Search

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper’s interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.

The structural effects of zinc replacement by xenobiotic metal ions have been widely studied in several eukaryotic and prokaryotic zinc-finger-containing proteins. The prokaryotic zinc finger, that presents a bigger &beta, &beta, &alpha, domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interaction with metallo-proteins. Several studies have been conducted on Ros87, the DNA binding domain of the prokaryotic zinc finger Ros, and have demonstrated that the domain appears to structurally tolerate Ni(II), albeit with important structural perturbations, but not Pb(II) and Hg(II), and it is in vitro functional when the zinc ion is replaced by Cd(II). We have previously shown that Ros87 unfolding is a two-step process in which a zinc binding intermediate converts to the native structure thorough a delicate downhill folding transition. Here, we explore the folding/unfolding behaviour of Ros87 coordinated to Co(II), Ni(II) or Cd(II), by UV-Vis, CD, DSC and NMR techniques. Interestingly, we show how the substitution of the native metal ion results in complete different folding scenarios. We found a two-state unfolding mechanism for Cd-Ros87 whose metal affinity Kd is comparable to the one obtained for the native Zn-Ros87, and a more complex mechanism for Co-Ros87 and Ni-Ros87, that show higher Kd values. Our data outline the complex cross-correlation between the protein&ndash, metal ion equilibrium and the folding mechanism proposing such an interplay as a key factor in the proper metal ion selection by a specific metallo-protein.

Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56–142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-‘like’ gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1–55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.

Acyclovir (ACV) is one of the most used antiviral drugs for the treatment of herpes simplex virus infections and other relevant mucosal infections caused by viruses. Nevertheless, the low water solubility of ACV limits both its bioavailability and antiviral performance. The combination of block copolymer micelles and cyclodextrins (CDs) may result in polypseudorotaxanes with tunable drug solubilizing and gelling properties. However, the simultaneous addition of various CDs has barely been investigated yet. The aim of this work was to design and characterize ternary combinations of Pluronic&reg, F127 (PF127), &alpha, CD and &beta, CD in terms of polypseudorotaxane formation, rheological behavior, and ACV solubilization ability and controlled release. The formation of polypseudorotaxanes between PF127 and the CDs was confirmed by FT-IR spectroscopy, X-ray diffraction, and NMR spectroscopy. The effects of &alpha, CD/&beta, CD concentration range (0&ndash, 7% w/w) on copolymer (6.5% w/w) gel features were evaluated at 20 and 37 &deg, C by rheological studies, resulting in changes of the copolymer gelling properties. PF127 with &alpha, CD improved the solubilization of ACV, maintaining the biocompatibility (hen&rsquo, s egg test on the chorio-allantoic membrane). In addition, the gels were able to sustain acyclovir delivery. The formulation prepared with similar proportions of &alpha, CD provided a slower and more constant release. The results obtained suggest that the combination of Pluronic with &alpha, CD mixtures can be a valuable approach to tune the rheological features and drug release profiles from these supramolecular gels.

Zinc ion binding is a principal event in the achievement of the correct fold in classical zinc finger domains since the motif is largely unfolded in the absence of metal. In the case of a prokaryotic zinc finger, the larger βββαα domain contributes to the folding mechanism with a larger hydrophobic core. For these reasons, following the great amount of attention devoted to unveiling the effect of xenobiotic metal ion replacement in zinc fingers and in zinc-containing proteins in general, the prokaryotic zinc finger domain appears to be an interesting model for studying metal ion interaction with metalloproteins. Here, we explore the binding of Ni(II), Hg(II), and Pb(II) to Ros87, the DNA binding domain of the prokaryotic zinc finger protein Ros. We measured Ros87-metal ion dissociation constants and monitored the effects on the structure and function of the domain. Interestingly, we found that the protein folds in the presence of Ni(II) with important structural perturbations, while in the presence of Pb(II) and Hg(II) it does not appear to be significantly folded. Accordingly, an overall strong reduction in the DNA binding capability is observed for all of the examined proteins. Our data integrate and complement the information collected in the past few years concerning the functional and structural effects of metal ion substitution in classical zinc fingers in order to contribute to a better comprehension of the toxicity of these metals in biological systems.

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the -30 amino acid eukaryotic zinc finger motif in which a beta-sheet and an or-helix are clustered around a zinc ion by various combinations of four ligands. The prokaryotic zinc finger domain (found in the Ros protein from Agrobacterium tumefaciens) is different from the eukaryotic counterpart as it consists of 58 amino acids arranged in a beta beta beta alpha alpha topology stabilized by a 15-residue hydrophobic core. Also, this domain tetrahedrally coordinates zinc and unfolds in the absence of the metal ion.The characterization of proteins belonging to the Ros homologs family has however shown that the prokaryotic zinc finger domain can overcome the metal requirement to achieve the same fold and DNA-binding activity. In the present work, two zinc-lacking Ros homologs (MI4 and MI5 proteins) have been thoroughly characterized using bioinformatics, biochemical and NMR techniques.We show how in these proteins a network of hydrogen bonds and hydrophobic interactions surrogate the zinc coordination role in the achievement of the same functional fold.(c) 2013 Published by Elsevier B.V.

Cyclodextrins are natural macrocyclic oligosaccharides able to form inclusion complexes with a wide variety of guests, affecting their physicochemical and pharmaceutical properties. In order to obtain an improvement of the bioavailability and solubility of 5-fluorouracil, a pyrimidine analogue used as chemotherapeutic agent in the treatment of the colon, liver, and stomac cancers, the drug was complexed with alpha- and beta-cyclodextrin. The inclusion complexes were prepared in the solid state by kneading method and characterized by Fourier transform-infrared (FT-IR) spectroscopy and X-ray powder diffractometry. In solution, the 1:1 stoichiometry for all the inclusion complexes was established by the Job plot method and the binding constants were determined at different pHs by UV-VIS titration. Furthermore, the cytotoxic activity of 5-fluorouracil and its complexation products were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on MCF-7 (breast cancer cell line), Hep G2 (hepatocyte carcinoma cell line), Caco-2 (colon adenocarcinoma cell line), and A-549 (alveolar basal epithelial carcinoma cell line). The results showed that both inclusion complexes increased the 5-fluorouracil capability of inhibiting cell growth. In particular, 5-fluorouracil complexed with beta-cyclodextrin had the highest cytotoxic activity on MCF-7; with alpha-cyclodextrin the highest cytotoxic activity was observed on A-549. The IC50 values were equal to 31 and 73 µM at 72 h, respectively. Our results underline the possibility of using these inclusion complexes in pharmaceutical formulations for improving 5-fluorouracil therapeutic efficacy.

An inclusion complex of hydroxymethylferrocene (FeMeOH) with β-cyclodextrin (β-CD) was prepared in the solid state by different techniques such as physical mixture, coprecipitation, kneading and freeze-drying. The formation of the inclusion complex was confirmed by X-ray Powder Diffractometry and Fourier Transform-Infrared spectroscopy. In aqueous solution, the 1:1 stoichiometry was established by a Job plot. The inclusion complex formation was also investigated by NMR and the stability constant (Kb) of the complex was determined to be 478 M−1, which is in agreement with that obtained with UV-Vis tritation (Kb = 541.3 M−1). The phase solubility study showed a diagram classified as BS type and that the solubility of FeMeOH was slightly increased in the presence of β-CD. Furthermore, utilizing phase solubility diagram data, the Kb was estimated to be equal to 528.0 M−1. The cytotoxic activity of FeMeOH and its complexation product with β-CD was determined using the MTT-assay on MDA-MB-231 cell line, showing that the inclusion complex has a higher capability of inhibiting cell growth compared to that of pure FeMeOH.

Pipemidic acid (HPPA) is a quinolone antibacterial agent used mostly to treat gram-negative infections of the urinary tract, but its therapeutic use is limited because of its low solubility. Thus, to improve drug solubility, natural cyclodextrins (CDs) are used for their ability of including guest molecules within their cavities. The aim of this work was to evaluate the antibacterial activity and the preliminary anticancer activity of HPPA included into Heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB) as a possible approach for a new innovative formulation. The inclusion complex of HPPA with TRIMEB was prepared in solid state by the kneading method and confirmed by FT-IR and powered X-ray diffraction. The association in aqueous solutions of pipemidic acid with TRIMEB was investigated by UV-Vis spectroscopy. Job’s plots have been drawn by UV-visible spectroscopy to confirm the 1:1 stoichiometry of the host–guest assembly. The antibacterial activity of HPPA, TRIMEB and of their complex was tested on Escherichia coli, Pseudomonas aeruginosa, and Staphilococcus aureus. The complex was able to increase 47.36% of the median antibacterial activity of the free HPPA against E. coli (IC50 = 249 µM vs. 473 µM). Furthermore, these samples were tested on HepG-2 and MCF-7. After 72 h, the median tumoral cytotoxicity exerted by the complex was increased by 78.08% and 94.27% for HepG-2 and MCF-7 respectively, showing a stronger bioactivity of the complex than the single HAPPA.

The conjugates of β-cyclodextrins with R- or with S-Etodolac were characterized by NMR spectroscopy, and S-Etodolac alone was characterized by X-ray diffraction analysis. In solution, the R-Etodolac conjugate is soluble in water; the other epimer shows a very low solubility. The NMR characterization of the R-Etodolac conjugate in D2O shows that, in aqueous solution, the Edotolac moiety is self-included in the cavity, while the NMR characterization in MeOH of both conjugates underlines that, in this solvent, the Etodolac moiety is not included in the CD cavity. The X-ray structure determination of the S-Etodolac conjugate reveals a “sleeping swan”-like shape, with the covalently bonded Etodolac moiety being folded with the 8-ethyl group inserted inside the hydrophobic cavity of the β-CD ring. The terminal methyl group of the 8-ethyl group enters the centre of cavity from the side of the primary hydroxyl groups and is buried inside the β-CD macrocycle. The terminal methyl group is positioned at a distance of 1.06 A from the O(4) plane in the side of the primary hydroxyl groups. In addition to van der Waals interactions between the hydrophobic ethyl group and the β-CD cavity, the folded conformation is further stabilized by one intramolecular H-bond involving the indole N–H group and the primary hydroxyl group of the glucose unit 7. Along the b axis, the β-CD molecules are arranged in columns; the macrocycles form a herring bone pattern, so that the cavity of each β-CD molecule is closed at each end by neighboring molecules. Within the layers, the β-CD macrocycles are held together by a complicated intermolecular hydrogen bond network, in which numerous water molecules and hydroxyl groups are involved. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1227–1235, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Carexanes are unusual secondary metabolites isolated from the herbaceous plant Carex distachya. Four new carexanes have been isolated and characterized from the methanolic leaf extract of the plant. All of the structures have been elucidated on the basis of spectroscopic data. The stereochemistry of all the carexane metabolites isolated from the plant, as well as their 1H and 13C NMR spectroscopic assignment, has been revised on the basis of X-ray analysis of carexane D, chemical correlations, and NOESY/ROESY experiments. All the data suggested a cis junction between B and C rings. © 2008 Elsevier Ltd. All rights reserved.

Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. β-cyclodextrins (β-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the β-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the β-CD.

Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique β,γ-dichlorinated proline [Pro(Cl 2], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the β,γ-dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear. With the aim at improving our knowledge of the conformational properties influencing the bioactivity in this class of compounds, new astin-related cyclopeptides were synthesized differing from the natural products by the presence of some non-proteinogenic amino acid residues: Aib, Abu, -(S)β3-hPhe and a peptide bond surrogate (-SO2-NH-). The analogues prepared c(-Pro-Thr-Aib-β3-Phe-Abu-), c[Pro-Thr-Aib-(S β3-hPhe-Abu], c[Pro-Abu-Ser-(S β3-hPheΨ(CH2-SO2-NH)-Abu] and c[Pro-Thr-Aib-(S β3-hPheΨ(CH2-SO2-NH)-Abu] were synthesized by classical methods in solution and tested for their antitumour effect. These molecules were studied by crystal-state x-ray diffraction analysis and/or solution NMR and MD techniques. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cyclodecane-1-carboxylic acid (Ac10c), an alpha-amino acid conformationally constrained through a medium-ring Calphai Calphai cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac10c monomer and homo-dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT-IR absorption and 1H NMR techniques. Furthermore, the molecular structures of two derivatives (Z-Ac10c-OH and Fmoc-Ac10c-OH) and two peptides (the dipeptide ester Z-Ac10c-L-Phe-OMe and the tripeptide ester Z-Aib-Ac10c-Aib-OtBu) were determined in the crystal state using X-ray diffraction. The experimental results support the view that beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac10c, the third largest cycloaliphatic C(alpha,alpha)-disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1-amino-cycloalkane-1-carboxylic acid (Ac(n)c, with n = 3-12) series, which represents the prerequisite for our recent proposal of the 'Ac(n)c scan' concept.

The synthesis, solution NMR investigation and solid-state structural characterization of a new difunctionalized β-cyclodextrin (β-CD) are reported. 6I,6II-Dideoxy-6I,6II-bis[2-(2-pyridyl)ethylamino]-β-cyclomaltoheptaose is synthesized for the first time using a regioselective synthetic procedure. On the basis of an aqueous solution NMR investigation, the intramolecular interaction of the two pyridine rings with the upper rim of the cavity is proposed. 6I,6II-Dideoxy-6I,6II-bis[2-(2-pyridyl)ethylamino]-β-cyclomaltoheptaose, C56H86N4O33, crystallizes in the monoclinic P21 space group with cell dimension a = 26.303(5), b = 15.670(5), c = 8.276(2) A and β = 103.60(2)°, and 5.5 molecules of water for each independent β-cyclodextrin molecule. The structure refines to R = 0.103 for 2270 observed reflections [I ≥ 3σ(I)] and represents the first example of a complete structural characterization of a branched difunctionalized β-cyclodextrin. In the solid state, the macrocycle structure maintains an approximate seven-fold symmetry with only small changes occurring in the cyclic carbohydrate conformation where two consecutive primary hydroxy groups are substituted with bulky moieties. The two aminoethylpyridine groups linked to contiguous glucosidic units show different behaviour, with one group extending away from the cavity of the β-CD, the other remaining in the proximity of the ‘mouth’ of the cavity. However, in the crystal the aminoethylpyridine group extending away from the cavity of the β-CD is deeply inserted into the cavity of the adjacent β-CD molecule translated along the c axis, giving rise to long rows of β-CD molecules stabilized by these host–guest interactions. The resulting polymeric arrangement has already been observed in crystal structures of other monosubstituted β-CDs.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.

A solid state analysis of the cyclic octapeptide c(-Pro(1)-Pro-Phe-Phe-Ac(6)c-Ile-D-Ala-Val(8)-) (C8-CLA), containing the Pro-Pro-Phe-Phe sequence, followed by the bulky helicogenic C(alpha,alpha)-dialkylated 1-aminocyclohexane-1-carboxylic acid (Ac(6)c) residue and a D-Ala residue in position 7, has been carried out by x-ray diffraction. The crystals, grown from a DMSO solution, are monoclinic, space group P2(1) with a = 13.458(3) A, b = 19. 404(5) A, c = 21.508(4) A, and beta = 90.83(6) degrees, with two independent cyclic molecules in the asymmetric unit, two DMSO molecules, and three water molecules. The structure has been solved using the half and bake procedure by Sheldrick, and refined to final R1 and wR2 indices of 0.0613 and 0.1534 for 9867 reflections with I > 2sigma(I). This cyclic peptide, a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A [c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), CLA] has been designed to study the influence of the ring size reduction on the conformational behavior of CLA and more in general to obtain structural information on asymmetric cyclic octapeptides. The compound exhibits, in the solid state, a "banana-twisted" conformation with a cis peptide bond located between the two proline residues. Five intramolecular H bonds stabilize the structure: one type VIa beta-turn, two consecutive type III/I beta-turns, one gamma-turn, and one C(16) bend. The structure has also been compared with either the solution structure previously reported by us and obtained by nmr and computational analysis, and with solid state structural data reported in the literature on cyclic octapeptides.

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.

Two complete series of N-protected, monodispersed oligopeptide esters to the pentamer level from 1-aminocyclododecane-1-carboxylic acid (Ac(12)c), an alpha-amino acid conformationally constrained through C(alpha)(i) C(alpha)(i) cyclization, and either L-Ala or Aib residues, along with the N-protected Ac(12)c homopeptide alkylamide series from monomer to trimer, have been synthesized by solution methods and fully characterized. The solution-preferred conformations of these peptides have been assessed by Fourier transform ir absorption and (1)H-nmr techniques. Moreover, the molecular structures of one derivative (Z-Ac(12)c-OH) and three peptides [the tripeptide ester Z-L-Ala-Ac(12)c-L-Ala-OMe, the tripeptide alkylamide Z-(Ac(12)c)(3)-NHiPr, and the tetrapeptide ester Z-(Aib)(2)-Ac(12)c-Aib-OtBu (Aib, alpha-aminoisobutyric acid)] have been determined in the crystal state by x-ray diffraction. The results obtained point to the conclusion that beta-bends and 3(10)-helices are preferentially adopted by peptides based on Ac(12)c, the largest cycloaliphatic C-disubstituted glycine known. A comparison with the structural tendencies extracted from published works on peptides from Aib, the prototype of C-disubstituted glycines, and the other extensively studied members of the class of 1-aminocycloalkane-1-carboxylic acids (Ac(n) c, with n = 3-9), is made and the implications for the use of the Ac(12)c residue in the Ac(n) c scan approach of conformationally restricted analogues of bioactive peptides are briefly discussed.

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cycloundecane-1-carboxylic acid (Ac11c), an α-amino acid conformationally constrained through a medium-ring Cαi↔Cαi cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac11c monomer and homo-dimer alkylamides, have been synthesized by solution methods and fully characterized. The preferred conformation of these model peptides has been assessed in deuterochloroform solution by FT-IR absorption and 1H-NMR techniques. Furthermore, the molecular structures of one derivative (Z-Ac11c-OH) and two peptides (the tripeptide ester Z-Aib-Ac11c-Aib-OtBu and the pentapeptide ester Z-Ac11c-(Aib)2-Ac11c-Aib-OtBu) have been determined in the crystal state by X-ray diffraction. The experimental results support the view that β-bends and 310-helices are preferentially adopted by peptides rich in Ac11c, the second largest cycloaliphatic Cα,α-disubstituted glycine known. This investigation has allowed the authors to approach the completion of a detailed conformational analysis of the whole 1-amino-cycloalkane-1-carboxylic acid (Acnc, with n=3–12) series, which represents the prerequisite for their recent proposal of the ‘Acnc scan’ concept. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

The synthesis and conformational properties of a series of dipeptide taste ligands, differing from the commercial sweetener aspartame by the presence of a methylene group between the Cα and the C′ carbon atoms (as in homo-β-residues) in either the L-Asp or the L-Phe residues, are described. Homo-β-residues such as homo-β-aspartic acid, homo-β-phenylglycine and homo-β-phenylalanine, obtained by homologation of the corresponding proteinogenic α-amino acids, have been used in the solution peptide synthesis of the following aspartame analogues in protected and unprotected forms: NH2-homo-β-(L or D)-Asp-L-Phe-OMe, NH2-L-Asp-homo-β-L-Phg-OMe and NH2-L-Asp-homo-β-L-Phe-OMe. Lengthening of the peptide skeleton at the L-Asp site results in a drastic loss of sweetness with the production of tasteless compounds; on the other hand, lengthening of the skeleton at the C-terminal L-Phe site partially mantains the sweet taste in both NH2-L-Asp-homo-β-L-Phe-OMe and NH2-L-Asp-homo-β-L-Phg-OMe.The solution conformation of the synthesized dipeptide taste ligands was investigated by NMR and circular dichroism techniques. The analysis of NMR data combined with restrained molecular dynamics calculations shows that all peptides are fairly flexible and they do not assume a preferred conformation in DMSO and methanol. The peptides containing homo-β-L-Phe and homo-β-L-Phg do adopt a discrete number of conformations among which mainly extended and ‘L-shaped’ conformation are represented. The circular dichroism spectra are consistent with the NMR results, indicating a significant flexibility for these compounds. Copyright © 1999 John Wiley & Sons, Ltd.

The molecular and crystal structures of three terminally blocked (Aib) n /(L-Xxx) m (n = 3, 4; m = 1,2; n + m = 5) pentapeptides [pBrBz-L-Ala-(Aib)4-OtBu (1), pBrBz-L-Val-(Aib)4-OtBu (2), pBrBz-L-Ala-(Aib)3-L-Ala-OMe (3)] were determined by X-ray diffraction. The three compounds are characterized by the following parameters: (1) orthorhombic, P212121, a = 33.833(3) Å, b = 11.776(2) Å, c = 9.317(1) Å, and Z = 4; (2) triclinic, P1, a = 10.316(6) Å, b = 10.580(5) Å, c = 18.735(6) Å, α = 95.46(4)°, β = 90.08(6)°, γ = 111.92(4)°, and Z = 2; (3) monoclinic, P21, a = 9.002(1) Å, b = 19.242(3) Å, c = 9.668(1) Å, β = 113.69(1)°, and Z = 2. The structures were solved by direct methods. The least-squares refinements led to R values of 0.042, 0.060, and 0.046 for (1), (2), and (3), respectively. The three pentapeptides are folded in a 310-helical structure. However, the structure of (1) is left-handed helical from residue 1, whereas that of (3) begins with a non-helical type-II β-bend that is followed by a left-handed helix. The two independent molecules, A and B, in the asymmetric unit of (2) are diastereomeric, the former being right-handed helical from residue 1, while the latter is left-handed.

The reaction of 3′,5′-di-O-acetylthymidine with [Pt(PPh3)4] and KCl yielded a platinum(II) complex where the platinum is co-ordinated to the nucleobase through the N3 atom. In a similar reaction 1-methylthymine (1-MeThy) furnished the complex cis-[PtCl(1-MeThy)(PPh3)2], whose structure was determined by spectroscopic data and single crystal X-ray diffraction. When 1-MeThy was treated with [Pt(PPh3)4] in the absence of chloride ions the complex trans-[Pt(OH)(1-MeThy)(PPh3)2] was obtained.

A variety of N α-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid residues and one chiral L-valine or C α-methyl-L-valine residue in the N-terminal, internal or C-terminal position of the sequence, have been synthesized by solution methods and fully characterized. The results of a solution conformational analysis, performed by using FTIR absorption and 1H NMR techniques, favour the conclusion that all of the pentapeptides examined fold into a 310-helical structure. In addition, a CD study of the N α-para-bromobenzoylated peptides strongly supports the view that the prevailing screw sense of the 310-helical structure that is formed is strongly dependent upon the position in the sequence of the single chiral C α-tri- or C α-tetrasubstituted α-amino acid.

A dipeptide taste ligand L-aspartyl-D-2-aminobutyric acid-(S)-α-ethylbenzylamide was found to be about 2000 times more potent than sucrose. To investigate the molecular basis of its potent sweet taste, we carried out conformational analysis of this molecule and several related analogues by NMR spectroscopy, computer simulations and X-ray crystallographic studies. The results of the studies support our earlier model that an ‘L’-shape molecular array is essential for eliciting sweet taste. In addition, we have identified an aromatic group located between the stem and the base of the ‘L-shape’, which is responsible for enhancement of sweetness potency. In this study, we also assessed the optimal size of the essential hydrophobic group (X) and the effects of the chirality of the second residue toward taste. ©1997 European Peptide Society and John Wiley & Sons, Ltd.

A complete series of N- and C-blocked, monodispersed homo-oligopeptides to the pentamer level from 1-aminocycloheptane-1-carboxylic acid (Ac7c), an α-amino acid conformationally restricted through Ciα ←→ Ciα cyclization, and three tripeptides with Ac7c combined with Ala, Leu, and Val residues have been synthesized by solution methods and fully characterized. The solution conformational preferences have been determined by IR absorption and 1H NMR spectroscopy. In addition, the molecular structures of three derivatives (Ac7c hydantoin, ClCH2CO-Ac7c-OH, and Z-Ac7c-OH; Z = benzyloxycarbonyl) and four peptides [the dipeptide Z-Ac7c-L-Ala-OMe, the tripeptides Z-Ac7c-(L-Ala)2-OMe and Z-(Ac7c)3-OBut, the tetrapeptide Z-(Ac7c)4-OBut, and the pentapeptide Z-(Ac7c)5-OBut] have been assessed in the crystal state by X-ray diffraction. The results obtained confirm the tentative conclusions put forward on the basis of our previous preliminary study, namely that β-bends and 310-helices are preferentially adopted by Ac7c-based peptides. A comparison with the structural tendencies extracted from published work on peptides from α-aminoisobutyric acid, the prototype of Cα,α-dialkylated glycines, and the other extensively investigated members of the class of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3–6, 8, 9) is made and the implications for the use of the Ac7c residue in conformationally constrained analogues of bioactive peptides are briefly discussed.

The conformational preferences of the alicyclic Cα,α-disubstituted glycines Acnc (1-amino-1-cycloalkane-carboxylic acid; n = 4, 7, 9, 12) were assessed in selected model compounds, including homopeptides and Ala (or Aib, α-aminoisobutyric acid)/Acnc peptides containing a small total number of residues, by Fourier transform ir absorption, 1H-nmr, and x-ray diffraction analyses. The results obtained indicate that β-turn and 310-helical structures are preferentially adopted by short peptides rich in these cycloaliphatic α-amino acids. © 1997 John Wiley & Sons, Inc. Biopoly 40: 519–522, 1996

The conjugates of beta-cyclodextrins with R- or with S-Etodolac were characterized by NMR spectroscopy, and S-Etodolac alone was characterized by X-ray diffraction analysis. In solution, the R-Etodolac conjugate is soluble in water; the other epimer shows a very low solubility. The NMR characterization of the R-Etodolac conjugate in D(2)O shows that, in aqueous solution, the Edotolac moiety is self-included in the cavity, while the NMR characterization in MeOH of both conjugates underlines that, in this solvent, the Etodolac moiety is not included in the CD cavity. The X-ray structure determination of the S-Etodolac conjugate reveals a "sleeping swan"-like shape, with the covalently bonded Etodolac moiety being folded with the 8-ethyl group inserted inside the hydrophobic cavity of the beta-CD ring. The terminal methyl group of the 8-ethyl group enters the centre of cavity from the side of the primary hydroxyl groups and is buried inside the beta-CD macrocycle. The terminal methyl group is positioned at a distance of 1.06 A from the O(4) plane in the side of the primary hydroxyl groups. In addition to van der Waals interactions between the hydrophobic ethyl group and the beta-CD cavity, the folded conformation is further stabilized by one intramolecular H-bond involving the indole N-H group and the primary hydroxyl group of the glucose unit 7. Along the b axis, the beta-CD molecules are arranged in columns; the macrocycles form a herring bone pattern, so that the cavity of each beta-CD molecule is closed at each end by neighboring molecules. Within the layers, the beta-CD macrocycles are held together by a complicated intermolecular hydrogen bond network, in which numerous water molecules and hydroxyl groups are involved.

Carexanes are unusual secondary metabolites isolated from the herbaceous plant Carex distachya . Four new carexanes have been isolated and characterized from the methanolic leaf extract of the plant. All of the structures have been elucidated on the basis of spectroscopic data. The stereochemistry of all the carexane metabolites isolated from the plant, as well as their 1 H and 13 C NMR spectroscopic assignment, has been revised on the basis of X-ray analysis of carexane D, chemical correlations, and NOESY/ROESY experiments. All the data suggested a cis junction between B and C rings.

Acid-catalysed condensation of tryptophan with different α-amino aldehyde derivatives has been explored as a useful route to the synthesis of novel amino acid derived heterocycles and peptidomimetic scaffolds. By this approach, compounds containing a tetrahydro-β-carboline and a novel octahydropyrrolo[3′,2′:3,4]pyrrolo[2,3-b]indole system have been efficiently synthesized. Here we report the characterization of these new compounds and preliminary studies of the reactivity of the tetrahydro-β-carboline system.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

A novel antioxidant prenylated stilbenoid, distachyasin, has been isolated from the leaves of Carex distachya. Its structure has been elucidated on the basis of the spectroscopic characteristics. Bidimensional NMR, and crystallographic data and computational calculations have furnished important data useful for the characterization and the stereochemistry of the molecule. The compound has a tetracyclic skeleton derived from carexane. The compound has been assayed, for the antioxidant activity, by measuring its capacity to scavenge the H2O2, nitric oxide, superoxide radical and to inhibit formation of TBARS (thiobarbituric acid reactive species). © 2006 Elsevier Ltd. All rights reserved.

b-Cyclodextrins (b-CD) have received considerable attention for their suitability to serve as relatively low molecular weight models for drug delivery and for artificial enzymes. In fact, b -CD shows remarkable ability to form inclusion complexes with various natural and synthetic molecules that fit inside the b -CD cavity. The inclusion process is influenced by the interaction between the guest molecules and the cavity, and also by the shape and size of the guest. This process can change the chemical and physical properties of the guest. In particular, pharmacological properties, such as stability, solubility, and toxicity, can be improved. Then, the rational design of functionalized b -CDs with bioactive moieties can represent an important step for the development of new drugs. In this work, we report a detailed conformational analysis at atomic resolution by x-ray diffraction data and computational techniques on several functionalized b-CDs to understand the structural requirement to modulate the binding properties and basic phenomena governing the inclusion process for b-CDs.

Oxidative addition reactions of Pd(PPh3)4 on the pyrimidine nucleosides 3′,5′-di-O-acetylthymidine and 3′,5′-di-O-acetyl-4-chlorothymidine and on the nucleobase 1-methylthymine have been investigated. N3 and C4 metal coordinated complexes 3 and 7 were isolated and characterized by spectroscopic techniques. Moreover, the crystal structur of the trans-[PdCl(1-methyl thymine)(PPh3) 2]·H2O (4) is reported. © 2005 American Chemical Society.

Three new resorcin[4]arene-capped porphyrins (3, 5 and 7) different in the porphyrin skeleton, in the linking arms and in the cavity dimensions, have been synthesised. Molecular modelling calculations explored the conformations and the cavity size of the three compounds and showed that their hydrophobic pockets can accommodate one molecule of water or methane (3 and 5), or benzene (7) without any distortion. Notably, the capped porphyrin 5 was able to inhibit the oxidation of Co(II) to Co(III), whereas compound 7 did it only partially.

The cyclopentapeptides of the astin family contain several uncoded amino acids, namely (-)-(3S,4R)-dichloroproline, β3-homo-phenylalanine, α-aminobutyric acid (Abu), and allo-threonine. The astins, isolated from the plant roots Aster tataricus, exhibit antitumor activity (Sarcoma 180 A), but the potential applications of this biological behaviour remain to be further investigated. Conformational studies of astin A, B and C were carried out by a combination of X-ray, NMR and computational techniques [1]. The results indicate that the backbone conformation of astin A and C., with lower activity, is different from that of astin B. Therefore, both the backbone conformation and a cis linkage shown by the 3,4-dichlorinated proline residue, were considered to play an important role in the antitumor activity of these molecules [2]. The development of efficient synthetic procedures for preparation of β3-H residues and of cis-3,4-dihydroxyproline, as well as (-)-(3S,4R)-dichloroproline [3], has brought new interest in the structure-activity relationships of astin analogs. With the aim to further understand the structural and conformational requirements for activity and possibly the mechanism of action of these molecules, we have focused our attention on several cyclic pentapeptide related to astin G [4], which presents in its 16-membered ring the sequence Pro-allo-Thr-Ser-β3-HPhe-Abu. The structural results of two synthetic astin G related cyclopentapeptides c-(Pro-Thr-Aib-β3-HPhe-Abu) (I) and c-(Pro-Thr-Aib-β3-HPhg-Abu) (II) are reported.

A complete series of Nα-protected, monodispersed homooligopeptide esters to the pentamer level from L-Cα-methyl,Cα-allylglycine (L-Mag) have been synthesized step-by-step in solution and fully characterized. The solution preferred conformation of these homooligomers has been assessed by FT-IR absorption and 1H NMR techniques. Moreover, the molecular structures of the homedimer and trimer have been determined in the crystal state by X-ray diffraction, and the conformational energy map of the homotrimer has been computed. The results obtained point to the conclusion that right-handed, single, or multiple β-bends are preferentially adopted by the conformationally restricted L-Mag homooligomers. In particular, 310-helices are formed by the longest homooligomer (pleionomer). The implications for the use of the Mag residue in designing conformationally constrained peptide substrates for reactions involving the side-chain C=C functionality are briefly discussed.

The crystal-state preferred conformations of six Nα-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid (Aib) residues followed by one chiral L-valine (L-Val) or Cα-methyl-L-valine [(αMe)Val] residue at the C-terminus, have been assessed by x-ray diffraction analysis. In all of the compounds the - (Aib)4 - sequence is folded in a regular 310-helical conformation. In the four pentapeptides characterized by the L-(αMe)Val residue two conformationally distinct molecules occur in the asymmetric unit. Conversely, only one molecule is observed in the asymmetric unit of two pentapeptides with the C-terminal L-Val residue. In the L-Val based peptides the helical screw sense of the - (Aib)4 - sequence is right-handed, whereas in the L - (αMe)Val - analogues both right- and left-handed helical screw senses concomitantly occur in the two crystallo graphic ally independent molecules. © 1998 John Wiley & Sons, Inc. Biopoly 46: 433-443, 1998.

A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined.

Abstract: An X-ray diffraction analysis of the C-terrninal, fully block...d pentapeptide segment of the antibiotic efrapeptin C Z-L-Pip-Aib-Gly-L-Leu-Aib-NHMe monohydrate showed that its secondary structure is characterized by three non-helical -bend conformations located at the -L-Pip-Aib-, -Aib-Gly-, and -L-Leu-Aib- sequences, respectively .