Synthesis and NMR studies of (13)C-labeled vitamin D metabolites

Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar solutions as compared to that in the solid state is most easily rationalized on the basis that the 5(10)-torsion angle in 58 is decreased in micellar solutions as compared to that in the solid state.     

4,4-Disubstituierte Imidazol-Derivate aus der Umsetzung von 3-Amino-2H-azirinen mit Salicylamid

4,4-Disubstituted Imidazole Derivatives from the Reaction of 3-Amino-2H-azirines with Salicylamide Reaction of 3-amino-2H-azirines 1a–c with salicylamide ( 7 ) in MeCN leads to imidazoles 10 and 11 in different rates, depending on the conditions. In the case of 1a and 1b, 11a and 11b , respectively, have been obtained as the main product at 50°; in reactions at 80°, 10a and 10b are the favored products (Tables 1 and 2). 2,2-Dimethyl-3-(N-methyl-N-phenylamino)-2H-azirine ( 1c ) reacts with 7 in MeCN mainly to 2-(2-hydroxyphenyl)-5,5-dimethyl-3,5-dihydroimidazol-4-one ( 10a ); in boiling toluene, 11c is formed with low preference (Table 3). The structure of the products has been established by spectroscopic means, and in the case of 10b and 11c , by X-ray crystallography. Two different reaction mechanisms for the formation of the products are discussed (Scheme 2).     

Tuning the singlet oxygen quantum yield of near-IR-absorbing porphyrazines

We report the quantum yields for singlet oxygen production by a series of porphyrazines (pz) of the form M[pz(An;B4-n)] (Scheme 1), where the peripheral substituent A is [S-R]2 with R = (CH2CH2O)3H, B is a fused alpha,alpha'-dialkoxybenzo group and M = 2H, Mg or Zn. These compounds show intense near-IR absorbance/emission (longest wavelength emission, approximately 830 nm). Their solubilities vary with R, whereas their optical properties do not. We show that singlet oxygen sensitization by these luminescent compounds can be "tuned" from essentially off to on by varying n and selection among M = 2H, Mg or Zn. The quantum yields vary ca 60-fold within the set of compounds studied, from phidelta = 0.007 for compound 3 to phidelta = approximately 0.4 for compound 11.     

NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins

NMR spectroscopy and X-ray crystallography, the two primary experimental methods for protein structure determination at high resolution, have different advantages and disadvantages in terms of sample preparation and data collection and analysis. It is therefore of interest to assess their complementarity when applied to small proteins. Structural genomics/proteomics projects provide an ideal opportunity to make such comparisons as they generate data in a systematic manner for large enough numbers of proteins to allow firm conclusions to be drawn. Here we report a comparison for 263 unique proteins screened by both NMR spectroscopy and X-ray crystallography in our structural proteomics pipeline. Only 21 targets (8%) were deemed amenable to both methods based on an initial 2D 15N-HSQC NMR spectrum and optimized crystallization trials. However, the use of both methods in the pipeline increased the total number of targets amenable to structure determination to 107, with 43 amenable to NMR only and 43 amenable to X-ray crystallographic methods only. We did not observe a correlation between 15N-HSQC spectral quality and the success of the same protein in crystallization screens. Similar results were found for an independent set of 159 proteins as reported in the accompanying paper by Snyder et al. Thus, we conclude that both methods are highly complementary, and in order to increase the number of proteins suited for structure determination, we suggest that both methods be used in parallel in screening of all small proteins for structure determination.     

Controlling electron delocalisation in constrained N,N′-dimethyl-4,4′-bipyridinium dications

The synthesis is described of two N,N′-dimethyl-4,4′-bipyridinium salts for which the two pyridinium rings are connected via varying length alkoxy chains attached at the 3,3′-locations.     

Steric effects—III: Composition of the E's parameter. Variation of alkyl steric effects with substitution. Role of conformation in determining sterically active and inactive sites

The steric effect of alkyl groups as parameterized by E'_s covers a range of over seven powers of ten. In this article an attempt is made to portray in mechanism terms the manifold nature of the steric effect as a function of substitution, i.e. the composition of the steric effect. One most consider the contribution of the topological carbon sites as a function of the true coordinates of these atoms. To this end, we evaluate the geometry of these alkyl groups in carboxylic acid indirectly by a search for the probable conformations of these acids. The starting point in this study is the determination of the minimum energy conformations in carboxylic acids using the empirical force field method based on analogous alkylmethyl ketone models.This approach to the ground state behaviour of these acids leads directly to a division of the complete set of alkyl groups into two subsets within which a Conformational sequence or filitation is associated with the substitution process which generates the successive alkyl groups of that subset. It is shown that eclipsed conformations are preferred in the case of secondary and tertiary groups bearing Et and Me sub-groups, giving way to preferred bisected conformations when t-Bu and i-Pr sub-groups are present. For further elucidation of steric perturbation terms these two subsets must be considered independently. These important observations lead to an overall coherent interpretation of steric effects.Three regions of distinct behaviour as a function of substitution have been identified. In Region I (the so-called “mormal” region, where the contribution of the introduction of successive methyl groups to the overall E'_s increases monotonically), besides excellent topological site correlations, an excellent hybrid correlation is proposed. For a conformationally homogeneous (i.e. eclipsed) set of alkyl groups the overall E'_s of groups is well expressed by a linear combination of the weighted contribution of sub-groups and the Newman six-number. This correlation leads to an interpretation of the composition of the overall steric effect of an alkyl group, as measured by the E'_s parameter, in terms of conformationally defined sites and stresses the steric importance of certain privileged sites. This result is consistent with the molecular mechanics viewpoint of preferred conformations but at variance with previous assumptions.The levelling effect (Region II), in the light of conformational information, is understood in terms of sterically active and inactive sites, while the inversion effect (Region III) is considered to be the result of molecular distortion brought about by relief of local internal steric strain within the alkyl groups. The X-ray structure of i-Pr₃CCO₂H illustrates this point.     

Temperature weighted histogram analysis method, replica exchange, and transition paths

We analyzed the data from a replica exchange molecular dynamics simulation using the weighted histogram analysis method to combine data from all of the temperature replicas (T-WHAM) to obtain the room-temperature potential of mean force of the G-peptide (the C-terminal beta-hairpin of the B1 domain of protein G) in regions of conformational space not sampled at room temperature. We were able to determine the potential of mean force in the transition region between a minor alpha-helical population and the major beta-hairpin population and identify a possible transition path between them along which the peptide retains a significant amount of secondary structure. This observation provides new insights into a possible mechanism of formation of beta-sheet secondary structures in proteins. We developed a novel Bayesian statistical uncertainty estimation method for any quantity derived from WHAM and used it to validate the calculated potential of mean force. The feasibility of estimating regions of the potential of mean force with unfavorable free energy at room temperature by T-WHAM analysis of replica exchange simulations was further tested on a system that can be solved analytically and presented some of the same challenges found in more complex chemical systems.     

Electrokinetic molecular separation in nanoscale fluidic channels

This report presents a study of electrokinetic transport in a series of integrated macro- to nano-fluidic chips that allow for controlled injection of molecular mixtures into high-density arrays of nanochannels. The high-aspect-ratio nanochannels were fabricated on a Si wafer using interferometric lithography and standard semiconductor industry processes, and are capped with a transparent Pyrex cover slip to allow for experimental observations. Confocal laser scanning microscopy was used to examine the electrokinetic transport of a negatively charged dye (Alexa 488) and a neutral dye (rhodamine B) within nanochannels that varied in width from 35 to 200 nm with electric field strengths equal to or below 2000 V m-1. In the negatively charged channels, nanoconfinement and interactions between the respective solutes and channel walls give rise to higher electroosmotic velocities for the negatively charged dye than for the neutral dye, towards the negative electrode, resulting in an anomalous separation that occurs over a relatively short distance (<1 mm). Increasing the channel widths leads to a switch in the electroosmotic transport behavior observed in microscale channels, where neutral molecules move faster because the negatively charged molecules are slowed by the electrophoretic drag. Thus a clear distinction between "nano-" and "microfluidic" regimes is established. We present an analytical model that accounts for the electrokinetic transport and adsorption (of the neutral dye) at the channel walls, and is in good agreement with the experimental data. The observed effects have potential for use in new nano-separation technologies.     

The transfer between instruments of a reflectance near-infrared assay for paracetamol in intact tablets

This study compares several correction methods to facilitate the transfer of a validated near-infrared (NIR) assay for paracetamol in intact tablets between two reflectance NIR instruments of the same type. Transfer was defined as the ability to accurately predict the true assay value of a sample measured on a NIR system using an assay developed on a different system, and was assessed using a comprehensive set of statistical tests. Direct electronic transfer of the calibration models, representing the NIR assay, was not possible as a result of a definite residual spectrum between instruments. The use of a correction method based on the standardisation of the material used to record the reference spectrum also proved ineffective. Two methods investigated did succeed, the first employed a response surface calculated between the reflectance values of a set of six certified photometric standards measured on both instruments, with all full range partial least square (PLS) regression models subsequently transferred. The next was correction of the spectra from the second instrument utilising the residual spectrum between the mean sample of the validation set measured on both instruments. Through this approach all PLS regression models and also a single multiple linear regression (MLR) model were transferred. As an outcome of this study guidelines are suggested for the transfer of NIR assays along with the criteria deemed necessary to conclusively prove transfer and justify any correction method utilised. The significant criteria were determined to be the paired t-test with both the UV reference assay data and the original NIR assay data, and comparison of the coefficient of multiple determinations.     

The fast multipole boundary element method for molecular electrostatics: An optimal approach for large systems

We propose a fast implementation of the boundary element method for solving the Poisson equation, which approximately determines the electrostatic field around solvated molecules of arbitrary shape. The method presented uses computational resources of order O(N) only, where N is the number of elements representing the dielectric boundary at the molecular surface. The method is based on the Fast Multipole Algorithm by Rokhlin and Greengard, which is used to calculate the Coulombic interaction between surface elements in linear time. We calculate the solvation energies of a sphere, a small polar molecule, and a moderately sized protein. The values obtained by the boundary element method agree well with results from finite difference calculations and show a higher degree of consistency due to the absence of grid dependencies. The boundary element method can be taken to a much higher accuracy than is possible with finite difference methods and can therefore be used to verify their validity. © 1995 by John Wiley & Sons, Inc.