Synthesis and circular dichroism characterization of L-azetidine-2-carboxylic acid cyclic peptides

Several cyclic hemopeptides containing L -azetidine-2-carboxylix acid (Aze)—an imino acid homologous with proline but containing one less methylene group in its cyclic side chain—have been prepared. The peptides reported include (Aze)₂, cyclo(Aze)₃, and cyclo(Aze)₆. The synthesis and spectral characterization of these cyclic peptides are described, and the results discussed in terms of the rigidity and steric constraints attributable to Aze-containing peptides. CD spectra of these materials in several solvents are reported and compared with those of proline analogs; the similarity between the CD spectra of cyclo(Aze)₃ and cyclo (Pro)_3, is noted.     

Incorporation of L-azetidine-2-carboxylic acid into hemoglobin in rabbit reticulocytes

L-Azetidine-2-car?ylic acid (AZA), the lower homologue of proline, was incorporated into hemoglobin in rabbit reticulocytes in vitro. Hydrolysis of [¹⁴C] AZA-hemolobin with 3 N p-toluenesulfonic acid and subsequent amino acid analysis resulted in recovery of 90% of the initial radioactivity in fractions containing added free AZA standard. An additional 6% of the radioactivity eluted with homoserine, a known degradation product of AZA. After tryptic digestion of [¹⁴C] AZA-hemoglobin, less than 9% of radioactivity eluted near added free AZA standard, while 91% of radioactivity was located in tryptic peptides elsewhere on the chromatogram. These data provide the first demonstration of AZA incorporation into a mammalian protein.     

Effects of L-azetidine-2-carboxylic acid on tooth morphogenesis and cytodifferentiation in mice

The effect of L-azetidine-2-carboxylic acid on tooth differentiation was studied in mouse embryos. This proline analogue produced reversible delay of morphogenesis and cytodifferentiation.     

Amino acid-azetidine chimeras: synthesis of enantiopure 3-substituted azetidine-2-carboxylic acids.

Azetidine-2-carboxylic acid (Aze) analogs possessing various heteroatomic side chains at the 3-position have been synthesized by modification of 1-9-(9-phenylfluorenyl) (PhF)-3-allyl-Aze tert-butyl ester (2S,3S)-1. 3-Allyl-Aze 1 was synthesized by regioselective allylation of alpha-tert-butyl beta-methyl N-(PhF)aspartate 13, followed by selective omega-carboxylate reduction, tosylation, and intramolecular N-alkylation. Removal of the PhF group and olefin reduction by hydrogenation followed by Fmoc protection produced nor-leucine-Aze chimera 2. Regioselective olefin hydroboration of (2S,3S)-1 produced primary alcohol 23, which was protected as a silyl ether, hydrogenated and N-protected to give 1-Fmoc-3-hydroxypropyl-Aze 26. Enantiopure (2S,3S)-3-(3-azidopropyl)-1-Fmoc-azetidine-2-carboxylic acid tert-butyl ester 3 was prepared as a Lys-Aze chimera by activation of 3-hydroxypropyl-Aze 26 as a methanesulfonate and displacement with sodium azide. Moreover, orthogonally protected azetidine dicarboxylic acid 4 was synthesized as an alpha-aminoadipate-Aze chimera by oxidation of alcohol 26. These orthogonally protected amino acid-Aze chimeras are designed to serve as tools for studying the influence of conformation on peptide activity.     

Synthesis and peptide bond orientation in tetrapeptides containing L-azetidine-2-carboxylic acid and L-proline

The role of the amino acid proline in influencing the secondary and tertiary structure of proteins and polypeptides has been an area of active study for many years. We have investigated this problem by incorporating the four-membered ring amino acid, azetidine-2-carboxylic acid, into some proline polypeptides. An adjunct to the synthesis of the peptides was the synthesis of azetidine-2-carboxylic acid and its resolution. We developed an improved synthesis of N-benzhydryl-2-carbobenzyloxy azetidine, an essential intermediate required for the synthesis of L-azetidine-2-carboxylic acid. This amino acid was subsequently obtained via the partial hydrogenation of the N-benzhydryl compound, under mild conditions. Our ability to isolate the intermediate N-benzhydryl-2-carboxylic acid demonstrated that the rate of cleavage of the O-benzyl ester group in this molecule is faster than the cleavage of the N-benzhydryl group. The tetrapeptides, Boc-(L-Pro)3-L-Aze-Opcp, and Boc-(L-Aze-L-Pro)2-Opcp (Boc: t-butoxycarbonyl; Pro: proline; Aze: azetidine-2-carboxyl acid; Opcp: pentachlorophenyl), were prepared using traditional solution peptide synthesis. They were characterized by direct chemical ionization-mass spectrometry, CD spectra, and 13C- and 1H-nmr spectroscopy. The assessment of the secondary structure of the two peptides using the methods noted above has led us to conclude that the compound Boc-(L-Aze-L-Pro)2-Opcp, in trifluoroethanol, has an all-cis peptide bond conformation with phi and psi torsion angles compatible with a left-handed helix. The secondary structure assessment of the peptide Boc-(L-Pro)3-L-Aze-Opcp, in chloroform or trifluoroethanol, leads to an assignment of both cis and trans peptide bonds as being present in the peptide. We have interpreted this latter finding as indicating that the introduction of the azetidine group into a peptide containing three consecutive proline residues in a linear sequence perturbs the normal proline peptide secondary structure in this tetrapeptide.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. II. Homopolymers and copolymers

The alteration of polymer conformational properties caused by the replacement of L-proline by L-azetidine-2-carboxylic acid (Aze) has been studied by means of conformational energy computations. In addition to poly(Aze), two sequential copolymers, poly(Pro-Aze) and poly(Aze3-Pro3), have been investigated. All polymers containing Aze are more flexible than poly(Pro). This is a consequence of an increased number of permitted conformational states for the Aze residue, as compared to Pro, when they are incorporated into a polypeptide, as well as of a lessened cooperativity of the trans-cis transition. The results of the computation can be used to interpret the observed physical properties of poly(Aze) and of its copolymers.     

Corneal development. IV. Interruption of collagen excretion into the primary stroma of the cornea with L-azetidine-2-carboxylic acid

The primary stroma of the cornea of the chick embryo contains a cell-free orthogonal ply of collagen fibrils which is delineated clearly by Gomori's silver stain for reticulin and has, in miniature, the same fibrous architecture as the mature stroma. The collagen of this matrix is synthesized by the basal cells of the corneal epithelium and deposited beneath them a layer at a time.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. III. Collagen-like poly(tripeptide)s

The chemical and biological properties of collagen are altered by the biosynthetic substitution of the L-azetidine-2-carboxylic acid(Aze) residue in the place of proline. The reasons for this alteration have been studied by means of conformational energy computations on single- and triple-stranded structures formed by poly(Gly-X-Y) poly(tripeptide)s, where X and Y can be Pro or Aze. The most stable triple helix formed by Poly(Gly-Pro-Aze) is collagen-like, but all low-energy triple helices that can be formed by poly(Gly-Aze-Pro) and poly(Gly-Aze-Aze) are very different from collagen. Thus, the regular substitution of Aze for Pro in position X is not compatible with the collagen structure. In the absence of solvent effects, all of these triple helices are stable, relative to the statistical coil, but the substitutions reduce the stability of the collagen-like triple helix, as compared with poly(Gly-Pro-Pro).     

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 © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and biological relationships of 3',6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives as antimitotic agents

As part of a continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, 3',6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives and their salts were synthesized and evaluated. Preliminary screening showed that carboxylic acid analogs containing a m-fluoro substituted 2-phenyl group displayed the highest in vitro anticancer activity. Activity decreased significantly if a chlorine or methoxy group replaced the fluorine atom. 3'-Fluoro-6-methoxy-2-phenyl-4-quinolone-3-carboxylic acid (68) had the highest in vitro cytotoxic activity among all tested carboxylic acid derivatives and their salts. The mechanism of action may be similar, but not identical, to that of tubulin binding drugs, such as navelbine and taxol. Compound 68 merits further investigation as a novel hydrophilic antimitotic agent.     

Asymmetric synthesis of 2-substituted 4-chromanones using enzyme-catalyzed reactions

2-Substituted 4-chromanones were synthesized in their optically active forms. The chiral intermediates were obtained via lipase-catalyzed enantioselective reactions. Lipase and esterase were also used for the hydrolysis of ester moieties of the precursors of the target compounds under mild conditions.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. I. Conformations of the residue and of dipeptides

The L-azetidine-2-carboxylic acid (Aze) residue can be incorporated into proteins in the place of L-proline, of which it is the lower homologue. This substitution alters the properties of proteins, especially of collagen. Conformational constraints in N-acetyl-Aze-N'-methylamide and in several dipeptides containing Aze have been analyzed by means of energy computations. They have been compared with peptides containing Pro. The overall conformational preferences of Aze and Pro are similar, but several significant differences occur between them. In general, peptides containing Aze are somewhat more flexible than corresponding peptides containing Pro, because of a decrease in constraints caused by repulsive nonconvalent interactions of the atoms of the ring with neighboring residues. This results in an entropic effect that lessens the stability of ordered polypeptide conformations with respect to the disordered statistical coil. The collagen-like near-extended conformation is energetically less favorable for Aze than for Pro in the single residue and in dipeptides. This effect also contributes to a destabilization of the collagen triple helix. The influence of Aze on the conformation of polypeptides is discussed in the accompanying papers.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. IV. Local substitutions in the collagen triple helix

The properties of collagen are affected by the replacement of Pro by imino acid analogues. The structural effect of the low-level local substitution of L -azetidine-2-carboxylic acid (Aze) has been analyzed by computing the energy of CH₃CO-(Gly-Pro-Pro)₄-NHCH₃ triple helices in which a single residue of one strand has been replaced by Aze. When Aze is in position Y of a (Gly-X-Y) unit, low-energy local deformations are introduced in the triple helix, i.e., it becomes more flexible. On the other hand, the flexibility of the triple helix is not increased with Aze in position X. The energy of the triple helix to coil transition is not changed significantly by this amount of substitution. In an earlier study, we have demonstrated that the regular substitution of Aze in every tripeptide distorts or destabilizes the triple helix to a large extent [A. Zagari, G. Némethy, & H. A. Scheraga (1990) Biopolymers, Vol. 30, pp. 967–974 ]. Thus, it appears that a high level of substitution is required to cause the observed chemical and biological effects of Aze on collagen. © 1994 John Wiley & Sons, Inc.     

Constrained phenylalanine analogues. Preferred conformation of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue.

Three Tic-containing (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) model peptides were synthesized to assess the tendency of this constrained Phe analogue to fold into a beta-bend and a helical structure, and to adopt a preferred side-chain disposition. The results of the solution conformational analysis, performed by using Fourier transform infrared absorption and 1H nuclear magnetic resonance, indicate that in chloroform the -Aib-D-Tic-Aib-, -(Aib)2-D-Tic-(Aib)2-, and -L-Pro-D-Tic- sequences fold into intramolecularly H-bonded forms to a great extent. An X-ray diffraction analysis on p-BrBz-(Aib)2-DL-Tic-(Aib)2-OMe monohydrate and p-BrBz-L-Pro-D-Tic-NHMe allows us to conclude that, while the pentapeptide methylester forms an incipient (distorted) 3(10)-helix, the dipeptide methylamide adopts a type-II beta-bend conformation. In both cases, the D-Tic side-chain conformation is D, gauche(-). The implications for the use of the Tic residue in designing conformationally restricted analogues of bioactive peptides are briefly discussed.