Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep.

Ethane is the second most abundant component of natural gas in addition to methane, and-similar to methane-is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps1-3, and through ethane-dependent sulfate reduction in slurries4-7. Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown8. Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name 'Candidatus Argoarchaeum ethanivorans'; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography-tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by 'Candidatus Syntrophoarchaeum'9. Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO2 occurs through the oxidative Wood-Ljungdahl pathway. The identification of an archaeon that uses ethane (C2H6) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (CnH2n+2) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps10-12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps.

Photo-Driven Iron-Induced Non-Oxidative Coupling of Methane to Ethane.

Photo-driven CH4 conversion to multi-carbon products and H2 is attractive but challenging, and the development of efficient catalytic systems is critical. Herein, we construct a solar-energy-driven redox cycle for combining CH4 conversion and H2 production using iron ions. A photo-driven iron-induced reaction system was developed, which is efficient at selective coupling of CH4 as well as conversion of benzene and cyclohexane under mild conditions. For CH4 conversion, 94 % C2 selectivity and a C2 H6 formation rate of 8.4 µmol h-1 is achieved. Mechanistic studies reveal that CH4 coupling is induced by hydroxyl radical, which is generated by photo-driven intermolecular charge migration of an Fe3+ complex. The delicate coordination structure of the [Fe(H2 O)5 OH]2+ complex ensures selective C-H bond activation and C-C coupling of CH4 . The produced Fe2+ can be used to reduce the potential for electrolytic H2 production, and then turns back into Fe3+ , forming an energy-saving and sustainable recyclable system.

Ethane-Bridged Hybrid Monolithic Column with Large Mesopores for Boosting Top-Down Proteomic Analysis.

Top-down proteomics is challenged by the high complexity of biological samples. The coelution of intact proteins results in overlapped mass spectra, and hence, an increased peak capacity for protein separation is needed. Herein, ethane-bridged hybrid monoliths with well-defined large mesopores were successfully prepared based on the sol-gel condensation of 1,2-bis(trimethoxysilyl)ethane and tetramethoxysilane, followed by two-step base etching of the Si-O-Si domain while maintaining the Si-C-C-Si domain in the structure. Relatively homogeneous macropores of 1.1 µm and large mesopores of 24 nm were obtained, permitting fast mass transfer of large molecules and efficient diffusion without obstruction. The use of less hydrophobic C1 ligand further sharpened the peak shape and improved peak capacity. A 120 cm-long capillary column was used for top-down proteomic analysis of E. coli lysates under low backpressure with 16 MPa. High peak capacity of 646 was achieved within 240 min gradient. With MS/MS analysis, 959 proteoforms corresponding to 263 proteins could be unambiguously identified from E. coli lysates in a single run. Furthermore, to illustrate the separation performance for large proteoforms, such monoliths were applied to top-down analysis of the SEC fraction of E. coli lysates with Mw ranging from 30 to 70 kDa. With highly effective separation, 347 large proteoforms with Mw higher than 30 kDa were detected in the single 75 min run. These results showed great potential for top-down proteomic analysis in complex samples.

Twofold and Threefold Sinusoidal Patterns in Coupled Molecular Motions of 184,025 Structures of Phenylethane, Nitroethane, and Carboxylate Derivatives.

Scatter plot analyses for 14,169 phenylethanes of the substructure Cß-CαH2-Ph with three open coordination positions at Cß and 150,568 phenylethanes of Cß-CαHX-Ph with an additional open coordination position X at Cα have been performed, based on searches in the Cambridge Structural Database. The correlation of rotation angle ψ = Cß-Cα-Ci-Co with a pyramidalization angle θ = Co-Co'-Ci-Cα in a 360° rotation about the bond Cα-Ci reveals a sinusoidal pattern with three maxima and minima, whereas the correlation of rotation angle ψ with bond angle ω = Cß-Cα-Ci and bond length d = Cß-Cα results in sinusoidal patterns with two maxima and minima. A total of 3993 nitro derivatives of the substructure Cß-CαHX-NO2 confirm the results and show that atoms Ci/Co/Co' in the phenyl compounds can be replaced by atoms N/O/O' without any change in the two- and threefold patterns. In 15,295 methyl acetates of the substructure Cß-CαHX-C'(═O)OMe, pyramidalization of the group CαC'(═O)OMe results in a chiral flat tetrahedron with four different corners. (Rθ)/(Sθ) selectivity in the configuration of the tetrahedron is induced by the bonds Cα-Cß, Cα-H, and Cα-X, emanating from the tetrahedral center Cα. It is surprising that bonds as different as Cα-Cß, Cα-H, and Cα-X (X = H, C, N, O, S, etc.) give almost the same induction intensities.

Large changes in biomass burning over the last millennium inferred from paleoatmospheric ethane in polar ice cores.

Biomass burning drives changes in greenhouse gases, climate-forcing aerosols, and global atmospheric chemistry. There is controversy about the magnitude and timing of changes in biomass burning emissions on millennial time scales from preindustrial to present and about the relative importance of climate change and human activities as the underlying cause. Biomass burning is one of two notable sources of ethane in the preindustrial atmosphere. Here, we present ice core ethane measurements from Antarctica and Greenland that contain information about changes in biomass burning emissions since 1000 CE (Common Era). The biomass burning emissions of ethane during the Medieval Period (1000-1500 CE) were higher than present day and declined sharply to a minimum during the cooler Little Ice Age (1600-1800 CE). Assuming that preindustrial atmospheric reactivity and transport were the same as in the modern atmosphere, we estimate that biomass burning emissions decreased by 30 to 45% from the Medieval Period to the Little Ice Age. The timing and magnitude of this decline in biomass burning emissions is consistent with that inferred from ice core methane stable carbon isotope ratios but inconsistent with histories based on sedimentary charcoal and ice core carbon monoxide measurements. This study demonstrates that biomass burning emissions have exceeded modern levels in the past and may be highly sensitive to changes in climate.

Ethyl esters production catalyzed by immobilized lipases is influenced by n-hexane and ter-amyl alcohol as organic solvents.

Lipase stability in organic solvent is crucial for its application in many biotechnological processes as biocatalyst. One way to improve lipase's activity and stability in unusual reaction medium is its immobilization on inert supports. Here, lipases from different sources and immobilized through weak chemical interactions on hydrophobic and ionic supports had their transesterification ability dramatically dependent on the support and also on the solvent that had been used. The ethanolysis of sardine oil was carried out at the presence of cyclohexane and tert-amyl alcohol, in which Duolite A568-Thermomyces lanuginosa lipase derivative achieved 49% of ethyl esters production after 24 h in cyclohexane. The selectivity of immobilized lipases was also studied and, after 3 h of synthesis, the reaction with Duolite A568-Thermomyces lanuginosa derivative in cyclohexane produced 24% ethyl ester of eicosapentaenoic acid and 1.2% ethyl ester of docosahexaenoic acid, displaying a selectivity index of 20 times the ethyl ester of eicosapentaenoic acid. Different derivatives of Candida antarctica lipases fraction B (CALB) and phospholipase Lecitase® Ultra (Lecitase) were also investigated. Along these lines, a combination between these factors may be applied to improve the activity and selectivity of immobilized lipases, decreasing the total cost of the process.

Selective Oxidation of Ethane to Acetic Acid Catalyzed by a C-Scorpionate Iron(II) Complex: A Homogeneous vs. Heterogeneous Comparison.

The direct, one-pot oxidation of ethane to acetic acid was, for the first time, performed using a C-scorpionate complex anchored onto a magnetic core-shell support, the Fe3O4/TiO2/[FeCl2{κ3-HC(pz)3}] composite. This catalytic system, where the magnetic catalyst is easily recovered and reused, is highly selective to the acetic acid synthesis. The performed green metrics calculations highlight the "greeness" of the new ethane oxidation procedure.

1-Aryl-2-(triisopropylsilyl)ethane-1,2-diones: Toward a New Class of Visible Type I Photoinitiators for Free Radical Polymerization of Methacrylates.

1-Aryl-2-(triisopropylsilyl)ethane-1,2-diones (SEDs) are proposed here as a new class of visible Type I photoinitiators (PIs) for free radical polymerization under air upon exposure to blue (@455 nm) and green (@520 nm) LEDs. Remarkably, these new systems present good polymerization performances and excellent bleaching properties compared to camphorquinone-based systems, and transparent polymers are obtained upon visible light irradiation. Real-time Fourier transform infrared spectroscopy is used to monitor the polymerization profiles. Molecular orbital calculations are also carried out for a better understanding of the structure/reactivity relationship of the photoinitiators.

Competitive aptasensor with gold nanoparticle dimers and magnetite nanoparticles for SERS-based determination of thrombin.

It is known that the intensity of surface-enhanced Raman scattering (SERS) of monomeric gold nanoparticles (GNPs) is insufficient for ultrasensitive analysis. The authors describe dimeric GNPs for use in a competitive SERS and aptamer based assay for thrombin. The reagent 1,2-bis(4-pyridyl) ethylene serves as both the coupling agent and the Raman reporter on the GNP dimers. In the presence of thrombin, the hybridization of two aptamers, one attached to the GNP dimers, the other to magnetic nanoparticles, is competitively prevented. This method takes advantage of the unique "hot spots" of the GNP dimers to amplify the Raman signal. This results in an ultra-sensitive thrombin assay when compared to assays using GNP monomers. The limit of detection is as low as 1 fM of thrombin. The Raman intensity, best measured at 1612 cm-1, increases linearly in the 1 fM to 10 nM thrombin concentration range. The method was applied to the determinaiton of thrombin in spiked simulated body fluid and human serum. Graphical abstract This method takes advantage of the unique "hot spots" of the gold nanoparticle dimers to amplify the Raman signal. The dimers are linked to the magnetic nanoparticles via an aptamer. The use of both competitive displacement and magnetic separation greatly improves the sensitivity of the thrombin assay.

Structural Transformations in the Thermal Dehydration of [Cu2(bpa)(btec)(H2O)4]n Coordination Polymer.

Reactions between pyridinic ligands such as 1,2-bis(4-pyridyl)ethane (bpa) and transition metal cations are a very widespread technique to produce extended coordination polymers such as Metal-Organic Frameworks. In combination with a second ligand these systems could present different topologies and behaviors. In this context, the use of 1,2,4,5-benzenetetracarboxylic acid (H4btec) gave us a novel 2D compound, [Cu2(bpa)(btec)(H2O)4]n (1), which was prepared by microwave-assisted synthesis and structurally characterized by means of single crystal X-ray diffraction. Its thermal behavior was analyzed through thermogravimetric analysis and variable temperature powder X-ray diffraction, concluding that thermal stability is influenced by the coordination water molecules, allowing two sequential thermochromic phase transformations to take place. These transformations were monitored by electronic paramagnetic resonance spectroscopy and magnetic susceptibility measurements. In addition, the crystal structure of the anhydrous compound [Cu2(bpa)(btec)]n (1.ah) was determined. Finally, a topological study was carried out for the bpa ligand considering all the structures deposited in the Cambridge Structural Databased. More than 1000 structures were analyzed and classified into 17 different topologies, according to the role of the ligand.

Encapsulation of Upconversion Nanoparticles in Periodic Mesoporous Organosilicas.

(1) Background: Nanomedicine has recently emerged as a promising field, particularly for cancer theranostics. In this context, nanoparticles designed for imaging and therapeutic applications are of interest. We, therefore, studied the encapsulation of upconverting nanoparticles in mesoporous organosilica nanoparticles. Indeed, mesoporous organosilica nanoparticles have been shown to be very efficient for drug delivery, and upconverting nanoparticles are interesting for near-infrared and X-ray computed tomography imaging, depending on the matrix used. (2) Methods: Two different upconverting-based nanoparticles were synthesized with Yb3+-Er3+ as the upconverting system and NaYF4 or BaLuF5 as the matrix. The encapsulation of these nanoparticles was studied through the sol-gel procedure with bis(triethoxysilyl)ethylene and bis(triethoxysilyl)ethane in the presence of CTAB. (3) Results: with bis(triethoxysilyl)ethylene, BaLuF5: Yb3+-Er3+, nanoparticles were not encapsulated, but anchored on the surface of the obtained mesoporous nanorods BaLuF5: Yb3+-Er3+@Ethylene. With bis(triethoxysilyl)ethane, BaLuF5: Yb3+-Er3+ and NaYF4: Yb3+-Er3+nanoparticles were encapsulated in the mesoporous cubic structure leading to BaLuF5: Yb3+-Er3+@Ethane and NaYF4: Yb3+-Er3+@Ethane, respectively. (4) Conclusions: upconversion nanoparticles were located on the surface of mesoporous nanorods obtained by hydrolysis polycondensation of bis(triethoxysilyl)ethylene, whereas encapsulation occurred with bis(triethoxysilyl)ethane. The later nanoparticles NaYF4: Yb3+-Er3+@Ethane or BaLuF5: Yb3+-Er3+@Ethane were promising for applications with cancer cell imaging or X-ray-computed tomography respectively.

Ethane-Bridged Bisporphyrin Conformational Changes As an Effective Analytical Tool for Nonenzymatic Detection of Urea in the Physiological Range.

Conformational switching induced in ethane-bridged bisporphyrins was used as a sensitive transduction method for revealing the presence of urea dissolved in water via nonenzymatic approach. Bisporphyrins were deposited on solid quartz slides by means of the spin-coating method. Molecular conformations of Zn and Ni monometalated bis-porphyrins were influenced by water solvated urea molecules and their fluorescence emission was modulated by the urea concentration. Absorption, fluorescence and Raman spectroscopies allowed the identification of supramolecular processes, which are responsible for host-guest interaction between the active layers and urea molecules. A high selectivity of the sensing mechanism was highlighted upon testing the spectroscopic responses of bis-porphyrin films to citrulline and glutamine used as interfering agents. Additionally, potential applicability was demonstrated by quantifying the urea concentration in real physiological samples proposing this new approach as a valuable alternative analytical procedure to the traditionally used enzymatic methods.

Crystal structure of hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 in complex with nitroethane as a nitroalkane substrate.

Nitroalkane oxidase (NAO) and nitronate monooxygenase (NMO) are two different types of nitroalkane oxidizing flavoenzymes identified in nature. A previous study suggested that the hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 is NMO and utilizes only anionic nitronates. However, the structural similarity between the PA4202 protein and Streptomyces ansochromogenes NAO has motivated investigation for what features of the two enzymes differentiate between the NAO and NMO activities. Herein, we report the crystal structure of PA4202 in a ternary complex with a neutral nitroethane (NE) and flavin mononucleotide (FMN) cofactor to elucidate the substrate recognition mechanism using a site-directed mutagenesis. The ternary complex structure indicates that the NE is bound with an orientation, which is poised for the proton transfer to H183 (which is the essential first catalytic step with nitroalkanes), and subsequent reactions with FMN. Moreover, a kinetic study reveals that the catalytic reactions of the wild type and H183 mutants PA4202s with nitroalkane substrates may yield the products of hydrogen peroxide and nitrite that are specified to NAO, although they show a low catalytic efficiency. Our results provide the first structure-based molecular insight into the substrate binding property of the hypothetical protein PA4202, including the interactions with neutral nitroalkanes as the substrate.

1,2-Diphenoxiethane salts as potent antiplasmodial agents.

In this article we present a series of non-cytotoxic potent human choline kinase (CK) inhibitors that exhibit nanomolar antiplasmodial activity in vitro. The most active antiplasmodial compounds, 10a-b, bearing a pyridinium cationic head were inactive against CK, while compounds 10g and 10j with a quinolinium moiety exhibit moderate inhibition of both the parasite and the enzyme. The results point towards an additional mechanism of action unrelated to CK inhibition that remains to be established.

Long-term decline of global atmospheric ethane concentrations and implications for methane.

After methane, ethane is the most abundant hydrocarbon in the remote atmosphere. It is a precursor to tropospheric ozone and it influences the atmosphere's oxidative capacity through its reaction with the hydroxyl radical, ethane's primary atmospheric sink. Here we present the longest continuous record of global atmospheric ethane levels. We show that global ethane emission rates decreased from 14.3 to 11.3 teragrams per year, or by 21 per cent, from 1984 to 2010. We attribute this to decreasing fugitive emissions from ethane's fossil fuel source--most probably decreased venting and flaring of natural gas in oil fields--rather than a decline in its other major sources, biofuel use and biomass burning. Ethane's major emission sources are shared with methane, and recent studies have disagreed on whether reduced fossil fuel or microbial emissions have caused methane's atmospheric growth rate to slow. Our findings suggest that reduced fugitive fossil fuel emissions account for at least 10-21 teragrams per year (30-70 per cent) of the decrease in methane's global emissions, significantly contributing to methane's slowing atmospheric growth rate since the mid-1980s.

Sorption, Structure and Dynamics of CO2 and Ethane in Silicalite at High Pressure: A Combined Monte Carlo and Molecular Dynamics Simulation Study.

Silicalite is an important nanoporous material that finds applications in several industries, including gas separation and catalysis. While the sorption, structure, and dynamics of several molecules confined in the pores of silicalite have been reported, most of these studies have been restricted to low pressures. Here we report a comparative study of sorption, structure, and dynamics of CO2 and ethane in silicalite at high pressures (up to 100 bar) using a combination of Monte Carlo (MC) and molecular dynamics (MD) simulations. The behavior of the two fluids is studied in terms of the simulated sorption isotherms, the positional and orientational distribution of sorbed molecules in silicalite, and their translational diffusion, vibrational spectra, and rotational motion. Both CO2 and ethane are found to exhibit orientational ordering in silicalite pores; however, at high pressures, while CO2 prefers to reside in the channel intersections, ethane molecules reside mostly in the sinusoidal channels. While CO2 exhibits a higher self-diffusion coefficient than ethane at low pressures, at high pressures, it becomes slower than ethane. Both CO2 and ethane exhibit rotational motion at two time scales. At both time scales, the rotational motion of ethane is faster. The differences observed here in the behavior of CO2 and ethane in silicalite pores can be seen as a consequence of an interplay of the kinetic diameter of the two molecules and the quadrupole moment of CO2.

Continuous Separation of Light Olefin/Paraffin Mixtures on ZIF-4 by Pressure Swing Adsorption and Membrane Permeation.

In this study, two zeolitic imidazolate frameworks (ZIFs) called ZIF-4 and ZIF-zni (zni is the network topology) were characterized by sorption studies regarding their paraffin/olefin separation potential. In particular, equilibrated pure and mixed gas adsorption isotherms of ethane and ethene were recorded at 293 K up to 3 MPa. ZIF-4 exhibits selectivities for ethane in the range of 1.5-3, which is promising for continuous pressure swing adsorption (PSA). ZIF-4 shows high cycle stability with promising separation potential regarding ethane, which results in purification of the more industrial desired olefin. Furthermore, both ZIF materials were implemented in Matrimid to prepare a mixed matrix membrane (MMM) and were used in the continuous separation of a propane/propene mixture. The separation performance of the neat polymer is drastically increased after embedding porous ZIF-4 crystals in the Matrimid matrix, especially at higher feed pressures (3-5 barg). Due to the smaller kinetic diameter of the olefin, the permeability is higher compared to the paraffin.

Pre-treatment of anodic inoculum with nitroethane to improve performance of a microbial fuel cell.

Methanogenic substrate loss is reported to be a major bottleneck in microbial fuel cell (MFC), which significantly reduces the power production capacity and coulombic efficiency (CE) of this system. Nitroethane is found to be a potent inhibitor of hydrogenotrophic methanogens in rumen fermentation process. Influence of nitroethane pre-treated sewage sludge inoculum on suppressing the methanogenic activity and enhancing the electrogenesis in MFC was evaluated. MFC inoculated with nitroethane pre-treated anodic inoculum demonstrated a maximum operating voltage of 541 mV, with CE and maximum volumetric power density of 39.85% and 20.5 W/m3, respectively. Linear sweep voltammetry indicated a higher electron discharge on the anode surface due to enhancement of electrogenic activity while suppressing methanogenic activity. A 63% reduction in specific methanogenic activity was observed in anaerobic sludge pre-treated with nitroethane, emphasizing the significance of this pre-treatment for suppressing methanogenesis and its utility for enhancing electricity generation in MFC.

Short chain nitrocompounds as a treatment of layer hen manure and litter; effects on in vitro survivability of Salmonella, generic E. coli and nitrogen metabolism.

The current study was conducted to assess the bactericidal effectiveness of several nitrocompounds against pathogens in layer hen manure and litter. Evidence from an initial study indicated that treatment of layer hen manure with 12 mM nitroethane decreased populations of generic E. coli and total coliforms by 0.7 and 2.2 log10 colony forming units (CFU) g-1, respectively, after 24 h aerobic incubation at ambient temperature when compared to untreated populations. Salmonella concentrations were unaffected by nitroethane in this study. In a follow-up experiment, treatment of 6-month-old layer hen litter (mixed with 0.4 mL water g-1) with 44 mM 2-nitroethanol, 2-nitropropanol or ethyl nitroacetate decreased an inoculated Salmonella typhimurium strain from its initial concentration (3 log10 CFU g-1) by 0.7 to 1.7 log10 CFU g-1 after 6 h incubation at 37°C in covered containers. After 24 h incubation, populations of the inoculated S. Typhmiurium in litter treated with 44 mM 2-nitroethanol, 2-nitropropanol, ethyl nitroacetate or nitroethane were decreased more than 3.2 log10 CFU g-1 compared to populations in untreated control litter. Treatment of litter with 44 mM 2-nitroethanol, 2-nitropropanol, ethyl nitroacetate decreased rates of ammonia accumulation more than 70% compared to untreated controls (0.167 µmol mL-1 h-1) and loses of uric acid (< 1 µmol mL-1) were observed only in litter treated with 44 mM 2-nitropropanol, indicating that some of these nitrocompounds may help prevent loss of nitrogen in treated litter. Results warrant further research to determine if these nitrocompounds can be developed into an environmentally sustainable and safe strategy to eliminate pathogens from poultry litter, while preserving its nitrogen content as a nutritionally valuable crude protein source for ruminants.

Correcting for the free energy costs of bond or angle constraints in molecular dynamics simulations.

BACKGROUND: Free energy simulations are an important tool in the arsenal of computational biophysics, allowing the calculation of thermodynamic properties of binding or enzymatic reactions. This paper introduces methods to increase the accuracy and precision of free energy calculations by calculating the free energy costs of constraints during post-processing. The primary purpose of employing constraints for these free energy methods is to increase the phase space overlap between ensembles, which is required for accuracy and convergence. METHODS: The free energy costs of applying or removing constraints are calculated as additional explicit steps in the free energy cycle. The new techniques focus on hard degrees of freedom and use both gradients and Hessian estimation. Enthalpy, vibrational entropy, and Jacobian free energy terms are considered. RESULTS: We demonstrate the utility of this method with simple classical systems involving harmonic and anharmonic oscillators, four-atomic benchmark systems, an alchemical mutation of ethane to methanol, and free energy simulations between alanine and serine. The errors for the analytical test cases are all below 0.0007kcal/mol, and the accuracy of the free energy results of ethane to methanol is improved from 0.15 to 0.04kcal/mol. For the alanine to serine case, the phase space overlaps of the unconstrained simulations range between 0.15 and 0.9%. The introduction of constraints increases the overlap up to 2.05%. On average, the overlap increases by 94% relative to the unconstrained value and precision is doubled. CONCLUSIONS: The approach reduces errors arising from constraints by about an order of magnitude. Free energy simulations benefit from the use of constraints through enhanced convergence and higher precision. GENERAL SIGNIFICANCE: The primary utility of this approach is to calculate free energies for systems with disparate energy surfaces and bonded terms, especially in multi-scale molecular mechanics/quantum mechanics simulations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.