Interrogating Aptamer Chemical Space Through Modified Nucleotide Substitution Facilitated by Enzymatic DNA Synthesis.

Chemical modification of aptamers is an important step to improve their performance and stability in biological media. This can be performed either during their identification (mod-SELEX) or after the in vitro selection process (post-SELEX). In order to reduce the complexity and workload of the post-SELEX modification of aptamers, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden often associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity. In addition, while bioisosteres often improve the potency of small molecule drugs, this does not extend to chemically modified aptamers. Overall, this versatile method can be applied for the post-SELEX modification of other aptamers and functional nucleic acids.

9-Azahomocubane.

Homocubane, a highly strained cage hydrocarbon, contains two very different positions for the introduction of a nitrogen atom into the skeleton, e. g., a position 1 exchange results in a tertiary amine whereas position 9 yields a secondary amine. Herein reported is the synthesis of 9-azahomocubane along with associated structural characterization, physical property analysis and chemical reactivity. Not only is 9-azahomocubane readily synthesized, and found to be stable as predicted, the basicity of the secondary amine was observed to be significantly lower than the structurally related azabicyclo[2.2.1]heptane, although similar to 1-azahomocubane.

seco-1-Azacubane-2-carboxylic Acid: Derivative Scope and Comparative Biological Evaluation.

The unusual and sterically constrained amino acid, seco-1-azacubane-2-carboxylic acid, was incorporated into a range of bioactive chemical templates, including enalaprilat, perindoprilat, endomorphin-2 and isoniazid, and subjected to biological testing. The endomorphin-2 derivative displayed increased activity at the δ opioid receptor, but a loss in activity was observed in the other cases, although human normal cell line evaluation suggests limited cytotoxic effects.

seco-1-Azacubane-2-carboxylic acid-Amide Bond Comparison to Proline.

seco-1-Azacubane-2-carboxylic acid, an unusual and sterically constrained amino acid, was found to undergo amide bond formation at both the N- and C-termini using proline based bioactive molecule templates as a concept platform.

Evolution of abiotic cubane chemistries in a nucleic acid aptamer allows selective recognition of a malaria biomarker.

Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.

A Relay Strategy Actuates Pre-Existing Trisubstituted Olefins in Monoterpenoids for Cross-Metathesis with Trisubstituted Alkenes.

A retrosynthetic disconnection-reconnection analysis of epoxypolyenes-substrates that can undergo cyclization to podocarpane-type tricycles-reveals relay-actuated Δ6,7-functionalized monoterpenoid alcohols for ruthenium benzylidene catalyzed olefin cross-metathesis with homoprenyl benzenes. Successful implementation of this approach provided several epoxypolyenes as expected (E/Z, ca. 2-3:1). The method is further generalized for the cross-metathesis of pre-existing trisubstituted olefins in other relay-actuated Δ6,7-functionalized monoterpenoid alcohols with various other trisubstituted alkenes to form new trisubstituted olefins. Epoxypolyene cyclization of an enantiomerically pure, but geometrically impure, epoxypolyene substrate provides an enantiomerically pure, trans-fused, podocarpane-type tricycle (from the E-geometrical isomer).

Rearrangement-Free Hydroxylation of Methylcubanes by a Cytochrome P450: The Case for Dynamical Coupling of C-H Abstraction and Rebound.

The highly strained cubylmethyl radical undergoes one of the fastest radical rearrangements known (reported k = 2.9 × 1010 s-1 at 25 °C) through scission of two bonds of the cube. The rearrangement has previously been used as a mechanistic probe to detect radical-based pathways in enzyme-catalyzed C-H oxidations. This paper reports the discovery of highly selective cytochrome P450-catalyzed methylcubane oxidations which notionally proceed via cubylmethyl radical intermediates yet are remarkably free of rearrangement. The bacterial cytochrome P450 CYP101B1 from Novosphingobium aromaticivorans DSM 12444 is found to hydroxylate the methyl group of a range of methylcubane substrates containing a regio-directing carbonyl functionality at C-4. Unlike other reported P450-catalyzed methylcubane oxidations, the designed methylcubanes are hydroxylated with high efficiency and selectivity, giving cubylmethanols in yields of up to 93%. The lack of cubane core ring-opening implies that the cubylmethyl radicals formed during these CYP101B1-catalyzed hydroxylations must have very short lifetimes, of just a few picoseconds, which are too short for them to manifest the side reactivity characteristic of a fully equilibrated P450 intermediate. We propose that the apparent ultrafast radical rebound can be explained by a mechanism in which C-H abstraction and C-O bond formation are merged into a dynamically coupled process, effectively bypassing a discrete radical intermediate. Related dynamical phenomena can be proposed to predict how P450s may achieve various other modes of reactivity by controlling the formation and fate of radical intermediates. In principle, dynamical ideas and two-state reactivity are each individually able to explain apparent ultrashort radical lifetimes in P450 catalysis, but they are best considered together.

Cyclooctatetraenes through Valence Isomerization of Cubanes: Scope and Limitations.

The scope and limitations of Eaton's rhodium(I)-catalyzed valence isomerization of cubane to cyclooctatetraene (COT) were investigated in the context of functional group tolerability, multiple substitution modes and the ability of cubane-alcohols to undergo one-pot tandem Ley-Griffith Wittig reactions in the absence of a transition metal catalyst.

Synthesis of the seco-Limonoid BCD Ring System Identifies a Hsp90 Chaperon Machinery (p23) Inhibitor.

D-Ring-seco-limonoids (tetranortriterpenoids), such as gedunin and xylogranin B display anti-cancer activity, acting via inhibition of Hsp90 and/or associated chaperon machinery (e.g., p23). Despite this, these natural products have received relatively little attention, both in terms of an enabling synthetic approach (which would allow access to derivatives), and as a consequence their structure-activity relationship (SAR). Disclosed herein is a generally applicable synthetic route to the BCD ring system of the seco-D-ring double bond containing limonoids. Furthermore, cell based assays revealed the first skeletal fragment that exhibited inhibition of the p23 enzyme at a level which was equipotent to that of gedunin, despite being much less structurally complex.

Cyclooctatetraene: A Bioactive Cubane Paradigm Complement.

Cubane was recently validated as a phenyl ring (bio)isostere, but highly strained caged carbocyclic systems lack π character, which is often critical for mediating key biological interactions. This electronic property restriction associated with cubane has been addressed herein with cyclooctatetraene (COT), using known pharmaceutical and agrochemical compounds as templates. COT either outperformed or matched cubane in multiple cases suggesting that versatile complementarity exists between the two systems for enhanced bioactive molecule discovery.

Determining the necessity of phenyl ring π-character in warfarin.

Despite the difficulty in administering a safe dose regimen and reports of emerging resistance, warfarin (1) remains the most widely-used oral anticoagulant for the prevention and treatment of thrombosis in humans globally. Systematic substitution of the warfarin phenyl ring with either 1,3,5,7-cyclooctatetraene (COT) (2), cubane (3), cyclohexane (4) or cyclooctane (5) and subsequent evaluation against the target enzyme, vitamin K epoxide reductase (VKOR), facilitated interrogation of both steric and electronic properties of the phenyl pharmacophore. The tolerance of VKOR to further functional group modification (carboxylate 14, PTAD adduct 15) was also investigated. The results demonstrate the importance of both annulene conferred π-interactions and ring size in the activity of warfarin.

Enantioselective synthesis of (R)-2-cubylglycine including unprecedented rhodium mediated C-H insertion of cubane.

The first enantioselective synthesis of (R)-2-cubylglycine, an analogue of (R)-2-phenylglycine in which the phenyl ring has been replaced by cubane, is disclosed. The key step was a telescoped Strecker reaction using (S)-2-amino-2-phenylethanol as a chiral auxiliary. Exploration of an alternative synthetic approach resulted in unprecedented cubane C-H insertion.

The cubane paradigm in bioactive molecule discovery: further scope, limitations and the cyclooctatetraene complement.

The cubane phenyl ring bioisostere paradigm was further explored in an extensive study covering a wide range of pharmaceutical and agrochemical templates, which included antibiotics (cefaclor, penicillin G) and antihistamine (diphenhydramine), a smooth muscle relaxant (alverine), an anaesthetic (ketamine), an agrochemical instecticide (triflumuron), an antiparasitic (benznidazole) and an anticancer agent (tamibarotene). This investigation highlights the scope and limitations of incorporating cubane into bioactive molecule discovery, both in terms of synthetic compatibility and physical property matching. Cubane maintained bioisosterism in the case of the Chagas disease antiparasitic benznidazole, although it was less active in the case of the anticancer agent (tamibarotenne). Application of the cyclooctatetraene (COT) (bio)motif complement was found to optimize benznidazole relative to the benzene parent, and augmented anticancer activity relative to the cubane analogue in the case of tamibarotene. Like all bioisosteres, scaffolds and biomotifs, however, there are limitations (e.g. synthetic implementation), and these have been specifically highlighted herein using failed examples. A summary of all templates prepared to date by our group that were biologically evaluated strongly supports the concept that cubane is a valuable tool in bioactive molecule discovery and COT is a viable complement.

Total synthesis of a biotinylated rocaglate: Selective targeting of the translation factors eIF4AI/II.

The total synthesis of a biotinylated derivative of methyl rocaglate is described. This compound was accessed from synthetic methyl rocaglate (2) via formation of the propargyl amide and subsequent click reaction with a biotin azide. Affinity purification revealed that biotinylated rocaglate (8) and methyl rocaglate (2) bind with high specificity to translation factors eIF4AI/II. This remarkable selectivity is in line with that found for the more complex rocaglate silvestrol (3).

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

The high-throughput synthesis and phase characterisation of amphiphiles: a sweet case study.

A new method for the discovery of amphiphiles by using high-throughput (HT) methods to synthesise and characterise a library of galactose- and glucose-containing amphiphilic compounds is presented. The copper-catalysed azide­alkyne cycloaddition (CuAAC) "click" reaction between azide-tethered simple sugars and alkyne-substituted hydrophobic tails was employed to synthesise a library of compounds with systematic variations in chain length and unsaturation in a 24-vial array format. The liquid­crystalline phase behaviour was characterised in a HT manner by using synchrotron small-angle X-ray scattering (SSAXS). The observed structural variation with respect to chain parameters, including chain length and degree of unsaturation, is discussed, as well as hydration effects and degree of hydrogen bonding between head groups. The validity of our HT screening approach was verified by resynthesising a short-chain glucose amphiphile. A separate phase analysis of this compound confirmed the presence of numerous lyotropic liquid­crystalline phases.

Design, synthesis and binding properties of a fluorescent α9ß1/α4ß1 integrin antagonist and its application as an in vivo probe for bone marrow haemopoietic stem cells.

The α9ß1 and α4ß1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4ß1 integrin have been thoroughly investigated with respect to HSC function, the role of α9ß1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9ß1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9ß1 integrin with potent cross-reactivity against α4ß1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9ß1 and α4ß1 integrins, which showed faster binding to α4ß1 integrin relative to α9ß1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9ß1 and α4ß1 integrins; (2) investigating the biochemical properties of α9ß1 and α4ß1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.

The search for new amphiphiles: synthesis of a modular, high-throughput library.

Amphiphilic compounds are used in a variety of applications due to their lyotropic liquid-crystalline phase formation, however only a limited number of compounds, in a potentially limitless field, are currently in use. A library of organic amphiphilic compounds was synthesised consisting of glucose, galactose, lactose, xylose and mannose head groups and double and triple-chain hydrophobic tails. A modular, high-throughput approach was developed, whereby head and tail components were conjugated using the copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction. The tails were synthesised from two core alkyne-tethered intermediates, which were subsequently functionalised with hydrocarbon chains varying in length and degree of unsaturation and branching, while the five sugar head groups were selected with ranging substitution patterns and anomeric linkages. A library of 80 amphiphiles was subsequently produced, using a 24-vial array, with the majority formed in very good to excellent yields. A preliminary assessment of the liquid-crystalline phase behaviour is also presented.

Fragment-based drug discovery for disorders of the central nervous system: designing better drugs piece by piece.

Fragment-based drug discovery (FBDD) has emerged as a powerful strategy to confront the challenges faced by conventional drug development approaches, particularly in the context of central nervous system (CNS) disorders. FBDD involves the screening of libraries that comprise thousands of small molecular fragments, each no greater than 300 Da in size. Unlike the generally larger molecules from high-throughput screening that limit customisation, fragments offer a more strategic starting point. These fragments are inherently compact, providing a strong foundation with good binding affinity for the development of drug candidates. The minimal elaboration required to transition the hit into a drug-like molecule is not only accelerated, but also it allows for precise modifications to enhance both their activity and pharmacokinetic properties. This shift towards a fragment-centric approach has seen commercial success and holds considerable promise in the continued streamlining of the drug discovery and development process. In this review, we highlight how FBDD can be integrated into the CNS drug discovery process to enhance the exploration of a target. Furthermore, we provide recent examples where FBDD has been an integral component in CNS drug discovery programs, enabling the improvement of pharmacokinetic properties that have previously proven challenging. The FBDD optimisation process provides a systematic approach to explore this vast chemical space, facilitating the discovery and design of compounds piece by piece that are capable of modulating crucial CNS targets.

The (±)-5-Aza[1.0]triblattane Skeleton via Azetine Cycloaddition.

The first synthesis of the 5-aza[1.0]triblattane skeleton was achieved through a [4 + 2] cycloaddition approach using a suitably protected azetine and cyclopentadiene. A series of azetines were synthesized to explore both stability and suitable N-protection. The key step following cycloaddition utilized a noninitiated protonated aminyl radical cyclization to install the final 5-azatriblattane bond, but it was found to be considerably more unstable than the 6-aza isomer under acidic conditions.