NMR conformational analysis of a hydroxyethylamine peptide isostere developed as an aspartic protease inhibitor shows that it is a flexible architecture. Cyclization to form pyrrolidines, ...piperidines, or morpholines results in a preorganization of the whole system in solution. The resulting conformation is similar to the conformation of the inhibitor in the active site of BACE-1. This entropic gain results in increased affinity for the enzyme when compared with the acyclic system. For morpholines 27 and 29, the combination of steric and electronic factors is exploited to orient substituents toward S1, S1′, and S2′ pockets both in the solution and in the bound states. These highly preorganized molecules proved to be the most potent compounds of the series. Additionally, the morpholines, unlike the pyrrolidine and piperidine analogues, have been found to be brain penetrant BACE-1 inhibitors.
A general methodology for assigning the configuration of chiral mono- and polyfunctional compounds by NMR is presented. The approach is based on the use of polystyrene-bound chiral derivatizing ...agents (CDA-resins) specifically designed to achieve the high-yield formation of the covalent linkages (amide or ester bonds) between the substrate and the chiral auxiliary within the NMR tube, without the need for other manipulations, on a microscale level and in a short time. The deuterated NMR solvents (CDCl3, CD3CN, CS2/CD2Cl2) are also the reaction solvents and separations, purifications or workups of any kind are not necessary prior to recording the spectra. The CDA-resins prepared included MPA, 9-AMA, BPG, MTPA, and 2-NTBA as auxiliary agents incorporated either as single enantiomers or as mixed combinations of the (R)- and the (S)-enantiomers at unequal and known ratios. The high versatility of these systems was successfully demonstrated in a variety of ways based on double and single derivatization, low temperature experiments, or the formation of metal complexes. The approach allowed the absolute configurations of chiral primary amines, primary and secondary alcohols, cyanohydrins, thiols, diols, triols, and amino alcohols to be determined. Extensive high-resolution magic angle spinning (HR-MAS) NMR experiments allowed the characterization of the new CDA-resins and enabled the study of their stability and regioselectivity.
Simply successful: A proton‐selective HSQMBC‐TOCSY experiment can be used to measure small proton–carbon (nJCH; n>1) coupling constants on both protonated and non‐protonated carbon atoms (see ...spectrum). The method combines in a single pulse scheme all the benefits of the widely used HSQMBC and HSQC‐TOCSY experiments. The magnitude and the sign of nJCH can be determined simply with excellent accuracy.
A general review of novel NMR methods to measure heteronuclear long-range proton-carbon coupling constants ((n)JCH; n>1) in small molecules is made. NMR experiments are classified in terms of NMR ...pulse scheme and cross-peak nature. A discussion about simplicity, general applicability and accuracy for each particular NMR experiment is presented and exemplified. Important aspects such as the sign determination and measurement of very small coupling values involving protonated and non-protonated carbons as well as the complementarity between different experiments are also discussed. Finally, a compilation of applications in structural and conformational analysis of different types of molecules since 2000 is surveyed.
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential ...HDX-MS include the characterization of protein–ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test–retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
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► Spin-state selection in HSQMBC experiments. ► Measurement of proton-carbon coupling constants from spin-state selective multiplets. ► Measurement of proton-carbon coupling ...constants in protonated and non-protonated centers.
A new NMR approach is proposed for the measurement of long-range heteronuclear coupling constants (
n
J
XH
,
n
>
1) in natural abundance molecules. Two complementary in-phase (IP) and anti-phase (AP) data are separately recorded from a modified HSQMBC experiment and then added/subtracted to provide spin-state-selective
α/
β–HSQMBC spectra. The magnitude of
n
J
XH
can be directly determined by simple analysis of the relative displacement between
α- and
β-cross-peaks. The robustness of this IPAP–HSQMBC experiment is evaluated experimentally and by simulation using a variety of different conditions. Important aspects such as signal intensity dependence and presence of unwanted cross-talk effects are discussed and examples on the measurement of small proton–carbon (
n
J
CH
) and proton–nitrogen (
n
J
NH
) coupling constants are provided.
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► A selective HSQMBC experiment to generate pure-phase multiplets without undesired proton–proton coupling modulation. ► Long-range proton–carbon coupling constants directly measured ...from relative cross-peak displacement of
α/
β multiplets. ► Accurate measurement of proton–carbon coupling constants over a wide range of experimental variables. ► Reliable validation of proton–carbon coupling constants measurements by three different analysis.
A simple proton-selective
α/
β-HSQMBC experiment is proposed for the accurate measurement of long-range proton–carbon coupling constants (
n
J
CH) in small molecules without need for an individualized and time-consuming post-processing fitting procedure. The method acquires two pure-phase In-phase (IP) and Anti-phase (AP) multiplets completely free of any phase distortion due to the absence of
J
HH evolution. Accurate
n
J
CH values can be directly measured analyzing the relative displacement of the resulting IPAP cross-peaks. Discussion about signal intensity dependence and cross-talk is made for a range of experimental conditions. The robustness of the method is evaluated by comparing the
n
J
CH value measured from the analysis of the three available IP, AP and IPAP multiplet patterns. Multiple-frequency and region-selective versions of the method can also be efficiently recorded provided that excited protons are not mutually coupled.
Carbohydrates are thought to be especially difficult to model because of their highly polar functionality, their flexibility, and their differences in electronic arrangements that occur during ...conformational and configurational changes, such as the anomeric, exo-anomeric and gauche effects. These issues have been addressed in recent years, yielding several contributions to set up some relevant parameterizations that would account for these specific features of carbohydrates. Within the framework of a workshop involving the participation of 11 research groups active in the field, several commonly used molecular mechanics force fields and special carbohydrate parameter sets have been considered. The application of 20 force fields and/or sets of parameters to a series of seven test cases provided a fairly general picture of the potentiality of these parameter sets for giving a consistent image of structure and energy of carbohydrate molecules. The results derived from a chemometric analysis (principal component analysis, PCA) give a global view of the performances of the force fields and parameter sets for carbohydrates. The present analysis (i) provides an identification of the parameter sets which differ from the bulk, (ii) helps to establish the relationship that exists between the different parameter sets, (iii) provides indications for selecting different parameter sets to explore the force field dependency (or the lack of thereof) of a given molecular modeling study. Through the PCA, we have created a force field landscape on which the different force fields are related to each other on a relative scale. New carbohydrate force fields can easily be inserted into this landscape (PCA model) and related to the performance of existing force fields.
Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and ...apolipoprotein(a) (apo(a))
. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (K
) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs.
). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) K
7-8. We identify compounds that bind to apo(a) K
7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).
