The 1,3‐diyne linker as a rigid “i,i+7” staple for α‐helix stabilization: Stereochemistry at work

Short alphahelical peptide sequences were stabilized through Glaser‐Hay couplings of propargylated l ‐ and/or d ‐serine residues at positions i and i+7. NMR analysis confirmed a full stabilization of the helical structure when a d ‐Ser (i), l ‐Ser (i+7) combination was applied. In case two l ‐Ser residues were involved in the cyclization, the helical conformation is disrupted outside the peptide's macrocycle.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on l‐lactate

Campylobacter jejuni, a major food‐borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and l ‐ and d ‐lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on l ‐lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, l ‐lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD‐independent l ‐LDHs; a non‐flavin iron–sulfur containing three subunit membrane‐associated enzyme (Cj0075c‐73c), and a flavin and iron–sulfur containing membrane‐associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on l ‐lactate, as single mutants in each system grew as well as wild‐type on this substrate, while a cj0075c cj1585c double mutant showed no l ‐lactate oxidase activity and did not utilize or grow on l ‐lactate; d ‐lactate‐dependent growth was unaffected. Orthologues of Cj0075c‐73c (LldEFG/LutABC) and Cj1585c (Dld‐II) were recently shown to represent two novel families of l ‐ and d ‐lactate oxidases; this is the first report of a bacterium where both enzymes are involved in l ‐lactate utilization only. The cj0075c‐73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for l ‐lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use l ‐ and d ‐lactate as both electron‐donor and carbon source.     

Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.     

Comparative studies of adhesion peptides based on l‐ or d‐amino acids

Detailed studies comparing solid‐supported l ‐ or d ‐amino acid adhesion peptides based on the sequence KLHRIRA were performed. Stability towards proteases and levels of cellular adhesion to the otherwise inert surface of PEGA resin were compared by using fluorescently labelled peptides. A clear difference in the peptide stability towards cleavage by subtilisin, trypsin, or papain was observed. However, all of the on‐bead peptides provided an optimal surface for cell adhesion and proliferation. In long‐term experiments, these properties were still found to be similar on the resins modified either with l ‐ or with d‐ amino acids and unaffected by the nature of their fluorescence labelling at either terminus. These results support that the more accessible l ‐amino acids can be utilized for cell adhesion experiments and confirm the nonspecific interaction mechanism of cell binding to these peptides on the bead surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial,anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d ‐Tyr‐d ‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d ‐Tyr‐d ‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d ‐Tyr‐d ‐Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d ‐Tyr‐d ‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d ‐Tyr‐d ‐Phe). Flow cytometry analysis showed that the cyclo(d ‐Tyr‐d ‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d ‐Tyr‐d ‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d ‐Tyr‐d ‐Phe) with synthetic cyclo(d ‐Tyr‐d ‐Phe) and cyclo(l ‐Tyr‐l ‐Phe). Natural and synthetic cyclo(d ‐Tyr‐d ‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded significant activity than natural and synthetic cyclo(d ‐Tyr‐d ‐Phe). The results of the present study reveals that cyclo(d ‐Tyr‐d ‐Phe) is more bioactive than cyclo(l ‐Tyr‐l ‐Phe). To the best of our knowledge, this is the first time that cyclo(d ‐Tyr‐d ‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d ‐Tyr‐d ‐Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular enantiorecognition of l‐glucose and d‐glucose in whole blood samples

In the past years, enantioanalysis became very important for clinical analysis; biomarkers/substances of biomedical importance with chiral structure should be analyzed and their presence correlated with the specific disorder. Therefore, we developed a method for the assay of l ‐ and d ‐glucose, based on molecular recognition of l ‐ and d ‐glucose. While for d ‐glucose there are many methods to assess its quantity, the l ‐enantiomer is not routinely detected by standard methods. Two stochastic microsensors based on the immobilization of Copper(II)phthalocyanine and Ni(II)phthalocyanine, in natural diamond powder, were proposed for the enantioanalysis of glucose. The proposed methods proved to have high sensitivities and were able to be used for determination of concentrations as low as 2.5 pg mL^?1 for d ‐glucose and as low as 2.5 fg mL^?1 for l ‐glucose. The enatioanalysis was performed with good results in whole blood samples collected from diabetic patients.     

Chiral single‐walled carbon nanotubes as chiral selectors in multimode enantioselective sensors

Single‐walled carbon nanotube‐(7,6) chirality was used for the design of multimode enantioselective sensors using different carbon matrices such as graphene paste, graphite paste, and carbon nanopowder‐based paste. l ‐ and d ‐malic acids were used as model analytes. The responses of the multimode sensors were evaluated for potentiometric and differential pulse voltammetry (DPV) modes. When carbon nanopowder was used as matrix, the multimode sensor was enantioselective for d ‐malic acid in the concentration range 10^?3 to 10^?15 mol/L for the potentiometric mode and 10^?5 to 10^?8 mol/L for the DPV mode. The graphite paste‐based sensor was enantioselective for l ‐malic acid in the ranges: 10^?10 to 10^?13 for the potentiometric mode and 10^?4 to 10^?7 mol/L for the DPV mode. The sensors based on graphene and chiral single‐walled carbon nanotubes were enantioselective for d ‐malic acid, and a response was obtained only in the DPV mode. Accordingly, the matrix influenced both the enantioselectivity and the sensitivity of the measurements. The application of the sensors was for the enantioanalysis of malic acid in wines and apple juice samples. The proposed method is fast and reliable and allows the quantification of l ‐ and d ‐malic acids using electrochemical methods based on different principles, from the real samples after a buffering of the samples. The enantioanalysis of malic acid in wine and juice samples was performed with high recoveries (higher than 90.00%) and low relative standard deviation (RSD) (%) values (lower than 1.00%).     

Novel Electrochemical Method for the Characterization of the Degree of Chirality in Chiral Polyaniline

A novel method to indicate the degree of chirality in polyaniline (PANI) was developed. The ( d ‐camphorsulfonic acid)‐ and (HCl)‐PANI‐based electrodes exhibited significantly different electrochemical performances in d ‐ and l ‐Alanine (Ala) aqueous solution, respectively, which can be used for the characterization the optical activity of chiral PANI. Cyclic voltammogram, tafel, and open circuit potential of PANI‐based electrodes were measured within d ‐ and l ‐Ala electrolyte solution, respectively. The open circuit potentials under different reacting conditions were analyzed by Doblhofer model formula, in which [C⁺]_poly1/[C⁺]_poly2 was used as a parameter to characterize the degree of chirality in chiral PANI. The results showed that [C⁺]_poly1/[C⁺]_poly2 can be increased with increasing concentrations of (1S)‐(+)‐ and (1R)‐(?)‐10‐camphorsulfonic acid. In addition, we detected that appropriate response time and lower temperature are necessary to improve the degree of chirality. Chirality 25:39‐42, 2013.© 2012 Wiley Periodicals, Inc.     

Antimicrobial activity and stability of protonectin with D‐amino acid substitutions

The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D ‐amino acids. Both the D ‐enantiomer of protonectin (D ‐prt) and D ‐Lys‐protonectin (D ‐Lys‐prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D ‐prt showed strong stability against trypsin, chymotrypsin and the human serum, while D ‐Lys‐prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D ‐Lys‐prt still kept typical α‐helical structure in the membrane mimicking environment, while D ‐prt showed left hand α‐helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D ‐amino acid substitution or partially D ‐amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D ‐prt and D ‐Lys‐prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography

The 50‐residue snake venom protein L ‐omwaprin and its enantiomer D ‐omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L ‐ and D ‐omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L ‐amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L ‐ and D ‐omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2₁/c and its structure was determined at atomic resolution (1.33 Å) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high‐resolution X‐ray structures by direct methods.     

D-Aspartate as a putative cell-cell signaling molecule in the Aplysia californica central nervous system

The content, synthesis and transport of d ‐aspartate (d ‐Asp) in the CNS of Aplysia californica is investigated using capillary electrophoresis (CE) with both laser‐induced fluorescence and radionuclide detection. Millimolar concentrations of d ‐Asp are found in various regions of the CNS. In the cerebral ganglion, three adjacent neuronal clusters have reproducibly different d ‐Asp levels; for example, in the F‐ and C‐clusters, up to 85% of the free Asp is present in the d ‐form. Heterogeneous distribution of d ‐Asp is also found in the individual identified neurons tested, including the optical ganglion top‐layer neurons, metacerebral cells, R2 neurons, and F‐, C‐ and G‐cluster neurons. The F‐cluster neurons have the highest percentage of d ‐Asp (～58% of the total Asp), whereas the lowest value of ～8% is found in R2 neurons. In pulse‐chase experiments with radiolabeled d ‐Asp, followed by CE with radionuclide detection, the synthesis of d ‐Asp from l ‐aspartate (l ‐Asp) is confirmed. Is d ‐Asp in the soma, or is it transported to distantly located release sites? d ‐Asp is clearly detected in the major nerves of A. californica, including the pleuroabdominal and cerebrobuccal connectives and the anterior tentacular nerves, suggesting it is transported long distances. In addition, both d ‐Asp and l ‐Asp are transported in the pleuroabdominal connectives in a colchicine‐dependent manner, whereas several other amino acids are not. Finally, d ‐Asp produces electrophysiological effects similar to those induced by l ‐Asp. These data are consistent with an active role for d ‐Asp in cell‐to‐cell communication.     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Computational studies on the water‐catalyzed stereoinversion mechanism of glutamic acid residues in peptides and proteins

In contrast with the common belief that all the amino acid residues in higher organisms are l ‐forms, d ‐amino acid residues have been recently detected in various aging tissues. Aspartic acid (Asp) residues are known to be the most prone to stereoinvert via cyclic imide intermediate. Although the glutamic acid (Glu) is similar in chemical structure to Asp, little has been reported to detect d ‐Glu residues in human proteins. In this study, we investigated the mechanism of the Glu‐residue stereoinversion catalyzed by water molecules using B3LYP/6‐31+G(d,p) density functional theory calculations. We propose that the Glu‐residue stereoinversion proceeds via a cyclic imide intermediate, i.e., glutarimide (GI). All calculations were performed by using a model compound in which a Glu residue was capped with acetyl and methylamino groups on the N‐ and C‐termini, respectively. We found that two water molecules catalyze the three steps involved in the GI formation: iminolization, cyclization, and dehydration. The activation energy required for the Glu residue to form a GI intermediate was estimated to be 32.3 kcal mol^?1, which was higher than that of the experimental Asp‐residue stereoinversion. This calculation result suggests that the Glu‐residue stereoinversion is not favored under the physiological condition.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.