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DOI: 10.1055/s-0036-1591518
Synthesis of 1,5-Anhydro-d-glycero-d-gluco-heptitol Derivatives as Potential Inhibitors of Bacterial Heptose Biosynthetic Pathways
Publikationsverlauf
Received: 01. September 2017
Accepted after revision: 12. Oktober 2017
Publikationsdatum:
08. November 2017 (online)
Abstract
A series of 1,5-anhydro-d-glycero-d-gluco-heptitol derivatives have been prepared from 3-O-benzyl-1,2-O-isopropylidene-d-glycero-d-gluco-heptofuranose via conversion into anomeric bromide and thiophenyl derivatives, followed by glycal formation and reductive desulfurization, respectively. Global deprotection of the protected intermediates afforded the 1,5-anhydro derivatives of the d-glycero-d-gluco- and 1,2-dideoxy-d-altro- configuration as well as the 1,5-anhydro-2-deoxy-d-altro-hept-1-enitol. In addition, the 7-O-phosphorylated d-glycero-d-gluco-heptose and its 1,5-anhydro analogue were prepared in good yields utilizing phosphoramidite chemistry. A novel heptitol analogue based on a 1-deoxynojirimycin scaffold was also elaborated via a Wittig-type chain elongation followed by dihydroxylation, separation of the resulting epimers, and global deprotection. The target compounds, however, were not active as inhibitors of the bacterial sedoheptulose-7-phosphate isomerase GmhA.
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Key words
heptose - carbohydrates - inhibitor - anhydro-sugar - C6 chain elongation - phosphorylationBiographical Sketches
Markus Blaukopf obtained his Ph.D. degree in organic chemistry from the University of Natural Resources and Life Sciences, Vienna in 2011 under the supervision of Prof. Paul Kosma. He continued to work in this group for a postdoctoral study on the development of novel potential heptose-based antibacterial agents. Supported by a FWF Schroedinger Fellowship he relocated to Vancouver in 2013 to work on the structure–activity relationship of LPS biosynthetic pathway enzymes in the group of S. G. Withers. In 2015, he returned to the University of Natural Resources and Life Sciences, Vienna where his research activities center around the synthesis of carbohydrate based compounds and their use as glycosyl transferase substrates.
Dmytro Atamanyuk holds a Ph.D. in medicinal chemistry from Lviv National Medical University, Ukraine under the supervision of Prof. Roman Lesyk. He has 9 years of experience in biotech (Mutabilis) and CRO companies (Enamine) in the infectious diseases and oncology as project leader and team leader. Currently he is a project leader in Medicinal Chemistry of AB Science, a Paris-based pharmaceutical company.
Nuno M. Xavier obtained a dual Ph.D. degree in organic chemistry from the University of Lisbon and from the National Institute of Applied Sciences of Lyon in 2011 under the supervision of Prof. Amélia Rauter and Dr. Yves Queneau, respectively. In a postdoctoral study, he worked on the development of novel potential heptose-based antibacterial agents in the group of Paul Kosma at the University of Natural Resources and Life Sciences, Vienna. In 2012, he returned to the University of Lisbon. His research activities – supported by an Investigator Starting Grant from the Portuguese Foundation for Science and Technology (FCT) – are the design and synthesis of original carbohydrate derivatives and nucleoside and nucleotide analogues of biological interest.
Vincent Gerusz holds a Ph.D. degree in chemistry from Stanford University and has 20 years of experience in drug discovery in both large pharmaceutical and biotech companies. He has led various research teams and is co-inventor of several NCEs put in development in the fields of infectious diseases and oncology. Currently he is heading the Medicinal Chemistry of Debiopharm, a Swiss-based global biopharmaceutical group.
Paul Kosma obtained a Ph.D. degree in organic chemistry at the University of Technology in Vienna. Ensuing postdoctoral experience was obtained at the SANDOZ-research Institute in Vienna and the N. D. Zelinsky Institute of Organic Chemistry in Moscow. Since 1992 he holds a chair of Organic Chemistry at the University of Natural Resources and Life Sciences, Vienna. His main research interests are focused on the synthesis of nucleotide-activated sugars, triterpene glycosides, and complex glycans related to biomedically relevant cell-surface glycans from bacteria, parasites, and viruses.
A threat to global health is presently associated with the increase of multidrug-resistant bacteria, for several of which common antibiotics are not effective anymore.[4] Novel approaches are therefore urgently needed to identify bacterial targets and to develop appropriate compounds with effective and specific modes of action.[5] Among these targets, the lipopolysaccharide (LPS) of Gram-negative bacteria is of significant importance. LPS is located in the outer membrane of the cell envelope, and harbors non-mammalian, higher-carbon sugars, which fulfill important functions within the bacterial membrane, but are nonetheless involved in a multitude of interactions with components of the innate and adaptive immune system.[6] In structural terms, the LPS may divided into three domains, corresponding to the endotoxic Lipid A, the core region, and the O-antigenic polysaccharide.[7] In addition to LPS, many bacterial surfaces are covered by capsular polysaccharides serving as an additional barrier, which may also contain these higher-carbon sugars as constituents of their repeating units.[8] In particular, the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) forms the linkage of the inner-core region to the Lipid A anchor and is further extended by several units of l-glycero-d-manno-heptose (LD-Hep) as well as less frequently by its 6-epimeric form.[9] Moreover, other heptose variants of different configuration and their 6-deoxy derivatives have been found on capsular polysaccharides of important pathogens such as Campylobacter jejuni, Yersinia tuberculosis, or Burkholderia pseudomallei.[10] Recently, both epimeric forms of glycero-d-manno-heptose linked to serine residues have been identified in bacterial glycoproteins associated with bacterial adhesion.[11]
The biosynthesis of heptoses and their nucleotide-activated forms has been elucidated, starting form sedoheptulose-7-phosphate (1), which is isomerized by GmhA[12c] to d-glycero-d-manno-heptose-7-phosphate (2), followed by a kinase step catalyzed by HldE or HddA,[12c] respectively, leading to either the β- or α-anomeric form of the resulting d-glycero-d-manno-heptose 1,7-bisphosphates 3 and 4, respectively (Scheme [1]).[12] This intermediate has very recently been shown to act as potent inducer of an innate immune response in particular in the context of Neisseria meningitidis infections.[13] The biosynthetic pathways diverge from compound 2 leading to the biosynthesis of the unstable ADP-l-glycero-β-d-manno-heptose (ADP: adenosine 5′-diphosphate) serving as the substrate for the inner-core bacterial heptosyl transferases involved in the assembly of LPS-units (3 → 5 → 6 → 7).[14] On the other hand, the glycosyl α-phosphate 4 is converted into the corresponding GDP-d-glycero-α-d-manno sugar (GDP: guanosine 5′-diphosphate) 9 (4 → 8 → 9) involved in the biosynthesis of capsular polysaccharides. The GDP heptose may then undergo further transformations such as deoxygenation and epimerization (9 → 10 → 11 or 12).[10a] The main enzymes involved in these biosynthetic steps have meanwhile been characterized and several crystal structures of the apo-forms and the liganded forms have been published.[15] Blocking these enzymes, which are involved in the early transformations leads, for example, to deep rough type bacterial mutants of the Re chemotype with impaired barrier functions, which are rapidly cleared from serum by the immune system. Heptose-deficient strains from Escherichia coli, Salmonella enterica, Shigella flexneri, Burkholderia, and Neisseria are known to be avirulent.[16] As a consequence, suitable inhibitors of the heptose biosynthetic pathways hold promise as broad-band drug candidates, which would not harm mammalian enzymes, would not damage the gut microbiome, and could serve as potent antivirulence agents.[5a]
Inhibition of the first enzymatic steps catalyzed by the sedoheptulose-7-phosphate isomerase GmhA and anomeric kinases HldE and HddA, respectively, would affect both biosynthetic pathways involved in LPS and CPS (capsular polysaccharide) assembly.[17] Previously, the group of Vincent has carried out the preparation of a library of d-glycero-d-manno-heptose 7-phosphates with manifold structural variations at the exocyclic side chain.[18] Notably the 2-epimeric derivative 13, corresponding to a d-gluco-configured d-glycero-heptose-7-phosphate, was found to inhibit both GmhA and HldE with IC50 values in the low micromolar range. In order to further investigate the impact of an equatorial 2-hydroxy group, we have set out to prepare additional d-glycero-d-gluco- derivatives with modifications in the vicinity of the anomeric center, including 1-deoxy derivatives, which were also envisaged to potentially inhibit the ensuing kinase reactions HldE and HddA, respectively. Furthermore, the biological activity of related analogues obtained by replacement of the ring oxygen by nitrogen should also be evaluated.
The syntheses of the target compounds were based on transformations starting from known 3-O-benzyl-1,2-O-isopropylidene-d-glycero-α-d-gluco-heptofuranose (14).[19] Acid hydrolysis of the acetal group using 50% aqueous TFA followed by per-O-acetylation afforded the penta-O-acetyl derivative 15 in 73% yield (Scheme [2]). The coupling constant J 5,6 (3.0 Hz) was in agreement with related derivatives of d-glycero-d-manno-heptose, thereby securing the assignment of the d-glycero-configuration at C-6.[20] Compound 15 was subjected to hydrogenation on Pd/C to give the alcohol 16 in 92% yield. Compound 16 was fully deprotected via Zemplén transesterification to give the d-glycero-d-gluco-heptose 18 in nearly theoretical yield as 1:1.9 α,β-anomeric mixture.[19]
Prior to the preparation of the 1-deoxy derivatives, alcohol 16 was converted into the per-O-acetylated heptose 17 in 97% yield. Introduction of the phenyl-1-thio group was achieved in a moderate yield by reaction of 17 with thiophenol in the presence of SnCl4 as Lewis acid promoter. The reaction could not be forced for full conversion, since only the β-anomeric acetate was reactive, while unreacted α-anomer could be recovered from the reaction mixture in 39% yield. Subsequently, reductive desulfurization[21] of the thioglycoside 19 was accomplished by hydrogenation with Raney-nickel under microfluidic conditions for an extended reaction time (32 h) at 40 °C, to give 20 in 55% yield. Its subsequent de-O-acetylation produced the 1,5-anhydro compound 21 in 92% yield.
Next, 1,2-dideoxy derivatives were generated from glycal 22, prepared in 75% yield via conversion of 17 into the corresponding anomeric bromide by treatment with HBr in acetic acid, followed by elimination using zinc in buffered acetic acid (Scheme [3]). Glycal 22 was deprotected to furnish tetraol glycal 23 in 90% yield. In addition, the double bond was hydrogenated to afford the 1,2-dideoxy derivative 24 in 85% yield followed by Zemplén de-O-acetylation to give 25 in 98% yield.
Proceeding toward the 7-O-phosphorylated derivatives, the 1,5-anhydro derivative 21 was converted into the 7-O-triisopropylsilyl derivative 26 by reaction with TIPS-chloride/imidazole in THF in modest yield followed by benzylation with NaH/benzyl bromide in DMF, which gave the tetra-O-benzyl derivative 27 in 54% yield (Scheme [4]). Next, the TIPS ether was smoothly cleaved by the action of TBAF to produce the primary alcohol 28 in 82% yield, which was then subjected to phosphoramidite-based phosphitylation with ensuing oxidation by mCPBA to give phosphotriester 29 in 85% yield.[22] De-O-benzylation of 29 by hydrogenolysis on Pd/C gave the 7-O-phosphorylated 1,5-anhydro-d-glycero-d-gluco-derivative 30 in near quantitative yield.
Along similar lines, albeit in improved yields, compound 14 was converted into the reducing d-glycero-d-gluco-heptose 7-O-phosphate 36. Regioselective silylation at position 7 by reaction with TIPS-chloride in THF/DABCO gave triisopropylsilyl derivative 31 in 71% yield (Scheme [5]). To selectively address the primary alcohol for phosphorylation, the remaining hydroxyl groups were benzylated. Similar to 27, compound 32 was isolated in only 44% yield, when basic conditions (NaH and BnBr in DMF) were used, due to the base lability of the silyl ether group. The yield, however, could be considerably improved (85%) using benzyl trichloroacetimidate[23] in the presence of triflic acid. Cleavage of the TIPS ether was carried out by the action of TBAF to produce the primary alcohol 33 in 60% yield. The ensuing phosphorylation of 33 with N,N-diisopropyl-di-O-benzylphosphorane in the presence of 1H-tetrazole afforded the intermediate phosphite-triester which was then oxidized with mCPBA to give the phosphotriester derivative 34 in 78% yield. Deprotection of 34 was performed in two steps by first removing the benzyl protecting groups by hydrogenolysis in the presence of Pd/C giving 35, followed by cleavage of the 1,2-acetonide by the action of 10% aqueous TFA to provide the 7-O-phosphoryl-d-glycero-d-gluco-heptose derivative 36 in 99% yield (Scheme [5]).
For the synthesis of the 5-iminoheptitol derivative 49, 1-deoxynojirimycin hydrochloride (37) was used as a starting material and first protected as the known N-Cbz derivative 38 (Scheme [6]).[24] In order to address carbon 6 for chain elongation, the primary alcohol group was converted into the 6-O-dimethoxytrityl derivative followed by per-O-benzylation and acidic hydrolysis of the DMTr protecting group to 39 in 57% yield (three steps). For the intended preferential formation of the d-glycero-configured compound, Wittig olefination of an intermediate 6-aldehyde – obtained via Swern oxidation of 39 – followed by catalytic dihydroxylation according to Kishi’s rule was carried out.[25] Reaction of the aldehyde with methyl triphenylphosphonium bromide/n-BuLi gave olefin 40 in 62% yield (2 steps). Catalytic osmylation then afforded an excellent yield of the diols 41a and 41b, albeit in poor diastereoselectivity (1.6:1),[26] presumably due to steric congestion exerted by the N-benzyloxycarbonyl appendix. A similar low diastereoselectivity (2:1 ratio) has previously been observed for dihydroxylation of a Boc-protected 1-deoxy-manno-nojirimycin derivative.[27]
Separation of the isomers had to rely on a regioselective 7-O-silylation to produce the 7-O-TBDPS derivatives 42 and 44 in a combined 95% yield. Assignment of the new stereocenter at C-6 was not straightforward – complicated by the presence of Cbz-rotamers – and was first tried upon formation of the (R)-MPTA Mosher ester derivatives 43 and 45.[28] In order to overcome severe line broadening, NMR spectra had to be recorded in toluene-d 8 at 75 °C. Minor differences in the chemical shift data and the presence of rotamers (42: H-5 at 4.45, H-6 at 5.92 ppm, 44: H-5 at 4.59 and 4.54, H-6 at 5.84 and 5.80 ppm) did not allow for an unambiguous assignment of the stereochemistry at C-6. A conclusive assignment, however, could be achieved upon formation of the bicyclic derivatives 46–48, respectively. Attempted benzylation of 42 with NaH/benzyl bromide in DMF furnished the 7-O-silyl ether 46 in 38% yield and the perbenzylated carbamate 47 in 47% yield with concomitant loss of the benzyloxy protecting group (Scheme [7]). Facile formation of the 1,3-oxazolidinone ring from Cbz-carbamates under basic conditions has previously been described.[29] NOESY experiments recorded for 47 and 48 in CDCl3 and benzene-d 6, respectively, showed the interaction between H-4 and H-7 in compound 47 versus H-4 and H-6 in compound 48, thus establishing the d-glycero configuration of the major isomer 42 and the l-glycero form for the minor isomer 44.
Deprotection was performed for 44 and comprised removal of the 6-O-TBDPS group with HF-pyridine, which gave 41b in near theoretical yield. Hydrogenolysis of the Cbz and benzyl was not straightforward and THF had to be used as solvent, since the reaction in methanol led to formation of the corresponding N-methyl derivative. The resulting amine was converted into the hydrochloride form followed by desalting on Sephadex G-10 to give 49 in 52% yield. The stereochemical assignment of both 6-epimers was further substantiated by the chemical shift of C-6 for the l-glycero isomer 49 (69.8 ppm), which is consistent with previously published NMR data of the related l-glycero-d-manno-heptose derivative[20] as well as 1,5-dideoxy-1,5-imino-glycero-d-manno-heptitol derivatives.[27] [30]
The inhibitory properties of compounds 21, 23, 25, 30, 36, and 49 were tested according to the literature.[31] None of the compounds, however, acted as effective inhibitors, thus substantiating the importance of the anomeric hydroxyl group for the interaction with GmhA. Compound 49, however, is also of interest as a potential glycosidase inhibitor[32] complementing the previously prepared series of 1,5-dideoxy-1,5-iminoheptitols of the l- and d-glycero-d-manno-, l-glycero-d-altro- and d-glycero-l-gulo- configuration.[27] [30] [33] The synthesis and activity of more potent inhibitors targeting the Zn-ion of GmhA will be published in due course.
All purchased chemicals were used without further purification, unless stated otherwise. The promotor BF3·OEt2 was used as a solution in Et2O (≥46% according to the manufacturer). Solvents were dried over activated 3Å (acetone, Et2O) or 4Å (CH2Cl2, DMF, pyridine) molecular sieves. Anhyd MeOH (Merck) and anhyd THF (Sigma-Aldrich) were purchased. Cation exchange resin DOWEX 50 H+ was regenerated by consecutive washing with HCl (3 M), H2O, and anhyd MeOH. Aqueous solutions of salts were saturated unless stated otherwise. Concentration of organic solutions was performed under reduced pressure <40 °C. Optical rotations were measured with a PerkinElmer 243 B Polarimeter. [α]D 20 values are given in units of 10–1 deg·cm2·g–1. TLC was performed on Merck precoated plates: generally on 5 × 10 cm, layer thickness 0.25 mm, Silica Gel 60F254; alternatively on HPTLC plates with 2.5 cm concentration zone (Merck). Spots were detected by dipping reagent (anisaldehyde/H2SO4) or 5% ethanolic phosphomolybdic acid. For column chromatography silica gel (0.040–0.063 mm) was used. HP-column chromatography was performed on pre-packed columns (YMC-Pack SIL-06, 0.005 mm, 250 × 10 mm and 250 × 20 mm). Desalting after ester saponification was performed on pre-packed PD-10 columns (GE Healthcare, SephadexTM G-25 M). NMR spectra were recorded with Bruker Avance III 300, 400 and 600 instruments using standard Bruker NMR software. 1H spectra were referenced to 7.26 (CDCl3) and 0.00 (D2O, external calibration to 2,2-dimethyl-2-silapentane-5-sulfonic acid) ppm unless stated otherwise. 13C spectra were referenced to 77.00 (CDCl3), 49.00 (CD3OD) and 67.40 (D2O, external calibration to 1,4-dioxane) ppm. 31P spectra were referenced to 0.00 ppm (orthophosphoric acid) for solutions in D2O and according to ref.[34] for solutions in CDCl3. ESI-MS data were obtained on a Waters Micromass Q-TOF Ultima Global instrument.
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1,2,4,6,7-Penta-O-acetyl-3-O-benzyl-d-glycero-d-gluco-heptopyranose (15)
A solution of 14 (583 mg, 1.71 mmol) in 50% aq TFA (10 mL) was stirred at r.t. for 16 h. The reaction mixture was concentrated and co-evaporated with toluene (3 × 10 mL). The remaining slightly red oil was taken up in pyridine (4 mL), cooled to 0 °C, and Ac2O (4 mL) was added dropwise at 0 °C. The mixture was stirred at r.t. for 12 h, then cooled to 0 °C, and MeOH (5 mL) was added dropwise. The mixture was then diluted with CHCl3 (15 mL), sat. aq NaHCO3 (10 mL) was added, and stirred for 15 min. The layers were separated, the aqueous layer was reextracted with CHCl3 (10 mL) and the combined organic layers were dried (MgSO4) and concentrated. The residue was purified by column chromatography (eluent: hexane/EtOAc, 3:1 → EtOAc) to afford the title compound 15 (645 mg, 73%) as a colorless amorphous solid.
1H NMR (600 MHz, CDCl3): δ (α anomer) = 7.36–7.21 (m, 5 H, ArH), 6.29 (d, J 1,2 = 3.5 Hz, 1 H, H-1), 5.13 (t, J 4,3 = J 4,5 = 9.8 Hz, 1 H, H-4), 5.15–5.09 (m, 1 H, H-6), 5.00 (dd, J 2,3 = 9.9 Hz, J 1,2 = 3.5 Hz, 1 H, H-2), 4.68 (d, J= 11.8 Hz, 1 H, CH 2Ph), 4.60 (d, J = 11.8 Hz, 1 H, CH 2Ph), 4.32 (dd, J 7a,7b = 12.0 Hz, J 7a,6 = 3.9 Hz, 1 H, H-7a), 4.20 (dd, J 7b,6 = 7.5 Hz, 1 H, H-7b), 4.04 (dd, J 5,6 = 2.5 Hz, J 4,5 =10.5 Hz, 1 H, H-5), 3.92 (app t, J = 9.6 Hz, H-3), 2.14, 2.06, 2.04, 2.01, 1.98 (5 s, each 3 H, CH3).
13C NMR (150 MHz, CDCl3): δ = 170.09, 169.58, 169.56, 169.07 (C=O), 128.46, 127.88, 127.56 (ArCH), 89.23 (C-1), 77.2 (C-3), 74.84 (CH2Ph), 71.71 (C-5), 71.32, 69.91, 69.83 (C-2, C-4, C-6), 61.27 (C-7), 20.90–20.67 (5 × CH3).
1H NMR for (600 MHz, CDCl3): δ (β anomer) = 7.36–7.21 (m, 5 H, ArH), 5.61 (d, J 1,2 = 7.9 Hz, 1 H, H-1), 5.16–5.08 (m, 3 H, H-2, H-4, H-6), 4.60 (s, 2 H, CH 2Ph), 4.29 (dd, J 7a,6 = 4.0 Hz, J 7a,7b = 12.0 Hz, 1 H, H-7a), 4.25 (dd, J 7b,6 = 7.1 Hz, J 7a,7b = 12.0 Hz, 1 H, H-7b), 3.77 (dd, J 5,6 = 2.9 Hz, J 4,5 = 9.8 Hz, 1 H, H-5), 3.71 (t, J 3,4 = J 2,3 = 8.9 Hz, 1 H, H-3), 2.10, 2.07, 2.05, 2.02, 1.97 (5 s, each 3 H, CH3).
13C NMR (150 MHz, CDCl3): δ = 170.09, 169.58, 169.56, 169.07 (C=O), 128.51, 127.98, 127.84 (ArCH), 92.00 (C-1), 79.88 (C-3), 74.39 (C-5), 74.21 (CH2Ph), 71.36 (C-2), 69.85, 69.83 (C-4, C-6), 61.25 (C-7), 20.90, 20.84, 20.78, 20.72, 20.67 (5 × CH3).
HRMS (ESI): m/z [M + COOH]+ calcd for C25H31O14: 555.1719; found: 555.1714.
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1,2,4,6,7-Penta-O-acetyl-d-glycero-d-gluco-heptopyranose (16)
A solution of 15 (50 mg, 0.098 mmol) in MeOH (33 mL) was hydrogenated in an H-Cube (H-Cube SS, cartridge Pd/C 10%, 33 mm, flow rate 0.2 mL/min, H2 mode: full) at 25 °C. The column was washed with 10 mL MeOH until no more product was detectable by TLC. After one run, TLC indicated complete consumption of the starting material. Evaporation of the solution gave 40 mg of crude product, which was purified by flash chromatography (toluene/EtOAc, 1:1) to give 38 mg (92%) of product 16 as a syrup.
1H NMR (600 MHz, CDCl3): δ (α anomer) = 6.29 (d, J 1,2 = 3.7 Hz, 1 H, H-1), 5.21–5.17 (m, 1 H, H-6), 5.07–5.00 (m, 1 H, H-4), 4.94 (dd, J 2,3 = 10.5 Hz, 1 H, H-2), 4.36 (dd, J 7a,7b = 12.0 Hz, J 7a,6 = 3.9 Hz, 1 H, H-7a), 4.17 (dd, J 7b,6 = 7.4 Hz, 1 H, H-7b), 4.09 (dd, J 5,4 = 10.4 Hz, J 5,6 = 2.4 Hz, 1 H, H-5), 4.01 (t, J 3,4 = 9.6 Hz, 1 H, H-3), 2.18, 2.14, 2.09, 2.08, 2.05 (5 s, each 3 H, CH3).
13C NMR (150 MHz, CDCl3): δ = 170.73–170.01 (q, 5 × C=O), 89.11 (C-1), 71.63 (C-2), 71.28 (C-4), 71.16 (C-5), 70.17 (C-3), 69.89 (C-6), 61.51 (C-7), 20.91–20.56 (5 × CH3).
1H NMR (600 MHz, CDCl3): δ (β anomer) = 5.63 (d, J 1,2 = 8.2 Hz, 1 H, H-1), 5.21–5.17 (m, 1 H, H-6), 5.07–5.00 (m, 1 H, H-4), 4.97 (dd, J 2,3 = 9.3 Hz, J 1,2 = 8.2 Hz, 1 H, H-2), 4.32 (dd, J 7a,7b = 12.0 Hz, J 7a,6 = 4.0 Hz, 1 H, H-7a), 4.21 (dd, J 7b,6 = 7.2 Hz, 1 H, H-7b), 3.80 (dd, J 5,4 = 10.0 Hz, J 5,6 = 2.8 Hz, 1 H, H-5), 3.74 (t, J 3,4 = 9.3 Hz, 1 H, H-3), 2.16, 2.12, 2.10, 2.09, 2.05 (5 s, each 3 H, CH3).
13C NMR (150 MHz, CDCl3): δ = 170.73–170.01 (q, 5 × C=O), 91.69 (C-1), 73.99 (C-5), 73.88 (C-3), 72.68 (C-2), 71.28 (C-4), 69.75 (C-6), 61.44 (C-7), 20.91–20.56 (5 × CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C17H24O12Na: 443.1160; found: 443.1156.
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d-Glycero-d-gluco-heptose (18)
A solution of 16 (38 mg, 0.09 mmol) in MeOH (3 mL) was stirred with 0.1 M NaOMe (0.2 mL) for 12 h at r.t. The solution was made neutral by adding Dowex 50 H+ resin, filtered, and the filtrate was concentrated. The residue was passed over a PD-10 column and eluted with HPLC-grade H2O. The eluate was lyophilized to give 19 mg (quant.) of 18 as an amorphous solid; [α]D 20 –47.2 (c 0.26, H2O).
1H NMR (600 MHz, D2O): δ = 5.16 (d, J 1,2 = 3.7 Hz, H-1α), 4.56 (d, J 1,2 = 7.9 Hz, H-1β), 3.96–3.94 (m, 1 H, H-6), 3.86 (dd, J 5,4 = 10.1 Hz, J 5,6 = 2.3 Hz, H-5α), 3.74 (dd, J 7a,7b = 11.9 Hz, J 7a,6 = 3.1 Hz, 1 H, H-7a), 3.66 (dd, J 7b,6 = 7.4 Hz, 1 H, H-7b), 3.65 (app t, H-3α), 3.52–3.44 (m, H-2α, H-5β, H-4α), 3.42 (dd, J 3,4 = 9.1 Hz, H-3β), 3.18 (app t, J 2,3 = 8.9 Hz, H-2β).
13C NMR (150 MHz, D2O): δ (α anomer) = 92.80 (C-1), 73.81 (C-3), 72.68 (C-6), 72.08 (C-5), 71.16 (2 C, C-2, C-4), 62.68 (C-7).
13C NMR (150 MHz, D2O): δ (β anomer) = 96.91 (C-1), 77.00 (C-5), 76.74 (C-3), 74.78 (C-2), 72.49 (C-6), 71.16 (C-4), 62.43 (C-7).
HRMS (ESI): m/z [M – H]– calcd for C7H14O7: 209.0667; found: 209.0665.
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1,2,3,4,6,7-Hexa-O-acetyl-d-glycero-d-gluco-heptopyranose (17)
Compound 15 (633 mg, 1.24 mmol) was dissolved in MeOH (24 mL) and hydrogenated in an H-Cube for 12 h (H-Cube SS; cartridge: 10% Pd/C 33 mm; solvent: MeOH; flow rate: 0.2 mL; H2-mode: full; temperature: 50 °C). The reaction mixture was concentrated (540 mg) and dissolved in pyridine (2 mL). Ac2O (0.5 mL) and a catalytic amount of DMAP were added and the mixture was stirred at r.t. for 12 h. The mixture was cooled to 0 °C, MeOH (1 mL) was added, stirred for 10 min, and then diluted with CH2Cl2 (5 mL). The organic phase was washed with sat aq NaHCO3 (2 × 5 mL), dried (MgSO4), and concentrated. The residue was purified by column chromatography (toluene/EtOAc, 4:1 → 1:1) to give compound 16 (557 mg, 1.20 mmol, 97%) as a colorless amorphous solid. The reaction mixture was evaporated to dryness (540 mg), taken up in pyridine (2 ml), Ac2O (500 µl), a catalytic amount of DMAP and the reaction was stirred at room temperature for 12h. The reaction mixture was cooled to 0°C, MeOH (1 ml) was added and the reaction mixture was stirred for 10 minutes, diluted with DCM (5 ml), washed with sat. aqu. NaHCO3 (2 × 5 ml), dried (MgSO4), evaporated to dryness and directly purified via column chromatography (silica gel 60, T/EtOAc4/1 ->T/EE 1/1), to give the title compound 17 (557 mg, 1.20 mmol, 97%) as white solid.
1H NMR (600 MHz, CDCl3): δ (α anomer) = 6.31 (d, J 1,2 = 3.7 Hz, 1 H, H-1), 5.43 (t, J = 9.6 Hz, 1 H, H-3), 5.18–5.15 (m, 2 H, H-4, H-6), 5.04 (dd, J 1,2 = 3.7 Hz, J 2,3 = 10.6 Hz, 1 H, H-2), 4.32 (dd, J 7a,6 = 4.3 Hz, J 7a,7b = 12.0 Hz, 1 H, H-7a), 4.16 (dd, J 5,4 = 10.5 Hz, J 5,6 = 2.8 Hz, 1 H, H-5), 4.14 (dd, J 7b,6 = 7.1 Hz, 1 H, H-7b), 2.17, 2.16, 2.08, 2.08, 2.05, 2.02 (6 s, each 3 H, 6 × CH3).
13C NMR (150 MHz, CDCl3): δ = 88.78 (C-1), 70.96 (C-5), 69.92 (C-3), 69.75 (C-6), 69.01 (C-2), 68.81 (C-4), 61.38 (C-7), 20.85–20.51 (6 × CH3).
1H NMR (600 MHz, CDCl3): δ (β anomer) = 5.68 (d, J 1,2 = 8.1 Hz, 1 H, H-1), 5.21 (t, J 3,2 = J 3,4 = 9.2 Hz, 1 H, H-3), 5.18–5.15 (m, 1 H, H-6), 5.13 (app t, J = 9.6 Hz, 1 H, H-4), 5.08 (dd, J 1,2 = 8.4 Hz, J 2,3 = 9.1 Hz, 1 H, H-2), 4.29 (dd, J 7a,6 = 4.3 Hz, J 7a,7b = 11.9 Hz, 1 H, H-7a), 4.19 (dd, J 7b,6 = 7.0 Hz, 1 H, H-7b), 3.88 (dd, J 5,4 = 9.9 Hz, J 5,6 = 3.1 Hz, 1 H, H-5), 2.11, 2.08, 2.07, 2.05, 2.03, 2.01 (6 s, each 3 H, 6 × CH3).
13C NMR (150 MHz, CDCl3): δ = 91.69 (C-1), 73.87 (C-5), 72.79 (C-3), 70.05 (C-2), 69.65 (C-6), 68.78 (C4), 61.28 (C-7), 20.85–20.51 (6 × CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C19H26O13SNa: 485.1266; found: 485.1268.
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Phenyl 2,3,4,6,7-Penta-O-acetyl-1-thio-d-glycero-α,β-d-gluco-heptopyranoside (19)
A solution of 17 (557 mg, 1.20 mmol) in anhyd CH2Cl2 (5 mL) was stirred under argon at 0 °C. Thiophenol (143 μL, 1.20 mmol) was added followed by dropwise addition of a 1 M solution of SnCl4 in CH2Cl2 (663 μL) and the solution was stirred at r.t. for 12 h. The reaction mixture was diluted with CH2Cl2 (5 mL), washed with sat. aq NaHCO3 (5 mL) and the aqueous phase was reextracted with CH2Cl2 (5 mL). The combined organic phases were dried (MgSO4), concentrated, and the residue was purified by column chromatography (toluene/EtOAc, 7:1) to afford an anomeric mixture (α/β = 1:2) of 19 (266 mg, 0.51 mmol, 43%) as a colorless oil; Rf = 0.65 (EtOAc/hexane, 1:1).
1H NMR (600 MHz, CDCl3): δ (α anomer) = 7.51–7.15 (m, 5 H, ArH), 6.27 (d, J 1,2 = 4.1 Hz, 1 H, H-1), 5.51 (t, J 3,2 = J 3,4 = 9.6 Hz, 1 H, H-3), 5.23–5.20 (m, 1 H, H-6), 5.19–5.17 (m, 1 H, H-4), 4.96 (dd, J 1,2 = 4.0 Hz, J 2,3 = 10.3 Hz, 1 H, H-2), 4.35 (dd, J 5,4 = 10.4 Hz, J 5,6 = 2.3 Hz, 1 H, H-5), 4.29 (dd, J 7a,6 = 4.6 Hz, J 7a,7b = 12.0 Hz, 1 H, H-7a), 4.17 (dd, J 7b,6 = 7.2 Hz, 1 H, H-7b), 2.09 (s, 9 H), 2.06 (s, 3 H), 2.03 (5 s, each 3 H, 5 × CH3).
1H NMR (600 MHz, CDCl3): δ (β anomer) = 7.51–7.15 (m, 5 H, ArH), 5.20–5.13 (m, 2 H, H-3, H-6), 5.04 (t, J 4,3 ≅ J 4,5 = 10.0 Hz, 1 H, H-4), 4.92 (app t, J 2,1 = 10.0 Hz, J 2,3 = 9.6 Hz, 1 H, H-2), 4.64 (d, J 1,2 = 10.0 Hz, 1 H, H-1), 4.32 (dd, J 7a,6 = 4.2 Hz, J 7a,7b = 11.8 Hz, 1 H, H-7a), 4.21 (dd, J 7b,6 = 7.2 Hz, 1 H, H-7b), 3.74 (dd, J 5,4 = 10.2 Hz, J 5,6 = 2.7 Hz, 1 H, H-5), 2.09, 2.07, 2.05, 2.03, 1.98 (5 s, each 3 H, 5 × CH3).
13C NMR (150 MHz, CDCl3): δ = 170.47, 170.08, 169.81, 169.77, 169.40, 169.23 (C=O), 133.78 (SPh, CH), 131.01 (SPh, Cq), 128.92 (SPh, CH), 128.66 (SPh, CH), 89.65 (C-1α), 85.55 (C-1β), 77.17 (C-5β), 74.11 (C-3β), 71.46 (C-5α), 70.54 (C-2α), 69.90 (C-6β), 69.78 (C-2β), 69.56 (C-3α), 69.36 (C-6α), 68.83 (C-4β), 68.39 (C-4α), 61.38 (C-7α, 7β), 20.84, 20.77, 20.72, 20.68, 20.66, 20.59, 20.58, 20.55 (CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C23H28O11SNa: 535.1245; found: 535.1247.
Further elution of the column (toluene/EtOAc, 1:1) afforded 219 mg (39%) of 17 (α-anomer); Rf = 0.56 (EtOAc/hexane, 1:1).
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2,3,4,6,7-Penta-O-acetyl-1,5-anhydro-d-glycero-d-gluco-heptitol (20)
Compound 19 (53 mg, 103 μmol) was dissolved in EtOH (50 mL) and dethionated in an H-Cube for 32 h (H-Cube SS; cartridge: Raney-Ni 33 mm; solvent: EtOH; flow rate: 0.2 mL; H2-mode: full; temperature: 40 °C). The reaction mixture was concentrated and the residue was purified by column chromatography (toluene/acetone, 14:1) to give 20 (23 mg, 57 μmol, 55%) as a colorless oil; [α]D 20 +37.3 (c 1.2, CHCl3). The reaction mixture was concentrated to dryness and directly purified by column chromatography (silica gel, toluene/acetone 14/1) to give the title compound (23 mg, 57 µmol, 55%) as colorless oil.
1H NMR (600 MHz, CDCl3): δ = 5.18–5.13 (m, 2 H, H-3, H-6), 5.04 (t, J 4,5 = J 3,4 = 9.9 Hz, 1 H, H-4), 4.95 (dt, J 2,1b = 10.9 Hz, J 2,1b = 5.5 Hz, J 2,3 = 9.9 Hz, 1 H, H-2), 4.31 (dd, J 7a,6 = 4.1 Hz, J 7a,7b = 11.9 Hz, 1 H, H-7a), 4.17 (dd, J 7b,6 = 7.5 Hz, 1 H, H-7b), 4.14 (dd, J 1a,1b = 11.2 Hz, J 2,1a = 5.6 Hz, 1 H, H-1a), 3.62 (dd, J 5,4 = 10.1 Hz, J 5,6 = 2.5 Hz, 1 H, H-5), 3.26 (t, J 1a,1b = 10.9 Hz, J 1b,2 = 10.9 Hz, 1 H, H-1b), 2.09, 2.07, 2.04, 2.02, 2.02 (5 s, each 3 H, 5 × CH3).
13C NMR (150 MHz, CDCl3): δ = 170.55, 170.24, 169.92, 169.75, 169.57 (5 × C=O), 77.96 (C-5), 73.68 (C-3), 69.95 (C-6), 69.03 (C-4), 68.74 (C-2), 66.77 (C-1), 61.41 (C-7), 20.90–20.65 (5 × CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C17H24O11Na: 427.1211; found: 427.1214.
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1,5-Anhydro-d-glycero-d-gluco-heptitol (21)
A solution of NaOMe in MeOH (100 μL, 0.1 M) was added to a solution of 20 (23.6 mg, 58 μmol) in MeOH (2 mL) at r.t. and the reaction mixture was stirred for 4 h. The mixture was then neutralized by addition of Dowex resin (50 H+-form), filtered and the filtrate was concentrated. The residue was taken up in HPLC grade H2O and purified over a short PD-10 column (Sephadex G-25, 1.45 × 5.0 cm, 8.3 mL column volume, eluent: H2O). Product containing fractions were pooled and lyophilized to give 21 (10.5 mg, 92%) as an amorphous solid; [α]D 20 +28.2 (c 0.5, H2O).
1H NMR (600 MHz, D2O): δ = 3.96 (dt, J 6,7a = 3.4 Hz, J 6,7b = 7.5 Hz, 1 H, H-6), 3.92 (dd, J 1a,1b = 11.0 Hz, J 1a,2 = 5.4 Hz, 1 H, H-1a), 3.72 (dd, J 7a,7b = 12.0 Hz, 1 H, H-7a), 3.63 (dd, J 7a,7b = 12.0 Hz, J 7a,6 = 7.6 Hz, 1 H, H-7b), 3.53 (ddd, J 2,3 ≅ 10.5 Hz, 1 H, H-2), 3.43 (t, J 4,3 = J 4,5 = 9.3 Hz, 1 H, H-4), 3.39–3.35 (m, 2 H, H-3, H-5), 3.19 (t, J 1b,2 = 10.9 Hz, 1 H, H-1b).
13C NMR (150 MHz, D2O): δ = 80.62 (C-5), 77.56 (C-3), 71.59 (C-6), 70.13 (C-4), 69.05 (C-2), 68.91 (C-1), 61.43 (C-7).
HRMS (ESI): m/z [M + Na]+ calcd for C7H14O6Na: 217.0683; found: 217.0682.
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3,4,6,7-Tetra-O-acetyl-1,5-anhydro-2-deoxy-d-altro-hept-1-enitol (22)
Compound 17 (500 mg, 0.43 mmol) was dissolved in HBr (33% in AcOH, 2.5 mL) and stirred at r.t. for 12 h. When according to TLC all starting materials had been converted to a higher running spot, NaOAc (2.3 g, 28 mmol) was slowly added at 0 °C. The mixture was diluted with AcOH (3 mL) and placed in an ice-cold ultrasonic bath. Zn dust (500 mg, 7.7 mmol) was added slowly and the mixture was allowed to react under sonication at 0 °C for 30 min (toluene/EtOAc, 3:1). The mixture was diluted with AcOH (5 mL) and CH2Cl2 (20 mL) and the Zn dust was filtered off. The filter was washed with CH2Cl2 and the filtrate was diluted with CH2Cl2 (20 mL) and washed with H2O. The organic phase was concentrated and solid NaHCO3 was added. The residue was dissolved in CH2Cl2, washed with H2O, dried (MgSO4), and concentrated. Flash chromatography through a short plug of silica gel (2 g, Isolute Si II, toluene/EtOAc, 1:1) followed by HPLC (column: YMC Pack SIL 06, 20 × 250, toluene/EtOAc, 8:1, flow rate 5 mL/m, fraction size 5 mL) gave 22 (290 mg, 75%) as a colorless oil; [α]D 20 –35.5 (c 2.1, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 6.48 (dd, J 1,2 = 6.2 Hz, J 1,3 = 0.8 Hz, 1 H, H-1), 5.37 (ddd, J 6,5 = 8.1 Hz, J 6,7a = 3.0 Hz, J 6,7b = 4.9 Hz, 1 H, H-6), 5.22 (ddd, J 4,5 = 4.3 Hz, J 4,3 = 3.5 Hz, 1 H, H-4), 5.07 (ddt, J 3,2 = 4.4 Hz, 1 H, H-3), 4.97 (ddd, J 2,1 = 6.2 Hz, J 2,3 = 4.4 Hz, J 2,4 = 1.1 Hz, 1 H, H-2), 4.48 (dd, J 7a,7b = 12.3 Hz, J 7a,6 = 2.9 Hz, 1 H, H-7a), 4.36 (ddd, J 5,4 = 8.3 Hz, J 5,6 = 4.4 Hz, J 5,3 = 1.4 Hz, 1 H, H-5), 4.13 (dd, J 7b,6 = 4.8 Hz, J 7a,7b = 12.3 Hz, 1 H, H-7b), 2.10, 2.08, 2.06, 1.98 (4 s, each 3 H, 4 × CH3).
13C NMR (75 MHz, CDCl3): δ = 170.55, 169.95, 169.48, 169.43 (q, 4 × C=O), 145.28 (C-1), 98.53 (C-2), 72.45 (C-5), 67.62 (C-6), 66.74 (C-4), 64.56 (C-3), 61.75 (C-7), 20.86, 20.85, 20.78, 20.68 (4 × CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C15H20O9Na: 367.1000; found: 367.0997.
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1,5-Anhydro-2-deoxy-d-altro-hept-1-enitol (23)
A 0.1 M solution of methanolic NaOMe (58 μL) was added to a cooled (0 °C) solution of 22 (20 mg, 58 μmol) in MeOH (1 mL) and stirred at 0 °C for 3 h. The reaction mixture was neutralized (Dowex H+), filtered, and flashed through a short plug of reversed phase (RP) silica gel (500 mg, eluent: H2O). The product containing fractions were lyophilized to give the target compound 23 (9.2 mg, 90%) as a colorless amorphous solid; [α]D 21 –14 (c 0.9, H2O).
1H NMR (600 MHz, D2O): δ = 6.40 (dd, J 1,2 = 6.0 Hz, J 1,3 = 1.5 Hz, 1 H, H-1), 4.81 (dd, J 2,1 = 6.0 Hz, J 2,3 = 2.7 Hz, 1 H, H-2), 4.20–4.18 (m, 1 H, H-3), 4.11 (quin, J = 3.7 Hz, J 7,6b = 7.5 Hz, H-6), 3.94 (dd, J 5,4 = 8.8 Hz, J 5,6 = 4.3 Hz, 1 H, H-5), 3.79–3.75 (m, 2 H, H-7a, H-4), 3.69 (dd, J 7b,7a = 12.0 Hz, J 7b,6 = 7.2 Hz, 1 H, H-7b).
13C NMR (150 MHz, D2O): δ = 144.72 (C-1), 103.35 (C-2), 78.89 (C-5), 71.34 (C-6), 69.94 (C-4), 68.85 (C-3), 62.55 (C-7).
HRMS (ESI): m/z [M – H]– calcd for C7H12O5: 175.0612; found: 175.0615.
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3,4,6,7-Tetra-O-acetyl-1,5-anhydro-1,2-dideoxy-d-altro-heptitol (24)
Pd/C (10%, 2 mg) was added to a solution of compound 22 (21 mg, 61 mmol) in THF (1 mL) and stirred under H2 atmosphere (1 bar) for 12 h at r.t. The reaction mixture was filtered through a syringe filter and the syringe filter was washed with THF (5 mL). The filtrates were combined and concentrated, flashed through a short plug of silica gel (2 g Isolute SI II), and purified via HPLC (hexane/EtOAc, 2:1) to give 24 (18 mg, 85%) as a colorless oil; [α]D 20 +29.8 (c 1.8, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 5.17 (ddd, J 6,7b = 7.8 Hz, J 6,7a = 3.7 Hz, J 6,5 = 2.4 Hz, H-6), 4.95–4.90 (m, 2 H, H-3, H-4), 4.34 (dd, J 7a,7b = 12.0 Hz, J 7a,6 = 3.7 Hz, 1 H, H-7a), 4.20 (dd, J 7a,7b = 12.0 Hz, J 7b,6 = 7.8 Hz, 1 H, H-7b), 4.01 (ddd, J 1a,1b = 12.0 Hz, J 1a,2a = 4.9 Hz, J 1a,2b = 1.9 Hz, 1 H, H-1a), 3.56–3.51 (m, 1 H, H-5), 3.45 (td, J = 12.2, 12.2, 2.3 Hz, 1 H, H-1b), 2.09–2.05 (m, 1 H, H-2a), 2.09, 2.08, 2.03, 2.02 (4 s, each 3 H, 4 × CH3), 1.79–1.71 (m, 1 H, H-2b).
13C NMR (150 MHz, CDCl3): δ = 170.61, 170.30, 170.02, 169.87 (q, 4 × C=O), 78.10 (C-5), 72.23 (C-4), 70.20 (C-6), 69.64 (C-3), 65.21 (C-1), 61.72 (C-7), 30.70 (C-2), 20.91, 20.73, 20.70 (4 × CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C15H22O9Na: 369.1156; found: 369.1155.
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1,5-Anhydro-1,2-dideoxy-d-altro-heptitol (25)
A solution of methanolic 0.1 M NaOMe (52 μL) was added to a cooled (0 °C) solution of 24 (18 mg, 52 μmol) in MeOH (1 mL) and stirred at 0 °C for 3 h. The reaction mixture was neutralized (Dowex H+), filtered, and flashed through a short plug of RP silica gel (500 mg, eluent: H2O). Product containing fractions were lyophilized to give 25 (9.0 mg, 98%) as a colorless amorphous solid; [α]D 20 +9.5 (c 0.9, H2O).
1H NMR (600 MHz, D2O): δ = 3.98 (dt, J = 3.0, 3.0 Hz, J 6,7b = 7.5 Hz, 1 H, H-6), 3.93 (ddd, J 1a,1b = 11.8 Hz, J 1a,2a = 5.0 Hz, J 1a,2b = 1.6 Hz, 1 H, H-1a), 3.74 (dd, J 7a,7b = 11.9 Hz, J 7a,6 = 3.4 Hz, 1 H, H-7a), 3.65 (dd, J 7b,6 = 7.5 Hz, J 7a,7b = 11.9 Hz, 1 H, H-7b), 3.61 (ddd, J 3,2b = 11.4 Hz, J 3,4 = 8.2 Hz, J 3,2a = 5.0 Hz, 1 H, H-3), 3.45 (td, J 1b,1a = J 1b,2b = 12.2 Hz, J 1b,2a = 1.9 Hz, 1 H, H-1b), 3.34 (dd, J 4,3 = 8.2 Hz, J 4,5 = 9.8 Hz, 1 H, H-4), 3.31 (dd, J 5,6 = 2.8 Hz, J 5,4 = 9.8 Hz, 1 H, H-5), 1.95 (ddt, J 2a,2b = 13.1 Hz, J 2a,3 = 5.1 Hz, J 2a,1b = 1.8 Hz, 1 H, H-2a), 1.61–1.54 (ddd, J = 5.0 Hz, J = 11.6 Hz, J = 12.8 Hz, 1 H, H-2b).
13C NMR (150 MHz, D2O): δ = 81.64 (C-5), 73.21 (C-3), 72.85 (C-4), 72.69 (C-6), 66.57 (C-1), 62.61 (C-7), 33.66 (C-2).
HRMS (ESI): m/z [M – H]– calcd for C7H14O5: 177.0768; found: 177.0767.
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7-O-Triisopropylsilyl-1,5-anhydro-d-glycero-d-gluco-heptitol (26)
TIPSCl (39 μL, 0.184 mmol) was added dropwise to a solution of 21 (34 mg, 0.175 mmol) and imidazole (36 mg, 0.526 mmol) in freshly distilled THF (5 mL) at 0 °C under argon. After 2 h, additional TIPSCl (13 μL) was added and the reaction mixture was stirred for further 12 h. Additional reagents were added portionwise every 60 min (in total 60 μL TIPSCl and 24 mg imidazole) and the mixture was stirred for further 12 h, when TLC showed complete consumption of the starting material. The mixture was concentrated, and partitioned between CH2Cl2 (10 mL) and sat. aq NH4Cl (6 mL). The aqueous phase was reextracted with CH2Cl2 (2 × 5 mL) and the combined organic phase was washed with brine (10 mL), dried (MgSO4), and concentrated. Purification of the residue on silica gel (EtOAc/toluene, 3:1) and HPLC (CHCl3/EtOH, 25:1) gave 26 as a colorless oil (20 mg, 32%); [α]D 20 +7.7 (c 1.0, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 4.49 (d, J 4,OH = 1.0 Hz, 1 H, OH-4), 4.14 (br s, 1 H, OH-3), 3.93 (dd, J 1e,1a = 11.1 Hz, J 1e,2 = 5.4 Hz, 1 H, H-1e), 3.86 (dd, J 7a,7b = 12.1 Hz, J 6,7a = 3.4 Hz, 1 H, H-7a), 3.81–3.75 (m, 2 H, H-6, H-7b), 3.65 (dddd, J 2,1a = 10.4 Hz, J 2,3 = 9.0 Hz, 1 H, H-2), 3.58 (br t, J 4,3 = 9.0 Hz, 1 H, H-4), 3.53 (d, J = 2.6 Hz, 1 H, OH-2), 3.50 (br t, J = 8.9 Hz, 1 H, H-3), 3.19 (dd, J 5,6 = 5.2 Hz, J 5,4 = 8.5 Hz, 1 H, H-5), 3.17 (m, 2 H, H-1a, OH-6), 1.17–1.09 [m, 3 H, 3 × SiCH(CH3)2], 1.08–1.05 [m, 18 H, 3 × SiCH(CH 3)2].
13C NMR (150 MHz, CDCl3): δ = 78.55 (C-3), 77.26 (C-5), 74.68 (C-6), 73.95 (C-4), 69.49 (C-2), 69.16 (C-1), 63.77 (C-7), 17.88, 17.88, 17.86 [SiCH(CH3)2], 11.85 [Si(CH(CH3)2].
HRMS (ESI): m/z [M + Na]+ calcd for C16H34O6SiNa: 373.2017; found: 373.2015.
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2,3,4,6-Tetra-O-benzyl-7-O-triisopropylsilyl-1,5-anhydro-d-glycero-d-gluco-heptitol (27)
NaH (60% in mineral oil, 18 mg, 0.457 mmol) was added to a stirred solution of 26 (20 mg, 57 μmol) in anhyd DMF (3 mL) under argon. After 20 min, BnBr (33 μL, 0.274 mmol) was added at 0 °C and the mixture was stirred at r.t. for 15 h. Additional BnBr (0.8 equiv, 5.5 μL) was then added at 0 °C and stirring was continued for 1.5 h. MeOH (20 μL) was added dropwise at 0 °C and the mixture was warmed to r.t. The solution was diluted with Et2O (10 mL), and washed with H2O (5 mL) and sat aq NaHCO3 (5 mL). The aqueous layers were extracted once more with Et2O (10 mL), the combined organic layers were dried (MgSO4), filtered, and the solvent was removed under vacuum. The residual oil was purified by HPLC (hexane/EtOAc, 20:1) to give 27 as a colorless oil (22 mg, 54%); [α]D 20 +3.2 (c 1.1, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.36–7.14 (m, 20 H, 4 × ArH), 4.97–4.61 (m, 8 H, 4 × OCH2Ph), 4.02 (dd, J 1e,1a = 11.2 Hz, J 1e,2 = 5.0 Hz, 1 H, H-1e), 3.89 (dd, J 7a,7b = 8.8 Hz, J 7a,6 = 4.0 Hz, 1-H, H-7a), 3.87–3.80 (m, 2 H, H-7b, H-6), 3.66 (app t, J = 8.8 Hz, 1 H, H-4), 3.62 (t, J 3,4 = J 3,2 = 8.6 Hz, 1 H, H-3), 3.58 (ddd, J 2,3 = 8.6 Hz, J 2,1e = 5.2 Hz, 1 H, H-2), 3.49 (br d, J 5,4 = 9.6 Hz, 1 H, H-5), 3.17 (app t, J = 10.6 Hz, 1 H, H-1a), 1.07–0.97 [m, 21 H, 3 × SiCH(CH 3)2].
13C NMR (150 MHz, CDCl3): δ = 138.81, 138.77, 138.36 (q, ArC), 128.46, 128.38, 128.29, 128.22, 127.84, 127.81, 127.78, 127.70, 127.66, 127.56, 127.43, 127.35 (ArC), 86.81 (C-3), 80.71 (C-6), 80.57 (C-5), 78.66 (C-2), 78.30 (C-4), 75.56, 74.79, 73.22, 72.99 (4 × OCH2Ph), 68.17 (C-1), 63.95 (C-7), 18.01 [Si(CH(CH3)2], 11.88 [Si(CH(CH3)2].
HRMS (ESI): m/z [M + Na]+ calcd for C44H58O6SiNa: 733.3895; found: 733.3894.
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2,3,4,6-Tetra-O-benzyl-1,5-anhydro-d-glycero-d-gluco-heptitol (28)
Compound 27 (22 mg, 31 μmol) was dissolved in anhyd THF (3 mL), TBAF (124 μL of a 1 M solution in THF) was added and the reaction mixture was stirred at r.t. for 17 h. The solution was diluted with Et2O (10 mL) and successively washed with sat. aq NH4Cl (5 mL) and H2O (5 mL). The aqueous layers were reextracted with Et2O (10 mL), the combined organics were dried (MgSO4), concentrated, and the remaining oil was directly purified by HPLC (toluene/EtOAc, 6:1) to give 28 (14 mg, 82%) as a colorless oil; [α]D 20 +18.1 (c 0.7, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.34–7.14 (m, 20 H, 4 × ArH), 4.90, 4.74, 4.66, 4.63 (8 H, 4 × OCH 2Ph), 4.02 (dd, J 1e,1a = 11.3 Hz, J 1e,2 = 5.2 Hz, 1 H, H-1e), 3.72 (ddd, J 6,5 = 1.2 Hz, J 6,7b = 4.2 Hz, J 6,7a = 6.4 Hz, 1 H, H-6), 3.67 (ddd, J 7a,7b = 11.9 Hz, J 7a,OH = 2.3 Hz, 1 H, H-7a), 3.64 (dd, J 3,4 = 8.3 Hz, J 3,2 = 9.0 Hz, 1 H, H-3), 3.59 (ddd, J 2,1a = 10.3 Hz, J 2,1e = 5.2 Hz, J 2,3 = 8.3 Hz, 1 H, H-2), 3.59–3.55 (m, 1 H, H-7b), 3.51 (dd, J 5,4 = 10.0 Hz, J 6,5 = 1.2 Hz, 1 H, H-5), 3.46 (dd, J 4,5 = 10.0 Hz, J 4,3 = 8.3 Hz, 1 H, H-4), 3.15 (dd, J 1a,2 = 10.4 Hz, 1 H, H-1a), 2.04 (dd, J = 9.0 Hz, J = 2.3 Hz, 1 H, CH2OH).
13C NMR (150 MHz, CDCl3): δ = 138.52, 138.15, 138.11, 137.78 (q, 4 × ArC), 128.59–127.69 (ArCH), 86.72 (C-3), 80.81 (C-5), 78.61 (C-2), 78.51 (C-6), 77.51 (C-4), 75.65, 74.86, 73.26, 71.97 (4 × OCH2Ph), 68.25 (C-1), 61.41 (C-7).
HRMS (ESI): m/z [M + Na]+ calcd for C35H38O6Na: 577.2561; found: 577.2559.
#
2,3,4,6-Tetra-O-benzyl-7-O-[bis(benzyloxy)phosphoryl]-1,5-anhydro-d-glycero-d-gluco-heptitol (29)
Compound 28 (14 mg, 25 μmol) was twice co-evaporated with toluene and dried under vacuum for 12 h. The material was dissolved in anhyd CH2Cl2 (3 mL), dibenzyl-N,N-diisopropylaminophosphoramidite reagent (8 μL, 25 µmol) was added, and the mixture was stirred at r.t. for 20 min. 1H-Tetrazole (0.45 M in MeCN, 56 μL, 25 μmol) was added slowly and the solution was stirred at r.t. for 20 min. Additional reagent was added portionwise every 20 min until the starting material was consumed (in total 12 μL = 36 mmol amidite reagent and 79 μL of tetrazole solution were used). The solution was cooled to –78 °C and a solution of mCPBA (70%, 16 mg, 63 μmol) in CH2Cl2 (1 mL) was added slowly to the reaction mixture. After 60 min, Et3N (8.8 μL, 63 mmol) was added and the mixture was allowed to warm up to r.t. The solution was diluted with CH2Cl2 (10 mL), washed with sat. aq NaHCO3 (2 × 5 mL), dried (MgSO4), and concentrated. The residue was purified by HPLC (toluene/EtOAc, 8:1, containing 0.5% Et3N) to give 29 as a colorless oil (18 mg, 22 mmol, 85%); [α]D 20 +4.2 (c 0.9, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.34–7.12 (m, 30 H, ArH), 5.03–4.56 (m, 12 H, 6 × OCH 2Ph), 4.15 (dd, J 7,P = 6.8 Hz, J 7a or 7b/6 = 5.8 Hz, 2 H, H-7a, H-7b), 3.97 (dd, J 1e,1a = 11.3 Hz, J 1e,2 = 5.2 Hz, 1 H, H-1e), 3.92 (dt, J 6/7a or 7b = 5.8 Hz, 1 H, H-6), 3.59 (t, J 3,4 = J 3,2 = 8.7 Hz, 1 H, H-3), 3.54 (dd, J 2,1a = 10.5 Hz, 1 H, H-2), 3.49 (dd, J 4,5 = 9.8 Hz, 1 H, H-4), 3.42 (br d, J 4,5 = 10.0 Hz, 1 H, H-5), 3.13 (t, J= 11.0 Hz, Hz, 1 H, H-1a).
13C NMR (150 MHz, CDCl3): δ = 128.58–128.22 and 127.96–127.57 (ArC), 86.59 (C-3), 80.02 (C-5), 75.51 (C-2), 78.26 (J P,6 = 7.3 Hz, C-6), 77.68 (C-4), 75.54, 74.83, 73.23, 72.84 (4 × OCH2Ph), 69.33–69.18 (POCH2Ph), 68.10 (C-1), 67.49 (J P,7 = 5.8 Hz, C-7).
31P NMR (242.94 MHz, CDCl3): δ = –0.93.
HRMS (ESI): m/z [M + H]+ calcd for C49H51O9P: 815.3343; found: 815.3345.
#
1,5-Anhydro-d-glycero-d-gluco-heptitol-7-phosphate Triethylammonium Salt (30)
A suspension of 29 (18 mg, 0.022 mmol), and 10% Pd/C (2 mg) in 5:3:2 EtOH/EtOAc/H2O (3 mL) was stirred under H2 atmosphere (1 bar) at r.t. for 48 h. Fresh catalyst (2 mg) was added after 16 h and 32 h. The reaction mixture was then filtered through a short plug of Celite and the Celite bed was successively washed with H2O (20 mL). The filtrate was passed through a short gel column (PD-10 prepacked, 8.3 mL G-25, H2O). Et3N (6 μL) was added and the solution was concentrated. The residue was dissolved in H2O (3 mL), aliquoted into three batches of 1 mL, filtered through a syringe filter, and lyophilized to give three batches of compound 30 (1.9, 2.8, and 2.1 mg, respectively, in total 6.8 mg, 99%) as a colorless amorphous solid; [α]D 20 +35.2 (c 0.4, H2O).
1H NMR (600 MHz, D2O): δ = 4.12 (dt, J 6,7a = 3.8 Hz, J 6,7b = 7.3 Hz, 1 H, H-6), 3.95 (dd, J 1e,1a = 11.0 Hz, J 1e,2 = 5.3 Hz, 1 H, H-1e), 3.94 (br d, J 7a,7b = 11.9 Hz, J 7a,6 = 3.8 Hz, 1 H, H-7a), 3.82 (dd, J 7b,7a = 11.4 Hz, J 7b,P = 7.3 Hz, 1 H, H-7b), 3.58 (ddd, J 2,3 = 9.1 Hz, J 1e,2 = 5.5 Hz, 1 H, H-2), 3.53 (br t, J 4,3 = 9.4 Hz, J 4,5 = 9.8 Hz, 1 H, H-4), 3.41 (dd, J 5,6 = 2.5 Hz, J 5,4 = 9.8 Hz, 1 H, H-5), 3.39 (br t, J 2,3 = J 3,4 = 9.1 Hz, 1 H, H-3), 3.22 (t, J 1a,2 = 10.3 Hz, J 1a,1e = 11.0 Hz, 1 H, H-1a), 3.18 (q, J= 7.3 Hz, 2.6 H, NCH2), 1.26 (t, J= 7.3 Hz, 4 H, NCH2CH 3).
13C NMR (150 MHz, D2O): δ = 81.52 (C-5), 78.25 (C-3), 71.59 (J 6,P = 6.0 Hz, C-6), 70.58 (C-4), 69.91 (C-2), 69.74 (C-1), 64.84 (J 7,P = 4.4 Hz, C-7), 47.29 (NCH2), 8.85 (NCH2 CH3).
31P NMR (242 MHz, D2O): δ = 3.95.
HRMS (ESI): m/z [M + H]+ calcd for C7H15O9P: 275.0526; found: 275.0525.
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3-O-Benzyl-1,2-O-isopropylidene-7-O-triisopropylsilyl-d-glycero-α-d-gluco-heptofuranose (31)
TIPSCl (165 μL, 772 μmol) was added dropwise to a solution of triol 14 (250 mg, 735 μmol) and DABCO (247 mg, 2.2 mmol) in freshly distilled THF (10 mL) at 0 °C under argon. The following reagents were added until the starting material was completely consumed: TIPSCl (165 μL, 0.772 mmol) and DABCO (247 mg, 726 μmol) after 18 h, and TIPSCl (41 μL, 192 mmol) and DABCO (62 mg, 18 μmol) after 42 h. The reaction mixture was concentrated to dryness, taken up in CH2Cl2 (10 mL) and sat. aq NH4Cl (6 mL) was added. The aqueous phase was reextracted with CH2Cl2 (2 × 5 mL) and the combined organic phases were washed with brine (10 mL), dried (MgSO4), and concentrated. The residue was purified on silica gel (toluene/EtOAc, 10:1) to give 31 as a colorless oil (260 mg, 71%); [α]D 20 –26.4 (c 1.1, CDCl3).
1H NMR (600 MHz, CDCl3): δ = 7.37–7.28 (m, 5 H, C6H5), 5.95 (d, J 1,2 = 3.7 Hz, 1 H, H-1), 4.69 (d, J = 11.5 Hz, 1 H, OCH 2Ph), 4.63 (d, J = 11.5 Hz, 1 H, OCH 2Ph), 4.59 (d, J 2,1 = 3.7 Hz, 1 H, H-2), 4.26 (dd, J 4,3 = 3.0 Hz, J 4,5 = 7.5 Hz, 1 H, H-4), 4.20 (d, J 3,4 ≅ 3.0 Hz, 1 H, H-3), 4.11 (q, J 5,4 = J 5,6 = 7.0 Hz, 1 H, H-5), 4.00 (dd, J 7a,7b = 10.2 Hz, J 7a,6 = 4.9 Hz, 1 H, H-7a), 3.93 (dd, J 7b,6 = 4.0 Hz, 1 H, H-7b), 3.75–3.70 (m, 1 H, H-6), 3.49 (d, J = 7.1 Hz, 1 H, 5-OH), 2.98 (d, J = 5.4 Hz, 1 H, 6-OH), 1.48 (s, 3 H, CH3), 1.32 (s, 3 H, CH3), 1.17–1.10 [m, 3 H, 3 × SiCH(CH3)3], 1.09–1.05 [m, 18 H, 3 × SiCH(CH 3)2].
13C NMR (150 MHz, CDCl3): δ = 137.22 (q, ArC), 128.54, 128.06, 127.83 (ArC), 111.77 [C(CH3)2], 105.11 (C-1), 82.56 (C-3), 82.10 (C-2), 80.46 (C-4), 72.51 (OCH2Ph), 71.80 (C-6), 70.63 (C-5), 65.47 (C-7), 26.77, 26.26 [CH(CH3)2], 17.91 [SiCH(CH3)2], 11.78 [SiCH(CH3)2].
HRMS (ESI): m/z [M + H]+ calcd for C26H44O7Si: 497.2929; found: 497.2923.
#
3,5,6-Tri-O-benzyl-1,2-O-isopropylidene-7-O-triisopropylsilyl-d-glycero-α-d-glucofuranose (32)
Triflic acid (0.25 μL, 2.8 μmol) was added to a solution of diol 31 (26 mg, 60 μmol) and benzyl 2,2,2-trichloroacetimidate (39 μL, 242 μmol) in anhyd CH2Cl2 (3 mL) at 0 °C under argon. After 2 h, additional benzyl 2,2,2-trichloroacetimidate (20 μL, 124 μmol) was added and the mixture stirred for 30 min. Sat. aq NaHCO3 (2 mL) was added, the phases were separated and the aqueous phase was reextracted with CH2Cl2 (2 mL). The combined organic phases were dried (MgSO4), concentrated, and the residue was purified by chromatography (silica gel 60, hexane/EtOAc, 15:1) to give 32 (30 mg, 85%) as a syrup.
1H NMR (300 MHz, CDCl3): δ = 7.45–7.18 (m, 15 H, 3 × ArH), 5.89 (d, J 1,2 = 3.7 Hz, 1 H, H-1), 4.87–4.57 (m, 4 H, 2 × OCH 2Ph), 4.55 (d, J 2,1 = 3.7 Hz, 1 H, H-2), 4.48–4.39 (m, 2 H, OCH 2Ph), 4.32 (dd, J 4,3 = 3.0 Hz, J 4,5 = 9.5 Hz, 1 H, H-4), 4.17 (dd, J 5,6 = 0.8 Hz, 1 H, H-5), 4.08 (d, J = 3.0 Hz, 1 H, H-3), 4.04–3.93 (m, 3 H, H-6, H-7a, H-7b), 1.44 and 1.29 [C(CH3)2], 1.13–1.00 [m, 21 H, 3 × Si(CH(CH3)2].
13C NMR (75 MHz, CDCl3): δ = 139.39, 138.83, 137.70 (q, ArC), 128.51, 128.37, 128.26, 128.20, 128.09, 127.70, 127.69, 127.49, 127.41, 127.35, 127.08 (ArC), 111.65 [C(CH3)2], 105.24 (C-1), 82.24 (C-3), 81.76 (C-6), 81.50 (C-2), 79.06 (C-4), 76.89 (C-5), 73.22, 73.13, 71.91 (3 × OCH2Ph), 63.61 (C-7), 26.78, 26.38 [C(CH3)2], 18.04 [Si(CH(CH3)2], 11.92 [Si(CH(CH3)2].
LC-MS: m/z [M + NH4]+ calcd for C40H60NO7Si: 694.4; found: 694.8.
#
3,5,6-Tri-O-benzyl-1,2-O-isopropylidene-d-glycero-α-d-gluco-heptofuranose (33)
A 1 M solution of TBAF in THF (126 μL) was added to a solution of 32 (30 mg, 42 μmol) in anhyd THF (3 mL) and the mixture was stirred at r.t. for 5 h. The solution was diluted with Et2O (10 mL) and successively washed with sat. aq NH4Cl (5 mL) and H2O (5 mL). The aqueous layers were extracted once more with Et2O (10 mL), the combined organic phases were dried (MgSO4), concentrated, and the residual oil was purified by chromatography (hexane/EtOAc, 3:1) to give 33 as a colorless oil (14 mg, 60%); [α]D 20 –30 (c 1.2, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.37–7.20 (m, 15 H, ArH), 5.90 (d, J 1,2 = 3.7 Hz, 1 H, H-1), 4.88 (d, J = 11.1 Hz, 1 H, OCH 2Ph), 4.67 (d, J = 11.7 Hz, 1 H, OCH 2Ph), 4.66 (d, J = 11.7 Hz, 1 H, OCH 2Ph), 4.62 (d, J = 11.5 Hz, 1 H, OCH 2Ph), 4.58 (d, J 2,1 = 3.7 Hz, 1 H, H-2), 4.45 (d, J = 11.5 Hz, 2 H, OCH 2Ph), 4.28 (dd, J 4,3 = 3.0 Hz, J 4,5 ≅ 9.0 Hz, 1 H, H-4), 4.22 (dd, J 4,5 ≅ 9.0 Hz, J 5,6 = 1.4 Hz, 1 H, H-5), 4.10 (d, J 3,4 = 3.0 Hz, 1 H, H-3), 3.95–3.85 (m, 3 H, H-6, H-7a, H-7b), 2.43 (dd, J = 2.8 Hz, J = 8.6 Hz, 1 H, 7-OH), 1.47 [s, 3 H, C(CH3)2], 1.30 [s, 3 H, C(CH3)2].
13C NMR (150 MHz, CDCl3): δ = 138.53, 138.41, 137.38 (q, ArC), 128.52, 128.46, 128.40, 128.35, 127.98, 127.70, 127.69, 127.60, 127.45 (ArCH), 111.96 [C(CH3)2], 105.16 (C-1), 81.99 (C-3), 81.44 (C-2), 79.99 (C-6), 78.91 (C-4), 76.92 (C-5), 74.07, 71.99, 71.99 (3 × OCH2Ph), 61.35 (C-7), 26.87 and 26.36 [C(CH3)2].
HRMS (ESI): m/z [M + Na]+ calcd for C31H36O7Na: 543.2353; found: 543.2357.
#
3,5,6-Tri-O-benzyl-7-O-[bis(benzyloxy)phosphoryl]-1,2-O-isopropylidene-d-glycero-α-d-gluco-heptofuranose (34)
Alcohol 33 (12 mg, 23 μmol) was twice co-evaporated with toluene, dried in vacuo for 12 h, and taken up in anhyd CH2Cl2 (3 mL). Dibenzyl-N,N-diisopropylaminophosphoramidite (8 μL, 25 μmol) was added and the mixture was stirred at r.t. for 20 min. 1H-Tetrazole [(0.3 M in MeCN (3 wt%), 75 μL, 25 μmol] was added very slowly and the solution was stirred at r.t. Additional reagent was added portionwise (0.4 equiv each) until the starting material was consumed (22 μL, 69 μmol of phosphoramidite and 230 μL of tetrazole, 69 μmol). The reaction mixture was stirred for further 30 min, cooled to –78 °C, and a solution of mCPBA (70%, 26 mg, 105 μmol) in CH2Cl2 (1 mL) was added slowly. The reaction mixture was stirred for 30 min. Et3N (9.7 μL, 70 µmol) was added slowly and the mixture was warmed to r.t. The mixture was diluted with CH2Cl2 (10 mL), washed with sat. aq NaHCO3 (2 × 5 mL), dried (MgSO4), and concentrated. The residue was purified by chromatography (toluene/EtOAc, 6:1 containing 0.5% Et3N) to give 34 as a colorless oil (14 mg, 18 μmol, 78%); [α]D 21 –22.4 (c 1.4, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.32–7.15 (m, 25 H, ArH), 5.87 (d, J 1,2 = 3.7 Hz, 1 H, H-1), 5.03–4.96 [m, 4 H, P(OCH 2Ph)2], 4.75 (d, J = 11.4 Hz, 1 H, OCH 2Ph), 4.66 (s, 2 H, OCH 2Ph), 4.59 (d, J = 11.5 Hz, 1 H, OCH 2Ph), 4.55 (d, J 2,1 = 3.7 Hz, 1 H, H-2), 4.42 (d, J = 11.5 Hz, 1 H, OCH 2Ph), 4.39 (d, J = 11.4 Hz, 1 H, OCH 2Ph), 4.37–4.30 (m, 2 H, H-7a, H-7b), 4.29 (dd, J 4,3 = 3.1 Hz, J 4,5 = 9.4 Hz, 1 H, H-4), 4.11 (dd, J 5,4 = 9.4 Hz, J 5,6 = 1.2 Hz, 1 H, H-5), 4.09 (ddd, J 6,5 = 1.3 Hz, J 6,7 = 4.4, 6.6 Hz, 1 H, H-6), 4.06 (br d, J 3,4 = 3.1 Hz, 1 H, H-3), 1.43 [s, 3 H, C(CH3)2], 1.28 [s, 3 H, C(CH3)2].
13C NMR (150 MHz, CDCl3): δ = 138.54, 138.44, 137.43 (q, ArC), 136.00, 135.96 (q, POCH2ArC), 128.46, 128.45, 128.32, 128.29, 128.25, 128.19, 127.88, 127.87, 127.79, 127.60, 127.51, 127.44, 127.37, 127.30 (ArCH), 111.86 [C(CH3)2], 105.19 (C-1), 82.06 (C-3), 81.50 (C-2), 79.28 (d, J P,C = 7.7 Hz, C-6), 78.72 (C-4), 76.50 (C-5), 73.43, 72.86, 72.00 (3 × OCH2Ph), 69.12, 69.10 (2 d, J P,C = 5.3 Hz, POCH2Ph), 67.46 (d, J P,C = 5.7 Hz, C-7), 26.83 and 26.37 [C(CH3)2].
31P NMR (242.94 MHz, CDCl3): δ = –0.98.
HRMS (ESI): m/z [M + Na]+ calcd for C45H49O10PNa: 803.2956; found: 803.2959.
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d-Glycero-d-gluco-heptopyranose 7-Phosphate (36)
A suspension of furanose 34 (14 mg, 18 μmol) and 10% Pd/C (2 mg) in EtOH/EtOAc/H2O (1.5:0.9:0.6, 3 mL) was stirred under H2 atmosphere (1 bar) at r.t. for 16 h. The reaction mixture was filtered through a syringe filter and the filter was additionally washed with HPLC grade H2O (10 mL). Et3N (10 μL, 72 μmol) was added to the filtrate and the filtrate was concentrated until no solvent except H2O were present. The remaining solution was passed through a short PD-10 column (HPLC grade H2O). Product-containing fractions were pooled and lyophilized to give the 1,2-acetonide 35 as the triethylammonium salt; colorless solid (9.6 mg, 99%). Further processing was performed in two batches.
Batch 1: The phosphate 35 (4 mg, 7.9 μmol) was dissolved in deionized H2O (2.6 mL) and rinsed through a short column of Dowex H+. TFA (260 μL) was added to the filtrate and the solution was stirred for 12 h at r.t. The solution was co-evaporated with H2O three times until no more TFA was present. Et3N (20 μmol, 2.8 μL) was added and the mixture was again co-evaporated with H2O (2 × 5 mL). The residue was taken up in H2O (0.5 mL) and purified via gel chromatography (P-2, H2O). Product-containing fractions were pooled and lyophilized to give the target compound as the free acid; white foam (2.5 mg, 99%).
Batch 2: The previous phosphate (4.7 mg, 9.3 μmol) was dissolved in deionized H2O (3 mL) and rinsed through a short column of Dowex H+, TFA (300 μL) was added and the reaction mixture was stirred for 12 h. The mixture was co-evaporated with H2O three times until no more TFA was present. Et3N (20 μmol, 2.8 μL) was added and the mixture was again co-evaporated with H2O (2 × 5 mL). The residue was taken up in H2O (0.5 mL) and purified via gel chromatography (P-2, H2O). Product-containing fractions were pooled and lyophilized to give the target compound 36 as the free acid; white foam (2.7 mg, 99%). Both batches were combined (5.2 mg); [α]D 21 +17.6 (c 0.5, H2O).
1H NMR (600 MHz, D2O): δ (α-anomer) = 5.18 (d, J 1,2 = 3.8 Hz, 1 H, H-1α), 4.08 (ddd, J 7b,6 = 7.4 Hz, J 7a,6 = 3.6 Hz, 1 H, H-6), 3.95 (ddd, J 7a,7b = 11.7 Hz, J 7a,P = 5.0 Hz, J 7a,6 = 3.9 Hz, 1 H, H-7a), 3.89 (dd, J 5,4 = 10.1 Hz, J 5,6 = 3.0 Hz, 1 H, H-5), 3.82 (ddd, J 7b,P = 2.3 Hz, J 7b,6 = 7.2 Hz, J 7a,7b = 11.7 Hz, 1 H, H-7b), 3.65 (app t, J 3,2 ≅ J 3,4 ≅ J = 9.4 Hz, 1 H, H-3α), 3.56 (dd, J 3,4 = 8.9 Hz, J 4,5 = 10.2 Hz, 1 H, H-4), 3.52 (dd, J = 3.8, 9.8 Hz, 1 H, H-2),
1H NMR (600 MHz, D2O): δ (β-anomer) = 4.58 (d, J ≅ 8 Hz, 1 H, H-1 β), 4.11 (ddd, J 7b,6 = 7.1 Hz, J 7a,6 = 3.7 Hz, 1 H, H-6), 3.94 (ddd, J 7a,7b = 11.7 Hz, J 7a,P = 4.6 Hz, 1 H, H-7a), 3.83 (ddd, J 7b,P = 2.3 Hz, 1 H, H-7b), 3.57 (dd, J 3,4 = 8.9 Hz, J 4,5 = 10.0 Hz, 1 H, H-4), 3.51 (dd, J 6,5 = 3.1 Hz, J 5,4 = 10.1 Hz, 1 H, H-5), 3.43 (t, J = 9.0 Hz, 1 H, H-3), 3.22 (dd, J 2,3 = 9.5 Hz, J 1,2 = 7.9 Hz, 1 H, H-2).
13C NMR (150 MHz, D2O): δ = 96.96 (C-1β), 92.82 (C-1α), 76.97 (C-5β), 76.66 (C-3β), 74.88 (C-2β), 73.74 (C-3α), 72.23 (C-2α), 72.15 (C-5α), 72.04 (J P,C = 6.6 Hz, C-6α), 71.73 (J P,C = 6.4 Hz, C-6β), 71.09 (C-4α), 70.97 (C-4β), 65.23 (J P,C = 5.1 Hz, C-7α), 64.94 (J P,C = 5.1 Hz, C-7β).
31P NMR (242 MHz, D2O): δ = 4.30.
LC-MS: m/z [M – H]– calcd for C7H15O10P: 289.033; found: 289.0334.
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N-Benzyloxycarbonyl-1,5-dideoxy-1,5-imino-d-glucitol (38)
To a solution of deoxynojirimycin hydrochloride (37; 1,5-dideoxy-1,5-imino-d-glucitol hydrochloride, 780 mg, 4 mmol) in MeOH/H2O (3:2, 55 mL) at 0 °C was added NaHCO3 (1.8 g, 0.02 mol) and then benzyl chloroformate (0.7 mL, 4.8 mmol) was added dropwise. The mixture was allowed to warm to r.t. and was stirred until complete conversion (2 h) as indicated by TLC (MeOH/EtOAc, 1:1). Detection was performed by spraying with ninhydrin stain followed by heating at 200 °C. The solvents were evaporated and the residue was partitioned between H2O (60 mL) and EtOAc (40 mL). The organic phase was separated and the aqueous phase was reextracted with EtOAc (3 × 25 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated. The residue was chromatographed on silica gel (EtOAc → EtOAc/MeOH, 1:1) to give 38 as a colorless syrup (763 mg, 67%).
1H NMR data were in agreement with those reported.[23]
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2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-d-glucitol (39)
To a solution of 38 (757 mg, 2.55 mmol) in anhyd pyridine (27 mL) were added DMTrCl (1.55 g, 4.59 mmol) and DMAP (27 mg, 0.2 mmol) under argon and the mixture was stirred at r.t. for 5 h. Then EtOAc (3 mL) was added and the solution was washed with sat. aq NaHCO3 (30 mL) and H2O (30 mL). The aqueous phase was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried (MgSO4) and concentrated. The residue was dried under vacuum and then dissolved in anhyd DMF (45 mL). The solution was cooled to 0 °C (ice/water bath) and NaH (60% in mineral oil, 0.816 g, 0.02 mol) was added. After a few minutes, benzyl bromide (2.44 mL, 0.02 mol) was added dropwise over a 10 min period. The mixture was stirred for 2 h at r.t. under argon, and then cold H2O (20 mL) was added. The mixture was extracted with Et2O (3 × 5 mL). The combined organic phases were washed with H2O, dried (MgSO4), filtered, and concentrated. A solution of the residue in 80% aq AcOH (30 mL) was stirred at r.t. for 2.5 h. After concentration and coevaporation with toluene (3 ×), the residue was chromatographed on silica gel (EtOAc/hexane, 1:9 → 3:7) to afford 39 (820 mg, 57%) as a colorless oil; [α]D 21 –0.9 (c 0.7, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.35–7.26 (m, 15 H, ArH), 5.11 (br s, 2 H, CH2, Cbz), 4.76–4.59 (m, 5 H, OCH 2Ph), 4.50 (d, part B of AB system, H-b, 1 H, J a,b = 11.7, OCH 2Ph), 4.00–3.79 (m, 3 H, H-5, H-6a, H-6b), 3.76–3.58 (m, 5 H, H-2, H-3, H-4, H-1a, H-1b).
13C NMR (100 MHz, CDCl3): δ = 157.8 (C=O), 138.2, 138.1, 136.4 (q, ArC), 128.6, 128.6, 128.5, 128.5, 128.2, 128.1, 128.0, 127.9, 127.9, 127.9, 127.9, 127.8 (ArCH), 82.3 (C-3), 77.6 (C-4), 75.6 (C-2), 73.6, 73.4, 71.4 (3 × ΟCH2Ph), 67.6 (CH2, Bn, Cbz), 61.6 (C-6), 59.1 (C-5), 43.1 (C-1).
HRMS (ESI): m/z [M + H]+ calcd for C35H37NO6: 568.2694; found: 568.2641.
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2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1,5,6,7-tetradeoxy-1,5-imino-d-gluco-hept-6-enopyranose (40)
DMSO (1.38 mmol, 0.1 mL) was added dropwise to a solution of oxalyl chloride (1.15 mmol, 0.57 mL, 2 M in CH2Cl2) in CH2Cl2 (6 mL) at –70 °C. After stirring for 10 min, a solution of 39 (650 mg, 1.15 mmol) in CH2Cl2 (6 mL) was added dropwise. The mixture was stirred for 1 h at –70 °C and then Et3N (0.35 mL, 2.53 mmol) was added dropwise. After 5 min, the reaction was left to warm up to r.t. and stirred until complete conversion (ca. 1 h). Then H2O was added and the aqueous layer was extracted with EtOAc (3 ×). The combined organic layers were washed with H2O, dried (MgSO4), and concentrated. The crude aldehyde was dried in vacuum.
n-BuLi (2.76 mmol, 1.1 mL) was added to a suspension of methyltriphenylphosphonium bromide (2.87 mmol, 1.03 g) in THF (4 mL) under argon at 0 °C and the mixture was then stirred at r.t. for 1 h. A solution of the crude aldehyde in THF (4 mL) was added dropwise at –70 °C. The solution was stirred at –60 °C for 1 h, then allowed to warm to r.t., and stirred overnight. After cooling to 0 °C, H2O was added, and the aqueous layer was extracted with EtOAc (3 ×). The combined organic phases were washed with brine, dried (MgSO4), and concentrated. The residue was purified by chromatography on silica gel (EtOAc/hexane, 1:9) to afford the alkene 40 (400 mg, 62%) as a colorless oil; [α]D 22 +22.4 (c 1.0, CHCl3).
1H NMR (400 MHz, CDCl3): δ = 7.40–7.22 (m, 15 H, ArH), 5.88 (ddd, J 6,5 = 5.3 Hz, J 6,7a = 9.9 Hz, J 6,7b = 16.7 Hz, 1 H, H-6), 5.22–5.10 (m, 4 H, H-7a, H-7b, CH2, Cbz), 4.76 (dd, J 6,5 = 5.3 Hz, J 5,4 = 4.7 Hz, 1 H, H-5), 4.71–4.46 (m, 6 H, 3 × OCH 2Ph), 3.95 (dd, J 1a,1e = 13.9 Hz, J 1a,2 = 6.0 Hz, 1 H, H-1a), 3.78–3.67 (m, 2 H, H-2, H-3), 3.58 (br t, J 3,4 = 4.4 Hz, J 4,5 = 4.7 Hz, 1 H, H-4), 3.44 (dd, J 1e,2 = 2.9 Hz, 1 H, H-1e).
13C NMR (100 MHz, CDCl3): δ = 156.3 (C=O), 138.3, 138.1, 136.9 (q, ArC), 134.9 (C-6), 128.5, 128.5, 128.5, 128.0, 127.9, 127.9, 127.8, 127.7 (ArCH), 116.1 (C-7), 80.5 (C-3), 78.4 (C-4), 77.4 (C-2), 72.8, 72.4, 71.2 (OCH2Ph), 67.4 (OCH2Ph, Cbz), 57.5 (C-5), 40.7 (C-1).
HRMS (ESI): m/z [M + H]+ calcd for C36H37NO5: 564.2744; found: 564.2724.
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2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-d/l-glycero-d-gluco-heptitol (41a, 41b)
To a solution of alkene 40 (158 mg, 0.28 mmol) in 2:1 THF/H2O (9 mL) was added N-methylmorpholine N-oxide (65 mg, 0.55 mmol). After stirring at r.t. for 10 min, OsO4 (cat. amount) was added and the mixture was stirred for 1 h until complete conversion as observed by TLC (EtOAc/hexane, 1:1). Then sat. aq Na2S2O5 (10 mL) was added. The mixture was extracted with EtOAc (3 × 10 mL). The organic phase was washed with 1 M aq HCl (10 mL), sat. aq NaHCO3 (10 mL) and brine (15 mL), and dried (MgSO4). After filtration and evaporation of volatiles, the crude was purified by chromatography on silica gel (hexane/EtOAc, 1:1) to afford the diol mixture 41a/41b as a colorless oil (162 mg, 97%).
1H NMR (300 MHz, CDCl3): δ = 7.38–7.18 (m, 20 H, ArH), 5.21–5.02 (m, 2 H, CH2, Cbz), 4.76–4.40 (m, 6 H, OCH 2Ph), 4.29 (br t, J = 4.0 Hz, H-5), 4.25–4.16 (m, 1.5 H, H-5, H-6), 3.95–3.43 (m, 6.5 H, H-1a, H-2, H-3, H-4, H-6, H-7a, H-7b), 3.33 (dd, J 1a,1b = 13.4 Hz, J 1b,2 = 2.9 Hz, 1 H, H-1b).
HRMS (ESI): m/z [M + HCOO]– calcd for C36H39NO7: 642.2709; found: 642.2722.
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2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-7-O-(tert-butyldiphenylsilyl)-1,5-dideoxy-1,5-imino-d-glycero-d-gluco-heptitol (42) and 2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-7-O-(tert-butyldiphenylsilyl)-1,5-dideoxy-1,5-imino-l-glycero-d-gluco-heptitol (44)
To a solution of 41a and 41b (0.162 g, 0.27 mmol) in anhyd CH2Cl2 (3.5 mL) was added imidazole (60 mg, 0.87 mmol). The solution was cooled to 0 °C and TBDPSCl (0.13 mL, 0.49 mmol) was added. The reaction mixture was stirred at 0–10 °C for 2 h, then diluted with CH2Cl2, and washed with H2O. The aqueous phase was extracted with CH2Cl2 (3 ×). The combined organic layers were dried (MgSO4). After filtration and evaporation of volatiles, the crude was purified by chromatography on silica gel (hexane/EtOAc, 7:1) to afford the 7-O-silylated derivatives 42 and 44 as colorless oils (216 mg, 95%, ratio 1.6:1).
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Major Diastereoisomer 42
Rf = 0.18 (EtOAc/hexane, 1:7); [α]D 22 +2.0 (c 1.0, CHCl3).
1H NMR (600 MHz, toluene-d 8, 75 °C): δ = 7.74–7.70 (m, 4 H, ArH), 7.24–7.00 (m, 26 H, ArH), 5.06 (d, part A of AB system, J = 12.4 Hz, 1 H, OCH2, Cbz), 4.99 (d, part B of AB system, J = 12.4 Hz, 1 H, OCH2, Cbz), 4.67–4.38 (m, 6 H, H-5, 2 × OCH 2Ph, 1 × OCH 2Ph), 4.36–4.24 (m, 2 H, H-6, 1 × OCH 2Ph), 4.20 (t, J 3,4 = J 4,5 = 4.3 Hz, 1 H, H-4), 4.14–4.01 (m, 1 H, H-1a), 3.98–3.85 (m, J 6,7a = 3.9 Hz, J 6,7b = 7.7 Hz, J 7a,7b = 10.7 Hz, 2 H, H-7a, H-7b), 3.83 (t, J 2,3 = 4.3 Hz, J 3,4 = 4.3 Hz 1 H, H-3), 3.56–3.51 (m, 1 H, H-2), 3.27 (br d, J 1a,1b = 13.9 Hz, 1 H, H-1b), 1.14 [s, 9 H, C(CH3)3].
13C NMR (150 MHz, toluene-d 8, 75 °C): δ = 156.6 (C=O), 139.4, 139.3, 139.0, 137.8, 136.1, 134.1 (q, ArC), 130.0, 129.2, 128.6, 128.6, 128.5, 128.5, 128.3, 128.2, 128.1, 128.0, 127.5, 127.5 (ArCH), 80.2 (C-3), 76.9 (C-2), 74.3 (C-4), 73.2 (CH2Ph), 72.3 (CH2Ph, C-6), 71.3 (CH2Ph), 67.5 (CH2Ph), 67.1 (C-7), 57.5 (C-5), 41.7 (C-1), 27.4 [C(CH3)3], 19.6 [C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C52H57NO7Si: 836.3977; found: 836.3955.
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Minor Diastereoisomer 44
Rf = 0.10 (EtOAc/hexane, 1:7); [α]D 20 +1.7 (c 0.7, CHCl3).
1H NMR (600 MHz, toluene-d 8, 75 °C): δ = 7.77–7.70 (m, 4 H, C6H5), 7.28–6.93 (m, 26 H, C6H5), 5.02 (d, part A of AB system, J = 12.5 Hz, 1 H, CH2, Cbz), 4.94 (d, part B of AB system, 1 H, CH2, Cbz), 4.66 (s, 2 H, OCH 2Ph), 4.56–4.43 (m, 4 H, H-5, 1 × OCH 2Ph, 1 × OCH 2Ph), 4.29 (d, J = 11.6 Hz, 1 H, OCH 2Ph), 4.21–4.14 (m, 1 H, H-6), 3.98 (dd, J 6,7a = 4.0 Hz, J 7a,7b = 10.7 Hz, 1 H, H-7a), 3.88 (dd, J 6,7b = 6.0 Hz, 1 H, H-7b), 3.82 (t, J 3,4 = J 4,5 = 6.0 Hz, 1 H, H-4), 3.72 (dd, J 2,3 = 5.1 Hz, J 3,4 = 6.0 Hz, 1 H, H-3), 3.62–3.53 (m, 3 H, H-2, H-1a, H-1b), 1.13 [s, 9 H, C(CH3)3].
13C NMR (150 MHz, toluene-d 8, 75 °C): δ = 157.3 (C=O), 139.4, 139.3, 139.2, 137.8, 134.4, 134.2 (q, ArC), 130.1, 129.3, 128.7, 128.6, 128.5, 128.5, 128.3, 128.3, 127.8, 127.6 (ArCH), 82.6 (C-3), 78.6 (C-2), 76.8 (C-4), 73.9 (CH2Ph), 73.6 (CH2Ph), 71.9 (C-6), 71.5 (CH2Ph), 67.7 (CH2Ph, Cbz), 66.9 (C-7), 58.7 (C-5), 43.7 (C-1 ), 27.4 [C(CH3)3] and 19.7 [C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C52H57NO7Si: 836.3977; found: 836.3945.
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Mosher’s Esters 43 and 45
To a solution of (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride (0.034 mmol, 6 μL) in anhyd pyridine (0.7 mL) and CCl4 (0.5 mL) was added a solution of alcohol 44 (minor diastereoisomer, 13 mg, 0.016 mmol) in anhyd pyridine (1.2 mL). Then DMAP (cat. amount) was added and the mixture was stirred at 60 °C under argon for 2 d. Et2NH (0.05 mol, 5 μL) was added and the mixture was partitioned between Et2O (2 mL) and H2O (6 mL). The organic layer was separated, washed with sat. aq NH4Cl, and dried (MgSO4). After filtration and evaporation of volatiles, the crude was purified by chromatography on silica gel (hexane/EtOAc, 7:1) to afford the MTPA-derivative 45 as a colorless oil (6 mg, 38%) along with recovered starting material 44 (5.6 mg, 43%).
1H NMR (600 MHz, CDCl3): δ (2 rotamers) = 7.69–6.88 (m, 29 H, ArH), 5.84 and 5.80 (ddd, 1 H, H-6 rotamers), 4.95–4.31 (m, 8 H, OCH 2Ph, Cbz), 4.59 and 4.54 (m, 1 H, H-5 rotamers), 4.27 and 3.97 (dd, H-1a rotamers), 3.95 and 3.85 (m, 2 H, H-7a, H-7b rotamers), 3.65–3.54 (m, H-3 rotamers), 3.59 and 3.52 (s, 3 H, OCH3 rotamers), 3.34 and 3.23 (br ddd, 1 H, H-2 rotamers), 3.32–3.27 (m, 1 H, H-4 rotamers), 2.83 and 2.77 (br d, 1 H, H-1e rotamers), 1.04 and 0.97 [s, 9 H, C(CH3)3, rotamers].
A similar procedure was applied to the major diastereoisomer 42 (13 mg), which gave 4 mg of the ester 43 (25%) with recovery of starting material 42 (7 mg, 65%).
1H NMR (600 MHz, CDCl3): δ = 7.63–7.02 (m, 29 H, ArH), 5.92 (ddd, J = J = 4.0 Hz, J = 7.3 Hz, J = 7.3 Hz, 1 H, H-6), 5.05 (d, part A of AB system, J a,b = 12.2 Hz, 1 H, CH2, Cbz), 4.96 (d, part B of AB system, 1 H, OCH 2Ph), 4.62–4.28 (m, 7 H, OCH 2Ph, H-5), 4.04 (br d, 1 H, H-1a), 3.88–3.74 (m, 2 H, H-7a, H-7b), 3.69 (t, J 2,3 = J 3,4 = 4.1 Hz, 1 H, H-3), 3.63 (t, J 3,4 = J 4,5 = 4.8 Hz, 1 H, H-4), 3.47 (br ddd, 1 H, H-2), 3.37 (s, 3 H, OCH3), 2.83 (br d, 1 H, H-1b), 0.99 [s, 9 H, C(CH 3)3].
#
2,3,4-Tri-O-benzyl-7-O-(tert-butyldiphenylsilyl)-5-N,6-O-carbonyl-1,5-dideoxy-1,5-imino-d-glycero-d-gluco-heptitol (46) and 2,3,4,7-Tetra-O-benzyl-5-N,6-O-carbonyl-1,5-dideoxy-1,5-imino-d-glycero-d-gluco-heptitol (47)
NaH (60% suspension in mineral oil; 31 mg, 0.08 mmol) was added to a solution of 42 (30 mg, 0.04 mmol) in anhyd DMF (1 mL) at 0 °C. After a few min, benzyl bromide (0.02 mL, 0.17 mmol) was added dropwise. The mixture was stirred overnight at r.t. under argon, and then cold H2O (2 mL) was added. The mixture was extracted with Et2O (3 × 1 mL). The combined organic phases were washed with H2O and dried (MgSO4). After filtration and concentration, the residue was subjected to column chromatography (EtOAc/hexane, 1:11 → 1:6) to give 46 (10 mg, 38%) and 47 (10 mg, 47%) as a syrup.
#
46
Rf = 0.2 (EtOAc/hexane, 1:3); [α]D 20 +19 (c 0.2, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.71–7.61 (m, 4 H, C6H5), 7.44–7.13 (m, 19 H, C6H5), 6.92–6.85 (m, 2 H, C6H5), 5.01 (d, part A of AB system, J = 10.8 Hz, 1 H, OCH 2Ph), 4.86 (d, part A of AB system, J = 11.1 Hz, 1 H, OCH 2Ph), 4.75–4.59 (m, 4 H, H-6, 1 × OCH 2Ph, 1 × OCH 2Ph), 4.15 (dd, J 1a,2 = 4.8 Hz, J 1a,1b = 12.8 Hz, 1 H, H-1a, 1 × OCH 2Ph), 4.10–4.02 (m, J 6,7a = 2.5 Hz, J 7a,7b = 11.8 Hz, 2 H, H-7a), 3.84–3.65 (m, 3 H, J 6,7b = 4.4 Hz, H-4, H-5, H-7b), 3.63–3.50 (m, 2 H, H-2, H-3), 2.74 (dd, J 1a,1b = 12.8 Hz, J 1b,2 = 9.8 Hz, H-1b), 1.05 [s, 9 H, C(CH3)3].
13C NMR (75 MHz, CDCl3): δ = 156.6 (C=O, Cbz), 86.8 (C-3), 77.5 (C-2), 76.7, 76.2 (C-4, C-6), 75.6 (CH2Ph), 74.2 (CH2Ph), 73.1 (CH2Ph), 62.6 (C-7), 58.4 (C-5), 43.0 (C-1 ), 27.0 [C(CH3)3], 19.3 [C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C45H49NO6Si: 728.3402; found: 728.3403.
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47
1H NMR (300 MHz, CDCl3): δ = 7.40–7.26 (m, 18 H, C6H5), 7.14–7.06 (m, 2 H, C6H5), 5.02 (d, part A of AB system, J = 10.8 Hz, 1 H, OCH 2Ph), 4.98 (d, part A of AB system, J = 11.1 Hz, 1 H, OCH 2Ph), 4.77–4.62 (m, 4 H, H-6, 1 × OCH 2Ph, 1 × OCH 2Ph), 4.54 (d, part A of AB system, J = 12.2 Hz, 1 H, OCH 2Ph), 4.48 (d, part B of AB system, 1 H, OCH 2Ph), 4.38 (d, part B of AB system, 1 H, OCH 2Ph), 4.15 (dd, J 1a,2 = 4.9 Hz, J 1a,1e = 12.9 Hz, 1 H, H-1a), 3.83–3.66 (m, J 6,7a = 3 Hz, J 7a,7b = 11.3 Hz, 3 H, H-5, H-4, H-7a), 3.64–3.50 (m, 3 H, H-2, H-3, H-7b), 2.74 (dd, J 1a,1b = 12.9 Hz, J 1b,2 = 10.1 Hz, 1 H, H-1b).
13C NMR (75 MHz, CDCl3): δ = 156.5 (C=O), 138.3, 138.1, 137.8, 137.5 (Cq, C6H5), 128.7, 128.7, 128.6, 128.6, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.6 (CH, C6H5), 86.9 (C-3), 77.6 (C-2), 76.4 (C-4), 75.8 (CH2Ph), 75.3 (C-6), 74.2 (CH2Ph), 74.0 (CH2Ph), 73.2 (CH2Ph), 68.0 (C-7), 58.5 (C-5), 43.0 (C-1).
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2,3,4-Tri-O-benzyl-7-O-(tert-butyldiphenylsilyl)-5-N,6-O-carbonyl-1,5-dideoxy-1,5-imino-l-glycero-d-gluco-heptitol (48)
NaH (60% suspension in mineral oil; 0.8 mg, 0.002 mmol) was added to a solution of 44 (9 mg, 0.01 mmol) in anhyd DMF (2 mL) at 0 °C. After a few min, benzyl bromide (0.005 mL, 0.04 mmol) was added dropwise. The mixture was stirred overnight at r.t. under argon. Additional NaH (1 mg) and benzyl bromide (5 μL) were added and the reaction stopped after 3 additional h. Cold H2O (2 mL) was added, the mixture extracted with Et2O (3 × 2 mL). The combined organic phases were washed with H2O and dried (MgSO4). After filtration and concentration, the residue was subjected to column chromatography (EtOAc/hexane, 1:10 → 1:5) to give 48 (2.6 mg, 37%) as a syrup; Rf = 0.34 (EtOAc/hexane, 1:3); [α]D 23 +0.5 (c 1.0, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.65–7.61 (m, 4 H, C6H5), 7.45–7.13 (m, 21 H, C6H5), 5.00 (d, J = 10.6 Hz, 1 H, CH 2Ph), 4.89 (d, J = 11.6 Hz, 1 H, CH 2Ph), 4.82 (d, J = 10.6 Hz, 1 H, CH 2Ph), 4.72–4.66 (m, 2 H, CH 2Ph), 4.59 (d, J = 11.6 Hz, 1 H, CH 2Ph), 4.17 (dd, J 2,1e = 5.3 Hz, J 1a,1e = 13.2 Hz, 1 H, H-1e), 3.94 (dd, J = 7.1 Hz, 3.5 Hz, 1 H, H-6), 3.79 (dd, J 6,7a = 3.0 Hz, J 7b,7a = 11.5 Hz, 1 H, H-7a), 3.61–3.53 (m, 3 H, H-2, H-3, H-7b), 3.49 (dd, J 6,5 = 4.2 Hz, J 4,5 = 9.6 Hz, 1 H, H-5), 3.32 (app t, J 3,4 ~ J 4,5 = 9.6 Hz, 1 H, H-4), 2.75 (dd, J 2,1a = 1.9 Hz, J 1a,1e = 13.2 Hz, 1 H, H-1a), 1.02 [s, 9 H, C(CH3)3].
13C NMR (150 MHz, CDCl3): δ = 156.03 (C=O), 138.23, 137.72, 137.69 (Cq, C6H5), 135.67, 135.55 (CH, C6H5), 132.95, 132.57 (Cq, TBDPS), 129.89, 129.87, 128.56, 128.54, 128.45, 128.14, 128.11, 127.97, 127.93, 127.83, 127.81, 127.78 (CH, C6H5), 85.99 (C-3), 79.66 (C-4), 77.74 (C-6), 77.64 (C-2), 75.88, 74.97, 73.17 (CH2Ph), 64.25 (C-7), 57.56 (C-5), 42.60 (C-1), 26.66 [C(CH3)3], 19.23 [C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C45H49NO6Si: 728.3402; found: 728.3412.
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2,3,4-Tri-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-l-glycero-d-gluco-heptitol (41b)
HF·pyridine (70% solution, 1.2 mL) was added to a solution of 44 (30 mg, 0.065 mmol) in THF (2.5 mL) at r.t. The solution was stirred for 5 h, then the reaction was quenched with sat. aq NaHCO3 and extracted with EtOAc (3 ×). The combined organic phases were dried (MgSO4) and concentrated. Purification of the residue on silica gel (hexane/EtOAc, 1:1) afforded 41b (21 mg, 98%) as a colorless oil.
1H NMR (300 MHz, CDCl3): δ = 7.37–7.17 (m, 20 H, ArH), 5.19–5.04 (m, 2 H, OCH2, Cbz), 4.77–4.52 (m, 5 H, 2 × OCH 2Ph, 1 × OCH 2Ph, Cbz), 4.44 (d, J = 11.9 Hz, 1 H, OCH 2Ph), 4.30 (br t, J = 3.7 Hz, 1 H, H-5), 4.02 (br d, J = 13.6 Hz, 1 H, H-1a), 3.95–3.85 (m, 1 H, H-6), 3.82–3.64 (m, 3 H, J 3,4 = 7.3 Hz, J 4,5 = 3.4 Hz, H-2, H-3, H-4), 3.63–3.38 (m, 3 H, H-1b, H-7a, H-7b).
13C NMR (75 MHz, CDCl3): δ = 79.9 (C-3), 76.6 (C-4), 75.7 (C-2), 72.6 (C-6), 63.4 (C-7), 56.5 (C-5), 42.5 (C-1).
HRMS (ESI): m/z [M + HCOO]– calcd for C36H39NO7: 642.2709; found: 642.2722.
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1,5-Dideoxy-1,5-imino-l-glycero-d-gluco-heptitol Hydrochloride (49)
The benzylated iminosugar 41b (22 mg, 0.037 mmol) was dissolved in anhyd THF (2.5 mL). Then a catalytic amount (spatula tip) of 10% Pd/C was added and the suspension was stirred under H2 atmosphere for 48 h. The suspension was then filtered, washed with MeOH (3 ×) and concentrated in vacuo. The residue was dissolved in H2O (HPLC grade) and subjected to gel filtration using a PD-10 Sephadex G 25 column (H2O). The eluate was lyophilized. The solid residue was dissolved in H2O and applied on a column of Dowex 50W-X8 resin (H+-form). The column was washed with H2O, and the product was eluted with aq 1 M NH4OH. Product-containing fractions were concentrated, and then lyophilized.
1H NMR (300 MHz, MeOD): δ = 3.95 (td, J 5,6 = 2.4 Hz, J 6,7a ~ J 6,7b = 5.8 Hz, 1 H, H-6), 3.69–3.63 (m, 2 H, H-7a, H-7b), 3.57 (ddd, J 2,3 = 8.9 Hz, J 2,1a = 10.6 Hz, J 2,1e = 5.1 Hz, 1 H, H-2), 3.36 (dd, J 3,4 = 9.0 Hz, J 4,5 = 9.6 Hz, 1 H, H-4), 3.20 (t, J 2,3 = J 3,4 = 8.9 Hz, 1 H, H-3), 3.06 (dd, part A of ABX, J 1e,2 = 5.1 Hz, J 1a,1e = 12.7 Hz, 1 H, H-1e), 2.45 (dd, J 4,5 = 9.6 Hz, J 5,6 = 2.4 Hz, 1 H, H-5), 2.4 (dd, part B of ABX, J 1a,2 = 10.6 Hz, J 1a,1e = 12.8 Hz, 1 H, H-1a).
H2O (1 mL) and aq 1 M HCl (0.5 mL) were added to the freeze-dried material and the solution was stirred for 2 h at r.t. and then concentrated. The residue was applied on a column of Sephadex G-10 and the product was eluted with H2O. After lyophilization, the target compound 49 was obtained as a white amorphous solid (4.4 mg, 52% overall yield); [α]D 22 +3 (c 0.2, H2O).
1H NMR (600 MHz, D2O): δ = 4.07 (ddd, J 5,6 = 2.0 Hz, J 6,7a = 7.4 Hz, J 6,7b = 5.0 Hz, 1 H, H-6), 3.74–3.69 (m, J 6,7a = 7.4 Hz, J 6,7b = 5.1 Hz, J 7a,7b = 11.7 Hz, 2 H, H-7a, H-7b), 3.51 (ddd, J 1e,2 = 5.1 Hz, J 2,3 = 9.0 Hz, J 1a,2 = 11.0 Hz, 1 H, H-2), 3.44–3.34 (m, 2 H, H-3, H-4), 3.12 (dd, J 1a,1b = 12.7 Hz, J 1e,2 = 5.0 Hz, 1 H, H-1e), 2.55 (dd, J 4,5 = 9.6 Hz, J 5,6 = 1.9 Hz, 1 H, H-5), 2.44 (dd, J 1a,1e = 12.8 Hz, J 1a,2 = 10.9 Hz, 1 H, H-1a).
13C NMR (data from HSQC spectra, D2O): δ = 78.4 (C-3), 71.4 (C-4), 71.0 (C-2), 69.1 (C-6), 63.9 (C-7), 60.0 (C-5), 48.3 (C-1).
HRMS (ESI): m/z [M + H]+ calcd for C7H15NO5: 194.1023; found: 194.1023; [M + Na]+ calcd for C7H15NO5: 216.0842; found: 216.0841.
#
#
Acknowledgment
The authors gratefully acknowledge financial support of this work by Mutabilis. Dr. Andreas Hofinger-Horvath is thanked for recording the NMR spectra and Hedda Drexler for measuring the HRMS data.
Supporting Information
- Supporting information for this article is available online at https://doi.org/10.1055/s-0036-1591518.
- Supporting Information
-
References
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- 14 Zamyatina A. Gronow S. Oertelt C. Puchberger M. Brade H. Kosma P. Angew. Chem. Int. Ed. 2000; 39: 4150
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- 16 Coleman WG. Leive L. J. Bacteriol. 1979; 139: 899
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- 17b Desroy N. Denis A. Oliveira C. Atamanyuk D. Briet S. Faivre F. Le Fralliec G. Bonvin Y. Oxoby M. Escaich S. Floquet S. Drocourt E. Vongsouthi V. Durant L. Moreau F. Verhey TB. Lee T.-W. Junop MS. Gerusz V. J. Med. Chem. 2013; 56: 1418
- 17c Lee T. Verhey TB. Antiperovitch PA. Atamanyuk D. Desroy N. Oliveira C. Denis A. Gerusz V. Drocourt E. Loutet SA. Hamad MA. Stanetty C. Andres SN. Sugiman-Marangos S. Kosma P. Valvano M. Moreau F. Junop MS. J. Med. Chem. 2013; 56: 1405
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- 18 Durka M. Tikad A. Périon R. Bosco M. Andaloussi M. Floquet S. Malacain E. Moreau F. Oxoby M. Gerusz V. Vincent SP. Chem. Eur. J. 2011; 17: 11305
- 19 Brimacombe JS. Kabir AK. M. S. Carbohydr. Res. 1986; 150: 35
- 20 Zamyatina A. Puchberger M. Graziani A. Gronow S. Hofinger A. Kosma P. Carbohydr. Res. 2003; 338: 2571
- 21 Richtmyer NK. Carr CJ. Hudson CS. J. Am. Chem. Soc. 1943; 65: 1477
- 22 Bannwarth W. Trzeciak A. Helv. Chim. Acta 1987; 70: 175
- 23 Wessel H.-P. Iversen T. Bundle DR. J. Chem. Soc., Perkin Trans. 1 1985; 2247
- 24 Khanna IK. Koszyk FJ. Stealey MA. Weier RM. Julien J. Mueller RA. Rao SN. Swenton L. J. Carbohydr. Chem. 1995; 14: 843
- 25 Cha JK. Christ WJ. Kishi Y. Tetrahedron 1984; 40: 2247
- 26a Van Straten NC. R. Kriek NM. A. J. Timmers CM. Wigchert SC. M. van der Marel GA. van Boom JH. J. Carbohydr. Chem. 1997; 16: 947
- 26b Crich D. Banerjee A. Org. Lett. 2005; 7: 1395
- 27 Chan T.-H. Chang Y.-F. Hsu J.-J. Cheng W.-C. Eur. J. Org. Chem. 2010; 5555
- 28 Dale JA. Mosher HS. J. Am. Chem. Soc. 1973; 95: 512
- 29 Lauritsen A. Madsen R. Org. Biomol. Chem. 2006; 4: 2898
- 30 Lundt I. Madsen R. Synthesis 1995; 787
- 31a Desroy N. Moreau F. Briet S. Le Fralliec G. Floquet S. Durant L. Vongsouthi V. Gerusz V. Denis A. Escaich S. Bioorg. Med. Chem. 2009; 17: 1276
- 31b Gerusz V. Vincent S. Oxoby M. Atamanyuk D. Moreau F. Andaloussi M. Tikad A. Patent PCT Int. Appl. WO 2012/073214 A2, 2012
- 32a Stütz AE. Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond. Wiley-VCH; Weinheim: 1999
- 32b Zou W. Curr. Top. Med. Chem. 2005; 5: 1363
- 32c Asano N. Glycobiol. 2003; 13: 93R
- 33 Dhavale DD. Kumar KA. A. Chaudhari VD. Sharma T. Sabharwal SG. Reddy JP. Org. Biomol. Chem. 2005; 3: 3720
- 34 Harris RK. Becker ED. Cabral de Menezes SM. Goodfellow R. Granger P. Pure Appl. Chem. 2001; 73: 1795
For example, see:
See, for example:
-
References
- 1 New address: D. Atamanyuk, AB-Science, 3 Avenue George V, 75008, Paris, France.
- 2 New address: N. M. Xavier, Center of Chemistry and Biochemistry, DQB, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, 2º Piso, Campo Grande 1749-016 Lisboa, Portugal.
- 3 New address: V. Gerusz, Debiopharm, Rue du Levant 146, CP368, 1920 Martigny, Switzerland.
- 4a Maxson T. Mitchell DA. Tetrahedron 2016; 72: 3609
- 4b Chellat MF. Raguž L. Riedl R. Angew. Chem. Int. Ed. 2016; 55: 6600
- 4c Walsh C. Nat. Rev. Microbiol. 2013; 1: 65
- 4d Schäberle TF. Hack IM. Trends Microbiol. 2014; 22: 165
- 4e Taylor PL. Wright GD. Anim. Health Res. 2008; 9: 237
- 5a Escaich S. Curr. Opin. Chem. Biol. 2008; 12: 400
- 5b Kuo C.-J. Chen J.-W. Chiu H.-C. Teng C.-H. Hsu T.-I. Lu P.-J. Syu W.-J. Wang S.-T. Chou T.-C. Chen C.-S. Frontiers Cell. Infect. Microbiol 2016; 6: 82 ; doi: 10.3389/fcimb.2016.00082
- 5c Loutet SA. Flannagan RS. Kooi C. Sokol PA. Valvano M. J. Bacteriol. 2006; 188: 2073
- 6a Reinhardt A. Wehle M. Geissner A. Crouch E. Kang Y. Yang Y. Anish C. Santer M. Seeberger PH. J. Struct. Biol. 2016; 195: 387
- 6b Gomery K. Müller-Loennies S. Brooks CL. Brade L. Kosma P. Di Padova F. Brade H. Evans SV. Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 20877
- 6c Wang H. Head J. Kosma P. Brade H. Müller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry 2008; 47: 710
- 6d Marchetti R. Malinovska L. Lameignère E. Adamova L. De Castro C. Cioci G. Stanetty C. Kosma P. Molinaro A. Wimmerova M. Imberty A. Silipo A. Glycobiol. 2012; 22: 1387
- 7 Raetz CR. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635
- 8 Holst O. In Bacterial lipopolysaccharides . Knirel YA. Valvano M. Springer; Wien: 2011: 21
- 9 Kosma P. Curr. Org. Chem. 2008; 12: 1021
- 10a Butty F. Aucoin M. Morrison L. Ho N. Shaw G. Creuzenet C. Biochemistry 2009; 48: 7764
- 10b McCallum M. Shaw SD. Shaw GS. Creuzenet C. J. Biol. Chem. 2012; 287: 29776
- 11a Lu Q. Yao Q. Xu Y. Li L. Li S. Liu Y. Gao W. Niu M. Sharon M. Ben-Nissan G. Zamyatina A. Liu X. Chen S. Shao F. Cell Host Microbe 2014; 16: 351
- 11b Yao Q. Lu Q. Wan X. Song F. Xu Y. Hu M. Zamyatina A. Liu X. Huang N. Zhu P. Shao F. eLife 2014; 3: e03714 ; doi: 10.7554/eLife.03714
- 11c Benz I. Schmidt MA. Mol. Microbiol. 2001; 40: 1403
- 12a Eidels L. Osborn MJ. Proc. Natl. Acad. Sci. U.S.A. 1971; 68: 1673
- 12b Valvano M. Messner P. Kosma P. Microbiol. 2002; 148: 1979 ; and references cited therein
- 12c For the expansion of the abbreviations, see: Kneidinger B. Marolda C. Graninger M. Zamyatina A. McArthur F. Kosma P. Valvano M. Messner P. J. Bacteriol. 2002; 184: 363
- 13 Gaudet RG. Sintsova A. Buckwalter CM. Leung N. Cochrane A. Li J. Cox A. Moffat J. Gray-Owen SD. Science 2016; 348: 1251
- 14 Zamyatina A. Gronow S. Oertelt C. Puchberger M. Brade H. Kosma P. Angew. Chem. Int. Ed. 2000; 39: 4150
- 15a Yu C.-K. Wang C.-J. Chew Y. Wang P.-C. Yin H.-S. Kao M.-C. Biochem. Biophys. Res. Commun. 2016; 477: 794
- 15b Do H. Yun J.-S. Lee CW. Choi YJ. Kim H.-Y. Kim Y.-J. Park H. Chang JH. Lee JH. Mol. Cells 2015; 38: 1086
- 15c Taylor PL. Blakeley KM. de Leon GP. Walker JR. McArthur F. Evdokimova E. Zhang K. Valvano MA. Wright GD. Junop MS. J. Biol. Chem. 2008; 283: 2835
- 15d Harmer NJ. Mol. Biol. 2010; 400: 379
- 15e Vivoli M. Isupov MN. Nocholas R. Hill A. Scott AE. Kosma P. Prior JL. Harmer NJ. Chem. Biol. 2015; 22: 1622
- 16 Coleman WG. Leive L. J. Bacteriol. 1979; 139: 899
- 17a Taylor PL. Blakeley KM. de Leon GP. Walker JR. McArthur F. Evdokimova E. Zhang K. Valvano MA. Wright GD. Junop MS. J. Biol. Chem. 2008; 283: 2835
- 17b Desroy N. Denis A. Oliveira C. Atamanyuk D. Briet S. Faivre F. Le Fralliec G. Bonvin Y. Oxoby M. Escaich S. Floquet S. Drocourt E. Vongsouthi V. Durant L. Moreau F. Verhey TB. Lee T.-W. Junop MS. Gerusz V. J. Med. Chem. 2013; 56: 1418
- 17c Lee T. Verhey TB. Antiperovitch PA. Atamanyuk D. Desroy N. Oliveira C. Denis A. Gerusz V. Drocourt E. Loutet SA. Hamad MA. Stanetty C. Andres SN. Sugiman-Marangos S. Kosma P. Valvano M. Moreau F. Junop MS. J. Med. Chem. 2013; 56: 1405
- 17d De Leon GP. Elowe NH. Koteva KP. Valvano MA. Wright GD. Chem. Biol. 2006; 13: 437
- 18 Durka M. Tikad A. Périon R. Bosco M. Andaloussi M. Floquet S. Malacain E. Moreau F. Oxoby M. Gerusz V. Vincent SP. Chem. Eur. J. 2011; 17: 11305
- 19 Brimacombe JS. Kabir AK. M. S. Carbohydr. Res. 1986; 150: 35
- 20 Zamyatina A. Puchberger M. Graziani A. Gronow S. Hofinger A. Kosma P. Carbohydr. Res. 2003; 338: 2571
- 21 Richtmyer NK. Carr CJ. Hudson CS. J. Am. Chem. Soc. 1943; 65: 1477
- 22 Bannwarth W. Trzeciak A. Helv. Chim. Acta 1987; 70: 175
- 23 Wessel H.-P. Iversen T. Bundle DR. J. Chem. Soc., Perkin Trans. 1 1985; 2247
- 24 Khanna IK. Koszyk FJ. Stealey MA. Weier RM. Julien J. Mueller RA. Rao SN. Swenton L. J. Carbohydr. Chem. 1995; 14: 843
- 25 Cha JK. Christ WJ. Kishi Y. Tetrahedron 1984; 40: 2247
- 26a Van Straten NC. R. Kriek NM. A. J. Timmers CM. Wigchert SC. M. van der Marel GA. van Boom JH. J. Carbohydr. Chem. 1997; 16: 947
- 26b Crich D. Banerjee A. Org. Lett. 2005; 7: 1395
- 27 Chan T.-H. Chang Y.-F. Hsu J.-J. Cheng W.-C. Eur. J. Org. Chem. 2010; 5555
- 28 Dale JA. Mosher HS. J. Am. Chem. Soc. 1973; 95: 512
- 29 Lauritsen A. Madsen R. Org. Biomol. Chem. 2006; 4: 2898
- 30 Lundt I. Madsen R. Synthesis 1995; 787
- 31a Desroy N. Moreau F. Briet S. Le Fralliec G. Floquet S. Durant L. Vongsouthi V. Gerusz V. Denis A. Escaich S. Bioorg. Med. Chem. 2009; 17: 1276
- 31b Gerusz V. Vincent S. Oxoby M. Atamanyuk D. Moreau F. Andaloussi M. Tikad A. Patent PCT Int. Appl. WO 2012/073214 A2, 2012
- 32a Stütz AE. Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond. Wiley-VCH; Weinheim: 1999
- 32b Zou W. Curr. Top. Med. Chem. 2005; 5: 1363
- 32c Asano N. Glycobiol. 2003; 13: 93R
- 33 Dhavale DD. Kumar KA. A. Chaudhari VD. Sharma T. Sabharwal SG. Reddy JP. Org. Biomol. Chem. 2005; 3: 3720
- 34 Harris RK. Becker ED. Cabral de Menezes SM. Goodfellow R. Granger P. Pure Appl. Chem. 2001; 73: 1795
For example, see:
See, for example: