One-Pot Peptide Ligation–Oxidative Cyclization Protocol for the Preparation of Short-/Medium-Size Disulfide Cyclopeptides

Native chemical ligation (NCL) employing the <i>N</i>-methylbenzimidazolinone (MeNbz) linker readily provided the linear precursor of a 16-mer peptide that is difficult to obtain by stepwise solid-phase peptide synthesis. NCL and the workup conditions were improved toward a protocol that allows for quantitative removal of the 4-hydroxymercaptophenol additive and subsequent formation of the disulfide bridge in the NCL cocktail by oxidation in air, tolerated by the presence of tris­(hydroxypropyl)­phosphine

Imidazole-1-sulfonyl Azide-Based Diazo-Transfer Reaction for the Preparation of Azido Solid Supports for Solid-Phase Synthesis

An efficient, standard, mild, and copper-free imidazole-1-sulfonyl azide hydrochloride-based diazo-transfer method was implemented in a set of four resins that cover a broad range of hydrophobicity. The imidazole-1-sulfonyl azide hydrochloride is easily prepared/commercially available, stable upon storage at 4 °C, and proved to be a suitable alternative to triflyl azide for diazo-transfer reactions in amine functionalized resins. We have successfully applied the azido resins for the conjugation of a TFA-labile Wang-type linker using Click Chemistry

Chemical Protein Synthesis Using a Second-Generation <i>N</i>‑Acylurea Linker for the Preparation of Peptide-Thioester Precursors

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C<i>-</i>terminal peptide-thioesters, key intermediates in NCL. We have developed an <i>N-</i>acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into <i>N</i>-acylurea peptides. Here, we introduce a new <i>N-</i>acylurea linker bearing an <i>o-</i>amino­(methyl)­aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired <i>N-</i>acyl-<i>N</i>′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer

Chemical Protein Synthesis Using a Second-Generation <i>N</i>‑Acylurea Linker for the Preparation of Peptide-Thioester Precursors

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C<i>-</i>terminal peptide-thioesters, key intermediates in NCL. We have developed an <i>N-</i>acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into <i>N</i>-acylurea peptides. Here, we introduce a new <i>N-</i>acylurea linker bearing an <i>o-</i>amino­(methyl)­aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired <i>N-</i>acyl-<i>N</i>′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer

Multimodal Characterization of a Linear DNA-Based Nanostructure

Designer DNA structures have garnered much interest as a way of assembling novel nanoscale architectures with exquisite control over the positioning of discrete molecules or nanoparticles. Exploiting this potential for a variety of applications such as light-harvesting, molecular electronics, or biosensing is contingent on the degree to which various nanoarchitectures with desired molecular functionalizations can be realized, and this depends critically on characterization. Many techniques exist for analyzing DNA-organized nanostructures; however, these are almost never used in concert because of overlapping concerns about their differing character, measurement environments, and the disparity in DNA modification chemistries and probe structure or size. To assess these concerns and to see what might be gleaned from a multimodal characterization, we intensively study a single DNA nanostructure using a multiplicity of methods. Our test bed is a linear 100 base-pair double-stranded DNA that has been modified by a variety of chemical handles, dyes, semiconductor quantum dots, gold nanoparticles, and electroactive labels. To this we apply a combination of physical/optical characterization methods including electrophoresis, atomic force microscopy, transmission electron microscopy, dynamic light scattering, Förster resonance energy transfer, voltammetry, and structural modeling. In general, the results indicate that the differences among the techniques are not so large as to prevent their effective use in combination, that the data tend to be corroborative, and that differences observed among them can actually be quite informative

Cytotoxicity of Quantum Dots Used for <i>In Vitro</i> Cellular Labeling: Role of QD Surface Ligand, Delivery Modality, Cell Type, and Direct Comparison to Organic Fluorophores

Interest in taking advantage of the unique spectral properties of semiconductor quantum dots (QDs) has driven their widespread use in biological applications such as <i>in vitro</i> cellular labeling/imaging and sensing. Despite their demonstrated utility, concerns over the potential toxic effects of QD core materials on cellular proliferation and homeostasis have persisted, leaving in question the suitability of QDs as alternatives for more traditional fluorescent materials (e.g., organic dyes, fluorescent proteins) for <i>in vitro</i> cellular applications. Surprisingly, direct comparative studies examining the cytotoxic potential of QDs versus these more traditional cellular labeling fluorophores remain limited. Here, using CdSe/ZnS (core/shell) QDs as a prototypical assay material, we present a comprehensive study in which we characterize the influence of QD dose (concentration and incubation time), QD surface capping ligand, and delivery modality (peptide or cationic amphiphile transfection reagent) on cellular viability in three human cell lines representing various morphological lineages (epithelial, endothelial, monocytic). We further compare the effects of QD cellular labeling on cellular proliferation relative to those associated with a panel of traditionally employed organic cell labeling fluorophores that span a broad spectral range. Our results demonstrate the important role played by QD dose, capping ligand structure, and delivery agent in modulating cellular toxicity. Further, the results show that at the concentrations and time regimes required for robust QD-based cellular labeling, the impact of our in-house synthesized QD materials on cellular proliferation is comparable to that of six commercial cell labeling fluorophores. Cumulatively, our results demonstrate that the proper tuning of QD dose, surface ligand, and delivery modality can provide robust <i>in vitro</i> cell labeling reagents that exhibit minimal impact on cellular viability

Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium­(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD–A647 donor–acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD

Proteolytic Activity at Quantum Dot-Conjugates: Kinetic Analysis Reveals Enhanced Enzyme Activity and Localized Interfacial “Hopping”

Recent studies show that polyvalent, ligand-modified nanoparticles provide significantly enhanced binding characteristics compared to isolated ligands. Here, we assess the ability of substrate-modified nanoparticles to provide enhanced enzymatic activity. Energy transfer assays allowed quantitative, real-time measurement of proteolytic digestion at polyvalent quantum dot-peptide conjugates. Enzymatic progress curves were analyzed using an integrated Michaelis–Menten (MM) formalism, revealing mechanistic details, including deviations from classic MM-behavior. A “hopping” mode of proteolysis at the nanoparticle was identified, confirming enhanced activity

Complex Förster Energy Transfer Interactions between Semiconductor Quantum Dots and a Redox-Active Osmium Assembly

The ability of luminescent semiconductor quantum dots (QDs) to engage in diverse energy transfer processes with organic dyes, light-harvesting proteins, metal complexes, and redox-active labels continues to stimulate interest in developing them for biosensing and light-harvesting applications. Within biosensing configurations, changes in the rate of energy transfer between the QD and the proximal donor, or acceptor, based upon some external (biological) event form the principle basis for signal transduction. However, designing QD sensors to function optimally is predicated on a full understanding of all relevant energy transfer mechanisms. In this report, we examine energy transfer between a range of CdSe–ZnS core–shell QDs and a redox-active osmium(II) polypyridyl complex. To facilitate this, the Os complex was synthesized as a reactive isothiocyanate and used to label a hexahistidine-terminated peptide. The Os-labeled peptide was ratiometrically self-assembled to the QDs <i>via</i> metal affinity coordination, bringing the Os complex into close proximity of the nanocrystal surface. QDs displaying different emission maxima were assembled with increasing ratios of Os–peptide complex and subjected to detailed steady-state, ultrafast transient absorption, and luminescence lifetime decay analyses. Although the possibility exists for charge transfer quenching interactions, we find that the QD donors engage in relatively efficient Förster resonance energy transfer with the Os complex acceptor despite relatively low overall spectral overlap. These results are in contrast to other similar QD donor–redox-active acceptor systems with similar separation distances, but displaying far higher spectral overlap, where charge transfer processes were reported to be the dominant QD quenching mechanism

Probing the Quenching of Quantum Dot Photoluminescence by Peptide-Labeled Ruthenium(II) Complexes

Charge transfer processes with semiconductor quantum dots (QDs) have generated much interest for potential utility in energy conversion. Such configurations are generally nonbiological; however, recent studies have shown that a redox-active ruthenium­(II)–phenanthroline complex (Ru²⁺-phen) is particularly efficient at quenching the photoluminescence (PL) of QDs, and this mechanism demonstrates good potential for application as a generalized biosensing detection modality since it is aqueous compatible. Multiple possibilities for charge transfer and/or energy transfer mechanisms exist within this type of assembly, and there is currently a limited understanding of the underlying photophysical processes in such biocomposite systems where nanomaterials are directly interfaced with biomolecules such as proteins. Here, we utilize redox reactions, steady-state absorption, PL spectroscopy, time-resolved PL spectroscopy, and femtosecond transient absorption spectroscopy (FSTA) to investigate PL quenching in biological assemblies of CdSe/ZnS QDs formed with peptide-linked Ru²⁺-phen. The results reveal that QD quenching requires the Ru²⁺ oxidation state and is not consistent with Förster resonance energy transfer, strongly supporting a charge transfer mechanism. Further, two colors of CdSe/ZnS core/shell QDs with similar macroscopic optical properties were found to have very different rates of charge transfer quenching, by Ru²⁺-phen with the key difference between them appearing to be the thickness of their ZnS outer shell. The effect of shell thickness was found to be larger than the effect of increasing distance between the QD and Ru²⁺-phen when using peptides of increasing persistence length. FSTA and time-resolved upconversion PL results further show that exciton quenching is a rather slow process consistent with other QD conjugate materials that undergo hole transfer. An improved understanding of the QD–Ru²⁺-phen system can allow for the design of more sophisticated charge-transfer-based biosensors using QD platforms