A versatile o-aminoanilide linker for native chemical ligation.

Chemical protein synthesis (CPS) is a consolidated field founded on the high chemospecificity of amide-forming reactions, most notably the native chemical ligation (NCL), but also on new technologies such as the Ser/Thr ligation of C-terminal salicylaldehyde esters and the α-ketoacid-hydroxylamine (KAHA) condensation. NCL was conceptually devised for the ligation of peptides having a C-terminal thioester and an N-terminal cysteine. The synthesis of C-terminal peptide thioesters has attracted a lot of interest, resulting in the invention of a wide diversity of different methods for their preparation. The N-acylurea (Nbz) approach relies on the use of the 3,4-diaminobenzoic (Dbz-COOH) and the 3-amino-(4-methylamino)benzoic (MeDbz-COOH) acids; the latter disclosed to eliminate the formation of branching peptides. Dbz-COOH has been also used for the development of the benzotriazole (Bt)-mediated NCL, in which the peptide-Dbz-CONH2 precursor is oxidized to a highly acylating peptide-Bt-CONH2 species. Here, we have brought together the Nbz and Bt approaches in a versatile linker, the 1,2-diaminobenzene (Dbz). The Dbz combines the robustness of MeDbz-COOH and the flexibility of Dbz-COOH: it can be converted into the Nbz or Bt C-terminal peptides. Both are ligated in high yields, and the reaction intermediates can be conveniently characterized. Our results show that the Bt precursors have faster NCL kinetics that is reflected by a rapid transthioesterification (<5 min). Taking advantage of this major acylating capacity, peptide-Bt can be transselenoesterified in the presence of selenols to afford peptide selenoesters which hold enormous potential in NCL.

On the Utility of Chemical Strategies to Improve Peptide Gut Stability.

Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of (i) the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and (ii) medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract.

Native Chemical Ligation via N-Acylurea Thioester Surrogates Obtained by Fmoc Solid-Phase Peptide Synthesis.

Native chemical ligation (NCL) enables the direct chemical synthesis and semisynthesis of proteins of different sizes and compositions, streamlining the access to proteins containing posttranslational modifications (PTMs). NCL assembles peptide fragments through the chemoselective reaction of a C-terminal α-thioester peptide, prepared either by chemical synthesis or via intein-splicing technology, and a recombinant or synthetic peptide containing an N-terminal Cys. Whereas the generation of C-terminal α-thioester proteins can be achieved via the recombinant fusion of the sequence of interest to an intein domain, chemical methods can also be used for synthetically accessible proteins. The use of Fmoc solid-phase peptide synthesis (Fmoc-SPPS) to obtain α-thioester peptides requires the development of novel strategies to overcome the lability of the thioester bond toward piperidine Fmoc-removal conditions. These new synthetic methods enable the easy introduction of PTMs in the thioester fragment. In this chapter, we describe an approach for the synthesis and use of C-terminal α-N-acylbenzimidazolinone (Nbz) and α-N-acyl-N'-methylbenzimidazolinone (MeNbz) peptides in NCL. Following stepwise peptide elongation, acylation with p-nitrophenylchloroformate and cyclization affords the Nbz/MeNbz peptides. The optimization of the coupling conditions allows the chemoselective incorporation of the C-terminal amino acid (aa) on the 3,4-diaminobenzoyl (Dbz) and prevents undesired diacylations of the resulting o-aminoanilide. Following synthesis, these Nbz/MeNbz peptides undergo NCL straightforwardly at neutral pH catalyzed by the presence of arylthiols. Herein, we apply the Nbz technology solid phase synthesis, NCL-mediated cyclization and folding of the heterodimeric RTD-1 defensin, an antimicrobial peptide isolated from the rhesus macaque leukocytes.

DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi.

Besides being regulated by G-protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11, or G12/13), and promoting nucleotide exchange in vitro However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq Unlike the nucleotide-exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq These findings indicate that GBA motifs have versatility in their G-protein-modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.

Peptide Ligations by Using Aryloxycarbonyl-o-methylaminoanilides: Chemical Synthesis of Palmitoylated Sonic Hedgehog.

A simple procedure for C-terminal activation of peptides in solution and its application in native chemical ligation and protein synthesis is described. This method involves a mild thioesterification based on the conversion of an aryloxy-o-methylaminoanilide to thioester under aqueous conditions and in situ ligation with an N-terminal cysteine peptide. The versatility is shown in pH-controlled sequential ligations. To illustrate the usefulness of this methodology, we synthesized the palmitoylated N-terminal domain of human Sonic Hedgehog, a morphogen protein that binds the transmembrane receptor Patched and activates the Hedgehog signaling pathway, involved in embryonic development and in the proliferation of multiple tumors. This approach extends the chemical toolset of chemical protein synthesis based on o-aminoanilide and o-methylaminoanilide peptides.

A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins.

Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein-coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif-containing proteins.

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

Native chemical ligation (NCL) employing the N-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.

Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein.

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

The Gαi-GIV binding interface is a druggable protein-protein interaction.

Heterotrimeric G proteins are usually activated by the guanine-nucleotide exchange factor (GEF) activity of GPCRs. However, some non-receptor proteins are also GEFs. GIV (a.k.a Girdin) was the first non-receptor protein for which the GEF activity was ascribed to a well-defined protein sequence that directly binds Gαi. GIV expression promotes metastasis and disruption of its binding to Gαi blunts the pro-metastatic behavior of cancer cells. Although this suggests that inhibition of the Gαi-GIV interaction is a promising therapeutic strategy, protein-protein interactions (PPIs) are considered poorly "druggable" targets requiring case-by-case validation. Here, we set out to investigate whether Gαi-GIV is a druggable PPI. We tested a collection of >1,000 compounds on the Gαi-GIV PPI by in silico ligand screening and separately by a chemical high-throughput screening (HTS) assay. Two hits, ATA and NF023, obtained in both screens were confirmed in secondary HTS and low-throughput assays. The binding site of NF023, identified by NMR spectroscopy and biochemical assays, overlaps with the Gαi-GIV interface. Importantly, NF023 did not disrupt Gαi-Gßγ binding, indicating its specificity toward Gαi-GIV. This work establishes the Gαi-GIV PPI as a druggable target and sets the conceptual and technical framework for the discovery of novel inhibitors of this PPI.

Molecular mechanism of Gαi activation by non-GPCR proteins with a Gα-Binding and Activating motif.

Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation.

Concurrent Modulation of Quantum Dot Photoluminescence Using a Combination of Charge Transfer and Förster Resonance Energy Transfer: Competitive Quenching and Multiplexed Biosensing Modality.

An emerging trend with semiconductor quantum dots (QDs) is their use as scaffolds to assemble multiple energy transfer pathways. Examples to date have combined various competitive and sequential Förster resonance energy transfer (FRET) pathways between QDs and fluorescent dyes, luminescent lanthanide complexes, and bioluminescent proteins. Here, we show that the photoluminescence (PL) of QD bioconjugates can also be modulated by a combination of FRET and charge transfer (CT), and characterize the concurrent effects of these mechanistically different pathways using PL measurements at both the ensemble and the single particle level. Peptides were distally labeled with either a fluorescent dye that quenched QD PL through FRET or a ruthenium(II) phenanthroline complex that quenched QD PL through electron transfer. The labeled peptides were assembled around a central CdSe/ZnS QD at different ratios, tuning the relative rates of FRET and CT, which were competitive quenching pathways. The concurrent effects of FRET and CT were predictable from a rate analysis that was calibrated to the isolated effects of each of these pathways. Notably, the dye/QD PL intensity ratio reflected changes in the relative rate of FRET but was approximately independent of CT. In turn, the sum of the QD and dye PL intensities, when adjusted for quantum yields, reflected changes in the relative rate of CT quenching, approximately independent of FRET. The capacity for multiplexed sensing of protease activity was demonstrated using these two orthogonal detection channels. Combined CT-FRET configurations with QDs are thus promising for applications in bioanalysis, sensing, and imaging, and may prove useful in other photonic applications.

Chemical Protein Synthesis Using a Second-Generation N-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-terminal peptide-thioesters, key intermediates in NCL. We have developed an N-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 N-acylurea peptides. Here, we introduce a new N-acylurea linker bearing an o-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 N-acyl-N'-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.

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.

Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates.

Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

Cytotoxicity of quantum dots used for in vitro 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 in vitro 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 in vitro 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 in vitro cell labeling reagents that exhibit minimal impact on cellular viability.

Site-specific cellular delivery of quantum dots with chemoselectively-assembled modular peptides.

Modular peptides displaying both quantum dot bioconjugation motifs and specific subcellular targeting domains were constructed using a chemoselective aniline-catalyzed hydrazone coupling chemistry. Peptides were ratiometrically assembled onto quantum dots to facilitate their specific delivery to either the plasma membrane, endosomes, the cytosol or the mitochondria of target cells.

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.

Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials.

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide's ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

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 via 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.