Bioconjugate Chem. 2005, 16, 1045−1048
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The First Total Synthesis of r-D-Glucopyranosyl-(1f2)-β-D-galactopyranosyl-O-pyridinoline, an Important Hallmark of Human Joint Diseases† Pietro Allevi,* Riccardo Cribiu`, Elios Giannini, and Mario Anastasia Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita` di Milano, via Saldini 50, I-20133 Milano, Italy. Received April 5, 2005; Revised Manuscript Received June 16, 2005
The first total synthesis of R-D-glucopyranosyl-(1f2)-β-D-galactopyranosyl-O-pyridinoline using a short “one-pot” assembly of its 1,4,5-trisubstituted 3-hydroxypyridinium ring is reported.
INTRODUCTION
This paper describes the first total synthesis of the optically pure R-D-glucopyranosyl-(1f2)-β-D-galactopyranosyl-O-pyridinoline (Glc-Gal-PYD, 1; Figure 1), a natural glycosylated analogue of the collagen cross-linked pyridinoline 2a, recently found in human synovium by Gineyts et al. (1). Glc-Gal-PYD, 1, appears to be specific of human synovium, and its levels are considered strongly predictive markers of joint destruction progression in various diseases, including rheumatoid arthritis (1-4). Moreover, Glc-Gal-PYD, 1, with its attractive and challenging structure, rich in stereogenic centers in both the amino acidic and the glycosidic portions, represents an attractive goal for organic chemists. Although several different strategies for the synthesis of pyridinoline 2a, the aglycone portion of Glc-Gal-PYD (1), and of its congener deoxypyridinoline 2b have been published (5, 6), starting from simple amino acids, no attempt was reported until now to synthesize the important glycoconjugate 1, which, in principle, could be obtained by glycosylation of an appropriate aglycone such as 3 (6, 7) with a glucosyl-galactosyl donor or, in a longer route, with a galactosyl donor having a participating and orthogonally cleavable protective group at position 2, which should allow a successive extension of the glycosylation in the 2 direction of galactose. Moreover, herein we report a completely different procedure for the synthesis of Glc-Gal-PYD, 1, adopting, as a key step, the “one-pot protocol” for the assembly of its 1, 4, 5-trisubstituted 3-hydroxypyridinium ring, originally reported by us (5, 8, 9). EXPERIMENTAL PROCEDURES
General Methods. Nuclear magnetic resonance spectra were recorded at 303 K on Bruker AM-500 spectrometer operating at 500.13 MHz for 1H and 125.76 MHz for 13 C. Chemical shifts are reported in parts per million (ppm, δ units) and are referenced to residual CHCl3 (δH ) 7.24 ppm) and to CDCl3 (δC ) 77.0 ppm) for solutions in CDCl3 or to internal CH3OD (δH ) 3.30 ppm and δC ) 49.0 ppm) for solutions in D2O. 1H NMR data are tabulated in the following order: number of protons, † This paper is dedicated to Prof. Guido Tettamanti on the occasion of his 70th birthday. * To whom correspondence should be addressed. Fax: +039 02503 16040. E-mail:
[email protected].
multiplicity (s, singlet; d, doublet; bs, broad singlet; m, multiplet), coupling constant(s) in hertz, assignment of proton(s). The 1H and 13C resonances were assigned by 1 H decoupling, 1H-1H COSY, and 1H-13C correlation experiments. Optical rotations were taken on a Perkin-Elmer 241 polarimeter, and [R]D values are given in 10-1 deg cm2 g-1. Mass spectra were obtained using a Finnigan LCQdeca (ThermoQuest) ion trap mass spectrometer fitted with an electrospray source (ESI). The spectra were collected in continuous flow mode by connecting the infusion pump directly to the ESI source. Solutions of compounds were infused at a flow rate of 5 mL/min. The spray voltage was set at 5.0 kV in the positive and at 4.5 kV in the negative ion mode with a capillary temperature of 220 °C. Full-scan mass spectra were recorded by scanning a m/z range of 100-2000. All reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60 F254) using UV light, 50% sulfuric acid or 0.2% ninhydrin in ethanol, and heat as developing agents. E. Merck 230-400 mesh silica gel was used for flash column chromatography (10). Preparation of tert-Butyl (2S,5R)-6-Amino-2-benzyloxycarbonylamino-5-[(2,3,4,6-tetra-O-benzyl-r-Dglucopyranosyl)-(1f2)-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)]hexanoate, 5. To a stirred solution of anhydrous SnCl2 (240 mg, 1.27 mmol) in anhydrous tetrahydrofuran (8 mL), PhSH (496 µL, 4.84 mmol) and Et3N (4.96 µL, 3.56 mmol) were added. Then, tert-butyl (2S,5R)-6-azido-2-benzyloxycarbonylamino-5-[(2,3,4,6tetra-O-benzyl-R-D-glucopyranosyl)-(1f2)-(3,4,6-tri-Obenzyl-β-D-galactopyranosyl)]hexanoate, 4 (1.00 g, 0.75 mmol), was added. After the reaction was stirred at 25 °C for 1 h, the solvent was evaporated, and the crude residue was purified by rapid chromatography with elution of the byproducts with dichloromethane-methanol (100:8, v/v) and the glycosylated amine 5 (834 mg; Y ) 85%) with dichloromethane-methanol (100:10, v/v). The amine 5, an oil, showed the following: [R]20D +30.5 (CHCl3, c 1). 1H NMR δH (500 MHz, CDCl3): 5.60 (1H, d, J 3.3 Hz, Hglu-1), 4.56 (1H, d, J 7.7 Hz, Hgal-1), 4.16 (1H, dd, J 10.2 and 3.3 Hz, Hgal-2), 4.09 (1H, m, Hlys-2), 2.84 (1H, dd, J 13.3 and 2.1 Hz, Hlys-6a), 2.59 (1H, dd, J 13.3 and 6.1 Hz, Hlys-6b). 13C NMR δC (CDCl3): 171.1 (CO2tBu), 155.9 (NHCO2tBu), 101.7 (Cgal-1), 95.7 (Cglu-1), 53.9(Clys-2), 44.9 (Clys-6). ESI-MS (positive) m/z: 1331.6
10.1021/bc050107h CCC: $30.25 © 2005 American Chemical Society Published on Web 06/25/2005
1046 Bioconjugate Chem., Vol. 16, No. 4, 2005
Technical Notes
Figure 1. Chemical structures of glycosylated and free collagen cross-links.
(37%), 1330.6 (82%), 1329.6 (100%; M + Na+), 1307.6 (23%; M + H+). Preparation of Completely Protected r-D-Glucopyranosyl-(1f2)-β-D-galactopyranosyl-O-pyridinoline, 7. To a solution of the tert-butyl (2S,5R)-6-amino2-benzyloxycarbonylamino-5-[(2,3,4,6-tetra-O-benzyl-R-Dglucopyranosyl)-(1f2)-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)]hexanoate, 5 (600 mg, 0.46 mmol), in CH3CN (30 mL) containing Na2CO3 (900 mg, 8.5 mmol), the protected bromoketone 6 (436 mg, 1.15 mmol) was added, and the mixture was stirred under nitrogen for 6 h. At this time, the disappearance of both the starting glycosylated amine 5 and the initially formed monoalkylated product was observed (TLC, CH2Cl2/MeOH 100/5 v/v; Rf ) 0.28 and 0.72, respectively). Then, the solvent was evaporated under vacuum, and the crude residue was recovered with MeOH (30 mL) and was vigorously shaken under a slight pressure of oxygen (1.3 atm) at room temperature for 60 h. At this time, the mixture was diluted with dichloromethane (50 mL) and filtered on a pad of Celite. Evaporation of the solvent afforded a crude residue, which was purified by rapid chromatography on silica. Elution with dichloromethane-methanol (100:4, v/v) afforded the protected glycosylated pyridinoline 7 (537 mg; Y ) 62%) as a resinous material: [R]20D +28.2 (CHCl3, c 1). UV λmax(EtOH)/nm (/dm3 mol-1 cm-1): 258.5 (5175), 340 (5962). 1H NMR δH (500 MHz, CDCl3): 5.41 (1H, d, J 3.5 Hz, Hglu-1), 4.54 (1H, d, J 7.4 Hz, Hgal-1), 4.12-4.06 (4H, overlapping, H1chain-1a, H1chain-1b, H4chain-2, H5chain-3), 3.26 (1H, dd, J 11.7 and 11.7 Hz, H4chain-1a), 2.85 (1H, dd, J 11.7 and 2.5 Hz, H4chain-1b). 13 C NMR δC (CDCl3): 171.2, 171.2, 170.5 (3 × CO2tBu), 156.0 (C-3), 143.6, 140.7, 136.3, 130.2 (pyridinium ring carbons), 103.3 (Cgal-1), 96.0 (Cglu-1), 56.6, 53.7, 53.6 (3 × amino acidic R-carbons). ESI-MS (positive) m/z: 1911.0 (24%), 1910.0, (55%), 1909.2 (100%), 1908.5 (91%; M + Na+), 1888.5 (13%), 1887.5 (21%), 1886.5 (17%; M + H+). Preparation of r-D-Glucopyranosyl-(1f2)-β-D-galactopyranosyl-O-pyridinoline, 1, by Successive Regeneration of the Protected Functionalities. i. Preparation of the Partially Protected Glc-Gal-PYD 8. The completely protected Glc-Gal-PYD 7 (400 mg; 0.212 mmol) was dissolved in triflouroacetic acid-water (8 mL, 95:5, v/v), and the resulting solution was stirred at 25 °C for 1.5 h. Evaporation of the mixture afforded the partially protected Glc-Gal-PYD 8 as its tetrafluoroacetate salt (389 mg; Y ) 93%) as a glass: [R]20D +33.4 (CHCl3, c 1). 1H NMR δH (500 MHz, CD3OD): 8.25, 8.21 (2H, 2 × bs, pyridinium ring protons), 5.43 (1H, d, J 2.9 Hz, Hglu-1), 4.46 (1H, d, J 8.0 Hz, Hglu-1), 4.12 (1H, m, H1chain-2), 4.02 (1H, dd, J 8.8 and 8.8 Hz, H1chain-5), 3.783.74 (2H, overlapping, Hgal-1 and H5chain-3). 13C NMR δC (CD3OD): 175.2, 171.2, 170.5 (3 × CO2tBu), 161.3 (q, J 37.5 Hz, CF3COO), 157.0 (C-3), 141.8, 141.4, 138.2, 130.0 (pyridinium ring carbons), 117.4 (q, J 298 Hz, CF3COO),
103.2 (Cgal-1), 97.4 (Cglu-1), 54.5, 53.3, 52.2 (3 × amino acidic R-carbons). ESI-MS (positive) m/z: 1519.8 (41%), 1518.8 (90%), 1517.8 (100%; M + H+), 1540.7 (16%), 1539.6 (16%; M + Na+). ii. Preparation of the R-D-Glucopyranosyl-(1f2)-β-Dgalactopyranosyl-O-pyridinoline, 1. The partially protected Glc-Gal-PYD 8 (390 mg, 0.20 mmol) dissolved in aqueous HCl (100 mL, 10-3 M) was hydrogenated in the presence of PdCl2 (100 mg) at 25 °C and atmospheric pressure. When the absorption of hydrogen was completed, the mixture was filtered over a pad of Celite, and the solution was lyophilized to afford the expected R-Dglucopyranosyl-(1f2)-β-D-galactopyranosyl-O-pyridinoline, 1 (170 mg; Y ) 92%), as its tetrahydrochloride salt dihydrate (C29H46N4O18‚4HCl‚2H2O), an amorphous solid: [R]20D +47.6.0 (H2O, c 1). 1H NMR δH (500 MHz, D2O): 8.31, 8.29 (2H, 2 × bs, pyridinium ring protons), 5.32 (1H, d, J 3.8 Hz, Hglu-1), 4.51 (1H, d, J 7.7 Hz, Hgal-1), 4.33 (1H, m, H1chain-2), 4.15 (1H, dd, J 6.3 and 6.3 Hz, H5chain-3), 4.08 (1H, dd, J 6.3 and 6.2 Hz, H1chain-5), 3.81 (1H, d, J 3.5 and
