The synthesis of C-9 modified derivatives of the α-methyl glycoside of kdn methyl ester

Article abstract of DOI:10.1081/CAR-100103960

The synthesis of a range of C-9 substituted derivatives of the α-methyl glycoside of KDN methyl ester (2) is reported. 9-Deoxy-9-iodo, 9-deoxy-9-thioacetyl and 9-azido-9-deoxy derivatives (compounds 10, 12 and 13) were all prepared from the corresponding

Full text of DOI:10.1081/CAR-100103960

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THE SYNTHESIS OF C-9 MODIFIED DERIVATIVES OF
THE α-METHYL GLYCOSIDE OF KDN METHYL ESTER
Adele K. Norton a , Gaik B. Kok a & Mark von Itzstein b
a Department of Medicinal Chemistry , Monash University , 381 Royal Parade, Parkville,
Victoria, 3052, Australia
b Griffith University (Gold Coast Campus) , Centre for Biomolecular Science and Drug
Discovery, 50 Gold Coast Mail Centre, Queensland, 9726, Australia
Published online: 16 Aug 2006.
To cite this article: Adele K. Norton , Gaik B. Kok & Mark von Itzstein (2001) THE SYNTHESIS OF C-9 MODIFIED DERIVATIVES
OF THE α-METHYL GLYCOSIDE OF KDN METHYL ESTER, Journal of Carbohydrate Chemistry, 20:2, 227-238, DOI: 10.1081/
CAR-100103960
To link to this article: http://dx.doi.org/10.1081/CAR-100103960
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J. CARBOHYDRATE CHEMISTRY, 20(2), 227–238 (2001)
THE SYNTHESIS OF C-9 MODIFIED
DERIVATIVES OF THE ꢀ-METHYL GLYCOSIDE
OF KDN METHYL ESTER
Adele K. Norton,1 Gaik B. Kok,1 and Mark von Itzstein1,2,*
1Department of Medicinal Chemistry, Monash University,
381 Royal Parade, Parkville 3052, Victoria, Australia
2Centre for Biomolecular Science and Drug Discovery,
Griffith University (Gold Coast Campus),
50 Gold Coast Mail Centre, Queensland 9726, Australia
ABSTRACT
The synthesis of a range of C-9 substituted derivatives of the ꢀ-methyl
glycoside of KDN methyl ester (2) is reported. 9-Deoxy-9-iodo, 9-deoxy-9-
thioacetyl and 9-azido-9-deoxy derivatives (compounds 10, 12 and 13) were all
prepared from the corresponding 9-O-tosylate 8. Catalytic hydrogenation of 10
and 13 results in reduction to give the 9-deoxy and 9-amino-9-deoxy deriva-
tives 20 and 15, respectively. Further derivatisation of the amine 15 into com-
pounds 17 and 18 was achieved via standard acylation procedures.
INTRODUCTION
Sialic acids are a family of more than 40 different sugars which have a 3-de-
oxy-2-nonulosonic acid structure.1 The most commonly occurring sialic acids are
N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) with
structural diversity arising from substitutions (e.g., O-acetylation, O-methylation
and O-sulfonylation) of the various hydroxyl groups of these systems. As terminal
*Corresponding author. E-mail: m.vonitzstein@mailbox.gu.edu.au
227
Copyright © 2001 by Marcel Dekker, Inc.
www.dekker.com

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NORTON, KOK, AND VON ITZSTEIN
carbohydrate units of many glycoproteins and glycolipids, sialic acids take part in
a myriad of biological functions including molecular recognition, cell adhesion and
inflammation.1 In 1986, 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid
(KDN, 1), a unique C-5 deaminated member of the sialic acid family, was discov-
ered from the membrane polysialoglycoproteins (PSGP) of Salmo gaidneri (rain-
bow trout) eggs.2 Studies have since shown that only sialic acid residues with an
acylamino group at C-5 are cleaved by bacterial sialidases.3 Therefore, it is likely
that as a terminal unit of the PSGP chains, KDN protects the membrane from bac-
terial degradation. Subsequently, the natural presence of KDN has been reported
for a wide variety of organisms ranging from bacteria and lower vertebrates to
higher vertebrates including mammals,4–6 suggesting its wide occurrence in nature.
Recently, Li and coworkers reported new sialidases that have unique roles in
KDN metabolism. For example, the deaminoneuraminic acid residue-cleaving en-
zyme, KDNase, specifically hydrolyses the ketosidic linkages of KDN but not N-
acylneuraminyl linkages and was first isolated from the liver of the loach (Misgu-
runus fossilis).7 The KDN-cleaving enzyme designated KDNase Sm has been
induced and purified from Sphingobacterium multivorum.8 Moreover this protein
has been shown9 to be a retaining enzyme not unlike the sialidases of bacterial and
viral origin.
A large amount of work has been directed towards the synthesis of analogues
and derivatives of naturally occurring sialic acids both for their use as probes for
the study of sialic acid recognising proteins, and for investigating substrate speci-
ficities of the enzymes involved in sialic acid metabolism.10,11 Further study of the
biological roles of KDN requires the synthesis of more complex analogues and
derivatives of 1. We report here the synthesis of some C-9 modified analogues of
the ꢀ-methyl glycoside of KDN methyl ester 2 that may be useful as chemical
probes to elucidate the sub-structural requirements for substrate/inhibitor recogni-
tion by the enzymes involved in KDN metabolism.
RESULTS AND DISCUSSION
As KDN-bearing glycoconjugates are ꢀ-linked, we have chosen the ꢀ-methyl
glycoside of KDN methyl ester 2 as the key starting material for this study. The
synthesis of this compound has previously been reported in the literature. Hence,
treatment12 of the methyl ester of KDN 3, using Fischer’s methyl glycosidation
method under controlled conditions (70°C for 15 h), resulted in a mixture of both
ꢀ and ꢁ-methyl glycosides (2 and 4) of which the latter was the major product
(74%). The ꢀ-glycoside 2 was obtained in only 4% yield. Given that there is prece-
dent in the literature for the stereoselective synthesis of the ꢀ-methyl glycoside of
Neu5Ac methyl ester from the 2-ꢁ-chloride, methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-2-chloro-2,3,5-trideoxy-D-glycero-ꢁ-D-galacto-2-nonulopyranosonate
(5),13 we thought it reasonable to explore this methodology for the synthesis of 2.
Accordingly, exposure of the methyl ester of KDN 3 to acetyl chloride gave the 2-
ꢁ-chloride, methyl 4,5,7,8,9-penta-O-acetyl-2-chloro-2,3-dideoxy-D-glycero-ꢁ-D-

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229
galacto-2-nonulopyranosonate (6). Subsequent treatment of the 2-ꢁ-chloride (6)
with methanol at room temperature for one hour led to the formation of 7 in good
yield (87%, 2 steps). The ꢀ-methyl glycoside 2 was obtained as the only product
following de-O-acetylation of 7 under standard Zemplen conditions. Both com-
pounds 2 and 7 were found to be identical to known materials.12
The 9-deoxy-9-iodo, 9-azido-9-deoxy and 9-deoxy-thioacetyl derivatives of
2 can be accessed via nucleophilic displacement of a tosyl group. Selective tosyla-
tion14 and tritylation15 of N-acetylneuraminic acid derivatives has been previously
reported. Selective tosylation at C-9 of the KDN derivative 2 was achieved in high
yield (96%) by the reaction with p-toluenesulfonyl chloride initially at 0°C (2.5 h)
and then at 5°C (20 h) to produce the 9-O-tosylate 8, which was fully characterised
as the peracetate 9. Reaction of 8 with sodium iodide in acetone under reflux pro-
ceeded uneventfully to produce iodide 10 in high yield (96%). The appearance of
a resonance at ꢂ 15.9 ppm for C-9 in the 13C NMR spectrum is consistent with sub-
stitution of an iodo moiety at this position.16 The iodide 10 was further charac-
terised as the peracetate 11. Introduction of sulfur at C-9 via the reaction of 8 with
potassium thioacetate in acetone proceeded with some concomitant de-O-acetyla-
tion, as was evident by TLC analysis of the crude reaction mixture. Therefore, for
ease of workup, the crude reaction mixture was acetylated and then purified by
chromatography which afforded the peracetate 12 in good yield (66%, 2 steps).
The introduction of a nitrogen functionality at C-9 via the azide was achieved
by treatment of the 9-O-tosylate 8 with lithium azide in dimethylformamide at
50°C, resulting in the formation of the 9-azide 13 in high yield (91%). An attempt
to synthesise this compound directly from the methyl ketoside of KDN methyl es-
ter 2 using well-known Mitsunobu chemistry (TPP, DIAD, TMSN3, DMF) also
gave 13, albeit in moderate yield (54%). This is in comparison to an overall yield
of 87% for the two-step procedure via the 9-O-tosylate 8. The 9-azide 13 was fur-
ther characterised as the peracetate 14.
The introduction of an azido moiety functionally allowed access to a range of
other nitrogen-substituted compounds for structure-activity relationship studies.
Hence, hydrogenation of the 9-azide 13 in the presence of 10% palladium on car-
bon in MeOH produced the 9-amine 15 in quantitative yield. This compound was
fully characterised as the peracetate 16 that was then de-O-acetylated to produce
the 9-acetamide 17 in good yield (72%). Acylation of the amine 15 with benzoyl
chloride in the presence of triethylamine provided the 9-benzamido-9-deoxy com-
pound 18 in 68% yield. This compound was further characterised as the peracetate
19.
Finally, under hydrogenolysis conditions (50 psi H2, 10% Pd/C), the 9-iodide
10 was converted into the corresponding 9-deoxy compound 20 in quantitative
yield. Further characterisation of this compound involved converting it to the per-
acetate 21.
In conclusion, we have synthesised a range of novel C-9 substituted deriva-
tives of the methyl ketoside of KDN methyl ester. These systems should be useful
as probes for the study of enzymes involved in KDN/sialic acid metabolism.

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NORTON, KOK, AND VON ITZSTEIN

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231
EXPERIMENTAL
General Methods. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra
(in ꢂ ppm) were recorded on a Bruker AMX300 spectrometer and were referenced
using solvent residues; J-values are in hertz (Hz). Both low resolution (LR) and
high resolution (HR) fast atom bombardment (FAB) mass spectra were obtained
using a JEOL JMS-DX 300 spectrometer. Optical rotations were measured using a
JASCO DIP-370 polarimeter with a path length 50 mm; [ꢀ]D-values are in units of
10ꢃ1 deg cm2 gꢃ1; concentrations are quoted in 10ꢃ2 deg cmꢃ3. IR spectra were
recorded using a Hitachi 270-30 infrared spectrophotometer. Column chromatog-
raphy was performed on Merck silica gel 60 (0.040–0.063 mm). Reactions were
monitored by TLC on Kieselgel 60 F254 plate (Merck 5554) and spots were de-
tected under ultraviolet (UV) irradiation and by spraying with a 95% aq. ethanol
solution containing 5% H2SO4 and charring at ca. 180°C. Microanalyses were per-
formed by the Chemical and Microanalytical Service, Essendon, Victoria. KDN
was prepared by aldolase (EC 4.1.3.3) condensation of D-mannose and pyruvate
according to the literature.17 Methanolysis of 1 and treatment of the resulting
methyl ester 3 with neat acetyl chloride (rt for 2.5 days) afforded the 2-ꢁ-chloride,
methyl 4,5,7,8,9-penta-O-acetyl-2-chloro-2,3-dideoxy-D-glycero-ꢁ-D-galacto-2-
nonulopyranosonate (6) (95%) which was used in subsequent step without further
purification. Spectral data for 2-ꢁ-chloride (6) were consistent with the literature.18
General Procedure for Acetylation. Approximately 50–100 mg of the
compound to be acetylated was dissolved in pyridine (1 mL) and the solution
treated with Ac2O (0.5 mL) and DMAP (ca. 5 mg) at rt for 16 h. The mixture was
then concentrated to dryness and the residue chromatographed on silica using the
solvent(s) specified.
Methyl (Methyl 3-Deoxy-D-glycero-ꢀ-D-galacto-2-nonulopyranosid)onate
(2). A solution of the crude 2-ꢁ-chloride, methyl 4,5,7,8,9-penta-O-acetyl-2-
chloro-2,3-dideoxy-D-glycero-ꢁ-D-galacto-2-nonulopyranosonate (6) (7.14 g, 13.98
mmol) in anhydrous methanol was stirred at rt for 1 h. The reaction mixture was then
concentrated under reduced pressure which gave methyl (methyl 4,5,7,8,9-penta-O-
acetyl-3-deoxy-D-glycero-ꢀ-D-galacto-2-nonulopyranosid)onate (7) as a colourless
foam, 6.17 g (87% from 3). Subsequent Zemplen de-O-acetylation of the peracetate
7 (5.96 g, 11.77 mmol) under standard conditions (NaOMe/MeOH) furnished the ti-
tle compound 2 as a colourless foam, 2.07 g (59%).
7: mp 112–116°C (lit.12 115–116°C); 1H NMR (CDCl3) data were consistent
with the literature12; 13C NMR (CDCl3) ꢂ 20.6, 20.8, 21.0 (OCOCH3 ꢄ 5), 37.4
(C-3), 52.5, 52.7 (OCH3, COOCH3), 62.2 (C-9), 66.7, 67.8, 67.9, 69.3, 71.2 (C-4,
C-5, C-6, C-7, C-8), 98.9 (C-2), 167.7, 169.8, 170.1, 170.6 (carbonyls); LRMS m/z:
507 (MHꢅ, 2%), 474 (25), 446 (60), 414 (100); IR (vmax, KBr): 1750, 1736, 1370,
1232, 1066 cmꢃ1
.
2: 1H NMR (D2O) and IR data were consistent with the literature12; 13C NMR
(D2O) ꢂ 41.0 (C-3), 54.3 (OCH3), 56.0 (COOCH3), 65.8 (C-9), 70.7, 71.8, 72.3,

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NORTON, KOK, AND VON ITZSTEIN
73.6, 76.7 (C-4, C-5, C-6, C-7, C-8), 101.9 (C-2), 172.7 (carbonyl); LRMS m/z:
297 (MHꢅ, 3%), 265 (100), 229 (50), 169 (33), 144 (23).
Methyl (Methyl 3-Deoxy-9-O-p-toluenesulfonyl-D-glycero-ꢀ-D-galacto-2-
nonulopyranosid)onate (8). To a stirred solution of 2 (427 mg, 1.44 mmol) in
pyridine (9 mL) at 0°C, was added p-TsCl (426 mg, 2.23 mmol). After stirring at
the same temperature for 2.5 h, additional p-TsCl (82 mg, 0.43 mmol) was added
and stirring maintained for a further 20 h at 5°C. The reaction mixture was allowed
to warm up to rt before MeOH (9 mL) was added. After 1 h, the volatiles were re-
moved under reduced pressure and the residue purified by chromatography
(EtOAc) which provided the 9-O-tosylate 8 as a colourless solid (620 mg; 96%):
[ꢀ]D –13.2° (c 3.25, CHCl3); 1H NMR (D2O) ꢂ 1.63 (1 H, dd, J3ax,3eq 12.3, J3ax,4
12.9, H-3ax), 2.35 (3 H, s, PhCH3), 2.52 (1 H, dd, J3eq,4 4.5, H-3eq), 3.10 (3H, s,
OCH3), 3.40 (1 H, dd, J5,4 9.2, J5,6 9.7, H-5), 3.50 (1 H, m, H-4), 3.56 (1 H, pseudo
d, H-6), 3.76 (3 H, s, COOCH3),. 3.91 (2 H, m, J8,9ꢆ 6.8, H-7, H-8), 4.16 (1 H, dd,
J9,9ꢆ 10.4, H-9), 4.34 (1 H, pseudo d, H-9ꢆ), 7.39–7.75 (4 H, m; Ph); 13C NMR
(D2O) ꢂ 23.4 (Ph-CH3), 41.2 (C-3), 54.3, 55.9 (OCH3, COOCH3), 70.7, 71.7, 71.9,
72.2, 76.8 (C-4, C-5, C-6, C-7, C-8), 75.3 (C-9), 102.0 (C-2), 130.5, 132.9, 138.1,
149.2 (aromatic carbons), 172.5 (carbonyl); LRMS m/z: 451 (MHꢅ, 3%), 418 (77),
382 (42), 351 (23), 211 (25), 185 (26), 173 (29) no base peak was observed; IR
(vmax, KBr): 3480, 2956, 1742, 1588, 1452, 1356, 1290, 1172, 1094, 974 cmꢃ1
.
Methyl (Methyl 4,5,7,8-Tetra-O-acetyl-3-deoxy-9-O-p-toluenesulfonyl-
D-glycero-ꢀ-D-galacto-2-nonulopyranosid)onate (9). Acetylation of 8 (30 mg,
0.07 mmol) according to the general conditions gave, after chromatography
(EtOAc/Hex, 1:1), 9 as a colourless solid (39 mg; 96%): [ꢀ]D –10.4° (c 6.12,
CHCl3); 1H NMR (CDCl3) ꢂ 1.87 (1 H, dd, J3ax,3eq 13.0, J3ax,4 12.4, H-3ax), 1.99 (6
H, s, OAc ꢄ 2), 2.03, 2.09 (each 3 H, s, OAc ꢄ 2), 2.44 (3 H, s, PhCH3), 2.63 (1
H, dd, J3eq,4 4.5, H-3eq), 3.26 (3H, s, OCH3), 3.81 (3 H, s, COOCH3),. 4.05 (1 H,
dd, J9,8 3.9, J9,9ꢆ 12.1, H-9), 4.09 (1 H, dd, J6,5 9.8, J6,7 3.0, H-6), 4.32 (1 H, dd, J9ꢆ,8
2.2, H-9ꢆ), 4.80 (1 H, pseudo t, J5,4 9.5, H-5), 4.90 (1 H, m, H-4), 5.37 (2 H, m, J7,8
8.3, H-7, H-8), 7.33–7.77 (4 H, m, Ph); 13C NMR (CDCl3) ꢂ 20.5, 20.7, 20.8, 20.9
(OCOCH3 ꢄ 4), 21.6 (PhCH3), 37.3 (C-3), 52.4, 52.8 (OCH3, COOCH3), 66.6,
67.4, 67.8, 69.3, 71.3 (C-4, C-5, C-6, C-7, C-8), 67.4 (C-9), 98.9 (C-2), 128.0,
129.8, 132.8, 144.9 (aromatic carbons), 167.6, 169.5, 170.0, 170.6 (carbonyls);
LRMS m/z: 610 (MHꢅ, 2%), 559 (29), 527 (100), ; IR (vmax, KBr): 3508, 1748,
1598, 1444, 1368, 1218 cmꢃ1
.
Anal. Calcd for C26H34O15S: C, 50.48; H, 5.54. Found: C 50.49; H 5.60.
Methyl (Methyl 3,9-Dideoxy-9-iodo-D-glycero-ꢀ-D-galacto-2-nonulopy-
ranosid)onate (10). Sodium iodide (259 mg, 1.73 mmol) was added to a solution
of 8 (259 mg, 0.58 mmol) in acetone (25 mL) and the mixture heated under reflux
for 2.5 days. After cooling and removal of volatiles under reduced pressure, flash
chromatography (EtOAc) gave the 9-iodide 10 as an amorphous mass (255 mg;

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233
96%): [ꢀ]D –10.7° (c 6.26, CHCl3); 1H NMR (D2O) ꢂ 1.78 (1 H, dd, J3ax,3eq 12.6,
J3ax,4 12.4, H-3ax), 2.66 (1 H, dd, J3eq,4 4.6, H-3eq), 3.40 (3H, s, OCH3), 3.55 (2 H,
m, H-4, H-5), 3.69 (3 H, m, H-6, H-7, H-8), 3.83 (2 H, m, H-9, H-9ꢆ), 3.92 (3 H, s,
COOCH3); 13C NMR (D2O) ꢂ 15.9 (C-9), 41.0 (C-3), 54.3, 56.0 (OCH3,
COOCH3), 71.7, 71.8, 72.4, 73.4, 76.6 (C-4, C-5, C-6, C-7, C-8), 101.9 (C-2),
172.6 (carbonyl); LRMS m/z: 407 (MHꢅ, 2%), 375 (100), 339 (47); IR (vmax
,
KBr): 3448, 1734, 1632, 1438, 1290, 1202, 1070, 1036 cmꢃ1
.
Methyl (Methyl 4,5,7,8-Tetra-O-acetyl-3,9-dideoxy-9-iodo-D-glycero-ꢀ-
D-galacto-2-nonulopyranosid)onate (11). Acetylation of 10 (121 mg, 0.30
mmol) according to the general conditions gave, after chromatography
(EtOAc/Hex, 1:1), 11 as a colourless solid (148 mg; 86%): [ꢀ]D –10.5° (c 7.54,
CHCl3); 1H NMR (CDCl3) ꢂ 1.91 (1 H, dd, J3ax,3eq 12.6, J3ax,4 12.2, H-3ax), 2.01 (6
H, s, OAc ꢄ 2), 2.14, 2.19 (each 3 H, s, OAc ꢄ 2), 2.66 (1 H, dd, J3eq,4 4.1, H-3eq),
3.21 (1 H, dd, J9,9ꢆ 11.2, J9,8 6.2, H-9), 3.34 (3 H, s, OCH3), 3.58 (1 H, dd, J9ꢆ,8 3.5,
H-9ꢆ), 3.84 (3 H, s, COOCH3), 4.18 (1 H, pseudo d, H-6), 4.86 (1 H, pseudo t, J5,4
9.3, H-5), 4.93 (1 H, m, H-4), 5.16 (1 H, m, J8,7 7.5, H-8), 5.28 (1 H, m, H-7); 13
C
NMR (CDCl3) ꢂ 3.8 (C-9), 20.6, 20.7, 21.0 (OCOCH3 ꢄ 4), 37.3 (C-3), 52.4, 52.8
(OCH3, COOCH3), 67.9, 69.2, 69.3, 69.6, 71.5 (C-4, C-5, C-6, C-7, C-8), 99.0 (C-
2), 167.7, 169.8, 169.9 (carbonyls); LRMS m/z: 515 (MHꢅ – OAc, 46%), 483
(100), 357 (34), 321 (22); IR (vmax, KBr): 1750, 1436, 1370, 1218, 1144, 1088,
1056 cmꢃ1
.
Anal. Calcd for C19H27O12I: C, 39.74; H, 4.74. Found: C, 39.85; H 4.75.
Methyl (Methyl 4,5,7,8-Tetra-O-acetyl- 3,9-dideoxy-9-thioacetyl-D-glyc-
ero-ꢀ-D-galacto-2-nonulopyranosid)onate (12). A mixture of the 9-O-tosylate
8 (99 mg, 0.22 mmol), KSAc (30 mg, 0.40 mmol) and acetone (20 mL) was
stirred at rt for 36 h. A further portion of KSAc (60 mg; 0.79 mmol) was added
and the mixture stirred for 5 days. The mixture was filtered and the filtrate con-
centrated. The resulting residue was acetylated according to the general condi-
tions. After chromatography (EtOAc/Hex, 1:1), the title compound 12 was ob-
tained as a colourless foam (76 mg; 66%): [ꢀ]D ꢅ8.2° (c 4.13, CHCl3); 1H NMR
(CDCl3) ꢂ 1.86 (1 H, dd, J3ax,3eq 12.9, J3ax,4 12.5, H-3ax), 1.96, 1.97, 2.08, 2.10
(each 3 H, s, OAc ꢄ 4), 2.28 (3 H, s, SCOCH3), 2.60 (1 H, dd, J3eq,4 4.4, H-3eq),
2.93 (1 H, dd, J9,9ꢆ 14.6, J9,8 5.7, H-9), 3.29 (3H, s, OCH3), 3.42 (1 H, dd, J9ꢆ,8
3.6, H-9ꢆ), 3.79 (3 H, s, COOCH3), 4.14 (1 H, dd, J6,5 10.0, J6,7 2.1, H-6), 4.82
(1 H, pseudo t, J5,4 9.5, H-5), 4.86 (1 H, m, H-4), 5.25 (2 H, m, J7,8 8.8, H-7)
5.40 (1 H, m, H-8); 13C NMR (CDCl3) ꢂ 20.8, 20.9, 21.0, 21.2 (OCOCH3 ꢄ 4),
29.9 (C-9), 30.5 (SCOCH3), 37.6 (C-3), 52.7, 52.9 (OCH3, COOCH3), 68.2, 68.3,
68.4, 69.5, 71.5 (C-4, C-5, C-6, C-7, C-8), 99.1 (C-2), 167.9, 170.0, 170.1, 170.2,
170.3, 194.7 (carbonyls); LRMS m/z: 523 (MHꢅ, 6%), 495 (31), 463 (31), 431
(100), 389 (54); IR (vmax, KBr): 3668, 2964, 1754, 1698, 1436, 1370, 1220,
1108, 1088, 1054 cmꢃ1
Anal. Calcd for C21H30O13S: C, 48.27; H, 5.79. Found: C, 48.52; H 5.95.
.

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NORTON, KOK, AND VON ITZSTEIN
Methyl (Methyl 9-Azido-3,9-dideoxy-D-glycero-ꢀ-D-galacto-2-nonulopy-
ranosid)onate (13).
Procedure A. LiN3 (0.85 g, 13.25 mmol) was added to a solution of the 9-
tosylate 8 (1.99 g, 4.42 mmol) in DMF (50 mL). After stirring at 50°C for 3 days,
the reaction mixture was cooled and then concentrated under reduced pressure. The
resulting residue was subjected to column chromatography (CH2Cl2/acetone, 1:1)
which furnished 13 as a colourless amorphous mass (1.30 g; 91%).
Procedure B. A solution of triphenylphosphine (207 mg, 0.79 mmol) in
DMF (2 mL) was cooled to 0°C prior to the addition of DIAD (0.16 mL, 0.81 mmol).
The temperature was maintained at 0°C for 15 min before azidotrimethylsilane
(0.06 mL, 0.45 mmol) was added dropwise. The resulting solution was allowed to
warm to rt over 2 h and stirred for 1 h. The solution was then re-cooled to 0°C and
a solution of methyl (methyl 3-deoxy-D-glycero-ꢀ-D-galacto-2-nonulopyra-
nosid)onate (2) (117 mg, 0.40 mmol) in DMF (4 mL) added. The resulting solution
was slowly warmed to rt and stirred at rt for 3 days. The residue obtained after sol-
vent removal was purified by chromatography as described above to give 13 (69 mg;
54%): [ꢀ]D –21.0° (c 3.28, CHCl3); 1H NMR (D2O) ꢂ 1.83 (1 H, dd, J3ax,3eq 12.8,
J3ax,4 12.4, H-3ax), 2.70 (1 H, dd, J3eq,4 4.5, H-3eq), 3.44 (3H, s, OCH3), 3.59 (2 H,
m, H-9, H-9ꢆ), 3.72 (2 H, m, H-4, H-5), 3.83 (1 H, m, H-7), 3.96 (4 H, m, H-6,
COOCH3), 4.10 (1 H, m, H-8); 13C NMR (D2O) ꢂ 46.2 (C-3), 59.5, 61.1 (OCH3,
COOCH3), 71.9 (C-9), 76.4, 77.0, 77.5, 77.6, 81.8 (C-4, C-5, C-6, C-7, C-8), 107.1
(C-2), 177.8 (carbonyl); LRMS m/z: 322 (MHꢅ, 8%), 290 (100); IR (vmax, KBr):
3460, 2932, 2856, 2104, 1738, 1444, 1382, 1288, 1198, 1066, 1032 cmꢃ1
.
Methyl (Methyl 4,5,7,8-Tetra-O-acetyl-9-azido-3,9-dideoxy-D-glycero-ꢀ-
D-galacto-2-nonulopyranosid)onate (14). Acetylation of the azide 13 (55 mg;
0.17 mmol) according to the general conditions provided, after chromatography
(EtOAc/Hex, 1:1), 14 as a colourless amorphous mass (80 mg; 95%): [ꢀ]D –5.0°
(c 2.09, CHCl3); 1H NMR (CDCl3) ꢂ 1.87 (1 H, dd, J3ax,3eq 13.3, J3ax,4 11.4, H-3ax),
1.96, 1.97, 2.09, 2.15 (each 3 H, s, OAc ꢄ 4), 2.62 (1 H, dd, J3eq,4 4.0, H-3eq), 3.24
(1 H, dd, J9,9ꢆ 13.9, J9,8 4.8, H-9), 3.28 (3H, s, OCH3), 3.57 (1 H, dd, J9ꢆ,8 2.2, H-
9ꢆ), 3.79 (3 H, s, COOCH3), 4.14 (1 H, dd, J6,5 8.8, J6,7 1.3, H-6), 4.83 (1 H, pseudo
t, J5,4 9.7, H-5), 4.86 (1 H, m, H-4), 5.32 (2 H, m, J8,7 7.2, H-7, H-8); 13C NMR
(CDCl3) ꢂ 20.7, 20.9, 21.1 (OCOCH3 ꢄ 4), 37.5 (C-3), 51.0 (C-9), 52.6, 52.9
(OCH3, COOCH3), 67.6, 68.0, 69.2, 69.3, 71.6 (C-4, C-5, C-6, C-7, C-8), 99.1 (C-
2), 167.8, 170 (carbonyls); LRMS m/z: 490 (MHꢅ, 2%), 464 (42), 430 (43), 328
(76), 268 (100); IR (vmax, KBr): 2100, 1744, 1444, 1370, 1282, 1220, 1152, 1114,
1088, 1054, 1002 cmꢃ1
Anal. Calcd for C19H27O12N3: C, 46.63; H, 5.56; N, 8.59. Found: C, 46.87; H
6.00; N, 8.21.
.
Methyl (Methyl 9-Amino-3,9-dideoxy-D-glycero-ꢀ-D-galacto-2-nonu-
lopyranosid)onate (15). Treatment of a mixture of the 9-azide 13 (1.14 g, 3.53
mmol), glacial acetic acid (6 drops) and MeOH (40 mL) under hydrogenolysis

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235
conditions (50 psi, 10% Pd/C, 3.5 days) gave, after chromatography (EtOAc/
MeOH/H2O, 25:15:1), the 9-amine 13 as a colourless amorphous mass (1.04 g;
1
99%): [ꢀ]D –20.8° (c 5.72, MeOH); H NMR (D2O) 1.65 (1 H, dd, J3ax,3eq 12.8,
J3ax,4 12.4, H-3ax), 2.52 (1 H, dd, J3eq,4 4.6, H-3eq), 2.97 (1 H, dd, J9,9ꢆ 13.2, J9,8 9.1,
H-9), 3.25 (3 H, s, OCH3), 3.33 (1 H, dd, J9ꢆ,8 3.1, H-9ꢆ), 3.41 (1 H, pseudo t, J5,4
9.5, J5,6 9.5, H-5), 3.52 (2 H, m, J7,8 6.7, H-7, H-8), 3.65 (1 H, pseudo d, H-6), 3.77
(3 H, s, COOCH3), 3.94 (1 H, m, H-4); 13C NMR (D2O) ꢂ 41.0 (C-3), 45.2 (C-9),
54.4, 56.0 (OCH3, COOCH3), 70.9, 71.7, 72.2, 72.5, 76.7 (C-4, C-5, C-6, C-7, C-
8), 102.0 (C-2), 175.2 (carbonyl); LRMS m/z: 296 (MHꢅ, 100%), 264 (42), 185
(35); IR (vmax, KBr): 3368, 2948, 1740, 1570, 1412, 1342, 1290, 1198, 1068, 1034,
652 cmꢃ1. HRMS: Calcd for C11H21NO8:[Mꢅ ꢅ 1], 296.1346. Found: 296.1342.
Methyl (Methyl 9-Acetamido-4,5,7,8-tetra-O-acetyl-3,9-dideoxy-D-glyc-
ero-ꢀ-D-galacto-2-nonulopyranosid)onate (16). Acetylation of the amine 15
(31 mg, 0.11 mmol) according to the general conditions gave, after chromatogra-
phy (EtOAc), the 9-acetamide 16 as a colourless syrup (36 mg; 68%): [ꢀ]D –17.7°
(c 3.41, CHCl3); 1H NMR (CDCl3) ꢂ 1.91 (1 H, dd, J3ax,3eq 12.9, J3ax,4 11.9, H-3ax),
1.98, 2.01, 2.02, 2.15, 2.17 (each 3 H, s, OAc ꢄ 4, NHCOCH3), 2.67 (1 H, dd, J3eq,4
4.4, H-3eq), 2.99 (1 H, dd, J9,8 4.6, J9,9ꢆ 9.1, J9,NH 6.0, H-9), 3.33 (3 H, s, OCH3),
3.82 (3 H, s, COOCH3), 3.94 (1 H, ddd, J9ꢆ,8 2.9, J9ꢆ,NH 7.4, H-9ꢆ), 4.16 (1 H, dd,
J6,5 9.9, J6,7 2.1, H-6), 4.93 (1 H, m, H-4), 4.98 (1 H, pseudo t, J5,4 10.5, H-5), 5.15
(1 H, dd, J7,8 9.5, H-7), 5.25 (1 H, m, H-8), 5.95 (1 H, br t, NHCOCH3); 13C NMR
(CDCl3) ꢂ 20.6, 20.7, 21.0 (OCOCH3 ꢄ 4), 23.2 (NHCOCH3), 37.4 (C-3), 38.5 (C-
9), 52.4, 52.7 (OCH3, COOCH3), 67.2, 68.0, 68.3, 69.2, 70.9 (C-4, C-5, C-6, C-7,
C-8), 98.8 (C-2), 169.8, 170.2, 171.2 (carbonyls); LRMS m/z: 506 (MHꢅ, 65%),
446 (37), 414 (100), 372 (62), 312 (31); IR (vmax, KBr): 1752, 1678, 1438, 1370,
1220, 1112, 1088, 1052 cmꢃ1
.
Anal. Calcd for C21H31NO13.CHCl3: C, 42.32; H, 5.13; N, 2.25. Found: C,
42.32; H, 5.17; N, 2.20.
Methyl (Methyl 9-Acetamido-3,9-dideoxy-D-glycero-ꢀ-D-galacto-2-
nonulopyranosid)onate (17). Treatment of a solution of peracetylated 9-ac-
etamide 16 (125 mg; 0.370 mmol) in MeOH under standard Zemplen de-O-acety-
lation conditions (NaOMe, 30 min) followed by conventional workup gave the title
compound 17 as a colourless amorphous mass (60 mg; 72%): [ꢀ]D –30.6° (c 6.03,
MeOH); 1H NMR (D2O) 1.65 (1 H, dd, J3ax,3eq 12.8, J3ax,4 12.4, H-3ax), 1.94 (3 H,
s, NHCOCH3), 2.52 (1 H, dd, J3eq,4 4.6, H-3eq), 3.24 (1 H, dd, J9,9ꢆ 14.3, J9,8 7.3,
H-9), 3.26 (3 H, s, OCH3), 3.44 (1 H, dd, J5,4 9.3, J5,6 9.6, H-5), 3.55 (2 H, m, J6,7
0.8, J9ꢆ,8 2.8, H-6, H-9ꢆ), 3.65 (1 H, dd, J7,8 6.0, H-7), 3.68 (1 H, m, H-8), 3.78 (3
H, s, COOCH3), 3.83 (1 H, m, H-4); 13C NMR (D2O) ꢂ 24.6 (NHCOCH3), 41.0 (C-
3), 45.3 (C-9), 54.4, 56.0 (OCH3, COOCH3), 71.7, 71.8, 72.1, 72.3, 76.7 (C-4, C-
5, C-6, C-7, C-8), 102.0 (C-2), 171.3 (carbonyl); LRMS m/z: 338 (MHꢅ, 96%),
306 (100); IR (vmax, KBr): 3348, 2948, 1738, 1638, 1562, 1438, 1374, 1292, 1198,
1072, 984, 880, 782, 650, 454 cmꢃ1. HRMS: Calcd for C13H23NO9:[Mꢅ ꢅ 1],
338.1451. Found: 338.1443.

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NORTON, KOK, AND VON ITZSTEIN
Methyl (Methyl 9-Benzamido-3,9-dideoxy-D-glycero-ꢀ-D-galacto-2-
nonulopyranosid)onate (18). A solution of the 9-amine 15 (91 mg; 0.308
mmol) and freshly distilled Et3N (1.2 mL) in MeOH (10 mL) was cooled to ꢃ70°C
prior to the addition of benzoyl chloride (0.5 mL). After 35 min, the solution was
warmed to rt and concentrated. Chromatography (EtOAc) gave the title compound
1
18 (84 mg; 68%): [ꢀ]D –10.4° (c 7.08, MeOH); H NMR (D2O) 1.88 (1 H, dd,
J3ax,3eq 12.9, J3ax,4 11.9, H-3ax), 2.74 (1 H, dd, J3eq,4 3.5, H-3eq), 2.97 (1 H, dd, J9,8
9.1, J9,9ꢆ 13.2, H-9), 3.48 (3 H, s, OCH3), 3.72 (4 H, m, H-5, H-6, H-9, H-9ꢆ), 3.94
(2 H, m, H-7, H-8), 3.99 (3 H, s, COOCH3), 4.17 (1 H, m, H-4), 7.66 – 7.73 (5 H,
m, Ph); 13C NMR (D2O) ꢂ 41.4 (C-3), 46.2 (C-9), 54.7, 56.3 (OCH3, COOCH3),
72.2, 72.3, 72.6, 72.7, 77.1 (C-4, C-5, C-6, C-7, C-8), 102.3 (C-2), 130.1, 131.8,
135.1, 136.6 (aromatic carbons), 173.0 (carbonyl); LRMS m/z: 400 (MHꢅ, 11%),
367 (79) no base peak was observed; IR (vmax, KBr): 3420, 2948, 1732, 1632,
1576, 1544, 1480, 1450, 1290, 1196, 1070, 984, 780, 710, 656 cmꢃ1. HRMS:
Calcd for C18H25NO9:[Mꢅ ꢅ 1], 400.1608. Found: 400.1613.
Methyl (Methyl 4,5,7,8-Tetra-O-acetyl-9-benzamido-3,9-dideoxy-D-glyc-
ero-ꢀ-D-galacto-2-nonulopyranosid)onate (19). According to the general con-
ditions, acetylation of 18 (84 mg, 0.21 mmol) gave, after chromatography (EtOAc),
the title compound 19 as a colourless solid (71 mg; 92%): [ꢀ]D ꢅ4.2° (c 4.35,
CHCl3); 1H NMR (CDCl3) ꢂ 1.91 (1 H, dd, J3ax,3eq 12.9, J3ax,4 12.1, H-3ax), 2.01 (6
H, s, OAc ꢄ 2), 2.01, 2.15 (each 3 H, s, OAc ꢄ 2), 2.69 (1 H, dd, J3eq,4 4.6, H-3eq),
3.12 (1 H, dd, J9,8 8.7, J9,9ꢆ 15.0, J9,NH 4.5, H-9), 3.33 (3 H, s, OCH3), 3.81 (3 H, s,
COOCH3), 4.17 (1 H, dd, J6,5 9.7, J6,7 2.2, H-6), 4.24 (1 H, ddd, J9ꢆ,8 2.9, J9ꢆ,NH 7.5,
H-9ꢆ), 4.92 (1 H, m, H-4), 4.98 (1 H, pseudo t, J5,4 9.6, H-5), 5.22 (1 H, dd, J7,8 9.7,
H-7), 5.36 (1 H, m, H-8), 6.86 (1 H, br dd, NH), 7.46 – 7.79 (5 H, m, Ph); 13C NMR
(CDCl3) ꢂ 20.8, 20.9, 21.0, 21.3 (OCOCH3 ꢄ 4), 37.7 (C-3), 39.2 (C-9), 52.6, 52.9
(OCH3, COOCH3), 67.8, 68.3, 68.6, 69.5, 71.2 (C-4, C-5, C-6, C-7, C-8), 99.1 (C-
2), 127.1, 128.7, 131.6, 134.7 (aromatic carbons), 167.6, 167.8, 170.1, 170.7, 171.8
(carbonyls); LRMS m/z: 569 (MHꢅ, 20%), 507 (48), 475 (100); IR (vmax, KBr):
1756, 1636, 1536, 1436, 1372, 1246, 1116, 1054, 990, 702 cmꢃ1
.
Anal. Calcd for C26H33NO13.1.5 H2O: C, 52.51; H, 6.11; N, 2.35. Found: C,
52.38; H, 5.85; N, 2.05.
Methyl (Methyl 3,9-Dideoxy-D-glycero-ꢀ-D-galacto-2-nonulopyra-
nosid)onate (20). A solution of the 9-iodide 10 (23 mg, 0.058 mmol) and glacial
acetic acid (2 drops) in MeOH (2 mL) was subjected to hydrogenolysis conditions
(50 psi H2, 10% Pd/C) until complete disappearance of starting material (as moni-
tored by TLC; EtOAc/MeOH, 10:1). The solids were then filtered off and filtrate
concentrated. The resulting residue was chromatographed (EtOAc/MeOH, 20:1)
which gave the title compound 20 (16 mg; 99%): [ꢀ]D –39.8° (c 2.82, CHCl3); 1H
NMR (D2O) ꢂ 1.25 (3 H, d, J9,8 6.3, H-9 ꢄ 3), 1.69 (1 H, dd, J3ax,3eq 12.9, J3ax,4
12.4, H-3ax), 2.57 (1 H, dd, J3eq,4 4.6, H-3eq), 3.30 (3H, s, OCH3), 3.50 (4 H, m, H-
5, H-6, H-7, H-8), 3.82 (3 H, s, COOCH3), 3.92 (1 H, m, H-4); 13C NMR (D2O) ꢂ
25.0 (C-9), 44.4 (C-3), 57.7, 59.4 (OCH3, COOCH3), 73.4, 75.3, 75.9, 78.1, 80.2

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237
(C-4, C-5, C-6, C-7, C-8), 105.3 (C-2), 176.1 (carbonyl); LRMS m/z: 281 (MHꢅ,
4%), 249 (100); IR (vmax, KBr): 3508, 2944, 1736, 1644, 1448, 1290, 1200, 1078
cmꢃ1
.
Methyl (Methyl 4,5,7,8-Tetra-O-acetyl-3,9-dideoxy-D-glycero-ꢀ-D-
galacto-2-nonulopyranosid)onate (21). Acetylation of 20 (47 mg, 0.17 mmol)
according to the general conditions gave, after chromatography (EtOAc/hexane,
1:1) 21 as a colourless solid (64 mg; 86%): [ꢀ]D ꢃ17.2° (c 5.42, CHCl3); 1H NMR
(CDCl3) ꢂ 1.20 (3 H, d, J9,8 6.2, H-9 ꢄ 3), 1.88 (1 H, dd, J3ax,3eq 12.6, J3ax,4 11.9,
H-3ax), 1.99 (6 H, s, OAc ꢄ 2), 2.10, 2.11 (each 3 H, s, OAc ꢄ 2), 2.65 (1 H, dd,
J3eq,4 3.6, H-3eq), 3.31 (3 H, s, OCH3), 3.81 (3 H, s, COOCH3), 4.15 (1 H, dd, J6,5
8.2, J6,7 1.7, H-6), 4.91 (2 H, m, J4,5 8.3, H-4, H-5), 5.14 (1 H, dd, J7,8 8.7, H-7),
5.27 (1 H, m, H-8); 13C NMR (CDCl3) ꢂ 16.6 (C-9), 20.6, 20.7, 21.4 (OCOCH3 ꢄ
4), 37.4 (C-3), 52.3, 52.6 (OCH3, COOCH3), 66.8, 68.1, 69.3, 70.2, 71.1 (C-4, C-
5, C-6, C-7, C-8), 98.8 (C-2), 170.0 (carbonyls); LRMS m/z: 449 (MHꢅ, 4%), 389
(37), 357 (100); IR (vmax, KBr): 1750, 1370, 1236, 1092 cmꢃ1
.
Anal. Calcd for C19H28O12: C, 50.89; H, 6.29. Found: C, 50.70; H, 6.30.
ACKNOWLEDGMENTS
This work was supported by an Australian Postgraduate Award to AKN and
by the Australian Research Council.
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Received October 10, 2000
Accepted November 25, 2000

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