Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides

The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability. They have a wide range of biological activities that make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (M?bius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure-activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity: derivatives in which the Trp residue was modified showed low effect, demonstrating the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the M?bius cyclotides. Despite these differences between cyclotide subfamilies, a systematic test of mixtures of cyclotides revealed that they act in an additive way.     

Isolation and Characterization of Bioactive Cyclotides from Viola labridorica

Many Violaceae plants contain cyclotides, which are plant cyclopeptides distinguished by a cyclic cystine knot motif with 28–37 amino acid residues. In the current study, four new cyclotides, vila A–D ( 1 – 4 , resp.), together with a known cyclotide, varv D ( 5 ), were isolated from Viola labridorica (Violaceae). A chromatography‐based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and 2D‐NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, i.e., U251, MDA‐MB‐231, A549, DU145, and BEL‐7402, with vila A and B ( 1 and 2 , resp.) being the most cytotoxic. The isolated cyclotides showed no antibacterial activity against Staphyloccocus aureus and Candida albicans. Homology modeling of the cyclotide structures was used to analyze structure–activity relationships.     

A liquid chromatography-electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation

Cyclotides are plant-produced, bioactive, cyclic mini-proteins with interesting pharmaceutical and agricultural applications. A reverse phase liquid chromatography electrospray ionization mass spectrometry (RP-LC-ESI-MS) method for analysis of cyclotides in plant materials with a minimum of sample pre-treatment is presented. Three exemplary cyclotides (kalata B1, kalata B2 and cycloviolacin O2) were used as reference substances for the method development. Linearity (r(2)>0.99) was achieved in the concentration range 0.05-10 mg/L and the limit of detection was 1.7-4.0 μg/L. The present study is the first to demonstrate that cyclotides dissolved in water sorb to glass vials, but the addition of 15% of acetonitrile or 40 mg/L of bovine serum albumin is sufficient to keep the cyclotides in solution. Cyclotides were extracted from candied violets, violet tea, and the plants Oldenlandia affinis and Viola odorata using 70% methanol containing 0.1% formic acid (v/v). The plant content was determined to be 23.5-14,200 μg/g (dry weight). The highest content of cyclotide was found in wild Danish V. odorata, and it is the highest content of cyclotide in a plant reported hitherto. Candied violets contained 0.00-8.66 μg/g (dry weight), while no cyclotides were detected in commercial violet tea.     

meso‐Aryl [20]π Homoporphyrin: The Simplest Expanded Porphyrin with the Smallest Möbius Topology

An unstable conjugated homoporphyrin was successfully stabilized by introducing meso ‐aryl substitutents. It was evident from the moderate diatropic ring current found by NMR analysis that the newly formed 20π conjugated free base and its protonated form exhibited Möbius aromatic character. Furthermore, complexation as a ligand with an Rh^I ion afforded a unique binding mode and retained the Möbius aromaticity. Overall, these compounds are the smallest Möbius aromatic molecules, as confirmed by spectral and crystal‐structure analysis and supported by theoretical studies.     

Novel strategies for isolation and characterization of cyclotides: the discovery of bioactive macrocyclic plant polypeptides in the Violaceae

This review focuses on the discovery of cyclotides in the Violaceae, their isolation and their anti-cancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solvent-solvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.     

Bond-alternating Hückel-Möbius and related,twisted linear,cyclic and helical systems—their molecular orbitals,energies and phase correlation upon dissociation

Cyclic group formalism and screw symmetry operation are used to clarify and generalize the definition of Hückel and Möbius systems. It is shown that the Möbius ring system has half-integral pseudo-angular momentum similar to that of spin space, and that applications of Möbius electronics to chemical reactions have been based on truncated single-circle Möbius rings which have unique beginning and end (Sect. 2). This concept is illustrated by application to the [1, 7] antarafacial hydrogen shift (Appendix A and Figs. A1–A3). Definition of a Hüickel versus Möbius ring system for in-plane and out-of-plane π, δ and φ orbitals as well as the appropriate relative angle of twists are given (Sect. 2 and Table 1). Using the concept of the compatibility of the twist (screw) angle and rotation around a ring, we also derive the proper phase coherence and energy correlation between a parent cyclic (Hückel or Möbius) molecule and its dissociated linear fragments (Sect. 4). The concept of parentage in diabatic fragmentation is discussed. Forfinite, open, helical chain molecules, an exact periodic boundary condition based on the compatibility of twist angle and number of turns in a helical ring parent molecule is applied to derive their analytic wave functions (Sect. 5 and Table 2). Forbond-alternating “linear” and cyclic Hückel and Möbius systems we also derive the explicit LCAO-MO wavefunctions, energies, their degeneracies and their exact corresponding quantum members for even and odd atom systems at highest bonding and lowest antibonding levels (Sect. 3, Figs. 1–3). The corresponding wavefunctions and energies for uniform-bond systems are given for comparison and for completeness (Sect. 3).     

Viability of Möbius Topologies in [26]‐ and [28]Hexaphyrins

Recently, hexaphyrins have emerged as a promising class of π‐conjugated molecules that display a range of interesting electronic, optical, and conformational properties, including the formation of stable Möbius aromatic systems. Besides the Möbius topology, hexaphyrins can adopt a variety of conformations with Hückel and twisted Hückel topologies, which can be interconverted under certain conditions. To determine the optimum conditions for viable Möbius topologies, the conformational preferences of [26]‐ and [28]hexaphyrins and the dynamic interconversion between the Möbius and Hückel topologies were investigated by density functional calculations. In the absence of meso substituents, [26]hexaphyrin prefers a planar dumbbell conformation, strongly aromatic and relatively strain free. The Möbius topology is highly improbable: the most stable tautomer is 33 kcal mol^?1 higher in energy than the global minimum. On the other hand, the Möbius conformer of [28]hexaphyrin is only 6.5 kcal mol^?1 higher in energy than the most stable dumbbell conformation. This marked difference is due to aromatic stabilization in the Möbius 4n electron macrocycle as opposed to antiaromatic destabilization in the 4n+2 electron system, as revealed by several energetic, magnetic, structural, and reactivity indices of aromaticity. For [28]hexaphyrins, the computed activation barrier for interconversion between the Möbius aromatic and Hückel antiaromatic conformers ranges from 7.2 to 10.2 kcal mol^?1, in very good agreement with the available experimental data. The conformation of the hexaphyrin macrocycle is strongly dependent on oxidation state and solvent, and this feature creates a promising platform for the development of molecular switches.     

Fragmentation reactions of deprotonated peptides containing proline. The proline effect

The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the 'b(2) ion; further evidence is presented that the 'b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline.     

[32]π Fused Core‐Modified Heptaphyrin with Möbius Aromaticity

A new fused core‐modified 32π heptaphyrin with Möbius aromatic character is reported. The ¹H NMR data indicated a weak Möbius aromaticity at 298 K; however, at 213–183 K, the molecule predominates [4n]π Möbius conformation with strong diatropic ring current, which was further confirmed by X‐ray analysis. The protonation experiment led to preservation of the Möbius aromaticity at 298 K. Nevertheless, the experimental results were further supported by theoretical studies. Overall, this study represents the first example of Möbius aromatic fused core‐modified expanded porphyrin.     

ESI‐MS/MS of expanded porphyrins: a look into their structure and aromaticity

Electrospray mass spectrometry/mass spectrometry was used to investigate the gas‐phase properties of protonated expanded porphyrins, in order to correlate those with their structure and conformation. We have selected five expanded meso‐pentafluorophenyl porphyrins, respectively, a pair of oxidized/reduced fused pentaphyrins (22 and 24 π electrons), a pair of oxidized/reduced regular hexaphyrins (26 and 28 π electrons) and a regular doubly N‐fused hexaphyrin (28 π electrons). The gas‐phase behavior of the protonated species of oxidized and reduced expanded porphyrins is different. The oxidized species (aromatic Hückel systems) fragment more extensively, mainly by the loss of two HF molecules. The reduced species (Möbius aromatic or Möbius‐like aromatic systems) fragment less than their oxidized counterparts because of their increased flexibility. The protonated regular doubly fused hexaphyrin (non‐aromatic Hückel system) shows the least fragmentation even at higher collision energies. In general, cyclization through losses of HF molecules decreases from the aromatic Hückel systems to Möbius aromatic or Möbius‐like aromatic systems to non‐aromatic Hückel systems and is related to an increase in conformational distortion. Copyright © 2016 John Wiley & Sons, Ltd.     

Aromaticity Reversal in the Lowest Excited Triplet State of Archetypical Möbius Heteroannulenic Systems

The aromaticity reversal in the lowest triplet state (T₁) of a comparable set of Hückel/Möbius aromatic metalated expanded porphyrins was explored by optical spectroscopy and quantum calculations. In the absorption spectra, the T₁ states of the Möbius aromatic species showed broad, weak, and ill‐defined spectral features with small extinction coefficients, which is in line with typical antiaromatic expanded porphyrins. In combination with quantum calculations, these results indicate that the Möbius aromatic nature of the S₀ state is reversed to Möbius antiaromaticity in the T₁ state. This is the first experimental observation of aromaticity reversal in the T₁ state of Möbius aromatic molecules.     

Enumeration of Möbius type cyclic polyazulenoids

A Möbius type cyclic polyazulenoid is a kind of nonalternant conjugated hydrocarbon, consisting of a cyclic series of alternatingly fused five membered rings and seven membered rings, which forms a “Möbius belt of rings” or say, “Möbius belt of azulenes”, more precisely. As a sequel of the recent paper (Deng and Zhang in J Math Chem 54(2):416–427, 2016), which counts “belt type” cyclic polyazulenoid, this article studies the enumeration problem of Möbius type cyclic polyazulenoid. We obtain the exact counting formula for the number of Möbius type cyclic polyazulenoids with n azulene units, by a method based on coding technique and a generalization of ‘Burnside’ lemma. And we give the numerical results for n not larger than 20, which may contribute to the fully synthesis of Möbius type cyclic polyazulenoids.     

The Extension of Baird's Rule to Twisted Heteroannulenes: Aromaticity Reversal of Singly and Doubly Twisted Molecular Systems in the Lowest Triplet State

We have investigated the aromaticity of singly twisted Möbius aromatic and doubly twisted Hückel antiaromatic bis(palladium(II)) [36]octaphyrins in the lowest triplet state (T₁) by spectroscopic measurements and quantum calculations. The T₁ state of the singly twisted Möbius [36]octaphyrin shows broad and weak absorption spectral features that are analogous to those of antiaromatic expanded porphyrins while the T₁ state of the doubly twisted Hückel [36]octaphyrin exhibits intense and distinct spectral features, indicating the aromatic nature. These results along with theoretical calculations support the hypothesis that the aromaticity is reversed in the T₁ state. Furthermore, we show that the degree of structural smoothness affects the aromaticity reversal in the T₁ state.     

Probing the relationship between spin contamination and first hyperpolarizability: Open‐shell Möbius anion

Möbius strip has attracted extensive interest due to its one‐side structure, special physical and chemical properties. A new extended porphyrin with Möbius structure has been synthesized (St?pień et al., Angew. Chem. Int. Ed. 2007, 46, 7869). In this work, an electron is injected into Möbius to form Möbius anion. For Möbius, four methods (B3LYP, BHandHLYP, CAM‐B3LYP, and LC‐BLYP) obtain the similar first hyperpolarizability (β₀). However, for Möbius anion, four methods obtain very different β₀ values (ranging from 9.85 × 10³ to 8.33 × 10⁵ au). Moreover, the β₀ values of Möbius anion are larger than those of Möbius. Why? The considerable variation (ranging from 0.8162 to 1.7630) of spin contamination (S²) of open‐shell Möbius anion captures our attention. To probe the relationship between S² and β₀, the β₀ values of Möbius anion were calculated using 23 methods. Interestingly, the Hartree‐Fock (HF) composition increases from 0 to 50, the S² value ranges from 0.7522 to 1.3372, and the β₀ value increases from 4.3 × 10³ to 7.36 × 10⁴ au. Further, methods are “dissected” to investigate the effect of HF composition on the S². © 2014 Wiley Periodicals, Inc.     

Optical Physical Mechanisms of Helicene Carbon Nanohoop with Möbius Topology

Optical and spectral properties of carbon nanohoop with Möbius topology is of great interest in nano-science and nano-technology. And it can be imagined that it has a lot of unexpected potential application prospects. However, theoretical calculations based on some figure-of-eight helicene carbon nanohoop with Möbius topology are still insufficient. Therefore, in this paper, we theoretically study the optical and spectral properties of figure-of-eight helicene carbon nanohoop with Möbius topology. Optical and spectral properties are analyzed with visualization method of transition density matrix and charge density difference, which reveal the unique characterization of carbon nanohoop with Möbius topology. Our results can not only deepen the understanding of the optical physical mechanisms of the nanorings with mobius carbons, but also provide deeper insight on optical properties and potential design on optical nanodevices.     

Anti-HIV cyclotides

The cyclotides are a recently discovered, structurally unique family of bioactive plant peptides. Their discovery spawned a series of structural analyses, synthetic efforts, and studies to define the biosynthesis and biological properties of these novel peptide metabolites. Cyclotides have a head-to-tail cyclized amino acid backbone and a conserved cystine knot motif that provides an extremely stable structural framework. They all share a common global fold and are highly resistant to denaturation and to cleavage by proteolytic enzymes. However, these macrocyclic peptides are quite permissive to amino acid substitutions or additions in several peripheral loop regions, since changes in these loops do not alter the core cyclotide structure. These features make cyclotides attractive templates for incorporating desired amino acid sequences and then delivering these peptide sequences in a well defined, highly stable framework. Cyclotides likely function in a defensive role in the source plants since they exhibit a broad spectrum of antimicrobial activity and are detrimental to the growth and survival of herbivorous insects. Cyclotides are gene-encoded polypeptides that are cleaved from larger precursor proteins and then cyclized. This review summarizes research done on a subset of cyclotides that were discovered due to their HIV inhibitory properties. It details the isolation and characterization of these compounds and describes this work in the context of our current state of knowledge of the entire cyclotide family.     

Hückel and möbius boundary conditions for solids and orbital symmmetry correlation in the rotational phase transition of molecular crystals

A new cyclic boundary condition which corresponds to a Möbius strip representation of a one-dimensional crystal is introduced. It is compared with the usual Bloch and Born—von Karman boundary condition which is shown to be a Hückel condition in the sense of LCAO MO treatment of a ring structure. The potential relevance of this Möbius condition to one-dimensional molecular and liquid crystals in which the relative molecular orientation changes during phase transition is alluded to. A comparison of the energies for the twisted and non-twisted form of the linear crystal is derived in the LCAO approximation. The orbital symmetry correlation in the concerted twist of the atomic or molecular orbitals atom! the linear backbone during a rotational polymorphic structural transition is also derived.     

Topology Switching in [32]Heptaphyrins Controlled by Solvent,Protonation, and meso Substituents

The switching of topology between “figure‐eight”, Möbius, and untwisted conformations in [32]heptaphyrins(1.1.1.1.1.1.1) has been investigated by using density functional theory calculations. Such a change is achieved by variation of one internal dihedral angle and, if properly controlled, can provide access to molecular switches with unique optical and magnetic properties. In this work, we have explored different conformational control methods, such as solvent, protonation and meso substituents. Despite its antiaromatic character, most of the [32]heptaphyrins (R=H, CH₃, CF₃, Ph, C₆F₅) adopt a figure‐eight conformation in the neutral state, owing to their more‐effective hydrogen‐bonding interactions. The aromatic Möbius topology is only preferred with dichlorophenyl groups, which minimize the steric hindrance that arises from the bulky chlorine atoms. The conformational equilibrium is sensitive to the solvent, so polar solvents, such as DMSO, further stabilize the Möbius conformation. Protonation induces a conformational change into the Möbius topology, irrespective of the meso‐aryl groups. In the triprotonated species, the conformational switch is blocked and a non‐twisted conformer becomes much more stable than the figure‐eight conformation. We have shown that the relative energies of the protonated [32]heptaphyrins are dominated by aromaticity. Importantly, this topology switching induces a dramatic change in the magnetic properties and reactivity of the macrocycles, as revealed by several energetic, magnetic, structural, and reactivity indices of aromaticity.     

The chemistry of cyclotides

Cyclotides are head-to-tail cyclic peptides that contain a cystine knot motif built from six conserved cysteine residues. They occur in plants of the Rubiaceae, Violaceae, Cucurbitaceae, and Fabaceae families and, aside from their natural role in host defense, have a range of interesting pharmaceutical activities, including anti-HIV activity. The variation seen in sequences of their six backbone loops has resulted in cyclotides being described as a natural combinatorial template. Their exceptional stability and resistance to enzymatic degradation has led to their use as scaffolds for peptide-based drug design. To underpin such applications, methods for the chemical synthesis of cyclotides have been developed and are described here. Cyclization using thioester chemistry has been instrumental in the synthesis of cyclotides for structure-activity studies. This approach involves a native chemical ligation reaction between an N-terminal Cys and a C-terminal thioester in the linear cyclotide precursor. Since cyclotides contain six Cys residues their syntheses can be designed around any of six linear precursors, thus providing flexibility in synthesis. The ease with which cyclotides fold, despite their topologically complex knot motif, as well as the ability to introduce combinatorial variation in the loops, makes cyclotides a promising drug-design scaffold.