A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Expedient Synthesis and Characterization of π-Extended Luciferins.

Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.

Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging.

Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

Modified norcyanines enable ratiometric pH imaging beyond 1000 nm.

Activatable fluorophores with emission beyond 1000 nm have the potential to enable high contrast imaging in complex in vivo settings. However, there are few scaffolds that can be applied to this challenge. Here we detail the synthesis and evaluation of benzo[c,d]indole-substituted norcyanines that enable pH responsive fluorescence imaging in the long wavelength (>1150 nm) range. A key component of our molecular design is the installation of a hydrophilic substituted quaternary amine in the central dihydropyridine ring system. A compound with a C4'-phenyl substituent, but not the C4'-protio homologue, exhibits absorbance maxima of 740 nm and 1130 nm in basic and acidic media, respectively, with evidence of J-aggregate-like properties. These two distinct absorbances enabled ratiometric imaging of probe internalization in a tumor model. Overall, these studies provide a new class of activatable long-wavelength responsive fluorophores with promising photophysical properties.

Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry.

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitatively assess the cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits a dramatically increased cellular signal due to an improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ∼50× brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or nonhindered disulfides, an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

Coumarins to Cyanines: Synthesis of Hemicyanines.

Near-infrared (NIR) emitting fluorophores are powerful tools for optical imaging. However, there are only a handful of broadly employed NIR-emitting scaffolds, and the synthetic methods to prepare these molecules are often problematic. Here, we describe a novel, three-step synthesis of chromene-containing hemicyanine probes exhibiting large Stokes shifts and NIR emissions. We develop a pH-activatable probe for visualizing lysosomal trafficking of mAb conjugates. These studies provide a concise approach to hemicyanines with promising properties.

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging.

Multi-component bioluminescence imaging requires an expanded collection of luciferase-luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging.

Elucidation of Spirodactylone, a Polycyclic Alkaloid from the Sponge Dactylia sp., and Nonenzymatic Generation from the Co-metabolite Denigrin B.

Spirodactylone (1), a hexacyclic indolizidone alkaloid possessing a novel spiro ring system, was isolated from the marine sponge Dactylia sp. The structure was elucidated by extensive spectroscopic methods including application of the LR-HSQMBC NMR pulse sequence. Oxidative cyclization of denigrin B (2), an aryl-substituted 2-oxo-pyrroline derivative that was also isolated from the sponge extract, provided material identical to spirodactylone (1). This confirmed the assigned structure and provides insight into the probable biogenesis of 1.

A Cyanine Photooxidation/ß-Elimination Sequence Enables Near-infrared Uncaging of Aryl Amine Payloads.

Uncaging strategies that use near-infrared wavelengths can enable the highly targeted delivery of biomolecules in complex settings. Many methods, including an approach we developed using cyanine photooxidation, are limited to phenol-containing payloads. Given the critical role of amines in diverse biological processes, we sought to use cyanine photooxidation to initiate the release of aryl amines. Heptamethine cyanines substituted with an aryl amine at the C4' position undergo only inefficient release, likely due electronic factors. We then pursued the hypothesis that the carbonyl products derived from cyanine photooxidation could undergo efficient ß-elimination. After examining both symmetrical and unsymmetrical scaffolds, we identify a merocyanine substituted with indolenine and coumarin heterocycles that undergoes efficient photooxidation and aniline uncaging. In total, these studies provide a new scheme-cyanine photooxidation followed by ß-elimination-through which to design photocages with efficient uncaging properties.

Heterogeneous Catalytic Oxidation of Amides to Imides by Manganese Oxides.

Herein, we report a one-step peroxide mediated heterogeneous catalytic oxidation of amides to imides utilizing a series of manganese oxides. Among them, Cs/Mn2O3 was found to be the most active catalyst for the selective partial oxidation of N-benzylbenzamide to diphenyl imide. We have been able to apply an optimized oxidation method to other aromatic substrates. The feasibility of using air as an oxidant, the heterogeneous nature, inexpensive catalytic materials, respectable turnover numbers, and chemoselectivity to imides make this methodology an attractive choice for functional group transformations of amides to imides.

Rh-Catalyzed Conjugate Addition of Aryl and Alkenyl Boronic Acids to α-Methylene-ß-lactones: Stereoselective Synthesis of trans-3,4-Disubstituted ß-Lactones.

A one-step preparation of 3,4-disubstituted ß-lactones through Rh-catalyzed conjugate addition of aryl or alkenyl boronic acids to α-methylene-ß-lactones is described. The operationally simple, stereoselective transformation provides a broad range of ß-lactones from individual α-methylene-ß-lactone templates. This methodology allowed for a direct, final-step C-3 diversification of nocardiolactone, an antimicrobial natural product.

Rhodium-Catalyzed Addition of Aryl Boronic Acids to 2,2-Disubstituted Malononitriles.

ß-Ketonitriles bearing a quaternary carbon at the 2-position were prepared through Rh-catalyzed addition of aryl boronic acids to 2,2-disubstituted malononitriles. In contrast to the previously described transnitrilative cyanation of aryl boronic acids with dialkylmalononitriles, the present reaction avoids retro-Thorpe collapse of the intermediate addition product through the use of a milder base. The reaction was amenable to a variety of aryl boronic acids and disubstituted malononitriles, providing a diverse array of ß-ketonitriles. The products could be further derivatized to valuable chiral α,α-disubstituted-ß-aminonitriles through addition reactions to the corresponding N-tert-butanesulfinyl imines.

Pd-Catalyzed Acyl C-O Bond Activation for Selective Ring-Opening of α-Methylene-ß-lactones with Amines.

A Pd-catalyzed ring-opening of ß-lactones with various types of amines (primary, secondary, and aryl) to provide ß-hydroxy amides with excellent selectivity toward acyl C-O bond cleavage is reported. The utility of this protocol is demonstrated in an asymmetric kinetic resolution providing enantioenriched α-methylene-ß-lactones.

General and Stereoselective Method for the Synthesis of Sterically Congested and Structurally Diverse P-Stereogenic Secondary Phosphine Oxides.

A general and efficient method for the synthesis of bulky and structurally diverse P-stereogenic chiral secondary phosphine oxides (SPOs) by using readily available chiral amino alcohol templates is described. These chiral SPOs could be used as chiral building blocks for the synthesis of difficult-to-access bulky P-stereogenic phosphine compounds or ligands for organic catalysis.