On-DNA Mannich Reaction for DNA-Encoded Library Synthesis.

The β-amino ketones produced through the Mannich reaction hold significant potential as candidates for various drugs. In this study, we optimized on-DNA Mannich reaction conditions and applied them to investigate the reactions of DNA-conjugated aldehydes with various amine and ketone building blocks. The developed on-DNA Mannich reaction preserved the DNA integrity and established viable routes for library production.

Groebke-Blackburn-Bienaymé Reaction for DNA-Encoded Library Technology.

This study presents a DNA-compatible synthesis of diverse 5-arylimidazo[1,2-]pyridin-3-amine derivatives using the Suzuki-Miyaura reaction, followed by a Groebke-Blackburn-Bienaymé (GBB) reaction. The GBB reaction demonstrates a wide substrate scope, mild one-pot reaction conditions, and compatibility with subsequent enzymatic ligation, highlighting its potential in DNA-encoded library technology.

The linkers in fluorene-labeled 2'-deoxyuridines affect fluorescence discriminating phenomena upon duplex formation.

Three fluorene-labeled 2'-deoxyuridines that differ in terms of their linkers-U (without linker), U (with ethynyl linker), and U (with diethynyl linker)-have been introduced at the central positions of oligodeoxynucleotides to examine the effects that their linkers have on the fluorescence emission properties upon duplex formation with fully matched and single-base-mismatched targets. Here, we describe the influence of the linkers on the emission behavior, the intramolecular electron transfer between the fluorene moiety and the uracil base after photoexcitation, and the structural stability upon duplex formation. The probe containing the U residue (with an ethynyl linker) and cytosine residues as flanking bases exhibited the greatest fluorescence turn-on selective behavior toward the perfectly matched target.

DNA-Encoded Library Screening as Core Platform Technology in Drug Discovery: Its Synthetic Method Development and Applications in DEL Synthesis.

DNA-encoded library technology (DELT) was introduced to our medicinal chemistry society more than 20 years ago. The application of DELT in the development of clinical candidates has been actively reported in the literature recently. A few representative examples include RIP1K inhibitors for inflammatory diseases and sEH inhibitors for endothelial dysfunction or abnormal tissue repair, among many others.

Synthesis and Biological Evaluation of BODIPY-PF-543.

Sphingosine-1-phosphate (S1P) regulates the proliferation of various cells and promotes the growth of cancer cells. Sphingosine kinase (SK), which transforms sphingosine into S1P, has two isotypes: SK1 and SK2. To date, both isotypes are known to be involved in the proliferation of cancer cells.

Synthesis of dansyl labeled sphingosine kinase 1 inhibitor.

PF-543 is a non-sphingosine analogue with inhibitory effect against SK1, based on a Ki of 4.3 nM and 130-fold selectivity for SK1 over SK2. Since the development of PF-543, animal studies demonstrated its valuable role in multiple sclerosis, myocardial infarction, and colorectal cancer.

5-Bromo-4',5'-bis(dimethylamino)fluorescein: Synthesis and Photophysical Studies.

In this study, three new fluorescein derivatives-5-bromo-4',5'-dinitrofluorescein (), 5-bromo-4',5'-diaminofluorescein (), and 5-bromo-4',5'-bis(dimethylamino)fluorescein ()-were synthesized and their pH-dependent protolytic equilibria were investigated. In particular, exhibited pH-dependent fluorescence, showing strong emission only at pH 3-6. bears a bromine moiety and thus, can be used in various cross-coupling reactions to prepare derivatives and take advantage of its unique emission properties.

Single-Labeled Oligonucleotides Showing Fluorescence Changes Upon Hybridization with Target Nucleic Acids.

Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs) is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection.

Catalytic enantioselective synthesis of carboxy-substituted 2-isoxazolines by cascade oxa-Michael-cyclization.

An efficient quinidine-based phase-transfer-catalyzed enantioselective cascade oxa-Michael-cyclization reaction of hydroxylamine with various β-carboxy-substituted α,β-unsaturated ketones has been achieved for the preparation of chiral carboxy-substituted 2-isoxazolines. This cascade reaction provided the desired products in good yields (up to 98%) with excellent enantioselectivities (91-96% ee). In addition, the cascade reaction was effectively applied to the first catalytic asymmetric synthesis of the herbicide (S)-methiozolin.

pH-Responsive quencher-free molecular beacon systems containing 2'-deoxyuridine units labeled with fluorene derivatives.

Three fluorescent nucleosides-U, U, and U, containing fluorene, 2-aminofluorene, and 2-dimethylaminofluorene units, respectively, covalently attached to 2'-deoxyuridine-have been incorporated into the central positions of oligodeoxynucleotides (ODNs) to examine the effects of their flanking bases (FBs) and pH on the emission properties upon hybridization with fully matched and single-base-mismatched targets. The ODN containing U and cytosine-FBs in the pH range from 5.5 to 8.

Highly efficient quencher-free molecular beacon systems containing 2-ethynyldibenzofuran- and 2-ethynyldibenzothiophene-labeled 2'-deoxyuridine units.

We have prepared two fluorescent DNA probes--UDBF and UDBT, containing 2-ethynyldibenzofuran and 2-ethynyldibenzothiophene moieties, respectively, covalently attached to the base dU--and incorporated them in the central positions of oligodeoxynucleotides (ODNs) so as to develop new types of quencher-free linear beacon probes and investigate the effect of functionalization of the fluorene scaffold on the photophysical properties of the fluorescent ODNs. The ODNs containing adenine flanking bases (FBs) displayed a selective fluorescence "turn-off" response to mismatched targets with guanine bases; this suggests that these probes could be used as base-discriminating fluorescent nucleotides. On the other hand, we observed a "turn-on" response to matched targets when the UDBF and UDBT units of ODNs containing pyrimidine-based FBs were positioned opposite the four natural nucleobases.

Synthesis and photophysical study of 2'-deoxyuridines labeled with fluorene derivatives.

We examined microenvironment-sensitive fluorescent 2'-deoxyuridines labeled with fluorene derivatives that exhibited solvent-dependent photophysical properties. The high sensitivity of the fluorescence shift and the nucleoside intensity dependence on solvent polarity provided information useful for estimating the polarity of the environment surrounding the fluorescent nucleoside.

Solution structure, mechanism of replication, and optimization of an unnatural base pair.

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge-on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue dMMO2, which forms an unnatural base pair with the nucleotide analogue d5SICS.

The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation.

In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context.

Efforts toward developing probes of protein dynamics: vibrational dephasing and relaxation of carbon-deuterium stretching modes in deuterated leucine.

The spectral position of C-D stretching absorptions in the so-called "transparent window" of protein absorption (1800-2300 cm(-1)) makes them well suited as probes of protein dynamics with high temporal and structural resolution. We have previously incorporated single deuterated amino acids into proteins to site-selectively follow protein folding and ligand binding by steady-state FT IR spectroscopy. Ultimately, our goal is to use C-D bonds as probes in time-resolved IR spectroscopy to study dynamics and intramolecular vibrational energy redistribution (IVR) in proteins.

Optimization of an unnatural base pair toward natural-like replication.

Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of Escherichia coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature.

Unnatural substrate repertoire of A, B, and X family DNA polymerases.

As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examined the ability of five different polymerases to replicate DNA containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees.

Cholesterol-linked pyrene excimer molecular beacon with enhanced cell permeability.

Covelently labeled pyrene excimer molecular beacon (MB) with cholesterol moiety has been developed for enhanced the cellular delivery of MB.(1) Pyrene units were covalently attached into adenosine and incorporated to oligonucleotides at the complementary locations in opposite strands in the middle positions of hairpin stems. The system behaves as an effective MB that changes color from green to blue upon duplex formation.

Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet.

DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addition of an unnatural base pair, promise to expand the biotechnological applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase.

Polymerase recognition and stability of fluoro-substituted pyridone nucleobase analogues.

Recently much effort has been focused on designing unnatural base pairs that are stable and replicated by DNA polymerases with high efficiency and fidelity. This work has helped to identify a variety of nucleobase properties that are capable of mediating the required interbase interactions in the absence of Watson-Crick hydrogen-bonding complementarity. These properties include shape complementarity, the presence of a suitably positioned hydrogen-bond donor in the developing minor groove, and fluorine substitution.

Pyrene-labeled deoxyuridine and deoxyadenosine: fluorescent discriminating phenomena in their duplex and hairpin oligonucleotides.

Pyrene-labeled deoxyuracil and deoxyadenine units are useful unnatural nucleobases. These fluorescent nucleobase analogues allow strong interstrand stacking interactions to compensate for a loss of hydrogen bonding and exhibit a range of different emission intensities when they form duplexes with one another. These findings may provide new insights into the design of new probes and nucleobase analogues for applications in molecular biology.

Cholesterol-linked fluorescent molecular beacons with enhanced cell permeability.

We appended pyrene units covalently onto adenosine (forming A(P) units) and then incorporated them into oligonucleotides such that they were positioned in complementary locations in opposite strands in the middle positions of hairpin stems. System 1 (A(P)A(P)) behaves as an effective molecular beacon (MB) that changes color from green to blue upon duplex formation. In addition, we attached a cholesterol unit to a free terminus of one of these hairpins; this approach enhanced the cellular delivery of the modified MB relative to those encountered when using conventional transfection methods.