Tirayut Vilaivan

Organocatalyzed Asymmetric α-Oxidation, α-Aminoxylation and α-Amination of Carbonyl Compounds

Organocatalytic asymmetric α-oxidation and amination reactions of carbonyl compounds are highly useful synthetic methodologies, especially in generating chiral building blocks that previously have not been easily accessible by traditional methods. The concept is relatively new and therefore the list of new catalysts, oxidizing and aminating reagents, as well as new substrates, are expanding at an amazing rate. The scope of this review includes new reactions and catalysts, mechanistic aspects and synthetic applications of α-oxidation, hydroxylation, aminoxylation, amination, hydrazination, hydroxyamination and related α-heteroatom functionalization of aldehydes, ketones and related active methylene compounds published during 2005–2009.

Malarial dihydrofolate reductase as a paradigm for drug development against a resistance-compromised target

Malarial dihydrofolate reductase (DHFR) is the target of antifolate antimalarial drugs such as pyrimethamine and cycloguanil, the clinical efficacy of which have been compromised by resistance arising through mutations at various sites on the enzyme. Here, we describe the use of cocrystal structures with inhibitors and substrates, along with efficacy and pharmacokinetic profiling for the design, characterization, and preclinical development of a selective, highly efficacious, and orally available antimalarial drug candidate that potently inhibits both wild-type and clinically relevant mutated forms of Plasmodium falciparum (Pf) DHFR. Important structural characteristics of P218 include pyrimidine side-chain flexibility and a carboxylate group that makes charge-mediated hydrogen bonds with conserved Arg122 (PfDHFR-TS amino acid numbering). An analogous interaction of P218 with human DHFR is disfavored because of three species-dependent amino acid substitutions in the vicinity of the conserved Arg. Thus, P218 binds to the active site of PfDHFR in a substantially different fashion from the human enzyme, which is the basis for its high selectivity. Unlike pyrimethamine, P218 binds both wild-type and mutant PfDHFR in a slow-on/slow-off tight-binding mode, which prolongs the target residence time. P218, when bound to PfDHFR-TS, resides almost entirely within the envelope mapped out by the dihydrofolate substrate, which may make it less susceptible to resistance mutations. The high in vivo efficacy in a SCID mouse model of P. falciparum malaria, good oral bioavailability, favorable enzyme selectivity, and good safety characteristics of P218 make it a potential candidate for further development.

Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti-malarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

Electrochemical detection of human papillomavirus DNA type 16 using a pyrrolidinyl peptide nucleic acid probe immobilized on screen-printed carbon electrodes

An electrochemical biosensor based on an immobilized anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe was successfully developed for the selective detection of human papillomavirus (HPV) type 16 DNA. A 14-mer acpcPNA capture probe was designed to recognize a specific 14 nucleotide region of HPV type 16 L1 gene. The redox-active label anthraquinone (AQ) was covalently attached to the N-terminus of the acpcPNA probe through an amide bond. The probe was immobilized onto a chitosan-modified disposable screen-printed carbon electrode via a C-terminal lysine residue using glutaraldehyde as a cross-linking agent. Hybridization with the target DNA was studied by measuring the electrochemical signal response of the AQ label using square-wave voltammetric analysis. The calibration curve exhibited a linear range between 0.02 and 12.0 µM with a limit of detection and limit of quantitation of 4 and 14 nM, respectively. This DNA sensing platform was successfully applied to detect the HPV type 16 DNA from a PCR amplified (240 bp fragment of the L1 gene) sample derived from the HPV type 16 positive human cancer cell line (SiHa), and failed to detect the HPV-negative c33a cell line. The sensor probe exhibited very high selectivity for the complementary 14 base oligonucleotide over the non-complementary oligonucleotides with sequences derived from HPV types 18, 31 and 33. The proposed sensor provides an inexpensive tool for the early stage detection of HPV type 16, which is an important biomarker for cervical cancer.

Comparison of DNA, aminoethylglycyl PNA and pyrrolidinyl PNA as probes for detection of DNA hybridization using surface plasmon resonance technique

Pyrrolidinyl peptide nucleic acid bearing a D-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) has been evaluated as a new sensing probe for detection of DNA hybridization. In this study, the biotinylated acpcPNA was immobilized on surface plasmon resonance (SPR) sensor chips via biotin-streptavidin interactions for solid-phase DNA hybridization. A critical comparison between acpcPNA, DNA and conventional peptide nucleic acid (aegPNA) probes of the same sequence was made by means of SPR on various important aspects. These include the effect of ionic strength on hybridization efficiency, the specificity to detect the mismatch(es) in target DNAs, the direction of binding (parallel or antiparallel) to target DNAs, and the effect of target DNA concentration on hybridization efficiency. Results indicated that the immobilized acpcPNA probe possesses distinct hybridization properties relative to aegPNA (and/or DNA) counterparts, including a higher single-base mismatch sensitivity, antiparallel selectivity and low ionic strength dependence of target hybridization. These properties substantiate the acpcPNA applicability as sensor probes for clinical and diagnostic applications. With a proper selection of regeneration conditions (10 mM NaOH, 2 min exposure), the sensor can be reused for multiple cycles of hybridization with as little as 1.3% loss in hybridization activity per regeneration cycle.

Phenylene-ethynylene trication as an efficient fluorescent signal transducer in an aptasensor for potassium ion.

A tricationic phenylene-ethynylene (N(3+)) fluorophore is investigated as a fluorescent transducer in homogeneous aptasensing system for potassium ion (K(+)) assay in aqueous media. The enhancement of the fluorescent signal of N(3+) by three K(+) aptamers consisting of 12, 15, and 21 nucleotides are observed and used for the determination of N(3+)-aptamer binding affinities. The binding affinities increase with the length of the aptameric oligonucleotides and are proven to be important to the sensitivity and selectivity of the aptasensors. The enhanced fluorescent signal of each N(3+)-aptamer solution is selectively quenched by K(+) due to the ability of K(+) in stabilizing the G-quadruplex structure of the aptamer. Among three aptamers, the 15-base aptamer provides optimal sensitivity and selectivity over other ions such as Li(+), Na(+), NH(4)(+), Mg(2+), Ca(2+) and Sr(2+). The sensing system shows the detection limit of 1 μM of K(+) in clean buffered solution and 30 μM of K(+) in the solution containing 4800-fold excess of Na(+), with wide linear dynamic ranges of micro- to millimolar concentration. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K(+) in urine samples.

N-Salicyl-β-aminoalcohols as a new class of ligand for catalytic asymmetric Strecker reactions

An enantioselective Strecker synthesis employing novel chiral titanium complex catalysts derived from structurally simple chiral N-salicyl-β-amino alcohols is described. Reactions of N-benzylidenebenzylamine with trimethylsilyl cyanide in the presence of the catalyst (10 mol%) gave the corresponding α-aminonitrile in good to excellent yields, along with relatively high enantioselectivity (up to 86% ee). Similar reactions with various imines derived from aromatic aldehydes resulted in moderate to good enantioselectivity (44–81% ee).

Indium mediated Barbier-type allylation of aldimines in alcoholic solvents

Abstract Barbier-type allylation of unactivated aldimines with allyl bromides in the presence of indium powder took place rapidly in alcoholic solvents to give homoallylic amines in fair to good yields.

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance.

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI-TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 degrees C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 microM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.

Synthesis of Solution-Phase Combinatorial Library of 4,6-Diamino-1,2-dihydro-1,3,5-triazine and Identification of New Leads Against A16V+S108T Mutant Dihydrofolate Reductase of Plasmodium falciparum

An efficient method to synthesize solution-phase combinatorial library of 1-aryl-4,6-diamino-1,2-dihydro-1,3,5-triazine was developed. The strategy involved an acid-catalyzed cyclocondensation between arylbiguanide hydrochlorides and carbonyl compounds in the presence of triethyl orthoacetate as water scavenger. A 96-membered combinatorial library was constructed from 6 aryl biguanides and 16 carbonyl compounds. Screening of the library by iterative deconvolution method revealed two candidate leads which are equally active against wild-type Plasmodium falciparum dihydrofolate reductase, but are about 100-fold more effective against the A16V+S108T mutant enzyme as compared to cycloguanil.