Enzymatic synthesis of a bicyclobutane fatty acid by a hemoprotein–lipoxygenase fusion protein from the cyanobacterium Anabaena PCC 7120
- Claus Schneider*,
- Katrin Niisuke*,
- William E. Boeglin*,
- Markus Voehler†,
- Donald F. Stec†,
- Ned A. Porter†, and
- Alan R. Brash*,‡
- Departments of *Pharmacology and
- †Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved October 5, 2007 (received for review July 30, 2007)
Abstract
Biological transformations of polyunsaturated fatty acids often lead to chemically unstable products, such as the prostaglandin endoperoxides and leukotriene A4 epoxide of mammalian biology and the allene epoxides of plants. Here, we report on the enzymatic production of a fatty acid containing a highly strained bicyclic four-carbon ring, a moiety known previously only as a model compound for mechanistic studies in chemistry. Starting from linolenic acid (C18.3ω3), a dual function protein from the cyanobacterium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.3ω3 in a lipoxygenase domain, then a catalase-related domain converts the 9R-hydroperoxide to two unstable allylic epoxides. We isolated and identified the major product as 9R,10R-epoxy-11trans-C18.1 containing a bicyclo[1.1.0]butyl ring on carbons 13–16, and the minor product as 9R,10R-epoxy-11trans,13trans,15cis-C18.ω3, an epoxide of the leukotriene A type. Synthesis of both epoxides can be understood by initial transformation of the hydroperoxide to an epoxy allylic carbocation. Rearrangement to an intermediate bicyclobutonium ion followed by deprotonation gives the bicyclobutane fatty acid. This enzymatic reaction has no parallel in aqueous or organic solvent, where ring-opened cyclopropanes, cyclobutanes, and homoallyl products are formed. Given the capability shown here for enzymatic formation of the highly strained and unstable bicyclobutane, our findings suggest that other transformations involving carbocation rearrangement, in both chemistry and biology, should be examined for the production of the high energy bicyclobutanes.
The ability of lipoxygenase (LOX) enzymes to oxygenate polyunsaturated fatty acids to specific fatty acid hydroperoxides is used throughout the eukaryotic world for the production of signaling molecules and other complex products (1–4). The initial hydroperoxy fatty acid product is often further transformed to a highly unstable biosynthetic intermediate. Thus, plants express specialized cytochrome P450 enzymes of the CYP74 family that convert hydroperoxy-C18 fatty acids to allene oxides, the best characterized of which is an intermediate in biosynthesis of the hormone jasmonic acid (5). In the leukocytes of higher animals, the 5-LOX enzyme forms the initial 5-hydroperoxy-C20.4 product and converts it into the highly unstable epoxide leukotriene A4 (LTA4), from which the other leukotriene family members arise (6). As yet another facet of this theme, marine corals express a natural fusion protein (7) in which a LOX domain converts arachidonic acid to its 8R-hydroperoxide and a catalase-related domain effects a further transformation to an unstable allene oxide, a potential intermediate in formation of marine prostanoids (8). This catalase-related domain of the coral fusion protein is structurally similar to true catalases (9) yet quite distinct in function. Based on the knowledge that the plant CYP74 enzyme family exhibits a spectrum of catalytic reactions, including formation of allene oxides, aldehydes, or vinyl ethers (10, 11), there is the possibility that the catalase-related allene oxide synthase (AOS) is prototypical of an enzyme family that also has diversified functions. Accordingly, the unusual catalytic activity of the catalase-related coral AOS provided the impetus for the present investigation, namely to explore other possible occurrences of catalase-related proteins with novel functions in the biotransformation of polyunsaturated fatty acids.
By using BLAST searches for sequences similar to the coral catalase-related domain, one of the top matching hits besides other coral homologues was identified in the cyanobacterium Anabaena sp. strain PCC 7120. The Anabaena genus of cyanobacteria are photosynthetic prokaryotes that grow in long strings or filaments and that can develop a nitrogen-fixing ability in specialized heterocysts. They are studied as a model for prokaryotic developmental biology (12). Anabaena PCC 7120 has a genome of 6.4 Mb, and the cells also contain several large plasmids. The novel gene resides on the 102 kb gamma plasmid. Enticingly, this small catalase-related sequence was found in the same ORF as a LOX-like sequence, albeit a highly unusual one, much smaller than any previously known member of the LOX superfamily. In a separate study, we show that this C-terminal domain of the fusion protein is a catalytically complete lipoxygenase that specifically forms 9R-hydroperoxides from C18 polyunsaturated fatty acid substrates (Y. Zheng, W.E.B., C.S., A.R.B., unpublished data). Here, we report characterization of the catalytic activities of the N terminus of the fusion protein, the heme-containing domain with sequence similarity to catalase. This unusual enzyme utilizes the 9R-hydroperoxylinolenic acid (C18.3ω3) product of the LOX domain as a substrate and converts it to two epoxy fatty acids, the major one of which contains a bicyclic four-carbon ring. Its synthesis has important implications for the possible existence of novel carbocation rearrangements in both chemistry and biology.
Results
Protein Sequences and Alignments.
The novel hemoprotein from Anabaena and the AOS domain from the coral Plexaura homomalla share an overall 35% amino acid identity. Particularly significant matches are conserved around the distal heme His residue and the distal heme Asn [supporting information (SI) Fig. 5]. A very significant mismatch occurs around the heme proximal ligand, which is a Tyr in the coral AOS, as is characteristic of all catalase family members, yet by alignment this residue is replaced by His in Anabaena. Remarkably, therefore, the Anabaena sequence appears to represent a His-ligated heme in the context of a catalase-related protein framework. We should note that, whereas Anabaena is a prokaryotic cyanobacterium and other cyanobacteria are found as symbionts in corals (13), the P. homomalla AOS–LOX is unambiguously eukaryotic based on the presence of multiple introns in the DNA (unpublished observations). Nonetheless, such coexistence could have provided the opportunity for an earlier gene transfer one way or the other.
Expression and Purification of the Anabaena Fusion Protein.
We expressed the whole Anabaena fusion protein as well as the isolated LOX domain in Escherichia coli and partially purified the proteins by nickel affinity chromatography by using N-terminal His6-tags; (expression of the catalase-related domain by itself gave protein containing no heme and exhibiting no catalytic activity). Anabaena contains abundant polyunsaturated fatty acids and is particularly rich in linolenic acid (C18.3ω3) (14–16). We found that this fatty acid is oxygenated by the LOX domain to the corresponding 9R-hydroperoxide (a contrast with plant 9-LOX enzymes, which have S stereospecificity) (5). The lipoxygenase is not involved in further metabolism, a point we established by using the separately expressed LOX domain. However, the catalase-related domain of the fusion protein avidly metabolizes the 9R-hydroperoxide to a complex spectrum of stable end products, including triols, diols, and epoxyalcohols (data not shown). It appeared likely that the primary enzymic product of the catalase-like hemoprotein is an unstable epoxide or epoxides, and we reasoned that if this could be analyzed directly it would greatly simplify the product profile and help clarify the fundamental mechanism of biosynthesis.
Isolation of Two Allylic Epoxides Formed by the Catalase-Related Domain.
We explored conditions under which the Anabaena enzyme reacted with pure 9R-hydroperoxylinolenic acid in a biphasic hexane/pH 8 aqueous system that would simultaneously extract the hydroperoxy substrate from the hexane into the water, allow enzymic metabolism, and then instantly extract the less polar epoxide product(s) back into the hexane, thus affording protection from hydrolysis, an approach similar to the one we developed for isolation of allene oxides (17). After 2 min of vortex mixing at 0°C, UV spectroscopy of the hexane showed disappearance of substrate and appearance of new chromophores, one near 200 nm and the other with λmax at 278 nm characteristic of the leukotriene A class of allylic epoxides (Fig. 1 A) (18). The resulting hexane extract was treated with diazomethane for 10 s at 0°C to form the methyl ester derivative (19) and the fatty acid derivatives subsequently analyzed by reversed-phase HPLC using conditions adapted from a method for analysis of synthetic leukotriene A4 (Fig. 1 B) (20). HPLC analysis showed near quantitative conversion to two products, present in a 2:1 ratio as determined by using a 14C substrate. The more prominent product 1 displays a UV spectrum with end absorbance extending beyond 230 nm, and product 2 has the spectrum of a conjugated triene, λmax 278 nm (Fig. 1 C). LC-MS analysis using positive ion electrospray ionization revealed that the two products have the same molecular weight (306 for the methyl ester) as indicated by their identical adduct ions with sodium, potassium, and triethylamine.
Preparation, purification, and UV analysis of unstable epoxides. An ice-cold solution of 9R-hydroperoxylinolenic acid (90 μM) in 10 ml of ice-cold hexane was vortex-mixed for 2 min with Anabaena catalase–LOX enzyme (0.8 nmol) in 0.2 ml of phosphate buffer, pH 8; reaction with the same sample of enzyme was repeated twice more using fresh substrate in ice-cold hexane. The combined hexane phases were evaporated to ≈2 ml under a strong stream of nitrogen, treated with diazomethane and 1% ethanol for 10 s at 0°C, and then evaporated to dryness and stored in hexane at −70°C. (A) UV spectrum of the hydroperoxy substrate in hexane before reaction and the hexane phase after mixing with enzyme. (B) Reversed-phase HPLC analysis of the product methyl esters with UV detection at 205 and 270 nm. A Waters Symmetry C18 column (25 × 0.46 cm) was eluted with methanol/20 mM aqueous triethylamine at pH 8 [80:20 (vol/vol)] at a flow rate of 1 ml/min. (C) Normalized UV spectra of the two main products.
Identification of the Major Allylic Epoxide Product.
The 1H-NMR spectrum of product 1 methyl ester, together with the COSY analysis of cross-couplings, outlined a structure of a 9,10trans-epoxy-11trans-C18.1 derivative (Fig. 2 and SI Dataset 1). With only one double bond and yet the same molecular weight as the leukotriene A-type epoxide, it follows that the structure of 1 must contain two rings. The arrangement of the carbon atoms was further investigated through conventional proton–proton decoupling experiments, heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC) to identify the carbon–proton couplings, the distortionless enhancement polarization transfer (DEPT) experiment to confirm the CH2, CH, and C carbons, and NOESY for through-space couplings (SI Figs. 6–9). The key structural data for establishing the bicyclobutane ring was the fact that all four carbons of C13 through C16 appeared as CH signals in the HSQC and DEPT experiments, suggesting that, given their chemical shift values, each of these carbons is bound to three other carbons and one proton. The C14 and C15 carbons, which exhibit almost identical chemical shifts at 9.3 ppm, are clearly resolved in the carbon spectrum at 800 MHz (SI Fig. 8). The COSY analysis showed strong correlation among the proton signals of H14, H15, and H16 of the ring and extending to H17 and H18 of the ω-carbon chain. H13 (a doublet at 2.36 ppm) couples only to H12 on the double bond; its couplings with the neighboring protons H14 and H15 thus must be very small, indicating that H14 and H15 are held at almost right angles in the bicyclic ring system. The through space cross-peaks in the NOESY spectrum confirm the covalent structure and confirmed assignment of the configuration of the side chains (C1–C12, C17, and C18) as exo–endo (Fig. 3). Thus, the complete covalent structure of product 1 was established as 9R,10Rtrans-epoxyoctadeca-11trans-(13,14,15,16-bicyclo[1.1.0]butyl)-enoic acid.
NMR analysis of product 1. Spectra were recorded in d6-benzene at 283 K using a Bruker 600 MHz spectrometer equipped with a cryoprobe. The two-dimensional H,H COSY spectrum is shown below, with an expanded view of two areas of the spectrum depicted above. On the COSY spectrum, the correlations are clear from H8 through the trans epoxide (H9, H10, J = 2.0 Hz) to the trans double bond (H11, H12, J = 15.5 Hz) to H13 of the bicyclobutane ring, a doublet at 2.36 ppm. H14 and H15 are in a very similar chemical environment and, therefore, have similar chemical shifts in the proton (1.29–1.36 ppm) and carbon (9.2 and 9.3 ppm) spectra (SI Fig. 8). Both H14 and H15 are coupled to H16, which extends the correlation into the ethyl substituent (H17 and H18). Although the correlations through H13 are not evident here, H12 shows a three-bond correlation to both C14 and C15 in the HMBC spectrum (SI Fig. 7).
Configuration of the bicyclobutane ring of product 1. The NOESY NMR spectrum of product 1 was recorded in d6-benzene at 600 MHz. The partial chemical structure illustrates the through-space couplings of the single protons at H13 and H16, the two ends of the bicyclobutyl ring. The 13-exo,16-endo configuration of the ring can be deduced from the observation that the coupling of H13 to H14/15 is weak, whereas H16-H14/15 is strong, and that the coupling of H13 to H17 is strong, whereas there is no detectable NOE between H12 and H16 (SI Fig. 9).
Identification of Product 2 as a LTA4 Analogue.
The 1H-NMR and H,H-COSY spectra of product 2 established the structure of an allylic epoxide with a conjugated triene double bond system (SI Dataset 2). Based on the coupling constants, the configuration of the 11,12, 13,14, and 15,16 double bonds is defined as trans,trans,cis (J = 15.2, 14.8, and 11.0 Hz, respectively). Considering also that the configuration of C9 should not change during the transformation of the 9Rhydroperoxide to the 9,10-epoxide, product 2 was identified as a leukotriene A-type epoxide, 9R,10R-trans-epoxyoctadeca-11E,13E,15Z-trienoic acid. This structure is directly analogous to the structure of the mammalian 5-LOX product, LTA4, a 5S,6S-trans-epoxy-20.4ω6 fatty acid with a trans,trans,cis (7E,9E,11Z) conjugated triene. Interestingly, biosynthetically produced LTA4 has not been subject to a complete and direct structural analysis. Knowledge of its structure rests on comparison of its biotransformation and hydrolytic reactions with synthetic LTA4, and on an understanding of its mechanism of biosynthesis by the leukocyte 5-LOX enzyme (21, 22).
Discussion
Bicyclobutane is the simplest bicyclic hydrocarbon (Scheme 1). It has a bond strain energy of 66 kcal/mol, more than double the value of either cyclopropane or cyclobutane (≈26 kcal/mol) (23). Extensive efforts have been made during the past 40 years to develop synthetic approaches to molecules containing a bicyclobutane subunit and to understand the chemistry of this structure (24–27). Although bicyclobutanes have been the focus of many fundamental studies in organic chemistry, this structure is, to our knowledge, heretofore unknown in nature. We report here on the isolation and identification of a substituted bicyclobutane formed in biosynthesis from a fatty acid hydroperoxide precursor. The proposed biosynthetic mechanism proceeds through the rearrangements of carbocations and is completed with an enzymatic step of deprotonation of a bicyclobutonium ion yielding a bicyclobutane ring via a route not observed in solution chemistry.
Bicyclo [1.1.D] butane.
Transformation of the linolenate hydroperoxide to the allylic epoxides 1 and 2 can be understood by a mechanism involving intermediate carbocations (Fig. 4). The conversion of conjugated diene hydroperoxides to allyl carbocations like 3 has ample precedent, being implicated in the enzymatic synthesis of allene oxides, vinyl ethers, and other fatty acid derivatives (28), as well as related transformations of prostaglandin endoperoxides to prostacyclin and thromboxane A2 (29). Removal of a proton from the carbocation 3 gives the leukotriene-type epoxide, product 2. Although the structure of 2 is directly analogous to that of the leukocyte 5-LOX product LTA4, its mechanism of biosynthesis is quite different. Whereas epoxide 2 is formed via initial activation of the hydroperoxide and subsequent rearrangements of an epoxy allylic carbocation with a final elimination of H+ (Fig. 4), the biosynthesis of LTA4 involves an initial LOX-catalyzed hydrogen abstraction from the carbon chain, followed by radical rearrangements that lead to cleavage of the hydroperoxide and epoxide formation at the ultimate step.
Transformation of 9R-hydroperoxy-C18.3ω3 by the Anabaena catalase-related enzyme.
Carbocation 3 is homoallylic and, as such, provides access to the cyclopropylcarbinyl and bicyclobutonium ions (30), two of which are shown in Fig. 4. The nature of these carbocation intermediates has been an important chapter in the “classical–nonclassical” ion debate (31, 32). It is generally agreed that the most stable carbocation intermediates present are separated by low-energy barriers (33) and that cyclopropanes, cyclobutanes, and homoallylic structures are the predictable set of products that form from these intermediates and the equivalent alcohol derivatives in aqueous media (34). Such reactions occur in enzymatic transformations to a wide array of natural products (e.g., refs. 28 and 35–38). In the case of the Anabaena catalase-related protein, we suggest that generation of bicyclobutonium ion 5 in the vicinity of an enzymatic base provides an additional product-forming route, deprotonation to give the bicyclobutane, product 1. This is a most unusual outcome that is not reproduced in solution chemistry. The enzyme must provide an environment in which proton abstraction from the bicyclobutonium ion facilitates production of the high-energy bicyclobutyl moiety, a species we then recovered by its immediate extraction into hexane. Notably, a potential epoxide product arising via proton abstraction from the cyclopropylcarbinyl ion 4 was not detected, and we isolated a single bicyclobutyl isomer, arguing for stringent reaction control in the enzyme active site.
Historically, the catalase gene family is known for its role in protection from oxidative stress via the breakdown of hydrogen peroxide, yet clearly the Anabaena gene has a biosynthetic function. A role in biosynthesis is precedented by the catalase-related domain of the coral AOS–LOX fusion protein that converts 8R-hydroperoxy-arachidonic acid to an allene oxide, a reaction equivalent to that of the P450 AOS of plants (10). With the recent successful x-ray structural analysis of the coral catalase-related domain, its modest sequence similarity to true catalases evolved into remarkable parallels in three-dimensional structure (9). The heme-binding pocket and surrounding protein network of true catalases are well conserved, whereas the structural features involved in melding together the catalase homotetramer are absent. The coral AOS domain is 43 kDa in size and crystallizes as a dimer (9), whereas true catalases usually are tetrameric or hexameric with subunits of 55–69 kDa (“small”) or 75–84 kDa (“large”) (39). The Anabaena hemoprotein domain is ≈41 kDa in size, with strong homology to the coral AOS, except near the end of the sequence where the proximal heme ligand appears to be substituted with a histidine (SI Fig. 5). The 9R-hydroxy metabolites of linolenic and linoleic acids have been detected in ≈5:1 ratio in extracts of a species of Anabaena (40), but these organisms have yet to be examined for the more complex products that might be expected to arise from the epoxy-bicyclobutyl linolenate. The Anabaena hemoprotein–lipoxygenase fusion protein resides on the γ-plasmid in Anabaena PCC 7120, and, by analogy with the plasmid-encoded antibiotic resistance genes, it may have a specialized role and confer an advantage in a selected environment. Other small catalase analogues reside in the genomes of many microorganisms and have the potential for additional functions in the metabolism of natural peroxides.
Given the capability shown here for enzymatic transformation to the highly strained and unstable bicyclobutane, other enzymic transformations involving carbocation rearrangements should be examined for the production of the high-energy bicyclobutanes. Immediate extraction of unstable reaction products may also be applicable to purely chemical transformations and may allow for the isolation of products believed not to be existent or considered too unstable for characterization. We show that, with appropriate methodology, even previously intractable biological products, such as the leukotriene A-type epoxide, and novel ones, like the bicyclobutane, are amenable to recovery and structural analysis.
Materials and Methods
Materials.
Fatty acids was purchased from NuChek Prep. [1-14C]Linolenic acid was purchased from NEN Life Science Products.
Cloning, Expression, and Purification of Anabaena Enzyme Constructs.
The cDNA for the full-length fusion protein was cloned by PCR from Anabaena sp. strain PCC 7120 genomic DNA, a kind gift from James W. Golden (Texas A&M University, College Station, TX). Sequencing confirmed the identity to the published sequence in the National Center for Biotechnology Information database (NP_478445) and at CyanoBase (http://bacteria.kazusa.or.jp/cyanobase). Several different cDNA expression constructs were prepared: each listed below was cloned into pET17b for expression in E. coli. Full-length constructs included the native cDNA sequence (amino acid code MDLNTY—LMMSINI.) and the same sequence with a His6 tag on the N terminus (MHHHHHHDLNTY—LMMSINI.). These constructs expressed with similar heme content (with the main Soret band observed at 406 nm) and were used for enzyme quantification assuming ε = 100,000. The affinity-purified His-tagged expression construct was used throughout these experiments. The proteins were expressed in E. coli BL21 (DE3) cells (Novagen) using methodology we described previously (41), and the His6-tagged protein was purified on Ni-NTA agarose (Qiagen) according to the manufacturer's instructions. The lipoxygenase-only domain (starting at amino acid 344 with the amino acid sequence KDDLPGK… and comprising the last 430 aa of the full-length construct) was expressed with an N-terminal His6 tag and purified by nickel affinity chromatography. We also attempted to express several constructs encoding the N-terminal domain only (amino acids 1–344). These constructs had the His tag placed at either the N or C terminus (or no His tag) and included constructs with the C terminus extended a further 20 aa along the fusion protein. However, these constructs expressed with no heme and exhibited no catalytic activity.
Preparation of Hydroperoxides.
9R-Hydroperoxy-C18.3ω3 was prepared by using the LOX domain of the Anabaena enzyme in pH 7.5 Tris buffer according to the methods described previously for other fatty acid hydroperoxides (42). The product was extracted into dichloromethane and purified by SP-HPLC (Beckman 5μ silica column; 25 × 0.46 cm) using a solvent system of hexane/isopropanol/acetic acid 100:1:0.1 by volume (flow rate of 1 ml/min), with detection of the product by UV detection at 235 nm (42). The product was quantified by UV spectroscopy (ε = 23,000 at 235 nm) and stored at −20°C in ethanol.
Activity Assays.
Small-scale incubations with the purified enzymes were typically conducted in a 0.5-ml UV cuvette and analyzed by UV spectrometry in an incubation buffer (50 mM Tris, 150 mM NaCl, pH 7.5). Enzyme activity was monitored by repetitive scanning in the range 350–200 nm or by monitoring disappearance of the signal at 235 nm in the time-drive mode. To measure the rate of reaction at a higher concentration of substrate (100–250 μM), reactions were conducted in a 2-mm path-length microcuvette.
Extraction and HPLC Analysis of Unstable Products.
Enzyme reactions were conducted at 0°C, with the substrate initially in hexane (10 ml, ≈100 μM 9-hydroperoxide) layered over the Anabaena enzyme (0.8 nmol) in 200 μl of phosphate buffer, pH 8. The reaction was initiated by vigorous vortex mixing of the two phases, which was continued for 2 min; then the test tube was placed back on ice. The hexane phase was scanned from 200–350 nm in UV light by using a Perkin-Elmer Lambda-35 spectrophotometer, and, if all substrate was consumed (as in Fig. 1 A), the reaction was repeated with fresh substrate in hexane mixed with the same batch of enzyme. The combined hexane phases were evaporated to ≈2 ml by using a vigorous stream of nitrogen. The sample was then treated with ethanol (20 μl) and ethereal diazomethane for 10 s at 0°C and then rapidly evaporated to dryness and stored in hexane at −20°C until further analysis. The same procedures but omitting the methylation step gave samples of the free acids.
The presence of an excess of alcohol over water in a slightly basic solution greatly prolongs the half-life of allylic epoxides, such as leukotriene A4, allowing their analysis by reversed phase HPLC at room temperature (20). The hexane extracts were analyzed and purified by using a Waters Symmetry C18 5-μm column (0.46 × 25 cm) eluted at a flow rate of 1 ml/min with methanol/20 mM potassium phosphate, pH 8 (replaced with triethylamine for LC-MS analysis; see below), in the proportions 80:20 (vol/vol), with UV light detection at 205, 220, 235, and 270 nm using an Agilent 1100 series diode array detector. The main products were recovered by extraction with cold hexane followed by evaporation to dryness under a strong stream of nitrogen.
LC-MS and NMR analysis.
LC-MS of the allylic epoxides was performed using a Thermo Finnigan LC Quantum instrument. A Waters Symmetry C18 column (0.2 × 15 cm) was eluted with methanol/20 mM aqueous triethylamine adjusted to pH 8 with acetic acid [80:20 (vol/vol)] at 0.2 ml/min. The heated capillary ion lens was operated at 220°C. Nitrogen was used as a nebulization and desolvation gas. The electrospray potential was held at 4 kV. Source-induced dissociation was set at −10 eV. Mass spectra were acquired over the mass range m/z 100–500 at 2 s per scan. Collision-induced dissociation was performed at −15 eV.
NMR spectra were recorded on a Bruker 800-MHz (13C and DEPT) or 600-MHz instrument at 283 K. The samples were dissolved in d6-benzene, and the chemical shifts are reported relative to the benzene signal (δ 7.16 ppm for hydrogen and 128.0 ppm for carbon). Both instruments were equipped with a Bruker TCI cryoprobe.
Acknowledgments
We thank Dr. Thomas M. Harris for careful review of the NMR data. This work was supported by National Institutes of Health Grant GM-74888.
Footnotes
- ‡To whom correspondence should be addressed at: Department of Pharmacology, Vanderbilt University School of Medicine, 23rd Avenue South at Pierce, Nashville, TN 37232-6602. E-mail: alan.brash{at}vanderbilt.edu
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Author contributions: A.R.B. designed research; C.S., K.N., W.E.B., M.V., and D.F.S. performed research; C.S., K.N., W.E.B., M.V., D.F.S., N.A.P., and A.R.B. analyzed data; and C.S., N.A.P., and A.R.B. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0707148104/DC1.
- © 2007 by The National Academy of Sciences of the USA
