Letter pubs.acs.org/OrgLett
Total Synthesis of Repeating Unit of O‑Polysaccharide of Providencia alcalifaciens O22 via One-Pot Glycosylation Ananda Rao Podilapu and Suvarn S. Kulkarni* Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India S Supporting Information *
ABSTRACT: The first total synthesis of the phosphorylated trisaccharide repeating unit of Providencia alcalifaciens O22 is reported. The trisaccharide contains rare deoxyamino sugar AAT at the reducing end and D-glyceramide 2-phosphate at the other end. The efficient synthesis involves one-pot assembly of trisaccharide and late-stage phosphorylation as key steps.
Providencia is a genus of Gram-negative, rod-shaped bacteria belonging to the family of Enterobacteriaceae. It is subdivided into eight species including P. alcalifaciens, P. stuartii, P. rettgeri, P. rustigianii, P. heimbachae, P. vermicola, P. sneebia, and P. burhodogranariea.1 These species have been identified and isolated from sputum, urine, perineum, axilla, stool, blood, and wound specimens of humans as well as from other animals and from soil and water sources.2 Medically important species, including P. alcalifaciens, P. rustigianii, P. stuartii, and P. rettgeri, are mostly associated with urinary tract infections and enteric diseases.3 These opportunistic pathogens have also been implicated in pericarditis,4 endocarditis,5 meningitis,6 and ocular7 infections. P. alcalifaciens is particularly known to cause diarrhea in children and travelers.3,8 Studies have shown that some strains of P. alcalifaciens are invasive to intestinal mucosa and other cell types in eukaryotes.9 Thus, accurate and speedy diagnosis and effective vaccines are urgently required to combat this pathogen. In this regard, the O-polysaccharides (Oantigens) present on the cell surface of Gram-negative bacteria which define serospecificity of strains and are used for the serotyping of bacteria are important. Recently, Ovchinnikova and co-workers10 isolated a new phosphorylated O-polysaccharide of the S-form lipopolysaccharide (LPS) from P. alcalifaciens O22. The trisaccharide repeating unit consists of two unusual components, 2acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N, AAT) and D-glyceramide 2-phosphate (D-GroAN-2-P), the latter being identified for the first time in bacterial polysaccharides. The structure of the trisaccharide repeating unit of the O-polysaccharide was established to be -4)-(DGroAN-2-P-3-)-β-D-GalNAc-(1−4)-β-D-Gal-(1−3)-β-D-FucNAc4N-(1-. Notably, the phosphate group and the free amino group of the AAT residue imparts a zwitterionic character to the O-polysaccharide. Over past few years, there has been great interest in harnessing the immunological potential of bacterial zwitterionic polysaccharides (ZPS) for vaccine development. Such ZPS have been shown to directly activate T-cells without © 2017 American Chemical Society
having to be conjugated to a carrier protein and elicit a stronger immune response.11 Thus, the phosphorylated trisaccharide repeating unit of P. alcalifaciens O22 is an important synthetic target. Our laboratory has been involved in the synthesis of rare sugar-containing bacterial glycoconjugates.12 Herein, we report the first total synthesis of the repeating unit of P. alcalifaciens O22 1 via one-pot glycosylation. The challenges encountered in the synthesis of phosphorylated trisaccharide 1 are the procurement of the orthogonally protected rare sugar AAT and phosphorylation of the secondary alcohol adjacent to the amide functionality in the D-glyceramide unit. Our retrosynthetic scheme is shown in Figure 1. The target molecule 1 could be synthesized by global deprotection of fully functionalized phosphorylated trisaccharide 2. At the very outset, it was envisioned that the assembly of the trisaccharide 3 could be carried out in a one-pot manner. This would demand a careful selection of the protecting groups not only to maintain the orthogonality but also to fine-tune the electronic properties of the glycosyl donors and acceptors.13 Accordingly, a 9-fluorenylmethyloxycarbonyl (Fmoc) group was employed as a temporary protecting group which could potentially be selectively removed post glycosylation in the same pot to obtain the 3-OH′′ trisaccharide 3, which could be coupled with H-phosphonate 4 to obtain 2. The Hphosphonate 4 could in turn be obtained from D-mannitol, whereas the rare AAT building block 5 could be accessed starting from cheaply available D-mannose via a one-pot double serial SN2 displacement of the corresponding D-rhamnosyl 2,4bistriflate.12a Lastly, building blocks 6 and 7 could be obtained by regioselective protection of D-galactose and D-glucosamine. The β-selectivity in glycosylation would be controlled by placement of participating groups (OBz, NHTroc, NHTCA) at the C2 position in all of the building blocks 5−7. The NH2 Received: September 6, 2017 Published: September 26, 2017 5466
DOI: 10.1021/acs.orglett.7b02791 Org. Lett. 2017, 19, 5466−5469
Letter
Organic Letters
Scheme 1. Synthesis of Rare Sugar AAT Building Block 5
group of 11 using NaBrO3 and Na2S2O417 furnished the key AAT building block 5 (76%). Our synthesis of the orthogonally protected β-thiogalactoside donor 6 is outlined in Scheme 2. The easily accessible 2,3-diol Figure 1. Retrosynthetic analysis.
Scheme 2. Synthesis of 4-OH Thiogalactoside Donor 6
group in 7 would be protected as NHTroc to enhance the reactivity14 of O3 position, whereas TCA would be used to mask the NH2 group in 5, as Troc would not be stable under oxidative debenzylation conditions. Similarly the NH2 group of D-glyceramide would be protected as N(Bn)2 so as to oblate the hydrogen bonding and enhance the reactivity of the secondary OH for phosphorylation. With these considerations we began the total synthesis of 1. Our synthesis started with procurement of the orthogonally protected rare deoxy amino sugar building block 5 from Dmannose along the lines of our earlier established protocol (Scheme 1).12a Accordingly, the known D-rhamnosyl 2,4-diol 812g was first converted to the corresponding 2,4-bis-triflate by treatment with Tf2O in pyridine. The crude triflate obtained after a brief aqueous workup was subjected to a regioselective C2-OTf displacement with a stoichiometric amount of tetrabutylammonium azide (TBAN3)12,15 at −30 °C. After complete conversion of the starting material as observed from TLC analysis, benzyl carbamate was added in the same pot at rt to displace the remaining C4-OTf to afford the desired orthogonally protected AAT building block 9 in 61% yield over three steps, after a single chromatographic purification. The regioselectivity observed in the one-pot reaction can be nicely explained by invoking Richardson−Hough rules for SN2 displacements of pyranosidic aryl and alkyl sulfonates, which were recently updated for pyranoside and furanoside O-triflates by Hale and co-workers.16 Reduction of azide in 9 to amine using PPh3 followed by trichloroacetyl protection furnished thioglycoside donor 10 in 78% yield over two steps. Glycosylation of donor 10, with 2-propanol using NIS, AgOTf as a promoter at −40 °C gave β-linked O-isopropyl glycoside 11 in 86% yield. Oxidative removal of the benzyl
12, was first converted to the corresponding tin ketal by treatment with dibutyltin oxide in toluene at 110 °C and further reacted with benzyl bromide and tetrabutylammonium bromide (TBAB) at 60 °C to afford the 3-OBn derivative 13, in high regioselectivity, as a single isomer in 81% yield over two steps. Benzoylation of the remaining 2-OH group in 13 using benzoyl chloride and pyridine afforded the corresponding fully protected β-thiophenyl galactoside which upon reductive benzylidene ring opening using TFA and Et3SiH furnished 4OH thiogalactoside donor 6 in 81% yield. Since D-galactosamine is expensive, we opted for its C4 epimer D-glucosamine as a cheap and abundant starting material (Scheme 3). First, commercially available D-glucosamine was rapidly converted to the known NHTroc derivative 14, following the procedure reported by Galan.18 Regioselective ring opening of 14 using Et3SiH and TFA furnished 4-OH derivative 1518 in 82% yield. Triflation of 15 using Tf2O in 5467
DOI: 10.1021/acs.orglett.7b02791 Org. Lett. 2017, 19, 5466−5469
Letter
Organic Letters
donor 7 in the same pot in the presence of NIS/TMSOTf promoter led to the formation of the corresponding fully protected trisaccharide. Et3N was added to the reaction mixture to quench the TfOH and simultaneously remove the Fmoc group to afford 3-OH′′ trisaccharide 3 in 72% yield over three steps, in a one-pot manner. The structural assignment of 3 was performed using 1H NMR, 13C NMR, 1H−13C HSQC, and 1 H−1H COSY analysis (see the SI). Prior to conducting the one-pot glycosylation, the corresponding disaccharide and trisaccharide intermediates were isolated and characterized by spectral means and then used as TLC standards during the onepot glycosylation. For the synthesis of D-glyceramide building block 20, we started with readily accessible dicyclohexylidene D-mannitol (Scheme 5).19 Oxidation of diol using TEMPO and PhI(OAc)2
Scheme 3. Synthesis of D-Galactosamine Donor 7
Scheme 5. Synthesis of D-Glyceramide Derivative 20
pyridine, followed by C4 inversion via intramolecular displacement of 4-OTf by 3-OAc furnished the 3-OH D-galactosamine derivative 16 in 91% yield over two steps. In this watermediated transformation, the C3-OAc migrates to C4-position via orthoester type transition state. Fmoc protection of the remaining 3-OH group in 16 using FmocCl and pyridine afforded the corresponding fully protected β-thio galactosamine donor 7 in 90% yield. All of the monosaccharide building blocks were thoroughly characterized by in-depth 2D NMR spectral analysis (see the Supporting Information). With all the desired building blocks 5, 6 and 7 in hand, the stage was set for one-pot glycosylation (Scheme 4). Accordingly, a highly regioselective glycosylation of 4-OH thiophenyl galactoside donor 6 and 3-OH AAT acceptor 5 using NIS/TMSOTf as the promoter cleanly afforded the corresponding 4-OH′ disaccharide in 1 h. Addition of glycosyl Scheme 4. One-Pot Assembly of Trisaccharide 3
afforded cyclohexylidene-protected D-glyceric acid, which upon brief workup was as such coupled with benzylamine using DCC and HOBt to obtain amide 18 in 62% yield over two steps. NBenzylation of 18 using NaH and BnBr followed by removal of the cyclohexylidene group using 80% AcOH gave diol 19 (80%). Selective monobenzylation of the diol was achieved by first converting it to the corresponding tin ketal by treatment with dibutyltinoxide in toluene at 110 °C and further reacting with benzyl bromide and TBAB at 60 °C to afford the requisite 2-OH D-glyceramide derivative 20, with high regioselectively, as a single isomer in 90% yield over two steps. As anticipated, phosphorylation of the D-glyceramide turned out to be a difficult task. As shown in Scheme 6, the Dglyceramide derivative 20 could be successfully phosphorylated using imidazole, PCl3, and Et3N to afford H-phosphonate 4 in 89% yield, which was further coupled with trisaccharide 3 in the presence of pivaloyl chloride and pyridine followed by oxidation with I2 to furnish the phosphorylated trisaccharide 2 in 64% yield over two steps. Global deprotection of 2 was done in three steps. First, NHTCA and NHTroc were converted to NHAc by using Zn, AcOH, and Ac2O, followed 5468
DOI: 10.1021/acs.orglett.7b02791 Org. Lett. 2017, 19, 5466−5469
Organic Letters
■
ACKNOWLEDGMENTS We thank the Department of Science and Technology (Grant No. EMR/2014/000235). A.R.P. thanks CSIR-New Delhi for a fellowship.
Scheme 6. Phosphorylation and Global Deprotection
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02791. Experimental details and procedures, compound characterization data, and 1H and 13C spectra for all new compounds (PDF)
■
REFERENCES
(1) Giammanco, G. M.; Grimont, P. A.-D.; Grimont, F.; Lefevre, M.; Giammanco, G.; Pignato, S. Int. J. Syst. Evol. Microbiol. 2011, 61, 1638−1644. (2) O’Hara, C. M.; Brenner, F. W.; Miller, J. M. Clin. Microbiol. Rev. 2000, 13, 534−536. (3) Yoh, M.; Matsuyama, J.; Ohnishi, M.; Takagi, K.; Miyagi, H.; Mori, K.; Park, K.-S.; Ono, T.; Honda, T. J. Med. Microbiol. 2005, 54, 1077−1082. (4) Simon, C.; Dieli, M.; Brucato, A.; Pedrotti, P.; Brambilla, P.; Curri, S. F.; Senni, M.; Pericotti, S.; Suter, F.; Ferrazzi, P. Circulation 2010, 122, e401−e403. (5) Krake, P. R.; Tandon, N. South. Med. J. 2004, 97, 1022−1023. (6) Sipahi, O. R.; Bardak-Ozcem, S.; Ozgiray, E.; Aydemir, S.; Yurtseven, T.; Yamazhan, T.; Tasbakan, M.; Ulusoy, S. J. Clin. Microbiol. 2010, 48, 4667−4668. (7) Koreishi, A. F.; Schechter, B. A.; Karp, C. L. Ophthalmology 2006, 113, 1463−1466. (8) Larson, P. S.; Shah, M. M.; Odoyo, E.; Apondi, E.; Kathiiko, C.; Miringu, G.; Nakashima, M.; Ichinose, Y. Am. J. Trop. Med. Hyg. 2015, 93, 497−500. (9) Janda, J. M.; Abbott, S. L.; Woodward, D.; Khashe, S. Curr. Micrbiol. 1998, 37, 159−165. (10) Ovchinnikova, O. G.; Kocharova, N. A.; Bialczak-Kokot, M.; Shashkov, A. S.; Rozalski, A.; Knirel, Y. A. Eur. J. Org. Chem. 2012, 3500−3506. (11) (a) Tzianabos, A. O.; Onderdonk, A. B.; Rosner, B.; Cisneros, R. L.; Kasper, D. L. Science 1993, 262, 416−419. (b) Mazmanian, S. K.; Kasper, D. L. Nat. Rev. Immunol. 2006, 6, 849−858. (c) Pragani, R.; Seeberger, P. H. J. Am. Chem. Soc. 2011, 133, 102−107. (d) Shi, M.; Kleski, K. A.; Trabbic, K. R.; Bourgault, J.-P.; Andreana, P. R. J. Am. Chem. Soc. 2016, 138, 14264−14272. (e) Ghosh, S.; Nishat, S.; Andreana, P. R. J. Org. Chem. 2016, 81, 4475−4484. (f) Christina, A. E.; van den Bos, L. J.; Overkleeft, H. S.; van der Marel, G. A.; Codée, J. D. C. J. Org. Chem. 2011, 76, 1692−1706. (12) (a) Emmadi, M.; Kulkarni, S. S. Nat. Protoc. 2013, 8, 1870− 1889. (b) Emmadi, M.; Kulkarni, S. S. Org. Biomol. Chem. 2013, 11, 4825−4830. (c) Emmadi, M.; Kulkarni, S. S. Org. Biomol. Chem. 2013, 11, 3098−3102. (d) Emmadi, M.; Kulkarni, S. S. Nat. Prod. Rep. 2014, 31, 870−879. (e) Emmadi, M.; Kulkarni, S. S. Carbohydr. Res. 2014, 399, 57−63. (f) Sanapala, S. S.; Kulkarni, S. S. J. Am. Chem. Soc. 2016, 138, 4938−4947. (g) Podilapu, A. R.; Kulkarni, S. S. Org. Lett. 2014, 16, 4336−4339. (h) Clark, E. L.; Emmadi, M.; Krupp, K. L.; Podilapu, A. R.; Helble, J. D.; Kulkarni, S. S.; Dube, D. H. ACS Chem. Biol. 2016, 11, 3365−3373. (13) Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 734−753. (14) Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819−6825. (15) Oberli, M. A.; Bindschädler, P.; Werz, D. B.; Seeberger, P. H. Org. Lett. 2008, 10, 905−908. (16) (a) Pyranoside triflate SN2 displacement rules: Hale, K. J.; Hough, L.; Manaviazar, S.; Calabrese, A. Org. Lett. 2014, 16, 4838− 4841. (b) Furanoside triflate SN2 displacement rules: Hale, K. J.; Hough, L.; Manaviazar, S.; Calabrese, A. Org. Lett. 2015, 17, 1738− 1741. (17) Adinolfi, M.; Guariniello, L.; Iadonisi, A.; Mangoni, L. Synlett 2000, 1277−1278. (18) Galan, M. C.; Tran, A. T.; Whitaker, S. Chem. Commun. 2010, 46, 2106−2108. (19) Sugiyama, T.; Sugawara, H.; Watanabe, M.; Yamashita, K. Agric. Biol. Chem. 1984, 48, 1841−1844.
by hydrolysis of esters using Et3N, MeOH, H2O at 60 °C and concomitant hydrogenolysis using H2 (1 atm), 20 wt % Pd(OH)2/C, and a drop of AcOH in MeOH to give target molecule 1 in 64% yield over three steps. The crude compound was purified using reversed-phase HPLC on a C18 column using pure deionized water as eluent. The target molecule was characterized satisfactorily using 1H, 13C, 31P NMR and HRMS data (see the SI). In conclusion, total synthesis of the phosphorylated trisaccharide repeating unit of P. alcalifaciens O22 has been achieved for the first time. The key features of the synthesis are efficient synthesis of the appropriately functionalized rare sugar AAT building block and its further elaboration into the target trisaccharide via a one-pot glycosylation and phosphorylation of glyceramide. The zwitterionic trisacccharide 1 is now available for bioevaluation to assess its immunological potential.
■
Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Suvarn S. Kulkarni: 0000-0003-2884-876X Notes
The authors declare no competing financial interest. 5469
DOI: 10.1021/acs.orglett.7b02791 Org. Lett. 2017, 19, 5466−5469
