Absolute Configurations of Naturally Occurring - ACS Publications

Article Cite This: J. Nat. Prod. 2018, 81, 2654−2666

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Absolute Configurations of Naturally Occurring [5]- and [3]Ladderanoic Acids: Isolation, Chiroptical Spectroscopy, and Crystallography Vijay Raghavan,† Jordan L. Johnson,† Donald F. Stec,† Bongkeun Song,‡ Grzegorz Zajac,§ Malgorzata Baranska,§,⊥ Constance M. Harris,† Nathan D. Schley,† Prasad L. Polavarapu,*,† and Thomas M. Harris†,‡ Downloaded via IOWA STATE UNIV on January 11, 2019 at 19:44:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States Department of Biological Sciences, Virginia Institute of Marine Science, Gloucester Point, Virginia 23062, United States § Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland ⊥ Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland ‡

S Supporting Information *

ABSTRACT: We have isolated mixtures of [5]- and [3]ladderanoic acids 1a and 2a from the biomass of an anammox bioreactor and have separated the acids and their phenacyl esters for the first time by HPLC. The absolute configurations of the naturally occurring acids and their phenacyl esters are assigned as R at the site of side-chain attachment by comparison of experimental specific rotations with corresponding values predicted using quantum chemical (QC) methods. The absolute configurations for 1a and 2a were independently verified by comparison of experimental Raman optical activity spectra with corresponding spectra predicted using QC methods. The configurational assignments of 1a and 2a and of the phenacyl ester of 1a were also confirmed by X-ray crystallography.

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than typical phospholipid membranes, but reduced permeability to hydroxylamine and hydrazine has not been demonstrated.9,10 [While this article was under review, a paper by Moss et al. (Moss, F. R., III; Shuken, S. R.; Mercer, J. A. M.; Cohen, C. M.; Weiss, T. M.; Boxer, S. G.; Burns, N. Z. Proc. Nat. Acad. Sci. USA 2018, 115, 9098−9103) appeared that disputed the proposal that the ladderane membranes reduce permeability of hydrazine.] The phospholipid membrane of the anammoxosome is structurally remarkable, containing fatty acids and analogous alcohols having multiple concatenated cyclobutane rings6 known as ladderanes. The two major ladderane components of the anammoxosome are [5]-ladderanoic acid 1a (n = 7) and [3]-ladderanoic acid 2a (n = 7) (Figure 1). Both 1a and 2a are eicosanoids, with [5]-ladderanoic acid 1a containing five linearly fused cyclobutane rings and [3]-ladderanoic acid 2a having three cyclobutane rings fused to a cyclohexane ring. Lesser quantities of longer and shorter chain analogues (n = 1, 3, 5, 9, and 11) have also been observed.3,11−14 Acids 1 and 2 occur in the anammoxosome membrane both as the free acids and as phospholipid esters. In addition, analogous [5]- and [3]-ladderanols 3 and 4 (Figure 1) are major constituents and

mmonium ion is essential to all living organisms, being the source of nitrogen in proteins, nucleic acids, and other cellular metabolites. It is produced from atmospheric N2 by nitrogen-fixing bacteria and in the atmosphere by lightning discharges. In addition, more than 200 million tons of ammonia are produced from N2 annually using the Haber process, the majority for use as fertilizer. Nature maintains homeostasis via enzymatic pathways of denitrification and anaerobic ammonium oxidation (anammox) to convert ammonia back to N2.1,2 Anammox is mediated by bacteria in the Planctomycetes phylum,3 which can oxidize ammonium while reducing nitrite to produce N2. Anammox bacteria are ubiquitously dispersed throughout the world, being found in terrestrial and aquatic ecosystems, and are used extensively in wastewater treatment plants (WWTPs) to remove ammonium ion from effluent. The anammox pathway, comprising enzymatic reactions for reduction of nitrite to nitric oxide (NO), synthesis of hydroxylamine and hydrazine from ammonium ion and NO, and oxidation of hydrazine to N2,2,4,5 occurs in an intracytoplasmic vesicle called the anammoxosome. A phospholipid membrane surrounding the anammoxosome is thought to be unusually dense, thereby retarding escape of the strongly nucleophilic hydroxylamine and hydrazine, which would otherwise react with cellular constituents and thus interfere with essential metabolism.6−8 The membrane has been shown to be less permeable to dyes © 2018 American Chemical Society and American Society of Pharmacognosy

Received: June 8, 2018 Published: December 18, 2018 2654

DOI: 10.1021/acs.jnatprod.8b00458 J. Nat. Prod. 2018, 81, 2654−2666

Journal of Natural Products

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Figure 1. Naturally occurring ladderane acids and alcohols 1−5. Structures are depicted for enantiomers having an R configuration at the site of attachment of the functionalized side chain. ACs of natural 4a and 5 have been established as R by Mercer et al.15 The ACs of natural 1a and 2a are established in the present work.

are present as ethers in the phospholipids. Other larger and smaller arrays of concatenated cyclobutanes and combinations of cyclobutanes with cyclohexanes have not, as yet, been found. The remarkable structure of the ladderane lipids has inspired significant interest in their chemistry and biology, as evident in the review by Nouri and Tantillo16 and in a patent on use for drug delivery.17 However, multiple chemical and biological problems have impeded the study of ladderane lipids. Anammox bacteria live in oxygen-minimum environments with slow growth rates leading to doubling times of 2 weeks or more. In no case have the anammox bacteria been obtained in pure culture, and it is likely that they are, in fact, dependent on associated bacteria for, as yet unidentified, essential nutrients. The ladderanoic acids are not readily obtained in pure form. The methyl esters cannot be fully separated from each other by liquid chromatography,6,9,10,15,18 and their analysis by gas chromatography is made challenging by the thermal instability of acid 1a, which has a half-life of only 1 h at 140 °C.19 As a result, the shortage of pure ladderanoic acids has been a major hindrance to their study. Throughout this article, the stated AC refers to that at the site of attachment of the functionalized side chain: R configuration at the site of attachment refers to the structures as depicted in Figures 1 and 2, while S configuration at the site of attachment refers to mirror images of the structures depicted in Figures 1 and 2. An enantioselective synthesis of (S)-1a was accomplished by Mascitti and Corey via resolution and configurational assignment of a cyclopentanone precursor.19 They were unable to establish the AC of the natural acid due to inability to obtain an authentic sample with which to compare specific rotation.20 This inability to gain access to a small reference sample of the acid exemplifies the experimental hurdles associated with isolation and purification of the individual ladderanes. Recently, enantioselective syntheses of (R)-[3]-ladderanol 4a and its glyceryl ether 5 were reported by Mercer et al. starting from a lipase-resolved cyclohexenediol.15 The AC of natural 4a was established to be R at the site of chain attachment by comparison of the specific rotations of synthetic and natural glyceryl ether 5. The synthetic strategy was

Figure 2. Phenacyl esters of ladderanoic acids 6 and 7. Structures are depicted for enantiomers having an R configuration at the site of attachment of the functionalized side chain.

extended to preparation of (R)-[5]-ladderanoic acid 1a. They were unable to compare their synthetic (R)-1a with the natural acid due to lack of an authentic sample of the latter. Configurational assignments are challenging due to the difficulties in separation of the [3]- and [5]-ladderanes and of their derivatives and also due to conformational mobility, which complicate both the calculations and production of crystals suitable for X-ray crystallography. These difficulties are successfully overcome in this work through a multipronged approach. Our initial attempts led to chromatographic separations of the respective phenacyl esters 6a and 7a (see Figure 2). Later, we were able to separate acids 1a and 2a themselves. In order to establish the ACs, we employed multiple chiroptical spectroscopic methods21,22 to overcome the inherent limitations associated with individual chiroptical spectroscopic methods. The ACs of 6a and 7a were established by comparison of their experimental specific rotations with those calculated using quantum chemical (QC) methods and confirmed by the X-ray crystal structure of 6a. The ACs of 1a 2655



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Figure 3. HSQC spectrum of a crude mixture of 1a and 2a. Key ladderane signals are enclosed in the dashed box. Assignments are based on Damsté et al.6 The signals for methylene and methine protons have opposing signs; the methylene signals have been colored blue and the methines red.

carboxylic acids. This gave fatty acid mixtures in which the ladderanoic acids were highly concentrated. Initially we had difficulty locating the ladderanes in the complex mixtures, but it was discovered that HSQC NMR spectra were uniquely useful for distinguishing the [3]- and [5]-ladderanoic acids from the other lipid components and from each other (Figure 3). The ladderane protons fall in a narrow range, such that much overlap occurs and the signals are strongly non-first-order at 600 MHz. Two-dimensional HSQC spectra improved the spectral dispersion. The inherently high sensitivity of HSQC spectra allows spectra to be acquired using small samples, but the usefulness of the HSQC spectra is due in no small part to the fact that the carbon−hydrogen pairs of methines of the concatenated cyclobutanes fall in a region of the two-dimensional spectrum that is relatively free of contributions from other components of the lipid mixtures. The situation is exemplified by the 1H and HSQC spectra (Figure 3) of a partially purified mixture of 1a and 2a that still contained substantial amounts of other fatty acids and lipophilic substances. Important ladderane signals are assigned in the HSQC spectrum. The key ladderane protons have signals ranging between 1.5 and 2.8 ppm with the associated carbon signals lying between 24 and 50 ppm. The HSQC spectrum reveals good differentiation between 1a and 2a. Preparation of Phenacyl Esters 6a and 7a. A crude mixture of 1a and 2a was obtained by CH2Cl2 extraction of biomass from the anammox process at the WWTP. With awareness that separation of the methyl esters of acids 1a and 2a had previously been problematic,6,15,18 we turned to phenacyl esters. The phenacyl esters have a strong UV signature, which facilitates detection during chromatography. Mixed phenacyl esters 6a and 7a were readily prepared by the method of Borch27 and found to be separable by HPLC on C8

and 2a were determined by comparison of experimental and QC predictions of specific rotations as well as vibrational Raman optical activity spectra. The ACs deduced in this manner for 1a and 2a were further confirmed by respective single-crystal structures.

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RESULTS AND DISCUSSION Isolation of the Ladderanoic Acids. For isolation of ladderane metabolites, previous investigators found it expeditious to employ laboratory cultures in which the content of Candidatus brocadia sp. had been significantly enriched.4,6,15,18,23−25 Our isolation was undertaken using biomass obtained from the anammox process at a WWTP where the C. brocadia abundance was no more than 2.5% based on 16S sequence analysis. This source of biomass is readily available and obviates the need for laboratory cultures; however the use of this material presents challenges due to the small fraction of C. brocadia sp. present in the microbial community and the presence of large quantities of nonladderane fatty acids and other lipophilic compounds. Two problems were apparent: separation of the ladderanoic acids from the other lipids and separation of the [3]- and [5]ladderanoic acids from one another. We developed a relatively simple isolation procedure involving acid treatment of biomass to convert ladderane esters to the free acids followed by collection of the resulting cell debris by centrifugation and then lyophilization. Soxhlet extraction with CH2Cl2 was found to be superior to methods that included MeOH, which extracted large quantities of more polar materials.6,18,24−26 Further purification was obtained by open-column chromatography on flash silica gel, which was best achieved using solvent mixtures to which trifluoroacetic acid (TFA) had been added to minimize tailing of the 2656



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Figure 4. HSQC spectrum of the phenacyl ester 6a. Assignments are based on Damsté et al.6 The signals for methylene and methine protons have opposing signs; the methylene signals have been colored blue and the methines red.

Figure 5. HSQC spectrum of phenacyl ester 7a. Assignments are based on Damsté et al.6 The signals for methylene and methine protons have opposing signs; the methylene signals have been colored blue and the methines red. 2657



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interpret the VCD and ROA spectra, along with associated vibrational absorption (VA) and Raman spectra. Prior to successful isolation of 1a and 2a, we focused our efforts on 6a and 7a. We measured the discrete wavelengthresolved specific rotations for 6a and 7a and analyzed them with corresponding QC predictions on 6b−e and 7b−e. The limited availability of 6a and 7a did not permit VCD and ROA measurements. After the successful isolation of 1a and 2a, we measured the discrete wavelength-resolved specific rotations and VCD and ROA spectra for 1a and 2a and analyzed them with corresponding QC predictions. The results obtained for 6a and 7a are presented first, followed by those for 1a and 2a. Specific Rotations and ACs of Phenacyl Esters 6a and 7a. The observed specific rotations in chloroform at 589 nm, i.e., [α]D values, of 6a and 7a are −2 ± 1 and +7 ± 4, respectively (Figure 6), and these magnitudes are not much

columns; 6a and 7a elute after the phenacyl esters of other aliphatic fatty acids, and 6a elutes ahead of 7a. The esters were identified by HSQC (Figures 4 and 5) and mass spectra. Isolation and Purification of Ladderanoic Acids 1a and 2a. In an attempt to obtain further confirmation of the configurational assignments (vide infra), the phenacyl esters of 1a and 2a were converted to the free acids. Phenacyl esters are resistant to acid hydrolysis, but are readily saponified with dilute KOH. However, isolation of the free acids in quantities sufficient for measurement of specific rotations was laborious, requiring purification by preparative HPLC first of the phenacyl esters and then of the free acids. Attempted scaleup caused partial overlap of the HPLC peaks for the phenacyl esters, leading to the acids becoming cross-contaminated. Nevertheless, the partially purified acids were useful as standards for development of an HPLC method to isolate 1a and 2a directly from the biomass. On thin-layer chromatograms (TLCs), the acids tailed significantly using mixtures of ethyl acetate and hexanes for elution, but addition of 1% TFA to the solvent mixture gave symmetrical spots, although only minutely separated from each other. Adequate separation of the two acids occurred on HPLC when 0.1% TFA was added to the acetonitrile−water eluent, permitting isolation of the two acids in quantities and purities needed for further characterization. Two groups have now published robust synthetic methods for synthesis of the ladderanes,15,19 but investigators wanting to work with the compounds will frequently not have in-house expertise to carry out the published syntheses. Although the anammox biomass employed in the present study was a complex mixture of microorganisms in which anammox bacteria accounted for only 2.5% of the total community, it served as a convenient “low-tech” source of the ladderanoic acids. Moreover, it avoided the technical difficulties of enriching anammox bacteria in a laboratory setting. Large quantities of the biomass can be obtained from WWTPs. At this point we have only explored the isolation of the [5]- and [3]-ladderanoic acids, but it is likely that wastewater treatment biomass could also be used to produce useful quantities of conjugates of the ladderane alcohols. Chiroptical Spectroscopy. Four methods, namely, electronic circular dichroism (ECD),28 discrete wavelength resolved specific rotation [commonly referred to as optical rotatory dispersion (ORD)29], vibrational circular dichroism (VCD), 22,30,31 and vibrational Raman optical activity (ROA),22,31,32 fall under the umbrella of chiroptical spectroscopy.21,22 Each of these methods possesses characteristic, inherent limitations. ECD is not suitable for ladderanoic acids, as there are no visible chromophores attached to chiral centers that can report on the AC. Specific rotations can be used to establish the AC by comparing the signs of measured specific rotations with those obtained using QC predictions. The limitation in these comparisons is that the QC-predicted magnitudes may for various reasons differ from those observed experimentally, restricting the focus to signs of observed and predicted rotations. VCD and ROA spectroscopies are sensitive to configuration and conformational details, but these spectral measurements require higher concentrations, where dimer formation is possible. Since QC predictions for dimers of molecules as large as 1a and 2a cannot be undertaken, it was hoped that QC predictions for monomer structures would capture the essential details needed to

Figure 6. Discrete wavelength-resolved experimental specific rotations of 6a and 7a and their comparison with predicted specific rotations for 6b−e and 7b−e. Concentrations used are 6 mg/mL for 6a and 1 mg/mL for 7a in CHCl3. Population-weighted specific rotations were derived from 4, 9, 15, and 18 lowest energy conformers respectively for 6b−e and 15, 17, 33, and 53 conformers respectively for 7b−e, at the B3LYP/6-311++G(2d,2p) level using the polarizable continuum model (PCM)34,35 representing chloroform solvent. Individual numerical values are given in the SI. Calculations were undertaken for mirror images of structures depicted in Figure 2, and the specific rotations so obtained were multiplied by −1 for comparison with the experimental data.

greater than the experimental errors. Specific rotations are commonly measured and reported only at 589 nm, although, based on Drude’s equation33 for specific rotation, larger specific rotations can be expected at shorter wavelengths. Shorter wavelengths offer advantages for substances that have inherently small rotations or for which the supply is limited. For this reason, specific rotations were also measured at shorter wavelengths: 546, 436, 405, and 365 as well as at 633 nm. The magnitudes of specific rotations of both phenacyl esters were greater at shorter wavelengths; for example, [α] values at 365 nm of 6a and 7a are respectively −7 ± 1 and +35 2658



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± 6 (Figure 6). The experimental specific rotations for 6a are negative, while those for 7a are positive at all six wavelengths measured. Although a quantum mechanical equation for calculation of specific rotation had been proposed by Rosenfeld36 in 1929, it could not be put to practical use as a predictive tool until recently. Remarkable advances in quantum chemical methods37−44 have rendered these predictions reliable enough for a confident assignment of absolute configurations in numerous cases. Beratan, Wipf, and co-workers using the BP-86/aug-ccpVDZ level of theory (with the RI-J approximation) calculated gauge-independent [α]D values for a series of model [5]ladderanes bearing linear alkyl chains ranging from methyl to pentyl on a terminal methylene group.41 The calculations gave positive specific rotations with [α]D values ranging from 35 to 51 when the site of attachment had an S configuration. They acknowledged that this procedure might be expected to give inflated values of specific rotations but judged that the method was sufficiently accurate to be able to predict the correct sign of the specific rotation. They did not carry out calculations on 1a itself but, on the basis of the results obtained for the alkyl series, predicted that (S)-1a would be dextrorotatory. However, this prediction needs to be verified. The relative contributions of the multitude of conformers of the long alkyl chain will need to be taken into consideration since they will influence the sign and magnitude of specific rotations.42−44 The presence of functionality at the terminus of the chain is an additional complication to be reckoned with. Conformational analysis and specific rotation calculations are not practical for 6a and 7a, due to the large size of these molecules. Our strategy then involved calculating specific rotations, at the six wavelengths enumerated above, for a homologous series of phenacyl esters 6 in which the number of methylene groups separating the carboxyl group from the ladderane moiety was increased in a stepwise fashion. Conflex45 was used to identify a set of initial conformations, giving 60, 91, 304, and 860 respectively for 6b−e. These were progressively reduced to 4, 9, 15, and 18 based on relative energies obtained at the B3LYP/6-311++G(2d,2p) level using the Gaussian09 program.46 For all these calculations, mirror images of structures depicted in Figure 2 were used. The specific rotations so obtained were multiplied by −1 and compared to the experimental specific rotations in Figure 6. The calculations could not be carried beyond n = 3 because the number of conformers grew unwieldy. Nevertheless, even at n = 3 the trend for 6 is clear that, as Zuber et al. had recognized,41 the sign of the specific rotation is determined by the absolute configuration of the carbon atom to which the chain is attached; increasing chain length from n = 1 to 3 had no influence on the sign of the specific rotation and had diminished effect on its magnitude. The results of these calculations (Figure 6) show that the observed negative specific rotation for the phenacyl ester 6a is indicative of an R configuration. The computational strategy employed for the homologous series of [5]-ladderanoate esters 6 was repeated for [3]ladderanoate esters 7, without imposing any constraints on the cyclohexane ring conformation. Again, the series was restricted to n = 0−3 due to the increasing number of starting conformations. The numbers of initially generated conformations, 145, 338, 1694, and 6015 respectively for 7b−e, using the Conflex program,45 were progressively reduced to 15, 17, 33, and 53 based on relative energies at the B3LYP/6-311+

+G(2d,2p) level using the Gaussian 09 program.46 Among the lowest energy conformers of 7e, those with the cyclohexane ring in chair conformation contributed 61%, and those in boat conformation contributed 39% to the total population. From the specific rotations obtained for 7b−e, the trend is clear that R substituents in 7 lead to positive rotations. On the basis of this finding it can be inferred (see Figure 6) that the configuration of phenacyl ester 7a derived from natural material is R. It should be noted that for the [5] series of phenacyl esters the calculated magnitudes of rotations are larger than those observed for 6a by an order of magnitude. For the [3] series, the differences between calculated and experimentally determined values are less but still large. As pointed out above, Zuber et al. had observed that computation overestimated the magnitude of specific rotations but accurately predicted the signs. Unrecognized errors in the experimental measurements could also contribute to the differences in magnitude of computed and experimental values. Phenacyl esters 6a and 7a were purified by separation on a semipreparative HPLC column where the ratio of retention times was only 1.048. Cross-contamination would lead to diminished rotations because the esters have optical rotations of opposite sign. However, sufficient cross-contamination to produce the observed values would have been readily observed in the HSQC spectra (Figures 4 and 5). Contamination by other substance(s), either chiral or achiral, can likewise be ruled out by the absence of spurious NMR signals of sufficient intensity to account for the discrepancy. Specific rotations calculated by Zuber et al.41 for the alkyl[5]-ladderanes and by us for 6e are also significantly larger than experimental values reported by Mascitti and Corey19 and by Mercer et al.,15 on synthetic samples of 1a and its methyl ester. Likewise, the magnitude of specific rotation calculated for 7e is significantly larger than that reported by Mercer et al.15 for glyceryl ether 5. As alluded to earlier, there are several possible sources for the disagreement between calculated and experimental magnitudes: (a) Computational limitations make it impossible to extend the calculations to full-length side chains, although this is unlikely to resolve the issue completely (see predictions for full-length acids 1a and 2a below). (b) The solvent influence in the experimental measurements was represented by the polarizable continuum model (PCM),34,35 and explicit inclusion of solvent molecules in the calculations is not possible for molecules of the size considered here. (c) Vibrational contributions to calculated specific rotations are known to be important,47−49 and these contributions can only be incorporated for small molecules. (d) Aggregation at the concentrations employed might occur via stacking of concatenated cyclobutanes, but this cannot be realistically modeled in our calculations. Precedents for aggregation altering the experimental chiroptical properties can be found in studies of chiral surfactants.50 Despite these limitations, the larger predicted magnitudes of specific rotations are comforting in the sense that they are less prone to have reversed sign when calculations are repeated at a different level of theory. Absolute Configurations of Acids 1a and 2a. Conformational analysis and chiroptical spectroscopic studies were undertaken for these two ladderanoic acids with R configuration. Calculations were undertaken for the full-size molecules (without any truncation, unlike for phenacyl esters 6 and 7). For simplicity, these calculations assume a monomer structure for the acids, although carboxylic acids are known to 2659



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of 2 of the corresponding experimental specific rotation magnitudes (see Figure 7). The experimental specific rotations of 2a are positive at all wavelengths measured, and [α]D is +10 ± 1. No synthesis of 2a has been reported, but the sign of the specific rotation is the same as that of the phenacyl ester reported above and that of the glyceryl ether reported by Mercer et al.15 QC-predicted specific rotations for the R configuration calculated at all six wavelengths are positive, indicating that naturally occurring 2a also has an R configuration. The QC-predicted specific rotation magnitudes are 4- to 5-fold larger compared to the corresponding experimental magnitudes. Among the lowest energy conformers, those with the cyclohexane ring in the chair conformation contributed 66% of the total population, with the boat conformation contributing the remaining 34%. Vibrational Raman Optical Activity Spectra. Experimental vibrational Raman and ROA spectra were measured for 1a and 2a in chloroform solvent. The vibrational Raman scattering from the solvent interfered in the following regions: ∼1275−1175 cm−1, ∼800−600 cm−1, and 1000 cm−1) and about the same in the lower wavenumber region (33 mg/ mL. The concentration used for 2a was 96 mg/mL. Experimental conditions used were as follows: 500 mW laser power and 20 h data collection for 1a; 500 mW laser power and 14 h data collection for 2a. For the spectra presented, solvent spectra were subtracted and baseline corrected. Experimental VA and VCD Spectra. The experimental VA and VCD spectra were measured for CDCl3 solutions using a ChiralIR spectrometer (BioTools). Concentrations used: 24 mg/mL for 1a and 120 mg/mL for 2a. Measurements were made in fixed path length BaF2 cells of 500, 200, and 100 μm pathlengths, with 3 h as well as 1 h data collection times. For the spectra presented, solvent spectra were subtracted. Crystallographic Structure Determinations of Phenacyl Ester 6a and Acids 1a and 2a. Crystals of phenacyl ester 6a were formed by slow cooling of a solution in MeOH. A crystal was selected having approximate dimensions of 0.194 × 0.061 × 0.009 mm and mounted in a nylon loop in immersion oil. All measurements were made on a Rigaku Oxford Diffraction Supernova diffractometer with filtered Cu Kα radiation at a temperature of 100 K. Using Olex2,61 the structure was solved with the ShelXS structure solution program using direct methods and refined with the ShelXL refinement package using least squares minimization. The absolute stereochemistry was determined on the basis of the absolute structure parameter and by analysis using likelihood methods.54−56 CCDC deposition number: 1552002. A crystal of 1a was selected having approximate dimensions of 0.259 × 0.068 × 0.012 mm. The structure and AC were determined as for 6a. CCDC deposition number: 1552003. Crystals of acid 2a were formed by evaporation of a CH2Cl2− MeOH mixture to dryness using a 60 °C oil bath. A small bundle of

Table 1. Conformers Optimized for (R)-5-Ladderanoic Acid theoretical level

starting number of conformers

PM6

7698

B3LYP/6-31G*

5673

B3LYP/6-311+ +G(2d,2p)/ PCM

1383

number of unique conformers 5679 within 4378 kcal/mol 4489 within 11.78 kcal/mol 1336 within 6.11 kcal/mol

number of conformers carried on to the next level 5673 within 10.58 kcal/mol 1383 within 5 kcal/mol 135 within 2 kcal/mol

Table 2. Conformers Optimized for (R)-3-Ladderanoic Acid starting number of conformers

number of unique conformers

PM6

24 004

B3LYP/6-31G*

10 303

B3LYP/6-311+ +G(2d,2p)/ PCM

307

16 427 within 11.92 kcal/mol 6692 within 10.22 kcal/mol 305

theoretical level

number of conformers carried on to the next level 10 303 within 5 kcal/mol 307 within 5 kcal/mol 185 within 2 kcal/mol

Out of the 135 conformers optimized at the B3LYP/6-311+ +G(2d,2p)/PCM level for 1a, two conformers containing imaginary vibrational frequencies were discarded and the remaining 133 conformers of 1a were used for specific rotation, ROA, and VCD calculations, which were carried out with appropriate Boltzmann population weighting. Out of the final 185 conformers optimized at the B3LYP/6-311+ +G(2d,2p) level for 2a, five conformers containing imaginary vibrational frequencies were discarded and the remaining 180 conformers of 2a were used for specific rotation, ROA, and VCD calculations, with appropriate Boltzmann population weighting. 2664



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Specific rotations, VCD, and ROA were predicted using the Gaussian 09 program.46 Specific rotations for 1a and 2a with R configuration were calculated at six wavelengths, namely, 365, 405, 436, 546, 589, and 633 nm. Vibrational ROA spectra were predicted using a 532 nm excitation wavelength. Specific rotation calculations for phenacyl esters 6b−e and 7b−e used the S configuration. We had insufficient amounts of 6a and 7a to carry out ROA and VCD spectral measurements, and accordingly the corresponding ROA and VCD calculations were not undertaken. Crystallographic data reported in this paper have been deposited with the Cambridge Crystallographic Data Centre. Copies can be obtained free of charge at https://www.ccdc.cam.ac.uk/ by referencing deposition numbers 1552002, 1552003, and 1555318.

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(3) Rattray, J. E.; van de Vossenberg, J.; Hopmans, E. C.; Kartal, B.; van Niftrik, L.; Rijpstra, W. I. C.; Strous, M.; Jetten, M. S. M.; Schouten, S.; Damsté, J. S. S. Arch. Microbiol. 2008, 190, 51−66. (4) Jetten, M. S. M.; van Niftrik, L.; Strous, M.; Kartal, B.; Keltjens, J. T.; Op den Camp, H. J. M. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 65−84. (5) Kartal, B.; Keltjens, J. T. Trends Biochem. Sci. 2016, 41, 998− 1011. (6) Damsté, J. S. S.; Strous, M.; Rijpstra, W. I. C.; Hopmans, E. C.; Geenevasen, J. A. J.; van Duin, A. C. T.; van Niftrik, L. A.; Jetten, M. S. M. Nature 2002, 419, 708−712. (7) Niftrik, L. A.; Fuerst, J. A.; Damsté, J. S. S.; Kuenen, J. G.; Jetten, M. S. M.; Strous, M. FEMS Microbiol. Lett. 2004, 233, 7−13. (8) Dietl, A.; Ferousi, C.; Maalcke, W. J.; Menzel, A.; de Vries, S.; Keltjens, J. T.; Jetten, M. S. M.; Kartal, B.; Barends, T. R. M. Nature 2015, 527, 394−397. (9) Boumann, H. A.; Longo, M. L.; Stroeve, P.; Poolman, B.; Hopmans, E. C.; Stuart, M. C. A.; Damsté, J. S. S.; Schouten, S. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 1444−1451. (10) Boumann, H. A.; Stroeve, P.; Longo, M. L.; Poolman, B.; Kuiper, J. M.; Hopmans, E. C.; Jetten, M. S. M.; Damsté, J. S. S.; Schouten, S. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 1452− 1457. (11) Boumann, H. A.; Hopmans, E. C.; van de Leemput, I.; Op den Camp, H. J. M.; Van De Vossenberg, J.; Strous, M.; Jetten, M. S. M.; Damsté, J. S. S.; Schouten, S. FEMS Microbiol. Lett. 2006, 258, 297− 304. (12) Rattray, J. E.; van de Vossenberg, J.; Jaeschke, A.; Hopmans, E. C.; Wakeham, S. G.; Lavik, G.; Kuypers, M. M. M.; Strous, M.; Jetten, M. S. M.; Schouten, S.; Damsté, J. S. S. Appl. Environ. Microbiol. 2010, 76, 1596−1603. (13) Rush, D.; Jaeschke, A.; Hopmans, E. C.; Geenevasen, J. A. J.; Schouten, S.; Damsté, J. S. S. Geochim. Cosmochim. Acta 2011, 75, 1662−1671. (14) Rush, D.; Hopmans, E. C.; Wakeham, S. G.; Schouten, S.; Damsté, J. S. S. Biogeosciences 2012, 9, 2407−2418. (15) Mercer, J. A. M.; Cohen, C. M.; Shuken, S. R.; Wagner, A. M.; Smith, M. W.; Moss, F. R., III; Smith, M. D.; Vahala, R.; GonzalezMartinez, A.; Boxer, S. G.; Burns, N. Z. J. Am. Chem. Soc. 2016, 138, 15845−15848. (16) Nouri, D. H.; Tantillo, D. J. Curr. Org. Chem. 2006, 10, 2055− 2074. (17) Burns, N.; Shuken, S. R.; Mercer, J. A. M.; Cohen, C. M. Ladderane lipid compounds and liposomes and methods of preparing and using the same. WO 2018045094, 2018. (18) Damsté, J. S. S.; Rijpstra, W. I. C.; Geenevasen, G. J. A.; Strous, M.; Jetten, M. S. M. FEBS J. 2005, 272, 4270−4283. (19) Mascitti, V.; Corey, E. J. J. Am. Chem. Soc. 2006, 128, 3118− 3119. (20) In a personal communication by J. S. S. Damsté to E. J. Corey cited by Mascitti and Corey (ref 19). (21) Berova, N.; Polavarapu, P. L.; Nakanishi, K.; Woody, R. W. Comprehensive Chiroptical Spectroscopy; Wiley, 2012; Vols. 1−2. (22) Polavarapu, P. L. Chiroptical Spectroscopy: Fundamentals and Applications; CRC Press: Boca Raton, FL, 2016. (23) Neumann, S.; Wessels, H. J. C. T.; Rijpstra, W. I. C.; Damsté, J. S. S.; Kartal, B.; Jetten, M. S. M.; van Niftrik, L. Mol. Microbiol. 2014, 94, 794−802. (24) Hopmans, E. C.; Kienhuis, M. V. M.; Rattray, J. E.; Jaeschke, A.; Schouten, S.; Damsté, J. S. S. Rapid Commun. Mass Spectrom. 2006, 20, 2099−2103. (25) Rattray, J. E.; Geenevasen, J. A. J.; van Niftrik, L.; Rijpstra, W. I. C.; Hopmans, E. C.; Strous, M.; Schouten, S.; Jetten, M. S. M.; Damsté, J. S. S. FEMS Microbiol. Lett. 2009, 292, 115−122. (26) Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911−917. (27) Borch, R. F. Anal. Chem. 1975, 47, 2437−2439.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00458. HPLC chromatograms of 1a and 2a and of 6a and 7a; NMR spectra of 1a and 2a and their phenacyl and 4phenylphenacyl esters; HSQC spectra of 1a and 2a and their 4-phenylphenacyl esters; computed specific rotations of 6b−e, 7b-e, 1a, and 2a; experimental specific rotations for 1a, 2a, 6a, and 7a; X-ray crystallographic structures and absolute configurations of 1a, 2a, and 6a (PDF) X-ray crystallographic data of 1a (CIF) X-ray crystallographic data of 2a (CIF) X-ray crystallographic data of 6a (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1-615322-2836. ORCID

Nathan D. Schley: 0000-0002-1539-6031 Prasad L. Polavarapu: 0000-0001-6458-0508 Thomas M. Harris: 0000-0002-3062-344X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Dr. Charles B. Bott and his staff at the York River wastewater treatment facility in Seaford, Virginia, for their assistance and for the generous gift of biomass from the anammox process. This work was conducted in part using the resources of the Advanced Computing Center for Research and Education (ACCRE), the Center for Innovative Technology and the Small Molecule NMR Facility at Vanderbilt University. We acknowledge funding by the National Institutes of Health (S10 RR019022) for purchase of the 600 MHz NMR spectrometer and accessories. We thank Drs. H.-Y. Kim, J. May, and S. Condreanu for mass spectra and Prof. C. J. Rizzo for use of research facilities. Financial support by the National Science Foundation (CHE-1464874, OCE1658135, and EAR-132984), the Virginia Institute of Marine Science, and Vanderbilt University is gratefully acknowledged.

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REFERENCES

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