Discovery of unusual dimeric piperazyl cyclopeptides encoded by a Lentzea flaviverrucosa DSM 44664 biosynthetic supercluster.

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC50 values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs.

An Integrated ANN-PRA/DP4+ Tandem Computational Approach Contributing to the Ordering of the Heliannuol Family.

Heliannuols are a unique class of sesquiterpenes isolated mostly from Helianthus annuus, commonly known as sunflower. The interesting allelopathic properties, combined with their unprecedented carbon skeletons, have drawn wide attention to phytochemistry and synthetic groups. So far, 14 heliannuols (heliannuols A-N) have been described in the literature, although some of them have not yet been validated by total synthesis. Moreover, the structural proposal of some compounds was based on the similarity of NMR data reported for previously isolated analogues (which in many instances turned out to be incorrect), coupled with little or no stereochemical analysis. Consequently, the structural reassignment is a recurring theme in heliannuol's family. Through a rigorous and comprehensive quantum chemical simulation of NMR parameters, encompassing an integrated ANN-PRA/DP4+ tandem approach, we intended to advance unexplored directions regarding the structure of the entire heliannuol family. Furthermore, we found that the size of the fused ring significantly influences the signals corresponding to the aromatic ring, making this discovery an excellent diagnostic tool for quickly determining the core structure of these compounds.

Study of heavy atom influence on poly-halogenated compounds using DP4/MM-DP4+/DP4+: insights and trends.

Nuclear magnetic resonance (NMR) spectroscopy complemented by density functional theory (DFT) calculations is a crucial tool for structural elucidation. Nevertheless, the precision of NMR predictions is influenced by the 'heavy atom effect', wherein heavy atoms affect the shielding values of neighboring light atoms (HALA effect). Standard practice in the field involves removing the conflicting signals. However, in the case of polyhalogenated molecules, this is challenging due to the significant amount of information that ends up being lost. In this study the HALA is thoroughly investigated in the context of three leading probability methods: DP4, MM-DP4+, and DP4+. The results show that DP4+ is more sensitive to C-Cl or C-Br signals, which is a consequence of the longer bond lengths computed with DFT. Removing conflicting signals is highly effective in DP4+, but has an uncertain outcome in methods based on molecular mechanics geometries, such as DP4 and MM-DP4+. A detailed investigation of the effect of bond distance on the corresponding chemical shifts has also been conducted.

Breaking the DFT Energy Bias Caused by Intramolecular Hydrogen-Bonding Interactions with MESSI, A Structural Elucidation Method Inspired by Wisdom of Crowd Theory.

The use of quantum-based NMR methods to complement and guide the connectivity and stereochemical assignment of natural and unnatural products has grown enormously. One of the unsolved problems is related to the improper calculation of the conformational landscape of flexible molecules that have functional groups capable of generating a complex network of intramolecular H-bonding (IHB) interactions. Here the authors present MESSI (Multi-Ensemble Strategy for Structural Identification), a method inspired by the wisdom of the crowd theory that breaks with the traditional mono-ensemble approach. By including independent mappings of selected artificially manipulated ensembles, MESSI greatly improves the sense of the assignment by neutralizing potential energy biases.

Road Map Toward Computer-Guided Total Synthesis of Natural Products. The Dysiherbol A Case Study: What if Serendipity Hadn't Intervened?

We present a computational study inspired by the story of dysiherbol A, a natural product whose putative structure was found incorrect through synthesis by a completely fortuitous event. While the carbon connectivity and chemical environment between both structures remain similar, the real dysiherbol A has a different molecular weight than that reported for the natural product. Had the synthesis groups not been favored by fortune, it could be speculated that a substantial amount of time and effort would have been required to solve the structural puzzle. Within the realm of computer-guided total synthesis of natural products, the question arose whether a synthesis group could have in silico reassigned the structure before embarking on the experimental adventure. To address this query, we evaluated some state-of-the-art computational procedures based on their computational demand and ease of implementation for nonexpert users with basic skills in computational chemistry (including HOSE, CASCADE, ANN-PRA, ML-J-DP4, DP4, and DP4+). While discussing the strengths and limitations of these methods, this case study provides a roadmap of what could be done before venturing into complex and time-demanding total synthesis projects.

E/Z configurational determination of oximes and related derivatives through quantum mechanics NMR calculations: scope and limitations of the leading probabilistic methods.

Oximes and related derivatives featuring a CN double bond are important in many areas of chemistry. Different methods for the determination of the E/Z configuration have been developed, each with its own scope and limitations. While some cannot be used when only one isomer is available, others require special NMR experiments. Here, three popular computational methodologies (DP4, DP4+, and ML-J-DP4) have been thoroughly studied using a challenging test set. Although DP4+ provides the best confidence, its computational cost might be high. On the other hand, ML-J-DP4 shows excellent performance in most cases in a fraction of CPU time. A detailed analysis of the structural factors affecting the NMR prediction and sense of the assignment is also provided.

InCl3-catalyzed intramolecular carbonyl-olefin metathesis.

An efficient and novel synthetic strategy for the generation of different carbocyclic moieties by ring closing carbonyl-olefin metathesis is reported. Herein, we describe a sustainably attractive protocol for one of the most powerful carbon-carbon bond-forming reactions, based on solvent-reduction, use of InCl3 catalyst, and microwave irradiation, affording target compounds with yields up to 96%.

DP4+App: Finding the Best Balance between Computational Cost and Predictive Capacity in the Structure Elucidation Process by DP4+. Factors Analysis and Automation.

DP4+ is one of the most popular methods for the structure elucidation of natural products using NMR calculations. While the method is simple and easy to implement, it requires a series of procedures that can be tedious, coupled with the fact that its computational demand can be high in certain cases. In this work, we made a substantial improvement to these limitations. First, we deeply explored the effect of molecular mechanics architecture on the DP4+ formalism (MM-DP4+). In addition, a Python applet (DP4+App) was developed to automate the entire process, requiring only the Gaussian NMR output files and a spreadsheet containing the experimental NMR data and labels. The script is designed to use the statistical parameters from the original 24 levels of theory (employing B3LYP/6-31G* geometries) and the new 36 levels explored in this work (over MMFF geometries). Furthermore, it enables the development of customizable methods using any desired level of theory, allowing for a free choice of test molecules.

A critical review on the use of DP4+ in the structural elucidation of natural products: the good, the bad and the ugly. A practical guide.

Covering: 2015 up to the end of 2020Even in the golden age of NMR, the number of natural products being incorrectly assigned is becoming larger every day. The use of quantum NMR calculations coupled with sophisticated data analysis provides ideal complementary tools to facilitate the elucidation process in challenging cases. Among the current computational methodologies to perform this task, the DP4+ probability is a popular and widely used method. This updated version of Goodman's DP4 synergistically combines NMR calculations at higher levels of theory with the Bayesian analysis of both scaled and unscaled data. Since its publication in late 2015, the use of DP4+ to solve controversial natural products has substantially grown, with several predictions being confirmed by total synthesis. To date, the structures of more than 200 natural products were determined with the aid of DP4+. However, all that glitters is not gold. Besides its intrinsic limitations, on many occasions it has been improperly used with potentially important consequences on the quality of the assignment. Herein we present a critical revision on how the scientific community has been using DP4+, exploring the strengths of the method and how to obtain optimal results from it. We also analyze the weaknesses of DP4+, and the paths to by-pass them to maximize the confidence in the structural elucidation.

BOPHY-Fullerene C60 Dyad as a Photosensitizer for Antimicrobial Photodynamic Therapy.

A novel BOPHY-fullerene C60 dyad (BP-C60 ) was designed as a heavy-atom-free photosensitizer (PS) with potential uses in photodynamic treatment and reactive oxygen species (ROS)-mediated applications. BP-C60 consists of a BOPHY fluorophore covalently attached to a C60 moiety through a pyrrolidine ring. The BOPHY core works as a visible-light-harvesting antenna, while the fullerene C60 subunit elicits the photodynamic action. This fluorophore-fullerene cycloadduct, obtained by a straightforward synthetic route, was fully characterized and compared with its individual counterparts. The restricted rotation around the single bond connecting the BOPHY and pyrrolidine moieties led to the formation of two atropisomers. Spectroscopic, electrochemical, and computational studies disclose an efficient photoinduced energy/electron transfer process from BOPHY to fullerene C60 . Photodynamic studies indicate that BP-C60 produces ROS by both photomechanisms (type I and type II). Moreover, the dyad exhibits higher ROS production efficiency than its individual constitutional components. Preliminary screening of photodynamic inactivation on bacteria models (Staphylococcus aureus and Escherichia coli) demonstrated the ability of this dyad to be used as a heavy-atom-free PS. To the best of our knowledge, this is the first time that not only a BOPHY-fullerene C60 dyad is reported, but also that a BOPHY derivative is applied to photoinactivate microorganisms. This study lays the foundations for the development of new BOPHY-based PSs with plausible applications in the medical field.

Chemical Stability of Petrichorins.

The structure of petrichorin C1 (4) converted from petrichorin C (3) was determined using NMR spectroscopy and X-ray crystallography. The chemical stability of petrichorins A and C (1 and 3) was investigated by NMR spectroscopy, X-ray crystallography, and calculations.

May the Force (Field) Be with You: On the Importance of Conformational Searches in the Prediction of NMR Chemical Shifts.

NMR data prediction is increasingly important in structure elucidation. The impact of force field selection was assessed, along with geometry and energy cutoffs. Based on the conclusions, we propose a new approach named mix-J-DP4, which provides a remarkable increase in the confidence level of complex stereochemical assignments-100% in our molecular test set-with a very modest increment in computational cost.

NMR Calculations with Quantum Methods: Development of New Tools for Structural Elucidation and Beyond.

Structural elucidation is an important and challenging stage in the discovery of new organic molecules. Single-crystal X-ray analysis provides the most unquestionable results, though in practice the availability of suitable crystals limits its broad use. On the other hand, NMR spectroscopy has become the leading and universal technique to accomplish the task. Despite continuous advances in the field, the misinterpretation of NMR data is commonplace, evidenced by the large number of erroneous structures being published in top journals. Quantum calculations of NMR chemical shifts and scalar coupling constants emerged as ideal complements to facilitate the elucidation process when experimental NMR data is inconclusive. Since seminal reports demonstrated that affordable DFT methods provide NMR predictions accurate enough to differentiate among closely related isomers, the discipline has experienced substantial growth. The impact has been felt in different areas, and nowadays the results of such calculations are routinely seen in high impact literature.This Account describes our investigations in the field of quantum NMR calculations, focusing on the development of tools for structural elucidation and practical applications. We pioneered the use of artificial intelligence methods in the development of novel strategies of structural validation. Our first generation of trained artificial neural networks (ANNs) showed excellent ability to identify mistakes at the atom connectivity level, whereas the use of multidimensional pattern recognition pushed the performance to the stereochemical limit. In a conceptually different approach, we developed DP4+, an updated version of the DP4 probability used to determine the most likely structure among two or more candidates when one set of experimental data is available. Increasing the level of theory in NMR calculations and including unscaled data in the formalism improved the performance of the method, further validated to settle the configuration of challenging motifs such as spiroepoxides or Mosher's derivatives. One of the limitations of DP4+ is related to the relatively large computational cost involved in obtaining DFT-optimized geometries, which led to the development of a fast variant including the valuable information provided by coupling constants (J-DP4 method).These tools were explored to suggest the most probable structure of controversial natural or unnatural products originally misassigned, with some predictions further validated by synthesis (as in the case of pseudorubriflordilactone B). The possibility of predicting the structure of a natural product without requiring authentic sample was investigated in collaboration with Prof. Pilli (UNICAMP, Brazil) in the computer-guided total synthesis and stereochemical revisions of several natural products. Despite these advances, there remain considerable challenges, such as the case of configurational assessment of polar systems featuring multiple intramolecular hydrogen bonding interactions because of the poor energy predictions provided by most DFT methods. In our latest work, we tackle this problem by averaging the results provided by randomly generated ensembles, paving the way for a new paradigm in quantum NMR-assisted structural elucidation.

Sensitivity Analysis of DP4+ with the Probability Distribution Terms: Development of a Universal and Customizable Method.

DP4+ is a popular tool for structural elucidation using GIAO NMR calculations. The method was built with 16 statistical parameters [µ,σ,ν], which depend on the level of theory. Herein, we deeply analyzed the sensitivity of DP4+ when using improper [µ,σ,ν] sets, a common situation found in the literature. The results led us to develop a customizable DP4+ methodology that allows preliminary calculations at any desired level of theory using a small set of training molecules.

BF3·OEt2-Catalyzed Unexpected Stereoselective Formation of 2,4-trans-Diallyl-2-methyl-6-aryltetrahydro-2H-pyrans with Quaternary Stereocenters.

The present manuscript describes a convenient, mild, and highly stereoselective method for the allylation of δ-hydroxy-α,ß-unsaturated ketones having a benzylic hydroxyl group at the δ-position using allyltrimethylsilane mediated by BF3·OEt2, leading to 2,4-diallyl-2-methyl-6-aryltetrahydro-2H-pyran ring systems with quaternary carbon stereogenic centers. This represents the first example of a tandem isomerization followed by one C-O and two C-C bond-forming reactions in one pot. The isolation of TMS-protected lactol as an intermediate from the reaction strongly supports the proposed mechanistic pathway.

Thermal decomposition of hexamethylenetetramine: mechanistic study and identification of reaction intermediates via a computational and NMR approach.

In a joint DFT and chemometrics study applied to NMR spectra, we disclose the structure of the main decomposition products of hexamethylenetetramine. The combination of these techniques enabled us to propose the structures of near-identical intermediates of the process and to unveil the structure of the main decomposition product of this priviliged structure.

In Silico Reassignment of (+)-Diplopyrone by NMR Calculations: Use of a DP4/J-DP4/DP4+/DIP Tandem to Revise Both Relative and Absolute Configuration.

The structural revision of natural (+)-diplopyrone (ND) was achieved by quantum NMR calculations. A DP4/J-DP4/DP4+ tandem suggested 3 as the most likely structure, but ECD calculations did not match the experimental values. The second more probable structure (6epi-1) showed the right ECD spectrum and high DP4/DP4+ probabilities obtained after fitting. However, further analysis of the MTPA-ND derivatives by DP4+/DIP calculations demonstrated that the absolute configuration at C-9 had been incorrectly assigned. Then, the structure of ND was proposed as ent-3.

Re-Engineering Organocatalysts for Asymmetric Friedel-Crafts Alkylation of Indoles through Computational Studies.

The discovery of efficient organocatalysts is generally carried out by thorough experimental screening of different candidates. We recently reported an efficient organocatalyst for iminium-ion-based asymmetric Diels-Alder reactions following a rational design approach. This result encouraged us to test this optimal catalyst in the mechanistically related Friedel-Crafts alkylation of indoles, but to our surprise, almost null enantioselectivity was observed. The results did not significantly improve with structurally related catalysts, and a totally unexpected facial selectivity inversion was also noticed. Using DFT calculations by modeling the competing transition structures with ONIOM, we could unravel the origins of those findings, further employed to predict the most efficient catalyst for this new transformation. The computational results were validated experimentally (up to 92:8 er), providing another successful example of a general strategy to accelerate catalyst development which still remains underexplored.

Design, synthesis and evaluation of novel levoglucosenone derivatives as promising anticancer agents.

A series of levoglucosenone-derived 1,2,3-triazoles and isoxazoles featuring a flexible spacer between the heteroaromatic and anhydropyranose cores have been designed and synthesized following an hetero Michael // 1,3-dipolar cycloaddition path. The use of a design of experiments approach allowed the optimization of the oxa-Michael reaction with propargyl alcohol as nucleophile, a key step for the synthesis of the target compounds. All of the compounds were tested for their anticancer activity on MDA-MB-231 cells, featuring mutant p53. The results highlighted the importance of the introduction of the flexible spacer as well as the higher activity of oxa-Michael isoxazole-derivatives. The most prominent compounds also showed anti-proliferative activities against lung and colon cancer cell lines. The compounds showed enhanced cytotoxic effects in the presence of mutant p53, determined both by endogenous mutant p53 knock down (R280K) and by reintroducing p53 R280K in cells lacking p53 expression.

Looking at the big picture in activation strain model/energy decomposition analysis: the case of the ortho-para regioselectivity rule in Diels-Alder reactions.

The regioselectivity of the Diels-Alder reaction is predicted by the ortho-para rule which has been explained from FMO theory. Using DFT calculations, the activation-strain model and energy decomposition analysis we studied the reaction of methyl acrylate with four unsymmetrical dienes. We found that if the analysis is carried out considering the TS structures, the selectivity would not be explained by the interaction energy as expected considering the FMO arguments. However, a thorough analysis along the reaction path revealed that the interaction energy is responsible for the regioselectivity. A deeper analysis with the EDA model showed that the decisive term that accounts for the HOMO-LUMO interactions favors the ortho and para paths, as predicted by FMO arguments.