Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties.

Nile Red is a benzo[a]phenoxazone dye containing a diethylamino substituent at the 9-position. In recent years, it has become a popular histological stain for cellular membranes and lipid droplets due to its unrivaled fluorescent properties in lipophilic environments. This makes it an attractive lead for chemical decoration to tweak its attributes and optimize it for more specialized microscopy techniques, e.g., fluorescence lifetime imaging or two-photon excited fluorescence microscopy, to which Nile Red has never been optimized. Herein, we present synthesis approaches to a series of monosubstituted Nile Red derivatives (9-diethylbenzo[a]phenoxazin-5-ones) starting from 1-naphthols or 1,3-naphthalenediols. The solvatochromic responsiveness of these fluorophores is reported with focus on how the substituents affect the absorption and emission spectra, luminosity, fluorescence lifetimes, and two-photon absorptivity. Several of the analogues emerge as strong candidates for reporting the polarity of their local environment. Specifically, the one- and two-photon excited fluorescence of Nile Red turns out to be very responsive to substitution, and the spectroscopic features can be finely tuned by judiciously introducing substituents of distinct electronic character at specific positions. This new toolkit of 9-diethylbenzo[a]phenoxazine-5-ones constitutes a step toward the next generation of optical molecular probes for advancing the understanding of lipid structures and cellular processes.

Computational and photophysical characterization of a Laurdan malononitrile derivative.

The malononitrile group is considered one of the strongest natural electron-withdrawing groups in a chemist's arsenal. However, surprisingly little is known about how this group functions in push-pull fluorophores. In a recent computational study, we reported that replacing the ketone group of the traditional push-pull dye Laurdan with a malononitrile group significantly improves the optical properties while retaining the membrane behavior of the parent molecule Laurdan. Motivated by these results, we report here the synthesis and photophysical characterization of the said compound, 6-(1-undecyl-2,2-dicyanovinyl)-N,N-dimethyl-2-naphthylamine (CN-Laurdan). To our surprise, this new CN-Laurdan probe is found to be much less bright than the parent Laurdan due to a large drop in the fluorescence quantum yield. Using computational methods, we determine that the origin of this low quantum yield is related to the existence of a non-radiative decay pathway related to a rotation of the malononitrile moiety, suggesting that the molecule could nonetheless function very well as a molecular rotor. We confirm experimentally that CN-Laurdan functions as a molecular rotor by measuring the quantum yield in methanol/glycerol mixtures of increasing viscosity. Specifically, we found a consistent increase in the quantum yield across the entire range of tested viscosities.

One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues.

The solvatochromic fluorophore Nile Red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is one of the most commonly used stains to enhance contrast of lipid-rich areas of microscopic biosamples. Quite surprisingly, relatively little is known about the spectrally-resolved two-photon absorption (2PA) properties of this dye despite its promising features for two-photon microscopy of biological matter. For this reason, the two-photon solvatochromism of Nile Red still remains an uncharted territory as well. Also, no study has yet reported on how electron-withdrawing substituents attached to the Nile Red backbone affect its solvatochromic properties and two-photon brightness. In this paper, we demonstrate how solvent polarity influences the one- and two-photon absorption spectra of Nile Red as well as its fluorescence parameters, and we present new analogues that contain -CF3, -F and -Br substituents on its eastern side. Two-photon excited fluorescence experiments in a broad spectral range (780-1240 nm) and electronic structure calculations show that both the nature and location of the substituent have particular influence on the strength of 2PA, peaking in all cases at approx. 860 and 1050 nm. 2PA cross sections are higher at 1050 nm than at 860 nm, which suggests that Nile Red and its analogues are best suited for two-photon imaging employing excitation in the NIR-II optical transparency window of biological tissues.

Double-headed nucleotides as xeno nucleic acids: information storage and polymerase recognition.

Xeno nucleic acids (XNAs) are artificial genetic systems based on sugar-modified nucleotides. Herein, we investigate double-headed nucleotides as a new XNA. A new monomer, AT, is presented, and together with previous double-headed nucleotide monomers, new nucleic acid motifs consisting of up to five consecutive A·T base pairs have been obtained. Sections composed entirely of double-headed nucleotides are well-tolerated within a DNA duplex and can condense the genetic information. For instance, a 13-mer duplex is condensed to an 11-mer modified duplex containing four double-headed nucleotides while simultaneously improving duplex thermal stability with +14.0 °C. Also, the transfer of information from double-headed to natural nucleotides by DNA polymerases has been examined. The first double-headed nucleoside triphosphate was prepared but could not be recognized and incorporated by the tested DNA polymerases. On the other hand, it proved possible for Therminator DNA polymerase to transfer the information of a double-headed nucleotide in a template sequence to natural DNA under controlled conditions.

Base-Pairing Properties of Double-Headed Nucleotides.

Nucleotides that contain two nucleobases (double-headed nucleotides) have the potential to condense the information of two separate nucleotides into one. This presupposes that both bases must successfully pair with a cognate strand. Here, double-headed nucleotides that feature cytosine, guanine, thymine, adenine, hypoxanthine, and diaminopurine linked to the C2'-position of an arabinose scaffold were developed and examined in full detail. These monomeric units were efficiently prepared by convergent synthesis and incorporated into DNA oligonucleotides by means of the automated phosphoramidite method. Their pairing efficiency was assessed by UV-based melting-temperature analysis in several contexts and extensive molecular dynamics studies. Altogether, the results show that these double-headed nucleotides have a well-defined structure and invariably behave as functional dinucleotide mimics in DNA duplexes.

Photophysical investigation of two emissive nucleosides exhibiting gigantic stokes shifts.

We report spectroscopic characterization of two emissive 2'-deoxycytidine analogues: 5-(5-phenylfuran-2-yl)-2'-deoxycytidine and 5-(1-phenyl-1H-pyrazol-3-yl)-2'-deoxycytidine. Their fluorescent properties were examined using a combined experimental and theory/simulation approach, where the latter was based on Born-Oppenheimer molecular dynamics and time-dependent density functional theory. The analogues were found to exhibit unusually large Stokes shifts in polar media (>100 nm), moderate fluorescence quantum yields, and their emissions were found to be very sensitive to the local dielectric environment. These two analogues of 2'-deoxycytidine thus hold a promising potential as probes in chemical biology. In addition, the accuracy of the theoretical models for determining the optical properties is validated, which opens up for a convenient way of assessing the potential of future probes.

A double-headed nucleotide with two cytosines: DNA with condensed information and improved duplex stability.

Double-headed nucleotide monomers are capable of condensing the genetic information of DNA. Herein, a double-headed nucleotide with two cytosine bases (CC) is constructed. The additional cytosine is connected through a methylene linker to the 2'-position of arabinocytidine. The nucleotide is incorporated into oligonucleotides and its effect on duplex stability is studied. For single incorporations, a thermal stabilization of 4.0 °C is found as compared to the unmodified duplex and it is shown that both nucleobases of CC participate in Watson-Crick base pairing. In combination with the previously published UT monomer, it is also shown that multiple incorporations are tolerated. For instance, a 16-mer sequence is targeted by a 13-mer oligonucleotide by using one CC and two UT monomers without compromising the overall duplex stability. Finally, the potential of double-headed nucleotides in triplex-forming oligonucleotides is studied, however, with the conclusion that the present design is not well-suited for this function.

Synthesis of new C-5-triazolyl-functionalized thymidine analogs and their ability to engage in aromatic stacking in DNA : DNA and DNA : RNA duplexes.

1-Phenyl-1,2,3-triazole scaffolds on the 5-position of pyrimidine nucleosides have previously shown to enhance nuclease stability and increase the duplex thermal stability (Tm) by engaging in duplex stacking interactions. In this study, we have introduced two new derivatives of this scaffold in DNA : DNA and DNA : RNA duplexes in order to explore the thermal effects of (1) using a 1,5-triazole instead of the usual 1,4-triazole, and (2) replacing the apolar phenyl substituent with a polar uracil-5-yl substituent.

Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis, duplex and triplex studies.

In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2'-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2'-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker.

Increasing the Stability of DNA:RNA Duplexes by Introducing Stacking Phenyl-Substituted Pyrazole, Furan, and Triazole Moieties in the Major Groove.

Consecutive incorporations of our previously published thymidine analogue, 5-(1-phenyl-1H-1,2,3-triazol-4-yl)-2'-deoxyuridine monomer W in oligonucleotides, has demonstrated significant duplex-stabilizing properties due to its efficient staking properties in the major groove of DNA:RNA duplexes. The corresponding 2'-deoxycytidine analogue is not as well-accommodated in duplexes, however, due to its clear preference for the ring-flipped coplanar conformation. In our present work, we have used ab initio calculations to design two new building blocks, 5-(5-phenylfuran-2-yl)-2'-deoxycytidine monomer Y and 5-(1-phenyl-1H-pyrazol-3-yl)-2'-deoxycytidine monomer Z, that emulate the conformation of W. These monomers were synthesized by Suzuki-Miyaura couplings, and the pyrazole moiety was obtained in a cycloaddition from N-phenylsydnone. We show that the novel analogues Y and Z engage in efficient stacking either with themselves or with W due to a better overlap of the aromatic moieties. Importantly, we demonstrate that this translates into very thermally stable DNA:RNA duplexes, thus making Y and especially Z good candidates for improving the binding affinities of oligonucleotide-based therapeutics. Since we now have both efficiently stacking T and C analogues in hand, any purine rich stretch can be effectively targeted using these simple analogues. Notably, we show that the introduction of the aromatic rings in the major groove does not significantly change the helical geometry.

High-affinity RNA targeting by oligonucleotides displaying aromatic stacking and amino groups in the major groove. Comparison of triazoles and phenyl substituents.

Three 5-modified 2'-deoxyuridine nucleosides were synthesized and incorporated into oligonucleotides and compared with the previously published 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine monomer W. The introduction of an aminomethyl group on the phenyl group led to monomer X, which was found to thermally stabilize a 9-mer DNA:RNA duplex, presumably through the partial neutralization of the negative charge of the backbone. By also taking advantage of the stacking interactions in the major groove of two or more of the monomer X, an extremely high thermal stability was obtained. A regioisomer of the phenyltriazole substituent, that is the 5-(4-phenyl-1,2,3-triazol-1-yl)-2'-deoxyuridine monomer Y, was found to destabilize the DNA:RNA duplex significantly, but stacking in the major groove compensated for this when two to four monomers were incorporated consecutively. Finally, the 5-phenyl-2'-deoxyuridine monomer Z was incorporated for comparison, and it was found to give a more neutral influence on duplex stability indicating less efficient stacking interactions. The duplexes were investigated by CD spectroscopy and MD simulations.

Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

Double-headed nucleic acids condense the molecular information of DNA to half the number of nucleotides.

Nucleotide monomers that hold two nucleobases each, i.e. double-headed nucleotides, have been shown to form two sets of functional Watson-Crick base pairs when incorporated into dsDNA, and they hereby behave as dinucleotides. To form the basis for fully modified double-headed nucleic acids (DhNA), we have prepared three new DhNA monomers and can now demonstrate that the molecular information of 10 Watson-Crick base pairs can be condensed to highly stable 5-mer DhNA duplexes.

Computational Characterization of Novel Malononitrile Variants of Laurdan with Improved Photophysical Properties for Sensing in Membranes.

Fluorescent probes are powerful tools for improving our understanding of cellular membranes and other complex biological environments. Using simulations, we gain atomistic and electronic insights into the effectiveness of the probes. In the current work, we have used various computational approaches to comprehensively investigate the properties of the fluorescent probe Laurdan and two Laurdan-like probes: AADAL and ECL. In addition, we propose the development of their corresponding novel malononitrile variants, which are computationally characterized herein. For the candidate probes, electronic structure calculations were used to rationalize their optical properties, including their ability for two-photon activation, and molecular dynamics simulations were used to unravel atomistic details of their functioning within lipid bilayers. While Laurdan, AADAL, and ECL were found to have very similar optical and membrane partitioning profiles, their malononitrile variants were found to show significantly improved optical properties, especially in regard to two-photon cross sections, and they appear to retain the desired membrane characteristics of the parent Laurdan molecule.

Rational Design of Nile Red Analogs for Sensing in Membranes.

Development of next-generation fluorescent probes is a key element in the quest for a greater understanding of complex biological environments (e.g., membranes) by bioimaging. Such fluorescence-based techniques rely on specialized small molecules that possess excellent fluorescent properties but also do not perturb the native biological environment in which they reside. Herein we present a theoretical/computational strategy for the design of novel optical probes for sensing in membranes based on the parent chromophore Nile Red. Using a combination of time-dependent density functional theory (TD-DFT) and molecular dynamics (MD), we have studied the optical properties and accommodation in a model membrane of Nile Red and eight analogs. Special attention has been given to the design of probes with improved solvatochromism and two-photon absorption (2PA) without altering the membrane properties. Of the eight studied analogs, two probes were found to possess attractive probe features and are hence suggested to be taken forward to chemical synthesis and experimental exploration.

Condensing the information in DNA with double-headed nucleotides.

A normal duplex holds as many Watson-Crick base pairs as the number of nucleotides in its constituent strands. Here we establish that single nucleotides can be designed to functionally imitate dinucleotides without compromising binding affinity. This effectively allows sequence information to be more compact and concentrated to fewer phosphates.