6-Strand to Stable 10/12 Helix Conformational Switch by Incorporating Flexible ß-hGly in the Homooligomers of Camphor Derived ß-Amino Acid: NMR and X-Ray Crystallographic Evidence.

Rational design of unnatural amino acid building blocks capable of stabilizing predictable secondary structures similar to protein fragments is pivotal for foldamer chemistry/catalysis. Here, we introduce novel ß-amino acid building blocks: [1S,2R,4R]exoCDA and [1S,2S,4R]endoCDA, derived from the abundantly available R(+)-camphor, which is traditionally known for its medicinal value. Further, we demonstrate that the homooligomers of exoCDA adopt 6-strand conformation, which switches to a robust 10/12-helix simply by inserting flexible ß-hGly spacer at alternate positions (1 : 1 ß-hGly/exoCDA heterooligomers), as evident by DFT-calculations, solution-state NMR spectroscopy and X-ray crystallography. To the best of our knowledge, this is the first example of crystalline-state structure of left-handed 10/12-mixed helix, that is free from the conventional approach of employing ß-amino acids of either alternate chirality or alternate ß2/ß3 substitutions, to access the 10/12-helix. The results also show that the homooligomers of heterochiral exoCDA don't adopt helical fold, instead exhibit banana-shaped strands, whereas the homodimers of the other diastereomer endoCDA, nucleate 8-membered turns. Furthermore, the homo-exoCDA and hetero-[ß-hGly-exoCDA] oligomers are found to exhibit self-association properties with distinct morphological features. Overall, the results offer new possibilties of constructing discrete stable secondary and tertiary structures based on CDAs, which can accommodate flexible residues with desired side-chain substitutions.

Prion-derived tetrapeptide stabilizes thermolabile insulin via conformational trapping.

Unfolding followed by fibrillation of insulin even in the presence of various excipients grappled with restricted clinical application. Thus, there is an unmet need for better thermostable, nontoxic molecules to preserve bioactive insulin under varying physiochemical perturbations. In search of cross-amyloid inhibitors, prion-derived tetrapeptide library screening reveals a consensus V(X)YR motif for potential inhibition of insulin fibrillation. A tetrapeptide VYYR, isosequential to the ß2-strand of prion, effectively suppresses heat- and storage-induced insulin fibrillation and maintains insulin in a thermostable bioactive form conferring adequate glycemic control in mouse models of diabetes and impedes insulin amyloidoma formation. Besides elucidating the critical insulin-IS1 interaction (R4 of IS1 to the N24 insulin B-chain) by nuclear magnetic resonance spectroscopy, we further demonstrated non-canonical dimer-mediated conformational trapping mechanism for insulin stabilization. In this study, structural characterization and preclinical validation introduce a class of tetrapeptide toward developing thermostable therapeutically relevant insulin formulations.

Rapid elucidation of chemical shift correlations in complex NMR spectra of organic molecules: Two-dimensional Hadamard pure shift NMR spectroscopy.

Novel two dimensional Hadamard encoding/decoding based pure shift NMR acquisition techniques (TOCSY and HSQC) have been developed, which provide chemical shift information at ultra high resolution in very short spectrometer times. The efficacy of these methods for rapid assignment of chemical shifts in complex NMR spectra of organic molecules/natural products has been demonstrated. This would be of great help for rapid analysis of samples during separation of complex mixtures.

Selective measurement of 1 H-1 H scalar couplings from crowded chemical shift regions: Combined pure shift and spin-echo modulation approach.

JHH scalar couplings carry rich structural information and their measurements are fundamental in the 1 H NMR based elucidation of small and medium molecules, which, however, are hampered in the presence of large J-coupling network. Further, enhanced spectral resolution is often essential for precise determination of a specific set of 1 H-1 H J-couplings among the complex J-multiplets. In the light of the recent advancements in homodecoupling pure shift strategies, here, we report absorption mode, band-selective refocused pure shift spin-echo method, which helps in determining 1 H-1 H J-couplings from crowded spectral regions. The importance of the present band-selective refocused pure shift spin-echo experiment is exemplified for 2 steroid molecules, estradiol and testosterone.

Real-time homonuclear broadband decoupled pure shift COSY.

The present manuscript reports development and applications of real-time homonuclear broadband decoupled pure shift version of in-phase zero-quantum filtered COSY (PS-IPZF-COSY) and clean in-phase COSY (PS-CLIP-COSY) pulse schemes. In contrast to the conventional COSY schemes, these pure shift versions provide enhanced spectral resolution and simplify the chemical shift correlation analysis of scalar coupled spins in complex organic molecules, which are exemplified for erythromycin A, estradiol, and a mixture of estradiol and testosterone.

Band-selective excited ultrahigh resolution PSYCHE-TOCSY: fast screening of organic molecules and complex mixtures.

Precise assignments of (1) H atomic sites and establishment of their through-bond COSY or TOCSY connectivity are crucial for molecular structural characterization by using (1) H NMR spectroscopy. However, this exercise is often hampered by signal overlap, primarily because of (1) H-(1) H scalar coupling multiplets, even at typical high magnetic fields. The recent developments in homodecoupling strategies for effectively suppressing the coupling multiplets into nice singlets (pure-shift), particularly, Morris's advanced broadband pure-shift yielded by chirp excitation (PSYCHE) decoupling and ultrahigh resolution PSYCHE-TOCSY schemes, have shown new possibilities for unambiguous structural elucidation of complex organic molecules. The superior broadband PSYCHE-TOCSY exhibits enhanced performance over the earlier TOCSY methods, which however warrants prolonged experimental times due to the requirement of large number of dwell increments along the indirect dimension. Herein, we present fast and band-selective analog of the broadband PSYCHE-TOCSY, which is useful for analyzing complex organic molecules that exhibit characteristic yet crowded spectral regions. The simple pulse scheme relies on band-selective excitation (BSE) followed by PSYCHE homodecoupling in the indirect dimension. The BSE-PSYCHE-TOCSY has been exemplified for Estradiol and a complex carbohydrate mixture comprised of six constituents of closely comparable molecular weights. The experimental times are greatly reduced viz., ~20 fold for Estradiol and ~10 fold for carbohydrate mixture, with respect to the broadband PSYCHE-TOCSY. Furthermore, unlike the earlier homonuclear band-selective decoupling, the BSE-PSYCHE-decoupling provides fully decoupled pure-shift spectra for all the individual chemical sites within the excited band. The BSE-PSYCHE-TOCSY is expected to have significant potential for quick screening of complex organic molecules and mixtures at ultrahigh resolution. Copyright © 2015 John Wiley & Sons, Ltd.

Precise Determination of Enantiomeric Excess by a Sensitivity Enhanced Two-Dimensional Band-Selective Pure-Shift NMR.

Unambiguous identification and precise quantification of enantiomers in chiral mixtures is crucial for enantiomer specific synthesis as well as chemical analysis. The task is often challenging for mixtures with high enantiomeric excess and for complex molecules with strong (1)H-(1)H scalar (J) coupling network. The recent advancements in (1)H-(1)H decoupling strategies to suppress the J-interactions offered new possibilities for NMR based unambiguous discrimination and quantification enantiomers. Herein, we discuss a high resolution two-dimensional pure-shift zCOSY NMR method with homonuclear band-selective decoupling in both the F1 and F2 dimensions (F1F2-HOBS-zCOSY). This advanced method shows a sharp improvement in resolution over the other COSY methods and also eliminates the problems associated with the overlapping decoupling sidebands. The efficacy of this method has been exploited for precise quantification of enantiomeric excess (ee) ratio (R/S) up to 99:1 in the presence of very low concentrations of chiral lanthanide shift reagents (CLSR) or chiral solvating agents (CSA). The F1F2-HOBS-zCOSY is simple and can be easily implemented on any modern NMR spectrometers, as a routine analytical tool.

Chemical shift assignments of zinc finger domain of methionine aminopeptidase 1 (MetAP1) from Homo sapiens.

Methionine aminopeptidase Type I (MetAP1) cleaves the initiator methionine from about 70 % of all newly synthesized proteins in almost every living cell. Human MetAP1 is a two domain protein with a zinc finger on the N-terminus and a catalytic domain on the C-terminus. Here, we report the chemical shift assignments of the amino terminal zinc binding domain (ZBD) (1-83 residues) of the human MetAP1 derived by using advanced NMR spectroscopic methods. We were able to assign the chemical shifts of ZBD of MetAP1 nearly complete, which reveal two helical fragments involving residues P44-L49 (α1) and Q59-K82 (α2). The protein structure unfolds upon complex formation with the addition of 2 M excess EDTA, indicated by the appearance of amide resonances in the random coil chemical shift region of (15)NHSQC spectrum.

Solid-state Hadamard NMR spectroscopy: simultaneous measurements of multiple selective homonuclear scalar couplings.

Unambiguous measurement of homonuclear scalar couplings (J) in multi-spin scalar network systems is not straightforward. Further, the direct measurement of J-couplings is obscured in solid-state samples due to the dipolar and chemical shift anisotropy (CSA)-dominated line broadening, even under the magic angle spinning (MAS). We present a new multiple frequency selective spin-echo method based on Hadamard matrix encoding, for simultaneous measurement of multiple homonuclear scalar couplings (J) in the solid-state. In contrast to the Hadamard encoded selective excitation schemes known for the solution-state, herein the selectivity is achieved during refocusing period. The Hadamard encoded refocusing scheme concurrently allows to create the spin-spin commutation property between number of spin-pairs of choice in uniformly labelled molecules, which, therefore avoids (1) the repetition of the double selective refocusing experiments for each spin-pair and (2) the synthesis of expensive selective labelled molecules. The experimental scheme is exemplified for determining (1)JCC and (3)JCC values in (13)C6l-Histidine.HCl molecule, which are found to be in excellent agreement with those measured in conventional double frequency selective refocusing mode as well as in the solution-state. This method can be simply extended to 2D/3D pulse schemes and be applied to small bio-molecular solids.

A concise diastereoselective approach to enantioenriched substituted piperidines and their in vitro cytotoxicity evaluation.

A library of diversely stereo-oriented, highly substituted 2,6-cis piperidine derivatives were synthesized, and evaluated for their anticancer activity in cancer cells that included A549 (lung cancer, CCL-185), MCF7 (breast cancer (HTB-22), DU145 (prostate cancer (HTB-81), and HeLa (cervical cancer, CCL-2). One stereo-variant emerged as a promising candidate for further design based structure-activity studies.

Real-time homonuclear broadband and band-selective decoupled pure-shift ROESY.

Unambiguous spectral assignments in (1)H solution-state NMR are central, for accurate structural elucidation of complex molecules, which is often hampered by signal overlap, primarily because of scalar coupling multiplets, even at typical high magnetic fields. The recent advances in homodecoupling methods have shown powerful means of achieving high resolution pure-shift (1)H spectra in 1D and also in 2D J-correlated experiments, by effectively collapsing the multiplet structures. The present work extends these decoupling strategies to through-space correlation experiments as well and describes two new pure-shift ROESY pulse schemes with homodecoupling during acquisition, viz., homodecoupled broadband (HOBB)-ROESY and homodecoupled band-selective (HOBS)-ROESY. Furthermore, the ROESY blocks suppress the undesired interferences of TOCSY cross peaks and other offsets. Despite the reduced signal sensitivity and prolonged experimental times, the HOBB-ROESY is particularly useful for molecules that exhibit an extensive scalar coupling network spread over the entire (1)H chemical shift range, such as natural/synthetic organic molecules. On the other hand, the HOBS-ROESY is useful for molecules that exhibit well-separated chemical shift regions such as peptides (NH, Hα and side-chain protons). The HOBS-ROESY sensitivities are comparable with the conventional ROESY, thereby saves the experimental time significantly. The power of these pure-shift ROESY sequences is demonstrated for two different organic molecules, wherein complex conventional ROE cross peaks are greatly simplified with high resolution and sensitivity. The enhanced resolution allows deriving possibly more numbers of ROEs with better accuracy, thereby facilitating superior means of structural characterization of medium-size molecules.

Enantioselective synthesis of δ-/γ-alkoxy-ß-hydroxy-α-alkyl-substituted Weinreb amides via DKR-ATH: application to the synthesis of advanced intermediate of (-)-brevisamide.

A method of preparing stereodefined δ-/γ-alkoxy-ß-hydroxy-α-alkyl-substituted Weinreb amides containing two successive hydroxyl-alkyl stereocenters has been developed. Further, this strategy coupled with organo-catalyzed asymmetric epoxidation culminates in the synthesis of a critical intermediate of (-)-brevisamide and its diastereomers.

Highly enantioselective carbon-carbon bond formation by Cu-catalyzed asymmetric [2,3]-sigmatropic rearrangement: application to the syntheses of seven-membered oxacycles and six-membered carbocycles.

A concise route for the syntheses of enantioenriched functionalized scaffolds of medium-sized oxacycles and carbocycles employing the chiral auxiliary-mediated Cu-catalyzed ylide formation/[2,3]-sigmatropic rearrangement as a key step was developed.

Pseudoguaianolides from the flowers of Parthenium hysterophorus.

Chemical investigation on the flowers of Parthenium hysterophorus has resulted in the isolation of four new pseudoguaianolides, hysterones A-D along with the known compounds, parthenin, coronopilin, 2beta-hydroxycoronopilin and tetraneurin-A. The structures of the new compounds were established by interpretation of their spectral (1D and 2D NMR) data. The X-ray crystallographic analysis of hysterones A and C was also carried out.