Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties.

Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.

Orthogonal Photoswitching with Norbornadiene.

Orthogonal photoswitching is a convenient but challenging way of controlling multiple functions in a system by selective photoisomerization of one unit before the other in any arbitrarily chosen sequence. Here, we present this concept for the norbornadiene/quadricyclane (NBD/QC) photo/thermo-switch in the presence or absence of a coordinated metal ion. Thus, introducing two pyridyl ligands via ethyne-1,2-diyl bridges provides a system that by chelation of metal ions, such as PdII , has altered optical and switching properties. Mixing the PdII complex with its free ligand furnishes a four-state system where NBD-to-QC photoisomerizations for complexed and uncomplexed species are controlled by the irradiation wavelength and can occur orthogonally, that is, the sequence of photoisomerizations can be swapped. Studies on AgI and PbII complexes, being less stable than the PdII complex, are also presented; these exhibit like the PdII complex significantly red-shifted NBD absorptions.

Establishing linear-free-energy relationships for the quadricyclane-to-norbornadiene reaction.

The kinetics of the thermal quadricyclane-to-norbornadiene (QC-to-NBD) isomerization reaction was studied for a large selection of derivatives where the one NBD double bond contains a cyano and aryl substituent of either electron-withdrawing or -donating character. While the kinetics data did not satisfy a linear-free-energy-relationship for all the derivatives based on Hammett σ values, we found individual linear relationships for derivatives containing either electron-withdrawing or electron-donating para substituents on the aryl group; with the most electron-witdrawing substituent in the one series and with the most electron-donating substituent in the other providing the fastest reaction (corresponding to opposite slopes of the Hammett plots). All data were well described, however, by a linear relationship when using Creary radical values; the correlation could be slightly improved by using a combination of σ and values (used in ratio of 0.104 : 1). The results imply a combination of polar and free radical effects for the isomerization reaction of this specific class of derivatives, with the latter playing the most significant role. The NBD derivatives were prepared by Diels-Alder cycloaddition reactions between cyclopentadiene and 3-arylpropiolonitriles, and in the case of bromophenyl derivatives further cyanation and Sonogashira coupling reactions were performed.

Donor-Acceptor Substituted Benzo-, Naphtho- and Phenanthro-Fused Norbornadienes.

The photochromic norbornadiene/quadricyclane (NBD/QC) couple has found interest as a molecular solar thermal energy (MOST) system for storage of solar energy. To increase the energy difference between the two isomers, we present here the synthesis of a selection of benzo-fused NBD derivatives that contain an aromatic unit, benzene, naphthalene or phenanthrene, fused to one of the NBD double bonds, while the carbon atoms of the other double bond are functionalized with donor and acceptor groups. The synthesis protocols involve functionalization of benzo-fused NBDs with bromo/chloro substituents followed by a subjection of these intermediates to a cyanation reaction (introducing a cyano acceptor group) followed by a Sonogashira coupling (introducing an arylethynyl donor group, -CCC6H4NMe2 or -CCC6H4OMe). While the derivatives have good absorption properties in the visible region (redshifted relative to parent system) in the context of MOST applications, they lack the ability to undergo NBD-to-QC photoisomerization, even in the presence of a photosensitizer. It seems that loss of aromaticity of the fused aromatics is too significant to allow photoisomerization to occur. The concept of destroying aromaticity of a neighboring moiety as a way to enhance the energy density of the NBD/QC couple thus needs further structural modifications, in the quest for optimum MOST systems.

Norbornadiene-dihydroazulene conjugates.

The introduction of various photochromic units into the same molecule is an attractive approach for the development of novel molecular solar thermal (MOST) energy storage systems. Here, we present the synthesis and characterisation of a series of covalently linked norbornadiene/dihydroazulene (NBD/DHA) conjugates, using the Sonogashira coupling as the key synthetic step. Generation of the fully photoisomerized quadricyclane/vinylheptafulvene (QC/VHF) isomer was found to depend strongly on how the two units are connected - by linear conjugation (a para-phenylene bridge) or cross-conjugation (a meta-phenylene bridge) or by linking to the five- or seven-membered ring of DHA - as well as on the electronic character of another substituent group on the NBD unit. When the QC-VHF system could be reached, the QC-to-NBD back-reaction occurred faster than the VHF-to-DHA back-reaction, while the latter could be promoted simply by the addition of Cu(i) ions. The absence or presence of Cu(i) can thus be used to control whether heat releases should occur on different or identical time scales. The experimental findings were rationalized in a computational study by comparing natural transition orbitals (NTOs). Moreover, the calculations revealed an energy storage capacity of 106-110 kJ mol-1 of the QC-VHF isomers, which is higher than the sum of the capacities of the individual, separate units. The major contribution to the energy storage relates to the energetic QC form, while the major contribution to the absorption of visible light originates from the DHA photochrome; some of the NBD-DHA conjugates had absorption onsets at 450 nm or beyond.

Dithiafulvene derivatized donor-acceptor norbornadienes with redshifted absorption.

Photoisomerization of norbornadiene (N) to its metastable isomer quadricyclane (Q) has attracted interest as a strategy for harvesting and storing solar energy. For this strategy to mature the absorption maximum of N has to be moved from the UV to the visible region. Here we show that functionalization of the system with dithiafulvene (DTF) electron donors causes remarkable redshifts of various N derivatives. Thus, some derivatives were found to absorb light with an absorption onset up to 556 nm. The incorporation of DTF units comes, however, with a drawback with regard to achieving reversible N-to-Q and Q-to-N isomerizations. For some derivatives, the photoisomerization was completely quenched. The compounds were subjected to a computational study to shed light on the underlying reason for this reluctance to undergo photoisomerization. The computational study revealed that in these systems, the first excited state (S1) is positioned close to or lower than the transition state for photoconversion, effectively blocking a possible conversion to Q, thus revealing a practical challenge for the future design of N-Q energy storage systems with an improved solar spectrum match.

Norbornadiene-Based Photoswitches with Exceptional Combination of Solar Spectrum Match and Long-Term Energy Storage.

Norbornadiene-quadricyclane (NBD-QC) photoswitches are candidates for applications in solar thermal energy storage. Functionally, they rely on an intramolecular [2+2] cycloaddition reaction, which couples the S0 landscape on the NBD side to the S1 landscape on the QC side of the reaction and vice-versa. This commonly results in an unfavourable correlation between the first absorption maximum and the barrier for thermal back-conversion. This work demonstrates that this correlation can be counteracted by using steric repulsion to hamper the rotational motion of the side groups along the back-conversion path. It is shown that this modification reduces the correlation between the effective back-conversion barrier and the first absorption maximum and also increases the back-conversion entropy. The resulting molecules exhibit exceptionally long half-lives for their metastable forms without significantly affecting other properties, most notably solar spectrum match and storage density.

Heteroaryl-linked norbornadiene dimers with redshifted absorptions.

Development of Molecular Solar Thermal (MOST) systems for harvesting and storing solar energy is based on molecular photoswitches that undergo photoisomerizations to metastable isomers. One challenge is to achieve low-molecular weight molecules that absorb at sufficiently long wavelengths to match the solar spectrum. Here we show that this can be achieved by linking two norbornadiene (NBD) photoswitches to a central heterocycle, thiophene or carbazole, via alkyne appendages. In this approach, the same heteroaryl is used to tune the properties of two photoswitches at the same time, thereby keeping the molecular weight as low as possible. A series of NBD dimers was prepared by Sonogashira coupling reactions, and these compounds showed remarkable redshifted absorptions, with onsets of absorption as high as 468 nm, and thermal half-lives ranging from 44 seconds to 16 hours.

Tetraceno[2,1,12,11-opqra]tetracene-extended tetrathiafulvalene - redox-controlled generation of a large PAH core.

Two tetraceno[2,1,12,11-opqra]tetracene-extended tetrathia-fulvalenes were prepared and found to undergo reversible conversion into their planar polycyclic aromatic hydrocarbons (PAHs) upon electrochemical oxidation - at potentials probing the best valence bond representations.

Synthesis and Single-Molecule Conductances of Neutral and Cationic Indenofluorene-Extended Tetrathiafulvalenes: Kondo Effect Molecules.

Development of molecules that can switch between redox states with paired and unpaired electrons is important for molecular electronics and spintronics. In this work, a selection of redox-active indenofluorene-extended tetrathiafulvalenes (IF-TTFs) with thioacetate end groups was prepared from a readily obtainable dibromo-functionalized IF-TTF building block using palladium-catalyzed cross-coupling reactions, such as the Suzuki reaction. The end groups served as electrode anchoring groups for single-molecule conductance studies, and the molecules were subjected to mechanically controlled break-junction measurements with gold contacts and to low-bias charge transport measurements in gated three-terminal electromigration junctions. The neutral molecules showed clear conductance signatures, and somewhat surprisingly, we found that a meta-meta anchoring configuration gave a higher conductance than a para-meta configuration. We explain this behavior by "through-space" coupling between the gold electrode and the phenyl on which the anchoring group is attached. Upon charging the molecule in a gated junction, we found reproducibly a Kondo effect (zero-bias conductance) attributed to a net spin. Ready generation of radical cations was supported by cyclic voltammetry measurements, revealing stepwise formation of radical cation and dication species in solution. The first oxidation event was accompanied by association reactions as the appearance of the first oxidation peak was strongly concentration dependent.

Imparting Stability to Organic Photovoltaic Components through Molecular Engineering: Mitigating Reactions with Singlet Oxygen.

One key challenge in the development of viable organic photovoltaic devices is to design component molecules that do not degrade during combined exposure to oxygen and light. Such molecules should thus remain comparatively unreactive towards singlet molecular oxygen and not act as photosensitizers for the generation of this undesirable species. Here, novel redox-active chromophores that combine these two properties are presented. By functionalizing indenofluorene-extended tetrathiafulvalenes (IF-TTFs) with cyano groups at the indenofluorene core using Pd-catalyzed cyanation reactions, we find that the reactivity of the exocyclic fulvene carbon-carbon double bonds towards singlet oxygen is considerably reduced. The new cyano-functionalized IF-TTFs were tested in non-fullerene acceptor based organic photovoltaic proof-of-principle devices, revealing enhanced device stability.

Solar Energy Storage by Molecular Norbornadiene-Quadricyclane Photoswitches: Polymer Film Devices.

Devices that can capture and convert sunlight into stored chemical energy are attractive candidates for future energy technologies. A general challenge is to combine efficient solar energy capture with high energy densities and energy storage time into a processable composite for device application. Here, norbornadiene (NBD)-quadricyclane (QC) molecular photoswitches are embedded into polymer matrices, with possible applications in energy storing coatings. The NBD-QC photoswitches that are capable of absorbing sunlight with estimated solar energy storage efficiencies of up to 3.8% combined with attractive energy storage densities of up to 0.48 MJ kg-1. The combination of donor and acceptor units leads to an improved solar spectrum match with an onset of absorption of up to 529 nm and a lifetime (t 1/2) of up to 10 months. The NBD-QC systems with properties matched to a daily energy storage cycle are further investigated in the solid state by embedding the molecules into a series of polymer matrices revealing that polystyrene is the preferred choice of matrix. These polymer devices, which can absorb sunlight and over a daily cycle release the energy as heat, are investigated for their cyclability, showing multicycle reusability with limited degradation that might allow them to be applied as window laminates.

Molecular solar thermal energy storage in photoswitch oligomers increases energy densities and storage times.

Molecular photoswitches can be used for solar thermal energy storage by photoisomerization into high-energy, meta-stable isomers; we present a molecular design strategy leading to photoswitches with high energy densities and long storage times. High measured energy densities of up to 559 kJ kg-1 (155 Wh kg-1), long storage lifetimes up to 48.5 days, and high quantum yields of conversion of up to 94% per subunit are demonstrated in norbornadiene/quadricyclane (NBD/QC) photo-/thermoswitch couples incorporated into dimeric and trimeric structures. By changing the linker unit between the NBD units, we can at the same time fine-tune light-harvesting and energy densities of the dimers and trimers so that they exceed those of their monomeric analogs. These new oligomers thereby meet several of the criteria to be met for an optimum molecule to ultimately enter actual devices being able to undergo closed cycles of solar light-harvesting, energy storage, and heat release.