Hydration of biologically relevant tetramethylammonium cation by neutron scattering and molecular dynamics.

Neutron scattering and molecular dynamics studies were performed on a concentrated aqueous tetramethylammonium (TMA) chloride solution to gain insight into the hydration shell structure of TMA, which is relevant for understanding its behavior in biological contexts of, e.g., properties of phospholipid membrane headgroups or interactions between DNA and histones. Specifically, neutron diffraction with isotopic substitution experiments were performed on TMA and water hydrogens to extract the specific correlation between hydrogens in TMA (HTMA) and hydrogens in water (HW). Classical molecular dynamics simulations were performed to help interpret the experimental neutron scattering data. Comparison of the hydration structure and simulated neutron signals obtained with various force field flavors (e.g. overall charge, charge distribution, polarity of the CH bonds and geometry) allowed us to gain insight into how sensitive the TMA hydration structure is to such changes and how much the neutron signal can capture them. We show that certain aspects of the hydration, such as the correlation of the hydrogen on TMA to hydrogen on water, showed little dependence on the force field. In contrast, other correlations, such as the ion-ion interactions, showed more marked changes. Strikingly, the neutron scattering signal cannot discriminate between different hydration patterns. Finally, ab initio molecular dynamics was used to examine the three-dimensional hydration structure and thus to benchmark force field simulations. Overall, while neutron scattering has been previously successfully used to improve force fields, in the particular case of TMA we show that it has only limited value to fully determine the hydration structure, with other techniques such as ab initio MD being of a significant help.

Why do polyarginines adsorb at neutral phospholipid bilayers and polylysines do not? An insight from density functional theory calculations and molecular dynamics simulations.

Adsorption of cell-penetrating peptides (CPPs) at cellular membranes is the first and necessary step for their subsequent translocation across cellular membranes into the cytosol. It has been experimentally shown that CPPs rich in arginine (Arg) amino acid penetrate across phospholipid bilayers more effectively than their lysine (Lys) rich counterparts. In this work, we aim to understand the differences in the first translocation step, adsorption of Arg9 and Lys9 peptides at fully hydrated neutral phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid bilayers and evaluate in detail the energetics of the process using molecular dynamics (MD) simulations and free energy calculations of adsorption of the single peptide. We show that the adsorption of Arg9 is energetically feasible, with the free energy of adsorption being ∼-5.0 kcal mol-1 at PC and ∼-5.5 kcal mol-1 at PE bilayers. In contrast, adsorption of Lys9 is not observed at PC bilayers, and their adsorption at PE bilayers is very weak, being ∼-0.5 kcal mol-1. We show by energy decomposition and analysis of peptide hydration along the membrane that significantly stronger electrostatic interactions of Arg9 with lipid phosphate groups, together with the greater loss of peptide hydration (and in turn stronger hydrophobic interactions) along the membrane translocation path, are the main driving factors governing the adsorption of Arg-rich peptides at neutral lipid bilayers in contrast to Lys-rich peptides. Finally, we also compare the energetics in lipid/bilayer systems with the density functional theory (DFT) calculations of the corresponding model systems in the continuum water model and reveal the energetic differences in different environments.

FA Sliding as the Mechanism for the ANT1-Mediated Fatty Acid Anion Transport in Lipid Bilayers.

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

FTIR spectroscopy and molecular level insight of diluted aqueous solutions of acetic acid.

Aqueous solutions of acetic acid (AA) have been intensively explored for decades with a particular attention addressed to the hydrogen bond network generated by COOH group at different concentrations. In majority of studies conducted so far the envelope originated from νCO is decomposed into two bands assigned to differently hydrated monomers: the one presumably to AA···H2O, and another one to AA···(H2O)2. In order to examine if species other than the mentioned monomers produce this spectral signature, we performed computational and FTIR spectroscopic study of AA in aqueous solutions. Dilute solutions of deuterated acetic acid (CD3COOD) in D2O and in C2Cl4 as a reference were prepared (c0 = 0.001, 0.01 and 0.1 mol dm-3) as well as of deuterated sodium acetate (CD3COONa) in D2O. CD3COOD in 0.1 mol dm-3 solution in D2O displays a feature that separated in two signals with maxima at 1706 cm-1 and 1687 cm-1. A combined DFT and molecular dynamics study performed in this work showed the assignation of those spectral bands to be a more complex problem than previously thought, with syn-anti isomerism and hydration contributing to the experimentally observed broad νCO envelope.

Stealthy Player in Lipid Experiments? EDTA Binding to Phosphatidylcholine Membranes Probed by Simulations and Monolayer Experiments.

Ethylenediaminetetraacetic acid (EDTA) is frequently used in lipid experiments to remove redundant ions, such as Ca2+, from the sample solution. In this work, combining molecular dynamics (MD) simulations and Langmuir monolayer experiments, we show that on top of the expected Ca2+ depletion, EDTA anions themselves bind to phosphatidylcholine (PC) monolayers. This binding, originating from EDTA interaction with choline groups of PC lipids, leads to the adsorption of EDTA anions at the monolayer surface and concentration-dependent changes in surface pressure as measured by monolayer experiments and explained by MD simulations. This surprising observation emphasizes that lipid experiments carried out using EDTA-containing solutions, especially of high concentrations, must be interpreted very carefully due to potential interfering interactions of EDTA with lipids and other biomolecules involved in the experiment, e.g., cationic peptides, that may alter membrane-binding affinities of studied compounds.

Interaction of guanidinium and ammonium cations with phosphatidylcholine and phosphatidylserine lipid bilayers - Calorimetric, spectroscopic and molecular dynamics simulations study.

The ability of arginine-rich peptides to cross the lipid bilayer and enter cytoplasm, unlike their lysine-based analogues, is intensively studied in the context of cell-penetrating peptides. Although the experiments have not yet reconstructed their internalization mechanism, the computational studies have shown that the type or charge of lipid polar groups is one of the crucial factors in their translocation. In order to gain more detailed insight into the interaction of guanidinium (Gdm+) and ammonium (NH4+) cations, as important building blocks in arginine and lysine amino acids, with lipid bilayers, we conducted the experimental and computational study that tackles this phenomenon. The adsorption of Gdm+ and NH4+ on lipid bilayers prepared from a zwitterionic (DPPC) and an anionic (DPPS) lipid was examined by thermoanalytic and spectroscopic techniques. Using temperature-dependent UV-Vis spectroscopy and DSC calorimetry we determined the impact of Gdm+ and NH4+ on the thermotropic properties of lipid bilayers. FTIR data, along with molecular dynamics simulations, unraveled the molecular-level details on the nature of their interactions, showing the proton transfer between NH4+ and DPPS, but not between Gdm+ and DPPS. The findings originated from this work imply that Gdm+ and NH4+ form qualitatively different interactions with lipids of different charge which is reflected in the physico-chemical interactions that arginine-and lysine-based peptides establish at a complex and chemically heterogeneous environment such as the biological membrane.

Membrane Lipid Reshaping Underlies Oxidative Stress Sensing by the Mitochondrial Proteins UCP1 and ANT1.

Oxidative stress and ROS are important players in the pathogenesis of numerous diseases. In addition to directly altering proteins, ROS also affects lipids with negative intrinsic curvature such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE adducts potentiates the protonophoric activity of mitochondrial uncoupling proteins, but the molecular mechanism remains unclear. Here, we linked the ROS-mediated change in lipid shape to the mechanical properties of the membrane and the function of uncoupling protein 1 (UCP1) and adenine nucleotide translocase 1 (ANT1). We show that the increase in the protonophoric activity of both proteins occurs due to the decrease in bending modulus in lipid bilayers in the presence of lysophosphatidylcholines (OPC and MPC) and PE adducts. Moreover, MD simulations showed that modified PEs and lysolipids change the lateral pressure profile of the membrane in the same direction and by the similar amplitude, indicating that modified PEs act as lipids with positive intrinsic curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. We demonstrated that UCP1 and ANT1 sense SCES and proposed a novel regulatory mechanism for the function of these proteins. The new findings should draw the attention of the scientific community to this important and unexplored area of redox biochemistry.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

Mitochondrial Uncoupling Proteins (UCP1-UCP3) and Adenine Nucleotide Translocase (ANT1) Enhance the Protonophoric Action of 2,4-Dinitrophenol in Mitochondria and Planar Bilayer Membranes.

2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.

ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport.

Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.

Molecular Dynamics Simulations of Mitochondrial Uncoupling Protein 2.

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

Chemistry and Reactivity of 4-hydroxy-2-nonenal (HNE) in Model Biological Systems.

Among many reactive oxygen species (ROS), which are constantly generated during oxidative stress in cellular membranes, the formation and subsequent reactivity of ubiquitous 4-hydroxy-2- nonenal (HNE) with nearby amino acids and lipids represent one of the main research targets in cell physiology in the last decades. Starting from the first synthesis of HNE in 1967, the chemistry and reactivity of HNE are constantly under intense scrutiny. This review shows recent advances in the field, which are discussed with the special emphasis on revealing intricate details of numerous reaction mechanisms of HNE with lipids and amino acids, with the goal of understanding the reactivity of HNE at the molecular level.

Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT Peptide and Bis(monoacylglycero)phosphate.

Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Here, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of CPP's membrane leakage activity. These results therefore support a model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.

Desaturase specificity is controlled by the physicochemical properties of a single amino acid residue in the substrate binding tunnel.

Membrane fatty acyl desaturases (mFAD) are ubiquitous enzymes in eukaryotes. They introduce double bonds into fatty acids (FAs), producing structurally diverse unsaturated FAs which serve as membrane lipid components or precursors of signaling molecules. The mechanisms controlling enzymatic specificity and selectivity of desaturation are, however, poorly understood. We found that the physicochemical properties, particularly side chain volume, of a single amino acid (aa) residue in insect mFADs (Lepidoptera: Bombyx mori and Manduca sexta) control the desaturation products. Molecular dynamics simulations of systems comprising wild-type or mutant mFADs with fatty acyl-CoA substrates revealed that the single aa substitution likely directs the outcome of the desaturation reaction by modulating the distance between substrate fatty acyl carbon atoms and active center metal ions. These findings, as well as our methodology combining mFAD mutational screening with molecular dynamics simulations, will facilitate prediction of desaturation products and facilitate engineering of mFADs for biotechnological applications.

Surface Propensity of Aqueous Atmospheric Bromine at the Liquid-Gas Interface.

Multiphase reactions of halide ions in aqueous solutions exposed to the atmosphere initiate the formation of molecular halogen compounds in the gas phase. Their photolysis leads to halogen atoms, which are catalytic sinks for ozone, making these processes relevant for the regional and global tropospheric ozone budget. The affinity of halide ions in aqueous solution for the liquid-gas interface, which may influence their reactivity with gaseous species, has been debated. Our study focuses on the surface properties of the bromide ion and its oxidation products. In situ X-ray photoelectron spectroscopy carried out on a liquid jet combined with classical and first-principles molecular dynamics calculations was used to investigate the interfacial depth profile of bromide, hypobromite, hypobromous acid, and bromate. The simulated core electron binding energies support the experimentally observed values, which follow a correlation with bromine oxidation state for the anion series. Bromide ions are homogeneously distributed in the solution. Hypobromous acid, a key species in the multiphase cycling of bromine, is the only species showing surface propensity, which suggests a more important role of the interface in multiphase bromine chemistry than thought so far.

Covalent modification of phosphatidylethanolamine by 4-hydroxy-2-nonenal increases sodium permeability across phospholipid bilayer membranes.

Reactive aldehydes (RAs), such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), produced by cells under conditions of oxidative stress, were shown to react with phosphatidylethanolamine (PE) in biological and artificial membranes. They form RA-PE adducts, which affect the function of membrane proteins by modifying various biophysical properties of the membrane. The ratio of protein to lipid in biological membranes is different, but can reach 0.25 in the membranes of oligodendrocytes. However, the impact of RA-PE adducts on permeability (P) of the neat lipid phase and molecular mechanism of their action are poorly understood. In this study, we showed that HNE increased the membrane P for ions, and in particular for sodium. This effect depended on the presence of DOPE, and was not recorded for the more toxic compound, ONE. Molecular dynamics simulations suggested that HNE-PE and ONE-PE adducts anchored different positions in the lipid bilayer, and thus changed the membrane lipid area and bilayer thickness in different ways. Sodium permeability, calculated in the presence of double HNE-PE adducts, was increased by three to four orders of magnitude when compared to PNa in adduct - free membranes. A novel mechanism by which HNE alters permeability of the lipid membrane may explain the multiple toxic or regulative effects of HNE on the function of excitable cells, such as neurons, cardiomyocytes and neurosensory cells under conditions of oxidative stress.

Vibrational spectroscopy combined with molecular dynamics simulations as a tool for studying behavior of reactive aldehydes inserted in phospholipid bilayers.

Vibrational Fourier-transform infrared (FTIR) spectroscopy aided with molecular dynamics (MD) simulations is used for studying the interaction of several reactive aldehydes (RAs), nonanal (NA), 2-nonenal (NE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The results obtained by the combination of these two techniques, supported also by electron paramagnetic resonance (EPR) spectroscopy, show that NA has the strongest stabilization in the bilayer, followed by less stabilized NE, HNE and ONE. We also revealed that HNE readily makes hydrogen bonds to carbonyl groups of POPC (but not to phosphate groups), in contrast to other RAs which are hydrogen bond acceptors and do not make hydrogen bonds with lipids. A combination of FTIR spectroscopy and MD simulations is sensitive to small chemical changes in the structures of RAs, thus making it a valuable tool for studying the weak interactions between compounds inserted to phospholipid bilayers.

Calculation of apparent pKa values of saturated fatty acids with different lengths in DOPC phospholipid bilayers.

We performed all-atom molecular dynamics simulations and calculated free energy profiles and apparent pKa values for neutral and anionic forms of single myristic (C14:0), palmitic (C16:0) and stearic (C18:0) fatty acid embedded in a DOPC bilayer and explicit water solvent. We showed that the neutral forms of the fatty acids are stabilized inside the bilayer by hydrogen bonding of a fatty acid carboxylic group with DOPC phosphate and carbonyl groups. In contrast to the neutral form, the anionic forms of the fatty acids are shifted towards the water-membrane interface and are instead stabilized by hydrogen bonding to interfacial water. By using umbrella sampling simulations, we calculated free energies of stabilization and revealed that the free energy of stabilization inside the bilayer increases with the chain length for both the neutral and deprotonated forms. On the other hand, the free energies of flip-flop of both the neutral and anionic forms are constant upon the prolongation of the fatty acid. Based on the free energy curves, we also calculated apparent fatty acid pKa,app values in the bilayer, which are 7.0, 7.2 and 6.3 for myristic, palmitic and stearic acid and are increased by several pKa units compared to the corresponding pKa values in water. By further analysis of the calculated curves we found that spontaneous protonation of fatty acid anions takes place in the bilayer interior at ca. 1.4 nm from the bilayer center for all studied fatty acids.

Revisited Mechanism of Reaction between a Model Lysine Amino Acid Side Chain and 4-Hydroxynonenal in Different Solvent Environments.

We revisit the mechanism of reaction between a model lysine side chain and reactive aldehyde 4-hydroxynonenal in different solvents with an increasing water content. We show by model organic reactions and qualitative spectrometric analysis that a nonpolar pyrrole adduct is dominantly formed in non-aqueous solvents dichloromethane and acetonitrile. On the other hand, in aqueous acetonitrile and neat water, other polar products are also isolated, including Michael adducts, hemiacetal adducts, and pyridinium salt adducts, at the same time as the ratio of nonpolar products to polar products is decreasing. The experiments are supported by detailed quantum chemical calculations of the reaction mechanism with different computational setups showing that the pyrrole adduct is the most thermodynamically stable product compared to Michael adducts and hemiacetal adducts and also indicating that water molecules released along the reaction pathway are catalyzing reaction steps involving proton transfer. Finally, we also identify the mechanism of the pyridinium salt adduct that is formed only in aqueous solutions.

Photocyclization of Tetra- and Pentapeptides Containing Adamantylphthalimide and Phenylalanines: Reaction Efficiency and Diastereoselectivity.

A series of tetrapeptides and pentapeptides was synthesized bearing a phthalimide chromophore at the N-terminus. The C-terminus of the peptides was strategically substituted with an amino acid, Phe, Phe(OMe), or Phe(OMe)2 characterized by different oxidation potentials. The photochemical reactivity of the peptides was investigated by preparative irradiation and isolation of photoproducts, as well as with laser flash photolysis. Upon photoexcitation, the peptides undergo photoinduced electron transfer (PET) and decarboxylation, followed by diastereoselective cyclization with the retention of configuration for tetrapeptides or inversion of configuration for pentapeptides. Molecular dynamics (MD) simulations and NOE experiments enabled assignment of the stereochemistry of the cyclic peptides. MD simulations of the linear peptides disclosed conformational reasons for the observed diastereoselectivity, being due to the peptide backbone spatial orientation imposed by the Phe amino acids. The photochemical efficiency for the decarboxylation and cyclization is not dependent on the peptide length, but it depends on the oxidation potential of the amino acid at the C-terminus. The results described herein are particularly important for the rational design of efficient photochemical reactions for the preparation of cyclic peptides with the desired selectivity.