Rosa Iacovino

Preferred conformation of peptides rich in alicyclic Cα,α-disubstituted glycines

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.

Conformational Characterization of the 1-Aminocyclobutane-1-carboxylic Acid Residue in Model Peptides

A series of N‐ and C‐protected, monodispersed homo‐oligopeptides (to the dodecamer level) from the small‐ring alicyclic Cα,α‐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 τ(N–Cα–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 β‐turn and helix former. A comparison with the structural propensities of α‐aminoisobutyric acid, the prototype of Cα,α‐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.

Helical screw sense of peptide molecules: The pentapeptide system (Aib)4/L-Val[L-(αMe)Val] in the crystal state

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 crystallographically independent molecules.

Conformational restriction through C?i ? C?i cyclization: Ac12c, the largest cycloaliphatic C?,?- disubstituted glycine known

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.

Solid state structural analysis of the cyclooctapeptide cyclo- (Pro1-Pro-Phe-Phe-Ac6c-Ile-D-Ala-Val8).

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.

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

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 β-sheet and an α-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 βββαα 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 (Ml4 and Ml5 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.

X-ray structures of new dipeptide taste ligands.

The molecular basis of sweet taste was investigated by carrying out the crystal state conformational analysis by X‐ray diffraction of the following dipeptide taste igands:N‐3,3‐dimethylbutyl‐aspartyl‐phenylalanine methyl ester,I(N‐DMB‐Asp‐Phe‐OMe), its sodium salt (N‐DMB‐Asp‐Phe‐ONa),II, aspartyl‐D‐2‐aminobutyric acid‐(S)‐α‐ethylbenzylamide,III(Asp‐D‐Abu‐(S)‐α‐ethylbenzylamide), aspartyl‐N′‐((2,2,5,5‐tetramethylcyclopentanyl)‐carbonyl)‐(R)‐1,1‐diamino‐ethane,IV(Asp‐(R)‐gAla‐TMCP), and aspartyl‐D‐valine‐(R)‐α‐methoxymethylbenzyl amide,V(Asp‐D‐Val‐(R)‐α‐methoxymethylbenzylamide). With the exception of the sodium saltII, all compounds are sweet‐tasting, showing in some cases considerable potency enhancement with respect to sucrose. The results of this study confirm the earlier model that an ‘L‐shape’ molecular array is essential for eliciting sweet taste for dipeptide‐like ligands. In addition, it was established that (i) substitution of the N‐terminal group does not inhibit sweet taste, if its zwitterionic character is maintained; (ii) a hydrophobic group located between the stem and the base of the L‐shape could be responsible for sweetness potency enhancement, as found inI, IIIandIV; in fact, the extraordinary potency of the N‐alkylated analogueIwould support a model with an additional hydrophobic binding domain above the base of the ‘L’; (iii) removal of the methyl ester at the C‐terminus of compoundIwith the salt formation gives rise to the tasteless compoundII; (iv) for the first time all possible side‐chain conformers (g−,g+andt) for the N‐substituted aspartyl residue were observed; and (v) a retro‐inverso modification, incorporated at position 2 of the dipeptide chain, confers greater flexibility to the molecule, as demonstrated by the contemporary presence of six conformationally distinct independent molecules in the unit cell and yet sweet taste properties are maintained, as found inIV.

Conformational analysis of the dipeptide taste ligandL-aspartyl-D-2-aminobutyric acid-(S)-α-ethylbenzylamide and its analogues by NMR spectroscopy, computer simulations and X-ray diffraction studies

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.

Alpha- and Beta-Cyclodextrin Inclusion Complexes with 5-Fluorouracil: Characterization and Cytotoxic Activity Evaluation

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.

Linear oligopeptides. Part 406.1 Helical screw sense of peptide molecules: the pentapeptide system (Aib)4/L-Val[L-(αMe)Val] in solution

A variety of N n α-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid residues and one chiral L-valine or C n 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 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 n α-tri- or C n α-tetrasubstituted α-amino acid.