Amino Acid Units Research Articles

A prelude to building mathematical models for polypeptide folding: analysis on the conformational potential energy hypersurface cross-sections of N-acetyl-glycyl-glycine-N′-methylamide

Finding a relationship on how a three-dimensional protein folds from its linear amino acid chain gets more complex with increasing chain length, so working on a smaller peptide conformational problem can provide initial ideas on what are the main molecular forces and how these influence the folding process. Following the study of conformations of amino acid units entering the proteins to understand the secondary structure of small peptides, this paper proposes mathematical models for the several two-rotor cross-sections of the five-dimensional N-acetyl-glycyl-glycine-N′-methylamide potential energy hypersurface (PEHS). These cross-sections are extracted along the first glycine subunit, with its coordinates fixed at the five energy minima of the glycine diamide. The resulting mathematical models yield an average RMSE of 1.36 kJ mol−1 and an average R2 of 0.9923 with respect to energy values obtained from DFT calculations. The minima geometries obtained from these models are also in good agreement with DFT-optimized energy minima conformers. An important aspect of this study also tackles the relationship between the PEHS of the glycyl-glycine diamide and its glycine subunits. It has been observed that there are deviations up to 28.35 kJ mol−1 and 29.52 kJ mol−1 between the PEHS cross-sections along γL and γD conformations, respectively, in the first glycine subunit. This may suggest that there are significant backbone–backbone intermolecular forces acting on the dipeptide. The abovementioned findings can help in developing more complex mathematical models for polypeptide folding from amino acid subunits.

A pivotal peptide (Val-Ser-Glu-Glu) from duck egg white promotes calcium uptake and structure-activity relationship study

The effects of desalted duck egg white peptides (DPs) and Val-Ser-Glu-Glu (VSEE) on calcium absorption were investigated in calcium-deficient diets rat model. Six-week oral administration results indicated DPs + CaCO3 and VSEE + CaCO3 were significantly higher than CaCO3 group in calcium absorption, serum osteocalcin, bone calcium content and max load, but lower in serum bone alkaline phosphatase level (P < 0.05). Additionally, in the 0.5% calcium diets groups, the rats fed with DPs showed notably increases in calcium absorption, length of tibias and bone max load than those without DPs. The structure-activity relationship results demonstrated the importance of amino acid units like Glu-Glu and Ser-Ser. Homology modeling and molecular docking results provided two possible active sites of TRPV6 in the promotion of calcium uptake by DPs. The present study expands the understanding of the mechanism of calcium uptake in vivo by DPs, as well as highlights the structure-activity relationship and the potential active sites of TRPV6.

A general strategy for synthesis of cyclophane-braced peptide macrocycles via palladium-catalysed intramolecular sp3 C-H arylation.

New methods capable of effecting cyclization, and forming novel three-dimensional structures while maintaining favourable physicochemical properties are needed to facilitate the development of cyclic peptide-based drugs that can engage challenging biological targets, such as protein-protein interactions. Here, we report a highly efficient and generally applicable strategy for constructing new types of peptide macrocycles using palladium-catalysed intramolecular C(sp3)-H arylation reactions. Easily accessible linear peptide precursors of simple and versatile design can be selectively cyclized at the side chains of either aromatic or modified non-aromatic amino acid units to form various cyclophane-braced peptide cycles. This strategy provides a powerful tool to address the long-standing challenge of size- and composition-dependence in peptide macrocyclization, and generates novel peptide macrocycles with uniquely buttressed backbones and distinct loop-type three-dimensional structures. Preliminary cell proliferation screening of the pilot library revealed a potent lead compound with selective cytotoxicity toward proliferative Myc-dependent cancer cell lines.

Polymorphisms analysis of the Plasmodium ovale tryptophan-rich antigen gene (potra) from imported malaria cases in Henan Province

BackgroundPlasmodium ovale has two different subspecies: P. ovale curtisi and P. ovale wallikeri, which may be distinguished by the gene potra encoding P. ovale tryptophan-rich antigen. The sequence and size of potra gene was variable between the two P. ovale spp., and more fragment sizes were found compared to previous studies. Further information about the diversity of potra genes in these two P. ovale spp. will be needed.MethodsA total of 110 dried blood samples were collected from the clinical patients infected with P. ovale, who all returned from Africa in Henan Province in 2011–2016. The fragments of potra were amplified by nested PCR. The sizes and species of potra gene were analysed after sequencing, and the difference between the isolates were analysed with the alignment of the amino acid sequences. The phylogenetic tree was constructed by neighbour-joining to determine the genetic relationship among all the isolates. The distribution of the isolates was analysed based on the origin country.ResultsTotally 67 samples infected with P. o. wallikeri, which included 8 genotypes of potra, while 43 samples infected with P. o. curtisi including 3 genotypes of potra. Combination with the previous studies, P. o. wallikeri had six sizes, 227, 245, 263, 281, 299 and 335 bp, and P. o. curtisi had four sizes, 299, 317, 335 and 353 bp, the fragment sizes of 299 and 335 bp were the overlaps between the two species. Six amino acid as one unit was firstly used to analyse the amino acid sequence of potra. Amino acid sequence alignment revealed that potra of P. o. wallikeri differed in two amino acid units, MANPIN and AITPIN, while potra of P. o. curtisi differed in amino acid units TINPIN and TITPIS. Combination with the previous studies, there were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The phylogenetic tree showed that 11 isolates were divided into two clusters, P. o. wallikeri which was then divided into five sub-clusters, and P. o. curtisi which also formed two sub-clusters with their respective reference sequences. The genetic relationship of the P. ovale spp. mainly based on the number of the dominant amino acid repeats, the number of MANPIN, AITPIN, TINPIN or TITPIS. The genotype of the 245 bp size for P. o. wallikeri and that of the 299 and 317 bp size for P. o. curtisi were commonly exiting in Africa.ConclusionThis study further proved that more fragment sizes were found, P. o. wallikeri had six sizes, P. o. curtisi had four sizes. There were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The genetic polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era.

Determination of the activity of telomerase in cancer cells by using BSA-protected gold nanoclusters as a fluorescent probe.

Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650nm) on photoexcitation with ultraviolet light (365nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity. Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.

Investigations on the Effect of Fatty Acid Additives on Casein Micelles: Role of Ethylenic Unsaturation on the Interaction and Structural Diversity

Casein, one of the major constituent of milk protein, is considered to be a good candidate for oral drug delivery system. Also, milk transports various essential fatty acid to blood through dietary supplements. In this study, we have explored the alteration in the structural characteristic in terms of the modulations in the microenvironment of the protein in the presence of different types of fatty acids. Herein, we have observed that the unsaturation of fatty acids mostly affects the structure of casein micelles (CMs) by impinging upon the hydrophobic force of interaction following a decrease in the electrostatic interaction of various amino acid unit. Alteration of such forces is responsible for the increase in the aggregate size, modification in the protein secondary structure, and different morphology of CMs. Fluorescence behavior of 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran indicates that the rigidity of the microenvironment is the main characteristic of the fatty acid binding, and the binding constant increases with the fatty acid chain length for saturated fatty acid or with the introduction of unsaturation onto it. Fluorescence lifetime imaging microscopy study indicates that the microstructure of CMs becomes more compact in the presence of unsaturated fatty acids, and this is also responsible for the increase in the diffusion time of the probe. Moreover, decrease in the fluorescence of extrinsic probe 8-anilinonaphthalene-1-sulfonate with the addition of unsaturated fatty acid reveals that these fatty acids alter the electrostatic interaction between casein units, more specifically in case of the surface-bound κ-casein. Therefore, this study provides a very useful information on the binding of fatty acids and helps to evaluate other fatty acid, as well as different small molecules binding in the applicative medicinal purpose.

Synthesis and antimicrobial activity of amino acid and peptide derivatives of mycophenolic acid

The series of 16 novel amino acid and peptide mycophenolic acid (MPA) derivatives was obtained as potential antibacterial agents. Coupling of MPA with respective amines was optimized with condensing reagents such as EDCI/DMAP and T3P/TEA. Amino acid analogs were received both as methyl esters and also with the free carboxylic group. The biological activity of the products was tested on five references bacterial strains: Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus aureus MSSA ATCC 25923. Peptide derivatives proved to be the most versatile ones, their MIC values relative to most strains was lower than MPA alone. It has been noted that the activity of amino acid derivatives depends on the configuration at the chiral center in the amino acid unit and methyl esters indicated better antimicrobial activity than analogs with free carboxylic group.

Dimerization of Phenylalanine: An Approach to Thiazoles and Oxazoles Involved S/O‐Insertion

AbstractWe herein describe the development of a dimerization procedure for amino acids to prepare 2,5‐disubstituted thiazoles and oxazoles in the presence of Na2S⋅9H2O and H2O, respectively. These approaches enabled the direct formation of five‐membered ring systems bearing two different heteroatoms from two amino acid units. Mechanically, decarboxylation, deamination, S/O insertion, cyclization and gradient oxidation processes were involved in the oligomer formation.magnified image

Structure-informed detection and quantification of peptides in food and biological fluids.

Peptides with biological properties, that is, bioactive peptides, are a class of biomolecules whose health-promoting properties are increasingly being exploited in food and health products. However, research on targeted techniques for the detection and quantification of these peptides is still in its infancy. Such information is needed in order to enhance the biological and chemometric characterization of peptides and their subsequent application in the functional food and pharmaceutical industries. In this review, the role of classic techniques such as electrophoretic, chromatographic, and peptide mass spectrometry in the structure-informed detection and quantitation of bioactive peptides are discussed. Prospects for the use of aptamers in the characterization of bioactive peptides are also discussed. PRACTICAL APPLICATIONS: Although bioactive peptides have huge potential applications in the functional foods and health area, there are limited techniques in enhancing throughput detection, quantification, and characterization of these peptides. This review discusses state-of-the-art techniques relevant in complementing bioactive detection and profiling irrespective of the small number of amino acid units. Insights into challenges, possible remedies and prevailing areas requiring thorough research in the extant literature for food chemists and biotechnologists are also presented.

Nicrophorusamides A and B, Antibacterial Chlorinated Cyclic Peptides from a Gut Bacterium of the Carrion Beetle Nicrophorus concolor.

Nicrophorusamides A and B (1 and 2) were discovered from a rare actinomycete, Microbacterium sp., which was isolated from the gut of the carrion beetle Nicrophorus concolor. The structures of the nicrophorusamides were established as new chlorinated cyclic hexapeptides bearing uncommon amino acid units mainly based on 1D and 2D NMR spectroscopic analysis. The absolute configurations of the amino acid residues 5-chloro-l-tryptophan, d-threo-β-hydroxyasparagine/d-asparagine, l-ornithine, l-allo-isoleucine, d-leucine, and d-valine were determined using Marfey's method and chemical derivatization with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate followed by LC/MS analysis. Nicrophorusamide A (1) showed antibacterial activity against several Gram-positive bacteria.

Biosynthetic study of &amp;beta;-methyl amino acid building blocks involved in natural products

Natural products are an important source of pharmaceuticals or drug leads for the human lifes. The biosynthetic studies on these small chemical molecules can offer the possibilities to improve the titers and produce a lot of non-natural natural products with enhanced biological activity. Amino acids are a type of important building blocks of natural products, and apart from twenty proteinogenic amino acids, about five hundred nonproteinogenic amino acids were found to be involved into the biosynthesis of natural products. These nonproteinogenic amino acid units in natural products have been confirmed to play crucial roles in their stabilities and biological activities. β-methyl amino acid is one kind of nonproteinogenic amino acids and has been found in many bioactive secondary metabolites, including daptomycin, streptonigrin, indolemycin and so on, and as well as several primary metabolites such as 3-methyl-glutamate in fermentation process of several Clostridium sp. The β-carbon of amino acids is not an active position for methylation, therefore the methylation at the β-position is still chemically very challenging. Recently, the biosynthetic studies of microbial natural products revealed many novel enzymatic reactions or pathways for amino acid modifications including the β-methylation of amino acids. Up to now, three different biosynthetic pathways for β-methyl amino acids have been reported, including B12-dependent glutamate aminomutase, combination of a methyltransferase and an aminotranferase, and B12-dependent radical SAM methyltransferase. B12-dependent glutamate aminomutase catalyzes the rearrangement of glutamate to generate β-methyl aspartic acid, a building black of Nikkomycins and other natural products. Combination of a methyltransferase and an aminotranferase uses the aminotransferase to generate α-keto acids as the active substrates for the methylation catalzyed by the methyltransferase from amino acids while B12-dependent radical SAM methyltransferase can catalyze the direct methylation at the β-carbon of amino acids via a radical manner. There have been many reviews that summarized the enzymatic modifications of amino acids, but there has been none of literatures covering this type of intereting and useful nonproteinogenic amino acids. This review summarized the recent advances in the biosynthesis of β-methyl amino acids as the building blocks of several natural products and discussed the biosynthetic or enzymatic mechanisms involved in the β-methylation of amino acids in detail. Basesd on the mechanisms of the biosynthesis of these β-methyl amino acids, the application of β-methyl amino acids was proposed in this review. This study may provide new strategies and useful information for improving the yields of the valuable natural products containing β-methyl amino acids and introducing these building blocks into more chemical scaffolds by metabolic engineering, synthetic biology and enzyme engineering methods.

Co-Catalyzed C(sp3)-H Oxidative Coupling of Glycine and Peptide Derivatives.

Cobalt-catalyzed selective α-alkylation and α-heteroarylation processes of α-amino esters and peptide derivatives are described. These cross-dehydrogenative reactions occur under mild conditions and allow for the rapid assembly of structurally diverse α-amino carbonyl compounds. Unlike enolate chemistry, these methods are distinguished by their site-specificity, occur without racemization of the existing chiral centers, and exhibit total selectivity for aryl glycine motifs over other amino acid units, hence providing ample opportunities for peptide modifications.

Increased Biological Activity of Aneurinibacillus migulanus Strains Correlates with the Production of New Gramicidin Secondary Metabolites

The soil-borne gram-positive bacteria Aneurinibacillus migulanus strain Nagano shows considerable potential as a biocontrol agent against plant diseases. In contrast, A. migulanus NCTC 7096 proved less effective for inhibition of plant pathogens. Nagano strain exerts biocontrol activity against some gram-positive and gram-negative bacteria, fungi and oomycetes through the production of gramicidin S (GS). Apart from the antibiotic effects, GS increases the rate of evaporation from the plant surface, reducing periods of surface wetness and thereby indirectly inhibiting spore germination. To elucidate the molecular basis of differential biocontrol abilities of Nagano and NCTC 7096, we compared GS production and biosurfactant secretion in addition to genome mining of the genomes. Our results proved that: (i) Using oil spreading, blood agar lysis, surface tension and tomato leaves wetness assays, Nagano showed increased biosurfactant secretion in comparison with NCTC 7096, (ii) Genome mining indicated the presence of GS genes in both Nagano and NCTC 7096 with two amino acid units difference between the strains: T342I and P419S. Using 3D models and the DUET server, T342I and P419S were predicted to decrease the stability of the NCTC 7096 GS synthase, (iii) Nagano produced two additional GS-like molecules GS-1155 (molecular weight 1155) and GS-1169 (molecular weight 1169), where one or two ornithine residues replace lysine in the peptide. There was also a negative correlation between surface tension and the quantity of GS-1169 present in Nagano, and (iv) the Nagano genome had a full protein network of exopolysaccharide biosynthesis in contrast to NCTC 7096 which lacked the first enzyme of the network. NCTC 7096 is unable to form biofilms as observed for Nagano. Different molecular layers, mainly gramicidin secondary metabolite production, account for differential biocontrol abilities of Nagano and NCTC 7096. This work highlighted the basis of differential biological control abilities between strains belonging to the same species and demonstrates techniques useful to the screening of effective biocontrol strains for environmentally friendly secondary metabolites that can be used to manage plant pathogens in the field.

Toward the Synthesis and Pharmacological Screening of a Natural Cycloheptapeptide of Plant Origin

The solution-phase synthesis of a proline and glycine-rich plant-derived cyclic heptapeptide, gypsophin E (8), is reported via coupling of a tetrapeptide unit Glycyl-L-leucyl-L-valyl-L-proline-OMe with a tripeptide unit Boc-L-isoleucyl-glycyl-L-proline-OH, followed by cyclization of the linear fragment having seven amino acid units. The structure of the newly synthesized cycloheptapeptide was confirmed by means of chemical and spectroscopic methods. The newly synthesized cyclopolypeptide displayed potent antifungal and anthelmintic activities against the pathogenic yeast Candida albicans, the dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at the 6 μg/mL level, and the earthworms Megascoplex konkanensis, Pontoscotex corethruses and - - Eudrilus eugeniea at a concentration of 2 mg/mL.

Sphaericin, a Lasso Peptide from the Rare Actinomycete Planomonospora sphaerica

A lasso peptide named sphaericin was isolated from the ISP2 agar medium culture of the rare actinomycete species Planomonospora sphaerica. The analysis of the amino acid composition of sphaericin gave a total of 15 amino acid units (three units each of Gly, Ile, and Pro; one unit each of Arg, Glu, Leu, Phe, Ser, and Tyr). In addition to these amino acids, NMR spectroscopy experiments indicated the presence of three Trp units. The structure of sphaericin was determined to be a peptide comprising 18 amino acids, and an additional peptide bond between the amino group of Gly‐1 and the γ‐carboxyl group of Glu‐9 forms a macrolactam ring, as demonstrated by analyses of the TOF MS/MS and NMR spectra. The analysis of the solution three‐dimensional structural through NOE experiments revealed that sphaericin possesses a typical lasso structure. From the genome data, the biosynthetic gene cluster of sphaericin was determined to consist of four genes, namely sphA, sphB1, sphB2, and sphC. Sphaericin showed a specific antibacterial activity against Micrococcus luteus at a dosage of 50 µg/disk.

SERS assay for pyrophosphate based on its competitive binding to Cu(II) ion on silver nanoparticles modified with cysteine and rhodamine 6G

The authors have developed a surface-enhanced Raman spectroscopic (SERS) method for the determination of pyrophosphate ion (PPi). It is based on the competitive coordination of Cu(II) between cysteine (Cys) and PPi using silver nanoparticles (AgNPs) modified with Cys and Rhodamine 6G (R6G). Cys was attached to the surface of the AgNPs via Ag−S bond, and the amino acid unit in Cys bind to Cu(II) to form a chelate complex. This results in the aggregation of AgNPs and a strong SERS signal for the probe R6G. However, in the presence of PPi, the aggregated AgNPs are solubilized because of the stronger affinity between PPi and Cu(II). This leads to a decrease of the SERS signal and forms the basis for the quantitation of PPi. The amount of AgNPs, the concentration of Cu(II), and the mixing time were optimized. The method displays a linear response in the 0.1 to 80 μM PPi concentration range, and the limit of detection is as low as 20 nM. The method was applied to the determination of PPi in spiked serum samples and urine samples, with recoveries between 95.2 to 100.5% and relative standard deviations of <4.4%.

Synthesis of Amino‐Acid–Nucleoside Conjugates

AbstractRepresentative derivatives of uridine‐conjugated amino acids that have been suitably protected for Fmoc solid‐phase chemistry have been prepared through efficient procedures that use “click” reactions as key steps, including thiol–ene radical reactions and copper‐catalyzed azide alkyne cycloaddition (CuAAC) reactions. Several linkers between the amino acid and nucleoside units, including alkyl chains and a triazole ring, have been successfully employed. Alkyl chains offer retention of flexibility, to allow the bisubstrate analogues to adopt an appropriate orientation, whilst the triazole ring can promote additional interactions at the active sites of target enzymes. Furthermore, a neutral surrogate of the pyrophosphate unit has been prepared by using a Staudinger–Vilarrasa reaction as a key step. Neutral analogues are promising surrogates for avoiding difficulties owing to cell‐membrane permeability.

Synthesis and chiroptical properties of novel helical polyacetylenes containing fluorene pendant groups in the side chains

A series of novel substituted acetylene monomers bearing fluorene pendant groups (C13H9COCH2CH2CO-R-CH2CCH, R=NH (IV1), R=O (IV2), R=NHCH(CH3)CONH (IV3), R=NHCH(CH3)COO (IV4)) have been synthesized and subsequently polymerized with [Rh(nbd)Cl]2 as a catalyst to obtain the corresponding polyacetylenes (Poly(IV1–4)). The 1H NMR spectra demonstrated that all the obtained polyacetylenes had high cis-stereoregular structures. The results of CD (circular dichroism) and UV–vis spectra showed that Poly(IV3,4) took a tight helical structure, while Poly(IV1,2) did not. The chiral amino acid units in the side chains of Poly(IV3,4) induced the main chain to form the helical conformation. In addition, owing to the large steric repulsion and π-π interaction between the bulky fluorene groups, Poly(IV3,4) showed good helical stability at various temperatures (−10–60°C) and in strong polar solvents. Especially, the CD intensity of Poly(IV3) was enhanced with increasing the solvent polarity. Moreover, Poly(IV1–4) also showed good photoluminescent (PL) properties. The nonconjugated aliphatic spacer and the twisting of the polymer main chain were favorable to improve the PL efficiency.

Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity.

Spider aciniform (wrapping) silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units). In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR) spectroscopy-based 15N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps–ns timescale in the context of the single W unit (W1) and the two unit concatemer (W2). Unambiguous mapping of backbone dynamics throughout W2 was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W1 and W2 reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre.

Enantioselective recognition of sodium carboxylates by an 1,8-diaminoanthracene based ion pair receptor containing amino acid units

An anthracene based ion pair receptor containing two amino acid units supported by cation and anion binding domains has been synthesized and shown to exhibit enhanced anion binding affinities in the presence of sodium cations. The receptor’s ability to recognize enantiomers was studied using chiral carboxylates derived from 2-phenylbutyric acid, mandelic acid, and three Boc-protected amino acids. Sodium cation coordination does not influence chiral recognition but does affect the strength of anion binding. The greatest enhancement of anion binding in the presence of sodium cations was found for halides, and the highest enantiodiscrimination was found for Boc-N-tryptophan. Comparative anion and salt binding studies revealed that the simultaneous action of multiple binding domains in the structure of receptor 1 is responsible for its stronger salt association and better enantioselectivity than in the case of mono-supported receptor 2.