Akiko Asano

The structure of an endomorphin analogue incorporating 1-aminocyclohexane-1-carboxlylic acid for proline is similar to the β-turn of Leu-enkephalin

Endomorphin (EM2, Tyr-Pro-Phe-Phe-NH(2)) can assume various conformations related to cis/trans-rotamers of the amide linkage of Tyr-Pro. To control isomerization, restricted or flexible components have been introduced at the Pro position. We focused on [Chx(2)]EM2, an EM2 analogue substituting 1-aminocyclohexane-1-carboxlylic acid (Chx) for Pro. X-ray diffraction analysis revealed that [Chx(2)]EM2 is folded into the trans-form of Tyr-Chx. The manner of folding resembled that seen in D-TIPP, an EM analogue incorporating tetrahydroisoquinoline carboxylic acid, as well as the beta-turn of Leu-enkephalin. Selectivity for the opioid mu-receptor was fairly well conserved by [Chx(2)]EM, suggesting that the folded form is important for mu-selectivity.

Effects of amino acids and chirality for molecular folding of desoxazoline‐ascidiacyclamide derivatives: X‐ray crystal structures of four cyclic octapeptides including unusual amino acids, cyclo(–Ile–aThr–D‐Val–Thz–)2, cyclo(–Ala–aThr–D‐Val–Thz–Ile–aThr–D‐Val–Thz–), cyclo(–Val–aThr–D‐Val–Thz–Ile–aThr–D‐Val–Thz–), and cyclo(–Ile–aThr–Val–Thz–Ile–aThr–D‐Val–Thz–)

Desoxazoline derivative of ascidiacyclamide (1), cyclo(-L-Ile-L-allo-threonine-D-Val-thiazole-)(2), was modified to disturb the C(2)-symmetry. An Ile(1) residue of 1 was replaced for Ala (2) or Val (3), and the D-Val(3) residue was replaced for Val (4). The crystal structures of 1-4 were analyzed by x-ray diffraction methods. The molecules of all compounds were folded and this type of structure was not observed in x-ray structures of ascidiacyclamide derivatives so far except for patellamide D. The folding patterns of 1-4 were similar to each other and resembled that of patellamide. The asymmetric modifications at position 1 caused the conformational changes at local area, and these were related with the peptide-peptide and peptide-solvent interactions. Despite the diverse backbone conformation by the epimeric modification at position 3, the entire molecule of 4 was folded. These results mean that (1) the desoxazoline-ascidiacyclamides favored the folded structures and (2) the modifications of the side chain size at position 1 and the chirality at position 3 brought the local conformational changes to derivatives, suggesting that (3) the lack of the oxazoline block leads to conformational flexibility of 1-4, which accepts the conformational change with no drastic change on the entire structure.

A flat squared conformation of an ascidiacyclamide derivative caused by chiral modification of an oxazoline residue

We designed a deoxazoline-ascidiacyclamide (dASC), cyclo(-L-Ile-L-allo-Thr-D-Val-thiazole-)(2), diastereomer having 10S, 11R, 37R, and 38S configurations ([SR,RS]dASC) and a corresponding product having 10S, 11S, 37R, and 38R configurations ([SS,RR]ASC) with the aim of understanding better the relationship between conformational behaviour and chirality. X-ray diffraction analysis revealed that [SR,RS]dASC is folded in a manner similar to other dASC analogues. By contrast, [SS,RR]ASC is a novel, flat conformer that is larger than the major square and folded ASC conformers and contains a cavity created by the flat peptide ring. In addition, [SS,RR]ASC retains approximately 60% of the cytotoxicity of the parent molecule.

The square conformation of phenylglycine-incorporated ascidiacyclamide is stabilized by CH/π interactions between amino acid side chains.

We designed a phenylglycine (Phg)-incorporated ascidiacyclamide (ASC) analogue, cyclo(-Phg-oxazoline-d-Val-thiazole-Ile-oxazoline-d-Val-thiazole- ([Phg]ASC), with the aim of stabilizing the square conformation of ASC through interactions between amino acid side chains. X-ray diffraction analysis showed that [Phg]ASC has a square structure, similar to ASC, in which the sec-butyl group of Ile and the benzene ring of Phg are in close proximity. Consistent with that finding, ¹H NMR experiments revealed significant high-field shifts in the sec-butyl group of Ile, which suggests a potential for CH/π interactions between the sec-butyl group of Ile and the benzene ring of Phg. The CD spectra of [Phg]ASC were less affected by TFE titration or increasing temperature than those of ASC. In addition, [Phg]ASC showed approximately three times greater toxicity toward HL-60 cells than ASC. Thus the potently cytotoxic conformation of [Phg]ASC may be stabilized by CH/π interactions between the side chains of the Ile and Phg residues.

Cyclo(-Cha-Oxz-D-Val-Thz-Ile-Oxz-D-Val-Thz-) N,N-dimethylacetamide dihydrate: a square form of cyclohexylalanine-incorporated ascidiacyclamide having the strongest cytotoxicity.

The title compound, 1-cyclohexylmethyl-1-de(1-methylpropyl)ascidiacyclamide N,N-dimethylacetamide dihydrate, C(39)H(56)N(8)O(6)S(2) x C(4)H(9)NO x 2H(2)O, a cyclohexylalanine-incorporated ascidiacyclamide analogue ([Cha]ASC), shows a square form similar to natural ASC. On the other hand, CD (circular dichroism) spectra showed [Cha]ASC to have a folded structure in solution, making it the second known analogue to show a discrepancy between its crystal and solution structures. Moreover, the cytotoxicity of [Cha]ASC (ED(50) = 5.6 micro g ml(-1)) was approximately two times stronger than that of natural ASC or a related phenylalanine-incorporated analogue, viz. cyclo(-Phe-Oxz-D-Val-Thz-Ile-Oxz-D-Val-Thz-) ([Phe]ASC), and was confirmed to be associated with the square form. However, [Phe]ASC was previously shown to be folded in the crystal structure, which suggests that the difference between the aromatic and aliphatic rings affects the molecular folding of the ASC molecule.

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1-naphthylalanine or 2-naphthylalanine

We designed five ascidiacyclamide analogues [cyclo(‐Xxx1‐oxazoline2‐d‐Val3‐thiazole4‐l‐Ile5‐oxazoline6‐d‐Val7‐thiazole8‐)] incorporating l‐1‐naphthylalanine (l‐1Nal), l‐2‐naphthylalanine (l‐2Nal), d‐phenylalanine (d‐Phe), d‐1‐naphthylalanine (d‐1Nal) or d‐2‐naphthylalanine (d‐2Nal) into the Xxx1 position of the peptide. The conformations of these analogues were then examined using 1H NMR, CD spectroscopy, and X‐ray diffraction. These analyses suggested that d‐enantiomer‐incorporated ASCs [(d‐Phe), (d‐1Nal), and (d‐2Nal)ASC] transformed from the folded to the open structure in solution more easily than l‐enantiomer‐incorporated ASCs [(l‐Phe), (l‐1Nal), and (l‐2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1‐naphthyl isomer induced a more stable open structure than the 2‐naphthyl isomer. In particular, [d‐1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL‐60 cells. Copyright

Modulating the structure of phenylalanine-incorporated ascidiacyclamide through fluorination

We designed four fluorinated Phe‐incorporated ascidiacyclamide ([Phe]ASC) analogs, (cyclo(−Xxx1‐oxazoline2‐d‐Val3‐thiazole4‐Ile5‐oxazoline6‐d‐Val7‐thiazole8‐)), [(4‐F)Phe]ASC (Xxx1: 4‐fluorophenylalanine), [(3,5‐F2)Phe]ASC (Xxx1: 3,5‐difluorophenylalanine), [(3,4,5‐F3)Phe]ASC (Xxx1: 3,4,5‐trifluorophenylalanine) and [(F5)Phe]ASC (Xxx1: pentafluorophenylalanine), to modulate the π‐electron density of the aromatic ring of the Phe residue. X‐ray diffraction analysis, 1H NMR and CD spectra all suggested that the interactions between the benzene ring of the Xxx1 residue and the alkyl groups of oxazoline2 contribute to the stability of the folded structure of these analogs. Substituting fluorines for the hydrogens progressively weakened those interactions through reducing the π‐electron density, thereby mediating transformation from the folded to square structure. As a result, [(F5)Phe]ASC preferred the square form more than the other analogs did. Also contributing to the preference for the square form may be the hindrance of the rotation around the Cα–Cβ bond by the two ortho‐fluoro substituents of [(F5)Phe]ASC. These findings demonstrate that the structure of ASC can be modulated by using fluorine as an electron‐withdrawing group. Copyright

Caged and clustered structures of endothelin inhibitor BQ123, cyclo(-d-Trp-d-Asp−-Pro-d-Val-Leu-)\cdotNa+, forming five and six coordination bonds between sodium ions and peptides

BQ123 is a cyclic pentapeptide and a potent endothelin-1 inhibitor. The crystal structure of the BQ123 sodium salt was determined as the first example of an endothelin inhibitor. Four independent molecules and many solvent molecules were found in the asymmetric unit; the total weight was about 3000 Da. The precise structure including the solvent molecules was determined using high-resolution data collected on a synchrotron source. Sodium ions formed unique structures with five and six coordination bonds and their forms were distinguished into three classes. An ion was sandwiched by two BQ123 molecules. This peptide-sodium (2:1) complex showed a cage-like structure and octahedral coordination was observed. Sodium ions also formed a cluster composed of hydrated water molecules and peptides. Two sodium ions were contained in this cluster, making five coordination bonds. Despite having the same coordination numbers, these ions were distinguishable by differences in the polyhedra. One was trigonal bipyramidal (having six planes) and the other was square pyramidal (having five planes). Both shapes were very similar to each other, although the synchrotron data clearly revealed slight geometrical differences.

A dimer model of human calcitonin13-32 forms an α-helical structure and robustly aggregates in 50% aqueous 2,2,2-trifluoroethanol solution

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13‐32 aggregated to a greater degree than native hCT under aqueous 2,2,2‐trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine‐T binding assays and atomic force microscopy suggest that the α‐helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β‐sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α‐helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright

Boc–Pro–Hyp–Gly–OBzl and Boc–Ala–Hyp–Gly–OBzl, two repeating triplets found in collagen

The protected tripeptides benzyl N-{2-[N-(tert-butoxycarbonyl)prolyl]-4-hydroxyprolyl}glycinate or Boc-Pro-Hyp-Gly-OBzl, C24H33N3O7, and benzyl N-{2-[N-(tert-butoxycarbonyl)alanyl]-4-hydroxyprolyl}glycinate or Boc-Ala-Hyp-Gly-OBzl, C22H31N3O7, are the minimum repeating triplets found in collagen. Within the crystal structure of each are two independent peptide molecules with similar structures. The peptides are arranged antiparallel to one another and interact through hydrogen bonds involving the main chains and the 4-hydroxyprolyl groups. The structures exhibit characteristics of a triple helix, but the peptides tend to assume a sheet-like structure.