Enabling synthesis in fragment-based drug discovery (FBDD): microscale high-throughput optimisation of the medicinal chemist's toolbox reactions.

Miniaturised high-throughput experimentation (HTE) is widely employed in industrial and academic laboratories for rapid reaction optimisation using material-limited, multifactorial reaction condition screening. In fragment-based drug discovery (FBDD), common toolbox reactions such as the Suzuki-Miyaura and Buchwald-Hartwig cross couplings can be hampered by the fragment's intrinsic heteroatom-rich pharmacophore which is required for ligand-protein binding. At Astex, we are using microscale HTE to speed up reaction optimisation and prevent target down-prioritisation. By identifying catalyst/base/solvent combinations which tolerate unprotected heteroatoms we can rapidly optimise key cross-couplings and expedite route design by avoiding superfluous protecting group manipulations. However, HTE requires extensive upfront training, and this modern automated synthesis technique largely differs to the way organic chemists are traditionally trained. To make HTE accessible to all our synthetic chemists we have developed a semi-automated workflow enabled by pre-made 96-well screening kits, rapid analytical methods and in-house software development, which is empowering chemists at Astex to run HTE screens independently with minimal training.

Fragment-to-Lead Medicinal Chemistry Publications in 2019.

Fragment-based drug discovery (FBDD) has grown and matured to a point where it is valuable to keep track of its extent and details of application. This Perspective summarizes successful fragment-to-lead stories published in 2019. It is the fifth in a series that started with literature published in 2015. The analysis of screening methods, optimization strategies, and molecular properties of hits and leads are presented in the hope of informing best practices for FBDD. Moreover, FBDD is constantly evolving, and the latest technologies and emerging trends are summarized. These include covalent FBDD, FBDD for the stabilization of proteins or protein-protein interactions, FBDD for enzyme activators, new screening technologies, and advances in library design and chemical synthesis.

Total Synthesis of Lycopalhine A.

The total synthesis of lycopalhine A has been accomplished. The synthesis features construction of the tricyclic system via cleavage of a cyclopropane ring and an ensuing intramolecular Michael addition, stereoselective introduction of a 2-aminoethyl moiety via a reaction of allyltrimethylsilane to a sulfonyliminium ion, and a stereoselective intramolecular aldol reaction.

Simple SNP typing assay using a base-discriminating fluorescent probe.

We have developed a new concept involving a single-step homogeneous method for single-nucleotide polymorphism (SNP) typing. In this method, a probe containing base-discriminating fluorescent (BDF) bases is added to a sample solution. BDF base-containing DNA usually shows only a weak fluorescence, but emits a strong blue fluorescence when it recognizes a target base at a specific site in a hybridized strand. By utilizing this feature, a simple mix-and-read SNP typing assay was achieved without any tedious probe-designing or washing processes for exclusion of hybridization error or any addition of DNA-modifying enzymes. This is very different from conventional methods. We simultaneously analyzed a number of samples with ease, with a high accuracy, using our BDF assay.

Modulation of base selectivity for a base-discriminating fluorescent nucleobase by addition of mercury ion.

We altered the fluorescence emission selectivity of a base-discriminating fluorescent base, (Py)U, from A-selective to T-selective by the addition of mercury ion. The strong fluorescence from a duplex containing the (Py)U/T base pair was specific to the mercury ion among divalent metal ions, providing a unique method for sensing mercury ions in aqueous solutions.

Fluorometric sensing of the salt-induced B-Z DNA transition by combination of two pyrene-labeled nucleobases.

We have developed a new fluorescent DNA sensor containing two pyrene-labeled nucleobases, (Pet)G and (Py)C, and the fluorescence color was altered by the salt-induced B-Z DNA transition.

SNPs typing by base-discriminating fluorescence DNA probe.

For the development of a novel SNP typing method using BDF (base-discriminating fluorescent) nucleosides in biological samples, we examined the detection of the single base alteration in BRCA1 gene with PCR products amplified by an asymmetric PCR. A combination of PyU- and PyC-containing BDF probes clearly facilitates the discrimination of not only A/G homozygous samples but also heterozygous samples. The present SNP typing method with BDF probes is a very powerful homogeneous assay that does not require a special device or time-consuming steps.

Fluorescent probe for the detection of DNA conformational transition.

Two pyrene-labeled bases, (py)C and (8py)G, have been synthesized and incorporated into d(CG)n. In the CD spectra, structural transition of a modified oligodeoxynucleotide (ODN-Py) from B-form to Z-form has been observed under different salt concentrations. The conformational transition of ODN-Py was monitored by the change of emission wavelengths based on exciplex and monomer fluorescence of (py)C and (8py)G.

Design of base-discriminating fluorescent (BDF) nucleobase for SNP typing.

We have devised novel pyrene-labeled BDF nucleosides, (Py)U, (Py)C, (Py)deazaA, and (Py)A. BDF probes containing these fluorescent nucleosides selectively emit fluorescence only when the base opposite the BDF base is a target base. Oligonucleotides containing these BDF nucleosides act as effective reporter probes for homogeneous SNP typing of DNA samples.

DNA nanomotor using duplex-quadruplex conformational transition.

For DNA nanomotor, we synthesized 8-Py-dG and incorporated it into d(T3G2)4 which can form quadruplex. We measured CD spectra for this ODN. The result suggested that this ODN was changed from quadruplex to duplex by the hybridization with the complementary strand. Further, this ODN had a very interesting property of fluorescence. The fluorescent intensity could be controlled whether the complementary strand was existed, or not.