Modelling, synthesis and biological evaluation of novel glucuronide-based probes of Vibrio cholerae sialidase

Bioorganic & Medicinal Chemistry 14 (2006) 1518–1537

Modelling, synthesis and biological evaluation of novel

glucuronide-based probes of Vibrio cholerae sialidase

Maretta C. Mann, Robin J. Thomson, Jeﬀrey C. Dyason,

Sarah McAtamney and Mark von Itzstein^*

Institute for Glycomics, Griﬃth University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Qld 9726, Australia

Received 31 August 2005; revised 3 October 2005; accepted 3 October 2005

Available online 4 November 2005

Abstract—The development of sialidase inhibitors is an area of continuing interest due to their potential use as therapeutic agents to

combat viral and bacterial infections. Herein, we report our studies involving the sialidase from the pathogen Vibrio cholerae,

through the modelling, synthesis and biological evaluation of mimetics of 5-acetamido-2,6-anhydro-3,5-dideoxy-^D-glycero-D-galac-

to-non-2-enonic acid (Neu5Ac2en, 1), a naturally occurring sialidase inhibitor. These mimetics are O- and S-glycosides of N-acetyl-

D-glucosaminuronic acid in which the aglycone portion eﬀectively replaces the C-6 glycerol side chain of Neu5Ac2en (1). The choice

of aglycones was aided by use of the X-ray crystal structure of V. cholerae sialidase complexed with Neu5Ac2en (1). All Neu5Ac2en

mimetics tested were found to inhibit V. cholerae sialidase as determined using a standard ﬂuorometric assay.

1. Introduction

sialidase inhibition.⁵In comparison, there has been little

research eﬀort directed towards the development of bac-

terial sialidase inhibitors.⁶

Sialic acids are a family of nine-carbon sugars, which are

often found as the terminal components of cell-surface

glycoconjugates. As such, they are well positioned for

involvement in many molecular and cellular recognition

events, generally by either acting as receptors or by eﬀec-

tively masking recognition sites.^1,2The biological impor-

tance of sialic acids has been conﬁrmed by the discovery

of a wide range of proteins that recognise them. Sialid-

ases form a widely studied group of sialic acid-recognis-

ing proteins, which catalyse the release of sialic acids

from glycoconjugates by hydrolysis of the sialosyl glyco-

sidic bond.³Sialidases from mammalian sources are

important for catabolism of sialic acid-containing mole-

cules and modiﬁcation of receptors to alter cellular func-

tion. A number of pathogenic microorganisms produce

sialidases, despite the fact that they do not biosynthesise

sialic acids themselves.^3,4This has led to the discovery of

their role in the pathogenesis of various microbial dis-

eases. In recent years, sialidases from viral sources have

been the subject of intense research, leading to the struc-

ture-based discovery of therapeutic agents that act by

Vibrio cholerae sialidase is considered a potential drug

target for therapeutic agents against cholera. The siali-

dase, secreted only by epidemic strains of the bacterium

V. cholerae,⁷is believed to play an important role in

infection. As part of a multi-enzyme mucinase complex,⁸

the sialidase may facilitate the cholera toxinꢀs entry into

epithelial cells⁹by reducing the viscosity of the gastroin-

testineꢀs protective mucosal coat.¹⁰V. cholerae sialidase

also cleaves sialic acids from higher order gangliosides

to form GM₁,¹¹the receptor for cholera toxin on epithe-

lial cells,¹²thereby increasing the number of available

receptor sites to which the toxin may bind. V. cholerae

sialidase is readily expressed¹³and puriﬁed,¹⁴and the

X-ray crystal structure with 5-acetamido-2,6-anhydro-

3,5-dideoxy-^D-glycero-D-galacto-non-2-enonic

acid

(Neu5Ac2en, 1), a natural inhibitor, bound in the cata-

15

˚

lytic site, has been solved to 1.9 A resolution. Thus,

V. cholerae sialidase is an ideal representative bacterial

sialidase for the development of inhibitors.

Research within our group has been recently directed to-

wards the preparation of carbohydrate-based mimetics

of Neu5Ac2en for sialidase inhibition. We have

described in preliminary reports^16,17the synthesis of

Keywords: Sialidase; Carbohydrate; Glucuronide; Inhibitors; Vibrio

cholerae.

*

Corresponding author. Tel.: +61 7 5552 7025; fax: +61 7 5552

9040; e-mail: m.vonitzstein@griﬃth.edu.au

doi:10.1016/j.bmc.2005.10.004

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1519

C-6 ether mimetics of Neu5Ac2en of the general struc-

ture 2 from N-acetylglucosamine, that contain diﬀerent

O-linked alkyl groups that eﬀectively replace the glycer-

ol side chain of Neu5Ac2en (1). During the preparation

of Neu5Ac2en mimetics represented by 2, we have devel-

oped an eﬃcient synthetic strategy that allows for rapid

access to a wide range of mimetics from a common, late

stage intermediate.¹⁶In a preliminary screen ethyl and

3-pentyl derivatives 2a and 2b, respectively, prepared

by this method were shown to inhibit V. cholerae siali-

dase.¹⁶We have now completed a more extensive inves-

tigation of a wider range of both C-6 ether and thioether

Neu5Ac2en mimetics 2 and 3, respectively, and their

biological evaluation against V. cholerae sialidase using

a ﬂuorometric enzyme assay.

H

OH

COOH

O

HO

R

H

HO

OH

1 Neu5Ac2en; R = NHAc

COOH

O

R

AcHN

X

OH

Figure 1. Neu5Ac2en (1) in the active site of V. cholerae sialidase

generated by energy-minimisation¹⁸of the X-ray crystal structure.¹⁵

2 X = O; R = alkyl

2a X = O; R = CH₂Me

2b X = O; R = CH(CH₂Me)₂

beyond the end of the glycerol side-chain pocket and

could potentially form favourable interactions, particu-

larly with hydrophobic groups that are of similar or

slightly larger size compared with the glycerol side chain

of Neu5Ac2en (1). This preliminary molecular model-

ling study suggested that Neu5Ac2en mimetics that con-

tain either hydrophobic or hydrophilic aglycones could

be accommodated in the active site of V. cholerae siali-

dase. With this information in hand, we aimed to pre-

pare a range of glucuronide-based derivatives 2 and 3

that contained diﬀerent functional groups (R), to probe

the glycerol side-chain pocket of V. cholerae sialidase.

3 X = S; R = alkyl

2. Results and discussion

2.1. Vibrio cholerae sialidase active site

The X-ray crystal structure¹⁵of the complex between

Neu5Ac2en (1) and V. cholerae sialidase was subjected

to an energy-minimisation protocol as previously de-

scribed.¹⁸Neu5Ac2en (1) and selected active site resi-

dues in the energy-minimised complex are depicted in

Figure 1. The interactions observed in the energy-mini-

mised complex suggested that there was little change

in the relative positions of the catalytic site amino acids

and Neu5Ac2en (1) compared with those reported¹⁵for

the X-ray crystal structure. The C-5 N-acetyl group of

Neu5Ac2en (1) is located in a hydrophobic pocket

formed by Trp311, Asn318, Asn545, Gln317 and

Pro251 residues. The hydrophobic residues Phe638 and

Leu580 encase the C-8/C-9 region, whilst Asp637 has

the potential to form a hydrogen bond to O-8 of the

glycerol side chain of 1. The triarginyl cluster (residues

Arg224, Arg635 and Arg712) is well placed for strong

interactions with the carboxyl group of Neu5Ac2en

(1). Hydrogen bonding is possible between the ring

hydroxyl group and both Arg245 and Asp250.

2.2. Chemistry

The general approach used for the synthesis of both C-6

ether and thioether Neu5Ac2en mimetics 2 and 3,

respectively, is outlined in retrosynthetic terms in

COOH

O

R

AcHN

O

X

HO

R

NHAc

OH

2 X = O; R = alkyl

3 X = S; R = alkyl

OH

O

COOMe

O

PivO

HO

In the region of the glycerol side chain, active site resi-

dues including Asp637, Ser618, Asn318, Glu619 and

Gln317 have the potential to form hydrogen bonds with

hydroxyl groups of Neu5Ac2en mimetics containing

hydrophilic glycerol side-chain mimics. Alternatively,

hydrophobic residues Phe638 and Leu580 are located

OH

NHAc

6 (GlcNAc)

XR

PivO

NHAc

4 X = O; R = alkyl

5 X = S; R = alkyl

Scheme 1.

1520

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

Table 1. b-Glycosidation of 7 with various alcohols

Scheme 1. O- and S-Glycosides 4 and 5, respectively, of

protected N-acetylglucosaminuronic acid were prepared

from readily available N-acetylglucosamine (GlcNAc, 6).

Product

Aglycone

Isolated yield

of 4 (%)

Yield (%) based

on recovered

a-7 and 8

4a^c

4b^c

64

80

2.3. O-linked Neu5Ac2en mimetics

O

The method used for the preparation of C-6 ether

Neu5Ac2en mimetics 2 is shown in Scheme 2. The piva-

loylated glucosaminuronate 7 was made in 5 steps and

58% yield from GlcNAc (6) (see Section 3 for full de-

tails).¹⁶A series of 11 O-glycosides 4a–4k (Table 1)

was prepared by TMSOTf-promoted glycosidation of

the pivaloylated glucosaminuronate 7 in 1,2-dichloro-

ethane (DCE). Conversion, determined from recovered

starting material 7 (a-anomer) and oxazoline 8, ranged

from 65% to 100%. While isolated yields were in the

range of 42–78%, the corrected yields, based on recov-

ered starting material 7 (a-anomer) and oxazoline 8,

ranged from 60% to 94%. In reactions in which the

acceptor alcohol contained an isopropylidene-protecting

group (i.e., formation of 4i and 4j), TLC analysis indi-

cated the presence of polar by-products, suggesting that

there may have been some partial loss of the isopropy-

lidene-protecting groups in the presence of TfOH.

78^a

94

O

4c^c

4d^c

4e

53

77

44

82

88

61

O

OH

O

OH

4f

42

66

OH

9

>

4g

>

=

O

63^b

64

70

81

>

;

OH

4h

4i^c

O

OH

OR

O

a

HO

PivO

OH

NHAc

6 (GlcNAc)

OPiv

NHAc

R = Tr

O

b

4j

47

60

68

R = H

O

c, d

O

4k

COOMe

O

COOMe

O

PivO

^aSome co-elution with unreacted a-7 during chromatographic

puriﬁcation.

^bCombined yield of a 3:2 mixture of 4g and 4h.

f

PivO

OPiv

O

NHAc

N

^cRef. 16.

7

8

Me

e

g

Conversion of 7 to the isobutyl b-glycoside 4d was

also carried out over two steps with isolation of the

intermediate oxazoline 8 (Scheme 2). Oxazoline 8

was isolated in 90% yield (based on recovered starting

material) by reaction of 7 with TMSOTf¹⁹over 3 d,

followed by quenching of the reaction with triethyl-

amine. O-Glycosidation of oxazoline 8 with isobutyl

alcohol was carried out in the presence of catalytic

TMSOTf²⁰at 60 ꢁC to give the b-isobutyl glycoside

4d in 72% yield. The 65% yield of this glycoside over

two steps from 7 was signiﬁcantly lower than the 88%

yield (based on recovered a-7 and 8) from the one-pot

reaction.

COOR'

O

COOMe

O

h

PivO

OR

NHAc

4 R = alkyl

4d R = CH₂CHMe₂

R''O

NHAc

9 R = alkyl; R' = Me; R'' = Piv

2 R = alkyl; R', R'' = H

i

Piv = trimethylacetyl (pivaloyl)

Scheme 2. Reagents and conditions: (a) i—TrCl, pyridine, ꢀ100 ꢁC,

20 min; ii—PivCl, DMAP, 0 ꢁC ! rt, 6 d (83%); (b) 80% aq AcOH,

65 ꢁC, 1 h (92%); (c) TEMPO, KBr, Bu₄NBr, 10–15% aq NaOCl, aq

NaHCO₃, aq NaCl, CH₂Cl₂, rt, 75 min; (d) SOCl₂, CH(OMe)₃,

MeOH, rt, 30 min (76% over 2 steps); (e) i—TMSOTf, DCE, 50 ꢁC, 3

O-Glycosidation of 7 with propan-1,2-diol gave a 3:2

mixture of the primary alcohol and the secondary alco-

hol glycosidation products 4g and 4h, respectively, in

70% yield based on recovered a-7. These compounds

proved to be inseparable by ﬂash chromatography,

and so selective derivatisation of 4h in the mixture was

˚

d; ii—3 A molecular sieves, rt, 30 min; iii—ROH, 24 h, rt (60–94%,

based on recovered a-7 or 8); (f) TMSOTf, DCE, 50 ꢁC, 3 d (90%,

based on recovered a-7); (g) i-BuOH, TMSOTf, DCE, 60 ꢁC, 24 h

(72%); (h) DBU, CH₂Cl₂, rt, 18 h (80–99%); (i) 50% aq MeOH, aq

NaOH, pH 13, 18 h (70–87%).

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1521

carried out. Selective acetylation of the primary hydrox-

yl group of 4h was accomplished using AcCl and i-

Pr₂EtN in CH₂Cl₂to give 4h-Ac in 95% yield. The

COOH

O

21

OR

HO

acetylated derivative 4h-Ac was easily separated from

unreacted 4g by chromatography. Other glycosides con-

taining a free hydroxyl group in their aglycone portion

(4e, 4f and 4g) were also acetylated, due to complica-

tions experienced with treatment of an unprotected

derivative with DBU in the subsequent b-elimination

step. Acetylation was achieved using Ac₂O in pyridine

to give the corresponding acetates 4e-Ac, 4f-Ac and

4g-Ac.

NHAc

2a R = CH₂Me

2b R = CH(CH₂Me)₂

2c R = CHMe₂

2d R = CH₂CHMe₂

2e R = CH₂CH₂OH

2f R = CH₂CH₂CH₂OH

2g R = CH₂CH(OH)Me

2h R = CH(Me)CH₂OH

2i R = CH₂CH(OH)CH₂OH

COOMe

O

PivO

OR

2.4. S-linked Neu5Ac2en mimetics

NHAc

4e-Ac R = CH₂CH₂OAc

We have had a long-standing interest in the synthesis of

thioglycosides of various carbohydrates.^22,23We thought

it of value to utilise this expertise to investigate the possi-

ble inﬂuence of replacement of oxygen with sulfur on the

biological activity of these mimetics. Synthesis of thioe-

ther mimetics of the general structure 3 (Scheme 3) was

achieved via the intermediate b-S-glucosaminuronides

5, which in turn were prepared from the b-thiolacetate

derivative 10. Treatment of oxazoline 8 with thiolacetic

acid in DMF at 80 ꢁC²⁴provided the thiolacetate 10 in

64% yield after chromatography. A characteristic ano-

meric S-acetyl resonance was observed at d2.34 in the

¹H NMR spectrum of 10, and the J_1,2coupling

(10.3 Hz) was consistent with a b-1-thiolacetate of a 1,2-

trans-conﬁgured sugar.²²

4f -Ac R = CH₂CH₂CH₂OAc

4g-Ac R = CH₂CH(OAc)Me

4h-Ac R = CH(Me)CH₂OAc

The ﬁnal stages in the preparation of C-6 ether Neu5-

Ac2en mimetics 2 involved b-elimination of the O-glyco-

sides 4 to give the 4,5-unsaturated derivatives 9,

followed by deprotection (Scheme 2). Treatment of com-

pounds 4a-4d, 4e-Ac-4h-Ac and 4i with DBU in CH₂Cl₂

aﬀorded the 4,5-unsaturated derivatives 9a–9i in 80–99%

1

yield after chromatography. H NMR spectroscopy of

9a–9i showed oleﬁnic H-4 resonances at d ꢀ 6.2 and

the absence of resonances for H-5.

With the b-1-thiolacetate 10 in hand, the synthesis of

thioglycosides was attempted using HNEt₂to generate

COOMe

O

OR

NHAc

9a R = CH₂Me

COOMe

O

PivO

COOMe

O

COOMe

O

NHAc

9i

PivO

a

PivO

SAc

9b R = CH(CH₂Me)₂

9c R = CHMe₂

NHAc

O

N

10

8

9d R = CH₂CHMe₂

9e R = CH₂CH₂OAc

9f R = CH₂CH₂CH₂OAc

9g R = CH₂CH(OAc)Me

9h R = CH(Me)CH₂OAc

Me

b

COOMe

O

PivO

9i-OH R = CH₂CH(OH)CH₂OH

SR

NHAc

5

Deprotection of the 4,5-unsaturated derivatives 9 was

then carried out to give a series of C-6 ether Neu5A-

c2en mimetics 2. Acid-catalysed hydrolysis of the iso-

propylidene group in 9i (50% aq TFA, 0 ꢁC) aﬀorded

9i-OH in 78% yield after chromatography. Base-cataly-

sed deacylation and de-esteriﬁcation of each of the

unsaturated derivatives 9a–9h and 9i-OH were

achieved at pH 13 using aqueous NaOH, with TLC

analysis indicating a clean conversion to the ﬁnal prod-

ucts within 18 h (Scheme 2). Puriﬁcation by HPLC

aﬀorded the ﬁnal derivatives 2a–2i in 70–87% yield.

Thus, a series of C-6 ether Neu5Ac2en mimetics 2a–

2i were prepared in 14–41% yield over 8–9 steps from

GlcNAc (6).

c

COOR'

O

COOMe

O

SR

R''O

PivO

NHAc

AcHN

SR

α

β-11 R = alkyl; R' = Me; R'' = Piv

3 R = alkyl; R', R'' = H

-11 R = alkyl

d

Scheme 3. Reagents and conditions: (a) HSAc, DMF, 80 ꢁC, 18 h

(64%); (b) alkyl halide, HNEt₂, DMF, ꢁ20 ꢁC-rt, 4–48 h (57–82%); (c)

DBU, CH₂Cl₂, rt, 18 h (90–100%, based on recovered b-5); (d) 50% aq

MeOH, aq NaOH, pH 13, rt, 18 h (70–85%).

1522

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

Table 2. Diethylamine-mediated coupling between b-1-thiolacetate 10 and diﬀerent alkyl halides (R-X) to give thioglycosides 5

Entry

Alkyl halide coupling partner

Reaction conditions

Temperature (ꢁC) Time (h)

Thioglycoside product

S–R Yield (%)

R-X

X

Equivalent

Product

a/b ratio

1

2

3

4

Br

1.5

5

rt

4

48

4

78

57

69

74

1:1

1:3

ꢁ20

rt

X

OH

S

OH

5a

1:8

Only b

5

4

24

5

6

Br

1.5

5

rt

4

24

82

76

1:1

1:8

5b

5c

OH

7

8

Br

I

5

4

24

77

73

1:1

1:9

X

S

9

10

11

Br

I

1.5

5

rt

4

65

72

73

Only a

1:2

5d

I

24

the intermediate 1-thiolate anion (Scheme 3).²³Origi-

nally developed for the synthesis of thioglycosides of

N-acetylneuraminic acid,²³this method has since been

ondary alkyl halide, the use of 5 equivalents of 2-

iodopropane at lower temperature was required to pro-

duce any of the b-thioglycoside 5d (Entry 11).

used to prepare thioglycosides of mannopyranose,²⁵

26

lactose

and various glycosylsulfenamides.²⁷The

The formation of anomeric mixtures of thioglycosides

during the one-pot de-S-acetylation and coupling of a

b-1-thiolacetate has only been reported for reactions

of mannose.²⁵Consistent with our observations, Bun-

dle and co-workers²⁵obtained anomeric mixtures of

thiomannopyranosides from HNEt₂-mediated coupling

between a b-1-thiolacetate of mannose and various

acceptors when the reactions were done at room tem-

perature. Lowering the reaction temperature and using

a more reactive coupling partner increased the forma-

tion of b-thiomannopyranosides. In the present work,

it is presumed that the presence of the electronegative

C-6 ester moiety in the glucuronic acid precursor 10

enhances the thermodynamic preference for the forma-

tion (by mutarotation) of the a-anomer of the inter-

mediate thiolate compared with the equivalent

glucose derivative. However, lowering the reaction

temperature, increasing the concentration of coupling

partner, or increasing the reactivity of the coupling

partner drives the formation of the kinetically fa-

voured b-thioglycoside product (b-5).

method has been reported to be consistently stereo-

selective, except in the synthesis of thiomannopyrano-

sides where anomeric mixtures of the products were

observed.²⁵According to the published procedure,²³

a

solution of b-1-thiolacetate 10 in DMF containing 1.5

equivalents of 3-bromopropan-1-ol was treated with

HNEt₂at room temperature and monitored by TLC.

Work-up of the reaction mixture after 4 h gave the

thioglycoside product 5a in 78% yield after chromatog-

raphy (Table 2, Entry 1). Unexpectedly, analysis by

NMR spectroscopy revealed that the product was a

mixture of two components in approximately 1:1 ratio.

The two components were identiﬁed as the a- and b-

anomers of the 3-hydroxypropyl thioglycoside 5a. In

the ¹H NMR spectrum of the mixture, doublets at

d4.58 (J 10.3 Hz) and at d5.48 (J 5.1 Hz) were assigned

to H-1 of the b- and a-anomers of 5a, respectively

(conﬁrmed by a COSY experiment). The signal for

H-5 of the a-anomer at d4.70 (d, J 9.0 Hz) was down-

ﬁeld compared to the H-5 signal for the b-anomer at

d4.01 (d, 9.5 Hz), which is consistent with an anomeric

mixture of 2-deoxy-2-N-thioglucopyranosides.²⁸Analy-

sis by LRMS showed a single ion (m/z 514) corre-

sponding to [M+Na]⁺of 5a. In subsequent

investigations (Table 2), it was found that lowering

the reaction temperature (Entry 2), or increasing the

amount of coupling partner (Entry 3), increased the

proportion of b-thioglycoside b-5a. The combination

of reduced temperature (4 ꢁC) and increased amount

of coupling partner (5 equivalents) produced the de-

sired result (Entry 4), giving pure b-thioglycoside prod-

uct 5a. Similar results were obtained during the

synthesis of 5b from coupling between 10 and 2-bro-

moethan-1-ol (Entries 5 and 6). Interestingly, reaction

of 5 equivalents of isobutyl bromide with the b-1-thio-

lacetate 10 at 4 ꢁC (Entry 7) gave a 1:1 mixture of ano-

mers. Use of the more reactive isobutyl iodide was

required to produce a higher ratio of the desired isobu-

tyl b-thioglycoside b-5c (Entry 8). In the case of a sec-

Thioglycosides 5 were treated with DBU in CH₂Cl₂

(Scheme 3) to give the corresponding unsaturated

derivatives 11 in 75–90% isolated yield or higher yield

(90–100%) based on recovered thioglycoside. The

b-thioglycosides, b-5, appeared to be less reactive than

the a-thioglycosides, a-5, as seen by the recovery of a

small amount of b-thioglycoside b-5 after chromatogra-

phy. The hydroxyl groups of compounds 5a and 5b were

acetylated (Ac₂O/pyridine), to give 5a-Ac and 5b-Ac,

respectively, prior to b-elimination, due to complica-

tions that were encountered after treating an unprotect-

ed derivative with DBU. The a- and b-anomers of the

4,5-unsaturated thioglycoside products 11a–11d were

easily separated from each other by ﬂash chromatogra-

phy due to a diﬀerence in R_fof ꢀ0.2. In each case, the

1

least mobile component was identiﬁed using H NMR

spectroscopy to be the desired unsaturated b-thioglyco-

side. Base-catalysed deprotection of each of the b-ano-

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1523

Table 3. Inhibitory activity of Neu5Ac2en mimetics determined

against V. cholerae sialidase

COOMe

O

COOMe

O

PivO

% Inhibition^a,b(1 mM) K_iestimate (M)

SR

Mimetic X-R

Neu5Ac2en

O

PivO

NHAc

1

97

71

3 · 10^ꢁ5

4 · 10^ꢁ4

11a R = CH₂CH₂CH₂OAc

11b R = CH₂CH₂OAc

11c R = CH₂CHMe₂

11d R = CHMe₂

5a-Ac R = CH₂CH₂CH₂OAc

5b-Ac R = CH₂CH₂OAc

2a

O

2b

2c

3d

2d

3c

2e

3b

3a

2g

2h

2i

90

75

85

82

75

64

70

55

79

57

1 · 10^ꢁ4

3 · 10^ꢁ4

2 · 10^ꢁ4

3 · 10^ꢁ4

6 · 10^ꢁ4

4 · 10^ꢁ4

8 · 10^ꢁ4

3 · 10^ꢁ4

8 · 10^ꢁ4

COOH

O

S

SR

HO

NHAc

3a R = CH₂CH₂CH₂OH

3b R = CH₂CH₂OH

3c R = CH₂CHMe₂

3d R = CHMe₂

O

S

mers of the unsaturated thioglycosides 11a–11d was car-

ried out in a manner similar to the deprotection of the

O-glycosides to give the ﬁnal derivatives 3a–3d in 70–

85% yield after HPLC puriﬁcation.

O

2.5. Biological evaluation

OH

Sialidase activity was assessed using a ﬂuorometric

assay, which was based upon a method developed by

Potier et al.²⁹and measures the hydrolysis of 4-methyl-

S

OH

umbelliferyl

5-acetamido-3,5-dideoxy-^D-glycero-a-D-

S

OH

galacto-non-2-ulopyranosidonic acid (MUN).^29,30To

investigate the nature of the inhibition of the Neu5-

Ac2en mimetics, Neu5Ac2en (1) and the S-isopropyl

derivative 3d were, separately, pre-incubated with V.

cholerae sialidase at 37 ꢁC for 0, 30 or 80 min prior to

the addition of MUN (at a ﬁnal concentration of

50 lM), followed by a further 20 min incubation period.

It was found that the maximum inhibition was achieved

when the enzyme and inhibitor were incubated for

30 min prior to MUN addition. This suggested that both

Neu5Ac2en (1) and the S-isopropyl derivative 3d may

have slow-binding characteristics.³¹Neu5Ac2en mimet-

ics 2a–2e, 2g–2i and 3a–3d were assessed for inhibition

against V. cholerae sialidase (see Section 3 for details),

and the results are shown in Table 3. The K_iestimates

were calculated based on inhibition observed at an

inhibitor concentration of 1 mM (i.e., [I] = 1 mM), using

the following equation:³²

OH

O

OH

O

OH

^aAll assays were performed in duplicate or triplicate.

^bNeu5Ac2en (1) was included in every assay for comparison.

(1) in the present study is comparable to that reported

in the literature.^17,33–37All Neu5Ac2en mimetics tested

showed similar inhibitory activity, being approximately

one order of magnitude less potent compared with

Neu5Ac2en (1). Interestingly, derivatives that contain

a hydrophobic side chain were generally found to be

more potent compared to derivatives with more hydro-

philic side chains. The S-linked derivatives 3c (i-Bu),

3d (i-Pr) and 3b (2-OH-Et) were of similar potency to

the O-linked derivatives containing the same aglycone

moiety 2d (i-Bu), 2c (i-Pr) and 2e (2-OH-Et), respective-

ly. This observed similarity in potency is not surprising

considering that the thioether and ether derivatives

adopt a similar ring conformation in solution. This ring

conformation is diﬀerent to the conformation adopted

by Neu5Ac2en (1)^38,39in which all substituents are in

an equatorial position.^38,39A Neu5Ac2en-like ring con-

formation is required for binding to both inﬂuenza virus

sialidase⁴⁰and V. cholerae sialidase.¹⁵The magnitude of

%Inhibition ¼ ½Iꢂ=f½Iꢂ þ K_ið1 þ ½Sꢂ=K_mÞg;³²

where [I] is the inhibitor concentration, K_iis the inhibi-

tion constant, [S] is the substrate concentration and K_m

is the Michaelis–Menten constant.

In previous studies, inhibition for Neu5Ac2en mimetics

and derivatives was determined to be of a linear compet-

itive type.^17,33The K_mfor V. cholerae sialidase using

MUN as the substrate was determined to be 1.5 mM.

The ﬂuorometric assay represented a ﬁrst screen of our

series of C-6 modiﬁed Neu5Ac2en mimetics and

provided an estimation of inhibitory activity against

V. cholerae sialidase compared with Neu5Ac2en (1).

The estimated K_ivalue (3 · 10^ꢁ5M) for Neu5Ac2en

1

the coupling constants in the H NMR spectra of the

1524

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

presently described series of C-6 modiﬁed Neu5Ac2en

mimetics, and in particular their small J_2,3values, is

characteristic⁴¹of a ¹H₂conformation in which the sub-

stituents are quasi-axial. It is possible that there is an

energy penalty incurred on binding of the C-6 ether

and C-6 thioether Neu5Ac2en mimetics to V. cholerae

sialidase in the appropriate Neu5Ac2en-like half-chair

conformation, which results in weaker inhibitory activi-

ty compared with Neu5Ac2en (1). A similar rationale

was proposed by Smith et al.⁴²to explain the marked

diﬀerences in inhibition potency between structurally

similar 4-amino-4-deoxy-Neu5Ac2en mimetics.⁴²

3.2. Methyl 2-acetamido-2-deoxy-1,3,4-tri-O-pivaloyl-a,b-

D-glucopyranuronate (7) from GlcNAc (6)

A stirred suspension containing anhyd GlcNAc (6)

(4.0 g, 18 mol), TrCl (6.0 g, 22 mmol) and DMAP

(50 mg, 0.5 mmol) in anhyd pyridine (50 mL) under an

atmosphere of N₂was warmed to 100 ꢁC and monitored

by TLC analysis (EtOAc/MeOH/H₂O 7:2:1). After

30 min, the reaction mixture was allowed to cool to rt,

followed by addition of PivCl (8.0 mL, 65 mmol). The

reaction was monitored by TLC analysis (EtOAc/hex-

ane 3:5). After 6 d, the reaction mixture was quenched

with MeOH and concentrated under reduced pressure.

The residue was taken up in CH₂Cl₂(200 mL), washed

with water (2· 200 mL), dried (Na₂SO₄), ﬁltered and

concentrated. Puriﬁcation of the crude residue by ﬂash

chromatography (EtOAc/hexane 1:4 ! 1:1) gave an

anomeric mixture (a/b 2:3) of 2-acetamido-2-deoxy-6-

O-triphenylmethyl-1,3,4-tri-O-pivaloyl-a,b-^D-glucopyr-

anose as a white foam (10.8 g, 83%). R_f= 0.29 (EtOAc/

In summary, we have successfully synthesised an extend-

ed array of Neu5Ac2en mimetics that are useful probes

of V. cholerae sialidase. Our preliminary molecular

modelling study suggested that alternative, more hydro-

phobic, functionalities could be accommodated in the

glycerol side-chain pocket of the active site and the pre-

sented inhibition data support this premise. The chemis-

try developed in this study provides a useful starting

point for the synthesis of designed mimetics, which

may signiﬁcantly inhibit this important enzyme.

1

hexane 3:5); H NMR (CDCl₃) a-anomer: d 0.88, 1.14,

1.36 (3· 9H, 3· s, 3· OPiv), 1.92 (3H, s, NAc), 3.05

(1H, dd, J_6a,6b10.2, J_6a,51.8 Hz, H-6a), 3.13 (1H, dd,

J_6b,6a10.2, J_6b,56.0 Hz, H-6b), 3.98–4.06 (1H, m, H-

5), 4.57 (1H, ddd, J_2,310.8, J_2,NH9.0, J_2,13.6 Hz, H-

2), 5.18–5.29 (2H, m, H-3, H-4), 5.43 (1H, br d, J_NH,2

9.0 Hz, NH), 6.33 (1H, d, J_1,23.6 Hz, H-1), 7.17–7.48

(15H, m, OTr); b-anomer: d 0.86, 1.14, 1.27 (3· 9H,

3· s, 3· OPiv), 1.90 (3H, s, NAc), 3.07 (1H, dd, J_6a,6b

10.2, J_6a,55.1 Hz, H-6a), 3.16 (1H, dd, J_6b,6a10.2, J_6b,5

1.8 Hz, H-6b), 3.77 (1H, ddd, J_5,49.6, J_5,6a5.1, J_5,6b

1.8 Hz, H-5), 4.53 (1H, ddd, J_2,310.6, J_2,NH10.2, J_2,1

9.0 Hz, H-2), 5.13 (1H, dd, J_3,210.6, J_3,49.6 Hz, H-3),

5.31 (1H, dd, J_4,3= J_4,59.6 Hz, H-4), 5.69 (1H, d, J_1,2

9.0 Hz, H-1), 5.72 (1H, br d, J_NH,210.2 Hz, NH),

7.16–7.47 (15H, m, OTr); ¹³C NMR (CDCl₃) d 23.6,

23.7 (NC(O)Me a/b), 27.4, 27.6, 27.7, 27.8 (3·

OC(O)CMe₃a/b), 39.1, 39.5, 39.6, 40.0 (3· OC(O)CMe₃

a/b), 52.5 (C-2 a), 53.5 (C-2 b), 62.5 (C-6 a/b), 67.8, 67.9

(C-4 a/b), 71.2 (C-3 a), 72.6 (C-5 a), 73.1 (C-3 b), 75.1

(C-5 b), 87.0 (OCPh₃a/b), 91.2 (C-1 a), 93.4 (C-1 b),

127.6, 128.4, 129.4, 144.2 (OCPh₃a/b), 170.1, 170.3,

176.4, 176.5, 176.6, 177.8, 179.6, 180.2 (NC(O)Me a/b,

3· OC(O)CMe₃a/b). LRMS m/z 738 ([M+Na]⁺, 30%),

243 ([Ph₃C]⁺, 100). HRMS calcd for C₄₂H₅₃NNaO₉

[M+Na] 738.3618. Found 738.3601. A solution of 2-

acetamido-2-deoxy-6-O-triphenylmethyl-1,3,4-tri-O-piva-

loyl-a,b-^D-glucopyranose (628 mg, 0.88 mmol) in dilute

AcOH (80%, 30 mL) was stirred at 60 ꢁC. After 1 h,

the solvent was evaporated under reduced pressure.

The residue was puriﬁed by ﬂash chromatography

(EtOAc/hexane 3:5 ! 1:1) to give 2-acetamido-2-de-

oxy-1,3,4-tri-O-pivaloyl-a,b-^D-glucopyranose as an

amorphous mass (383 mg, 92%). Recrystallisation of

the product from EtOAc/hexane gave pure a-anomer.

3. Experimental section

3.1. General

Reactions were monitored by TLC using Merck silica gel

plates 60 F₂₅₄. Detection was typically eﬀected under UV

light where applicable, followed by treatment with H₂SO₄

in EtOH (5% v/v) and charring at ꢀ180 ꢁC. Puriﬁcation

by ﬂash chromatography was achieved with Merck silica

gel 60 (0.040–0.063 mm). HPLC was performed using an

Agilent HP1100 instrument, and ChemStation for LC 3D

software (revision A.09.01 [1206]). Analytical HPLC was

˚

carried out using a Phenomenex Aqua 5 l C18 124 A col-

umn (250 · 4.60 mm). Semi-preparative chromatography

˚

was performed using a Phenomenex Aqua 5 l C18 124 A

column (250 · 10.00 mm). ¹H and ¹³C NMR spectra were

recorded using a Bruker Avance 300 spectrometer. All ¹H

¨

NMR spectra were recorded at 300 MHz, and all ¹³C

NMR spectra were recorded at 75.5 MHz. Chemical

shifts are expressed as parts per million (ppm, d) and

are relative to the solvent as an internal reference (CDCl₃:

d7.27 for ¹H; d77.0 for ¹³C; CD₃OD: d4.78 for ¹H; d49.0

1

for ¹³C; D₂O: d4.67 for H). In the NMR assignments,

(⁰) and (⁰⁰) refer to the atoms of the aglycone unit. Where

(#) is used, the assignment is tentative, while (*) desig-

nates the minor component or diastereomer in a mixture.

Two-dimensional COSY and HMQC experiments were

recorded in order to assist with spectral assignment.

LRMS were recorded in electrospray ionisation mode

on a Bruker Esquire 3000 spectrometer using Bruker Es-

¨

1

quire Control software (version 5.0). All spectra were

recorded in positive ion mode at a concentration of

0.1 mg/mL using 0.1% AcOH. HRMS and elemental

analyses were recorded at the Department of Chemistry

at the University of Queensland, Australia. All HRMS

were run in positive ion electrospray ionisation mode.

All solvents were distilled prior to use or were of analyti-

cal grade.

R_f= 0.15 (EtOAc/hexane 3:5); H NMR (CDCl₃) a-an-

omer: d 1.15, 1.19, 1.31 (3· 9H, 3· s, 3· OPiv), 1.90 (3H,

s, NAc), 2.34 (1H, dd, J_OH,6a8.7, J_OH,6b5.7 Hz, OH),

3.52 (1H, ddd, J_6b,6a12.6, J_6b,OH5.7, J_6b,55.1 Hz,

H-6b), 3.65 (1H, ddd, J_6a,6b12.6, J_6a,OH8.7, J_6a,5

2.1 Hz, H-6a), 3.77 (1H, ddd, J_5,49.9, J_5,6b5.1, J_5,6a

2.1 Hz, H-5), 4.50 (1H, ddd, J_2,310.8, J_2,NH9.0, J_2,1

3.6 Hz, H-2), 5.20 (1H, dd, J_4,5= J_4,39.9 Hz, H-4),

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1525

5.32 (1H, dd, J_3,210.8, J_3,49.9 Hz, H-3), 5.46 (1H, br d,

J_NH,29.0 Hz, NH), 6.17 (1H, d, J_1,23.6 Hz, H-1); b-an-

omer: d 1.14, 1.17, 1.20 (3· 9H, 3· s, 3· OPiv), 1.87 (3H,

s, NAc), 2.45 (1H, br s, OH), 3.54 (1H, dd, J_6a,6b12.3,

J_6a,54.8 Hz, H-6a), 3.64 (1H, ddd, J_5,49.0, J_5,6a4.8,

J_5,6b2.1 Hz, H-5), 3.71 (1H, dd, J_6b,6a12.3, J_6b,5

2.1 Hz, H-6b), 4.41 (1H, ddd, J_2,310.5, J_2,NH9.9, J_2,1

9.0 Hz, H-2), 5.12 (1H, dd, J_4,39.3, J_4,59.0 Hz, H-4),

5.20 (1H, dd, J_3,210.5, J_3,49.3 Hz, H-3), 5.60 (1H, br

d, J_NH,29.9 Hz, NH), 5.63 (1H, d, J_1,29.0 Hz, H-1);

¹³C NMR (CDCl₃) d 23.3 (NC(O)Me a/b), 27.1, 27.2,

27.3, 27.4 (3· OC(O)CMe₃a/b), 39.1, 39.2, 39.3, 39.7

(3· OC(O)CMe₃a/b), 52.0 (C-2 a), 53.1 (C-2 b), 61.3,

61.5 (C-6 a/b), 67.6 (C-4 a), 68.1 (C-4 b), 70.2 (C-3 a),

72.3 (C-3 b), 72.8 (C-5 a), 75.6 (C-5 b), 90.9 (C-1 a),

93.0 (C-1 b), 169.8, 170.0, 176.5, 177.5, 177.6, 177.7,

179.1, 179.9 (NC(O)Me a/b, 3· OC(O)CMe₃a/b).

LRMS m/z 496 ([M+Na]⁺, 100%). HRMS calcd for

C₂₃H₃₉NNaO₉[M+Na] 496.2522. Found 496.2531.

Anal. Calcd for C₂₃H₃₉NO₉: C, 58.33; H, 8.30; N,

2.96. Found: C, 58.25; H, 8.42; N, 2.75. To a solution

of 2-acetamido-2-deoxy-1,3,4-tri-O-pivaloyl-a,b-^D-glu-

copyranose (6.3 g, 13 mmol) and TEMPO (21 mg,

0.13 mmol) in CH₂Cl₂(37 mL) was added a solution

of saturated aq NaHCO₃(25 mL) containing KBr

(141 mg, 1.3 mmol) and Bu₄NBr (216 mg, 0.67 mmol).

The biphasic mixture was stirred vigorously at rt, while

a solution of aq NaOCl (10–15%, 32 mL), containing

saturated aq NaHCO₃(14 mL) and saturated aq NaCl

(27 mL), was added over 15 min. After 45 min, a further

portion of aq NaOCl (10–15%, 32 mL) was added. After

a further 15 min, the reaction mixture was adjusted to

pH 2 using dilute HCl (4 M) and diluted with CHCl₃

(50 mL). The layers were separated, and the organic

layer was washed with water (2· 150 mL), dried

(Na₂SO₄), ﬁltered and concentrated. To the residue in

anhyd MeOH (40 mL) was added CH(OMe)₃(2.1 mL,

19 mmol), followed by cautious addition of SOCl₂

(0.7 mL, 9.6 mmoL) and the solution was stirred under

N₂at rt for 30 min and then concentrated. The crude

product was puriﬁed by ﬂash chromatography

(EtOAc/hexane 2:3) to give the title compound 7 as a

white foam (5.10 g, 76%). R_f= 0.25 (EtOAc/hexane

2:3); ¹H NMR (CDCl₃) a-anomer: d 1.16, 1.17, 1.32

(3· 9H, 3· s, 3· OPiv), 1.90 (3H, s, NAc), 3.73 (3H, s,

OMe), 4.25 (1H, d, J_5,49.6 Hz, H-5), 4.57 (1H, ddd,

J_2,310.8, J_2,NH9.3, J_2,13.6 Hz, H-2), 5.27–5.38 (2H,

m, H-3, H-4), 5.40 (1H, br d, J_NH,29.3 Hz, NH), 6.25

(1H, d, J_1,23.6 Hz, H-1); b-anomer: d 1.15, 1.20 (3·

9H, 3· s, 3· OPiv), 1.88 (3H, s, NAc), 3.73 (3H, s,

OMe), 4.16 (1H, d, J_5,49.3 Hz, H-5), 4.46 (1H, ddd,

J_2,310.2, J_2,NH9.9, J_2,18.7 Hz, H-2), 5.20 (1H, dd,

J_3,210.2, J_3,49.3 Hz, H-3), 5.29 (1H, dd, J_4,3= J_4,5

9.3 Hz, H-4), 5.44 (1H, br d, J_NH,29.9 Hz, NH), 5.67

(1H, d, J_1,28.7 Hz, H-1); ¹³C NMR (CDCl₃): d 23.3,

23.4 (NC(O)Me a/b), 27.1, 27.3, 27.4 (3· OC(O)CMe₃

a/b), 39.1, 39.3, 39.4, 39.7 (3· OC(O)CMe₃a/b), 51.6

(C-2 a), 52.8 (C-2 b), 53.2, 53.3 (CO₂Me a/b), 68.6 (C-

4 a), 69.1 (C-4 b), 69.7 (C-3 a), 71.3 (C-5 a), 71.6 (C-3

b), 73.9 (C-5 b), 90.6 (C-1 a), 92.8 (C-1 b), 167.2,

167.6, 169.7, 169.9, 176.0, 176.8, 179.8, 177.4, 178.3,

178.9 (CO₂Me a/b, NC(O)Me a/b, 3· OC(O)CMe₃a/

b). LRMS m/z 524 ([M+Na]⁺, 100%). Anal. Calcd for

C₂₄H₃₉NO₁₀: C, 57.47; H, 7.84; N, 2.79. Found: C,

56.92; H, 8.01; N, 2.79.

3.3. General procedure for the synthesis of 4a–4k

TMSOTf (99 lL, 0.55 mmol) was added to a stirred solu-

tion of 7 (a/b ꢀ2:3) (250 mg, 0.50 mmol) in anhyd DCE

(2.5 mL) under Ar. The clear yellow solution was warmed

to 50 ꢁC. After 3 d, TLC analysis (EtOAc/hexane 1:3)

indicated that the starting material was nearly all con-

sumed. The resulting brown reaction mixture was cooled

˚

to rt, and 3 A molecular sieves were added. After 30 min,

anhyd alcohol (1.50 mmol) was added and the reaction

mixture was stirred at rt under Ar for 24 h. NEt₃was add-

ed to adjust to pH 9, the reaction mixture was ﬁltered

through Celite^ꢂ, the residue was washed with CHCl₃/

MeOH 10:1 (75 mL) and the ﬁltrate was concentrated to

give a brown gum. Puriﬁcation of the crude product by

ﬂash chromatography aﬀorded 4a–4k (60–94%, based

on recovered a-7 and 8).

3.4. Methyl (ethyl 2-acetamido-2-deoxy-3,4-di-O-pivaloyl-

b-^D-glucopyranosid)uronate (4a)

Prepared from reaction between 7 and EtOH in 64%

yield after chromatography (EtOAc/hexane 1:1 ! 3:2)

as a clear colourless gum. Starting material 7 (a-anomer)

(7%) and oxazoline 8 (13%) were also isolated. R_f= 0.13

(EtOAc/hexane 1:1); ¹H NMR (CDCl₃): d 1.12, 1.13 (2·

9H, 2· s, 2· OPiv), 1.17 (3H, t, J_{2 ;1}7.1 Hz, H-2⁰), 1.91

0

(3H, s, NAc), 3.57 (1H, dd, J_{1 a;1 b}9.7, J_{1 a;2}7.1 Hz, H-

1⁰a), 3.90 (1H, dd, J_{1 b;1 a}9.7, J_{1 b;2}7.1 Hz, H-1⁰b), 3.71

(3H, s, OMe), 3.93 (1H, ddd, J_2,310.4, J_2,NH9.0, J_2,1

8.1 Hz, H-2), 4.09 (1H, d, J_5,49.7 Hz, H-5), 4.73 (1H,

d, J_1,28.1 Hz, H-1), 5.21 (1H, dd, J_4,59.7, J_4,39.5 Hz,

H-4), 5.38 (1H, dd, J_3,210.4, J_3,49.5 Hz, H-3), 5.92

(1H, br d, J_NH,29.0 Hz, NH); ¹³C NMR (CDCl₃): d

14.9 (C-2⁰), 23.1 (NC(O)Me), 27.0 (2· OC(O)CMe₃),

38.6, 38.8 (2· OC(O)CMe₃), 52.6 (CO₂Me), 54.2 (C-2),

65.3 (C-1⁰), 69.4 (C-4), 71.2 (C-3), 72.9 (C-5), 100.6

(C-1), 167.6, 169.9, 176.5, 178.2 (CO₂Me, NC(O)Me,

2· OC(O)CMe₃). LRMS m/z 468 ([M+Na]⁺, 100%).

HRMS calcd for C₂₁H₃₆NO₉[M+H] 446.2390. Found

446.2402. Anal. Calcd for C₂₁H₃₅NO₉Æ0.5H₂O: C,

55.49; H, 7.98; N, 3.08. Found C, 55.77; H, 8.04; N, 3.08.

0

3.5. Methyl (3-pentyl 2-acetamido-2-deoxy-3,4-di-O-piva-

loyl-b-^D-glucopyranosid)uronate (4b)

Prepared from reaction between 7 and pentan-3-ol in

78% yield after chromatography (EtOAc/hexane

1:3 ! 2:3) as a white amorphous mass. Starting material

7 (a-anomer) (12%) and oxazoline 8 (5%) were also iso-

lated. R_f= 0.28 (EtOAc/hexane 2:3); ¹H NMR (CDCl₃):

d 0.76–0.85 (6H, m, H-3⁰, H-3⁰⁰), 1.08, 1.10 (2· 9H, 2· s,

2· OPiv), 1.37–1.58 (4H, m, H-2⁰, H-2⁰⁰), 1.85 (3H, s,

NAc), 3.43 (1H, tt, J_{1 ;2}¼ J_{1 ;2}5.7 Hz, H-1⁰), 3.67

(3H, s, OMe), 3.91 (1H, ddd, J_2,310.6, J_2,NH9.1, J_2,1

8.3 Hz, H-2), 4.06 (1H, d, J_5,49.9 Hz, H-5), 4.74 (1H,

d, J_1,28.3 Hz, H-1), 5.15 (1H, dd, J_4,3= J_4,59.7 Hz, H-

4), 5.40 (1H, dd, J_3,210.6, J_3,49.5 Hz, H-3), 6.40 (1H,

br d, J_NH,29.1 Hz, NH); ¹³C NMR (CDCl₃): d 8.9, 9.4

(C-3⁰, C-3⁰⁰), 23.0 (NC(O)Me), 25.6, 26.7 (C-2⁰, C-2⁰⁰),

0

00

1526

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

26.9, 27.0 (2· OC(O)CMe₃), 38.6, 38.8 (2· OC(O)CMe₃),

52.8 (CO₂Me), 54.7 (C-2), 69.4 (C-4), 71.2 (C-3), 72.6 (C-

5), 82.9 (C-1⁰), 100.2 (C-1), 167.6, 169.9, 176.5, 178.2

(CO₂Me, NC(O)Me, 2· OC(O)CMe₃). LRMS m/z 510

([M+Na]⁺, 100%). HRMS calcd for C₂₄H₄₁NNaO₉

[M+Na] 510.2679. Found 510.2686.

dried (Na₂SO₄), ﬁltered and concentrated. Puriﬁcation

of the crude product by ﬂash chromatography

(EtOAc ! EtOAc/MeOH 10:1) gave 4e [R_f= 0.23

(EtOAc); 44%] as a clear yellow syrup. Starting material

7 (a-anomer) (28%) was also isolated. To 4e (212 mg,

0.51 mmol) dissolved in anhyd pyridine (3 mL) was add-

ed Ac₂O (0.1 mL, 1 mmol) and the solution was stirred

at rt overnight under N₂. The reaction was quenched

using MeOH (0.5 mL) and then concentrated. The crude

product was dissolved in EtOAc (15 mL), washed with

dilute HCl (1 M, 2· 15 mL), water (15 mL), dried

(Na₂SO₄), ﬁltered and concentrated. Puriﬁcation of the

crude product by ﬂash chromatography (EtOAc/hexane

3:2 ! 3:1) gave the title compound 4e-Ac (211 mg, 91%)

as an amorphous mass. R_f= 0.21 (EtOAc/hexane 3:2);

¹H NMR (CDCl₃): d 1.14, 1.15 (2· 9H, 2· s, 2· OPiv),

1.93 (3H, s, NAc), 2.07 (3H, s, OAc), 3.74 (3H, s,

3.6. Methyl (isopropyl 2-acetamido-2-deoxy-3,4-di-O-piva-

loyl-b-^D-glucopyranosid)uronate (4c)

Prepared from reaction between 7 and i-PrOH in 53%

yield after chromatography (EtOAc/hexane 2:3 ! 1:1)

as a white amorphous mass. Starting material 7 (a-ano-

mer) (32%) and oxazoline 8 (3%) were also isolated.

R_f= 0.20 (EtOAc/hexane 1:1); ¹H NMR (CDCl₃): d

1.13 (3H, d, J_{2 ;1}6.3 Hz, H-2⁰), 1.22 (3H, d, J_{2 ;1}

0

00

0

6.3 Hz, H-2⁰⁰), 1.13, 1.14 (2· 9H, 2· s, 2· OPiv), 1.92

(3H, s, NAc), 3.73 (3H, s, OMe), 3.79 (1H, ddd, J_2,3

10.4, J_2,1= J_2,NH8.4 Hz, H-2), 3.96 (1H, sept,

0

OMe), 3.80 (1H, ddd, J_{1 a;1 b}11.5, J_{1 a;2 a}6.5, J_{1 a;2 b}

3.2 Hz, H-1⁰a), 3.92–4.06 (2H, m, H-1⁰b, H-2), 4.09

J_{1 ;2}¼ J_{1 ;2}6.3 Hz, H-1⁰), 4.09 (1H, d, J_5,49.8 Hz, H-

5), 4.87 (1H, d, J_1,28.2 Hz, H-1), 5.21 (1H, dd,

J_4,5= J_4,39.6 Hz, H-4), 5.47 (1H, dd, J_3,210.0, J_3,4

9.5 Hz, H-3), 5.65 (1H, br d, J_NH,28.5 Hz, NH); ¹³C

(1H, d, J_5,49.6 Hz, H-5), 4.13 (1H, ddd, J_{2 a;2 b}12.1,

0

00

0

J_{2 a;1 a}6.5, J_{2 a;1 b}3.0 Hz, H-2⁰a), 4.33 (1H, ddd, J_{2 b;2 a}

0

12.1, J_{2 b;1 b}6.6, J_{2 b;1 a}3.2 Hz, H-2⁰b), 4.80 (1H, d, J_1,2

8.1 Hz, H-1), 5.24 (1H, dd, J_4,59.6, J_4,39.3 Hz, H-4),

5.36 (1H, dd, J_3,210.3,J_3,49.3 Hz, H-3), 5.57 (1H, br d,

0

NMR (CDCl₃):

d

21.7, 23.0 (C-2⁰, C-2⁰⁰), 23.2

(NC(O)Me), 27.0, 27.1 (2· OC(O)CMe₃), 38.6, 38.8

(2· OC(O)CMe₃), 52.6 (CO₂Me), 54.9 (C-2), 69.5 (C-

4), 71.7 (C-3), 72.4 (C-1⁰), 72.8 (C-5), 99.3 (C-1),

167.6, 170.1, 176.5, 178.1 (CO₂Me, NC(O)Me, 2·

OC(O)CMe₃). LRMS m/z 482.5 ([M+Na]⁺, 100%).

Anal. Calcd for C₂₂H₃₇NO₉: C, 57.50; H, 8.12; N,

3.05. Found: C, 57.34; H, 8.30; N, 3.03.

J_NH,29.0 Hz, NH); ¹³C NMR (CDCl₃):

d 20.9

(NC(O)Me), 23.2 (OC(O)Me), 27.0 (2· OC(O)CMe₃),

38.7, 38.9 (2· OC(O)CMe₃), 52.7 (CO₂Me), 54.0 (C-2),

62.8 (C-2⁰), 67.0 (C-1⁰), 69.2 (C-4), 70.9 (C-3), 73.0 (C-

5), 100.3 (C-1), 167.4, 169.9, 171.0, 176.5, 178.1 (CO₂Me,

OC(O)Me, NC(O)Me, 2· OC(O)CMe₃). LRMS m/z 526

([M+Na]⁺, 100%). Anal. Calcd for C₂₃H₃₇NO₁₁: C,

54.86; H, 7.41; N, 2.78. Found: C, 54.62; H, 7.48; N, 2.68.

3.7. Methyl (2-methylpropyl 2-acetamido-2-deoxy-3,4-di-

O-pivaloyl-b-^D-glucopyranosid)uronate (4d)

3.9. Methyl (3-O-acetyl-3-hydroxypropyl 2-acetamido-2-

deoxy-3,4-di-O-pivaloyl-b-^D-glucopyranosid)uronate (4f-Ac)

Prepared from reaction between 7 and i-BuOH in 77%

yield after chromatography (EtOAc/hexane 1:3 ! 1:1)

as a white foam. Starting material 7 (a-anomer) (6%)

and oxazoline 8 (6%) were also isolated. R_f= 0.24

Prepared from reaction between 7 and propan-1,3-diol.

To remove excess propan-1,3-diol, the crude product

was diluted with EtOAc and washed twice with water,

dried (Na₂SO₄), ﬁltered and concentrated. The crude

product was puriﬁed by ﬂash chromatography

(EtOAc ! EtOAc/MeOH 10:1) to give 4f [R_f= 0.21

(EtOAc); 42%] as a clear colourless residue. Starting

material 7 (a-anomer) (36%) was also isolated. Ac₂O

(0.3 mL, 3 mmol) was added to a solution of 4f

(432 mg, 0.91 mmol) in anhyd pyridine (4 mL) under

N₂. The solution was stirred at rt overnight. The reac-

tion was quenched with MeOH (0.5 mL), and then the

solvent was evaporated under reduced pressure. The res-

idue was dissolved in EtOAc (15 mL), washed with di-

lute HCl (1 M, 2· 15 mL), water (15 mL), dried

(Na₂SO₄), ﬁltered and concentrated. The crude product

was puriﬁed by ﬂash chromatography (EtOAc/hexane

3:2) to give the title compound 4f-Ac (447 mg, 95%) as

1

(EtOAc/hexane 2:3); H NMR (CDCl₃): d 0.87 (3H, d,

J_{3 ;2}6.7 Hz, H-3⁰), 0.88 (3H, d, J_{3 ;2}6.7 Hz, H-3⁰⁰), 1.14,

1.15 (2· 9H, 2· s, 2· OPiv), 1.85 (1H, m, H-2⁰), 1.91

0

00

0

(3H, s, NAc), 3.20 (1H, dd, J_{1 a;1 b}9.3, J_{1 a;2}7.1 Hz, H-

1⁰a), 3.70 (1H, dd, J_{1 b;1 a}9.3, J_{1 b;2}7.1 Hz, H-1⁰b), 3.72

(3H, s, OMe), 3.99 (1H, ddd, J_2,310.3, J_2,NH9.0, J_2,1

8.1 Hz, H-2), 4.07 (1H, d, J_5,49.6 Hz, H-5), 4.68 (1H, d,

J_1,28.1 Hz, H-1), 5.23 (1H, dd, J_4,5= J_4,39.4 Hz, H-4),

5.36 (1H, dd, J_3,210.3, J_3,49.4 Hz, H-3), 5.59 (1H, br d,

J_NH,29.0 Hz, NH); ¹³C NMR (CDCl₃): d 18.9, 19.1 (C-

3⁰, C-3⁰⁰), 23.1 (NC(O)Me), 27.0 (2· OC(O)CMe₃), 28.3

(C-2⁰), 38.7, 38.9 (2· OC(O)CMe₃), 52.7 (CO₂Me), 54.4

(C-2), 69.3 (C-4), 71.7 (C-3), 73.0 (C-5), 76.6 (C-1⁰),

101.2 (C-1), 167.6, 169.8, 176.5, 178.3 (CO₂Me,

NC(O)Me 2· OC(O)CMe₃). LRMS m/z 496 ([M+Na]⁺,

100%). HRMS calcd for C₂₃H₂₉NNaO₉[M+Na]

496.2523. Found 496.2530.

0

1

an amorphous mass. R_f= 0.24 (EtOAc/hexane 3:2); H

NMR (CDCl₃): d 1.04, 1.06 (2· 9H, 2· s, 2· OPiv),

1.72–1.87 (2H, m, H-2⁰, H-2⁰⁰), 1.83 (3H, s, NAc), 1.95

0

3.8. Methyl (2-O-acetyl-2-hydroxyethyl 2-acetamido-2-de-

oxy-3,4-di-O-pivaloyl-b-^D-glucopyranosid)uronate (4e-Ac)

(3H, s, OAc), 3.46 (1H, ddd, J_{1 a;1 b}9.9, J_{1 a;2 a}6.9,

J_{1 a;2 b}6.0 Hz, H-1⁰a), 3.63 (3H, s, OMe), 3.89 (1H,

0

ddd, J_{1 b;1 a}9.9, J_{1 b;2 a}5.7, J_{1 b;2 b}5.4 Hz, H-1⁰b), 3.91–

0

Prepared from reaction between 7 and ethylene glycol.

To remove excess ethylene glycol, the crude product

was diluted with EtOAc and washed twice with water,

4.04 (2H, m, H-2, H-3⁰a), 4.03 (1H, d, J_5,49.9 Hz, H-

0

5), 4.12 (1H, ddd, J_{3 b;3 a}10.8, J_{3 b;2 a}¼ J_{3 b;2 a}6.6 Hz,

H-3⁰b), 4.64 (1H, d, J_1,28.1 Hz, H-1), 5.11 (1H, dd,

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1527

J_4,5= J_4,39.6 Hz, H-4), 5.32 (1H, dd, J_3,210.5, J_3,4

9.6 Hz, H-3), 6.49 (1H, br d, J_NH,29.3 Hz, NH); ¹³C

NMR (CDCl₃): d 20.7 (OC(O)Me), 22.8 (NC(O)Me),

26.8 (2· OC(O)CMe₃), 28.5 (C-2⁰), 38.4, 38.6 (2·

OC(O)CMe₃), 52.5 (CO₂Me), 53.6 (C-2), 60.8 (C-3⁰),

65.9 (C-1⁰), 69.3 (C-4), 71.0 (C-3), 72.5 (C-5), 100.7 (C-

1), 167.5, 170.1, 171.0, 176.4, 177.8 (CO₂Me, NC(O)Me,

OC(O)Me, 2· OC(O)CMe₃). LRMS m/z 540 ([M+Na]⁺,

100%). Anal. Calcd for C₂₄H₃₉NO₁₁: C, 55.69; H, 7.60;

N, 2.71. Found: C, 55.70; H, 7.72; N, 2.66.

(C-2⁰), 67.0 (C-2⁰*), 69.3 (C-4), 70.6 (C-3*), 71.1 (C-3),

72.8, 73.8 (C-5*, C-1⁰*), 72.9, 73.6 (C-5, C-1⁰), 99.2

(C-1*), 99.8 (C-1), 167.4, 169.9, 171.0, 176.5, 178.0

(NC(O)Me, OC(O)Me, CO₂Me, 2· OC(O)CMe₃), *mi-

nor diastereomer. LRMS m/z 540 ([M+Na]⁺, 100%).

HRMS calcd for C₂₄H₃₉NNaO₁₁[M+Na] 540.2421.

Found 540.2428. Acetic anhydride (0.2 mL, 2 mmol)

was added to a solution of a 1:1 diastereomeric mixture

of 4g (337 mg, 0.71 mmol) in anhyd pyridine (3 mL).

The solution was stirred at rt overnight under N₂.

MeOH (1 mL) was added and the solution was concen-

trated under reduced pressure. The crude product was

dissolved in EtOAc (15 mL) and then washed with dilute

HCl (1 M, 2· 15 mL), water (2· 15 mL), saturated aq

NaCl (15 mL), dried (Na₂SO₄), ﬁltered and concentrat-

ed. The crude product was puriﬁed by ﬂash chromatog-

raphy (EtOAc/hexane 3:2) to give a 1:1 diastereomeric

mixture of 4g-Ac as an amorphous mass (285 mg,

78%). Compound 4g-Ac: R_f= 0.24 (EtOAc/hexane 3:2);

¹H NMR (CDCl₃): d 1.13, 1.14 (2· 18H, 2· s, 4· OPiv),

3.10. Methyl (1-O-acetyl-1-hydroxyisopropyl 2-acetamido-

2-deoxy-3,4-di-O-pivaloyl-b-^D-glucopyranosid)uronate

(4h-Ac) and methyl (2-O-acetyl-2-hydroxypropyl 2-acet-

amido-2-deoxy-3,4-di-O-pivaloyl-b-^D-glucopyranosid)uro-

nate (4g-Ac)

Prepared from reaction between 7 and propan-1,2-diol.

To remove excess propan-1,2-diol, the crude product

was diluted with EtOAc and washed twice with water,

dried (Na₂SO₄), ﬁltered and concentrated. The crude

product was puriﬁed by ﬂash chromatography

(EtOAc/hexane 1:3 ! EtOAc/MeOH 5:1) to give a 3:2

mixture of 4g and 4h [R_f= 0.20 (EtOAc)] as a clear col-

ourless residue in 63% yield. Starting material 7 (a-ano-

mer) (10%) was also isolated. i-Pr₂EtN (26 lL,

0.37 mmol) was added to a 3:2 mixture of 4g and 4h

(85 mg, 0.18 mmol) in anhyd CH₂Cl₂(0.88 mL) under

Ar. The solution was cooled to ꢁ78 ꢁC, and AcCl

(5.0 mL, 0.11 mmol) was added dropwise. The reaction

mixture was stirred for 2.5 h at ꢁ78 ꢁC and then

warmed to rt. After a further 1 h at rt, the reaction mix-

ture was diluted with CHCl₃(5 mL), washed with dilute

HCl (1 M, 5 mL), water (5 mL), saturated aq NaCl

(5 mL), dried (Na₂SO₄), ﬁltered and concentrated under

reduced pressure to give a pale yellow residue. Puriﬁca-

tion of the crude product by ﬂash chromatography

(EtOAc ! EtOAc/MeOH 5:1) aﬀorded a 5:1 diastereo-

meric mixture of 4h-Ac (36 mg, 95% from 4h) as a col-

1.19 (3H, d, J_{3 ;2}6.6 Hz, H-3⁰), 1.20 (3H, d, J_{3 ;2}6.6 Hz,

H-3⁰), 1.91 (2· 3H, 2· s, 2· NAc), 2.04 (2· 3H, 2· s, 2·

0

OAc), 3.57 (1H, dd, J_{1 a;1 b}10.8, J_{1 a;2}6.3 Hz, H-1⁰a),

0

3.67 (1H, dd, J_{1 a;1 b}11.7, J_{1 a;2}3.6 Hz, H-1⁰a), 3.73

0

(6H, s, 2· OMe), 3.76 (1H, dd, J_{1 b;1 a}11.7, J_{1 b;2}

4.5 Hz, H-1⁰b), 3.85 (1H, dd, J_{1 b;1 a}10.8, J_{1 b;2}4.5 Hz,

H-1⁰b), 3.94 (1H, ddd, J_2,310.2, J_2,NH9.0, J_2,18.4 Hz,

H-2), 4.05 (1H, ddd, J_2,310.2, J_2,NH9.0, J_2,18.4 Hz, H-

2), 4.06 (1H, d, J_5,49.6 Hz, H-5), 4.07 (1H, d, J_5,4

9.6 Hz, H-5), 4.73 (1H, d, J_1,28.4 Hz, H-1), 4.76 (1H,

d, J_1,28.4 Hz, H-1), 4.96-5.07 (1H, m, H-2⁰), 5.08–5.18

(1H, m, H-2⁰), 5.21 (1H, dd, J_4,59.6, J_4,39.3 Hz, H-4),

5.21 (1H, dd, J_4,59.6, J_4,39.3 Hz, H-4), 5.31 (1H, dd,

J_3,210.2, J_3,49.3 Hz, H-3), 5.38 (1H, dd, J_3,210.2, J_3,4

9.3 Hz, H-3), 5.64 (1H, br d, J_NH,29.0 Hz, NH), 5.67

(1H, br d, J_NH,29.0 Hz, NH); ¹³C NMR (CDCl₃): d

16.3, 16.5 (2· C-3⁰), 21.2, 21.3 (2· OC(O)Me), 23.1 (2·

NC(O)Me), 27.0 (4· OC(O)CMe₃), 38.7, 38.8 (4·

OC(O)CMe₃), 52.7 (2· CO₂Me), 53.7, 54.2 (2· C-2),

67.8, 69.2, 69.3 (2· C-2⁰, 2· C-4), 70.7, 71.2 (2· C-3),

70.9, 71.4 (2· C-1⁰), 72.9, 73.0 (2· C-5), 99.8, 100.8 (2·

C-1), 167.4, 169.8, 170.5, 170.9, 176.5, 178.1 (2· CO₂Me,

2· NC(O)Me, 2· OC(O)Me, 4· OC(O)CMe₃). LRMS m/

0

ourless residue. A 1:1 diastereomeric mixture of

unreacted 4g (35 mg, 66% from 4g) was also isolated.

1

Compound 4h-Ac: R_f= 0.43, 0.50* (EtOAc); H NMR

(CDCl₃) including partial assignment of minor diaste-

reomer: d 1.13 (9H, s, OPiv), 1.14 (9H, s, OPiv*), 1.23

z

540.5 ([M+Na]⁺, 100%). HRMS calcd for

(3H, d, J_{3 ;1}7.5 Hz, H-3⁰*), 1.25 (3H, d, J_{3 ;1}6.3 Hz,

H-3⁰), 1.91 (3H, s, NAc), 1.92 (3H, s, NAc*), 2.07

(3H, s, OAc*), 2.08 (3H, s, OAc), 3.71 (3H, s, OMe*)

C₂₄H₃₉NNaO₁₁[M+Na] 540.2421. Found 540.2421.

0

3.11. Methyl (2,3-dihydroxy-2,3-O-isopropylidenepropyl 2-

acetamido-2-deoxy-3,4-di-O-pivaloyl-b-^D-glucopyranosid)

uronate (4i)

0

3.73 (3H, s, OMe), 3.88 (1H, dd, J_{2 a;2 b}11.7, J_{2 a;1}

5.7 Hz, H-2⁰a), 3.90 (1H, ddd, J_2,39.9, J_2,NH9.0, J_2,1

0

8.4 Hz, H-2), 4.04 (1H, ddd, J_{1 ;3}6.3, J_{1 ;2 a}5.7, J_{1 ;2 b}

3.3 Hz, H-1⁰), 4.07 (1H, d, J_5,49.9 Hz, H-5), 4.07 (1H,

Prepared from reaction between 7 and 2,3-dihydroxy-

2,3-O-isopropylidenepropanol. After addition of 2,3-di-

hydroxy-2,3-O-isopropylidenepropanol the reaction

mixture was stirred for 48 h before work-up by the usual

method, to give a 3:2 diastereomeric mixture of the title

compound 4i in 64% yield after chromatography

(EtOAc/hexane 3:2) as a white amorphous mass. Start-

ing material 7 (a-anomer) (14%) and oxazoline 8 (7%)

were also isolated. R_f= 0.20 (EtOAc/hexane 3:2); ¹H

NMR (CDCl₃): d 1.13, 1.14 (2· 18H, 2· s, 2· OPiv,

2· OPiv*), 1.33, 1.34, 1.40, 1.41 (4· 3H, 4· s,

O₂CMe₂, O₂CMe₂*), 1.91, 1.92 (2· 3H, 2· s, NAc,

0

d, J_5,49.9 Hz, H-5*), 4.26 (1H, dd, J_{2 b;2 a}11.7, J_{2 b;1}

3.3 Hz, H-2⁰b), 4.88 (1H, d, J_1,28.4 Hz, H-1), 4.93

(1H, d, J_1,28.4 Hz, H-1*), 5.20 (1H, dd, J_4,59.9, J_4,3

9.6 Hz, H-4), 5.39 (1H, dd, J_3,29.9, J_3,49.6 Hz, H-3),

5.50 (1H, dd, J_3,29.9, J_3,49.6 Hz, H-3*), 5.62 (1H, br

d, J_NH,29.0 Hz, NH), 5.65 (1H, br d, J_NH,29.0 Hz,

NH*); ¹³C NMR (CDCl₃) including partial assignment

of minor diastereomer: d 16.7 (C-3⁰), 18.1 (C-3⁰), 20.8

(OC(O)Me*), 20.9 (OC(O)Me), 23.1 (NC(O)Me,

NC(O)Me*), 26.9, 27.0, 27.1 (2· OC(O)CMe₃, 2·

OC(O)CMe₃*), 38.7, 38.8 (2· OC(O)CMe₃), 52.6

(CO₂Me*), 52.7 (CO₂Me), 54.5 (C-2), 55.2 (C-2*), 66.2

0

NAc*), 3.63 (1H, dd, J_{1 a;1 b}10.8, J_{1 a;2}5.4 Hz,

1528

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

#

H-1⁰a ), 3.64 (1H, dd, J_{1 a;1 b}10.5, J_{1 a;2}6.6 Hz, H-

(a-anomer) (26%) and 8 (5%) were also isolated.

R_f= 0.12 (EtOAc/hexane 2:3); ¹H NMR (CDCl₃): d

1.12, 1.13 (2· 9H, 2· s, 2· OPiv), 1.91 (3H, s, NAc),

3.72 (3H, s, OMe), 4.00–4.13 (2H, m, H-2, H-1⁰a),

0

#

1⁰a* ), 3.67 (1H, dd, J_{3 a;3 b}8.4, J_{3 a;2}6.3 Hz, H-3⁰a ),

0

3.72 (6H, s, OMe, OMe*), 3.80 (1H, dd, J_{3 a;3 b}8.1,

#

J_{3 a;2}6.0 Hz, H-3⁰a* ), 3.84 (1H, dd, J_{1 b;1 a}10.8, J_{1 b;2}

0

#

5.4 Hz, H-1⁰b ), 3.89 (1H, dd, J_{1 b;1 a}10.5, J_{1 b;2}

4.07 (1H, d, J_5,49.6 Hz, H-5), 4.35 (1H, ddd, J_{1 b;1 a}

0

4.5 Hz, H-1⁰b*^#), 3.97-4.15 (4H, m, H-2, H-2*, H-

3⁰b^#, H-3⁰b*^#), 4.06 (1H, d, J_5,49.3 Hz, H-5), 4.07

(1H, d, J_5,49.6 Hz, H-5*), 4.20-4.30 (2H, m, H-2⁰, H-

2⁰*), 4.77 (1H, d, J_1,28.4 Hz, H-1), 4.77 (1H, d, J_1,2

8.1 Hz, H-1*), 5.21 (1H, dd, J_4,5= J_4,39.3 Hz, H-4),

5.21 (1H, dd, J_4,59.6, J_4,39.3 Hz, H-4*), 5.31 (1H, dd,

J_3,210.5, J_3,49.3 Hz, H-3), 5.31 (1H, dd, J_3,210.2, J_3,4

9.3 Hz, H-3*), 5.70 (1H, br d, J_NH,29.0 Hz, NH), 5.71

(1H, br d, J_NH,29.0 Hz, NH*); ¹³C NMR (CDCl₃): d

23.1, 23.2 (2· NC(O)Me), 25.1, 25.4, 26.6, 26.8 (2·

O₂CMe₂), 27.0 (4· OC(O)CMe₃), 38.7, 38.8 (4·

OC(O)CMe₃), 52.7 (2· CO₂Me), 53.8 (2· C-2), 66.0,

13.1, J_{1 b;2}4.9, J_{1 b;3}1.4 Hz, H-1⁰b), 4.71 (1H, d, J_1,2

8.1 Hz, H-1), 5.15–5.38 (4H, m, H-3, H-4, H-3⁰), 5.76–

5.90 (2H, m, NH, H-2⁰); ¹³C NMR (CDCl₃): d 23.2

(NC(O)Me), 27.0 (2· OC(O)CMe₃), 38.7, 38.9 (2·

OC(O)CMe₃), 52.7 (CO₂Me), 54.0 (C-2), 69.3 (C-4),

69.9 (C-1⁰), 71.1 (C-3), 72.9 (C-5), 99.8 (C-1), 117.9

(C-3⁰), 133.4 (C-2⁰), 167.5, 169.9, 176.5, 178.3 (CO₂Me,

NC(O)Me, 2· OC(O)CMe₃). LRMS m/z 480

([M+Na]⁺, 100%). HRMS calcd for C₂₂H₃₅NNaO₉

[M+Na] 480.2210. Found 480.2204.

0

3.14. 2-Methyl-4,5-dihydro-(methyl 1,2-dideoxy-3,4-di-O-

pivaloyl-a-^D-glucopyranuronato)[2,1-d]-1,3-oxazole (8)

#

66.5 (2· C-3⁰), 69.1 (C-1⁰), 69.1 (2· C-4), 70.6 (C-

#

1⁰), 71.1, 71.2 (2· C-3), 72.9, 73.0 (2· C-5), 74.3, 74.9

(2· C-2⁰), 101.0, 101.1 (2· C-1), 109.3, 109.6 (2·

O₂CMe₂), 167.4, 169.9, 170.0, 176.5, 178.2 (2· CO₂Me,

2· NC(O)Me, 4· OC(O)CMe₃), *minor diastereomer,

^#assignments tentative. LRMS m/z 555 ([M+Na]⁺,

100%). Anal. Calcd for C₂₅H₄₁NO₁₁: C, 56.48; H,

7.77; N, 2.63. Found: C, 56.10; H, 7.81; N, 2.38.

TMSOTf (198 lL, 1.1 mmol) was added to a solution of

7 (a/b 2:3) (500 mg, 1.0 mmol) in anhyd DCE (5 mL).

The clear yellow solution was stirred under Ar at

50 ꢁC and monitored by TLC analysis (toluene/acetone

8:1). After 3 d, NEt₃was added to the brown reaction

mixture to adjust to pH 9, and the solvent was evaporat-

ed under reduced pressure. The crude product was puri-

ﬁed by ﬂash chromatography (EtOAc/hexane 1:3 ! 1:1)

to give the title compound 8 as a colourless syrup

3.12. Methyl (1,2:3,4-di-O-isopropylidene-a-^D-galactopyr-

anosyl 2-acetamido-2-deoxy-3,4-di-O-pivaloyl-b-^D-gluco-

pyranosid)uronate (4j)

(273 mg, 69%). Starting material

7

(a-anomer)

(117 mg, 23%) was also isolated. R_f= 0.38 (toluene/ace-

tone 8:1); R_f= 0.33 (EtOAc/hexane 2:3); ¹H NMR

(CDCl₃): d 1.15, 1.16 (2· 9H, 2· s, 2· OPiv), 2.04

(3H, d, J_Me,21.5 Hz, NAc), 3.71 (3H, s, OMe), 3.97

(1H, d, J_5,47.8 Hz, H-5), 4.07–4.09 (1H, m, H-2), 5.05

(1H, ddd, J_4,57.8, J_4,32.9, J_4,21.2 Hz, H-4), 5.21 (1H,

dd, J_3,2= J_3,42.9 Hz, H-3), 6.03 (1H, d, J_1,27.1 Hz, H-

1); ¹³C NMR (CDCl₃): d 13.6 (NC(O)Me), 26.7, 26.8

(2· OC(O)CMe₃), 38.4, 38.5 (2· OC(O)CMe₃), 52.6

(CO₂Me), 64.9 (C-2), 68.2 (C-4), 68.7 (C-3), 69.4 (C-5),

98.6 (C-1), 166.0, 168.5, 176.4, 176.5 (CO₂Me,

NC(O)Me, 2· OC(O)CMe₃). LRMS m/z 422

([M+Na]⁺, 100%). HRMS calcd for C₁₉H₂₉NNaO₈

[M+Na] 422.1791. Found 422.1799.

Prepared from reaction between 7 and 1,2:3,4-di-O-iso-

propylidene-a-^D-galactopyranose^43,44in 47% yield after

chromatography (EtOAc/hexane 2:3 ! EtOAc) as an

amorphous mass. Starting material

7 (a-anomer)

(16%) and oxazoline 8 (5%) were also isolated.

R_f= 0.15 (EtOAc/hexane 1:1); ¹H NMR (CDCl₃) GlcN-

AcA unit: d 1.14, 1.15 (2· 9H, 2· s, 2· OPiv), 1.94 (3H,

s, NAc), 3.74 (3H, s, OMe), 4.06 (1H, d, J_5,49.6 Hz,

H-5^#), 4.19 (1H, ddd, J_2,39.6, J_2,NH9.3, J_2,18.4 Hz,

H-2), 4.74 (1H, d, J_1,28.4 Hz, H-1), 5.17-5.26 (2H, m,

H-3, H-4), 5.47 (1H, br d, J_NH,29.3 Hz, NH); Gal unit:

1.31, 1.33, 1.45, 1.51 (4· 3H, 4· s, 4· Me), 3.77 (1H, dd,

J_6a,6b12.9, J_6a,59.3 Hz, H-6a), 3.92-4.01 (2H, m, H-5,

H-6b), 4.14 (1H, dd, J_4,37.8, J_4,51.5 Hz, H-4), 4.31

(1H, dd, J_2,15.1, J_2,32.4 Hz, H-2), 4.58 (1H, dd, J_3,4

7.8, J_3,22.4 Hz, H-3), 5.53 (1H, d, J_1,25.1 Hz, H-1);

¹³C NMR (CDCl₃) GlcNAcA unit: d 23.2 (NC(O)Me),

26.9 (2· OC(O)CMe₃), 38.6, 38.8 (2· OC(O)CMe₃),

52.6 (CO₂Me), 53.3 (C-2), 69.3 (C-4), 71.7 (C-3), 73.0

(C-5), 101.9 (C-1), 167.4, 169.9, 176.4, 178.2 (CO₂Me,

NC(O)Me, 2· OC(O)CMe₃); Gal unit: d 24.1, 24.9,

25.9, 26.0 (4· Me), 68.3 (C-5), 69.0 (C-6), 70.1 (C-2),

70.5 (C-3), 70.9 (C-4), 96.1 (C-1), 108.5, 109.2 (2·

O₂CMe₂), ^#assignments tentative. LRMS m/z 682

([M+Na]⁺, 100%). Anal. Calcd for C₃₁H₄₉NO₁₄: C,

56.44; H, 7.49; N, 2.12. Found: C, 56.20; H, 7.64; N, 2.11.

3.15. General procedure for the synthesis of 9a–9i

DBU (150 lL, 0.76 mmol) was added to a solution of

compound 4a–4d, 4e–Ac—4h–Ac or 4i (0.38 mmol) in

anhyd CH₂Cl₂(2 mL) under N₂. The pale yellow solu-

tion was stirred at rt and monitored by TLC analysis.

After 18 h, the reaction mixture was concentrated under

reduced pressure to give a clear yellow syrup. Puriﬁca-

tion of the crude product by ﬂash chromatography

aﬀorded 9a–9i (80–99%).

3.16. Methyl (ethyl 2-acetamido-2,4-dideoxy-3-O-pivaloyl-

a-^L-threo-hex-4-enopyranosid)uronate (9a)

3.13. Methyl (allyl 2-acetamido-2-deoxy-3,4-di-O-pivaloyl-

b-^D-glucopyranosid)uronate (4k)

Prepared from 4a in 99% yield after chromatography

(EtOAc/hexane 3:2) as a colourless syrup. R_f= 0.23

1

Prepared from reaction between 7 and allyl alcohol in

47% yield after chromatography (EtOAc/hexane

2:3 ! 1:1) as a clear colourless gum. Compounds 7

(EtOAc/hexane 3:2); H NMR (CDCl₃): d 1.15 (3H, t,

J_{2 ;1}7.1 Hz, H-2⁰), 1.16 (9H, s, OPiv), 1.96 (3H, s,

0

NAc), 3.55 (1H, dq, J_{1 a;1 b}9.4, J_{1 a;2}7.1 Hz, H-1⁰a),

0

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1529

3.79 (1H, dq, J_{1 b;1 a}9.4, J_{1 b;2}7.1 Hz, H-1⁰b), 3.81 (3H,

s, OMe), 4.38 (1H, dddd, J_2,NH9.0, J_2,33.5, J_2,11.9, J_2,4

1.3 Hz, H-2), 4.95 (1H, ddd, J_3,44.9, J_3,23.5, J_3,10.6 Hz,

H-3), 5.17 (1H, dd, J_1,21.9, J_1,30.6 Hz, H-1), 5.90 (1H,

br d, J_NH,29.0 Hz, NH), 6.19 (1H, dd, J_4,34.9, J_4,2

J_{2 ;1 a}¼ J_{2 ;1 b}6.6 Hz, H-2⁰), 1.99 (3H, s, NAc), 3.33

0

(1H, dd, J_{1 a;1 b}9.3, J_{1 a;2}6.6 Hz, H-1⁰a), 3.56 (1H, dd,

0

J_{1 b;1 a}9.3, J_{1 b;2}6.6 Hz, H-1⁰b), 3.86 (3H, s, OMe),

4.46 (1H, dddd, J_2,NH9.3, J_2,13.9, J_2,31.8, J_2,41.2 Hz,

H-2), 5.00 (1H, ddd, J_3,44.8, J_3,21.8, J_3,10.9 Hz, H-

3), 5.18 (1H, dd, J_1,22.1, J_1,30.6 Hz, H-1), 5.44 (1H,

br d, J_NH,29.0 Hz, NH), 6.27 (1H, dd, J_4,34.8, J_4,2

0

13

1.3 Hz, H-4); C NMR (CDCl₃): d 14.8 (C-2⁰), 23.0

(NC(O)Me), 26.9 (OC(O)CMe₃), 38.7 (OC(O)CMe₃),

48.4 (C-2), 52.6 (CO₂Me), 64.2 (C-3), 65.0 (C-1⁰), 98.1

(C-1), 107.5 (C-4), 142.2 (C-5), 162.6, 170.0, 177.5

(CO₂Me, NC(O)Me, OC(O)CMe₃). LRMS m/z 366

([M+Na]⁺, 100%). HRMS calcd for C₁₆H₂₅NNaO₇

[M+Na] 366.1529. Found 366.1532.

13

1.2 Hz, H-4); C NMR (CDCl₃): d 19.0, 19.1 (C-3⁰,

C-3⁰⁰), 23.1 (NC(O)Me), 27.0 (OC(O)CMe₃), 28.3 (C-

2⁰), 38.7 (OC(O)CMe₃), 48.7 (C-2), 52.6 (CO₂Me), 64.4

(C-3), 76.1 (C-1⁰), 98.3 (C-1), 107.9 (C-4), 142.2 (C-5),

162.6, 170.0, 177.6 (CO₂Me, NC(O)Me, OC(O)CMe₃).

LRMS m/z 394 ([M+Na]⁺, 100%). HRMS calcd for

C₁₈H₂₉NNaO₇[M+Na] 394.1842. Found 394.1848.

Anal. Calcd for C₁₈H₂₉NO₇Æ0.5H₂O: C, 56.83; H, 7.95;

N, 3.68. Found: C, 56.95; H, 8.08; N, 3.63.

3.17. Methyl (3-pentyl 2-acetamido-2,4-dideoxy-3-O-piva-

loyl-a-^L-threo-hex-4-enopyranosid)uronate (9b)

Prepared from 4b in 82% yield after chromatography

(EtOAc/hexane 3:2) as a colourless syrup. R_f= 0.20

3.20. Methyl (2-O-acetyl-2-hydroxyethyl 2-acetamido-2,4-

dideoxy-3-O-pivaloyl-a-^L-threo-hex-4-enopyranosid)uronate

(9e)

1

(EtOAc/hexane 1:1); H NMR (CDCl₃): d 0.80 (3H, t,

J_{3 ;2}7.4 Hz, H-3⁰), 0.89 (3H, t, J_{3 ;2}7.4 Hz, H-3⁰⁰), 1.19

(9H, s, OPiv), 1.37–1.58 (4H, m, H-2⁰, H-2⁰⁰), 1.98 (3H,

s, NAc), 3.57–3.65 (3H, s, OMe), 4.41 (1H, dddd, J_2,NH

9.0, J_2,11.8, J_2,41.5, J_2,30.9 Hz, H-2), 4.97 (1H, ddd,

J_3,45.1, J_3,20.9, J_3,1< 1 Hz, H-3), 5.27 (1H, dd, J_1,2

1.8, J_1,3< 1 Hz, H-1), 5.59 (1H, br d, J_NH,29.0 Hz,

NH), 6.26 (1H, dd, J_4,35.1, J_4,21.5 Hz, H-4); ¹³C

NMR (CDCl₃): d 9.2, 9.6 (C-3⁰, C-3⁰⁰), 23.2 (NC(O)Me),

25.9, 26.6 (C-2⁰, C-2⁰⁰), 27.0 (OC(O)CMe₃), 38.7

(OC(O)CMe₃), 48.6 (C-2), 52.6 (CO₂Me), 64.2 (C-3),

81.7 (C-1⁰), 96.9 (C-1), 107.5 (C-4), 142.5 (C-5), 162.7,

169.5, 177.5 (CO₂Me, NC(O)Me, OC(O)CMe₃). LRMS

m/z 408 ([M+Na]⁺, 100%). HRMS calcd for

C₁₉H₃₁NNaO₇[M+Na] 408.1998. Found 408.1995.

0

00 00

Prepared from 4e-Ac in 87% yield after chromatogra-

phy (EtOAc/hexane 3:2 ! 3:1) as a colourless syrup.

R_f= 0.33 (EtOAc/hexane 3:1); ¹H NMR (CDCl₃): d

1.16 (9H, s, OPiv), 1.97 (3H, s, NAc), 2.01 (3H, s,

0

OAc), 3.77 (1H, ddd, J_{1 a;1 b}11.2, J_{1 a;2 a}6.6, J_{1 a;2 b}

3.8 Hz, H-1⁰a), 3.83 (3H, s, OMe), 3.93 (1H, ddd,

J_{1 b;1 a}11.2, J_{1 b;2 a}5.3, J_{1 b;2 b}3.4 Hz, H-1⁰b), 4.12–

4.20 (2H, m, H-2⁰a, H-2⁰b), 4.43 (1H, dddd, J_2,NH

8.9, J_2,12.4, J_2,32.0, J_2,41.3, Hz, H-2), 5.00 (1H,

ddd, J_3,44.8, J_3,22.0, J_3,10.7 Hz, H-3), 5.22 (1H, dd,

J_1,22.4, J_1,30.7 Hz, H-1), 5.75 (1H, br d, J_NH,2

8.9 Hz, NH), 6.22 (1H, dd, J_4,34.8, J_4,21.3 Hz, H-4);

¹³C NMR (CDCl₃): d 20.7 (OC(O)Me), 23.0 (NC(O)Me),

26.9 (OC(O)CMe₃), 38.7 (OC(O)CMe₃), 48.4 (C-2), 52.6

(CO₂Me), 63.0 (C-2⁰), 64.1 (C-3), 66.9 (C-1⁰), 98.0 (C-1),

107.7 (C-4), 142.1 (C-5), 162.3, 170.0, 170.1, 177.5

(CO₂Me, OC(O)Me, NC(O)Me, OC(O)CMe₃). LRMS

m/z 424 ([M+Na]⁺, 100%), 322 (30). Anal. Calcd for

C₁₈H₂₇NO₉ÆH₂O: C, 51.55; H, 6.97; N, 3.34. Found: C,

51.54; H, 6.78; N, 3.21.

0

3.18. Methyl (isopropyl 2-acetamido-2,4-dideoxy-3-O-piva-

loyl-a-^L-threo-hex-4-enopyranosid)uronate (9c)

Prepared from 4c in 80% yield after chromatography

(EtOAc/hexane 3:2) as a colourless syrup. R_f= 0.22

1

(EtOAc/hexane 3:2); H NMR (CDCl₃): d 1.15 (3H, d,

J_{2 ;1}6.5 Hz, H-2⁰), 1.17 (3H, d, J_{2 ;2}6.5 Hz, H-2⁰⁰),

0

00

0

1.19 (9H, s, OPiv), 1.97 (3H, s, NAc), 3.83 (3H, s,

OMe), 3.98 (1H, sept, J_{1 ;2}¼ J_{1 ;2}6.2 Hz, H-1⁰),

4.33–4.40 (1H, m, H-2), 4.97 (1H, br dd, J_3,45.1,

J_3,21.5 Hz, H-3), 5.28 (1H, br d, J_1,22.4 Hz, H-1),

5.64 (1H, br d, J_NH,29.0 Hz, NH), 6.23 (1H, dd, J_4,3

5.1, J_4,21.2 Hz, H-4); ¹³C NMR (CDCl₃): d 21.6, 23.1

(C-2⁰, C-2⁰⁰), 23.2 (NC(O)Me), 26.9 (OC(O)CMe₃),

38.7 (OC(O)CMe₃), 48.8 (C-2), 52.6 (CO₂Me), 64.3

(C-3), 71.6 (C-1⁰), 96.6 (C-1), 107.5 (C-4), 142.3 (C-5),

162.6, 169.6, 177.5 (CO₂Me, NC(O)Me, OC(O)CMe₃).

LRMS m/z 380 ([M+Na]⁺, 100%), 278 (56). HRMS

calcd for C₁₇H₂₇NNaO₇[M+Na] 380.1685. Found

380.1692.

3.21. Methyl (3-O-acetyl-3-hydroxypropyl 2-acetamido-2,4-

dideoxy-3-O-pivaloyl-a-^L-threo-hex-4-enpopyranosid)uro-

nate (9f)

0

00

Prepared from 4f-Ac in 99% yield after chromatography

(EtOAc/hexane 3:1) as a colourless syrup. R_f= 0.16

1

(EtOAc/hexane 3:2); H NMR (CDCl₃): d 1.15 (9H, s,

OPiv), 1.78–1.92 (2H, m, H-2⁰), 1.96 (3H, s, NAc),

0

2.00 (3H, s, OAc), 3.60 (1H, dt, J_{1 a;1 b}9.3, J_{1 a;2}

6.0 Hz, H-1⁰a), 3.81 (3H, s, OMe), 3.84 (1H, dt, J_{1 b;1 a}

0

9.3, J_{1 b;2}6.0 Hz, H-1⁰b), 4.07 (2H, t, J_{3 ;2}6.3 Hz, H-

3⁰), 4.39 (1H, dddd, J_2,NH9.0, J_2,33.6, J_2,12.1, J_2,4

1.2 Hz, H-2), 4.98 (1H, ddd, J_3,44.8, J_3,21.2, J_3,1

0.8 Hz, H-3), 5.16 (1H, dd, J_1,22.1, J_1,30.8 Hz, H-1),

5.91 (1H, br d, J_NH,29.0 Hz, NH), 6.19 (1H, dd, J_4,3

4.8, J_4,21.2 Hz, H-4); ¹³C NMR (CDCl₃): d 20.8

(OC(O)Me), 22.9 (NC(O)Me), 26.9 (OC(O)CMe₃),

28.6 (C-2⁰), 38.6 (OC(O)CMe₃), 48.5 (C-2), 52.6

(CO₂Me), 60.9 (C-3⁰), 64.3 (C-3), 65.4 (C-1⁰), 98.0 (C-

1), 107.8 (C-4), 142.0 (C-5), 162.4, 169.6, 170.9, 177.5

(CO₂Me, NC(O)Me, OC(O)Me, OC(O)CMe₃). LRMS

0

3.19. Methyl (2-methylpropyl 2-acetamido-2,4-dideoxy-3-

O-pivaloyl-a-L-threo-hex-4-enopyranosid)uronate (9d)

Prepared from 4d in 95% yield after chromatography

(EtOAc/hexane 4:3) as a colourless syrup. R_f= 0.17

1

(EtOAc/hexane 1:1); H NMR (CDCl₃): d 0.88 (3H, d,

J_{3 ;2}6.9 Hz, H-3⁰), 0.89 (3H, d, J_{3 ;2}6.9 Hz, H-3⁰⁰),

0

00

0

00

1.21 (9H, s, OPiv), 1.84 (1H, septt, J_{2 ;3}¼ J_{2 ;3}6.9,

1530

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

m/z 438.5 ([M+Na]⁺, 100%). HRMS calcd for

C₁₉H₂₉NNaO₉[M+Na] 438.1740. Found 438.1743.

3.24. Methyl (2,3-dihydroxy-2,3-O-isopropylidenepropyl 2-

acetamido-2,4-dideoxy-3-O-pivaloyl-a-^L-threo-hex-4-eno-

pyranosid)uronate (9i)

3.22. Methyl (2-O-acetyl-2-hydroxypropyl 2-acetamido-2,4-

dideoxy-3-O-pivaloyl-a-^L-threo-hex-4-enopyranosid)uro-

nate (9g)

Prepared as a 3:2 diastereomeric mixture from 4i in 99%

yield after chromatography (EtOAc/hexane 3:1) as a col-

1

ourless syrup. R_f= 0.27 (EtOAc/hexane 3:1); H NMR

Prepared as a 1:1 diastereomeric mixture from 4g-Ac

in 81% yield after chromatography (EtOAc/hexane

3:2 ! 3:1) as a colourless syrup. R_f= 0.31 (EtOAc/hex-

(CDCl₃): d 1.19 (18H, s, 2· OPiv), 1.33, 1.39 (2· 6H,

2· s, 4· O₂CMe₂), 1.98 (6H, s, 2· NAc), 3.59 (1H, dd,

J_{1 a;1 b}10.2, J_{1 a;2}6.3 Hz, H-1⁰a), 3.65 (1H, dd, J_{3 a;3 b}

0

1

0

ane 3:1); H NMR (CDCl₃): d 1.11, 1.12 (2· 9H, 2· s,

8.4, J_{3 a.2}6.0 Hz, H-3⁰a), 3.67 (1H, dd, J_{1 a;1 b}10.2,

2· OPiv), 1.13 (3H, d, J_{3 ;2}6.6 Hz, H-3⁰), 1.14 (3H, d,

J_{1 a;2}6.0 Hz, H-1⁰a), 3.72 (1H, dd, J_{1 b;1 a}10.2, J_{1 b;2}

0

J_{3 ;2}6.6 Hz, H-3⁰), 1.92 (2· 3H, s, 2· NAc), 1.93, 1.94

6.0 Hz, H-1⁰b), 3.80 (1H, dd, J_{3 a;3 b}8.4, J_{3 a;2}6.0 Hz,

0

(2· 3H, 2· s, 2· OAc), 3.55 (1H, dd, J_{1 a;1 b}10.5, J_{1 a;2}

H-3⁰a), 3.84 (1H, dd, J_{1 b;1 a}10.2, J_{1 b;2}6.3 Hz, H-1⁰b),

0

6.6 Hz, H-1⁰a), 3.58 (1H, dd, J_{1 a;1 b}10.8, J_{1 a;2}4.2 Hz,

3.84 (6H, s, 2· OMe), 3.98 (1H, dd, J_{3 b;3 a}8.4, J_{3 b;2}

0

H-1⁰a), 3.68 (1H, dd, J_{1 b;1 a}10.5, J_{1 b;2}3.9 Hz, H-1⁰b),

6.3 Hz, H-3⁰b), 4.01 (1H, dd, J_{3 b;3 a}8.4, J_{3 b;2}6.3 Hz,

H-3⁰b), 4.16–4.26 (2H, m, 2· H-2⁰), 4.42–4.49 (2H, m,

2· H-2), 4.99–5.04 (2H, m, 2· H-3), 5.22–5.26 (2H, m,

2· H-1), 5.60 (1H, br d, J_NH,29.0 Hz, NH), 5.60 (1H,

br d, J_NH,29.0 Hz, NH), 6.21–6.26 (2H, m, 2· H-4);

¹³C NMR (CDCl₃): d 22.9 (2· NC(O)Me), 25.2, 26.7

(2· O₂CMe₂), 26.7, 26.9 (2· OC(O)CMe₃), 38.7 (2·

OC(O)CMe₃), 48.4 (2· C-2), 52.6, 52.7 (2· CO₂Me),

64.2, 64.3 (2· C-3), 66.5, 66.8 (2· C-3⁰), 69.4, 69.9 (2·

C-1⁰), 73.9, 74.0 (2· C-2⁰), 98.0, 98.3 (2· C-1), 107.9

(2· C-4), 109.3, 109.5 (2· O₂CMe₂), 141.9, 142.0 (2·

C-5), 162.3, 169.6, 177.5 (2· CO₂Me, 2· NC(O)Me, 2·

OC(O)CMe₃). LRMS m/z 452.5 ([M+Na]⁺, 100%).

HRMS calcd for C₂₀H₃₁NNaO₉[M+Na] 452.1897.

Found 452.1900.

0

3.71 (1H, dd, J_{1 b;1 a}10.8, J_{1 b;2}5.1 Hz, H-1⁰b), 3.77

(6H, s, 2· OMe), 4.31–4.38 (2H, m, 2· H-2), 4.86–

4.99 (4H, m, 2· H-3, 2· H-2⁰), 5.13–5.16 (2H, m, 2·

H-1), 6.08–6.17 (4H, m, 2· H-4, 2· NH); ¹³C NMR

(CDCl₃): d 16.3 (2· C-3⁰), 20.9 (2· OC(O)Me), 22.8

(2· NC(O)Me), 26.8 (2· OC(O)CMe₃), 38.5, 38.6 (2·

OC(O)CMe₃), 48.4, 48.5 (2· C-2), 52.5 (2· CO₂Me),

64.0, 64.2 (2· C-3), 68.7, 69.0 (2· C-2⁰), 70.6, 71.1

(2· C-1⁰), 97.6, 98.0 (2· C-1), 107.8, 107.9 (2· C-4),

141.8, 141.9 (2· C-5), 162.3, 169.7, 170.2, 177.4 (2·

CO₂Me, 2· NC(O)Me, 2· OC(O)Me, 2· OC(O)-

CMe₃). LRMS m/z 438 ([M+Na]⁺, 100%). HRMS

calcd for C₁₉H₂₉NNaO₉[M+Na] 438.1740. Found

438.1744.

0

3.23. Methyl (1-O-acetyl-1-hydroxyisopropyl 2-acetami-

do-2,4-dideoxy-3-O-pivaloyl-a-^L-threo-hex-4-enopyrano-

sid)uronate (9h)

3.25. Methyl (2,3-dihydroxypropyl 2-acetamido-2,4-dide-

oxy-3-O-pivaloyl-a-^L-threo-hex-4-enopyranosid)uronate

(9i-OH)

Prepared as a 5:1 diastereomeric mixture from 4h-Ac in

80% yield after chromatography (EtOAc/hexane

3:2 ! 3:1) as a colourless syrup. R_f= 0.11 (EtOAc/hex-

ane 1:1); ¹H NMR (CDCl₃) including partial assignment

Compound 9i (147 mg, 0.342 mmol) was dissolved in

TFA/H₂O 1:1 (2 mL) at 0 ꢁC. The solution was stirred

at 0 ꢁC and monitored by TLC analysis (EtOAc/MeOH

20:1). After 2 h, the reaction mixture was diluted with

toluene (0.5 mL) and then concentrated in vacuo. The

crude product was puriﬁed by ﬂash chromatography

(EtOAc/MeOH 20:1) to give a 3:2 diastereomeric

mixture of the title compound 9i-OH (104 mg, 78%) as

an amorphous mass. R_f= 0.22 (EtOAc/MeOH 20:1);

¹H NMR (CDCl₃): d 1.18 (18H, s, 2· OPiv), 1.99 (6H,

s, 2· NAc), 2.99 (4H, br s, 4· OH), 3.52–3.82 (10H,

m, 4· H-1⁰, 2· H-2⁰, 4· H-3⁰), 3.87 (6H, s, 2· OMe),

4.39–4.48 (2H, m, 2· H-2), 5.12–5.21 (2H, m, 2· H-3),

5.22–5.28 (2H, m, 2· H-1), 6.21–6.20 (4H, 2· NH, 2·

H-4); ¹³C NMR (CDCl₃): d 23.1 (2· NC(O)Me), 27.0

(2· OC(O)CMe₃), 38.8 (2· OC(O)CMe₃), 49.1, 49.2

(2· C-2), 52.8 (2· CO₂Me), 63.3 (2· C-3⁰), 65.1, 65.2

(2· C-3), 70.4, 70.6 (2· C-2⁰), 70.8 (2· C-1⁰), 98.8, 98.9

(2· C-1), 108.1, 108.2 (2· C-4), 142.2, 142.3 (2· C-5),

162.3, 162.4, 170.4, 177.8 (2· CO₂Me, 2· NC(O)Me,

2· OC(O)CMe₃). LRMS m/z 412 ([M+Na]⁺, 100%).

HRMS calcd for C₁₇H₂₇NNaO₉[M+Na] 412.1584.

Found 412.1584.

0

of the minor diastereomer: d 1.12 (3H, d, J_{3 ;1}6.3 Hz, H-

3⁰), 1.15 (18H, s, OPiv, OPiv*), 1.95 (3H, s, NAc), 2.06

(3H, s, OAc), 3.81 (6H, s, OMe, OMe*), 3.94 (1H, dd,

J_{2 a;2 b}12.1, J_{2 a;1}7.8 Hz, H-2⁰a), 4.04 (1H, dd, J_{2 b;2 a}

0

12.1, J_{2 b;1}3.3 Hz, H-2⁰b), 4.04 (1H, ddd, J_{1 ;2 a}7.8,

0

J_{1 ;3}6.3, J_{1 ;2 b}3.3 Hz, H-1⁰), 4.37 (1H, dddd, J_2,NH

9.0, J_2,12.4, J_2,31.5, J_2,41.2 Hz, H-2), 4.95 (1H, ddd,

J_3,44.8, J_3,21.5, J_3,1< 1 Hz, H-3), 5.27 (1H, dd, J_1,2

2.1, J_1,3< 1 Hz, H-1*), 5.33 (1H, dd, J_1,22.4,

J_1,3< 1 Hz, H-1), 5.80 (1H, br d, J_NH,29.0 Hz, NH),

5.85 (1H, br d, J_NH,29.0 Hz, NH*), 6.20 (1H, dd, J_4,3

4.8, J_4,51.2 Hz, H-4), 6.21 (1H, m, H-4*); ¹³C NMR

(CDCl₃): d 16.1 (C-3⁰*), 17.9 (C-3⁰), 20.5 (OC(O)Me*),

20.7 (OC(O)Me), 23.0 (NC(O)Me, NC(O)Me*), 26.8

(OC(O)CMe₃), 27.0 (OC(O)CMe₃*), 38.6 (OC(O)CMe₃,

OC(O)CMe₃*), 48.5 (C-2), 48.6 (C-2*), 52.5 (CO₂Me),

52.6 (CO₂Me*), 64.0 (C-3), 64.1 (C-3*), 67.1 (C-2⁰*),

67.2 (C-2⁰), 71.8 (C-1⁰*), 73.6 (C-1⁰), 95.6 (C-1*), 97.8

(C-1), 107.6 (C-4, C-4*), 142.1 (C-5, C-5*), 162.4,

162.5, 169.5, 169.6, 170.6, 170.8 177.4 (NC(O)Me,

NC(O)Me*, OC(O)Me, OC(O)Me*, CO₂Me, CO₂Me*,

OC(O)CMe₃, OC(O)CMe₃*), *minor diastereomer.

LRMS m/z 438 ([M+Na]⁺, 100%). HRMS calcd for

C₁₉H₂₉NNaO₉[M+Na] 438.1740. Found 438.1747.

0

3.26. General procedure for the synthesis of 2a–2i

A solution of compound 9a–9h or 9i–OH (ꢀ0.4 mmol)

in aq MeOH (50%, 5 mL) was adjusted to pH 13 using

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1531

aq NaOH (0.5 M). The solution was stirred at rt and

monitored by TLC analysis (EtOAc/MeOH/H₂O

7:2:1). After 18 h, Amberlite^ꢂIR-120 (H⁺) resin was

added to adjust to pH 3, the reaction mixture was ﬁl-

tered, the resin was washed with MeOH/H₂O 1:1

(30 mL) and the ﬁltrate was concentrated to dryness. Pi-

vOH was then removed by evaporation under reduced

pressure (ꢀ1 mmHg) at 40 ꢁC for 3 h. The residue was

dissolved in water (5 mL), aq NaOH was added to ad-

just to pH 7.3 and the solution was lyophilised to aﬀord

an amorphous solid. The crude product was puriﬁed by

HPLC and then lyophilised to give 2a–2i (70–87%).

(C-2), 65.0 (C-3), 73.0 (C-1⁰), 97.5 (C-1), 107.2 (C-4),

145.1 (C-5), 169.3, 174.3 (CO₂Na, NC(O)Me). LRMS

m/z 282 ([M+H]⁺, 100%). HRMS calcd for

C₁₁H₁₆NNa₂O₆[M+Na] 304.0773. Found 304.0772.

3.30. Sodium (2-methylpropyl 2-acetamido-2,4-dideoxy-a-

L-threo-hex-4-enopyranosid)uronate (2d)

Prepared from 9d in 87% yield after HPLC (5%

CH₃CN in water) as an amorphous solid. R_f= 0.20

(EtOAc/MeOH/H₂O 7:2:1); ¹H NMR (D₂O): d 0.68

(3H, d, J_{3 ;2}6.6 Hz, H-3⁰), 0.69 (3H, d, J_{3 ;2}6.6 Hz,

0

00

0

H-3⁰⁰), 1.68 (1H, septt, J_{2 ;1}6.9, J_{2 ;3}¼ J_{2 ;3}6.6 Hz,

0

00

3.27. Sodium (ethyl 2-acetamido-2,4-dideoxy-a-^L-threo-hex-

4-enopyranosid)uronate (2a)

H-2⁰), 1.84 (3H, s, NAc), 3.25 (1H, dd, J_{1 a;1 b}9.9,

0

J_{1 a;2}6.9 Hz, H-1a⁰), 3.48 (1H, dd, J_{1 b;1 a}9.9, J_{1 b;2}

0

6.9 Hz, H-1⁰b), 3.90 (1H, br dd, J_2,1= J_2,35.4 Hz, H-

2), 4.04 (1H, br dd, J_3,25.4, J_3,43.9 Hz, H-3), 4.91

(1H, br d, J_1,25.4 Hz, H-1), 5.69 (1H, br d, J_4,3

Prepared from 9a in 86% yield after reverse-phase

HPLC (1% CH₃CN in water) as a creamy-coloured

13

amorphous mass. R_f= 0.14 (EtOAc/MeOH/H₂O

0

3.9 Hz, H-4); C NMR (CD₃OD): d 19.5 (C-3⁰, C-

1

0

7:2:1); H NMR (D₂O): d 1.04 (3H, t, J_{2 ;1}7.2 Hz, H-

3⁰⁰), 22.6 (NC(O)Me), 29.4 (C-2⁰), 49.9 (C-2), 66.0 (C-

3), 77.3 (C-1⁰), 100.7 (C-1), 113.0 (C-4), 142.6 (C-5),

165.8, 173.4 (CO₂Na, NC(O)Me). LRMS m/z 296

([M+H]⁺, 100%). Anal. Calcd for C₁₂H₁₈NNaO₆ÆH₂O:

C, 46.01; H, 6.43; N, 4.47. Found: C, 45.46; H, 6.42;

N, 4.16.

2⁰), 1.87 (3H, s, NAc), 3.54 (1H, dq, J_{1 a;1 b}10.2, J_{1 a;2}

0

7.2 Hz, H-1⁰a), 3.71 (1H, dq, J_{1 b;1 a}10.2, J_{1 b;2}7.2 Hz,

H-1⁰b), 3.94 (1H, ddd, J_2,14.8, J_2,34.2, J_2,40.9 Hz, H-

2), 4.01 (1H, ddd, J_3,2= J_3,44.2, J_3,10.6 Hz, H-3), 5.01

(1H, dd, J_1,24.8, J_1,30.6 Hz, H-1), 5.75 (1H, dd, J_4,3

0

13

4.2, J_4,20.9 Hz, H-4); C NMR (D₂O): d 15.1 (C-2⁰),

22.9 (NC(O)Me), 53.0 (C-2), 65.1 (C-3), 66.9 (C-1⁰),

99.7 (C-1), 113.2 (C-4), 141.6 (C-5), 166.4, 175.2

(CO₂Na, NC(O)Me). LRMS m/z 268 ([M+H]⁺, 100%).

HRMS calcd for C₁₀H₁₄NNa₂O₆[M+Na] 290.0617.

Found 290.0615.

3.31. Sodium (2-hydroxyethyl 2-acetamido-2,4-dideoxy-a-

L-threo-hex-4-enopyranosid)uronate (2e)

Prepared from 9e in 80% yield after reverse-phase

HPLC (1% CH₃CN in water) as a creamy-coloured

amorphous mass. R_f= 0.09 (EtOAc/MeOH/H₂O

3.28. Sodium (3-pentyl 2-acetamido-2,4-dideoxy-a-^L-threo-

hex-4-enopyranosid)uronate (2b)

1

7:2:1); H NMR (D₂O): d 1.80 (3H, s, NAc), 3.48–3.59

(3H, m, H-1⁰, H-2⁰a), 3.63–3.73 (1H, m, H-2⁰b), 3.92

(1H, ddd, J_3,44.2, J_3,23.6, J_3,10.6 Hz, H-3), 3.96 (1H,

ddd, J_2,13.9, J_2,33.6, J_2,40.9 Hz, H-2), 5.00 (1H, dd,

J_1,23.9, J_1,30.6 Hz, H-1), 5.80 (1H, dd, J_4,34.2, J_4,2

0.9 Hz, H-4); ¹³C NMR (D₂O): d 22.8 (NC(O)Me),

52.7 (C-2), 61.5 (C-2⁰), 65.0 (C-3), 71.6 (C-1⁰), 99.4 (C-

1), 109.0 (C-4), 144.8 (C-5), 169.4, 175.1 (CO₂Na,

NC(O)Me). LRMS m/z 306 ([M+Na]⁺, 53%), 284

([M]⁺, 100), 261 (38). HRMS calcd for C₁₀H₁₄NNa₂O₇

[M+Na] 306.0566. Found 306.0564.

Prepared from 9b in 75% yield after reverse-phase

HPLC (18% CH₃CN in water) as a creamy-coloured

amorphous mass. R_f= 0.19 (EtOAc/MeOH/H₂O

0

1

0

7:2:1); H NMR (D₂O): d 0.70 (3H, t, J_{3 ;2}7.5 Hz, H-

3⁰), 0.75 (3H, t, J_{3 ;2}7.5 Hz, H-3⁰⁰), 1.30–1.48 (4H, m,

00 00

H-2⁰, H-2⁰⁰), 1.88 (3H, s, NAc), 3.43–3.58 (1H, m, H-

1⁰), 3.96–4.02 (2H, m, H-3, H-2), 5.10 (1H, br d, J_1,2

4.5, H-1), 5.78 (1H, dd, J_4,34.2, J_4,20.9 Hz, H-4); ¹³C

NMR (CD₃OD):

d

9.6, 10.1 (C-3⁰, C-3⁰⁰), 22.6

(NC(O)Me), 27.1, 27.8 (C-2⁰, C-2⁰⁰), 53.7 (C-2), 65.5

(C-3), 83.9 (C-1⁰), 99.5 (C-1), 112.8 (C-4), 142.5 (C-5),

165.7, 173.4 (CO₂Na, NC(O)Me). LRMS m/z 310

([M+H]⁺, 100%). HRMS calcd for C₁₃H₂₀NNa₂O₆

[M+Na] 332.1086. Found 332.1084.

3.32. Sodium (3-hydroxypropyl 2-acetamido-2,4-dideoxy-

1-a-^L-threo-hex-4-enopyranosid)uronate (2f)

Prepared from 9f in 85% yield after reverse-phase HPLC

(5% CH₃CN in water) as a creamy-coloured amorphous

1

mass. R_f= 0.08 (EtOAc/MeOH/H₂O 7:2:1); H NMR

3.29. Sodium (isopropyl 2-acetamido-2,4-dideoxy-a-^L-threo-

(D₂O): d 1.65 (2H, m, H-2⁰), 1.84 (3H, s, NAc), 3.41-

hex-4-enopyranosid)uronate (2c)¹⁷

3.54 (2H, m, H-3⁰), 3.56 (1H, dt, J_{1 a;1 b}10.2, J_{1 a;2}

0

6.0 Hz, H-1⁰a), 3.76 (1H, dt, J_{1 b;1 a}10.2, J_{1 b;2}6.0 Hz,

H-1⁰b), 3.92 (1H, ddd, J_3,24.5, J_3,43.9, J_3,10.6 Hz, H-

3), 3.99 (1H, ddd, J_2,34.5, J_2,14.2, J_2,40.6 Hz, H-2),

4.96 (1H, dd, J_1,24.2, J_1,30.6 Hz, H-1), 5.73 (1H, dd,

J_4,33.9, J_4,20.6 Hz, H-4); ¹³C NMR (CD₃OD): d 22.6

(NC(O)Me), 33.2 (C-2⁰), 53.6 (C-2), 60.1 (C-3⁰), 65.9

(C-3), 67.8 (C-1⁰), 100.2 (C-1), 111.4 (C-4), 143.7 (C-

5), 167.1, 173.4 (CO₂Na, NC(O)Me). LRMS m/z 298

([M+H]⁺, 100%). HRMS calcd for C₁₁H₁₆NNa₂O₇

[M+Na] 320.0722. Found 320.0721.

0

Prepared from 9c in 80% yield after reverse-phase HPLC

(1% CH₃CN in water) as a creamy-coloured amorphous

1

mass. R_f= 0.15 (EtOAc/MeOH/H₂O 7:2:1); H NMR

(D₂O): d 1.03 (3H, d, J_{2 ;1}6.3 Hz, H-2⁰), 1.05 (3H, d,

0

J_{2 ;1}6.3 Hz, H-2⁰⁰), 1.87 (3H, s, NAc), 3.87 (1H, ddd,

J_2,15.4, J_2,35.1, J_2,40.6 Hz, H-2), 3.93 (1H, sept,

00

0

J_{1 ;2}¼ J_{1 ;2}6.3 Hz, H-1⁰), 4.05 (1H, ddd, J_3,25.1, J_3,4

3.9, J_3,10.6 Hz, H-3), 5.06 (1H, dd, J_1,25.4, J_1,30.6 Hz,

H-1), 5.71 (1H, dd, J_4,33.9, J_4,20.6 Hz, H-4); ¹³C NMR

(D₂O): d 21.0, 21.9 (C-2⁰, C-2⁰⁰), 22.3 (NC(O)Me), 52.5

0

00

1532

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

3.33. Sodium (2-hydroxypropyl 2-acetamido-2,4-dideoxy-

a-^L-threo-hex-4-enopyranosid)uronate (2g)

174.1 (CO₂Na, CO₂Na*, NC(O)Me, NC(O)Me*), *mi-

nor diastereomer. LRMS m/z 314 ([M+H]⁺, 100%).

Anal. Calcd for C₁₁H₁₆NNaO₈ÆH₂O: C, 39.88; H, 5.48;

N, 4.23. Found: C, 40.28; H, 5.69; N, 4.10.

Prepared as a 1:1 diastereomeric mixture from 9g in 85%

yield after reverse-phase HPLC (5% CH₃CN in water)

as a creamy-coloured amorphous mass. R_f= 0.08

(EtOAc/MeOH/H₂O 7:2:1); ¹H NMR (CD₃OD): d

3.36. Methyl 2-acetamido-1-S-acetyl-2-deoxy-3,4-di-O-piva-

loyl-1-thio-b-D-glucopyranuronate (10)

0.97 (6H, d, J_{3 ;2}6.6 Hz, 2· H-3⁰), 1.81 (6H, s, 2·

0

NAc), 3.31 (1H, dd, J_{1 a;1 b}10.5, J_{1 a;2}7.2 Hz, H-1⁰a),

Thiolacetic acid (133 lL, 1.86 mmol) was added to a

solution of oxazoline 8 (248 mg, 0.622 mmol) in anhyd

DMF (2.5 mL). The solution was stirred at 80 ꢁC under

N₂. After 18 h, the dark brown reaction mixture was

concentrated in vacuo. Puriﬁcation of the crude product

by ﬂash chromatography (EtOAc/hexane 1:1) aﬀorded

10 as an amorphous mass (189 mg, 64%). For analytical

purposes, recrystallisation from EtOAc/hexane gave 10

as ﬁne white needles. R_f= 0.16 (EtOAc/hexane 1:1);

¹H NMR (CDCl₃): d 1.13, 1.14 (2· 9H, 2· s, 2· OPiv),

1.88 (3H, s, NAc), 2.34 (3H, s, SAc), 3.70 (3H, s, OMe),

4.18 (1H, d, J_5,49.6 Hz, H-5), 4.47 (1H, ddd, J_2,1= J_2,3

10.3, J_2,NH9.9 Hz, H-2), 5.22–5.32 (3H, m, H-1, H-3, H-

4), 5.96 (1H, br d, J_NH,29.9 Hz, NH); ¹³C NMR

(CDCl₃): d 23.0 (NC(O)Me), 27.0 (2· OC(O)CMe₃),

30.7 (SC(O)Me), 38.7, 38.9 (2· OC(O)CMe₃), 51.5 (C-

2), 52.8 (CO₂Me), 68.9 (C-4), 72.7 (C-3), 76.8 (C-5),

81.6 (C-1), 167.0, 170.0, 176.5, 178.6, 193.3 (CO₂Me,

NC(O)Me, 2· OC(O)CMe₃, SC(O)Me). LRMS m/z

498 ([M+Na]⁺, 100%). Anal. Calcd for C₂₁H₃₃NO₉S:

C, 53.04; H, 6.99; N, 2.95. Found: C, 53.02; H, 7.10;

N, 2.93.

0

3.37 (1H, dd, J_{1 a;1 b}9.9, J_{1 a;2}3.9 Hz, H-1⁰a), 3.60

0

(1H, dd, J_{1 b;1 a}9.9, J_{1 b;2}6.9 Hz, H-1⁰b), 3.59 (1H, dd,

0

J_{1 b;1 a}10.5, J_{1 b;2}3.3 Hz, H-1⁰b), 3.79–3.80 (2H, m, 2·

H-2⁰), 3.84–3.89 (2H, m, 2· H-3), 4.01–4.06 (2H, m,

2· H-2), 4.98–5.03 (2H, m, 2· H-1), 6.00–6.04 (2H, m,

0

13

2· H-4); C NMR (CD₃OD): d 19.3, 19.4 (2· C-3⁰),

22.5 (2· NC(O)Me), 53.5 (2· C-2), 65.4 (2· C-3), 67.1,

67.4 (2· C-2⁰), 76.0, 76.1 (2· C-1⁰), 100.1, 100.5 (2· C-

1), 112.6 (2· C-4), 142.5 (2· C-5), 165.9, 166.0, 173.4

(2· CO₂Na, 2· NC(O)Me). LRMS m/z 298 ([M+H]⁺,

100%). HRMS calcd for C₁₁H₁₆NNa₂O₇[M+Na]

320.0722. Found 320.0725.

3.34. Sodium (1-hydroxyisopropyl 2-acetamido-2,4-dide-

oxy-a-^L-threo-hex-4-enopyranosid)uronate (2h)

Prepared as a 5:1 diastereomeric mixture from 9h in 78%

yield after reverse-phase HPLC (5% CH₃CN in water)

as a creamy-coloured amorphous mass. R_f= 0.12

1

(EtOAc/MeOH/H₂O 7:2:1); H NMR (D₂O) including

partial assignment of the minor diastereomer: d 1.05

(3H, d, J_{3 ;1}6.3 Hz, H-3⁰), 1.07 (3H, d, J_{3 ;1}6.3 Hz,

0

H-3⁰*), 1.91 (3H, s, NAc), 1.93 (3H, s, NAc*), 3.42

3.37. General procedure for the synthesis of 5a–5d

(1H, m, H-2⁰a*), 3.43 (1H, dd, J_{2 a;2 b}12.0, J_{2 a;1}

0

6.6 Hz, H-2⁰a), 3.53 (1H, dd, J_{2 b;2 a}12.0, J_{2 b;1}3.6 Hz,

Alkyl halide (2.1 mmol) was added to a stirred solution

of 10 (200 mg, 0.42 mmol) in anhyd DMF (2 mL) con-

˚

taining 3 A molecular sieves under Ar. The solution

0

H-2⁰b), 3.89 (1H, ddd, J_{1 ;2 a}6.6, J_{1 ;3}6.3, J_{1 ;2 b}3.6 Hz,

H-1⁰), 3.98 (1H, m, H-3*), 4.01 (1H, br dd, J_3,44.2,

J_3,23.6 Hz, H-3), 4.08 (1H, br dd, J_2,1= J_2,33.6 Hz,

H-2), 4.10 (1H, m, H-2*), 5.14 (1H, br d, J_1,25.1 Hz,

H-1*), 5.22 (1H, br d, J_1,23.6 Hz, H-1), 5.80 (1H, br

d, J_4,33.9 Hz, H-4*), 5.85 (1H, br d, J_4,34.5 Hz, H-4);

¹³C NMR (D₂O): d 15.0 (C-3⁰*), 16.6 (C-3⁰), 21.8

(NC(O)Me, NC(O)Me*), 51.8 (C-2), 52.3 (C-2*), 63.7

(C-3), 64.2 (C-3*), 64.8 (C-2⁰, C-2⁰*), 76.5 (C-1⁰*), 77.8

(C-1⁰), 97.5 (C-1*), 98.7 (C-1), 111.7 (C-4, C-4*), 140.8

(C-5), 141.0 (C-5*), 165.6, 174.3 (CO₂Na*, NC(O)Me*),

165.7, 174.2 (CO₂Na, NC(O)Me), *minor diastereomer.

LRMS m/z 298 ([M+H]⁺, 100%). HRMS calcd for

C₁₁H₁₆NNa₂O₇[M+Na] 320.0722. Found 320.0724.

0

was cooled to 4 ꢁC, and then HNEt₂(0.9 mL, 8 mmol)

was added. After 24 h, the reaction mixture was concen-

trated to remove HNEt₂. The residue was diluted with

EtOAc (15 mL), and then ﬁltered. The ﬁltrate was

washed with dilute HCl (1 M, 15 mL), water (15 mL),

saturated aq NaCl (15 mL), dried (Na₂SO₄), ﬁltered

and concentrated. Puriﬁcation of the crude product by

ﬂash chromatography aﬀorded 5a–5d (73–76%).

3.38. Methyl (3-hydroxypropyl 2-acetamido-2-deoxy-3,4-

di-O-pivaloyl-1-thio-b-^D-glucopyranosid)uronate (b-5a)

Prepared from reaction between 10 and 3-bromopro-

pan-1-ol in 74% yield after chromatography (EtOAc/

hexane 3:1) as an amorphous mass. R_f= 0.20 (EtOAc/

hexane 3:1); ¹H NMR (CDCl₃): d 1.13 (18H, s, 2·

OPiv), 1.72–2.02 (2H, m, H-2⁰), 1.94 (3H, s, NAc),

2.52–2.59 (1H, m, OH) 2.82–2.99 (2H, m, H-1⁰), 3.67–

3.78 (2H, m, H-3⁰), 3.72 (3H, s, OMe), 4.01 (1H, d,

J_5,49.5 Hz, H-5), 4.29 (1H, ddd, J_2,110.3, J_2,310.0,

J_2,NH9.7 Hz, H-2), 4.58 (1H, d, J_1,210.3 Hz, H-1),

5.18–5.29 (2H, m, H-3, H-4), 6.07 (1H, br d, J_NH,2

9.7 Hz, NH); ¹³C NMR (CDCl₃): d 23.0 (NC(O)Me),

25.5 (C-1⁰), 26.9 (2· OC(O)CMe₃), 31.6 (C-2⁰), 38.6,

38.8 (2· OC(O)CMe₃), 52.2 (C-2), 52.7 (CO₂Me), 59.8

(C-3⁰), 69.1 (C-4), 72.3 (C-3), 76.2 (C-5), 84.3 (C-1),

167.5, 170.5, 176.5, 178.3 (CO₂Me, NC(O)Me, 2·

3.35. Sodium (2,3-dihydroxypropyl 2-acetamido-2,4-dide-

oxy-a-^L-threo-hex-4-enopyranosid)uronate (2i)

Prepared as a 3:2 diastereomeric mixture from 9i-OH in

70% yield after reverse-phase HPLC (5% CH₃CN in

water) as an amorphous solid. R_f= 0.04 (EtOAc/

1

MeOH/H₂O 7:2:1); H NMR (D₂O): d 1.84 (6H, s, 2·

NAc), 3.33-3.77 (10H, m, 2· H-1⁰, 2· H-2⁰, 2· H-3⁰),

3.93–4.01 (4H, m, 2· H-2, 2· H-3), 4.98–5.02 (2H, m,

2· H-1), 5.74–5.78 (2H, m, 2· H-4); ¹³C NMR (D₂O):

d 21.7 (NC(O)Me, NC(O)Me*), 51.6 (C-2, C-2*), 62.1

(C-3⁰), 62.2 (C-3⁰*), 63.6 (CO₂Me, CO₂Me*), 69.9 (C-

2*), 70.2 (C-2⁰), 70.3 (C-1⁰*), 70.6 (C-1⁰), 98.6 (C-1*),

99.1 (C-1), 112.2 (C-4, C-4*), 140.2 (C-5, C-5*), 165.1,

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1533

OC(O)CMe₃). LRMS m/z 513.5 ([M+Na]⁺, 100%).

Anal. Calcd for C₂₂H₃₇NO₉S: C, 53.75; H, 7.59; N,

2.85. Found: C, 53.57; H, 7.65; N, 2.65.

br d, J_NH,29.3 Hz, NH b); ¹³C NMR (CDCl₃): d 21.6,

21.7, 22.1 (C-3⁰a/b, C-3⁰⁰a/b), 23.0 (NC(O)Me a/b),

26.9, 27.0 (2· OC(O)CMe₃a/b), 28.5 (C-2⁰b), 28.6 (C-

2⁰a), 38.5 (C-1⁰b), 38.6, 38.8 (2· OC(O)CMe₃a/b),

40.4 (C-1⁰a), 51.6 (C-2 a), 52.6 (C-2 b, CO₂Me b),

52.7 (CO₂Me a), 68.8 (C-4 a), 69.2, 72.6 (C-3 b, C-4

b), 69.7, 69.8 (C-3 a, C-5 a), 76.5 (C-5 b), 85.0 (C-1 a/

b), 167.2, 168.0, 169.6, 169.7, 176.3, 178.5, 176.4, 178.6

(CO₂Me a/b, NC(O)Me a/b, 2· OC(O)CMe₃a/b).

LRMS m/z 512.5 ([M+Na]⁺, 100%). Anal. Calcd for

C₂₃H₃₉NO₈S: C, 56.42; H, 8.03; N, 2.86. Found: C,

56.40; H, 8.26; N, 2.73.

3.39. Methyl (2-hydroxyethyl 2-acetamido-2-deoxy-3,4-di-

O-pivaloyl-1-thio-a,b-^D-glucopyranosid)uronate (5b)

Prepared as an anomeric mixture (a/b 1:8) from reaction

between 10 and 2-bromoethan-1-ol in 76% yield after

chromatography (EtOAc/MeOH 10:1) as an amorphous

mass. R_f= 0.20 (EtOAc/MeOH 10:1); ¹H NMR

(CDCl₃) b-anomer: d 1.11 (18H, s, 2· OPiv), 1.92 (3H,

0

s, NAc), 2.04 (1H, br s, OH), 2.77 (1H, ddd, J_{1 a;1 b}

14.0, J_{1 a;2 a}6.3, J_{1 a;2 b}4.7 Hz, H-1⁰a), 3.04 (1H, dt,

3.41. Methyl (isopropyl 2-acetamido-2-deoxy-3,4-di-O-piva-

loyl-1-thio-a,b-^D-glucopyranosid)uronate (5d)

0

J_{1 b;1 a}14.0, J_{1 b;2}5.4 Hz, H-1⁰b), 3.7 (3H, s, OMe),

3.75–3.83 (2H, m, H-2⁰), 4.15 (1H, d, J_5,49.9 Hz, H-5),

4.26 (1H, ddd, J_2,1= J_2,3= J_2,NH9.9 Hz, H-2), 4.74

(1H, d, J_1,210.2 Hz, H-1), 5.19 (1H, dd, J_4,3= J_4,5

9.9 Hz, H-4), 5.30 (1H, dd, J_3,2= J_3,49.9 Hz, H-3),

6.47 (1H, br d, J_NH,29.6 Hz, NH); a-anomer: d 1.13,

1.18 (2· 9H, 2· s, 2· OPiv), 1.95 (3H, s, NAc), 2.81

0

Prepared as an anomeric mixture (a/b 1:2) from reaction

between 10 and 2-iodopropane in 73% yield after chro-

matography (EtOAc/hexane 2:3) as an amorphous mass.

Crystallisation of the product from EtOAc/hexane aﬀor-

ded pure b-anomer of 5d. Compound a-5d: R_f= 0.23

(EtOAc/hexane 2:3); ¹H NMR (CDCl₃): d 1.14, 1.17

(1H, dt, J_{1 a;1 b}14.0, J_{1 a;2}5.8 Hz, H-1⁰a), 2.94 (1H, dt,

0

J_{1 b;1 a}14.0, J_{1 b;2}6.0 Hz, H-1⁰b), 3.74 (3H, s, OMe),

3.76–3.87 (2H, m, H-2⁰), 4.59 (1H, ddd, J_2,310.2,

J_2,NH9.0, J_2,15.0 Hz, H-2), 4.73 (1H, d, J_5,49.1 Hz,

H-5), 5.12 (1H, dd, J_3,210.2, J_3,48.4 Hz, H-3), 5.26

(1H, dd, J_4,59.1, J_4,38.4 Hz, H-4), 5.55 (1H, d, J_1,2

5.0 Hz, H-1), 5.90 (1H, br d, J_NH,29.0 Hz, NH); ¹³C

NMR (CDCl₃): d 23.0 (NC(O)Me a/b), 26.9, 27.0 (2·

OC(O)CMe₃a/b), 33.6 (C-1⁰b), 34.6 (C-1⁰a), 38.6,

38.8 (2· OC(O)CMe₃a/b), 51.4 (C-2 a), 52.5 (C-2 b),

52.7 (CO₂Me a/b), 61.4 (C-2⁰a), 62.1 (C-2⁰b), 68.6

(C-4 a), 69.0 (C-4 b), 69.6 (C-3 a), 72.3 (C-3 b), 69.9

(C-5 a), 76.0 (C-5 b), 84.5 (C-1 a/b), 167.3, 168.0,

169.9, 170.3, 176.4, 178.4, 176.5, 178.2 (CO₂Me a/b,

NC(O)Me a/b, 2· OC(O)CMe₃a/b). LRMS m/z 500

([M+Na]⁺, 100%). Anal. Calcd for C₂₁H₃₅NO₉SÆ1/

3H₂O: C, 52.16; H, 7.43; N, 2.90. Found: C, 52.28; H,

7.46; N, 2.70.

(2· 9H, 2· s, 2· OPiv), 1.32 (3H, d, J_{2 ;1}6.8 Hz, H-

0

2⁰), 1.34 (3H, d, J_{2 ;1}6.8 Hz, H-2⁰⁰), 1.94 (3H, s, NAc),

00

0

3.12 (1H, sept, J_{1 ;2}¼ J_{1 ;2}6.8 Hz, H-1⁰), 3.74 (3H, s,

OMe), 4.61 (1H, ddd, J_2,310.7, J_2,NH9.4, J_2,15.2 Hz,

H-2), 4.72 (1H, d, J_5,49.4 Hz, H-5), 5.11 (1H, dd, J_3,2

10.7, J_3,48.9 Hz, H-3), 5.25 (1H, dd, J_4,59.4,J_4,3

8.9 Hz, H-4), 5.51 (1H, d, J_1,25.2 Hz, H-1), 5.66 (1H,

br d, J_NH,29.2 Hz, NH); ¹³C NMR (CDCl₃): d 23.1

(NC(O)Me), 23.6, 23.8 (C-2⁰, C-2⁰⁰), 27.0 (2·

OC(O)CMe₃), 36.4 (C-1⁰), 38.7, 38.9 (2· OC(O)CMe₃),

51.6 (C-2), 52.7 (CO₂Me), 68.8 (C-4), 70.0 (C-3, C-5),

83.4 (C-1), 168.1, 169.5, 176.4, 178.6 (CO₂Me,

NC(O)Me, 2· OC(O)CMe₃). LRMS m/z 498

([M+Na]⁺, 100%), 476 (66), 153 (82). Compound b-5d:

R_f= 0.23 (EtOAc/hexane 2:3); ¹H NMR (CDCl₃): d

0

00

0

1.14, 1.15 (2· 9H, 2· s, 2· OPiv), 1.27 (3H, d, J_{2 ;1}

6.8 Hz, H-2⁰), 1.32 (3H, d, J_{2 ;1}6.8 Hz, H-2⁰⁰), 1.93

00

0

00

(3H, s, NAc), 3.23 (1H, sept, J_{1 ;2}¼ J_{1 ;2}6.8 Hz, H-

1⁰), 3.73 (3H, s, OMe), 4.05 (1H, d, J_5,49.6 Hz, H-5),

4.17 (1H, ddd, J_2,110.2, J_2,NH9.6, J_2,310.5 Hz, H-2),

4.71 (1H, d, J_1,210.2 Hz, H-1), 5.23–5.31 (2H, m, H-3,

H-4), 5.38 (1H, br d, J_NH,29.6 Hz, NH); ¹³C NMR

(CDCl₃): d 23.2 (NC(O)Me), 23.5, 24.2 (C-2⁰, C-2⁰⁰),

27.0 (2· OC(O)CMe₃), 34.8 (C-1⁰), 38.7, 38.9 (2·

OC(O)CMe₃), 52.7 (CO₂Me), 53.0 (C-2), 69.1 (C-4),

72.6 (C-3), 76.5 (C-5), 84.4 (C-1), 167.2, 169.7, 176.4,

178.5 (CO₂Me, NC(O)Me, 2· OC(O)CMe₃). LRMS

m/z 498 ([M+Na]⁺, 100%), 476 (66), 153 (82). Anal.

Calcd for C₂₂H₃₇NO₈S: C, 55.56; H, 7.84; N, 2.95.

Found: C, 53.34; H, 7.95; N, 2.90.

3.40. Methyl (isobutyl 2-acetamido-2-deoxy-3,4-di-

O-pivaloyl-1-thio-a,b-^D-glucopyranosid)uronate (5c)

Prepared as an anomeric mixture (a/b 1:9) from reaction

between 10 and isobutyl iodide in 73% yield after chro-

matography (EtOAc/hexane 2:3) as an amorphous mass.

R_f= 0.26 (EtOAc/hexane 2:3); ¹H NMR (CDCl₃): d 0.98

(3H, d, J_{3 ;2}6.6 Hz, H-3⁰b), 0.99 (3H, d, J_{3 ;2}6.6 Hz, H-

0

00

0

3⁰⁰b), 1.00 (3H, d, J_{3 ;2}6.6 Hz, H-3⁰a), 1.01 (3H, d, J_{3 ;2}

0

00

0

6.6 Hz, H-3⁰⁰a), 1.14, 1.15, 1.17 (36H, 3· s, 4· OPiv a/

b), 1.74–1.91 (2H, m, H-2⁰a/b), 1.94 (3H, s, NAc b),

0

1.95 (3H, s, NAc a), 2.50 (1H, dd, J_{1 a;1 b}12.9, J_{1 a;2}

7.2 Hz, H-1⁰a a), 2.61 (1H, dd, J_{1 b;1 a}12.9, J_{1 b;2}

0

7.2 Hz, H-1⁰b a), 2.59 (1H, dd, J_{1 a;1 b}9.3, J_{1 a;2}

3.42. General procedure for the synthesis of 11a–11d

0

6.6 Hz, H-1⁰a b), 2.65 (1H, dd, J_{1 b;1 a}9.3, J_{1 b;2}6.6 Hz,

H-1⁰b b), 3.73 (3H, s, OMe b), 3.74 (3H, s, OMe a),

4.04 (1H, d, J_5,49.6 Hz, H-5 b), 4.23 (1H, ddd, J_2,1

10.5, J_2,39.9, J_2,NH9.3 Hz, H-2 b), 4.54 (1H, d, J_1,2

10.5 Hz, H-1 b), 4.59 (1H, ddd, J_2,310.5, J_2,NH9.3,

J_2,15.1 Hz, H-2 a), 4.71 (1H, d, J_5,49.3 Hz, H-5 a),

5.13 (1H, dd, J_3,210.5, J_3,49.0 Hz, H-3 a), 5.18–5.28

(3H, m, H-4 a, H-3 b, H-4 b), 5.40 (1H, d, J_1,25.1 Hz,

H-1 a), 5.68 (1H, br d, J_NH,29.3 Hz, NH a), 5.72 (1H,

0

DBU (150 lL, 0.76 mmol) was added to a solution of

an anomeric mixture of compound 5a–Ac, 5b–Ac, 5c,

or 5d (0.38 mmol) in anhyd CH₂Cl₂(2 mL) under N₂.

The pale yellow solution was stirred at rt and moni-

tored by TLC analysis. After 18 h, the reaction mix-

ture was concentrated under reduced pressure to give

a clear yellow syrup. Puriﬁcation of the crude prod-

uct by ﬂash chromatography aﬀorded 11a–11d

1534

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

[90–100%, based on recovered starting material (b-

anomer)].

Puriﬁcation of the crude product by ﬂash chromatogra-

phy (EtOAc/hexane 3:2) aﬀorded 5b-Ac [R_f= 0.30

(EtOAc/hexane 3:2); 174 mg, 97%] as an amorphous

mass. Treatment of an anomeric mixture of 5b-Ac (a/

b 3:2) with DBU aﬀorded pure b-anomer of 11b

(37% from 5b-Ac) after chromatography (EtOAc/hex-

ane 3:2 ! 3:1) as a colourless syrup. The a-anomer

of 11b (53% from 5b-Ac) was also isolated. Compound

a-11b: R_f= 0.22 (EtOAc/hexane 3:2); ¹H NMR

(CDCl₃): d 1.20 (9H, s, OPiv), 1.99 (3H, s, NAc),

3.43. Methyl (3-O-acetyl-3-hydroxypropyl 2-acetamido-

2,4-dideoxy-3-O-pivaloyl-1-thio-a-^L-threo-hex-4-enopyr-

anosid)uronate (11a)

Acetic anhydride (0.1 mL, 1 mmol) was added to a solu-

tion of an anomeric mixture of 5a (162 mg, 0.33 mmol)

in anhyd pyridine (3 mL). The solution was stirred over-

night at rt under N₂. The reaction was quenched by

addition of MeOH (0.5 mL) and then concentrated.

The residue was diluted with EtOAc (10 mL), washed

with dilute HCl (0.1 M, 10 mL), water (2· 10 mL), dried

(Na₂SO₄), ﬁltered and concentrated. Puriﬁcation of the

crude product by ﬂash chromatography (EtOAc/hexane

3:2) aﬀorded 5a-Ac [R_f= 0.24 (EtOAc/hexane 1:1);

155 mg, 88%] as an amorphous mass. Treatment of an

anomeric mixture of 5a–Ac (a/b 2:3) with DBU aﬀorded

pure b-anomer of 11a (40% from 5a-Ac) after chroma-

tography (EtOAc/hexane 2:1) as a colourless syrup. A

mixture containing recovered starting material 5a–Ac

(b-anomer) (20% from 5a-Ac) and the a-anomer of

11a (40% from 5a-Ac) was also isolated. Compound a-

0

2.07 (3H, s, OAc), 2.94 (1H, dt, J_{1 a;1 b}14.1,

J_{1 a;2 a}¼ J_{1 a;2 b}6.6 Hz, H-1⁰a), 3.08 (1H, dt, J_{1 b;1 a}

0

14.1, J_{1 b;2 a}¼ J_{1 b;2 b}6.6 Hz, H-1⁰b), 3.83 (3H, s,

0

OMe), 4.23 (1H, dt, J_{2 a;2 b}11.4, J_{2 a;1 a}¼ J_{2 a;1 b}

6.6 Hz, H-2⁰a), 4.40 (1H, dt, J_{2 b;2 a}11.4,

0

J_{2 b;1 a}¼ J_{2 b;1 b}6.6 Hz, H-2⁰b), 4.58 (1H, ddd, J_2,NH

8.4, J_2,36.9, J_2,13.6 Hz, H-2), 5.33 (1H, dd, J_3,26.9,

J_3,43.6 Hz, H-3), 5.51 (1H, d, J_1,23.6 Hz, H-1), 5.71

(1H, br d, J_NH,28.4 Hz, NH), 6.12 (1H, d, J_4,3

3.6 Hz, H-4); ¹³C NMR (CDCl₃): d 20.7 (OC(O)Me),

22.9 (NC(O)Me), 26.9 (OC(O)CMe₃), 30.1 (C-1⁰),

38.9 (OC(O)CMe₃), 49.5 (C-2), 52.5 (CO₂Me), 63.3

(C-2⁰), 65.6 (C-3), 83.9 (C-1), 108.5 (C-4), 143.7 (C-

5), 161.7, 170.0, 170.7, 178.0 (CO₂Me, NC(O)Me,

OC(O)CMe₃, OC(O)Me). LRMS m/z 440 ([M+Na]⁺,

100%). Compound b-11b: R_f= 0.11 (EtOAc/hexane

3:2); ¹H NMR (CDCl₃): d 1.23 (9H, s, OPiv), 1.99

(3H, s, NAc), 2.07 (3H, s, OAc), 2.86 (1H, ddd,

0

1

11a: R_f= 0.37 (EtOAc/hexane 2:1); H NMR (CDCl₃)

distinguishable signals in a mixture containing b-5a-

Ac: d 1.21 (9H, s, OPiv), 1.95–2.19 (2H, m, H-2⁰), 1.99

(3H, s, NAc), 2.07 (3H, s, OAc), 2.79–2.92 (2H, m, H-

1⁰), 3.84 (3H, s, OMe), 4.12–4.20 (2H, m, H-3⁰), 4.58

(1H, ddd, J_2,NH8.4, J_2,37.5, J_2,13.3 Hz, H-2), 5.36

(1H, dd, J_3,27.5, J_3,43.6 Hz, H-3), 5.70 (1H, br d,

J_NH,28.4 Hz, NH), 6.12 (1H, d, J_4,33.6 Hz, H-4). Com-

J_{1 a;1 b}14.1, J_{1 a;2 a}7.2, J_{1 a;2 b}6.0 Hz, H-1⁰a), 3.02

0

(1H, ddd, J_{1 b;1 a}14.1, J_{1 b;2 a}¼ J_{1 b;2 b}7.2 Hz, H-1⁰b),

0

3.85 (3H, s, OMe), 4.21 (1H, ddd, J_{2 a;2 b}11.4,

J_{2 a;1 a}¼ J_{2 a;1 b}7.2 Hz, H-2⁰a), 4.36 (1H, ddd, J_{2 b;2 a}

0

1

11.4, J_{2 b;1 a}6.0, J_{2 b;1 b}7.2 Hz, H-2⁰b), 4.52 (1H, dddd,

J_2,NH8.7, J_2,1= J_2,31.8, J_2,41.2 Hz, H-2), 4.98 (1H,

ddd, J_3,44.8, J_3,21.8, J_3,10.9 Hz, H-3), 5.56 (1H, dd,

J_1,21.8, J_1,30.9 Hz, H-1), 5.69 (1H, br d, J_NH,2

8.7 Hz, NH), 6.31 (1H, dd, J_4,34.8, J_4,21.2 Hz, H-4);

0

pound b-11a: R_f= 0.20 (EtOAc/hexane 2:1); H NMR

(CDCl₃): d 1.24 (9H, s, OPiv), 1.95–2.08 (2H, m, H-

2⁰), 1.99 (3H, s, NAc), 2.06 (3H, s, OAc), 2.76 (1H, dt,

J_{1 a;1 b}13.5, J_{1 a;2}7.2 Hz, H-1⁰a), 2.83 (1H, dt, J_{1 b;1 a}

0

13.5, J_{1 b;2}7.2 Hz, H-1⁰b), 3.85 (3H, s, OMe), 4.14

0

(2H, t, J_{3 ;2}6.2 Hz, H-3⁰), 4.52 (1H, dddd, J_2,NH9.0,

J_2,3= J_2,1= 1.8, J_2,41.2 Hz, H-2), 4.98 (1H, ddd, J_3,4

5.1, J_3,21.5, J_3,11.2 Hz, H-3), 5.47 (1H, dd, J_1,22.1,

J_1,31.2 Hz, H-1), 5.59 (1H, br d, J_NH,29.0 Hz, NH),

6.32 (1H, dd, J_4,35.1, J_4,21.2 Hz, H-4); ¹³C NMR

(CDCl₃): d 20.9 (OC(O)Me), 23.0 (NC(O)Me), 27.0

(OC(O)CMe₃), 28.7 (C-2⁰), 29.4 (C-1⁰), 38.9

(OC(O)CMe₃), 49.5 (C-2), 52.6 (OMe), 62.8 (C-3⁰),

64.3 (C-3), 83.2 (C-1), 107.2 (C-4), 143.0 (C-5), 162.1,

169.3, 170.1, 177.3 (CO₂Me, NC(O)Me, OC(O)Me,

OC(O)CMe₃). LRMS m/z 454.5 ([M+Na]⁺, 100%).

HRMS calcd for C₁₉H₂₉NNaSO₈[M+Na] 454.4909.

Found 454.1522.

¹³C NMR (CDCl₃):

d

20.7 (OC(O)Me), 23.0

0

(NC(O)Me), 27.0 (OC(O)CMe₃), 31.1 (C-1⁰), 38.9

(OC(O)CMe₃), 49.3 (C-2), 52.7 (CO₂Me), 63.3 (C-2⁰),

64.3 (C-3), 83.2 (C-1), 107.2 (C-4), 142.9 (C-5), 162.1,

169.3, 170.7, 177.2 (CO₂Me, NC(O)Me, OC(O)Me,

OC(O)CMe₃). LRMS m/z 440 ([M+Na]⁺, 100%).

HRMS calcd for C₁₈H₂₇NNaSO₈[M+Na] 440.1355.

Found 440.1358.

3.45. Methyl (2-methylpropyl 2-acetamido-2,4-dideoxy-3-

O-pivaloyl-1-thio-a-^L-threo-hex-4-enopyranosid)uronate

(11c)

Treatment of anomeric mixture of 5c (a/b 2:3) with

DBU aﬀorded pure b-anomer of 11c (40%) after

chromatography (EtOAc/hexane 1:1) as a colourless

syrup. A mixture of recovered starting material 5c

(b-anomer) (18%) and the a-anomer of 11c (35%)

was also isolated. Compound a-11c: R_f= 0.40

(EtOAc/hexane 1:1); ¹H NMR (CDCl₃) distinguish-

able signals in a mixture containing b-5c: d 0.95–

1.01 (6H, m, H-3⁰, H-3⁰⁰), 1.74–1.91 (1H, m, H-2⁰),

1.93 (3H, s, NAc), 2.55–2.72 (2H, m, H-1⁰), 3.72

(3H, s, OMe), 4.56 (1H, ddd, J_2,NH8.7, J_2,37.2,

J_2,13.3 Hz, H-2), 5.37 (1H, dd, J_3,27.2, J_3,43.3 Hz,

H-3), 5.41 (1H, d, J_1,23.3 Hz, H-1), 5.75 (1H, br

3.44. Methyl (2-O-acetyl-2-hydroxyethyl 2-acetamido-

2,4-dideoxy-3-O-pivaloyl-1-thio-a-^L-threo-hex-4-enopyr-

anosid)uronate (11b)

Acetic anhydride (0.1 mL, 1 mmol) was added to a

solution of an anomeric mixture of 5b (165 mg,

0.35 mmol) in anhyd pyridine (3 mL). The solution

was stirred overnight at rt under N₂. The reaction

was quenched by addition of MeOH (0.5 mL) and then

concentrated. The residue was diluted with EtOAc

(10 mL), washed with dilute HCl (0.1 M, 10 mL), water

(2· 10 mL), dried (Na₂SO₄), ﬁltered and concentrated.

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1535

d, J_NH,28.7 Hz, NH), 6.10 (1H, d, J_4,33.3 Hz, H-4).

3.47. General procedure for the synthesis of 3a–3d

LRMS m/z 410 ([M+Na]⁺, 80%). Compound b-11c:

R_f= 0.22 (EtOAc/hexane 1:1); ¹H NMR (CDCl₃): d

A solution of 11a–11d (ꢀ0.4 mmol) in aq MeOH (50%,

5 mL) was adjusted to pH 13 using aq NaOH (0.5 M).

The solution was stirred at rt and monitored by TLC

analysis (EtOAc/MeOH/H₂O 7:2:1). After 18 h, Amber-

lite^ꢂIR-120 (H⁺) resin was added to adjust to pH 3, the

reaction mixture was ﬁltered, the resin was washed with

MeOH/H₂O 1:1 (30 mL), and the ﬁltrate was concen-

trated to dryness. PivOH was then removed by evapora-

tion under high vacuum (ꢀ1 mmHg) at 40 ꢁC for 3 h.

The residue was dissolved in water (5 mL), aq NaOH

was added to adjust to pH 7.3 and the solution was lyo-

philised to aﬀord an amorphous solid. The crude prod-

uct was puriﬁed by HPLC and then lyophilised to give

3a–3d (70–85%).

0.98 (3H, d, J_{3 ;2}6.6 Hz, H-3⁰), 0.99 (3H, d, J_{3 ;2}

0

00

0

6.6 Hz, H-3⁰⁰), 1.24 (9H, s, OPiv), 1.88 (1H, septt,

J_{2 ;1 a}¼ J_{2 ;1 b}6.9, J_{2 ;3}¼ J_{2 ;3}6.6 Hz, H-2⁰), 1.99

0

00

0

(3H, s, NAc), 2.57 (1H, dd, J_{1 a;1 b}12.6, J_{1 a;2}

6.9 Hz, H-1⁰a), 2.64 (1H, dd, J_{1 b;1 a}12.6, J_{1 b;2}

0

6.9 Hz, H-1⁰b), 3.85 (3H, s, OMe), 4.53 (1H, dddd,

J_2,NH9.0, J_2,1= J_2,31.8, J_2,41.2 Hz, H-2), 4.97 (1H,

ddd, J_3,44.8, J_3,21.8, J_3,11.2 Hz, H-3), 5.47 (1H,

dd, J_1,21.8, J_1,31.2 Hz, H-1), 5.65 (1H, br d, J_NH,2

9.0 Hz, NH), 6.32 (1H, dd, J_4,34.8, J_4,21.2 Hz, H-

13

4); C NMR (CDCl₃): d 21.8, 21.9 (C-3⁰, C-3⁰⁰),

23.2 (NC(O)Me), 27.0 (OC(O)CMe₃), 28.5 (C-2⁰),

38.9 (OC(O)CMe₃), 41.8 (C-1⁰), 49.5 (C-2), 52.7

(CO₂Me), 64.4 (C-3), 83.6 (C-1), 107.2 (C-4), 143.0

(C-5), 162.3, 169.1, 177.3 (CO₂Me, NC(O)Me,

OC(O)CMe₃). LRMS m/z 410 ([M+Na]⁺, 100%).

HRMS calcd for C₁₈H₂₉NNaSO₆[M+Na] 410.1613.

Found 410.1609.

3.48. Sodium (3-hydroxypropyl 2-acetamido-2,4-dideoxy-

1-thio-a-^L-threo-hex-4-enopyranosid)uronate (3a)

Prepared from b-11a in 85% yield after reverse-phase

HPLC (5% CH₃CN in water) as a creamy-coloured

amorphous solid. R_f= 0.09 (EtOAc/MeOH/H₂O 7:2:1);

¹H NMR (D₂O): d 1.67–1.82 (2H, m, H-2⁰), 1.95 (3H,

3.46. Methyl (isopropyl 2-acetamido-2,4-dideoxy-3-O-

pivaloyl-1-thio-a-^L-threo-hex-4-enopyranosid)uronate

(11d)

0

s, NAc), 2.64 (1H, dt, J_{1 a;1 b}13.2, J_{1 a;2}7.2 Hz, H-

1⁰a), 2.74 (1H, dt, J_{1 b;1 a}13.2, J_{1 b;2}7.2 Hz, H-1⁰b),

0

3.55 (2H, t, J_{3 ;2}6.3 Hz, H-3⁰), 4.00 (1H, ddd, J_2,15.4,

J_2,34.5, J_2,40.3 Hz, H-2), 4.10 (1H, ddd, J_3,24.5, J_3,4

3.9, J_3,10.6 Hz, H-3), 5.28 (1H, dd, J_1,25.4, J_1,3

0.6 Hz, H-1), 5.84 (1H, dd, J_4,33.9, J_4,20.3 Hz, H-4);

¹³C NMR (D₂O): d 21.9 (NC(O)Me), 27.8 (C-1⁰), 31.3

(C-2⁰), 52.4 (C-2), 60.0 (C-3⁰), 64.7 (C-3), 82.4 (C-1),

107.6 (C-4), 145.7 (C-5), 168.4, 173.9 (CO₂Na,

NC(O)Me). LRMS m/z 336 ([M+Na]⁺, 100%). Anal.

Calcd for C₁₁H₁₆NNaO₆SÆH₂O: C, 39.88; H, 5.48; N,

4.23. Found: C, 39.70; H, 5.47; N, 4.05.

0

Treatment of an anomeric mixture of 5d (a/b 2:3) with

DBU aﬀorded pure b-anomer of 11d in 47% yield after

chromatography (EtOAc/hexane 1:1 ! 3:2) as a col-

ourless syrup. A mixture of recovered starting material

5d (b-anomer) (12%) and the a-anomer of 11d (41%)

was also isolated. Compound a-11d: R_f= 0.31

1

(EtOAc/hexane 1:1); H NMR (CDCl₃) distinguishable

signals in a mixture containing b-5d: d 1.19 (9H, s,

OPiv), 1.34 (3H, d, J_{2 ;1}6.8 Hz, H-2⁰), 1.35 (3H, d,

0

J_{2 ;1}6.8 Hz, H-2⁰⁰), 1.97 (3H, s, NAc), 3.25 (1H, sept,

00

0

J_{1 ;2}¼ J_{1 ;2}6.8 Hz, H-1⁰), 3.82 (3H, s, OMe), 4.56

(1H, ddd, J_2,NH8.5, J_2,37.2, J_2,13.5 Hz, H-2), 5.33

(1H, dd, J_3,27.2, J_3,43.5 Hz, H-3), 5.49 (1H, d, J_1,2

3.5 Hz, H-1), 5.76 (1H, br d, J_NH,28.5 Hz, NH), 6.09

(1H, d, J_4,33.5 Hz, H-4); ¹³C NMR (CDCl₃) distin-

guishable signals in a mixture containing b-5d: d 23.1

0

00

3.49. Sodium (2-hydroxyethyl 2-acetamido-2,4-dideoxy-1-

thio-a-^L-threo-hex-4-enopyranosid)uronate (3b)

Prepared from b-11b in 79% yield after reverse-phase

HPLC (1% CH₃CN in water) as a creamy-coloured

amorphous solid. R_f= 0.12 (EtOAc/MeOH/H₂O 7:2:1);

¹H NMR (D₂O): d 1.89 (3H, s, NAc), 2.72 (1H, dt,

(NC(O)Me),

23.6,

23.9

(C-2⁰,

C-2⁰⁰),

27.0

(OC(O)CMe₃), 36.2 (C-1⁰), 38.8 (OC(O)CMe₃), 49.7

(C-2), 52.5 (CO₂Me), 66.0 (C-3), 82.8 (C-1), 108.5 (C-

4), 144.0 (C-5), 161.9, 169.9, 178.1 (CO₂Me, NC(O)Me,

OC(O)CMe₃). LRMS m/z 396 ([M+Na]⁺, 100%). Com-

J_{1 a;1 b}14.1, J_{1 ;2}6.3 Hz, H-1⁰a), 2.84 (1H, dt, J_{1 b;1 a}

0

14.1, J_{1 b;2}6.3 Hz, H-1⁰b), 3.64 (1H, dt, J_{2 a;2 b}11.4,

0

J_{2 a;1}6.3 Hz, H-2⁰a), 3.70 (1H, dt, J_{2 b;2 a}11.4, J_{2 b;1}

0

1

pound b-11d: R_f= 0.19 (EtOAc/hexane 1:1); H NMR

6.3 Hz, H-2⁰b), 4.01 (1H, ddd, J_2,15.1, J_2,34.5, J_2,4

0.6 Hz, H-2), 4.08 (1H, ddd, J_3,24.5,J_3,43.9, J_3,1

0.6 Hz, H-3), 5.32 (1H, dd, J_1,25.1, J_1,30.6 Hz, H-1),

5.84 (1H, dd, J_4,33.9, J_4,20.6 Hz, H-4); ¹³C NMR

(D₂O) d 21.8 (NC(O)Me), 33.7 (C-1⁰), 52.4 (C-2), 60.7

(C-2⁰), 64.6 (C-3), 82.2 (C-1), 107.5 (C-4), 145.7 (C-5),

168.6, 173.9 (NC(O)Me, CO₂Na). LRMS m/z 300

([M+H]⁺, 100%). Anal. Calcd for C₁₀H₁₄NNaO₆SÆH₂O:

C, 37.86; H, 5.08; N, 4.41. Found: C, 37.88; H, 5.26; N,

4.29.

0

(CDCl₃): d 1.23 (9H, s, OPiv), 1.32 (3H, d, J_{2 ;1}

6.8 Hz, H-2⁰), 1.34 (3H, d, J_{2 ;1}6.8 Hz, H-2⁰⁰), 1.99

00

0

00

(3H, s, NAc), 3.15 (1H, sept, J_{1 ;2}¼ J_{1 ;2}6.8 Hz, H-

1⁰), 3.84 (3H, s, OMe), 4.50 (1H, dddd, J_2,NH8.7,

J_2,4= J_2,1= J_2,31.8 Hz, H-2), 4.96 (1H, ddd, J_3,45.1,

J_3,2= J_3,11.8 Hz, H-3), 5.56–5.59 (1H, m, H-1), 5.72

(1H, br d, J_NH,28.4 Hz, NH), 6.31 (1H, dd, J_4,35.1,

J_4,21.5 Hz, H-4); ¹³C NMR (CDCl₃):

(NC(O)Me), 23.4, 23.8

(C-2⁰, C-2⁰⁰),

d

23.2

27.0

(OC(O)CMe₃), 36.9 (C-1⁰), 38.9 (OC(O)CMe₃), 49.7

(C-2), 52.6 (CO₂Me), 64.4 (C-3), 82.1 (C-1), 107.1 (C-

4), 143.1 (C-5), 162.3, 169.2, 177.3 (CO₂Me, NC(O)Me,

OC(O)CMe₃). LRMS m/z 396 ([M+Na]⁺, 100%), 294

(30). HRMS calcd for C₁₇H₂₇NNaSO₆[M+Na]

396.1457. Found 396.1461.

3.50. Sodium (isobutyl 2-acetamido-2,4-dideoxy-1-thio-a-

L-threo-hex-4-enopyranosid)uronate (3c)

Prepared from b-11c in 80% yield after reverse-phase

HPLC (15% CH₃CN in water) as a creamy-coloured

1536

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

amorphous mass. R_f= 0.31 (EtOAc/MeOH/H₂O 7:2:1);

0

Acknowledgments

¹H NMR (D₂O): d 0.82 (3H, d, J_{3 ;2}6.6 Hz, H-3⁰), 0.82

0

(3H, d, J_{3 ;2}6.6 Hz, H-3⁰⁰), 1.62–1.80 (1H, m, H-2⁰), 2.48

We gratefully acknowledge the ﬁnancial support of the

Australian Research Council (MvI, Federation Fellow-

ship) and the National Health and Medical Research

Council of Australia. MCM thanks Griﬃth University

for the award of a Postgraduate Research Scholarship

and the Institute for Glycomics, Griﬃth University,

for an Institute Postgraduate Award. The Alexander

von Humboldt Stiftung is gratefully acknowledged for

the award of a von Humboldt Forschungspreis to MvI.

00

0

(1H, dd, J_{1 a;1 b}12.6 Hz, J_{1 a;2}7.2, H-1⁰a), 2.57 (1H, dd,

0

J_{1 b;1 a}12.6, J_{1 b;2}6.9 Hz, H-1⁰b), 4.00 (1H, br dd, J_2,1

5.7, J_2,34.5 Hz, H-2), 4.11 (1H, br dd, J_3,24.5, J_3,4

3.9 Hz, H-3), 5.26 (1H, br d, J_1,25.7 Hz, H-1), 5.98

(1H, br d, J_4,33.9 Hz, H-4); ¹³C NMR (D₂O): d 21.6,

21.7 (C-3⁰, C-3⁰⁰), 22.5 (NC(O)Me), 28.6 (C-2⁰), 40.8

(C-1⁰), 53.1 (C-2), 65.3 (C-3), 83.7 (C-1), 110.6 (C-4),

144.4 (C-5), 167.2, 174.5 (CO₂Na, NC(O)Me). LRMS

m/z 312 ([M+H]⁺, 100%). HRMS calcd for

C₁₂H₁₈NNa₂SO₅[M+Na] 334.0701. Found 334.0701.

0

References and notes

3.51. Sodium (isopropyl 2-acetamido-2,4-dideoxy-1-thio-

a-^L-threo-hex-4-enopyranosid)uronate (3d)

1. Schauer, R.; Kamerling, J. P. Chemistry, Biochemistry

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Microbiol. 1986, 22, 325.

Prepared from b-11d in 70% yield after reverse-phase

HPLC (5% CH₃CN in water) as an amorphous crea-

my-coloured solid. R_f= 0.24 (EtOAc/MeOH/H₂O

0

1

0

7:2:1); H NMR (D₂O): d 1.10 (3H, d, J_{2 ;1}6.6 Hz, H-

2⁰), 1.14 (3H, d, J_{2 ;1}6.6 Hz, H-2⁰⁰), 1.84 (3H, s, NAc),

00

0

3.06 (3H, sept, J_{1 ;2}¼ J_{1 ;2}6.6 Hz, H-1⁰), 3.92 (1H, br

dd, J_2,15.1, J_2,34.5 Hz, H-2), 4.02 (1H, br dd, J_3,24.5,

J_3,43.9 Hz, H-3), 5.31 (1H, br d, J_1,25.1 Hz, H-1),

5.73 (1H, br d, J_4,33.9 Hz, H-4); ¹³C NMR (D₂O) d

21.8 (NC(O)Me), 22.8 (C-2⁰, C-2⁰⁰), 36.2 (C-1⁰), 52.5

(C-2), 64.3 (C-3), 81.9 (C-1), 111.7 (C-4), 142.3 (C-5),

165.3, 174.0 (CO₂Na, NC(O)Me). LRMS m/z 320

([M+Na]⁺, 100%), 298 ([M+H]⁺, 39), 265 (33), 229

(56). Anal. Calcd for C₁₁H₁₆NO₅SNa 1.5H₂O: C,

40.74; H, 5.90; N, 4.32. Found: C, 40.96; H, 5.58; N,

4.03.

0

00

9. Middlebrook, J. L.; Dorland, R. B. Microbiol. Rev. 1984,

48, 199.

10. Ohyashiki, T.; Taka, M.; Mohri, T. Biochim. Biophys.

Acta 1987, 905, 57.

3.52. Sialidase assay

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Vibrio cholerae sialidase was expressed and puriﬁed

using previously published methods.^13,45Sialidase activ-

ity was assayed using a modiﬁed ﬂuorometric assay³³

developed by Potier et al..²⁹The substrate used for the

enzyme assay, MUN, was prepared using published

methods.³⁰Solutions were prepared in Eppendorf tubes

containing sialidase (6.2 · 10⁵U,⁴⁶10 lL) in the pres-

ence or absence of inhibitor (1 mM ﬁnal concentration,

10 lL). Samples were made up to 95 lL using 2-(N-mor-

pholino)ethanesulfonic acid (MES) buﬀer (50 mM, pH

5.6, containing 6 mM CaCl₂). The samples were incu-

bated at 37 ꢁC for 30 min with shaking, prior to the

addition of MUN (50 lM ﬁnal concentration, 5 lL).

The reactions were stopped after a further 20 min incu-

bation period at 37 ꢁC with shaking using glycine solu-

tion (0.25 M, pH 10, 2.4 mL). Fluorescence was

measured using a TD-700 Fluorometer (Turner Design,

CA, USA) at emission and excitation wavelengths of

400 and 365 nm, respectively. The assay was performed

at least in duplicate. Inhibition was measured as a per-

centage of the control (incubations performed in the ab-

sence of inhibitor). Sample measurements were

corrected for background ﬂuorescence that was not pro-

duced by the enzyme-catalysed hydrolysis of the sub-

strate, by subtracting a blank sample that contained

MUN in MES buﬀer. Neu5Ac2en (1) was included in

every assay as a comparison.

M. C. Mann et al. / Bioorg. Med. Chem. 14 (2006) 1518–1537

1537

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Article Doi

Modelling, synthesis and biological evaluation of novel glucuronide-based probes of Vibrio cholerae sialidase

DOI: 10.1016/j.bmc.2005.10.004

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