Cyclic Pseudopeptide Research Articles

Cyclohexamer [-(d-Phe-azaPhe-Ala)2-]: good candidate to formulate supramolecular organogels.

Molecular self-assembly is a fascinating process which has become an area of great interest in supramolecular chemistry, as it leads in certain cases to molecular gels. Organogels formulated from low molecular weight compounds (LMWOGs) have attracted much interest in the past decades due to their applications as new soft materials. Herein, we report on the ability of the cyclic pseudopeptide cyclo-[-(d-Phe-azaPhe-Ala)2-] (2) to self-assemble in some aromatic solvents and to form organogels driven by non-covalent forces, mainly hydrogen bonding and π-stacking interactions. Comprehensive FTIR and NMR studies emphasized that this cyclic aza-peptide adopts a β-turn conformation at low concentration in toluene, while an equilibrium between the monomeric states (intramolecular forces) and the supramolecular structures (intra- and intermolecular forces) is established at high concentration (gel state). Rheological investigations of the organogels highlight the dependence of their stiffness (up to ∼4 kPa) and sol/gel transition temperatures (up to 100 °C) as a function of the solvent and concentration of gelator used. The formulation of fibrous structures confirmed the phenomenon of self-assembly. Finally, we found that cyclo-[-(d-Phe-azaPhe-Ala)2-] is an effective organogelator for application in phase selective gelation (PSG) of organic solvents from aqueous/organic mixtures with recovery percents up to 96%.

CO2 Capture via Crystalline Hydrogen-Bonded Bicarbonate Dimers

Summary Limiting global temperature rises is increasingly dependent on the development of energy-efficient carbon-capture methods. Here, we report a simple CO2-separation cycle using an aqueous bis(iminoguanidine) (BIG) sorbent that reacts with CO2 and crystallizes into an insoluble bicarbonate salt. X-ray diffraction analysis of the bicarbonate crystals revealed “anti-electrostatic” hydrogen-bonded (HCO3−)2 dimers, stabilized by guanidinium cations and water. Mild heating of the crystals releases the CO2 and regenerates the BIG sorbent quantitatively, thereby closing the CO2-separation cycle. Experimental and computational investigations support a CO2-release mechanism consisting of surface-initiated low-barrier proton transfer from guanidinium groups to bicarbonate anions with the formation of carbonic acid dimers, followed by CO2 and H2O release in the rate-limiting step, with a measured activation energy of 102 ± 12 kJ/mol. The minimum energy required for sorbent regeneration is 151.5 kJ/mol CO2, which is 24% lower than the regeneration energy of monoethanolamine, a benchmark industrial sorbent.

A neutral halogen bonding macrocyclic anion receptor based on a pseudocyclopeptide with three 5-iodo-1,2,3-triazole subunits.

The converging arrangement of iodine atoms along its confined cavity causes a cyclic pseudopeptide with three 5-iodo-1,2,3-triazole subunits to interact with halides, in particular with chloride, in 2.5 vol% water/DMSO.

Efficient stabilisation of a dihydrogenphosphate tetramer and a dihydrogenpyrophosphate dimer bya cyclic pseudopeptide containing 1,4-disubstituted 1,2,3-triazole moieties.

A cyclic pseudooctapeptide 2 is described containing 1,4-disubstituted 1,2,3-triazole moieties. This compound features eight converging hydrogen bond donors along the ring, namely four amide NH and four triazole CH groups, which enable 2 to engage in interactions with anions. While fully deprotonated sulfate anions exhibit only moderate affinity for 2, protonated anions such as dihydrogenpyrophosphate and dihydrogenphosphate anions are strongly bound. Complexation of the phosphate-derived anions involves sandwiching of a dihydrogenpyrophosphate dimer or a dihydrogenphosphate tetramer between two pseudopeptide rings. X-ray crystallography provided structural information, while 1H NMR spectroscopy, mass spectrometry, and isothermal titration calorimetry demonstrated that these complexes are stable in solution (2.5 vol% water/DMSO) and can even be transferred without decomposition into the gas phase. The observed high thermodynamic stabilities are attributed to the mutual reinforcement of the interactions between the individual complex components, namely, hydrogen-bonding between the anions, multiple hydrogen bonding interactions between the anion aggregates and the triazole CH and NH hydrogen bond donors of 2, and potential dispersive interactions between the closely arranged pseudopeptide rings. Pseudopeptide 2 thus represents a promising lead for the construction of phosphate receptors, whose binding selectivity makes use of the unique ability of certain anions to assemble into higher aggregates.

Oxoanion binding to a cyclic pseudopeptide containing 1,4-disubstituted 1,2,3-triazole moieties.

A macrocyclic pseudopeptide 3 is described featuring three amide groups and three 1,4-disubstituted 1,2,3-triazole units along the ring. This pseudopeptide was designed such that the amide NH groups and the triazole CH groups converge toward the cavity, thus creating an environment well suited for anion recognition. Conformational studies in solution combined with X-ray crystallography confirmed this preorganisation. Solubility of 3 restricted binding studies to organic media such as 5 vol% DMSO/acetone or DMSO/water mixtures with a water content up to 5 vol%. These binding studies demonstrated that 3 binds to a variety of inorganic anions in DMSO/acetone including chloride, nitrate, sulfate, and dihydrogenphosphate anions. In the more competitive DMSO/water mixtures, only affinity to the more strongly coordinating oxoanions is retained. Quantitative binding studies showed that dihydrogen phosphate complexation in DMSO/water involves the dimer of the H2PO4- anion. By contrast, sulfate and hydrogenpyrophosphate complexation involves a stepwise process comprising formation of a 1 : 1 complex followed by a 2R : 1A complex in which two molecules of 3 (R) bind to a single anion (A). While the second binding equilibrium is associated with a much smaller stability constant in comparison with the first one in the case of sulfate complexation, the two binding constants are of similar magnitude in the case of the hydrogenpyrophosphate anion. Formation of the 2R : 1A complex was attributed to the fact that the cavity size and rigidity of 3 prevents saturation of all hydrogen acceptor sites on the anionic guests.

Anion-Binding Properties of a Cyclic Pseudohexapeptide Containing 1,5-Disubstituted 1,2,3-Triazole Subunits

A C(3) symmetric cyclic pseudohexapeptide containing 2-aminopicoline-derived subunits and 1,5-disubstituted 1,2,3-triazole rings is introduced as a potent anion receptor. This macrocycle was designed to mimic both the conformation and the receptor properties of a previously described cyclic hexapeptide containing alternating L-proline and 6-aminopicolinic acid subunits. Conformational analyses demonstrate that the cyclic peptide and the cyclic pseudopeptide are structurally closely related. Most importantly, both exhibit a converging arrangement of the NH groups, hence a good preorganization for anion binding. As a consequence, the pseudopeptide also very efficiently interacts with halide and sulfate ions, and this is the case even in competitive aqueous solvent mixtures. However, there are clear differences in the structures of both compounds, which translate into characteristic differences in receptor properties. Specifically, (i) the pseudopeptide possesses an anion affinity intrinsically higher than that of the cyclopeptide, (ii) the pseudopeptide is well preorganized for anion binding in a wider range of solvents from aprotic to protic, (iii) anion affinity in aprotic solvents is very high and associated with complexation equilibria that are slow on the NMR time-scale, (iv) the propensity of the pseudopeptide to form sandwich-type 2:1 complexes with two receptor molecules surrounding one anion is significantly lower than that of the cyclopeptide. A solvent-dependent calorimetric characterization of the binding equilibria of both compounds provided clear evidence for the stabilizing effect of hydrophobic interactions between the receptor subunits in such 2:1 complexes. The pseudopeptide thus represents the first member of a new family of anion receptors whose properties may be fine-tuned by varying the side chains in the periphery of the cavity.

Synthesis of a cyclic pseudopeptide containing a flexible β-Alaψ[CH 2NH]unit

The target cyclic pseudopeptide, cyclo(Tyr-Gly-β-Alaψ[CH 2NH]Ala-Phe-Leu), was efficiently synthesized using a methodology combining solid phase synthesis of the linear pseudopeptide and solution phase cyclization of the linear pseudopeptide.

Cyclic RGD peptide by ring-closing metathesis

A cyclic pseudopeptide containing the RGD sequence has been synthesized in high yield by ring-closing metathesis using a Grubbs’ catalyst in tetrahydrofuran. Conformational analysis has been correlated to structure modeling and preliminary biological activity as an integrin binding inhibitor has been evaluated.

A new cyclic pseudopeptide receptor for Li+ from a dynamic combinatorial library.

A new cyclic pseudopeptide receptor for Li+ from a dynamic combinatorial library.

Identification and Isolation of a Receptor for N-Methyl Alkylammonium Salts: Molecular Amplification in a Pseudo-peptide Dynamic Combinatorial Library.

A cyclic pseudo-peptide receptor for acetylcholine has been amplified and isolated from a dynamic combinatorial library by virtue of templated stabilization under thermodynamic control (see scheme, TFA=trifluoroacetic acid). This is a demonstration of significant molecular amplification in dynamic systems to evolve a novel receptor.

Neurotensin Modulates the Electrical Activity of Frog Pituitary Melanotropes via Activation of a G-Protein-Coupled Receptor Pharmacologically Related to Both the NTS1 and nts2 Receptors of Mammals

The primary structure of frog neurotensin (fNT) has recently been determined and it has been shown that fNT is a potent stimulator of α-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of fNT on the electrical activity of cultured frog melanotropes by using the patch-clamp technique and we have determined the pharmacological profile of the receptors mediating the effect of fNT. In the cell-attached configuration, fNT (10^–7 M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, fNT (10^–7 M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10^–10 to 10^–6 M) of fNT or the C-terminal hexapeptide NT(8–13) caused a dose-dependent increase in the frequency of action potentials with EC₅₀ of 2 × 10^–8 and 5 × 10^–9 M, respectively. The stimulatory effect of fNT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp¹¹]NT(8–13) > Boc-[D-Trp¹¹]NT(8–13) > Boc-[Lys^8,9, Nal¹¹]NT(8–13) > Boc-[Ψ11,12]NT(8–13). In contrast, the cyclic pseudopeptide analogs of NT(8–13), Lys-Lys-Pro-D-Trp-Ile-Leu and Lys-Lys-Pro-D-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10^–5 M) stimulated the spike discharge but did not block the response to fNT. In contrast, SR 142948A (10^–5 M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of fNT. The specific nts2 receptor ligand levocabastine (10^–6 M) had no effect on the basal electrical activity and the response of melanotropes to fNT. In cells which were dialyzed with guanosine-5′-O-(3-thiotriphosphate) (10^–4 M), fNT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5′-O-(2-thiodiphosphate) (10^–4 M) completely blocked the effect of fNT. Pretreatment of cells with cholera toxin (1 µg/ml) or pertussis toxin (0.2 µg/ml) did not affect the electrical response to fNT. Intracellular application of the G_o/i/s protein antagonist GPAnt-1 (3 × 10^–5 M) had no effect on the fNT-evoked stimulation. In contrast, dialysis of melanotropes with the G_q/11 protein antagonist GPAnt-2A (3 × 10^–5 M) abrogated the response to fNT. The present data demonstrate that fNT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by fNT can be accounted for by activation of a G_q/11-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.

A New Cyclic Pseudopeptide Composed of (l)-Proline and 3-Aminobenzoic Acid Subunits as a Ditopic Receptor for the Simultaneous Complexation of Cations and Anions

The synthesis and receptor properties of a cyclic pseudopeptide composed of (l)-proline and the nonnatural amino acid 3-aminobenzoic acid in an alternating sequence are described. The structure of cyclo[(l)Pro-AB]3 was determined in the solid state by X-ray crystallography and in solution by one- and two-dimensional NMR techniques and FT-IR spectroscopy. The cyclic peptide preferentially adopts conformations comparable with the cone conformation of calixarenes. Similar to calixarenes, cyclo[(l)Pro-AB]3 is able to bind cations by cation−π interactions with its aromatic subunits. In some complexes, the peptide NH groups interact additionally with anions and the cyclic peptide thus behaves as a ditopic receptor. The structure of the ternary complex between cyclo[(l)Pro-AB]3 and N-methylquinuclidinium iodide was determined by X-ray crystallography. Spectroscopic investigations show that this complex has a similar geometry in solution. Stability constants of complexes of the cyclopeptide with various ion pairs...

Synthesis and characterization of cyclic pseudopeptide libraries containing thiomethylene and thiomethylene-sulfoxide amide bond surrogates.

We describe the first examples of a series of cyclic pseudopeptide libraries that have been prepared in a systematic approach in order to facilitate both synthesis and subsequent deconvolution attempts. Our synthetic strategy involved the attachment of a trifunctional amino acid (Asp, Asn or Glu) to a polystyrene resin via its side chain, and stepwise chain elongation using either protected amino acids or a pseudodipeptide building block. Head to tail cyclic peptides were formed by removal of the temporary N- and C-terminal protecting groups followed by ring closure by amide formation. Cyclization of the hexa, hepta, and octapseudopeptides on the resin avoided dimer formation, as monitored by mass spectrometry. We utilized a 'psi-scan' approach in which a second fixed position was serially addressed by stepping a dipeptide surrogate, Pro psi [CH2S]Gly around the rings to generate a group of cyclic pseudopeptide sub-libraries. Oxidation of psi [CH2S] to psi [CH2SO] helped validate the synthesis and also provides a strategy for forming a new set of pseudopeptide libraries (previously described as 'libraries from libraries'). Our results suggest that libraries of cyclic pseudopeptides are an efficient method of preparing and assaying these synthetically more challenging entities as potential drug leads.

Pseudopeptide synthesis of a pentaazamacrocycle containing two trans-fused cyclohexane rings

The synthesis of a bis-cyclohexyl-fused pentaazamacrocycle via head-to-tail pseudopeptide cyclization chemistry is described. The bis-cyclohexyl-fused cyclic pseudopeptide 11 displays intramolecular hydrogen bonding which has been characterized by variable temperature NMR spectroscopy and X-Ray Crystallography.

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated ileum and colon.

The affinities of the monocyclic pseudopeptides MEN10,508, MEN10,573, MEN10,581, MEN10,612, MEN10,619 and MEN10,677, and the bicyclic peptides MEN10,627, MEN10,692, MEN10,771, MEN10,882 and MEN10,993 were evaluated at the tachykinin NK2 receptors of the human isolated ileum and colon circular muscle preparations, by using [betaAla8]neurokinin A(4-10) as an agonist. All of the antagonists tested produced a concentration-dependent and competitive antagonism of [betaAla8]neurokinin A(4-10)-mediated contractions in both preparations. MEN10,612 (pKB = 8.1) and MEN10,627 (pKB = 8.4-8.8) were among the most potent analogs within their chemical classes. In general, the bicyclic peptide antagonists were more potent than the monocyclic peptide compounds, showing a nanomolar affinity for the human NK2 receptor. By comparing the affinities shown by the antagonists under study at NK2 receptors of the human gut with the affinities measured at NK2 receptors of the rabbit isolated pulmonary artery and hamster isolated trachea, a high degree of pharmacological homology was found between human and rabbit NK2 receptors. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN10,627, as candidates for testing in pathological conditions characterized by exaggerated gut motility, in which tachykinins might play a role as non-cholinergic excitatory neurotransmitters.

Structure-based design of new constrained cyclic agonists of the cholecystokinin CCK-B receptor.

New constrained cyclic pseudopeptide cholecystokinin-B (CCK-B) agonists have been designed on the basis of conformational characteristics of the potent and selective CCK-B agonist Boc-Trp-(NMe)Nle-Asp-Phe-NH2 (Ki = 0.8 nM, selectivity ratio CCK-A/CCK-B > 6000) (Goudreau et al. Biopolymers, 1994, 34, 155-169). These compounds are among the first successful examples of macrocyclic constrained CCK4 analogs endowed with agonist properties and as such may be of value for the development of nonpeptide CCK-B agonists. The affinities and selectivities of these compounds for CCK-B and CCK-A receptors have been determined in vitro by measuring the displacement of [3H]pCCK8 binding to guinea pig cortex and pancreas membranes, respectively. The most potent compound, 8b, N-(cycloamido)-alpha-Me(R)Trp-[(2S)-2-amino-9- ((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH2, has a Ki value of 15 +/- 1 nM for guinea pig cortex membranes with a good CCK-B selectivity ratio (CCK-A/CCK-B = 147). Furthermore, 8b behaved as a potent and full agonist in a functional assay which measures the stimulation of inositol phosphate accumulation in CHO cells transfected with the rat CCK-B receptor (EC50 = 7 nM). The in vivo affinity of 8b for mouse brain CCK-B receptors was determined following intracerebroventricular injection (ID50 approximately 29 nmol/kg). 8b was also shown to cross the blood-brain barrier (0.16%), after intravenous administration in mice. 8b also increased gastric acid secretion measured in anesthetized rats after intravenous injection. Therefore, 8b appears to be an interesting pharmacological tool and is currently under investigation as a lead for further development of nonpeptide CCK-B agonists.

A systematic approach to the solid-phase synthesis of linear and cyclic pseudopeptide libraries containing psi[CH2NH] amide bond surrogates.

A systematic approach has been adopted for the synthesis and characterization of a series of linear and cyclic pseudopeptide mixtures containing the psi[CH2NH] amide replacement. The parent structures were based on biologically relevant compounds including an enkephalin analog, H-Tyr-D-Ala-Gly-Phe-Leu-OH, and an Arg-Gly-Asp peptide sequence. The linear mixtures containing 4 and 64 pseudopeptide components with 1, 2 or 3 amide bond surrogates were synthesized using Boc-SPPS. The amount of desired linear pseudopeptides in the mixtures ranged from 67 to 90% as determined by integration of HPLC peak areas. Comparative studies indicated: (i) racemization is not a problem in the synthesis of pseudopeptide mixtures containing the psi[CH2NH] surrogate; and (ii) protection of the psi[CH2NH] surrogate with a benzyloxycarbonyl group during the synthesis is beneficial. Cyclic mixtures containing 4 and 256 cyclic pseudopeptide components with a single amide bond surrogate were synthesized using a resin-bound cyclization approach featuring side-chain attachment of Boc-Asp-OFm to the solid support. Cyclization kinetic studies revealed that the newly developed HATU coupling reagent provided a fast cyclization rate for a pseudopeptide mixture and that the position of the reduced peptide bond within a peptide mixture had only a small effect on the cyclization rates of the mixture. Pseudopeptide libraries permit the more efficient bioassay of complex structures and can also be used to reveal more rapidly trends in physicochemical variables. For example, we observed that the expected increase in hydrophilicity with psi[CH2NH] substitutions during RP-HPLC analysis did not continue with several such replacements.

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated urinary bladder.

1. We have studied several tachykinin NK2 receptor antagonists, bearing a monocyclic pseudopeptide (MEN 10,508, MEN 10,573, MEN 10,581, MEN 10,612, MEN 10,619 and MEN 10,677), or bicyclic peptide (MEN 10,627, MEN 10,692, MEN 10,771, MEN 10,882 and MEN 10,993) structure, on the human isolated urinary bladder detrusor muscle against neurokinin A as an agonist, and compared their affinities in this preparation with those for NK2 receptors expressed in the rabbit isolated pulmonary artery and hamster isolated trachea. 2. In the human bladder, all the antagonists tested produced a concentration-dependent and competitive antagonism of neurokinin A-mediated contractions: among the cyclic pseudopeptides MEN 10,677 (pKB = 8.0) was the most potent antagonist, while among the bicyclic analogues it was MEN 10,993 (pKB = 8.8). 3. In general, the bicyclic peptide antagonists tested were more potent than the monocyclic pseudopeptide compounds, either in the human urinary bladder or in the rabbit pulmonary artery or hamster trachea, showing a nanomolar affinity for the human NK2 receptor. 4. A highly significant correlation was found between the estimated pKB values of all the antagonists tested in the human urinary bladder and rabbit pulmonary artery (r2 = 0.94, n = 12, P < 0.01), whereas no linear correlation was found between pKB values measured in the human urinary bladder and hamster trachea (r2 = 0.52, n = 12, P > 0.05): these observations provide further pharmacological evidence for receptor homology between the human and rabbit NK2 receptor. 5. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN 10,627, as candidates for testing in pathological conditions, such as bladder hyperactivity, for which preclinical evidence indicates that a therapeutic effect could result from the block of the tachykinin NK2 receptor.

A ?-turn mimic and a thiomethylene dipeptide surrogate employed in the study of cyclic peptide RGD and RCD cell-adhesion inhibitors

During an inflammatory response, leukocytes adhere to the blood-vessel wall and to underlying extracellular matrix-protein fibronectin via noncovalent interaction through two distinct cell-surface integrins, α5β1 and α4β1. An Arg-Gly-Asp tripeptide in the cell-binding domain of fibronectin has been demonstrated to be the major site that mediates cell attachment. Our group has previously disclosed Arg-Gly-Asp- and Arg-Cys-Asp-containing cyclic peptide cell-adhesion inhibitors, which are potentially useful as anti-inflammatory agents, particularly in the treatment of asthma and rheumatoid arthritis. Subsequently, we have incorporated a β-turn dipeptide mimic and a suitably protected Arg-Gly thiomethylene dipeptide surrogate into our lead peptides by solid-phase synthesis. The preparation of the surrogates, and the structure-activity studies of the surrogate-containing peptides are described in this paper. Complete loss of cell-adhesion inhibition activity was observed in the case of incorporation of the conformationally constrained β-turn dipeptide into a cyclic Arg-Cys-Asp peptide inhibitor. Incorporation of an Arg-Gly thiomethylene dipeptide surrogate into cyclic Arg-Gly-Asp and Arg-Cys-Asp cyclic disulfide peptide inhibitors was tolerated, resulting in cyclic pseudopeptide cell-adhesion inhibitors lacking the Arg-Gly peptide amide bond.

P-C3-07 Ion-channels of alamethicins assembled with a cyclic pseudopeptide: Asami K, 1 Matsubara A, 1 Akagi A, 2 Nishino N. 2 1 Inst. Chem. Res., Kyoto University, 2 Dept. of Applied Chemistry, Kyushu Institute of Technology

P-C3-07 Ion-channels of alamethicins assembled with a cyclic pseudopeptide: Asami K, 1 Matsubara A, 1 Akagi A, 2 Nishino N. 2 1 Inst. Chem. Res., Kyoto University, 2 Dept. of Applied Chemistry, Kyushu Institute of Technology