Synthesis of potassium (2R)-2-O-α-d -glucopyranosyl-(1→6)-α-d -glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute

Carbohydrate Research 344 (2009) 2073–2078

Contents lists available at ScienceDirect

Carbohydrate Research journal homepage: www.elsevier.com/locate/carres

Note

Synthesis of potassium (2R)-2-O-a-D-glucopyranosyl-(1?6)a-D-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute Eva C. Lourenço a, Christopher D. Maycock a,b,*, M. Rita Ventura a a b

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780-901 Oeiras, Portugal Faculdade de Ciências da Universidade de Lisboa, Departamento de Química e Bioquímica, 1749-016 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 7 August 2008 Received in revised form 19 June 2009 Accepted 21 June 2009 Available online 27 June 2009 Keywords: a-Glucosylation Hypersolutes Glucosyl glycerate Thioglycosides Glyceric acid

a b s t r a c t Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-D-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-a-D-glucopyranosyl-(1?6)-a-D-glucopyranosyl-2,3-dihydroxypropanoic acid (a-D-glucosyl-(1?6)-a-D-glucosyl-(1?2)-D-glyceric acid, GGG), a recently isolated compatible solute. The a-anomer was by far the major product of both glycosylation reactions using NIS/TfOH as activator. Ó 2009 Elsevier Ltd. All rights reserved.

Halotolerant and moderately halophilic microorganisms accumulate compatible solutes to counter fluctuations in the water activity of their environment.1 Hyperthermophiles (thriving optimally above 80 °C) isolated from marine sources also use the same general strategy. A superior thermoprotecting ability was soon ascribed to these ‘hypersolutes’, and confirmed by in vitro protein stabilisation experiments.2,3 In contrast to the solutes more commonly found in mesophiles, hypersolutes are generally negatively charged, and most fall into two categories: glyceric acid glycosides such as a-D-mannosyl-D-glycerate (MG) and a-D-glucosyl-D-glycerate (GG) 1 (Fig. 1), and polyol-phosphodiesters such as di-myo-inositol phosphate (DIP). From the hyperthermophile Persephonella marina the new trisaccharide a-D-glucosyl-(1?6)-a-D-glucosyl(1?2)-D-glycerate (GGG) 2 has recently been isolated (Fig 1).4 These compounds are available in very small quantities from their natural sources and chemical synthesis was considered the best route to obtain GG 1 and GGG 2 in larger amounts for testing and to confirm the (R) stereochemistry of the glycerate moiety. Glucosyl glycerate (GG)-terminated oligosaccharides have been studied previously5 and a synthesis by chemical degradation of leucrose has been reported with the characterisation and confirmation of the structure and stereochemistry a salient feature of this work. In fact, the oligosaccharides studied appear to have a glucosyl-(1?6)-glucosyl-(1?2)-glycerate terminal. This motif appears to be widely dispersed in nature. We found that, in our * Corresponding author. Tel.: +351 21 4469775; fax: +351 21 4469789. E-mail address: [email protected] (C.D. Maycock). 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.06.037

hands, the chemical degradation process was difficult and unsuitable for large-scale preparation and so we opted for a stepwise glycosylation strategy. 13C-Labelled GG was prepared photosynthetically, in low yield, using a blue-green alga and [13C] enriched carbon dioxide as a carbon source.6 The main challenge was to form the a-glucosidic bonds selectively,7 for which there are good procedures but still no general method. We opted for a thioglycoside donor protocol and the activation method developed by Crich and co-workers.8,9 The glucosyl donor, S-phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-deoxy-1thio-b-D-glucopyranoside 3, was activated with 1-benzenesulfinyl piperidine (BSP) and Tf2O, in the presence of a hindered base (2,6-di-tert-butyl-4-methylpyridine, DTBMP). The intermediates (perhaps 1-triflates)9 thus formed were rapidly converted to the glucoside 4 upon treatment with methyl 3-O-tert-butyldiphenylsilylglycerate 9, obtained from D-serine10 in good yield (72%), and no b-anomer was detected by NMR (Scheme 1). Even though this was a good method for our purposes, we experienced some difficulties with the lability of the benzylidene-protecting group and for the GGG synthesis an important aspect was the need to have the 6-OH group free for the second glycosylation reaction. Removal of the 4,6-benzylidene-protecting group at this stage would release two hydroxyl groups and opened up the possibility of side reactions during subsequent glycosylation processes, reducing the overall efficiency. Thus, we looked for another suitable glucosyl donor and for our purposes a simple glucose derivative having a differentially protected 6-OH group was needed. 6-Hydroxysugars, that is sugars having all other positions protected, have been used

2074

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078

tate, hydrolytic removal of the acetates, followed by perbenzylation of the remaining hydroxyls and finally acidolysis of the trityl group and acetylation of the 6-OH group. Treatment of methyl tetra-O-benzylglucopyranoside with a mixture of sulfuric acid, acetic acid and acetic anhydride affords directly the diacetate 7 in 91% yield.16 This could then be converted to thioglycoside 8, as a mixture of anomers, by acid catalysed thiolysis. Roy14 and more recently Iadonisi and co-workers17 have reported highly a selective glycosylation reactions of acetate 8. Although the 6-O-acetyl group could disarm the system slightly, this group could also participate in the stabilisation of the intermediate carbocation and favour formation of the a-glycoside.18 Uronic acid ester glycosylation has shown that anomer selectivity is considerably reduced19 indicating that the carbonyl group of similar compounds does not participate. The use of large glucosyl acceptors may have the effect of increasing a-selectivity.20 Applying the Crich protocol for the glycosylation of donor 8 (2.7:1, a:b),17 with glycerate acceptor 9 afforded 10 in 76% yield; however, the simpler NIS–TfOH21 mixture afforded predominantly (>10:1) the a-anomer in 96% yield and the product mixture was easily separated. The major isomer showed a doublet at 5.15 ppm (J = 3.2 Hz) in the 1H NMR corresponding to the a-anomer.

OH HO HO

OH O

HO HO

OH O 1

O OH O O

HO HO

OH

OH O

CO2K 2

OH CO2K

Figure 1. Two natural glycerate solutes.

for a variety of applications. The connection to resins11 for solidsupported reactions and directed glycosidations through tethers and templates12 at the 6-OH have also been reported. They are, obviously, also useful for the preparation of uronic acids and 6-deoxysugars.13 The methods available for the preparation of these useful molecules have normally involved an extended sequence of protection and deprotection reactions.14 Thus 6-acetoxy perbenzylated glucose 8 (b-anomer) has previously been prepared by the selective tritylation of the 6-OH group of 1-ethylthio-b-Dglucopyranoside, obtained by the thiolysis15 of glucosepentaace-

OH TBDPSO Ph

O

O

O BnO

SPh

OBn

3

9

CO2Me

Ph

a

O O BnO

O 4

BnO

OTBDPS

O

CO2Me Scheme 1. Reagents and conditions: (a) PhSONC5H10, DTBMP, Tf2O, MS, CH2Cl2, 72%.

OH

OAc

OBn O

HO HO

a

O

BnO BnO

OH OMe

b

OBn OAc

OBn OMe

5

6

7

OAc c

O

BnO BnO

OAc O

BnO BnO

d

OBn SEt 8

OH TBDPSO 9

BnO BnO

CO2Me

BnO

O

10

OTBDPS CO2Me

OH

OH e

f

O

BnO BnO

O

BnO

BnO BnO O

11

O BnO

OTBDPS 12

CO2Me

O

OH CO2Me

OH g

O

HO HO

h

1 OH O 13

OH CO2Me

Scheme 2. Reagents and conditions: (a) BnBr, NaH, DMF, 0 °C/rt, 88%; (b) Ac2O, AcOH, H2SO4, 0 °C, 91%; (c) EtSH, BF3OEt2, CH2Cl2, 0 °C, 80%; (d) NIS, TfOH, CH2Cl2, 96%; (e) Na, MeOH, 0 °C, 94%; (f) TBAF, THF, 90%; (g) H2, Pd, AcOEt/EtOH, 35 psi, 99%; (h) KOH 2 M, H2O, rt.

2075

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078

OAc

OAc 8

+ 11

O

BnO BnO

a

BnO

O

BnO BnO

b

BnO

O O

BnO BnO

BnO

14

O O

BnO BnO O

OTBDPS

BnO 15

O

OH c

OH O

BnO BnO

OH CO2Me

CO2Me

BnO

d

HO HO

O O

BnO BnO

BnO

e

O OH O O

HO HO O

16

2

OH O

OH 17

CO2Me

OH CO2Me

Scheme 3. Reagents and conditions: (a) NIS–TfOH, CH2Cl2, 0 °C, 89%; (b) TBAF, THF, 88%; (c) Na, MeOH, 0 °C, 95%; (d) H2, Pd, 35 psi, 99%; (e) KOH 2 M, H2O rt.

Table 1 Comparison of 13C NMR chemical shifts for the potassium salt of GG 1 with data from the literature GG

Glucosyl moiety

Glyceryl moiety

13

C-1

C-2

C-3

C-4

C-5

C-6

C-1

C-2

C-3

NH4 þ salt5 K+ salt

99.0 99.0

73.7 73.8

74.9 74.8

71.0 71.1

73.2 73.2

62.1 62.1

178.5 178.6

80.6 80.7

64.7 64.7

C, d ppm

Methanolysis of the 6-O-acetyl group of fully protected GG 10 furnished alcohol 11 (Scheme 2) which was used for the second glycosylation reaction (Scheme 3). Removal of the silyl ether, followed by hydrogenolysis of the benzyl groups and finally hydrolysis of the methyl ester afforded the potassium salt of GG 1, which was purified by ion exchange chromatography. The NMR data (Table 1) of the synthetic product and those of an authentic sample of the natural product were identical and corresponded well to the data for the salts.5 For the GGG synthesis, thioglycoside 8 was again the glycosyl donor and the acceptor was alcohol 11. This glycosylation reaction failed under Crich conditions, but was successfully carried out using NIS–TfOH, affording predominantly the a-anomeric dissacharide 14 (>10:1 a:b, 89% yield). Successive deprotection of the silyl ether, of the acetate at 6-OH, the benzyl groups and hydrolysis of the methyl ester afforded GGG 2 as the potassium salt (Scheme 3). The NMR data of the synthesised product were identical to those of a sample of natural GGG4 (Table 2), confirming the D-glycerate stereochemical assignment for this solute. In conclusion, the efficient syntheses of two natural solutes, GG and GGG, have been achieved. A readily available simple glucosyl donor was applied to afford a-glucosides with high selectivity, and offers a very easy method for preparing 1,6-linked di and higher saccharides. Table 2 Comparison of

13

1. Experimental 1.1. General 1 H NMR spectra were obtained at 400 MHz in CDCl3 or D2O with chemical shift values (d) in ppm downfield from tetramethylsilane in the case of CDCl3, and 13C NMR spectra were obtained at 100.61 MHz in CDCl3 or D2O. Assignments are supported by 2D correlation NMR studies. Medium pressure preparative column chromatography: Silica Gel Merck 60 H. Analytical TLC: Aluminium-backed Silica Gel Merck 60 F254. Specific rotations (½a20 D ) were measured using an automatic polarimeter. Reagents and solvents were purified and dried according to Ref. 22. All the reactions were carried out in an inert atmosphere (argon), except when the solvents were undried. Compounds 1 and 2 were purified by anion exchange chromatography using a QAE-Sephadex A25 column eluted with sodium bicarbonate buffer (pH 9.8) gradient (5 mM to 1 M). The combined relevant fractions were lyophilised and passed through a H+ Dowex 50W-X8 column eluting with water. Again the relevant fractions were combined and the pH adjusted to 4.5–5.0 with ultra-pure potassium hydroxide solution and concentrated to dryness.

1.2. 1,6-Di-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-glucopyranoside 7 Concentrated sulfuric acid (1 mL) was added dropwise to a solution of 6 (5.4 g, 9.8 mmol) in acetic acid/acetic anhydride (1:1, 50 mL) at 0 °C. After complete conversion of the starting material (TLC) saturated aqueous NaHCO3 (50 mL) was added and the mixture was extracted with AcOEt (3  80 mL), the combined organic phases were dried (MgSO4), filtered and the solvent removed. The crude product was purified by medium pressure column chromatography (20/80 AcOEt/hexane) to afford the product 7

C NMR chemical shifts for the synthetic Potassium salt of GGG 2 with data from the natural product

a(1?6)glucosyl moiety

GGG

a(1?2)glucosyl moiety

Glyceryl moiety

13

C, d ppm

C-1

C-2

C-3

C-4

C-5

C-6

C-1

C-2

C-3

C-4

C-5

C-6

C-1

C-2

C-3

Natural Synthesised

98.3 98.4

72.0 72.0

73.5 73.7

70.0 70.1

72.3 72.4

60.8 61.0

98.0 98.0

71.9 72.0

73.9 74.0

69.9 70.0

71.1 71.3

66.0 66.9

177.3 177.5

79.7 79.5

63.6 63.7

2076

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078

(mixture of anomers) as a viscous colourless foam (4.71 g, 91%). mmax (film): 1744 (C@O) cm1. 1H NMR (CDCl3): d 7.36–7.24 (m), 6.32 (d, J = 3.5 Hz), 5.62 (d, J = 8.2 Hz), 5.01–4.55 (m), 4.31–4.21 (m), 4.01–3.92 (m), 3.67 (dd, J = 9.7 Hz, J = 3.5 Hz), 3.60–3.53 (m), 2.15 (s), 2.05 (s), 2.04 (s), 2.02 (s). 13C NMR (CDCl3): d 170.6, 169.3, 138.4, 137.5, 137.4, 128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.7, 89.6, 81.6, 78.8, 76.6, 75.7, 75.2, 75.0, 73.2, 71.0, 62.6, 21.0, 20.8. Anal. Calcd for C31H34O8: C, 69.65; H, 6.41. Found: C, 69.80; H, 6.42. 1.3. Ethyl 6-O-acetyl-2,3,4-tri-O-benzyl-1-thio-a/b-Dglucopyranoside 8 To a solution of diacetate 7 (4.3 g, 8.0 mmol) in CH2Cl2 (20 mL) at 0 °C was added ethanethiol (2.7 mL, 24.2 mmol), followed by BF3OEt2 (1.54 mL, 12.1 mmol). After 3 h at 0 °C, the reaction was quenched with saturated aqueous NaHCO3 (20 mL), extracted with CH2Cl2 (3  20 mL), the combined organic phases were dried (MgSO4) and concentrated. Purification by medium pressure column chromatography (10/90 AcOEt/hexane) afforded the thioglycoside 8 (mixture of anomers) as a viscous colourless foam (3.43 g, 80%). mmax (film): 1742 (C@O) cm1. 1H NMR (CDCl3): d 7.33–7.26 (m), 5.37 (d, J = 5.3 Hz), 4.99–4.54 (m), 4.47 (d, J = 9.8 Hz), 4.34–4.20 (m), 3.91–3.79 (m), 3.56–3.44 (m), 2.80– 2.66 (m), 2.62–2.44 (m), 2.03 (s), 2.01 (s), 1.34–1.26 (m). 13C NMR (CDCl3): d 170.7, 170.6, 138.5, 138.3, 137.9, 137.8, 137.7, 137.6, 128.5–127, 86.6, 85.2, 83.0, 82.4, 81.7, 79.5, 77.6, 77.1, 76.9, 75.8, 75.7, 75.5, 75.1, 75.0, 72.3, 68.9, 63.4, 63.1, 25.2, 23.7, 20.9, 20.8, 15.1, 14.8. Anal. Calcd for C31H36O6S: C, 69.38; H, 6.76; S, 5.97. Found: C, 69.13; H, 6.44; S, 5.97. 1.4. Methyl (2R)-3-O-tert-butyldiphenylsilyl-2,3dihydroxypropanoate 9 To a solution of methyl (2R)-2,3-dihydroxypropanoate8 (3.8 g, 28.3 mmol) in pyridine (19 mL) at rt was added TBDPSCl (8.0 mL, 30.5 mmol), followed by a catalytic amount (about 5 mg) of DMAP. After 16 h at rt, the reaction was quenched with H2O (20 mL), extracted with CH2Cl2 (3  20 mL) and the combined organic phases were dried (MgSO4) and concentrated. Purification by medium pressure column chromatography (5/95 AcOEt/hexane) afforded the product 9 as viscous colourless oil (9.10 g, 79%). ½a20 D 22.2 (c 1.0, CHCl3). FT-IR (film): 3510, 1745 (C@O). 1H NMR (CDCl3): d 7.67–7.61 (4H, m), 7.46–7.36 (6H, m), 4.24 (1H, t, J = 2.9 Hz), 3.98 (1H, dd, J = 10.6 Hz, J = 2.9 Hz), 3.91 (1H, dd, J = 10.6 Hz, J = 2.9 Hz), 3.79 (3H, s), 1.04 (9H, s). 13C NMR (CDCl3): d 173.3, 135.6, 135.5, 133.0, 132.9, 129.8, 127.8, 127.7, 71.9, 65.8, 52.4, 26.7, 19.3. Anal. Calcd for C20H26O4Si: C, 67.00; H, 7.31. Found: C, 67.12; H, 7.45. 1.5. Methyl 3-O-tert-butyldiphenylsilyl-(2R)-2-O-(6-O-acetyl2,3,4-tri-O-benzyl-a-D-glucopyranosyl)-2,3dihydroxypropanoate 10 A suspension of thioglycoside 8 (2.38 g, 4.4 mmol), glycerate 9 (1.56 g, 4.4 mmol) and 4 Å MS in CH2Cl2 (15 mL) was stirred at rt for 1 h. N-Iodosuccinimide (1.25 g, 5.6 mmol) and TfOH (0.026 mL) were added, at 0 °C. After 30 min, 10% Na2S2O3 aqueous solution (20 mL) and saturated NaHCO3 aqueous solution (10 mL) were added and the mixture was extracted with CH2Cl2 (3  20 mL), the combined organic phases were dried (MgSO4), filtered and the solvent removed under vacuum. The crude product was purified by medium pressure column chromatography (3/7 AcOEt/hexane) to afford 3.23 g of glycoside 10 (90%) as a colourless viscous foam (12:1 mixture of a- and b-anomers). a-Anomer ½a20 D +45.2 (c 1.45, CH2Cl2). FT-IR (film) 1744 cm1 (C@O). 1H NMR

(CDCl3): d 7.38–7.37 (5H, m), 7.37–7.22 (25H, m), 5.16 (1H, d, J = 3.6 Hz, H-1), 5.02 (1H, d, J = 10.6 Hz), 4.89 (2H, dd, J = 11.9 Hz, J = 3.1 Hz), 4.76 (1H, d, J = 10.5 Hz), 4.70 (1H, d, J = 11.8 Hz), 4.54 (1H, d, J = 11.2 Hz), 4.49 (1H, dd, J = 6.3 Hz; J = 4.3 Hz), 4.15–3.94 (6H, m), 3.73 (3H, s), 3.60 (1H, dd, J = 9.7 Hz, J = 3.5 Hz), 3.48 (1H, t, J = 9.5 Hz), 1.99 (3H, s), 1.03 (9H, s). 13C NMR (CDCl3): d 170.6 (C@O), 170.2 (C@O), 138.7, 138.1, 138.0, 135.6, 135.5, 133.0, 132.8, 129.8, 129.8, 128.4, 128.3, 128.2, 128.2, 128.1, 127.8, 127.7, 127.6, 94.8 (anomeric-C), 81.6, 75.8, 74.8, 72.0, 71.9, 69.0, 65.8, 64.6, 62.9, 51.9, 26.7, 20.8, 19.1. HR-MS: calcd for C49H56O10Si [M]+: 559.2130; found: 559.2125. 1.6. Methyl 3-O-tert-butyldiphenylsilyl-(2R)-2-O-(2,3,4-tri-Obenzyl-a-D-glucopyranosyl)-2,3-dihydroxypropanoate 11 A solution (7.6 mL) of Na (0.046 g) in MeOH (10 mL) was added to a stirred solution of the acetate 10 (2.0 g, 2.4 mmol) in MeOH (7.5 mL) at 0 °C. After 30 min saturated aqueous NH4Cl (15 mL) was added. The aqueous phase was extracted with AcOEt (3  20 mL) and the combined organic extracts were dried (MgSO4), filtered and the solvent was removed. Purification by medium pressure column chromatography (10/90 to 20/80 AcOEt/hexane) afforded the glyceryl a-glycoside 11 as a viscous colourless foam (1.79 g, 94%). ½a20 D +17.2 (c 1.49, CH2Cl2). FT-IR (film) 1753 cm1 (C@O) 1H NMR (CDCl3): d 7.70–7.67 (5H, m), 7.44–7.24 (25H, m), 5.14 (1H, d, J = 3.6 Hz, H-1), 4.99 (1H, d, J = 10.8 Hz), 4.90 (1H, d, J = 11.5 Hz), 4.88 (1H, d, J = 11.2 Hz), 4.78 (1H, d, J = 10.1 Hz), 4.71 (1H, d, J = 11.7 Hz), 4.62 (1H, d, J = 11.3 Hz), 4.46 (1H, dd, J = 6.1 Hz, J = 4.3 Hz), 4.13–3.95 (3H, m), 3.35–3.79 (1H, m), 3.74 (3H, s), 3.70–3.45 (5H, m), 1.03 (9H, s). 13 C NMR (CDCl3): d 170.3 (C@O), 138.8, 138.4, 138.2, 135.6, 135.5, 133.0, 132.8, 129.7, 128.3, 128.1, 128.0, 127.7, 127.6, 127.5, 95.0 (anomeric-C), 81.5, 79.3, 76.9, 75.7, 74.8, 74.7, 72.1, 71.2, 64.6, 61.6, 51.9, 26.7, 19.1. HR-MS: calcd for C47H54O9Si [M]+: 790.3531; found: 790.3500. 1.7. Methyl (2R)-2-O-(2,3,4-tri-O-benzyl-a-D-glucopyranosyl)2,3-dihydroxypropanoate 12 To a solution of 11 (3.00 g, 3.8 mmol) in THF (15 mL) at rt was added Bu4NF (1.20 g, 4.6 mmol). The reaction mixture was stirred for 3 h and then water was added. The mixture was extracted with AcOEt (3  20 mL), dried (MgSO4) and concentrated to furnish a yellow viscous residue. Purification by medium pressure column chromatography (50/50 to 100% AcOEt) afforded the product 12 as a very viscous colourless foam (1.89 g, 90%). 1 ½a20 D +49.5 (c 1.16, CH2Cl2). H NMR (CDCl3): d 7.43–7.26 (18H, m), 5.20 (1H, d, J = 3.6 Hz, H-1), 5.00 (1H, d, J = 10.8 Hz), 4.92– 4.80 (3H, m), 4.70 (1H, d, J = 11.4 Hz), 4.62 (1H, d, J = 10.8 Hz), 4.36 (1H, t, J = 4.3 Hz), 4.03 (1H, t, J = 9.3 Hz), 3.93 (2H, d, J = 4.5 Hz), 3.76 (3H, s), 3.75–3.52 (4H, m), 2.09 (2H, br s). 13C NMR (CDCl3): d 170.1, 138.6, 137.9, 137.7, 128.5, 128.4, 128.3, 127.9, 94.9 (anomeric-C), 81.3, 79.4, 77.1, 75.7, 75.1, 74.6, 72.4, 71.6, 63.3, 61.7, 52.1. HR-MS: calcd for C31H36O9 [M]+: 552.2354; found: 552.2333. 1.8. Potassium (2R)-2-O-(a-D-glucopyranosyl)-2,3dihydroxypropanoate 1 Benzyl ether 12 (1.89 g, 3.4 mmol) in AcOEt/EtOH (20 mL/ 10 mL) was hydrogenated at 35 psi in the presence of a catalytic amount of Pd/C 10% (0.05 equiv). After 3 h, the reaction mixture was filtered, the solvent was evaporated and the residue dried under vacuum to afford 13 as a water soluble viscous colourless foam (0.95 g, 99%). This material was sufficiently pure to proceed to the next step.

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078

To a solution of 13 (0.95 g, 3.4 mmol) in H2O (10 mL) was added a 2 M KOH aqueous solution (1.65 mL). The reaction was stirred overnight at rt. The solution was neutralised with HCl 10% and the solvent evaporated to afford a very viscous colourless foam. This was purified by ion exchange chromatography as described in the general procedures to form the potassium salt 1 (0.51 g, 49%). Its NMR data were identical to those of the natural sample4 and comparable to those found by Walton et al.5 for ammonium 1 (C@O). salt (Table 1). ½a20 D +97.6 (c 2.08, H2O). FT-IR 1605 cm 1 H NMR (D2O): d 5.00 (1H, d, J = 3.7 Hz, H-1), 4.39 (1H, t, J = 3.5 Hz), 3.89–3.87 (2H, m), 3.78–3.62 (4H, m), 3.49 (1H, dd, J = 9.8 Hz, J = 3.9 Hz), 3.34 (1H, t, J = 9.1 Hz). 13C NMR (D2O): d 178.6 (C@O), 99.0 (C-1), 80.7 (C-2 glycerate), 74.8 (C-3), 73.8 (C2), 73.2 (C-5), 71.1 (C-4), 64.7 (C-3 glycerate), 62.1 (C-6). 1.9. Methyl 3-O-tert-butyldiphenylsilyl-(2R)-2-O-[6-O-acetyl2,3,4-tri-O-benzyl-a-D-glucopyranosyl-(1?6)-2,3,4-tri-Obenzyl-1-O-a-D-glucopyranosyl]-2,3-dihydroxypropanoate 14 A suspension of 8 (1.74 g, 3.2 mmol), 11 (2.38 g, 3.0 mmol) and 4 Å MS in CH2Cl2 (35 mL) was stirred at rt for 1 h. A solution of Niodosuccinimide (0.96 g, 4.2 mmol) and TfOH (0.020 lL) in 10 mL of CH2Cl2 was added at 0 °C. After 30 min, 10% Na2S2O3 aqueous solution (20 mL) and saturated NaHCO3 aqueous solution (20 mL) were added and the mixture was extracted with CH2Cl2 (3  30 mL), the combined organic phases were dried (MgSO4), filtered and the solvent was removed under vacuum. The crude product was purified by medium pressure column chromatography (10/ 90 AcOEt/hexane) to afford 3.47 g of 14 (84%) as a colourless vis1 cous foam. ½a20 D +21.1 (c 1.39, CH2Cl2). H NMR (CDCl3): d 7.68 (5H, br s), 7.35–7.20 (35H, m), 5.15 (1H, s), 5.00–4.74 (8H, m), 4.65–4.51 (6H, m), 4.15–3.89 (7H, m), 3.79–3.71 (2H, m), 3.71 (3H, s) 3.61–3.43 (5H, m), 1.95 (3H, s), 1.02 (9H, s). 13C NMR (CDCl3): d 170.7 (C@O), 170.3 (C@O), 138.9, 138.6, 138.3, 138. 0, 135.6, 135.5, 133.1, 132.9, 129.7, 128.3, 128.2, 128.1, 127.8, 127.5, 127.4, 97.1 (anomeric-C), 94.8 (anomeric-C), 81.6, 79.9, 79.5, 75.6, 74.9, 74.6, 72.1, 71.0, 68.7, 64.7, 63.0, 51.9, 26.7, 20.8, 19.2. HR-MS: calcd for C76H84O15Si [M]+: 1264.5574; found: 1264.5556. 1.10. Methyl (2R)-2-O-[6-O-acetyl-2,3,4-tri-O-benzyl-a-Dglucopyranosyl-(1?6)-2,3,4-tri-O-benzyl-1-O-a-Dglucopyranosyl]-2,3-dihydroxypropanoate 15 To a solution of 14 (3.4 g, 2.7 mmol) in THF (10 mL) at rt was added anhydrous Bu4NF 1 M in THF (3.2 mL, 3.2 mmol). The reaction mixture was stirred for 2 h and then water (15 mL) was added. The mixture was extracted with AcOEt (3  10 mL), dried (MgSO4) and concentrated under vacuum to furnish a viscous yellow residue. Purification by medium pressure column chromatography (20/80 AcOEt/hexane) afforded the alcohol 15 as a colourless foam 1 (2.4 g, 88%). ½a20 D +5.9 (c 1.06, CH2Cl2). H NMR (CDCl3): d 7.39–7.29 (30H, m), 5.12 (1H, d, J = 3.2 Hz), 5.02–4.43 (14H, m), 3.74 (3H, s), 4.20–3.46 (14H, m), 1.98 (3H, s). HR-MS: calcd for C60H66O15 [M]+: 1026.4396; found: 1026.4371. 1.11. Methyl (2R)-2-O-2,3,4-tri-O-benzyl-a-D-glucopyranosyl(1?6)-2,3,4-tri-O-benzyl-a-D-glucopyranosyl-2,3dihydroxypropanoate 16 A solution (4.6 mL) of Na (0.046 g) in MeOH (5 mL) was added to a stirred solution of the acetate 15 (1.6 g, 1.6 mmol) in MeOH (4.6 mL) at 0 °C. After 30 min saturated aqueous NH4Cl (10 mL) was added. The aqueous phase was extracted with AcOEt (3  15 mL) and the combined organic extracts were dried (MgSO4), filtered and the solvent removed. Filtration by silica gel afforded the

2077

product 16 as a viscous colourless foam (1.47 g, 95%). ½a20 D +77.0 (c 1.13, CH2Cl2). 1H NMR (CDCl3): d 7.37–7.25 (30H, m), 5.10 (1H, br s), 5.02–4.44 (14H, m), 4.11–3.93 (6H, m), 3.74 (3H, s), 3.66–3.50 (8H, m). 13C NMR (CDCl3): d 170.2 (C@O), 138.7, 138.6, 138.1, 138. 0, 137.9, 137.8, 128.4–127.6, 97.4 (anomeric-C), 95.0 (anomeric-C), 81.9, 81.8, 79.9, 79.5, 77.8, 77.3, 75.7, 75.6, 75.2, 75.1, 75.0, 73.0, 72.4, 70.9, 70.6, 66.5, 63.4, 61.8, 52.1. HR-MS: calcd for C58H64O14 [M]+: 984.4290; found: 984.4275. 1.12. Potassium (2R)-2-O-a-D-glucopyranosyl-(1?6)-a-Dglucopyranosyl-2,3-dihydroxypropanoate 2 Benzyl ether 16 (1.47 g, 1.5 mmol) in AcOEt/EtOH (20/10 mL) was hydrogenated at 50 psi in the presence of Pd/C 10% (0.25 equiv). After 3 h, the reaction mixture was filtered and the solvent was evaporated to afford ester 17 as a very viscous colourless foam (0.640 g, 99%). To a solution of ester 17 (0.50 g, 1.12 mmol) in H2O (5 mL) was added 2 M KOH (1.05 mL). After all the starting material had been consumed (3 h), the pH was adjusted to 7 with HCl 10% and the solvent evaporated to afford crude 2 as a viscous colourless foam which was purified by ion exchange chromatography as indicated in the general procedures and afforded 2 (0.33 g, 63%) which was detected homogeneous by NMR. Its NMR data were identical to those of a natural sample4 (Table 2). ½a20 D +105.3 (c 0.83, H2O). FT-IR: 1604 cm1 (C@O). 1H NMR (D2O): d 5.04 (1H, d, J = 3.5 Hz, H-1), 4.96 (1H, d, J = 3.3 Hz), 4.20 (1H, dd, J = 5.0 Hz, J = 3.2 Hz), 3.99–3.71 (10H, m), 3.60–3.40 (4H, m). 13C NMR (D2O): d 177.5 (C@O), 98.4 (anomeric-C), 98.0 (anomeric-C), 79.5, 74.0, 73.7, 72.4, 72.0, 71.3, 70.1, 70.0, 66.9, 63.7, 61.0. HR-MS: calcd for C15H25O14 [M]: 429.12498; found: 429.13007. Acknowledgements We wish to acknowledge the analysis department at the ITQB for providing elemental analyses. We also acknowledge the generous financial support provided by FCT POCI/BIA-PRO/57263/2004 and CRAFT Hotsolutes Project (FP6-2002-SME-1). We also thank the Mass spectrometry unit at the Faculty of Sciences at the University of Lisbon for HRMS. References 1. Da Costa, M. S.; Santos, H.; Galinski, E. A. Adv. Biochem. Eng. Biotechnol. 1998, 61, 117–153. 2. Santos, H.; da Costa, M. S. Environ. Microbiol. 2002, 4, 501–509. 3. Ramos, A.; Raven, N. D. H.; Sharp, R. J.; Bartolucci, S.; Rossi, M.; Cannio, R.; Lebbink, J.; Van Der Oost, J.; De Vos, W. M.; Santos, H. Appl. Environ. Microbiol. 1997, 63, 4020–4025. 4. Prof. H. Santos, ITQB/UNL, Portugal. Private communication. We also thank her for providing NMR spectra of the natural compound 2. Faria, T. Q.; Mingote, A.; Siopa, F.; Ventura, R.; Maycock, C.; Santos, H. Carbohydr. Res. 2008, 343, 3025– 3033. 5. Hunter, B. K.; Mowbray, S. L.; Walton, D. J. Biochemistry 1979, 18, 4458–4465. 6. Kollman, V. H.; Hanners, J. L.; London, R. E.; Adama, E. G.; Walker, T. E. Carbohydr. Res. 1979, 73, 193–202. 7. Demchenko, A. V. General Aspects of the Glycosidic Bond Formation. In Handbook of Chemical Glycosylation; Demchenko, A. V., Ed.; Wiley-VCH: Weinheim, 2008; pp 1–28. 8. Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020. 9. Crich, D.; Cai, W. J. Org. Chem. 1999, 64, 4926–4930. 10. Lok, C. M.; Ward, J. P.; Dorp, D. A. Chemistry and Physics of Lipids 1976, 16, 115– 122. TBDMS ether formation was not nearly as selective. 11. For example: Zhu, T.; Boons, G.-J. Angew. Chem., Int. Ed. 1998, 37, 1898–1900. 12. Tennant-Eyles, R. J.; Davis, B. G.; Fairbanks, A. J. Tetrahedron: Asymmetry 2000, 11, 231–243. and references therein; Burt, J.; Dean, T.; Warriner, S. Chem. Commun. 2004, 454–455. 13. For a recent approach: Crich, D.; Bowers, A. A. J. Org. Chem. 2006, 71, 3452– 3463. 14. Ray, A. K.; Roy, N. Carbohydr. Res. 1990, 196, 95–100; See also: Koto, S.; Uchida, T.; Zen, S. Bull. Chem. Soc. Jpn. 1973, 46, 2520–2523. 15. Lemieux, R. U. Can. J. Chem. 1951, 29, 1079–1091.

2078

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078

16. Shingu, Y.; Nishida, Y.; Dohi, H.; Matsuda, K.; Kobayashi, K. J. Carbohydr. Chem. 2002, 21, 605–611. and references therein. 17. Valerio, S.; Iadonisi, A.; Adolfini, M.; Ravida, A. J. Org. Chem. 2007, 72, 6097–6106. 18. Koto, S.; Morishima, N.; Kihara, Y.; Suzuki, H.; Kosugi, S.; Zen, S. Bull. Chem. Soc. Jpn. 1983, 56, 188–191. 19. van den Bos, L. J.; Dinkelaar, J.; Overkleeft, H. S.; van der Marel, G. A. J. Am. Chem. Soc. 2006, 128, 13066–13067. 20. Transglycosylation of thioglycoside 8 with methyl lactate produced the glucosyl lactate with a a:b ratio of only 4:1. Similarly, glycosylation with

methyl (2S)-3-O-tert-butyldiphenylsilylglycerate afforded a a:b ratio of about 9:1 indicating that double stereodifferentiation was probably active but not very important. 21. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 1331–1334; Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313–4316. 22. Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory Chemicals, 5th ed.; Elsevier, 2003.