Structural versatility of peptides containing C-dialkylated glycines. An X-ray diffraction study of six 1-aminocyclopropane-1-carboxylic acid rich peptides

The molecular and crystal structures of six fully blocked, Ac₃c-rich peptides to the tetramer level were determined by X-ray diffraction. The peptides are Fmoc-(Ac₃c)₂-OMe·CH₃OH, Ac-(Ac₃c)₂-OMe, t-Boc-Ac₃c-l-Phe-OMe, pBrBz-(Ac₃c)₃-OMe·H₂O, Z-Gly-Ac₃c-Gly-OTmb·(CH₃₂CO, andt-Boc-(Ac₃c)₄-OMe·2H₂O. Type-I (I′) β-bends and distorted 3₁₀-helices were found to be typical of the tri- and tetrapeptides, respectively. In the dipeptides, too short to form β-bend conformations, other less common structural features may be observed. The average geometry of the cyclopropyl moiety of the Ac₃c residue is asymmetric and the N-C^α-C′ bond angle is significantly expanded from the regular tetrahedral value. A comparison with the structural preferences of other extensively investigated C^α,α-dialkylated α-amino acids is made and the implications for the use of the Ac₃c residue in conformational design are examined.     

Structural versatility of peptides containing C-dialkylated glycines: conformational energy computations, i.r. absorption and H n.m.r. analysis of 1-aminocyclopropane-1-carboxylic acid homopeptides

Conformational energy computations on the 1-aminocyclopropane-1-carboxylic acid mono-, di-, and tripeptide amides, (Ac-(Ac₃c)_n---NHMe (n=1−3), indicate that this C^,-dialkylated, cyclic -amino acid residue is conformally restricted and that type-I(I′) β-bends and distorted 3₁₀-helices are particularly stable conformations for the di- and tripeptide amides, respectively. The results of the theoretical analysis are in agreement with those obtained in an i.r. absorption and ¹H n.m.r. investigation in chloroform solution of A.c.₃c-rich tri- and tetrapeptide esters. A comparisons is also made with the conclusions extracted from our previous work on peptides rich in Aib (-aminoisobutyric acid), Ac₅c(1-aminocyclopentane-1-carboxylic acid), and Ac₆c (1-aminocyclohexane-1-carboxylic acid).     

Preferred conformation of peptides based on cycloaliphatic C(alpha,alpha)-disubstituted glycines: 1-amino-cycloundecane-1-carboxylic acid (Ac11c).

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cycloundecane-1-carboxylic acid (Ac11c), an alpha-amino acid conformationally constrained through a medium-ring C(i)alpha<-->C(i)alpha 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 beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac11c, the second largest cycloaliphatic C(alpha,alpha)-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 (Ac(n)c, with n = 3-12) series, which represents the prerequisite for their recent proposal of the 'Ac(n)c scan' concept.     

Synthesis and X-ray study of the 6-(N-pyrrolyl)purine and thymine derivatives of 1-aminocyclopropane-1-carboxylic acid.

The novel purine and pyrimidine derivatives of 1-aminocyclopropane-1-carboxylic acid 1 and 2 were obtained by alkylation of 6-(N-pyrrolyl)purine and thymine with methyl 1-benzamido-2-chloromethylcyclopropanecarboxylate. X-ray crystal structure analysis shows that the cyclopropane rings in 1 and 2 posses Z-configuration. The cyclopropane ring atoms and attached atoms of the benzamido and methoxycarbonyl moiety of both molecules are disposed perpendicularly to each other. The carbonyl oxygen of the methoxycarbonyl moiety adopts in both compounds a synperiplanar conformation with respect to the midpoint of the distal bond of the cyclopropane ring. The torsion angles Phi and psi for the 1-aminocyclopropane-1-carboxylic acid residue in 1 and 2 correspond to a folded conformation, while the torsion angles omega define antiperiplanar conformation. Intermolecular hydrogen bonds connect the molecules of 1 into dimers. Each dimer is hydrogen-bonded with four ethanol molecules, thus forming discrete unit. On the contrary, intermolecular hydrogen bonds link the molecules of 2 generating three-dimensional network.     

Inhibition of apple 1-aminocyclopropane-1-carboxylic acid oxidase, by cyclopropane-1,1-dicarboxylic acid and trans-2-phenylcyclopropane-1-carboxylic acid

Cyclopropane-1,1-dicarboxylic acid (CDA) and trans-2-phenylcyclopropane-1-carboxylic acid (PCCA) are the main representatives of a group of compounds that are structural analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) and have been proved to have an inhibitory effect on the wound ethylene produced by Lycopersicum esculentum fruit discs. During the experiments, that were carried out in this work the inhibition pattern of PCCA and CDA were studied when tested on partially purified apple ACO and their Ki values were determined. A mechanistic proposal was given, in order to explain the kinetic behaviour of the inhibitors. The common feature of these molecules is their cyclopropane ring, with different substitutes mainly at the positions C1 and C2. Two other compounds with similar structure where also tested as inhibitors, in order to clarify the relationship between structure and activity. These compounds are: 2-methyl cyclopropanecarboxylic acid (MCA), and cyclopropanecarboxylic acid (CCA).     

{alpha}-Aminoisobutyric acid : A probable competitive inhibitor of conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

Of 16 compounds related to 1-aminocyclopropane-1-carboxylicacid (ACC), aminoisobutyric acid (AIB) inhibited the productionof endogenous ethylene in the cotyledonary segments of cocklebur(Xanthium pennsylvanicum Wallr.) seeds most strongly. AIB at4 mM inhibited the formation of ethylene by about 50%, althoughthe O₂ uptake of the segments was not affected even at 20 mM.AIB also inhibited ethylene formation in the stem segments ofetiolated pea (Pisum sativum L. cv. Alaska) seedlings. Kineticanalysis with cell free extracts from etiolated pea shoots revealedthat AIB competitively inhibits the conversion of ACC into ethylene. (Received May 26, 1980; )     

Conformations of beta-amino acid residues in peptides: X-ray diffraction studies of peptides containing the achiral residue 1-aminocyclohexaneacetic acid, beta3,3Ac6c

The conformational preferences of the 3,3-disubstituted beta-amino acid residue, 1-aminocyclohexaneacetic acid (beta3,3Ac6c) have been investigated by determining the crystal structures of the parent amino acid, the hydrochloride derivative, 10 protected derivatives and di and tripeptides. The symmetrical cyclohexyl substituent at the beta-position restricts the values of the torsion angles phi (N--C(beta)) and theta (C(beta)--C(alpha)) to approximately gauche values (+/-60 degrees ). Relatively few intramolecularly hydrogen bonded conformations are observed. In the dipeptide Boc-beta(3,3)Ac6c-beta(3,3)Ac6c-NHMe a C6 hydrogen bond is observed. In Piv-Pro-beta(3,3)Ac6c-NHMe a C11 hydrogen bonded hybrid alphabeta turn is characterized. In a majority of cases the amino group occupies the axial position in the cyclohexane ring. The conformations observed are compared with crystallographically observed structures for other beta-residues, including beta(2,2)Ac6c.     

Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c).

The crystal structures of four peptides incorporating 1-aminocycloheptane-1-carboxylic acid (Ac7c) are described. Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe adopt beta-turn conformations stabilized by an intramolecular 4----1 hydrogen bond, the former folding into a type-I/III beta-turn and the latter into a type-II beta-turn. In the dipeptide esters, Boc-Aib-Ac7c-OMe and Boc-Pro-Ac7c-OMe, the Ac7c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac7c-OMe found in the asymmetric unit. The cycloheptane ring of Ac7c residues adopts a twist-chair conformation in all the peptides studied. 1H-NMR studies in CDCl3 and (CD3)2SO and IR studies in CDCl3 suggest that Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe maintain the beta-turn conformations in solution.     

Structural versatility of peptides from Cα,αdialkylated glycines: Linear Ac3c homo-oligopeptides

The crystal-state molecular structures of five linear Ac₃c homo-oligopeptides to the tetramer were determined by x-ray diffraction. The oligomers are H-(Ac₃c)₂-OMe, Fmoc-(Ac₃c)₂-OMe MeOH, Ac-(Ac₃c)₂-OMe, pBrBz-(Ac₃c)₃-OMe · H₂O, and t-Boc-(Ac₃c)₄-OMe · 2H₂O. The results indicate the propensity of the tri- and tetrapeptides to fold into type I β-bends and distorted 3₁₀-helices, respectively, in partial contrast to Aib, Ac₅c, and Ac₆c homo-peptides of comparable main-chain length, where regular type III β-bends and 3₁₀-helical structures were found. When the influence of the constraints produced by the intramolecular H bonds of the C₁₀-type is absent, other less common structural features may be observed. The average geometry of the cyclopropyl group of the Ac₃c residue is found to be asymmetric and the N? C^α? C′ bond angle significantly expanded from the regular tetrahedral value.     

The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, in peach fruit softening

Ethylene promotes fruit ripening, including softening. The fruit of melting-flesh peach (Prunus persica (L). Batsch) cultivar 'Akatsuki' produces increasing levels of ethylene, and the flesh firmness softens rapidly during the ripening stage. On the other hand, the fruit of stony hard peach cultivars 'Yumyeong', 'Odoroki', and 'Manami' does not soften and produces little ethylene during fruit ripening and storage. To clarify the mechanism of suppression of ethylene production in stony hard peaches, the expression patterns of four ethylene biosynthesis enzymes were examined: ACC synthases (Pp-ACS1, Pp-ACS2, and Pp-ACS3) and ACC oxidase (Pp-ACO1). In the melting-flesh cultivar 'Akatsuki', Pp-ACS1 mRNA was dramatically induced after harvesting, and a large amount of ethylene was produced. On the other hand, in stony hard peaches, Pp-ACS1 mRNA was not induced during the ripening stage, and ethylene production was inhibited. Since Pp-ACS1 mRNA was induced normally in senescing flowers, wounded leaves, and wounded immature fruit of 'Yumyeong', Pp-ACS1 was suppressed only at the ripening stage, and was not a defect in Pp-ACS1. These results indicate that the suppression of fruit softening in stony hard peach cultivars was caused by a low level of ethylene production, which depends on the suppressed expression of Pp-ACS1.     

Structural versatility of peptides from Cα,α-dialkylated glycines. II. An IR absorption and 1H-nmr study of homo-oligopeptides from Cα,α-diethylglycine

The conformational preferences of the N-trifluoroacetylated homo-peptides of C^α,α-diethylglycine from monomer to pentamer in chloroform solution were determined by using ir absorption and ¹H-nmr. Intramolecular hydrogen bonding was found to be the dominant factor for all NH groups. The likely absence of a conformational transition upon increasing main-chain length, and the remarkable stability to dilution, heating, and addition of perturbing agents, are additional relevant findings of this study. These results are in agreement with those of the fully extended, C₅-conformation-forming homo-peptides from the higher homolog C^α,α-di-n-propylglycine, but contrast dramatically to those of the homo-peptides from the lower homolog C^α,α-dimethylglycine, which have been shown to adopt the 3₁₀-helical structure.     

Crystallographic characterization of conformation of 1-aminocyclopropane-1-carboxylic acid residue (Ac3c) in simple derivatives and peptides

The molecular and crystal structures of the C alpha,alpha-dialkylated alpha-amino acid residue 1-aminocyclopropane-1-carboxylic acid hemihydrate (H2+-Ac3c-O-.1/2 H2O) and nine derivatives and dipeptides have been determined by X-ray diffraction. The derivatives are pBrBz-Ac3c-OH, Piv-Ac3c-OH, Z-Ac3c-OH, the alpha-and beta-forms of t-Boc-Ac3c-OH, Z-Ac3c-OMe, and the 5(4H)-oxazolone from pBrBz-Ac3c-OH; the dipeptides are H-(Ac3c)-OMe and c(Ac3c)2. The values determined for the torsion angles about the N-C alpha (phi) and C alpha-C' (psi) bonds for the single Ac3c residue of Piv-Ac3c-OH, the alpha- and beta-forms of t-Boc-Ac3-OH and Z-Ac3c-OMe, and the C-terminal Ac3c residue of H-(Ac3c)2-OMe correspond to folded conformations in the bridge region of the Ramachandran map. The structures of pBrBz-Ac3c-OH and Z-Ac3c-OH, however, are unusual in having a semi-extended conformation for the phi, psi angles. The N-terminal Ac3c residue of H-(Ac3c)2-OMe adopts a novel type of C5 conformation, characterized inter alia by an (amino) N. . .H-N (peptide) intramolecular hydrogen bond. While the acyl N alpha-blocking groups form trans amides (pBrBz-Ac3c-OH and Piv-Ac3c-OH), the urethane groups may adopt either the trans [Z-Ac3c-OH and t-Boc-Ac3c-OH (alpha-form)] or the cis amide conformations [t-Boc-Ac3c-OH(beta-form) and Z-Ac3c-OMe]. The five- and six-membered rings of the 5(4H)-oxazolone and the 2,5-dioxopiperazine, respectively, are planar. The four independent molecules in the asymmetric unit of the free alpha-amino acid are zwitterionic.     

An X-ray diffraction study of poly-L-homoarginine hydrochloride.

An X-ray study of the synthetic polypeptide poly(L -homoarginine hydrochloride) has been made to investigate whether, like the chemically related polypeptides poly(L -lysine hydrochloride), poly(L -arginine hydrochloride), and poly(L -ornithine hydrobromide), it can undergo conformational transitions merely from variations in its degree of hydration. X-ray photographs of powder and oriented specimens containing one to 15 molecules of water per L -homoarginine hydrochloride residue showed that this polymer forms only a β-pleated-sheet structure. The pleated sheets, formed by antiparallel polypeptide chains hydrogen-bonded to each other, are piled up along the b axis in an alternating sequence (“sandwich structure”). This structure did not appreciably change with variations of the degree of hydration, and the observed reflections at 56% relative humidity (1.8 molecules of water per residue) could be indexed satisfactorily in terms of a monoclinic unit cell, of space group P2₁, with a = 9.34 Å, b = 40.07 Å, c = 6.94 Å, and γ = 106°. These dimensions are shown by models to be compatible with the proposed structure, and the calculated density of 1.27 g/cm³ agrees well with the experimental value of 1.29 g/cm³. Removal of the last molecule of water results in a very diffuse pattern, while specimens containing 20 molecules of water per residue show only reflections due to water.     

Preferred solution conformation of peptides rich in the lipophilic, chiral, C(alpha)-methylated alpha-amino acid (alpha Me)Aoc.

The lipophilic, chiral, C(alpha)-methylated alpha-amino acid L-(alphaMe)Aoc (2-methyl-2-amino-octanoic acid) was prepared using a chemo-enzymatic approach. Two series of terminally protected model peptides, from dimer through to hexamer, containing L-(alphaMe)Aoc in combination with either Gly or Aib, were synthesized by solution methods and were fully characterized. A solution conformational analysis, based on FT-IR absorption, 1H-NMR and circular dichroism (CD) techniques, was performed with the aim at determining the preferred conformation of this novel amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that L-(alphaMe)Aoc favours the formation of the right-handed 3(10)-helical conformation.     

Azospirillum lipoferum strain AZm5 containing 1-aminocyclopropane-1-carboxylic acid deaminase improves early growth of tomato seedlings under nitrogen deficiency

In this study we evaluated the ability of two wild strains of Azospirillum, A. lipoferum AZm5 and A. brasilense VS9, to produce ACC deaminase. We tested the effects of a deficiency and medium doses of nitrogenous fertilizers on the growth and physiology of tomato plants (Lycopersicon esculentum Mill cv. ACE VF55) inoculated with both Azospirillum strains independently. Tomato plants were evaluated by root elongation assay and grown in pot soil culture with different nitrogen levels (0 kg N ha^–1 and 170 kg N ha^–1). The root:shoot ratio (R:S) and some ecophysiological traits were determined after 42 days of plant growth. Results showed very different physiological characteristics in both strains. We found three relevant aspects related to the AZm5 strain: it produces high amounts of cytokinins, it contains the gene acdS, which encodes ACC deaminase, and it promotes plant growth. We conclude that AZm5 maybe useful to increase N uptake in N-deficient soil by production of cytokinins and the promotion of ACC deaminase activity, which favored leaf expansion and higher leaf N investment. Therefore, for tomato culture, a simultaneous biofertilization with AZm5 and a relatively low fertilization with N (170 kg N ha^–1) to promote AZm5 activity could be advantageous.     

Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.