Conjugate Position Research Articles

The Impact of Conjugation Position and Linker Chemistry on the Lymphatic Transport of a Series of Glyceride and Phospholipid Mimetic Prodrugs

Drug delivery to the lymphatic system is gaining increasing attention, particularly in fields such as immunotherapy where drug access to lymphocytes is central to activity. We have previously described a prodrug strategy that facilitates the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA) via incorporation into intestinal triglyceride transport pathways. The current study explored a series of structurally related glyceride and phospholipid mimetic prodrugs of MPA in an attempt to enhance lymph targeting and to better elucidate the design criteria for lipid mimetic prodrugs. MPA was conjugated to a glyceride or phospholipid backbone at various positions using different spacers employing ester, ether, carbonate and amide bonds. Patterns of prodrug hydrolysis were evaluated in rat digestive fluid, and lymphatic transport and plasma pharmacokinetics were assessed in lymph duct cannulated rats. Prodrugs with different spacers between MPA and the glyceride backbone resulted in up to 70-fold differences in gastrointestinal stability. MPA conjugation at the 2 position of the glyceride backbone and via an ester bond were most effective in promoting lymphatic transport. Phospholipid prodrug derivatives, or glyceride derivatives with MPA attached at the 1 position or when linked via ether, carbonate or amide bonds were poorly incorporated into lymphatic transport pathways.

Defect edge steepness dependence of multiple nonlinear hot-image formation from a single phase defect.

Nonlinear hot image is one of the key elements that limit the output performance of high-power laser systems. In most hot-image researches, only one hot image peak is observed in the conjugate position for a single defect. Generally, multiple hot image peaks occur for multiple defects or cascaded nonlinear media. However, a new phenomenon is found by numerical simulation in our work: one defect can also afford two hot-image peaks near the conjugate position when considering the defect edge steepness. The super-Gaussian defect model is employed to mimic the defect edge steepness. When the super-Gaussian order is higher than one, there could be two hot image peaks under certain conditions. The formation of the double hot image peaks is primarily due to the co-effect of the hard-edge diffraction and the self-focusing effect. The influence of different factors, including the super-Gaussian order, defect size, modulation depth, and Kerr medium thickness, on the double hot image peaks intensity and location is systematically investigated. The results show that with the increase in the super-Gaussian order, the intensity of the double hot image peaks increases gradually. The defect size has a great influence on the position of the two hot image peaks. The modulation depth and thickness of the Kerr medium influence the intensity of the two hot image peaks; however, they have less impact on the peak location. Importantly, the defect edge steepness and size dependences of multiple nonlinear hot-image formation from a single-phase defect are further discussed in this paper. The two hot image peaks are fatal to optical components in high-power laser systems; in particular, the hot image peak behind the conjugate position is totally unexpected for a single defect. This research provides insights into basic physical images and hot-image formation laws. It also provides important guidance for optical defect specification evaluation and optical component layout design, as well as for beam quality control, in high-power laser systems.

Identification of Novel Low-Weight Sulfhydryl Conjugates of Oxidized 5-O- and 6-O-Substituted Betanidin Pigments.

The formation of conjugates of oxidized betacyanin pigments with selected low-weight sulfhydryl scavengers was studied. Short-lived quinonoids, quinone methides, and aminochromes derived from oxidized betacyanins are able to form adducts with different efficiencies. In this report, mass spectrometric and NMR identifications of CS-linked conjugates of cysteine, cysteamine, N-acetylcysteine, and dl-dithiolthreitol with quinonoid forms generated through oxidation of betanidin, betanin, and gomphrenin is presented. An adduct that formed between cysteine and quinonoid generated from betanin by its oxidation and decarboxylation (2-decarboxy-xanbetanin) was detected and reported for the first time. The most stable gomphrenin CS-conjugate, N-acetylcysteinylated gomphrenin, was isolated by semipreparative chromatography and its structure was established by NMR analysis. This enabled to confirm the conjugation position at carbon C-4 and to indicate the presence of a dopachromic intermediate during oxidation of gomphrenin. Conjugation of betacyanins with thiol-bearing moieties may generate new molecules with modified chemical and biological properties. Obtained results confirm that gomphrenin is capable of forming CS-conjugates with higher efficiency than betanin.

Three-dimensional laser damage positioning by a deep-learning method.

A holographic and deep learning-based method is presented for three-dimensional laser damage location. The axial damage position is obtained by numerically focusing the diffraction ring into the conjugate position. A neural network Diffraction-Net is proposed to distinguish the diffraction ring from different surfaces and positions and obtain the lateral position. Diffraction-Net, which is completely trained by simulative data, can distinguish the diffraction rings with an overlap rate greater than 61% which is the best of results reported. In experiments, the proposed method first achieves the damage pointing on each surface of cascade slabs using diffraction rings, and the smallest inspect damage size is 8µm. A high precision result with the lateral positioning error less than 38.5µm and axial positioning error less than 2.85mm illustrates the practicability for locating the damage sites at online damage inspection.

Proof of site-specificity of antibody-drug conjugates produced by chemical conjugation technology: AJICAP first generation

Antibody-drug conjugates (ADCs) have become major biopharmaceutical drugs in the field of oncology. Traditional ADCs possess a stochastic distribution of cytotoxic payloads linked to several different amino acid residues of the antibody. This heterogeneous nature of stochastic ADCs results in a complex conjugation-site characterization. To improve upon traditional ADC technology, we have developed a chemical conjugation platform, termed AJICAP™, for site-specific modification of native antibodies using a class of IgG Fc-affinity reagents (Yamada et al., 2019). Here, we report further investigation focusing on peptide mapping of the AJICAP™-ADC to confirm the exact conjugation position of the first generation AJICAP™-ADC. Neutral pH pretreatment for peptide mapping prevented undesired PTMs such as succinimide ring hydrolysis. Mirroring comparison using the purified ADC visibly indicated that Lys248 in the Fc region was conjugated to the drug-linker. MS/MS analysis also provided evidence to support Lys248 conjugation. Finally, extracted ion-chromatogram methodology suggested the site-specificity of AJICAP™ conjugation. Purified ADCs by preparative HIC-HPLC showed clear visual results and more than 93% sequence coverage by a single enzymatic digestion. The analytical strategy described herein demonstrated a robust analytical methodology for revealing the conjugation site of ADCs.

Site Directed Disulfide PEGylation of Interferon-β-1b with Fork Peptide Linker.

The attachment of PEG to biopharmaceuticals has been applied for enhancement of bioavailability and improved stability. The PEG polymer is highly hydrated; thus effective attachment to inaccessible sites could be hindered. We have devised a scheme to address this issue by introducing a considerable distance between PEG and protein by addition of a linear peptide, appended to long chained reactive linkers. Second, the position of PEG conjugation directly affects biological activity. Accordingly, a disulfide bond could be considered as an ideal choice for site directed PEGylation; but reactivity of both thiol moieties to bridging reagent is critical for maintenance of protein structure. In our design, a forked structure with two arms provides essential flexibility to account for dissociation of reduced cysteines. An efficient yield for disulfide PEGylation of IFN-β1b was attained and specificity, biophysical characterization, biological activity, and pharmacokinetics were surveyed.

A study on regulating the conjugate position of NLO chromophores for reducing the dipole moment and enhancing the electro-optic activities of organic materials

Adjust the dipole moment of the chromophore by tuning its shape to efficiently transform microscopic nonlinear properties into macroscopic electro-optic activity.

Quantum mechanics as a calculus for estimation under epistemic restriction

Consider a statistical model with an epistemic restriction such that, unlike in classical mechanics, the allowed distribution of positions is fundamentally restricted by the form of an underlying momentum field. Assume an agent (observer) who wishes to estimate the momentum field given information on the conjugate positions. We discuss a classically consistent, weakly unbiased, best estimation of the momentum field minimizing the mean squared error, based on which the abstract mathematical rules of quantum mechanics can be derived. The results suggest that quantum wave function is not an objective agent-independent attribute of reality, but represents the agent's best estimation of the momentum, given the positions, under epistemic restriction. Quantum uncertainty and complementarity between momentum and position find their epistemic origin from the trade-off between the mean squared errors of simultaneous estimations of momentum field and mean position, with the Gaussian wave function represents the simultaneous efficient estimations, achieving the Cram\'er-Rao bounds of the associated mean squared errors. We then argue that unitary time evolution and wave function collapse in measurement are normative rules for an agent to update her/his estimation given information on the experimental settings.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule.

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

Diazocine-functionalized TATA platforms.

Recently, it has been shown that the thermochemical cis→trans isomerization of azobenzenes is accelerated by a factor of more than 1000 by electronic coupling to a gold surface via a conjugated system with 11 bonds and a distance of 14 Å. The corresponding molecular architecture consists of a platform (triazatriangulenium (TATA)) which adsorbs on the gold surface, with an acetylene spacer standing upright, like a post in the middle of the platform and the azobenzene unit mounted on top. The rate acceleration is due to a very peculiar thermal singlet–triplet–singlet mechanism mediated by bulk gold. To investigate this mechanism further and to examine scope and limitation of the “spin-switch catalysis” we now prepared analogous diazocine systems. Diazocines, in contrast to azobenzenes, are stable in the cis-configuration. Upon irradiation with light of 405 nm the cis-configuration isomerizes to the trans-form, which slowly returns back to the stable cis-isomer. To investigate the thermal trans→cis isomerization as a function of the conjugation to the metal surface, we connected the acetylene spacer in meta (weak conjugation) and in para (strong conjugation) position. Both isomers form ordered monolayers on Au(111) surfaces.

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI-MS/MS after conversion to MS/MS-fragmentable derivatives.

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D3 [25(OH)D3 ] and 24,25-dihydroxyvitamin D3 [24,25(OH)2 D3 ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D3 -3- and-25-glucuronides, or 24,25(OH)2 D3 -3-, -24- and-25-glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D3 and 24,25(OH)2 D3 , respectively. The 3-glucuronide was not present for 24,25(OH)2 D3 , unlike 25(OH)D3 , thus we found that selective glucuronidation occurs at the C24-hydroxy group for 24,25(OH)2 D3 .

A huge diversity of metopids (Ciliophora, Armophorea) in soil from the Murray River floodplain, Australia. III. Morphology, ontogenesis and conjugation of Metopus boletus nov. spec., with implications for the phylogeny of the SAL supercluster

The morphology, ontogenesis, conjugation, and phylogenetic position of Metopus boletus nov. spec. were studied using live observation, various silver impregnation methods, scanning electron microscopy, morphometry, and the 18S rRNA gene sequence. The new species is outstanding in having a mushroom-like appearance; a globular to broadly ellipsoid macronucleus in anterior body half; 5–10 elongated caudal cilia; 4–6 dikinetids curved rightwards in the anterior portion of the first postoral kinety; and an adoral zone composed of an average of 28 small polykinetids. Ontogenesis of M. boletus follows the metopid mode and the species-specific vegetative morphology is obtained after division. Its conjugation is temporary, isogamic and the partners unite ventral-to-dorsal, forming strongly arched to almost rod-like pairs, which indicates a heteropolar arrangement. There are only two maturation divisions and a single synkaryon division in exconjugants. The conjugation data corroborate a sister group relationship of the classes Armophorea and Litostomatea within the SAL (Spirotrichea + Armophorea + Litostomatea) supercluster in that the partners unite ventral-to-dorsal and the main body axes are antiparallel. On the other hand, the last common ancestor of the spirotricheans very likely had a ventral-to-ventral and homopolar conjugation mode with the main body axes oriented in parallel.

Application of Heisenberg uncertainty relation for the optimal modeling of surface diffraction.

The modeling of the scattering of a plane wave at a rough aperiodic surface-as well as its diffraction by a microstructured surface-is possible only by limiting the infinite surface to a window of finite width D. We show that the scattering spectrum at infinity in the Fraunhofer zone can be obtained from the diffraction modeling of a grating of period D whose surface profile coincides with the aperiodic surface in this window. This is justified by adopting the corpuscular representation of light and resorting to Heisenberg's uncertainty relation applied to the photon's canonically conjugate variables momentum and position. This approach gives a deep and comprehensive representation of scattering phenomena, and also the limit of what can be meaningfully calculated and measured. Numerical examples of grating profiles demonstrate that results obtained under the widely used Beckmann-Kirchoff approximation are matched. The described approach can solve scattering problems that usual methods cannot, or face difficulties, such as when there is significant roughness with respect to the wavelength.

Homogeneous Antibody-Drug Conjugates via Glycoengineering.

Conventional antibody-drug conjugates (ADCs) randomly assemble small-molecule drugs onto Lys or Cys residues of a tumor-targeting antibody, featured with heterogeneity in payload numbers and conjugation positions. Glycosite-specific ADCs (gsADCs) link payload drugs onto IgG Fc N-glycans with high homogeneity that facilitates structural optimization and quality control for ADC drug development. In this protocol, we report two strategies for preparation of homogeneous ADCs via chemoenzymatic glycoengineering. First, an azido-tagged unnatural N-glycan substrate is transferred onto Fc glycosites of a therapeutic antibody through Endo-S-catalyzed glycoremodeling, followed by click reaction with an alkyne-tagged payload drug to give a well-defined gsADC. In an alternative way, glycoengineering of antibody with a natural sialylated N-glycan and successive selective oxidation of sialic acid moieties using sodium periodate provided an aldehyde handle on the glycans for conjugation with an aminooxy-assembled payload. These two strategies both enable gsADCs with high homogeneity in their conjugation sites, payload numbers, and glycoforms, which are characterized of a single mass under mass-spectral detection.

Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0.

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.

Synthetic Approaches toward Vitamin B12 Conjugates

AbstractThe ability to conjugate functional molecules to vitamin B12 (cobalamin) is an important strategy for the construction of hybrid molecules with various applications in medicinal chemistry, diagnostics, and biological sciences. Cobalamin, as an exogenous compound, reaches mammalian cells via a system of transport proteins and therefore the position of conjugation must be selected in such a way that the recognition process is not disturbed. The complex structure of vitamin B12 provides many routes for the synthesis of conjugates containing cargos appended at different sites, however, only the e‐propionamide chain, β‐axial position and R5′‐OH are found to meet the aforementioned criteria. Over the years, a number of synthetic approaches leading to cobalamin conjugates have been developed and, herein, we summarize those leading to conjugates with a cargo tethered at the central cobalt ion, macrocyclic core, ribonucleotide moiety, or a combination thereof.

Gaussian Quantum Trajectories for the Variational Simulation of Open Quantum-Optical Systems

We construct a class of variational methods for the study of open quantum systems based on Gaussian ansatzes for the quantum trajectory formalism. Gaussianity in the conjugate position and momentum quadratures is distinguished from Gaussianity in density and phase. We apply these methods to a driven-dissipative Kerr cavity where we study dephasing and the stationary states throughout the bistability regime. Computational cost proves to be similar to the Truncated Wigner Approximation (TWA) method, with at most quadratic scaling in system size. Meanwhile, strong correspondence with the numerically-exact trajectory description is maintained so that these methods contain more information on the ensemble constitution than TWA and can be more robust.

Nonpupil adaptive optics for visual simulation of a customized contact lens.

We present a method for determining the deformable mirror profile to simulate the optical effect of a customized contact lens in the central visual field. Using nonpupil-conjugated adaptive optics allows a wider field simulation compared to traditional pupil-conjugated adaptive optics. For a given contact lens, the mirror shape can be derived analytically using Fermat's principle of the stationary optical path or numerically using optimization in ray-tracing programs. An example of an aspheric contact lens simulation is given to illustrate the method, and the effect of eye misalignment with respect to the deformable mirror position is investigated. The optimal deformable mirror conjugation position is found to be near the posterior corneal surface. Chromatic aberration analysis is also presented, and our findings indicate that the polychromatic simulation quality is similar to that of the monochromatic case, even though the mirror is a reflective component. The limitations of a single continuous surface deformable mirror to mimic a contact lens are outlined, with some recommendations for improving the quality of simulation.

Effect of Double Bond Position on 2-Phenyl-benzofuran Antioxidants: A Comparative Study of Moracin C and Iso-Moracin C.

Two 2-phenyl-benzofurans, moracin C {2-[3′,5′-dihydroxy-4′-(3-methlbut-2-enyl)phenyl]-6-hydroxybenzofuran} and its isomer iso-moracin C{2-[3′,5′-dihydroxy-4′-(3-methlbut-1-enyl)phenyl]-6-hydroxybenzofuran}, were comparatively studied using redox-related antioxidant assays and non-redox antioxidant assays. Moracin C always resulted in higher IC50 values than iso-moracin C in the redox-related antioxidant assays, including •O2−-inhibition, Cu2+-reducing power, DPPH•-inhibition, and ABTS+•-inhibition assays. In the non-redox antioxidant assay, moracin C and iso-moracin C underwent similar radical-adduct-formation (RAF), evidenced by the peaks at m/z 704 and m/z 618 in HPLC-MS spectra. In conclusion, both moracin C and iso-moracin C can act as 2-phenyl-benzofuran antioxidants; their antioxidant mechanisms may include redox-related ET and H+-transfer, and non-redox RAF. A double bond at the conjugation position can enhance the redox-related antioxidant potential, but hardly affects the RAF potential.

Opening of the central Atlantic Ocean: Implications for geometric rifting and asymmetric initial seafloor spreading after continental breakup

AbstractStudy of the deep structure of conjugate passive continental margins combined with detailed plate kinematic reconstructions can provide constraints on the mechanisms of rifting and formation of initial oceanic crust. In this study the central Atlantic conjugate margins are compared based on compilation of wide‐angle seismic profiles from NW Africa Nova Scotian and U.S. passive margins. The patterns of volcanism, crustal thickness, geometry, and seismic velocities in the transition zone suggest symmetric rifting followed by asymmetric oceanic crustal accretion. Conjugate profiles in the southern central Atlantic image differences in the continental crustal thickness. While profiles on the eastern U.S. margin are characterized by thick layers of magmatic underplating, no such underplate was imaged along the African continental margin. In the north, two wide‐angle seismic profiles acquired in exactly conjugate positions show that the crustal geometry of the unthinned continental crust and the necking zone are nearly symmetric. A region including seismic velocities too high to be explained by either continental or oceanic crust is imaged along the Canadian side, corresponding on the African side to an oceanic crust with slightly elevated velocities. These might result from asymmetric spreading creating seafloor by faulting the existing lithosphere on the Canadian side and the emplacement of magmatic oceanic crust including pockets of serpentinite on the Moroccan margin. After isochron M25, a large‐scale plate reorganization might then have led to an increase in spreading velocity and the production of thin magmatic crust on both sides.