1-Methyl 1′-cyclopropylmethyl: An acid labile O-protecting group for polymer-supported oligosaccharide synthesis

TETRAHEDRON

Pergamon

Tetrahedron 57 ,2001) 6679±6693

1-Methyl 1⁰-cyclopropylmethyl: an acid labile O-protecting group

for polymer-supported oligosaccharide synthesis

Eva Eichler, Fengyang Yan, Jennifer Sealy and Dennis M. Whit®eld^p

National Research Council, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ont., Canada K1A0R6

Received 18 April 2001; accepted 22 May 2001

AbstractÐR,S-1-Methyl 1⁰-cyclopropylmethanol can be converted to its trichloroacetimidate derivative. Lewis acid catalyzed etheri®cation

can then be used to prepare methyl cyclopropylmethyl ,MCPM) ethers. Thus, ethyl 2,3,4-tri-O-benzyl-6-O-,R,S-1-methyl 1⁰-cyclopropyl-

methyl)-1-thio-a/b-d-glucopyranoside was prepared and the linker polymer combination ,MPEG),DOX)OH glycosylated. The MCPM

group was cleaved with 10% tri¯uoroacetic acid in CH₂Cl₂and the resulting alcohol glycosylated. After protecting group manipulations

and cleavage the peracetylated disaccharide GlcNAc,b1!6)GlcOAc was isolated. Similarly the 4,6-O-phenylboronate diester of ethyl

1-thio-b-d-galactopyranoside was regioselectively etheri®ed at O-3 and after boronate cleavage the sugar benzoylated to give ethyl

2,4,6-tri-O-benzoyl-3-O-,R,S-1-methyl 1⁰-cyclopropylmethyl)-1-thio-b-d-galactopyranoside. Through a similar sequence of glycosylation

of ,MPEG),DOX)OH, MCPM deprotection, glycosylation, functional group manipulation and cleavage the peracylated disaccharide

GlcNAc,b1!3)Gal,b1!)DOXOAc was prepared. Finally, the donor 2,6-di-O-benzoyl-4-O-levulinoyl-3-O-,R,S-1-methyl 1⁰-cyclopropyl-

methyl)-1-thio-b-d-galactopyranoside was prepared and elaborated into the trisaccharide GlcNAc,b1!3)[Glc,b1!4)]Gal,b1!)DOXOH.

This trisaccharide was elaborated into the pentasaccharide Neu5Ac,a2!3)Gal,b1!4)GlcNAc,b1!3) [Glc,b1!4)]Gal,b1!)DOXOH

using GalE-LgtB fusion and CMP-Neu5Ac synthetase/sialyltransferase fusion enzymes. This pentasaccharide is a single repeat unit of

1. Introduction

presence of acetic anhydride to yield easily puri®able pera-

cetylated sugar-DOXOAc products.¹¹

Improved methods for the preparation of oligosaccharides

are necessary to meet the growing demand for glycothera-

peutics.¹To this end our research group is investigating

methods to simplify oligosaccharide synthesis.²In particu-

lar, we are interested in preparing oligosaccharides that can

be used as antibacterial vaccines.³One appealing approach

is to use polymer-supported methods that largely avoid the

need for expensive chromatography since the experi-

mentalist needs to purify the same polymeric support and

not the variable sugar at each synthetic step.⁴Although, a

number of supports⁵and linkers⁶have been tested we have

concentrated on the combination of the soluble polymer

,MPEG), CH₃O±,CH₂CH₂O)_n±H⁷and the linker dioxy-

xylene ,DOX), ±,O)-CH₂-Ph-CH₂-,O)±.⁸This combination

,MPEG),DOX) is stable to most reaction conditions used in

oligosaccharide chemistry⁹and has been used with sialidase

enzymes to synthesize sialylated oligosaccharides.¹⁰The

polymer bound compounds can be puri®ed by precipitation

with tert-butyl methylether ,TBME) and reprecipitation

from absolute ethanol. The ,MPEG),DOX) bound products

can be released by hydrogenation⁸or by Sc,OTf)₃in the

For implementation of a chemistry based ,MPEG),DOX)

synthetic strategy, a suite of protecting groups with different

cleavage conditions is necessary. If acyl groups adjacent to

the anomeric center are used to control the stereoselectivity

of the glycosylation reactions then protecting groups which

can be cleaved in the presence of both the linker DOX and

these acyl groups are necessary. Also, the protecting groups

must be absolutely stable to glycosylation conditions. For

example, experimentation with tert-butyl dimethylsilyl

ether protected glycosyl donors showed that some silyl

ether was cleaved during glycosylation resulting in unde-

sirable extraneous oligosaccharides.¹²To date isopropyl-

idene,¹³benzylidene,⁸phenylboronate diesters,¹⁴3-iodo-4-

methoxybenzyl¹⁵and levulinoyl have been successfully

used with the ,MPEG),DOX) combination for O-protection.

Especially, if branched oligosaccharides are to be synthe-

sized, more protecting groups would be bene®cial.

Recently, a number of protecting groups have been

examined in conjunction with linkers different from DOX

for use in MPEG supported oligosaccharide synthesis.¹⁶

Besides the intrinsic properties of ease of preparation and

cleavage other useful properties of protecting groups for

glycosyl donors are: ,1) small steric size, ,2) electron

donating ,activating) potential, ,3) easily characterized by

¹H NMR spectroscopy, ,4) cleavable by reagents that are not

Keywords: polymer support; protecting groups; thioglycosides; cyclopro-

panes; glycosidation.

Corresponding author. Tel.: 1613-993-5265; fax: 1613-952-9092;

e-mail: dennis.whit®eld@nrc.ca

p

PII: S0040-4020,01)00582-8

6680

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

Scheme 3.

for polymer-supported oligosaccharide synthesis. To this

end we etheri®ed the primary OH-6 of the 1-thioglucose

derivative 1ab²²to give the anomeric mixture 2ab ,75%

yield) using standard alkoxide displacement of a primary

halide ,Scheme 1). Either anomer or in general the anomeric

mixture could be used under standard N-iodosuccinimide/

tri¯uoromethanesulfonic acid ,NIS/TfOH) conditions²³to

glycosylate ,MPEG),DOX)OH, 3, to yield a 65:35a:b

anomeric mixture of polymer bound glycosides 4ab. As

judged by ¹H NMR of 4ab the glycosylation reaction

went to completion. By testing various concentrations of

tri¯uoroacetic acid ,TFA) in dichloromethane it was deter-

mined that 40±60% TFA was suf®cient to affect cleavage at

room temperature. However, under these conditions the

DOX linker was partly ,40% TFA) or mostly cleaved

,60% TFA) too. By conducting the cleavage reaction at

58C for 16 h in 40% TFA it was possible to isolate alcohol

5ab accompanied by only a small amount of DOX

cleavage. Thus, cyclopropylmethyl ethers could be used

for oligosaccharide synthesis if glycosides more acid stable

than DOX are used.

Scheme 1.

very context sensitive, ,5) contains few if any electronega-

tive functional groups which may coordinate Lewis acid

promotors used in glycosylation reactions. To this end we

were intrigued by the reported use of the acid cleavable

cyclopropylmethyl ether for the protection of the hydroxyl

groups of serine and threonine for solid phase peptide syn-

thesis¹⁷since this group appears to satisfy all ®ve criteria

listed above. ,1) It has only four carbons. ,2) Ethers are

electron donating. In fact, electronic energy calculations

suggest that the cyclopropyl group is as ef®cient an electron

donor as phenyl.¹⁸,3) The characteristic high ®eld ¹H NMR

resonances of cyclopropyl resonate in a region where few

sugar resonances are expected. ,4) It is acid cleavable which

should not be very sensitive to steric effects. ,5) It has very

little tendencies to coordinate Lewis acids. Cyclopropyl-

methyl ether has been used as an O-protecting group for

phenols too.¹⁹This communication reports the development

of an analogue, 1-methyl 1⁰-cyclopropylmethyl ether²⁰as an

O-protecting group for polymer-supported oligosaccharide

synthesis of a branched trisaccharide. This oligosaccharide

is the branchpoint of a single repeat unit of the capsular

polysaccharide of Group B Streptococcus type 1A²¹and

can be converted to the pentasaccharide single repeat unit

by the use of bacterial glycosyl transferases.²

However, these conditions are unlikely to be general. We

therefore decided to examine 1-methyl substituted 1⁰-cyclo-

propylmethyl ,MCPM) ethers since these should be more

acid labile. There is one brief report of the use of this group

in peptide synthesis.²⁰Since the site of attachment is now a

secondary center we thought it unlikely that halide dis-

placement reactions would be ef®cient. Therefore, we

prepared the trichloroacetimidate 7 ,MCPMTCI) of racemic

alcohol 6 ,Scheme 2). It was necessary to use 1 equiv. of the

strong base sodium hydride for the reaction to proceed to

completion ,see Section 4). The resulting reagent is unstable

in solution but is stable at 2208C as the pure compound. At

room temperature, it is a liquid that can be handled by

routine syringe methods.

2. Results and discussion

We ®rst tested cyclopropylmethyl as an O-protecting group

A variety of Lewis acids were tested for their ability to

activate 7. The best results were obtained with boron

tri¯uoride etherate ,BF₃´OEt₂) and silver tri¯uoromethane-

sulfonate ,AgOTf) in chlorinated solvents. Thus, 1 could

be etheri®ed in a reasonable yield of 56% to give the

anomeric mixture 8ab ,Scheme 3). The yield was lowered

Scheme 2.

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

6681

Scheme 4.

by concomitant formation of 2,3,4-tri-O-benzyl-1,6-

anhydro-b-d-glucopyranose. MCPM ethers 8ab are also

inseparable diastereomers. In this case, except for the H-6

are reported. Thus, yields from cleavage reactions which

are based on puri®ed compounds and therefore include all

losses due to on polymer steps with less than 100% conver-

sion. In this case eight on polymer steps were used and so

each step proceeded on average in just over 80% yield

,80%⁸17%). The benzyl ethers and the DOX glycoside

of 13ab were removed by hydrogenation and after acetyl-

ation and puri®cation the peracetylated disaccharide 14ab

was isolated.

1

and MCPM CH resonances, the H NMR resonances were

not distinguishable ,see Section 4). Thioglycosides 8ab

were readily activated to glycosylate 3 to yield a

65:35a:b anomeric mixture of polymer bound glycosides

1

9ab. Experiments, monitored by H NMR, at increasing

volume percentages of TFA in CH₂Cl₂showed that the

MCPM ether could be cleaved with as little as 7.5% TFA

in CH₂Cl₂to yield 5ab. Thus, MCPM ethers are stable to

5% TFA. This contrasts with p-methoxybenzyl ethers which

are partly cleaved with 5% TFA. Routinely, we use 10%

TFA for 1 h at room temperature to effect cleavage. The

polymer is collected by precipitation with TBME and

further puri®ed by reprecipitation from absolute ethanol.

In the cases examined to date, the DOX linker is stable to

these conditions. Interestingly under the Sc,OTf)₃cleavage

conditions the MCPM group is also cleaved and the position

acetylated cf. 9ab to 10ab. It should be noted that in the

absence of PEG sugars are unstable towards the Sc,OTf)₃

cleavage conditions.¹¹

Next, the secondary O-3 position of d-galactose was selec-

tively protected as its O-3 MCPM ether 16ab by taking

advantage of the easily prepared 4,6-phenylboronate diester

of ethyl 1-thio-b-d-galactopyranoside, 15 ,Scheme 5).²⁷

The boronate is easily cleaved with IRA-743 resin to give

triol 17ab in 46% yield from 15. The MCPM diastereomers

1

were separately identi®able in the H and ¹³C NMR but

could not be separated. In this and all subsequent

compounds the diastereomer with the most up®eld

MCPM-CH resonance is arbitrarily assigned as a. Attempts

to selectively benzoylate 17ab at the 2,6-positions were

unsuccessful as the only diesters isolated were the diastereo-

meric 4,6-dibenzoates 18a and 18b in low yields of 5 and

4%, respectively. Attempts to assign the absolute con®gu-

ration at the MCPM chiral carbon were unsuccessful. The

tribenzoate 19ab was also prepared. This donor was easily

activated to form glycosides 20ab and the MCPM group

readily cleaved with 10% TFA to give alcohol 21. Glyco-

sylation at 2208C with known donor 22²⁸to give 23 was

necessary to prevent migration of the O-2benzoate which

occurred at higher temperatures. Subsequent protecting

group manipulations readily led to disaccharide 24.¹⁴

Surprisingly, the Galp O-2benzoate was not cleaved during

this sequence.

In order to demonstrate the usefulness of this process, alco-

hol 5ab was glycosylated with glucosamine derivative 11²⁴

under triethylsilyltri¯uoromethanesulfonate ,TESOTf) acti-

vation conditions²⁵to cleanly yield disaccharide 12ab

,Scheme 4). One major advantage of polymer-supported

chemistry is the ease of functional group manipulation.

Thus, 12ab was partly deprotected and cleaved from the

polymer in a two step procedure using ethylenediamine²⁶to

cleave the phthalimide group followed by the combination

of Sc,OTf)₃and acetic anhydride to both O- and N-acetylate

as well as effect cleavage from the polymer. The major

product 13ab was isolated and puri®ed in 20% yield. For

polymer-supported reactions only gravimetric recoveries

Next, a branched trisaccharide was synthesized using the

6682

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

Scheme 5.

MCPM group ,Scheme 6). Readily available diol 25¹⁸was

selectively etheri®ed with MCPMTCI to give 26ab in a

moderate yield of 38%. Subsequent levulinoylation

produced 27ab in 71% yield. The MCPM diastereomers

of 26ab or 27ab were not completely separable but were

frequently isolated enriched in one diastereomer thus allow-

NMR were completely assigned and are entirely consistent

with the expected regio- and stereochemistry.

The MCPM group thus allowed for an ef®cient synthesis of

trisaccharide 36 which is a key building block for the syn-

thesis of the pentasaccharide representing a single repeating

unit of the type 1A Group B Streptococcus capsular poly-

saccharide. Trisaccharide 36 was deacylated to 37 and then

enzymatically glycosylated to yield the type 1A Group B

Streptococcus single repeat pentasaccharide, 39 ,Scheme 7).

Trisaccharide 37 was treated with gluco-galacto epimerase/

b-d-galactose transferase fusion enzyme²and uridinedi-

phospho-glucose ,UDP-Glc) to generate tetrasaccharide

38. Subsequent treatment with cytidinemonophospho-

neuraminic acid synthetase/a-d-neuraminic acid transferase

fusion enzyme,²⁹cytidinetriphosphate ,CTP) and sialic acid

,to generate CMP-Neu5Ac) yielded 39. After puri®cation

by C-18 reverse phase chromatography 39 was charac-

1

ing the assignment of the H- and ¹³C NMR spectra ,see

Section 4). Glycosylation of the polymer led to 28ab.

Previous experience showed that glycosylation at O-3

followed by O-4 is preferred.¹³Thus, the MCPM ether

was cleaved to give 29 and the O-3 alcohol was glycosylated

as for 23 to give 30. To prove the 1,3-regiochemistry disac-

charide 30 was cleaved from the polymer to give known

b1,3-linked disaccharide 31.¹⁵The levulinoyl group of 30

was cleaved with buffered hydrazine to give polymer bound

Galp OH-4 disaccharide 32 which was subsequently glyco-

sylated with glucose donor 33 to yield trisaccharide 34.

Trisaccharide 34 could be cleaved to known 35¹⁵or treated

as for 23 to 24 to give peracylated trisaccharide 36 in 10%

1

terized by H NMR and MS. As well, its properties were

compared to other synthetic single repeat units.³⁰

1

yield ,almost 80% per step; 80%¹⁰11%). The H and ¹³C

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

6683

Scheme 6.

3. Conclusions

glycosylation reactions are done in the presence of only

very weak nucleophiles. In our studies some glycosylation

conditions did lead to MCPM cleavage. These all involved

reactions at high concentrations of Lewis acids ,.1 equiv.

to ,MPEG)) such as TESOTf in the absence of molecular

sieves or added bases. This is comparable to the sensitivity

of 4-methoxybenzyl during glycosylation reactions.³³As

exempli®ed in this work standard NIS/TfOH glycosylation

conditions can be used with MCPM in the presence of

molecular sieves. Many plausible combinations of

glycosyl donors and protecting group strategies need to be

tested to fully evaluate the potential of the MCPM group.

However, it is clear that MCPM can be successfully used for

A number of recent publications suggest orthogonal protect-

ing group strategies for polymer-supported oligosaccharide

synthesis.³¹Although most remain to be tested in an actual

target oriented synthesis. Our reasoning was that one

plausible reagent for cleavage of a protecting group in the

presence of base labile esters and O-benzyl ethers is mild

acid. Since some glycosylation reactions involve strong acid

conditions it is necessary to carefully choose the acid sensi-

tivity of the proposed protecting group.³²Most acid

promoted cleavage reactions also require a nucleophile

and this requirement allows some differentiation since

6684

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

Scheme 7.

polymer-supported oligosaccharide chemistry. In fact this

report demonstrates its use to synthesize a primary linked

disaccharide, a secondary linked disaccharide and a

branched trisaccharide.

4. Experimental

4.1. Materials and general methods

Silica gel ,230±400 mesh) was used for ¯ash chroma-

tography. All starting materials were dried overnight in

vacuo ,10²³mmHg) over KOH or P₂O₅prior to use, and

the solvents were distilled from appropriate drying agents.

Optical rotations were measured ,l589 nm) at room

This trisaccharide represents the branchpoint of type 1A

Group B Streptococcus capsular polysaccharide and could

be converted to the single repeat pentasaccharide using

enzymes. The availability of fusion enzymes derived from

bacterial enzymes allows the use of relatively inexpensive

donors such as UDP-Glc or the in situ generation from

relatively inexpensive compounds like CTP and Neu5Ac.

The chemical synthesis required only ®ve silica gel

chromatographic steps including the three building blocks

and the enzymatic synthesis only two C-18 reverse phase

puri®cations which is a great simpli®cation over the tra-

ditional stepwise chemical synthesis. With further optimi-

zations the feasibility of commercial production of entirely

synthetic oligosaccharide based vaccines becomes more

realistic.

1

temperature in a 10 cm 1 mL cell. The H and ¹³C NMR

spectra were recorded in deuteriochloroform solution at

500.1 or 600.2MHz and 125.8 or 150.9 MHz, respectively.

¹H NMR spectra in CDCl₃, CD₃OD, C₆D₆or D₂O were

referenced to residual CHCl₃at 7.26 ppm, CHD₂OD at

3.31 ppm, C₆HD₅at 7.26 ppm or were referenced to internal

acetone 2.225 ppm. ¹³C NMR spectra in CDCl₃, CD₃OD,

C₆D₆or D₂O were referenced to the central peaks at 77.0,

35.0 and 129.0 ppm or to internal acetone at 31.07 ppm.

1

Assignments were made by standard H±¹H-COSY and

¹³C±¹H-COSY experiments. ¹H chemical shifts are reported

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

6685

to two decimal places and ¹³C shifts to one. In the case of

closely separated resonances an additional ®gure is added to

show that they are separately identi®able. The sugar residue

nomenclature is shown in Scheme 7. For polymer bound

samples the MPEG methylenes were saturated and quanti-

tation was made by comparing integrals to the terminal

methyl of the MPEG. Advantage was also taken of gradient

enhanced 1D-selective TOCSY and NOESY experiments.

Typically 64 transients were used for TOCSY spectra with

mixing times from 20 to 150 ms and 6 k transients for

NOESY spectra with mixing times of 500 ms.

H-6ba), 3.76 ,2H, m, H-6aa, H-6b), 3.85 ,2H, m, H-2a,

H-3a), 4.18 ,1H, brd, J8.8 Hz, H-5a), 4.47 ,1H, d, J_1,2

10.3 Hz, H-1b), 4.61 ,1H, d, J11.2Hz, CH HAr), 4.69

,1H, d, J12.2 Hz, CHHAr), 4.70 ,2H, d, J10.8 Hz,

CHHAr), 4.76 ,1H, d, J12.2 Hz, CHHAr), 4.80 ,2H, d,

J10.8 Hz, CHHAr), 4.91 ,1H, d, J11.2Hz, C HHAr),

4.93 ,2H, d, J10.8 Hz, CHHAr), 4.98 ,2H, d, J

10.8 Hz, CHHAr), 5.41 ,1H, d, J_1,24.8 Hz, H-1a), 7.31

,26H, m, ArH), 7.39 ,4H, m, ArH); ¹³C NMR ,CDCl₃) d

1.0 ,CH₂-Cp), 3.0 ,CH₂-Cp), 3.2, CH₂-Cp), 10.4 ,CH-Cp),

14.8 ,SCH₂CH₃a), 15.1 ,SCH₂CH₃b), 23.7 ,SCH₂CH₃a),

25.1 ,SCH₂CH₃b), 68.8 ,C-6a), 69.5 ,C-6b), 70.5 ,C-5a),

72.4, 75.0, 75.7 ,CH₂Ar), 76.1 ,OCH₂Cp), 77.2,C-4 b), 77.5

,C-4a), 79.2,C-5 b), 79.5 ,C-3a), 81.8 ,C-2b), 82.6 ,C-2a),

83.1 ,C-1a), 85.1 ,C-1b), 86.7 ,C-3b), 127.0, 128.0, 137.86,

137.95, 138.5, 138.8 ,ArC); MS-FAB1ve 547.3 ,M2H¹);

HRMS C₃₃H₃₉O₅S ,M2H¹, 547.2504) Calcd 547.2519;

Anal. Calcd for C₃₃H₄₀O₅S ,548.7446): C 72.23, H 7.34,

found: C 72.32, H 7.79.

The mass spectral analysis was done on a forward mass

spectrometer. Fast atom bombardment ,FAB) MS was

performed using Xenon atom at 6 kV as source. Thio-

glycerol or a mixture of glycerol and thioglycerol were

used as FAB matrix. Typically, 10±15 full range, low reso-

lution MS scans were averaged to yield a low resolution

mass spectrum. For high resolution MS, the electric sector

was scanned over the range of interest. Typically polyethyl-

ene glycol or polypropylene glycol was used as an internal

mass standard and between 75 and 150 scans were averaged.

MALDI-MS spectra were taken on a Voyager-DE STR

Biochemistry Workstation. 2,5-Dihydroxybenzoic acid

was used as MALDI matrix. Microanalyses were performed

by Guelph Chemical Laboratories Ltd., Guelph, Ontario.

4.2.3. )MPEG))DOX)yl 2,3,4-tri-O-benzyl-6-O-cyclo-

propylmethyl-a/b-d-glucopyranoside 4ab. Donor 2ab

,500 mg, 0.92mmol), ,MPEG),DOX)OH , 3, 3.2g,

Ê

0.62mmol) and powdered molecular sieves 4 A ,about

1 g) were dried together in vacuo. The ¯ask was opened

to an argon atmosphere and dichloromethane ,17 mL) was

added and the mixture cooled in an ice bath. To this mixture

was added NIS ,910 mg, 4.61 mmol) followed by TESOTf

,104 mL, 0.46 mmol). After stirring for 2h the reaction was

quenched with diisopropylethylamine ,DIEPA, 200 mL)

and standard worked up afforded 4ab ,3.1 g; 97%): partial

¹H NMR ,CDCl₃) d 0.20 ,m, CHH-Cp), 0.51 ,m, CHH-Cp),

1.06 ,m, CH-Cp), 4.4±5.0 ,m, CH₂Ar, H-1a, H-1b),7.30

,m, ArH).

4.2. )MPEG) general work-up procedure

,MPEG) bound substrates were worked up by precipitation,

by adding TBME ,10±20 volumes) to the reaction mixtures

cooled in an ice bath. The polymer was recovered by ®l-

tration on a coarse sintered glass frit, rinsed with TBME,

and reprecipitated from absolute ethanol ,about 75 mL

per g). The white precipitate was collected by ®ltration

through a medium glass frit, rinsed with ethanol then

Et₂O, and taken up in CH₂Cl₂and ®ltered through the

same glass frit. The ®ltrate was concentrated and dried in

vacuo to afford the polymer-bound product.

4.2.4. R,S-1-Methyl-1⁰-cyclopropyl-methyl-O-trichloro-

acetimidate 7. A three-necked RB ¯ask charged with

,^)-a-methyl cyclopropylmethanol ,6, 5.0 mL, 51 mmol),

trichloroacetonitrile ,25 mL, 10 equiv.), benzene ,50 mL)

was cooled in an ice bath under an atmosphere of argon.

To this solution was added solid 95% NaH ,1.35 g,

53 mmol) through the central opening to avoid NaH sticking

to the sides of the ¯ask. After 3 h of stirring the reaction was

carefully quenched by the dropwise addition of cold water.

After the effervescence ceased the mixture was transferred

to a separatory funnel and the organic layers separated and

then washed again with water. After drying with MgSO₄and

®ltration the mixture was concentrated. The residue was

taken up in cyclohexane, ®ltered, concentrated and dried

in vacuo to yield a colorless oil 7 ,8.4 g, 71%): d

4.2.1. )MPEG))DOX)OH )3). This compound was synthe-

sized according to Ref. 34.

4.2.2. Ethyl 2,3,4-tri-O-benzyl-6-O-cyclopropylmethyl-1-

thio-a/b-d-glucopyranoside 2ab. Ethyl 2,3,4-tri-O-ben-

zyl-1-thio-a/b-d-glucopyranoside²²

,1ab,

750 mg,

1.52mmol) was dissolved in dry THF ,6.0 mL). Then

95% NaH ,214 mg, 7.58 mmol) was added. Subsequently,

cyclopropylmethyl bromide ,441 mL, 4.6 mmol) and

sodium iodide ,636 mg, 4.55 mmol) were added as a THF

,6.0 mL) solution. The mixture was heated to 408C for 1 h.

After concentration the residue was puri®ed by MPLC

eluting with hexanes/ethyl acetate 4:1 to yield 2ab

,621 mg, 75%) a:b 65:35 a small portion was further puri-

®ed by preparative TLC to yield a small amount of pure

a-isomer 2a: [a]_D30.4 ,c 8.3, CHCl₃); ¹H NMR

,CDCl₃) d 0.18 ,4H, m, CHH-Cp), 0.50 ,4H, m, CHH-

Cp), 0.89 ,2H, m, CH-Cp), 1.29 ,3H, t, J7.3 Hz,

SCH₂CH₃b), 1.34 ,3H, t, J7.3 Hz, SCH₂CH₃a), 2.56

,2H, m, SCH₂CH₃a), 2.75 ,2H, m, SCH₂CH₃b), 3.19 ,1H,

dd, J7.4 Hz, J10.3 Hz, OCHHCpa), 3.29 ,1H, dd, J

7.3 Hz, J10.3 Hz, OCHHCpb), 3.40 ,4H, m, CHHCpab,

H-2b, H-5b), 3.65 ,5H, m, H-3b, H-4b, H-6bb, H-5a,

1

1.336 g mL²¹; H NMR ,C₆D₆) d 0.01 ,1H, m, CHH-Cp),

0.22 ,1H, m, CHH-Cp), 0.29 ,1H, m, CHH-Cp), 0.37 ,1H,

m, CHH-Cp), 0.86 ,1H, m, CH-Cp), 1.22 ,3H, d, J5.9 Hz,

OCHCH₃), 4.52,1H, dq, J7.3 Hz, J5.9 Hz, OCHCH₃),

8.17 ,1H, s, NH); ¹³C NMR ,C₆D₆) d 2.8 ,CH₂-Cp), 3.9

,CH₂-Cp), 16.7 ,CH-Cp), 19.3 ,CH₃), 80.5 ,OCHCH₃),

93.0 ,CCl₃), 162.8 ,CvN).

4.2.5. Ethyl 6-O-)R,S-1-methyl 1⁰-cyclopropylmethyl)-

2,3,4-tri-O-benzyl-1-thio-a/b-d-glucopyranosides 8ab.

Alcohol ,1, 970 mg, 1.96 mmol) was dissolved in dichloro-

methane ,25 mL) under an atmosphere of argon. To this

solution was added AgOTf ,502mg, 1.96 mmol) and the

6686

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

mixture cooled in an ice salt bath to near 08C. To the

mixture was added imidate ,7, 0.68 mL, 3.92mmol) and

after stirring for 45 min the reaction was quenched with

excess solid NH₄HCO₃. The solids were removed by ®ltra-

tion through a bed of celite. The ®ltrate was concentrated

and puri®ed by MPLC eluting with TBME/dichloromethane

1:50 to yield a diastereomieric mixture of anomers 8ab

,620 mg, 56%). A small amount was puri®ed by preparative

dichloromethane was added ,30 mL) and cooled in an

ice bath. To this was added NIS ,400 mg, 1.77 mmol)

followed by tri¯uoromethanesulfonic acid ,TfOH, 55 mg,

0.21 mmol). After stirring for 20 min the reaction was

quenched with DIPEA and after standard workup to give

9ab ,3.0 g, 99%): partial ¹H NMR ,CDCl₃) d 0.04 ,2H, m,

CH-Cp), 0.32,4H, m, C H-Cp), 0.42,4H, m, C H-Cp), 0.55

,4H, m, CH-Cp), 0.81 ,2H, m, CH-Cp), 0.86 ,8H, m, CH-

Cp), 1.21, 1.22, 1.24, 1.25 ,12H, 4d, CH₃-Cp), 2.78, 2.84

,4H, m, OCH-Cp), 3.86 ,brd), 3.94 ,brd), 4.01 ,brt), 4.5±5.0

,m, H-1ab, CH₂Ar), 7.30 ,m, ArH).

1

TLC to yield pure R,S-8a: [a]_D68.8 ,c 1.7, CHCl₃); H

NMR ,CDCl₃) d 0.04 ,2H, m, CHH-Cp), 0.32,2H, m,

CHH-Cp), 0.44 ,2H, m, CHH-Cp), 0.54 ,1H, m, CHH-

Cp), 0.59 ,1H, m, CHH-Cp), 0.84 ,2H, m, CH-Cp), 1.25

,3H, d, J6.0 Hz, OCHCH₃), 1.26 ,3H, d, J6.0 Hz,

OCHCH₃), 1.31 ,6H, brt, SCH₂CH₃), 2.55 ,2H, m,

SCH₂CH₃), 2.67 ,1H, m, OCHCH₃-a), 2.67 ,2H, m,

SCH₂CH₃), 2.76 ,1H, dq, J7.3 Hz, J6.0 Hz, OCHCH₃-

4.2.7. )MPEG))DOX)yl 2,3,4-tri-O-benzyl-a/b-d-gluco-

pyranoside 5ab. Glycosides ,9ab, 3.0 g, 0.54 mmol)

were dissolved in dichloromethane ,22.5 mL) under an

atmosphere of argon and tri¯uoroacetic acid ,TFA, 2.5 mL)

was added. After stirring for 1 h the reaction was cooled in an

ice bath and processed by the standard worked to give 5ab

b), 3.29 ,2H, m, H-2), 3.60 ,1H, dd, J_56b3.9 Hz, J_6a6b

10.8 Hz, H-6b-a), 3.68 ,2H, brt, H-3), 3.71 ,1H, m, H-6b-

b), 3.85 ,2H, brt, H-3), 3.87 ,1H, m, H-6a-a), 3.94 ,1H, dd,

J,1 Hz, J10.8 Hz, H-6a-b), 4.19 ,2H, m, H-5), 4.61

,1H, d, J11.2Hz, CH HAr), 4.69 ,1H, d, J12.2 Hz,

CHHAr), 4.70 ,2H, d, J10.8 Hz, CHHAr), 4.76 ,1H, d,

J12.2 Hz, CHHAr), 4.80 ,2H, d, J10.8 Hz, CHHAr),

4.91 ,1H, d, J11.2Hz, C HHAr), 4.93 ,2H, d, J

10.8 Hz, CHHAr), 4.98 ,2H, d, J10.8 Hz, CHHAr), 5.44

,2H, d, J_1,24.9 Hz, H-1), 7.32,26H, m, Ar H), 7.41 ,4H, m,

ArH); ¹³C NMR ,CDCl₃) d 0.6 ,CH₂-Cp), 1.0 ,CH₂-Cp), 4.9

,CH₂-Cp), 5.1 ,CH₂-Cp), 14.8 ,SCH₂CH₃), 15.9 ,CH-Cp),

16.4 ,CH-Cp), 20.2 ,CH₃), 20.7 ,CH₃), 23.7 ,SCH₂CH₃),

66.7, 67.1 ,C-6), 70.55, 70.62,C-5), 7.23, 74.9, 75.7

,CH₂Ar), 77.5, 77.4 ,C-4),79.5 ,C-3), 80.6 ,OCHCH₃),

81.1 ,OCHCH₃), 82.7 ,C-2), 83.0 ,C-1), 127.5, 127.6,

127.7, 127.8, 128.3, 128.0, 137.9, 138.6, 138.7 ,ArC);

MS-FAB1ve 569.1 ,M1Li¹), 561.2,M 2H¹); HRMS

C₃₄H₄₂O₅SK ,M1K¹, 601.2396) Calcd 601.2390; Anal.

Calcd for C₃₄H₄₂O₅S ,562.7715): C 72.57, H 7.52, found:

C 72.03, H 7.75. and R,S-8b: [a]_D6.5 ,c 0.6, CHCl₃); ¹H

NMR ,CDCl₃) d 0.02,2H, m, C HH-Cp), 0.29 ,2H, m,

CHH-Cp), 0.36 ,2H, m, CHH-Cp), 0.48 ,1H, m, CHH-

Cp), 0.76 ,2H, m, CH-Cp), 1.07 ,6H, d, J6.0 Hz,

OCHCH₃), 1.29 ,6H, brt, SCH₂CH₃), 2.73 ,4H, m,

SCH₂CH₃), 2.73 ,2H, m, OCHCH₃), 3.39 ,4H, m, H-2,

H-5), 3.49 ,1H, dd, J_56b5.5 Hz, J_6a6b10.6 Hz, H-6b-a),

3.53, 3.55 ,2H, 2£brt, H-4), 3.64 ,2H, brt, H-3), 3.68 ,2H,

brs, H-6a-a, H-6b-b), 3.89 ,1H, dd, J,1 Hz, J10.6 Hz,

H-6a-b), 4.42, 4.41 ,2H, 2£d, J_1,29.9 Hz, H-1), 4.60 ,1H,

d, J10.6 Hz, CHHAr), 4.66 ,1H, d, J11.0 Hz, CHHAr),

4.70 ,2H, d, J10.3 Hz, CHHAr), 4.80 ,4H, m, CHHAr),

4.87 ,4H, m, CHHAr), 7.26 ,26H, m, ArH), 7.33 ,4H, m,

ArH); ¹³C NMR ,CDCl₃) d 0.8 ,CH₂-Cp), 0.9 ,CH₂-Cp), 4.5

,CH₂-Cp), 15.3 ,SCH₂CH₃), 16.2, CH-Cp), 16.3 ,CH-Cp),

20.2 ,CH₃), 20.3 ,CH₃), 25.0 ,SCH₂CH₃), 67.3 ,C-6), 74.9,

75.5, 75.8 ,CH₂Ar), 78.2, 78.1 ,C-4), 79.3 ,C-2), 80.2

,OCHCH₃), 80.3 ,OCHCH₃), 81.8 ,C-5), 86.7 ,C-3), 84.9

,C-1), 127.6, 127.8, 127.9, 128.3, 128.4, 138.0, 138.2, 138.5

,ArC); MS-FAB1ve 569.1 ,M1Li¹), 561.3 ,M2H¹);

HRMS C₃₄H₄₁O₅S ,M2H¹, 561.2640) Calcd 561.2674.

1

,2.9 g, 97%): Partial H NMR ,CDCl₃) d 4.03 ,brt, H-3a),

4.5±5.0 ,m, H-1a, H-1b, CH₂Ar), 7.30 ,m, ArH).

4.2.8. )4-O-Acetoxymethyl)-benzyl 6-O-acetyl-2,3,4-tri-

O-benzyl-a/b-d-glucopyranoside 10ab. A portion of

glycosides ,9ab, 247 mg, 44 mmol) were dissolved in

dichloromethane ,2mL) and acetic anhydride ,2mL). To

this mixture was added Sc,OTf)₃,11 mg, 22 mmol) and

the stirring continued for 16 h. The polymer was worked

up as usual and the combined ®ltrates concentrated to

dryness. The residue was puri®ed by preparative TLC

eluting with hexanes/ethyl acetate 50:50 to yield 10ab

1

,5 mg, 17%): [a]_D127.5 ,c 0.38, CHCl₃); a:b 65:35; H

NMR ,CDCl₃) d 2.01, 2.04, 2.09 ,3£s, CH₃CO), 3.49 ,m,

H-2b, H-4b, H-5b, H-2a), 3.50 ,brt, J_4,59.9 Hz, H-4a),

3.64 ,brt, J8.9 Hz, H-3b), 3.84 ,dd, J_5,6a4.4 Hz, H-5a),

4.04 ,brt, J_3,49.2Hz, H-3 a), 4.15 ,dd, J_5,6b2.0 Hz, H-

6ba), 4.22 ,dd, J_5,6b4.8 Hz, H-6bb), 4.24 ,dd, J_6a,6b

12.0 Hz, H-6aa), 4.35 ,dd, J_6a,6b12.0 Hz, J_5,6a2.0 Hz,

H-6ab), 4.48 ,d, J12.8 Hz, CHHAr), 4.48 ,d, J_1,2

7.8 Hz, H-1b), 4.53 ,d, J13.6 Hz, CHHAr), 4.54 ,d,

J12.3 Hz, CHHAr), 4.55 ,d, J10.9 Hz, CHHAr), 4.64

,d, J12.0 Hz, CHHAr), 4.66 ,d, J12.1 Hz, CHHAr),

4.68 ,d, J12.0 Hz, CHHAr), 4.71 ,d, J10.9 Hz,

CHHAr), 4.77 ,d, J11.1 Hz, CHHAr), 4.78 ,d, J_1,2

4.0 Hz, H-1a), 4.79 ,d, J9.9 Hz, CHHAr), 4.83 ,d,

J11.4 Hz, CHHAr), 4.84 ,d, J10.9 Hz, CHHAr), 4.91

,d, J12.0 Hz, CHHAr), 4.92,d, J10.9 Hz, CHHAr),

4.93 ,d, J10.9 Hz, CHHAr), 5.00 ,d, J10.8 Hz,

CHHAr), 5.09 ,s, CH₂Ar-DOX), 7.30 ,m, ArH); ¹³C NMR

,CDCl₃) d 20.9, 21.0 ,CH₃CO), 63.4 ,C-6a), 63.6 ,C-6b),

66.4 ,CH₂Ar-DOX), 69.3 ,C-5a), 69.4 ,CH₂Ar), 71.1

,CH₂Ar), 73.2,C-2 b), 73.5 ,CH₂Ar), 75.3 ,CH₂Ar), 75.35

,CH₂Ar), 75.4 ,CH₂Ar), 76.1 ,CH₂Ar), 76.2, CH₂Ar), 77.6

,C-2b), 77.8 ,C-5a), 80.5 ,C-4a), 82.3 ,C-3a), 82.6 ,C-4b),

85.1 ,C-3b), 95.7 ,C-1a), 102.5 ,C-1b), 127,0, 128.0 136.2,

137.4, 137.6, 137.7, 137.8, 138.0, 138.2, 138.4, 138.6

,ArC), 170.7, 170.8 ,CH₃CO); MS-FAB1ve 653.3 ,M2

H¹); HRMS C₃₉H₄₂O₉Na ,M1Na¹, 677.2781) Calcd

677.2727.

4.2.6. )MPEG))DOX)yl 6-O-)R,S-1-methyl 1⁰-cyclopro-

pylmethyl)-2,3,4-tri-O-benzyl-a/b-d-glucopyranoside

9ab. ,MPEG),DOX)OH ,3, 3.0 g, 0.59 mmol) and ,8ab,

600 mg, 1.06 mmol) were dried together overnight in vacuo

over P₂O₅. The ¯ask was opened to an argon atmosphere,

4.2.9. )MPEG))DOX)yl 6-O-[3,4,6-tri-O-acetyl-2-deoxy-

2-N-phthalimido-b-d-glucopyranosyl]-2,3,4-tri-O-benzyl-

a/b-d-glucopyranoside 12ab. Alcohol ,5ab, 3.0 g,

Ê

0.54 mmol), powdered molecular sieves 4 A and 3,4,6-tri-

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

6687

O-acetyl-2-deoxy-2-N-phthalimido-b-d-glucopyranosyl-O-

trichloroacetimidate²⁴,11, 630 mg, 1.36 mmol) were dried

together overnight in vacuo. The ¯ask was opened to an

argon atmosphere, dichloromethane ,22.5 mL) was added

and the mixture cooled in an ice bath. Then TESOTf

,96 mL, 0.42mmol) was added by syringe. After stirring

for 2h, the reaction was quenched with DIPEA ,240 mL)

and worked up as usual to give 12ab ,3.08 g, 99%): Partial

¹H NMR ,CDCl₃) d 2.00, 2.04, 2.06, 2.11 ,4£s, CH₃CO),

5.16 ,brt, H-4^II), 5.32,d, J8.8 Hz, H-1^IIa), 5.34 ,brt,

H-4^II), 5.45 ,d, J8.8 Hz, H-1^II), 5.76 ,,brt, H-3^II), 7.02

,m, ArH-DOX), 7.49±7.76 ,m, ArH-Phth).

MS-FAB1ve 964.1 ,M1Na¹), 330 ,TriAcHexNAc¹);

HRMS C₅₁H₅₉NO₁₆Na ,M1Na¹, 964.3667) Calcd

964.3600; Anal. Calcd for C₅₁H₅₉NO₁₆´,H₂O)_2.5

,987.0674): C 62.06, H 6.54, N 1.42 found: C 62.08, H

6.69, N 1.30.

4.2.11. 6-O-[3,4,6-Tri-O-acetyl-2-deoxy-2-acetamido-b-

d-glucopyranosyl]-1,2,3,4-tetra-O-acetyl-a/b-d-gluco-

pyranose 14ab. Disaccharide ,13ab, 56 mg, 59 mmol)

was dissolved in acetic acid ,10 mL) and hyrdrogenated in

a Parr apparatus at 50 psi of H₂for 16 h in the presence of

10% Pd on charcoal. The catalyst was removed by ®ltration

through a bed of celite. TLC analysis of the ®ltrate revealed

that the hydrogenation was not complete and so the pro-

cedure was repeated. The ®ltrate was concentrated and the

residue was dissolved in pyridine ,4 mL) and acetic

anhydride ,1 mL) was added. After stirring for 16 h at rt

the mixture was concentrated and the residue co-concen-

trated with toluene. The residue was puri®ed by MPLC

eluting with hexanes/ethyl acetate 3:1 followed by pure

ethyl acetate to yield pure 14ab ,14 mg, 35%): [a]_D

4.2.10. )4-O-Acetoxymethyl)-benzyl 6-O-[3,4,6-tri-O-

acetyl-2-deoxy-2-acetamido-b-d-glucopyranosyl]-2,3,4-

tri-O-benzyl-a/b-d-glucopyranoside 13ab. To disacchar-

ide ,12ab, 2.1 g, 0.35 mmol) was added ethanol ,48 mL)

followed by ethylenediamine ,3.0 mL) and the mixture

heated to 708C and stirred under an atmosphere of argon

for 2h. The mixture was diluted with ethanol ,150 mL) and

cooled to 2208C to precipitate the polymer. The precipitate

was recovered by ®ltration, rinsed with ethanol followed by

diethyl ether and then dissolved in dichloromethane, ®ltered

and concentrated. The residue ,2.15 g) was dissolved in

dichloromethane ,12mL) cooled in an ice bath under an

atmosphere of argon and acetic anhydride ,12.0 mL)

followed by Sc,OTf)₃,100 mg, 0.2mmol) were added.

The polymer was worked up as usual and the combined

®ltrates were concentrated to dryness. The residue was puri-

®ed by MPLC eluting with CH₂Cl₂/iC₃H₇OH 20:1 to yield

1

14.2, c 0.82, CHCl₃); a:b 1:1.19; H NMR ,CDCl₃) d

1.94, 1.981, 1.984, 1.987, 1.989, 1.992, 1.998, 2.00,2),

2.01, 2.02, 2.04, 2.06, 2.07, 2.15, 2.16 ,16s, CH₃CO), 3.44

,dd, J_6a,6b11.6 Hz, J_5,6b4.9 Hz, H-6b^Ia), 3.61 ,m,

H-6b^Ib, H-6a^Ia), 3.63 ,m, H-5^IIab), 3.75 ,brt, H-5^Ib),

3.84 ,brd, J_6a,6b12.8 Hz, J_5,6a,1.0 Hz, H-6a^Ib), 3.92

,brq, J_2,310.4 Hz, J_1,28.6 Hz, J_2,NH9.8 Hz, H-2^IIa),

3.99 ,brt, H-5^Ia), 4.11 ,m, H-2^IIb), 4.12,brd, J_6a,6b

12.8 Hz, J_5,6a,1.0 Hz, H-6b^IIab), 4.21 ,m, H-6a^IIab),

4.50 ,d, J8.6 Hz, H-1^IIb), 4.55 ,d, J8.6 Hz, H-1^IIa),

4.93 ,brt, J_4,59.8 Hz, J_3,49.8 Hz, H-4^Ib), 4.98 ,brt, J

9.8 Hz, H-3^IIb), 5.019 ,dd, J_1,23.7 Hz, J_2,39.8 Hz,

1

13ab ,66 mg, 20%):a:b 1:0.75; H NMR ,CDCl₃) d 1.82,

1.86, 1.98, 1.99, 2.00, 2.08 ,6s, CH₃CO), 3.48 ,m, H-5^Ib,

H-4^Ib, H-2^Ib), 3.50 ,m, H-4^Ia), 3.51 ,m, H-2^Ia), 3.60 ,m,

H-5^IIab), 3.61 ,brt, J8.8 Hz, H-3^Ib), 3.68 ,m, H-6b^Ia,

H-6b^Ib), 3.80 ,m, H-5^Ia), 3.85 ,m, H-2^IIb), 3.87 ,m,

H-2^IIa), 4.01 ,m, H-3^Ia, H-6a^Ia), 4.07 ,m, H-6a^Ib), 4.10

,m, H-6b^IIab), 4.20 ,m, H-6a^IIab), 4.47 ,d, J8.2Hz,

H-1^Ib), 4.49 ,d, J12.0 Hz, CHHAr), 4.52,d, J12.4 Hz,

CHHAr), 4.56 ,2d, J10.9 Hz, CHHAr), 4.64 ,d, J

8.1 Hz, H-1^IIa), 4.65 ,d, J11.0 Hz, CHHAr), 4.66 ,d,

J12.4 Hz, CHHAr), 4.67 ,d, J12.0 Hz, CHHAr), 4.69

,d, J10.9 Hz, CHHAr), 4.70 ,d, J8.3 Hz, H-1^IIb), 4.75

,d, J11.0 Hz, CHHAr), 4.78 ,d, J10.9 Hz, CHHAr), 4.79

,d, J4.2Hz, H-1 ^Ia), 4.81 ,d, J10.9 Hz, CHHAr), 4.82,d,

J10.9 Hz, CHHAr), 4.89 ,d, J11.0 Hz, CHHAr), 4.912

,d, J10.9 Hz, CHHAr), 4.915 ,d, J11.0 Hz, CHHAr),

4.98 ,d, J10.9 Hz, CHHAr), 5.03 ,brt, J9.6 Hz,

H-4^IIa), 5.04 ,brt, J9.6 Hz, H-4^IIb), 5.08 ,s, CH₂Ar-

DOX), 5.09 ,s, CH₂Ar-DOX), 5.24 ,brt, J9.4 Hz,

H-3^IIa), 5.25 ,brt, J9.4 Hz, H-3^IIb), 5.29 ,d, NH^IIb),

5.34 ,d, NH^IIa), 7.24 ,12H, m, ArH), 7.31 ,14H, m, ArH),

7.38 ,4H, m, ArH); ¹³C NMR ,CDCl₃) d 20.6, 20.7, 21.0

,CH₃COO), 23.4 ,CH₃CON), 54.5 ,C-2^IIa), 54.7 ,C-2^IIb),

62.2 ,C-6^IIab), 66.04 ,CH₂Ar-DOX), 66.06 ,CH₂Ar-DOX),

67.5 ,C-6^Ib), 67.7 ,C-6^Ia), 68.62,C-4 ^IIab), 68.60 ,CH₂Ar),

69.9 ,C-5^Ia), 70.7 ,CH₂Ar), 72.0 ,C-5^IIab), 72.3 ,C-3^IIb),

72.4 ,C-3^IIa), 73.1 ,CH₂Ar), 74.4 ,C-2^Ib), 74.7 ,CH₂Ar),

74.8 ,CH₂Ar), 74.9 ,CH₂Ar), 75.7 ,2CH₂Ar), 77.4 ,C-5^Ib),

77.9 ,C-4^Ib), 79.9 ,C-2^Ia), 82.0 ,C-3^Ia), 82.2 ,C-4^Ia), 84.6

,C-3^Ib), 95.6 ,C-1^Ia), 100.51 ,C-1^IIa), 100.58 ,C-1^IIb),

102.4 ,C-1^Ib), 127.0, 128.0, 135.5, 135.6, 137.3, 137.6,

138.1, 138.2, 138.3, 138.4, 138.5, 138.9 ,ArC), 169.38,

169.41, 169.45, 169.5, 169.9, 170.7, 170.8, 170.85 ,CO);

II

H-2^Ia), 5.038, 5.062,m, H-4 a, H-4^IIb), 5.080 ,dd, J_1,2

8.6 Hz, J_2,3 9.8 Hz, H-2^Ib), 5.085 ,dd, J_3,49.7 Hz, J_4,5

10.4 Hz, H-4^Ia), 5.194 ,brt, J_2,310.4 Hz, J_3,49.8 Hz,

H-3^IIa), 5.206 ,brt, J_2,39.8 Hz, J_3,49.8 Hz, H-3^Ib), 5.44

,brt, J_2,39.8 Hz, J_3,49.7 Hz, H-3^Ia), 5.49 ,d, J8.6 Hz,

H-1^Ib), 5.75 ,d, J_2,NH8.6 Hz, NH^IIb), 6.20 ,d, J_2,NH

9.8 Hz, NH^IIa), 6.25 ,d, J3.7 Hz, H-1^Ia); ¹³C NMR

,CDCl₃) d 20.4±21.0 ,CH₃COO), 22.9, 23.1 ,CH₃CON),

II

53.3 ,C-2^IIb), 54.2,C-2 a), 62.0 ,C-6^IIab), 66.8 ,C-6^Ia),

66.9 ,C-6^Ib), 67.9 ,C-4^Ib), 68.1 ,C-4^Ia), 68.4 ,C-4^IIab),

69.1 ,C-2^Ia), 69.8 ,C-3^Ia), 69.9 ,C-2^Ib), 70.7 ,C-5^Ia),

71.9 ,C-5^IIab), 72.6 ,C-3^IIa, C-3^Ib), 72.9 ,C-3^IIb), 75.9

,C-5^Ib), 89.1 ,C-1^Ia), 92.3 ,C-1^Ib), 101.1 ,C-1^IIa), 101.6

,C-1^IIb), 169.2, 169.4, 169.5, 169.6, 169.8, 170.0, 170.1,

170.3, 170.4, 170.6, 170.8, 170.9, 171.0,2), 171.1,2)

1

,CO); H NMR ,CDCl₃) d MS-FAB1ve 678.2,M 1H¹),

700.1 ,M1Na¹); HRMS C₂₈H₄₀NO₁₈,M1H¹, 678.2259)

Calcd 678.2244.

4.2.12. Ethyl 4,6-O-phenylboronate-b-d-galactothio-

pyranoside 15. A solution of phenylboronic acid ,560 mg,

4.58 mmol) in benzene ,100 mL) was re¯uxed in a 2-neck

¯ask ®tted with a dropping funnel and Dean±Stark trap. A

solution of ethyl 1-thio-b-d-galactopyranoside ,1.00 g,

4.58 mmol) in methanol ,5 mL) was added dropwise from

the funnel and this solution re¯uxed for 1/2h, periodically

draining the trap and replacing the volume with benzene.

The solution was allowed to cool and on standing overnight

at room temperature the product crystallized. Three crops

were collected totaling ,1.31 g, 94%): mp148.5±149.28C;

6688

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

¹H NMR ,CDCl₃) d 1.22 ,t, J7.4 Hz, 3H, SCH₂CH₃), 2.65

,m, 2H, SCH₂CH₃), 2.75 ,brs, 1H, OH), 2.95 ,brs, 1H, OH),

3.60 ,m, 2H, H-2, H-3), 3.81 ,brs, 1H, H-5), 4.12 ,d, J

12.2 Hz, 1H, H-6b), 4.20 ,d, J12.3 Hz, 1H, H-6a), 4.33

,m, 2H, H-1, H-4), 7.26 ±7.37 ,m, 3H, ArH), 7.75 ,brd, 2H,

ArH); ¹³C NMR ,CDCl₃): d 15.1 ,SCH₂CH₃), 23.8

,SCH₂CH₃), 64.7 ,C-6), 69.8, 70.4, 72.2, 74.4, ,C-2, C-3,

C-4, C-5), 85.7 ,C-1), 127.6 ,2ArCH), 128.3 ,ArCB), 131.1

,ArCH), 133.9 ,2ArCH).

1.33 ,3H, t, J7.6 Hz, SCH₂CH₃), 2.65 ,1H, brs, OH),

2.79 ,2H, m, SCH₂CH₃), 3.17 ,1H, dq, J6.1 Hz, J

8.3 Hz, OCHCH₃), 3.84 ,1H, brt, J_1,29.8 Hz, J_2,3

9.2Hz, H-2), 3.97 ,1H, dd, J_2,39.2Hz, J_3,43.4 Hz, H-

3), 4.08 ,1H, brt, H-5), 4.35 ,1H, dd, J_5,6b5.8 Hz, J_6a,6b

11.3 Hz, H-6b), 4.51 ,1H, dd, J_5,6a7.0 Hz, J_6a,6b11.3 Hz,

H-6a), 4.55 ,1H, d, J_1,28.2Hz, H-1), 5.72,1H, brd, H-4),

7.44 ,4H, m, Bz), 7.55 ,2H, m, Bz), 8.00 ,2H, d, J7.1 Hz,

Bz), 8.05 ,2H, d, J7.0 Hz, Bz); ¹³C NMR ,CDCl₃) d 0.1

,CH₂-Cp), 5.6 ,CH₂-Cp), 15.2, CH-Cp), 15.9 ,SCH₂CH₃),

20.9 ,CH₃), 24.6 ,SCH₂CH₃), 63.0 ,C-6), 67.3 ,C-4), 69.2

,C-2), 75.1 ,C-5), 77.3 ,C-3), 78.1 ,OCHCH₃), 86.0 ,C-1),

128.4, 128.5, 129.4, 129.6, 129.7, 129.9, 133.2, 133.3 ,ArC-

Bz), 165.7, 166.2, CO, Bz); 18b low R_f[a]_D225.7 ,c

4.2.13. Ethyl 3-O-)R,S-1-methyl 1⁰-cyclopropylmethyl)-

1-thio-b-d-galactopyranoside 17ab. Ethyl 4,6-O-

phenylboronate-1-thio-b-d-galactopyranoside ,15, 1.8 g,

5.8 mmol) was dissolved in, freshly distilled from CaH₂,

chloroform ,15 mL). This solution was cooled to 2208C

under an atmosphere of argon and covered in aluminum

foil. Then, 7 ,1.75 mL, 10.1 mmol) was added followed

by AgOTf ,651 mg, 2.5 mmol). After stirring for 2 h, the

reaction was diluted with cyclohexane ,100 mL), ®ltered

through a bed of celite and concentrated to yield 16ab.

The residue was shaken overnight with IRA-743 resin

,about 5 g), which had been previously well washed with

acetonitrile then methanol, and methanol ,about 100 mL).

After ®ltration and evaporation the residue was puri®ed by

MPLC eluting with CH₂Cl₂/CH₃OH 93:7 to yield 17ab

,0.78 g, 46%): [a]_D214.6 ,c 0.12, CHCl₃); ¹H NMR

,CD₃OD) d 0.14 ,2H, m, CHH-Cp), 0.44 ,4H, m, CHH-

Cp), 0.55 ,2H, m, CHH-Cp), 0.90 ,2H, m, CH-Cp), 1.28

,12H, m, OCHCH₃, SCH₂CH₃), 2.74 ,4H, m, SCH₂CH₃),

3.04 ,1H, m, OCHCH₃-b), 3.16 ,1H, m, OCHCH₃-a), 3.41

,1H, dd, J_2,39.2Hz, J_3,43.2Hz, H-3-b), 3.50 ,2H, brd, H-

5-a,b), 3.56 ,1H, J_1,28.2Hz, J_2,39.2Hz, H-2-b), 3.57

,1H, dd, J_2,39.2Hz, J_3,43.2Hz, H-3-a), 3.61 ,1H, J_1,2

8.2Hz, J_2,39.2 Hz, H-2-a), 3.68 ,2H, dd, J_5,6b5.5 Hz,

J_6a,6b11.5 Hz, H-6b-a,b), 3.74 ,2H, dd, J_5,6a6.4 Hz,

J_6a,6b11.5 Hz, H-6a-a,b), 3.98 ,1H, brd, H-4-a), 4.05 ,1H,

brd, H-4-b), 4.33, 4.34 ,2H, 2£d, J_1,28.2Hz, H-1-b, H-1-

a); ¹³C NMR ,CD₃OD) d 0.6 ,CH₂-Cp), 1.2, CH₂-Cp), 4.3

,CH₂-Cp), 14.5 ,SCH₂CH₃), 16.5 ,CH-Cp), 17.3 ,CH-Cp),

19.8 ,CH₃), 20.3 ,CH₃), 23.8 ,SCH₂CH₃), 61.6 ,C-6-a,b),

67.3 ,C-4-b), 68.2,C-4-a), 69.7 ,C--a2,b), 78.9

,OCHCH₃-a), 79.5 ,C-5-a,b), 80.1 ,C-3-a), 80.9

,OCHCH₃-b), 81.3 ,C-3-b), 85.5 ,C-1-a,b); MS-FAB1ve

293.2 ,M1H¹), 231.2 ,M2SEt¹); HRMS C₁₃H₂₅O₅S

,M1H¹, 293.1414) Calcd 293.1423.

1

0.30, CHCl₃); H NMR ,CDCl₃) d 0.07 ,1H, m, CHH-

Cp), 0.26 ,1H, m, CHH-Cp), 0.39 ,2H, m, CHH-Cp), 0.88

,1H, m, CH-Cp), 1.22 ,3H, d, J6.1 Hz OCHCH₃), 1.32

,3H, t, J7.3 Hz, SCH₂CH₃), 2.54 ,1H, brs, OH), 2.78

,2H, m, SCH₂CH₃), 3.06 ,1H, dq, J6.1 Hz, J8.2Hz,

OCHCH₃), 3.65 ,1H, dd, J_2,39.2Hz, J_3,43.1 Hz, H-3),

3.88 ,1H, brt, J_1,29.8 Hz, J_2,39.2Hz, H-2), 4.06 ,1H,

brt, H-5), 4.35 ,1H, dd, J_5,6b5.8 Hz, J_6a,6b11.3 Hz,

H-6b), 4.48 ,1H, dd, J_5,6a7.0 Hz, J_6a,6b11.3 Hz, H-6a),

4.52,1H, d, J_1,28.2Hz, H-1), 5.80 ,1H, brd, H-4), 7.42

,4H, m, Bz), 7.54 ,2H, m, Bz), 8.01 ,2H, d, J7.3 Hz, Bz),

8.07 ,2H, d, J7.3 Hz, Bz); ¹³C NMR ,CDCl₃) d 1.8

,CH₂-Cp), 4.1 ,CH₂-Cp), 15.2, CH-Cp), 17.0 ,SCH₂CH₃),

19.7 ,CH₃), 24.6 ,SCH₂CH₃), 63.0 ,C-6), 68.2,C-4), 69.3

,C-2), 75.3 ,C-5), 78.0 ,C-3), 79.1 ,OCHCH₃), 86.2,C-1),

128.39, 128.42, 129.5, 129.6, 129.7, 130.0, 133.2, 133.3

,ArC-Bz), 165.7, 166.2, CO, Bz); MS-FAB1ve 539.2

,M1K¹), 439.2,M 2SEt¹); HRMS C₂₇H₃₂O₇SNa ,M1

Na¹, 523.1783) Calcd 523.1766; Anal. Calcd for

C₂₇H₃₂O₇S ,500.6132): C 64.78, H 6.44, found: C 65.24,

H 6.76.

4.2.15. Ethyl 2,4,6-tri-O-benzoyl-3-O-)R,S-1-methyl 1⁰-

cyclopropylmethyl)-1-thio-b-d-galactopyranoside 19ab.

Triol ,17ab, 0.78 g, 2.6 mmol) was dissolved in pyridine

,10 mL) and cooled in an ice bath under an atmosphere of

argon. To this solution was added benzoyl chloride

,1.81 mL, 6 eq.) and the reaction mixture was left to

warm to rt with stirring for 16 h. Methanol was added to

quench the reaction. After evaporation, the residue was puri-

®ed by MPLC eluting with hexanes/ethyl acetate/CH₂Cl₂

8:1:1 to yield 19ab ,1.41 g, 90%): [a]_D32.4 ,c 0.54,

1

4.2.14. Ethyl 4,6-di-O-benzoyl-3-O-)R,S-1-methyl 1⁰-

cyclopropylmethyl)-1-thio-b-d-galactopyranoside 18ab.

Triol ,17ab, 287 mg, 0.98 mmol) was dissolved in pyridine

under an atmosphere of argon and cooled in an ice salt bath

to 08C. To this solution was added benzoyl chloride

,0.285 mL, 2.4 mmol). After 5 h at 08C the reaction was

quenched with methanol and concentrated. The residue

was dissolved in dichloromethane and washed 3£ with

cold 0.05 M HCl_aqfollowed by aqueous NaHCO₃and

water. The organic layers were dried with MgSO₄, ®ltered

and concentrated. The residue was puri®ed by MPLC

eluting with hexanes/ethyl acetate 3:1 to yield ,19ab,

147 mg, 25%) followed by 18a ,24 mg, 5%) and 18b

,20 mg, 4%): 18a high R_f[a]_D215.3 ,c 0.33, CHCl₃);

¹H NMR ,CDCl₃) d 0.08 ,1H, m, CHH-Cp), 0.34 ,1H, m,

CHH-Cp), 0.42,1H, m, C HH-Cp), 0.64 ,1H, m, CHH-Cp),

0.80 ,1H, m, CH-Cp), 1.09 ,3H, d, J6.1 Hz OCHCH₃),

CHCl₃); H NMR ,CDCl₃) d 20.06 ,2H, m, CHH-Cp),

0.22 ,3H, m, CHH-Cp), 0.33 ,2H, m, CHH-Cp), 0.46 ,1H,

m, CHH-Cp), 0.54 ,1H, m, CH-Cp), 0.78 ,1H, m, CH-Cp),

1.00 ,6H, d, J6.1 Hz, OCHCH₃), 1.27 ,3H, t, J7.3 Hz,

SCH₂CH₃), 2.77 ,4H, m, SCH₂CH₃), 2.93 ,1H, dq,

J6.1 Hz, J8.2Hz, OC HCH₃-a), 3.04 ,1H, dq, J

6.1 Hz, J8.2Hz, OC HCH₃-b), 3.91 ,1H, dd, J_2,39.5 Hz,

J_3,43.3 Hz, H-3-b), 4.14 ,2H, brt, H-5-a,b), 4.22 ,1H, dd,

J_2,39.5 Hz, J_3,43.3 Hz, H-3-a), 4.39 ,2H, dd, J_5,6b

7.0 Hz, J_6a,6b11.6 Hz, H-6b-a,b), 4.53 ,1H, dd, J_5,6a

5.8 Hz, J_6a,6b11.6 Hz, H-6a-b), 4.56 ,1H, dd, J_5,6a

5.8 Hz, J_6a,6b11.6 Hz, H-6a-a), 4.71 ,1H, d, J_1,2

10.1 Hz, H-1-b), 4.74 ,1H, d, J_1,210.1 Hz, H-1-a), 5.47

,1H, brt, J_1,29.5 Hz, J_2,310.1 Hz, H-2-a), 5.51 ,1H, brt,

J_1,29.5 Hz, J_2,310.1 Hz, H-2-b), 5.80 ,1H, brd, H-4-a),

5.91 ,1H, brd, H-4-b), 7.45 ,12H, m, Bz), 7.56 ,6H, m,

Bz), 8.03 ,8H, m, Bz), 8.11 ,4H, m, Bz); ¹³C NMR

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

6689

,CDCl₃) d 0.0 ,CH₂-Cp), 1.4 ,CH₂-Cp), 4.1 ,CH₂-Cp), 5.3

,CH₂-Cp), 15.0 ,SCH₂CH₃), 15.9 ,CH-Cp), 16.9 ,CH-Cp),

20.0 ,CH₃), 20.8 ,CH₃), 24.3 ,SCH₂CH₃), 62.95, 62.96 ,C-

6-a,b), 67.9 ,C-4-b), 69.0 ,C-4-a), 70.0 ,C-2-a), 70.2 ,C-2-

b), 75.3 ,C-5-b), 75.4 ,C-5-a), 75.7 ,C-3-b), 76.9 ,C-3-a),

78.4 ,OCHCH₃-a), 79.9 ,OCHCH₃-b), 84.3 ,C-1-a,b),

128.3, 128.41, 128.42, 128.44, 128.51, 128.52, 129.5,

129.9, 129.70, 129.74, 130.0, 130.1, 133.04, 133.07,

133.20, 133.23, 133.27, 133.31 ,ArC-Bz), 165.26,2),

165.89, 165.92, 166.1, 166.2 ,CO-Bz); MS-FAB1ve

643.2,M 1K¹), 543.2,M 2SEt¹); HRMS C₃₄H₃₆O₈SNa

,M1Na¹, 627.2075) Calcd 627.2038; Anal. Calcd for

C₃₄H₃₆O₈S ,604.7216): C 67.53, H 5.33 found: C 67.63, H

5.55.

added. After stirring for 1.5 h the reaction was quenched

with DIPEA ,105 mL) and worked up as usual to give 23

,3.6 g, 99%): partial ¹H NMR ,CDCl₃) d 1.68 ,3H, s,

CH₃CO), 1.94 ,6H, s, CH₃CO), 4.03 ,m, H-5^II), 4.12,m,

H-2^II), 4.19 ,m, H-6b^II), 4.45 ,m, H-6a^II), 4.64 ,m, H-1^I),

4.69 ,d, J11.7 Hz, CHHAr-DOX), 4.81 ,d, J11.7 Hz,

CHHAr-DOX), 5.02,brt, J9.8 Hz, H-4^II), 5.16 ,brt,

J9.8 Hz, H-2^I), 5.50 ,d, J_1,27.8 Hz, H-1^II), 5.57 ,brt,

J9.8 Hz, H-3^II), 5.78 ,brd, J2.0 Hz, H-4^I); 7.03 ,4H,

brs, ArH-DOX), 7.44 ,6H, m, ArH-Bz), 7.58 ,3H, m,

ArH-Bz), 7.72,H2 , m, Ar H-Phth), 7.75 ,1H, m, ArH-

Phth), 7.83 ,1H, m, ArH-Phth), 8.05, 8.06, 8.07 ,6H, 3£

brd, ArH-Bz).

4.2.19. )4-Acetoxymethyl)-benzyl 3-O-[3,4,6-tri-O-acetyl-

2-deoxy-2-acetamido-b-d-glucopyranosyl]-4,6-di-O-acetyl-

2-O-benzoyl-b-d-galactopyranoside 24. Disaccharide ,23,

3.61 g, 0.59 mmol) was dissolved in methanol ,30 mL) by

gentle heating with a heat gun and after cooling DBU ,30

drops) was added and the mixture left to stir for 16 h. The

polymer was worked up as usual. To the solid was added

ethanol ,35 mL) followed by ethylenediamine ,410 mL,

6.1 mmol) and the mixture heated at 708C for 2h. Then

ethanol ,75 mL) was added and the mixture cooled at

2208C to achieve precipitation. The solid was collected

by ®ltration, rinsed with ethanol and diethyl ether. Then it

was dissolved in dichloromethane, ®ltered, concentrated

and dried in vacuo. The solid was dissolved in dichloro-

methane ,15 mL) followed by acetic anhydride ,15 mL)

and cooled in an ice bath. To this mixture was added

Sc,OTf)₃,150 mg, 0.30 mmol) and the mixture left to stir

and warm to rt for 16 h. The polymer was worked up as

usual and the combined ®ltrates were concentrated to

dryness. The residue was puri®ed by MPLC eluting with

CH₂Cl₂/iC₃H₇OH 25:1 to yield slightly impure 24 which

was further puri®ed on Sephadex LH-20 eluting with

CHCl₃/CH₃OH 4:3 to yield pure 24 ,94 mg, 19%):

4.2.16. )MPEG))DOX)yl 3-O-)R,S-1-methyl 1⁰-cyclopro-

pylmethyl)-2,4,6-tri-O-benzoyl-b-d-galactopyranoside

20ab. ,MPEG),DOX)OH ,3, 4.23 g, 0.82 mmol), powdered

Ê

molecular sieves 3 A ,about 2g) and thioglycoside , 19ab,

1.0 g, 1.65 mmol) were dried in vacuo overnight over P₂O₅.

The ¯ask was opened to an argon atmosphere and dichloro-

methane ,25 mL) was added. The reaction mixture was

cooled in an ice bath and NIS ,558 mg, 2.48 mmol)

followed by TESOTf ,73 mL, 0.32mmol) were added.

After stirring for 1 h the reaction was quenched with

DIPEA ,200 mL) and worked up as usual to give 20ab

1

,4.4 g, 99%): partial H NMR ,CDCl₃) d 20.05 ,2H, m,

CHH-Cp), 0.20 ,3H, m, CHH-Cp), 0.31 ,2H, m, CHH-

Cp), 0.41 ,1H, m, CHH-Cp), 0.52,1H, m, C H-Cp), 0.76

,1H, m, CH-Cp), 0.98 ,6H, d, J6.1 Hz, OCHCH₃), 2.88

,1H, m, OCHCH₃S), 3.01 ,1H, m, OCHCH₃R), 3.82,1H, m,

H-3S), 4.13 ,1H, m, H-3R), 4.62,1H, d, J_1,28.1 Hz, H-1S),

4.65 ,1H, d, J_1,28.1 Hz, H-1R), 4.88 ,1H, d, J12.4 Hz,

CHHAr-DOX), 4.89 ,1H, d, J12.8 Hz, CHHAr-DOX),

5.52 ,2H, m, H-2), 5.74 ,1H, brd, H-4S), 5.83 ,1H, brd,

H-4R), 7.12,8H, m, Ar H-DOX), 7.46 ,12H, m, ArH-Bz),

7.58 ,6H, m, ArH-Bz), 7.99 ,4H, brd, ArH-Bz), 8.06 ,4H,

brd, ArH-Bz), 8.15 ,4H, brd, ArH-Bz).

1

[a]_D38.5 ,c 0.18, CHCl₃); H NMR ,CDCl₃) d 1.43,

1.95, 1.98, 2.10, 2.11, 2.12, 2.17 ,21H, 7£s, CH₃CO),

3.51 ,1H, brq, J_1,28.1 Hz, J_NH,28.1 Hz, J_2,310.2Hz,

4.2.17. )MPEG))DOX)yl 2,4,6-tri-O-benzoyl-b-d-galacto-

pyranoside 21. Glycoside ,20ab, 3.59 g, 0.63 mmol) was

treated with 10% TFA as for 9ab±5 to afford 21 ,3.54 g,

99%): partial ¹H NMR ,CDCl₃) d 2.82 ,1H, brs, OH), 4.12

,1H, dd, J_2,38.8 Hz, J_3,43.0 Hz, H-3), 4.46 ,1H, dd,

J_5,6b5.9 Hz, J_6a,6b11.7 Hz, H-6b), 4.53 ,2H, s, CH₂Ar-

DOX), 4.64 ,1H, dd, J_5,6a7.3 Hz, J_6a,6b11.7 Hz, H-6a),

4.71 ,1H, d, J_1,27.3 Hz, H-1), 4.72, 4.92 ,2H, 2£d, J

12.2 Hz, CHHAr-DOX), 5.44 ,1H, brt, H-2), 5.77 ,1H,

brd, H-4), 7.21 ,4H, m, ArH-DOX), 7.49 ,6H, m, ArH-

Bz), 7.62,3H, m, Ar H-Bz), 8.01 ,2H, brd, ArH-Bz), 8.08

,2H, brd, ArH-Bz), 8.19 ,2H, brd, ArH-Bz).

H-2^II), 3.60 ,1H, ddd, J_4,59.9 Hz, J_5,6a2.2 Hz, J_5,6b

4.0 Hz, H-5^II), 3.85 ,1H, brt, H-5^I), 3.92,1H, dd, J_2,3

9.9 Hz, J_3,43.3 Hz, H-3^I), 4.06 ,1H, dd, J_5,6b4.0 Hz,

J_6a,6b12.1 Hz, H-6b^II), 4.15 ,1H, dd, J_5,6b7.0 Hz, J_6a,6b

11.4 Hz, H-6b^I), 4.22 ,1H, dd, J_5,6a5.9 Hz, J_6a,6b11.4 Hz,

H-6a^I), 4.23 ,1H, dd, J_5,6a2.2 Hz, J_6a,6b12.1 Hz, H-6a^I),

4.54 ,1H, d, J8.1 Hz, H-1^I), 4.64 ,1H, d, J12.8 Hz,

CHHAr-DOX), 4.86 ,1H, d, J12.8 Hz, CHHAr-DOX),

4.88 ,1H, d, J8.2Hz, H-1 ^II), 5.00 ,1H, brt, J_3,49.9 Hz,

J_4,59.9 Hz, H-4^II), 5.04 ,2H, s, CH₂Ar-DOX), 5.14 ,1H, d,

NH), 5.24 ,1H, brt, J_2,310.2Hz, J_3,49.9 Hz, H-3^II), 5.50

,1H, brt, J_1,28.1 Hz, J_2,39.9 Hz, H-2^I), 7.15 ,4H, m, ArH-

DOX), 7.50 ,2H, brt, J7.7 Hz, ArH-Bz), 7.65 ,1H, brt,

J7.3 Hz, ArH-Bz), 8.00 ,2H, brd, J7.3 Hz, ArH-Bz);

¹³C NMR ,CDCl₃) d 20.6, 20.7, 21.0 ,CH₃COO), 22.6

,CH₃CON), 55.3 ,C-2^II), 61.4 ,C-6^II), 62.3 ,C-6^I), 65.9

,CH₂Ar-DOX), 68.5 ,C-4^II), 69.4 ,C-4^I), 69.8 ,CH₂Ar-

4.2.18.

)MPEG))DOX)yl

3-O-[3,4,6-tri-O-acetyl-2-

deoxy-2-N-phthalimido-b-d-glucopyranosyl]-2,4,6-tri-

O-benzoyl-b-d-galactopyranoside 23. The polymer bound

alcohol ,21, 3.38 g, 0.60 mmol) and phenyl 3,4,6-tri-O-ace-

tyl-2-deoxy-2-N-phthalimido-1-thio-b-d-glucopyranoside²⁸

,22, 494 mg, 0.94 mmol) were dried in vacuo for 16 h. The

¯ask was opened to an atmosphere of argon and dichloro-

methane ,15 mL) followed by powdered molecular sieves

I

DOX), 71.2,C-2 ), 71.3 ,C-3^II), 71.5 ,C-5^I), 71.6 ,C-5^II),

99.5 ,C-1^I), 100.8 ,C-1^II), 128.0, 128.2 ,ArCH-DOX),

128.7 ,ArCH-Bz), 129.4 ,ArCC-Bz), 129.8, 133.6 ,ArCH-

Bz), 135.6, 136.7 ,ArCC-DOX),165.0 ,CO-Bz), 169.3,

169.8, 170.2, 170.5, 170.6, 170.7, 171.6 ,CO-Ac); MS-

FAB1ve 860.3 ,M1H¹), 882.2 ,M1Na¹); HRMS

Ê

3 A ,about 2g) were added. The mixture was cooled to

2208C and NIS ,561 mg, 2.49 mmol) followed by tri¯uoro-

methanesulfonic acid ,TfOH, 40 mL, 0.45 mmol) were

6690

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

C₄₁H₄₉NO₁₉Na ,M1Na¹, 882.2823) Calcd 882.2796; Anal.

Calcd for C₄₁H₄₉NO₁₉,859.8370): C 57.27, H 5.74, N 1.63

found: C 56.80, H 5.71, N 1.62.

with dichloromethane/ethyl acetate 92:8 to yield 27ab

,400 mg, 71%): [a]_D20.9 ,c 0.34, CHCl₃); ¹H NMR

,CDCl₃) d 0.02,1H, m, C HH-Cp-a), 0.08 ,1H, m, CHH-

Cp-b), 0.29 ,1H, m, CHH-Cp-b), 0.35 ,1H, m, CHH-Cp-a),

0.54 ,2H, m, CHH-Cp-a,b), 0.55 ,1H, m, CHH-Cp-b), 0.60

,1H, m, CHH-Cp-a), 0.90 ,1H, m, CHC-Cp-a), 0.97 ,1H, m,

CHC-Cp-b), 1.12,3H, d, J6.2Hz, C H₃-a), 1.15 ,3H, d,

J6.2Hz, C H₃-b), 1.34 ,6H, t, J7.3 Hz, CH₃CH₂S-a,b),

2.26 ,6H, s, CH₃CO-Lev), 2.88 ,13H, m, CH₂CH₂-Lev-a,b,

OCHCH₃-b, CH₃CH₂S-a,b), 3.05 ,1H, m, OCHCH₃-a), 3.78

,1H, dd, J_2,39.8 Hz, J_3,43.0 Hz, H-3-a), 4.02,2H, brt,

H-5-a,b), 4.07 ,1H, dd, J_2,39.8 Hz, J_3,43.0 Hz, H-3-b),

4.35 ,2H, dd, J_5,6a5.9 Hz, J_6a,6b11.7 Hz, H-6a-a,b), 4.51

,2H, m, H-6b-a,b), 4.62 ,1H, d, J_1,29.8 Hz, H-1-a), 4.64

,1H, d, J_1,29.8 Hz, H-1-b), 5.33 ,1H, dd, J_1,29.8 Hz,

J_2,39.8 Hz, H-2-b), 5.35 ,1H, dd, J_1,29.8 Hz, J_2,3

9.8 Hz, H-2-a), 5.52 ,1H, brd, J_3,43.0 Hz, H-4-b), 5.65

,1H, brd, J_3,43.0 Hz, H-4-a), 7.43 ,8H, brt, ArH), 7.55

,4H, brt, ArH), 8.02,8H, m, Ar H); ¹³C NMR ,CDCl₃) d

0.2, CH₂-Cp-b), 1.4 ,CH₂-Cp-a,b), 4.4 ,CH₂-Cp-a), 5.4

,CH₂-Cp-b), 15.0 ,CH₃CH₂S-a,b), 16.0 ,CHC-Cp-b), 16.8

,CHC-Cp-a), 20.1 ,CH₃-a), 20.8 ,CH₃-b), 24.4 ,CH₃CH₂S-

a,b), 28.1 28.2 ,CH₂CH₂-Lev-a,b), 29.8 ,CH₃CO-Lev-a,b),

62.7 ,C-6-a,b), 67.5 ,C-4-b), 68.7 ,C-4-a), 69.9 ,C-2-b),

70.1 ,C-2-a), 74.9 ,C-5-b), 75.1 ,C-5-a), 75.4 ,C-3-b),

76.8 ,C-3-a), 78.6 ,OCHCH₃-b), 80.5 ,OCHCH₃-a), 84.1

,C-1-a,b), 128.4, 129.6, 129.7 ,ArCH), 129.8, 129.9

,ArCC), 133.0, 133.1 ,ArCH), 165.2, 166.1 ,CO-Bz),

171.9 ,COO-Lev), 206.1 ,CH₃CO-Lev); MS-FAB1ve

637.2,M 1K¹), 537.2,M 2SEt¹); HRMS C₃₂H₃₈O₉SNa

,M1Na¹, 621.2157) Calcd 621.2134; Anal. Calcd for

C₃₂H₃₈O₉S ,598.7148): C 64.20, H 6.40 found: C 64.26, H

6.71.

4.2.20. Ethyl 2,6-di-O-benzoyl-3-O-)R,S-1-methyl 1⁰-

cyclopropylmethyl)-1-thio-b-d-galactopyranoside 26ab.

Ethyl

2,6-di-O-benzoyl-1-thio-b-d-galactopyranoside¹⁵

,25, 2.0 g, 4.6 mmol) was dissolved in, freshly distilled

from CaH₂, chloroform ,15 mL). This solution was cooled

to 2208C under an atmosphere of argon and covered in

aluminum foil. Then, R,S-1-methyl-1-cyclopropylmethyl-

O-trichloroacetimidate ,1.4 mL, 8.1 mmol) was added

followed by AgOTf ,1.18 g, 4.6 mmol). The reaction was

quenched after 1 h with solid NH₄HCO₃, ®ltered through a

bed of celite and concentrated. The residue was taken up in

dichloromethane, ®ltered and concentrated. The residue was

puri®ed by MPLC eluting with CH₂Cl₂/CH₃OH 50:1 to

yield 26ab ,875 mg, 38%): [a]_D25.3 ,c 0.23, CHCl₃);

¹H NMR ,CDCl₃) high R_fd 0.02,1H, m, C HH-Cp), 0.23

,1H, m, CHH-Cp), 0.44 ,1H, m, CHH-Cp), 0.50 ,1H, m,

CHH-Cp), 0.82,1H, m, C HC-Cp), 1.04 ,3H, d, J5.9 Hz,

CH₃), 1.18 ,3H, t, J7.3 Hz, CH₃CH₂S), 2.68 ,2H, m,

CH₃CH₂S), 2.75 ,1H, brs, OH), 2.76 ,1H, m, OCHCH₃),

3.67 ,1H, dd, J_2,39.5 Hz, J_3,43.3 Hz, H-3), 3.87 ,1H,

brt, H-5), 4.11 ,1H, brd, J_3,43.3 Hz, H-4), 4.52,1H, d,

J_1,210.3 Hz, H-1), 4.57 ,1H, dd, J_5,6a5.1 Hz, J_6a,6b

11.4 Hz, H-6a), 4.65 ,1H, dd, J_5,6ab7.3 Hz, J_6a,6b

11.4 Hz, H-6b), 5.38 ,1H, dd, J_1,210.3 Hz, J_2,39.5 Hz,

H-2), 7.41 ,4H, brt, ArH), 7.53 ,2H, brt, ArH), 8.01 ,4H,

m, ArH); low R_fd 0.02,1H, m, C HH-Cp), 0.22 ,1H, m,

CHH-Cp), 0.37 ,1H, m, CHH-Cp), 0.46 ,1H, m, CHH-Cp),

0.78 ,1H, m, CHC-Cp), 1.28 ,6H, m, CH₃, CH₃CH₂S), 2.81

,2H, m, CH₃CH₂S), 2.78 ,1H, brs, OH), 3.08 ,1H, m,

OCHCH₃), 3.97 ,1H, dd, J_2,39.2Hz, J_3,43.3 Hz, H-3),

3.99 ,1H, brt, H-5), 4.19 ,1H, brd, J_3,43.3 Hz, H-4), 4.66

,1H, d, J_1,210.3 Hz, H-1), 4.70 ,1H, dd, J_5,6a5.1 Hz,

4.2.22. )MPEG))DOX)yl 2,6-di-O-benzoyl-4-O-levulinoyl-

3-O-)R,S-1-methyl 1⁰-cyclopropylmethyl)-b-d-galacto-

pyranoside 28ab. ,MPEG),DOX)OH ,3, 796 mg,

0.16 mmol) and ,27ab, 186 mg, 0.32mmol) were dried

together in vacuo over P₂O₅. The ¯ask was opened to an

argon atmosphere, dichloromethane ,4 mL) was added and

the mixture cooled to 2208C. To this mixture was added

NIS ,105 mg, 0.48 mmol) followed by TfOH ,10 mL,

0.11 mmol). After stirring for 1 h the reaction was quenched

with DIPEA and after standard workup afforded 28ab

J_6a,6b11.4 Hz, H-6a), 4.78 ,1H, dd, J_5,6ab7.0 Hz, J_6a,6b

11.4 Hz, H-6b), 5.52,1H, dd, J_1,210.3 Hz, J_2,39.2Hz,

H-2), 7.53 ,4H, brt, ArH), 7.66 ,2H, brt, ArH), 8.15 ,4H,

m, ArH); ¹³C NMR ,CDCl₃) high R_fd 2.0 ,CH₂-Cp), 4.6

,CH₂-Cp), 14.9 ,CH₃CH₂S), 17.3 ,CHC-Cp), 20.3 ,CH₃),

24.0 ,CH₃CH₂S), 64.0 ,C-6), 67.8 ,C-4), 69.7 ,C-2), 76.1

,C-5), 78.6 ,C-3), 81.2,O CHCH₃), 83.6 ,C-1), 128.4, 129.7

,ArCH), 129.89, 129.93 ,ArCC), 133.0, 133.1 ,ArCH),

165.3, 166.4 ,CO); low R_fd 1.1 ,CH₂-Cp), 4.4 ,CH₂-Cp),

14.9 ,CH₃CH₂S), 16.5 ,CHC-Cp), 21.2 ,CH₃), 23.9

,CH₃CH₂S), 63.9 ,C-6), 67.7 ,C-4), 69.7 ,C-2), 76.1

,C-5), 77.8 ,C-3), 79.9 ,OCHCH₃), 83.6 ,C-1), 128.4,

129.7 ,ArCH), 129.7, 130.0 ,ArCC), 133.0, 133.1 ,ArCH),

165.3, 166.5 ,CO); MS-FAB1ve 523.2 ,M1Na¹); HRMS

C₂₇H₃₂O₇SNa ,M1Na¹, 523.1791) Calcd 523.1766; Anal.

Calcd for C₂₇H₃₂O₇S ,500.6132): C 64.78, H 6.44 found: C

64.62, H 6.43.

1

,736 mg, 92%): partial H NMR ,CDCl₃) d-0.07 ,1H, m,

CHH-Cp), 20.01 ,1H, m, CHH-Cp), 0.17 ,1H, m, CHH-

Cp), 0.24 ,2H, m, CHH-Cp), 0.40 ,1H, m, CHH-Cp), 0.44

,1H, m, CHH-Cp), 0.56 ,1H, m, CHC-Cp), 0.77 ,1H, m,

CHC-Cp), 1.02,3H, d, J6.0 Hz, CH₃), 1.06 ,3H, d, J

6.0 Hz, CH₃), 2.24 ,6H, s, CH₃CO-Lev), 2.84 ,8H, m,

CH₂CH₂-Lev), 2.87, 2.93 ,2H, 2£m, OCHCH₃), 3.67 ,1H,

dd, J_2,39.7 Hz, J_3,43.0 Hz, H-3), 3.97 ,2H, brt, H-5), 4.42

,1H, dd, J_5,6b5.9 Hz, J_6a,6b10.8 Hz, H-6b), 4.56, 4.58

,2H, 2£d, J_1,28.8 Hz, H-1), 4.62,4H, m, H-6a, CH HAr-

DOX), 4.87 ,2H, d, CHHAr-DOX), 5.43 ,2H, brt, J_1,2

8.8 Hz, J_2,39.7 Hz, H-2), 5.50, 5.62 ,2H, 2£brd, J_3,4

3.0 Hz, H-4), 7.13 ,8H, m, ArH-DOX), 7.48 ,8H, brt,

ArH-Bz), 7.61 ,4H, brt, ArH-Bz), 8.01 ,4H, m, ArH-Bz),

8.09 ,4H, m, ArH-Bz).

4.2.21. Ethyl 2,6-di-O-benzoyl-4-O-levulinoyl-3-O-)R,S-

1⁰-cyclopropylmethyl)-1-thio-b-d-galacto-

1-methyl

pyranoside 27ab. Alcohols ,26ab, 470 mg, 0.94 mmol)

were dissolved in dry THF under an atmosphere of argon.

To this was added levulinic acid ,0.2mL, 2.0 mmol)

followed by dicyclohexylcarbodiimide ,387 mg, 1.9

mmol) and DMAP ,about 10 mg). The mixture was left to

stir for 16 h and ®ltered through a bed of celite. After

evaporation the residue was puri®ed by MPLC eluting

4.2.23. )MPEG))DOX)yl 2,6-di-O-benzoyl-4-O-levulinoyl-

b-d-galactopyranoside 29. Glycosides ,28ab, 736 mg,

0.13 mmol) were dissolved in dichloromethane ,9 mL)

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

6691

under an atmosphere of argon and TFA ,1 mL) was added.

After stirring for 1 h the mixture was cooled in an ice bath

and the polymer precipitated with TBME ,100 mL). The

polymer was worked up as usual to give 29 ,657 mg,

2-O-benzoyl-b-d-galactopyranoside 36. Disaccharide ,31,

0.77 g, 0.13 mmol) was glycosylated with 2,3,4,6-tetra-O-

acetyl-b-d-glucopyranosyl-O-trichloroacetimidate³⁶,33) as

described in Ref. 13 to yield 34 and then cleaved with

Sc,OTf)₃as described above for 10 to yield known tri-

saccharide 35 after puri®cation by preparative TLC eluting

with CH₂Cl₂/iC₃H₇OH 25:1 followed by Sephadex LH-20

chromatography eluting with chloroform/methanol 4:3.

Alternatively 34 ,0.93 g, 0.16 mmol) was treated as

described for 23 to 24 to yield after MPLC eluting with

1

89%): partial H NMR ,CDCl₃) d 2.24 ,3H, s, CH₃CO-

Lev), 2.73, 2.90 ,4H, m, CH₂CH₂-Lev), 3.94 ,1H, brd,

J_2,3 9.8 Hz, H-3), 4.02,1H, brt, H-5), 4.40 ,1H, dd,

J_5,6b 6.8 Hz, J_6a,6b11.7 Hz, H-6b), 4.52,2H, s, C H₂Ar-

DOX), 4.59 ,1H, d, J_1,27.8 Hz, H-1), 4.60 ,1H, dd,

J_5,6a7.9 Hz, J_6a,6b11.7 Hz, H-6a), 4.67, 4.87 ,2H, 2£d,

J12.6 Hz, CHHAr-DOX), 5.33 ,1H, brt, J_1,27.8 Hz,

J_2,39.8 Hz, H-2), 5.56 ,1H, brd, J_3,42.9 Hz, H-4), 7.18

,4H, m, ArH-DOX), 7.48 ,4H, brt, ArH-Bz), 7.61 ,2H,

brt, ArH-Bz), 8.01 ,2H, m, ArH-Bz), 8.09 ,2H, m, ArH-Bz).

1

hexanes/ethyl acetate/ethanol 6:6:1 36 ,18 mg, 10%): H

NMR ,CDCl₃) d 1.42,3H, s, C H₃CON), 1.96, 1.99,

2.016, 2.021, 2.077, 2.084, 2.088, 2.11, 2.19 ,27H, 9£s,

CH₃COO), 3.61 ,1H, m, H-5^III), 3.67 ,2H, m, H-5^I, H-2^II),

3.75 ,1H, m, H-5^II), 3.86 ,1H, dd, J_2,310.4 Hz, J_3,4

3.1 Hz, H-3^I), 4.00 ,1H, brd, J_6a,6b12.2 Hz, H-6b^I), 4.14

,1H, dd, J_5,6b3.7 Hz, J_6a,6b12.2 Hz, H-6b^III), 4.22 ,3H,

m, H-6b^I, H-6a^III, H-4^I), 4.34 ,1H, dd, J_5,6b4.3 Hz,

4.2.24.

)MPEG))DOX)yl

3-O-[3,4,6-tri-O-acetyl-2-

deoxy-2-N-phthalimido-b-d-glucopyranosyl]-2,6-di-O-

benzoyl-4-O-levulinoyl-b-d-galactopyranoside 30. The

polymer bound alcohol ,29, 1.17 g, 0.21 mmol) and

J_6a,6b11.6 Hz, H-6a^I), 4.39 ,1H, dd, J_5,6b4.9 Hz, J_6a,6b

powdered molecular sieves 4 A ,about 500 mg) were dried

12.2 Hz, H-6a^II), 4.43 ,1H, d, J_1,28.0 Hz, H-1^I), 4.60 ,1H,

d, J12.8 Hz, CHHAr-DOX), 4.78 ,1H, d, J12.8 Hz,

CHHAr-DOX), 4.81 ,1H, d, J_1,28.0 Hz, H-1^II), 4.93

,1H, dd, J_1,28.0 Hz, J_2,39.8 Hz, H-2^III), 5.01 ,2H, s,

CH₂Ar-DOX), 5.02,1H, m, H-4 ^II), 5.11 ,1H, brt,

J_3,49.8 Hz, J_4,59.8 Hz, H-4^III), 5.16 ,3H, m, NH, H-1^III,

H-3^II), 5.41 ,2H, brt, H-2^I, H-3^III), 7.12,4H, m, Ar H-DOX),

7.49 ,2H, brt, J7.3 Hz, ArH-Bz), 7.63 ,1H, brt, J7.3 Hz,

ArH-Bz), 7.97 ,2H, brd, J7.2Hz, Ar H-Bz); ¹³C NMR

,CDCl₃) d 20.6±21.0 ,CH₃COO), 22.5 ,CH₃CON), 55.4

,C-2^II), 61.8 ,C-6^III), 61.9 ,C-6^II), 63.8 ,C-6^I), 65.9

,CH₂Ar-DOX), 68.3 ,C-4^II), 68.8 ,C-4^III), 68.9 ,CHHAr-

DOX), 71.0 ,C-2^I), 71.3 ,C-5^III), 71.5 ,C-3^II), 71.9 ,C-2^III),

72.1 ,C-5^I, C-5^II), 72.8 ,C-3^III), 73.8 ,C-4^I),79.6 ,C-3^I), 98.9

,C-1^I), 99.3 ,C-1^III), 101.3 ,C-1^II), 128.0, 128.1 ,ArCH-

DOX), 128.7, 129.5, 129.7 ,ArCH-Bz), 133.7 ,ArCC-Bz),

135.3, 137.1 ,ArCC-DOX), 164.8 ,CO-Bz), 169.36, 169.45,

169.9, 170.25, 170.36,2), 170.5, 170.66, 170.7 ,COO,

CON); MS-FAB1ve 1148.3 ,M1H¹), 968 ,M2DOX¹),

331 ,Ac₄Hex¹), 330 ,Ac₃HexNAc¹); HRMS C₅₃H₆₆O₂₇N

,M1H¹, 1148.4047) Calcd 1148.3803.

Ê

at 408C in vacuo for 16 h. After cooling to rt ,22, 224 mg,

0.42mmol) was added under an atmosphere of argon

followed by dichloromethane ,8 mL). The mixture was

cooled to 2208C and NIS ,190 mg, 0.85 mmol) followed

by TfOH ,21 mL, 0.24 mmol) were added. After stirring for

1 h the reaction was quenched with DIPEA and worked up

as usual to give 30 ,1.15 g; 98%): partial ¹H NMR ,CDCl₃)

d 1.78, 2.00, 2.06 ,9H, 3£s, CH₃COO), 2.26 ,3H, s,

CH₃CO-Lev), 2.74, 2.94 ,4H, m, CH₂CH₂-Lev), 4.67, 4.87

,2H, 2£d, J12.7 Hz, CHHAr-DOX), 5.14 ,1H, brt, H-4^II),

I

5.32,1H, brt, H-2 ), 5.41 ,1H, d, J_1,28.0 Hz, H-1^II), 5.60

,1H, brd, J_3,42.9 Hz, H-4^I), 5.66 ,1H, brt, H-3^II), 6.80, 6.92

,2£2H, brd, J7.8 Hz, ArH-DOX), 7.47 ,brm, ArH), 7.60

,brm, ArH), 8.02,m, Ar H-Bz), 8.08 ,m, ArH-Bz).

4.2.25. )MPEG))DOX)yl 3-O-[3,4,6-tri-O-acetyl-2-deoxy-

2-N-phthalimido-b-d-glucopyranosyl]-2,6-di-O-benzoyl-

b-d-galactopyranoside 31. Disaccharide ,30, 0.94 g,

0.16 mmol) was dissolved in pyridine ,12mL) and cooled

in an ice bath. To this solution was added sequentially acetic

acid ,3.0 mL) followed by hydrazine hydrate ,300 mL).³⁵

After 30 min the polymer was worked up as usual except

that the ethanol solution was concentrated to dryness and the

residue reprecipitated from fresh ethanol to give 31 ,0.77 g,

82%): partial ¹H NMR ,CDCl₃) d 1.78, 2.02, 2.10 ,9H, 3£s,

CH₃COO), 4.86 ,1H, d, J11.7 Hz, CHHAr-DOX), 5.11

,1H, brt, H-4^II), 5.41 ,1H, brt, H-2^I), 5.56 ,1H, d, J_1,2

8.0 Hz, H-1^II), 5.65 ,1H, brt, H-3^II), 6.95, 7.00 ,2£2H,

brd, J7.8 Hz, ArH-DOX), 7.47 ,brm, ArH), 7.60 ,brm,

ArH), 8.00 ,m, ArH-Bz), 8.09 ,m, ArH-Bz).

4.2.28. )4-O-Acetoxymethyl)-benzyl 3-O-)2-deoxy-2-N-

acetamido-b-d-glucopyranosyl-)-4-O-[b-d-glucopyrano-

syl]-b-d-galactopyranoside 37. Trisaccharide ,36, 16 mg,

14 mmol) was dissolved in methanol ,5 mL) and 1 M

sodium methoxide ,0.5 mL) was added. After stirring for

16 h the mixture was neutralized with RexynH¹resin,

®ltered and concentrated to yield 37 ,9 mg, 97%): [a]_D

233.1 ,c 0.25, CH₃OH); ¹H NMR ,D₂O) d 2.01 ,CH₃CON),

3.29 ,1H, dd, J_1,28.6 Hz, J_2,39.2Hz, H-2 ^III), 3.41 ,1H,

brt, J_3,49.8 Hz, J_4,59.8 Hz, H-4^III), 3.43 ,1H, m, H-5^III),

3.45 ,1H, m, H-5^II), 3.50 ,1H, brt, J_2,38.6 Hz, J_3,49.2Hz,

H-3^III), 3.52,1H, brt, J_3,49.2Hz, J_4.59.2Hz, H-4 ^II), 3.56

,1H, brt, J_3,49.2Hz, J_4,59.9 Hz, H-3^II), 3.67 ,1H, brt,

J6.1 Hz, H-5^I), 3.68 ,1H, m, H-2^I), 3.69 ,1H, m,

H-6b^III), 3.70 ,1H, m, H-3^I), 3.71 ,1H, m, H-2^II), 3.73

,1H, m, H-6b^II), 3.78 ,2H, m, H-6a^I, H-6b^I), 3.92,H2 ,

brdd, H-6a^II, H-6a^III), 4.37 ,1H, dd, J_3,42.4 Hz, H-4^I),

4.45 ,1H, d, J_1,27.9 Hz, H-1^I), 4.65 ,2H, s, CH₂Ar-

DOX), 4.68 ,1H, d, J_1,27.9 Hz, H-1^II), 4.74 ,1H, d, J

11.6 Hz, CHHAr-DOX), 4.88 ,1H, d, J_1,27.9 Hz, H-1^III),

4.92,1H, d, J11.6 Hz, CHHAr-DOX), 7.42,2H, d, J

7.9 Hz, ArH-DOX), 7.47 ,2H, d, J7.9 Hz, ArH-DOX);

4.2.26. )4-O-Acetoxymethyl)-benzyl 3-O-[3,4,6-tri-O-ace-

tyl-2-deoxy-2-N-phthalimido-b-d-glucopyranosyl]-2,6-

di-O-benzoyl-4-O-levulinoyl-b-d-galactopyranoside 32.

A portion of 30 ,170 mg, 28 mmol) was cleaved with

Sc,OTf)₃as for 10. The residue was puri®ed by preparative

TLC eluting with hexanes/ethyl acetate 50:50 to yield

known 32¹⁸,5 mg, 17%): [a]_D12.9 ,c 0.18, CHCl₃);

MS-FAB1ve 886.2,M 2DOX¹).

4.2.27. )4-O-Acetoxymethyl)-benzyl 3-O-)3,4,6-tri-O-ace-

tyl-2-deoxy-2-N-acetamido-b-d-glucopyranosyl-)-4-O-

[2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl]-6-O-acetyl-

6692

E. Eichler et al. / Tetrahedron 57 )2001) 6679±6693

¹³C NMR ,D₂O) d 23.1 ,CH₃CON), 56.6 ,C-2^II), 61.4 ,C-6^I),

61.6 ,C-6^II, C-6^III), 64.5 ,CH₂Ar-DOX), 70.4 ,C-4^III), 70.7 ,C-

3^III), 71.0 ,C-2^I), 71.9 ,CHHAr-DOX), 74.5 ,C-2^III, C-3^II),

75.0 ,C-5^I), 75.7 ,C-4^I), 76.5 ,C-5^III), 76.7 ,C-4^II, C-5^II),

82.9 ,C-3^I), 102.6 ,C-1^I), 102.9 ,C-1^III), 103.9 ,C-1^II),

128.5, 129.8 ,ArCH-DOX), 136.9, 141.1 ,ArCC-DOX),

175.7 ,CH₃CON); MS-FAB1ve 688.2,M 1Na¹).

1^III), 4.93 ,1H, d, J12.0 Hz, CHHAr-DOX), 7.42,2H, d,

J7.5 Hz, ArH-DOX), 7.47 ,2H, d, J7.5 Hz, ArH-DOX);

¹³C NMR ,D₂O) d 22.8, 23.0 ,2£CH₃CON), 40.4 ,C-3^V),

52.4 ,C-5^V), 56.1 ,C-2^II), 61.0 ,C-6^II), 61.3 ,C-6^I), 61.6

,C-6^III), 61.8 ,C-6^IV), 63.3 ,C-9^V), 64.3 ,CH₂Ar-DOX),

68.2,C-4 ^IV), 68.9 ,C-7^V), 69.1 ,C-4^V), 70.1 ,C-2^IV), 70.6

,C-4^III), 70.8 ,C-2^I), 71.8 ,CHHAr-DOX), 72.5 ,C-8^V), 72.9

,C-3^II), 73.6 ,C-6^V), 74.3 ,C-2^III),74.9 ,C-5^IV), 75.49 ,C-5^II),

75.50 ,C-4^I), 75.9 ,C-5^I), 76.2,C-3 ^IV), 76.4 ,C-5^III), 76.6

,C-3^III), 78.9 ,C-4^II), 83.1 ,C-3^I), 100.6 ,C-2^V), 102.5

,C-1^I), 102.8 ,C-1^III), 103.3 ,C-1^IV), 103.9 ,C-1^II), 128.4,

129.7 ,ArCH-DOX), 136.9, 141.0 ,ArCC-DOX), 174.6

,C-1^V), 175.6, 175.8 ,2£CH₃CON); MS-MALDI1ve

1141.4 ,MH1Na¹); HRMS C₄₅H₆₉N₂O₃₀Na₂,M12Na¹,

1163.3792) Calcd 1163.3809.

4.2.29. Sodium )4-O-acetoxymethyl)-benzyl )5-aceta-

mido-3,5-dideoxy-d-glycero-a-d-galacto-nonulopyrano-

sylonate-3-O-b-d-galactopyranosyl-4-O-2-deoxy-2-N-

acetamido-b-d-glucopyranosyl-)-4-O-[-3-O-b-d-gluco-

pyranosyl]-b-d-galactopyranoside 39. A fusion enzyme

UDP-galactose 4-epimerase/b ,1!4)Galactosyltransferase

,GalE-LgtB, 0.5 units, 3000 mmL) was added to a solution

of 2mM acceptor trisaccharide , 37, 9.0 mg, 136 mL of

10 mM), 10 mM MnCl₂,70 mL of 1 M), and 1 mM UDP-

Glc ,340 mL of 20 mM) in 50 mM HEPES buffer ,350 mL

of 1 M, pH 7.5). The reaction was performed at 378C for a

total of 2h while two additional portions of 1 mM UDP-Glc

,340 mL of 20 mM) were added in 30 min intervals. The

formation of 38 ,R_f0.45) was monitored by TLC ,MeOH/

CHCl₃/0.5%CaCl₂; 45:45:10). Chromatography of the crude

reaction mixture on a C-18 reverse-phase column ,water/

MeOH 95:5 to 90:10) afforded 38 which was used directly.

The reaction was performed in a total volume of 5.0 mL,

and the following reagents were added sequentially: accep-

tor 38, CTP ,14.4 mg) and sialic acid ,8.5 mg). The pH was

adjusted to 7.0 with 1N NaOH, and 100 mM HEPES

,350 mL of 1 M, pH 7.4), 20 mM MgCl₂,700 mL of

100 mM), and 0.2mM dithiothreitol ,14 mL of 100 mM)

were added. The reaction was allowed to proceed at 378C

after the addition of the CMP-Neu5Ac synthetase/sialyl-

transferase fusion enzyme ,1.6 units, 250 mL). The reaction

progress, i.e. the formation of material having R_f0.1, was

monitored by TLC ,MeOH/CHCl₃/0.5%CaCl₂; 45:45:10).

After a total reaction time of 3 h, the crude product was

chromatographed on a C-18 reverse-phase column by

elution with water followed by 99:1 water/MeOH to yield

pure pentasaccharide 39 ,5.8 mg, 38%). Before spectro-

scopic analysis an aqueous solution of the pentasaccharide

was passed through a short column of Chelex-100 ,Na¹

form) and lyophilized, to insure the uniformity of cations

Acknowledgements

The author would like to thank K. Chan for measuring FAB

MS spectra, D. Krajcarski for measuring the MALDI MS

spectra and J. R. Brisson for advice with the NMR spectra.

This is NRC paper #42437.

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Article Doi

1-Methyl 1′-cyclopropylmethyl: An acid labile O-protecting group for polymer-supported oligosaccharide synthesis

DOI: 10.1016/S0040-4020(01)00582-8

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