Novel interleukin-5 inhibitors based on hydroxyethylaminomethyl-4H-chromen-4-one scaffold.

Hydroxyethylaminomethyl-4H-chromenones were previously discovered as fairly strong IL-5 inhibitor. For determination of detail structure activity relationship, N-substituted hydroxyethylaminomethylchromenones 4a-n were prepared and evaluated for their IL-5 inhibitory activity. Shifting the hydrophobic group to nitrogen from 1-position of hydroxyethylamino moiety of hydroxyethylaminomethyl-4H-chromenones enhances the activity. The increment in bulkiness or hydrophobicity of alkyl side chain at amino group increases the activity. The same level of activity of 5-(cyclohexylmethoxy)-3-(N-benzyl-2-hydroxyethylaminomethyl)-4H-chromenone analogs regardless of hydrophobic or hydrophilic substituents at 4th position of phenyl ring might infer the existence of tunnel structure in the putative receptor for accepting these side chains.

Structure-activity relationship study of arylsulfonylimidazolidinones as anticancer agents.

In an effort to find novel N-arylsulfonylimidazolidinones as highly potent anticancer agent, the structure-activity relationship of ethyl 2-methyl-4-(2-oxo-4-phenylimidazolidin-1-ylsulfonyl)phenylcarbamate was explored through synthesis and evaluation of in vitro cytotoxicity of its analogs against HCT116, A549 and NCL-H460 cancer cell lines. Among the synthesized derivatives, the carbamate analogs (4a-f and 4k-p) exhibited superior cytotoxicity to doxorubicin for all cancer cell lines. The SAR studies of these derivatives confirm that the intact 4-phenyl-l-benzenesulfonylimidazolidinone has a pivotal role as a basic pharmacophore and hydrophobic substitutions only at 2-position of 1-aminobenzenesulfonyl moiety are beneficial for the enhancement of the activity.

Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase.

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N'-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.

Photolytic control and infrared probing of amide I mode in the dipeptide backbone-caged with the 4,5-dimethoxy-2-nitrobenzyl group.

Alanine dipeptide analog 1 backbone-caged with a photolabile linker, 4,5-dimethoxy-2-nitrobenzyl (DmNb), was synthesized. UV-pulse-induced photochemical reaction of 1 was monitored by Fourier transform IR absorption spectroscopy under a steady-state condition or in a fast-scan mode. Upon photolysis of 1, the amide I band is changed from a doublet to a singlet with concomitant line shape changes of several IR bands. The change of the amide I band is directly associated with the photocleavage of the covalent N-C bond connecting the backbone amide of 2 to DmNb. Therefore, IR spectroscopy is useful for directly probing the photocleavage of backbone-caged peptide 1 and the concurrent release of native peptide 2. In contrast, UV-vis spectroscopy probing the irradiation-induced structural change of the 2-nitrobenzyl moiety itself may not provide a clue directly relevant to the photocleavage of such N-C bond. Time-resolved IR spectra recorded in a fast-scan mode after pulsed UV irradiation of 1 reveal that such photocleavage occurs at least faster than a few seconds of our instrumental time resolution.

Site-selective intramolecular hydrogen-bonding interactions in phosphorylated serine and threonine dipeptides.

To study the phosphorylation effect on the peptide conformation, we carried out nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform (FT)-IR, and vibrational circular dichroism (VCD) experiments with serine and threonine dipeptides (SD and TD) and their phosphorylated ones (pSD and pTD). It is found that both unphosphorylated and phosphorylated serine and threonine dipeptides adopt two conformations, polyproline II (P(II)) and beta-strand. The pH-dependent NMR study shows that the side-chain dianionic phosphoryl group can form direct intramolecular hydrogen bonds with the backbone amide protons at both the acetyl and amide ends of pTD, but only at the acetyl end of pSD. Temperature- and pH-dependent CD studies reveal that, unlike pSD, pTD undergoes conformational transition from P(II) to beta-strand upon double ionization of the phosphoryl group. The subtle but distinct differences between pTD and pSD in site-selective intramolecular hydrogen-bonding interaction and charge-dependent conformational transition may sometimes become significant when choosing between serine and threonine for the conformational control of peptides and proteins by phosphorylation.

Beta-azidoalanine as an IR probe: application to amyloid Abeta(16-22) aggregation.

Beta-azidoalanine dipeptide 1 was synthesized, and its azido stretching vibration in H2O and dimethyl sulfoxide (DMSO) was studied by using Fourier transform (FT) IR spectroscopy. The dipole strength of the azido stretch mode is found to be about 19 and 5 times larger than those of the CN and SCN stretch modes, respectively, which have been used as local environmental IR sensors. The azido stretch band in H2O is blue-shifted by about 14 cm(-1) in comparison to that in DMSO, indicative of its sensitivity to the electrostatic environment. To test the utility of beta-azidoalanine as an IR probe of the local electrostatic environment in proteins, azidopeptide 4 was prepared by its incorporation into Abeta(16-22) peptide of the Alzheimer's disease amyloid beta-protein at position Ala21. The amide I IR spectrum of 4 in D2O suggests that the azidopeptide thus modified forms in-register beta-sheets in aggregates as observed for normal Abeta(16-22). The azido peak frequency of 4 in aggregates is almost identical to that in DMSO, indicating that the azido group is not exposed to water but to the hydrophobic environment. We believe that beta-azidoalanine will be used as an effective IR probe for providing site-specific information about the local electrostatic environments of proteins.

Structure of N-acetylproline amide in liquid water: experimentally measured and numerically simulated infrared and vibrational circular dichroism spectra.

A few experimental and theoretical studies on the molecular structure of N-acetylproline amide (AP) in D2O solution have been reported recently. However, there is no consensus of the precise structure of AP in D2O because spectroscopically determined structures and a theoretically simulated one have been found to be different from one another. To determine its aqueous solution structure, IR and vibrational circular dichroism spectra of both L- and D-form AP solutions were measured. Molecular dynamics simulations with two different force fields and density functional theory calculations for the trans and cis rotamers of AP were performed to numerically simulate those spectra. Comparisons between experimentally measured and computationally simulated spectra directly suggest that the AP in water adopts a polyproline II-like conformation and that the force field parameter ff03 in the AMBER 8 suite of programs is more realistic and reliable in predicting molecular structure of AP in water than the ff99 in AMBER 7.

Infrared probing of 4-azidoproline conformations modulated by azido configurations.

4-Azidoproline (Azp) can tune the stability of the polyproline II (P(II)) conformation in collagen. The azido group in the 4R and 4S configurations stabilizes and destabilizes the P(II) conformation, respectively. To obtain insights into the dependence of the conformational stability on the azido configuration, we carried out Fourier transform (FT) IR experiments with four 4-azidoproline derivatives, Ac-(4R/S)-Azp-(NH/O)Me. We found that the amide I and azido IR spectra are different depending on the azido configuration and C-terminal structure. The origin of such spectral differences between 4R and 4S configurations and between C-terminal methylamide and ester ends was elucidated by quantum chemistry calculations in combination with (1)H NMR and time- and frequency-resolved IR pump-probe spectroscopy. We found that the azido configurations and C-terminal structures affect intramolecular interactions, which are responsible for the ensuing conformational and thereby IR spectral differences. Consequently, 4-azidoproline conformations modulated by azido configurations can be probed by IR spectroscopy. These findings suggest that 4-azidoproline can be both a structure-control and -probing element, which enables the infrared tracking of proline roles in protein structure, function, and dynamics.

Azido gauche effect on the backbone conformation of ß-azidoalanine peptides.

To study the azido gauche effect on the backbone conformation of ß-azidoalanine (Aza) dipeptide (AAD, Ac-Aza-NHMe) and tripeptide (AAT, Ac-Aza-Aza-NH(2)), we used spectroscopic methods in combination with quantum chemistry calculations and molecular dynamics (MD) simulations. From the (1)H NMR coupling constants and (1)H,(1)H NOESY experimental data, we found that AAD in water mainly adopts a seven-membered cyclic (C(7)) rather than polyproline II (P(II)) backbone conformation and prefers the gauche- (g(-)) side-chain conformer. From the amide I IR absorption and circular dichroism (CD) spectra, the backbone conformation of AAD in water is found to deviate from P(II) but is rather close to C(7). Thus, the backbone conformation of AAD differs from that of alanine dipeptide (AD, Ac-Ala-NHMe), which is mainly P(II) in water. The underlying origin of the backbone conformational difference between AAD and AD in water was elucidated by quantum chemistry calculations with density functional theory (DFT). It was found that the C(7)/g(-) conformer is the lowest energy structure of an isolated AAD. Here, the ß-azido group forms intramolecular electrostatic interactions with two neighboring peptide bonds, which are facilitated by the azido gauche effect. Thus, the ß-azido group appears to be responsible for directing the peptide backbone conformation toward the C(7) structure. The quantum mechanical/molecular mechanical (QM/MM) MD simulations show that AAD in water adopts neither P(II) nor right-handed α-helix (α(R)) and prefers the g(-) conformer. Thus, the intramolecular electrostatic interactions between the ß-azido group and two nearby peptide bonds are also found even in the aqueous solution structure of AAD. Consequently, the ß-azido group appears to be an effective C(7)-conformation-directing element, which may also be useful for tuning the structures of other amino acids and polypeptides.

Phosphorylation effect on the GSSS peptide conformation in water: infrared, vibrational circular dichroism, and circular dichroism experiments and comparisons with molecular dynamics simulations.

The phosphorylation effect on the small peptide conformation in water has not been clearly understood yet, despite the widely acknowledged notion that control of protein activity by phosphorylation works mainly by inducing conformational change. To elucidate the detailed mechanism, we performed infrared (IR) absorption and vibrational and electronic circular dichroism studies of both unphosphorylated and phosphorylated tetrapeptides, GSSS 1 and GSSpS 2. The solution structure of the tetrapeptide is found to be little dependent on the presence of the neutral or negatively charged phosphoryl group, and to be a mixture of extended structures including polyproline II (PII) and beta-sheet conformations. The additional band at 1598 cm(-1) in the amide I IR spectrum of the phosphorylated peptide GSSpS at neutral pD appears to be clear spectroscopic evidence for direct intramolecular hydrogen-bonding interaction between the side chain dianionic phosphoryl group and the backbone amide proton. On the basis of amide I IR band analyses, the authors found that the probability of finding the phosphoryl group strongly H bonded to the backbone proton in GSSpS is about 43% at pD 7.0 and 37 degrees C. Such a H-bonding interaction in GSSpS has the biological standard enthalpy and entropy of -15.1 kJ/mol and -51.2 J/K mol, respectively. Comparisons between the experimentally measured IR and VCD spectra and the numerically simulated ones suggested that the currently available force field parameters need to be properly modified. The results in this paper may shed light on an unknown mechanism of controlling the peptide conformation by phosphorylation.

Dipeptide structure determination by vibrational circular dichroism combined with quantum chemistry calculations.

The solution structure and the local solvation environments of alanine dipeptide (AD, 1 a) and its isotopomer (AD*, 1 b, 13C on the acetyl end C==O) are studied by using infrared (IR) spectroscopy and vibrational circular dichroism (VCD). From the amide I IR spectra of AD* in various protic solvents, it is found that each of the two carbonyl groups is fully H-bonded to two water molecules. However, the number of alcohol molecules H-bonded to each C==O varies from one to two, and the local solvation environments are asymmetric around the two peptides of AD* in alcohol solutions. The amide I VCD spectra of AD and AD* in D2O are also measured, and a series of density functional theory (DFT, B3LYP/6-311++G**) calculations are performed to obtain the amide I normal-mode rotational strengths of AD and the intrinsic rotational strengths of its two peptide fragments. By combining the VCD-measurement and DFT-calculation results and employing a coupled oscillator theory, we show that the aqueous-solution structure of the dipeptide can be determined. We believe that the present method will be of use in building up a library of dipeptide solution structures in water.