Facile Peptide Macrocyclization and Multifunctionalization via Cyclen Installation.

Cyclen-peptide bioconjugates are usually prepared in multiple steps that require individual preparation and purification of the cyclic peptide and hydrophilic cyclen derivatives. An efficient strategy is discovered for peptide cyclization and functionalization toward lanthanide probe via three components intermolecular crosslinking on solid-phase peptide synthesis with high conversion yield. Multifunctionality can be conferred by introducing different modular parts or/and metal ions on the cyclen-embedded cyclopeptide. As a proof-of-concept, a luminescent Eu3+ complex and a Gd3+-based contrasting agent for in vitro optical imaging and in vivo magnetic resonance imaging, respectively, are demonstrated through utilizing this preparation of cyclen-embedded cyclic arginylglycylaspartic acid (RGD) peptide.

Structural Variations in Carboxylated Bispidine Ligands: Influence of Positional Isomerism and Rigidity on the Conformation, Stability, Inertness and Relaxivity of their Mn2+ Complexes.

Mn2+ complexes of 2,4-pyridyl-disubstituted bispidine ligands have emerged as more biocompatible alternatives to Gd3+ -based MRI probes. They display relaxivities comparable to that of commercial contrast agents and high kinetic inertness, unprecedented for Mn2+ complexes. The chemical structure, in particular the substituents on the two macrocyclic nitrogens N3 and N7, are decisive for the conformation of the Mn2+ complexes, and this will in turn determine their thermodynamic, kinetic and relaxation properties. We describe the synthesis of four ligands with acetate substituents in positions N3, N7 or both. We evidence that the bispidine conformation is dependent on N3 substitution, with direct impact on the thermodynamic stability, kinetic inertness, hydration state and relaxivity of the Mn2+ complexes. These results unambiguously show that (i) solely a chair-chair conformation allows for favorable inertness and relaxivity, and (ii) in this family such chair-chair conformation is accessible only for ligands without N3-appended carboxylates.

Direct Construction of Corrole-Peptide Conjugates by Controllable Bilane Formation on Resin.

Corroles have attracted increasing research interests in recent decades owing to their unique properties over porphyrins. However, the relatively inefficient and tedious synthetic procedures of corrole building blocks with functional groups for bioconjugation hindered their bioapplications. Herein, we report a highly efficient protocol to synthesize corrole-peptide conjugates with good yields (up to 63 %) without using prepared corrole building blocks. By condensing two -COOH-bearing-dipyrromethane molecules onto an aldehyde group on resin-bound peptide chains in a controllable manner, a series of desired products with long (up to 25 residues) and bioactive peptide chains were obtained with at most one chromatographic purification. The synthesized compounds exhibited potential applications as chelators for metal ions for biomedical applications, as building blocks for supramolecular materials, as well as targeted fluorescent probes.

Energy Transfer Mechanism and Quantitative Modeling of Rate from an Antenna to a Lanthanide Ion.

The excitation energy transfer (ET) pathway and mechanism from an organic antenna to a lanthanide ion has been the subject of discussion for many decades. In the case of europium (Eu3+), it has been suggested that the transfer originates from the ligand singlet state or a triplet state. Taking the lanthanide complex Eu(TTA)3(H2O)2 as an example, we have investigated the spectra and luminescence kinetics, mainly at room temperature and 77 K, to acquire the necessary experimental data. We put forward an experimental and theoretical approach to measure the energy transfer rates from the antenna to different Eu3+ levels using the Dexter formulation. We find that transfer from the ligand singlet state to Eu3+ may account for the ET pathway, by combined electric dipole-electric dipole (ED-ED) and ED-electric quadrupole (EQ) mechanisms. The contributions from the triplet state by these mechanisms are very small. An independent systems rate equation approach can effectively model the experimental kinetics results. The model utilizes the cooperative processes that take place on the metal ion and ligand and considers S0, S1, and T1 ligand states in addition to 7F0,1, 5D0, 5D1, and 5DJ (=5L6, 5D3, 5D2 combined) Eu3+ states. The triplet exchange ET rate is estimated to be of the order 107 s-1. The observation of a nanosecond risetime for the Eu3+ 5D1 level does not enable the assignment of the ET route or the mechanism. Furthermore, the 5D1 risetime may be contributed by several processes. Observation of its temperature dependence and also that of the ground-state population can supply useful information concerning the mechanism because the change in metal-ion internal conversion rate has a greater effect than changes in singlet or triplet nonradiative rates. A critical comparison is included for the model of Malta employed in the online software LUMPAC and JOYSpectra. The theoretical treatment of the exchange mechanism and its contribution are now being considered.

Upconversion in a d-f [RuYb3] Supramolecular Assembly.

We have prepared a hetero-tetrametallic assembly consisting of three ytterbium ions coordinated to a central [Ru(bpm)3]2+ (bpm = 2,2'-bipyrimidine) motif. Irradiation into the absorption band of the peripheral ytterbium ions at 980 nm engenders emission of the 3MLCT state of the central [Ru(bpm)3]2+ core at 636 nm, which represents the first example of f → d molecular upconversion (UC). Time-resolved measurements reveal a slow rise of the UC emission, which was modeled with a mathematical treatment of the observed kinetics according to a cooperative photosensitization mechanism using a virtual Yb centered doubly excited state followed by energy transfer to the Ru centered 1MLCT state.

Versatile Synthesis of Multivalent Porphyrin-Peptide Conjugates by Direct Porphyrin Construction on Resin.

Multifunctional porphyrin-peptide conjugates with different propensities for self-assembly into various supramolecular nanoarchitectures play important roles in advanced materials and biomedical research. However, preparing prefunctionalized core porphyrins by traditional low-yielding statistical synthesis and purifying them after peptide ligation through many rounds of HPLC purification is tedious and unsustainable. Herein, we report a novel integrated solid-phase synthetic protocol for the construction of porphyrin moieties from simple aldehydes and dipyrromethanes on resin-bound peptides directly to form mono-, cis/trans-di-, and trivalent porphyrin-peptide conjugates in a highly efficient and controllable manner; moreover, only single final-stage HPLC purification of the products is needed. This efficient strategy enables the rapid, greener, and substrate-controlled diversity-oriented synthesis of multivalent porphyrin-(long) peptide conjugate libraries for multifarious biological and materials applications.

Thermally Activated Photophysical Processes of Organolanthanide Complexes in Solution.

The effect of temperature upon the lanthanide luminescence lifetime and intensity has been investigated in toluene solution for the complexes LnPhen(TTA)3 (Ln = Eu, Sm, Nd, Yb; Phen = 1,10-phenanthroline; TTA = thenoyltrifluoroacetonate). Thermally excited back-transfer to a charge transfer state was found to occur for Ln = Eu and can be explained by lifetime and intensity back-transfer models. The emission intensity and lifetime were also quenched with increasing temperature for Ln = Sm, and the activation energy for nonradiative decay is similar to that for the thermal population of Sm3+ excited states. Unusual behavior for lifetime and intensity was found for both Ln = Nd, Yb. The usually assumed equivalence of τ/τ0 = I/I0 (where τ is lifetime and I is intensity) does not hold for these cases. We infer that for these lanthanide systems the intensity decreases with temperature in the stage prior to population of the luminescent state. The lifetime changes are discussed.

Charging and ultralong phosphorescence of lanthanide facilitated organic complex.

Emission from the triplet state of an organo-lanthanide complex is observed only when the energy transfer to the lanthanide ion is absent. The triplet state lifetime under cryogenic conditions for organo-lanthanide compounds usually ranges up to tens of milliseconds. The compound LaL1(TTA)3 reported herein exhibits 77 K phosphorescence observable by the naked eye for up to 30 s. Optical spectroscopy, density functional theory (DFT) and time-dependent DFT techniques have been applied to investigate the photophysical processes of this compound. In particular, on-off continuous irradiation cycles reveal a charging behaviour of the emission which is associated with triplet-triplet absorption because it shows a shorter rise lifetime than the corresponding decay lifetime and it varies with illumination intensity. The discovery of the behaviour of this compound provides insight into important photophysical processes of the triplet state of organo-lanthanide systems and may open new fields of application such as data encryption, anti-counterfeiting and temperature switching.

Orbital transitions: insight into energy transfer through an antenna for an organo-lanthanide complex.

Supported by experimental work, wavefunction theory (WFT) calculations and density functional theory (DFT) calculations employing a range of functionals have been performed for two lanthanide complexes to investigate, in gas and solution phases, the representations of frontier orbitals and the orbital transitions between singlet states. The orbital transitions calculated using CASSCF/NEVTP2 served as reference. Functionals with a higher proportion of Hartree-Fock exchange gave better agreement with WFT. The choice of functional is therefore important for understanding the nature of orbital transitions and this is especially relevant in formulating antenna-metal ion energy transfer (ET) mechanisms.

Lanthanide-Based Peptide-Directed Visible/Near-Infrared Imaging and Inhibition of LMP1.

A lanthanide-based peptide-directed bioprobe LnP19 (Ln = Eu or Yb) is designed as an impressive example of a small molecule-based dual-functional probe for the EBV oncoprotein LMP1. The peptide P19 (Pra-KAhx-K-LDLALK-FWLY-K-IVMSDKW-K-RrRK) is designed to selectively bind to LMP1 by mimicking its TM1 region during oligomerization in lipid rafts while signal transduction is significantly suppressed. Immunofluorescence imaging and Western blotting results reveal that P19 can effectively inactivate the oncogenic cellular pathway nuclear factor κB (NF-κB) and contribute to a selective cytotoxic effect on LMP1-positive cells. By conjugation with cyclen-based europium(III) and ytterbium(III) complexes, EuP19 and YbP19 were constructed to offer visible and near-infrared LMP1-targeted imaging and cancer monitoring. In addition to the ability to target and inhibit LMP1 and to selective inhibit LMP1-positive cells, selective growth inhibition toward the LMP1-positive tumor by LnP19 is also demonstrated.

Solid-Phase Peptide Macrocyclization and Multifunctionalization via Dipyrrin Construction.

We introduce a new and highly efficient synthetic protocol towards multifunctional fluorescent cyclopeptides by solid-phase peptide macrocyclization via dipyrrin construction, with full scope of proteinogenic amino acids and different ring sizes. Various bicyclic peptides can be created by dipyrrin-based crosslinking and double dipyrrin-ring formation. The embedded dipyrrin can be either transformed to fluorescent BODIPY and then utilized as cancer-selective targeted protein imaging probe in vitro, or directly employed as a selective metal sensor in aqueous media. This work provides a valuable addition to the peptide macrocyclization toolbox, and a blueprint for the development of multifunctional dipyrrin linkers in cyclopeptides for a wide range of potential bioapplications.

Solid-phase fluorescent BODIPY-peptide synthesis via in situ dipyrrin construction.

Traditional fluorescent peptide chemical syntheses hinge on the use of limited fluorescent/dye-taggable unnatural amino acids and entail multiple costly purifications. Here we describe a facile and efficient protocol for in situ construction of dipyrrins on the N-terminus with 20 natural and five unnatural amino acids and the lysine's side chain of selected peptides/peptide drugs through Fmoc-based solid-phase peptide synthesis. The new strategy enables the direct formation of boron-dipyrromethene (BODIPY)-peptide conjugates from simple aldehyde and pyrrole derivatives without pre-functionalization, and only requires a single-time chromatographic purification at the final stage. As a model study, synthesized EBNA1-targeting BODIPY1-Pep4 demonstrates intact selectivity in vitro, responsive fluorescence enhancement, and higher light cytotoxicity due to the photo-generation of cytotoxic singlet oxygen. This work offers a novel practical synthetic platform for fluorescent peptides for multifaceted biomedical applications.

Reactivation of Epstein-Barr virus by a dual-responsive fluorescent EBNA1-targeting agent with Zn2+-chelating function.

Epstein-Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein-Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn2+-responsive probe (ZRL5P4) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn2+ chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL5P4 can respond independently to its interactions with Zn2+ and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization and oriP-enhanced transactivation of EBNA1. ZRL5P4 can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL5P4 alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL5P4 is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

Synthesis of the Tricyclic Picrotoxane Motif by an Oxidative Cascade Cyclization.

An oxidative cascade cyclization of ß-keto esters has been developed for the construction of the tricyclic picrotoxane motif in a single step, and DFT calculations suggested a possible cationic cyclization mechanism. This cascade cyclization can be operated on a 20 g scale to obtain a 77% total yield of the tricyclic products, which in turn can be converted to versatile intermediates for further elaboration to picrotoxanes and their structurally related compounds.

Substrate Sequence Determines Catalytic Activities, Domain-Binding Preferences, and Allosteric Mechanisms in Pin1.

Pin1 is a unique phosphorylation-dependent peptidyl-prolyl isomerase that regulates diverse subcellular processes and an important potential therapeutic target. Functional mechanisms of Pin1 are complicated because of the two-domain structural organization: the catalytic domain both binds the specific pSer/Thr-Pro motif and catalyzes the cis/trans isomerization, whereas the WW domain can only bind the trans configuration and is speculated to be responsible for substrate-binding specificity. Numerous studies of Pin1 have led to two divergent conclusions on the functional role of the WW domain. One opinion states that the WW domain is an allosteric effector, and substrate binding to this domain modulates the binding and catalysis in the distal catalytic domain. The other opinion, however, argues that the WW domain does not have any allosteric role. Here, using molecular dynamics and binding free-energy calculations, we examine catalysis and allosteric mechanisms in Pin1 under various substrate- and WW-binding conditions. Our results reveal a strong substrate sequence dependency of catalysis, domain-binding preferences, and allosteric outputs in Pin1. Importantly, we show that the different opinions about the WW domain can be unified in one framework, in which substrate sequences determine whether a positive, negative, or neural allosteric effect will be elicited. Our work further elucidates detailed mechanisms underlying the sequence-dependent allostery of Pin1 and finds that interdomain contacts are key mediators of intraprotein allosteric communications. Our findings collectively provide new insights into the function of Pin1, which may facilitate the development of novel therapeutic drugs targeting Pin1 in the future.