Synthesis and NMR spectroscopy of nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids

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Carbohydrate Research 335 (2001) 221–243

Synthesis and NMR spectroscopy of nine stereoisomeric

5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids

Yury E. Tsvetkov,^a,* Alexander S. Shashkov,^aYuriy A. Knirel,^aUlrich Za¨hringer^b

^aN.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospeckt 47,

119991 Moscow, Russian Federation

^bForschungszentrum Borstel, Zentrum fu¨r Medizin und Biowissenschaften, Parkallee 22, D-23845 Borstel, Germany

Received 28 June 2001; accepted 15 August 2001

Abstract

Derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids are essential constituents of some bacterial

polysaccharides and glycoproteins. In order to establish reliably the conﬁguration of the natural sugars, nine

stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids were synthesized, including di-N-acetyl-legion-

aminic and -pseudaminic acids (the

isomers at C-4, C-5, C-7, and C-8 having the

tro, -glycero- -altro, -glycero- -manno, and

sation of 2,4-diacetamido-2,4,6-trideoxy- -gulose, -

D

-glycero-

-mannose, -

D

-galacto and

-galacto,

-gluco conﬁgurations. Synthesis was performed by conden-

-talose, and - -allose with oxalacetic acid under

L

-glycero-

L

-manno isomers, respectively) and their

L

D

-glycero-

D

-talo, -glycero- -talo, -glycero- -al-

L

D

L

D

L

D

L

basic conditions, the reaction of the last two precursors being accompanied by epimerisation at C-2. The H and ¹³C

NMR data of the synthetic compounds are discussed. Acetylated methyl esters of the C-7 and C-8 isomeric

nonulosonic acids were prepared and used for analysis of the side-chain conformation by NMR spectroscopy.

1

Keywords: 5,7-Diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids, synthesis; 2,4-Diacetamido-2,4,6-trideoxyhexoses; Legion-

aminic acid; Pseudaminic acid; Lipopolysaccharide components

peculiar physicochemical properties, and are

likely involved in bacterial virulence. Whereas

the relative conﬁguration within the confor-

mationally rigid pyranose ring (C-4,5,6) of the

higher sugars could be easily established using

¹H NMR and NOE spectroscopy, reliable de-

termination of the conﬁguration in the ﬂexible

side chain (C-7,8) required data from model

compounds. To solve this problem, we synthe-

sised, for the ﬁrst time, nine stereoisomeric

5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulo-

sonic acids. Synthesis of four of these sugars

has been reported in preliminary communi-

cations.^7,8

1. Introduction

N-Acyl and O-acetyl derivatives of various

5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic

acids are known as components of glycopoly-

mers of Gram-negative bacteria, including

lipopolysaccharides,^1–3a capsular polysaccha-

ride,⁴and glycoproteins.^5,6They play a role in

immunospeciﬁcity, endow the cell surface with

* Corresponding author. Tel.: +7-95-9383686; fax: +7-

95-1355328.

E-mail address: tsvetkov@ioc.ac.ru (Y.E. Tsvetkov).

PII: S0008-6215(01)00235-X

222

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

Since introduction of an azido group (as a

2. Results and discussion

precursor of the acetamido function) is ac-

companied by inversion of the conﬁguration,

azidation at positions 2 and 4 would lead

directly to the desirable conﬁgurations of C-2

and C-4. Hence, one additional inversion at

C-3 has to be performed to achieve the target

L

-gulo conﬁguration.

The prerequisite for successful S_N2 substitu-

tion of an axial sulfonate group at C-2 is that

10

the substituent at C-1 is equatorial (see Ref.

and references cited therein). Therefore, ben-

acetamido-2,4,6-trideoxy-

talose (25), - -mannose (32), and -

(48) were used as the C₆precursors (See

Schemes 1–4). They possess the same,

conﬁguration at C-2 and C-3 (C-5 and C-6 in

the expected C₉acids), whereas the conﬁgura-

tions at C-4 and C-5 vary, thus adopting all

possible stereochemical combinations at C-7

L

-gulose (15),

-

D

-

zyl b- -rhamnopyranoside was thought to be

L

D

L

-allose

the precursor of choice. This was prepared by

Bu₂SnO-mediated benzylation of 1,2-diol 1¹¹

(Scheme 1). As expected,¹²the reaction oc-

curred in a regio- and stereoselective manner,

though it was accompanied by migration of

benzoyl protecting groups. As a result, a mix-

ture of benzyl b-rhamnopyranoside diben-

zoates 2-4 was obtained in total yield of

90–95% and 2:3:4 ratios of ꢀ4:3:1. These

compounds were separated and their struc-

L L

,

and C-8 in the higher sugars (

and ; respectively).

D

,L

;

L

,D

;

D

,D;

L,L

The 2,4-diacetamido-2,4,6-trideoxyhexoses

1

15, 25, 32, and 48 were synthesized as follows.

tures proved by H NMR spectroscopy. Most

L

-Rhamnose, the most readily available 6-de-

importantly, the b-conﬁguration of 2–4 was

demonstrated by a relatively high-ﬁeld posi-

oxyhexose, served as the progenitor of 15.

Scheme 1. Reagents and conditions: i, Bu₂SnO, benzene, reﬂux; ii, BnBr, Bu₄NBr, benzene, reﬂux; iii, MeONa, MeOH; iv,

trimethyl orthoacetate, PTSA, MeCN, rt; v, Ac₂O, pyridine, rt; vi, 80% aq AcOH, rt; vii, Tf₂O, pyridine, CH₂Cl₂, 0 °C; viii, NaN₃,

DMF, rt; ix, NaN₃, NH₄Cl, aq EtOH, reﬂux; x, H₂, Pd(OH)₂/C, MeOH, 30°C; xi, Ac₂O, MeOH, rt.

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

223

Scheme 2. Reagents and conditions: i, pyridinium perchlorate, aq MeCN, 80 °C; ii, TsCl, pyridine, 0°Crt; iii, NaI, MeCN,

80 °C; iv, H₂, Pd(OH)₂/C, i-Pr₂NEt, MeOH, 30 °C; v, Ac₂O, MeOH, rt; vi, oxalyl chloride, DMSO, i-Pr₂NEt, CH₂Cl₂, −60 °C;

vii, NH₂OH^.HCl, pyridine, CH₂Cl₂, rt; viii, NaBH₄, NiCl₂^.6H₂O, MeOH, −35 °C; ix, H₂, Pd(OH)₂/C, aq MeOH, rt.

Scheme 3. Reagents and conditions: i, Bu₂SnO, benzene, reﬂux; ii, BzCl, benzene, 0 °C rt; iii, Tf₂O, pyridine, CH₂Cl₂, 0 °C;

iv, Bu₄NN₃, toluene, 60100 °C; v, MeONa, MeOH, rt; vi, H₂, Pd(OH)₂/C, MeOH, 30 °C; vii, Ac₂O, MeOH, rt.

2,4-diacetate 6. Low-ﬁeld positions of the sig-

tion at l 3.5–3.8 of the signal for H-5 (in

a-rhamnopyranosides H-5 would resonate at l

4.1–4.3¹³). The positions of the benzoyl

groups in 2–4 were inferred from the low-ﬁeld

chemical shifts of the signals for H-3,4, H-2,4,

and H-2,3, respectively. Debenzoylation of the

mixture of 2–4 with sodium methoxide in

methanol yielded 5.

Treatment of 5 with trimethyl orthoacetate

in the presence of TsOH, followed by acetyla-

tion of OH-4 and hydrolytic opening of the

orthoester ring in the 2,3-orthoester, yielded

1

nals for H-2 and H-4 in the H NMR spec-

trum proved the location of the acetyl groups

in 6. Reaction of 6 with triﬂic anhydride in the

presence of pyridine led to triﬂate 7, which

readily gave the 3,4-anhydro- -altroside 8 on

L

treatment with sodium methoxide in

methanol. The structure of 8, particularly the

position of the epoxy group, was conﬁrmed by

NMR analysis of 8 and its acetate 9. The

transformation of 8 into 9 resulted in a

downﬁeld shift of the signal for H-2 (l 4.02

224

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

Scheme 4. Reagents and conditions: i, 2,2-dimethoxypropane, PTSA, acetone, rt; ii, oxalyl chloride, DMSO, i-Pr₂NEt, CH₂Cl₂,

−60 °C; iii, NaBH₄, aq EtOH, rt; iv, 80% aq AcOH, 40 °C; v, Bu₂SnO, benzene, reﬂux; vi, BzCl, benzene, 0 °Crt; vii, Tf₂O,

pyridine, CH₂Cl₂, 0 °C; viii, Bu₄NN₃, toluene, 60 °C; ix, NaN₃, DMF, dibenzo-18-crown-6, rt; x, NaN₃, DMF, rt; xi, LiAlH₄,

THF, 0 °Crt; xii, Ac₂O, MeOH, rt; xiii, MsCl, pyridine, CH₂Cl₂, 0 °Crt; xiv, AcONa, aq 2-methoxyethanol, reﬂux; xv, H₂,

Pd(OH)₂/C, aq MeOH, 35 °C.

J_3,4, and J_4,5coupling constant values in the

¹H NMR spectra of 12 and the derived acetate

5.25), thus indicating the position of the hy-

droxy group in 8. This ﬁnding excluded con-

13 were in accordance with the b- -gulo

L

version of the 3,4-

2,3-

L

-altro-epoxide into the

conﬁguration. Chemical shifts for H-2 (l

3.69), H-3 (l 5.33), and H-4 (l 3.48) in the

spectrum of 13 demonstrated that the azido

groups were at C-2 and C-4. Hydrogenolysis

of 12 over Pd(OH)₂/C reduced the azido

groups, whereas the benzyl group remained

intact (compare published data¹⁶). Follow-

ing N-acetylation, hydrogenolysis of the di-

acetamido derivative 14 proceeded smoothly

providing the target sugar 15 in high yield.

L

-manno-isomer that might proceed under

basic conditions.¹⁴Compound 8 had the nec-

essary conﬁguration of C-3, a free OH-2

group required for subsequent introduction of

an azido group, and a 3,4-epoxy function

suitable for introduction of the second azido

group at position 4.

As anticipated, conversion of 8 into triﬂate

10 and subsequent reaction with sodium azide

in DMF resulted in a high yield of azide 11. A

large J_1,2coupling constant value of 7.6 Hz in

1

According to H NMR data, 15 exists in

1

aqueous solution in the pyranose form as a

mixture of a- and b-anomers in a ratio of

ꢀ1:5.

the H NMR spectrum of 11 showed that the

azido group was pseudo-equatorial. Opening

of the epoxide ring in 11 by treatment with

sodium azide in the presence of ammonium

chloride in boiling aqueous ethanol¹⁵fur-

nished diazide 12. Large J_1,2and small J_2,3,

2-Azido-2-deoxy derivative 16¹⁷was used as

a starting compound for preparation of 2,4-di-

acetamido-2,4,6-trideoxy- -talose (25). De-

D

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

225

Table 1

NMR data for 2,4-diacetamido-2,4,6-trideoxy-

D

-talose 25 (D₂O, 30 °C)

Form

¹H NMR data (chemical shift, l; coupling constant, Hz) ¹³C NMR data (chemical shifts, l)

(content,%)

H-1

H-2

H-3

H-4

H-5

H-6

C-1

C-2

C-3

C-4

C-5

C-6

(J_1,2

)

(J_2,3

)

(J_3,4

)

(J_4,5

)

(J_5,6)

a-25p

5.18

(2.0)

4.97

(1.9)

5.38

(5.3)

5.47

(6.3)

5.51

(5.6)

5.58

(6.2)

4.12

(4.6)

4.36

(4.2)

4.51

(5.0)

4.38

(4.8)

4.45

(4.9)

4.38

(4.3)

4.24

(4.2)

4.12

—

4.29

(B1)

4.25

(B1)

4.31

(B1)

4.25

(B1)

4.18

(2.8)

4.12

(B1)

4.09

(6.2)

3.85

(8.9)

4.19

(5.1)

3.98

(7.9)

4.43

(6.6)

3.91

(6.4)

4.04

(6.3)

3.91

(6.2)

4.16

(6.5)

3.77

(6.1)

1.18

1.24

1.33

1.11

1.25

94.2

94.6

86.9

84.9

82.0

80.0

52.8

53.4

60.9

59.9

55.0

53.4

66.7

69.6

71.6

72.1

71.3

72.8

52.7

52.0

70.6

73.9

70.5

71.9

66.7

71.7

69.0

68.7

67.0

67.9

17.0

17.1

20.5

20.6

19.2

20.0

(18)

b-25p

(32)

a-25f major

(20)

a-25f minor

(14)

b-25f major

(13.5)

b-25f minor

(2.5)

manno conﬁguration. No well-resolved spec-

trum could be obtained for 23 even at elevated

temperatures that could be accounted for by a

restricted internal rotation of spatially close

axial acetamido groups at C-2 and C-4 and

the 3-O-benzyl group. Nevertheless, based on

the line width for H-3 and H-5, it was con-

cluded that J_2,3and J_3,4coupling constant

values did not exceed 4 Hz and J_4,5was less

than 2 Hz. These data were in good agreement

with the expected talo conﬁguration of 23.²¹

Hydrogenolysis of 23 over Pd(OH)₂/C af-

forded the target hexose 25.

oxygenation at C-6 and introduction of an

amino function with inversion of the conﬁgu-

ration at C-4 had to be carried out to obtain

the desired structure. To this aim, the benzyli-

dene group in 16 was removed by treatment

with pyridinium perchlorate¹⁸in aqueous ace-

tonitrile (Scheme 2). Selective tosylation of the

resulting diol 17, followed by substitution of

the tosyloxy group in 18 with iodide, afforded

the 6-iodo derivative, 19. Simultaneous reduc-

tion of the azido group and deiodination with-

out affecting the benzyl protective groups

occurred on hydrogenation over Pd(OH)₂/C in

the presence of N,N-diisopropylethylamine.

Following N-acetylation, the 2-acetamido-2,6-

dideoxy derivative 20 was obtained in 84%

yield. The use of LiAlH₄in THF for reduction

of 19 was less effective giving only 49% of 20.

A direct S_N2 substitution at C-4 in the manno

series is known to be complicated;¹⁹therefore,

the reaction sequence oxidation-oximation-re-

duction was applied to introduce the second

amino group into 20. Swern oxidation of 20

gave the ketone 21, which was converted,

without isolation, into the oxime 22. Reduc-

tion of the latter with NaBH₄–NiCl₂in

methanol at −35 °C²⁰and subsequent N-

acetylation gave a mixture of talo and manno

isomers 23 and 24 in a ratio of 8:1. The

The behaviour of 25 in aqueous solution is

1

13

noteworthy. The H and C NMR spectra of

25, which were assigned using 2D COSY,

TOCSY, and HSQC techniques (Table 1),

contained six anomeric signals. Comparison

with published data for talopyranose and its

derivatives, including ¹³C NMR chemical

shifts and coupling constant values,^21,22en-

abled identiﬁcation of a- and b-pyranoses a-

25p and b-25p. The four other signals

belonged to a- and b-furanoses a-25f and

b-25f, as followed from the coupling constant

values (compare published data²³) and charac-

teristic changes in the ¹³C NMR chemical

shifts, especially those for C-1 and C-4, com-

pared to the data of talofuranose²²and 25p

(Table 1). Each of a-25f and b-25f existed as

two stereoisomers (E and Z) at the 4-acetam-

ido group,²⁴which were not assigned, and

designated as major and minor (Table 1). The

1

structures of 23 and 24 were proved by H

NMR spectroscopy. Large J_3,4and J_4,5cou-

pling constant values (each of ꢀ10 Hz) and a

small J_2,3value (4.2 Hz) showed 24 to have the

226

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

¹H NMR signals for the stereoisomers with-

in each anomeric pair coalesced when

the spectrum was run at 90 °C. Therefore,

isopropylidene group was removed and the

resulting triol 36 was selectively benzoylated

via dibutylstannylene derivative to yield

monobenzoate 37. However, instead of the

expected diazide 39, reaction of 2,4-ditriﬂate

2,4-diacetamido-2,4,6-trideoxy- -talose (25)

D

represents a rare example of a 4-acylamino-4-

deoxyhexose that exists in aqueous solution to

a signiﬁcant extent in the furanose form. Most

likely, the reason for this unusual behavior is

destabilization of the pyranose form by unfa-

vourable 1,3-diaxial interaction of the acetam-

ido groups.

38

obtained

from

37

with

tetra-

butylammonium azide gave a complex mix-

ture of products that was not further

investigated. Therefore, another approach

based on a consecutive introduction of azido

groups to positions 4 and 2 was exploited.

Azidation of 4-triﬂate 40 with sodium azide in

DMF in the presence of a crown ether²⁸fur-

nished azide 41 with the necessary conﬁgura-

tion of C-4. To liberate HO-2 for introduction

of the second azido group, 41 was subjected to

deacetalation followed by Bu₂SnO-mediated

selective 3-O-benzoylation of diol 42.

Monobenzoate 43 thus obtained was con-

verted into triﬂate 44, which was then con-

verted to the target diazide 39 by reaction

with sodium azide in DMF. Large values of

2,4-Diacetamido-2,4,6-trideoxy-

D

-mannose

(32) was prepared from benzyl b-

D

-fucopyran-

oside (26)²⁵using at the key step the known

approach to b-mannosides based on simulta-

neous substitution with inversion at positions

2 and 4 in b-galactosides²⁶(Scheme 3).

Bu₂SnO-mediated selective benzoylation of 26

gave 3-benzoate 27, as followed from a low-

1

ﬁeld position of the signal for H-3 in the H

NMR spectrum. Conversion of 27 into ditri-

ﬂate 28 and its subsequent reaction with tetra-

butylammonium azide in toluene resulted in

diazide 29 in high yield. Large J_3,4and J_4,5

coupling constant values (each of ꢀ10 Hz)

and small J_1,2and J_2,₁₃values (B1 and 2.5 Hz,

respectively) in the H NMR spectrum con-

ﬁrmed the manno conﬁguration of 29. Con-

ventional debenzoylation of 29 afforded 30.

Further transformations of 30 were similar to

those described above in synthesis of 15 and

included reduction of the azido groups by

hydrogenation, N-acetylation (3031), and

removal of the benzyl protecting group (31

32). According to NMR data, the hexose 32

exists in aqueous solution exclusively in the

pyranose form (a,b ratio ꢀ1:1).

1

all coupling constants in the H NMR spec-

trum proved unambiguously the

L

-gluco

conﬁguration of 39. LiAlH₄reduction of azido

groups in 39 with concomitant removal of the

benzoyl group and subsequent N-acetylation

resulted in the diacetamido derivative 45. Me-

sylation of 45 afforded mesylate 46. Inversion

of the conﬁguration at C-3 mediated by par-

ticipation of

a

neighbouring acetamido

group(s) was achieved by heating of 46 in

aqueous 2-methoxyethanol in the presence of

sodium acetate¹⁶to give the

-allo derivative

L

47 in high yield. The conﬁguration of 47 was

conﬁrmed by large J_1,2and J_4,5coupling con-

stant values (8.6 and 10.2 Hz) and small J_2,3

and J_3,4values (2.8 and 2.3 Hz, respectively).

Conventional removal of the anomeric benzyl

group afforded hexose 48, which occurred in

the pyranose form (a,b ratio of 1:3.3, NMR

data).

Condensation of compounds 15, 25, 32, and

48 with oxalacetic acid was performed in the

presence of sodium tetraborate at pH 10.5⁹

(Scheme 5). Acidic reaction products were iso-

lated by anion exchange chromatography,

and then the individual isomers of 5,7-

diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic

acids were separated by reversed-phase C₁₈

HPLC.

Benzyl b-

as the starting compound for preparation of

2,4-diacetamido-2,4,6-trideoxy- -allose (48).

To provide the proper conﬁguration at C-4,

which would lead to the necessary conﬁgu-

ration on azidation, the -rhamnoside 5 was

converted into -taloside 35 (Scheme 4). Con-

L

-rhamnopyranoside 5 was chosen

L

D

L

ventional isopropylidenation of 5 gave 33,

which was subjected to the known²⁷oxida-

tion–reduction procedure to afford, via ke-

tone 34,

L

-taloside 35 in nearly quantitative

yield. In an attempt to apply the same ap-

proach that was successively used for the syn-

thesis of the manno isomer 32, the

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

227

Scheme 5. Reagents and conditions: i, oxalacetic acid, Na₂B₄O₇, pH 10.5, rt Only main isomers of acids 49–57 with an equatorial

carboxyl group are shown.

Compounds 15 and 32 with the threo

conﬁguration of the fragment C-3ꢀC-4 af-

forded pairs of epimers at C-4 having the

They demonstrated the pyranose form of all

sugars, which existed in aqueous solution as

mixtures of anomers with a high predomi-

nance of the anomer having an equatorial

carboxyl group (b for 49, 50, 53, and 54, and

a for 51, 52, 55–57). The ratios of the free

acids anomers are given in Table 2. The

conﬁguration within the pyranose ring fol-

L

-glycero-

D

-galacto/

D

-talo (49/50) and -glyc-

D

ero- -galacto/

D

-talo (53/54) conﬁgurations,

respectively, in a nearly 1:1 ratio. Compounds

25 and 48 with the erythro conﬁguration of

the fragment C-3ꢀC-4 gave the equatorial

3

HO-4 epimers having the

D

-glycero-

L

-altro

lowed from the J_H,Hcoupling constant values

1

(51) and -glycero-

L

-altro (55) conﬁguration,

determined from the H NMR spectra. Large

respectively, with no axial HO-4 epimers. The

reason for this unexpected stereoselectivity is

not clear. In addition to 51 and 55, com-

pounds 25 and 48 afforded, as minor prod-

ucts, isomeric nonulosonic acids with an axial

J_3a,4, J_4,5, and J_5,6values for 49, 51, 53, and 55

indicated equatorial substituents at C-4, C-5,

and C-6. Large J_5,6and small J_3a,4and J_4,5

values for 50 and 54 showed that the OH-4

group is axial. In contrast, large J_3a,4and

small J_4,5and J_5,6values for 52 and 56 con-

ﬁrmed the axial orientation of the AcNH-5

group. Finally, small J_3a,4, J_4,5, and J_5,6values

for 57 demonstrated that both OH-4 and

AcNH-5 are axial.

AcNH-5 group having the

(52), -glycero- -manno( 56), and

-gluco (57) conﬁguration. They evidently re-

D

-glycero-

L

-manno

L

-glycero-

L

sulted from epimerisation at C-2 in the start-

ing monosaccharides prior to condensation.

The structures of the synthesised acids were

proved by ESIMS and NMR spectral data.

Major peaks corresponding to either [M+

Na]⁺(positive mode) or [M−H]⁻(negative

mode) pseudomolecular ions were observed in

the ESIMS spectra of 49–57. The data of the

¹H and ¹³C NMR spectra of both free acids

and sodium salts are given in Tables 2 and 3.

Four of the synthesised isomers occur in

nature. These are the

D

-glycero-

D

-galacto

called

and

L

-glycero- -manno isomers

L

legionaminic^3,8and pseudaminic¹acid, respec-

tively, as well as C-4 and C-8 epimers of

legionaminic acid having the

D

-glycero- -talo

D

and -glycero- -galacto conﬁguration (4- and

L

D

228

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

Table 2

¹H NMR data for 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids (l_H; J_H,H, Hz; D₂O, 30 °C)

Compound

a:b

Form

H-3e

(J_3,3

H-3a

H-4

(J_3e,4

H-5

(J_4,5

H-6

(J_5,6

H-7

(J_6,7

H-8

(J_7,8

H-9

(J_8,9)

CH₃CON

1.98, 2.02

ratio

)

(J_3a,4

)

49a

49b

1:19

H

2.69

(13.0)

2.62

(12.7)

2.32

(13.1)

2.20

1.71

(12.0)

1.61

(12.0)

1.86

(11.5)

1.79

3.82

(4.9)

3.83

(4.7)

3.95

(4.8)

3.89

(4.8)

3.67

(10.4)

3.63

(10.1)

3.73

(10.2)

3.70

3.85

(10.4)

3.82

(10.1)

4.16

(10.3)

4.06

3.93

4.00

1.20

(B2)

3.90

(1.7)

3.95

(2.0)

3.89

(1.3)

(6.4)

3.98

(6.4)

3.91

(6.4)

3.90

(6.3)

1.17

(6.5)

1.18

(6.2)

1.15

(5.8)

Na

H

1.96, 2.00

1.97, 2.01

Na

(13.0)

(11.5)

(10.2)

(10.3)

50a

50b

1:8

H

2.65

(14.4)

2.40

(15.0)

2.18 ^a

(14.9)

2.10 ^a

(14.8)

1.94

(2.7)

2.03

4.08

(3.6)

3.97

(2.7)

4.11

(2.9)

4.09

(2.9)

3.84

(2.7)

3.84

(2.6)

3.89

(2.8)

3.88

(2.6)

4.47

(10.5)

4.26

(10.4)

4.48

(10.6)

4.42

3.89

4.08

3.95

3.96

1.28

(6.3)

1.18

(6.3)

1.21

(5.7)

1.20

(6.3)

1.96, 2.04

1.96, 2.02

1.97, 2.01

Na

H

3.87

(2.0)

3.95

(1.3)

3.90

(1.9)

2.13 ^a

(3.3)

Na

2.06 ^a

(3.1)

3.98

(6.5)

(10.6)

51b

13.3:1

H

Na

2.71

2.70

(12.5)

2.34

(13.2)

2.24

(13.1)

2.22

(13.1)

1.75

1.68

(11.1)

1.93

(11.6)

1.86

(11.5)

1.91

(11.5)

3.82

3.75

(4.5)

3.99

(4.8)

3.95

3.81

3.79

(9.8)

3.86

(9.4)

3.83

3.56

3.42

(9.8)

3.90

(10.1)

3.83

3.91

3.93

(5.1)

3.92

(2.8)

3.89

4.40

4.41

(1.8)

4.42

(B1)

4.43

(B1)

4.39

(B1)

1.08

1.05

(6.3)

1.08

(6.4)

1.05

(6.5)

1.06

(6.5)

51a

H

2.06

2.05

51a

Na

51a (333 K)

3.93

(4.9)

3.82

(9.5)

3.85

(10.6)

3.88

(B1)

52b

52a

12.5:1

1:18

H

2.50

(13.2)

2.03

(13.4)

1.99

(13.2)

1.64

(12.8)

1.82

(12.3)

1.79

(12.3)

4.10

(4.9)

4.25

(5.0)

4.18

(4.8)

4.22

(4.5)

4.29

(4.3)

4.24

(4.0)

4.14

(2.1)

4.27

(1.8)

4.19

(1.0)

3.86

(10.4)

3.82

(10.1)

3.77

(10.2)

4.25

(1.6)

4.12

(1.2)

4.12

(B2)

1.10

(6.4)

1.09

(6.6)

1.07

(6.6)

H

Na

1.98, 1.99

53a

53b

H

2.73

(12.9)

2.75

(12.9)

2.31

(13.1)

2.19

1.71

(11.9)

1.61

(11.8)

1.87

(11.7)

1.82

3.82

(4.7)

3.66

(3.9)

3.98

(4.8)

3.94

(4.8)

3.68

3.67

3.93

(10.3)

3.77

3.94

3.93

1.16

1.97

Na

H

3.82

1.95

3.72

(10.3)

3.70

4.31

(10.5)

4.23

3.91

(1.9)

3.85

(B2)

3.85

(8.9)

3.85

1.16

(6.2)

1.15

1.99, 2.00

Na

(12.9)

(12.2)

(10.7)

(10.5)

54a

54b

55b

1:5.4

8.3:1

H

2.69

1.94

(3.5)

1.95

(3.6)

2.14

(3.4)

2.07

(3.2)

1.72

(12.2)

4.10

(3.0)

4.02

(2.9)

4.13

(3.0)

4.10

(2.7)

3.79

(4.5)

3.86

(2.9)

3.85

(2.8)

3.90

(2.9)

3.88

(2.9)

3.85

(10.7)

4.55

(10.8)

4.42

(10.7)

4.63

(10.8)

4.56

(10.8)

3.57

(10.7)

3.88

(2.3)

3.82

(2.4)

3.92

4.00

(8.6)

3.93

(8.9)

3.92

1.20

(6.4)

1.16

1.96, 2.00

1.96

(14.4)

2. 54

(14.7)

2.19

(14.9)

2.11

(14.8)

2.70

(12.8)

Na

H

1.18

(5.5)

1.17

(6.1)

1.21

(6.3)

1.98, 1.99

Na

H

3.86

(B2)

4.13

(3.3)

3.91

(8.7)

4.07

(6.0)

55b

Na

2.61

(12.9)

1.61

(11.2)

3.81

(4.3)

3.82

3.54

(10.1)

4.08

(3.4)

4.06

1.19

(6.3)

2.03, 2.05

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

229

Table 2 (Continued)

Compound

a:b

ratio

Form

H-3e

(J_3,3

H-3a

H-4

(J_3e,4

H-5

(J_4,5

H-6

(J_5,6

H-7

(J_6,7

H-8

(J_7,8

H-9

(J_8,9)

CH₃CON

)

(J_3a,4

)

55a

H

2.32

(13.3)

2.21

1.93

(12.5)

1.89

3.94

(4.4)

3.89

(4.3)

3.91

(10.2)

3.88

3.94

(10.2)

3.84

4.16

4.06

1.18

2.04, 2.08

2.02, 2.07

(2.8)

4.13

(3.0)

(5.8)

4.08

(6.0)

(6.4)

1.16

(6.3)

Na

(13.2)

(11.3)

(11.0)

56b

56a

7.5:1

H

2.48

(13.0)

2.41

(13.1)

2.01

(12.8)

1.92

1.62

(12.9)

1.55

(12.2)

1.80

(12.0)

1.78

4.08

(4.7)

4.08

(4.9)

4.20

(4.6)

4.16

(5.2)

4.29

(3.6)

4.26

(3.7)

4.27

(3.7)

4.24

(3.7)

3.96

(2.4)

3.75

(B2)

4.08

(B2)

4.02

(1.0)

4.15

(10.5)

4.01

(10.5)

4.17

(10.7)

4.13

4.18

(3.4)

4.04

(3.4)

4.10

(3.3)

4.11

(3.3)

1.12

Na

H

1.125

1.10

(6.5)

1.11

(6.5)

1.90, 2.01

1.97, 2.00

Na

(13.2)

(12.0)

(10.7)

57b

4:1

H

2.48

(14.8)

2.22

1.95

(15.2)

1.85

1.92

(2.9)

1.97

2.13

4.00

(3.1)

3.90

4.00

(3.7)

3.97

(3.6)

3.85

(2.6)

3.82

3.91

(3.3)

3.88

(3.3)

4.36

(2.2)

4.16

4.42

(2.1)

4.42

(2.1)

4.15

(10.3)

4.16

4.22

(10.5)

4.17

4.24

(4.0)

4.08

4.16

(3.5)

4.17

1.21

(6.5)

1.11

1.15

(6.6)

1.14

(6.3)

1.95, 1.99

57b

57a

Na

H

1.97, 1.98

Na

2.09

(3.6)

57a

(15.2)

(10.5)

^aAssignment could be interchanged.

8-epilegionaminic acid, respectively). Using

the synthetic models 53, 54, and 49, the

conﬁgurations of legionaminic, 4- and 8-epile-

gionaminic acids were conﬁrmed in some bac-

terial polysaccharides and revised in others.⁸

manno isomers, a signiﬁcant difference was

observed for the C-9 signal, which appeared at

20.0 ppm in the

16.7 ppm in the

D

epimer (52) at C-8 but at

epimer (56).

L

The chemical shifts are inﬂuenced also by

the anomeric conﬁguration. In the sugars with

an equatorial OH-4 (49, 51–53, 55, 56), the

1

The H and ¹³C NMR spectroscopy and spe-

ciﬁc optical rotation data of the compound 56

ﬁtted reasonably well those of pseudaminic

acid derivatives isolated from bacterial

1

difference between the H NMR resonances

for H-3_axand H-3_eqwas 0.86–1.02 ppm for

the anomer with an axial carboxyl group but

only 0.21–0.46 ppm for the other anomer,

independently of whether AcNH-5 is axial or

equatorial. When OH-4 was axial and AcNH-

5 equatorial (50, 54), a typical difference of

0.71–0.75 or 0.05 ppm was observed for the

anomers with an axial or an equatorial car-

boxyl group, respectively. In the ¹³C NMR

spectra, the clearest dependence was shown by

the C-3 and C-6 resonances. Compared to the

other anomer, in the anomer with an axial

carboxyl group they both appeared upﬁeld by

0.8–1.3 and 1.6–2.9 ppm when OH-4 was

equatorial (49, 51–53, 55, 56) or by 2.3–2.6

and 3.8–4.7 ppm, respectively, when OH-4

was axial (50, 54, 57).

polysaccharides^4,29, thus conﬁrming the

L

-glyc-

-manno conﬁguration of the natural

monosaccharide.

ero-L

Comparison of the NMR data of various

isomers (Tables 2 and 3, free acids) revealed

several regularities, which can be useful for

determination of the conﬁguration of natu-

rally occurring compounds of this class. Thus,

the J_6,7coupling constant is dependent on the

conﬁguration at C-5: it is small (1.3–3.3 Hz)

when AcNH-5 is equatorial (49–51, 53–55)

and large (10.1–10.7 Hz) when it is axial (52,

56, 57), indicating the syn (gauche)- and trans-

like relationship for H-6 and H-7, respectively.

In the

had the

D

-galacto and

D

-talo isomers, when C-8

D

conﬁguration (53, 54), the C-6 and

C-8 signals appeared upﬁeld by 2.0–2.3 and

1.4–2.0 ppm, respectively, compared to the

For conformational studies by NMR spec-

troscopy, the

L

-glycero-

D

-galacto (49),

D

-glyc-

corresponding

L

epimers (49, 50). In the

L

-

ero- -altro (51),

L

D

-glycero-

D

-galacto (53), and

230

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

Table 3

¹³C NMR data of 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids (l_C; D₂O, 30 °C)

Form

C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

C-9

CH₃CON

49a

49b

H

Na

H

173.1

176.7

174.3

177.3

97.3

98.2

96.5

97.5

41.2

41.7

40.4

40.8

68.9

69.7

68.3

68.6

53.7

54.2

54.1

54.2

75.2

72.9

54.4

69.4

70.1

69.3

69.7

19.8

23.1, 23.4

175.1, 175.2

175.1, 175.3

Na

50a

50b

H

Na

H

40.0

38.9

37.7

37.8

66.6

66.7

66.9

67.4

50.1

50.4

50.0

50.1

72.6

72.2

68.5

68.7

54.9

54.7

69.7

19.9

176.1

174.5 ^a

177.3

96.5

96.1

97.1

69.2

69.7

23.0, 23.1

174.6^a, 175.2

174.5, 175.2

Na

51b

51a

H

Na

H

172.9

175.6

173.7

177.1

97.2

97.4

96.1

97.2

41.1

41.7

39.9

40.4

68.7

69.5

67.6

68.1

54.7

55.4

54.7

54.9

77.3

76.0

75.7

75.9

54.3

54.9

53.9

53.5

66.5

66.6

66.5

20.0

20.2

20.3

23.1, 23.3

175.2, 175.8

Na

52b

52a

H

Na

173.9

174.7

177.6

96.0

96.8

97.8

36.8

35.5

36.0

68.0

66.1

66.7

49.1

49.9

50.1

73.0

70.3

70.2

54.4

54.6

67.4

66.0

20.0

23.1, 23.2

22.3, 22.3

175.0, 175

174.2, 175.0

53a

53b

H

Na

H

97.2

98.6

96.6

97.6

41.4

41.9

40.3

40.8

69.2

70.1

68.4

68.8

53.4

53.9

54.0

73.2

72.8

70.9

70.7

54.4

54.8

54.4

54.5

68.0

68.2

67.5

67.7

20.4

176.6

174.4 ^a

177.6

23.0, 23.4

175.0 ^a, 175.2

175.0, 175.1

Na

54a

54b

H

Na

H

40.2

39.5

37.6

37.8

66.9

67.1

67.6

49.7

50.1

49.7

49.9

70.3

69.9

66.5

66.2

55.0

54.7

54.8

68.2

67.8

67.2

67.4

19.8

20.4

177.0

174.7^a

177.6

96.7

96.3

97.3

23.0, 23.1

22.9, 23.1

174.7^a, 175.0

174.5, 174.9

Na

55b

55a

H

Na

H

173.0

175.4

173.7

177.1

97.0

98.0

96.1

97.1

41.2

41.7

39.9

40.4

69.1

69.8

68.2

68.9

54.9

55.2

55.3

55.7

76.0

75.8

73.7

73.8

55.8

55.9

55.5

55.2

67.8

67.6

19.7

19.8

19.7

19.8

23.3, 23.4

23.2, 23.5

175.0, 175.9

174.9, 175.8

Na

56b

56a

H

Na

H

35.0

35.9

35.6

36.0

67.3

67.0

66.1

66.5

49.9

50.1

49.9

50.1

72.1

72.2

71.4

53.8

54.2

54.0

54.2

68.2

68.3

68.1

16.7

16.6

16.7

16.6

174.9

177.4

97.0

97.7

23.2, 23.3

23.1, 23.3

175.0, 175.9

Na

57b

57a

H

Na

H

93.5

35.7

34.8

33.3

33.5

67.4

67.6

67.1

67.6

48.5

49.5

48.9

49.0

71.8

71.5

67.1

67.0

54.4

53.8

54.1

68.6

67.9

68.1

17.0

16.7

174.6

174.9

96.4

97.3

23.0, 23.2

175.1

Na

^aAssignment could be interchanged.

L

-glycero-

L

-altro (55) isomers were converted

and 60 in the conﬁguration of C-7, gave pre-

dominantly a-acetates (a:b ratios were 13:1 and

6:1 for 63 and 65, respectively). The anomeric

conﬁgurations in 62–65 were assigned based

to methyl esters 58–61 and then acetylated to

2,4,8-tri-O-acetyl derivatives 62–65, respec-

tively. These compounds were more convenient

to analyze than the free acids because of a

wider range of H NMR chemical shifts and

easier observation of signals for NH protons.

1

on the H and ¹³C NMR data (Table 4) by

1

analogy with anomers of methyl N-acetylneu-

3

raminate pentaacetate.^30,31The J_H,Hcoupling

The

L

- and

D

-glycero-

D

-galacto esters 58 and

constant values in the acetates 62–65 and the

parent free acids were essentially the same, and,

hence, O-acetylation did not signiﬁcantly

change the conformation of the molecules.

60 afforded mixtures of a- and b-acetates, 62

and 64, in a 1:1 ratio. The -and -glycero-

altro esters 59 and 61, which differed from 58

D

L

-

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

231

Table 4

¹H and ¹³C NMR data of methyl (5,7-diacetamido-2,4,8-tri-O-acetyl-3,5,7,9-tetradeoxynon-2-ulopyranos)onates (l_H; l_C; J_H,H, Hz;

CDCl₃, rt)

Conﬁguration

H-3eq

H-3ax H-4

H-5

H-6

H-7

H-8

H-9

NH-5

NH-9

(compound)

(J_3eq,3ax

)

(J_3ax,4

)

(J_3eq,4

)

(J_4,5

)

(J_5,6

)

(J_6,7

)

(J_7,8

)

(J_8,9

)

(J_NH,5

)

(J_NH,7)

a-

b-

a-

b-

a-

b-

D

-glycero-

D

-galacto (a-64)

-galacto (b-64)

2.71

(13.5)

2.55

(13.3)

2.72

(13.6)

2.61

(13.2)

2.39

(13.2)

2.48

(13.3)

2.65

(13.4)

2.11

5.28

(5.1)

5.66

(5.1)

5.44

(5.1)

5.82

(5.0)

5.18

(5.0)

5.21

(4.9)

5.05

(4.8)

3.85

(10.0)

3.45

(10.6)

3.59

(9.5)

3.21

(10.2)

4.42

(10.3)

4.14

(10.4)

4.07

(10.5)

4.53

(10.7)

4.34

(10.5)

4.61

(10.0)

4.53

(10.3)

3.69

(10.9)

3.83

(11.0)

4.15

(9.7)

4.38

(1.4)

4.40

4.88

(ꢀ7) (6.4)

4.86 1.21

1.23

5.73

(9.4)

5.94

(8.4)

5.81

(9.3)

5.99

(7.6)

5.86

(9.0)

6.11

(9.4)

6.25

(8.8)

5.83

(10.0)

6.88

(10.0)

5.87

(9.9)

6.73

(9.7)

6.38

(9.2)

6.40

(9.7)

6.77

(9.8)

D

1.80

(11.0)

2.07

(ꢀ1) (ꢀ7) (6.5)

L

-glycero-

D

-galacto (a-62)

-galacto (b-62)

-altro (a-63)

4.23

(ꢀ1) (7.2)

4.29 5.10

(ꢀ1) (7.0)

4.06 5.81

5.14

1.31

(6.4)

1.17

(6.4)

1.19

1.77

(11.2)

1.94

(12.7)

1.95

D

-glycero-

L

(ꢀ1) (ꢀ1) (6.4)

L

-glycero-

L

-altro (a-65)

-altro (b-65)

4.59

(2.5)

4.53

5.01

(5.0)

4.89

1.29

(6.4)

1.19

(6.3)

2.03

(ꢀ2) (6.8)

C-1

C-2

C-3

C-4

C-5

C-6

C-7

C-8

C-9

a-

b-

a-

b-

a-

b-

D

-glycero-

D

-galacto (a-64)

-galacto (b-64)

96.5

96.8

96.5

96.6

96.9

97.3

36.2

36.8

36.2

36.6

36.1

36.7

36.1

68.3

67.1

67.5

66.3

68.3

68.7

50.6

51.7

51.5

52.6

50.3

50.7

50.9

72.5

70.6

73.4

72.0

75.9

73.9

75.1

50.6

52.5

51.8

51.2

50.0

52.3

69.5

69.8

70.5

70.2

65.9

68.8

69.1

16.8

17.1

17.5

19.1

16.2

166.7

167.9

167.2

166.5

166.7

L

-glycero-

D

-galacto (a-62)

-galacto (b-62)

-altro (a-63)

D

-glycero-

L

-glycero-

L

-altro (a-65)

-altro (b-65)

Fig. 1. NOE correlations and conformation of the side chain in methyl (5,7-diacetamido-2,4,8-tri-O-acetyl-3,5,7,9-tetradeoxynon-

2-ulopyranos)onates (62-65). Only correlations designated in Table 5 as strong are shown.

3

large J_H-7,H-8values of ꢀ7 Hz for 62 (

L

-glyc-

Small J_H-6,H-7coupling constant values (1–

ero- -galacto) and 64 ( -glycero- -galacto)

showed the trans-like orientation of H-7 and

H-8 in the predominant conformer. Almost

D

2.5 Hz) were observed for all O-acetylated

compounds, thus indicating the gauche orien-

tation of H-6 and H-7 (Fig. 1). Relatively

232

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

Table 5

NOE correlations for H-9, H-8, H-7 and NH-7 in methyl (5,7-diacetamido-2,4,8-tri-O-acetyl-3,5,7,9-tetradeoxynon-2-

ulopyranos)onates (relative intensity in parentheses; s, strong; m, medium; w, weak)

Conﬁguration

H-9

H-8

H-7

NH-7

(compound)

D

-glycero-

(64)

D

-galacto H-8 (s), H-7 (s), NH-7

(m)

H-7 (w), H-6 (s), NH-7 (m) H-6 (s), NH-5 (s), NH-7

H-5 (m)

H-6 (w)

(m)

L

-glycero-

D

-galacto H-8 (s), H-7 (m), H-6

(s)

H-7 (w), H-6 (m), NH-7 (m) H-6 (s), NH-5 (s), NH-7

(m)

(62)

-glycero-

D

L

-altro

H-8 (s), H-7 (s), NH-7

H-7 (m), H-5 (s)

H-6 (s), NH-7 (m)

(63)

(w)

L

-glycero-

(65)

L

-altro

H-8 (s), H-7 (w), H-5

(w)

H-7 (s), H-5 (s), NH-5 (w)

H-6 (s), NH-5 (w)

H-9 (m), H-7

(m)

the same large value (5–7 Hz, depending on

the anomeric conﬁguration) was observed for

relations in the NOESY spectra of 63 and 65.

The most populated rotamers around the C-

7ꢀC-8 bond are characterized by strong H-

9,H-7 and H-8,H-5 correlations for 63 and

strong H-8,H-5 and weak H-9,H-5 correla-

tions for 65 (Table 5, Fig. 1(c) and (d)). The

presence of an intense H-7,H-8 correlation

peak in the NOESY spectrum of 65 seems to

65 (

ero-

Hz).

Correlations between H-7 and both H-6

and NH-5 in the - and -glycero- -galacto

L

-glycero-

L

-altro), whereas in 63 ( -glyc-

D

L

-altro) it was signiﬁcantly smaller (ꢀ1

L

D

isomers (62 and 64) that were revealed by a

NOESY experiment deﬁned the predominant

rotamer around the C-6ꢀC-7 bond (Table 5,

Fig. 1(a) and (b)). This conformation was in

3

be inconsistent with a relatively large J_H-7,H-8

value (5–7 Hz). This contradiction could be

accounted for by a signiﬁcant contribution of

rotamers with a small H-7ꢀC-7ꢀC-8ꢀH-8 dihe-

dral angle. The predominant conformers of 63

and 64 have cis,trans and cis,cis side-chain

orientation, respectively.

3

agreement with a small J_H-6,H-7value (1–1.4

Hz). The NOESY spectra of both 62 and 64

showed only a weak H-8,H-7 correlation that,

3

together with a relatively large J_H-7,H-8value

(ꢀ7 Hz), indicated the trans-like orientation

of H-7 and H-8. Strong H-9,H-7 and medium

H-9,NH-7 correlations for 64 showed a spatial

proximity of H-9 and H-7, and a strong H-

8,H-6 correlation demonstrated that these

protons are close to each other (Fig. 1(b)). In

62, H-9 is more close to H-6 than to H-7 as

followed from a stronger H-9,H-6 correlation

compared to a H-9,H-7 correlation (Fig. 1(a)).

Therefore, in 62 and 64, predominant are the

trans,trans and trans,cis side-chain (C-7ꢀC-

8ꢀC-9) conformers, respectively.

3. Experimental

NMR spectra were recorded on Bruker

DRX-500 and Bruker AM-300 instruments.

Spectra of hexose derivatives were measured

for solutions in CDCl₃unless otherwise stated,

1

and H NMR chemical shifts were referenced

to a residual solvent signal. Spectra of 5,7-di-

acetamido-3,5,7,9-tetradeoxynon-2-ulosonic

acids were measured for solutions in D₂O

using acetone (l_H2.225, l_C31.45) as internal

standard. Sodium salts were obtained by pass-

ing aqueous solutions of the acids through a

short column of Amberlite IR-420 (Na⁺

form). A mixing time of 300 or 900 ms was

used in NOESY experiments with methyl (5,7-

diacetamido-2,4,8-tri-O-acetyl-3,5,7,9-tetra-

deoxynon-2-ulopyranos)onates.

An HMBC experiment optimized for a cou-

pling constant of 8 Hz revealed a strong

H-6,C-8 correlation for the

D

- and -glycero-

L

-altro isomers (63 and 65), which corre-

3

sponded to a J_H-6,C-8coupling constant of

\5 Hz. This ﬁnding showed the trans-like

orientation of H-6 and C-8 in the predomi-

nant rotamer around the C-6ꢀC-7 bond,³²

which was conﬁrmed by a strong H-7,H-6

correlation with no H-8,H-6 and H-9,H-6 cor-

Melting points were determined with a

Koﬂer apparatus and are uncorrected. Optical

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

233

2,4-Dibenzoate 3: mp 123–125 °C (EtOAc–

rotation values were measured on a JASCO

DIP-360 polarimeter at 2292 °C. TLC was

performed on Kieselgel 60 F₂₅₄plates (E.

Merck), and visualization was accomplished

using UV light or by charring with 10%

H₂SO₄. Column chromatography was carried

out on Silpearl silica gel (Chemapol) in a

medium pressure mode. Preparative reversed-

phase C₁₈HPLC was performed on a column

(250×24 mm) of 7.5 mm Silasorb C₁₈(Czech

Republic) in 0.05% aq CF₃CO₂H at 10 mL/

min using a Knauer 98.00 refractometer for

monitoring. ESIMS data were recorded with a

Micromass Quattro system. All reactions in-

volving air- or moisture-sensitive reagents

were carried out in dry solvents under dry

argon.

1

hexane); [h]_D+91° (c 2). H NMR: l, 1.44

(d, 3 H, J_5,66.1, H-6), 3.71 (dq, 1 H, H-5),

4.02 (dd, 1 H, J_3,49.8 Hz, H-3), 4.70, 4.93 (2

d, 2 H, J_gem12.4 Hz, PhCH₂), 4.73 (s, 1 H,

H-1), 5.23 (t, 1 H, J_4,59.6 Hz, H-4), 5.72 (d, 1

H, J_2,33.6 Hz, H-2), 7.28–8.21 (m, 15 H, 3

Ph). Anal. Calcd for C₂₇H₂₆O₇: C, 70.12; H,

5.67. Found: C, 70.13; H, 5.61.

2,3-Dibenzoate 4 had mp 135–137 °C

(EtOAc–hexane), [h]_D+82° (c 1.7). ¹H

NMR: l, 1.54 (d, 3 H, J_5,66.1, H-6), 2.43 (d,

1 H, J_4,OH4.6 Hz, OH), 3.52 (dq, 1 H, H-5),

3.92 (dt, 1 H, J_4,59.2 Hz, H-4), 4.71, 4.92 (2 d,

2 H, J_gem12.3 Hz, PhCH₂), 4.78 (s, 1 H, H-1),

5.17 (dd, 1 H, J_3,49.7 Hz, H-3), 5.83 (d, 1 H,

J_2,33.1 Hz, H-2), 7.29—8.15 (m, 15 H, 3 Ph).

Anal. Calcd for C₂₇H₂₆O₇: C, 70.12; H, 5.67.

Found: C, 69.71; H, 5.65.

Benzyl 3,4-di-O-benzoyl-i-

oside (2), benzyl 2,4-di-O-benzoyl-i-

rhamnopyranoside (3), and benzyl 2,3-di-O-

benzoyl-i- -rhamnopyranoside (4).—A mix-

L

-rhamnopyran-

L

-

Benzyl i- -rhamnopyranoside (5).—A solu-

L

tion of the above mixture of 2–4 (9.44 g, 20.4

mmol) in MeOH (50 mL) was treated with 2

M CH₃ONa (1 mL) for 5 h at 40 °C. The

mixture was neutralized with Amberlite IR-

120 (H⁺), ﬁltered, and the ﬁltrate was concen-

trated. The residue was chromatographed (1:1

toluene–acetone) to yield 5 (4.40 g, 85%): mp

107–108 °C (EtOAc–hexane); [h]_D+103° (c

L

ture of 1 (7.8 g, 21 mmol) and Bu₂SnO (5.49 g,

22 mmol) in benzene (150 mL) was boiled

with azeotropic removal of water for 2 h,

whereupon a crystalline precipitate of the

stannylene

derivative

formed.

Tetra-

butylammonium bromide (7.08 g, 22 mmol)

and benzyl bromide (5 mL, 42 mmol) were

added, and the mixture was boiled under

reﬂux for 4 h. After cooling, the solution was

washed thoroughly with water and concen-

trated. Column chromatography of the

residue (95:5 toluene–EtOAc) gave a mixture

of 2–4 (8.95 g, 92%) in ratios of ꢀ4:3:1,

which was used in the next step without

separation.

1

1.7). H NMR (CDCl₃+D₂O): l, 1.33 (d, 3

H, J_5,66.0 Hz, H-6), 3.16 (dq, 1 H, H-5), 3.34

(dd, 1 H, J_3,49.5 Hz, H-3), 3.42 (t, 1 H, J_4,5

8.9 Hz, H-4), 3.89 (d, 1 H, J_2,33.0 Hz, H-2),

4.36 (s, 1 H, H-1), 4.55, 4.84 (2 d, 2 H, J_gem

11.9 Hz, PhCH₂), 7.23–7.34 (m, 5 H, Ph).

Anal. Calcd for C₁₃H₁₈O₅: C, 61.40; H, 7.14.

Found: C, 61.43; H, 7.04.

Benzyl 2,4-di-O-acetyl-i- -rhamnopyran-

L

A small portion of the above mixture was

subjected to column chromatography (85:15

light petroleum–EtOAc) to give, in order of

elution, pure compounds 2, 3, and 4.

oside (6).—p-Toluenesulfonic acid monohy-

drate (190 mg, 1 mmol) was added to a

solution of 5 (8.86 g, 34.9 mmol) and

trimethyl orthoacetate (13.2 mL, 105 mmol) in

CH₃CN (100 mL), and the mixture was stirred

for 30 min. Py (5 mL) was added, and the

solvent was evaporated. The residue was dis-

solved in py (60 mL) and treated with Ac₂O

(20 mL) overnight. The excess of Ac₂O was

destroyed by adding water at 0 °C, the result-

ing mixture was diluted with CHCl₃, washed

successively with water, M HCl, satd aq

NaHCO₃, and water. The solvent was evapo-

rated, and the residue was treated with 80%

3,4-Dibenzoate 2: mp 172–174 °C (EtOAc–

1

hexane); [h]_D+129° (c 2). H NMR: l, 1.42

(d, 3 H, J_5,66.2 Hz, H-6), 2.53 (br. s, 1 H,

OH), 3.73 (dq, 1 H, H-5), 4.37 (dd, 1 H, J_2,3

3.2 Hz, H-2), 4.74, 4.99 (2 d, 2 H, J_gem11.9

Hz, PhCH₂), 4.75 (d, 1 H, J_1,21.1 Hz, H-1),

5.28 (dd, 1 H, J_3,410.8 Hz, H-3), 5.67 (t, 1 H,

J_4,59.7 Hz, H-4), 7.30–8.04 (m, 15 H, 3 Ph).

Anal. Calcd for C₂₇H₂₆O₇: C, 70.12; H, 5.67.

Found: C, 69.98; H, 5.59.

234

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

aq AcOH (50 mL) for 15 min. The mixture

was concentrated, and residual AcOH was

coevaporated with toluene. Column chro-

matography of the residue (1:1 toluene–

EtOAc) gave 6 (8.68g, 73.5%): mp 141–143 °C

Benzyl 3,4-anhydro-2-azido-2,6-dideoxy-i-

-allopyranoside (11).—Compound 8 (5.66 g,

L

24 mmol) was treated with Tf₂O (6.71 mL, 40

mmol) in the presence of py (5.82 mL, 72

mmol) in CH₂Cl₂(75 mL) as described in the

preparation of 7. The crude triﬂate 10 ob-

tained was dissolved in DMF (50 mL), sodium

azide (7.8 g, 120 mmol) was added, and the

mixture was stirred for 1 h at rt. The mixture

was diluted with EtOAc, washed thoroughly

with water, dried with MgSO₄and concen-

trated. Column chromatography of the

residue (85:15 light petroleum–EtOAc) gave

11 (5.22 g, 83%): mp 52–54 °C (hexane); [h]_D

+78° (c 1.1). ¹H NMR: l 1.44 (d, 3 H, J_5,66.8

Hz, H-6), 3.17 (d, 1 H, H-4), 3.45 (dd, 1 H,

J_3,44.2 Hz, H-3), 3.69 (dd, 1 H, J_2,32.2 Hz,

H-2), 4.08 (q, 1 H, H-5), 4.58 (d, 1 H, J_1,27.6

Hz, H-1), 4.62, 4.87 (2 d, 2 H, J_gem11.4 Hz,

PhCH₂), 7.29–7.41 (m, 5 H, Ph). Anal. Calcd

for C₁₃H₁₅N₃O₃: C, 59.76; H, 5.79; N, 16.08.

Found: C, 59.64; H, 5.80; N, 16.00.

1

(EtOAc–hexane); [h]_D+64.5° (c 1.8). H

NMR: l, 1.32 (d, 3 H, J_5,66.2 Hz, H-6), 2.12,

2.21 (2 s, 6 H, 2 CH₃CO), 2.48 (d, 1 H, J_3,OH

7.3 Hz, OH), 3.46 (dq, 1 H, H-5), 3.75 (ddd, 1

H, J_3,410.0 Hz, H-3), 4.57 (s, 1 H, H-1), 4.65,

4.90 (2 d, 2 H, J_gem12.0 Hz, PhCH₂), 4.84 (t,

1 H, J_4,59.5 Hz, H-4), 5.43 (d, 1 H, J_2,33.5

Hz, H-2), 7.29–7.38 (m, 5 H, Ph). Anal. Calcd

for C₁₇H₂₂O₇: C, 60.34; H, 6.55. Found: C,

60.43; H, 6.63.

Benzyl

3,4-anhydro-6-deoxy-i- -altropy-

L

ranoside (8).—A solution of triﬂuoromethane-

sulfonic anhydride (Tf₂O) (8.4 mL, 50 mmol)

in CH₂Cl₂(10 mL) was added dropwise at

0 °C to a solution of 6 (10.2 g, 30.2 mmol) and

py (7.27 mL, 90 mmol) in CH₂Cl₂(100 mL),

and the mixture was stirred at the same tem-

perature for 45 min. The solution was diluted

with CHCl₃, washed successively with ice-cold

water, M HCl, and water, and concentrated.

The crude triﬂate 7 was dissolved in MeOH

(70 mL) and treated with 2 M CH₃ONa (30

mL) for 30 min at rt. The solution was neu-

tralised with AcOH, taken to dryness, and the

residue was distributed between water and

CHCl₃. The organic layer was separated, and

the water layer was extracted twice with

CHCl₃. The combined organic solution was

concentrated, and the residue was subjected to

column chromatography (3:2 toluene–EtOAc)

to give 8 (5.66 g, 79%) as a syrup: [h]_D+87°

Benzyl 2,4-diazido-2,4,6-trideoxy-i- -gulo-

L

pyranoside (12).—A solution of sodium azide

(6.7 g, 103 mmol) and ammonium chloride

(5.51 g, 103 mmol) in water (25 mL) was

added to a solution of 11 (5.38 g, 20.6 mmol)

in EtOH (100 mL). The mixture was boiled

under reﬂux for 7 h, EtOH was distilled off,

and the remaining aqueous solution was ex-

tracted three times with CHCl₃. The combined

extract was concentrated, and the residue was

subjected to column chromatography (92:8

toluene–EtOAc) to yield 12 (5.40 g, 86%) as a

1

syrup: [h]_D+63° (c 2). H NMR: l 1.37 (d, 3

1

H, J_5,66.5 Hz, H-6), 2.47 (s, 1 H, OH), 3.47

(dd, 1 H, J_4,51.3 Hz, J_3,43.4 Hz, H-4), 3.66

(dd, 1 H, J_2,33.1 Hz, H-2), 4.14 (m, 2 H,

H-3,5), 4.67, 4.96 (2 d, 2 H, J_gem11.8 Hz,

PhCH₂), 4.77 (d, 1 H, J_1,28.1 Hz, H-1),

7.30–7.41 (m, 5 H, Ph). Anal. Calcd for

C₁₃H₁₆N₆O₃: C, 51.31; H, 5.30; N, 27.62.

Found: C, 51.44; H, 5.35; N, 27.63.

(c 2.6). H NMR: l 1.47 (d, 3 H, J_5,67.0 Hz,

H-6), 2.63 (br, s, 1 H, OH), 3.04 (d, 1 H, H-4),

3.44 (dd, 1 H, J_3,43.9 Hz, H-3), 4.02 (t, 1 H,

J_2,31.7 Hz, H-2), 4.07 (q, 1 H, H-5), 4.54 (d 1

H, J_1,21.6 Hz, H-1), 4.58, 4.89 (2 d, 2 H, J_gem

11.9 Hz, PhCH₂), 7.27–7.42 (m, 5 H, Ph).

Anal. Calcd for C₁₃H₁₆O₄: C, 66.08; H, 6.83.

Found: C, 66.13; H, 6.95.

Conventional acetylation of 12 with Ac₂O

Conventional acetylation of 8 with Ac₂O in

1

in py gave 3-acetate 13. H NMR: l 1.38 (d, 3

py afforded 2-acetate 9. H NMR: l 1.50 (d, 3

H, J_5,66.5 Hz, H-6), 2.05 (s, 3 H, CH₃CO),

3.48 (dd, 1 H, J_4,51.7 Hz, H-4), 3.69 (dd, 1 H,

J_2,33.4 Hz, H-2), 4.03 (dq, 1 H, H-5), 4.69,

4.96 (2 d, 2 H, J_gem11.8 Hz, PhCH₂), 4.79 (d,

1 H, J_1,28.1 Hz, H-1), 5.33 (t, 1 H, J_3,43.5 Hz,

H-3), 7.30–7.42 (m, 5 H, Ph).

H, J_5,67.5 Hz, H-6), 2.17 (s, 3 H, CH₃CO),

3.07 (d, 1 H, H-4), 3.39 (dd, 1 H, J_3,44.0 Hz,

H-3), 4.10 (q, 1 H, H-5), 4.57, 4.86 (2 d, 2 H,

J_gem13.2 Hz, PhCH₂), 4.64 (d, 1 H, J_1,22.1

Hz, H-1), 5.25 (t, 1 H, J_2,31.8 Hz, H-2),

7.25–7.38 (m, 5 H, Ph).

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

235

Benzyl

2-azido-3-O-benzyl-2-deoxy-i- -

D

Benzyl 2,4-diacetamido-2,4,6-trideoxy-i- -

L

gulopyranoside (14).—20% Pd(OH)₂/C (950

mg) was added to a solution of 12 (3.77 g,

12.4 mmol) in MeOH (60 mL), and the mix-

ture was stirred vigorously in a hydrogen at-

mosphere for 2.5 h at 30–32 °C. The catalyst

was ﬁltered through Celite, washed with

MeOH, and the combined ﬁltrate and wash-

ings were concentrated to a volume of ꢀ20

mL. Ac₂O (6 mL) was added, and the solution

was kept for 1 h and evaporated. Residual

Ac₂O was removed by coevaporation with

toluene, and the residue was chromatographed

(95:5 chloroform–MeOH) to give 14 (3.48 g,

84%) as an amorphous solid: [h]_D+46.5° (c

mannopyranoside (17).—Pyridinium perchlo-

rate (1.14 g, 6.34 mmol) was added to a

solution of 16 (3.00 g, 6.34 mmol) in 90% aq

CH₃CN (30 mL), and the mixture was heated

at 80 °C for 4 h. Py (0.5 mL) was added, and

the solvent was evaporated. The residue was

distributed between water (50 mL) and CHCl₃

(50 mL), the organic layer was separated, and

the water layer was extracted with CHCl₃

(3×50 mL). The combined extract was con-

centrated, and the residue was subjected to

column chromatography (3:2 toluene–EtOAc)

to give 17 (2.37 g, 97%) as a syrup: [h]_D

1

−106° (c 1, CHCl₃). H NMR: l 3.26 (ddd, 1

1

3). H NMR (CDCl₃+D₂O): l 1.17 (d, 3 H,

H, H-5), 3.46 (dd, 1 H, J_3,49.2 Hz, H-3), 3.82

(dd, 1 H, J_5,6a4.9 Hz, J_6a,6b12.1 Hz, H-6a),

3.87 (t, 1 H, J_4,59.5 Hz, H-4), 3.92 (dd, 1 H,

J_5,6b3.7 Hz, H-6b), 3.95 (d, 1 H, J_2,33.6 Hz,

H-2), 4.56 (br. s, 1 H, H-1), 4.61, 4.74 (2 d, 2

H, J_gem11.7 Hz, PhCH₂), 4.64, 4.94 (2 d, 2 H,

J_gem12.2 Hz, PhCH₂), 7.30–7.42 (m, 10 H, 2

Ph). Anal. Calcd for C₂₀H₂₃N₃O₅: C, 62.32; H,

6.02; N, 10.90. Found: C, 62.46; H, 5.92; N,

10.85.

J_5,66.5 Hz, H-6), 1.96, 2.02 (2 s, 6 H,

CH₃CO), 3.87 (t, 1 H, J_3,43.2 Hz, H-3), 3.98

(dd, 1 H, J_4,51.5 Hz, H-4), 4.05 (dd, 1 H, J_2,3

3.4 Hz, H-2), 4.20 (dq, 1 H, H-5), 4.56, 4.86 (2

d, 2 H, J_gem12.5 Hz, PhCH₂), 4.58 (d, 1 H,

J_1,28.6 Hz, H-1), 7.28–7.37 (m, 5 H, Ph).

Anal. Calcd for C₁₇H₂₄N₂O^.₅0.5 H₂O: C, 59.11;

H, 7.29; N, 8.11. Found: C, 59.28; H, 7.36; N,

8.45.

2,4-Diacetamido-2,4,6-trideoxy- -gulopyran-

L

Benzyl 2-azido-3-O-benzyl-2-deoxy-6-O-to-

ose (15).—A solution of 14 (4.45 g, 13.2

mmol) in MeOH (60 mL) was stirred with

20% Pd(OH)₂/C (1 g) under hydrogen for 4 h

at 32–34 °C, ﬁltered through Celite, (Caution:

Extreme ﬁre hazard) and concentrated.

Column chromatography of the residue (94:6

CHCl₃–MeOH) gave 15 (3.20g, 94%): mp

144–146 °C (MeOH–ether); [h]_D+6.3°

syl-i- -mannopyranoside (18).—p-Toluene-

D

sulfonyl chloride (1.72 g, 9.04 mmol) was

added at 0 °C to a solution of 17 (2.32 g, 6.02

mmol) in py (15 mL). The stirred mixture was

allowed to warm to rt for 1.5 h, and stirring

was continued for the next 2 h. The reaction

was quenched by adding water, the resulting

mixture was diluted with CHCl₃, washed suc-

cessively with water, M HCl, and water, and

concentrated. Column chromatography of the

residue (9:1 toluene–EtOAc) yielded 18 (2.86

g, 88%) as a syrup: [h]_D−79.6° (c 2, CHCl₃).

¹H NMR: l 2.41 (s, 3 H, CH₃C₆H₄), 2.47 (br.

s, 1 H, OH), 3.39 (dd, 1 H, J_3,49.1 Hz, H-3),

3.42 (ddd, 1 H, H-5), 3.70 (t, 1 H, J_4,59.8 Hz,

H-4), 3.94 (d, 1 H, J_2,33.4 Hz, H-2), 4.23 (dd,

1 H, J_5,6a6.6 Hz, J_6a,6b10.9 Hz, H-6a), 4.43

(dd, 1 H, J_5,6b1.7 Hz, H-6b), 4.48 (s, 1H,

H-1), 4.56 (d, 2 H, J_gem11.8 Hz, PhCH₂), 4.73

(d, 1 H, J_gem11.7 Hz, PhCH₂), 4.86 (d, 1 H,

J_gem12.0 Hz, PhCH₂), 7.30–7.84 (m, 14 H, 2

Ph, CH₃C₆H₄). Anal. Calcd for C₂₇H₂₉N₃O₇S:

C, 60.10; H, 5.42; N, 7.79. Found: C, 59.89;

H, 5.57; N, 8.03.

1

+78° (c 1.6, MeOH). H NMR (D₂O): 15a, l

1.16 (d, 3 H^†, J_5,66.6 Hz, H-6), 2.06, 2.07 (2 s,

6 H, 2 CH₃CO), 3.91 (t, 1 H, J_3,43.8 Hz, H-3),

3.95 (dd, 1 H, J_4,51.7 Hz, H-4), 4.11 (t, 1 H,

J_2,33.5 Hz, H-2), 4.62 (dq, 1 H, H-5), 5.15 (d,

1 H, J_1,24.0 Hz, H-1); 15b, l 1.18 (d, 3 H*,

J_5,66.5 Hz, H-6), 2.04, 2.08 (2 s, 6 H, 2

CH₃CO), 3.85 (dd, 1 H, J_2,33.2 Hz, H-2), 3.87

(dd, 1 H, J_4,51.6 Hz, H-4), 3.94 (t, 1 H, J_3,4

3.4 Hz, H-3), 4.29 (dq, 1 H, H-5), 4.94 (d, 1

H, J_1,28.9 Hz, H-1). The ratio 15a:15b :1:5.

Anal. Calcd for C₁₀H₁₈N₂O₅·0.25 H₂O: C,

47.60; H, 7.39; N, 11.10. Found: C, 47.59; H,

7.50; N, 11.49.

^†Integral intensities of signals for the compounds 15, 32,

and 48 are given within anomeric series.

236

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

Benzyl

2-acetamido-3-O-benzyl-2,6-dide-

Benzyl 2-azido-3-O-benzyl-2,6-dideoxy-6-

iodo-i- -mannopyranoside (19).—A solution

oxy-i- -lyxo-hexopyranoside-4-ulose, oxime

D

(22).—A solution of DMSO (0.97 mL, 13.6

mmol) in CH₂Cl₂(4 mL) was added at

−60 °C to a solution of oxalyl chloride (0.54

mL, 6.19 mmol) in CH₂Cl₂(15 mL), and the

mixture was stirred for 0.5 h while the temper-

ature gradually increased to −30 °C. After

cooling to −60 °C, a solution of 20 (1.59 g,

4.13 mmol) in CH₂Cl₂(15 mL) was added

dropwise, the mixture was stirred at −60 °C

for 45 min, and then N,N-diisopropylethyl-

amine (5.3 mL) was added. The mixture was

allowed to warm to −20 °C, diluted with

CHCl₃, washed with M HCl, water, and con-

centrated. The residue was passed through a

short column with silica gel in (85:15) tolu-

ene–acetone, and the eluate was concentrated

to afford 21. Hydroxylamine hydrochloride

(565 mg, 8.1 mmol) was added to a solution of

the ketone 21 in a mixture of CH₂Cl₂(12 mL)

and py (9 mL), and the solution was stirred at

rt overnight. The mixture was diluted with

CHCl₃, and washed with water, and the water

phase was extracted twice with CHCl₃. The

combined organic extract was concentrated,

and the residue was chromatographed (3:2

toluene–acetone) to give 22 (1.59 g, 97%) as a

mixture of isomers in a ratio ofꢀ8:1. Crystal-

lization from ether–light petroleum gave the

major isomer: mp 127–129 °C, [h]_D−35° (c

of 18 (2.76 g, 5.12 mmol) and sodium iodide

(3.84 g, 25.6 mmol) in CH₃CN (30 mL) was

heated with stirring at 80–85 °C for 7 h. The

solvent was evaporated, and a suspension of

the residue in CHCl₃was washed successively

with water, M Na₂S₂O₃, and water, and then

concentrated. The residue was chro-

matographed (95:5 toluene–EtOAc) to give 19

(2.42 g, 96%) as a syrup: [h]_D−77.3° (c 2,

1

CHCl₃). H NMR: l 3.22 (dt, 1 H, H-5), 2.46

(d, 1 H, J_4,OH1.5 Hz, OH), 3.30 (t, 1 H, J_5,6a

9.1 Hz, J_6a,6b10.4 Hz, H-6a), 3.39 (dd, 1 H,

J_3,49.0 Hz, H-3), 3.64 (dt, 1 H, J_4,58.9 Hz,

H-4), 3.65 (dd, 1 H, J_5,6b1.8 Hz, H-6b), 3.97

(d, 1 H, J_2,33.4 Hz, H-2), 4.53, 4.75 (2 d, 2 H,

J_gem11.6 Hz, PhCH₂), 4.54 (s, 1 H, H-1), 4.75,

5.02 (2 d, 2 H, J_gem12.1 Hz, PhCH₂), 7.33–

7.45 (m, 10 H, 2 Ph). Anal. Calcd for

C₂₀H₂₂IN₃O₄: C, 48.50; H, 4.48; N, 8.48.

Found: C, 48.23; H, 4.47; N, 8.18.

Benzyl

2-acetamido-3-O-benzyl-2,6-dide-

oxy-i- -mannopyranoside (20).—A solution

D

of 19 (2.50 g, 5.05 mmol) and N,N-diiso-

propylethylamine (1.75 mL, 10.1 mmol) in

MeOH (25 mL) was stirred with 20%

Pd(OH)₂/C (1 g) under hydrogen at 32 °C for

6 h. The catalyst was ﬁltered off through

Celite and washed with MeOH, and the com-

bined ﬁltrate and washings were treated with

Dowex 1×8 (HCO₃^–) anion-exchange resin,

ﬁltered, and concentrated. Ac₂O (2.5 mL) was

added to a solution of the residue in MeOH

(25 mL), and the mixture was kept overnight

at rt. The solvent was evaporated, Ac₂O was

removed by coevaporation with toluene, and

the residue was subjected to column chro-

matography (85:15 toluene–acetone) to give

20 (1.63 g, 84%) as a foam: [h]_D−116° (c 1,

1

1, CHCl₃). H NMR: l 1.64 (d, 3 H, J_5,67.1

Hz, H-6), 2.00 (s, 3 H, CH₃CO), 4.28 (d, 1 H,

J_1,24.9 Hz, H-1), 4.47 (ddd, 1 H, J_2,34.3 Hz,

H-2), 4.30, 5.01 (2 d, 2 H, J_gem12.0 Hz,

PhCH₂), 4.51, 4.67 (2 d, 2 H, J_gem11.9 Hz,

PhCH₂), 4.92 (d, 1 H, H-3), 5.29 (q, 1 H,

H-5), 6.45 (d, 1 H, J_NH,29.6 Hz, NH), 7.25–

7.38 (m, 10 H, 2 Ph). Anal. Calcd for

C₂₂H₂₆N₂O₅: C, 66.31; H, 6.58; N, 7.03.

Found: C, 66.40; H, 6.54; N, 6.88.

1

CHCl₃). H NMR: l 1.49 (d, 3 H, J_5,66.3 Hz,

Benzyl

trideoxy-i-

2,4-diacetamido-3-O-benzyl-2,4,6-

-talopyranoside (23) and benzyl

H-6), 2.08 (s, 3 H, CH₃CO), 3.27 (t, 1 H, J_4,5

9.1 Hz, H-4), 3.32 (dq, 1 H, H-5), 3.35 (dd, 1

H, J_3,49.1 Hz, H-3), 4.39, 4.93 (2 d, 2 H, J_gem

10.9 Hz, PhCH₂), 4.54 (s, 1 H, H-1), 4.63, 4.86

(2 d, 2 H, J_gem12.2 Hz, PhCH₂), 4.84 (dd, 1

H, J_2,34.0 Hz, H-2), 5.74 (d, 1 H, J_NH,29.5

Hz, NH), 7.27–7.39 (m, 10 H, 2 Ph). Anal.

Calcd for C₂₂H₂₇NO₅: C, 68.55; H, 7.06; N,

3.63. Found: C, 68.65; H, 7.11; N, 3.52.

D

2,4-diacetamido-3-O-benzyl-2,4,6-trideoxy-i-

-mannopyranoside (24).—Sodium borohy-

D

dride (1.55 g, 40.7 mmol) was added

portionwise to a solution of 22 (1.62 g, 4.07

mmol) and NiCl₂·6H₂O (1.94 g, 8.14 mmol) in

MeOH (50 mL) at −35 °C over 0.5 h. The

mixture was stirred at the same temperature

for 0.5 h and quenched by adding satd aq

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

237

(630 mg, 88%) as an amorphous solid: [h]_D

NaHCO₃(50 mL). The bulk of the MeOH

was evaporated, and the remaining aqueous

solution was extracted with CH₂Cl₂(3×50

mL). The combined extract was concentrated,

and the residue was acetylated with Ac₂O (1.5

mL) in MeOH (20 mL) overnight. The solvent

was evaporated, the Ac₂O was coevaporated

with toluene, and the residue was subjected to

column chromatography (95:5 CHCl₃–

MeOH) to yield a mixture of 23 and 24.

Individual 23 (1.39 g, 80%) and 24 (130 mg,

8%) were isolated by preparative HPLC on a

Zorbax SIL (250×21 mm) column (DuPont)

in 97:3 EtOAc–MeOH.

1

+23.6° +15.3° (c 1, water). For H and

¹³C NMR data see Table 1. Anal. Calcd for

C₁₀H₁₈N₂O₅: C, 48.77; H, 7.37; N, 11.38.

Found: C, 48.98; H, 7.57; N, 11.42.

Benzyl

3-O-benzoyl-i- -fucopyranoside

D

(27).—A mixture of 26 (2.22 g, 8.74 mmol)

and Bu₂SnO (2.29 g, 9.18 mmol) in benzene

(40 mL) was boiled with stirring and

azeotropic removal of water for 5 h, then 15

mL benzene was distilled off from the mix-

ture. The remaining solution was cooled to

0 °C and benzoyl chloride (1.12 mL, 9.61

mmol) was added. After stirring at 0–5 °C for

2 h, MeOH (0.5 mL) and py (0.5 mL) were

added to destroy the excess benzoyl chloride.

After being stirred for 0.5 h at rt, the mixture

was concentrated, and the residue was sub-

jected to column chromatography (9:1 tolu-

ene–EtOAc) to give 27 (1.90 g, 61%): mp

Compound 23: mp 158–159 °C (MeOH–

1

Et₂O), [h]_D−49.4° (c 1, CHCl₃). H NMR

(toluene-d₈, 333 K): l 1.50 (d, 3 H, J_5,66.3 Hz,

H-6), 1.98, 2.00 (2 s, 6 H, 2 CH₃CO), 3.53

(poorly resolved q, 1 H, H-5), 3.61 (poorly

resolved t, 1 H, J_2,3ꢀJ_3,4ꢀ2 Hz, H-3), 4.60

(s, 1 H, H-1), 4.62 (unresolved, 1 H, H-4),

4.69, 4.98 (2 d, 2 H, J_gem12.0 Hz, PhCH₂),

4.79 (unresolved, 1 H, H-2), 4.81 (s, 2 H

PhCH₂), 6.18 (unresolved, 1 H, NH), 6.44

(unresolved, 1 H, NH), 7.33–7.73 (m, 10 H, 2

Ph). Anal. Calcd for C₂₄H₃₀N₂O₅: C, 67.58; H,

7.09; N, 6.57. Found: C, 67.44; H, 7.07; N,

6.56.

97–98 °C (EtOAc–light petroleum); [h]_D

+

1

12.5° (c 1, CHCl₃). H NMR: l 1.38 (d, 3 H,

J_5,66.4 Hz, H-6), 2.11, 2.34 (2 br. s, 2 H, 2

OH), 3.76 (q, 1 H, H-5), 3.96 (d, 1 H, H-4),

4.03 (dd, 1 H, J_2,310.1 Hz, H-2), 4.47 (d, 1 H,

J_1,27.7 Hz, H-1), 4.65, 4.99 (2 d, 2 H, J_gem

11.7 Hz, PhCH₂), 5.07 (dd, 1 H, J_3,43.0 Hz,

H-3), 7.32–8.11 (m, 10 H, 2 Ph). Anal. Calcd

for C₂₀H₂₂O₆: C, 67.02; H, 6.19. Found: C,

66.92; H, 6.29.

Compound 24 was obtained as a foam: [h]_D

1

−109.5° (c 1, CHCl₃). H NMR (C₆D₆): l

1.37 (d, 3 H, J_5,66.2 Hz, H-6), 1.68, 1.70 (2 s,

6 H, 2 CH₃CO), 3.02 (dq, 1 H, H-5), 3.15 (dd,

1 H, J_3,410.3 Hz, H-3), 3.94 (q, 1 H, J_4,59.8

Hz, H-4), 4.35 (s, 1 H, H-1), 4.43, 4.84 (2 d, 2

H, J_gem12.2 Hz, PhCH₂), 4.57, 4.94 (2 d, 2 H,

J_gem12.2 Hz, PhCH₂), 4.86 (d, 1 H, J_NH,48.9

Hz, NH-4), 5.09 (dd, 1 H, J_2,34.2 Hz, H-2),

6.29 (d, 1 H, J_NH,29.7 Hz, NH-2), 7.08–7.39

(m, 10 H, 2 Ph). Anal. Calcd for C₂₄H₃₀N₂O₅:

C, 67.58; H, 7.09; N, 6.57. Found: C, 66.86;

H, 6.96; N, 6.69.

Benzyl 2,4-diazido-3-O-benzoyl-2,4,6-tride-

oxy-i- -mannopyranoside (29).—Tf₂O (3.10

D

mL, 18.5 mmol) was added dropwise at 0 °C

to a solution of 27 (2.20 g, 6.14 mmol) and py

(2.98 mL, 36.8 mmol) in CH₂Cl₂(40 mL). The

mixture was stirred at 0 °C for 1 h, diluted

with CHCl₃, washed successively with water,

M HCl, and water, and concentrated. The

crude ditriﬂate 28 obtained was dissolved in

toluene (40 mL) and tetrabutylammoniun

azide (10.45 g, 36.8 mmol) was added. The

mixture was stirred for 1 h at 65–70 °C and

then for 1.5 h at 100–105 °C, cooled, diluted

with toluene, washed twice with water, and

concentrated. Column chromatography of the

residue (7:3 toluene–light petroleum) gave 29

(2.12 g, 85%) as a syrup, [h]_D−46.1° (c 1,

2,4 - Diacetamido - 2,4,6 - trideoxy -

D

- talose

(25).—A solution of 23 (1.24 g, 2.91 mmol) in

MeOH (20 mL) and water (10 mL) was stirred

with 20% Pd(OH)₂/C (500 mg) under hydro-

gen for 6 h at rt. The catalyst was ﬁltered

through Celite, and washed with MeOH,

(Caution: Extreme ﬁre hazard), the combined

ﬁltrate and washings were concentrated, and

the residue was subjected to column chro-

matography (85:15 CHCl₃–MeOH) to give 25

1

CHCl₃). H NMR: l 1.59 (d, 3 H, J_5,66.1 Hz,

H-6), 3.40 (dq, 1 H, H-5), 3.77 (t, 1 H, J_4,59.7

Hz, H-4), 4.36 (d, 1 H, J_2,33.6 Hz, H-2), 4.77,

5.09 (2 d, 2 H, J_gem12.1 Hz, PhCH₂), 4.77 (s,

238

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

1 H, J_3,410.8 Hz, H-3), 4.47 (dd, 1 H, J_2,34.3

Hz, H-2), 4.96 (d, 1 H, J_1,21.3 Hz, H-1). The

ratio 32a:32b :55:45. Anal. Calcd for

C₁₀H₁₈N₂O₅·0.5H₂O: C, 47.05; H, 7.50; N,

10.97. Found: C, 47.21; H, 7.74; N, 11.12.

1 H, H-1), 5.14 (dd, 1 H, J_3,410.1 Hz, H-3),

7.40–8.23 (m, 10 H, 2 Ph). Anal. Calcd for

C₂₀H₂₀N₆O₄: C, 58.81; H, 4.94; N, 20.58.

Found: C, 59.18; H, 5.05; N, 20.61.

Benzyl 2,4-diacetamido-2,4,6-trideoxy-i- -

D

Benzyl 2,3-O-isopropylidene-i- -rhamnopy-

L

mannopyranoside (31).—A solution of 29

(2.12 g, 5.20 mmol) in MeOH (20 mL) was

treated with 2 M sodium methoxide in MeOH

(1 mL) for 1 h at rt. The solution was neu-

tralised by adding Amberlite IR-120 (H⁺

form) ion-exchange resin, and ﬁltered, and the

ﬁltrate was concentrated. A solution of crude

30 in MeOH (30 mL) was stirred with 20%

Pd(OH)₂/C (400 mg) at 30 °C under hydrogen

for 1.5 h. The catalyst was ﬁltered off through

Celite, washed with MeOH, and the combined

ﬁltrate and washings were concentrated to a

volume of ꢀ20 mL, then Ac₂O (2 mL) was

added. After being kept for 1 h at rt, the

mixture was evaporated to dryness, and the

residue was subjected to column chromatogra-

phy (97:3 CHCl₃–MeOH) to yield 31 (1.51 g,

ranoside (33).—p-Toluenesulfonic acid mono-

hydrate (190 mg, 1 mmol) was added to a

solution of 5 (6.20 g, 24.4 mmol) and 2,2-

dimethoxypropane (18.5 mL, 150 mmol) in

acetone (50 mL), and the mixture was kept for

1 h at rt. A few drops of Et₃N were added, the

solvent was evaporated, and a solution of the

residue in CHCl₃was washed twice with water

and concentrated. The residue was crystallized

from EtOAc–light petroleum to give 5.77 g of

33. An additional portion of the product (1.17

g) was obtained by column chromatography

(4:1 toluene–EtOAc) of the mother liquor.

Total yield of 33 was 6.49 g (97%), mp 97–

1

98 °C, [h]_D+142.6° (c 1, CHCl₃). H NMR:

l 1.38 (d, 3 H, J_5,66.1 Hz, H-6), 1.40, 1.58 (2

s, 6 H, isopropylidene), 2.61 (d, 1 H, J_OH,43.1

Hz, OH), 3.27 (dq, 1 H, H-5), 3.56 (ddd, 1 H,

J_4,59.8 Hz, H-4), 3.99 (t, 1 H, J_3,46.4 Hz,

H-3), 4.20 (dd, 1 H, J_2,35.6 Hz, H-2), 4.71 (d,

1 H, J_1,21.3 Hz, H-1), 4.75, 4.97 (2 d, 2 H,

J_gem12.5 Hz, PhCH₂), 7.29–7.40 (m, 5 H, Ph).

Anal. Calcd for C₁₆H₂₂O₅: C, 65.29; H, 7.53.

Found: C, 65.32; H, 7.50.

1

86%) as a foam: [h]_D−25.2° (c 1, CHCl₃). H

NMR (C₆D₆+CD₃OD): l 1.40 (d, 3 H, J_5,6

6.2 Hz, H-6), 1.86, 1.96 (2 s, 6 H, 2 CH₃CO),

3.32 (dq, 1 H, H-5), 3.83 (dd, 1 H, J_3,410.5

Hz, H-3), 3.95 (t, 1 H, J_4,510.1 Hz, H-4), 4.50,

4.80 (2 d, 2 H, J_gem12.0 Hz, PhCH₂), 4.54 (s,

1 H, H-1), 4.73 (d, 1 H, J_2,32.5 Hz, H-2),

7.07–7.43 (m, 5 H, Ph). Anal. Calcd for

C₁₇H₂₄N₂O₅: C, 60.70; H, 7.19; N, 8.33.

Found: C, 60.79; H, 7.24; N, 8.36.

Benzyl 6-deoxy-2,3-O-isopropylidene-i- -

L

talopyranoside (35).—A solution of DMSO

(4.60 mL, 64.7 mmol) in CH₂Cl₂(10 mL) was

added at −60 °C to a solution of oxalyl

chloride (2.57 mL, 29.4 mmol) in CH₂Cl₂(40

mL) and the mixture was stirred for 20 min

while the temperature gradually increased to

−40 °C. After cooling to −60 °C, a solution

of 33 (5.77 g, 19.6 mmol) in CH₂Cl₂(50 mL)

was added dropwise, the mixture was stirred

at −60 °C for 45 min, then N,N-diisopropy-

lethylamine (17.4 mL) was added. The mixture

was allowed to warm to −20 °C, diluted with

CHCl₃, washed with M HCl, water, and con-

centrated to yield ketone 34. NaBH₄(760 mg,

20 mmol) was added at 0 °C to a solution of

crude 34 in 80% aq EtOH (60 mL) and the

mixture was stirred for 20 min. The reduction

was quenched by adding acetone, the solvent

was evaporated, and a solution of the residue

in CHCl₃was washed with M HCl, water, and

2,4-Diacetamido-2,4,6-trideoxy- -mannopy-

D

ranose (32).—A mixture of 31 (1.35 g, 4.02

mmol) and 20% Pd(OH)₂/C (400 mg) in

MeOH (25 mL) was stirred at 32 °C in hydro-

gen atmosphere for 2 h. The catalyst was

ﬁltered through Celite, washed with MeOH,

(Caution: Extreme ﬁre hazard), and the com-

bined ﬁltrate and washings were concentrated.

The residue was chromatographed (85:15

CHCl₃–MeOH) to give 32 (840 mg, 85%) as

an amorphous solid: [h]_D+38.1° (c 1, water).

¹H NMR (D₂O): 32a, l 1.19 (d, 3 H, J_5,66.3

Hz, H-6), 2.04, 2.08 (2 s, 6 H, 2 CH₃CO), 3.78

(t, 1 H, J_4,510.2 Hz, H-4), 3.97 (dq, 1 H, H-5),

4.07 (dd, 1 H, J_3,410.8 Hz, H-3), 4.30 (dd, 1

H, J_2,34.6 Hz, H-2), 5.11 (d, 1 H, J_1,21.3 Hz,

H-1). 32b, l 1.23 (d, 3 H, J_5,66.2 Hz, H-6),

2.03, 2.12 (2 s, 6 H, 2 CH₃CO), 3.51 (dq, 1 H,

H-5), 3.67 (t, 1 H, J_4,510.0 Hz, H-4), 3.84 (dd,

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

239

concentrated. Column chromatography of the

residue (85:15 toluene–EtOAc) afforded 35

(5.44 g, 94%), mp 69–70 °C (ether–hexane),

Benzyl 4-azido-4,6-dideoxy-2,3-O-isopropy-

lidene-i- -mannopyranoside (41).—Tf₂O (4.87

L

mL, 29 mmol) was added dropwise at 0 °C to

a solution of 35 (4.27 g, 14.5 mmol) and py

(11.7 mL, 145 mmol) in CH₂Cl₂(40 mL), and

the mixture was stirred for 3 h at the same

temperature. After dilution with CHCl₃, the

solution was washed successively with water,

M HCl, and water, and concentrated to yield

crude triﬂate 40. Sodium azide (4.71g, 72.5

mmol) and dibenzo-18-crown-6 (130 mg, 0.36

mmol) were added to a solution of 40 in DMF

(40 mL) and the resulting mixture was stirred

overnight at rt. Most of the DMF was evapo-

rated, and a suspension of the residue in

EtOAc was washed twice with water, then

with satd aq NaCl solution. The organic solu-

tion was dried with MgSO₄, and concentrated,

and the residue was chromatographed (95:5

toluene–EtOAc) to give 41 (3.44 g, 74%): mp

95–96 °C (light petroleum); [h]_D+137.2° (c

1

[h]_D+108.6° (c 1, CHCl₃). H NMR: l 1.40

(d, 3 H, J_5,66.4 Hz, H-6), 1.42, 1.63 (2 s, 6 H,

isopropylidene), 2.81 (d, 1 H, J_OH,49.8 Hz,

OH), 3.50 (q, 1 H, H-5), 3.60 (dd, 1 H, H-4),

4.16 (dd, 1 H, J_2,36.3 Hz, H-2), 4.21 (t, 1 H,

J_3,45.6 Hz, H-3), 4.72 (d, 1 H, J_1,22.4 Hz,

H-1), 4.77, 4.97 (2 d, 2 H, J_gem12.3 Hz,

PhCH₂), 7.30–7.43 (m, 5 H, Ph). Anal. Calcd

for C₁₆H₂₂O₅: C, 65.29; H, 7.53. Found: C,

65.35; H, 7.47.

Benzyl 6-deoxy-i- -talopyranoside (36).—

L

A solution of 35 (925 mg, 3.15 mmol) in 80%

aq AcOH (10 mL) was heated at 40 °C for 1.5

h. The solvent was coevaporated several times

with toluene, and the residue was subjected to

column chromatography (4:1 toluene–ace-

tone) to give 36 (795 mg, 99%): mp 86–88 °C

(EtOAc–light petroleum), [h]_D+89.4° (c 1,

1

CHCl₃). H NMR: l 1.40 (d, 3 H, J_5,66.5 Hz,

1

1, CHCl₃). H NMR: l 1.38 (d, 3 H, J_5,66.0

H-6), 3.45 (q, 1 H, H-5), 3.48 (t, 1 H, J_3,43.4

Hz, H-3), 3.53 (d, 1 H, H-4), 3.95 (d, 1 H, J_2,3

3.2 Hz, H-2), 4.38 (s, 1 H, H-1), 4.68, 4.96 (2

d, 2 H, J_gem11.9 Hz, PhCH₂), 7.30–7.38 (m, 5

H, Ph). Anal. Calcd for C₁₃H₁₈O₅: C, 61.40;

H, 7.14. Found: C, 61.34; H, 7.04.

Hz, H-6), 1.40, 1.62 (2 s, 6 H, isopropylidene),

3.17 (dq, 1 H, H-5), 3.34 (dd, 1 H, J_4,510.3

Hz, H-4), 4.07 (dd, 1 H, J_3,47.6 Hz, H-3), 4.17

(dd, 1 H, J_2,35.5 Hz, H-2), 4.65 (d, 1 H, J_1,2

2.1 Hz, H-1), 4.75, 4.97 (2 d, 2 H, J_gem12.5

Hz, PhCH₂), 7.28–7.42 (m, 5 H, Ph). Anal.

Calcd for C₁₆H₂₁N₃O₄: C, 60.17; H, 6.63; N,

13.16. Found: C, 59.92; H, 6.60; N, 13.30.

Benzyl 3-O-benzoyl-6-deoxy-i- -talopyran-

L

oside (37).—A mixture of 36 (302 mg, 1.19

mmol) and Bu₂SnO (311 mg, 1.25 mmol) in

benzene (20 mL) was boiled with stirring and

azeotropic removal of water for 2.5 h, then 10

mL benzene was distilled from the mixture.

The remaining solution was cooled to 0 °C,

and benzoyl chloride (152 ml, 1.31 mmol) was

added. After stirring at 0–5 °C for 45 min,

MeOH (0.1 mL) and py (0.1 mL) were added

to destroy the excess of benzoyl chloride. Af-

ter being stirred for 0.5 h at rt, the mixture

was concentrated, and the residue was sub-

jected to column chromatography (85:15 tolu-

ene–EtOAc) to give 37 (334 mg, 78%): mp

Benzyl 4-azido-4,6-dideoxy-i- -mannopy-

L

ranoside (42).—A solution of 41 (3.37 g, 10.56

mmol) in 80% aq AcOH (30 mL) was heated

at 40 °C for 5 h. The solvent was evaporated

and coevaporated several times with toluene.

Column chromatography of the residue (7:3

toluene–EtOAc) gave 42 (2.79 g, 95%): mp

89–91 °C (Et₂O–light petroleum); [h]_D

+

1

85.3° (c 1, CHCl₃). H NMR: l 1.41 (d, 3 H,

J_5,66.1 Hz, H-6), 2.71 (br s, 1 H, OH-2), 2.80

(d, 1 H, J_3,OH8.4 Hz, OH-3), 3.15 (dq, 1 H,

H-5), 3.29 (t, 1 H, J_4,59.8 Hz, H-4), 3.54 (m,

1 H, J_3,49.6 Hz, H-3), 3.97 (d, 1 H, J_2,33.0

Hz, H-2), 4.44 (s, 1 H, H-1), 4.64, 4.92 (2 d, 2

H, J_gem11.8 Hz, PhCH₂), 7.33–7.39 (m, 5 H,

Ph). Anal. Calcd for C₁₃H₁₇N₃O₄: C, 55.90; H,

6.14; N, 15.05. Found: C, 56.02; H, 6.38; N,

15.24.

100–102 °C (ether–light petroleum); [h]_D

+

1

27.1° (c 1, CHCl₃). H NMR: l 1.43 (d, 3 H,

J_5,66.4 Hz, H-6), 3.60 (q, 1 H, H-5), 3.78 (d,

1 H, H-4), 4.19 (d, 1 H, J_2,33.1 Hz, H-2), 4.55

(s, 1H, H-1), 4.73, 4.99 (2 d, 2 H, J_gem11.9 Hz,

PhCH₂), 4.94 (t, 1 H, J_3,43.2 Hz, H-3), 7.34–

8.15 (m, 10 H, 2 Ph). Anal. Calcd for

C₂₀H₂₂O₆: C, 67.02; H, 6.19. Found: C, 67.19;

H, 6.06.

Benzyl 4-azido-3-O-benzoyl-4,6-dideoxy-i-

L

-mannopyranoside (43).—Diol 42 (3.25 g,

11.65 mmol) was subjected to Bu₂SnO-medi-

240

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

the aqueous layer was thoroughly extracted

with Et₂O. The combined organic solution

was dried with MgSO₄and concentrated. The

residue in MeOH (30 mL) was acetylated with

Ac₂O (5 mL) overnight at rt. The crystalline

precipitate was separated, washed with

MeOH, and dried to give 45 (1.28 g). Column

chromatography of the mother liquor (9:1

CHCl₃–MeOH) gave an additional portion of

the product (0.72 g). The total yield of 45 was

2.00 g (68%): mp 302–307 °C (EtOH); [h]_D

ated benzoylation as described for 37. After

column chromatography of the reaction mix-

ture (95:5 toluene–EtOAc), the monobenzoate

43 (3.98 g, 89%) was obtained as a syrup: [h]_D

+126° (c 1, CHCl₃). ¹H NMR: l 1.50 (d, 3 H,

J_5,66.1 Hz, H-6), 2.52 (broad s, 1 H, OH),

3.34 (dq, 1 H, H-5), 3.82 (t, 1 H, J_4,59.7 Hz,

H-4), 4.31 (broad s, 1 H, H-2), 4.59 (s, 1

H, H-1), 4.69, 4.94 (2 d, 2 H, J_gem11.9

Hz, PhCH₂), 5.00 (dd, 1 H, J_3,410.2 Hz,

J_2,32.6 Hz, H-3), 7.32–8.16 (m, 10 H, 2 Ph).

Anal. Calcd for C₂₀H₂₁N₃O₅: C, 62.65; H,

5.52; N, 10.96. Found: C, 62.74; H, 5.51; N,

11.00.

1

+55.1° (c 1, DMF). H NMR (CDCl₃+

CD₃OD): l 1.11 (d, 3 H, J_5,65.8 Hz, H-6),

1.79, 1.83 (2 s, 6 H, 2 CH₃CO), 3.32 (dq, 1 H,

H-5), 3.36 (t, 1 H, J_4,510.0 Hz, H-4), 3.38 (dd,

1 H, J_2,39.7 Hz, H-2), 3.49 (t, 1 H, J_3,49.9 Hz,

H-3), 4.42, 4.71 (2 d, 2 H, J_gem12.0 Hz,

PhCH₂), 4.42 (d, 1 H, J_1,27.5 Hz, H-1),

7.11–7.19 (m, 5 H, Ph). Anal. Calcd for

C₁₇H₂₄N₂O₅: C, 60.70; H, 7.19; N, 8.33.

Found: C, 60.60; H, 7.21; N, 8.37.

Benzyl

2,4-diazido-3-O-benzoyl-2,4,6-tri-

-glucopyranoside (39).—Tf₂O (3.36

deoxy-i-

L

mL, 20 mmol) was added at 0 °C to a solution

of 43 (3.84 g, 10.03 mmol) and py (4.04 mL,

40 mmol) in CH₂Cl₂(40 mL), and the mixture

was stirred at 0–5 °C for 1 h. The solution

was diluted with CHCl₃, washed successively

with water, M HCl, and water, and then

concentrated. Crude triﬂate 44 was dissolved

in DMF (30 mL), sodium azide (3.25 g, 50

mmol) was added, and the resulting mixture

was stirred at rt overnight. The mixture was

diluted with EtOAc, washed thoroughly with

water, satd aq NaCl solution, dried with

MgSO₄and concentrated. Column chro-

matography of the residue (9:1 light

petroleum–EtOAc) gave 39 (3.82 g, 93%): mp

91–92 °C (Et₂O–light petroleum); [h]_D−1.3°

(c 1, CHCl₃). ¹H NMR: l 1.47 (d, 3 H, J_5,66.1

Hz, H-6), 3.35 (t, 1 H, J_4,59.7 Hz, H-4), 3.44

(dq, 1 H, H-5), 3.63 (dd, 1 H, J_2,310.3 Hz,

H-2), 4.51 (d, 1 H, J_1,28.0 Hz, H-1), 4.73, 4.97

(2 d, 2 H, J_gem11.8 Hz, PhCH₂), 5.18 (t, 1 H,

J_3,49.8 Hz, H-3), 7.33–8.15 (m, 10 H, 2 Ph).

Anal. Calcd for C₂₀H₂₀N₆O₄: C, 58.81; H,

4.94; N, 20.58. Found: C, 58.91; H, 5.06; N,

20.61.

Benzyl 2,4-diacetamido-2,4,6-trideoxy-3-O-

mesyl-i- -glucopyranoside (46).—Methane-

L

sulfonyl chloride (1.68 mL, 21.7 mmol) was

added at 0 °C to a suspension of 45 (1.82 g,

5.42 mmol) in CH₂Cl₂(30 mL) and py (10

mL), and the mixture was stirred for 2 h at

0 °C, then overnight at rt. Water (100 mL)

was added, and the two-phase solution was

stirred for 2 h at rt. The organic layer was

separated, the aqueous layer was extracted

twice with CHCl₃, and the combined organic

solution was concentrated to give 0.72 g of

crude 46. The aqueous solution was concen-

trated to a volume of ꢀ30 mL and left at

5 °C overnight. The crystalline precipitate was

ﬁltered off and dried to give the second por-

tion of 46. Both portions of 46 were combined

and subjected to column chromatography

(95:5 CHCl₃–MeOH) to yield pure 46 (2.16 g,

96%): mp 187–189 °C (MeOH); [h]_D+43° (c

1

1, DMF). H NMR (CDCl₃+CD₃OD): l

Benzyl 2,4-diacetamido-2,4,6-trideoxy-i- -

L

1.15 (d, 3 H, J_5,65.7 Hz, H-6), 1.78, 1.83 (2 s,

6 H, 2 CH₃CO), 2.84 (s, 3 H, CH₃SO₂), 3.46–

3.59 (m, 3 H, H-2,4,5), 4.45, 4.73 (2 d, 2 H,

J_gem12.1 Hz, PhCH₂), 4.64 (d, 1 H, J_1,28.3

Hz, H-1), 4.85 (t, 1 H, J_2,3ꢀJ_3,4ꢀ9.8 Hz,

H-3), 7.13–7.21 (m, 5 H, Ph). Anal. Calcd for

C₁₈H₂₆N₂O₅S: C, 52.16; H, 6.32; N, 6.76.

Found: C, 52.13; H, 6.46; N, 6.47.

glucopyranoside (45).—Lithium aluminum hy-

dride (2.00 g, 52.8 mmol) was added

portionwise at 0 °C to a stirred solution of 39

(3.59 g, 8.80 mmol) in THF (60 mL). After

completion of the exothermic reaction, the

mixture was stirred for 1.5 h at rt. The excess

of LiAlH₄was destroyed by careful addition

of water, then 5 M NaOH solution (150 mL)

was added. The organic layer was separated,

Benzyl 2,4-diacetamido-2,4,6-trideoxy-i-

L

-

allopyranoside (47).—A mixture of 46 (2.10 g,

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

241

mL), and the pH of the solution was adjusted

to 10.5 by adding 5 M NaOH solution.

Sodium tetraborate decahydrate (190 mg, 0.5

mmol) was added, and the pH was adjusted to

10.5 again. Then solid 15 (308 mg, 1.25 mmol)

was added, and the resulting mixture was

stirred at rt while the pH was maintained at

10.5 as above. More oxalacetic acid (42 mg,

0.31 mmol) was added after 6, 24, and 48 h.

After being stirred for 72 h, the mixture was

neutralized with Amberlite 420 (H⁺), and

ﬁltered, and the ﬁltrate was concentrated to a

volume of 3–4 mL. The solution was applied

to a column of Dowex 1×8 (HCOO⁻), and

the column was washed ﬁrst with water to

elute neutral products, then with 0.3 M formic

acid. The appropriate fractions were pooled

and concentrated, and the residue was sub-

jected to preparative reversed-phase C₁₈

HPLC to give 49 (75 mg, 18%), t_R9.2 min,

and 50 (84 mg, 20%), t_R10.5 min, as amor-

phous solids. Acid 49 had [h]_D+15.4° (c 1.6,

water). ESIMS (+): Calcd for [M+Na]⁺

357.1. Found 356.8. Compound 50 had [h]_D

−19.2° (c 1.6, water). ESIMS (+): Calcd for

5.07 mmol) and AcONa (2.08 g, 25.4 mmol)

in 95% aq 2-methoxyethanol (50 mL) was

boiled under reﬂux for 2.5 h. The solvent was

evaporated and a suspension of the residue in

85:15 CHCl₃–MeOH was ﬁltered through a

layer of silica gel. The ﬁltrate was concen-

trated, and the residue was chromatographed

(92:8 CHCl₃–MeOH) to give 47 (1.62 g, 95%):

mp 301–305 °C (EtOH–Et₂O); [h]_D+47.6°

1

(c 1, DMF). H NMR (C₆D₆+CD₃OD): l

1.28 (d, 3 H, J_5,65.8 Hz, H-6), 1.83, 1.84 (2 s,

6 H, 2 CH₃CO), 3.92 (dq, 1 H, H-5), 3.97 (dd,

1 H, J_4,510.2 Hz, H-4), 4.02 (t, 1 H, J_3,42.3

Hz, H-3), 4.16 (dd, 1 H, J_2,32.8 Hz, H-2),

4.51, 4.82 (2 d, 2 H, J_gem12.0 Hz, PhCH₂),

4.87 (d, 1 H, J_1,28.6 Hz, H-1), 7.08–7.28 (m,

5 H, Ph). Anal. Calcd for C₁₇H₂₄N₂O₅: C,

60.70; H, 7.19; N, 8.33. Found: C, 60.53; H,

7.13; N, 8.29.

2,4-Diacetamido-2,4,6-trideoxy- -allopyran-

L

ose (48).—20% Pd(OH)₂/C (500 mg) was

added to a solution of 47 (1.40 g, 4.17 mmol)

in MeOH (50 mL), (Caution: Extreme ﬁre

hazard), and the mixture was stirred under

hydrogen at 35 °C for 1 h. Water (10 mL) was

added until complete dissolution of a white

precipitate and formation of a clear solution

over the catalyst, then hydrogenolysis was

continued for another 1 h. The catalyst was

ﬁltered off through Celite, and washed with

80% aq MeOH, and the combined ﬁltrate and

washings were concentrated. Crystallization

from water–EtOH–Et₂O gave 48 (890 mg,

87%): mp 235–242 °C; [h]_D−3.5° −15.7°

1

[M+Na]⁺357.1. Found 356.8. For H and

¹³C NMR data for 49 and 50, see Tables 2 and

3.

5,7-Diacetamido-3,5,7,9-tetradeoxy-

D

-glyc-

ero-

L

-altro- and -glycero- -manno-non-2-

D

L

ulosonic acids (51 and 52).—Hexose 25 (150

mg, 0.61 mmol) was allowed to react with

oxalacetic acid in the presence of sodium tet-

raborate as described for 15. Anion-exchange

chromatography and reversed-phase HPLC of

the condensation products afforded 52 (8 mg,

4%), t_R8.5 min, [h]_D−39.0° (c 0.5, water),

1

(c 1, water). H NMR (D₂O): 48a, l 1.20 (d, 3

H, J_5,66.2 Hz, H-6), 2.04, 2.06 (2s, 6 H, 2

CH₃CO), 3.79 (dd, 1 H, J_4,510.6 Hz, H-4),

3.96 (t, 1 H, J_3,42.8 Hz, H-3), 4.05 (t, 1 H, J_2,3

3.3 Hz, H-2), 4.18 (dq, 1 H, H-5), 5.15 (d, 1

H, J_1,23.8 Hz, H-1). 48b, l 1.20 (d, 3 H, J_5,6

6.2 Hz, H-6), 2.03, 2.04 (2 s, 6 H, 2 CH₃CO),

3.76 (dd, 1 H, J_4,510.4 Hz, H-4), 3.81 (dd, 1

H, J_2,32.9 Hz, H-2), 3.92 (dq, 1 H, H-5), 3.97

(t, 1 H, J_3,42.9 Hz, H-3), 4.94 (d, 1 H, J_1,28.8

Hz, H-1). The ratio 48a:48b :1:3.3. Anal.

Calcd for C₁₀H₁₈N₂O₅: C, 48.77; H, 7.37; N,

11.38. Found: C, 48.76; H, 7.41; N, 11.40.

and 51 (57 mg, 28%), t_R14.6 min, [h]_D

−

14.3° (c 1, water). ESIMS (+): Calcd for

[M+Na]⁺357.1. Found 357.0 for both 51

1

13

and 52. For H and C NMR data see Tables

2 and 3.

5,7-Diacetamido-3,5,7,9-tetradeoxy-

D

-glyc-

ero-D

-galacto- and

D

-glycero-

D

-talo-non-2-

ulosonic acids (53 and 54).—Condensation of

32 (375 mg, 1.52 mmol) with oxalacetic acid

was performed as described for 15. After an-

ion-exchange chromatography and reversed-

phase HPLC, compounds 53 (36 mg, 7%), t_R

11.2 min, [h]_D+27.2° (c 1, water), and 54 (50

mg, 10%), t_R13.0 min, [h]_D−12.5° (c 1,

water), were obtained. ESIMS (−): Calcd for

5,7-Diacetamido-3,5,7,9-tetradeoxy-

L

-glyc-

ero- -galacto- and -glycero- -talo-non-2-

D

L

D

ulosonic acids (49 and 50).—Oxalacetic acid

(165 mg, 1.25 mmol) was dissolved in water (4

242

Y.E. Ts6etko6 et al. / Carbohydrate Research 335 (2001) 221–243

[M−H]⁻333.1. Found 332.9 for 53 and

H, J_5,610.5 Hz, H-5), 3.83 (dq, 1 H, H-8), 3.86

(s, 3 H, CH₃O), 3.91 (dd, 1 H, J_7,88.8 Hz,

H-7), 3.97 (ddd, 1 H, J_4,510.2 Hz, H-4), 4.32

(dd, 1 H, J_6,72.1 Hz, H-6).

1

333.1 for 54. For H and ¹³C NMR data, see

Tables 2 and 3.

5,7-Diacetamido-3,5,7,9-tetradeoxy-

L

-glyc-

-gluco-non-2-ulosonic acids (55, 56, and

Compound 61: yield 49%; [h]_D−55.1° (c 1,

ero-

L

-altro-,

L

-glycero-

L

-manno-, and

L

1

water). H NMR: l 1.18 (d, 3 H, J_8,96.4 Hz,

ero-L

H-9), 1.94 (dd, 1 H, J_3ax,411.2 Hz, H-3ax),

2.03, 2.07 (2 s, 6 H, 2 CH₃CO), 2.30 (dd, 1 H,

J_3eq,44.6 Hz, J_3eq,3ax13.2 Hz, H-3eq), 3.84 (s, 3

H, CH₃O), 3.88 (t, 1 H, J_5,610.4 Hz, H-5),

3.94 (ddd, 1 H, J_4,511.1 Hz, H-4), 3.95 (dd, 1

H, J_6,73.2 Hz, H-6), 4.04 (quintet, 1 H, H-8),

4.15 (dd, 1 H, J_7,85.7 Hz, H-7).

57).—Reaction of 48 (308 mg, 1.25 mmol)

with oxalacetic acid, followed by anion-ex-

change chromatography and reversed-phase

HPLC, afforded 56 (12 mg, 3%), t_R8.5 min,

[h]_D−56.9° (c 1, water), 57 (5 mg, 1%), t_R

12.5 min, [h]_D−76.0° (c 0.5, water), and 55

(34 mg, 8%), t_R13.3 min, [h]_D−48.2° (c 1,

water). ESIMS (−): Calcd for [M−H]⁻

333.1. Found 333.4 for 55 and 333.3 for both

Esters 58–61 (5–10 mg of each) were acety-

lated with Ac₂O (0.2 mL) in py (0.4 mL) for

48 h at rt. After concentration and removal of

residual Ac₂O and py by coevaporation with

toluene, the residues were passed through a

Sep Pak Silica cartridge in 96:4 CHCl₃–

MeOH and the eluates were concentrated to

1

56 and 57. For H and ¹³C NMR data see

Tables 2 and 3.

Methyl (5,7-diacetamido-2,4,8-tri-O-acetyl-

3,5,7,9 - tetradeoxynon - 2 - ulopyranos)onates

(62–65).—Etheral diazomethane was added

to solutions of acids 49, 51, 53, and 55 (15–20

mg of each) in MeOH (0.5–0.7 mL) until a

yellow colour in the solutions persisted. A

drop of AcOH was added, and the mixtures

were taken to dryness. The residues were sub-

jected to reversed-phase C₁₈HPLC in aq

MeOH (6–8%) to give esters 58–61 as amor-

phous solids.

1

give acetates 62–65, respectively. For H and

¹³C NMR data, see Table 4.

Acknowledgements

This work was supported by grant 436 RUS

113/314/0 of the Deutsche Forschungsgemein-

schaft and grants 99-03-32955 and 00-04-

04009 of the Russian Foundation for Basic

Research. The authors thank Prof. L.V. Back-

inowsky for critical reading of the manuscript.

Compound 58: yield 52%; [h]_D+26.0° (c 1,

1

water). H NMR: l 1.15 (d, 3 H, J_8,96.3 Hz,

H-9), 1.89 (dd, 1 H, J_3ax,411.5 Hz, H-3ax),

1.98, 2.00 (2 s, 6 H, 2 CH₃CO), 2.30 (dd, 1 H,

J_3eq,44.9 Hz, J_3ax,3eq13.1 Hz, H-3eq), 3.71 (t, 1

H, J_5.610.5 Hz, H-5), 3.83 (dq, 1 H, H-8), 3.86

(s, 3 H, CH₃O), 3.91 (dd, 1 H, J_7,88.8 Hz,

H-7), 3.97 (ddd, 1 H, J_4,510.2 Hz, H-4), 4.32

(dd, 1 H, J_6,72.1 Hz, H-6).

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Article Doi

Synthesis and NMR spectroscopy of nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids

DOI: 10.1016/S0008-6215(01)00235-X

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Full text of DOI:10.1016/S0008-6215(01)00235-X

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