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Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch
Tetrahedron 68 (2012) 9405e9412
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Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch Guan-Nan Wang a, Pui Sai Lau b, c, Yingfu Li b, c, Xin-Shan Ye a, * a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Rd No. 38, Beijing 100191, China Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada c Department of Chemistry and Chemical Biology, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2012 Accepted 4 September 2012 Available online 10 September 2012
The glmS riboswitch is a ribozyme found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. Herein, we describe the efficient synthesis of three GlcN6P analogues and their evaluation on inducing self-cleavage of the glmS riboswitch from Bacillus subtilis. Our results provide valuable information for further elucidation of the structureeactivity relationships and drug design for glmS riboswitch antibiotics. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: glmS Riboswitch GlcN6P analogues Antibiotics Synthesis Carbohydrate
1. Introduction Riboswitches are structural mRNA domains that regulate gene expression in response to the intracellular concentration of specific metabolites. They control essential genes in many pathogenic bacteria, thus representing an intriguing class of RNA target for the development of antibiotics and chemicalebiological tools.1e3 Notably, the glmS riboswitch, which is located upstream of the gene encoding glucosamine-6-phosphate synthase (GlmS) in numerous Gram-positive bacteria, is unique in that it is the first example of a natural ribozyme that is also a riboswitch.4 As part of gene regulation, it binds to its small molecule metabolite, glucosamine-6phosphate (GlcN6P), which triggers self-cleavage targeting its own mRNA.5,6 Functionally, the glmS ribozyme controls the amount of GlmS to regulate cellular production of GlcN6P,7,4 which is an essential metabolite for the biosynthesis of bacterial cell walls and fungal cell wall chitin.8e11 Given the importance of GlcN6P, inhibition of the metabolite is lethal to microorganisms. Consequently, the glmS riboswitch is emerging as a new drug target, and small molecules that mimic GlcN6P to trigger riboswitch activity have the potential to become antibiotics. Although substantial structural and mechanistic information of the glmS riboswitch have been disclosed over recent years,5,6,12e15
* Corresponding author. Tel.: þ86 10 82805736; fax: þ86 10 82802724; e-mail address: [email protected] (X.-S. Ye). 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2012.09.015
few small molecule agonists and antagonists have been developed.16,17 Activators that have comparable or superior activity than natural GlcN6P are in great demand for a better understanding of the riboswitch and paving the way to the discovery of effective antibiotics. In this research, three GlcN6P analogues have been designed and synthesized (Fig. 1). Carba-sugar 1 was designed to address the role of the oxygen atom in pyranose ring for riboswitch activation. Compounds 2 and 3 were designed to add an extra hydroxyl group at the C-6 position with the consideration of providing extra binding sites around the pocket. Instead of 6-phosphate, the 6-phosphonate was introduced to the 6-position. It is known that the PeC bond makes compounds resistant to enzymatic hydrolysis18,19 and has conformational preferences different from those in phosphates.20 These analogues were subsequently investigated for their ability to induce self-cleavage of the glmS riboswitch from Bacillus subtilis.
Fig. 1. The structures of three GlcN6P analogues.
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2. Results and discussion The synthesis of carba-sugar 1 is shown in Scheme 1. Carbocyclic compound 5 was prepared from methyl glucopyranoside 4 according to the procedures described by Barton et al.21 Compound 5 was treated with dibenzyl N,N-diisopropylphosphoramidite, followed by oxidation with meta-chloroperoxybenzoic acid (m-CPBA) to give the fully protected phosphate 6 in 86% isolated yield. The chemical shift of compound 6 in 31P NMR spectroscopy is at d 3.14, indicating the formation of phosphate.22 Subsequently, basic opening of the oxazolidinone in 6 also resulted in the removal of one of the benzyl groups on phosphate, yielding compound 7.
Catalytic hydrogenolysis of 7 provided the phosphated carba-sugar 1 smoothly. The synthesis of compounds 2 and 3 started from glucosamine 8. As shown in Scheme 2, perbenzylation of 8 provided compound 9, and selective acetolysis of 9 afforded the 6-acetylated compound 10 in good yield. It was reported that the anomeric position is usually acetylated prior to the 6-position in substrates, such as perbenzylated glucose, galactose and mannose.23,24 However, in the case of compound 9, the dibenzylated amino group protected the anomeric position from acetolysis. Removal of the acetyl group in 10 led to compound 11, which was oxidized by DesseMartin periodinane followed by the Pudovik reaction25,26 with dibenzyl
Scheme 1. Synthesis of carba-sugar 1.
Scheme 2. Synthesis of GlcN6P analogues 2 and 3.
G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
phosphonate and triethylamine to yield the a-hydroxyphosphonate 12 in 93% isolated yield as an inseparable mixture of diastereoisomers (3:1). The separation of two diastereoisomers was achieved after benzoylation of the nascent hydroxyl groups. Deprotection of the benzoyl groups with methyl amine solution, which was followed by deprotection of the benzyl groups using catalytic hydrogenolysis, afforded the target compounds 2 and 3 in good yields, respectively. The absolute configurations of diastereoisomers 12a and 12b were assigned by the Mosher method (also known as Mosher ester analysis).27e29 As displayed in Scheme 3, each of the diastereomeric S- and R-MTPA esters of major diastereomer 12a was prepared from (R)-MTPAeCl and (S)-MTPAeCl, respectively (S-Mosher acid chloride gives rise to the R-Mosher ester due to relative priority: CF3 is lower than COCl but higher than COOR). The H-5 proton signal of each diastereomeric Mosher ester was assigned by 1He1H COSY experiments. The chemical shift of H-5 in S-MTPA ester is dS¼3.93, while the chemical shift of H-5 in R-MTPA ester is dR¼3.85. That is, the DdSR¼dSdR¼3.933.85¼0.08 was positive. According to the Mosher empirical rule, the absolute configuration of the 6-position in compound 12a was S-. This could be explained by the MTPA plane figures (Fig. 2). The H-5 of R-MTPA ester, in which H-5 and phenyl group reside are on the same side of the MTPA plane, is relatively more shielded (upfield in its spectrum) than H-5 in the SMTPA ester due to the magnetic shielding effect of phenyl group. Correspondingly, the minor diastereomer was assigned as Rconfiguration.
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H-5 proton of the major diastereomer 14a was downfield (d¼3.93 ppm) relative to that of the minor diastereomer 14b (d¼3.56 ppm). These confirmed that the configuration of major product 12a is S and the configuration of minor product 12b is R. The Pudovik reaction, one of the most versatile methods for construction of CeP bonds, involves the addition of organophosphorus compounds to unsaturated bonds. With the potential to synthesize biologically important a-hydroxyphosphonic acids derivatives,31e33 this reaction has received more and more attention to make the process highly enantioselective.34,35 The examples presented herein showed the versatility of the Mosher method as a convenient and alternative approach to the assignment of stereochemistry of Pudovik products especially when the crystallization of products is impossible. It is applicable to either a single enantiomer or a pair of enantiomers. The synthetic compounds 1, 2 and 3 were subsequently investigated for their ability to induce self-cleavage of 50 -32P-labelled glmS riboswitch from B. subtilis. As shown in Table 1 and Fig. 4, carba-sugar 1 showed moderate activation of self-cleavage of glmS riboswitch from B. subtilis. The percentage of riboswitch self-cleavage induced by compound 1 is around half to that by natural metabolite GlcN6P. At the same time during the preparation of this manuscript, Wittmann and Mayer et al. reported their research of carba-sugar 1 on glmS riboswitch of vancomycin-resistant Staphylococcus aureus.36 In their study, it was found that the potency of compound 1 for glmS riboswitch of S. aureus is similar to that of GlcN6P. They also mentioned the
Scheme 3. Synthesis of R- and S-Mosher esters from compound 12a.
Fig. 2. The MTPA plane of S- and R-MTPA esters.
To further confirm the assignment of the absolute configurations of 12a and 12b, the S-MTPA esters (14a and 14b) generated from both diastereoisomers 12a and 12b were compared (Fig. 3).27,30 The 1H NMR analysis of both products showed that the
activation by compound 1 in the glmS riboswitch of B. subtilis. The activation achieved by compound 1 in our experiments compared favourably with the data reported by them. It seems that different sources of glmS ribozyme might have different
Fig. 3. The MTPA plane of S-MTPA esters 14a and 14b made from 12a and 12b.
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Table 1 Effect of synthetic compounds on glmS riboswitch Sample
No reaction (NR) No magnesium (-Mg2þ) GlcN6P 1 2 3
% Cleavage 1 min
2 min
d d 67.50 29.08 0 0
d d 65.12 38.68 0.37 0.29
moderate self-cleavage of the glmS riboswitch from B. subtilis. The displacement of phosphate in GlcN6P by a-hydroxyphosphonate led to massive loss of activation. These results highlight the role of phosphate moiety in contributing to the binding of the glmS riboswitch. These insights are useful for further development of chemical agonists or antagonists for the glmS riboswitch. It seems that glmS riboswitch exhibits high level of discrimination against many closely related GlcN6P analogues. Our results provide a deeper understanding of the glmS riboswitch and further elucidation on the structureeactivity relationships of metabolite derivatives.
Fig. 4. Cleavage of glmS RNA with and without compounds. (a) Cleavage of glmS RNA after 1 min reaction time; (b) Cleavage of glmS RNA after 2 min reaction time. A 200 mM concentration of GlcN6P or a test compound was added to 0.5 pmol of 50 -32P labelled glmS RNA. Samples were incubated in 50 mM of HEPES (pH 7.5), 10 mM of MgCl2, 200 mM of KCl at room temperature for a duration of 1 or 2 min. Negative control samples included: i) addition of double-distilled water instead of compound, or ii) addition of GlcN6P, without Mg2þ in the reaction buffer. RNA products were separated by 10% denaturing polyacrylamide gel electrophoresis (PAGE).
sensitivities to GlcN6P analogues. On the other hand, the 6-phosphonate derivatives 2 and 3 did not exhibit activity under the tested assay conditions. It was determined that the oxygen ring and phosphate group are required for stabilization of the interaction of the riboswitch with GlcN6P rather than being involved in the catalytic reaction.37,38 The results herein show that the destabilization of the interaction can tolerate carba-sugar to some extent, whereas the 6-phosphate group is critical for interaction.16,38 The phosphate moiety forms the fixed network of Mg2þ-coordinated interactions with the various functional groups of the ligand binding pocket.15 The electrical properties of a-hydroxylphosphonate are slightly different from 6-phosphate. The change in electrical properties of 6-phosphonate might change the coordination with cations and further change the interaction with the binding pocket.13 The other factor may rely on the unfilled space within the ligand binding pocket in the absence of the bridging oxygen. 3. Conclusions Three GlcN6P analogues have been prepared to investigate their effect on the glmS riboswitch. The Pudovik reaction was applied to synthesize the a-hydroxyphosphonate substrates and the configurations of two diastereomeric products were assigned by the Mosher method. Among the three analogues, carba-sugar 1 induced
4. Experimental section 4.1. General procedures Air- and/or moisture-sensitive reactions were carried out under an atmosphere of argon using flame-dried glassware and standard syringe/septa techniques. All chemicals were purchased as reagent grade and used without further purification, unless otherwise noted. Dichloromethane (CH2Cl2) and pyridine were distilled over calcium hydride (CaH2). Methanol was distilled from magnesium. DMF was stirred with CaH2 and distilled under reduced pressure. Tetrahydrofuran (THF) was distilled over sodium/benzophenone. Reactions were monitored by analytical thin-layer chromatography on silica gel 60 F254 precoated on aluminium plates (E. Merck). Spots were detected under UV (254 nm) and/or by staining with acidic ceric ammonium molybdate. Column chromatography was performed on silica gel (200e300 mesh). 1H NMR, 13C NMR, and 31P NMR spectra were recorded on a JEOL AL-300, or Varian INOVA-500 spectrometers at 25 C. Chemical shifts (in parts per million) were referenced to tetramethylsilane (d¼0 ppm) in deuterated chloroform. 13C NMR spectra were obtained by using the same NMR spectrometers and were calibrated with CDCl3 (d¼77.00 ppm) or CD3OD (d¼49.00 ppm). 31P NMR spectra were reported parts per million (d) relative to H3PO4 (0.00 ppm) as an internal reference. Mass spectra were recorded using a PE SCIEX QSTAR spectrometer. Elemental analysis data were recorded on a Vario EL-Z elemental analyser.
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4.2. Dibenzyl((3aS,4R,5R,6R,7aS)-3-benzyl-4,5-bis(benzyloxy)2-oxooctahydrobenzo[d]oxazol-6-yl)methyl phosphate (6)
(121.5 MHz, D2O) d 2.84; HRMS: calcd for C7H16NO7P Na [MþNa]þ, 258.0737; found, 258.0733.
To a solution of dibenzyl N,N-diisopropylphosphoramidite (90%, 111 mL, 300 mmol) in CH2Cl2 (10 mL) was added 1H-tetrazole (42.5 mg, 607 mmol). The mixture was stirred at room temperature for 15 min under argon atmosphere, and then the CH2Cl2 solution (2 mL) of compound 5 (57.0 mg, 120 mmol) was added. The mixture was stirred for another 4 h at room temperature under argon atmosphere. The mixture was cooled to 0 C and m-CPBA (70%, 89.0 mg, 361 mmol) was added. After stirring for 45 min, the mixture was diluted with EtOAc (50 mL). The solution was washed with aqueous 10% Na2SO3 (220 mL), 1 M HCl (220 mL), saturated NaHCO3 (220 mL), and brine (320 mL). The organic layer was dried (MgSO4) and concentrated. Purification by column chromatography (petroleum ethereethyl acetate, 1:1) gave the phosphate triester 6 as an amorphous solid (76.0 mg, 86% yield). 1H NMR (300 MHz, CDCl3) d 1.65e1.73 (m, 1H), 1.89e1.94 (d, J¼14.4 Hz, 1H), 2.04 (brs, 1H), 3.41e3.43 (m, 1H), 3.56e3.59 (m, 1H), 3.67 (brs, 1H), 3.84e3.95 (m, 2H), 4.14 (brs, 1H), 4.40 (d, J¼11.7 Hz, 1H), 4.49 (s, 2H), 4.54 (brs, 1H), 4.66 (d, J¼12.3 Hz, 1H), 4.83 (d, J¼15.3 Hz, 1H), 5.02 (brs, 4H), 7.07e7.32 (m, 25H); 13C NMR (75 MHz, CDCl3) d 26.8, 35.7 (d, JPC¼8.0 Hz), 46.3, 55.9, 68.1 (d, JPC¼5.6 Hz), 69.5 (d, JPC¼3.8 Hz), 72.6, 72.9, 73.3, 77.2 (d, JPC¼4.4 Hz), 77.8, 127.6, 127.7, 127.9, 128.0, 128.2, 128.5, 128.6, 128.8, 135.5, 135.6, 137.4, 137.6, 158.0; 31P NMR (121.5 MHz, CDCl3) d 3.14. HRMS: calcd for C43H44NO8P Na [MþNa]þ, 756.2702; found, 756.2704.
4.5. (3R,4R,5S,6R)-N,N-Dibenzyl-2,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-2H-pyran-3-amine (9)
4.3. Sodium benzyl((1R,2R,3R,4S,5S)-4-(benzylamino)-2,3bis(benzyloxy)-5-hydroxy cyclohexyl)methyl phosphate (7) A solution of compound 6 (100.0 mg, 0.14 mmol) in ethanolicsodium hydroxide solution (10%) (v/v¼1:1, 5 mL) was heated under reflux overnight. The solvents were removed and the residue was purified by C-18 reversed-phase column chromatography (eluent: H2O to CH3OH/H2O, 1:1) to give 7 (80.0 mg, 92% yield) as an amorphous solid. 1H NMR (300 MHz, CD3OD) d 1.54 (t, J¼12.6 Hz, 1H), 1.87e1.91 (m, 1H), 2.11 (brs, 1H), 2.51 (dd, J¼2.4, 9.9 Hz, 1H), 3.46 (t, J¼10.2 Hz, 1H), 3.63 (t, J¼9.6 Hz, 1H), 3.65 (d, J¼13.2 Hz, 1H), 3.85 (d, J¼13.2 Hz, 1H), 3.92e3.96 (m, 1H), 4.06e4.07 (m, 1H), 4.12e4.18 (m, 1H), 4.63 (d, J¼11.4 Hz, 1H), 4.73e4.90 (m, 5H), 7.18e7.35 (m, 20H); 13C NMR (75 MHz, CD3OD) d 32.9, 39.2 (d, JPC¼8.6 Hz), 51.9, 63.9, 65.8, 66.4 (d, JPC¼5.6 Hz), 68.1 (d, JPC¼5.6 Hz), 75.8, 75.9, 83.1, 83.7, 128.15, 128.2, 128.4, 128.5, 128.6, 128.8, 128.9, 129.3, 129.4, 139.8, 139.9, 140.1 (d, JPC¼3.2 Hz), 140.2, 141.2; 31P NMR (121.5 MHz, CDCl3) d 2.69; HRMS: calcd for C35H40NO7PNa[MþNa]þ, 640.2440; found, 640.2440. 4.4. ((1R,2R,3R,4S,5S)-4-Amino-2,3,5-trihydroxycyclohexyl)methyl dihydrogen phosphate (1) A methanolic solution (8 mL) of compound 7 (50.0 mg, 0.078 mmol) was stirred at room temperature in the presence of 10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 48 h. The catalyst was then removed by filtration through Celite, and the filtrate was concentrated. In most cases, direct lyophilization gave the pure compound 1 (19.5 mg, 97% yield) as white amorphous solids. In the cases when further purification was needed, the water solution of residue was adjusted to pH¼2 by 1 N HCl, and purified by Dowex 50W8 (Hþ form, 200e400 mesh, eluent: H2O) to give 1 as white amorphous solids after lyophilization. 1H NMR (300 MHz, D2O) d 1.43 (t, J¼13.5 Hz, 1H), 1.71e1.80 (m, 2H), 2.96 (d, J¼11.1 Hz, 1H), 3.17 (t, J¼9.9 Hz, 1H), 3.48 (t, J¼10.5 Hz, 1H), 3.77e3.82 (m, 1H), 3.89e3.95 (m, 1H), 4.03 (s, 1H); 13C NMR (75 MHz, D2O) d 31.7, 37.4 (d, JPC¼7.4 Hz), 56.9, 65.6, 66.9 (d, JPC¼5.0 Hz), 71.7, 72.6; 31P NMR
To a suspension of D-glucosamine (5.0 g, 27.9 mmol) in anhydrous DMF (70 mL) was added NaH (3.7 g, 60% dispersion in mineral oil, 92.5 mmol) at 0 C. Benzyl bromide (8.1 mL, 66.6 mmol) was added dropwise over 5 min. After stirring at room temperature for 1 h, same quantities of NaH and benzyl bromide were added at 0 C. After stirring for another 1 h, the third portion of NaH (3.8 g, 95.0 mmol) and benzyl bromide (8.2 mL, 67.6 mmol) were added consecutively at 0 C and then the mixture was warmed to room temperature. The reaction mixture was stirred overnight, and then MeOH was added slowly to quench the excess of the NaH. DMF was removed under reduced pressure. The residue was dissolved in CH2Cl2 (150 mL) and washed with water (20 mL) and brine (20 mL). The organic layer was dried (Na2SO4), filtered, and evaporated to give a yellow oil. The residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 20:1 to 6:1) to give 9 as a colourless oil (17.8 g, 89% yield) as a mixture of anomers (a/b¼1:8). 1H NMR (300 MHz, CDCl3) d 3.03 (t, J¼8.4 Hz, 1H), 3.45 (brs, 1H), 3.54e3.86 (m, 8H), 3.92 (d, J¼14.1 Hz, 2H), 4.04e4.73 (m, 7H), 4.84 (d, J¼10.8 Hz, 1H), 4.98 (t, J¼10.8 Hz, 2H), 5.19 (d, J¼2.7 Hz, 0.1H, H-1a), 7.08e7.48 (m, 35H); 13C NMR (75 MHz, CDCl3) d 54.8 (b), 55.2 (a), 55.9 (a), 63.4 (b), 69.2 (b), 69.6 (a), 70.3 (b), 70.4 (a), 70.8 (a), 71.4 (a), 72.2 (a), 73.3 (a), 73.4 (b), 74.3 (b), 74.7 (b), 74.8 (b), 79.2 (b), 79.9 (a), 80.0 (a), 81.3 (b), 100.5 (b), 103.6 (a), 126.7, 127.0, 127.2, 127.6, 127.8, 128.0, 128.2, 128.3, 128.4, 128.6, 128.8, 137.5, 138.1, 139.0, 139.2, 139.7; Anal. Calcd for C48H49NO5: C, 80.08; H, 6.86; N, 1.95; found: C, 79.83; H, 6.80; N, 1.84; HRMS: calcd for C48H50NO5 [MþH]þ, 720.3684; found, 720.3673. 4.6. ((2R,3S,4R,5R,6R)-3,4,6-Tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)methyl acetate (10) To a solution of compound 9 (3.0 g, 4.2 mmol) in acetic anhydride and acetic acid (20 mL, v:v¼1:1), the 1.5% H2SO4 solution in acetic anhydride (1.0 mL) was added dropwise. The mixture was stirred at 60 C for 5 h under argon. Then the mixture was cooled down to room temperature and poured to ice water (80 mL). Sodium bicarbonate was added to the stirred solution in portions until no bubble occurred. The solution was extracted with CH2Cl2 (100 mL3) and the combined organic phase was dried over Na2SO4. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 6:1 to 4:1) to give 10 as a colourless oil (2.66 g, 95% yield) as major b-anomer. 1H NMR (300 MHz, CDCl3) d 2.03 (s, 3H), 3.03 (t, J¼8.4 Hz, 1H), 3.51e3.57 (m, 2H), 3.75e3.79 (m, 3H), 3.90e3.94 (m, 2H), 4.22 (dd, J¼4.2, 11.7 Hz, 1H), 4.35 (d, J¼11.7 Hz, 1H), 4.48 (d, J¼10.8 Hz, 1H), 4.58e4.67 (m, 2H), 4.76 (d, J¼10.8 Hz, 1H), 4.84 (d, J¼11.1 Hz, 1H), 4.93 (d, J¼11.4 Hz, 1H), 5.03 (d, J¼11.1 Hz, 1H), 7.12e7.48 (m, 25H); 13C NMR (75 MHz, CDCl3) d 20.8, 54.6, 63.2, 63.4, 70.6, 72.9, 74.4, 74.7, 78.8, 81.1, 100.2, 126.7, 127.2, 127.3, 127.8, 127.9, 128.0, 128.2, 128.3, 128.5, 128.7, 137.1, 137.7, 138.7, 139.5, 170.7; Anal. Calcd for C43H45NO6: C, 76.87; H, 6.75; N, 2.08; found: C, 76.60; H, 6.78; N, 1.87; HRMS: calcd for C43H46NO6 [MþH]þ, 672.3320; found, 672.3331. 4.7. ((2R,3S,4R,5R,6R)-3,4,6-Tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)methanol (11) Compound 10 (2.5 g, 3.7 mmol) was dissolved in methanol (20 mL), to which NaOMe (1 M solution in methanol, 1.0 mL, 1.0 mmol) was added and the resulting solution was stirred for 1 h
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at room temperature. Dowex 50w Hþ resin was added to neutralize the solution (pH¼7), after which the resin was removed by filtration and washed with ethyl acetate. The solvent was removed to afford 11 (2.3 g, quantitative) as an amorphous solid without further purification. 1H NMR (300 MHz, CDCl3) d 1.87 (t, J¼6.6 Hz, 1H), 2.98 (t, J¼9.6 Hz, 1H), 3.30e3.34 (m, 1H), 3.54 (t, J¼9.3 Hz, 1H), 3.64e3.71 (m, 1H), 3.77e3.82 (m, 4H), 3.93 (d, J¼13.8 Hz, 2H), 4.55e4.69 (m, 3H), 4.76 (d, J¼10.8 Hz, 1H), 4.86e4.93 (ABq, J¼11.4 Hz, 2H), 5.01 (d, J¼11.1 Hz, 1H), 7.18e7.48 (m, 25H); 13C NMR (75 MHz, CDCl3) d 54.9, 62.3, 63.3, 70.9, 74.3, 74.8, 75.1, 79.2, 81.0, 101.0, 126.8, 127.3, 127.8, 127.9, 128.1, 128.3, 128.4, 128.8, 137.3, 137.9, 138.9, 139.7; Anal. Calcd for C41H43NO5: C, 78.19; H, 6.88; N, 2.22; found: C, 77.89; H, 6.91; N, 2.05; HRMS: calcd for C41H44NO5 [MþH]þ, 630.3219; found, 630.3223. 4.8. (Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5(dibenzylamino) tetrahydro-2H-pyran-2-yl)methyl benzoate (12) A solution of 11 (60.0 mg, 0.10 mmol) in dichloromethane (5 mL) was added DesseMartin periodinane (65.0 mg, 0.15 mmol). After stirring at room temperature for 2 h, the reaction was quenched by adding saturated aqueous solutions of NaHCO3 (5 mL) and Na2S2O3 (5 mL). The mixture was diluted with diethyl ether (30 mL) and stirred at room temperature for 30 min. The aqueous layer was extracted with diethyl ether (20 mL3); the organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in dichloromethane (3 mL), triethylamine (0.1 mL) and dibenzyl phosphonate (25.0 mL, 0.15 mmol) were added. After stirring for 24 h at room temperature under argon, the reaction mixture was concentrated under reduced pressure. Purification by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1 to 4:1) followed by Sephedex (LH20) size-exclusion chromatography afforded compound 12 (79.0 mg, 93% yield) as a colourless oil, which is a mixture of diastereomers (3:1). Benzoyl chloride (65.0 mL, 0.55 mmol) was added dropwise to a solution of 12 (100.0 mg, 0.11 mmol) and Et3N (235.0 mL, 1.65 mmol) in dichloromethane (15 mL) under argon. The reaction was stirred at room temperature for 15 h, then diluted with dichloromethane (50 mL). The organic phase was washed by sodium bicarbonate solution, brine, dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 6:1 to 3:1) to afford 13a (79.0 mg) and 13b (24.0 mg) as colourless oil, in total yield of 93%. Data for compound 13a: 1H NMR (300 MHz, CDCl3) d 3.06 (t, J¼8.4 Hz, 1H), 3.51 (t, J¼9.0 Hz, 1H), 3.74e3.83 (m, 3H), 3.88e4.00 (m, 3H), 4.30 (d, J¼9.9 Hz, 1H), 4.66 (d, J¼10.8 Hz, 2H), 4.77e4.82 (m, 2H), 4.91 (d, J¼12.0 Hz, 1H), 5.06e5.16 (m, 5H), 6.18 (d, J¼11.4 Hz, 1H), 7.18e7.59 (m, 38H), 8.06 (d, J¼8.1 Hz, 2H); 13C NMR (75 MHz, CDCl3) d 54.5, 63.5, 66.4 (d, JPC¼165.1 Hz), 68.1 (d, JPC¼6.2 Hz), 68.5 (d, JPC¼6.8 Hz), 70.4, 73.1, 74.7 (d, JPC¼5.0 Hz), 77.4, 77.6, 82.0, 100.9, 126.6, 127.1, 127.3, 127.7, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 128.9, 130.0, 130.2, 133.6, 135.8 (d, JPC¼6.2 Hz), 137.3, 137.8, 138.6, 139.6, 165.1; 31P NMR (121.5 MHz, CDCl3) d 21.14; HRMS: calcd for C62H61NO9P [MþH]þ, 994.4078; found, 994.4093. Data for compound 13b: 1H NMR (300 MHz, CDCl3) d 3.08 (dd, J¼8.1, 9.6 Hz, 1H), 3.66e3.79 (m, 2H), 3.76 (d, J¼13.8 Hz, 2H), 3.89 (d, J¼13.8 Hz, 2H), 4.16 (t, J¼8.4 Hz, 1H), 4.56e4.65 (m, 2H), 4.76e4.81 (m, 3H), 4.85e5.05 (m, 4H), 5.05e5.22 (m, 2H), 6.06 (dd, J¼2.7, 11.1 Hz, 1H), 7.14e7.59 (m, 38H), 8.03e8.06 (m, 2H); 13C NMR (75 MHz, CDCl3) d 54.7, 63.0, 67.6 (d, JPC¼6.2 Hz), 68.0 (d, JPC¼165.0 Hz), 68.5 (d, JPC¼5.6 Hz), 70.1, 73.9, 74.5, 75.9, 77.2 (d, JPC¼5.0 Hz), 79.5, 81.4, 100.5, 126.7, 127.0, 127.1, 127.4, 127.6, 127.7, 127.8, 128.08, 128.15, 128.26, 128.35, 128.5, 128.7, 128.8, 129.4, 129.9, 133.4, 135.9 (d, JPC¼6.2 Hz), 136.5 (d,
JPC¼6.2 Hz), 136.9, 138.2, 138.9, 139.9, 164.8 (d, JPC¼6.2 Hz); 31P NMR (121.5 MHz, CDCl3) d 20.70; HRMS: calcd for C62H61NO9P [MþH]þ, 994.4078; found, 994.4079. 4.9. Dibenzyl (S)-hydroxy((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5-(dibenzylamino) tetrahydro-2H-pyran-2-yl)methylphosphonate (12a) Compound 13a (100.0 mg, 0.1 mmol) was treated with 33% methyl amine solution in ethanol (10 mL) at 0 C for 20 min. Then the mixture was diluted with dichloromethane. The mixture was concentrated and the residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1) to afford compound 12a (82.0 mg, 92% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 2.87e2.98 (m, 2H), 3.68e3.75 (m, 4H), 3.86 (d, J¼14.1 Hz, 2H), 4.21 (t, J¼11.1 Hz, 1H), 4.46 (d, J¼11.7 Hz, 1H), 4.58e4.66 (m, 1H), 4.65 (d, J¼8.4 Hz, 1H), 4.75e4.86 (m, 3H), 4.98 (d, J¼11.1 Hz, 1H), 5.05e5.09 (m, 4H), 7.16e7.38 (m, 35H); 13C NMR (75 MHz, CDCl3) d 54.6, 63.0, 66.6 (d, JPC¼159.5 Hz), 67.9 (d. JPC¼6.8 Hz), 68.1 (d, JPC¼6.8 Hz), 70.3, 73.6, 74.4, 74.7, 77.7 (d, JPC¼9.9 Hz), 80.9, 100.6, 126.7, 127.2, 127.7, 127.9, 128.0, 128.2, 128.3, 128.4, 128.6, 128.7, 136.0 (d, JPC¼5.0 Hz), 136.1 (d, JPC¼5.0 Hz), 137.1, 138.1, 138.8, 139.6; 31P NMR (121.5 MHz, CDCl3) d 25.57; HRMS: calcd for C55H57NO8P [MþH]þ, 890.3816; found, 890.3834. 4.10. Dibenzyl (R)-hydroxy((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5-(dibenzylamino) tetrahydro-2H-pyran-2-yl)methylphosphonate (12b) Compound 13b (50.0 mg, 0.05 mmol) was treated with 33% methyl amine solution in ethanol (5 mL) at 0 C for 2 h. Then the mixture was diluted with dichloromethane. The mixture was concentrated and the residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1) to afford compound 12b (39.0 mg, 88% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 3.05 (t, J¼8.7 Hz, 1H), 3.38 (brs, 1H), 3.73e3.81 (m, 4H), 3.90 (d, J¼13.5 Hz, 2H), 4.02 (t, J¼8.1 Hz, 1H), 4.26 (brs, 1H), 4.56e4.73 (m, 5H), 4.87 (t, J¼11.1 Hz, 2H), 5.00e5.13 (m, 4H), 7.13e7.39 (m, 35H); 13C NMR (75 MHz, CDCl3) d 54.8, 63.0, 67.7 (d, JPC¼6.8 Hz), 68.4 (d, JPC¼6.8 Hz), 69.0 (d, JPC¼158.3 Hz), 70.6, 73.5, 74.2, 76.2, 79.7 (d, JPC¼3.1 Hz), 81.3, 100.7, 126.7, 127.1, 127.3, 127.7, 127.8, 128.0, 128.1, 128.2, 128.4, 128.5, 128.8, 136.0 (d, JPC¼5.6 Hz), 136.4 (d, JPC¼6.2 Hz), 137.2, 137.8, 138.5, 139.6; 31P NMR (121.5 MHz, CDCl3) d 24.38; HRMS: calcd for C55H57NO8P [MþH]þ, 890.3816; found, 890.3823. 4.11. (1S)-((2S,3S,4R,5R)-5-Amino-3,4,6-trihydroxytetrahydro2H-pyran-2-yl)(hydroxy) methylphosphonic acid (2) A methanolic solution (3 mL) of compound 12a (50.0 mg, 0.056 mmol) was stirred at room temperature in the presence of 10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 2 days. The catalyst was then removed by filtration through Celite, and the filtrate was concentrated. The residue was purified by C-18 reversed-phase column chromatography (eluent: H2O) to give compound 2 (14.0 mg, a/b¼2:1, 96% yield) as white amorphous solids after lyophilization. 1H NMR (300 MHz, D2O) d 2.85 (t, J¼9.3 Hz, 0.5H), 3.11 (dd, J¼3.3, 10.5 Hz, 1H), 3.45e3.60 (m, 2.2H), 3.79 (t, J¼9.6 Hz, 1H), 3.88e4.00 (m, 2.8H), 4.79 (d, J¼8.7 Hz, 0.5H), 5.29 (d, J¼3.3 Hz, 1H); 13C NMR (75 MHz, D2O) data for both a and b anomers: d 54.8, 57.2, 66.5 (d, JPC¼155.8 Hz), 66.7 (d, JPC¼155.7 Hz), 69.4, 69.6 (d, JPC¼9.2 Hz), 70.4, 71.5, 72.6, 76.0, 89.8, 93.6; 31P NMR (121.5 MHz, CDCl3) d 19.25 (a), 19.62 (b); HRMS: calcd for C6H15NO8P [MþH]þ, 260.0530; found, 260.0533.
G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
4.12. (1R)-((2S,3S,4R,5R)-5-Amino-3,4,6trihydroxytetrahydro-2H-pyran-2-yl)(hydroxy) methylphosphonic acid (3) A methanolic solution (3 mL) of compound 12b (50.0 mg, 0.056 mmol) was stirred at room temperature in the presence of 10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 2 days. The catalyst was then removed by filtration through Celite, and the filtrate was concentrated. The residue was purified by C-18 reversed-phase column chromatography (eluent: H2O) to give compound 3 (13.5 mg, a/b¼2:1, 92% yield) as white amorphous solids after lyophilization. 1H NMR (300 MHz, D2O) d 2.86 (t, J¼8.7 Hz, 0.5H), 3.15 (dd, J¼3.0, 10.2 Hz, 1H), 3.49e4.00 (m, 6H), 4.76 (d, J¼8.4 Hz, 0.5H), 5.27 (d, J¼2.7 Hz, 1H); 13C NMR (75 MHz, D2O) data for both a and b anomers: d 56.6, 59.1, 70.1, 71.9 (d, JPC¼152.6 Hz), 71.8 (d, JPC¼151.4 Hz), 72.1, 72.2, 74.7, 75.1 (d, JPC¼7.2 Hz), 79.5 (d, JPC¼8.0 Hz), 91.7, 95.4; 31P NMR (121.5 MHz, CDCl3) d 18.50 (a), 18.10 (b); HRMS: calcd for C6H15NO8P [MþH]þ, 260.0530; found, 260.0531. 4.13. (S)-((S)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14a) chloride (R)-()-a-Methoxy-a-trifluoromethylphenylacetyl (4.3 mL, 22.4 mmol) was added dropwise to a solution of 12a (5.0 mg, 5.6 mmol), Et3N (6.4 mL, 45.0 mmol) and 4-dimethylaminopyridine (1.0 mg, 8.4 mmol) in dichloromethane (2 mL) under argon. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated. The residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1) to afford 14a as colourless oil (5.0 mg, 80% yield). 1H NMR (300 MHz, CDCl3) d 2.90 (dd, J¼8.1, 10.2 Hz, 1H), 3.22 (t, J¼9.0 Hz, 1H), 3.49 (s, 3H), 3.70e3.72 (m, 1H), 3.73 (d, J¼14.0 Hz, 2H), 3.88 (d, J¼14.0 Hz, 2H), 3.93 (dd, J¼4.2, 9.3 Hz, 1H, H-5), 4.32 (d, J¼10.5 Hz, 1H), 4.47 (d, J¼11.7 Hz, 1H), 4.64e4.75 (m, 4H), 4.81 (dd, J¼7.8, 12.0 Hz, 1H), 4.91e5.05 (m, 4H), 6.02 (d, J¼11.1 Hz, 1H), 7.06e7.40 (m, 38H), 7.57 (d, J¼7.8 Hz, 2H). 4.14. (R)-((S)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)3,4,6-tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (15a) Compound 15a was prepared from compound 12a and (S)(þ)-a-methoxy-a-trifluoromethylphenylacetyl chloride as described in the preparation of compound 14a, yielding 15a (84% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 2.81 (t, J¼9.0 Hz, 1H), 3.05 (t, J¼9.0 Hz, 1H), 3.43 (s, 3H), 3.65e3.73 (m, 3H), 3.83e3.87 (m, 3H, H-5 and NeCH2), 4.26 (d, J¼10.2 Hz, 1H), 4.44 (d, J¼12.0 Hz, 1H), 4.58e4.68 (m, 4H), 4.93e5.10 (m, 5H), 5.95 (d, J¼11.4 Hz, 1H), 7.09e7.41 (m, 38H), 7.57 (d, J¼7.5 Hz, 2H). 4.15. (S)-((R)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)3,4,6-tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14b) Compound 14b was prepared from compound 12b and (R)()-a-methoxy-a-trifluoromethylphenylacetyl chloride as described in the preparation of compound 14a, yielding 14b (85% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 2.99 (t, J¼8.7 Hz, 1H), 3.46 (s, 3H), 3.56 (d, J¼10.8 Hz, 1H, H-5), 3.70e3.76 (m, 3H), 3.88 (d, J¼13.5 Hz, 2H), 4.12 (t, J¼9.3 Hz, 1H), 4.27 (d, J¼11.4 Hz, 1H), 4.40 (d, J¼8.1 Hz, 1H), 4.56 (d, J¼12.0 Hz, 1H), 4.71e5.09 (m, 8H), 6.00 (d, J¼11.7 Hz, 1H), 7.18e7.39 (m, 38H), 7.54 (d, J¼7.8 Hz, 2H).
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4.16. Polymerase chain reaction The glmS DNA template has a sequence of 50 -GAATT CTAAT ACGAC TCACT ATAGG TCTTG TTCTT ATTTT CTCAA TAGGA AAAGA AGACG GGATT ATTGC TTTAC CTATA ATTAT AGCGC CCGAA CTAAG CGCCC GGAAA AAGGC TTAGT TGACG AGGAT GGAGG TTATC GAATT TTCGG CGGAT GCCTC CCGGC TGAGT GTGCA GATCA CAGCC GTAAG GATTT CTTCA AACCA AGGGG GTGAC TCCTT GAACA AAGAG AAATC ACATG ATCTT CCAAA AAACA TGTAG GAGGG GAC-30 , which was obtained through PCR amplification of a lab plasmid, originally derived from B. subtilis chromosomal DNA (1A40). DNA primers were obtained through automated synthesis using standard phosphoramidite chemistry (Integrated DNA Technologies, Iowa, USA). Before usage, the oligonucleotides were purified by 10% denaturing PAGE and quantified by absorbance at 260 nm. Primers used include the forward sequence 50 -GAATT CTAAT ACGAC TCACT ATAGG TCTTG TTCTT ATT-30 (T7 RNA polymerase promoter sequence is shown in italicized letters; the þ1 transcription initiation site and critical nucleotides for in vitro transcription are shown in bold letters), and the reverse sequence 50 -GTCCC CTCCT ACATG TTTTT TGG-30 . PCR was conducted in 75 mM TriseHCl (pH 9.0), 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, each primer at 0.5 mM, DNA plasmid at w3 nM, each dNTP at 0.5 mM, and 5 units of Taq DNA polymerase (Biotools, Madrid, Spain). Thermal cycling steps were: 94 C for 1 min, 20 cycles of 94 C to 50 C to 72 C (30 s for each temperature), and finally 72 C for 8 min.
4.17. RNA preparation In vitro transcription was conducted at 37 C for 3 h in 150 mL of 40 mM TriseHCl, pH7.9, 6 mM MgCl2, 10 mM DTT, 10 mM NaCl, 2 mM spermidine, 25 pmol PCR amplified DNA, 2.5 mM each of GTP, CTP, UTP, ATP, 1.07 units/mL RiboLock Ribonuclease Inhibitor and 1.33 units/mL T7 RNA polymerase (Fermentas, Canada). The transcription mixture was then treated with DNase I (3 units) in the presence of 0.2 mM CaCl2 at 37 C for 15 min (Fermentas, Canada). The transcribed RNA was subsequently concentrated through standard ethanol precipitation, purified by 10% denaturing PAGE and then quantified by absorbance at 260 nm. Afterwards, the 50 triphosphate of the RNA was dephosphorylated with calf intestine alkaline phosphatase following manufacturer’s protocol (Fermentas, Canada), and extracted with phenol/chloroform (1:1) (BioShop, Canada). RNA samples were concentrated through standard ethanol precipitation, before labelling the 50 -terminus with [g-32P]ATP (Perkin Elmer, Massachusetts, USA) using T4 polynucleotide kinase according to manufacturer’s protocol (Fermentas, Canada). 50 -32P labelled RNA was purified again by 10% denaturing PAGE before usage.
4.18. Ribozyme cleavage assays A 200 mM concentration of glucosamine-6-phosphate (GlcN6P) (Sigma, Oakville, Canada), or a test compound (1, 2 or 3) was added to 0.5 pmol 50 -32P labelled glmS RNA. Samples were incubated in 50 mM HEPES (pH7.5), 10 mM MgCl2, 200 mM KCl at room temperature for a duration of 1 or 2 min. Negative control samples included: i) addition of double-distilled water instead of compound, or ii) addition of GlcN6P, without Mg2þ in the reaction buffer. After the testing period, reactions were terminated by the addition of loading dye solution containing 100 mM EDTA and 7 M urea (Sigma, Oakville, Canada). RNA products were separated by 10% denaturing PAGE and visualized using a PhosphorImager and ImageQuant software. Percent cleavage for each reaction was calculated using the following equation:
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Percent Cleavage ¼
cleavage fragment cleavage fragment þ precursor glmS RNA background 100%
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21072014) and ‘973’ grant from the Ministry of Science and Technology of China (2012CB822100). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2012.09.015. References and notes 1. Winkler, W.; Nahvi, A.; Breaker, R. R. Nature 2002, 419, 952e956. 2. Coppins, R. L.; Hall, K. B.; Groisman, E. A. Curr. Opin. Microbiol. 2007, 10, 176e181. 3. Blount, K. F.; Breaker, R. R. Nat. Biotechnol. 2006, 24, 1558e1564. 4. Barrick, J. E.; Corbino, K. A.; Winkler, W. C.; Nahvi, A.; Mandal, M.; Collins, J.; Lee, M.; Roth, A.; Sudarsan, N.; Jona, I.; Wickiser, J. K.; Breaker, R. R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6421e6426. 5. Klein, D. J.; Ferre-D’Amare, A. R. Science 2006, 313, 1752e1756. 6. Cochrane, J. C.; Lipchock, S. V.; Strobel, S. A. Chem. Biol. 2007, 14, 97e105. 7. Winkler, W. C.; Nahvi, A.; Roth, A.; Collins, J. A.; Breaker, R. R. Nature 2004, 428, 281e286. 8. Milewski, S. Biochim. Biophys. Acta 2002, 1597, 173e192. 9. Wu, H. C.; Wu, T. C. J. Bacteriol. 1971, 105, 455e466. 10. Sarvas, M. J. Bacteriol. 1971, 105, 467e471. 11. Freese, E. B.; Cole, R. M.; Klofat, W.; Freese, E. J. Bacteriol. 1970, 101, 1046e1062.
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Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch Guan-Nan Wang a, Pui Sai Lau b, c, Yingfu Li b, c, Xin-Shan Ye a, * a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Rd No. 38, Beijing 100191, China Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada c Department of Chemistry and Chemical Biology, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2012 Accepted 4 September 2012 Available online 10 September 2012
The glmS riboswitch is a ribozyme found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. Herein, we describe the efficient synthesis of three GlcN6P analogues and their evaluation on inducing self-cleavage of the glmS riboswitch from Bacillus subtilis. Our results provide valuable information for further elucidation of the structureeactivity relationships and drug design for glmS riboswitch antibiotics. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: glmS Riboswitch GlcN6P analogues Antibiotics Synthesis Carbohydrate
1. Introduction Riboswitches are structural mRNA domains that regulate gene expression in response to the intracellular concentration of specific metabolites. They control essential genes in many pathogenic bacteria, thus representing an intriguing class of RNA target for the development of antibiotics and chemicalebiological tools.1e3 Notably, the glmS riboswitch, which is located upstream of the gene encoding glucosamine-6-phosphate synthase (GlmS) in numerous Gram-positive bacteria, is unique in that it is the first example of a natural ribozyme that is also a riboswitch.4 As part of gene regulation, it binds to its small molecule metabolite, glucosamine-6phosphate (GlcN6P), which triggers self-cleavage targeting its own mRNA.5,6 Functionally, the glmS ribozyme controls the amount of GlmS to regulate cellular production of GlcN6P,7,4 which is an essential metabolite for the biosynthesis of bacterial cell walls and fungal cell wall chitin.8e11 Given the importance of GlcN6P, inhibition of the metabolite is lethal to microorganisms. Consequently, the glmS riboswitch is emerging as a new drug target, and small molecules that mimic GlcN6P to trigger riboswitch activity have the potential to become antibiotics. Although substantial structural and mechanistic information of the glmS riboswitch have been disclosed over recent years,5,6,12e15
* Corresponding author. Tel.: þ86 10 82805736; fax: þ86 10 82802724; e-mail address: [email protected] (X.-S. Ye). 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2012.09.015
few small molecule agonists and antagonists have been developed.16,17 Activators that have comparable or superior activity than natural GlcN6P are in great demand for a better understanding of the riboswitch and paving the way to the discovery of effective antibiotics. In this research, three GlcN6P analogues have been designed and synthesized (Fig. 1). Carba-sugar 1 was designed to address the role of the oxygen atom in pyranose ring for riboswitch activation. Compounds 2 and 3 were designed to add an extra hydroxyl group at the C-6 position with the consideration of providing extra binding sites around the pocket. Instead of 6-phosphate, the 6-phosphonate was introduced to the 6-position. It is known that the PeC bond makes compounds resistant to enzymatic hydrolysis18,19 and has conformational preferences different from those in phosphates.20 These analogues were subsequently investigated for their ability to induce self-cleavage of the glmS riboswitch from Bacillus subtilis.
Fig. 1. The structures of three GlcN6P analogues.
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2. Results and discussion The synthesis of carba-sugar 1 is shown in Scheme 1. Carbocyclic compound 5 was prepared from methyl glucopyranoside 4 according to the procedures described by Barton et al.21 Compound 5 was treated with dibenzyl N,N-diisopropylphosphoramidite, followed by oxidation with meta-chloroperoxybenzoic acid (m-CPBA) to give the fully protected phosphate 6 in 86% isolated yield. The chemical shift of compound 6 in 31P NMR spectroscopy is at d 3.14, indicating the formation of phosphate.22 Subsequently, basic opening of the oxazolidinone in 6 also resulted in the removal of one of the benzyl groups on phosphate, yielding compound 7.
Catalytic hydrogenolysis of 7 provided the phosphated carba-sugar 1 smoothly. The synthesis of compounds 2 and 3 started from glucosamine 8. As shown in Scheme 2, perbenzylation of 8 provided compound 9, and selective acetolysis of 9 afforded the 6-acetylated compound 10 in good yield. It was reported that the anomeric position is usually acetylated prior to the 6-position in substrates, such as perbenzylated glucose, galactose and mannose.23,24 However, in the case of compound 9, the dibenzylated amino group protected the anomeric position from acetolysis. Removal of the acetyl group in 10 led to compound 11, which was oxidized by DesseMartin periodinane followed by the Pudovik reaction25,26 with dibenzyl
Scheme 1. Synthesis of carba-sugar 1.
Scheme 2. Synthesis of GlcN6P analogues 2 and 3.
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phosphonate and triethylamine to yield the a-hydroxyphosphonate 12 in 93% isolated yield as an inseparable mixture of diastereoisomers (3:1). The separation of two diastereoisomers was achieved after benzoylation of the nascent hydroxyl groups. Deprotection of the benzoyl groups with methyl amine solution, which was followed by deprotection of the benzyl groups using catalytic hydrogenolysis, afforded the target compounds 2 and 3 in good yields, respectively. The absolute configurations of diastereoisomers 12a and 12b were assigned by the Mosher method (also known as Mosher ester analysis).27e29 As displayed in Scheme 3, each of the diastereomeric S- and R-MTPA esters of major diastereomer 12a was prepared from (R)-MTPAeCl and (S)-MTPAeCl, respectively (S-Mosher acid chloride gives rise to the R-Mosher ester due to relative priority: CF3 is lower than COCl but higher than COOR). The H-5 proton signal of each diastereomeric Mosher ester was assigned by 1He1H COSY experiments. The chemical shift of H-5 in S-MTPA ester is dS¼3.93, while the chemical shift of H-5 in R-MTPA ester is dR¼3.85. That is, the DdSR¼dSdR¼3.933.85¼0.08 was positive. According to the Mosher empirical rule, the absolute configuration of the 6-position in compound 12a was S-. This could be explained by the MTPA plane figures (Fig. 2). The H-5 of R-MTPA ester, in which H-5 and phenyl group reside are on the same side of the MTPA plane, is relatively more shielded (upfield in its spectrum) than H-5 in the SMTPA ester due to the magnetic shielding effect of phenyl group. Correspondingly, the minor diastereomer was assigned as Rconfiguration.
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H-5 proton of the major diastereomer 14a was downfield (d¼3.93 ppm) relative to that of the minor diastereomer 14b (d¼3.56 ppm). These confirmed that the configuration of major product 12a is S and the configuration of minor product 12b is R. The Pudovik reaction, one of the most versatile methods for construction of CeP bonds, involves the addition of organophosphorus compounds to unsaturated bonds. With the potential to synthesize biologically important a-hydroxyphosphonic acids derivatives,31e33 this reaction has received more and more attention to make the process highly enantioselective.34,35 The examples presented herein showed the versatility of the Mosher method as a convenient and alternative approach to the assignment of stereochemistry of Pudovik products especially when the crystallization of products is impossible. It is applicable to either a single enantiomer or a pair of enantiomers. The synthetic compounds 1, 2 and 3 were subsequently investigated for their ability to induce self-cleavage of 50 -32P-labelled glmS riboswitch from B. subtilis. As shown in Table 1 and Fig. 4, carba-sugar 1 showed moderate activation of self-cleavage of glmS riboswitch from B. subtilis. The percentage of riboswitch self-cleavage induced by compound 1 is around half to that by natural metabolite GlcN6P. At the same time during the preparation of this manuscript, Wittmann and Mayer et al. reported their research of carba-sugar 1 on glmS riboswitch of vancomycin-resistant Staphylococcus aureus.36 In their study, it was found that the potency of compound 1 for glmS riboswitch of S. aureus is similar to that of GlcN6P. They also mentioned the
Scheme 3. Synthesis of R- and S-Mosher esters from compound 12a.
Fig. 2. The MTPA plane of S- and R-MTPA esters.
To further confirm the assignment of the absolute configurations of 12a and 12b, the S-MTPA esters (14a and 14b) generated from both diastereoisomers 12a and 12b were compared (Fig. 3).27,30 The 1H NMR analysis of both products showed that the
activation by compound 1 in the glmS riboswitch of B. subtilis. The activation achieved by compound 1 in our experiments compared favourably with the data reported by them. It seems that different sources of glmS ribozyme might have different
Fig. 3. The MTPA plane of S-MTPA esters 14a and 14b made from 12a and 12b.
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Table 1 Effect of synthetic compounds on glmS riboswitch Sample
No reaction (NR) No magnesium (-Mg2þ) GlcN6P 1 2 3
% Cleavage 1 min
2 min
d d 67.50 29.08 0 0
d d 65.12 38.68 0.37 0.29
moderate self-cleavage of the glmS riboswitch from B. subtilis. The displacement of phosphate in GlcN6P by a-hydroxyphosphonate led to massive loss of activation. These results highlight the role of phosphate moiety in contributing to the binding of the glmS riboswitch. These insights are useful for further development of chemical agonists or antagonists for the glmS riboswitch. It seems that glmS riboswitch exhibits high level of discrimination against many closely related GlcN6P analogues. Our results provide a deeper understanding of the glmS riboswitch and further elucidation on the structureeactivity relationships of metabolite derivatives.
Fig. 4. Cleavage of glmS RNA with and without compounds. (a) Cleavage of glmS RNA after 1 min reaction time; (b) Cleavage of glmS RNA after 2 min reaction time. A 200 mM concentration of GlcN6P or a test compound was added to 0.5 pmol of 50 -32P labelled glmS RNA. Samples were incubated in 50 mM of HEPES (pH 7.5), 10 mM of MgCl2, 200 mM of KCl at room temperature for a duration of 1 or 2 min. Negative control samples included: i) addition of double-distilled water instead of compound, or ii) addition of GlcN6P, without Mg2þ in the reaction buffer. RNA products were separated by 10% denaturing polyacrylamide gel electrophoresis (PAGE).
sensitivities to GlcN6P analogues. On the other hand, the 6-phosphonate derivatives 2 and 3 did not exhibit activity under the tested assay conditions. It was determined that the oxygen ring and phosphate group are required for stabilization of the interaction of the riboswitch with GlcN6P rather than being involved in the catalytic reaction.37,38 The results herein show that the destabilization of the interaction can tolerate carba-sugar to some extent, whereas the 6-phosphate group is critical for interaction.16,38 The phosphate moiety forms the fixed network of Mg2þ-coordinated interactions with the various functional groups of the ligand binding pocket.15 The electrical properties of a-hydroxylphosphonate are slightly different from 6-phosphate. The change in electrical properties of 6-phosphonate might change the coordination with cations and further change the interaction with the binding pocket.13 The other factor may rely on the unfilled space within the ligand binding pocket in the absence of the bridging oxygen. 3. Conclusions Three GlcN6P analogues have been prepared to investigate their effect on the glmS riboswitch. The Pudovik reaction was applied to synthesize the a-hydroxyphosphonate substrates and the configurations of two diastereomeric products were assigned by the Mosher method. Among the three analogues, carba-sugar 1 induced
4. Experimental section 4.1. General procedures Air- and/or moisture-sensitive reactions were carried out under an atmosphere of argon using flame-dried glassware and standard syringe/septa techniques. All chemicals were purchased as reagent grade and used without further purification, unless otherwise noted. Dichloromethane (CH2Cl2) and pyridine were distilled over calcium hydride (CaH2). Methanol was distilled from magnesium. DMF was stirred with CaH2 and distilled under reduced pressure. Tetrahydrofuran (THF) was distilled over sodium/benzophenone. Reactions were monitored by analytical thin-layer chromatography on silica gel 60 F254 precoated on aluminium plates (E. Merck). Spots were detected under UV (254 nm) and/or by staining with acidic ceric ammonium molybdate. Column chromatography was performed on silica gel (200e300 mesh). 1H NMR, 13C NMR, and 31P NMR spectra were recorded on a JEOL AL-300, or Varian INOVA-500 spectrometers at 25 C. Chemical shifts (in parts per million) were referenced to tetramethylsilane (d¼0 ppm) in deuterated chloroform. 13C NMR spectra were obtained by using the same NMR spectrometers and were calibrated with CDCl3 (d¼77.00 ppm) or CD3OD (d¼49.00 ppm). 31P NMR spectra were reported parts per million (d) relative to H3PO4 (0.00 ppm) as an internal reference. Mass spectra were recorded using a PE SCIEX QSTAR spectrometer. Elemental analysis data were recorded on a Vario EL-Z elemental analyser.
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4.2. Dibenzyl((3aS,4R,5R,6R,7aS)-3-benzyl-4,5-bis(benzyloxy)2-oxooctahydrobenzo[d]oxazol-6-yl)methyl phosphate (6)
(121.5 MHz, D2O) d 2.84; HRMS: calcd for C7H16NO7P Na [MþNa]þ, 258.0737; found, 258.0733.
To a solution of dibenzyl N,N-diisopropylphosphoramidite (90%, 111 mL, 300 mmol) in CH2Cl2 (10 mL) was added 1H-tetrazole (42.5 mg, 607 mmol). The mixture was stirred at room temperature for 15 min under argon atmosphere, and then the CH2Cl2 solution (2 mL) of compound 5 (57.0 mg, 120 mmol) was added. The mixture was stirred for another 4 h at room temperature under argon atmosphere. The mixture was cooled to 0 C and m-CPBA (70%, 89.0 mg, 361 mmol) was added. After stirring for 45 min, the mixture was diluted with EtOAc (50 mL). The solution was washed with aqueous 10% Na2SO3 (220 mL), 1 M HCl (220 mL), saturated NaHCO3 (220 mL), and brine (320 mL). The organic layer was dried (MgSO4) and concentrated. Purification by column chromatography (petroleum ethereethyl acetate, 1:1) gave the phosphate triester 6 as an amorphous solid (76.0 mg, 86% yield). 1H NMR (300 MHz, CDCl3) d 1.65e1.73 (m, 1H), 1.89e1.94 (d, J¼14.4 Hz, 1H), 2.04 (brs, 1H), 3.41e3.43 (m, 1H), 3.56e3.59 (m, 1H), 3.67 (brs, 1H), 3.84e3.95 (m, 2H), 4.14 (brs, 1H), 4.40 (d, J¼11.7 Hz, 1H), 4.49 (s, 2H), 4.54 (brs, 1H), 4.66 (d, J¼12.3 Hz, 1H), 4.83 (d, J¼15.3 Hz, 1H), 5.02 (brs, 4H), 7.07e7.32 (m, 25H); 13C NMR (75 MHz, CDCl3) d 26.8, 35.7 (d, JPC¼8.0 Hz), 46.3, 55.9, 68.1 (d, JPC¼5.6 Hz), 69.5 (d, JPC¼3.8 Hz), 72.6, 72.9, 73.3, 77.2 (d, JPC¼4.4 Hz), 77.8, 127.6, 127.7, 127.9, 128.0, 128.2, 128.5, 128.6, 128.8, 135.5, 135.6, 137.4, 137.6, 158.0; 31P NMR (121.5 MHz, CDCl3) d 3.14. HRMS: calcd for C43H44NO8P Na [MþNa]þ, 756.2702; found, 756.2704.
4.5. (3R,4R,5S,6R)-N,N-Dibenzyl-2,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-2H-pyran-3-amine (9)
4.3. Sodium benzyl((1R,2R,3R,4S,5S)-4-(benzylamino)-2,3bis(benzyloxy)-5-hydroxy cyclohexyl)methyl phosphate (7) A solution of compound 6 (100.0 mg, 0.14 mmol) in ethanolicsodium hydroxide solution (10%) (v/v¼1:1, 5 mL) was heated under reflux overnight. The solvents were removed and the residue was purified by C-18 reversed-phase column chromatography (eluent: H2O to CH3OH/H2O, 1:1) to give 7 (80.0 mg, 92% yield) as an amorphous solid. 1H NMR (300 MHz, CD3OD) d 1.54 (t, J¼12.6 Hz, 1H), 1.87e1.91 (m, 1H), 2.11 (brs, 1H), 2.51 (dd, J¼2.4, 9.9 Hz, 1H), 3.46 (t, J¼10.2 Hz, 1H), 3.63 (t, J¼9.6 Hz, 1H), 3.65 (d, J¼13.2 Hz, 1H), 3.85 (d, J¼13.2 Hz, 1H), 3.92e3.96 (m, 1H), 4.06e4.07 (m, 1H), 4.12e4.18 (m, 1H), 4.63 (d, J¼11.4 Hz, 1H), 4.73e4.90 (m, 5H), 7.18e7.35 (m, 20H); 13C NMR (75 MHz, CD3OD) d 32.9, 39.2 (d, JPC¼8.6 Hz), 51.9, 63.9, 65.8, 66.4 (d, JPC¼5.6 Hz), 68.1 (d, JPC¼5.6 Hz), 75.8, 75.9, 83.1, 83.7, 128.15, 128.2, 128.4, 128.5, 128.6, 128.8, 128.9, 129.3, 129.4, 139.8, 139.9, 140.1 (d, JPC¼3.2 Hz), 140.2, 141.2; 31P NMR (121.5 MHz, CDCl3) d 2.69; HRMS: calcd for C35H40NO7PNa[MþNa]þ, 640.2440; found, 640.2440. 4.4. ((1R,2R,3R,4S,5S)-4-Amino-2,3,5-trihydroxycyclohexyl)methyl dihydrogen phosphate (1) A methanolic solution (8 mL) of compound 7 (50.0 mg, 0.078 mmol) was stirred at room temperature in the presence of 10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 48 h. The catalyst was then removed by filtration through Celite, and the filtrate was concentrated. In most cases, direct lyophilization gave the pure compound 1 (19.5 mg, 97% yield) as white amorphous solids. In the cases when further purification was needed, the water solution of residue was adjusted to pH¼2 by 1 N HCl, and purified by Dowex 50W8 (Hþ form, 200e400 mesh, eluent: H2O) to give 1 as white amorphous solids after lyophilization. 1H NMR (300 MHz, D2O) d 1.43 (t, J¼13.5 Hz, 1H), 1.71e1.80 (m, 2H), 2.96 (d, J¼11.1 Hz, 1H), 3.17 (t, J¼9.9 Hz, 1H), 3.48 (t, J¼10.5 Hz, 1H), 3.77e3.82 (m, 1H), 3.89e3.95 (m, 1H), 4.03 (s, 1H); 13C NMR (75 MHz, D2O) d 31.7, 37.4 (d, JPC¼7.4 Hz), 56.9, 65.6, 66.9 (d, JPC¼5.0 Hz), 71.7, 72.6; 31P NMR
To a suspension of D-glucosamine (5.0 g, 27.9 mmol) in anhydrous DMF (70 mL) was added NaH (3.7 g, 60% dispersion in mineral oil, 92.5 mmol) at 0 C. Benzyl bromide (8.1 mL, 66.6 mmol) was added dropwise over 5 min. After stirring at room temperature for 1 h, same quantities of NaH and benzyl bromide were added at 0 C. After stirring for another 1 h, the third portion of NaH (3.8 g, 95.0 mmol) and benzyl bromide (8.2 mL, 67.6 mmol) were added consecutively at 0 C and then the mixture was warmed to room temperature. The reaction mixture was stirred overnight, and then MeOH was added slowly to quench the excess of the NaH. DMF was removed under reduced pressure. The residue was dissolved in CH2Cl2 (150 mL) and washed with water (20 mL) and brine (20 mL). The organic layer was dried (Na2SO4), filtered, and evaporated to give a yellow oil. The residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 20:1 to 6:1) to give 9 as a colourless oil (17.8 g, 89% yield) as a mixture of anomers (a/b¼1:8). 1H NMR (300 MHz, CDCl3) d 3.03 (t, J¼8.4 Hz, 1H), 3.45 (brs, 1H), 3.54e3.86 (m, 8H), 3.92 (d, J¼14.1 Hz, 2H), 4.04e4.73 (m, 7H), 4.84 (d, J¼10.8 Hz, 1H), 4.98 (t, J¼10.8 Hz, 2H), 5.19 (d, J¼2.7 Hz, 0.1H, H-1a), 7.08e7.48 (m, 35H); 13C NMR (75 MHz, CDCl3) d 54.8 (b), 55.2 (a), 55.9 (a), 63.4 (b), 69.2 (b), 69.6 (a), 70.3 (b), 70.4 (a), 70.8 (a), 71.4 (a), 72.2 (a), 73.3 (a), 73.4 (b), 74.3 (b), 74.7 (b), 74.8 (b), 79.2 (b), 79.9 (a), 80.0 (a), 81.3 (b), 100.5 (b), 103.6 (a), 126.7, 127.0, 127.2, 127.6, 127.8, 128.0, 128.2, 128.3, 128.4, 128.6, 128.8, 137.5, 138.1, 139.0, 139.2, 139.7; Anal. Calcd for C48H49NO5: C, 80.08; H, 6.86; N, 1.95; found: C, 79.83; H, 6.80; N, 1.84; HRMS: calcd for C48H50NO5 [MþH]þ, 720.3684; found, 720.3673. 4.6. ((2R,3S,4R,5R,6R)-3,4,6-Tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)methyl acetate (10) To a solution of compound 9 (3.0 g, 4.2 mmol) in acetic anhydride and acetic acid (20 mL, v:v¼1:1), the 1.5% H2SO4 solution in acetic anhydride (1.0 mL) was added dropwise. The mixture was stirred at 60 C for 5 h under argon. Then the mixture was cooled down to room temperature and poured to ice water (80 mL). Sodium bicarbonate was added to the stirred solution in portions until no bubble occurred. The solution was extracted with CH2Cl2 (100 mL3) and the combined organic phase was dried over Na2SO4. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 6:1 to 4:1) to give 10 as a colourless oil (2.66 g, 95% yield) as major b-anomer. 1H NMR (300 MHz, CDCl3) d 2.03 (s, 3H), 3.03 (t, J¼8.4 Hz, 1H), 3.51e3.57 (m, 2H), 3.75e3.79 (m, 3H), 3.90e3.94 (m, 2H), 4.22 (dd, J¼4.2, 11.7 Hz, 1H), 4.35 (d, J¼11.7 Hz, 1H), 4.48 (d, J¼10.8 Hz, 1H), 4.58e4.67 (m, 2H), 4.76 (d, J¼10.8 Hz, 1H), 4.84 (d, J¼11.1 Hz, 1H), 4.93 (d, J¼11.4 Hz, 1H), 5.03 (d, J¼11.1 Hz, 1H), 7.12e7.48 (m, 25H); 13C NMR (75 MHz, CDCl3) d 20.8, 54.6, 63.2, 63.4, 70.6, 72.9, 74.4, 74.7, 78.8, 81.1, 100.2, 126.7, 127.2, 127.3, 127.8, 127.9, 128.0, 128.2, 128.3, 128.5, 128.7, 137.1, 137.7, 138.7, 139.5, 170.7; Anal. Calcd for C43H45NO6: C, 76.87; H, 6.75; N, 2.08; found: C, 76.60; H, 6.78; N, 1.87; HRMS: calcd for C43H46NO6 [MþH]þ, 672.3320; found, 672.3331. 4.7. ((2R,3S,4R,5R,6R)-3,4,6-Tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)methanol (11) Compound 10 (2.5 g, 3.7 mmol) was dissolved in methanol (20 mL), to which NaOMe (1 M solution in methanol, 1.0 mL, 1.0 mmol) was added and the resulting solution was stirred for 1 h
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at room temperature. Dowex 50w Hþ resin was added to neutralize the solution (pH¼7), after which the resin was removed by filtration and washed with ethyl acetate. The solvent was removed to afford 11 (2.3 g, quantitative) as an amorphous solid without further purification. 1H NMR (300 MHz, CDCl3) d 1.87 (t, J¼6.6 Hz, 1H), 2.98 (t, J¼9.6 Hz, 1H), 3.30e3.34 (m, 1H), 3.54 (t, J¼9.3 Hz, 1H), 3.64e3.71 (m, 1H), 3.77e3.82 (m, 4H), 3.93 (d, J¼13.8 Hz, 2H), 4.55e4.69 (m, 3H), 4.76 (d, J¼10.8 Hz, 1H), 4.86e4.93 (ABq, J¼11.4 Hz, 2H), 5.01 (d, J¼11.1 Hz, 1H), 7.18e7.48 (m, 25H); 13C NMR (75 MHz, CDCl3) d 54.9, 62.3, 63.3, 70.9, 74.3, 74.8, 75.1, 79.2, 81.0, 101.0, 126.8, 127.3, 127.8, 127.9, 128.1, 128.3, 128.4, 128.8, 137.3, 137.9, 138.9, 139.7; Anal. Calcd for C41H43NO5: C, 78.19; H, 6.88; N, 2.22; found: C, 77.89; H, 6.91; N, 2.05; HRMS: calcd for C41H44NO5 [MþH]þ, 630.3219; found, 630.3223. 4.8. (Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5(dibenzylamino) tetrahydro-2H-pyran-2-yl)methyl benzoate (12) A solution of 11 (60.0 mg, 0.10 mmol) in dichloromethane (5 mL) was added DesseMartin periodinane (65.0 mg, 0.15 mmol). After stirring at room temperature for 2 h, the reaction was quenched by adding saturated aqueous solutions of NaHCO3 (5 mL) and Na2S2O3 (5 mL). The mixture was diluted with diethyl ether (30 mL) and stirred at room temperature for 30 min. The aqueous layer was extracted with diethyl ether (20 mL3); the organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in dichloromethane (3 mL), triethylamine (0.1 mL) and dibenzyl phosphonate (25.0 mL, 0.15 mmol) were added. After stirring for 24 h at room temperature under argon, the reaction mixture was concentrated under reduced pressure. Purification by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1 to 4:1) followed by Sephedex (LH20) size-exclusion chromatography afforded compound 12 (79.0 mg, 93% yield) as a colourless oil, which is a mixture of diastereomers (3:1). Benzoyl chloride (65.0 mL, 0.55 mmol) was added dropwise to a solution of 12 (100.0 mg, 0.11 mmol) and Et3N (235.0 mL, 1.65 mmol) in dichloromethane (15 mL) under argon. The reaction was stirred at room temperature for 15 h, then diluted with dichloromethane (50 mL). The organic phase was washed by sodium bicarbonate solution, brine, dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 6:1 to 3:1) to afford 13a (79.0 mg) and 13b (24.0 mg) as colourless oil, in total yield of 93%. Data for compound 13a: 1H NMR (300 MHz, CDCl3) d 3.06 (t, J¼8.4 Hz, 1H), 3.51 (t, J¼9.0 Hz, 1H), 3.74e3.83 (m, 3H), 3.88e4.00 (m, 3H), 4.30 (d, J¼9.9 Hz, 1H), 4.66 (d, J¼10.8 Hz, 2H), 4.77e4.82 (m, 2H), 4.91 (d, J¼12.0 Hz, 1H), 5.06e5.16 (m, 5H), 6.18 (d, J¼11.4 Hz, 1H), 7.18e7.59 (m, 38H), 8.06 (d, J¼8.1 Hz, 2H); 13C NMR (75 MHz, CDCl3) d 54.5, 63.5, 66.4 (d, JPC¼165.1 Hz), 68.1 (d, JPC¼6.2 Hz), 68.5 (d, JPC¼6.8 Hz), 70.4, 73.1, 74.7 (d, JPC¼5.0 Hz), 77.4, 77.6, 82.0, 100.9, 126.6, 127.1, 127.3, 127.7, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 128.9, 130.0, 130.2, 133.6, 135.8 (d, JPC¼6.2 Hz), 137.3, 137.8, 138.6, 139.6, 165.1; 31P NMR (121.5 MHz, CDCl3) d 21.14; HRMS: calcd for C62H61NO9P [MþH]þ, 994.4078; found, 994.4093. Data for compound 13b: 1H NMR (300 MHz, CDCl3) d 3.08 (dd, J¼8.1, 9.6 Hz, 1H), 3.66e3.79 (m, 2H), 3.76 (d, J¼13.8 Hz, 2H), 3.89 (d, J¼13.8 Hz, 2H), 4.16 (t, J¼8.4 Hz, 1H), 4.56e4.65 (m, 2H), 4.76e4.81 (m, 3H), 4.85e5.05 (m, 4H), 5.05e5.22 (m, 2H), 6.06 (dd, J¼2.7, 11.1 Hz, 1H), 7.14e7.59 (m, 38H), 8.03e8.06 (m, 2H); 13C NMR (75 MHz, CDCl3) d 54.7, 63.0, 67.6 (d, JPC¼6.2 Hz), 68.0 (d, JPC¼165.0 Hz), 68.5 (d, JPC¼5.6 Hz), 70.1, 73.9, 74.5, 75.9, 77.2 (d, JPC¼5.0 Hz), 79.5, 81.4, 100.5, 126.7, 127.0, 127.1, 127.4, 127.6, 127.7, 127.8, 128.08, 128.15, 128.26, 128.35, 128.5, 128.7, 128.8, 129.4, 129.9, 133.4, 135.9 (d, JPC¼6.2 Hz), 136.5 (d,
JPC¼6.2 Hz), 136.9, 138.2, 138.9, 139.9, 164.8 (d, JPC¼6.2 Hz); 31P NMR (121.5 MHz, CDCl3) d 20.70; HRMS: calcd for C62H61NO9P [MþH]þ, 994.4078; found, 994.4079. 4.9. Dibenzyl (S)-hydroxy((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5-(dibenzylamino) tetrahydro-2H-pyran-2-yl)methylphosphonate (12a) Compound 13a (100.0 mg, 0.1 mmol) was treated with 33% methyl amine solution in ethanol (10 mL) at 0 C for 20 min. Then the mixture was diluted with dichloromethane. The mixture was concentrated and the residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1) to afford compound 12a (82.0 mg, 92% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 2.87e2.98 (m, 2H), 3.68e3.75 (m, 4H), 3.86 (d, J¼14.1 Hz, 2H), 4.21 (t, J¼11.1 Hz, 1H), 4.46 (d, J¼11.7 Hz, 1H), 4.58e4.66 (m, 1H), 4.65 (d, J¼8.4 Hz, 1H), 4.75e4.86 (m, 3H), 4.98 (d, J¼11.1 Hz, 1H), 5.05e5.09 (m, 4H), 7.16e7.38 (m, 35H); 13C NMR (75 MHz, CDCl3) d 54.6, 63.0, 66.6 (d, JPC¼159.5 Hz), 67.9 (d. JPC¼6.8 Hz), 68.1 (d, JPC¼6.8 Hz), 70.3, 73.6, 74.4, 74.7, 77.7 (d, JPC¼9.9 Hz), 80.9, 100.6, 126.7, 127.2, 127.7, 127.9, 128.0, 128.2, 128.3, 128.4, 128.6, 128.7, 136.0 (d, JPC¼5.0 Hz), 136.1 (d, JPC¼5.0 Hz), 137.1, 138.1, 138.8, 139.6; 31P NMR (121.5 MHz, CDCl3) d 25.57; HRMS: calcd for C55H57NO8P [MþH]þ, 890.3816; found, 890.3834. 4.10. Dibenzyl (R)-hydroxy((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5-(dibenzylamino) tetrahydro-2H-pyran-2-yl)methylphosphonate (12b) Compound 13b (50.0 mg, 0.05 mmol) was treated with 33% methyl amine solution in ethanol (5 mL) at 0 C for 2 h. Then the mixture was diluted with dichloromethane. The mixture was concentrated and the residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1) to afford compound 12b (39.0 mg, 88% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 3.05 (t, J¼8.7 Hz, 1H), 3.38 (brs, 1H), 3.73e3.81 (m, 4H), 3.90 (d, J¼13.5 Hz, 2H), 4.02 (t, J¼8.1 Hz, 1H), 4.26 (brs, 1H), 4.56e4.73 (m, 5H), 4.87 (t, J¼11.1 Hz, 2H), 5.00e5.13 (m, 4H), 7.13e7.39 (m, 35H); 13C NMR (75 MHz, CDCl3) d 54.8, 63.0, 67.7 (d, JPC¼6.8 Hz), 68.4 (d, JPC¼6.8 Hz), 69.0 (d, JPC¼158.3 Hz), 70.6, 73.5, 74.2, 76.2, 79.7 (d, JPC¼3.1 Hz), 81.3, 100.7, 126.7, 127.1, 127.3, 127.7, 127.8, 128.0, 128.1, 128.2, 128.4, 128.5, 128.8, 136.0 (d, JPC¼5.6 Hz), 136.4 (d, JPC¼6.2 Hz), 137.2, 137.8, 138.5, 139.6; 31P NMR (121.5 MHz, CDCl3) d 24.38; HRMS: calcd for C55H57NO8P [MþH]þ, 890.3816; found, 890.3823. 4.11. (1S)-((2S,3S,4R,5R)-5-Amino-3,4,6-trihydroxytetrahydro2H-pyran-2-yl)(hydroxy) methylphosphonic acid (2) A methanolic solution (3 mL) of compound 12a (50.0 mg, 0.056 mmol) was stirred at room temperature in the presence of 10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 2 days. The catalyst was then removed by filtration through Celite, and the filtrate was concentrated. The residue was purified by C-18 reversed-phase column chromatography (eluent: H2O) to give compound 2 (14.0 mg, a/b¼2:1, 96% yield) as white amorphous solids after lyophilization. 1H NMR (300 MHz, D2O) d 2.85 (t, J¼9.3 Hz, 0.5H), 3.11 (dd, J¼3.3, 10.5 Hz, 1H), 3.45e3.60 (m, 2.2H), 3.79 (t, J¼9.6 Hz, 1H), 3.88e4.00 (m, 2.8H), 4.79 (d, J¼8.7 Hz, 0.5H), 5.29 (d, J¼3.3 Hz, 1H); 13C NMR (75 MHz, D2O) data for both a and b anomers: d 54.8, 57.2, 66.5 (d, JPC¼155.8 Hz), 66.7 (d, JPC¼155.7 Hz), 69.4, 69.6 (d, JPC¼9.2 Hz), 70.4, 71.5, 72.6, 76.0, 89.8, 93.6; 31P NMR (121.5 MHz, CDCl3) d 19.25 (a), 19.62 (b); HRMS: calcd for C6H15NO8P [MþH]þ, 260.0530; found, 260.0533.
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4.12. (1R)-((2S,3S,4R,5R)-5-Amino-3,4,6trihydroxytetrahydro-2H-pyran-2-yl)(hydroxy) methylphosphonic acid (3) A methanolic solution (3 mL) of compound 12b (50.0 mg, 0.056 mmol) was stirred at room temperature in the presence of 10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 2 days. The catalyst was then removed by filtration through Celite, and the filtrate was concentrated. The residue was purified by C-18 reversed-phase column chromatography (eluent: H2O) to give compound 3 (13.5 mg, a/b¼2:1, 92% yield) as white amorphous solids after lyophilization. 1H NMR (300 MHz, D2O) d 2.86 (t, J¼8.7 Hz, 0.5H), 3.15 (dd, J¼3.0, 10.2 Hz, 1H), 3.49e4.00 (m, 6H), 4.76 (d, J¼8.4 Hz, 0.5H), 5.27 (d, J¼2.7 Hz, 1H); 13C NMR (75 MHz, D2O) data for both a and b anomers: d 56.6, 59.1, 70.1, 71.9 (d, JPC¼152.6 Hz), 71.8 (d, JPC¼151.4 Hz), 72.1, 72.2, 74.7, 75.1 (d, JPC¼7.2 Hz), 79.5 (d, JPC¼8.0 Hz), 91.7, 95.4; 31P NMR (121.5 MHz, CDCl3) d 18.50 (a), 18.10 (b); HRMS: calcd for C6H15NO8P [MþH]þ, 260.0530; found, 260.0531. 4.13. (S)-((S)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-3,4,6tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14a) chloride (R)-()-a-Methoxy-a-trifluoromethylphenylacetyl (4.3 mL, 22.4 mmol) was added dropwise to a solution of 12a (5.0 mg, 5.6 mmol), Et3N (6.4 mL, 45.0 mmol) and 4-dimethylaminopyridine (1.0 mg, 8.4 mmol) in dichloromethane (2 mL) under argon. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated. The residue was purified by column chromatography on silica gel (petroleum ethereethyl acetate, 5:1) to afford 14a as colourless oil (5.0 mg, 80% yield). 1H NMR (300 MHz, CDCl3) d 2.90 (dd, J¼8.1, 10.2 Hz, 1H), 3.22 (t, J¼9.0 Hz, 1H), 3.49 (s, 3H), 3.70e3.72 (m, 1H), 3.73 (d, J¼14.0 Hz, 2H), 3.88 (d, J¼14.0 Hz, 2H), 3.93 (dd, J¼4.2, 9.3 Hz, 1H, H-5), 4.32 (d, J¼10.5 Hz, 1H), 4.47 (d, J¼11.7 Hz, 1H), 4.64e4.75 (m, 4H), 4.81 (dd, J¼7.8, 12.0 Hz, 1H), 4.91e5.05 (m, 4H), 6.02 (d, J¼11.1 Hz, 1H), 7.06e7.40 (m, 38H), 7.57 (d, J¼7.8 Hz, 2H). 4.14. (R)-((S)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)3,4,6-tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (15a) Compound 15a was prepared from compound 12a and (S)(þ)-a-methoxy-a-trifluoromethylphenylacetyl chloride as described in the preparation of compound 14a, yielding 15a (84% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 2.81 (t, J¼9.0 Hz, 1H), 3.05 (t, J¼9.0 Hz, 1H), 3.43 (s, 3H), 3.65e3.73 (m, 3H), 3.83e3.87 (m, 3H, H-5 and NeCH2), 4.26 (d, J¼10.2 Hz, 1H), 4.44 (d, J¼12.0 Hz, 1H), 4.58e4.68 (m, 4H), 4.93e5.10 (m, 5H), 5.95 (d, J¼11.4 Hz, 1H), 7.09e7.41 (m, 38H), 7.57 (d, J¼7.5 Hz, 2H). 4.15. (S)-((R)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)3,4,6-tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14b) Compound 14b was prepared from compound 12b and (R)()-a-methoxy-a-trifluoromethylphenylacetyl chloride as described in the preparation of compound 14a, yielding 14b (85% yield) as a colourless oil. 1H NMR (300 MHz, CDCl3) d 2.99 (t, J¼8.7 Hz, 1H), 3.46 (s, 3H), 3.56 (d, J¼10.8 Hz, 1H, H-5), 3.70e3.76 (m, 3H), 3.88 (d, J¼13.5 Hz, 2H), 4.12 (t, J¼9.3 Hz, 1H), 4.27 (d, J¼11.4 Hz, 1H), 4.40 (d, J¼8.1 Hz, 1H), 4.56 (d, J¼12.0 Hz, 1H), 4.71e5.09 (m, 8H), 6.00 (d, J¼11.7 Hz, 1H), 7.18e7.39 (m, 38H), 7.54 (d, J¼7.8 Hz, 2H).
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4.16. Polymerase chain reaction The glmS DNA template has a sequence of 50 -GAATT CTAAT ACGAC TCACT ATAGG TCTTG TTCTT ATTTT CTCAA TAGGA AAAGA AGACG GGATT ATTGC TTTAC CTATA ATTAT AGCGC CCGAA CTAAG CGCCC GGAAA AAGGC TTAGT TGACG AGGAT GGAGG TTATC GAATT TTCGG CGGAT GCCTC CCGGC TGAGT GTGCA GATCA CAGCC GTAAG GATTT CTTCA AACCA AGGGG GTGAC TCCTT GAACA AAGAG AAATC ACATG ATCTT CCAAA AAACA TGTAG GAGGG GAC-30 , which was obtained through PCR amplification of a lab plasmid, originally derived from B. subtilis chromosomal DNA (1A40). DNA primers were obtained through automated synthesis using standard phosphoramidite chemistry (Integrated DNA Technologies, Iowa, USA). Before usage, the oligonucleotides were purified by 10% denaturing PAGE and quantified by absorbance at 260 nm. Primers used include the forward sequence 50 -GAATT CTAAT ACGAC TCACT ATAGG TCTTG TTCTT ATT-30 (T7 RNA polymerase promoter sequence is shown in italicized letters; the þ1 transcription initiation site and critical nucleotides for in vitro transcription are shown in bold letters), and the reverse sequence 50 -GTCCC CTCCT ACATG TTTTT TGG-30 . PCR was conducted in 75 mM TriseHCl (pH 9.0), 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, each primer at 0.5 mM, DNA plasmid at w3 nM, each dNTP at 0.5 mM, and 5 units of Taq DNA polymerase (Biotools, Madrid, Spain). Thermal cycling steps were: 94 C for 1 min, 20 cycles of 94 C to 50 C to 72 C (30 s for each temperature), and finally 72 C for 8 min.
4.17. RNA preparation In vitro transcription was conducted at 37 C for 3 h in 150 mL of 40 mM TriseHCl, pH7.9, 6 mM MgCl2, 10 mM DTT, 10 mM NaCl, 2 mM spermidine, 25 pmol PCR amplified DNA, 2.5 mM each of GTP, CTP, UTP, ATP, 1.07 units/mL RiboLock Ribonuclease Inhibitor and 1.33 units/mL T7 RNA polymerase (Fermentas, Canada). The transcription mixture was then treated with DNase I (3 units) in the presence of 0.2 mM CaCl2 at 37 C for 15 min (Fermentas, Canada). The transcribed RNA was subsequently concentrated through standard ethanol precipitation, purified by 10% denaturing PAGE and then quantified by absorbance at 260 nm. Afterwards, the 50 triphosphate of the RNA was dephosphorylated with calf intestine alkaline phosphatase following manufacturer’s protocol (Fermentas, Canada), and extracted with phenol/chloroform (1:1) (BioShop, Canada). RNA samples were concentrated through standard ethanol precipitation, before labelling the 50 -terminus with [g-32P]ATP (Perkin Elmer, Massachusetts, USA) using T4 polynucleotide kinase according to manufacturer’s protocol (Fermentas, Canada). 50 -32P labelled RNA was purified again by 10% denaturing PAGE before usage.
4.18. Ribozyme cleavage assays A 200 mM concentration of glucosamine-6-phosphate (GlcN6P) (Sigma, Oakville, Canada), or a test compound (1, 2 or 3) was added to 0.5 pmol 50 -32P labelled glmS RNA. Samples were incubated in 50 mM HEPES (pH7.5), 10 mM MgCl2, 200 mM KCl at room temperature for a duration of 1 or 2 min. Negative control samples included: i) addition of double-distilled water instead of compound, or ii) addition of GlcN6P, without Mg2þ in the reaction buffer. After the testing period, reactions were terminated by the addition of loading dye solution containing 100 mM EDTA and 7 M urea (Sigma, Oakville, Canada). RNA products were separated by 10% denaturing PAGE and visualized using a PhosphorImager and ImageQuant software. Percent cleavage for each reaction was calculated using the following equation:
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Percent Cleavage ¼
cleavage fragment cleavage fragment þ precursor glmS RNA background 100%
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21072014) and ‘973’ grant from the Ministry of Science and Technology of China (2012CB822100). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2012.09.015. References and notes 1. Winkler, W.; Nahvi, A.; Breaker, R. R. Nature 2002, 419, 952e956. 2. Coppins, R. L.; Hall, K. B.; Groisman, E. A. Curr. Opin. Microbiol. 2007, 10, 176e181. 3. Blount, K. F.; Breaker, R. R. Nat. Biotechnol. 2006, 24, 1558e1564. 4. Barrick, J. E.; Corbino, K. A.; Winkler, W. C.; Nahvi, A.; Mandal, M.; Collins, J.; Lee, M.; Roth, A.; Sudarsan, N.; Jona, I.; Wickiser, J. K.; Breaker, R. R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6421e6426. 5. Klein, D. J.; Ferre-D’Amare, A. R. Science 2006, 313, 1752e1756. 6. Cochrane, J. C.; Lipchock, S. V.; Strobel, S. A. Chem. Biol. 2007, 14, 97e105. 7. Winkler, W. C.; Nahvi, A.; Roth, A.; Collins, J. A.; Breaker, R. R. Nature 2004, 428, 281e286. 8. Milewski, S. Biochim. Biophys. Acta 2002, 1597, 173e192. 9. Wu, H. C.; Wu, T. C. J. Bacteriol. 1971, 105, 455e466. 10. Sarvas, M. J. Bacteriol. 1971, 105, 467e471. 11. Freese, E. B.; Cole, R. M.; Klofat, W.; Freese, E. J. Bacteriol. 1970, 101, 1046e1062.
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