Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. 2018, 20, 3656−3660
Rhodopseudomonas palustris Strain CGA009 Produces an O‑Antigen Built up by a C‑4-Branched Monosaccharide: Structural and Conformational Studies Roberta Marchetti,*,† Emiliano Bedini,† Djamel Gully,‡ Rosa Lanzetta,† Eric Giraud,‡ Antonio Molinaro,† and Alba Silipo*,†
Downloaded via TUFTS UNIV on June 15, 2018 at 14:15:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant’Angelo, Università di Napoli Federico II, Via Cintia 4, I-80126 Napoli, Italy ‡ IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), UMR IRD/SupAgro/INRA/UM2/CIRAD, TA-A82/J Campus de Baillarguet ,Montpellier 34398 Cedex 5, France S Supporting Information *
ABSTRACT: Here, the analysis of the peculiar homopolymeric O-chain, isolated from the lipopolysaccharide (LPS) of Rhodopseudomonas palustris strain CGA009, is reported. The O-chain is built up of a novel 4-C-branched sugar (12-deoxy-4-C-(D-altro-5,7,8,9-tetrahydroxyhexyl))-3-O-methyl-D-galactopyranose)) whose structure, absolute configuration, and conformational features were deduced by 2D NMR spectroscopy, optical rotation measurements, and molecular dynamics simulations.
Rhodopseudomonas palustris is a purple photosynthetic bacterium that has developed the ability to grow and survive, even in anaerobic conditions, in different soil and water environments.1,2 The remarkable metabolically versatile character of this bacterium, its ability to switch between four different metabolic states, photoautotrophic, photoheterotrophic, chemoautotrophic, and chemoheterotrophic, results in a capacity to convert both light and a large panel of organic compounds into energy.3−5 Over the years, the peculiar properties of R. palustris have made it an attractive organism for industrial and biotechnological applications in the field of biofuel production and biodegradation of organic pollutants.6 In this regard, the correlation between its versatility and the properties of its cell envelope is fundamental. Here, we chose to go deeper into the structure and the conformational behavior of bacterial membrane glycoconjugates decorating the cell surface.7 Specifically, we were interested in studying the lipopolysaccharides (LPSs), the main component of the external leaflet of the outer membrane (OM) of Gram-negative bacteria. LPSs are essential for bacterial growth and survival and play pivotal roles in the structural and mechanical support of OM.8 Moreover, due to their location, they mediate a plethora of host−bacteria interaction events including adhesion, recognition, pathogenesis, and symbiosis.9 Structurally, LPSs are amphiphilic macromolecules comprised of a common structural motif characterized by a lipophilic moiety, named lipid A, covalently linked to a hydrophilic heteropolysaccharide, formed by a core oligosaccharide and an O-specific polysaccharide (or O-chain).10,11 Herein, we have examined the structural and conformational features of the O-chain region of the LPS isolated from R. palustris strain CGA009, by NMR spectroscopy and MD © 2018 American Chemical Society
simulations. The detailed experimental procedures are given in the Supporting Information. Specifically, a combination of homo- and heteronuclear 2D NMR experiments was executed on the fully O-deacylated LPS, the OS product, to assign the spin systems and define the polysaccharide repeating unit (Table S1, Figure 1, and Figure S1). The HSQC spectrum in D2O gave rise to 12 signals, with a single resonance in the anomeric region (δ 4.9/102.3 ppm), suggesting that the OS fraction was composed of a homopolymer. Additionally, signals assignable to methyl groups were present, likely suggesting the existence of a deoxy sugar unit. Besides the cross peak of the anomeric carbon (δ 102.3 ppm), there were signals corresponding to seven methine carbons (δ 66.8−80.4 ppm), two methyl groups (δ 14.6−15.0 ppm), one methylene group (δ 35.7 ppm), and one methoxyl carbon (δ 61.3 ppm). The anomeric configuration of the sugar unit was assigned on the basis of 3JH‑1,H‑2 (below 1 Hz) and 1JC,H (174 Hz) coupling constants, whereas the relative configuration of the sugar on the basis of the vicinal 3JH,H ring coupling constants. Altogether, the NMR data converged on the OS structure reported in Figure 2 (as fully described in the SI). Thus, the OS was a homopolymer composed of a 3-O-methyl 6-deoxyhexopyranose in the galacto configuration (sugar N) and further substituted by a side chain at position C-4. In order to define the relative and absolute configuration of the sugar N, we performed a methanolysis of the polymer to isolate the constituent monosaccharide. Two major products were isolated, the O-methyl glycoside S1 and the intramolecular Received: May 7, 2018 Published: June 6, 2018 3656
DOI: 10.1021/acs.orglett.8b01439 Org. Lett. 2018, 20, 3656−3660
Letter
Organic Letters
Figure 1. HSQC (blue), HMBC (green), and 1H NMR spectra of R. palustris CGA009 O-antigen homopolymer in D2O at 600 MHz, 310 K; [D4] (trimethylsilyl)propionic acid sodium salt (TSP, 10 mM) was used as the internal reference. The structure of the repeating unit of the OS is also shown.
proton H-5 and thus supported the bicyclic ring structure. In particular, NOE contacts between H-5 and H-11 and the CH3 at position 12 with H-2 (Figure 3b) were possible only by considering the bicycle depicted in Figure 3c. In order to assign the relative configurations of carbons 7−9, glycoside 2 was treated with 2,2-dimethoxypropane in DMF in the presence of an acid catalyst [(+)-10-camphorsulfonic acid] to give the isopropylidene derivative S2 (Figure S2). The obtained compound structure was defined by NMR analysis, 600 MHz in D2O (Table S4 and Figure S2). The signal at δ 3.02 ppm identified as H-8 appeared as a triplet with large coupling constants (3JH,H = 9.7 Hz), indicating the chair conformation of the dioxane ring and the trans diaxial orientation of H-8 with respect to H-9 and H-7. On the basis of the above data, the relative configuration of S1 has been defined. Furthermore, the NMR assignment of the O-methyl glycoside S1 (Table S5 and Figure S3) allowed us to confirm the relative configuration of the stereocenters of the ring moiety. In addition, the comparison of NMR chemical shifts of the α-O-methyl glycoside of N with those of the structurally related sugar α-caryophyllose (α-Car), further supported the altro configuration of the side chain (Figure 2).12,13All the above NMR data suggested that the new compound N (Figure 2) possessed a galacto configuration in the sugar ring and an altro configuration in the side chain. Finally, the absolute configuration of the sugar was estimated in detail since the sugar residue N had the same structure as the caryophyllose, except the C-3 methylene group of the latter was replaced by a C-3 methoxy group in the former, and since the absolute configuration of caryophyllose is known, we have compared their [α]D in order to tentatively indicate, by analogy, the absolute configuration of residue N (see also the SI). On
Figure 2. Structure (a) and Fischer projection (b) of sugar N, 3-Omethyl-12-deoxy-4-C-(D-altro-5,7,8,9-tetrahydroxyhexyl)-D-galactopyranose, constituting the repeating unit of R. palustris CGA009 OS. (c) Structure of caryophyllose, 3,12-dideoxy-4-C-(D-altro-5,7,8,9-tetrahydroxyhexyl)-D-xylo-hexopyranose.
glycoside 2 (Figure 3, Figures S3 and S4, and Table S3). Indeed, the involvement of position 5 in the formation of the intramolecular acetal and thus its axial orientation was evident from analysis of HMBC and NOE. The assessment of the relative configuration of the lateral chain was inferred by the analysis of product 2 (Figure 3). The long-range correlation of the anomeric position 1 with positions 5, 11, and 3 as well as the long-range correlation of 5 with positions 3, 4, 7, and 11 were unequivocally diagnostic of the intramolecular glycoside depicted in Figure 3c; the analysis of the intraresidue NOE contacts allowed us to define the relative configuration of 3657
DOI: 10.1021/acs.orglett.8b01439 Org. Lett. 2018, 20, 3656−3660
Letter
Organic Letters
Figure 3. (a) HSQC (blue), HMBC (green), and 1H NMR spectra and (b) NOESY (zoom) and 1H NMR spectra of the intramolecular glycoside 2 in D2O at 600 MHz, 310 K; [D4] (trimethylsilyl)propionic acid sodium salt (TSP, 10 mM) was used as the internal reference. (c) Structure and model of 2; the key NOE contacts are also illustrated.
Figure 4. (a) Scatter plots and trajectories of Φ vs Ψ values of the dodecasaccharide fragment. Since the conformational behavior of all glycosidic junctions of the dodecasaccharide during the MD simulation was almost comparable (Figure S5), the behavior of a representative Φ/Ψ couple is here presented. (b) View of representative structures and Connelly surface of CGA polymer.
the basis of the above data, the sugar N was identified as a 12deoxy-4-C-(D-altro-5,7,8,9-tetrahydroxyhexyl))-3-O-methyl-Dgalactopyranose. The conformational behavior of Rhodopseudomonas CGA009 polymer (CGA polymer) was investigated by molecular mechanics and dynamic calculations. Initially, the calculation of the potential energy surface of the CGA α-(1→5)
disaccharide repeating unit suggested that it mainly adopted conformations located around the global minimum at Φ= −54° and Ψ = 0° (see the SI) in which the glycosidic torsion adopted the exo-anomeric orientation. The conformational regions energetically accessible to the disaccharide fragment were further confirmed by an MD simulation (Figure S5) and basically predicted the existence of 3658
DOI: 10.1021/acs.orglett.8b01439 Org. Lett. 2018, 20, 3656−3660
Letter
Organic Letters
methyl groups which enhance the polysaccharide hydrophobic character, has already been described. It is, in fact, acknowledged that the genome of the bacterium Rhodopseudomonas exhibits a huge metabolic versatility that is responsible for its capacity to adapt to several external environments, thus attracting attention for its potential use for biotechnological applications. On the other side, carbohydrates with striking chemical structures do raise interest also in basic organic chemistry. Despite the complexity of higher organisms, the number of carbohydrate residues available in their cells is limited, i.e., about 13 different sugar residues are present in mammals. It is exactly the opposite for the microbes in which a huge number of sugar residues are produced (over one hundred residues are known and many more are yet to be discovered). Among these, particularly attractive for carbohydrate chemists are the C-4-branched sugars, either for their obscure role or for their completely unknown biosynthesis.26 As for the function of such unusual compounds, C-4-branched sugars are only found in prokaryotes, especially as components of their cell envelope, LPS, capsules, or exopolysaccharides. This has prompted several chemists to undertake the organic synthesis of these compounds and of their oligomers in order to understand their putative role in the microbial cell and in the interaction of it with the eukaryotic world.27 It has been found that they might have a role in the elicitation and/or suppression of the innate immune response in plants. As for their biosynthesis, very scarce information is available. In the case of presence of a methyl group branched chain at C-4, S-adenosylmethionine has been found to be the donor with the electrophilic methyl group, whereas the acceptor is a nucleotidyl hexulose. Nevertheless, it not reasonable to conceive S-adenosylmethionine as transporter of a long hydroxyl−alkyl electrophile chain;28 therefore, microbes must adopt completely new routes for the biosynthesis of such astounding molecules.
one main minimum. Subsequently, starting from the favorite global minimum, a dodecasaccharide fragment was built, and the conformational behavior of the oligosaccharide was studied by using molecular dynamic simulations (see the SI).14−19 The computational model obtained from the MD was then compared to the experimental results. Ensemble average interproton distances for the α-(1→5) repeating unit of the polysaccharide were extracted from the MD simulations and translated into NOE contacts according to a full-matrix relaxation approach. The corresponding average distances obtained for the simulation from ⟨r−6⟩ values were compared to those collected experimentally. An excellent accordance between the experimental and calculated data was found; i.e., the key H-1− H-5 distance, obtained by NMR data, was 2.23 Å, while the corresponding calculated averaged interglycosydic distance was 2.33 Å. Given the limited flexibility around the glycosidic linkages, the CGA polysaccharide adopted a defined shape. All data suggested that the polymer tends to adopt an extended conformation, characterized by a right handed helicoidal structure with a three-fold screw axis and a pitch of around 10 Å, with the lateral chain pointing outward from the helix and thus exposed to the external environment (Figure 4). Although the majority of homopolysaccharides, including starch and β-glucans, typically form helicoidal structures, the CGA polysaccharide chain adopted an uncommon conformation by using three monosaccharide units to complete one turn instead of six. The extended helicoidal structure of the O-antigen may influence the bacterial antigenicity as well as other biophysical properties. To summarize, the R. palustris strain CGA009 produces an O-polysaccharide chain structure composed of a unique repeating unit made of the novel C-4-branched sugar, 12deoxy-4-C-(D-altro-5,7,8,9-tetrahydroxyhexyl)-3-O-methyl-Dgalactopyranose. Interestingly, this sugar is structurally similar to the branched monosaccharide carrying a six-carbon-atom chain at C-4, reported for the first time in Pseudomonas (Burkholderia) caryophylli and named caryophyllose (Figure 2).12 The former differs in the presence of an additional Omethyl group at position 3, a feature never described so far. Some other Gram-negative bacteria, such as Burkholderia brasiliensis20 and Yersinia species,21 also produce 4-C-branched sugars, and all include a 3,6-dideoxyhexose core. Moreover, further studies have also reported the presence of unusual branched monosaccharides in the lipooligosaccharides isolated from mycobacteria, including M. gastri22 and M. marinum.23 The study of Gram-negative outer membrane glycoconjugates has a two-fold importance. From a microbiology and biotechnology point of view, it is extremely important to understand how the microbe is able to thrive in a hostile external environment and which strategies are chemically adopted to further shield the outer membrane. Indeed, it is known that chemical modifications to the repeating units of LPS O-antigen, including methylation and acetylation, are important for bacterial adaptation and adhesion since they influence some bacterial features. For example, they increase the viscosity and emulsifying properties.24 Therefore, this polysaccharide, which is characterized by a certain level of hydrophobicity, since it is composed by a deoxy sugar decorated by different methyl groups, may raise interest in the field of food and cosmetics. Notably, the structure of the lipopolysaccharide isolated from another strain of R. palustris, BISA53,25 that produces a polymer composed by a trisaccharide repeating unit characterized by the presence of acetyl and
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01439. NMR analysis, experimental details, Tables S1−S5, and Figures S1−S5 (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Emiliano Bedini: 0000-0003-4923-3756 Alba Silipo: 0000-0002-5394-6532 Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS A.S. and A.M. acknowledge the European Commission (H2020-MSCA- ETN-642157 TOLLerant project). REFERENCES
(1) Aprasad, E. V. V.; Sasikala, C.; Ramana, C. V. Descriptions of Rhodopseudomonas parapalustris sp. nov., Rhodopseudomonas harwoo-
3659
DOI: 10.1021/acs.orglett.8b01439 Org. Lett. 2018, 20, 3656−3660
Letter
Organic Letters diae sp. nov. and Rhodopseudomonas pseudopalustris sp. nov., and emended description of Rhodopseudomonas palustris. Int. J. Syst. Evol. Microbiol. 2012, 62, 1790−1798. (2) Oda, Y.; Larimer, F. W.; Chain, P. S.; Malfatti, S.; Shin, M. V.; Vergez, L. M.; Hauser, L.; Land, M. L.; Braatsch, S.; Beatty, J. T.; Pelletier, D. A.; Schaefer, A. L.; Harwood, C. S. Multiple genome sequences reveal adaptations of a phototrophic bacterium to sediment microenvironments. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18543−8. (3) Kanno, N.; Matsuura, K.; Haruta, S. Different Metabolomic Responses to Carbon Starvation between Light and Dark Conditions in the Purple Photosynthetic Bacterium. Rhodopseudomonas palustris Microbes Environ 2018, 33, 83−88. (4) Oda, Y.; Star, B.; Huisman, L. A.; Gottschal, J. C.; Forney, L. J. Biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris. Appl. Environ. Microbiol. 2003, 69, 5186−5191. (5) Kanno, N.; Matsuura, K.; Haruta, S. Differences in survivability under starvation conditions among four species of purple nonsulfur phototrophic bacteria. Microbes Environ 2014, 29, 326−328. (6) Larimer, F. W.; Chain, P.; Hauser, L.; Lamerdin, J.; Malfatti, S.; Do, L.; Land, M. L.; Pelletier, D. A.; Beatty, J. T.; Lang, A. S.; Tabita, F. R.; Gibson, J. L.; Hanson, T. E.; Bobst, C.; Torres, J. L.; Peres, C.; Harrison, F. H.; Gibson, J.; Harwood, C. S. Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nat. Biotechnol. 2004, 22, 55−61. (7) (a) Raetz, C. R.; Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 2002, 71, 635−700. (b) Silipo, A.; Molinaro, A.; Molteni, M.; Rossetti, C.; Parrilli, M.; Lanzetta, R. Full Structural Characterization of an Extracellular Polysaccharide Produced by the Freshwater Cyanobacterium Oscillatoria planktothrix FP1. Eur. J. Org. Chem. 2010, 2010, 5594−5600. (8) Silipo, A.; De Castro, C.; Lanzetta, R.; Parrilli, M.; Molinaro, A. Lipopolysaccharides. In Prokaryotic Cell Wall Compounds - Structure and Biochemistry; König, H., Claus, H., Varma, A., Eds.; Springer: Heidelberg, 2010. (9) De Castro, C.; Lanzetta, R.; Holst, O.; Parrilli, M.; Molinaro, A. Bacterial Lipopolysaccharides in Plant and Mammalian Innate Immunity. Protein Pept. Lett. 2012, 19, 1040−1044. (10) Molinaro, A.; Holst, O.; Di Lorenzo, F.; Callaghan, M.; Nurisso, A.; D’Errico, G.; Zamyatina, A.; Peri, F.; Berisio, R.; Jerala, R.; JiménezBarbero, J.; Silipo, A.; Martín-Santamaría, S. Chemistry of lipid A: at the heart of innate immunity. Chem. - Eur. J. 2015, 21, 500−519. (11) Molinaro, A.; Parrilli, M.; De Castro, C. Bacterial LPS, OPS, and Lipid A. Encyclopedia of Biophysics; Roberts, G.C.K., Ed.; SpringerVerlag: Berlin, 2012. (12) Molinaro, A.; De Castro, C.; Lanzetta, R.; Manzo, E.; Parrilli, M. Solvent effect on the isomeric equilibrium of carbohydrates: the superior ability of 2,2,2-trifluoroethanol for intramolecular hydrogen bond stabilization. J. Am. Chem. Soc. 2001, 123 (50), 12605−10. (13) Adinolfi, M.; Corsaro, M. M.; De Castro, C.; Evidente, A.; Lanzetta, R.; Mangoni, L.; Parrilli, M. The relative and absolute configurations of stereocenters in caryophyllose. Carbohydr. Res. 1995, 274, 223−232. (14) Bernardi, A.; Colombo, A.; Sánchez-Medina, I. Conformational analysis and dynamics of mannobiosides and mannotriosides using Monte Carlo/stochastic dynamics simulations. Carbohydr. Res. 2004, 339 (5), 967−73. (15) Bernardi, A.; Raimondi, L.; Zuccotto, F. Simulation of proteinsugar interactions: a computational model of the complex between ganglioside GM1 and the heat-labile enterotoxin of Escherichia coli. J. Med. Chem. 1997, 40 (12), 1855−62. (16) Rodríguez-Carvajal, M. A.; Bernabe, M.; Espartero, J. L.; TejeroMateo, P.; Gil-Serrano, A.; Jiménez-Barbero, J. Studies on the solution conformation and dynamics of a polysaccharide from Sinorhizobium f redii HH103 and its monosaccharide repeating unit. J. Mol. Graphics Modell. 2000, 18 (2), 135−42. (17) Iida-Tanaka, N.; Fukase, K.; Utsumi, H.; Ishizuka, I. Conformational studies on a unique bis-sulfated glycolipid using NMR spectroscopy and molecular dynamics simulations. Eur. J. Biochem. 2000, 267 (23), 6790−7.
(18) Jaud, S.; Tobias, D. J.; Brant, D. A. Molecular dynamics simulations of aqueous pullulan oligomers. Biomacromolecules 2005, 6 (3), 1239−51. (19) (a) Asensio, J. L.; Cañada, F. J.; Cheng, X.; Khan, N.; Mootoo, D. R. Jiménez-Barbero J. Conformational differences between O- and C-glycosides: the alpha-O-man-(1−1)-beta-Gal/alpha-C-Man-(1−1)beta-Gal case - a decisive demonstration of the importance of the exoanomeric effect on the conformation of glycosides. Chem. - Eur. J. 2000, 6, 1035−1041. (b) Asensio, J. L.; Jimenez-Barbero, J. The use of the AMBER force field in conformational analysis of carbohydrate molecules: determination of the solution conformation of methyl alpha-lactoside by NMR spectroscopy, assisted by molecular mechanics and dynamics calculations. Biopolymers 1995, 35, 55−75. (20) Mattos, K. A.; Todeschini, A. R.; Heise, N.; Jones, C.; Previato, J. O.; Mendonca-Previato, L. Nitrogen-fixing bacterium Burkholderia brasiliensis produces a novel yersiniose A-containing O-polysaccharide. Glycobiology 2005, 15, 313−321. (21) (a) Gorshkova, R. P.; Zubkov, V. A.; Isakov, V. V. Ovodov, IuS. A new branched-chain monosaccharide from the Yersinia enterocolitica serotype O:4.32 lipopolysaccharide. Bioorg. Khim. 1987, 13, 1146− 1147. (b) Gorshkova, R. P.; Zubkov, V. A.; Isakov, V. V.; Ovodov, Y. S. Yersiniose, a new branched-chain sugar. Carbohydr. Res. 1984, 126, 308−312. (22) Gilleron, M.; Vercauteren, J.; Puzo, G. Lipooligosaccharidic antigen containing a novel C4-branched 3, 6-dideoxy-alpha-hexopyranose typifies Mycobacterium gastri. J. Biol. Chem. 1993, 268, 3168− 3179. (23) Rombouts, Y.; Burguière, A.; Maes, E.; Coddeville, B.; Elass, E.; Guérardel, Y.; Kremer, L. Mycobacterium marinum lipooligosaccharides are unique caryophyllose-containing cell wall glycolipids that inhibit tumor necrosis factor-alpha secretion in macrophages. J. Biol. Chem. 2009, 284, 20975−88. (24) Lerouge, I.; Vanderleyden, J. O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. FEMS Microbiol. Rev. 2002, 26, 17−47. (25) Silipo, A.; Di Lorenzo, F.; De Felice, A.; Vanacore, A.; De Castro, C.; Gully, D.; Lanzetta, R.; Parrilli, M.; Giraud, E.; Molinaro, A. Structural and conformational study of the O-polysaccharide produced by the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris strain BisA53. Carbohydr. Polym. 2014, 114, 384−391. (26) (a) De Castro, C.; Molinaro, A.; Lanzetta, R.; Holst, O.; Parrilli, M. The linkage between O-specific caryan and core region in the lipopolysaccharide of Burkholderia caryophylli is furnished by a primer mono-saccharide. Carbohydr. Res. 2005, 340, 1802−7. (b) Adinolfi, M.; Corsaro, M. M.; De Castro, C.; Evidente, A.; Lanzetta, R.; Molinaro, A.; Parrilli, M. Caryose: a carbocyclic monosaccharide from Pseudomonas caryophylli. Carbohydr. Res. 1996, 284, 111−118. (c) Silipo, A.; Leone, M. R.; Erbs, G.; Lanzetta, R.; Parrilli, M.; Chang, W.-S.; Newman, M.-A.; Molinaro, A. A unique bicyclic monosaccharide from the Bradyrhizobium lipopolysaccharide and its role in the molecular interaction with plants. Angew. Chem., Int. Ed. 2011, 50, 12610−12612. (27) Li, W.; Silipo, A.; Gersby, L. B. A.; Newman, M.-A.; Molinaro, A.; Yu, B. Synthesis of Bradyrhizose Oligosaccharides Relevant to the Bradyrhizobium O-Antigen. Angew. Chem., Int. Ed. 2017, 56, 2092− 2096. (28) He, X. M.; Liu, H. Formation of unusual sugars: mechanistic studies and biosynthetic applications. Annu. Rev. Biochem. 2002, 71, 701−754.
3660
DOI: 10.1021/acs.orglett.8b01439 Org. Lett. 2018, 20, 3656−3660
