Using indirect covariance spectra to identify artifact responses in unsymmetrical indirect covariance calculated spectra

Several groups of authors have reported studies in the areas of indirect and unsymmetrical indirect covariance NMR processing methods. Efforts have recently focused on the use of unsymmetrical indirect covariance processing methods to combine various discrete two-dimensional NMR spectra to afford the equivalent of the much less sensitive hyphenated 2D NMR experiments, for example indirect covariance (icv)-heteronuclear single quantum coherence (HSQC)-COSY and icv-HSQC-nuclear Overhauser effect spectroscopy (NOESY). Alternatively, unsymmetrical indirect covariance processing methods can be used to combine multiple heteronuclear 2D spectra to afford icv-13C-15N HSQC-HMBC correlation spectra. We now report the use of responses contained in indirect covariance processed HSQC spectra as a means for the identification of artifacts in both indirect covariance and unsymmetrical indirect covariance processed 2D NMR spectra.     

Multistep correlations via covariance processing of COSY/GCOSY spectra: opportunities and artifacts

Long-range homonuclear coupling pathways can be observed in COSY or GCOSY spectra by the acquisition of spectra with larger numbers of increments of the evolution period, t(1), than would normally be used. Alternatively, covariance processing of COSY-type spectra acquired with modest numbers of t(1) increments, allows the observation of multistage correlations. In this work results obtained from covariance-processed GCOSY spectra are fully analyzed and compared to normally processed COSY and 80 ms TOCSY spectra. Multistage or 'RCOSY-type' correlations are observed when remote protons both exhibit correlations to the same coupling partner e.g. A --> B and B --> C gives rise to an A --> C correlation. In the strict sense, RCOSY-type responses are artifacts albeit providing useful information. Nonbeneficial artifact correlations are observed when protons couple to other protons that overlap or partially overlap. The origin of artifact responses is also analyzed.     

Dissecting heterogeneous molecular chaperone complexes using a mass spectrum deconvolution approach

Small heat-shock proteins (sHSPs) are molecular chaperones that prevent irreversible aggregation through binding nonnative target proteins. Due to their heterogeneity, these sHSP:target complexes remain poorly understood. We present a nanoelectrospray mass spectrometry analysis algorithm for estimating the distribution of stoichiometries comprising a polydisperse ensemble of oligomers. We thus elucidate the organization of complexes formed between sHSPs and different target proteins. We find that binding is mass dependent, with the resultant complexes reflecting the native quaternary architecture of the target, indicating that protection happens early in the denaturation. Our data therefore explain the apparent paradox of how variable complex morphologies result from the generic mechanism of protection afforded by sHSPs. Our approach is applicable to a range of polydisperse proteins and provides a means for the automated and accurate interpretation of mass spectra derived from heterogeneous protein assemblies.     

Structural Characterization of a Phenoxazine Analog Formed as a By‐product of the Synthesis of a 3‐Amino‐N,N‐dimethylsalicylamide

A highly colored impurity formed at low levels during the preparation of 3‐amino N,N‐dimethyl salicylamide was isolated and identified as 2‐amino‐N,N,N′,N′‐tetramethyl‐3‐oxo‐3H‐phenoxazin‐4,6‐dicarboxamide using mass spectrometry and two‐dimensional NMR methods that included long‐range ¹H‐¹⁵N heteronuclear chemical shift correlation data to assemble the proton‐deficient “right‐half” of the 2‐aminophenoxazin‐3‐one nucleus. The structure was independently confirmed using computer‐assisted structure elucidation methods and by synthesis.     

High-performance capillary electrophoretic separation of double-stranded oligonucleotides using a poly-(ethylpyrrolidine methacrylate-co-methylmethacrylate)-coated capillary

Here we describe a capillary electrophoretic method for the separation of double-stranded oligonucleotides (ds-ODNs) ranging from 16-20 bp with 2 bp resolution using a low concentration of poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) copolymer physically adsorbed to a capillary surface. Contrary to traditional DNA separations, we show that the ds-ODN with the highest molecular size eluted first and propose that this phenomena is due to a screening effect by the PEPyM-co-PMMA coating on the smaller ds-ODNs negative charge during elution. Key to the performance of this separation was a sample preparation time of less than 1 h and analysis time of 40 min. Repeatability of intraday migration time for the mixtures was typically < 1% relative standard deviation (n = 3). In addition, we demonstrate that the coating has an acceptable capillary lifetime of over 70 injections.     

The preparation of 3,3a,4,5‐tetrahydronaphth[1,2‐c]isoxazoles from dilithiated 1‐tetralone oxime and select aromatic aldehydes

Dilithiated 1‐tetralone oxime was condensed with several electron enriched aromatic aldehydes, such as 4‐methoxybenzaldehyde or lithiated 4‐hydroxybenzaldehyde, followed by acid cyclization to new tetrahydronaphthisoxazoles, 3,3a,4,5‐tetrahydronaphth[1,2‐c]isoxazoles, with a trans geometry of the C₃‐H and C_3a‐H protons that was confirmed by X‐ray single crystal analysis.