Protein-Loop Mimetics: A Diketopiperazine-Based Template to Stabilize Loop Conformations in Cyclic Peptides Containing the NPNA and RGD Motifs

We explore here an approach to mimic the structures and biological functions of protein loops in small synthetic molecules, by grafting the loop of interest onto an organic template comprising a bicyclic diketopiperazine, prepared by the formal coupling of (2S,4S)-4-aminoproline (Pro(NH₂)) and aspartic acid (Asp). The Fmoc-protected template 4 is used to prepare cyclo(-Ala¹-Asn²-Pro³-Asn⁴-Ala⁵- Ala⁶-Temp-) ( 5 ) and cyclo(-Ala¹-Arg²-Gly³-Asp⁴-Temp-) ( 6 ) (where Temp = template derived from 4 ), containing the Asn-Pro-Asn-Ala (NPNA) and Arg-Gly-Asp (RGD) motifs. The conformational properties of these molecules are studied in aqueous solution by NMR and simulated-annealing methods. The NPNA motif, an immunodominant epitope on the circumsporozoite surface protein of the malaria parasite Plasmodium falciparum, is shown to adopt a stable type-I β-turn in 5 . The template in 5 adopts a preferred conformation with Pro(NH₂)χ₁ ≈? ?35° and the Asp moiety χ₁ ≈? 70°. A different template conformation is inferred for 6 , with Pro(NH₂)χ₁ ≈? 0°, but the ARGD loop appears by NMR to undergo rapid conformational averaging. Solid-phase binding assays reveal that 6 displays modest antagonist activity towards both the integrin α_IIbβ₃ and α_vβ₃ receptors.     

Synthèse de la désamino1-Arg8-vasopressine et de la désamino1-Phe2-Arg8-vasopressine,deux analogues possédant une activité antidiurétique plus élevée et plus sélective que celle des vasopressines naturelles

Deamino¹-Arg⁸-vasopressin and deamino¹-Phe-²Arg⁸-vasopressin have been synthesized by condensation of L -glutaminyl-L -asparaginyl-S-benzyl-L -cysteinyl-L -prolyl-G-tosyl-L -arginyl-glycinamide with β-benzylthio-propionyl-L -tyrosyl-L -phenyl-alanylazide and β-benzylthio-propionyl-L -phenylalanyl-L -phenylalanylazide, respectively. After removal of the protecting groups and oxidation with hydrogen peroxide, the deamino-nonapeptides have been purified by counter-current distribution. Deamino¹-Arg⁸-vasopressin as well as deamino¹-Phe²-Arg⁸-vasopressin exhibit a higher antidiuretic activity (1300 IU/mg and 800 IU/mg, respectively) and a lower pressor activity than arginine-vasopressin. This confers to both analogues, and especially to the latter, a remarkable selectivity of the antidiuretic action.     

Sequence and three-dimensional structure of cycloreticulins A and B, new cyclooctapeptides from the seeds of Annona reticulata

Two cyclooctapeptides, cycloreticulin A, cyclo(Pro¹-Gly²-Asp³-Ile⁴-Ser⁵-Ile⁶-Tyr⁷-Tyr⁸) (1) and cycloreticulin B, cyclo(Pro¹-Mso²-Tyr³-Gly⁴-Thr⁵-Val⁶-Ala⁷-Val⁸) (2), have been isolated from the methanol extract of the seeds of Annona reticulata L. The sequences were elucidated on the basis of the MS/MS fragmentation using a QTOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-NMR. The solid state conformation of cycloreticulin A, carried out by X-ray study, is characterised by the presence of two β-turns (types II and III) and an inversed γ-turn. Its solution structure appeared quite similar to the crystal one. The cyclic backbone solution structure of cycloreticulin B, close to that of the cyclooctapeptide squamin A, from which its sequence only differs by a Val⁸/Ile⁸ substitution, involves three β-turns, two of type I and one of type III, being similar to the crystal structure of squamin A.     

Synthèse d'une sonde biotinylée à bras clivable allongé pour l'isolement des récepteurs de l'angiotensine II

Synthesis of a Biotinylated Probe with an Extended Cleavable Arm for Angiotensin II Receptors Purification We have synthesized a new biotinylated probe for angiotensin II receptors studies: biotinyl-NH(CH₂)₂? SS? (CH₂)₂? CO-Gly-? Ahx-[Ala¹, Phe(4N₃)⁸]angiotensin II ( 5 ). This molecule can be photoactivated through an arylazido group. ¹H-NMR studies suggest that it adopts an extended conformation which should allow a simultaneous recognition of both streptavidin and hormone receptor. It has a good affinity for receptors (K_d = 1 nM) and hence is a promising tool in their detection (autoradiography, gold-, ferritin-, enzyme-, or fluorescent streptavidin derivatives) and separation (cell sorting, affinity chromatography). It can be monoiodinated (°6) at its tyrosine residue without a significant loss of affinity. Its extended cleavable arm allows an easy recovery of the ‘probe-receptor’ complex from streptavidin. An HPLC monitoring of the synthesis is described, particularly of the segment coupling 1 + 2 in presence of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP). This method can be used as well for synthesis of the D -Phe⁸ derivative that has antagonist properties.     

Prolinoamino acids as a tool to stabilize β-turns with the side chain of natural amino acids

Molecular mechanics calculations, X-ray, FT-IR, and NMR analysis performed on Piv-d-Pro^c_l-l-Pro-NHMe (d-Pro^c_l=a proline/leucine chimera) show that it possesses a water stable type II^′ β-turn structure. The isopropyl side chain of d-Pro^c_l compares with the leucine side chain of a typical type I β-turn found in a protein (taken from the PDB). Thus cis-3-substituted prolines with the appropriate side chain can be used to mimic type I, II or II^′ β-turns and incorporate the side chain functionalities on both the i+1 and i+2 positions of β-turns.     

The Structure of the Antibiotic Hedamycin. I. Chemical,Physical and Spectral Properties

Interpretation of the chemical and spectral (IR., UV., ¹H- and ¹³C-NMR.) properties of the antitumor antibiotic hedamycin (C₄₁H₅₀N₂O₁₁) suggests that the molecule contains a methyl substituted 1-hydroxyanthraquinone nucleus, an α, β-unsaturated ketone, two sugar-like tetrahydropyran rings ( 4 and 8 ) and an aliphatic chain 2 , presumably with an epoxy group (see the Scheme).     

Desoxy-nitrozucker 14. Mitteilung 1-C-Nitroglycale. Herstellung und Umsetzung mit einigen Stickstoff-Nucleophilen

1-C-Nitroglycals. Preparation and Reaction with Some Nitrogen Nucleophiles Acetylation of the 1-deoxy-1-nitromannopyranoses 2 and 6 was accompagnied by spontanous β-elimination to give the 1-C-nitroglucals 3 and 7 , respectively, while acetylation of the gluco- and galacto-configurated 1-deoxy-1-nitropyranoses 8 and 14 gave the acetates 9 and 15 , respectively (Scheme 1). The acetylation of the ribo- and arabino-configurated 1-deoxy-1-nitrofuranoses 19 and 21 also occurred without β-elimination to give the acetates 20 and 22 , respectively (Scheme 2). Mild base treatment of the previously described O-acetylnitro-β-D -glucose 4 , the O-acetylnitro-β-D -pyranoses 9 and 15 , and the O-acetylnitro-β-D -furanoses 17 , 20 , and 22 gave the 1-C-nitroglycals 3 , 10 , 16 , 18 and 23 , respectively (Scheme 1 and 2). The previously obtained 1-C-nitroglucal 3 was deacetylated by treatment with MeOH in the presence of KCN or sodium m-nitrophenolate to give the free nitroglucal 5 . Deacetylation of the benzylidene protected 1-C-nitroglucal 10 (MeOH, NaOMe) gave the 4,6-O-benzylidene-1-C-nitroglucal 11 and traces of the 2-O-methyl-1-C-nitromannoses 12 and 13 . The UV, IR, ¹H-NMR and ¹³C-NMR spectra of the 1-C-nitroglycals are discussed. In solution, the 1-C-nitroglycals 1 , 5 , 7 , 10 , 11 , and 16 adopt approximately a ⁴H_5? and 3 a flattened ⁴H₅ conformation. The structure of 5 was established by X-ray analysis. In the solid state, 5 adopts a sofa conformation, which is stabilized by an intramolecular H-bond. The β-addition of NH₃ to the 1-C-nitroglucals 7 and 10 was followed by an O→ N acetyl migration to give exclusively anomeric pairs of the N-acetyl-1-nitromannosamine derivatives 24 / 25 and 26/27 , respectively (Scheme 3). The β-addition of methylamine, octadecylamine, and tryptamine to the 1-C-nitroglucal 11 also stereoelectronically controlled and gave the crystalline N-alkyl-1-nitromannosamines 28 , 29 , and 30 , respectively. The stereoelectronically controlled β-addition of NH₃ to the 1-C-nitrogalactal 16 , followed by acetylation, yielded exclusively the talosamine derivative 31 , while the reversible β-addition of azide ions to 16 gave the anomeric 2-azido-1-nitrogalactoses 32 and 33 . The β-addition of azide ions to the 1-C-nitroglucal 1 led to the 2-azido-1-nitromannose 34 . In the presence of excess formaldehyde, this addition was followed by a Henry reaction. Chromatography of the crude product was accompagnied by solvolytic removal of the NO₂ group to give the 3-azidomannoheptulose 35 in high yields (Scheme 4).     

3D-structure of cycloreticulin C and glabrin A, cyclopeptides from the seeds of Annona reticulata

Two cyclopeptides, the cycloheptapeptide cycloreticulin C, cyclo(Pro¹-Gly²-Gln³-Pro⁴-Pro⁵-Tyr⁶-Val⁷) (1), and the cyclohexapeptide glabrin A, cyclo(Pro¹-Gly²-Leu³-Val⁴-Ile⁵-Tyr⁶) (2), have been isolated from the methanol extract of the seeds of Annona reticulata. Their structures were elucidated on the basis of the MS/MS fragmentation using a Q-TOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-NMR. The solution conformation of cycloreticulin C involves two β-turns, one of type II with trans-Pro¹ and Gly² at the corners, another of type VIa with trans-Pro⁴ and cis-Pro⁵ at the corners, and followed by a β-bulge at the Tyr⁶-Val⁷ level. The solid state and solution conformations of glabrin A have been analyzed by X-ray and 2D-NMR studies, respectively, and are characterized by the presence of two β-turns, the first of type VIa and the second intermediary between types I and III at the solid state and a γ-turn in solution.     

Conformational Studies on Potential β-Turn-Forming Model Peptides

The conformational behavior of POE-bound model peptides Boc-(L -Ala)₂-X-Y-(L -Ala)₂-NHPOE (X – Y = L -Pro-Gly ( I ), Gly-L -Ile ( II ); NHPOE = aminopoly(oxyethylene)) as well as of the repetitive hexapeptide of elastin, Boc-L -Val-L -Ala-L -Pro-Gly-L -Val-Gly-A-NHPOE-M ( III ) (A = photosensitive 3-nitro-4-(bromomethyl)benzoyl group; NHPOE-M = aminopoly(oxyethylane) monomethyl ether) has been studied by means of ¹H-NMR and CD spectroscopy. Compounds I and III form a β-turn with Pro and Gly in positions i + 1 and i + 2, respectively, while an aggregated state for II , has been identified. The results are in good agreement with published prediction codes giving experimental evidence for the dominance of short-range interactions to establish secondary structure in solution.     

Angiotensin-II Analogues. I: Synthesis and incorporation of the halogenated amino acids 3-(4′-iodophenyl)alanine, 3-(3′,5′-dibromo-4′-chlorophenyl)alanine, 3-(3′,4′,5′-tribromophenyl)alanine,and 3-(2′,3′,4′,5′,6′-pentabromophenyl)alanine

The synthesis of the polyhalogenated phenylalanines Phe(3′,4′,5′-Br₃) ( 3 ), Phe(3′,5′-Br₂-4′-Cl) ( 4 ) and DL -Phe (2′,3′,4′,5′,6′-Br₅) ( 9 ) is described. The trihalogenated phenylalanines 3 and 4 are obtained stereospecifically from Phe(4′-NH₂) by electrophilic bromination followed by Sandmeyer reaction. The most hydrophobic amino acid 9 is synthesized from pentabromobenzyl bromide and a glycine analogue by phase-transfer catalysis. With the amino acids 4, 9 , Phe(4′-I) and D -Phe, analogues of [1-sarcosin]angiotensin II ([Sar¹]AT) are produced for structure-activity studies and tritium incorporation. The diastereomeric pentabromo peptides L - and D - 13 are separated by HPLC. and identified by catalytic dehalogenation and comparison to [Sar¹]AT ( 10 ) and [Sar¹, D -Phe⁸]AT ( 14 ).     

Stabilization of β-turn conformations in Pro-X sequences by disulphide bridging. Synthesis and solution conformations of five cyclic cystine peptides

Five cyclic peptide disulphides of the type

have been synthesized, where X = Gly (1), L-Ala (2), D-Ala (3), Aib (4) and L-Leu (5). ¹H NMR studies at 270 MHz in CDCl₃ and (CD₃)₂SO provide evidence of a Pro-X β-turn conformation, stabilized by a transannular 4→1 hydrogen bond involving the Cys(4) NH, in all the peptides. In addition peptides 2, 4 and 5 also possess a second intramolecular hydrogen bond involving the -NHMe group. The spectroscopic data are consistent with a consecutive type III β-turn conformation for peptides 2, 4 and 5, a type I(III) β-turn structure for 1 and a type II turn for 3.     

Supramolecular helix and β-sheet through self-assembly of two isomeric tetrapeptides in crystals and formation of filaments and ribbons in the solid state

Single crystal X-ray diffraction studies show that the β-turn structure of tetrapeptide I, Boc-Gly-Phe-Aib-Leu-OMe (Aib: α-amino isobutyric acid) self-assembles to a supramolecular helix through intermolecular hydrogen bonding along the crystallographic a axis. By contrast the β-turn structure of an isomeric tetrapeptide II, Boc-Gly-Leu-Aib-Phe-OMe self-assembles to a supramolecular β-sheet-like structure via a two-dimensional (a, b axis) intermolecular hydrogen bonding network and π-π interactions. FT-IR studies of the peptides revealed that both of them form intermolecularly hydrogen bonded supramolecular structures in the solid state. Field emission scanning electron micrographs (FE-SEM) of the dried fibrous materials of the peptides show different morphologies, non-twisted filaments in case of peptide I and non-twisted filaments and ribbon-like structures in case of peptide II.     

Nature et stabilité des complexes métalliques de cryptands dinucléants en solution II. Polythiamacrotricycles et monocycles apparentés

Nature and Stability of Some Metallic Complexes of Dinucleating Cryptands in Solution II. Polythiamacrotricycles and Related Monocyclic Subunits The stability constants of the Cu²⁺ and Ag⁺ complexes of the cylindrical macrotricycle 1a (1,7,13,19-tetraaza 4,16-dioxa 10,22,27,32-tetrathiatricyclo[17.5.5.5]tetratriacontane) have been determined by pH-metry, as well as those of the Cu²⁺, Co²⁺, Zn²⁺, Cd²⁺, Pb²⁺, and Ag⁺ complexes of the monocyclic subunit 2a (1,7-dimethyl-1,7-diaza 4,10-dithiacyclododecane), in aqueous solutions (NaClO₄) at 25°. In the Cu(II) systems, equilibria were reached slowly, and the results established by pH-metry were confirmed by UV/VIS spectrophotometric studies. The tricycle 1a forms dinuclear cryptates with copper and silver, with overall stability constants log β₂₁₀ (Cu₂- 1a )⁴⁺ = 18.5, log β_21-2 (Cu₂- 1a (OH)₂)²⁺ = 4.8, log β₂₁₀(Ag₂- 1a )²⁺ = 23.0. Ag⁺ also forms a mononuclear (Ag- 1a )⁺ complex, with log β₁₁₀ = 13.1, but no mononuclear species were detected in the Cu- 1a system. The absorption spectra of the bis-Cu(II) complexes of 1a and 2a in aqueous medium, MeOH and propylene carbonate (PC) are given, as well as those, in MeOH and PC, of the bis-copper complexes of the related monocycles 3 and 4 (1,7-diaza-4,10,13-trithiacyclopentadecane and 1.10-diaza 4,7,13,16-tetrathiacyclooctadecane, respectively), and tricycle 5 with two benzyl groups in the lateral chains. The complexing properties of the polyoxa- and polythia macrotricycles (Parts I and II of this series) are compared to those of other bis-chelating ligands, the bicyclic bis-tren and the monocyclic bis-dien.     

Cyclopropane analogue of valine: influence of side chain orientation on peptide folding

The cyclopropane analogue of valine (1-amino-2,2-dimethylcyclopropanecarboxylic acid, c₃Val) has been synthesised and incorporated into the model peptides ^tBuCO-l-Pro-l-c₃Val-NHⁱPr and ^tBuCO-l-Pro-d-c₃Val-NHⁱPr. In the solid state, both dipeptides accommodate a type II β-turn stabilised by an NHⁱPr to ^tBuCO hydrogen bond. Remarkably, the peptide incorporating l-c₃Val also exhibits a distorted γ-turn around the cyclopropane residue, with Pro-CO and NHⁱPr intramolecularly hydrogen-bonded.     

Synthesis of hydroxy- and methoxy-substituted octahydrobenzo[g]isoquinolines as potential ligands for serotonin receptors

In 7 steps, 6- or 9-hydroxylated or -methoxylated trans-octahydrobenzo[g]isoquinolines were efficiently synthesized starling from dimethoxynaphtbalenes (Scheme), as potential new selective ligands for serotonin receptors. The 6-substituted compounds had very little affinity to common neurotransmitter receptors, with the exception of adrenergic α₂. The 9-substituted compounds, while showing interesting affinity for 5HT_1a receptors, had comparable affinities for adrenergic α₁ and β₂, and in one case for dopamine D2 receptors.     

Cyclotetrapeptides with alternating d-Ala residues: synthesis and spectroscopic studies

Three cyclotetrapeptides, c[Leu-d-Ala-Xaa-d-Ala], where Xaa is Leu (P1), Lys (P2) and Glu (P3) were synthesized and studied by ¹H and ¹³C NMR and CD spectroscopy. These cyclotetrapeptides exhibit similar coupling constants, ³J_HNHα, in the range of 8.56-9.93 Hz, commonly observed for β-turn structures. All amide proton chemical shifts for P1, P2 and P3 exhibited linear dependence on temperature with moderate temperature coefficients ranging from −3.1 to −9.8 ppb/K. Amide proton signal broadening was observed for all residues in P1, P2 and P3, indicating that they are solvent accessible. The number of resonance observed for P1 was half of the total counts, indicating a C2 symmetric conformation. P2 and P3 exhibit similar CD in solvents of varying dielectric constants and dilutions, with characteristic positive CD bands at ca. 210 and 222 nm, which correspond to a β-turn type structure. Small CD/temperature effect was also observed with isodichroic points, consistent with conformational stability and a well-populated cyclotetrapeptide energy state. These heterochiral cyclotetrapeptides consisting of alternating d-Ala residues adopt stabilized open β-turn conformations and may be useful as a ligand template for further functionalization.     

Structural and energetic relations between β turns

A systematic quantum chemical study on the structure and stability of the major types of β-turn structures in peptides and proteins was performed at different levels of ab initio MO theory (MP2/6-31G*, HF/6-31G*, HF/3-21G) considering model turns of the general type Ac(SINGLE BOND)X_aa(SINGLE BOND)Y_aa(SINGLE BOND)NHCH₃ with the amino acids glycine, l - and d -alanine, aminoisobutyric acid, and l -proline. The influence of correlation effects, zero-point vibration energies, thermal energies, and entropies on the turn formation was examined. Solvent effects on the turn stabilities were estimated employing quantum chemical continuum approaches (Onsager's self-consistent reaction field and Tomasi's polarizable continuum models). The results provide insight into the geometry and stability relations between the various β-turn subtypes. They show some characteristic deviations from the widely accepted standard rotation angles of β turns. The stability order of the β-turn subtypes depends strongly on the amino acid type. Thus, the replacement of l -amino acids in the two conformation-determining turn positions by d - or α,α-disubstituted amino acid residues generally increases the turn formation tendency and can be used to favor distinct β-turn subtypes in peptide and protein design. The β-turn subtype preferences, depending on amino acid structure modifications, can be well illustrated by molecular dynamics simulations in the gas phase and in aqueous solution. © 1997 by John Wiley & Sons, Inc. J Comput Chem 18 : 1415–1430, 1997     

The Degradation Mechanism of an Oral Cephalosporin: Cefaclor

The degradation of cefaclor ( 1 ), an oral cephalosporin antibiotic, was studied at 37° in a neutral aqueous medium by HPLC and ¹H-NMR. Under these conditions, 1 underwent intramolecular aminolysis by the 7-side-chain NH₂ group on the β-lactam moiety to give a piperazine-2,5-dione. The most prominent peak in the HPLC profile of a degradation solution from 1 was isolated by prep. HPLC. Mechanistically, the formation of this degradation product cis- 11 from 1 involves the contraction from a six-membered cephem ring to a five-membered ring, which presumably takes place via a common episulfonium ion intermediate 9 (see Scheme). Loss of the Cl-atom from 3-chloro-3-cephem is a general reaction subsequent to β-lactam ring opening.     

Stabilité dans le méthanol et propriétés thermodynamiques de transfert des cryptates de certains cations de transition et de métaux lourds

Stability in Methanol and Thermodynamic Transfer Properties of the Cryptates of some Transition Cations and Heavy Metals The nature and stability of the macrocyclic and macrobicyclic complexes of Ag⁺, Cd²⁺, and Pb²⁺ (Mⁿ⁺) with 21, 22, 211, 221 and 222 in anhydrous methanol 0.05M in Et₄N⁺ClO^?₄, at 25° (see Scheme) have been determined by potentiometry and spectrophotometry. Binuclear complexes M₂L²ⁿ⁺ have been observed in all cases, besides the mononuclear MLⁿ⁺ complexes. The macrobicyclic 1:1 complexes MLⁿ⁺ exhibit an important ‘cryptate effect’ with Mⁿ⁺=Ag⁺, Pb²⁺ and Cd²⁺, but not with Cu²⁺ and Zn²⁺; their stability is in all cases maximum with 221. The applicability to our results of the recent extrathermodynamic hypothesis involving MLⁿ⁺ cryptates is examined.     

Cyclic Heptapeptides Axinastatin 2, 3, and 4: Conformational analysis and evaluation of the biological potential

The conformational analysis of naturally occurring cytostatic cyclic heptapeptides axinastatin 2, 3, and 4 was carried out by two-dimensional NMR spectroscopy in combination with distance-geometry (DG) and molecular-dynamics (MD) calculations in explicit solvents. The synthesized secondary metabolites were examined in (D₆)DMSO. Axinastatin 2 was also investigated in CD₃OH. In all structures, Pro² is in the i + 1 position of a βI turn and Pro⁶ occupies the i + 2 position of a βVIa turn about the cis amide bond between residue 5 and Pro^6. In all peptides, a bifurcated H-bond occurs between residue 4 CO and the amide protons of residue 1 and 7. For axinastatin 2 and 3, an Asn I_g turn was found about Asn¹ and Pro^2. We compared these structures with conformations of cyclic heptapeptides obtained by X-ray and NMR studies. A β-bulge motif with two β turns and one bifurcated H-bond is found as the dominating backbone conformation of cyclic all-L-heptapeptides. Axinastatin 2, 3, and 4 can be characterized by six trans and one cis amide bond resulting in a β/βVI(a)-turn motif, a conformation found for many cyclic heptapeptides. Detailed biological tests of the synthetic compounds in different human cancer cell lines indicates these axinastatins to be inactive or of low activity.