Renatus W. Sinkeldam

Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties and Applications

Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules. For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids. The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings. Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review. This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

Emissive RNA Alphabet

A fluorescent ribonucleoside alphabet consisting of highly emissive purine ((th)A, (th)G) and pyrimidine ((th)U, (th)C) analogues, all derived from thieno[3,4-d]pyrimidine as the heterocyclic nucleus, is described. Structural and biophysical analyses demonstrated that the emissive analogues are faithful isomorphic nucleoside surrogates. Photophysical analysis established that the nucleosides offer highly desirable qualities, including visible emission, high quantum yield, and responsiveness to environmental perturbations, traits entirely lacking in their native counterparts.

Emissive nucleosides as molecular rotors.

Naturally occurring nucleosides, the building blocks of nucleic acids, are characterized by two distinct molecular elements: a rigid aromatic nucleobase and a more flexible (2’-deoxy)-d-ribose moiety. The structural features of nucleosides and their corresponding nucleotides are context-dependent and are largely dictated by two major and distinct “degrees” of conformational freedom: (a) the sugar pucker, and (b) the syn/anti orientation of the nucleobase with respect to the ribose ring (Figure 1A).[1]

Polarity of Major Grooves Explored by Using an Isosteric Emissive Nucleoside

More than half a century after the double helical structure of DNA was revealed, our understanding of certain fundamental features of this magnificent macromolecular assembly is still lacking. In particular, the polarity of nucleic acid grooves remains rather illusive. Knowledge of the forces that operate in these cavities, where key biological-recognition events take place, is of fundamental as well as practical importance. A basic understanding of these unique environments, where multiple functional groups coalesce, is highly desirable as it can shed light on the interplay of weak molecular forces within confined spaces. Practically, information on the local polarity of specific cavities in nucleic acids can facilitate the design of low molecular weight ligands that impact the structure and biological function of these key biopolymers. 6] The polarity of DNA grooves has been experimentally interrogated by using environmentally sensitive fluorescent probes. This approach is informative, but bears several predicaments: a) any probe placed within the cavity to be assessed inherently modifies the molecular architecture of the native environment and therefore taints the readout; b) most studies have utilized dielectric constant (e, relative permittivity)—a parameter that defines bulk solvent property and not an anisotropic medium as its measure; c) relatively large fluorophores (e.g. , dansyl), which are frequently connected by flexible linkers, have been employed; these possibly populate multiple conformers each of which senses a different microenvironment. Collectively, these challenges are responsible, at least in part, for the dramatically different estimates so far reported for the dielectric constant of the major groove in nucleic acids, which range from about 40 to 70. For the most accurate readout of groove polarity by means of fluorescence spectroscopy, an “ideal” probe must meet the following requirements: 1) its size and shape must be such that only the groove is examined; a linker, if used, must be as short and rigid as possible; 2) its presence must not hamper Watson–Crick (WC) base pairing or native-helix formation; 3) its absorption maximum must allow for selective excitation; and 4) its fluorescence maximum must be sensitive to polarity changes while maintaining sufficient quantum yield under all conditions. To meet these requirements, we avoided the conjugation of large fluorophores, but rather developed new emissive nucleoside analogues in which a natural nucleobase fragment is an integral electronic element of the chromophore. In this fashion, small and minimally invasive probes, capable of engaging in normal WC base pairing within unaltered duplexes, were employed. Here, we report the application of an ACHTUNGTRENNUNGenvironmentally sensitive furan-containing deoxyuridine 1 for probing the groove microenvironment in B-, A-, and abasicduplex DNA. The emissive furan-containing nucleoside analogue 1 nicely fulfils the criteria listed above. It represents an isosteric nucleobase mimic of T that is capable of participating in WC base pairing with A to form stable duplexes (Figure 1). The direct

An Emissive C Analog Distinguishes between G, 8-oxoG and T

A minimally disruptive fluorescent dC analog provides a rapid and non-destructive method for in vitro detection of G, 8-oxoG, and T, the downstream transverse mutation product.

Visibly Emissive and Responsive Extended 6-Aza-Uridines

A family of extended 5-modified-6-aza-uridines was obtained via Suzuki coupling reactions with a common brominated precursor. Extending the conjugated-6-aza-uridines with substituted aryl rings increases the push–pull interactions yielding enhanced bathochromic shifts and solvatochromism compared to the parent nucleosides. For example, the methoxy substituted derivative 1d displays λmax abs around 375 nm, with visible emission maxima at 486 nm (Φ = 0.74) and 525 nm (Φ = 0.02) in dioxane and water, respectively.

Modified 6‐Aza Uridines: Highly Emissive pH‐Sensitive Fluorescent Nucleosides

Optimized facile syntheses and highly desirable spectroscopic properties of two isomorphic fluorescent pyrimidines, comprising a 1,2,4-triazine motif conjugated to a thiophene (1 a) or a furan (1 b), are described. Although structurally related to their 5-modified uridine counterparts, these modified 6-aza-uridines reveal dramatically improved fluorescence properties and a remarkable sensitivity to polarity and pH changes. The thiophene derivative 1 a has an absorption maximum around 335 nm, which upon excitation yields visible emission with a polarity-sensitive maximum and fluorescence quantum yield ranging from 415 nm (Φ=0.8) to 455 nm (Φ=0.2) in dioxane and water, respectively. Nucleoside 1 a also displays susceptibility to acidity. Correlating emission intensity and solution pH yields a pK(a) value of 6.7-6.9, reasonably close to physiological pH values. The results illustrate that highly sought-after fluorescence features (brightness and responsiveness) are not necessarily the trait of large fluorophores alone, but can be observed with probes that meet stringent isomorphic design criteria.

Enzymatic Interconversion of Isomorphic Fluorescent Nucleosides: Adenosine Deaminase Transforms an Adenosine Analogue into an Inosine Analogue

Adenosine deaminase, a major enzyme involved in purine metabolism, converts an isomorphic fluorescent analogue of adenosine (thA) to an isomorphic inosine analogue (thI), which possesses distinct spectral features, allowing one to monitor the enzyme-catalyzed reaction and its inhibition in real time. The utility of this sensitive fluorescently-monitored transformation for the high throughput detection and analysis of ADA inhibitors is demonstrated.

A Fluorescent Adenosine Analogue as a Substrate for an A-to-I RNA Editing Enzyme†

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Two-Photon-Induced Fluorescence of Isomorphic Nucleobase Analogs

Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the 2P excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both thiophene-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications.