Modular Conjugation Research Articles

Holographic thermal entropy from geodesic bit threads

The holographic bit threads are an insightful tool to investigate the holographic entanglement entropy and other quantities related to the bipartite entanglement in AdS/CFT. We mainly explore the geodesic bit threads in various static backgrounds, for the bipartitions characterized by either a sphere or an infinite strip. In pure AdS and for the sphere, the geodesic bit threads provide a gravitational dual of the map implementing the geometric action of the modular conjugation in the dual CFT. In Schwarzschild AdS black brane and for the sphere, our numerical analysis shows that the flux of the geodesic bit threads through the horizon gives the holographic thermal entropy of the sphere. This feature is not observed when the subsystem is an infinite strip, whenever we can construct the corresponding bit threads. The bit threads are also determined by the global structure of the gravitational background; indeed, for instance, we show that the geodesic bit threads of an arc in the BTZ black hole cannot be constructed.

Quantitative Evaluation of a Multimodal Aptamer-Targeted Long-Circulating Polymer for Tumor Targeting.

Aptamers are promising targeting agents for imaging and therapy of numerous diseases, including cancer. However, a significant shortcoming of aptamers is their poor stability and fast excretion, limiting their application in vivo. Common strategies to overcome these challenges is to chemically modify aptamers in order to increase their stability and/or to apply formulation technologies such as conjugating them to polymers or nanocarriers in order to increase their circulation half-life. This is expected to result in improved cellular uptake or retention to passively targeted nanomedicines. Herein, we report a modular conjugation strategy based on click chemistry between functionalized tetrazines and trans-cyclooctene (TCO), for the modification of high molecular weight hyperbranched polyglycerol (HPG) with sgc8 aptamer, fluorescent dyes, and 111In. Our data indicate strong affinity of sgc8 against a range of solid tumor-derived cell lines that have previously not been tested with this aptamer. Nevertheless, nonspecific uptake of scrambled ssDNA-functionalized HPG in cells highlights inherent challenges of aptamer-targeted probes that remain to be solved for clinical translation. We validate HPG-sgc8 as a nontoxic nanoprobe with high affinity against MDA-MB-468 breast and A431 lung cancer cells and show significantly increased plasma stability compared to free sgc8. In vivo quantitative SPECT/CT imaging indicates EPR-mediated tumor uptake of HPG-sgc8 and nontargeted or scrambled ssDNA-conjugated HPG but no statistically significant difference between these formulations in terms of total tumor uptake or retention. Our study emphasizes the need for stringent controls and quantification in the evaluation of aptamer-targeted probes. For this purpose, our versatile synthesis strategy provides a simple approach for the design and evaluation of long-circulating aptamer-conjugated nanoformulations.

Modular Design and Bonding Mechanism of Internal Boron–Nitrogen Coordinated Boronic Ester Hydrogels with Alkaline pH Responsiveness and Tunable Gelation pH

Boronic ester hydrogels have been widely used in biomedical fields for their stimuli responsiveness to multiple disease-related triggers. However, the restricted pH for gelation and the poor hydrolysis stability limit their application in variable physiological microenvironments. Here, we report a modular conjugation method for designing internal boron–nitrogen coordinated boronic ester (IBNCB) hydrogels by constructing polymers with phenylboronic acid or N,N-bis(2-hydroxyethyl) moieties based on amides. Eight distinct IBNCB hydrogel models composed of polymer/polymer or polymer/micromolecule were prepared, exhibiting a unique pH responsiveness in the alkaline environment, an enhanced hydrolysis stability, bidirectional pH-tunable mechanical properties, and a lowered and tunable pH for gelation. In addition, the IBNCB hydrogels maintained a sol–gel transitional responsiveness to reactive oxygen species (ROS), glucose, and temperature. Especially, we found that the pH required to form an ideal IBNCB hydrogel should be greater than the pKa of phenylboronic acid but lower than the pKa of N,N-bis(2-hydroxyethyl), and reducing the pKa of phenylboronic acid could lower the gelation pH. The pKa of the tertiary amine in N,N-bis(2-hydroxyethyl) was shown to be important in the creation of the B–N coordination bond, which influenced the formation of the IBNCB bond. Finally, we explored the effect of the main chain’s charge on gelability and proposed the dynamic equilibrium mechanism of IBNCB bonds in the hydrogel. We expect that the multi-stimuli-responsive IBNCB hydrogels will provide a new strategy for designing smart materials sensitive to physicochemical signals. Furthermore, the amide-based modular conjugation could be exploited to generate a theoretically limitless number of novel IBNCB materials including microgels, polymer vesicles, and micro–nano particles.

Modular conjugation for multicomponent regions

We consider a massless Dirac field in $1+1$ dimensions, and compute the Tomita-Takesaki modular conjugation corresponding to the vacuum state and a generic multicomponent spacetime region. We do it by analytic continuation from the modular flow, which was computed recently. We use our result to discuss the validity of Haag duality in this model.

Modular conjugations in 2D conformal field theory and holographic bit threads

We study the geometric action of some modular conjugations in two dimensional (2D) conformal field theories. We investigate the bipartition given by an interval when the system is in the ground state, either on the line or on the circle, and in the thermal Gibbs state on the line. We find that the restriction of the corresponding inversion maps to a spatial slice is obtained also in the gauge/gravity correspondence through the geodesic bit threads in a constant time slice of the dual static asymptotically AdS background. For a conformal field theory in the thermal state on the line, the modular conjugation suggests the occurrence of a second world which can be related through the geodesic bit threads to the horizon of the BTZ black brane background. An inversion map is constructed also for the massless Dirac fermion in the ground state and on the line bipartite by the union of two disjoint intervals.

Modular Site-Specific Conjugation of Nanobodies Using Two Co-Associating Tags.

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of VHH and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in Escherichia coli in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.

A Modular Operator Approach to Entanglement of Causally Closed Regions

Quantum entanglement is shown for causally separated regions along the radial direction by using a conformal quantum mechanical correspondence with conformal radial Killing fields of causal diamonds in Minkowski space. In particular, the theory of local von Neumann algebras and Tomita Takesaki modular operators is applied in the entanglement structure of causal diamonds in conformal quantum mechanics. The entanglement of local states in their respective causal regions is shown through the measures of concurrence and entanglement entropy using the Tomita Takesaki modular conjugation operator. A holographic entropy formula is derived for the conformal quantum mechanics causal diamond correspondence. A new connection is made between the thermal time flow defined by the modular group of automorphisms to the physical time flow in a causal diamond via the aforementioned correspondence. The thermal interpretation of these results via two-point thermal Green’s functions and modular group flow supports the idea of a possible emergent theory of spacetime.

Bivalent EGFR-Targeting DARPin-MMAE Conjugates.

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Bis‐ethynylphosphonamidates as an Modular Conjugation Platform to Generate Multi‐Functional Protein‐ and Antibody‐Drug‐Conjugates

AbstractBis‐ethynylphosphonamidates allow for a simple chemoselective addition of two thiol‐containing modules in a row. We describe four such bis‐electrophiles that carry different functional O‐substituents with tunable hydrophilicity and enable further subsequent conjugations, thus facilitating a simple protocol for constructing protein‐protein conjugates. An increased spacer length between the two ethynylphosphonamidates simplifies the formation of a conjugate from two bulky proteins. We apply these reagents to obtain homogeneous Antibody‐Drug‐Conjugates (ADCs) from DM1 and trastuzumab with excellent cytotoxicity and selectivity for the targeted cell line. Moreover, a bis‐ethynylphosphonamidate, carrying an additional alkyne for a chemoselective triple conjugation, has been subjected to fluorescent labeling of an ADC specifically at the drug site give an Antibody‐Drug‐Fluorophore‐Conjugate (ADFC), allowing for the observation of intracellular trafficking after ADC uptake into the targeted cell.

Modular operators and entanglement in supersymmetric quantum mechanics

The modular operator approach of Tomita–Takesaki to von Neumann algebras is elucidated in the algebraic structure of certain supersymmetric (SUSY) quantum mechanical systems. A von Neumann algebra is constructed from the operators of the system. An explicit operator characterizing the dual infinite degeneracy structure of a SUSY two dimensional system is given by the modular conjugation operator. Furthermore, the entanglement of formation for these SUSY systems using concurrence is shown to be related to the expectation value of the modular conjugation operator in an entangled bi-partite supermultiplet state thus providing a direct physical meaning to this anti-unitary, anti-linear operator as a quantitative measure of entanglement. Finally, the theory is applied to the case of two-dimensional Dirac fermions, as is found in graphene, and a SUSY Jaynes Cummings model.

Nets of standard subspaces on Lie groups

Let G be a Lie group with Lie algebra g, h∈g an element for which the derivation ad h defines a 3-grading of g and τG an involutive automorphism of G inducing on g the involution eπiadh. We consider antiunitary representations (U,H) of the Lie group Gτ=G⋊{idG,τG} for which the positive cone CU={x∈g:−i∂U(x)≥0} and h span g. To a real subspace E⊆H−∞ of distribution vectors invariant under exp⁡(Rh) and an open subset O⊆G, we associate the real subspace HE(O)⊆H, generated by the subspaces U−∞(φ)E, where φ∈Cc∞(O,R) is a real-valued test function on O. Then HE(O) generates the complex Hilbert space HE(G):=HE(G)+iHE(G)‾ for every non-empty open subset O⊆G (Reeh–Schlieder property).For the real standard subspace V⊆H, for which JV=U(τG) is the modular conjugation and ΔV−it/2π=U(exp⁡th) is the modular group, we obtain sufficient conditions to be of the form HE(S) for an open subsemigroup S⊆G. If g is semisimple with simple hermitian ideals of tube type, we verify these criteria and obtain nets of cyclic subspaces HE(O), O⊆G, satisfying the Bisognano–Wichmann property in a suitable sense. Our construction also yields such nets on simple Jordan space-times and compactly causal symmetric spaces of Cayley type. By second quantization, these nets lead to free quantum fields in the sense of Haag–Kastler on causal homogeneous spaces whose groups are generated by modular groups and conjugations.

Radio‐ and Fluorescent‐Labeling of Rituximab Based on the Inverse Electron Demand Diels‐Alder Reaction

AbstractThe bioorthogonal reaction between[1,2,4,5]tetrazines with trans‐cyclooctene through the inverse electron demand Diels‐Alder (IEDDA) has been described as powerful bioconjugation tool. In this work, we explore the IEDDA as a modular conjugation strategy for in vitro and in vivo labeling of Rituximab for the generation of radioactive and fluorescently label immunoconjugates. The strategy allowed the generation, in vitro and in vivo, of conjugated Rituximab with cyanine 5 and 7 and the gamma emmiter technetium‐99m.

Light-induced primary amines and o-nitrobenzyl alcohols cyclization as a versatile photoclick reaction for modular conjugation

The advent of click chemistry has had a profound impact on many fields and fueled a need for reliable reactions to expand the click chemistry toolkit. However, developing new systems to fulfill the click chemistry criteria remains highly desirable yet challenging. Here, we report the development of light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) as a photoclick reaction via primary amines as direct click handle, to rapid and modular functionalization of diverse small molecules and native biomolecules. With intrinsic advantages of temporal control, good biocompatibility, reliable chemoselectivity, excellent efficiency, readily accessible reactants, operational simplicity and mild conditions, the PANAC photoclick is robust for direct diversification of pharmaceuticals and biorelevant molecules, lysine-specific modifications of unprotected peptides and native proteins in vitro, temporal profiling of endogenous kinases and organelle-targeted labeling in living systems. This strategy provides a versatile platform for organic synthesis, bioconjugation, medicinal chemistry, chemical biology and materials science.

Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Advantages and Prospects of Tag/Catcher Mediated Antigen Display on Capsid-Like Particle-Based Vaccines.

Capsid-like particles (CLPs) are multimeric, repetitive assemblies of recombinant viral capsid proteins, which are highly immunogenic due to their structural similarity to wild-type viruses. CLPs can be used as molecular scaffolds to enable the presentation of soluble vaccine antigens in a similar structural format, which can significantly increase the immunogenicity of the antigen. CLP-based antigen display can be obtained by various genetic and modular conjugation methods. However, these vary in their versatility as well as efficiency in achieving an immunogenic antigen display. Here, we make a comparative review of the major CLP-based antigen display technologies. The Tag/Catcher-AP205 platform is highlighted as a particularly versatile and efficient technology that offers new qualitative and practical advantages in designing modular CLP vaccines. Finally, we discuss how split-protein Tag/Catcher conjugation systems can help to further propagate and enhance modular CLP vaccine designs.

Effect of modular conjugation strategy for N-acetylgalactosamine-targeted antisense oligonucleotides

The asialoglycoprotein receptor (ASGPr) and N-acetylgalactosamine (GalNAc) is one of the most reliable receptor-ligand combinations for delivering antisense oligonucleotides (ASOs) to the liver. Here, we show that a modular GalNAc conjugation strategy allows us to reinforce the activity of the parent, naked 2′,4′-BNA/LNA gapmer targeting apolipoprotein B. The conjugation partly reduced a possible hepatotoxicity of the parent ASO. The structure-activity study revealed the significance of the metabolic susceptibility of the GalNAc moiety to nucleolytic cleavage that results in exposure of the parent gapmer. The broad usefulness of our delivery strategy of ASOs to the liver has been demonstrated.

Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells.

Monitoring the assembly of macromolecules to design entities with novel properties can be achieved either chemically creating covalent bonds or by noncovalent connections using appropriate structural motifs. In this report, two self-associating peptides (named K3 and E3) that originate from p53 tetramerization domain were developed as tools for highly specific and noncovalent heterotetramerization of two biomolecules. The pairing/coupling preferences of K3 and E3 were first evaluated by molecular modeling data and confirmed using circular dichroism spectroscopy, size-exclusion chromatography, and biological assays. Regardless of the moieties fused to K3 and E3, these two peptides self-assembled into dimers of dimers to form bivalent heterotetrameric complexes that proved to be extremely stable inside living cells. The benefits of the multivalency in terms of avidity, specificity, and expanded functional activity were strikingly revealed when the proliferating cell nuclear antigen (PCNA), which is essential for DNA replication, was targeted using a heterotetramer presenting both an antibody fragment against PCNA and a specific PCNA binder peptide. In vitro heterotetramerization of these two known PCNA ligands increased their binding efficiencies to PCNA up to 80-fold compared to the best homotetramer counterpart. In cellulo, the heterotetramers were able to efficiently inhibit DNA replication and to trigger cell death. Altogether, we demonstrate that these two biselective self-assembling peptidic domains offer a versatile noncovalent conjugation method that can be easily implemented for protein engineering.

A Concise, Modular Antibody-Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry.

The synthesis of antibody-oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves "plug-and-play" antibody conjugation with succinimidyl-functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl-linked oligonucleotides are synthesized with 5'-amine-modified oligonucleotides and disuccinimidyl suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl- oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide-antibody and oligonucleotide-streptavidin conjugates retain potent and specific binding in activity-dependent proximity ligation imaging, and proximity ligation-mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein-oligonucleotide conjugates.

Quantum field theory in phase space

The tilde conjugation rule in thermofield dynamics, equivalent to the modular conjugation in a [Formula: see text]-algebra, is used to develop unitary representations of the Poincaré group, where the Hilbert space has the phase space content, a symplectic Hilbert space. The state is described by a quasi-amplitude of probability, which is a sort of wave function in phase space, associated with the Wigner function. The quantum field theory in phase space is then constructed, including the quantization rules for the Klein–Gordon and the Dirac fields, the derivation of the electrodynamics in phase space and elements of a relativistic quantum kinetic theory. Towards a physical interpretation of the theory, propagators are associated with the corresponding Wigner functions. The Feynman rules follow accordingly with vertices similar to those of usual non-Abelian quantum field theories.

Thermoadaptive Supramolecular α-Cyclodextrin Crystallization-Based Hydrogels via Double Hydrophilic Block Copolymer Templating.

Supramolecular hydrogels play a prominent role in contemporary research of hydrophilic polymers. Especially, hydrogels based on α-cyclodextrin/poly(ethylene glycol) (α-CD/PEG) complexation and crystal formation are studied frequently. Here, the effect of double hydrophilic block copolymers (DHBCs) on α-CD/PEG hydrogel properties is investigated. Therefore, a novel DHBC, namely poly(N-vinylpyrrolidone)-b-poly(oligo ethylene glycol methacrylate) (PVP-b-POEGMA), was synthesized via a combination of reversible deactivation radical polymerization and modular conjugation methods. In the next step, hydrogel formation was studied after α-CD addition. Interestingly, DHBC-based hydrogels showed a significant response to thermal history. Heating of the gels to different temperatures led to different mechanical properties after cooling to ambient temperature, i.e., gels with mechanical properties similar to the initial gels or weak flowing gels were obtained. Thus, the hydrogels showed thermoadaptive behavior, which might be an interesting property for future applications in sensing.