Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein

Abstract

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Dengue virus (DENV; genus Flavivirus, family Flaviviridae) is responsible for the major arthropod-borne viral human disease of the tropics (1). It is estimated that 50–100 million dengue cases occur annually, ranging from mild fever to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (2). The number of severe forms can exceed half a million per year and lead to tens of thousands deaths (1). DHF is associated with thrombocytopenia, coagulopathy, acute inflammation, frequent hepatomegaly, and, most importantly, plasma leakage to which the risk of fatal hypovolemic shock (DSS) is associated (3–5). It has been proposed that an inadequate immune response is the major cause of severe clinical manifestations (6–8). Soluble mediators produced during the acute phase of the disease likely play a pivotal role in vascular permeability, as suggested by the rapid recovery of most DHF patients (9–11).

There is accumulating evidence that flavivirus NS1, a 50-kDa nonstructural glycoprotein, contributes to different stages of the virus life cycle. Part of NS1 resides in virally induced intracellular organelles, where it plays an essential role in viral replication (12–16). The protein, possibly modified by GPI, also associates to lipid rafts at the plasma membrane and mediates a signaling pattern common to GPI-anchored proteins in the presence of specific antibodies (17–19). NS1 is eventually secreted by DENV-infected mammalian cells (20–22) and released in the blood stream of infected individuals (23, 24). The protein is detectable in plasma from the onset of fever up to the first days of convalescence at concentrations that can exceed several micrograms per milliliter (23–25). The amount of NS1 circulating in human sera appears to be significantly higher in patients who developed DHF rather than dengue fever (26), although it is not clear whether this effect is a cause or a consequence of plasma leakage. In vitro, the protein binds cell-surface glycosaminoglycans (27) and is targeted to late endosomes upon entry into target cells (28). Preincubation of hepatocytes with soluble NS1 enhances subsequent infection by a homologous strain of DENV (28). In addition, both soluble and cell-surface–associated NS1 are capable of modulating complement activation pathways through the formation of immune complexes or binding to host proteins, such as the regulatory protein factor H, complement factor C4, or clusterin (29–32).

In this article, we investigated the structure/function relationship of DENV NS1. We determined a low-resolution 3D reconstruction of the hexamer, which appears as an open barrel with a wide central channel. We identified that specific lipids—in particular triglycerides, cholesteryl esters, and phospholipids—associate to the NS1 particle and that the interaction with lipids is required for efficient assembly and egress of the NS1 hexamer (as summarized in Fig. S1). The nature of the NS1 lipid moiety points to striking similarities between DENV NS1 and high-density lipoproteins (HDLs) involved in vascular homeostasis.

Results

3D Organization of the DENV NS1 Hexamer.

To get insights into the NS1 structure/function relationship, we sought to characterize the 3D organization of the secreted hexamer. We analyzed an authentic NS1 protein purified from the extracellular medium of Vero cells infected with DENV serotype 1 (DENV-1). Numerous crystallization trials failed to yield diffraction-quality crystals. Using cryoelectron microscopy (cryo-EM), we obtained a 3D reconstruction of the DENV-1 NS1 hexamer. The resolution of the reconstruction (at ∼3.0 nm) is sufficient for visualization of the protein as an open barrel with a 32 point-symmetry, ∼10 nm in diameter, and 9 nm in height, featuring a prominent central channel running along the molecular threefold axis (Fig. 1). Three twofold-symmetric twisted rods, corresponding to the dimeric subunits, form the walls lining the channel. The lateral interactions between the dimeric building blocks take place along a fairly thin area representing, at most, 5 nm². Each rod is made of two ellipsoidal lobes, which most likely correspond to the individual protomers (Fig. 1 and Fig. S2). The central channel has an estimated volume of 80 nm³ with triangular openings of ∼9 nm² at each end, rotated by 40° about the threefold axis (Fig. 1C).

Fig. 1.

Cryo-EM analysis of DENV-1–secreted NS1. (A) Representative field showing a preparation of NS1 purified from DENV-1–infected Vero cell supernatants. (B) Characteristic class averages of NS1 used to produce an initial 3D reconstruction. (C) 3D reconstruction of NS1, in an isosurface representation. Upper Left displays a view down the threefold molecular axis. Lower Left is a view down the twofold axis, relating two dimeric subunits (90° rotation as indicated). Lower Right shows the NS1 particle further rotated by 180° about the threefold axis, down the twofold axis, relating protomers within a dimeric subunit. The intradimer contacts are much more extensive than the interdimer ones. Note the large central channel of ∼80 nm³ running along the threefold axis of the molecule.

DENV NS1 Dimeric Subunits Behave as Membranous Proteins in a Triton X-114 (TX-114) Detergent Phase-Partitioning Assay.

The very narrow interfaces observed between the dimeric subunits, together with the previously reported instability of the NS1 hexamer in nonionic detergents (20, 33) and its resistance to high molarities of salt or to chelating agents (Fig. S3), indicated that the dimers are essentially held together by weak hydrophobic interactions. Further elements localizing within the channel—for instance, amphiphilic molecules such as lipids—are likely to be necessary to hold the dimeric rods together, as inferred by the dual behavior of the protein in a TX-114 detergent phase-partitioning assay (Fig. 2). Whereas soluble and membranous proteins segregate in the aqueous and detergent phases, respectively, the detergent-treated NS1 partitions into both phases, with a higher proportion of protein retained in the detergent fraction (Fig. 2A). The DENV E protein, which contains a transmembrane anchor, remains exclusively in the detergent phase, as expected (Fig. 2A). NS1 recovered from the detergent-rich phase is essentially dimeric, as observed by treatment of the corresponding fraction with the chemical cross-linker dimethylsuberimidate (DMS) and analysis of the resulting products by SDS/PAGE and Coomassie blue staining or MS (Fig. 2 B and C, respectively). In contrast, NS1 from the aqueous phase maintains its characteristic hexameric pattern with DMS treatment (Fig. 2 B and C). Thus, the NS1 protein displays amphipathic properties and behaves as a membranous protein upon dissociation of the soluble hexamer into dimers, indicating that dimeric precursors likely interact with lipid membranes before hexamer assembly, possibly dragging lipids out of the membrane during the oligomeric transition.

Fig. 2.

The NS1 hexamer is composed of amphipathic dimeric subunits. (A) DENV-1 NS1 purified from infected Vero cell supernatants was treated with the nonionic detergent TX-114 at a 1% final concentration. (Left) NS1 proteins from the insoluble (I), aqueous (A), and detergent (D) fractions were analyzed by SDS/PAGE and Coomassie blue staining. (Right) The transmembrane DENV-1 envelope protein E, used as a control, was detected by immunoblotting. (B and C) The oligomeric state [hexameric (H), dimeric (D), or monomeric (M)] of NS1 was analyzed by SDS/PAGE and Coomassie blue staining (B) or surface-enhanced laser desorption ionization/time-of-flight (SELDI-TOF) MS (C) in the initial protein preparation (Tot), as well as the aqueous (Aq) and TX-114 detergent (Det) phases. NS1 protein was chemically cross-linked with DMS as described in SI Experimental Procedures. In C, asterisks indicate irrelevant peaks corresponding to double-charged species (2H).

NS1 Protein Is Secreted as a Lipoprotein Particle Rich in Triglycerides.

To investigate the presence of lipids in the DENV NS1 particle, we subjected native DENV-1 NS1 to treatment with organic solvent and separated the lipid moiety on TLC. A predominant species, well resolved on the TLC plate (Fig. 3A, arrow), was recovered and analyzed by NMR (Fig. 3B). The ¹H NMR spectrum displays the characteristic signals of triglycerides, including peaks at 5.17, 4.17, and 4.03 ppm related to protons of the glycerol moiety as well as the corresponding cross-peaks in 2D double quantum-filtered correlation spectroscopy (DQF-COSY) (Fig. 3B). These findings were corroborated by GLC analysis of NS1-associated triglycerides (Fig. 3C). Triglyceride molecules are formed by three fatty acid chains that can be different or all alike. Their length and degree of saturation is variable, although aliphatic chains with 16, 18, and 20 carbon atoms, which may contain one or two double bonds, are most frequently observed. GLC analysis of fatty acids derived from DENV-1 NS1 triglycerides shows two major peaks, corresponding to saturated palmitic acid (16:0) and unsaturated oleic acid (18:1), as well as minor peaks, polyunsaturated palmitoleic acid (16:1) and linoleic acid (18:2), and other peaks that likely correspond to background signals (Fig. 3C). Compared with authentic NS1 recovered from the supernatant of DENV-1–infected Vero cells, recombinant DENV-2 NS1 produced in Drosophila Schneider 2 (S2) cells also contains triglycerides that display a homogeneous fatty acid profile composed of palmitic acid and stearic acid (18:0) (Fig. 3C). Triglyceride molecules extracted from control HDL particles are formed by palmitic, oleic, and linoleic acids (Fig. 3C).

Fig. 3.

The NS1 hexamer carries a lipid cargo rich in triglycerides. (A) TLC of lipids extracted from native DENV-1 NS1, colored by iodine vapor. Lane 1, phosphatidycholine (PC; flash) and rhodamine-labeled phosphatidyl-ethanolamine (Rh-PE; pink) used as lipid standards (Std); lane 2, NS1-associated lipids (Lipid_NS1). One predominantly represented lipid species (arrow) was extracted for further analysis. (B) Identification of triglycerides by 600-MHz NMR 1D proton and 2D DQF-COSY spectra. Characteristic NMR signals and the corresponding protons of the triglyceride moiety are connected by arrows. The cross-peaks between the glycerol protons signals (at 5.17, 4.17, and 4.03 ppm) are circled, and the connectivities are traced. Vinylic to allylic proton correlations are boxed. Aliphatic and methyl protons are found near 1.2 and 0.8 ppm, respectively, as expected. (C) GLC analysis of fatty acid derivatives from transesterified triglycerides. Triglycerides associated with DENV-1 and DENV-2 recombinant NS1 are composed of three saturated and unsaturated aliphatic chains of 16 or 18 carbon atoms long. HDL is shown as a control.

NS1 Lipid Moiety Is Similar to the Lipid Cargo of HDLs.

In addition to triglycerides, we were able to isolate cholesteryl ester molecules from recombinant DENV-2 NS1 (Fig. S4). Sterol esters were isolated from purified NS1 preparations, separated on a TLC plate, and saponified. The corresponding sterol fractions were found to be identical to standard cholesterol by both GLC and MS (Fig. S4). Other lipid species were identified by TLC and GLC, including mono- and diacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. We estimated the number of lipid molecules and found that each NS1 hexamer binds 6 triglyceride molecules (i.e., 1 per protomer), twice as many mono- and diacylglycerol molecules, 16–33 cholesteryl esters, and 18–27 phospholipids (Table 1 and SI Experimental Procedures). Cholesterol and sphingomyelin were also observed on TLC but not quantified. Overall, the NS1 lipid composition is very similar to that of HDLs, although HDLs show a higher lipid:protein weight ratio (Table 1). Accordingly, the NS1 hexamer is denser than what would be expected for an HDL particle of similar size (1.20–1.23 g/mL for the NS1 hexamer in comparison with 1.063–1.12 g/mL for 9- to 12-nm-wide HDLs) and instead fits within the class of very HDLs (1.21–1.25 g/mL), close to soluble proteins (1.26–1.28 g/mL). Of note, we found that the hexameric organization and lipid content are identical for two different DENV serotypes of the NS1 protein (Table 1 and Fig. 3C).

Table 1.

Quantification by GLC of different lipid species associated with the DENV-1 NS1 hexamer

Lipoprotein	Triglyceride	Mono- and diacylglycerol	Cholesteryl ester	Phospholipid
DENV-1 NS1*	5.3–4.0	7.9–9.8	11.4–23.3	12.5–19.1
HDL	21.9	11.8	98	63.4

Data are expressed in nanomoles. Relative values were estimated by using a C17 fatty acid calibration standard. Lipids were recovered from 200-μg purified protein preparations (∼4.5 nmol of the 50-kDa NS1 protomer; SI Experimental Procedures).

*Values correspond to two purified protein preparations (recombinant NS1 produced in the presence or absence of FCS, respectively).

Modeling Lipid Organization Within the DENV NS1 Channel.

We estimated that ∼70 lipid molecules could be extracted from a single NS1 hexamer particle (Table 1 and SI Experimental Procedures), representing a total volume of ∼75 nm³ based on the specific volumes of the various lipid species (34, 35). These numbers do not take into account molecules of cholesterol and sphingomyelin that are part of the lipid cargo. Depending on the lipid composition, and the presence of cholesterol in particular (35), lipid compaction events can occur, suggesting that the whole NS1 lipid cargo can fit well into the 80-nm³ channel. Triglycerides and cholesteryl esters probably constitute the central lipid core, whereas charged lipids such as phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin occupy the outer layers of the NS1 channel. As described above, we estimate that there are between 18 and 27 phospholipids in total (i.e., 9–13 per channel opening). Considering an average surface area of 0.5 nm² per phospholipid (34, 35), these phospholipids can fill ∼60% of the 9-nm² triangular surface present at each end of the channel (Fig. 1), leaving extra space for other polar lipids, such as sphingolipids and glycolipids. Altogether, the number of lipids extracted from the NS1 particle is compatible with the dimensions of the central channel.

Chemical Lipid Inhibitors Affect NS1 Secretion.

To confirm that lipid interaction is an essential step in the process of hexamer formation and secretion, we made use of chemical inhibitors targeting lipid components of lipid droplets or lipid rafts. Niacin is an inhibitor of diacylglycerol acyltransferase 2, which prevents triglyceride synthesis (36). Methyl-β-cyclodextrin (MβCD) mobilizes cholesterol, and possibly phospholipids, thus modifying the composition of lipids in both raft and non-raft domains (37). d-Threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4) is an inhibitor of glucosylceramide synthase that leads to a marked reduction in the raft content of glycosphingolipids (38, 39). We first tested the effect of niacin on NS1 secretion in cells constitutively expressing DENV-1 NS1. Niacin reduced NS1 secretion by up to 3.7-fold in conditions that did not affect cell proliferation (Fig. 4 A and B). Levels of inhibition varied depending on the concentration of the inhibitor (Fig. 4A) as well as cell density (Fig. 4B). The two other compounds, MβCD and P4, used at non-toxic concentrations also had a dose-dependent inhibitory activity that reached 5.4- and 3.2-fold, respectively (Fig. 4C and Fig. S5). Interestingly, levels of intracellular NS1 remained unaffected upon treatment with any of the drugs tested (Fig. 4D and Fig. S5), indicating that reduced NS1 secretion did not result from altered protein synthesis or increased protein degradation. These results confirm that NS1 association with membrane microdomains, possibly at sites of nascent lipid droplets, is a prerequisite to the assembly and release of the viral lipoprotein particle.

Fig. 4.

Lipid inhibitors affect NS1 protein secretion. (A–C) NS1 secretion was monitored at increasing doses of niacin and different cell-seeding densities (A and B) or in the presence of various concentrations of MβCD (C). Data correspond to the mean value ± SEM (n = 3). (D) Levels of intracellular and extracellular NS1 were compared in HEK293 cells expressing recombinant DENV-1 NS1 cultured in the presence of 3 mM niacin or 0.4 mM MβCD. The amounts of NS1 were measured by ELISA after 3 d (A–C) or 4 d (D) of treatment. Cell proliferation was assessed by dehydrogenase activity measurement (SI Experimental Procedures).

Discussion

The flavivirus nonstructural protein NS1 has long been reported to undergo a complex maturation process. On the one hand, it is attached to intracellular membranes and the surface of infected cells; on the other, it is secreted in the extracellular medium and circulates in the serum of infected patients. In this study, we used a combination of biochemical and structural approaches to investigate the organization and composition of NS1 released by DENV-infected cells.

We obtained a cryo-EM reconstruction of the secreted form of NS1, which reveals a barrel-like hexameric particle of ∼10 nm in diameter, in which the three dimeric rods interact along narrow lateral surfaces and form a wide central channel (Fig. 1). The channel was a most unexpected finding, and we investigated its possible contribution to NS1 structure and function. Because the contact areas between the dimers appeared insufficient to maintain the hexameric state of NS1 in solution (40), and were not consistent with the high stability of the protein in an aqueous environment, we searched for the presence of stabilizing elements that would localize within the channel. The dual behavior of the NS1 protein in the TX-114 detergent phase-partitioning assay, where the hexamer and dimeric subunits partitioned, respectively, into the aqueous and detergent phases, indicated that amphiphilic molecules such as lipids could possibly be present. Lipids could indeed be isolated from purified hexamer preparations, and we observed by TLC a heterogeneous population with one predominant species identified by NMR as triglycerides (Figs. 2 and 3). Other NS1-associated lipid species included mono- and diacylglycerol, cholesterol, cholesteryl ester, phosphatidylcholine, phosphatidyl-ethanolamine, and sphingomyelin (Table 1 and Fig. S4), an overall lipid composition thus very close to that of endogenous HDLs circulating in plasma.

Despite notable homology in their lipid content, DENV NS1 and HDL particles have a fundamentally different protein organization. Whereas HDL particles are composed of narrow ribbons of apolipoprotein A1 that tie up a large lipid bundle (41–43), the NS1 hexamer consists of a thick protein shell organized as an open barrel that can only accommodate a much smaller lipid cargo compared with HDLs of similar size. Accordingly, the density of the NS1 hexamer (1.20–1.23 g/mL) corresponds to the smallest subclass of HDL particles (i.e., 7 nm in diameter). We estimate that >70 lipid molecules associate to an NS1 hexamer, in agreement with the number of lipids that could theoretically fit into the central channel (35). According to a model of lipid distribution in lipoprotein particles (34), we hypothesize that triglycerides, along with cholesteryl esters, preferentially constitute the central core of the lipid cargo, around which polar lipids (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, in particular) can pack, their charged heads facing the aqueous environment at either opening of the NS1 channel.

The mechanism by which NS1 acquires its lipid cargo appears to involve an initial interaction of NS1 dimers with intracellular membranes. Because recombinant NS1, lacking the putative GPI-anchor signal present in the downstream NS2A coding region (17, 19), shows no difference in lipid-binding capacity in comparison with native NS1, a GPI modification cannot account for NS1 attachment to membranes. Early studies following the folding process of NS1 in the endoplasmic reticulum (ER) indicated that the NS1 monomer is water-soluble and becomes membrane-associated once the protein dimerizes (44). We propose that lipid-binding sites form during the dimerization process itself, possibly at the dimer interface (i.e., between the two lobes of the dimer; Fig. 1). The 1:1 molar ratio of triglycerides to NS1 (i.e., six triglyceride molecules per NS1 hexamer; Table 1) suggests that NS1 dimers would bind specifically the hydrophilic head of the lipid. Because intracellular triglycerides essentially accumulate between the two leaflets of the ER membrane from which cytosolic lipid droplets arise (45, 46), one possibility is that NS1 dimers insert into the hemimembrane, thus gaining access to the neutral lipid pool. Such insertion would locally destabilize the organization of lipids, favoring the association of three NS1 dimers around a lipid cargo and its release from the membrane by pinching off, as pictured in Fig. S1. This overall scheme, which implies that an initial association of NS1 dimers with membranes is required before assembly of soluble hexamers, is supported by the fact that inhibitors targeting components of lipid droplets or lipid rafts (triglycerides and cholesterol, respectively) significantly impair NS1 secretion without reducing intracellular protein levels (Fig. 4).

The link between the maturation process of NS1 and lipid-droplet biogenesis within the infected cell corroborates a recent report indicating that the DENV capsid (C) protein interacts with lipid droplets and that this interaction is essential for viral replication and virus particle assembly (47). Another recent study reveals that DENV infection, through an interaction of NS3 with fatty acid synthase, increases biosynthesis of fatty acids, which are then recruited in viral replication complexes (48). The question as to whether the C protein, NS1, and NS3 cooperate in any way to hijack the host cell lipid machinery and support important molecular processes at different stages of the virus life cycle remains open.

The discovery that DENV NS1 carries lipids in the extracellular milieu (Fig. S1) also has important pathophysiological implications. Lipoproteins are known to play a key role in vascular homeostasis, and defects in lipoprotein functions can affect coagulation and predispose to vascular inflammation and thrombosis (49, 50). By diverting certain lipids from their original fate, NS1 has the potential to interfere with the biogenesis of endogenous lipoprotein particles (51) or to deregulate the intracellular lipid-sensing machinery during entry into target cells (52, 53). In line with our observations, several reports show that DENV-infected patients who developed DHF/DSS present a decrease of HDL and low-density lipoprotein content in plasma as well as altered levels of cholesterol and triglycerides (54–56). Moreover, the nutritional status of children seems to have an impact on the risk of developing fatal DHF/DSS (57, 58). The NS1 lipoprotein particle also has in common with HDLs the ability to bind factors of the complement system (29–32), and can thereby modulate host innate-immune response either as part of a viral escape mechanism or as a means to exacerbate inflammation (59, 60).

In conclusion, our study identifies a previously uncharacterized class of lipoprotein particle, exemplified by the barrel-like DENV NS1 protein. The organization of the NS1 hexamer around a “soft” lipid core explains, at least in part, the lack of success in the crystallization efforts and the limited resolution of the cryo-EM reconstruction. Maturation of the NS1 lipoprotein involves an initial interaction of NS1 dimers with lipid components of intracellular membranes, clustering of NS1 on the luminal border of nascent lipid droplets, packing of triglycerides and neighboring lipids during hexamer assembly, and, eventually, secretion of the soluble lipoprotein particle. The striking similarity between the lipid moieties of NS1 and HDLs observed for different DENV serotypes raises the possibility that NS1 may play a major role in acute vascular dysfunction and the associated life-threatening hypovolemic shock. These findings open promising alternative therapeutic avenues to fight dengue disease, such as interfering with NS1 secretion or targeting its hydrophobic channel.

Experimental Procedures

3D Reconstruction of the DENV NS1 Hexamer.

Cryo-EM analysis of the NS1 protein sample was carried out with a Philips CM12 transmission electron microscope with an LaB6 filament at 120 kV. Images were analyzed with the EMAN (61) and EM (62, 63) software packages. Characteristic class averages representing different orientations of the particles on the micrograph were used to calculate an initial 3D reconstruction, which was further refined (as described in SI Experimental Procedures) to obtain a final electron-density map to a resolution of ∼3 nm.

TX-114 Detergent Phase-Partitioning Assay.

TX-114 detergent phase partitioning was performed by using a precondensed preparation of TX-114 at a 1% final concentration. After an overnight incubation at 4 °C, potential aggregates were pelleted at high speed before separating the detergent from the aqueous phase at 30 °C. In a set of experiments, proteins from the different phases were cross-linked with DMS at 4 °C and analyzed by SDS/PAGE and Coomassie blue staining or by MS on a PBS II mass reader (Ciphergen Biosystems).

Characterization of the NS1 Lipid Moiety.

Potential lipid components were extracted with a standard solvent-extraction procedure. Lipids were analyzed by TLC and stained with iodine or dichlorofluorescein. Different lipid classes were transesterified for their characterization by GLC. Fatty acid methyl esters were analyzed on an Agilent Technologies model 6890 chromatograph equipped with a BPX70 fused-silica capillary column (60 m × 0.25 mm inner diameter, 0.25-μm film thickness). One of the predominant lipid species was also subjected to NMR (SI Experimental Procedures). NMR spectra were acquired on a Bruker 600-MHz spectrometer equipped with a triple-resonance z-axis gradient cryoprobe at 298K in deuterated chloroform (CDCl₃).

Full experimental procedures and associated references are available as SI Experimental Procedures.