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A method for the measurement of residual chemical shift anisotropy in one experiment using a biphasic isotropic/anisotropic lyotropic liquid crystalline medium based on poly-γ-benzyl-l-glutamate as the alignment medium is presented. This approach is demonstrated on the model compound strychnine and neotricone, a depsidone natural product with a questionable structural assignment based on comparison with the closely related excelsione and in-depth density functional theory calculations.

Determining the configuration of proton-deficient molecules is challenging using conventional NMR methods including nuclear Overhauser effect (NOE) and the proton-dependent J-based configuration analysis (JBCA). The problem is exacerbated when only one stereoisomer is available. Alternative methods based on the utilization of 13C NMR chemical shifts, 13C-13C homonuclear couplings measured at natural abundance, and residual chemical shift anisotropy measurements in conjunction with density functional theory calculations are illustrated with a proton-deficient model compound.

Thiopeptides are a broad class of macrocyclic, heavily modified peptide natural products that are unified by the presence of a substituted, nitrogen-containing heterocycle core. Early work indicated that this core might be fashioned from two dehydroalanines by an enzyme-catalyzed aza-[4 + 2] cycloaddition to give a cyclic-hemiaminal intermediate. This common intermediate could then follow a reductive path toward a dehydropiperidine, as in the thiopeptide thiostrepton, or an aromatization path to yield the pyridine groups observed in many other thiopeptides. Although several of the enzymes proposed to perform this cycloaddition have been reconstituted, only pyridine products have been isolated and any hemiaminal intermediates have yet to be observed. Here, we identify the conditions and substrates that decouple the cycloaddition from subsequent steps and allow interception and characterization of this long hypothesized intermediate. Transition state modeling indicates that the key amide-iminol tautomerization is the major hurdle in an otherwise energetically favorable cycloaddition. An anionic model suggests that deprotonation and polarization of this amide bond by TbtD removes this barrier and provides a sufficient driving force for facile (stepwise) cycloaddition. This work provides evidence for a mechanistic link between disparate cyclases in thiopeptide biosynthesis.

A novel approach to produce chiral diaryl sulfoxides from aryl benzyl sulfoxides and aryl bromides via an enantioselective arylation of aryl sulfenate anions is reported. A (JosiPhos)Pd-based catalyst successfully promotes the asymmetric arylation reaction with good functional group compatibility. A wide range of enantioenriched diaryl, aryl heteroaryl, and even diheteroaryl sulfoxides were generated. Many of the sulfoxides prepared herein would be difficult to prepare via classic enantioselective oxidation of sulfides, including Ph(Ph-d5)SO (90% ee, 95% yield). A DFT-based computational study suggested that chiral induction originates from two primary factors: (i) both a kinetic and a thermodynamic preference for oxidative addition that places the bromide trans to the JosiPhos-diarylphosphine moiety and (ii) Curtin-Hammett-type control over the interconversion between O- and S-bound isomers of palladium sulfenate species following rapid interconversion between re- and si-bound transmetalation products, re/si-Pd-OSPh (re/si-PdO-trans). Fil: Jia, Tiezheng. University of Pennsylvania; Estados Unidos Fil: Zhang, Mengnan. University of Pennsylvania; Estados Unidos Fil: McCollom, Samuel P.. University of Pennsylvania; Estados Unidos Fil: Bellomo Peraza, Ana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina Fil: Montel, Sonia. University of Pennsylvania; Estados Unidos Fil: Mao, Jianyou. Nanjing Tech University; República de China Fil: Dreher, Spencer D.. Merck & Company; Estados Unidos Fil: Welch, Christopher J.. Merck & Company; Estados Unidos Fil: Regalado, Erik L.. Merck & Company; Estados Unidos Fil: Williamson, R. Thomas. Merck & Company; Estados Unidos Fil: Manor, Brian C.. Merck & Company; Estados Unidos Fil: Tomson, Neil C.. University of Pennsylvania; Estados Unidos Fil: Walsh, Patrick J.. University of Pennsylvania; Estados Unidos

A novel technique that allows efficient measurement of the 35,37Cl isotope pattern for any 13C resonance has been developed. The band-selective CLIP-HSQMBC experiment is reliable and universally applicable for the indirect measurement of the 35,37Cl isotope shift of 13C resonances. The experiment provides advantages over conventional 1D 13C NMR and the recently developed bs-HSQC experiments. The utility and performance of the bs-CLIP-HSQMBC experiment is demonstrated for polyhalogenated synthons in a synthetic route and for a polyhalogenated marine natural product.

Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A's formation by this path, a six-electron oxidation, could be a response to oxidative stress triggered by bafilomycin C1.

Accurate measurement of residual dipolar couplings (RDCs) requires an appropriate degree of alignment in order to optimize data quality. An overly weak alignment yields very small anisotropic data that are susceptible to measurement errors, whereas an overly strong alignment introduces extensive anisotropic effects that severely degrade spectral quality. The ideal alignment amplitude also depends on the specific pulse sequence used for the coupling measurement. In this work, we introduce a practical strategy for the accurate measurement of one-bond 13C–1H RDCs up to a range of ca. −300 to +300 Hz, corresponding to an alignment that is an order of magnitude stronger than typically employed for small molecule structural elucidation. This strong alignment was generated in the mesophase of the commercially available poly-γ-(benzyl-L-glutamate) polymer. The total coupling was measured by the simple and well-studied heteronuclear two-dimensional J-resolved experiment, which performs well in the presence of strong anisotropic effects. In order to unequivocally determine the sign of the total coupling and resolve ambiguities in assigning total couplings in the CH2 group, coupling measurements were conducted at an isotropic condition plus two anisotropic conditions of different alignment amplitudes. Most RDCs could be readily extracted from these measurements whereas more complicated spectral effects resulting from strong homonuclear coupling could be interpreted either theoretically or by simulation. Importantly, measurement of these very large RDCs actually offers significantly improved data quality and utility for the structure determination of small organic molecules.

Even C/CH(2) and odd CH/CH(3) carbon-multiplicity information can be directly distinguished from the relative positive/negative phase of cross-peaks in a novel ME (Multiplicity-Edited)-selHSQMBC experiment. The method can be extended by a TOCSY propagation step, and it is fully compatible for the simultaneous and precise determination of long-range heteronuclear coupling constants. Broadband homonuclear decoupling techniques can also be incorporated to enhance sensitivity and signal resolution by effective collapse of J(HH) multiplets.

The absolute configuration of remisporine B was determined based on a comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Density functional theory (DFT) was used to calculate the ECD spectra varying the parameter controlling the number of calculated electronic transitions. Mapping the reaction surface provided support for the proposed Diels-Alder dimerization of remisporine A to form remisporine B.

Difficulties in isolating samples from complex biological matrices and sensitivity limitations have long stymied the utilization of heteronuclear 2D NMR for the characterization of drug metabolites. Small diameter cryogenic NMR probes have largely ameliorated sensitivity limitations and the recently reported pure shift HSQC 2D NMR pulse sequence offers a further and marked improvement in both resolution and sensitivity. Using a 7.4 μg sample of the commercially available metabolite 3-hydroxy carbamazepine dissolved in 30 μL of deuterated solvent and a 600 MHz NMR equipped with a 1.7 mm cryogenic NMR probe, it was possible to acquire high signal-to-noise pure shift HSQC data in just over 30 min. A conventional HSQC spectrum acquired with identical parameters had approximately half the signal-to-noise of the pure shift HSQC spectrum. Collapsing the vicinal homonuclear couplings in the pure shift HSQC spectrum also significantly improves resolution. A practical, real world application of the technique is illustrated with the chromatographically isolated metabolite 3-hydroxy amiodarone from incubation with CYP2J2 recombinant enzyme. High quality pure shift HSQC data were recorded in slightly over 14 h for a 3 μg sample of the metabolite.

Determination of relative configuration is frequently a rate-limiting step in the characterization of small organic molecules. Solution NMR-based nuclear Overhauser effect and scalar J-coupling constants can provide useful spatial information but often fail when stereocenters are separated by more than 4-5 Å. Residual dipolar couplings (RDCs) can provide a means of assigning relative configuration without limits of distance between stereocenters. However, sensitivity limits their application. Chemical shift is the most readily measured NMR parameter, and partial molecular alignment can reveal the anisotropic component of the chemical shift tensor, manifested as residual chemical shift anisotropy (RCSA). Hence, (13)C RCSAs provide information on the relative orientations of specific structural moieties including nonprotonated carbons and can be used for stereochemical assignment. Herein, we present two robust and sensitive methods to accurately measure and apply (13)C RCSAs for stereochemical assignment. The complementary techniques are demonstrated with five molecules representing differing structural classes.

A relatively unexplored and unexploited means of establishing molecular structure, stereochemistry, and probing vicinal bond angles is through the use of long-range (13)C-(13)C coupling constants. The measurement of these multifunctional, diagnostic (3)J(CC) couplings has not been reported on sample amounts viable for the practicing organic chemist. A generalized protocol for the measurement of (1)J(CC) and (3)J(CC) couplings using a 4.6 mg sample of strychnine as a model compound is described, and the utility of DFT calculations for the prediction of these useful molecular descriptors and the congruence of the calculated and experimental data is demonstrated.

Two new diketopiperazine dipeptides, cyclomarazines A and B, were isolated and characterized along with the new cyclic heptapeptide cyclomarin D from the marine bacterium Salinispora arenicola CNS-205. These structurally related cyclic peptides each contain modified amino acid residues, including derivatives of N-(1,1-dimethylallyl)-tryptophan and delta-hydroxyleucine, which are common in the di- and heptapeptide series. Stable isotope incorporation studies in Streptomyces sp. CNB-982, which was first reported to produce the cyclomarin anti-inflammatory agents, illuminated the biosynthetic building blocks associated with the major metabolite cyclomarin A, signifying that this marine microbial peptide is nonribosomally derived largely from nonproteinogenic amino acid residues. DNA sequence analysis of the 5.8 Mb S. arenicola circular genome and PCR-targeted gene inactivation experiments identified the 47 kb cyclomarin/cyclomarazine biosynthetic gene cluster (cym) harboring 23 open reading frames. The cym locus is dominated by the 23 358 bp cymA, which encodes a 7-module nonribosomal peptide synthetase (NRPS) responsible for assembly of the full-length cyclomarin heptapeptides as well as the truncated cyclomarazine dipeptides. The unprecedented biosynthetic feature of the megasynthetase CymA to synthesize differently sized peptides in vivo may be triggered by the level of beta oxidation of the priming tryptophan residue, which is oxidized in the cyclomarin series and unoxidized in the cyclomarazines. Biosynthesis of the N-(1,1-dimethyl-2,3-epoxypropyl)-beta-hydroxytryptophan residue of cyclomarin A was further illuminated through gene inactivation experiments, which suggest that the tryptophan residue is reverse prenylated by CymD prior to release of the cyclic peptide from the CymA megasynthetase, whereas the cytochrome P450 CymV installs the epoxide group on the isoprene of cyclomarin C post-NRPS assembly. Last, the novel amino acid residue 2-amino-3,5-dimethylhex-4-enoic acid in the cyclomarin series was shown by bioinformatics and stable isotope experiments to derive from a new pathway involving condensation of isobutyraldehyde and pyruvate followed by S-adenosylmethionine methylation. Assembly of this unsaturated, branched amino acid is unexpectedly related to the degradation of the environmental pollutant 3-(3-hydroxyphenyl)propionic acid.

The detection of long-range heteronuclear correlations presenting J(CH) coupling values smaller than 1-2Hz is a challenge in the structural analysis of small molecules and natural products. HSQMBC-COSY and HSQMBC-TOCSY pulse schemes are evaluated as complementary NMR methods to standard HMBC/HSQMBC experiments. Incorporation of an additional J(HH) transfer step in the basic HSQMBC pulse scheme can favor the sensitive observation of traditionally missing or very weak correlations and, in addition, facilitates the detection of a significant number of still longer-range connectivities to both protonated and non-protonated carbons under optimum sensitivity conditions. A comparative (1)H-(13)C study is performed using strychnine as a model compound and several examples are also provided including (1)H-(15)N applications.

[structure: see text] Herein we report a significant body of spectroscopic data that supports the originally proposed structure of medermycin/lactoquinomycin A. In addition, we demonstrate that these data are inconsistent with the revised structure reported recently in the literature.

Structural and biosynthetic studies have been conducted on the barbamide class of molluscicidal agent. Dechlorobarbamide was isolated from a Curacao collection of the marine cyanobacterium Lyngbya majuscula and its structure determined through spectroscopic analysis and comparisons with barbamide. The absolute stereochemistry of the dolaphenine moiety of barbamide was determined to be S , defining the absolute configuration of barbamide as 2 S ,7 S . Stable isotope feeding experiments conducted with cultured L. majuscula have provided clear evidence that barbamide biosynthesis involves chlorination of the unactivated pro - R methyl group of leucine. Experiments with l -[ 2 H 10 ]leucine demonstrated that chlorination of the pro- R methyl occurs without detectable activation via the leucine-catabolic pathway. Moreover, an extremely high level of incorporation of fed [2- 13 C]-5,5,5-trichloroleucine into barbamide indicates that leucine is the probable substrate for the chlorination reaction. Incorporations of [1,2- 13 C 2 ]acetate and [1- 13 C, 1- 18 O]acetate confirmed the origins of C-5 and C-6 whereas incorporation of l -[3- 13 C]phenylalanine supported the hypothesis that the phenyl group and its three carbon side-chain in barbamide (C-7, C-8 and C-10–C-16) arise from phenylalanine. The thiazole ring (C-17–C-18) of 1 was shown to likely arise from cysteine through a [2- 13 C, 15 N]glycine feeding experiment. Detection of intact 13 C– 15 N bond was observed by application of a new GHNMBC NMR experiment. Results from this latter feeding experiment also indicated that the N–CH 3 and O–CH 3 groups of 1 originate from the C 1 pool; this was supported by enrichment in these methyl groups when cultures were provided with l -[methyl- 13 C]methionine.

Mono- and bis 3-(hydroxypropanoyl) esters of long chain, unsaturated diols have been isolated and identified from two genera of the family Bruchidae, and have been shown to be responsible for the mitogenic activity observed on pea pods resulting from oviposition by the pea weevil, Bruchus pisorum . The mitogenic compounds have been characterized and synthesized.

A new NMR experiment is presented for the detection of intact 13C15N units in biosynthetic studies. Its use is demonstrated through a feeding experiment utilizing [2-13C, 15N] glycine which confirmed the origin of the thiazole ring in the marine cyanobacterial metabolite barbamide as originating from cysteine.

The application of 1H-15N PEP-HSQC-TOCSY at natural abundance is demonstrated here on a 13 mM sample (0.048 mM 15N/position) of the biologically active linear tetradecapeptide bombesin (MW=1619.9) as a way to expedite the structural characterization of peptidic natural products.

Establishing the carbon skeleton of a molecule greatly facilitates the process of structure elucidation, both manual and computer-assisted. Recent advances in the family of ADEQUATE experiments demonstrated their potential in this regard. 1,1-ADEQUATE, which provides direct 13 C– 13 C correlation via 1 J CC , and 1, n -ADEQUATE, which typically yields 3 J CC and 1 J CC correlations, are more sensitive and more widely applicable experiments than INADEQUATE and PANACEA. A recently reported modified pulse sequence that semi-selectively inverts 1 J CC correlations in 1, n -ADEQUATE spectra provided a significant improvement, allowing 1 J CC and n J CC correlations to be discerned in the same spectrum. However, the reported experiment requires a careful matching of the amplitude transfer function with 1 J CC coupling constants in order to achieve the inversion, and even then some 1 J CC correlations could still have positive intensity due to the oscillatory nature of the transfer function. Both shortcomings limit the practicality of the method. We now report a new, dual-optimized inverted 1 J CC 1, n -ADEQUATE experiment, which provides more uniform inversion of 1 J CC correlations across the range of 29–82 Hz. Unlike the original method, the dual optimization experiment does not require fine-tuning for the molecule’s 1 J CC coupling constant values. Even more usefully, the dual-optimized version provides up to two-fold improvement in signal-to-noise for some long-range correlations. Using modern, cryogenically-cooled probes, the experiment can be successfully applied to samples of ∼1 mg under favorable circumstances. The improvements afforded by dual optimization inverted 1 J CC 1, n -ADEQUATE experiment make it a useful and practical tool for NMR structure elucidation and should facilitate the implementation and utilization of the experiment.

Quantum chemical calculations of one-bond carbon-carbon coupling constants are demonstrated as potential probes for the configurational assignment of organic molecules. The stereochemical analysis of strychnine and its possible stereoisomers is presented as proof of concept.

The recently reported LR-HSQMBC experiment has been optimized for 1H–15N long-range heteronuclear couplings. Several previously unreported four-bond correlations, consistent with the predicted by DFT calculations (0.2–0.3 Hz 4JNH couplings), have been observed for strychnine using 2 Hz optimization of the LR-HSQMBC experiment. This experiment offers an advantage over accordion-optimized experiments such as IMPEACH and CIGAR for the observation of long-range 1H–15N correlations in that the experiment is refocused and employs a CLIP pulse sequence element to bring the long-range correlations into phase, allowing broadband X-decoupling to be employed during acquisition.

The muraymycins, a family of nucleoside-lipopeptide antibiotics, were purified from the extract of Streptomyces sp. LL-AA896. The antibiotics were purified by chromatographic methods and characterized by NMR spectroscopy, degradation studies, and mass spectrometry. The structures of 19 compounds were established. The muraymycins constitute a new antibiotic family whose core structure contains a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidylglycyl residue (epicapreomycidine) containing dipeptide that is further extended by a urea-valine moiety. Members of this family show broad-spectrum in vitro antimicrobial activity against a variety of clinical isolates (MIC 2 to >64 mug/mL). The muraymycins inhibited peptidoglycan biosynthesis. The fatty acid substituent and the presence or absence of the amino sugar play important roles in biological activity. One of the most active compounds, muraymycin A1, demonstrated protection in vivo against Staphylococcus aureus infection in mice (ED50 1.1 mg/kg).

Using the hemiasterlin analogs taltobulin (I, HTI-286), II, and III as model compounds, we demonstrate that relaxation-compensated STD-NMR can be used as an effective tool to efficiently provide a qualitative epitope map for microtubule destabilizing peptides. Due to the disparate relaxation behavior of the protons in these model compounds, it was essential to collect STD with very short saturation times to render an accurate picture of the binding interaction. The conformation of HTI-286 (I) in complex with the protein was determined from TRNOESY/ROESY experiments and is similar to the X-ray crystal structure conformation observed for hemiasterlin methyl ester in the absence of protein.

During a study on the chemistry and biological activity of Kuwaiti plants, new metabolites including 4,6-dihydroxy-3-[3'-methyl-2'-butenyl]-5-[4''-hydroxy-3''-methyl-2''-butenyl]-cinnamic acid (1), the 3R,8R stereoisomer of the C17 polyacetylene dehydrofalcarindiol (2) and a C10 polyacetylene glucoside (3) were characterised by spectroscopic means. Additionally, the previously characterised natural products 1,3R,8R-trihydroxydec-9-en-4,6-yne (4), spathulenol (5) and eriodyctiol-7-methyl ether (6) were also isolated. Compounds 2, 3, and 4 were evaluated for their ability to inhibit the enzyme 12-lipoxygenase and 3 and 4 showed moderate activity at 30 microg/ml. Compound 2 was evaluated against a panel of colorectal and breast cancer cell lines and IC50 values ranged from 5.8 to 37.6 microg/ml. Against a panel of fast-growing mycobacteria and a standard ATCC strain of Staphylococcus aureus, compound 6 exhibited minimum inhibitory concentrations in the range of 64-128 microg/ml.

[reaction: see text] Aldol reactions using bis-(chiral alpha-methylbenzyl)glycine esters with aldehydes gave excellent diastereoselectivity. Thus, an enantiopure ribosylglycine was prepared for the synthesis of analogues of the natural antibiotics muraymycin. This method was extended for formation of beta-hydroxyamino esters.

[reaction: see text] Herein we report a new set of silyl ether reagents for determining the enantiomeric purity and absolute stereochemistry of secondary alcohols. These derivatives are easily synthesized, provide straightforward spectroscopic results, and allow for facile recovery of the original chiral alcohol.

A general chiroptical protocol for determination of absolute configuration of secondary amines including acyclic and cyclic aliphatic amines, aromatic amines, amino acids, and amino alcohols is described. The chiral substrate is linked to the achiral carrier moiety (3-N-Boc-amino-propyl-N-Boc-amino)acetic acid 1 (BocHNCH(2)CH(2)CH(2)BocNCH(2)COOH), which after deprotection, yields a bidentate conjugate, capable of forming a 1:1 host/guest complex with dimeric zinc porphyrin host 2. As in the cases of primary amines and secondary alcohols reported earlier, the complexation of secondary amine conjugates to porphyrin tweezer host 2 represents a stereodifferentiating process, where the large (L) group at the stereogenic center (assigned on the basis of conformational energies A value) protrudes from the porphyrin binding pocket. This leads to formation of host/guest complexes with a preferred porphyrin helicity that exhibit intense exciton split CD spectra. It was found that the chiral sense of porphyrin twist is clearly controlled by the stereogenic center despite the Z/E conformational complexity around the tertiary amide bond of secondary amine conjugates that has greatly hampered previous configurational assignments. Thus, in cases where there is no ambiguity regarding the relative steric size of substituents, the observed CD couplet can be applied for straightforward assignment of absolute configurations. In addition, to extend the application to more difficult cases a molecular mechanics calculation approach using the Merck Molecular Force Field (MMFFs) was developed; this provides conformational information of host/guest complexes and leads to prediction of preferred porphyrin helicity independent of conformational A values. This chiroptical protocol in combination with molecular modeling represents a general method for configurational assignments of secondary amines.

A series of novel antibiotics with activity against methicillin-resistant staphylococci and vancomycin-resistant enterococci has been purified, and their structures have been characterized using spectroscopic analyses and chemical conversions. These antibiotics, designated mannopeptimycins alpha-epsilon (1-5), are glycosylated cyclic hexapeptides containing two stereoisomers of an unprecedented amino acid, alpha-amino-beta-[4'-(2'-iminoimidazolidinyl)]-beta-hydroxypropionic acid (Aiha), as a distinguishing feature. The cyclic peptide core of these antibiotics is attached to a mannosyl monosaccharide moiety in 2 and to mannosyl monosaccharide and disaccharide moieties in 1, 3, 4, and 5. The presence and position of an isovaleryl group in the terminal mannose (Man-B) in 3-5 are critical for retaining antibacterial potency.

The muraymycins, a family of nucleoside-lipopeptide antibiotics, were purified from the extract of Streptomyces sp. LL-AA896. The antibiotics were purified by chromatographic methods and characterized by NMR spectroscopy, degradation studies, and mass spectrometry. The structures of 19 compounds were established. The muraymycins constitute a new antibiotic family whose core structure contains a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidylglycyl residue (epicapreomycidine) containing dipeptide that is further extended by a urea-valine moiety. Members of this family show broad-spectrum in vitro antimicrobial activity against a variety of clinical isolates (MIC 2 to >64 mug/mL). The muraymycins inhibited peptidoglycan biosynthesis. The fatty acid substituent and the presence or absence of the amino sugar play important roles in biological activity. One of the most active compounds, muraymycin A1, demonstrated protection in vivo against Staphylococcus aureus infection in mice (ED50 1.1 mg/kg).