Immunogens related to the synthetic tetrasaccharide side chain of the Bacillus anthracis exosporium

Bioorganic & Medicinal Chemistry 15 (2007) 4283–4310

Immunogens related to the synthetic tetrasaccharide side chain

of the Bacillus anthracis exosporium

*

´

Rina Saksena, Roberto Adamo and Pavol Kovacˇ

NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA

Received 11 January 2007; revised 14 March 2007; accepted 18 March 2007

Available online 23 March 2007

Abstract—The known methyl 2-O-acetyl-3,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside (3) was converted to the corresponding

5-methoxycarbonylpentyl glycoside 4 which was deacetylated. The product 5 was used as the initial glycosyl acceptor to construct

two trirhamnoside glycosyl acceptors having HO-3^IIIﬂanked by either benzoyl or benzyl groups, compounds 10 and 29, respectively

[fully protected, except HO-3^III, a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-1-O-(CH₂)₅COOCH₃]. When these were glycosylated

with ethyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-bromoacetyl-1-thio-b-D-glucopyranoside (18), only the benzylated glycosyl acceptor

29 gave good yield of the desired tetrasaccharide 30. The a- and b-linked products, together with the corresponding orthoester 23,

were formed in almost equal amount when glycosylation of 10 was performed with the glycosyl donor carrying the 2-O-bromoacetyl

protecting group. Deprotection at O-2 of 30, followed by further functionalization of the molecule and global deprotection, gave the

5-methoxycarbonylpentyl glycoside of the title tetrasaccharide, b-Ant-(1 ! 3)-a-^L-Rha-(1 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha (35).

Except for diﬀerences due to presence of the anomeric 5-methoxycarbonylpentyl group, the fully assigned NMR spectra of glycoside

35 were found to be virtually identical to those reported for the parent tetrasaccharide isolated from Bacillus anthracis exosporium,

thus proving the correct structure assigned to the naturally occurring substance. All theoretically possible structural fragments of 35,

as well as analog of 35 lacking the 2-O-methyl group at the terminal 4,6-dideoxyglucosyl residue, compound 40, were also synthe-

sized. Tetrasaccharide 35, its b-linked and non-methylated analogs 2 and 40, respectively, as well as the trirhamnoside fragment of

35, glycoside 12, were further functionalized and conjugated to BSA using squaric acid chemistry, to give neoglycoconjugates with a

predetermined carbohydrate–protein ratio of ꢀ3 and ꢀ6.

Published by Elsevier Ltd.

1. Introduction

a spore, which may come to life in the right conditions.

Spores of B. anthracis are enclosed in a layer of exospo-

rium whose components are the ﬁrst to interact with the

host. An approach to control anthrax by targeting

spores with neutralizing antibodies has thus far not been

fully developed. This is partially due to our poor under-

standing of the structure of components of anthrax

spores. Daubenspeck and coworkers⁴have recently

determined the structure of the tetrasaccharide side

chain of the collagen-like region of the major glycopro-

tein of the B. anthracis exosporium (BclA). The tetrasac-

charide (1, Fig. 1) is unique in that it contains the newly

discovered sugar anthrose⁴[4,6-dideoxy-4-(3-hydroxy-3-

methylbutyramido)-2-O-methyl-^D-glucopyranose] as the

upstream⁵terminal moiety.

Anthrax is predominantly a disease of animals but hu-

mans can become infected by contact through a cut in

skin (cutaneous anthrax), by contaminated food or soil,

or by inhaling the spores (inhalational anthrax). Until

recently, the disease had not caused serious health prob-

lems in the civilized world, but new concerns regarding

anthrax have recently emerged in context with bioterror-

ism. Therefore, development of a potent vaccine for an-

thrax has become

a

pressing issue worldwide.

Approaches to developing a synthetic vaccine for an-

thrax^1–3have been focused mainly on the use of immu-

nogenic conjugates from capsular polypeptide

(polyglutamic acid) of Bacillus anthracis, which is the

etiologic agent of anthrax. The bacterium is housed in

Studies aimed at better understanding of the role of

BclA in the B. anthracis exosporium, preparation of

diagnostic probes, and, perhaps, vaccine for anthrax call

for further investigations involving the tetrasaccharide

whose isolation from the exosporium in larger quantities

is diﬃcult. More importantly, if prepared in this way,

Keywords: Oligosaccharides; Carbohydrates; Glycoconjugates; Syn-

thetic carbohydrate antigens; Conjugation by squaric acid chemistry.

*

Corresponding author. Tel.: +1 301 496 3569; e-mail: kpn@helix.

nih.gov

0968-0896/$ - see front matter Published by Elsevier Ltd.

doi:10.1016/j.bmc.2007.03.057

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R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

Figure 1. Structure of the tetrasaccharide side chain of the major glycoprotein of the Bacillus anihracis exosporium (1) and its methyl

6-hydroxyhexanoyl b-glycoside (2).

the tetrasaccharide would be obtained as a mixture of

anomers. Results of binding or inhibition studies with

material that does not completely imitate the structure

of the tetrasaccharide, including the anomeric conﬁgura-

tion at the site of attachment to the exosporium, would

likely be inconclusive. Studies of such nature require the

tetrasaccharide in a form where the anomeric conﬁgura-

tion is locked, as, for example, in a glycoside. Syntheses

of a-benzyl and a-pentenyl glycosides of 1 have been re-

ported.^6,7The a-pentenyl glycoside of 1 was used in

work toward generating monoclonal IgG antibodies

speciﬁc to B. anthracis endospores.⁸More recently,

Mehta et al.⁹described an independent approach to

the tetrasaccharide–BSA and tetrasaccharide–KLH

conjugates.

structural fragments (Fig. 1, A, AB, ABC, BC, BCD,

CD, and D) suitable for conjugation to carrier proteins.

To facilitate preparation of the large number of sub-

stances, we employed intermediates that were previously

found useful in preparation of rhamnooligosaccharides,

for example, Refs. 13–21. The initial glycosyl acceptor

for the synthesis of 35, monosaccharide 5, was readily

obtained (Scheme 1) by NIS/AgOTf-mediated reaction

of the known thioglycoside 3¹⁶with methyl 6-hydroxy-

hexanoate,²²to give 4 (along with a small amount of

´

the b-anomer 87) followed by deacetylation (Zemplen)

of the linker equipped rhamnoside 4 (Scheme 1). To

extend the oligosaccharide chain by two ^L-rhamnosyl

residues, alcohol 5 was coupled with the disaccharide

glycosyl donor 8, obtained by condensation of methyl

2,4-di-O-benzyl-1-thio-a-^L-rhamnopyranoside²³(6) with

2,4-di-O-benzoyl-3-O-bromoacetyl-a-^L-rhamnopyrano-

syl chloride (7), which was available in this laboratory

from previous work.¹⁸Subsequent debromoacetylation

of the trisaccharide 9 thus formed gave trisaccharide gly-

cosyl acceptor 10, and complete deprotection of 9 gave,

through benzoate 11, glycoside 12, the trirhamnoside

fragment of 35.

In order to obtain optimal immune response from neo-

glycoconjugates prepared from 1, the anomeric conﬁgu-

ration of the terminal, downstream⁵L-rhamnosyl

residue in the conjugates must be the same as in BclA.

With that information unknown,⁴our initial work to-

ward immunogens for anti-B. anthracis exosporium

antibodies involves synthesis of neoglycoconjugates

from each of the a- and b-forms of 1 and evaluation

of immunogenicity thereof. We have recently reported¹⁰

the synthesis of spacer-equipped b glycoside of 1 (com-

pound 2), and its structural fragments. Here we report

full details of the synthesis¹¹of the a glycoside of 1

(compound 35), and its conversion to BSA-conjugates.

Similar conjugates were prepared from b glycoside 2,

the non-methylated analog of 35 (glycoside 40), and tri-

rhamnoside fragment of 35 (compound 12). Syntheses of

other spacer-equipped structural fragments of 35 are

also described. Compounds 2, 35, and some fragments

thereof, were instrumental in developing photogenerat-

ed glycan arrays that are useful to identify immunogenic

sugar moieties of B. anthracis exosporium.¹²

Synthesis of the target tetrasaccharide required stereose-

lective construction of the b-anthrosyl linkage. Synthesis

of such linkage with high stereoselectivity could be prob-

lematic using a glycosyl donor derived from anthrose,

due to the presence of the nonparticipating methyl

group at O-2. Therefore, instead of using a glycosyl

donor derived from anthrose, or the known ethyl 4-azi-

do-3-O-benzyl-4,6-dideoxy-2-O-methyl-1-thio-b-^D-gluco-

pyranoside,²⁴we have prepared the latent anthrosyl

donor 18 (Scheme 2) from the known²⁴glucosides 13

or 19, or from thioglycoside 21. We presumed that the

bromoacetyl protecting group at O-2 in 18 would be

particularly advantageous because it can be selectively

removed in the presence of other acyl groups. Thus,

compound 18 could be used as a versatile glycosyl donor

in related syntheses involving presence or absence of

acyl groups in intermediates. Once attached, the termi-

nal b-^D-glucopyranosyl moiety would be transformed

into b-anthrosyl residue as described previously.²⁴

2. Results and discussion

The purpose of this work was to prepare spacer-

equipped a-glycoside of the title tetrasaccharide and its

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4285

Scheme 1. Synthesis of trirhamnoside 12 and tetrasaccharide 24. Reagents: (a) methyl 6-hydroxyhexanoate, NIS, AgOTf; (b) NaOMe, MeOH;

(c) AgOTf, DBMP; (d) NIS, AgOTf; (e) thiourea; (f) H₂Pd/C; (g) NaOMe, MeOH; (h) 18, NIS, AgOTf.

The silver triﬂate/NIS-mediated glycosylation of 10 with

18 (Scheme 1) was performed under base-deﬁcient con-

ditions.²⁵Chromatography of the crude product aﬀor-

ded orthoester 23, the desired b-linked tetrasaccharide

24 and the a-anomer 88, together with unidentiﬁed

byproducts. A considerable amount of unchanged gly-

cosyl acceptor was recovered. The problem of orthoester

formation was solved when the more reactive trisaccha-

ride 29 was used as the glycosyl acceptor for 18 (Scheme

3). To obtain 29, ^L-rhamnose was converted, through

methyl 1-thiopyranoside,¹⁶to methyl 3-O-acetyl-2,4-di-

O-benzyl-1-thio-a-^L-rhamnopyranoside^17,23(25). The

latter was treated with chlorine,²⁶and condensation of

glycosyl chloride 26 thus formed with di-O-benzyl deriv-

ative²³6 gave disaccharide glycosyl donor 27. Reaction

of donor 27 with 5 gave the linker-equipped trisaccha-

ride 28. When the latter was deacetylated, and the

formed glycosyl acceptor 29 was treated with 18, the de-

sired tetrasaccharide 30 was obtained in 84% yield. After

subsequent debromoacetylation of 30, further conver-

sion to the title tetrasaccharide 35 comprised a series

of conventional transformations established during the

synthesis of anthrose and its methyl glycosides.²⁴¹H

and ¹³C NMR spectra of glycoside 35, fully assigned

by 2D experiments, were found to be virtually identical

to those reported for the parent tetrasaccharide isolated

from B. anthracis exosporium (Tables 1 and 2), thus

proving the correct structure assigned to the naturally

occurring substance. The diﬀerences in chemical shifts

found for some nuclei (Tables 1 and 2) were due to pres-

ence of the anomeric 5-methoxycarbonylpentyl group.

Orthoester formation from alcohol 10 was less pro-

nounced when the same acceptor was glycosylated with

the benzoylated donor 22 (Scheme 4), prepared from 21

as shown in Scheme 2. The latter reaction aﬀorded the

fully protected product 36 in 67% yield with some a

product 89 (see Section 3). The latter was then converted

to the non-methylated analog of 35, tetrasaccharide 40,

by the same sequence of reactions as described for the

conversion 32 ! 35 (Scheme 4). The following conclu-

sions could be made from observations during glycosy-

lations leading to tetrasaccharides described above:

The prerequisite to obtaining a good yield of the desired

b product was the correct combination of coupling syn-

thons. Desired tetrasaccharides, that is, 30 or 36, could

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R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

be obtained in good to excellent yields from reactions

involving either 2-O-bromoacetylated or 2-O-benzoylat-

ed glycosyl donor when the hydroxyl group to be glycos-

ylated was ﬂanked by benzyloxy groups. An acceptable

yield of glycosylation could also be obtained from 2-O-

benzoylated glycosyl donor when the hydroxyl group to

be glycosylated was ﬂanked with benzoyl groups. On the

other hand, a- and b-linked products, together with the

corresponding orthoester 23, were formed in almost

equal amount when glycosylation of 10, whose hydroxyl

group was ﬂanked with benzoyl groups, was performed

with the glycosyl donor 18 carrying the 2-O-bromoace-

tyl protecting group. We have previously reported the

tendency of 2-O-chloroacetylated glycosyl donors to

form stable orthoesters.²⁷

Binding and inhibition studies with antibodies speciﬁc

for anthrax exosporium require a large repertoire of

substances related to tetrasaccharide 1. Synthesis of

the trirhamnoside fragment (ABC) of 35 (compound

12) has already been mentioned (Scheme 1), and synthe-

ses of structural fragments of the b-glycoside 2 have

been reported.¹⁰Syntheses of the remaining, theoreti-

cally possible partial structures of 35 (for alphabetical

Scheme 2. Synthesis of glycosyl donors 18 and 22. Reagents: (a) BrCH₂COBr, TMU; (b) Ac₂O, H₂SO₄; (c) EtSH, BF₃ÆEt₂O; (d) PhCOCI.

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4287

Scheme 3. Synthesis of the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium (35). Reagents: (a) Cl₂; (b) 6,

AgOTf, DBMP; (c) 5, NIS, AgOTf; (d) 18, NIS, AgOTf; (e) NaOMe, MeOH; (1) Mel, Ag₂O; (g) H₂S; (h) 3-hydroxy-3-methylbutyric acid, HATU;

(i) H₂, Pd/C.

Table 1. Comparison of ¹H chemical shifts^afor tetrasaccharide 35 with data reported for 1 in Ref. 4

Ring

I

H-1

H-2

H-3

H-4

H-5

H-6

4.566 [1.5]

4.870

3.634

3.601

3.775

3.783

3.872

3.880

2.845

2.850

3.530

3.615

3.572

3.592

3.745

3.760

3.281

3.301

3.155

3.144

3.325

3.339

3.402

3.404

3.402

3.401

3.373

3.592

3.487

3.504

3.636

3.655

3.275

3.278

1.134

1.114

1.101

1.103

1.132

1.142

1.074

1.082

II

4.842 [1.4]

4.848

III

IV

4.857 [1.4]

4.876

4.577 [7.9]

4.582

NH

7.706

7.754

CH₂

2.204

2.211

CH₃

1.155, 1.143

1.160, 1.148

OCH₃

3.519

3.526

Other^b,c

^aSpectra taken at 600 MHz for a solution in DMSO-d₆at 25 °C. Selected, structurally signiﬁcant coupling constants [Hz] are in brackets. Data for 1

(the second row for each ring) were taken from Ref. 4.

^bd_OH(disappear on deuteration): 4.995 (d, J 6.0 Hz, OH-2^IV), 4.945 (d, J 6.8 Hz, OH-4^II), 4.885 (d, J 5.3 Hz, OH-4^I), 4.875 (d, J 5.8 Hz, OH-3^I),

4.831 (s, OH-3⁰), 4.779 (d, J 4.9 Hz, OH-2^II), 4.718 (d, J 5.3 Hz, OH-4^III), 4.534 (d, J 4.6 Hz, OH-2^III). Not reported in Ref. 4.

^cd_Spacer: 3.57 (COOCH₃), 3.52 (H-1⁰⁰a), 3.33 (H-1⁰⁰b), 2.29 (t, J 7.4 Hz, H-5⁰⁰a,b), 1.56–1.45 (m, H-2⁰⁰a,b,4⁰⁰a,b), 1.36–1.26 (m, H-3⁰⁰a,b).

notation of sequences, see Fig. 1), saccharides A (42),

AB (45), BC (48), BCD (70), CD (62), and D (54), are

shown in Schemes 5 and 6. Methyl 6-hydroxyhexa-

noyl-a-L-rhamnopyranoside (42), which imitates the

reducing end in 1, was obtained (Scheme 5) by reaction

of 2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl bromide²⁸

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R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

Table 2. Comparison of ¹³C chemical shifts^afor tetrasaccharide 35 with data for 1 reported in Ref. 4

Ring

A

C-1

C-2

C-3

C-4

C-5

C-6

103.50; J_C,H160.7

103.4; J_C,H161

101.71; J_C,H171.3

101.6; J_C,H171

101.76; J_C,H170.6

101.6; J_C,H170

98.62; J_C,H167.6

92.8; J_C,H166

CO

84.36

84.3

72.77

72.7

56.42

56.4

70.34

70.3

18.18

18.1

B

69.67

69.6

79.48

79.4

71.65

71.6

68.31

68.2

17.73

17.6

C

69.97

69.9

76.28

76.2

71.37

71.4

72.22

69.04

68.8

17.81

17.9

D

76.66

77.7

70.56

70.1

CH₃

68.58

67.7

18.02

17.9

72.6

C⁰_quat

Other^b

CH₂

48.67

48.6

OCH₃

60.16

59.9

171.45

171.4

29.58, 29.48

29.5, 29.4

68.71

not reported

^aSpectra taken at 150 MHz for a solution in DMSO-d₆at 25 °C. Data for 1 (the second row for each ring) were taken from Ref. 4.

^bd_Spacer: 173.36 (CO), 66.32 (C-1⁰⁰), 51.25 (COOCH₃), 33.26 (C-5⁰⁰), 28.66 (C-2⁰⁰), 25.21 (C-3⁰⁰), 24.25 (C-4⁰⁰).

Scheme 4. Synthesis of tetrasaccharide 40, the non-methylated analog of the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis

exosporium. Reagents: (a) 22, AgOTf, NIS; (b) H₂S; (c) 3-hydroxy-3-methylbutyric acid, HATU; (d) NaOMe, MeOH; (e) H₂, Pd/C.

with methyl 6-hydroxyhexanoate (!41) followed by

debenzoylation. To obtain the same-spacer-equipped,

(1 ! 2)-linked dirhamnoside 45, alcohol 5 was treated

with the same glycosyl donor, and the formed disaccha-

ride 43 was subjected to a two-step deprotection

(Scheme 5). The analogous (1 ! 3)-linked disaccharide

48 was obtained by condensation of methyl 6-hydroxy-

hexanoate and the disaccharide glycosyl chloride 46,

which was available from previous work,¹⁸followed

by deprotection (Scheme 5). The yield of glycosylation

was high (78%), but a large amount of the correspond-

ing orthoester (90, 34%) was isolated by chromatogra-

phy along with the desired product 47 (44%). The

three, anthrose-containing partial structures of the title

tetrasaccharide 35 were synthesized as follows (Scheme

6). Reaction of methyl 6-hydroxyhexanoate with the

2-O-benzoylated glycosyl donor 22 gave the fully pro-

tected glycoside 49 in excellent yield of 87%, together

with a small amount of the a-anomer 91. After deben-

zoylation (!50) and methylation, the fully protected

azido compound 51 was subjected to the series of trans-

formations described for tetrasaccharide 32, to give

methyl 6-hydroxyhexanoyl b-anthroside (54). To obtain

compound 62 that mimics the anthrose-containing ter-

minal disaccharide of 1, thioglycoside donor 25^16,23

was treated with methyl 6-hydroxyhexanoate (Scheme

6) and the product 55 was deacetylated. Alcohol 56 thus

formed was condensed with latent anthrose precursor 18

to give fully protected disaccharide 57, which was trans-

formed to the desired structural fragment of 1 (com-

pound 62) as described above for conversions 30 ! 35.

Our original plan to prepare the terminal spacer-

equipped fragment of 1, trisaccharide 70, was based on

the use of disaccharide 47 which, after debromoacetyla-

tion, was expected to give a suitable glycosyl acceptor

for donor 22 (cf. glycosylation of that donor with accep-

tor 10, which gave a good yield of the expected b-linked

product). However, since glycosylation leading to 47

(Scheme 5) was low-yielding, a more eﬃcient route

was sought. This was found in a stepwise approach,

starting with condensation of glycosyl donor 25 with

alcohol 56. After deacetylation of the formed disaccha-

ride 63, the resulting alcohol 64 was treated with anth-

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4289

for by running conjugations at a higher concentration

only in the case of hapten 84 (Table 3). We have no

other explanation for the much slower rate of conjuga-

tion with haptens 2, 12, and 35, other than, as we

pointed out previously,²⁹that we may not be aware of

some parameters that aﬀect conjugation. Because the

reaction rate is not quite predictable, having a method

that allows monitoring of conjugation of synthetic car-

bohydrates to proteins in virtually real time is invalu-

able. The utility of the SELDI-TOF method for this

purpose by this technique^29,33is evident from the but

small diﬀerences between the targeted carbohydrate–

protein ratios and those observed in the products (Table

3). The conjugates were made according to a new proto-

col. Compared to our previously developed procedure, it

involved more eﬃcient puriﬁcation of products of trans-

formation of the ester function in the 5-methoxycarbon-

ylpentyl spacer molecule to the corresponding amines

and squaric acid derivatives. Also, puriﬁcation of the ﬁ-

nal glycoconjugates was done with the aid of ultraﬁltra-

tion devices (see Section 3) having vertical, rather than

horizontal, orientation of the membrane. As a result

of this conﬁguration, the centrifugal force pulls material

away from the membrane to the bottom of the device.

This, in turn, reduces axial pressure on the ﬁltration

membrane and minimizes losses due to adherence of

the glycoprotein to it, resulting consistently in high

yields of the ﬁnal product. Table 3 shows the content

of carbohydrates in the glycoconjugates prepared from

haptens 72, 76, 80, and 84, as well as reaction times

required to reach these carbohydrate–protein ratios.

Scheme 5. Synthesis of partial structures of tetrasaccharide 35;

saccharides A (42), AB (45), and BC (48). Reagents: (a) methyl

6-hydroxy-hexanoate, AgOTf, DBMP; (b) AgOTf, DBMP.

3. Experimental

3.1. General methods

Unless stated otherwise, optical rotations were mea-

sured at ambient temperature for solutions in CHCl₃,

with a Perkin-Elmer automatic polarimeter, Model

341. All reactions were monitored by thin-layer chroma-

tography (TLC) on Silica gel 60 coated glass slides. Col-

umn chromatography was performed by gradient

elution from columns of silica gel with the High Perfor-

mance Rapid Flash Chromatography System (RT

Scientiﬁc) or with the CombiFlash Companion Chro-

matograph (Isco, Inc.). Solvent mixtures less polar than

those used for TLC were used at the onset of separation.

FAB Mass spectra were obtained with Jeol SX 102 spec-

trometer. Liquid Chromatography–Electron Spray-Ion-

ization Mass Spectrometry (ESI-MS) was performed

with a Hewlett-Packard 1100 MSD spectrometer. Nu-

clear Magnetic Resonance (NMR) spectra were mea-

sured with a Varian Gemini or Varian Mercury at

300 MHz (¹H) and 75 MHz (¹³C), or with Bruker

Avance 600 spectrometers. J values are given in Hertz.

rose precursor 18. The b product 65 (82%) was then con-

verted to the desired trisaccharide 70 through the same

series of transformations (Scheme 6) as for 57 ! 62.

NMR and MS data fully conﬁrmed structures of sub-

stances described above.

For future immunological studies, glycoconjugates were

prepared from the title tetrasaccharide 35 (35 ! 73,

35 ! 74) and the corresponding trisaccharide lacking

the anthrose residue 12 (12 ! 77, 12 ! 78), by chemical

linking to BSA through squaric acid chemistry. To be

able to evaluate the importance of anomeric conﬁgura-

tion of the tetrasaccharide and the methyl group in the

anthrose residue, we also made similar neoglycoconju-

gates from the corresponding b-glycoside 2¹⁰(2 ! 81,

2 ! 82) and the analog of 35 lacking the 2-O-methyl

group in the anthrose residue, tetrasaccharide 40

(40 ! 85, 40 ! 86). Two conjugates from each oligosac-

charide were prepared. They were targeted to show car-

bohydrate–protein ratio of 3 and 6. To minimize waste

of the precious oligosaccharides, conjugations described

here were performed at a considerably lower initial hap-

ten/carrier ratio (20:1) than in our earlier work.^29–31The

expected slower reaction rate due to conducting the con-

jugation under these conditions³²was fully compensated

1

For measurements in D₂O, chemical shifts for H and

¹H

¹³C are reported relative to Me₂CO (d 2.218; d

33.0); for measurements in CDCl₃, chemical shifts for

¹³C

¹H and ¹³C are reported relative to Me₄Si (d 0; d

¹H

¹³C

0) and CDCl₃, d

NMR signals were made by homonuclear and heteronu-

77.0, respectively. Assignments of

¹³C

clear two-dimensional correlation spectroscopy, run

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R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

Scheme 6. Synthesis of partial structures of tetrasaccharide 35; saccharides D (54), CD (62), and BCD (70). Reagents and conditions: (a) methyl

6-hydroxyhexanoate, AgOTf, NIS; (b) NaOMe, MeOH; (c) Mel, Ag₂O, Me₂S; (d) H₂S; (e) 3-hydroxy-3-methylbutyric acid, HATU; (f) H₂, Pd/C;

(g) AgOTf, NIS.

with the software supplied with the spectrometers. When

reporting assignment of NMR signals, nuclei associated

with the N-amido side chain are denoted with a prime,

and those with the other aglycone (the linker) are de-

noted with a double prime even in cases when the prime

nuclei are absent. Sugar residues in oligosaccharides are

serially numbered, beginning with the one bearing the

aglycone, and are identiﬁed by a Roman numeral super-

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4291

Table 3. Reaction times, yields, and carbohydrate–protein ratios in

prepared neoglycoconjugates

the reaction was complete (0.5–3 h) and ﬁltered through

a Celite pad, and the ﬁltrate was partitioned between a

mixture of aq NaHCO₃and Na₂S₂O₃. After concentra-

tion of the organic phase, the material in the residue was

chromatographed. The ratio between the glycosyl donor

and the glycosyl acceptor was such that the less labor-

intensive material was kept at ꢀ1.3 equiv excess, to as-

sure complete consumption of the more labor-intensive

reactant. The amount of DBMP and AgOTf was 0.9

and 1.2 equiv, respectively, of that of the glycosyl halide,

to assure complete consumption of the halide. In spite of

these base deﬁcient conditions, which favor isomeriza-

tion of the intermediate orthoester, the latter was pres-

ent among the ﬁnal products of some reactions.

Hapten Reaction Conjugate (Targeted) and

time [h]

Yield

ﬁnal hapten/BSA mg (%)

ratio [M:M]

72

76

80

84

48

240

24

73

74

77

78

81

82

85

86

(3) 3.1

(6) 6.0

(3) 2.9

(6) 5.5

(3) 2.6

(6) 5.9

(3) 3.5

(6) 5.7

14.7 (94)

14.0 (86)

14.0 (92)

14.0 (88)

15.0 (96)

14.0 (86)

13.0 (83)

15.0 (93)

216

24

192

4

12

script in listings of signal assignments. NMR spectra of

amines 71, 75, 79, and 83 as well as those of squaric acid

derivatives 72, 76, 80, and 84 were very similar to those

of the corresponding esters. Spectra of amines showed

that, due to hindered rotation around the CN bond in

amides,³⁴each of these substances was a mixture of

two isomers, whose ratio varied with the structure.

The presence of isomers manifested itself by splitting

of signals not only of some nuclei in the linker but also

some ring protons and, mainly, carbons of some sugar

moieties. Due to the double bond nature of the vinylo-

gous amide group, the NMR spectra of squaric acid es-

ters 72, 76, 80, and 84 showed further splitting of some

signals.³⁵When reporting NMR data of these sub-

stances only resonances originating from the major iso-

mer are listed. Assignments denoted with an asterisk

may be reversed. Attempts have been made to obtain

correct analytical data for all new compounds. How-

ever, some compounds tenaciously retained traces of

solvents, despite exhaustive drying, and analytical ﬁg-

ures for carbon could not be obtained within 0.4%.

Structures of these compounds follow unequivocally

from the mode of synthesis, NMR data, and m/z values

found in their mass spectra, and their purity was veriﬁed

by TLC and NMR spectroscopy. Carbohydrate–protein

conjugates were rid of low-molecular mass materials by

ﬁltration through either Amicon Ultra-15 Centrifugal

Filter (Millipore Corporation) or similar Vivaspin 15

R (Sartorius Group) devices. Palladium-on-charcoal

catalyst (5%, ESCAT 103) was a product of Engelhard

Industries. HATU {N-[(dimethylamino)-1H-1,2,3-triaz-

olo-[4,5-b]-pyridin-1-ylmethylene]-N-methylmethana-

minium hexaﬂuorophosphate N-oxide} was purchased

from Applied Biosystems. 3-Hydroxy-3-methylbutyric

acid was purchased from Alfa Aesar Chemical Com-

pany. A stock solution of chlorine²⁶in CCl₄contained

1.06 mmol Cl₂/mL (determined by weighing). Solutions

in organic solvents were dried with anhydrous Na₂SO₄

and concentrated at 40 °C/2 kPa.

3.3. General method for glycosylation with thioglycosides

Except when stated otherwise, NIS (1.4 mmol/mmol of

thioglycoside) followed by AgOTf (0.4 mmol/mmol of

thioglycoside) was added to a mixture of thioglycoside,

˚

glycosyl acceptor, and 4 A molecular sieves (0.5 g/mmol),

which had been stirred at room temperature for 15 min.

For ratio between the glycosyl donor and the glycosyl

acceptor, see above. The mixture turned red and, after

ꢀ15 min, TLC showed that all glycosyl donor was con-

sumed. The mixture was ﬁltered through a Celite pad into

mixed aq solutions of NaHCO₃and Na₂S₂O₃. After

partitioning of solutes, the colorless organic phase was

concentrated, and the residue was chromatographed.

3.4. General procedure for the conversion of azides to amines

Water (ꢀ1 mL) was added dropwise and with stirring to

a solution of an azide (1 mmol) in pyridine (4 mL). If

slight turbidity developed a few drops of pyridine were

added. A slow stream of H₂S gas was passed for 1 h

through the resulting clear solution, which was then

kept overnight at 40 °C in a loosely closed ﬂask. After

concentration with co-evaporation of water, chromato-

graphy gave the corresponding amine.

3.5. General procedure for amidation with HATU

HATU (1 mmol) followed by N,N-diisopropylethyl-

amine (1 mmol) was added to a solution of amine

(1 mmol) and carboxylic acid (1.2 mmol) in CH₂Cl₂,

and the mixture was stirred at room temperature for

2 h. TLC then showed that virtually all amine was con-

sumed and that one faster moving product was formed.

The mixture was concentrated, and the residue was

chromatographed to give pure amide.

3.6. General procedure for deacetylation and

debenzoylation

3.2. General method for glycosylation with glycosyl halides

Methanolic (ꢀ1 M) NaOMe was added to a solution of

the starting material (1 mmol) in MeOH (5 mL) until the

solution was strongly alkaline to litmus, and the solu-

tion was kept at room temperature until the reaction

was complete (1–24 h TLC). After neutralization with

a cation-exchange resin (H⁺-form), ﬁltration, and con-

centration of the ﬁltrate, the residue was eluted from a

small column of silica gel.

2,6-Di-t-butyl-4-methylpyridine (DBMP) followed by

silver triﬂate (AgOTf, 1.2 mmol) was added to a mixture

of glycosyl acceptor (1 mmol), glycosyl halide

˚

(1.3 mmol), and 4 A molecular sieves (0.5 g/mmol) in

CH₂Cl₂(10 mL/mmol), which had been stirred for

15 min. The mixture was stirred until TLC showed that

4292

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

Scheme 7. Synthesis of neoglycoconjugates 73, 74, 77, 78, 81, 82, 85, and 86. Reagents and conditions: (a) ethylenediamine; (b) diethyl squarate,

pH 7; (c) BSA, pH 9.

3.7. General procedure for debenzylation

gen until the reaction was complete (ꢀ8–24 h). After ﬁl-

tration through a Celite pad and concentration of the

ﬁltrate, the material in the residue was passed through

a small silica gel column, to remove residual carbon. A

solution of the pure, deprotected compound in water

A mixture of benzylated compound (500 mg) and palla-

dium-on-charcoal catalyst (150–200 mg) in methanol

(ꢀ25 mL) was stirred at room temperature under hydro-

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4293

Scheme 7. (continued)

(HPLC grade) was ﬁltered through sterile syringe ﬁlter

(0.22 lm) and freeze-dried.

2, 12, 35, and 40 (after conversion to squaric acid mono-

ester derivatives 72, 76, 80, and 84 see text below, Table

3, and Scheme 7), applying a slightly modiﬁed one-pot

protocol.³⁰The initial hapten/BSA ratio of 20:1 was

chosen, to maintain reasonable reaction rate. To a solu-

tion of BSA (30 mg, 0.45 lmol, obtained by sonication)

in borate buﬀer, pH 9 (180 lL) was added squaric acid

monoester (9 lmol), to form a 50 mM solution with re-

spect to the squaric acid derivative. The mixture was

stirred at room temperature and the progress of conju-

gation was monitored³³by SELDI-TOF-MS. When

the desired carbohydrate ratio ꢀ3 was reached, as deter-

mined by SELDI-TOF-MS, 90 lL of the reaction mix-

ture was withdrawn and added with stirring into

borate buﬀer, pH 7 (5 mL), to terminate the reaction.

The rest of the mixture was allowed to react until the

next desired carbohydrate protein ratio was reached

and then processed as described below. The mixture that

was diluted with pH 7 buﬀer was transferred into a cen-

trifugal ﬁlter device and processed to remove salts. A

minimum of eight washes with aq 10 mM (NH₄)₂CO₃

were applied, to ensure complete removal of the buﬀer.

Freeze-drying aﬀorded conjugates (73, 74, 77, 78, 81,

82, 85, and 86) as white solids. Reaction times and

results are summarized in Table 3.

3.8. General procedure for methylation

Me₂S (2 drops) was added to a mixture of alcohol (0.1 g),

MeI (0.5 mL) and Ag₂O (0.2 g) in 1,2-dimethoxyethane

(2 mL), and the mixture was stirred in the dark at room

temperature until the conversion was complete. After ﬁl-

tration through a Celite pad, the ﬁltrate was concentrated,

and chromatography gave the desired product.

3.9. General procedure for functionalization of the spacer

(A) Conversion of esters to amides using 1,2-diaminoe-

thane: A solution of an ester (0.1 mmol) in freshly dis-

tilled 1,2-diaminoethane (3 mL) was kept at 50 °C

until all starting material was consumed (24–48 h), while

progress of the reaction was monitored by TLC. After

concentration and puriﬁcation by reverse-phase chro-

matography, the pure (TLC, NMR) product was

freeze-dried, to give white, amorphous material.

(B) Conversion of amides to squaric acid monoethyl es-

ters: A solution of amine (0.02 mmol) and diethyl squa-

rate (2.5 equiv, 0.05 mmol) in borate buﬀer (1 mL, pH

7.00) was stirred until the reaction was complete (3–

24 h, TLC). After concentration, the crude product

was adsorbed on silica gel and chromatographed. A

solution of the pure compound in water (HPLC grade)

was ﬁltered through 0.22 lm Millex sterile syringe ﬁlter

and freeze-dried, to give white amorphous solid.

3.11. 5-(Methoxycarbonyl)pentyl 2-O-acetyl-3,4-di-O-

benzyl-a-(4) and b-^L-rhamnopyranoside (87)

Starting with methyl 2-O-acetyl-3,4-di-O-benzyl-1-thio-

a-^L-rhamnopyranoside (3)¹⁶(470 mg, 1.12 mmol) and

methyl 6-hydroxyhexanoate²²(197 mg, 1.35 mmol), gly-

cosylation according to the general protocol gave, after

chromatography, ﬁrst the a-anomer 4 (420 mg, 81%);

3.10. General procedure for conjugation of squaric acid

derivatives to BSA

1

[a]_Dꢁ6° (c 1.0). H NMR (600 MHz, CDCl₃): d 5.35

(dd, 1H, J_1,21.8 Hz, J_2,33.4 Hz, H-2), 4.91–4.61 (2d,

2H, J 10.7 Hz, CH₂Ph), 4.70–4.53 (2d, 2H, J 11.2 Hz,

CH₂Ph), 4.69 (br s, 1H, H-1), 3.92 (dd, 1H, J_3,49.4

The conjugates, showing carbohydrate–protein ratio ꢀ3

and ꢀ6, were prepared from BSA and oligosaccharides

4294

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

Hz, H-3), 3.75–3.71 (m, 1H, H-5), 3.66 (s, 3H, OCH₃);

3.65–3.61 (m, 1H, H-1a⁰⁰), 3.43 (t, 1H, H-4), 3.39–3.35

(m, 1H, H-1b⁰⁰), 2.32 (t, J 7.5 Hz, 2H, H-5⁰⁰), 2.15 (s,

3H, CH₃CO), 1.66–1.61 (m, 4H, H-4⁰⁰), 1.58–1.52 (m,

2H, H-2⁰⁰), 1.38–1.34 (m, 2H, H-3⁰⁰), 1.33 (d, 1H, J_5,6

6.3 Hz, H-6); ¹³C NMR (150 MHz, CDCl₃): d 174.14,

170.46 (CO), 97.60 (C-1, J_C-1,H-1170.2 Hz), 80.04 (C-

4), 78.05 (C-3), 75.45 (CH₂Ph), 71.71 (CH₂Ph), 69.03

(C-2), 67.60 (C-5), 67.49 (C-1⁰⁰), 51.48 (OCH₃), 33.91

(C-5⁰⁰), 29.05 (C-2⁰⁰), 25.69 (C-3⁰⁰), 24.67 (C-4⁰⁰), 21.12

(CH₃CO), 17.94 (C-6). CIMS: m/z 537.2467 [M+Na]⁺.

Anal. Calcd for C₂₉H₃₈O₈: C, 67.68; H, 7.44. Found:

C, 67.96; H, 7.43.

CHCl₃); lit. [a]_Dꢁ82° (c 0.7). ¹H NMR (300 MHz,

CDCl₃): d 5.23 (br s, 1H, H-1), 4.91–4.51 (4d, 4H, J

11.3 Hz, 2 CH₂Ph), 4.05–3.96 (m, partially overlapped,

1H, H-5), 3.91 (ddd, partially overlapped, 1H, J_2,3

3.7 Hz, J_3,OH= J_3,49.3 Hz, H-3), 3.83 (dd, 1H, J_1,2

0.9, J_2,33.3 Hz, H-2), 3.35 (t, 1H, J 9.3 Hz, H-4), 2.35

(d, 1H, OH), 2.10 (s, 3H, SMe), 1.34 (d, 1H, J_5,6

6.0 Hz, H-6); ¹³C NMR (75 MHz, CDCl₃): d 82.42 (C-

4), 82.28 (C-1), 79.84 (C-2), 74.96, 72.28 (2CH₂Ph),

72.02 (C-3), 67.54 (C-5), 17.96 (C-6), 13.56 (SCH₃).

CIMS: m/z 397.1467 ([M+Na]⁺). Anal. Calcd for

C₂₁H₂₆O₄S: C, 67.35; H, 7.00. Found: C, 67.40; H, 7.05.

3.14. Methyl 2,4-di-O-benzoyl-3-O-bromoacetyl-a-^L-

rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-1-thio-a-^L-

rhamnopyranoside (8)

Second eluted was 5-(methoxycarbonyl)pentyl 2-O-

acetyl-3,4-di-O-benzyl-b-^L-rhamnopyranoside (87, 60 mg,

12%); [a]_D+47° (c 0.6). H NMR (600 MHz, CDCl₃):

d 5.61 (dd, 1H, J_1,2, J_2,33.4 Hz, H-2), 4.91–4.60 (d,

1

Reaction of alcohol 6 (93 mg, 0.24 mmol) and chloride

7,¹⁸as described in the general procedure, gave 8

(170 mg, 80%) as a white solid, after freeze drying its solu-

2H,

J 10.7 Hz, CH₂Ph), 4.76–4.49 (2d, 2H, J

11.2 Hz, CH₂Ph), 4.46 (d, 1H, H-1), 3.87–3.84 (m,

1H, H-1a⁰), 3.66 (s, 3H, COOCH₃), 3.61 (dd, 1H,

J_3,49.0 Hz, H-3), 3.48–3.45 (m, 1H, H-1b⁰), 3.42 (t,

1H, H-4), 3.39–3.35 (m, 1H, H-5), 2.31 (t, 2H, J

7.4 Hz, H-5⁰⁰), 2.20 (s, 3H, CH₃CO), 1.66–1.57 (m,

4H, H-4⁰⁰,2⁰⁰), 1.38–1.34 (m, partially overlapped, H-

3⁰⁰), 1.37 (d, 1H, J_5,66.0 Hz, H-6); ¹³C NMR

(150 MHz, CDCl₃): d 174.67, 170.62 (CO), 98.65 (C-

1, J_C-1,H-1152.9 Hz), 80.08 (C-3), 79.64 (C-4), 75.38

(CH₂Ph), 71.63 (C-5), 71.33 (CH₂Ph), 69.50 (C-2),

68.16 (C-1⁰), 51.42 (COOCH₃), 33.88 (C-5⁰⁰), 29.04

(C-2⁰⁰), 25.44 (C-3⁰⁰), 24.59 (C-4⁰⁰), 21.08 (CH₃CO),

17.87 (C-6). CIMS: m/z 537.2480 [M+Na]⁺. Anal.

Calcd for C₂₉H₃₈O₈: C, 67.68; H, 7.44. Found: C,

67.51; H, 7.42.

1

tion in benzene, [a]_Dꢁ0.5 (c 0.5). H NMR (CDCl₃,

600 MHz): d 5.71 (dd, 1H, J_2,33.3, J_3,410.0 Hz, H-3^II),

5.68 (dd, 1H, J_1,21.7 Hz, H-2^II), 5.46 (t, J 9.9 Hz, H-

4^II), 5.29 (d, 1H, J_1,21.3 Hz, H-1^I), 5.27 (d, 1H, H-1^II),

4.91, 4.88, 4.70, 4.60 (4d, 4H, 2 CH₂Ph), 4.10 (dd, partially

overlapped, J_2,33.3, J_3,49.5 Hz, H-3^I), 4.09–4.01 (2 m,

partially overlapped, H-5^II, 5^Iin that order), 3.92 (dd,

1H, H-2^I), 3.73 (t, 1H, H-4^I), 3.67, 3.62 (2d, 2H, J

12.4 Hz, CH₂Br), 2.13 (s, 3H, SCH₃), 1.36 (d, 3H, J_5,6

6.2 Hz, H-6^I), 1.23 (d, 3H, J_5,66.3 Hz, H-6^II); ¹³C NMR

(CDCl₃, 150 MHz): d 166.50, 165.52, 165.34 (3 CO),

99.44 (J_C-1,H-1173.5 Hz, H-1^II), 82.28 (J_C-1,H-1163.7 Hz,

H-1^I), 80.61 (H-4^I), 79.37 (br, C-3^I), 79.27 (C-2^I), 75.52,

71.48 (2 CH₂Ph), 71.26 (C-4^II), 71.12 (C-3^II), 70.19 (C-

2^II), 68.54 (C-5^I), 67.14 (C-5^II), 25.13 (CH₂Br), 17.96 (C-

6^I), 17.59 (C-6^II), 13.77 (SCH₃); ESI-MS: m/z 871.1764

([M+Na]⁺). Anal. Calcd for C₄₃H₄₅BrO₁₁S: C, 60.78; H,

5.34. Found: C, 60.90; H, 5.35.

3.12. 5-(Methoxycarbonyl)pentyl 3,4-di-O-benzyl-^L-

rhamnopyranoside (5)

This compound was obtained in virtually theoretical

yield by deacetylation of methyl 2-O-acetyl-3,4-di-O-

benzyl-1-thio-a-^L-rhamnopyranoside,¹⁶[a]_Dꢁ38.6° (c

3.15. 5-Methoxycarbonylpentyl 2,4-di-O-benzoyl-3-O-

bromoacetyl-a-^L-rhamnopyranosyl-(1 ! 3)-(2,4-di-O-

benzyl-a-^L-rhamnopyranosyl)-(1 ! 2)-3,4-di-O-benzyl-

a-^L-rhamnopyranoside (9)

1

0.9). H NMR (CDCl₃): d 4.88, 4.70, 4.64 (2d, s, 4H, J

11.1 Hz, 2 CH₂Ph), 4.70 (d, 1H, J_1,21.6 Hz, H-1),

4.03–4.01 (m, 1H, H-2), 3.84 (dd, 1H, J_2,33.3, J_3,4

9.0 Hz, H-3), 3.76–3.60 (m, 5H, H-5,1⁰⁰, incl., s, 3.66,

OCH₃), 3.45 (t, 1H, J 9.0 Hz, H-4), 3.40, 3.36 (2t, 1H,

J 6.4 Hz, H-1⁰⁰b), 2.52 (d, 1H, J_2,OH2.0 Hz, OH), 2.31

(t, 2H, J 7.4 Hz, H-5⁰⁰), 1.69–1.52 (m, 4H, H-

2⁰⁰a,b,4⁰⁰a,b), 1.41–1.30 (m, 5H, H-3⁰⁰, incl., d, 1.31, J_5,6

6.3 Hz, H-6); ¹³C NMR (CDCl₃): d 98.83 (C-1), 80.06

(C-3), 79.90 (C-4), 75.34, 71.90 (2 CH₂Ph), 68.51

(C-2), 67.21 (C-1⁰⁰), 67.13 (C-5), 51.39 (OCH₃), 33.85

(C-5⁰⁰), 29.01 (C-2⁰⁰), 25.64 (C-3⁰⁰), 24.61 (C-4⁰⁰), 17.79

(C-6); ESI-MS: m/z 473.2539 ([M+H]⁺). Anal. Calcd for

C₂₇H₃₆O₇: C, 68.62; H, 7.68. Found: C, 68.86; H, 7.76.

Reaction of alcohol 5 (1.2 g, 2.6 mmol) and disaccharide

thioglycoside 8, as described above, gave the fully pro-

1

tected trisaccharide 9 (2.9 g, 90%), [a]_D+17° (c 0.5). H

NMR (CDCl₃): d 5.69 (dd, 1H, J_1,21.7, J_2,33.4 Hz, H-

2^III), 5.66 (dd, 1H, J_3,410.1 Hz, H-3^III), 5.45 (t, 1H, J

9.8 Hz, H-4^III), 5.29 (bd, 1H, H-1^III), 5.18 (d, 1H, J_1,2

1.9 Hz, H-1^II), 4.91–4.36 (m, 9H, 4 CH₂Ph, incl., br s, par-

tially overlapped, H-1^I), 4.19 (dd, 1H, J_2,33.1, J_3,49.3 Hz,

H-3^II), 4.06–4.01 (m, 2H, H-5^III, incl., 4.04, dd, H-2^I), 3.85

(dd, partially overlapped, H-2^II), 3.85 (dd, partially over-

lapped, H-3^I), 3.83–3.78 (m, partially overlapped, 5^II),

3.68 (t, 1H, J 9.4 Hz, H-4^II), 3.65 (s, 3H, OCH₃), 3.64–

3.58 (m, 2H, H-5^I,1⁰⁰a), 3.52, 3.50 (2d, 2H, J 10.2 Hz,

CH₂Br), 3.38–3.33 (m, 2H, H-1⁰⁰b, incl., t, 3.37, J

9.5 Hz, H-4^I), 2.31 (t, 2H, J 7.6 Hz, H-5⁰⁰), 1.66–1.61 (m,

2H, H-4⁰⁰), 1.58–1.53 (m, 2H, H-2⁰⁰), 1.38–1.32 (m, 5H,

H-3⁰⁰, incl., d, J_5,66.2 Hz, H-6⁰⁰), 1.22 (d, 3H, J_5,66.3 Hz,

H-6^I), 1.15 (d, 3H, J_5,66.3 Hz, H-6^III); ¹³C NMR (CDCl₃):

d 174.02, 168.11, 165.59, 165.35 (4 CO), 99.20 (br, J_C-1,H-1

3.13. Methyl 2,4-di-O-benzyl-1-thio-a-^L-rhamnopyrano-

side (6)

This compound was prepared as described for the amor-

phous substance,²³and for which incomplete NMR data

were reported. The compound solidiﬁed on standing,

mp 51–52 °C (from i-Pr₂O–hexane), [a]_Dꢁ87° (c 0.5,

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4295

173 Hz, C-1^III), 98.79 (J_C-1,H-1169 Hz, C-1^I), 98.46 (br,

J_C-1,H-1169 Hz, C-1^II), 80.54 (C-4^II), 80.33 (C-4^I), 80.13

(C-3^I), 78.20 (br, C-3^II), 77.72 (C-2^II), 75.50, 75.32,

72.48, 71.61 (4 CH₂Ph), 74.80 (C-2^I), 71.31 (C-4^III),

70.87 (C-3^III) 70.33 (C-2^III), 68.66 (C-5^II), 67.79 (C-5^I),

67.16 (C-1⁰⁰), 67.04 (C-5^III), 51.45 (COOCH₃), 33.93

(C-5⁰⁰), 29.11 (C-2⁰⁰), 25.73 (C-3⁰⁰), 25. 17 (CH₂Br) 24.70

(C-4⁰⁰), 18.11 (C-6^II), 17.89 (C-6^I), 17.47 (C-6^III);

ESI-MS: m/z 1295.4218 ([M+Na]⁺). Anal. Calcd for

C₆₉H₇₇BrO₁₈: C, 65.04; H, 6.09. Found: C, 65.23; H, 6.16.

tially overlapped, J 9.6 Hz, H-4^III), 2.41 (t, 2H, J 7.3 Hz,

H-5⁰⁰a,b), 1.66–1.58 (m, H-2⁰⁰a,b,4⁰⁰a,b), 1.44–1.34 (m,

2H, H-3⁰⁰a,b), 1.30 (d, 6H, J_5,66.3 Hz, H-6^I,III), 1.28 (d,

J_5,66.3 Hz, H-6^II); ¹³C NMR (D₂O, 150 MHz): d 105.18

(C-1^III), 104.87 (C-1^II), 101.04 (C-1^I), 81.47 (C-2^I), 80.81

(C-3^II), 74.94 (C-4^I), 74.71 (C-4^III), 74.04 (C-4^II), 72.88

(C-2^III), 72.83 (2C, C-3^I,II), 72.61 (C-2^II), 72.10 (C-5^II),

71.90 (C-5^III), 71.55 (C-5^I), 70.58 (C-1⁰⁰), 54.86

(COOCH₃), 36.35 (C-5⁰⁰), 30.84 (C-2⁰⁰), 27.67 (C-3⁰⁰),

26.76 (C-4⁰⁰), 19.43 (C-6^II), 19.38 (2 C, C-6^I,III); ESI-MS:

607.2579 C₂₅H₄₄O₁₅Na requires 607.2578.

3.16. 5-Methoxycarbonylpentyl 2,4-di-O-benzoyl-a-^L-

rhamnopyranosyl-(1 ! 3)-(2,4-di-O-benzyl-a- ^L-rhamno-

pyranosyl)-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyrano-

side (10)

3.18. Methyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-

dideoxy-b-^D-glucopyranoside (14)

A solution of methyl 4-azido-3-O-benzyl-4,6-dideoxy-b-

D-glucopyranoside²⁴(13, 4 g, 13.6 mmol) and tetrameth-

ylurea (18 mL, 150 mmol) in CH₂Cl₂(70 mL) was

treated with bromoacetyl bromide (12 mL, 136 mmol),

and the mixture was stirred at room temperature over-

night. The mixture was poured into stirred aq NaHCO₃

solution and, after 3 h the product was extracted into

CH₂Cl₂. After concentration, chromatography gave 14

(5.6 g, 86%), mp 73–74 °C (from EtOH), [a]_D+75° (c

A solution of thiourea (626 mg, 8.24 mmol) in MeOH

(30 mL) was added to a solution of the foregoing trisac-

charide 9 (3.5 g, 2.75 mmol) in CH₂Cl₂(50 mL), and the

mixture was stirred at room temperature for 4 h. After

concentration, the residue was partitioned between

CH₂Cl₂and aq NaHCO₃. The organic phase was con-

centrated and chromatography gave 10 (3.1 g, 96%),

1

[a]_D+11° (c 0.5). H NMR (CDCl₃): d 5.54 (dd, 1H,

J_1,21.5, J_2,33.3 Hz, H-2^III), 5.31 (br s, 1H, H-1^III),

5.22 (t, 1H, J 9.8 Hz, H-4^III), 5.17 (d, 1H, J_1,21.9 Hz,

H-1^II), 4.94–4.35 (m, 9H, incl., d, 4.68, J_1,21.6 Hz, 4

CH₂Ph, H-1^I), 4.31 (ddd, 1H, J_3,410.0, J_3,OH7.8 Hz,

H-3^III), 4.19 (dd, 1H, J_2,33.1, J_3,49.1 Hz, H-3^II), 4.08–

4.04 (m, 2H, H-2^I,5^III), 3.86–3.79 (m, 3H, H-2^II,3^I,5^II),

3.67–3.59 (m, 6H, H-4^II,5^I,1⁰⁰a, incl., 3.65, s, OCH₃),

3.39 (t, 1H, J 9.5 Hz, H-4^I), 3.35, 3.34 (2t, 1H, J

6.8 Hz, H-1⁰⁰b), 2.46 (d, 1H, OH), 2.31 (t, 2H, J

7.4 Hz, H-5⁰⁰a,b), 1.66–1.61 (m, 2H, H-4⁰⁰a,b), 1.58–

1.53 (m, 2H, H-2⁰⁰a,b), 1.39–1.35 (m, partially over-

lapped, H-3⁰⁰), 1.32 (d, partially overlapped, J_5,6

6.1 Hz, H-6^II), 1.23 (d, 3H, J_5,66.5 Hz, H-6^I), 1.17 (d,

3H, H-6^III); ¹³C NMR (CDCl₃): d 98.95 (br, C-1^III),

98.89 (C-1^I), 98.75 (C-1^II), 80.69 (C-4^II), 80.50 (C-4^I),

80.17 (C-3^I), 78.10 (C-2^II), 77.88 (br, C-3^II), 75.49 (C-

4^III), 75.34 (2C, 2 CH₂Ph), 74.95 (C-2^I), 73.29 (C-2^III),

72.50, 71.79 (2 CH₂Ph), 69.10 (C-3^III), 68.59 (C-5^II),

67.87 (C-5^I), 67.19 (C-1⁰⁰), 66.58 (C-5^III), 51.42

(OCH₃), 33.94 (C-5⁰⁰), 29.12 (C-2⁰⁰), 25.74 (C-3⁰⁰), 24.71

(C-4⁰⁰), 18.17 (C-6^II), 17.91 (C-6^I), 17.53 (C-6^III); ESI-

MS: m/z 1174.4980 ([M+Na]⁺). Anal. Calcd for

C₆₇H₇₆O₁₇: C, 69.77; H, 6.64. Found: C, 69.80; H, 6.95.

0.7). H NMR (CDCl₃, 300 MHz): d 4.96 (dd, 1H, J_1,2

1

8.0, J_2,39.5 Hz, H-2), 4.83, 4.69 (2d, 2H, J 11.3 Hz,

CH₂Ph), 4.26 (d, 1H, H-1), 3.68, 3.64 (2d, 2H, J

12.3 Hz, CH₂Br), 3.56 (t, J 9.2 Hz, H-3), 3.46 (s, 3H,

OCH₃), 3.33–3.20 (m, 2H, H-4,5), 1.39 (d, 3H, J_5,6

5.8 Hz, H-6); ¹³C NMR (CDCl₃, 75 MHz): d 101.36

(C-1), 81.13 (C-3), 75.06 (CH₂Ph), 74.81 (C-2), 70.82

(C-5), 67.80 (C-4), 56.80 (OCH₃), 25.35 (CH₂Br), 18.28

(C-6). FAB MS: m/z 382.4 ([M+HꢁMeOH]⁺). Anal.

Calcd for C₁₆H₂₀BrN₃O₅: C, 46.39; H, 4.87; N, 10.14.

Found: C, 46.25; H, 4.82; N, 10.09.

3.19. Methyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-

dideoxy-a-^D-glucopyranoside (20)

Methyl 4-azido-3-O-benzyl-4,6-dideoxy-a-^D-glucopyr-

anoside²⁴(19, 2.08 g, 7.1 mmol) was converted to the ti-

tle compound in the manner described above for the b

anomer, to give amorphous 20 (2.6 g, 89.5%), [a]_D

1

+204° (c 0.5). H NMR (CDCl₃, 600 MHz): d 4.86 (d,

partially overlapped, 1H, J_1,23.7 Hz, H-1), 4.84 (dd,

partially overlapped, J_2,39.7 Hz, H-2), 4.84 (d, partially

overlapped, J 11.2 Hz, CHPh), 4.77 (d, 1H, CHPh), 3.87

(t, 1H, J 9.7 Hz, H-3), 3.74, 3.68 (2d, 2H, J 12.2,

CH₂Br), 3.62–3.57 (m, 1H, H-5), 3.35 (s, 3H, OCH₃),

3.17 (dd, 1H, J_4,510.1 Hz, H-4), 1.32 (d, 3H, J_5,6

6.2 Hz, H-6); ¹³C NMR (CDCl₃, 75 MHz): d 96.41 (C-

1), 78.05 (C-3), 75.38 (CH₂Ph), 75.21 (C-2), 67.92 (C-

4), 65.85 (C-5), 55.24 (OCH₃), 25.28 (CH₂Br), 18.17

(C-6); ESI-MS: m/z 436.0484 ([M+Na]⁺). Anal. Calcd

for C₁₆H₂₀BrN₃O₅: C, 46.39; H, 4.87; N, 10.14. Found:

C, 46.45; H, 4.81; N, 10.05.

3.17. 5-Methoxycarbonylpentyl a-^L-rhamnopyranosyl-

(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 2)-L-rhamnopyrano-

side (12)

Hydrogenolysis of compound 10 (300 mg) in MeOH

(10 mL) followed by deacylation with 1 M NaOMe in

MeOH gave amorphous 12 as a white solid (135 mg,

1

88%, over 2 steps), [a]_Dꢁ71° (c 0.5, H₂O). H NMR

(D₂O, 600 MHz): 5.04 (d, 1H, J_1,21.7 Hz, H-1^III), 4.93

(d, 1H, J_1,21.9 Hz, H-1^II), 4.89 (d, 1H, J_1,21.7 Hz, H-

1^I), 4.15 (dd, 1H, J_2,33.2 Hz, H-2^II), 4.06 (dd, 1H, J_2,3

3.4 Hz, H-2^III), 3.92 (dd, 1H, J_2,33.4 Hz, H-2^I), 3.86–

3.76 (m, 5H, H-3^I-III, 5^II,III), 3.72–3.66 (m, 5H, H-5^I,1⁰⁰a,

incl., 3.69, s, COOCH₃), 3.56–3.52 (m, 2H, H-4^II,1⁰⁰b),

3.48 (t, partially overlapped, J 9.7 Hz, H-4^I), 3.46 (t, par-

3.20. 1-O-acetyl-4-azido-3-O-benzyl-2-O-bromoacetyl-

4,6-dideoxy-a- (15) and b-^D-glucopyranose (16)

(a) From methyl 4-azido-3-O-benzyl-2-O-bromoacetyl-

4,6-dideoxy-a-^D-glucopyranoside (20): Compound 20

(1.4 g) was treated, at room temperature, with a mixture

4296

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

of 25:10:0.25 Ac₂O/AcOH/H₂SO₄(28 mL). After

30 min, TLC (4:1 hexane/EtOAc) showed that all start-

ing material was consumed and that two products were

formed, the faster moving of the two largely predomi-

nating. Solid NaOAc trihydrate was added, to neutralize

sulfuric acid, and the reaction mixture was poured into a

stirred aq NaHCO₃solution, to destroy excess Ac₂O.

After 3 h the mixture was partitioned between water

and CH₂Cl₂, the organic phase was dried and concen-

trated, to give a mixture of anomers (1.3 g, 87%),

a:b = ꢀ4.4 (NMR). The material was used for the next

step without further puriﬁcation. For characterization,

a portion was chromatographed to give ﬁrst the a-ano-

mer 15, mp 97–98 °C (from EtOH), [a]_D+162°, (c 0.5),

¹H NMR (CDCl₃, 600 MHz): d 6.24 (d, J_1,23.7 Hz,

H-1), 5.01 (dd, J_2,39.9 Hz, H-2), 4.86, 4.79 (2d, J

11.2 Hz, CH₂Ph), 3.87 (t, J 9.9 Hz, H-3), 3.74–3.63 (m,

H-5), 3.67, 3.65 (2 d, J 12.8 Hz, CH₂Br), 3.25 (t, J

9.9 Hz, H-4), 2.15 (s, COCH₃), 1.33 (d, J_5,66.2 Hz, H-

6); ¹³C NMR (CDCl₃, 150 MHz): d 89.04 (J_C-1,H-1

177.3 Hz, C-1), 77.77 (C-3), 75.46 (CH₂Ph), 73.59 (C-

2), 68.88 (C-5), 67.33 (C-4), 24.90 (CH₂Br), 20.91

(COCH₃), 18.33 (C-6); ESI-MS: m/z 448.03 ([M+Li]⁺).

Anal. Calcd for C₁₇H₂₀BrN₃O₆: C, 46.17; H, 4.56; N,

9.50. Found: C, 46.14; H, 4.38; N, 9.45.

CH₂CH₃); ¹³C NMR (CDCl₃, 150 MHz): d 82.82 (C-1),

82.28 (C-3), 75.17 (CH₂Ph), 75.09 (C-5), 73.31 (C-2),

67.68 (C-4), 25.40 (CH₂Br), 23.80 (CH₂CH₃), 18.55 (C-

6), 14.67 (CH₂CH₃); ESI-MS: m/z 466.0412 ([M+Na]⁺)

Anal. Calcd for C₁₇H₂₂BrN₃O₄S: C, 45.95; H, 4.99; N,

9.46. Found: C, 46.05; H, 4.91; N, 9.50.

(b) From a mixture of acetates 15 and 16. BF_3ÆEt₂O

(2.5 mL, 20 mmol) was added dropwise at 0 °C to a

solution of the starting acetates (6.1 g, 13.8 mmol) and

EtSH (3 mL, 40 mmol) in CH₂Cl₂(70 mL). After 3 h

at room temperature, when TLC showed that the reac-

tion was complete, the mixture was poured into aq

NaHCO₃, and the product was extracted with CH₂Cl₂.

After concentration of the extracts, chromatography

gave ﬁrst the ethyl 1-thio-a-glycoside (17, 1.6 g, 23%),

mp 31–32 °C (from hexane at 0 °C), [a]_D+266° (c 0.9).

¹H NMR (CDCl₃, 300 MHz): d 5.57 (d, 1H, J_1,2

5.7 Hz, H-1), 4.97 (dd, 1H, J_2,39.9 Hz, H-2), 4.83,

4.78 (2d, 2H, J 11.0 Hz, CH₂Ph), 4.04–4.94 (m, 1H,

H-5), 3.75 (t, 1H, partially overlapped, H-3), 3,73, 3.69

(2d, 2H, partially overlapped, J 12.6 Hz, CH₂Br), 3.18

(t, 1H, H-4), 2.59–2.46 (m, 2H, CH₂CH₃), 1.32 (d, 3H,

J_5,66.3 Hz, H-6), 1.24 (7, 3H, J 7.4 Hz, CH₂CH₃); ¹³C

NMR (CDCl₃): d 81.27 (J_C-1,H-1168 Hz, C-1), 78.60

(C-3), 75.41 (CH₂Ph), 75.21 (C-2), 67.89 (C-4), 66.39

(C-5), 25.24 (CH₂Br), 24.35 (CH₂CH₃), 18.11 (C-6),

14.77 (CH₃CH₂); ESI-MS: m/z 444.0593 ([M+H]⁺).

Anal. Calcd for C₁₇H₂₂BrN₃O₄S: C, 45.95; H, 4.99; N,

9.46. Found: C, 46.25; H, 4.86; N, 9.42.

Eluted next was the b-anomer 16, mp 61–62 °C (from

1

EtOH) [a]_D+83° (c 1.6). H NMR (CDCl₃): d 5.58 (d,

J_1,28.4 Hz, H-1), 5.07 (dd, J_2,39.4 Hz, H-2), 4.87, 4.66

(2d, J 11.5 Hz, CH₂Ph), 3.60 (t, J 9.6 Hz, H-3), 3.54,

3.51 (2d, CH₂Br), 3.45–3.40 (m, H-5), 3.26 (t, J

9.7 Hz, H-4), 2.17 (s, COCH₃), 1.38 (d, J_5,66.1 Hz, H-

6); ¹³C NMR (CDCl₃, 15 MHz): d 91.41 (J_C-1,H-1

164.4 Hz. C-1), 81.06 (C-3), 75.31 (CH₂Ph),), 73.44 (C-

2), 71.83 (C-5), 67.50 (C-4), 24.82 (CH₂Br), 20.85

(COCH₃), 18.20 (C-6); FAB: m/z 448.0696 ([M+Li]⁺).

Anal. Calcd for C₁₇H₂₀BrN₃O₆: C, 46.17; H, 4.56; N,

9.50. Found: C, 46.34; H, 4.60; N, 9.47.

Eluted later was ethyl 1-thio-b-glycoside 18 (4.1 g, 62%),

which was identical with the above described substance.

3.22. Ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-

1-thio-b-^D-glucopyranoside (22)

Benzoyl chloride (0.6 mL, 5.3 mmol) was added at 0 °C

to a solution of methyl 4-azido-3-O-benzyl-4,6-dideoxy-

1-thio-b-^D-glucopyranoside²⁴(21, 427 mg, 1.32 mmol)

in pyridine (10 mL) and the mixture was kept at room

temperature overnight. Conventional processing and

chromatography gave 22 (842 mg, 97%), mp 64–66 °C

(b) From methyl 4-azido-3-O-benzyl-2-O-bromoacetyl-

4,6-dideoxy-b-^D-glucopyranoside (14): Compound 14

(4.68 g) was treated as described above. After 2 h when

the reaction was complete, the mixture was processed as

described above to give an anomeric mixture of 15 and

16 (4.68 g, 94%).

1

(from MeOH), [a]_D+124° (c 0.9). H NMR (CDCl₃,

300 MHz): d 5.30 (dd, 1H, J_1,210.1, J_2,39.0 Hz, H-2),

4.75, 4.65 (2d, 2H, CH₂Ph), 4.50 (d, 1H, H-1), 3.71 (t,

1H, J 9.9 Hz, H-3), 3.40–3.26 (m, 2H, H-4,5), 2.78–

2.61 (m, 2H, CH₂CH₃), 1.41 (d, 1H, J_5,65.7 Hz, H-6),

1.22 (t, J 7.5 Hz, CH₂CH₃). ¹³C NMR (CDCl₃,

75 MHz): d 83.29 (C-1), 82.50 (C-3), 75.22 (C-5), 75.08

(CH₂Ph), 72.50 (C-2), 67.72 (C-4), 23.83 (CH₂CH₃),

18.67 (C-6), 14.73 (CH₂CH₃). ESI-MS 434.1713

([M+Li]⁺). Anal. Calcd for C₂₂H₂₅N₃O₄S: C, 61.81; H,

5.89; N, 9.83. Found: C, 61.99; H, 5.86; N, 9.76.

3.21. Ethyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-

dideoxy-1-thio-a- (17) and b-^D-glucopyranoside (18)

(a) From ethyl 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-b-

D-glucopyranoside (21). Methyl 4-azido-3-O-benzyl-4,6-

dideoxy-1-thio-b-^D-glucopyranoside (21)²⁴(313 mg,

1 mmol) in CH₂Cl₂(5 mL) was treated with tetramethyl-

urea (0.239 mL, 2.0 mmol) and bromoacetyl bromide

(0.13 mL, 1.5 mmol) in dichloromethane (5 mL) as de-

scribed for the preparation of 14, to give the b-compound

18 (0.260 g, 67%), mp 82–83 °C (from ethanol), [a]_D+60°

(c 0.5). ¹H NMR (CDCl₃, 600 MHz): d 5.01 (dd, 1H, J_1,2

10.0 Hz, J_2,39.0 Hz, H-2), 4.83, 4.71 (2d, 2H, J 11.2 Hz,

CH₂Ph), 4.34 (d, 1H, H-1), 3.69, 3.65 (2d, 2H, J

12.5 Hz, CH₂Br), 3.57 (t, J 9.1 Hz, H-3), 3.31–3.27 (m,

1H, H-5), 3.23 (bdd, 1H, H-4), 2.72–2.61 (m, 2H,

CH₂CH₃), 1.38 (d, 3H, J_5,66.0 Hz, H-6), 1.24 (t, 3H,

3.23. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-2-O-

bromoacetyl-4,6-dideoxy-b-D-glucopyranosyl-(1 ! 2)-(2,

4-di-O-benzoyl-a-^L-rhamnopyranosyl)-(1 ! 3)-(2,4-di-

O-benzyl-a-^L-rhamnopyranosyl)-(1 ! 2)-3,4-di-O-benzyl-

a- ^L-rhamnopyranoside (24)

A mixture of acceptor 10 (288 mg, 0.25 mmol), donor 18

˚

(111 mg, 0.25 mmol), and 4 A molecular sieves was

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4297

allowed to react as described in the general method for

glycosylation of thioglycosides and chromatography

gave ﬁrst orthoester 23 (60 mg, 16.2% and 26%, based

on the initial amount of the acceptor and that con-

sumed, respectively). ¹H NMR (CDCl₃, 600 MHz): d

5.93 (d, 1H, J_1,25.7 Hz, H-1^IV), 5.68 (dd, 1H, J_2,32.0,

J_3,43.3 Hz, H-2^III), 5.42 (t, 1H, J 9.9 Hz, H-4^III), 5.26

(d, 1H, J_1,21.7 Hz, H-1^III), 5.19 (d, 1H, J_1,21.8 Hz, H-

1^II), 4.96–4.43 (m, 12H, 5 CH₂Ph, incl., ꢀ4.69, d, over-

lapped, H-1^I, 4.50, dd, 1H, J_3,49.7 Hz, H-3^III), 4.26 (dd,

1H, J_2,34.2 Hz, H-2^IV), 4.21 (dd, 1H, J_2,33.1, J_3,4

9.4 Hz, H-3^II), 4.12–4.07 (m, partially overlapped, H-

5^III), 4.06 (dd, partially overlapped, H-2^I), 3.91 (d, 1H,

J_2,33.3 Hz, H-2^II), 3.86–3.78 (m, 3H, H-3^I,5^IV,3^IV, in

that order), 3.69 (t, partially overlapped, J 9.4 Hz, H-

4^II), 3.67 (m, partially overlapped, H-1⁰⁰a,CHaBr, incl.,

s, 3.64, OCH₃), 3.41 (t, 1H, J 9.4 Hz, H-4^I), 3.36, 3.34

(2 t, 1H, H-1⁰⁰b), 3.25 (d, 1H, J 11.7 Hz, CHbBr), 3.02

(dd, 1H, J_3,48.2, J_4,510.1 Hz, H-4^IV), 2.31 (t, 2H, J

7.4 Hz, H-5⁰⁰), 1.66–1.61 (m, 2H, H-4⁰⁰a,b), 1.59–1.54

(m, 2H, H-2⁰⁰a,b), 1.40–1.33 (m, partially overlapped,

H-3⁰⁰a,b), 1.34, 1.23, 1.21, 1.18 (4d, 3H each,

J_5,6ꢀ 6.1 Hz, H-6^II,I,IIIin that order); ¹³C NMR

(CDCl₃, 150 MHz): d 173.94, 165.56, 165.20 (3 CO),

118.27 (quart C, orthoester), 99.54 (C-1^III), 98.77 (C-

1^IV), 98.72 (C-1^I), 98.22 (C-1^II), 80.27 (C-4^I), 80.09 (C-

3^I), 80.07 (C-4^II), 79.80 (C-3^IV), 79.13 (C-3^II), 78.51

(C-2^II), 76.90 (C-2^IV), 75.49, 75.28, 72.46, 71.71 (4

CH₂Ph), 75.02 (C-2^I), 72.03 (C-4^III), 71.77 (C-2^III),

70.17 (C-3^III), 68.43 (C-5^II), 67.73 (C-5^I), 67.34 (C-5^IV),

67.25 (C-5^III), 67.08 (C-1⁰⁰), 64.38 (C-4^IV), 51.53

(COOCH₃), 33.96 (C-5⁰⁰), 31.56 (CH₂Br), 29.16 (C-2⁰⁰),

25.83 (C-3⁰⁰), 24.50 (C-4⁰⁰), 18.09 (C-6^II), 17.98 (C-6^IV),

17.82 (C-6^I), 17.62 (C-6^III). ESI-MS: m/z 1556.5332

([M+Na]⁺) C₈₂H₉₂BrN₃O₂₁Na requires 1556.5304.

(C-3^I), 78.25 (br, C-3^II), 77.93 (C-2^II), 77.31 (C-

3^IV),75.41, 75.36, 75.19 (3 CH₂Ph), 74.93 (C-2^I), 74.21

(C-2^IV), 72.70 (C-3^III), 72.45 (CH₂Ph), 72.41 (C-4^III),

71.83 (CH₂Ph), 69.02 (C-2^III), 68.58 (C-5^II), 67.80 (C-

5^I), 67.53 (C-4^IV), 67.19 (C-1⁰⁰), 67.14 (C-5^III), 66.70

(C-5^IV), 51.52 (COOCH₃), 34.00 (C-5⁰⁰), 29.17 (C-2⁰⁰),

25.76 (C-3⁰⁰), 25.43 (CH₂Br), 24.77 (C-4⁰⁰), 18.13 (C-

6^II), 17.90 (C-6^I), 17.88 (C-6^IV), 17.57 (C-6^III). ESI-

MS: m/z 1556.5303 ([M+Na]⁺) C₈₂H₉₂BrN₃O₂₁Na

requires 1556.5304.

Eluted next was the desired b-linked tetrasaccharide 24

(46 mg, 12.4% and 20%, based on the initial amount

of the acceptor and that consumed, respectively) ¹H

NMR (CDCl₃, 600 MHz): 5.53 (dd, 1H, J_1,21.9, J_2,3

3.4 Hz, H-2^III), 5.44 (t, 1H, J 9.4 Hz, H-4^III), 5.31 (d,

1H, J_1,21.6 Hz, H-1^III), 5.17 (d, 1H, J_1,22.0 Hz, H-

1^II), 5.00–4.28 (m, 10H, 5 CH₂Ph), 4.81 (dd, 1H, J_1,2

7.9, J_2,39.5 Hz, H-2^IV), 4.66 (d, J_1,21.7 Hz, H-1^I), 4.38

(d, partially overlapped, H-1^IV), 4.36 (dd, partially over-

lapped, J_2,33.5, J_3,49.9 Hz, H-3^III), 4.18 (dd, 1H, J_2,3

3.0, J_3,49.0 Hz, H-3^II), 4.11–4.06 (m, 1H, H-5^III), 4.03

(dd, 1H, J_1,22.0, J_2,33.0 Hz, H-2^I), 3.89 (dd, 1H, J_2,3

3.0 Hz, H-2^II), 3.84 (dd, partially overlapped, J_2,33.0,

J_3,49.2 Hz, H-3^I), 3.84–3.80 (m, partially overlapped,

H-5^II), 3.66–3.58 (m, 6H, H-5^I, 1⁰⁰a, incl., 3.65, s

OCH₃, 3.60, t, J 9.5 Hz, H-4^II), 3.40 (t, 1H, J 9.5 Hz,

H-4^I), 3.36–3.32 (m, 2H, H-1⁰⁰b, incl., 3.50, d, J

12.5 Hz, CHaBr), 3.23 (d, 1H, J 12.5 Hz, CHbBr),

3.17 (t, 1H, J 9.3 Hz, H-3^IV), 3.03–3.00 (m, 2H, H-

5^IV,4^IV), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.66–1.61 (m,

2H, H-4⁰⁰a,b), 1.58–1.53 (m, 2H, H-2⁰⁰a,b), 1.37–1.32

(m, partially overlapped, H-3⁰⁰a,b), 1.32 (d, partially

overlapped, J_5,66.1 Hz, H-6^II), 1.23 (d, 3H, J_5,6

6.1 Hz, H-6^I), 1.16 (d, 3H, J_5,66.3 Hz, H-6^III), 1.09 (d,

3H, J_5,65.9 Hz, H-6^IV); ¹³C NMR (CDCl₃, 150 MHz):

d 100.48 (C-1^IV), 99.07 (br, C-1^III), 98.79 (C-1^I), 98.31

(br, C-1^II), 80.57 (C-3^IV), 80.40 (C-4^I), 80.16 (C-4^II),

80.09 (C-3^I), 79.33 (C-3^II), 78.20 (C-2^II), 75.38 (2 C, 2

CH₂Ph), 75.05 (C-2^I), 74.65 (CH₂Ph), 74.53 (C-3^III),

74.34 (C-2^IV), 73.52 (C-4^III), 72.53 (CH₂Ph), 72.47 (C-

2^III), 71.57 (CH₂Ph), 70.78 (C-4^IV), 68.39 (C-5^II), 67.82

(C-5^I), 67.14 (2 C, C-5^IV,1⁰⁰), 66.59 (C-5^III), 51.47

(OCH₃), 33.96 (C-5⁰⁰), 31.56 (CH₂Br), 29.11 (C-2⁰⁰),

25.72 (C-3⁰⁰), 24.71 (C-4⁰⁰), 18.16 (C-6^II), 17.99 (C-6^IV),

17.85 (C-6^I), 17.52 (C-6^III); ESI-MS: m/z 1556.5385

([M+Na]⁺).

Eluted next was the a-linked tetrasaccharide 88 (75 mg,

20.2% and 32%, based on the initial amount of the

acceptor and that consumed, respectively). ¹H NMR

(CDCl₃, 600 MHz): d 5.60 (dd, 1H, J_1,21.9, J_2,3

3.0 Hz, H-2^III), 5.50 (t, 1H, J 9.8 Hz, H-4^III), 5.25 (d,

1H, J_1,21.5 Hz, H-1^III), 5.14 (d, 1H, J_1,21.8 Hz, H-

1^II), 5.04 (d, 1H, J_1,23.5 Hz, H-1^IV), 4.87–4.38 (m,

11H, 5 CH₂Ph, incl., 1.66, d, J_1,21.7 Hz, H-1^I), 4.72

(dd, 1H, J_2,310.0 Hz, H-2^IV), 4.34 (dd, J_2,33.1, J_3,4

9.7 Hz, H-3^III), 4.17 (dd, 1H, J_2,33.1, J_3,49.4 Hz, H-

3^II), 4.06 (m, 1H, H-5), 4.02 (dd, 1H, J_1,21.8, J_2,3

2.8 Hz, H-2^I), 3.85 (dd, partially overlapped, J_2,3

3.1 Hz, H-2^II), 3.84 (dd, partially overlapped, J_2,33.0,

J_3,49.5 Hz, H-3^I), 3.80 (m, H-5^II), 3.66–3.58 (m, 6H,

H-4^II,5^I, 1⁰⁰a, incl., 3.65, s OCH₃), 3.52 (t, 1H, J

9.6 Hz, H-3^IV), 3.41 (m, 1H, H-5^IV), 3.38–3.32 (m, 3H,

H-4^I,1⁰⁰b, incl., 3.35, d, CHaBr), 3.17 (d, 1H, ²J

12.9 Hz, CHbBr), 2.92 (dd, 1H, J_3,49.5, J_4,510.2 Hz,

H-4^IV), 2.31 (t, 2H, J 7.6 Hz, H-5⁰⁰a,b), 1.66–1.61 (m,

2H, H-4⁰⁰a,b), 1.58–1.53 (m, 2H, H-2⁰⁰a,b), 1.37–1.32

(m, partially overlapped, H-3⁰⁰a,b), 1.32 (d, partially

overlapped, J_5,66.3 Hz, H-6^II), 1.22 (d, 3H, J_5,6

6.2 Hz, H-6^I), 1.20 (d, 3H, J_5,66.3 Hz, H-6^III), 0.88 (d,

3H, J_5,65.9 Hz, H-6^IV); ¹³C NMR (CDCl₃): 99.38 (br,

J_C-1,H-1174 Hz, C-1^III), 98.79 (J_C-1,H-1169 Hz, C-1^I),

98.57 (br, J_C-1,H-1169 Hz, C-1^II), d 93.81 (J_C-1,H-1

171 Hz, C-1^IV), 80.49 (C-4^II), 80.44 (C-4^I), 80.08

Eluted last was the unchanged glycosyl acceptor 10

(109 mg, 38%).

3.24. Methyl 3-O-acetyl-2,4-di-O-benzyl-a-^L-rhamnopyr-

anosyl-(1 ! 3)-2,4-di-O-benzyl-1-thio-a-^L-rhamnopyrano-

side (27)

Methyl 1-thio-a-^L-rhamnopyranoside¹⁶was converted

to

3-O-acetyl-2,4-di-O-benzyl-1-thio-a-^L-rhamnopyr-

anoside (25) as described.²³A solution of 25 (1.77 g,

4.7 mmol) in CCl₄(6 mL) was treated with a stock solu-

tion²⁶of Cl₂in CCl₄(6 mL, ꢀ6 mmol). The resulting

yellow solution was concentrated after 3 min, when

NMR spectra of products of preliminary experiments

showed that the conversion was complete and that

4298

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

3-O-acetyl-2,4-di-O-benzyl-1-thio-a-L-rhamnopyranosyl

chloride (26) was stereospeciﬁcally formed. After con-

centration and co-evaporation with dry toluene, the

material in the residue was used for the next step with-

68.75 (C-5^II), 68.12 (C-5^III), 67.84 (C-5^I), 67.15 (C-1⁰⁰),

51.46 (COOCH₃), 33.92 (C-5⁰⁰), 29.11 (C-2⁰⁰), 25.72 (C-

3⁰⁰), 24.69 (C-4⁰⁰), 21.04 (COCH₃), 17.93 (C-6^II), 17.92 (C-

6^I), 17.89 (C-6^III); ESI-MS: m/z 1189.5475 ([M+Na]⁺).

Anal. Calcd for C₆₉H₈₂O₁₆: C, 70.99; H, 7.08. Found: C,

70.85; H, 7.13.

1

out further puriﬁcation. H NMR (CDCl₃, 300 MHz):

d 5.98 (d, 1H, J_1,21.6 Hz, H-1), 5.42 (dd, 1H, J_2,33.5,

J_3,49.8 Hz, H-3), 4.73, 4.65 (2 d, partially overlapped,

²J 11.1 Hz, CH₂Ph), 4.67, 4.56 (2 d, partially over-

3.26. 5-Methoxycarbonylpentyl 2,4-di-O-benzyl-a-^L-rha-

mnopyranosyl-(1 ! 3)-(2,4-di-O-benzyl-a-^L-rhamnopyr-

anosyl)-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyranoside

(29)

2

lapped, J 12.1 Hz, CH₂Ph), 4.10–4.00 (m, 2H, H-2,5),

3.68 (t, 1H, J 9.6 Hz, H-4), 1.96 (d, 3H, COCH₃), 1.36

(d, 3H, J_5,66.2 Hz, H-6); ¹³C NMR (CDCl₃,75 MHz):

d 90.55 (J_C-1,H-1182.8 Hz, C-1), 78.89 (C-2), 78.35 (C-

4), 75.06, 73.30 (2 CH₂Ph), 72.16 (C-3), 70.73 (C-5),

20.88 (COCH₃), 17.61 (C-6).

Deacetylation of 28 (880 mg) gave amorphous 29

(716 mg, 85%), [a]_Dꢁ7° (c 0.6). ¹H NMR (CDCl₃,

600 MHz): d 5.21 (br s, 1H, H-1^III), 5.11 (d, 1H, J_1,2

1.9 Hz, H-1^II), 4.89–4.08 (m, 13H, 6 CH₂Ph, incl., 4.70,

d, partially overlapped, J_1,21.8 Hz, H-1^I), 4.19 (dd, 1H,

J_2,33.0, J_3,49.6 Hz, H-3^II), 4.03 (dd, 1H, J_2,32.8 Hz, H-

2^I), 3.96 (ddd, 1H, H-3^III), 3.84 (dd, partially overlapped,

H-3^I), 3.82 (m, partially overlapped, H-5^II), 3.79 (dd, 1H,

H-2^II), 3.73–3.59 (m, 2H, H-2^III,5^III), 3.66 (m, partially

overlapped, H-5^I), 3.64 (s, partially overlapped,

COOCH₃), 3.65–3.59 (m, partially overlapped, H-

4^II,1⁰⁰a), 3.40 (t, 1H, J 9.6 Hz, H-4^I), 3.37, 3.35 (2t, 1H, J

6.5 Hz, H-1⁰⁰b), 3.29 (t, 1H, J 9.3 Hz, H-4^III), 2.31 (t,

2H, J 7.5 Hz, H-5⁰⁰a,b), 1.82 (s, 1H, OH), 1.66–1.61 (m,

2H, H-4⁰⁰), 1.59–1.54 (m, 2H, H-2⁰⁰), 1.38–1.32 (m, 2H,

H-3⁰⁰), 1.31 (d, 2H, J_5,66.3 Hz, H-6^II), 1.28 (d, 3H, J_5,6

6.2 Hz, H-6^I), 1.23 (d, 3H, J_5,66.3 Hz, H-6^III); ¹³C

NMR (CDCl₃, 125 MHz): d 98.93 (C-1^II), 98.82 (C-1^I),

98.34 (C-1^III), 82.01 (C-4^III), 80.89 (C-4^II), 80.40 (C-4^I),

79.93 (C-3^I), 78.98 (C-2^III), 77.65 (C-2^II), 76.57 (C-3^II),

75.35, 74.74, 74.51, 72.35, 72.27, 72.11 (6CH₂Ph), 74.46

(C-2^I), 71.54 (C-3^II), 68.68 (C-5^II), 67.79 (C-5^I), 67.49

(C-5^III), 67.08 (C-1⁰⁰), 51.38 (COOCH₃), 33.87 (C-5⁰⁰),

29.03 (C-2⁰⁰), 25.67 (C-3⁰⁰), 24.64 (C-4⁰⁰), 17.94 (C-6^II),

17.87 (C-6^I), 17.82 (C-6^III). ESI-MS: m/z 1147.5365

([M+Na]⁺). Anal. Calcd for C₆₇H₈₀O₁₅: C, 71.51; H,

7.17. Found: C, 71.69; H, 6.99.

A solution of the foregoing glycosyl chloride and 6

(1.2 g, 2.9 mmol) were allowed to react as described

above in the general procedure to give 27 (1.25 g,

1

52%), mp 84–85 ° (from MeOH), [a]_Dꢁ42° (c 0.5). H

NMR (CDCl₃, 600 MHz): d 5.27 (dd, 1H, J_2,33.2, J_3,4

9.4 Hz, H-3^II), 5.18, (d, 1H, J_1,21.4 Hz, H-1^I), 5.11, (d,

1H, J_1,21.9 Hz, H-1^II), 4.81–4.17 (8d, 8H, 4 CH₂Ph),

4.05 (dd, partially overlapped, J_2,33.1, J_3,49.6 Hz, H-

3^I), 4.04–3.99 (m, partially overlapped, H-5^I), 3.85–

3.84 (m, 2H, H-2^I,II), 3.80–3.76 (m, 1H, H-5^II), 3.66 (t,

1H, J 9.4 Hz, H-4^I), 3.60 (t, 1H, J 9.4 Hz, H-4^II), 2.11

(s, 3H, SCH₃), 1.93 (s, 3H, COCH₃), 1.30 (d, 3H, J_5,6

6.3 Hz, H-6^I), 1.24 (d, 3H, J_5,66.3 Hz, H-6^II); ¹³C

NMR (CDCl₃, 150 MHz): d 99.53 (C-1^II), 83.09 (C-1^I),

80.74 (C-4^I), 79.23 (C-2^I), 78.97 (C-4^II), 78.55 (C-3^I),

76.64 (C-2^II), 74.74, 74.66 (2 CH₂Ph), 73.45 (C-3^I),

68.62 (C-5^I), 68.28 (C-5^II), 21.05 (COOCH₃), 17.99 (C-

6^II), 17.85 (C-6^I), 13.82 (SCH₃); ESI-MS: m/z 765.3066

([M+Na]⁺). Anal. Calcd for C₄₃H₅₀O₉S: C, 69.52; H,

6.78. Found: C, 69.30; H, 6.74.

3.25. 5-Methoxycarbonylpentyl 3-O-acetyl-2,4-di-O-benzyl-

a-^L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-L-rhamno-

pyranosyl-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyranoside

(28)

3.27. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-b-^D-glucopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-

L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-L-rhamno-

pyranosyl-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyrano-

side (31)

Reaction of alcohol 5 (390 mg, 0.82 mmol) and glycosyl

donor 27 (798 mg, 1.07 mmol), as described in the general

procedure,

gave

amorphous

trisaccharide

28

(880 mg,94%), [a]_Dꢁ5° (c 0.9). ¹H NMR (CDCl₃,

600 MHz): d 5.29 (dd, 1H, J_2,33.3, J_3,49.6 Hz, H-3^III),

5.16 (d, 1H, J_1,21.7 Hz, H-1^III), 5.11 (d, 1H, J_1,21.9 Hz,

H-1^II), 4.87–4.35 (m, 12H, 11 CHPh, incl., d, 4.69, J_1,2

1.8 Hz, H-1^I), 4.17 (m, 2H, CHPh, H-3^II), 4.02 (dd, 1H,

J_1,21.9, J_2,33.0 Hz, H-2^I), 3.85 (dd, 1H, J_1,21.9, J_2,3

3.3 Hz, H-2^II), 3.83 (dd, 1H, J_2,33.0, J_3,49.4 Hz, H-

3^I),3.81–3.75 (m, 3H, H-2^II,5^II,5^III), 3.66 (s, 3H,

COOCH₃), 3.65–3.58 (m, 4H, H-4^II,4^III,5^I,H-1⁰⁰a), 3.39

(t, J 9.4 Hz, H-4^I), 3.48 (t, 1H, J 9.4 Hz, H-4^I), 3.36, 3.35

(2t, 1H, J 6.4 Hz, H-1⁰⁰b), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b),

1.94 (s, 3H, COCH₃), 1.66–1.61 (m, 2H, H-4⁰⁰a,b), 1.58–

1.53 (m, 2H, H-2⁰⁰a,b), 1.38–1.32 (m, H-3⁰⁰a,b), 1.29 (d,

J_5,66.2 Hz, H-6^II), 1.27 (d, 3H, J_5,66.2 Hz, H-6^I), 1.21 (d,

3H, J_5,66.2 Hz, H-6^III); ¹³C NMR (CDCl₃, 150 MHz): d

99.25 (C-1^III), 98.98 (C-1^II), 98.87 (C-1^I), 80.70 (C-4^III),

80.44 (C-4^I), 80.01 (C-3^I), 78.95 (C-4^II), 77.63 (C-2^II),

77.21(C-3^II), 76.73(C-2^III), 75.41, 74.67, 74.64(3CH₂Ph),

74.49 (C-2^I), 73.53 (C-3^III), 72.62, 72.33, 72.18 (3 CH₂Ph),

Alcohol 29 (580 mg, 0.5 mmol) was treated with thiogly-

coside 18 (271 mg, 0.612 mmol) as described above and

chromatography gave 5-methoxycarbonylpentyl 4-azi-

do-3-O-benzyl-2-O-bromoacetyl-4,6-dideoxy-b-^D-gluco-

pyranosyl-(1 ! 3)-2,4-di-O-benzyl-a- ^L-rhamnopyranosyl-

(1 ! 3)-2,4-di-O-benzyl-a- ^L-rhamnopyranosyl-(1 ! 2)-

3,4-di-O-benzyl-a- ^L-rhamnopyranoside (30, 630 mg,

84%), [a]_D+2.3° (c 0.8). ¹H NMR (CDCl₃, 600 MHz): d

5.10 (d, 1H, J_1,21.9 Hz, H-1^II), 5.09 (br s, 1H, H-1^III),

5.05 (dd, 1H, J_1,28.0 Hz, J_2,39.6 Hz, H-2^IV), 4.86–4.55

(m, 16H, 7 CH₂Ph, incl, ꢀ4.70, d, partially overlapped,

J_1,2ꢀ 8 Hz, H-1^IV, 4.66, partially overlapped, H-1^I),

4.12 (dd, 1H, J_2,33.1, J_3,49.4 Hz, H-3^II), 4.09 (dd, 1H,

J_2,33.0, J_3,49.7 Hz, H-3^III), 4.01 (dd, 1H, J_1,22.0, J_2,3

2.9 Hz, H-2^I), ꢀ3.84 (dd, 1H, H-2^II), 3.83 (dd, 1H, J_2,3

3.0, J_3,49.4 Hz, H-3^I), 3.82 (dd, 1H, H-2^III), 3.80–3.76

(m, 2H, H-5^II,III), 3.65 (s, 3H, COOCH₃), 3.64–3.50 (m,

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4299

5H, H-2^II, 4^II,III,5^I,1⁰⁰a), 3.40 (t, 2H, J 9.3 Hz, H-4^I,3^IV),

3.38 (m, H-1⁰⁰b), 3.35, 3.32 (2d, ²J 12.6 Hz, CH₂Br), 3.14

(dd, 1H, J_3,49.3, J_4,59.8 Hz, H-4^IV), 3.83 (m, 1H, H-

5^IV), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.66–1.61 (m, 2H,

H-4⁰⁰a,b), 1.58–1.53 (m, 2H, H-2⁰⁰a,b), 1.39–1.32 (m, 2H,

H-3⁰⁰a,b), 1.27 (d, 6H, J_5,66.2 Hz, H-6^I,II), 1.21 (d, J_5,6

6.3 Hz, H-6^III), 1.13 (d, J_5,65.9 Hz, H-6^IV). ¹³C NMR

(CDCl₃, 150 MHz): d 100.62 (C-1^IV), 102.23 (br, C-1^III),

98.87 (C-1^I), 98.75 (br, C-1^II), 81.06 (C-3^IV), 80.52 (C-

4^III), 80.51 (C-4^I), 80.45 (C-4^II), 80.06 (C-3^I), 78.87 (C-

3^III), 78.58 (C-2^II), 78.00 (2 C, C-2^III,3^II), 75.45 (CH₂Ph),

75.10 (C-2^IV), 75.04, 74.83 (2 CH₂Ph), 74.66 (C-2^I), 74.54,

73.32, 72.40, 71.96 (4 CH₂Ph), 70.61 (C-5^IV), 68.52

(C-5^II), 68.37 (C-5^III), 67.84 (C-5^I), 67.72 (C-4^IV), 67.14

(C-1⁰⁰), 51.48 (COOCH₃), 33.93 (C-5⁰⁰), 29.11 (C-2⁰⁰),

25.73 (C-3⁰⁰), 25.20 (CH₂Br), 24.70 (C-4⁰⁰), 18.21

(C-6^IV), 18.01, 17.90 (C-6^I,II), 17.85 (C-6^III). ESI-MS:

m/z 1550.5834 ([MꢁH+HCOOH]⁺) C₈₂H₉₆BrN₃O₁₉–

H+HCOOH requires 1550.5798.

600 MHz): d 5.11 (br s, 1H, H-1^III), 5.08 (d, 1H, J_1,2

1.8 Hz, H-1^II), 4.97–4.43 (m, 16H, 7CH₂Ph, incl, ꢀ4.66,

d, partially overlapped, J_1,2ꢀ 1.8 Hz, H-1^I, 4.61, d, par-

tially overlapped, J_1,28.0 Hz, H-1^IV), 4.13 (dd, 1H, J_2,3

3.0, J_3,49.4 Hz, H-3^II), 4.10 (dd, 1H, J_2,33.1, J_3,49.7 Hz,

H-3^III), 4.00 (dd, 1H, J_2,32.8 Hz, H-2^I), 3.88 (dd, 1H,

J_1,21.7 Hz, H-2^III), 3.82 (dd, partially overlapped, H-

3^I), 3.81–3.75 (m, 3H, H-2^II,5^II,III), 3.65–3.58 (m, 9H, H-

4^III,5^I,1⁰⁰a, incl., 3.65, s, COOCH₃, OCH₃-2), 3.56 (bt,

1H, H-4^II), 3.40 (t, 1H, J 9.5 Hz, H-4^I), 3.36–3.31 (m,

H-3^IV, 1⁰⁰b), 3.12 (dd, 1H, J_2,39.1 Hz, H-2^IV), 3.10–3.04

(m, 2H, H-4^IV,5^IV), 2.31 (t, 2H, J 7.4 Hz, H-5⁰⁰a,b),

1.66–1.61 (m, 2H, H-4⁰⁰a,b), 1.57–1.53 (m, 2H, H-2⁰⁰a,b),

1.37–1.32 (m, 2H, H-3⁰⁰a,b), 1.27 (d, partially overlapped,

J_5,66.3 Hz, H-6^III), 1.26 (d, partially overlapped, J_5,6

6.2 Hz, H-6^I), 1.25 (d, partially overlapped, J_5,66.2 Hz,

H-6^II), 1.10 (d, J_5,65.8 Hz, H-6^IV). ¹³C NMR (CDCl₃,

150 MHz): d 103.51 (J_C-1,H-1159 Hz, C-1^IV), 100.23 (br,

J_C-1,H-1167 Hz, C-1^III), 98.96 (br, J_1,2168 Hz, C-1^II),

98.90 (J_C-1,H-1170 Hz, C-1^I), 84.76 (C-2^IV), 82.66

(C-3^IV), 80.90 (C-4^III), 80.56 (C-4^I), 80.53 (C-4^II), 80.06

(C-3^I), 79.27 (C-2^III), 78.50 (C-3^III), 78.01 (C-2^II), 77.69

(C-3^II), 75.46, 75.40, 74.73, (3 CH₂Ph), 74.72 (C-2^I),

74.57, 73.30, 72.36, 72.09 (4 CH₂Ph), 70.18 (C-5^IV),

68.59 (C-5^II), 68.35 (C-5^III), 67.87 (C-5^I), 67.57 (C-4^IV),

67.16 (C-1⁰⁰), 60.72 (OCH₃), 51.46 (COOCH₃), 33.94

(C-5⁰⁰), 29.14 (C-2⁰⁰), 25.74 (C-3⁰⁰), 24.71 (C-4⁰⁰), 18.00

(C-6^IV), 17.99, 17.94, 17.92 (C-6^I,II,III). ESI-MS: m/z

1422.6631 ([M+Na]⁺). Anal. Calcd for C₈₁H₉₇N₃O₁₈: C,

69.46; H, 6.98; N, 3.00. Found: C, 69.73; H, 7.09; N, 3.00.

Deacylation of the foregoing fully protected tetrasaccha-

ride 30 (0.63 g) gave alcohol 31 (0.53g, 93%), [a]_D+10.5°

(c 0.5). ¹H NMR (CDCl₃, 600 MHz): d 5.11 (d, 1H, J_1,2

2.0 Hz, H-1^II), 5.08 (br s, 1H, H-1^III), 4.91–4.37 (m,

16H, 7 CH₂Ph, incl., ꢀ4.67 d, partially overlapped,

J_1,2ꢀ 1.7 Hz, H-1^I), 4.39, d, partially overlapped, J_1,2

7.7 Hz, H-1^IV, 4.11 (dd, 1H, J_2,33.0, J_3,49.3 Hz, H-

3^II), 4.04 (dd, 1H, J_2,33.1, J_3,49.6 Hz, H-3^III), 4.02

(dd, 1H, J_2,32.9 Hz, H-2^I), 3.86 (dd, 1H, H-2^III), 3.84

(dd, 1H, J_2,33.0, J_3,49.4 Hz, H-3^I), 3.80 (dd, 1H, H-

2^II), 3.78–3.74 (m, 2H, H-5^II,III), 3.67–3.59 (m, 6H, H-

4^III,5^I,1⁰⁰a, incl., 3.65, s, COOCH₃), 3.53–3.50 (m, 2H,

H-2^IV,4^II), 3.40 (t, 1H, J 9.2 Hz, H-4^I), 3.36–3.31 (m,

2H, H-3^IV,1⁰⁰b), 3.09–3.06 (m, 2H, H-4^IV,5^IV), 2.69 (br

s, 1H, OH), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.66–1.61

(m, 2H, H-4⁰⁰a,b), 1.58–1.53 (m, 2H, H-2⁰⁰a,b), 1.40–

1.32 (m, 2H, H-3⁰⁰a,b), 1.28 (d, partially overlapped,

J_5,6H-6^I), 1.25 (d, partially overlapped, J_5,66.1 Hz, H-

6^III), 1.24 (d, partially overlapped, J_5,66.1 Hz, H-6^II),

1.17 (d, J_5,65.8 Hz, H-6^IV). ¹³C NMR (CDCl₃,

150 MHz): d 103.70 (C-1^IV), 99.97 (br, C-1^III), 98.86

(C-1^I), 98.70 (br, C-1^II), 82.05 (C-3^IV), 80.64 (C-4^III),

80.56 (C-4^I), (C-3^I), 80.53 (C-4^II), 80.05 (C-3^I), 79.92

(C-3^III), 78.25 (C-2^III), 77.85 (C-2^II), 77.49 (C-3^II),

75.71 (C-2^IV), 75.47, 75.23, 74.81 (2 C) (4 CH₂Ph),

74.60 (C-2^I), 73.17, 72.42, 72.00 (3 CH₂Ph), 70.67 (C-

5^IV), 68.52 (C-5^II), 68.44 (C-5^III), 67.85 (C-5^I), 67.15

(C-1⁰⁰), 67.03 (C-4^IV), 51.48 (COOCH₃), 33.93 (C-5⁰⁰),

29.11 (C-2⁰⁰), 25.73 (C-3⁰⁰), 24.70 (C-4⁰⁰), 18.50 (C-6^IV),

18.01 (2 C, C-6^II,III), 17.93 (C-6^I). ESI-MS: m/z

1430.6578 ([MꢁH+HCOOH]⁺). Anal. Calcd for

C₈₅H₉₀N₃O₁₈: C, 69.29; H, 6.91; N, 3.03. Found: C,

69.51; H, 7.16; N, 2.92.

3.29. 5-Methoxycarbonylpentyl 3-O-benzyl-4,6-dideoxy-

4-(3-hydroxy-3-methylbutyramido)-2-O-methyl-b-^D-

glucopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamnopyr-

anosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamnopyranosyl-

(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyranoside (34)

Azide 32 (560 mg) was treated with hydrogen sulﬁde as

described in the general procedure for azide to amine

conversions, to give 5-methoxycarbonylpentyl 4-ami-

no-3-O-benzyl-4,6-dideoxy-2-O-methyl-b-^D-glucopyr-

anosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamnopyranosyl-

(1 ! 3)-2,4-di-O-benzyl-a- ^L-rhamnopyranosyl-(1 ! 2)-

3,4-di-O-benzyl-a-^L-rhamnopyranoside (33, 490 mg,

1

88%). H NMR (CDCl₃, 600 MHz): d 5.12 (br s, 1H,

H-1^III), 5.08 (d, 1H, J_1,21.9 Hz, H-1^II), 5.01–4.45 (m,

16H, 7 CH₂Ph, incl, 4.70, d, partially overlapped,

J_1,2ꢀ 7.7 Hz, H-1^IV, ꢀ4.66, d, overlapped, H-1^I), 4.17

(dd, 1H, J_2,33.1, J_3,49.6 Hz, H-3^III), 4.13 (dd, 1H, J_2,3

3.0, J_3,49.6 Hz, H-3^II), 3.99 (dd, 1H, J_2,32.8 Hz, H-

2^I), 3.92 (dd, 1H, J_1,21.7, J_2,33.0 Hz, H-2^III), 3.82 (dd,

partially overlapped, H-3^I), 3.82–3.75 (m, 3H, H-

2^II,5^II,III), 3.65–3.58 (m, 9H, H-4^III,5^I,1⁰⁰a, incl., 3.65, s,

COOCH₃, OCH₃-2), 3.56 (bt, 1H, H-4^II), 3.40 (t, 1H,

J 9.5 Hz, H-4^I), 3.46, 3.33 (2t, J 6.4 Hz, H-1⁰⁰b), 3.16–

3.12 (m, 2H, H-3^IV,2^IV), 3.05 (m, 1H, H-5^IV), 2.48 (m,

1H, H-4^IV), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.66–1.61

(m, 2H, H-4⁰⁰a,b), 1.57–1.52 (m, 2H, H-2⁰⁰a,b), 1.37–

1.32 (m, 2H, H-3⁰⁰a,b), 1.27 (d, partially overlapped,

J_5,66.3 Hz, H-6^III), 1.26 (d, partially overlapped, J_5,6

6.2 Hz, H-6^I), 1.24 (d, partially overlapped, J_5,6

6.3 Hz, H-6^II), 1.06 (d, J_5,66.1 Hz, H-6^IV). ¹³C NMR

(CDCl₃, 150 MHz): d 103.81 (C-1^IV), 100.37 (br,

3.28. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-2-O-methyl-b-^D-glucopyranosyl-(1 ! 3)-2,4-di-

O-benzyl-a-^L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-

L-rhamnopyranosyl-(1 ! 2)-3,4-di-O-benzyl-a-L-

rha-mnopyranoside (32)

Methylation of alcohol 31 (600 mg, 0.43 mmol), as de-

scribed above for similar conversions, gave amorphous

32 (579 mg, 96%), [a]_D+2° (c 1). ¹H NMR (CDCl₃,

4300

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

C-1^III), 99.03 (br, C-1^II), 98.90 (C-1^I), 85.36 (C-3^IV), 84.11

(C-2^IV), 81.00 (C-4^III), 80.55 (2 C, C-4^I,4^II), 80.05 (C-3^I),

79.37 (C-2^III), 78.11 (C-3^III), 77.97 (C-2^II), 77.53 (C-3^II),

75.45, 75.01 (2 C), (3 CH₂Ph), 74.73 (C-2^I), 74.52,

73.31, 72.35, 72.11 (4 CH₂Ph), 72.53 (C-5^IV), 68.63 (C-

5^II), 68.37 (C-5^III), 67.86 (C-5^I), 67.14 (C-1⁰⁰), 60.43

(OCH₃), 58.14 (C-4^IV), 51.46 (COOCH₃), 33.94 (C-5⁰⁰),

29.12 (C-2⁰⁰), 25.74 (C-3⁰⁰), 24.71 (C-4⁰⁰), 18.03 (C-6^IV),

17.96, 17.92 (2 C) (C-6^I,II,III). ESI-MS: m/z 1396.6710

([M+Na]⁺) C₈₁H₉₉NO₁₈Na requires 1396.6760.

1.8 Hz, H-1^II), 4.88 (d, 1H, J_1,21.4 Hz, H-1^I), 4.74 (d,

1H, J_1,27.9 Hz, H-1^IV), 4.28 (dd, 1H, J_2,33.2 Hz, H-

2^III), 4.15 (dd, 1H, J_2,33.1 Hz, H-2^II), 3.99 (dd, 1H,

J_3,49.6 Hz, H-3^III), 3.92 (dd, 1H, J_2,33.3 Hz, H-2^I),

3.86 (m, partially overlapped, H-5^III), 3.84 (dd, partially

overlapped, H-3^I,II), 3.79 (m, 1H, H-5^II), 3.72–3.66 (m,

2H, H-5^I,1⁰⁰a), 3.69 (s, partially overlapped, COOCH₃),

3.63 (t, partially overlapped, H-4^IV), 3.63 (s, partially

overlapped, COOCH₃), 3.62 (t, partially overlapped,

H-4^III), 3.58–3.51 (m, H-5^IV,1⁰⁰b, incl., 3.54, t, H-4^II,

3.53, t, H-3^IV), 3.48 (t, 1H, H-4^I), 3.13 (dd, 1H, J_2,3

9.1 Hz, H-2^IV), 2.47, 2.44 (2d, 2H, J 13.8 Hz, H-2⁰a,b),

2.41 (t, 2H, J 7.4 Hz, H-5⁰⁰), 1.66–1.58 (m, 4H, H-

2⁰⁰a,b,4⁰⁰a,b), 1.43–1.34 (m, 2H, H-3⁰⁰a,b), 1.32 (d, par-

tially overlapped, H-6^III), 1.31, 1.30 (2 s, partially over-

lapped, 2 CH⁰₃), 1.29 (d, partially overlapped, H-6^I), 1.28

(d, 3H, J_5,66.3 Hz, H-6^II), 1.22 (d, 3H, J_5,66.1 Hz, H-

6^IV); ¹³C NMR (D₂O, 150 MHz): d 106.38 (C-1^IV),

104.83 (C-1^II*), 104.82 (C-1^III*), 101.00 (C-1^I), 85.99

(C-2^IV), 82.32 (C-3^III), 81.43 (C-2^I), 80.69 (C-3^II), 75.56

(C-3^IV), 74.89 (C-4^I), 74.01 (C-4^II), 73.84 (C-4^III), 73.49

(C-5^IV), 72.93 (C-3⁰), 72.78 (C-3^I), 72.55 (C-2^II), 72.53

(C-2^III), 72.06 (C-5^II), 72.03 (C-5^III), 71.51 (C-5^I),

70.53 (C-1⁰⁰), 62.77 (OCH₃-2), 59.29 (C-4^IV), 54.81

(COOCH₃), 51.64 (C-2⁰), 36.30 (C-5⁰⁰), 31.00, 30.82 (2

CH₃⁰), 30.80 (C-2⁰⁰), 27.63 (C-3⁰⁰), 26.72 (C-4⁰⁰), 19.77

(C-6^IV), 19.40 (C-6^II), 19.35 (C-6^I), 19.34 (C-6^III); ESI-

MS: m/z 866.4018 ([M+Na]⁺) C₃₇H₆₅NO₂₀Na requires

866.3998.

A stirred solution of the foregoing amine (430 mg,

0.31 mmol) and 3-hydroxy-3-methylbutyric acid

(55 mg, 0.65 mmol) in CH₂Cl₂(15 mL) was treated with

N-ethyldiisopropylamine (60 lL, 0.46 mmol) and

HATU (176 mg, 0.65 mmol). After 1 h the mixture

was concentrated, and chromatography of the residue

gave amorphous 34 (445 mg, 97%), [a]_Dꢁ26° (c 0.8).

¹H NMR (CDCl₃, 600 MHz): d 5.32 (d, 1H, J_4,NH

9.3 Hz, NH), 5.09 (br s, 1H, H-1^III), 5.08 (d, 1H, J_1,2

2 Hz, H-1^II), 5.01–4.43 (m, 16H, 7 CH₂Ph, incl.,

ꢀ4.66, d, overlapped, H-1^I, ꢀ4,62, d, partially over-

lapped, H-1^IV), 4.12 (dd, partially overlapped, J_2,33.0,

J_3,49.1 Hz, H-3^II), 4.11 (dd, partially overlapped, J_2,3

3.0, J_3,4ꢀ 9.6 Hz, H-3^III), 4.00 (dd, 1H, J_1,22.0, J_2,3

2.8 Hz, H-2^I), 3.91 (dd, 1H, J_1,21.8, J_2,33.0 Hz, H-

2^III), 3.83–3.75 (m, 4H, H-3^I,2^II,5^III,5^II), 3.68–3.58 (m,

10H, H-4^IV,4^III,5^I,1⁰⁰a, incl., 3.65, s, COOCH₃, and

3.64, s, OCH₃-2), 3.56–3.50 (m, 1H, H-4^II), 3.40 (t,

1H, J 9.6 Hz, H-4^I), 3.35, 3.33 (2t, partially overlapped,

H-1⁰⁰b), 3.30 (t, partially overlapped, J 9.7 Hz, H-3^IV),

3.19–3.15 (m, 2H, H-2^IV,5^IV), 2.31 (t, 2H, J 7.5 Hz, H-

5⁰⁰a,b), 2.19, 2.08 (2d, J 14.9 Hz, H-2⁰a,b), 1.66–1.61

(m, 4H, H-4⁰⁰a,b, 2⁰⁰a,b), 1.38–1.30 (m, 2H, H-3⁰⁰a,b),

1.28 (d, partially overlapped, J_5,66.3 Hz, H-6^III), 1.27

(d, partially overlapped, J_5,66.3 Hz, H-6^I), 1.22, 1.18

(2 s, 6H, 2 CH⁰₃), 1.03 (d, 3H, J_5,66.1 Hz, H-6^IV). ¹³C

NMR (CDCl₃, 125 MHz): d 103.69 (C-1^IV), 100.34 (C-

1^III), 98.94 (C-1^II), 98.86 (C-1^I), 84.91 (C-2^IV), 80.83

(C-4^III), 80.51 (C-4^I), 80.47 (C-4^II), 80.02 (C-3^I), 79.97

(C-3^IV), 79.15 (C-2^III), 78.71 (C-3^III), 77.91 (br, 2 C, C-

2^II,3^II), 74.66 (C-2^I), 75.45, 74.69, 74.51, 73.70, 73.37,

72.31, 72.04 (7 CH₂Ph), 70.85 (C-5^IV), 69.39 (C-3⁰),

68.56 (C-5^II), 68.31 (C-5^III), 67.83 (C-5^I), 67.12 (C-1⁰⁰),

60.55 (OCH₃-2), 55.52 (C-4^IV), 51.47 (COOCH₃),

47.70 (C-2⁰), 33.93 (C-5⁰⁰), 29.30, 29.28 (2 CH⁰₃), 29.10

(C-2⁰⁰), 25.72 (C-3⁰⁰), 24.69 (C-4⁰⁰), 18.08 (C-6^IV), 17.99

(C-6^II), 17.92, 17.90 (C-6^I,III). ESI-MS: m/z 1496.7297

([M+Na]⁺). Anal. Calcd for C₈₆H₁₀₇NO₂₀: C, 70.04;

H, 7.31; N, 0.95. Found: C, 70.00; H, 7.43; N, 1.03.

3.31. 5-Methoxycarbonylpentyl 4-azido-2-O-benzoyl-3-

O-benzyl-4,6-dideoxy-b-^D-glucopyranosyl-(1 ! 2)-(2,4-

di-O-benzoyl-a-^L-rhamnopyranosyl)-(1 ! 3)-(2,4-di-O-

benzyl-a-^L-rhamnopyranosyl)-(1 ! 2)-3,4-di-O-benzyl-

a-^L-rhamnopyranoside (36)

Glycosyl acceptor 10 (576 mg, 0.49 mol) and glycosyl

donor 22 were treated as described in the general proce-

dure, and chromatography gave ﬁrst the desired b-

linked substance 36 (480 mg, 66.3%), [a]_D+39° (c 0.9).

¹H NMR (CDCl₃, 600 MHz): 5.58 (dd, 1H, J_1,21.9,

J_2,33.3 Hz, H-2^III), 5.39 (t, 1H, J 9.7 Hz, H-4^III), 5.31

(d, 1H, J_1,21.9 Hz, H-1^III), 5.14 (d, 1H, J_1,21.8 Hz,

H-1^II), 5.08 (dd, 1H, J_1,27.8, J_2,39.3 Hz, H-2^IV), 5.05–

4.29 (5 d, partially overlapped, 5 CH₂Ph), 4.66 (d, par-

tially overlapped, J_1,21.8 Hz, H-1^I), 4.56 (d, partially

overlapped, H-1^IV), 4.37 (dd, 1H, J_2,33.4, J_3,49.5 Hz,

H-3^III), 4.18 (dd, 1H, J_2,33.1, J_3,49.3 Hz, H-3^II), 4.03–

3.98 (m, 2H, H-2^I,5^III), 3.85 (dd, 1H, J_2,33.1 Hz,

H-2^II), 3.83–3.79 (m, 2H, H-3^I, 5^II), 3.64 (t, partially

overlapped, J 9.4 Hz, H-4^II), 3.65 (s, partially over-

lapped, COOCH₃), 3.64–3.55 (m, 3H, H-5^I, 1⁰⁰a), 3.42

(t, 1H, J 9.2 Hz, H-3^IV), 3.37 (t, 1H, J 9.5 Hz, H-4^I)

3.34, 3.32 (2t, 1H, J 6.5 Hz, H-1⁰⁰b), 3.13–3.06 (m, 2H,

H-5^IV,4^IV), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.65–1.60

(m, 2H, H-4⁰⁰a,b), 1.55–1.52 (m, 2H, H-2⁰⁰a,b), 1.37–

1.31 (m, partially overlapped, H-3⁰⁰a,b), 1.31 (d, partially

overlapped, J_5,66.2 Hz, H-6^II), 1.22 (d, 3H, J_5,66.3 Hz,

H-6^I), 1.09 (d, 3H, J_5,65.7 Hz, H-6^IV), 1.06 (d, 3H, J_5,6

6.3 Hz, H-6^III); ¹³C NMR (CDCl₃): d 100.76 (C-1^IV),

99.01 (br, C-1^III), 98.77 (C-1^I), 98.38 (C-1^II), 80.89

(C-3^IV), 80.39 (C-4^I), 80.21 (C-4^II), 80.12 (C-3^I), 78.97

(C-3^II), 78.11 (C-2^II), 75.38, 75.32 (2 CH₂Ph), 75.04

3.30. 5-Methoxycarbonylpentyl 4,6-dideoxy-4-(3-hydro-

xy-3-methylbutyramido)-2-O-methyl-b-^D-glucopyranosyl-

(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 3)-a-L-rhamnopyr-

anosyl-(1 ! 2)-a-^L-rhamnopyranoside (35)

Compound 34 (450 mg) was treated with hydrogen in

the presence of palladium-on charcoal catalyst

(150 mg), as described above for similar conversion, to

give, after freeze-drying, the title tetrasaccharide as a

white solid, (0.211 g, 83%); [a]_Dꢁ52.6° (c 0.5, H₂O);

1

[a]_Dꢁ67° (c 0.6, MeOH). H NMR (D₂O, 600 MHz):

d 5.04 (d, 1H, J_1,21.0 Hz, H-1^III), 4.94 (d, 1H, J_1,2

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4301

(C-2^I), 75.00 (C-3^III) 74.45 (CH₂Ph), 73.54 (C-2^IV), 72.98

(C-4^III), 72.47 (CH₂Ph), 72.25 (C-2^III), 71.59 (CH₂Ph),

70.67 (C-5^IV), 68.41 (C-5^II), 67.82 (C-5^I), 67.06 (C-1⁰⁰),

67.03 (C-4^IV), 66.79 (C-5^III), 51.43 (COOCH₃), 33.91

(C-5⁰⁰), 28.09 (C-2⁰⁰), 25.71 (C-3⁰⁰), 24.68 (C-4⁰⁰), 18.12

(C-6^II), 17.99 (C-6^I), 17.83 (C-6^IV), 17.51 (C-6^III); ESI-

MS: m/z 1540.6309 ([M+Na]⁺). Anal. Calcd for

C₈₇H₉₅N₃O₂₁: C, 68.80; H, 6.31; N, 2.77. Found: C,

68.76; H, 6.47; N, 2.74.

of the starting azide and showed the characteristic up-

ﬁeld shift of the signal for H-4^IV(d 2.56 vs 3.07 in 36).

The same could be concluded from the ¹³C NMR spec-

trum (CDCl₃, 75 MHz) showing upﬁeld shift of the sig-

nal for C-4^IV(d 57.38 vs 67.03), ESI-MS: m/z 1492.6567

([M+1]⁺) C₈₇H₉₈NO₂₁requires 1492.6631.

The foregoing amine (109 mg, 0.073 mmol) was pro-

cessed according to the general procedure for amidation

to give, after chromatography, 5-methoxycarbonylpen-

tyl 2-O-benzoyl-3-O-benzyl-4,6-dideoxy-4-(3-hydroxy-

3-methylbutyramido)-b-^D-glucopyranosyl-(1 ! 2)-(2,4-di-

O-benzoyl-a-^L-rhamnopyranosyl)-(1 ! 3)-(2,4-di-O-ben-

zyl-a-^L-rhamnopyranosyl)-(1 ! 2)-3,4-di-O-benzyl-a-L-

rhamnopyranoside (38, 90 mg, 78%). ¹H NMR (CDCl₃,

600 MHz): d 5.83 (d, 1H, J_4,NH8.3 Hz, NH), 5.62 (dd,

1H, J_1,22.0, J_3,43.3 Hz, H-2^III), 5.42 (t, 1H, J 9.5 Hz,

H-4^III), 5.33 (d, 1H J_1,21.9 Hz, H-1^III), 5.16–5.13 (m,

2H, H-1^II,2^IV), 5.07–4.30 (5 d, partially overlapped, 5

CH₂Ph), 4.67 (d, partially overlapped, J_1,27.7 Hz, H-

1^IV), 4.66 (d, partially overlapped, J_1,2ꢀ 2.0 Hz, H-1^I),

4.39 (dd, partially overlapped, J_2,33.5, J_3,49.6 Hz, H-

3^III), 4.18 (dd, 1H, J_2,33.1, J_3,49.2 Hz, H-3^II), 4.00 (m,

2H, H-2^I,5^III), 3.87 (t, partially overlapped, J 9.9 Hz,

H-3^IV), 3.86 (dd, partially overlapped, H-2^II), 3.83–

3.80 (m, 2H, H-3^I,5^II), 3.65 (t, partially overlapped, H-

4^II), 3.64 (s, partially overlapped, COOCH₃), 3.63–3.57

(m, 3H, H-5^I, 5^IV,1⁰⁰a), 3.38-3.32 (m, 3H, H-4^I,4^II,1⁰b),

2.30 (t, 2H,₀J 7.3 Hz, H-5⁰⁰a,b), 2.11 (dd, 2H, J

15.1 Hz, H-2 ), 1.66–1.60 (m, 2H, H-4⁰⁰a,b), 1.57–1.52

(m, 2H, H-2⁰⁰a,b), 1.37–1.32 (m, partially overlapped,

2H, H-3⁰⁰a,b), 1.31 (d, partially overlapped, J_5,66.2 Hz,

H-6^II), 1.22 (d, partially overlapped, J_5,66.3 Hz, H-6^I),

1.16, 1.14 (2 CH⁰₃), 1.06 (d, partially overlapped, J_5,6

6.3 Hz, H-6^III), 0.99 (d, J_5,66.4 Hz, H-6^IV). ¹³C NMR

(CDCl₃, 150 MHz): d 100.99 (C-1^IV), 99.09 (br, C-1^III),

98.76 (C-1^I), 98.44 (br, C-1^II), 80.39 (C-4^I), 80.29

(C-4^II), 80.10 (C-3^I), 78.91 (C-3^II), 78.12 (C-2^II), 77.97

(C-3^IV), 75.42, 75.33 (2 CH₂Ph), 75.22 (C-3^III), 75.03

(C-2^I), 73.43 (C-2^IV), 72.95 (C-4^III), 72.45 (CH₂Ph),

72.31 (C-2^III), 71.86, 71.61 (2 CH₂Ph), 70.05 (C-5^IV),

69.43 (C-3⁰), 68.42 (C-5^II), 67.82 (C-5^I), 67.10 (C-1⁰⁰),

66.82 (C-5^III), 56.38 (C-4^IV), 51.44 (COOCH₃), 47.70

(C-2⁰), 33.92 (C-5⁰⁰), 29.30, 29.26 (2 CH⁰₃), 29.09 (C-2⁰⁰),

25.71 (C-3⁰⁰), 24.69 (C-4⁰⁰), 18.11 (C-6^IV), 17.83 (C-6^I),

17.63 (C-6^III), 17.50 (C-6^IV). ESI-MS: m/z 1614.6956

([M+Na]⁺) C₉₂H₁₀₅NO₂₃Na requires 1614.6975.

Eluted next was a small amount of material whose

NMR data (CDCl₃, 600 MHz) showed that it was the

a-anomer, 5-methoxycarbonylpentyl 4-azido-2-O-ben-

zoyl-3-O-benzyl-4,6-dideoxy-b-^D-glucopyranosyl-(1 ! 2)-

(2,4-di-O-benzoyl-a-^L-rhamnopyranosyl)-(1 ! 3)-(2,4-di-

O-benzyl-a-^L-rhamnopyranosyl)-(1 ! 2)-3,4-di-O-benzyl-

a-^L-rhamnopyranoside (89): ¹H NMR (CDCl₃,

600 MHz): d 5.55 (dd, 1H, J_1,21.8, J_2,33.0 Hz, H-2^III),

5.45 (t, 1H, J 9.8 Hz, H-4^III), 5.28 (d, 1H, J_1,23.6 Hz,

H-1^IV), 5.15 (d, 1H, J_1,21.6 Hz, H-1^III), 5.04 (d, 1H,

J_1,21.7 Hz, H-1^II), 4.89 (dd, 1H, J_2,310.0 Hz, H-2^IV),

4.76–4.33 (5 d, partially overlapped, 5 CH₂Ph), 4.57

(d, partially overlapped, J_1,21.8 Hz, H-1^I), 4.32 (dd,

J_2,33.2, J_3,49.9 Hz, H-3^III), 4.07 (dd, 1H, J_2,33.1, J_3,4

9.3 Hz, H-3^II), 4.03–3.96 (m, 1H, H-5^III), 3.92 (dd, 1H,

J_2,33.0 Hz, H-2^I), 3.76–3.74 (m, 2H, H- 2^II,3^I), 3.71–

3.66 (m, H, H-5^II), 3.61 (t, J 9.7 Hz, H-3^IV), 3.57 (s,

3H, COOCH₃), 3.54–3.49 (m, 3H, H-5^I,4^II,1⁰⁰a), 3.45–

3.40 (m, 1H, H-5^IV), 3.27 (t, partially overlapped, J

9.5 Hz, H-4^I), 3.23 (m, partially overlapped, H-1⁰⁰b),

2.92 (dd, 1H, J_4,510.1 Hz, H-4^IV), 2.23 (t, 2H,

J

7.5 Hz, H-5⁰⁰a,b), 1.58–1.52 (m, 2H, H-4⁰⁰a,b),

1.49–1.46 (m, 2H, H-2⁰⁰a,b), 1.30–1.24 (m, partially over-

lapped, H-3⁰⁰a,b), 1.21 (d, partially overlapped, J_5,6

6.2 Hz, H-6^II), 1.22 (d, 6H, J_5,66.2 Hz, H-6^I,III), 0.90

(d, 3H, J_5,66.2 Hz, H-6^IV); ¹³C NMR (CDCl₃,

150 MHz): d 99.27 (br, C-1^III), 98.77 (C-1^I), 98.68 (C-

1^II), 93.72 (C-1^IV), 80.39 (C-4^I), 80.35 (C-4^II), 80.05

(C-3^I), 78.30 (C-3^II), 77.96 (C-2^II), 77.62 (C-3^IV), 75.47,

75.34, 75.24 (3 CH₂Ph), 74.96 (C-2^I), 73.52 (C-2^IV),

72.64 (C-4^III), 72.39 (CH₂Ph), 72.17 (C-3^III), 71.89

(CH₂Ph), 68.73 (C-2^III), 68.57 (C-5^II), 67.78 (C-5^I),

67.33 (C-4^IV), 67.15 (C-1⁰⁰), 67.08 (C-5^III), 66.51

(C-5^IV), 51.46 (COOCH₃), 33.94 (C-5⁰⁰), 29.11 (C-2⁰⁰),

25.74 (C-3⁰⁰), 24.71 (C-4⁰⁰), 18.08 (C-6^II), 17.99 (C-6^IV),

17.88, 17.60 (C-6^I,III); ESI-MS: m/z 1535.6843

([M+NH₄]⁺) C₈₇H₉₆N₄O₂₁requires 1535.6802.

3.32. 5-Methoxycarbonylpentyl 4,6-dideoxy-4-(3-hydro-

xy-3-methylbutyramido)-b-^D-glucopyranosyl-(1 ! 2)-

(a-^L-rhamnopyranosyl)-(1 ! 3)-(a-L-rhamnopyranosyl)-

(1 ! 2)-a-^L-rhamnopyranoside (40)

The foregoing tetrasaccharide 38 (90 mg) was debenzoy-

lated, to give alcohol 39 (50 mg, 92%), ESI-MS:

m/z 1302.6256 ([M+Na]⁺) C₇₁H₉₃NO₂₀Na requires

1302.6341.

Azide 36 (290 mg) was converted to the corresponding

amine, 5-methoxycarbonylpentyl 4-amino-2-O-benzoyl-

3-O-benzyl-4,6-dideoxy-b-^D-glucopyranosyl-(1 ! 2)-

(2,4-di-O-benzoyl-a-^L-rhamnopyranosyl)-(1 ! 3)-(2,4-

di-O-benzyl-a-^L-rhamnopyranosyl)-(1 ! 2)-3,4-di-O-

benzyl-a-^L-rhamnopyranoside (37, 109 mg, 55%) as

described in the general procedure; some unchanged

starting azide was recovered. The intended conversion

was conﬁrmed by the ¹H NMR spectrum (CDCl₃,

300 MHz) of the product, which was very similar to that

Debenzylation of 39, as described above for similar con-

versions, gave, after freeze-drying, the title tetrasaccha-

ride 40 as a white solid (23 mg, 72%), [a]_Dꢁ63° (c 0.5,

1.7 Hz, H-1^III), 4.93 (d, 1H, J_1,21.9 Hz, H-1^II), 4.88

(d, 1H, J_1,21.6 Hz, H-1^I), 4.70 (d, 1H, J_1,27.8 Hz, H-

1^IV), 4.28 (dd, 1H, J_2,33.3 Hz, H-2^III), 4.15 (dd, 1H,

J_2,33.2 Hz, H-2^II), 4.01 (dd, 1H, J_3,49.6 Hz, H-3^III),

3.91 (dd, 1H, J_2,33.3 Hz, H-2^I), 3.88 (m, H-5^III), 3.84

(dd, 2H, J_3,49.7 Hz, superimposed, H-3^I,II), 3.79 (m,

1

H₂O). H NMR (D₂O, 600 MHz): d 5.04 (d, 1H, J_1,2

4302

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

1H, H-5^II), 3.72–3.66 (m, 5H, H-5^I,1⁰⁰a, incl., s, 3.69,

COOCH₃), 3.64 (t, partially overlapped, H-4^IV), 3.63

(t, partially overlapped, H-4^III), 3.60 (m, partially over-

lapped, H-5^IV), 3.54 (m, partially overlapped, H-1⁰⁰b),

3.54 (t, partially overlapped, H-4^II), 3.52 (t, partially

overlapped, H-3^IV), 3.48 (t, 1H, H-4^I), 3.41 (dd, 1H,

J_2,39.0 Hz, H-2^IV), 2.48, 2.45 (2 d, 2H, J 13.8 Hz, H-

2⁰a,b), 2.41 (t, 2H, J 7.4 Hz, H-5⁰⁰), 1.66–1.56 (m, 4H,

H-2⁰⁰a,b, 4⁰⁰a,b), 1.44–1.33 (m, 2H, H-3⁰⁰), 1.31 (d, par-

tially overlapped, J_5,66.3 Hz, H-6^III), 1.31, 1.30 (2 s,

partially overlapped, 2 CH⁰₃), 1.29 (d, partially over-

lapped, H-6^I), 1.28 (d, 3H, J_5,66.3 Hz, H-6^II), 1.23 (d,

3H, J_5,66.0 Hz, H-6^IV); ¹³C NMR (D₂O, 150 MHz): d

106.27 (J_C-1,H-1162 Hz, C-1^IV), 104.86, 104.85 (C-1^II,

C-1^III), 101.04 (C-1^I), 82.36 (C-3^III), 81.46 (C-2^I), 80.75

(C-3^II), 76.82 (C-2^IV), 76.15 (C-3^IV), 74.95 (C-4^I), 74.05

(C-4^II), 73.99 (C-4^III), 73.75 (C-5^IV), 72.97 (C-3⁰), 72.84

(C-3^I), 72.59 (C-2^II), 72.56 (C-2^III), 72.10 (C-5^II), 71.77

(C-5^III), 71.56 (C-5^I), 70.59 (C-1⁰⁰), 59.33 (C-4^IV), 54.86

(COOCH₃), 51.66 (C-2⁰), 36.35 (C-5⁰⁰), 31.05, 30.88 (2

CH₃⁰), 30.83 (C-2⁰⁰), 27.67 (C-3⁰⁰), 26.75 (C-4⁰⁰), 19.86

(C-6^IV), 19.44 (C-6^II), 19.42 (C-6^I), 19.38 (C-6^III); ESI-

MS: m/z 852.3842 ([M+Na]⁺) C₃₆H₆₃NO₂₀requires

852.3841.

(COOCH₃), 34.67 (C-5⁰⁰), 30.26 (C-2⁰⁰), 26.88 (C-3⁰⁰),

25.76 (C-4⁰⁰), 18.02 (C-6). ESI-MS: m/z 315.1

([M+Na]⁺). Anal. Calcd for C₁₃H₂₄O₇: C, 53.41; H,

8.28. Found: C, 53.43; H, 8.31.

3.35. 5-Methoxycarbonylpentyl (2,3,4-tri-O-benzoyl-a-^L-

rhamnopyranosyl)-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamno-

pyranoside (43)

Compound 5 (380 mg, 0.8 mmol) was treated with 2,3,4-

tri-O-benzoyl-a-^L-rhamnopyranosyl bromide,²⁸as de-

scribed for preparation of 41. After processing and chro-

matography, disaccharide 43 (500 mg, 67%) had [a]_D

+9.8° (c 0.4). ¹H NMR (CDCl₃, 300 MHz): d 5.91–

5.86 (m, 2H, H-2^II,3^II), 5.68–5.61 (m, 1H, H-4^II), 5.22

(d, 1H, H-1^II), 4.97–4.67 (m, 5H, 2 CH₂Ph, incl., 4.81,

d, J_1,21.6 Hz, H-1^I), 4.34–4.25 (m, 1H, H-5^II), 4.03

(bt, 1H, H-2^I), 3.90 (dd, 1H, J_2,33.8, J_3,48.8 Hz, H-

3^I), 3.78–3.60 (m, 6H, H-4^I,5,1⁰a, incl., 3.66, s,

COOCH₃), 3.42, 3.86 (2 t, 1H, J 6.5 Hz, H-1⁰a), 2.32

(t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.71–1.55 (m, 4H, H-

4⁰⁰a,b,2⁰⁰a,b), 1.44–1.34 (m, partially overlapped, H-

3⁰⁰a,b), 1.39 (d, partially overlapped, J_5,65.9 Hz, H-6^I),

1.35 (d, partially overlapped, J_5,66.2 Hz, H-6^II); ¹³C

NMR (CDCl₃, 75 MHz): d 99.35 (C-1^II), 98.76 (C-1^I),

80.08 (C-4^I), 79.69 (C-3^I), 76.15 (C-2^I), 75.48, 72.42 (2

CH₂Ph), 71.80 (C-4^II), 70.52 (C-3^II), 69.88 (C-2^II),

68.08 (C-5^I), 67.23 (C-1⁰⁰), 66.99 (C-5^II), 51.39

(COOCH₃), 33.85 (C-5⁰⁰), 29.05 (C-2⁰⁰), 25.66 (C-3⁰⁰),

24.63 (C-4⁰⁰), 17.88 (C-6^I), 17.61 (C-6^II). ESI-MS: m/z

953.3739 ([M+Na]⁺). Anal. Calcd for C₅₄H₅₈O₁₄: C,

69.66; H, 6.28; N,. Found: C, 69.56; H, 6.34.

3.33. 5-Methoxycarbonylpentyl 2,3,4-tri-O-benzoyl-a-^L-

rhamnopyranoside (41)

Methyl 6-hydroxyhexanoate²²(0.64 g, 4.4 mmol) and

2,3,4-tri-O-benzoyl-a-^L-rhamnopyranosyl

bromide²⁸

(2 g, 3.6 mmol) were condensed as described above for

similar conversions, to give the amorphous glycoside

1

41 (1.88 g, 84%), [a]_D+121° (c 0.9). H NMR (CDCl₃,

300 MHz): d 5.83 (dd, 1H, J_2,33.2, J_3,410.1 Hz, H-3),

5.67 (t, partially overlapped, J 9.8 Hz, H-4), 5.65 (dd,

partially overlapped, J_1,21.8, J_2,33.6 Hz, H-2), 4.99

(d, 1H, H-1), 4.23–4.13 (m, 1H, H-5), 3.81, 3.79 (2 t,

1H, J 6.5 Hz, H-1⁰a), 3.69 (s, 3H, COOCH₃), 3.56,

3.52 (2 t, 1H, H-1⁰b), 2.38 (t, 2H, J 7.5 Hz, H-5⁰⁰),

1.77–1.66 (m, 4H, H-4⁰⁰a,b,2⁰⁰a,b), 1.53–1.43 (m, 2H,

H-3⁰⁰a,b), 1.36 (d, 3H, J_5,66.1 Hz, H-6). ¹³C NMR

(CDCl₃, 75 MHz): d 97.54 (C-1), 71.89 (C-4), 70.95 (C-

3), 70.05 (C-2), 68.11 (C-1⁰⁰), 66.63 (C-5), 51.48

(COOCH₃), 33.94 (C-5⁰⁰), 29.09 (C-2⁰⁰), 25.70 (C-3⁰⁰),

24.69 (C-4), 17.67 (C-6). ESI-MS: m/z 627.2211

([M+Na]⁺). Anal. Calcd for C₃₄H₃₆O₁₀: C, 67.54; H,

6.00. Found: C, 67.80; H, 5.94.

3.36. 5-Methoxycarbonylpentyl (2,3,4-tri-O-benzoyl-a-^L-

rhamnopyranosyl)-(1 ! 2)-a-^L-rhamnopyranoside (44)

Hydrogenolysis of the fully protected disaccharide 43

(450 mg) gave amorphous 44 (340 mg, 94%), [a]_D

1

+73° (c 0.6). H NMR (CDCl₃): d 5.86 (dd, 1H, J_1,2

1.9, J_2,33.3 Hz, H-2^II), 5.77 (dd, 1H, J_3,410.0 Hz, H-

3^II), 5.69 (t, 1H, H-4^II), 5.24 (d, 1H, H-1^II), 4.94 (d,

1H, J_1,21.7 Hz, H-1^I), 4.43–4.34 (m, 1H, H-5^II), 4.99

(dd, 1H, J_2,33.3 Hz, H-2^I), 3.92 (bdd, 1H, J_3,4

9.4 Hz, H-3^I), 3.74–3.64 (m, 6H, H-4^I,5^I,1⁰⁰a, incl.,

3.67, s, COOCH₃), 3.43, 3.40 (2 t, 1H, J 6.3 Hz, H-

1⁰⁰b), 2.33 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.71–1.56 (m,

4H, H-4⁰⁰a,b,2⁰⁰a,b), 1.45–1.34 (m, 8H, H-3⁰⁰a,b, incl.,

1.40, d, J_5,65.7 Hz, 6^I, incl., 1.35, d, J_5,61.35 Hz,

6^II). ¹³C NMR (CDCl₃): d 99.88 (C-1^II), 98.50 (C-1^I),

80.83 (C-2^I), 73.23 (C-4^I), 71.43 (C-4^II), 71.34 (C-3^I),

70.41, 70.38 (C-2^II,3^II), 68.27 (C-5^I), 67.29 (C-1⁰⁰),

67.05 (C-5^II), 51.38 (COOCH₃), 33.81 (C-5⁰⁰), 29.00

(C-2⁰⁰), 25.62 (C-3⁰⁰), 24.56 (C-4⁰⁰), 17.72, 17.55 (C-

3.34. 5-Methoxycarbonylpentyl a-^L-rhamnopyranoside (42)

Conventional debenzoylation of 41 gave the title rham-

noside 42 in virtually theoretical yield, mp 62–63 °C

1

(from EtOAc), [a]_Dꢁ58.6° (c 1.1). H NMR (CD₃OD,

600 MHz): d 4.65 (d, 1H, J_1,21.7 Hz, H-1), 3.78 (dd,

1H, J_2,33.4 Hz, H-2), 3.68, 3.65 (2 t, partially over-

lapped, J 6.5 Hz, H-1⁰a), 3.66 (s, partially overlapped,

COOCH₃), 3.63 (dd, 1H, J_3,49.5 Hz, H-3), 3.59–3.54

(m, 1H, H-5), 1.41, 1.38 (2, t, 1H, H-1⁰b), 3.36 (t, 1H,

J 9.5 Hz, H-4), 2.34 (t, 2H, J 7.6 Hz, H-5⁰⁰a,b), 1.66–

1.57 (m, 4H, H-4⁰⁰a,b,2⁰⁰a,b), 1.44–1.37 (m, 2H, H-

3⁰⁰a,b), 1.26 (d, 3H, J_5,66.3 Hz, H-6); ¹³C NMR

(CD₃OD, 150 MHz): d 101.58 (C-1), 73.93 (C-4), 72.41

(C-3), 72.28 (C-2), 69.71 (C-5), 68.23 (C-1⁰⁰), 51.99

6

^I,II). ESI-MS: m/z 773.2749 ([M+Na]⁺). Anal. Calcd

for C₄₀H₄₆O₁₄: C, 63.99; H, 6.18. Found: C, 63.85;

H, 6.23.

3.37. 5-Methoxycarbonylpentyl a-^L-rhamnopyranosyl-

(1 ! 2)-a-^L-rhamnopyranoside (45)

´

Debenzoylation (Zemplen) of 44 (280 mg), as described

above, gave the amorphous disaccharide glycoside 45

(157 mg, 97%), [a]_Dꢁ31° (c 0.5). ¹H NMR (D₂O,

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4303

600 MHz): d 4.94 (d, 1H, J_1,21.8 Hz, H-1^II), 4.87 (d, 1H,

J_1,21.6 Hz, H-1^I), 4.06 (dd, 1H, J_2,33.3 Hz, H-2^II), 3.89

(dd, 1H, J_2,33.5 Hz, H-2^I), 3.82 (dd, 1H, J_3,49.7 Hz, H-

3^I), 3.78 (dd, 1H, J_3,49.8 Hz, H-3^II), ꢀ3.74 (m, partially

overlapped, H-5^II), ꢀ3.70 (m, partially overlapped, H-

1⁰⁰a), 3.69 (s, partially overlapped, COOCH₃), ꢀ3.68

(m, partially overlapped, H-5^I), 3.53, 3.52 (2 t, 1H,

H-1⁰⁰a), 3.46, 3.44 (2 t, 2H, J ꢀ 9.6 Hz, H-4^I,IIin that

order), 2.40 (t, 1 H, J 7.4 Hz, H-5⁰⁰a,b), 1.65–1.56 (m,

4H, H-4⁰⁰a,b,2⁰⁰a,b), 1.43–1.34 (m, 2H, H-3⁰⁰a,b), 1.28

(d, 3H, J_5,66.3 Hz, H-6^I), 1.26 (d, 3H, J_5,66.3 Hz, H-

6^II); ¹³C NMR (CDCl₃, 150 MHz): d 105.06 (J_C-1,H-1

170.4 Hz, C-1^II), 101.08 (J_C-1,H-1171.0 Hz, C-1^I), 81.50

(C-2^I), 74.88 (C-4^I), 74.70 (C-4^II), 72.82 (C-3^I), 72.79

(C-3^II), 72.77 (C-2^II), 71.84 (C-5^II), 71.46 (C-5^I), 70.52

(C-1⁰⁰), 54.84 (COOCH₃), 36.33 (C-5⁰⁰), 30.85 (C-2⁰⁰),

27.68 (C-3⁰⁰), 26.76 (C-4⁰⁰), 19.40 (C-6^II), 19.37 (C-6^I).

ESI-MS: m/z 461.2007 ([M+Na]⁺) C₁₉H₃₄NO₁₁Na

requires 461.1999.

150 MHz): d 122.64 (C_quart, orthobenzoate), 99.53

(C-1^II), 97.35 (C-1^I), 78.47 (C-3^I), 78.01 (C-2^I), 71.84

(C-4^I), 71.30 (C-4^II), 70.49 (C-3^II), 70.26 (C-2^II), 69.94

(C-5^I), 67.42 (C-5^II), 63.60 (C-1⁰⁰), 51.34 (COOCH₃),

33.71 (C-5⁰⁰), 29.10 (C-2⁰⁰), 25.53 (C-3⁰⁰), 24.90 (CH₂Br),

24.48 (C-4⁰⁰), 17.76 (C-6^I), 17.30 (C-6^II); TOF-MS: m/z

997.1 ([M+Na]⁺) C₄₉H₅₁BrO₁₆Na requires 997.2.

3.39. 5-Methoxycarbonylpentyl a-^L-rhamnopyranosyl-

(1 ! 3)-a-^L-rhamnopyranoside (48)

Deacylation of 47 (248 mg) gave, after removal of

methyl benzoate by chromatography and freeze-drying,

disaccharide 48 (57 mg, 52%), [a]_Dꢁ62° (c 0.5). ¹H

NMR (D₂O, 600 MHz): d 5.02 (d, 1H, J_1,21.6 Hz, H-

1^II), 4.74 (d, 1H, J_1,21.7 Hz, H-1^I), 4.05 (dd, 1H, J_2,3

3.4 Hz, H-2^II), 3.99 (dd, 1H, J_2,33.4 Hz, H-2^I), 3.83

(dd, partially overlapped, J_3,49.8 Hz, H-3^II), ꢀ3.80 (m,

partially overlapped, H-5^II), 3.78 (dd, partially over-

lapped, J_3,49.8 Hz, H-3^I), 3.74–3.68 (m, 5H, H-5^I, 1⁰⁰a,

incl., 3.68, s, COOCH₃), 3.54–3.50 (m, 2H, H-4^I,1⁰⁰b),

3.45 (t, 1H, J 9.7 Hz, H-4^II), 2.40 (t, 2H, J 7.4 Hz, H-

5⁰⁰), 1.66–1.58 (m, 4H, H-2⁰⁰a,b,4⁰⁰a,b), 1.42–1.35 (m,

2H, H-3⁰⁰a,b), 1.28 (dd, partially overlapped, J_5,6

6.3 Hz, H-6^I), 1.27 (d, partially overlapped, J_5,6

6.3 Hz, H-6^II); ¹³C NMR (H₂O, 150 MHz): d 105.10

(J_C-1,H-1170. 7 Hz, C-1^II), 102.42 (J_C-1,H-1170. 7 Hz,

C-1^I), 80.89 (C-3^I), 74.78 (C-4^II), 74.18 (C-4^I), 72.91

(C-2^II), 72.87 (C-3^II), 72.71 (C-2^I), 71.81 (C-5^II), 71.50

(C-5^I), 70.46 (C-1⁰⁰), 54.87 (COOCH₃), 36.39 (C-5⁰⁰),

30.90 (C-2⁰⁰), 27.73 (C-3⁰⁰), 26.79 (C-4⁰⁰), 19.37, 19.36

3.38. 5-Methoxycarbonylpentyl 2,4-di-O-benzoyl-3-O-

bromoacetyl-a-^L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-

benzoyl-a-^L-rhamnopyranoside (47)

Glycosyl chloride¹⁸46 (500 mg, 0.58 mmol) was treated

with methyl 6-hydroxyhexanoate²²(130 mg, mmol), as

described for the preparation of 41. After processing,

chromatography gave ﬁrst the desired glycoside 47

(248 mg, 44%), [a]_D+99° (c 0.7). ¹H NMR (CDCl₃,

300 MHz): 5.56 (t, 1H, J 9.7 Hz, H-4^I), 5.48 (dd, 1H,

J_1,21.8, J_2,33.4 Hz, H-2^I), 5.41 (dd, 1H, J_2,32.7, J_3,4

10.0 Hz, H-3^II), 5.34 (t, partially overlapped, H-4^II),

5.16–5.15 (m, 2H, H-1^II,2^II), 4.98 (d, 1H, H-1^I), 4.45

(dd, 1H, J_3,49.9 Hz, H-3^I), 4.10–4.01 (m, 2H, H-5^I,^II),

3.76, 3.73 (2 t, 1H, J 6.5 Hz, H-1⁰a), 3.66 (s, 3H,

COOCH₃), 3.54–3.42 (m, partially overlapped, H-1⁰⁰b),

3.45, 3.50 (2 d, partially overlapped, J 12.2 Hz, CH₂Br),

2.36 (t, J 7.5 Hz, H-5⁰⁰a,b), 1.75–1.63 (m, 4H, H-4⁰⁰a,b,

2⁰⁰a,b, in that order), 1.49–1.39 (m, 2H, H-3⁰⁰), 1.34 (d,

3H, J_5,66.3 Hz, H-6^I), 1.14 (d, 3H, J_5,66.3 Hz, H-6^II);

¹³C NMR (CDCl₃, 75 Hz): d 99.13 (J_C-1,H-1172 Hz,

C-1^II), 97.25 (J_C-1,H-1172 Hz, C-1^I), 76.27 (C-3^I), 73.17

(C-4^I), 72.33 (C-2^I), 71.21 (C-3^II), 70.41 (C-4^II), 71.17

(C-2^II), 60.00 (C-1⁰⁰), 67.39 (C-5^II), 66.69 (C-5^I), 51.41

(COOCH₃), 33.88 (C-5⁰⁰), 29.05 (C-4⁰⁰), 25.69

(C-3⁰⁰), 24.88 (CH₂Br), 24.65 (C-2⁰⁰), 17.70 (C-6^I), 17.28

(C-6^II); ESI-MS: m/z 997.1 ([M+Na]⁺). Anal. Calcd

for C₄₉H₅₁BrO₁₆: C, 60.31; H, 5.27. Found: C, 60.24; H,

5.28.

(C-6^I,II);

C₁₉H₃₄O₁₁Na requires 461.1999.

ESI-MS:

m/z

461.1972

([M+Na]⁺)

3.40. 5-Methoxycarbonylpentyl 4-azido-2-O-benzoyl-3-

O-benzyl-4,6-dideoxy-b-^D-glucopyranoside (49)

Thioglycoside 22 (400 mg, 0.85 mmol) was treated with

methyl 6-hydroxynexanoate (190 mg, 1.1 mmol) as de-

scribed for the preparation of 4. After work-up, chroma-

tography gave ﬁrst 5-methoxycarbonylpentyl 4-azido-2-

O-benzoyl-3-O-benzyl-4,6-dideoxy-a-^D-glucopyranoside

(91, 21 mg, 3.5%), [a]_D+254° (c 0.5); ¹H NMR (CDCl₃,

600 MHz): d 5.08–5.05 (m, 2H, H-1,2), 4.83, 4.80 (2 d,

2H, J 10.7 Hz, CH₂Ph), 4.06–4.02 (m, 1H, H-3), 3.69–

3.64 (m, 5H, H-5,1⁰⁰a, incl., 3.64, s, COOMe), 3.37,

3.35 (2 t, 1H, J 6.4 Hz, H-1⁰⁰b), 3.23 (t, 1H, J 9.8 Hz,

H-4), 2.16 (t, 2 H, J 7.6 Hz, H-5⁰⁰a,b), 1.58–1.52 (m,

4H, H-2⁰⁰a,b, 4⁰⁰a,b), 1.33 (d, partially overlapped, J_5,6

6.2 Hz, H-6), 1.34–1.29 (m, partially overlapped, H-

3⁰⁰a,b); ¹³C NMR (CDCl₃, 150 MHz): d 95.95 (J_C-1,H-1

170 Hz, C-1), 78.27 (C-3), 75.41 (CH₂Ph), 74.31 (C-2),

68.04 (2 C, C-4,1⁰⁰), 66.06 (C-5), 51.43 (COOCH₃),

33.82 (C-5⁰⁰), 28.99 (C-2⁰⁰), 25.65 (C-3⁰⁰), 24.57 (C-4⁰⁰),

18.35 (C-6). ESI-MS: m/z 518.2479 ([M+Li]⁺). Anal.

Calcd for C₂₇H₃₃N₃O₇: C, 63.39; H, 6.50; N, 8.21.

Found: C, 53.56; H, 6.56; N, 8.23.

Eluted next was the orthoester 90 (190 mg, 34%, total

yield of glycosylation, 78%). ¹H NMR (CDCl₃,

600 MHz): d 5.64 (dd, 1H, J_2,33.4, J_3,410.1 Hz, H-

3^II), 5.58 (d, 1H, J_1,22.7 Hz, H-1^I), 5.49 (t, J 10.0 Hz,

H-4^II), 5.36 (t, 1H, J 9.4 Hz, H-4^I), 5.28 (dd, 1H, J_1,2

1.7, J_2,33.4 Hz, H-2^II), 5.19 (d, 1H, H-1^II), 4.87 (dd,

1H, J_2,34.2 Hz, H-2^I), 4.48–4.44 (m 1H, H-5^II), 4.19

(dd, 1H, J_3,49.7 Hz, H-3^I), 3.68–3.64 (m, 1H, H-5^I),

3.60 (s, 3H, COOCH₃), 3.55, 3.52 (2 d, 2H, J 12.2 Hz,

CH₂Br), 3.48–3.39 (m, 2H, H-1⁰⁰a,b), 2.16 (t, 2H, J

7.1 Hz, H-5⁰⁰a,b), 1.59–1.49 (m, 4H, H-2⁰⁰a,b, 4⁰⁰a,b),

1.34 (d, 3H, J_5,66.2 Hz, H-6^II), 1.32–1.26 (m, 2H, H-

3⁰⁰), 1.20 (d, 3H, J_5,66.2 Hz, H-6^I); ¹³C NMR (CDCl₃,

Eluted next was the b-anomer 49 (499 mg, 84%), [a]_D

+110° (c 0.9). ¹H NMR (CDCl₃, 600 MHz): d 5.24

(dd, 1H, J_1,28.1, J_2,39.4 Hz, H-2), 4.74, 4.65 (2 d, 2H,

CH₂Ph), 4.45 (d, 1H, H-1), 3.85, 3.83 (2 t, 1H, J

6.1 Hz, H-1⁰⁰a), 3.69 (t, 1H, J 9.1 Hz, H-3), 3.61 (s,

4304

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

3H, COOCH₃), 3.43–3.39 (m, 1H, H-1⁰⁰b), 3.35 (m, par-

tially overlapped, H-5), 3.29 (t, partially overlapped, H-

4), 2.07–1.95 (m, 2H, H-5⁰⁰a,b), 1.54–1.39 (m, 7H, H-

2⁰⁰a,b, 4⁰⁰, incl., d, 1.40, J_5,65.8 Hz, H-6), 1.24–1.11 (m,

2H, H-3⁰⁰). ¹³C NMR (CDCl₃, 150 MHz): d 100.88 (C-

1), 81.03 (C-3), 74.82 (CH₂Ph), 73.86 (C-2), 70.77 (C-

5), 69.48 (C-1⁰⁰), 67.59 (C-4), 51.33 (COOCH₃), 33.63

(C-5⁰⁰), 28.98 (C-2⁰⁰), 25.26 (C-3⁰⁰), 24.35 (C-4⁰⁰), 18.38

(C-6). ESI-MS: 518.2463 ([M+Li]⁺). Anal. Calcd for

C₂₇H₃₃N₃O₇: C, 63.39; H, 6.50; N, 8.21. Found: C,

63.53; H, 6.66; N, 8.01.

2H, CH₂Ph), 4.26 (d, 1H, J_1,27.7 Hz, H-1), 3.92, 3.90

(2 t, 1H, J 6.5 Hz, H-1⁰⁰a), 3.66 (s, 3H, COOCH₃),

3.60 (s, 3H, OCH₃-2), 3.54–3.48 (2 t, 1H, H-1⁰⁰b),

3.20–3.16 (m, 2H, H-3,5), 3.12 (dd, 1H, J_2,39.0 Hz, H-

2), 2.53 (t, 1H, J 9.3 Hz, H-4), 2.32 (t, 2H, J 7.4 Hz,

H-5⁰⁰a,b), 1.71–1.61 (m, 4H, H-2⁰⁰a,b, 4⁰⁰a,b), 1.48–1.41

(m, 2H, H-3⁰⁰), 1.40–1.32 (br s, 2H, NH₂), 1.26 (d, 3H,

J_5,66.2 Hz, H-6). ¹³C NMR (150 MHz, CDCl₃): d

103.55 (C-1), 84.72 (C-2), 84.05 (C-3), 74.96 (CH₂Ph),

72.57 (C-5), 69.57 (C-1⁰⁰), 60.26 (OCH₃), 57.84 (C-4),

51.41 (COOCH₃), 33.93 (C-5⁰⁰), 29.33 (C-2⁰⁰), 25.61 (C-

3⁰⁰), 24.65 (C-4⁰⁰), 17.95 (C-6). ESI-MS 418.2219

([M+Na]⁺) C₂₁H₃₃O₆Na requires 418.2206.

3.41. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-b-^D-glucopyranoside (50)

Condensation of the foregoing amine (340 mg,

0.86 mmol) with 3-hydroxy-3-methylbutyric acid

(152 mg, 1.3 mmol), as described above for similar con-

versions, gave amide 53 (324 mg, 76%), mp 80–81 °C

(from i-Pr₂O containing a few drops of EtOH), [a]_D

Debenzoylation of the foregoing benzoate 49 (520 mg)

gave alcohol 50 (380 mg, 92%), [a]_D+47° (c 1). ¹H

NMR (CDCl, 600 MHz): d 4.79, 4.83 (2 d, 2H, ²J

11.0 Hz, 2 CH₂Ph), 4.18 (d, 1H, J_1,27.7 Hz, H-1),

3.90, 3.88 (2 t, 1H, J 6.3 Hz, H-1⁰a), 3.66 (s, 3H,

COOCH₃), 3.53 (ddd, 1H, J_2,39.1, J_2,OH2.6 Hz, H-2),

3.48, 3.50 (2 t, 1H, H-1⁰⁰b), 3.22 (m, 1H, H-5), 3.14

(dd, 1H, J_3,49.6 Hz, H-4), 2.79 (d, J 2.6 Hz, 1H, OH),

2.32 (t, 2H, J 7.4 Hz, H-5⁰⁰a,b), 1.70–1.58 (m, 4H, H-

2⁰⁰a,b, 4⁰⁰a,b), 1.45–1.36 (m, 2H, H-3⁰⁰a,b), 3.34 (d, 3H,

J_5,66.1 Hz, H-6); ¹³C NMR (CDCl₃, 150 MHz): d

102.45 (C-1), 82.40 (C-3), 74.90 (C-2), 74.88 (CH₂Ph),

70.60 (C-5), 69.59 (C-1⁰⁰), 67.18 (C-4), 51.47 (COOCH₃),

33.71 (C-5⁰⁰), 28.88 (C-2⁰⁰), 25.33 (C-3⁰⁰), 24.32 (C-4⁰⁰),

18.38 (C-6). ESI-MS 430.1926 ([M+Na]⁺) Anal. Calcd

for C₂₀H₂₉NO₆: C, 58.95; H, 7.17; N, 10.31. Found:

C, 51.18; H, 7.34; N, 10.27.

1

ꢁ50° (c 0.7). H NMR (600 MHz, CDCl₃): d 5.68 (d,

1H, J_NH,48.8 Hz, NH), 4.85, 4.62 (2 d, 2H, J 11.8 Hz,

CH₂Ph), 4.25 (d, 1H, J_1,27.8 Hz, H-1), 4. (2 s, 1H,

OH), 3.91, 3.89 (2 t, 1H, J 6.4 Hz, H-1a⁰⁰), 3.66 (s, 3H,

COOCH₃), 3.60 (ddd, partially overlapped, H-4), 3.59

(s, partially overlapped, OCH₃-2), 3.50, 3.49 (2 t, par-

tially overlapped, H-1b⁰⁰), ꢀ3.46 (t, partially overlapped,

H-3), ꢀ3.44 (m, partially overlapped, H-5), 3.15 (dd, J_2,3

8.8 Hz, H-2), 2.32 (t, 2H, J 7.4 Hz, H-5⁰⁰a,b), 2.22, 2.13

(2 d, 2H, J 15.0 Hz, H-2⁰a,b), 1.70–1.62 (m, 4H, H-

2⁰⁰a,b, 4⁰⁰a,b), 1.48–1.37 (m, 2H, H-3⁰⁰a,b), 1.23, 1.20 (2

s, 6H, 2 CH⁰₃), 1.22 (d, partially overlapped, J_5,6

6.2 Hz, H-6); ¹³C NMR (150 MHz, CDCl₃): d 103.27

(C-1), 84.44 (C-2), 79.98 (C-3), 73.81 (CH₂Ph), 70.64

(C-5), 69.60 (C-3⁰), 69.41 (C-1⁰⁰), 60.38 (OCH₃-2),

56.02 (C-4), 51.45 (COOCH₃), 47.74 (C-2⁰), 33.95 (C-

5⁰⁰), 29.31 (3 C, 2 CH⁰₃, 2⁰⁰), 25.60 (C-3⁰⁰), 24.65 (C-4⁰⁰),

18.06 (C-6). ESI-MS 518.2760 ([M+Na]⁺) Anal. Calcd

for C₂₆H₄₁NO₈: C, 63.01; H, 8.34; N, 2.83. Found: C,

62.96; H, 8.40; N, 2.87.

3.42. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-2-O-methyl-b-^D-glucopyranoside (51)

Methylation of 50 (400 mg, 1 mmol), as described

above, gave the title methyl ether (390 mg, 97.5%),

1

[a]_D+38° (c 1). H NMR (600 MHz, CDCl₃): d 4.89,

4.79 (2 d, 2H, CH₂Ph), 4.21 (d, 1H, J_1,27.9 Hz, H-1),

3.89, 3.87 (2 t, 1H, J 6.4 Hz, H-1⁰⁰a), 3.66 (s, 3H,

COOCH₃), 3.59 (s, 3H, OCH₃-2), 3.49–3.48 (2 t, 1H,

H-1⁰⁰b), 3.38 (t. 1H, J 9.2 Hz, H-3), 3.18 (m, H-5),

3.12–3.08 (m, 2H, H-2,4), 2.32 (t, 2H, J 7.4 Hz, H-

5⁰⁰a,b), 1.70–1.59 (m, 4H, H-2⁰⁰,a,b, 4⁰⁰), 1.47–1.36 (m,

2H, H-3⁰⁰), 1.32 (d, 3H, J_5,66.1 Hz, H-6). ¹³C NMR

(150 MHz, CDCl₃): d 103.25 (C-1), 84.22 (C-2), 82.72

(C-3), 75.40 (CH₂Ph), 70.31 (C-5), 69.74 (C-1⁰⁰), 67.42

(C-4), 60.55 (OCH₃), 51.46 (COOCH₃), 33.94 (C-5⁰⁰),

29.30 (C-2⁰⁰), 25.59 (C-3⁰⁰), 24.65 (C-4⁰⁰), 18.41 (C-6).

ESI-MS: m/z 428.2354 ([M+Li]⁺). Anal. Calcd for

C₂₁H₃₁N₃O₆: C, 59.84; H, 7.41; N, 9.97. Found: C,

60.08; H, 7.59; N, 9.99.

3.44. 5-Methoxycarbonylpentyl 4,6-dideoxy-4-(3-hydro-

xy-3-methylbutyramido)-2-O-methyl-b-^D-glucopyrano-

side (54)

Compound 53 (315 mg) was deprotected as described in

the general procedure for debenzylation, to give amide

1

54 (250 mg, 76%), [a]_Dꢁ15° (c 0.6, H₂O). H NMR

(600 MHz, D₂O): d 4.43 (d, 1H, J_1,28.0 Hz, H-1),

3.91, 3.89 (2 t, 1H, J 6.3 Hz, H-1a⁰⁰), 3.68 (s, 3H,

COOCH₃), 3.65, 3.64 (2 t, partially overlapped, H-

1⁰⁰b), 3.61 (t, partially overlapped, H-4), 3.59 (s, partially

overlapped, OCH₃-2), ꢀ3.54 (m, partially overlapped,

H-5), 3.52 (t, partially overlapped, H-3), 3.03 (dd, J_2,3

9.1 Hz, H-2), 2.47, 2.44 (2 d, 2H, J 13.6 Hz, H-2⁰a,b),

2.40 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.66–1.61 (m, 4H, H-

2⁰⁰a,b, 4⁰⁰a,b), 1.44–1.35 (m, 2H, H-3⁰⁰a,b), 1.30, 1.29 (2

s, 6H, 2 CH⁰₃), 1.21 (d, J_5,66.1 Hz, H-6); ¹³C NMR

(150 MHz, D₂O): d 104.85 (C-1), 86.16 (C-2), 75.84

(C-3), 73.56 (C-5), 73.06 (C-1⁰⁰), 72.92 (C-3⁰), 63.06

(OCH₃-2), 59.33 (C-4), 54.82 (COOCH₃), 51.64 (C-2⁰),

36.35 (C-5⁰⁰), 31.19 (C-2⁰⁰), 31.04, 30.87 (2 CH⁰₃⁰), 27.53

(C-3⁰⁰), 26.76 (C-4⁰⁰), 19.72 (C-6). ESI-MS: m/z

428.2264 ([M+Na]⁺) C₁₉H₃₅NO₈Na requires 428.2260.

3.43. 5-Methoxycarbonylpentyl 4,6-dideoxy-3-O-benzyl-

4-(3-hydroxy-3-methylbutyramido)-2-O-methyl-b-^D-

glucopyranoside (53)

The foregoing azide (400 mg) was treated with H₂S as

described above for similar conversion, to give 5-meth-

oxycarbonylpentyl 4-amino-3-O-benzyl-4,6-dideoxy-2-

O-methyl-b-^D-glucopyranoside (52, 340 mg, 94%). ¹H

NMR (600 MHz, CDCl₃): d 4.97, 4.66 (2 d, J 11.5 Hz,

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4305

3.45. 5-Methoxycarbonylpentyl 3-O-acetyl-2,4-di-O-

benzyl-a-^L-rhamnopyranoside (55)

1.30 (m, partially overlapped, H-3⁰⁰a,b), 1.33 (d, partially

overlapped, J_5,65.9 Hz, H-6^II), 1.27 (d, 3H, J_5,66.1 Hz,

H-6^I); ¹³C NMR (CDCl₃, 125 MHz): d 100.68 (J_C-1,H-1

162 Hz, C-1^II), 98.36 (J_C-1,H-1168 Hz, C-1^I), 81.13 (C-

3^II), 80.46 (C-4^I), 79.22 (C-3^I), 78.04 (C-2^I), 75.16 (C-

2^II), 75.06, 74.82, 73.37 (3 CH₂Ph), 70.66 (C-5^II), 67.87

(C-5^I), 67.85 (C-4^II), 67.13 (C-1⁰⁰), 33.90 (C-5⁰⁰), 29.01

(C-2⁰⁰), 25.66 (C-3⁰⁰), 25.24 (CH₂Br), 24.64 (C-4⁰⁰),

18.32 (C-6^II), 17.81 (C-6^I). ESI-MS: m/z 876.2692

([M+Na]⁺). Anal. Calcd for C₄₂H₅₂N₃O₁₁: C, 59.02;

H, 6.13; N, 4.92. Found: C, 59.20; H, 6.15; N, 4.89.

Reaction of thioglycoside 25 (2 g, 4.8 mmol) with methyl

6-hydroxyhexanoate²²(1.05 g, 7.2 mmol), as described

in the general procedure, gave amorphous 55 (2.3 g,

1

96%), [a]_Dꢁ6.5° (c 1.3). H NMR (CDCl₃, 300 MHz):

d 5.18 (dd, 1H, J_2,33.3, J_3,49.5 Hz, H-3), 4.70 (d, par-

tially overlapped, J_1,21.8 Hz, H-1), 4.72–4.55 (4 d,

partially overlapped, 2 CH₂Ph), 3.83 (dd, 1H, H-2),

3.80–3.69 (m, 1H, H-5), 3.66–3.58 (m, 5H, H-4,1⁰a, incl.,

3.65, s, OCH₃), 3.70, 3.34 (2 t, 1H, H-1⁰b), 2.30 (t, 2H, J

7.6 Hz, H-5⁰⁰a,b), 1.96 (s, 3H, COOCH₃), 1.68–1.51 (m,

4H, H-4⁰⁰a,b,2⁰⁰a,b), 1.40–1.32 (m, partially overlapped,

H-3), 1.32 (d, partially overlapped, J_5,66.2 Hz, H-6);

¹³C NMR (CDCl₃, 75 MHz): d 97.66 (C-1), 79.19 (C-

4), 76.31 (C-2), 74.93, 73.06 (2 CH₂Ph), 73.76 (C-3),

73.06 (C -4), 67.61 (C-5), 67.34 (C-1⁰⁰), 51.40 (COOCH₃),

33.88 (C-5⁰⁰), 29.00 (C-2⁰⁰), 25.63 (C-3⁰⁰), 24.63 (C-4⁰⁰),

21.03 (COCH₃), 17.96 (C-6). ESI-MS: m/z 537.2

([M+Na]⁺). Anal. Calcd for C₂₉H₃₈O₈: C, 67.68; H,

7.44. Found: C, 67.84; H, 7.42.

3.48. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-b-^D-glucopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-

L-rhamnopyranoside (58)

´

Debromoacetylation (Zemplen) of 57 (900 mg) gave

1

alcohol 58 (693 mg, 88%), [a]_D+2° (c 0.8). H NMR

(CDCl₃, 600 MHz): d 4.92–4.63 (m, 6 d, partially over-

lapped, J ꢀ 11.5 Hz, 6 CH₂Ph), 4.59 (d, J_1,21.8 Hz, H-

1^I), 4.45 (d, partially overlapped, J_1,27.8 Hz, H-1^I),

3.99 (dd, 1H, J_2,33.2, J_3,49.1 Hz, H-3^I), 3.78 (dd, 1H,

H-2^I), 3.69–3.61 (m, 5H, H-4^I,5^I,1⁰⁰a, incl., 3.65 s,

COOCH₃), 3.59–3.55 (m, 2H, H-2^II,1⁰⁰), 3.40 (t, 1H, J

9.2 Hz, H-3^II), 3.30, 3.28 (2 t, 1H, J 6.4 Hz, H-1⁰⁰b),

3.27–3.23 (m, partially overlapped, H-5^II), 3.14 (dd,

J_4,59.8 Hz, 1H, H-4^II), 2.74 (d, 1H, J_2,OH2.2 Hz,

OH), 2.29 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.63–1.58 (m,

2H, H-4⁰⁰a,b), 1.53–1.48 (m, 2H, H-2⁰⁰), 1.35 (d, 3H,

J_5,66.0 Hz, H-6^I), 1.33 (d, 3H, J_5,66.1 Hz, H-6^II),1.33–

1.28 (m, partially overlapped, H-3⁰⁰a,b); ¹³C NMR

(CDCl₃, 125 MHz): d 103.83 (C-1^II), 98.36 (C-1^I),

82.17 (C-3^II), 80.68 (C-4^I), 80.53 (br, C-3^I), 77.57 (C-

2^I), 75.69 (C-2^II), 75.47, 74.81, 73.29 (3 CH₂Ph), 70.74

(C-5^II), 67.92 (C-5^I), 67.15 (C-1⁰⁰), 67.10 (C-4^II), 51.46

(COOCH₃), 33.87 (C-5⁰⁰), 28.97 (C-2⁰⁰), 25.63 (C-3⁰⁰),

24.62 (C-4⁰⁰), 18.59 (C-6^II), 18.04 (C-6^I). ESI-MS:

m/z 778.3537 ([M+HCOO]^ꢁ). Anal. Calcd for

C₄₀H₅₁N₃O₁₀: C, 65.47; H, 7.00; N, 5.73. Found: C,

65.55; H,7.02; N, 5.70.

3.46. 5-Methoxycarbonylpentyl 2,4-di-O-benzyl-a-^L-

rhamnopyranoside (56)

Conventional deacetylation of the foregoing acetate 55

(2 g) gave glycosyl acceptor 56 (1.6 g, 89%); [a]_D

1

ꢁ14.7° (c 0.8). H NMR (CDCl₃, 300 MHz): d 4.88,

4.74, 4.64, 4.59 (4 d, 4H, J 11.8 Hz, 2 CH₂Ph), 4.77 (d,

1H, J_1,21.3 Hz, H-1), 3.94, 3.92 (2 t, 1H, J 9.6 Hz, H-

3), 3.70 (dd, 1H, J_2,33.7 Hz, H-2), 3.68–3.59 (m, 5H,

H-5, 1⁰⁰a, incl., 3.65, s, COOCH₃), 3.40–3.28 (m, 2H,

H-4,1⁰⁰b), 2.30 (t, 2H, J 7.4 Hz, H-5⁰⁰), 1.67–1.49 (m,

4H, H-4⁰⁰a,b, 2⁰⁰,a,b), 1.39–1.29 (m, 5H, H-3⁰⁰, incl.,

1.32, d, J_5,66.3 Hz, H-6); ¹³C NMR (CDCl₃,

75 MHz): d 96.79 (C-1), 82.27 (C-4), 78.72 (C-2),

75.03, 72.96 (2 CH₂Ph), 71.63 (C-3), 67.16 (C-1⁰⁰),

67.05 (C-5), 51.40 (COOCH₃), 33.85 (C-5⁰⁰), 29.02 (C-

2⁰⁰), 25.65 (C-3⁰⁰), 24.60 (C-4⁰⁰), 17.96 (C-6). ESI-MS:

m/z 495.2338 ([M+Na]⁺). Anal. Calcd for C₂₇H₃₆O₇:

C, 67.54; H, 6.00. Found: C, 67.80; H, 5.94.

3.49. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-2-O-methyl-b-^D-glucopyranosyl-(1 ! 3)-2,4-di-

O-benzyl-a-^L-rhamnopyranoside (59)

3.47. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-2-O-

bromoacetyl-4,6-dideoxy-b-^D-glucopyranosyl-(1 ! 3)-

2,4-di-O-benzyl-a-^L-rhamnopyranoside (57)

When treated as described in the general procedure for

methylation, compound 58 (693 mg, 0.94 mmol) gave

1

Compound 18 (825 mg, 1.85 mmol) was treated with

alcohol 56 (800 mg, 1.69 mmol) according to the general

procedure, to aﬀord 57 (950 mg, 68%), [a]_D+0.3° (c 0.8).

¹H NMR (CDCl₃, 600 MHz): d 5.10 (dd, 1H, J_1,2

8.1 Hz, J_2,39.6 Hz, H-2^II), 4.82–4.56 (m, 6 d, partially

overlapped, J ꢀ 11 Hz, 6 CH₂Ph), 4.76 (d, partially

overlapped, J 8.1 Hz, H-1^II), 4.62 (d, partially over-

lapped, J_1,21.7 Hz, H-1^I), 4.02 (dd, 1H, J_2,33.1, J_3,4

9.5 Hz, H-3^I), 3.76 (dd, 1H, H-2^I), 3.66 (s, partially over-

lapped, COOCH₃), 3.66–3.56 (m, partially overlapped,

H-5^I,1⁰⁰a), 3.56 (t, partially overlapped, H-4^I), 3.49 (t,

1H, J 9.3 Hz, H-3^II), 3.38, 3.35 (2 d, 2H, J 12.6 Hz,

CH₂Br), 3.32 (m, partially overlapped, H-1⁰⁰b), 3.27

(m, partially overlapped, H-5^II), 3.22 (t, partially over-

lapped, H-4^II), 2.30 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.64–

1.59 (m, 2H, H-4⁰⁰a,b), 1.55–1.50 (m, 2H, H-2⁰⁰), 1.35–

methyl ether 59 (600 mg, 86%), [a]_Dꢁ1.3° (c 0.6). H

NMR (CDCl₃, 600 MHz): d 4.97–4.58 (m, 8H, 3

CH₂Ph, incl., 4.63, d, J_1,21.8 Hz, H-1^I; 4.62 d, J_1,2

7.8 Hz, H-1^II), 4.04 (d, 1H, J_2,33.2, J_3,49.3 Hz, H-3^I),

3.79 (d, 1H, H-2^I), 3.69–3.64 (m, partially overlapped,

H-5^I), 3.66 (s, partially overlapped, OCH₃-2), 3.64 (s,

partially overlapped, COOCH₃), 3.61 (t, partially over-

lapped, J 9.5 Hz, H-4^I), 3.59, 3.58 (2, t, partially over-

lapped, J 6.7 Hz, H-1⁰⁰a), 3.39 (t, 1H, J 9.2 Hz, H-3^II),

3.32, 3.30 (2, t, 1H, H-1⁰⁰b), 3.19–3.14 (m, 2H, H-2^II,5^II),

3.10 (bt, 1H, H-4^II), 2.28 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b),

1.63–1.58 (m, 2H, H-4⁰⁰a,b), 1.53–1.49 (m, 2H, H-

2⁰⁰a,b), 1.34 (d, J_5,66.1 Hz, partially overlapped, H-6^I),

1.35–1.30 (m, partially overlapped, H-3⁰⁰a,b), 1.27 (d,

3H, J_5,66.0 Hz, H-6^II); ¹³C NMR (CDCl₃): d 103.59

(C-1^II), 98.47 (C-1^I), 84.79 (C-2^II), 82.76 (C-3^II), 80.87

4306

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

(C-4^I), 78.86 (C-3^I), 78.68 (C-2^I), 75.41, 74.84, 73.41 (3

CH₂Ph), 70.20 (C-5^II), 67.81 (C-5^I), 67.69 (C-4^II),

67.08 (C-1⁰⁰), 60.72 (OCH₃-2), 51.43 (COOCH₃), 33.90

(C-5⁰⁰), 29.02 (C-2⁰⁰), 25.66 (C-3⁰⁰), 24.64 (C-4⁰⁰), 18.46

(C-6^II), 17.92 (C-6^I); ESI-MS 770.3636 ([M+Na]⁺).

Anal. Calcd for C₄₁H₅₃N₃O₁₀: C, 65.08; H, 7.14; N,

5.62. Found: C, 65.86; H, 7.22; 5.58N.

150 MHz): d 103.77 (J_C-1,H-1159.0 Hz, C-1^II), 98.60

(J 167.0 Hz, C-1^I), 84.98 (C-2^II), 80.82 (C-4^I), 80.21

(C-3^II), 79.09 (C-3^I), 78.59 (C-2^I), 74.82, 73.77, 73.49

(3 CH₂Ph), 70.87 (C-5^II), 69.42 (C-3⁰), 67.81 (C-5^I),

67.05 (C-1⁰⁰), 60.57 (OCH₃-2), 55.72 (C-4^II), 51.44

(COOCH₃), 47.77 (C-2⁰), 33.92 (C-5⁰⁰), 29.32 (2 C, 2

CH₃⁰), 29.02 (C-2⁰⁰), 25.66 (C-3⁰⁰), 24.64 (C-4⁰⁰), 18.12

(C-6^II), 17.93 (C-6^I). ESI-MS: 844.4289 ([M+Na]⁺)

Anal. Calcd for C₄₆H₆₃NO₁₂: C, 67.21; H, 7.73; N,

1.70. Found: C, 67.36; H, 7.66; N, 1.61.

3.50. 5-Methoxycarbonylpentyl 4,6-dideoxy-3-O-benzyl-

4-(3-hydroxy-3-methylbutyramido)-2-O-methyl-b-^D-

glucopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamnopyr-

anoside (61)

3.51. 5-Methoxycarbonylpentyl 4,6-dideoxy-4-(3-hydro-

xy-3-methylbutyramido)-2-O-methyl-b-^D-glucopyranosyl-

(1 ! 3)-a-^L-rhamnopyranoside (62)

Azide 59 was converted to 5-methoxycarbonylpentyl 4-

amino-3-O-benzyl-4,6-dideoxy-2-O-methyl-b-^D-gluco-

pyranosyl-2,4-di-O-benzyl-a-^L-rhamnopyranoside (60)

(500 mg, 93%) by treatment with H₂S as described

Hydrogenolysis of 61 (500 mg), as described above

for similar conversions, gave the deprotected disac-

charide 62 (335 mg, 85%), [a]_Dꢁ38° (c 0.6). ¹H

NMR (600 MHz, D₂O): d 4.76 (d, 1H, J_1,21.8 Hz,

H-1^I), 4.68 (d, 1H, J_1,27.9 Hz, H-1^II), 4.11 (dd,

1H, J_2,33.3 Hz, H-2^I), 3.82 (dd, 1H, J_3,49.6 Hz,

H-3^I), 3.69 (m, partially overlapped, H-5^I), 3.68 (s,

partially overlapped, COOCH₃), 3.63 (t, partially

overlapped, J 10.0 Hz, C-4^II), 3.62 (s, partially over-

lapped, OCH₃-2), 3.58 (t, 1H, J 9.6 Hz, H-4^I), 3.54

(m, partially overlapped, H-5^II), 3.53 (t, partially

overlapped, H-3^II), 3.13 (dd, 1H, J_2,39.2 Hz, H-2^II),

2.46, 2.43 (2 d, 2H, J 13.6 Hz, H-2⁰a,b), 2.40 (t,

1

above. H NMR (CDCl₃, 600MHz): d 5.01–4.58 (6 d,

partially overlapped, 3 CH₂Ph), 4.68 (d, partially over-

lapped, J_1,27.5 Hz, H-1^II), 4.63 (d, 1H, J_1,21.8 Hz, H-

1^I), 4.08 (dd, 1H, J_2,33.2, J_3,49.4 Hz, H-3^I), 3.83 (dd,

1H, H-2^I), 3.68 (m, partially overlapped, H-5^I), 3.67 (s,

partially overlapped, OCH₃-2), 3.65 (s, COOCH₃),

3.62 (t, partially overlapped, J 9.4 Hz, H-4^I), 3.60, 3.58

(2 t, partially overlapped, J 6.2 Hz, H-1⁰⁰a), 3.32, 3.30

(2 t, 1H, H-1⁰⁰b), ꢀ3.2 (t, partially overlapped, H-3^II),

ꢀ3.19 (t, partially overlapped, H-2^II), 3.16 (m, partially

overlapped, H-5^II), 2.53 (m, 1H, H-4^II), 2.28 (t, 2H, J

7.3 Hz, H-5⁰⁰a,b), 1.63–1.58 (m, 2H, H-4⁰⁰a,b), 1.54–

1.50 (m, 2H, H-2⁰⁰a,b), 1.47–1.29 (m, NH₂, H-3⁰⁰a,b,

incl., d, 1.35, J_5,66.2 Hz, H-6^I), 1.22 (d, J_5,66.2 Hz, H-

6^II). ¹³C NMR (CDCl₃, 150 MHz): d 103.86 (C-1^II),

98.58 (C-1^I), 85.37 (C-2^II), 84.24 (C-3^II), 80.96 (C-4^I),

78.74 (C-2^I), 78.51 (C-3^I), 75.02, 74.79, 73.41 (3 CH₂Ph),

72.51 (C-5^I), 67.82 (C-5^I), 76.05 (C-1⁰⁰), 60.23 (OCH₃-2),

58.22 (C-4^II), 51.42 (COOCH₃), 33.91 (C-5⁰⁰), 29.03 (C-

2⁰⁰), 25.65 (C-3⁰⁰), 24.64 (C-4⁰⁰), 18.06 (C-6^II), 17.93 (C-

6^I). ESI-MS: m/z 722.3863 C₄₁H₅₆NO₁₀+H requires

722.3904.

2H,

J

7.3 Hz, H-5⁰⁰a,b), 1.67–1.57 (m, 4H, H-

2⁰⁰a,b,4⁰⁰a,b), 1.43–1.34 (H-3⁰⁰a,b), 1.30, 1.29 (2 s,

6H, 2 CH⁰₃), 1.29 (d, 3H, J_5,66.4 Hz, H-6^I), 1.21

(d, 3H, J_5,66.2 Hz, H-6^II); ¹³C NMR (150 MHz,

D₂O): d 106.55 (C-1^II), 102.18 (C-1^I), 85.96 (C-2^III),

82.74 (C-3^I), 75.52 (C-3^II), 73.79 (C-4^I), 73.47 (C-

5^II), 72.89 (C-3⁰), 72.77 (C-2^I), 71.52 (C-5^I), 70.36

(C-1⁰⁰), 62.74 (OCH₃-2), 59.29 (C-4^II), 54.80

(COOCH₃), 51.60 (C-2⁰), 36.32 (C-5⁰⁰), 31.03 (CH⁰₃),

30.89 (CH⁰₃,2⁰⁰), 27.72 (C-3⁰⁰), 26.77 (C-4⁰⁰), 19.87 (C-

6^II), 19.38 (C-6^I); ESI-MS 574.2856 ([M+Na]⁺)

C₂₅H₄₅NO₁₂Na requires 574.2839.

The foregoing amine (500 mg, 0.69 mmol) was treated

with

3-hydroxy-3-methylbutyric

acid

(120 mg,

3.52. 5-Methoxycarbonylpentyl 3-O-acetyl-2,4-di-O-

benzyl-a-^L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-

a-^L-rhamnopyranoside (63)

1 mmol), HATU (395 mg, 1 mmol), and N-diisopro-

pylethylamine (0.13 mL, 1 mmol), as described in the

general procedure, to give amide 61 (495 mg, 90%),

1

[a]_Dꢁ60° (c 1). H NMR (CDCl₃, 600 MHz): d 5.49

Condensation of glycosyl acceptor 25 (600 mg,

1.27 mmol) with thioglycoside 56 (624 mg, 1.5 mmol)

according to the general procedure gave product 63

(860 mg, 86%), [a]_Dꢁ18° (c 0.7). ¹H NMR (CDCl₃,

600 MHz): d 5.31 (dd, 1H, J_2,33.3, J_3,49.6 Hz, H-

3^II), 5.15 (d, 1H, J_1,21.9 Hz, H-1^II), 4.80, 4.76, 4.74,

4.69, 4.47, 4.62, 3.36, 3.18 (8 d, partially overlapped,

J ꢀ 11.7 Hz, 4 CH₂Ph), 4.73 (d, partially overlapped,

J_1,21.8 Hz, H-1^I), 4.09 (dd, 1H, J_2,33.0, J_3,49.5 Hz,

H-3^I), 3.89–3.85 (m, 2H, H-5^II, incl., ꢀ3.87, dd, H-

2^II), 3.71 (dd, 1H, H-2^I), ꢀ3.67 (m, partially over-

lapped, H-5^I), 3.66–3.60 (m, 6H, H-4^I,II, H-1⁰⁰a, incl.,

3.65 s, COOCH₃), 3.39, 3.33 (2 t, 1H, J 6.4 Hz, H-

1⁰⁰b), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.94 (s, 3H,

COCH₃), 1.66–1.61 (m, 2H, H-4⁰⁰a,b), 1.56–1.52 (m,

2H, H-2⁰⁰a,b), 1.37–1.31 (m, 2H, H-3⁰⁰a,b), 1.29 (d,

3H, J_5,66.2 Hz, H-6^I), 1.26 (d, 3H, J_5,66.3 Hz, H-

6^II); ¹³C NMR (CDCl₃, 150 MHz): d 99.46 (J_C-1,H-1

(d, 1H, J_4,NH9.1 Hz, NH), 5.00–4.59 (m, 8H, 3

CH₂Ph, incl., 4.63, d, J_1,27.8 Hz, H-1^II, 4.62, d, J_1,2

1.6 Hz, H-1^I), 4.04 (dd, 1H, J_2,33.2, J_3,49.3 Hz, H-

3^I), 3.82 (dd, 1H, J_2,33.3 Hz, H-2^I), 3.72 (q, 1H, J

10.0 Hz, H-4^II), ꢀ3.68 (m, partially overlapped, H-

5^II), 3.66 (s, partially overlapped, OCH₃-2), 3.64 (s,

partially overlapped, COOCH₃), 3.62 (t, partially

overlapped, J 9.3 Hz, H-4^I), 3.59, 3.57 (2 t, partially

overlapped, J 6.6 Hz, H-1⁰⁰a), 3.39 (dd, 1H, J_2,3

8.9 Hz, H-3^II), 3.33 (m, partially overlapped, H-5^II),

3.31, 3.29 (2 t, partially overlapped, H-1⁰⁰b), 3.22

(dd, 1H, H-2^II), 2.27 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b),

2.23, 2.13 (2 d, 2H, J 15 Hz, CH₂Br), 1.62–1.57 (m,

2H, H-4⁰⁰a,b), 1.53–1.48 (m, 2H, H-2⁰⁰), 1.34 (d, par-

tially overlapped, J 6.2 Hz, H-6^I), 1.34–1.25 (m, par-

tially overlapped, H-3⁰⁰), 1.24, 1.21 (2 s, 6H, 2 CH⁰₃),

1.18 (d, 3H, J 6.2 Hz, H-6^I). ¹³C NMR (CDCl₃,

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4307

170.2 Hz, C-1^II), 97.57 (J_C-1,H-1168 Hz, C-1^I), 80.75

(C-4^II), 79.01 (C-4^I), 78.21 (C-3^I), 77.97 (C-2^I), 76.77

(C-2^II), 74.74, 74.64 (2 CH₂Ph), 73.52 (C3^II), 72.69,

72.58 (2 CH₂Ph), 68.23 (C-5^II), 68.22 (C-5^I), 67.24

(C-1⁰⁰), 51.43 (COOCH₃), 33.91 (C-5⁰⁰), 29.06 (C-2⁰⁰),

25.68 (C-3⁰⁰), 24.64 (C-4⁰⁰), 21.02 (COCH₃), 18.01 (C-

6^II), 17.88 (C-6^I); ESI-MS: m/z 863.3974 ([M+Na]⁺).

Anal. Calcd for C₄₉H₆₀O₁₂: C, 69.98; H, 7.19. Found:

C, 70.16; H, 7.29.

78.77 (C-3^II), 78.54 (C-3^I), 78.48 (C-2^II), 78.06 (C-2^I),

75.00 (C-2^III), 74.96, 74.88, 74.48, 73.16, 72.38 (5 CH₂Ph),

70.54 (C-5^III), 68.43 (C-5^II), 67.94 (C-5^I), 67.63 (C-4^III),

67.23 (C-1⁰⁰), 51.39 (COOCH₃), 33.82 (C-5⁰⁰), 28.99 (C-

2⁰⁰), 25.60 (C-3⁰⁰), 25.13 CH₂Br), 24.56 (C-4⁰⁰), 18.15 (C-

6^III), 17.86 (2 C, C-6^I,II); ESI-MS: m/z 1202.4205

([M+Na]⁺). Anal. Calcd for C₆₂H₇₄BrN₃O₁₅: C, 63.04;

H, 6.31; N, 3.96. Found: C, 63.28; H, 6.40; N, 4.52.

3.55. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-b-^D-glucopyranosyl-(1 ! 3)-(2,4-di-O-benzyl-a-

L-rhamnopyranosyl)-(1 ! 3)-2,4-di-O-benzyl-a-L-

rhamnopyranoside (66)

3.53. 5-Methoxycarbonylpentyl 2,4-di-O-benzyl-a-^L-

rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamno-

pyranoside (64)

´

Conventional deacetylation (Zemplen) of the foregoing

Deacylation (Zemplen) of 65 (1.02 g) gave 66 (800 mg,

acetate 63 (922 mg) gave alcohol 64 (850 mg, 96%),

[a]_Dꢁ26° (c 0.7). ¹H NMR (CDCl₃, 600 MHz): d

5.19 (d, 1H, J_1,21.4 Hz, H-1^II), 4.90–4.10 (m, 10H,

4 CH₂Ph, incl., 4.72, d, J_1,21.8 Hz, H-1^I, incl., 4.10,

dd, J_2,33.1, J_3,49.5 Hz, H-3^I), 3.99 (dt, 1H, H-3⁰⁰),

3.81–3.76 (m, 1H, H-5^I), 3.71 (dd, 1H, H-2^II),

ꢀ3.72–3.68 (m, overlapped, H-5^II), 3.69 (dd, partially

overlapped, H-2^I), 3.72–3.59 (m, 6H, H-4^I,5^II, H-1⁰⁰a,

incl., 3.64 s, COOCH₃), 3.35–3.29 (m, 2H, H-4^II,

1⁰⁰b), 2.30, (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.65–1.60 (m,

2H, H-4⁰⁰a,b), 1.58–1.53 (m, 2H, H-2⁰⁰a,b), 1.37–1.32

(m, 2H, H-3⁰⁰a,b), 1.30 (d, 3H, J_5,66.2 Hz, H-6^I),

1.27 (d, 3H, J_5,66.3 Hz, H-6^II); ¹³C NMR (CDCl₃,

150 MHz): d 98.65 (C-1^II), 97.53 (C-1^I), 82.12 (C-

4^II), 80.95 (C-4^I), 79.05 (C-2^II), 77.95 (C-2^I), 77.74

(C-3^I), 74.72, 74.65, 72.62, 72.35 (4 CH₂Ph), 71.52

(C-3^II), 68.17 (C-5^II), 67.65 (C-5^I), 67.22 (C-1⁰⁰),

51.38 (COOCH₃), 33.85 (C-5⁰⁰), 29.06 (C-2⁰⁰), 25.62

(C-3⁰⁰), 24.59 (C-4⁰⁰), 17.94 (C-6^I), 17.90 (C-6^II); ESI-

MS: m/z 821.3884 ([M+Na]⁺). Anal. Calcd for

C₄₇H₅₈O₁₁: C, 70.65; H, 7.32. Found: C, 70.63; H,

7.38.

88%), [a]_D+2.5° (c 0.7). ¹H NMR (CDCl₃, 600 MHz): d

5.07 (bd, 1H, J_1,2ꢀ 1.3 Hz, H-1^II), 4.91–4.44 (m, 11H, 5

CH₂Ph, incl., 4.74, d, 1H, J_1,21.8 Hz, H-1^I), 4.40 (d, 1H,

J_1,28.8 Hz, H-1^III), 4.09 (dd, 1H, J_2,33.1, J_3,49.5 Hz, H-

3^II), 4.02 (dd, 1H, J_2,33.2, J_3,49.4 Hz, H-3^I), 3.87 (dd,

1H, H-2^II), 3.86–3.81 (m, 1H, H-5^II), 3.70 (dd, 1H, H-

2^I), 3.62 (s, partially overlapped, COOCH₃), 3.65 (m, par-

tially overlapped, H-5^I), 3.64 (t, partially overlapped, H-

4^II), ꢀ3.53–3.50 (m, 2H, H-4^I,2^III), 3.35–3.30 (m, 2H, H-

3^III,1⁰⁰a), 3.12–3.05 (m, 3H, H-4^III,5^III,1⁰⁰b), 2.72 (s, 1H,

OH), 2.31 (t, 2H, J 7.5 Hz, H-5⁰⁰a,b), 1.66–1.60 (m, 2H,

H-4⁰⁰a,b), 1.56–1.52 (m, 2H, H-2⁰⁰a,b), 1.36–1.31 (m, 2H,

H-3⁰⁰a,b), 1.31 (d, 3H, J_5,66.2 Hz, H-6^II), 1.25 (d, 3H,

J_5,66.3 Hz, H-6^I), 1.17 (d, 3H, J_5,65.7 Hz, H-6^III). ¹³C

NMR (CDCl₃, 125 MHz): d 103.68 (C-1^III), 100.09 (C-

1^II), 97.20 (C-1^I), 82.07 (C-3^III), 80.67 (C-4^II), 80.58 (C-

4^I), 79.79 (C-3^II), 78.39 (C-3^I), 78.28 (C-2^II), 78.10 (C-

2^I), 75.68 (C-2^III), 75.23, 74.83 (2 C), 73.07, 72.53 (5

CH₂Ph), 70.67 (C-5^III), 68.57 (C-5^II), 68.00 (C-5^I), 67.32

(C-1⁰⁰), 67.04 (C-4^III), 51.50 (COOCH₃), 33.92 (C-5⁰⁰),

29.07 (C-2⁰⁰), 25.68 (C-3⁰⁰), 24.65 (C-4⁰⁰), 18.51 (C-6^III),

18.11 (C-6^II), 17.92 (C-6^I). ESI-MS: m/z 1082.4971

([M+Na]⁺). Anal. Calcd for C₆₀H₇₃N₃O₁₄: C, 67/97; H,

6/94; N, 6.96. Found: C, 68.08; H, 6.91; N, 3.98.

3.54. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-2-O-

bromoacetyl-4,6-dideoxy-b-^D-glucopyranosyl-(1 ! 3)-

2,4-di-O-benzyl-a-^L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-

benzyl-a-^L-rhamnopyranoside (65)

3.56. 5-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-

dideoxy-2-O-methyl-b-^D-glucopyranosyl-(1 ! 3)-2,4-di-

O-benzyl-a-^L-rhamnopyranosyl-(1 ! 3)-2,4-di-O-benzyl-

a-^L-rhamnopyranoside (67)

Reaction of 64 (877 mg, 1.09 mol) and 18 (533 mg,

1.2 mmol) gave trisaccharide 65 (1.05 g, 81%), [a]_Dꢁ1°

(c 0.7). ¹H NMR (CDCl₃, 600 MHz): d 5. (8 (d, partially

overlapped, J_1,2ꢀ 2 Hz, H-1^II), 5.06 (dd, partially over-

lapped, J_1,28.0, J_2,39.6 Hz, H-2^III), 4.79–4.41 (m, 12H,

5 CH₂Ph, incl., d, 4.74 for H-1^Iand d, 4.70, H-1^III), 4.13

(dd, 1H, J_2,33.0, J_3,49.7 Hz, H-3^II), 4.04 (dd, 1H, J_2,3

3.0, J_3,49.5 Hz, H-3^I), 3.87–3.82 (m, 2H, H-2^II,5^IIin that

order), 3.73 (dd, 1H, J_1,21.8, J_2,33.1 Hz, H-2^I), 3.69–3.54

(m, 7H, H-5^I,1⁰⁰a, 4^II,4^Iin that order, incl., s, 3.64,

COOCH₃), 3.40 (t, J 9.3 Hz, H-3^III), 3.34, 3.32 (2 d, par-

tially overlapped, J 12.6 Hz, CH₂Br), 1.32 (m, partially

overlapped, H-1⁰⁰a), 3.14 (dd, partially overlapped, J_4,5

9.9 Hz, H-4^III), 3.11 (m, partially overlapped, H-5^III),

2.30 (t, 2H, J 7.4 Hz, H-5⁰⁰a,b), 1.65–1.60 (m, 2H, H-

4⁰⁰a,b), 1.57–1.52 (m, 2H, H-2⁰⁰), 1.37–1.30 (m, 2H, H-

3⁰⁰a,b), 1.27 (d, 3H, J_5,66.2 Hz, H-6^I), 1.26 (d, 3H, J_5,6

6.2 Hz, H-6^II), 1.14 (d, 3H, J_5,65.9 Hz, H-6^III); ¹³C

NMR (CDCl₃, 150 MHz): d 100.56 (C-1^III), 100.26 (C-

1^II), 97.12 (C-1^I), 80.96 (C-3^III), 80.47, 80.46 (C-4^I,4^II),

Methylation of 66 (800 mg), as described above for sim-

ilar conversions, gave 67 (740 mg, 93%). [a]_Dꢁ3° (c 0.7).

¹H NMR (CDCl₃, 600 MHz): d 5.09 (d, 1H, J_1,21.6 Hz,

H-1^II), 4.98–4.45 (m, 12H, 5 CH₂Ph, incl., 4.72, d, J_1,2

1.8 Hz, H-1^I, incl., 4.61, d, J_1,27.9 Hz, H-1^III), 4.14

(dd, 1H, J_2,33.2, J_3,49.6 Hz, H-3^II), 4.04 (dd, 1H, J_2,3

3.1, J_3,49.5 Hz, H-3^I), 3.89 (dd, 1H, H-2^II), 3.86 (m,

1H, H-5^II), 3.72 (dd, 1H, H-2^I), 3.68–3.59 (m, 9H, H-

5^I, 4^II, 1⁰⁰a, in that order, incl., 3.65, s, COOCH₃, 3.64,

s, OCH₃-2), 3.55 (t, 1H, J 9.5 Hz, H-4^I), 3.35–3.30 (m,

2H, H-3^III,1⁰⁰b), 3.13 (dd, 1H, J_2,39.6 Hz, H-2^III),

3.06–3.02 (m, 2H, H-3^III,1⁰⁰a,b), 3.25 (dd, 1H, H-2^III),

3.04–3.02 (H-4^III,5^III), 2.30 (t, 2H, J 7.4 Hz, H-5⁰⁰a,b),

1.65–1.60 (m, 2H, H-4⁰⁰a,b), 1.56–1.52 (m, 2H, H-2⁰⁰),

1.38–1.31 (m, partially overlapped, H-3⁰⁰a,b), 1.31 (d,

partially overlapped, J_5,66.3 Hz, H-6^II), 1.25 (d, 3H,

J_5,66.2 Hz, H-6^I), 1.10 (d, 3H, J_5,65.8 Hz, H-6^III); ¹³C

NMR (CDCl₃, 150 MHz): d 103.54 (C-1^III), 100.41

4308

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

(C-1^II), 97.37 (C-1^I), 84.75 (C-2^III), 82.64 (C-3^III), 80.90

(C-4^II), 80.58 (C-4^I), 79.23 (C-2^II), 78.56 (C-3^I), 78.48

(C-3^II), 78.16 (C-2^I), 75.40, 74.92, 74.57, 73.21, 72.55

(5 CH₂Ph), 70.19 (C-5^III), 68.46 (C-5^II), 68.08 (C-5^I),

67.57 (C-4^III), 67.29 (C-1⁰⁰), 60.70 (OCH₃-2), 51.46

(COOCH₃), 33.92 (C-5⁰⁰), 29.07 (C-2⁰⁰), 25.69 (C-3⁰⁰),

24.66 (C-4⁰⁰), 18.40 (C-6^III), 18.01 (C-6^II), 17.89 (C-6^I);

ESI-MS: m/z 1080.5354 ([M+Li]⁺). Anal. Calcd for

C₆₁H₇₅N₃O₁₄: C, 68.20; H, 7.04; N, 3.91. Found: C,

68.44; H, 7.01; N, 3.90.

(s, partially overlapped, OCH₃-2), 3.64 (t, partially over-

lapped, H-4^II), 3.61, 3.60 (2 t, partially overlapped, J

6.5 Hz, H-1⁰⁰a), 3.53 (t, 1H, J 9.5 Hz, H-4^I), 3.32, 3.30

(2 t, partially overlapped, H-1⁰⁰b), 3.31 (dd, partially

overlapped, H-3^III), 3.19–3.18 (m, 2H, 2^III,5^III), 2.30 (t,

2H, J 7.6 Hz, H-5⁰⁰a,b), 2.19, 2.09 (2 d, 2H, J 14.9 Hz,

H-2⁰a,b), 1.36–1.31 (m, 5H, H-3⁰⁰a,b, incl., 1.31, d, J_5,6

6.2 Hz, H-6^II), 1.24 (d, 3H, J_5,66.2 Hz, H-6^I), 1.21,

1.18 (2 s, 6H, 2 CH⁰₃), 1.03 (d, J_5,66.2 Hz, H-6^III); ¹³C

NMR (CDCl₃): d 103.74 (C-1^III), 100.51 (C-1^II), 97.41

(C-1^I), 84.93 (C-2^III), 80.85 (C-4^II), 80.57 (C-4^I), 80.04

(C-3^III), 79.16 (C-2^II), 78.73 (C-3^II), 78.49 (C-3^I), 78.07

(C-2^I), 74.89, 74.52, 73.73, 73.29, 72.51 (5 CH₂Ph),

70.88 (C-5^III), 69.39 (C-3⁰), 68.45 (C-5^II), 68.09 (C-5^I),

67.27 (C-1⁰⁰), 60.55 (OCH₃-2), 55.57 (C-4^III), 51.45

(COOCH₃), 47.73 (C-2⁰), 33.92 (C-5⁰⁰), 29.31, 29.29 (2

CH₃⁰), 29.06 (C-2⁰⁰), 25.67 (C-3⁰⁰), 24.65 (C-4⁰⁰), 18.09

(C-6^III), 18.01 (C-6^II), 17.89 (C-6^I); ESI-MS 1148.5973

([M+H]⁺). Anal. Calcd for C₆₆H₈₅NO₁₆: C, 69.03; H,

7.46; N, 1.22. Found: C, 69.11; H, 7.43; N, 1.24.

3.57. 5-Methoxycarbonylpentyl 4,6-dideoxy-3-O-benzyl-

4-(3-hydroxy-3-methylbutyramido)-2-O-methyl-b-^D-

glucopyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamnopyr-

anosyl-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyranoside

(69)

Treatment of azide 67 (700 mg) with H₂S, as described

in the general procedure, gave 5-methoxycarbonylpentyl

4-amino-4,6-dideoxy-3-O-benzyl-2-O-methyl-b-^D-gluco-

pyranosyl-(1 ! 3)-2,4-di-O-benzyl-a-^L-rhamnopyrano-

syl-(1 ! 2)-3,4-di-O-benzyl-a-^L-rhamnopyranoside (68,

3.58. 5-Methoxycarbonylpentyl 4,6-dideoxy-4-(3-hydroxy-

3-methylbutanamido)-2-O-methyl-b-^D-glucopyranosyl-

(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 2)-a-L-rhamnopyr-

anoside (70)

1

510 mg, 75%). H NMR (600 MHz, CDCl₃): d 5.11 (d,

1H, J_1,21.7 Hz, H-1^II), 5.03–4.47 (m, 12H, 5 CH₂Ph,

incl, 4.71, d, J_1,21.9 Hz, H-1^I, ꢀ4.71, d, J_1,2ꢀ 7.6 Hz,

H-1^III), 4.21 (dd, 1H, J_2,33.1, J_3,49.6 Hz, H-3^II), 4.06

(dd, 1H, J_2,33.1, J_3,49.6 Hz, H-3^I), 3.94 (dd, 1H, J_2,3

3.2 Hz, H-2^II), 3.86 (m, 1H, H-5^II), 3.72 (dd, 1H, H-

2^I), 3.67 (s, partially overlapped, OCH₃-2), ꢀ3.65 (m,

partially overlapped, H-5^I), 3.65 (s, partially overlapped,

COOCH₃), 3.66 (t, partially overlapped, H-4^II), 3.61,

3.60 (2 t, partially overlapped, J 6.5 Hz, H-1⁰⁰a), 3.57

(t, J 9.4 Hz, H-4^I), 3.32, 3.30 (2 t, partially overlapped,

H-1⁰⁰b), 3.16 (m, 2H, H-2^III,3^III), 3.07 (m, 1H, H-5^III),

2.50–2.49 (m, 1H, H-4^III), 2.30 (t, 2H, J 7.4 Hz, H-

5⁰⁰a,b), 1.65–1.60 (m, 2H, H-4⁰⁰a,b), 1.56–1.52 (m, 4H,

H-2⁰⁰a,b), 1.37–1.31 (m, 5H, H-3⁰⁰a,b, incl., 1.32, d, J_5,6

6.2 Hz, H-6^II), 1.25 (d, 3H, J_5,66.3 Hz, H-6^I), 1.07 (d,

J_5,66.2 Hz, H-6^III); ¹³C NMR (150 MHz, CDCl₃): d

103.75 (C-1^III), 100.43 (C-1^II), 97.36 (C-1^I), 85.27 (C-

2^III), 83.97 (C-3^III), 80.91 (C-4^II), 80.53 (C-4^I), 79.24

(C-2^II), 78.33 (C-3^I), 78.00, 77.98 (C-2^I, 3^II), 74.94,

74.83, 74.44, 73.14, 72.47 (5 CH₂Ph), 72.46 (C-5^III),

68.39 (C-5^II), 68.01 (C-5^I), 67.18 (C-1⁰⁰), 60.35 (OCH₃-

2), 58.03 (C-4^II), 51.39 (COOCH₃), 33.84 (C-5⁰⁰), 29.00

(C-2⁰⁰), 25.61 (C-3⁰⁰), 24.58 (C-4⁰⁰), 17.98 (C-6^III), 17.93

(C-6^II), 17.81 (C-6^I); ESI-MS 1048.5409 ([M+H]⁺)

C₆₁H₇₈NO₁₄+H requires 1048.5422.

Hydrogenolysis of 69 (400 mg), as described above, gave

1

70 (220 mg, 90%), [a]_Dꢁ54° (c 0.6, H₂O). H NMR

(600 MHz, D₂O): d 5.03 (d, 1H, J_1,21.7 Hz, H-1^I), 4.75

(d, 1H, J_1,21.8 Hz, H-1^I), 4.73 (d, 1H, J_1,28.0 Hz, H-

1^III), 4.27 (dd, 1H, J_2,33.3 Hz, H-2^II), 4.00 (dd, partially

overlapped, H-2^I), 3.99 (dd, partially overlapped, H-3^II),

3.83 (m, 1H, H-5^II), 3.79 (dd, 1H, J_2,33.3 Hz, J_3,49.7 Hz,

H-3^I) 3.72 (m, partially overlapped, H-5^I), ꢀ3.70 (m, par-

tially overlapped, H-1⁰⁰a), 3.68 (s, partially overlapped,

COOCH₃), 3.64 (t, partially overlapped, J 10.0 Hz, H-

4^III), 3.60 (t, J 9.7 Hz, partially overlapped, H-4^II), 3.55

(m, partially overlapped, H-5^III), ꢀ3.53 (m partially over-

lapped, H-1⁰⁰b), 3.52 (m, partially overlapped, H-3^III,4^I),

3.13 (dd, 1H, J_2,39.2 Hz, H-2^III), 2.47, 2.44 (2 d, 2H, J

13.7 Hz, H-2⁰a,b), 2.41 (t, 2H, J 7.3 Hz, H-5⁰⁰), 1.67–1.57

(m, 4H, H-2⁰⁰a,b,4⁰⁰a,b), 1.45–1.33 (m, 2H, H-3⁰⁰), 1.31,

1.30 (2 s, partially overlapped, 2 CH⁰₃), 1.29 (d, partially

overlapped, J_5,66.3 Hz, H-6^II), 1.30 (d, 3H, J_5,66.3 Hz,

H-6^I), 1.22 (d, 3H, J_5,66.1 Hz, H-6^III); ¹³C NMR (D₂O,

150 MHz): d 106.42 (C-1^III), 104.80 (C-1^II), 102.41

(C-1^I), 86.02 (C-2^III), 82.39 (C-3^II), 80.80 (C-3^I), 75.60

(C-3^III), 74.17 (C-4^I), 73.91 (C-4^II), 73.52 (C-5^III), 72.97

(C-3⁰), 72.72 (C-2^I), 72.56 (C-2^II), 71.98 (C-5^II), 71.48 (C-

5^I), 70.44 (C-1⁰⁰), 62.81 (OCH₃-2), 59.32 (C-4^III), 54.85

(COOCH₃), 51.68 (C-2⁰), 36.36 (C-5⁰⁰), 31.04, 30.86 (2

OCH₃⁰), 30.90 (C-2⁰⁰), 27.72 (C-3⁰⁰), 26.78 (C-4⁰⁰), 19.81

(C-6^III), 19.36 (2 C, C-6^I,II), ESI-MS 720.3416 ([M+18]⁺)

C₃₁H₅₅NO₁₆Na requires 720.3419.

Treatment of the foregoing amine (455 mg, 0.43 mmol)

with

0.65 mmol), as described for similar conversions, gave

3-hydroxy-3-methylbutyric

acid

(76 mg,

1

amide 69 (424 mg, 86%), [a]_Dꢁ46° (c 0.6). H NMR

(600 MHz, CDCl₃): d 5.35 (d, 1H, J_4,NH9.1 Hz, NH),

5.08 (d, 1H, J_1,21.8 Hz, H-1^II), 5.02–4.46 (m,12H, 5

CH₂Ph, incl., 4.71, d, 1H, J_1,21.7 Hz, H-1^I, incl.,

ꢀ4.62, d, partially overlapped, J_1,2ꢀ 7.7 Hz, H-1^III),

4.15 (dd, 1H, J_2,33.1, J_3,49.6 Hz, H-3^II), 4.04 (dd, 1H,

J_2,33.1, J_3,49.5 Hz, H-3^I), 3.92 (dd, partially over-

lapped, H-2^II), 3.91 (s, partially overlapped, OH⁰), 3.86

(m, 1H, H-5^II), 3.71 (dd, 1H, H-2^I), ꢀ3.67 (t, partially

overlapped, H-4^III), 3.65 (s, partially overlapped,

COOCH₃), ꢀ3.65 (m, partially overlapped, H-5^I), 3.64

3.59. (2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-

(3-hydroxy-3-methylbutyramido)-2-O-methyl-b-^D-gluco-

pyranosyl-(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 3)-a-L-

rhamnopyranosyl-(1 ! 2)-a-^L-rhamnopyranoside (71)

Starting with ester 35 (86 mg) and following the general

procedure for this conversion gave amine 71 (72 mg,

80%). Characteristic NMR signals were at d 3.32–

¹H

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

4309

3.30 (m, H-6⁰⁰), 2.85–2.82 (m, H-7⁰⁰); d 43.34 (C-6⁰⁰),

CH₂CH₃), 1.45, 1.43 (2 t, 3H, CH₂CH₃); d

(CH₂CH₃), 17.89 (CH₂CH₃). ESI-MS: m/z 996.4769

73.50

¹³C

42.83 (C-7⁰⁰). ESI-MS: m/z 872.4615 ([M+H]⁺)

C₃₈H₇₀N₃O₁₉requires 872.4604.

([M+H]⁺) C₄₄H₇₄N₃O₂₂requires 996.4764.

3.60. 1-[2-Aminoethylamido]carbonylpentyl 4,6-dideoxy-

4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-b-^D-

glucopyranosyl-(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 3)-a-

L-rhamnopyranosyl-(1 ! 2)-a-L-rhamnopyranoside]-2-

ethoxycyclobutene-3,4-dione (72)

3.65. (2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-

(3-hydroxy-3-methylbutanamido)-b-^D-glucopyranosyl-

(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 3)-a-L-rhamnopyr-

anosyl-(1 ! 2)-a-^L-rhamnopyranoside (83)

Starting with ester 40 (18.5 mg) and following the gen-

eral procedure for this conversion gave amine 83

(14.6 mg, 79%). Characteristic NMR signals were at

Treatment of amine 71 (20 mg) with squaric acid diethyl

ester, as described in the general procedure for prepara-

tion of this class of compounds, gave monoester 72

(17 mg, 74%). Characteristic NMR signals were at d₁_H

4.80–4.69 (m, partially overlapped, CH₂CH₃), 1.46–

d

3.38 (t, H-6⁰⁰), 2.93 (m, H-7⁰⁰); d

41.94 (C-6⁰⁰),

¹³C

¹H

41.84 (C-7⁰⁰). ESI-MS: m/z 858.4444 ([M+H]⁺)

C₃₇H₆₈N₃O₁₉requires 858.4447.

1.42 (m, 3H, CH₂CH₃); d

(CH₂CH₃). ESI-MS: m/z

C₄₄H₇₄N₃O₂₂requires 996.4764.

70.63 (CH₂CH₃), 15.02

996.4748 ([M+H]⁺)

¹³C

3.66. 1-[2-Aminoethylamido]carbonylpentyl 4,6-dideoxy-

4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-b-^D-

glucopyranosyl-(1 ! 3)-(1 ! 3)-a-^L-rhamnopyranosyl-a-

L-rhamnopyranosyl-(1 ! 2)-a-L-rhamnopyranoside]-2-

ethoxycyclobutene-3,4-dione (84)

3.61. (2-Aminoethylamido)carbonylpentyl a-^L-rhamno-

pyranosyl-(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 2)-L-

rhamnopyranoside (75)

Treatment of amine 83 (14.0 mg) with squaric acid

diethyl ester, as described in the general procedure

for preparation of this class of compounds, gave

monoester 84 (14.0 mg, 90%). Characteristic NMR

Treatment of ester 12 (44 mg), as described for prepara-

tion of 71, gave 75 (32 mg, 70%). Characteristic NMR

signals were at d 3.25 (t, J 6.3 Hz, H-6⁰⁰), 2.74 (t, J

¹H

6.0, H-7⁰⁰); d 41.15 (C-6⁰⁰), 39.66 (C-7⁰⁰). ESI-MS: m/z

signals were at d 4.72–4.64 (m, partially overlapped

¹³C

¹H

613.3184 ([M+H]⁺) C₂₆H₄₉N₂O₁₄requires 613.3184.

with H-1^IV, CH₂CH₃), 1.43–1.38 (m, 3H, CH₂CH₃);

d

73.47 (CH₂CH₃), 17.87 (CH₂CH₃). ESI-MS:

¹³C

3.62. 1-[2-Aminoethylamido]carbonylpentyl a-^L-rhamno-

pyranosyl-(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 2)-L-

rhamnopyranoside]-2-ethoxycyclobutene-3,4-dione (76)

m/z 1004.4446 ([M+Na]⁺) C₄₃H₇₁N₃O₂₂requires

1004.4427.

Treatment of amine 75 (20 mg) with squaric acid diethyl

ester, as described for preparation of 72, gave 76 (15 mg,

63%). Characteristic NMR signals were at d₁_H4.70–4.63

Acknowledgment

This research was supported by the Intramural Research

Program of the NIH, NIDDK.

(m, 2H, CH₂CH₃), 1.41, 1.35 (2 t, 3H, CH₂CH₃); d

73.46 (CH₂CH₃), 17.78 (CH₂CH₃). ESI-MS: m/z

¹³C

735.3177 ([Mꢁ1]^ꢁ) C₃₂H₅₁N₂O₁₇requires 735.3188.

References and notes

3.63. (2-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-

(3-hydroxy-3-methylbutanamido)-2-O-methyl-b-^D-gluco-

pyranosyl-(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 3)-a-L-

rhamnopyranosyl-(1 ! 2)-b-^L-rhamnopyranoside (79)

1. Schneerson, R.; Kubler-Kielb, J.; Liu, T.-Y.; Dai, Z.-D.;

Leppla, S. H.; Yergey, A.; Backlund, P.; Shiloach, J.;

Majadly, F.; Robbins, J. B. Proc. Natl. Acad. Sci. U.S.A

2003, 100, 8945–8950.

2. Chabot, D. J.; Scorpio, A.; Tobery Steven, A.; Little

Stephen, F.; Norris Sarah, L.; Friedlander Arthur, M.

Vaccine 2004, 23, 43–47.

3. Wang, T.; Fellows Patricia, F.; Leighton Terrance, J.;

Lucas Alexander, H. FEMS Immunol. Med. Microbiol.

2004, 40, 231–237.

4. Daubenspeck, J. M.; Zeng, H.; Chen, P.; Dong, S.;

Steichen, C. T.; Krishna, N. R.; Pritchard, D. G.;

Turnbough, C. L. J. Biol. Chem. 2004, 279, 30945–

30953.

5. McNaught, A. D. Carbohydr. Res. 1997, 297, 1–92.

6. Werz, D. B.; Seeberger, P. H. Angew. Chem., Int. Ed. Engl.

2005, 44, 6315–6318.

7. Guo, H.; O’Doherty, G. A., De novo asymmetric synthesis

of antigen Bacillus anthracis tetrasaccharide. Abstracts

from the 232nd ACS National Meeting (ORGN-917), San

Francisco, CA, September 10–14, 2006.

8. Tamborrini, M.; Werz, D. B.; Frey, J.; Pluschke, G.;

Seeberger, P. H. Angew. Chem., Int. Ed. Engl. 2006, 45,

6581–6582.

Starting with ester 2 (86 mg) and following the general

procedure for this conversion gave amine 79 (40 mg,

44%). Characteristic NMR signals were at d 3.31 (t,

¹H

J 6.3 Hz, H-6⁰⁰), 2.84 (t, J 6.3, H-7⁰⁰); d 43.40 (C-6⁰⁰),

¹³C

42.60 (C-7⁰⁰). ESI-MS: m/z 872.4595 ([M+H]⁺)

C₃₈H₇₀N₃O₁₉requires 872.4604.

3.64. 1-[2-aminoethylamido]carbonylpentyl 4,6-dideoxy-

4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-b-^D-

glucopyranosyl-(1 ! 3)-a-^L-rhamnopyranosyl-(1 ! 3)-a-

L-rhamnopyranosyl-(1 ! 2)-b-L-rhamnopyranoside]-2-

ethoxycyclobutene-3,4-dione (80)

Treatment of amine 79 (20 mg) with squaric acid diethyl

ester, as described in the general procedure for prepara-

tion of this class of compounds, gave after chromatogra-

phy ester 80 (16 mg, 70%) Characteristic NMR signals

¹H

were at

d

4.75–4.68 (m, partially overlapped,

4310

R. Saksena et al. / Bioorg. Med. Chem. 15 (2007) 4283–4310

9. Mehta, A. S.; Saile, E.; Zhong, W.; Buskas, T.; Carlson,

R.; Kannenberg, E.; Reed, Y.; Quinn, C. P.; Boons, G.-J..

22. Solladie, G.; Ziani-Cherif, C. J. Org. Chem. 1993, 58,

2181–2185.

´

Chem. Eur. J. 2006, 12, 9136–9149.

10. Adamo, R.; Saksena, R.; Kovac, P. Helv. Chim. Acta

2006, 89, 1075–1089.

23. Liptak, A.; Szabo, L.; Harangi, J. J. Carbohydr. Chem.

1988, 7, 687–699.

´ˇ

24. Saksena, R.; Adamo, R.; Kovac, P. Carbohydr. Res. 2005,

´ˇ

11. Saksena, R.; Adamo, R.; Kovac, P. Bioorg. Med. Chem.

Lett. 2006, 16, 615–617.

12. Wang, D.; Carroll, G. T.; Turro, N. J. T.; Koberstein, J.;

340, 1591–1600.

25. Garegg, P. J.; Norberg, T. Acta Chem. Scand. 1979, B33,

116–118.

´ˇ

26. Kovac, P.; Lerner, L. Carbohydr. Res. 1988, 184, 87–112.

´ˇ

27. Adamo, R.; Kovac, P. Eur. J. Org. Chem. 2006, 2803–

2809.

28. Ness, R. K.; Fletcher, H. G.; Hudson, C. S. J. Am. Chem.

Soc. 1951, 73, 296–300.

´

Kovac, P.; Saksena, R.; Adamo, R.; Herzenberg, L. A.;

Herzenberg, L. A.; Steinman, L. Proteomics 2007, 7, 180–

184.

13. Liptak, A.; Szurmai, Z.; Nanasi, P. Carbohydr. Res. 1982,

´

99, 13–21.

´

´ˇ

14. Liptak, A.; Nanasi, P.; Neszmelyi, A.; Wagner, H.

Tetrahedron 1980, 36, 1261–1268.

29. Saksena, R.; Chernyak, A.; Karavanov, A.; Kovac, P. In

Conjugating Low Molecular Mass Carbohydrates to Pro-

teins. 1. Monitoring the Progress of Conjugation; Lee, Y. C.,

Lee, R., Eds.; Academic Press, 2003; Vol. 362, pp 125–139.

´

15. Liptak, A.; Imre, J.; Harangi, J. J.; Nanasi, P. Tetrahedron

1982, 38, 3721–3727.

´ˇ

30. Saksena, R.; Ma, X.; Kovac, P. Carbohydr. Res. 2003, 338,

2591–2603.

31. Ma, X.; Saksena, R.; Chernyak, A.; Kovac, P. Org.

16. Pozsgay, V.; Jennings, H. J. J. Org. Chem. 1988, 53, 4042–

4052.

´

17. Kerekgyarto, J.; Szurmai, Z.; Liptak, A. Carbohydr. Res.

´ˇ

1993, 245, 65–80.

´ˇ

Biomol. Chem. 2003, 1, 775–784.

32. Zhang, J.; Yergey, A.; Kowalak, J.; Kovac, P. Carbohydr.

´ˇ

18. Kovac, P.; Edgar, K. J. J. Org. Chem. 1992, 57, 2455–

2467.

Res. 1998, 313, 15–20.

33. Chernyak, A.; Karavanov, A.; Ogawa, Y.; Kovac, P.

Carbohydr. Res. 2001, 330, 479–486.

´ˇ

19. Bundle, D. R.; Josephson, S. Carbohydr. Res. 1980, 80,

75–85.

20. Wessel, H. P.; Bundle, D. R. Carbohydr. Res. 1983, 124,

301–311.

21. Palomino, J. C. C.; Rensoli, M. H.; Bencomo, V. V.

J. Carbohydr. Chem. 1996, 15, 137–146.

34. Friebolin, H. Basic One- and Two Dimensional NMR

Spectroscopy, 2nd ed.; Wiley: New York, 1993.

35. Tietze, L. F.; Fischer, R.; Guder, H. J.; Neumann, M.

Justus Liebigs Ann. Chem. 1987, 847–856.

Article Doi

Immunogens related to the synthetic tetrasaccharide side chain of the Bacillus anthracis exosporium

DOI: 10.1016/j.bmc.2007.03.057

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Full text of DOI:10.1016/j.bmc.2007.03.057

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