-induced cross-linking in peptides.
abstract: The aim of this study was to demonstrate, and to characterize by high-resolution mass spectrometry that it is possible to preferentially induce covalent cross-links in peptides by using high-energy femtosecond ultraviolet (UV) laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence.



Three peptides, xenopsin, angiotensin I, and interleukin, individually or in combination, were exposed to high-energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry.



High-resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters.



High-energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.

title: Tables of molecular vibrational frequencies. Consolidated volume II
abstract: The compilations of fundamental vibrational frequencies of molecules previously published as Tables of Molecular Vibrational Frequencies Part 5, Part 6, Part 7, and Part 8, which appeared in the Journal of Physical and Chemical Reference Data in 1972, 1973, and 1974, have been revised. This Consolidated Volume II includes data on a total of 212 molecules in addition to those on 223 molecules included in Volume 1 (NSRDS‐NBS 39). Selected values of the fundamental vibrational frequencies are given for each molecule, together with observed infrared and Raman spectral data and citations to the original literature. The selection of vibrational fundamentals has been based on careful studies of the spectral data and comprehensive normal‐coordinate analyses. An estimate of the accuracy of the selected values is included. The tables provide a convenient source of information for those who require vibrational energy levels and related properties in molecular spectroscopy, thermodynamics, analytical chemistry, and o...

title: Optics of Femtosecond Laser Pulses
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