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1.  Mechanism of chiral proofreading during translation of the genetic code 
eLife  2013;2:e01519.
The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs. The crystal structure of dimeric D-aminoacyl-tRNA deacylase (DTD) from Plasmodium falciparum in complex with a substrate-mimicking analog shows how it uses an invariant ‘cross-subunit’ Gly-cisPro dipeptide to capture the chiral centre of incoming D-aminoacyl-tRNA. While no protein residues are directly involved in catalysis, the unique side chain-independent mode of substrate recognition provides a clear explanation for DTD’s ability to act on multiple D-amino acids. The strict chiral specificity elegantly explains how the enriched cellular pool of L-aminoacyl-tRNAs escapes this proofreading step. The study thus provides insights into a fundamental enantioselection process and elucidates a chiral enforcement mechanism with a crucial role in preventing D-amino acid infiltration during the evolution of translational apparatus.
eLife digest
Amino acids are ‘chiral’ molecules that come in two different forms, called D and L, which are mirror images of each other, similar to how our left and right hands are mirror images of each other. However, only one of these forms is used to make proteins: the more abundant L-amino acids are linked together to make proteins, whereas the scarcer D-amino acids are not. This ‘homochirality’ is common to all life on Earth.
The molecular machinery inside cells that manufactures proteins involves many enzymes that carry out different tasks. Among these is an enzyme called DTD (short for D-aminoacyl-tRNA deacylase), which prevents D-amino acids being incorporated into proteins. To do this, DTD must be able to recognise and remove the D forms of many different amino acids before they are taken to the growing protein by transfer RNA molecules. However, the details of this process are not fully understood.
To investigate this mechanism, Ahmad et al. made crystals of the DTD enzyme in complex with a molecule that mimics a D-amino acid attached to a transfer RNA molecule. By studying this structure at a high resolution, Ahmad et al. were able to identify how the active site of DTD can specifically accommodate the ‘chiral centre’ of a complex made of a D-amino acid and a transfer RNA molecule.
DTD is able to recognize D-amino acids because of a critical dipeptide that is inserted from one subunit of the DTD into the active site of another subunit of the enzyme. The effect of this dipeptide is to generate a binding pocket that is a perfect fit for the chiral centre of a complex that contains a D-amino acid and a transfer RNA molecule. Moreover, this pocket specifically excludes complexes that contain an L-amino acid.
The crucial parts of DTD that form the binding pocket are highly conserved—that is, they are the same in a wide variety of organisms, from bacteria to mammals. This conservation suggests that DTD is crucial for ensuring homochirality throughout all forms of life. Intriguingly, DTD is particularly highly expressed in neurons which are abundant in D-amino acids: this indicates that the DTD enzyme has an important physiological role, which will certainly be the focus of future work.
PMCID: PMC3845328  PMID: 24302572
homochirality; proofreading; enzyme mechanism; translation; E. coli; Plasmodium falciparum
2.  Contrasting enantioselective DNA preference: chiral helical macrocyclic lanthanide complex binding to DNA 
Nucleic Acids Research  2012;40(16):8186-8196.
There is great interest in design and synthesis of small molecules which selectively target specific genes to inhibit biological functions in which particular DNA structures participate. Among these studies, chiral recognition has been received much attention because more evidences have shown that conversions of the chirality and diverse conformations of DNA are involved in a series of important life events. Here, we report that a pair of chiral helical macrocyclic lanthanide (III) complexes, (M)-Yb[LSSSSSS]3+ and (P)-Yb[LRRRRRR]3+, can enantioselectively bind to B-form DNA and show remarkably contrasting effects on GC-rich and AT-rich DNA. Neither of them can influence non-B-form DNA, nor quadruplex DNA stability. Our results clearly show that P-enantiomer stabilizes both poly(dG-dC)2 and poly(dA-dT)2 while M-enantiomer stabilizes poly(dA-dT)2, however, destabilizes poly(dG-dC)2. To our knowledge, this is the best example of chiral metal compounds with such contrasting preference on GC- and AT-DNA. Ligand selectively stabilizing or destabilizing DNA can interfere with protein–DNA interactions and potentially affect many crucial biological processes, such as DNA replication, transcription and repair. As such, bearing these unique capabilities, the chiral compounds reported here may shed light on the design of novel enantiomers targeting specific DNA with both sequence and conformation preference.
PMCID: PMC3439914  PMID: 22675072
3.  Polymeric Sulfated Amino Acid Surfactants: A New Class of Versatile Chiral Selectors for Micellar Electrokinetic Chromatography (MEKC) and MEKC-MS 
Analytical chemistry  2007;79(3):879-898.
In this work, three amino acids derived (L-leucinol, L-isoleucinol and L-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy (cryo-HRSEM). The polysodium N-undecenoyl-L-leucine sulfate (poly-L-SUCLS) shows distinct tubular structure, while polysodium N-undecenoyl-L-valine sulfate (poly-L-SUCVS) also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-L-isoleucine sulfate (poly-L-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantio-separation of broad range of structurally diverse racemic compounds at very acidic, neutral and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEA (e.g., (±)-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is ca 16 times better compared to pH 8.0 (LOD 5.2 µg/mL). Other broad range of chiral analytes (β-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, and benzodiazepinones) studied also provided improved chiral separation at low pH compared to high pH conditions. Among the three polymeric sulfated surfactants, poly-L-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS.
PMCID: PMC2569972  PMID: 17263313
4.  Dynamic Mirror-Symmetry Breaking in Bicontinuous Cubic Phases** 
Chiral segregation of enantiomers or chiral conformers of achiral molecules during self-assembly in well-ordered crystalline superstructures has fascinated chemists since Pasteur. Here we report spontaneous mirror-symmetry breaking in cubic phases formed by achiral multichain-terminated diphenyl-2,2′-bithiophenes. It was found that stochastic symmetry breaking is a general phenomenon observed in bicontinuous cubic liquid crystal phases of achiral rod-like compounds. In all compounds studied the ${{\it Im}\bar 3m}$ cubic phase is always chiral, while the ${Ia\bar 3d}$ phase is achiral. These intriguing observations are explained by propagation of homochiral helical twist across the entire networks through helix matching at network junctions. In the ${Ia\bar 3d}$ phase the opposing chiralities of the two networks cancel, but not so in the three-networks ${{\it Im}\bar 3m}$ phase. The high twist in the ${{\it Im}\bar 3m}$ phase explains its previously unrecognized chirality, as well as the origin of this complex structure and the transitions between the different cubic phases.
PMCID: PMC4501316  PMID: 25257551
chiral isotropic liquid; conglomerate; deracemization; polycatenar liquid crystal; spontaneous chiral induction
5.  Crystal Structure of Chiral γ PNA with Complementary DNA Strand—Insights into the Stability and Specificity of Recognition and Conformational Preorganization 
Journal of the American Chemical Society  2010;132(31):10717-10727.
We have determined the structure of a PNA-DNA duplex to 1.7 Å resolution by multiple-wavelength anomalous diffraction on a zinc derivative. This structure represents the first high-resolution view of a hybrid duplex containing a contiguous chiral PNA strand with complete γ-backbone modification (“γPNA”). Unlike the achiral counterpart, which adopts a random-fold, this particular γPNA is already preorganized into a right-handed helix as a single strand. The new structure illustrates the unique characteristics of this modified PNA, possessing conformational flexibility while maintaining sufficient structural integrity to ultimately adopt the preferred P-helical conformation upon hybridization with DNA. The unusual structural adaptability found in the γPNA strand is crucial for enabling the accommodation of backbone modifications while constraining conformational states. In conjunction with NMR analysis characterizing the structures and substructures of the individual building blocks, these results provide unprecedented insights into how this new class of chiral γPNA is preorganized and stabilized, before and after hybridization with a complementary DNA strand. Such knowledge is crucial for the future design and development of PNA for applications in biology, biotechnology and medicine.
PMCID: PMC2929025  PMID: 20681704
6.  Chirality- and sequence-selective successive self-sorting via specific homo- and complementary-duplex formations 
Nature Communications  2015;6:7236.
Self-recognition and self-discrimination within complex mixtures are of fundamental importance in biological systems, which entirely rely on the preprogrammed monomer sequences and homochirality of biological macromolecules. Here we report artificial chirality- and sequence-selective successive self-sorting of chiral dimeric strands bearing carboxylic acid or amidine groups joined by chiral amide linkers with different sequences through homo- and complementary-duplex formations. A mixture of carboxylic acid dimers linked by racemic-1,2-cyclohexane bis-amides with different amide sequences (NHCO or CONH) self-associate to form homoduplexes in a completely sequence-selective way, the structures of which are different from each other depending on the linker amide sequences. The further addition of an enantiopure amide-linked amidine dimer to a mixture of the racemic carboxylic acid dimers resulted in the formation of a single optically pure complementary duplex with a 100% diastereoselectivity and complete sequence specificity stabilized by the amidinium–carboxylate salt bridges, leading to the perfect chirality- and sequence-selective duplex formation.
The recognition and self-sorting of chiral molecules is a vital feature of many biomolecules. Here, the authors report chirality- and sequence-specific self-sorting of organic strands containing carboxylic acid or amidine groups, leading to selective duplex formation.
PMCID: PMC4468858  PMID: 26051291
7.  Chiral separation of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography with biphasic recognition 
Journal of chromatography. A  2010;1217(18):3044-3052.
This work concentrates on a novel chiral separation technology named biphasic recognition applied to resolution of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (−)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-β-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (−)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Various parameters involved in the chiral separation were investigated, namely the types of the chiral selector (CS); the concentration of each chiral selector; pH of the mobile phase; and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for each chiral selector. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both lipophilic and hydrophilic chiral selectors.
PMCID: PMC2854300  PMID: 20303497
Chiral separation; High-speed counter-current chromatography; Biphasic chiral recognition; α-Cyclohexylmandelic acid; Optical activity
8.  Mirrors in the PDB: left-handed α-turns guide design with D-amino acids 
Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids.
Propensities for amino acids to occur in contiguous αL helices correlate with published thermodynamic scales for incorporation of D-amino acids into αR helices. Two backbone rules for terminating a left-handed helix are found: an αR conformation is disfavored at the amino terminus, and a βR conformation is disfavored at the carboxy terminus. Helix capping sidechain-backbone interactions are found which are unique to αL helices including an elevated propensity for L-Asn, and L-Thr at the amino terminus and L-Gln, L-Thr and L-Ser at the carboxy terminus.
By examining left-handed α-turns containing L-amino acids, new interaction motifs for incorporating D-amino acids into right-handed α-helices are identified. These will provide a basis for de novo design of novel heterochiral protein folds.
PMCID: PMC2759939  PMID: 19772623
9.  Chiral plasmonics of self-assembled nanorod dimers 
Scientific Reports  2013;3:1934.
Chiral nanoscale photonic systems typically follow either tetrahedral or helical geometries that require four or more different constituent nanoparticles. Smaller number of particles and different chiral geometries taking advantage of the self-organization capabilities of nanomaterials will advance understanding of chiral plasmonic effects, facilitate development of their theory, and stimulate practical applications of chiroplasmonics. Here we show that gold nanorods self-assemble into side-by-side orientated pairs and “ladders” in which chiral properties originate from the small dihedral angle between them. Spontaneous twisting of one nanorod versus the other one breaks the centrosymmetric nature of the parallel assemblies. Two possible enantiomeric conformations with positive and negative dihedral angles were obtained with different assembly triggers. The chiral nature of the angled nanorod pairs was confirmed by 4π full space simulations and the first example of single-particle CD spectroscopy. Self-assembled nanorod pairs and “ladders” enable the development of chiral metamaterials, (bio)sensors, and new catalytic processes.
PMCID: PMC3678134  PMID: 23752317
10.  L-nucleotides and 8-methylguanine of d(C1m8G2C3G4C5LG6LC7G8C9G10)2 act cooperatively to promote a left-handed helix under physiological salt conditions 
Nucleic Acids Research  2003;31(23):6986-6995.
The structure and thermal stability of a hetero chiral decaoligodeoxyribonucleotide duplex d(C1m8 G2C3G4C5LG6LC7G8C9G10)d(C11m8G12C13G14C15LG16LC17G18C19G20) (O1) with two contiguous pairs of enantiomeric 2′-deoxy-l-ribonucleotides (C5LG6L/C15LG16L) at its centre and an 8-methylguanine at position 2/12 was analysed by circular dichroism, NMR and molecular modelling. O1 resolves in a left-handed helical structure already at low salt concentration (0.1 M NaCl). The central l2-sugar portion assumes a B* left-handed conformation (mirror-image of right-handed B-DNA) while its flanking d4-sugar portions adopt the known Z left-handed conformation. The resulting Z4–B2*–Z4 structure (left-handed helix) is the reverse of that of B4–Z2*–B4 (right-handed helix) displayed by the nearly related decaoligodeoxyribonucleotide d(mC1G2mC3G4C5L G6LmC7G8mC9G10)2, at the same low salt concentration (0.1 M NaCl). In the same experimental conditions, d(C1m8G2C3G4C5G6C7G8C9G10)2 (O2), the stereoregular version of O1, resolves into a right-handed B-DNA helix. Thus, both the 8-methylguanine and the enantiomeric step CLpGL at the centre of the molecule are needed to induce left-handed helicity. Remarkably, in the various heterochiral decaoligodeoxyribonucleotides so far analysed by us, when the central CLpGL adopts the B* (respectively Z*) conformation, then the adjacent steps automatically resolves in the Z (respectively B) conformation. This allows a good optimisation of the base–base stackings and base–sugar van der Waals interactions at the ZB*/B*Z (respectively BZ*/Z*B) junctions so that the Z4–B2*–Z4 (respectively B4–Z2*–B4) helix displays a Tm (∼65°C) that is only 5°C lower than the one of its homochiral counterpart. Here we anticipate that a large variety of DNA helices can be generated at low salt concentration by manipulating internal factors such as sugar configuration, duplex length, nucleotide composition and base methylation. These helices can constitute powerful tools for structural and biological investigations, especially as they can be used in physiological conditions.
PMCID: PMC290261  PMID: 14627831
11.  Intelligent Chiral Sensing Based on Supramolecular and Interfacial Concepts 
Sensors (Basel, Switzerland)  2010;10(7):6796-6820.
Of the known intelligently-operating systems, the majority can undoubtedly be classed as being of biological origin. One of the notable differences between biological and artificial systems is the important fact that biological materials consist mostly of chiral molecules. While most biochemical processes routinely discriminate chiral molecules, differentiation between chiral molecules in artificial systems is currently one of the challenging subjects in the field of molecular recognition. Therefore, one of the important challenges for intelligent man-made sensors is to prepare a sensing system that can discriminate chiral molecules. Because intermolecular interactions and detection at surfaces are respectively parts of supramolecular chemistry and interfacial science, chiral sensing based on supramolecular and interfacial concepts is a significant topic. In this review, we briefly summarize recent advances in these fields, including supramolecular hosts for color detection on chiral sensing, indicator-displacement assays, kinetic resolution in supramolecular reactions with analyses by mass spectrometry, use of chiral shape-defined polymers, such as dynamic helical polymers, molecular imprinting, thin films on surfaces of devices such as QCM, functional electrodes, FET, and SPR, the combined technique of magnetic resonance imaging and immunoassay, and chiral detection using scanning tunneling microscopy and cantilever technology. In addition, we will discuss novel concepts in recent research including the use of achiral reagents for chiral sensing with NMR, and mechanical control of chiral sensing. The importance of integration of chiral sensing systems with rapidly developing nanotechnology and nanomaterials is also emphasized.
PMCID: PMC3231122  PMID: 22163577
chiral sensing; supramolecular chemistry; interface; molecular recognition; nanotechnology; nanomaterial
12.  Origin of Homochirality in Biosystems 
Experimental data for a series of central and simple molecules in biosystems show that some amino acids and a simple sugar molecule have a chiral discrimination in favor of homochirality. Models for segregation of racemic mixtures of chiral amphiphiles and lipophiles in aqueous solutions show that the amphiphiles with an active isomerization kinetics can perform a spontaneous break of symmetry during the segregation and self-assembly to homochiral matter. Based on this observation it is argued that biomolecules with a sufficiently strong chiral discrimination could be the origin of homochirality in biological systems.
PMCID: PMC2672030  PMID: 19399249
Origin of chirality; Origin of Life; Prebiotic self-assambly
13.  Cyclodextrin Derivatives as Chiral Supramolecular Receptors for Enantioselective Sensing 
Sensors (Basel, Switzerland)  2006;6(6):593-615.
In view of the chiral nature of many bio-molecules (and all bio-macromolecules), most of therapeutically active compounds which target these molecules need to be chiral and “good handed” to be effective. In addition to asymmetric synthetic and separation methodologies, enantioselective chemical sensors, able to distinguish between two enantiomers of the same molecule, are of relevance. In order to design these sensing tools, two major classes of enantioselective layers have been developed. The first is based on molecularly imprinted polymers which are produced (polymerized) in the presence of their target, thus the polymeric material keep in “memory” the size and the shape of this molecule and the system could be used for sensing (not reviewed here). The second approach makes use of sensitive layers containing chiral macrocyclic receptors able of stereoselective molecular recognition; these receptors are mainly based on cyclodextrins. In this contribution, are reviewed achievements in the use of native or chemically modified cyclodextrins for chiral sensing purposes (at interfaces). Potentialities of other chiral macrocycles based on calixarenes, calix-resorcinarenes or crown-ethers as supramolecular receptors for enantioselective sensing are discussed.
PMCID: PMC3874827
Stereoselective Sensor; Macrocycle; Cyclodextrin; Calixarene; Crown-ether; Supramolecular Chemistry
14.  Growth of Optically Active Chiral Inorganic Films through DNA Self-Assembly and Silica Mineralisation 
Scientific Reports  2014;4:4866.
The circularly polarized reflection of nature is due to their distinct azimuthally twisted or helical character in the nanostructure of the surface films. Although many chiral inorganic powders have been successfully synthesised, the artificial synthesis of chiral inorganic films is rare. Herein, we reported a facile synthetic route for the growth of monolayered chiral film on the quaternary ammonium-modified silicon substrate. The films grew on the substrate surface because of the strong electrostatic interaction between positively charged quaternary ammonium groups and negatively charged phosphate groups of DNA, with subsequent growth to right-handed, vertically aligned, impeller-like helical architectures with left-handed two-dimensional square p4mm-structured DNA chiral packing. The DNA–silica composite films exhibited strong optical activity at 295 nm and in the range of 400–800 nm, corresponding to DNA chiral packing (absorption) and to the helical blade in the impeller (scattering), respectively. Upon removal of DNA templates, the pure inorganic impeller-like helical morphology was maintained; consequently, the scattering-based optical response was blue-shifted approximately 200 nm as a result of a decrease in the effective average refractive index. The hierarchical structures were reflected from the surfaces by cross-polarised light, which confirmed that the films were strongly birefringent, with long-range anisotropy.
PMCID: PMC4007082  PMID: 24784912
15.  Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer 
Nucleic Acids Research  2011;39(16):7329-7335.
Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ3T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ3T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.
PMCID: PMC3167616  PMID: 21586589
16.  Chiral Mutagenesis of Insulin. Foldability and Function Are Inversely Regulated by a Stereospecific Switch in the B Chain†,‡ 
Biochemistry  2005;44(13):10.1021/bi048025o.
How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis–comparison of corresponding d and l amino acid substitutions in the hormone–to define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T → R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, GlyB8 lies within a β-turn and exhibits a positive ϕ angle (like a d amino acid); in the alternative R state, GlyB8 is part of an α-helix and exhibits a negative ϕ angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 ϕ angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T → R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an “ambidextrous” glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin–and the β-cell–from protein misfolding.
PMCID: PMC3845378  PMID: 15794637
17.  Molecular Basis for Chiral Selection in RNA Aminoacylation 
The chiral-selective aminoacylation of an RNA minihelix is a potential progenitor to modern tRNA-based protein synthesis using l-amino acids. This article describes the molecular basis for this chiral selection. The extended double helical form of an RNA minihelix with a CCA triplet (acceptor of an amino acid), an aminoacyl phosphate donor nucleotide (mimic of aminoacyl-AMP), and a bridging nucleotide facilitates chiral-selective aminoacylation. Energetically, the reaction is characterized by a downhill reaction wherein an amino acid migrates from a high-energy acyl phosphate linkage to a lower-energy carboxyl ester linkage. The reaction occurs under the restriction that the nucleophilic attack of O, from 3′-OH in the terminal CCA, to C, from C=O in the acyl phosphate linkage, must occur at a Bürgi-Dunitz angle, which is defined as the O–C=O angle of approximately 105°. The extended double helical form results in a steric hindrance at the side chain of the amino acid leading to chiral preference combined with cation coordinations in the amino acid and the phosphate oxygen. Such a system could have developed into the protein biosynthetic system with an exclusively chiral component (l-amino acids) via (proto) ribosomes.
PMCID: PMC3155382  PMID: 21845109
homochirality; amino acid; RNA; aminoacylation; stereochemistry; extended double helix; origin of life
18.  Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size 
Cell & Bioscience  2014;4:30.
Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days.
Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection.
The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion.
PMCID: PMC4060578  PMID: 24940480
DNA translocase; DsDNA viruses; Viral DNA packaging motor; Viral assembly; Bacteriophage; DNA helicase; Revolution force; Phi29; FtsK; RecA; Nanomotor
19.  Chirality transfer from gold nanocluster to adsorbate evidenced by vibrational circular dichroism 
Nature Communications  2015;6:7117.
The transfer of chirality from one set of molecules to another is fundamental for applications in chiral technology and has likely played a crucial role for establishing homochirality on earth. Here we show that an intrinsically chiral gold cluster can transfer its handedness to an achiral molecule adsorbed on its surface. Solutions of chiral Au38(2-PET)24 (2-PET=2-phenylethylthiolate) cluster enantiomers show strong vibrational circular dichroism (VCD) signals in vibrations of the achiral adsorbate. Density functional theory (DFT) calculations reveal that 2-PET molecules adopt a chiral conformation. Chirality transfer from the cluster to the achiral adsorbate is responsible for the preference of one of the two mirror images. Intermolecular interactions between the adsorbed molecules on the crowded cluster surface seem to play a dominant role for the phenomena. Such chirality transfer from metals to adsorbates likely plays an important role in heterogeneous enantioselective catalysis.
The transfer of chirality from surfaces to molecular species may have implications in areas from the origin of homochirality to heterogeneous catalysis. Here, the authors show that a chiral gold cluster can transfer its inherent chirality to adsorbed, achiral molecules, causing them to adopt chiral conformations.
PMCID: PMC4432620  PMID: 25960309
20.  Enantioselective recognition at mesoporous chiral metal surfaces 
Nature Communications  2014;5:3325.
Chirality is widespread in natural systems, and artificial reproduction of chiral recognition is a major scientific challenge, especially owing to various potential applications ranging from catalysis to sensing and separation science. In this context, molecular imprinting is a well-known approach for generating materials with enantioselective properties, and it has been successfully employed using polymers. However, it is particularly difficult to synthesize chiral metal matrices by this method. Here we report the fabrication of a chirally imprinted mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and chiral template molecules. The porous platinum retains a chiral character after removal of the template molecules. A matrix obtained in this way exhibits a large active surface area due to its mesoporosity, and also shows a significant discrimination between two enantiomers, when they are probed using such materials as electrodes.
Chemical synthesis of chiral materials with enantioselective properties is an ongoing challenge. Here, the authors fabricate a chirally imprinted mesoporous metal from the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and chiral templating molecules.
PMCID: PMC3948375  PMID: 24548992
21.  Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences 
Nucleic Acids Research  2008;36(5):1464-1471.
Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this ‘double-duplex invasion’, a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the α-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G–C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.
PMCID: PMC2275137  PMID: 18203747
22.  Extension of UNRES force field to treat polypeptide chains with D-amino-acid residues 
Coarse-grained force fields for protein simulations are usually designed and parameterized to treat proteins composed of natural L-amino-acid residues. However, D-amino-acid residues occur in bacterial, fungal (e.g., gramicidins), as well as human-designed proteins. For this reason, we have extended the UNRES coarse-grained force field developed in our laboratory to treat systems with D-amino-acid residues. We developed the respective virtual-bond-torsional and double-torsional potentials for rotation about the Cα · · · Cα virtual-bond axis and two consecutive Cα · · · Cα virtual-bond axes, respectively, as functions of virtual-bond-dihedral angles γ. In turn, these were calculated as potentials of mean force (PMFs) from the diabatic energy surfaces of terminally-blocked model compounds for glycine, alanine, and proline. The potential-energy surfaces were calculated by using the ab initio method of molecular quantum mechanics at the Møller-Plesset (MP2) level of theory and the 6-31G(d,p) basis set, with the rotation angles of the peptide groups about Ci-1α⋯Ciα(λ(1)) and Ciα⋯Ci+1α(λ(2)) used as variables, and the energy was minimized with respect to the remaining degrees of freedom. The PMFs were calculated by numerical integration for all pairs and triplets with all possible combinations of types (glycine, alanine, and proline) and chirality (D or L); however, symmetry relations reduce the number of non-equivalent torsional potentials to 13 and the number of double-torsional potentials to 63 for a given C-terminal blocking group. Subsequently, one- (for torsional) and two-dimensional (for double-torsional potentials) Fourier series were fitted to the PMFs to obtain analytical expressions. It was found that the torsional potentials of the x-Y and X-y types, where X and Y are Ala or Pro, respectively, and a lowercase letter denotes D-chirality, have global minima for small absolute values of γ, accounting for the double-helical structure of gramicidin A, which is a dimer of two chains, each possessing an alternating D-Tyr-L-Tyr sequence, and similar peptides. The side-chain and correlation potentials for D-amino-acid residues were obtained by applying the reflection about the Ci-1α⋯Ciα⋯Ci+1α plane to the respective potentials for the L-amino-acid residues.
PMCID: PMC3982868  PMID: 24729761
23.  Synthesis, Characterization and Application of Novel Chiral Ionic Liquids and their Polymers in Micellar Electrokinetic Chromatography 
Analytical chemistry  2006;78(19):7061-7069.
Two amino acid derived (leucinol and N-methyl pyrrolidinol) chiral ionic liquids are synthesized and characterized both in monomeric and polymeric forms. Leucinol based chiral cationic surfactant is room a temperature ionic liquid (RTIL), and pyrrolidinol based chiral cationic surfactant melts at 30-35 °C to form ionic liquid (IL). The monomeric and polymeric ILs are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation and partial specific volume. Here in, we present the first enantioseparation using chiral IL as pseudostationary phase in capillary electrophoresis. Chiral separation of two acidic analytes, (±)-alpha-bromo-phenylacetic acid (±)-(α-BP-AA) and (±)-2-(2-chlorophenoxy)propanoic acid(±)-(2-PPA) can be achieved with both monomers and polymers of undecenoxy carbonyl-L-pryrrolidinol bromide (L-UCPB) and undecenoxy carbonyl-L-leucinol bromide (L-UCLB) at 25 mM surfactant concentration using phosphate buffer at pH 7.50. The chiral recognition seems to be facilitated by the extent of interaction of the acidic analytes with the cationic head group of chiral selectors. Polysodium N-undecenoxy carbonyl-L-leucine sulfate (poly-L-SUCLS) and polysodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL) were compared at high and low pH for the enantioseparation of (±)-(2-PPA). At pH 7.5, poly-L-SUCLS, poly-L-SUCL and ±-2-PPA are negatively charged resulting in no enantioseparation. However, chiral separation was observed for (±)-(2-PPA) using poly-L-SUCLS at low pH (pH 2.00) at which analyte is neutral. The comparison of chiral separation of anionic and cationic surfactants demonstrates that the electrostatic interaction between the acidic analyte and cationic micelle plays a profound role in enantioseparation.
PMCID: PMC2526052  PMID: 17007537
24.  Chirality Emergence in Thin Solid Films of Amino Acids by Polarized Light from Synchrotron Radiation and Free Electron Laser 
One of the most attractive hypothesis for the origin of homochirality in terrestrial bioorganic compounds is that a kind of “chiral impulse” as an asymmetric excitation source induced asymmetric reactions on the surfaces of such materials such as meteorites or interstellar dusts prior to the existence of terrestrial life (Cosmic Scenario). To experimentally introduce chiral structure into racemic films of amino acids (alanine, phenylalanine, isovaline, etc.), we irradiated them with linearly polarized light (LPL) from synchrotron radiation and circularly polarized light (CPL) from a free electron laser. After the irradiation, we evaluated optical anisotropy by measuring the circular dichroism (CD) spectra and verified that new Cotton peaks appeared at almost the same peak position as those of the corresponding non-racemic amino acid films. With LPL irradiation, two-dimensional anisotropic structure expressed as linear dichroism and/or linear birefringence was introduced into the racemic films. With CPL irradiation, the signs of the Cotton peaks exhibit symmetrical structure corresponding to the direction of CPL rotation. This indicates that some kinds of chiral structure were introduced into the racemic film. The CD spectra after CPL irradiation suggest the chiral structure should be derived from not only preferential photolysis but also from photolysis-induced molecular structural change. These results suggest that circularly polarized light sources in space could be associated with the origin of terrestrial homochirality; that is, they would be effective asymmetric exciting sources introducing chiral structures into bio-organic molecules or complex organic compounds.
PMCID: PMC2738911  PMID: 19742124
chirality; circularly polarized light; amino acids; solid films; synchrotron radiation; free electron laser; origin of terrestrial homochirality
25.  G-Quadruplex binding enantiomers show chiral selective interactions with human telomere 
Nucleic Acids Research  2014;42(6):3792-3802.
Chiral recognition of DNA molecules is important because DNA chiral transition and its different conformations are involved in a series of important life events. Among them, polymorphic human telomere DNA has attracted great interests in recent years because of its important roles in chromosome structural integrity. In this report, we examine the short-term effect of chiral metallo-supramolecular complex enantiomers treatment on tumor cells, and find that a zinc-finger-like alpha helical chiral metallo-supramolecular complex, [Ni2L3]4+-P enantiomer (NiP), can selectively provoke the rapid telomere uncapping, trigger DNA damage responses at telomere and degradation of G-overhang and the delocalization of telomeric protein from telomeres. Further studies indicate that NiP can induce an acute cellular apoptosis and senescence in cancer cells rather than normal cells. These results are further evidenced by the upregulation of p21 and p16 proteins. Moreover, NiP can cause translocation of hTERT from nuclear to cytoplasm through Tyr 707 phosphorylation. While its enantiomer, [Ni2L3]4+-M (NiM), has no such mentioned effects, these results clearly demonstrate the compound’s chiral selectivity in cancer cells. Our work will shed light on design of chiral anticancer drugs targeting G-quadruplex DNA, and developing telomere and telomerase modulation agents.
PMCID: PMC3973297  PMID: 24413564

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