Design and synthesis of inositolphosphoglycan putative insulin mediators

View Article Online / Journal Homepage / Table of Contents for this issue

A R T I C L E

Design and synthesis of inositolphosphoglycan putative insulin

mediators†

Javier Lo´pez-Prados, Fe´lix Cuevas, Niels-Christian Reichardt, Jose´-Luis de Paz,

Ezequiel Q. Morales and Manuel Mart´ın-Lomas*

Grupo de Carbohidratos, Instituto de Investigaciones Qu´ımicas, CSIC, Ame´rico Vespucio s/n,

41092, Sevilla, Spain. E-mail: manuel.martin-lomas@iiq.csic.es; Fax: +34 95 446 05 65;

Tel: +34 95 448 95 63

Received 29th November 2004, Accepted 23rd December 2004

First published as an Advance Article on the web 2nd February 2005

The binding modes of a series of molecules, containing the glucosamine (1→6) myo-inositol structural motif, into the

ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using

molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in

pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that

pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and

pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the

basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase

using the same intermediates has been explored. The results from the biological evaluation of these molecules provide

additional support to an insulin-mediated signalling system which involves the intermediacy of

inositolphosphoglycans as putative insulin mediators.

galactopyranosyl)-3-O-methyl-D-chiro-inositol 1 (Fig. 1) has

Introduction

been proposed in a recent study.⁴

An intracellular signalling mechanism which involves inosi-

tolphosphoglycans (IPGs) as intracellular mediators has been

postulated for insulin as well as for several growth factors,

classical hormones and cytokines.¹IPGs are incompletely

characterised molecules which are generated after the binding

of these agonists to their receptors and act as second messengers

which modulate the activity of a number of enzymes and mediate

a variety of cellular events. The precise structures of IPGs have

notbeen determinedbuttwo main groups have been proposed on

the basis of chemical composition and biological activity data.

Type-A IPGs inhibit cAMP-dependent protein kinase (PKA)

and contain myo-inositol and D-glucosamine,²while type-P

IPGs activate pyruvate dehydrogenase phosphatase (PDHPase)

and contain chiro-inositol and D-galactosamine.³Both groups

may additionally contain different sugars and phosphate groups.

For type-P IPGs a structure of 4-O-(2-amino-2-deoxy-b-D-

This molecule, which could be a member of a family of

closely related chiro-inositol containing oligosaccharides, acti-

vated PDHPase in vitro when chelated to Mn²⁺and decreased

in vivo blood glucose in diabetic rats in a dose dependent

manner.⁴For type-A IPGs a close structural similarity with the

glycosylphosphatidyl inositols (GPIs), which anchor proteins to

the outer face of cellular membranes (GPI anchors), has been

proposed.⁵However, we have previously synthesised compound

2,⁶which contains the conserved pseudopentasaccharide struc-

ture of the GPI anchors,⁷and found that this molecule does not

show signiﬁcant activity with regards to either inhibiting PKA or

activating PDHPase. Nevertheless, insulin-like activity has been

found for synthetic molecules containing the basic structural

motif D-glucosaminyl (1→6) myo-inositol. Thus, compound 3

stimulated both lipogenesis in rat adipocites⁸and proliferative

growth in the otic vesicle of chicken embryos.^9,10In contrast,

an extensive study with synthetic molecules containing the

conserved GPI anchor structure and a series of variations

thereof, such as 4, has shown different insulin mimetic activities

which included stimulation of lipogenesis, activation of glycogen

† Electronic supplementary information (ESI) available: Synthesis and

full characterisation of compounds 43–53, 58, 67, 68, 71–73. See

http://www.rsc.org/suppdata/dt/b4/b418041k/

Fig. 1 Compounds 1–4.

T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5

7 6 4

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

©

View Article Online

synthase and glycerol-3-phosphate acyltransferase, activation

of glucose transport, inhibition of lipolysis and induction of

tyrosine phosphorylation of insulin receptor substrate-1.¹¹All

these results support the existence of an intracellular insulin-

mediated signalling system, operating together with the tyrosine

kinase cascade mechanism, which involves the intermediacy

of putative insulin mediators.¹²They also indicate that these

mediators must contain some of the structural features present in

molecules 1–4, although their precise chemical structures remain

to be elucidated.

Further studies directed towards establishing the molecular

basis of this postulated alternative mechanism of intracellular

signal transduction have been seriously hampered by the limited

amount of precise structural information. Taking into consider-

ation that type-A IPGs are inhibitors of PKA, we reasoned that

computational docking methods could be used as an additional

tool to gain some insight into the structural requirements for

molecules containing the structural motif D-glucosaminyl (1→6)

myo-inositol to inhibit PKA. This information could be used to

design and synthesise compounds that may show some of the

biological activities which have been reported for type A-IPGs.

Thus, we have performed molecular docking studies of a series

of such compounds in the ATP binding site of the catalytic

subunit of PKA and, according to the docking predictions, we

have developed effective synthetic strategies to prepare a series

of IPG-like molecules whose insulin mimetic activity has been

tested. The results of this study are reported in this paper.

to achieve a good agreement between the calculated balanol

conformation and that reported for balanol in the complex.

With these modiﬁcations, calculations with a series of balanol

derivatives whose inhibition data have been reported were

subsequently carried out. The structures of these derivatives were

built with Sybyl³⁷and a good agreement between experimental

and calculated K_ivalues was found (data not shown). Following

this, the IPG-like pseudooligosaccharide structures 3,5–32 were

examined. For the pseudooligosaccharide docking runs, the

starting geometries were obtained from scratch building the

structures using Sybyl.³⁷References for good geometries were

obtained by comparing Sybyl calculated geometries with those

determined by NMR and molecular dynamics simulations for

the previously synthesised compounds 3,5,14.^10,34Docked ener-

gies and RMS deviations are given as supporting information†.

IPG-like molecules showing the structural motif GlcNH₂-

a-(1→6) myo-inositol lacking phosphate groups (compounds

5,10,12,13,28,29) docked in the ATP binding site of the catalytic

subunit of PKA. The docked geometry of these molecules

showed interesting similarities with that of 33. The cyclitol and

the D-glucosamine rings superimpose respectively with the 4-

hydroxybenzamide and the azepane ring of 33 and both the

phenolic OH group in 33 and the 3-OH group of the myo-

inositol unit bind to residues Glu121 and Val123 of the protein;

an interaction which also takes place with the adenine unit of

ATP (Fig. 4, bottom). Pseudotrisaccharides 10,12,13 seem to

have the optimum molecular size for effective docking and an a-

D-galactopyranosyl unit (i.e. 13) seems to provide effective shape

complementarity.

Results and discussion

The presence of phosphate groups in these structures strongly

determines the orientation into the binding site. When the

phosphate group is sitting on the myo-inositol unit (compounds

3,14,24,30) a strong interaction with Lys72 or Lys168 changes

the orientation of the molecule in the binding site. This is in

agreement with results obtained by calculating energy maps

using a phosphate anion probe by means of GRID program³⁸

(see supporting information†). For effective docking, the nega-

tive charge should be located either on the glucosamine moiety

(effective interaction with Lys72), on a pyranoid ring directly

linked to it (effective interaction with Lys168) or on both of

them (compounds 15–19,25,26,31). In a molecule like 26, the 3-

OH and 1-OH groups of the myo-inositol unit interact through

hydrogen bonding with Val123, Glu121 and Thr183 respectively;

the phosphate groups interact with Lys72 and Lys168; and OH-2

of the terminal hexopyranose unit interacts with Phe154. These

structural details are shown in Fig. 4 in comparison with the

experimental X-ray diffraction data for ATP and 33.

Molecules containing the GlcNH₂-b-(1→6) myo-inositol

structural motif (6,7,11,21,23,26, and 27) docked effectively in

the binding site when lacking phosphate groups (compounds 6

and 11). However, the presence of phosphate groups prevented

effective docking. Steric congestion appeared to hinder effective

docking of the phosphate group in these cases. In summary, a

myo-inositol unit carrying no phosphate group linked to a a-

conﬁgurated D-glucosaminyl moiety seems to be the minimum

structural requirement for a type-A IPG-like molecule docking

in the ATP binding site of the catalytic subunit of PKA. This

a-conﬁgurated linkage could attach the D-glucosaminyl unit to

any equatorially oriented OH group of the cyclitol ring. The

presence of a phosphate group on this D-glucosaminyl unit

seems to be important to orientate the molecule into the binding

site and this effect is most pronounced when an additional

hexopyranosyl unit carrying also a phosphate group is attached

to the D-glucosaminyl moiety. Such a pseudotrisaccharide

arrangement seems to provide the best shape complementarity.

Computational docking

Protein kinases are actually considered as major drug targets

and there is a growing interest in developing protein kinase

inhibitors.¹³PKA is a well characterised protein kinase.¹⁴

Inactive PKA holoenzyme consists of two regulatory and two

catalytic subunits that dissociate in response to elevated levels

of intracellular cAMP. The catalytic subunit shares with other

kinases a conserved catalytic core of approximately 260 amino

acids and 11 invariant residues¹⁵. Owing to this, PKA has been

used as a surrogate kinase to analyse inhibitor–kinase binding

properties.¹⁶Therefore, the analysis of the binding modes of a

set of IPG-like molecules to the catalytic subunit of PKA may

provide information on the structural requirements for IPGs

to display biological activity through interacting with protein

kinases.

On the basis of this working hypothesis we decided to

undertake studies of molecular docking, expecting that the

structure of the PKA catalytic subunit will act as a template

to provide structural information on IPGs. This is a similar

approach to that employed in lead discovery using molecular

docking.¹⁷

A considerable number of crystal structures of complexes

involving different inhibitors and the PKA catalytic subunit

have been published.^18–32Most of these inhibitors bind to the

catalytic domain and compete with ATP for binding.^33,34ATP

binds to the catalytic subunit of PKA within the active site

cleft, which is formed by a large, predominantly a-helical lobe,

connected by a short linker segment (residues 121–127). For the

docking studies in this catalytic site, a small library of 29 IPG-

like structures, some of which have been previously synthesised,

was used. (Fig. 2). These pseudooligosaccharides are relatively

ﬂexible ligands^6,10,34and require a thorough search of their

conformational space. In contrast, all the reported crystalline

structures of the catalytic subunit of PKA are consistently

similar. Therefore, the semi-ﬂexible Autodock programme³⁵was

used. To test the docking strategy, the docking of the natural

inhibitor balanol 33 (Fig. 3), whose three dimensional structure

complexed to the catalytic subunit of PKA has been previously

reported,³⁶was ﬁrst studied. A series of modiﬁcations were made

Synthesis

On the basis of the above results we set up to synthesise com-

pounds 8–10,13,17, and 34–36 (Fig. 5) in an effective manner.

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 6 5

View Article Online

Fig. 2 Docked structures 3,5–32.

7 6 6

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

synthesis of type-A IPG-like compounds^6,10,34we constructed the

GlcNH₂-a-(1→6)-D-myo-inositol moiety by regio- and stereose-

lective glycosylation of diol 37¹⁰with a conveniently protected 2-

azido-2-deoxy-D-glucopyranosyl trichloroacetimidate. Follow-

ing the same approach, we now have optimised this glycosy-

lation reaction using trichloroacetimidates 42^40,44and 45. Both

compounds have been prepared as shown in Scheme 1 from the

common intermediate 39,^41,42which has in turn been synthesised

from 38⁴³readily prepared from D-glucosamine.⁴⁴Conventional

benzylation of 39 gave 40,^41,44which was desilylated to yield

41⁴⁴and this in turn transformed in trichloroacetimidate 42.^40,44

Treatment of 39 with p-methoxybenzyl trichloroacetimidate

afforded 43 which was desilylated to yield 44 and this converted

into 45.

Fig. 3 Structure of balanol 33.

With the exception of 8, all these molecules have been synthe-

sised from the common precursor 52 (Fig. 6). In our previous

Fig. 4 Predicted binding modes of 26 into the ATP binding site of the catalytic subunit of PKA (top). Comparison of binding details in the X-ray

structures of PKA–ATP and PKA–33 complexes with the predicted binding of 26 (bottom).

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 6 7

View Article Online

Fig. 5 Compounds 8–10,13,17 and 34–36.

46 was converted in 52 which was transformed into 53⁴⁵by

regioselective reductive opening of the benzylidene ring with

NaCNBH₃–HCl.^46,47Attempts to direct the reductive opening

of the benzylidene ring in 52 to the formation of the alternative

48

6-OH 4-OBn derivative using BH₃·Me₂NH and BF₃·OEt₂led

to 54 and then 55 as a result of reductive cleavage of the dioxolane

ring as a ﬁrst step in the reaction.^49,50Under carefully controlled

experimental conditions however, the selective hydrolysis of the

six membered acetal ring in 52 could be achieved, giving rise to

56 which was regioselectively silylated to yield building block 57.

The synthesis of pseudodisaccharides 8 and 9 was carried

out from 47 as shown in Scheme 4. From 47, compound 58

was prepared in two steps. Removal of the p-methoxybenzyl

group in 58⁵¹gave 59 which was treated with N,N-diisopropyl

dibenzyl phosphoramidite⁵²and then with m-chloroperbenzoic

acid to afford 60. The acetals groups in 60 were removed to

give 61 which was submitted to catalytic hydrogenation. This

reaction gave 8 quantitatively when performed in a mixture of

methanol–water. In the presence of acetic acid, a rearrangement

of the phosphate group took place and compound 9 was isolated

also in quantitative yield. Similarly, pseudodisaccharide 34 was

prepared from 53 through 62 and 63 as shown in Scheme 5. Also

in this case, the catalytic hydrogenation of 63 in the presence of

acetic acid afforded 9 in quantitative yield.

Fig. 6 Compounds 37 and 52.

The synthesis of the pseudotrisaccharides 10,13,17 and 35 was

performed from the common precursor 53 as shown in Scheme 7

and 8. Trichloroacetimidates 68 and 73 were synthesised follow-

ing the same synthetic procedure, starting from the correspond-

ing pentacetate derivatives 64 and 69 respectively (Scheme 6).

For the synthesis of 10 and 17 (Scheme 7), trichloroacetimidate

68 was reacted with 53 in ether using TMSOTf as promoter to

give 74 in 93% yield. Acid hydrolysis transformed 74 into 75

from which pseudotrisaccharide 10 was obtained after catalytic

hydrogenation. According to previous experience,^6,39,53removal

of the silyl group in 74 required treatment with ten eq. of TBAF

for twenty four hours to afford 76. Phosphorylation of 76 gave

77. Removal of the ketal function as before gave 78 which was

quantitatively transformed into 17. A similar sequence from 53

Scheme 1 Reagents and conditions: a) MeONa, MeOH, 10 min; TfN₃,

DMAP, 48 h; PhCH(OMe)₂, pTsOH, DMF, 40 ^◦C, 48 h, 71%. b)

TBDMSCl, imidazol, CH₂Cl₂, −10 ^◦C, 2 h, 70%. c) NaH, BnBr, TBAI,

CH₂Cl₂, 24 h, 85%. d) AcOH, TBAF, THF, −40 ^◦C, 100%. e) CNCCl₃,

˚

DBU, CH₂Cl₂, 2 h, 95%. f) PMBOC(NH)CCl₃, BF₃·OEt₂, MS 4 A, from

0 ^◦C to rt, 18 h, 70%. g) AcOH, TBAF, −40 ^◦C, 2 h, 98%. h) CNCCl₃,

DBU, CH₂Cl₂, 2 h, 95%.

The glycosylation⁶of 42 with 37 was optimised affording,

as indicated in Scheme 2, a major pseudodisaccharide 46,⁶in

72% yield, accompanied by the b-(1→6) 48 (5%) and the a-

(1→5) 50 (6%) isomers. Similarly, the condensation of 45 with

37 yielded 47 (66%), 49 (4%) and 51 (14%). From 46, building

blocks 53⁴⁵and 57 were prepared as depicted in Scheme 3. First,

7 6 8

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

Scheme 2 Reagents and conditions: TMSOTf, Et₂O, MS 4 A, −40 ^◦C, 2 h; yielding 46 (72%), 48 (5%) and 50 (6%) from 42, and 47 (66%), 49 (4%)

and 51 (14%) from 45.

˚

Scheme 3 Reagents and conditions: a) TBAF, THF, 1 h; BnBr, NaH, DMF, 10 h, 95%. b) NaCNBH₃, HCl, THF, MS 3 A, 5 min, 90%. c) BH₃·NHMe₂,

BF₃·OEt₂, 10 min, 79% of 55. d) EtSH, BF₃·OEt₂, 0 ^◦C, 45 min, 98%. e) TBDMSCl, imidazol, DMF, −15 ^◦C, 2 h, 96%.

Scheme 4 Reagents and conditions: a) TBAF, THF, 1 h; BnBr, NaH, DMF, 10 h, 95%. b) DDQ, CH₂Cl₂–H₂O 9 : 1, 1 h, 90%. c) 1H-tetrazol,

(ⁱPr₂N)₂P(OBn)₂, CH₂Cl₂, 30 min; MCPBA, 0 ^◦C, 10 min, 96%. d) TFA, H₂O, CH₂Cl₂, 2 h, 82%. e) H₂, Pd/C, MeOH–H₂O 9 : 1, 5 h, 100%. f) H₂,

Pd/C, MeOH–H₂O–AcOH 9 : 1 : 0.1, 48 h, 100%.

Scheme 5 Reagents and conditions: a) 1H-tetrazol, (ⁱPr₂N)₂P(OBn)₂, CH₂Cl₂, 30 min; MCPBA, 0 ^◦C, 10 min, 95%. b) TFA, H₂O, CH₂Cl₂, 2 h, 83%.

c) H₂, Pd/C, MeOH–H₂O 9 : 1, 5 h, 100%. d) H₂, Pd/C, MeOH–H₂O–AcOH 9 : 1 : 0.1, 24 h, 100%.

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 6 9

View Article Online

In this synthetic approach, the common precursor 52 was

transformed in three steps into diol 88 which was attached to

a functionalised resin through OH-1 of the myo-inositol unit.

We also explored a different approach however, starting from

alcohol 57 and attaching the pseudodisaccharide building block

to the solid-support through 6-OH of the 2-azido-2-deoxy-a-D-

glucopyranosyl moiety. As a ﬁrst attempt, an amino Merriﬁeld-

type resin was functionalised with the benzylamino-type linker

90 prepared from 89 which was in turn obtained from 1-nitro-

4-triphenylmethyloxymethylbenzene (Scheme 10). Benzylamino

type linkers have been previously used in oligosaccharide

synthesis⁵⁴and can be easily cleaved under oxidative conditions

with DDQ.⁵⁴The trityl group in the functionalised resin 91

was removed⁵⁵and the benzylic function in 92 was activated

as trichloroacetimidate 93.⁵⁶Pseudodisaccharide 95, that was

prepared from 57 after levulination⁵⁷and removal of the silyl

group in the levulinated derivative 94, was immobilized⁵⁶on the

solid-support. The loading, determined by cleaving a preparative

sample of the functionalised resin 96, was 0.71 mmol g⁻¹.

Delevulination⁵⁸of 96 gave the resin-bound glycosyl acceptor 97.

Unfortunately, the attempted glycosylation with glycosyl

donor 68 did not afford the desired pseudotrisaccharide, most

likely as a result of steric hindrance due to the high resin

loading.⁵⁹The approach was therefore redesigned using Mer-

riﬁeld resins of decreased loading⁵⁹functionalised as in 93, with

a linker distribution of 0.2 mmol g⁻¹, on one hand, and changing

the solid-support to a Wang-chloride functionalised PEG-

grafted polystyrene resin on the other. Also, the attachment

of the glycosyl acceptor to the solid-support was simpliﬁed by

directly reacting diol 56 with the functionalised resin. In the case

of the low loading Merriﬁeld-type resin, 56 was react_◦ed with the

trichloroacetimidate functionalised support at −15 C and the

selectivity of the benzylation was determined by benzoylating

on solid-phase, cleaving and analysing the reaction mixture.

Under these conditions the selectivity was around 2 : 1 for

the 4-O-Bz versus the 6-O-Bz regioisomers. In the case of the

Wang-chloride functionalised PEG-grafted polystyrene resin, a

similar regioselectivity was observed in the attachment of diol

56 under basic conditions. Nevertheless, several glycosylation

Scheme 6 Reagents and conditions: a) PhSH, BF₃·OEt₂, CH₂Cl₂, 20 h,

83–100%. b) MeONa, MeOH, 10 min; TBDPSCl, imidazol, THF, 0 ^◦C,

20 h; NaH, BnBr, TBAI, THF, 24 h, 75%. c) H₂O, NBS, acetone, 0 ^◦C,

30 min, 95%. Pd/C, MeOH–H₂O 9 : 1, 5 h, 100%. d) CNCCl₃, DBU,

CH₂Cl₂, 2 h, 96%.

using 73 as a glycosyl donor led to 79 in 71% yield. Compound

79 was converted into 13 through 80 and into 35 through the

sequence 81,82 and 83. (Scheme 8).

For the synthesis of 36, compound 57 was glycosylated with

trichloroacetimidate 73 to afford 84 which without further

puriﬁcation was directly submitted to desilylation to obtain 85

in 45% overall yield from 57 (Scheme 9). Compound 85 was then

transformed into 36 through the sequence 86,87.

According to the docking predictions, these pseudotrisaccha-

ride structures present the best shape complementarity with the

ATP binding site of the catalytic subunit of PKA. In order to fa-

cilitate the synthetic process for searching for biologically active

synthetic IPG-like molecules, we also explored the feasibility of

constructing small pseudotrisaccharide libraries on solid-phase

using the same intermediates. We have already developed a step

by step solid-phase synthesis of GPI anchor precursors which

may allow for the synthesis of a variety of pseudotrisaccharide

structures.³⁹

◦

˚

Scheme 7 Reagents and conditions: a) TMSOTf, Et₂O, MS 4 A, 0 C, 30 min, 93%. b) TFA–H₂O 9 : 1, 30 min; MeONa, MeOH, 10 min, 90%. c) H₂,

Pd/C, MeOH–H₂O 9 : 1, 12 h, 100%. d) TBAF, THF, 48 h, 94%. e) 1H-tetrazol, (ⁱPr₂N)₂P(OBn)₂, CH₂Cl₂, 30 min; MCPBA, 0 ^◦C, 10 min, 95%. f)

TFA, H₂O, CH₂Cl₂, 2 h, 92%. g) H₂, Pd/C, MeOH–H₂O 9 : 1, 12 h, 100%.

7 7 0

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

Scheme 8 Reagents and conditions: a) TMSOTf, Et₂O, MS 4 A, 0 ^◦C, 1 h, 71%. b) TFA, H₂O, CH₂Cl₂, 2 h, 92%. c) H₂, Pd/C, MeOH–H₂O 9 : 1,

24 h, 100%. d) TBAF, THF, 4 h, 96%. e) 1H-tetrazol, (ⁱPr₂N)₂P(OBn)₂, CH₂Cl₂, 30 min; MCPBA, 0 ^◦C, 10 min, 96%. f) TFA, H₂O, CH₂Cl₂, 2 h,

90%. g) H₂, Pd/C, MeOH–H₂O 9 : 1, 12 h, 100%.

˚

i

˚

Scheme 9 Reagents and conditions: a) TMSOTf, Et₂O, MS 4 A, rt, 30 min. b) TBAF, THF, 6 h, 45% (two steps). c) 1H-tetrazol, ( Pr₂N)₂P(OBn)₂,

CH₂Cl₂, 30 min; MCPBA, 0 ^◦C, 10 min, 94%. d) TFA, H₂O, CH₂Cl₂, 2 h, 82%. e) H₂, Pd/C, MeOH–H₂O 9 : 1, 12 h, 100%.

reactions were studied using these regioisomeric mixtures of

functionalised Merriﬁeld resin with decreased loading and

of PEG-grafted functionalised resins with a loading around

0.15 mmol g⁻¹.

by regioselectively attaching diol 88 to the solid-support through

OH-1 of the myo-inositol moiety.

Biological activity

The results are summarised in Scheme 11, where only the

major regioisomers (98 and 99) are shown. In all cases the

glycosylations were monitored by MALDI-TOF MS and the

corresponding pseudotrisaccharides, which were released from

the resin either by treatment with DDQ (compound 102) or

after treatment with TFA⁶⁰(compounds 104 and 106), could

be isolated and the yields ranged from 71% (compound 104)

and 40% (compound 106). It was concluded that solid-phase

generation of small libraries of IPG-like structures is most

conveniently performed using our previously reported approach,

The insulin-like activity of the synthesised IPG-like molecules

was tested in vitro and some of the molecules were submitted to

in vivo assays using animal models of diabetes. A full account

of this work will be reported elsewhere. It is interesting to note

that only 8 inhibited PKA at lM concentrations and that this

molecule also activated PDHPase at lM concentrations. Com-

pounds 9,17 and 34 also activated PDHPase. Thus, contrary

to expectations, these results have not greatly contributed to

elucidate the molecular basis of this mechanism of intracellular

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 7 1

View Article Online

Scheme 10 Reagents and conditions: a) Glutaric anhydride, Py, 45 ^◦C, 2 h, 95%. b) PS-NH₂resin, DIC, DMF, 1-OH benzotriazol; Ac₂O, Py, DMAP,

98%. c) TFA, sec-butanol, 97%. d) Cl₃CCN, DBU, 0 ^◦C, 90%. e) LevOH, DCC, DMAP, CH₂Cl₂, 96%. f) HF·Py, AcOH, THF, −20 ^◦C to rt, 99%. g)

BF₃·Et₂O, C₆H₁₂, CH₂Cl₂. h) N₂H₄·AcOH, CH₂Cl₂, 18 h.

Scheme 11 Reagents and conditions: a) TMSOTf, CH₂Cl₂, 5 cycles, 60%. b) DDQ, CH₂Cl₂–H₂O 20 : 1. c) TMSOTf, CH₂Cl₂, 2 cycles, −20 ^◦C; TFA,

CH₂Cl₂, H₂O, 6 h, 71%. d) TMSOTf, CH₂Cl₂, 2 cycles, −20 ^◦C; TFA, CH₂Cl₂, H₂O, 6 h, 40%.

signal transduction. However, they provide additional proof that

cAMP-dependent and cAMP-independent protein kinases and

phosphoprotein phosphatases are intracellular targets for IPGs.

They also conﬁrm the capacity of these types of structures

to induce the phosphorylation and the dephosphorylation of

cellular proteins which are modiﬁed in response to insulin.

(1 : 9); or with anisaldehyde (25 mL) solution in sulfuric acid

(25 mL), ethanol (450 mL) and acetic acid (1 mL), followed by

heating to over 200 ^◦C. Column chromatography was carried

out on silica gel 60 (0.2 − 0.063 mm or 0.040 − 0.015 mm;

Merck). Optical rotations were determined with a Perkin-Elmer

1

341 polarimeter. H- and ¹³C-NMR spectra were acquired on

Bruker DPX-300, DRX-400 and DRX-500 spectrometers and

chemical shifts are given in ppm (d) relative to tetramethylsilane

as an internal reference or relative to D₂O. Elemental analyses

were performed with a Leco CHNS-932 apparatus, after drying

analytical samples under a vacuum over phosphorous pentoxide

for 24 h. High (HRMS) and low resolution (FAB-MS) fast

atom bombardment mass spectra were carried out by the

Mass Spectrometry Service, Facultad Qu´ımica, Seville, with a

Kratos MS-80 RFA spectrometer. Maldi-tof mass spectra were

recorded with a MALDITOF GSG System spectrometer. Gel

ﬁltration chromatography (Sephadex G-10 and Sephadex G-25;

Experimental

General

Thin layer chromatography (TLC) analyses were performed on

silica gel 60 F₂₅₄precoated on aluminium plates (Merck) and the

compounds were detected by staining with cerium(IV) sulfate

(13 g), phosphomolybdic acid (10 g), sulfuric acid (60 mL)

solution in water (1 L); with phosphomolybdic acid (40 g)

solution in water (1 L); with sulfuric acid–ethanol solution

7 7 2

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

Pharmacia) and ion-exchange chromatography (Amberlite

IRA-402 Cl⁻, Merck) were used in order to achieve puriﬁcation

of the ﬁnal products.

C₅₇H₆₇O₁₀N₃·H₂O: C 70.42%; H 7.15%; N 4.32%; found C

70.61%; H 7.14%; N 3.92%.

Data for intermediate 54. TLC (hexane–AcOEt, 4 : 1) R_f=

0.36. [a]²_D⁰= +43.1 (c = 0.7, CHCl₃). ¹H-NMR (CDCl₃,

500 MHz): d 7.44 (m, 2H, ArH), 7.41 − 7.19 (m, 23H, ArH),

5.51 (s, 1H, CH_benzylicbenzylidene), 5.35 (d, J_1,2= 3.5 Hz, 1H,

H_1b), 4.97 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.95 (d, J = 11.5 Hz,

1H, CH_benzylic), 4.88 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.77 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.74 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.72 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.71 (d, J = 11.5 Hz, 1H,

Docking calculations. All docking calculations were made

using the PKA structure obtained from 1BX6.pdb (Brookhaven

Data Bank) after removal of all substructures except the catalytic

subunit and adding all H atoms possible. Kollman type charges

for the PKA structure were obtained from Sybyl. A grid of

˚

22.5 A was used with a spacing of 0.375 A. The center of the grid

was the same as the calculated centroid for 33 before removal.

Obtention of the ligands was made as described in the main

text.³³All of non-cyclic bonds were marked as rotatables. The

root ring was in all cases the D-glucosamine ring. The docking

process was carried out mainly following the guidelines given in

Autodock User Guide 3.0.5. Afﬁnity maps were created for each

of the atoms present in each molecule (C, H, O, N, P). For each

compound, several conformational geometries were used as a

starting point for the docking calculations, obtaining equivalent

results. For each of these studies, 10 runs were calculated using

a population of 50 individuals with a limit of 250 000 energy

evaluations.

CH_benzylic), 4.64 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.05 (t, J_3,2

=

J_3,4= 9.5 Hz, 1H, H_3b), 4.05 (td, J_5,6= 4.5 Hz, J_5,4= J_5,6

ꢀ

10.5 Hz, 1H, H_5b), 3.91 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4a), 3.90

(t, J_6,1= J_6,5= 9.5 Hz, 1H, H_6a), 3.89 (br s, 1H, H_2a), 3.84 (dd,

J_6,5= 5.0 Hz, J_6,6= 10.5 Hz, 1H, H_6b), 3.67 (dd, J_A= 3.0 Hz,

ꢀ

J_B= 7.5 Hz, 1H, CH_aL-camphor), 3.66 (t, J_4,3= J_4,5= 9.5 Hz,

1H, H_4b), 3.59 (br ddd, J_1,2= 2.0 Hz, J_1,OHeq= 5.0 Hz, J_1,6

9.0 Hz, 1H, H_1a), 3.57 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H,

H_2b), 3.54 (d, J_OH,1= 5.0 Hz, 1H, OH_eq.), 3.53 (t, J_{6 ,5}= J_{6 ,6}

10.5 Hz, 1H, H_6b

), 3.38 (t, J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.34

=

ꢀ

=

ꢀ

(dd, J_3,2= 2.5 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 2.02 (m, 1H, L-

camphor), 1.63 (m, 3H, L-camphor), 1.49 (m, 1H, L-camphor),

1.06 (s, 3H, CH₃^L-camphor), 1.03 (m, 1H, L-camphor), 0.98 (s,

3H, CH₃^L-camphor), 0.95 (m, 1H, L-camphor), 0.81 (s, 3H, CH₃

L-camphor). ¹³C-NMR (CDCl₃, 125 MHz): d 138.52, 138.32,

138.15, 137.75, 137.39 (ArC), 128.90, 128.40, 128.31, 128.25,

128.21, 128.17, 128.01, 127.88, 127.65, 127.57, 127.47, 127.41,

126.05 (ArCH), 101.24 (CH_benzylicbenzylidene), 99.08 (C_1b), 89.49

(CH_aL-camphor), 82.80 (C_4b), 81.91 (C_4a), 81.83 (C_6a), 81.03

(C_5a), 80.30 (C_3a), 77.00 (C_3b), 76.75 (C_2a), 75.57 (CH_2benzylic),

75.03 (2 × CH_2benzylic), 74.01 (C_1a), 72.66 (CH_2benzylic), 68.62 (C_6b),

64.10 (C_2b), 63.11 (C_5b), 49.61, 46.60 (C L-camphor), 45.17 (CH

L-camphor), 39.56, 34.39, 27.30 (CH₂L-camphor), 20.46, 20.20,

12.68 (CH₃L-camphor). FAB-MS: m/z 974 (M + Na)⁺.

Compounds 43–53,58,67,68,71–73. The synthesis and the

full characterisation of these compounds are included as sup-

porting information†.

2-Azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1→6)-

3,4,5-tri-O-benzyl-2-O-(L-1,7,7-trimethyl-[2,2,1]-bicycloheptyl)-

D-myo-inositol 55. To a solution of 52 (350 mg, 0.368 mmol)

and BH₃·HNMe₂(87 mg, 1.477 mmol) in dry CH₂Cl_2.(7.4 mL)

under an argon atmosphere, Et₂O·BF₃(192 ll, 1.484 mmol)

was added dropwise. The solution was stirred for 10 min,

diluted with AcOEt (50 mL) and washed with sat. NaHCO₃

(2 × 50 mL) and sat. NaCl (3 × 50 mL) solutions. The organic

layer was washed over MgSO₄and concentrated in vacuo. The

residue was puriﬁed by ﬂash chromatography (hexane–AcOEt

9 : 1, 4 : 1) to yield 277 mg (79%) of 55 as a white foam. TLC

(hexane–AcOEt 4 : 1) R_f= 0.10. [a]²_D⁰= +50.6 (c = 1.3, CHCl₃).

¹H-NMR (CDCl₃, 500 MHz): d 7.35 − 7.25 (m, 18H, ArH),

7.24 − 7.18 (m, 7H, ArH), 5.38 (d, J_1,2= 3.5 Hz, 1H, H_1b),

5.00 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.91 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.88 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.86 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.82 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.731 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.728 (d, J = 11.0 Hz,

1H, CH_benzylic), 4.64 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.63 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.62 (d, J = 11.5 Hz, 1H, CH_benzylic),

2-Azido-3-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1→6)-1,2-

O-(L-1,7,7-trimethyl-[2,2,1]-bicyclohept-2-ylidene)-3,4,5-tri-O-

benzyl-D-myo-inositol 56. To a solution of 52 (450 mg,

0.474 mmol) in dry CH₂Cl₂(9.5 mL) under an argon

atmosphere, ethanethiol (526 ll, 7.102 mmol) and Et₂O·BF₃

(3 ll, 0.024 mmol) were added at 0 ^◦C. The solution was stirred

for 45 min, diluted with AcOEt (100 mL) and washed with

sat. NaHCO₃(125 mL) and sat. NaCl (3 × 125 mL) solutions.

The organic layer was dried over MgSO₄and concentrated

in vacuo. The residue was puriﬁed by ﬂash chromatography

(hexane–AcOEt 9 : 1, 4 : 1, 2 : 1) to yield 400 mg (98%) of

1

56 as a white foam. [a]_D²⁰= +41.3 (c = 1.3, CHCl₃). H-NMR

3.99 (t, J_3,2= J_3,4= 10.0 Hz, 1H, H_3b), 3.892 (t, J_4,3= J_4,5

=

(CDCl₃, 500 MHz): d 7.41 − 7.26 (m, 20H, ArH), 5.55 (d,

J_1,2= 3.5 Hz, 1H, H_1b), 4.89 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.82 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.81 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.76 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.75 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.70 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.69 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.62 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.29 (dd, J_2,3= 4.0 Hz, J_2,1= 6.0 Hz, 1H, H_2a), 4.03

9.5 Hz, 1H, H_4a), 3.887 (t, J_2,1= J_2,3= 2.5 Hz, 1H, H_2a), 3.87

(t, J_6,1= J_6,5= 9.5 Hz, 1H, H_6a), 3.85 (dt, J_5,6= J_5,6= 2.5 Hz,

ꢀ

J_5,4= 10.0 Hz, 1H, H_5b), 3.66 (dd, J_A= 3.5 Hz, J_B= 8.0 Hz,

1H, CH_a^L-camphor), 3.61 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.59

(ddd, J_1,2= 2.5 Hz, J_1,OHeq= 6.0 Hz, J_1,6= 9.0 Hz, 1H, H_1a),

3.46 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H, H_2b), 3.34 (t, J_5,4

=

J_5,6= 9.5 Hz, 1H, H_5a), 3.33 (dd, J_3,2= 2.0 Hz, J_3,4= 10.0 Hz,

1H, H_3a), 3.31 (d, J_OHeq,1= 6.0 Hz, 1H, OH_eq.), 3.30 (m, 2H,

(dd, J_1,2= 6.0 Hz, J_1,6= 7.0 Hz, 1H, H_1a), 3.99 (dd, J_6,1=

ꢀ

7.5 Hz, J_6,5= 10.0 Hz, 1H, H_6a), 3.86 (dt, J_5,6= J_5,6= 3.5 Hz,

H_6b+ H_6b

ꢀ

), 2.02 (m, 1H, L-camphor), 1.63 (m, 3H, L-camphor),

J_5,4= 10.0 Hz, 1H, H_5b), 3.83 (t, J_4,3= J_4,5= 8.0 Hz, 1H,

H_4a), 3.80 (dd, J_3,2= 4.0 Hz, J_3,4= 8.0 Hz, 1H, H_3a), 3.73

1.49 (m, 1H, L-camphor), 1.31 (br t, J_OH,6= J_OH,6

ꢀ

4.5 Hz, 1H,

OH_6b), 1.06 (s, 3H, CH₃L-camphor), 1.00 (m, 1H, L-camphor),

0.98 (s, 3H, CH₃^L-camphor), 0.94 (m, 1H, L-camphor), 0.81 (s,

3H, CH₃^L-camphor). ¹³C-NMR (CDCl₃, 125 MHz): d 138.47,

138.38, 138.12, 138.09, 137.76 (ArC), 128.45, 128.41, 128.32,

128.27, 128.08, 128.04, 127.88, 127.80, 127.76, 127.65, 127.59,

127.50, 127.45 (ArCH), 98.40 (C_1b), 89.64 (CH_aL-camphor),

81.78 (C_4a), 81.27 (C_5a), 80.89 (C_6a), 80.62 (C_3b), 80.37 (C_3a),

77.80 (C_4b), 77.16 (C_2a), 75.60, 75.52, 75.22, 74.91 (CH_2benzylic),

74.11 (C_1a), 72.73 (CH_2benzylic), 71.51 (C_5b), 64.39 (C_2b), 60.77

(C_6b), 49.64, 46.60 (C L-camphor), 45.15 (CH L-camphor),

39.70, 34.41, 27.28 (CH₂L-camphor), 20.45, 20.19, 12.71 (CH₃

L-camphor). FAB-MS: m/z 976 (M + Na)⁺. Anal. calcd. for

(dd, J_3,4= 9.0 Hz, J_3,2= 10.5 Hz, 1H, H_3b), 3.60 (br t, J_4,3=

ꢀ

J_4,5= 9.5 Hz, 1H, H_4b), 3.57 (m, 2H, H_6b+ H_6b

), 3.44 (dd,

J_5,4= 7.5 Hz, J_5,6= 10.0 Hz, 1H, H_5a), 3.26 (dd, J_2,1= 3.5 Hz,

J_2,3= 10.0 Hz, 1H, H_2b), 1.92 (m, 1H, L-camphor), 1.86 (m,

2H, L-camphor + OH_4b), 1.76 (m, 1H, L-camphor), 1.72 (m, 1H,

L-camphor), 1.56 (br s, 1H, OH_6b), 1.47 (d, J = 13.0 Hz, 1H,

L-camphor), 1.39 (m, 1H, L-camphor), 1.22 (m, 1H, L-camphor),

1.07 (s, 3H, CH₃^L-camphor), 0.87 (s, 6H, 2 × CH₃L-camphor).

¹³C-NMR (CDCl₃, 125 MHz): d 138.37 (ArC), 138.30 (2 ×

ArC), 138.05 (ArC), 128.62, 128.42, 128.38, 128.35, 128.10,

128.03, 128.00, 127.87, 127.81, 127.76, 127.73, 127.65 (ArCH),

118.11 (C_acetalicL-camphor), 95.59 (C_1b), 80.75 (C_5a), 80.65 (C_4a),

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 7 3

View Article Online

79.45 (C_3b), 77.94 (C_6a), 76.91 (C_3a), 76.04 (C_1a), 75.00, 74.80,

74.73 (CH_2benzylic), 73.90 (C_2a), 72.61 (CH_2benzylic), 71.61 (C_4b),

70.55 (C_5b), 62.88 (C_2b), 62.29 (C_6b), 51.58, 47.97 (C L-camphor),

45.14 (CH L-camphor), 44.79, 29.89, 26.96 (CH₂L-camphor),

20.60, 20.34, 9.77 (CH₃L-camphor). FAB-MS: m/z 884 (M +

Na)⁺. Anal. calcd. for C₅₀H₅₉O₁₀N₃·H₂O: C 68.31%; H 6.99%;

N 4.77%; found C 68.31%; H 6.99%; N 4.47%.

10.5 Hz, 1H, CH_benzylic), 4.70 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.67 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.29 (br t, J_2,1= J_2,3

=

4.5 Hz, 1H, H_2a), 4.19 (dd, J_6,5= 5.0 Hz, J_6,6

ꢀ

= 10.0 Hz, 1H,

H_6b), 4.16 (m, 1H, H_5b), 4.14 (td, J_OH,3= 2.5 Hz, J_3,2= J_3,4

10.0 Hz, 1H, H_3b), 4.03 (m, 2H, H_1a+ H_6a), 3.83 (t, J_4,3= J_4,5

=

8.5 Hz, 1H, H_4a), 3.81 (dd, J_3,2= 4.0 Hz, J_3,4= 8.0 Hz, 1H, H_3a),

3.66 (t, J_{6 ,5}

ꢀ

= J_{6 ,6}

ꢀ

= 10.0 Hz, 1H, H_6b), 3.50 (t, J_4,3= J_4,5=

ꢀ

9.5 Hz, 1H, H_4b), 3.45 (m, 1H, H_5a), 3.28 (dd, J_2,1= 3.5 Hz,

J_2,3= 10.0 Hz, 1H, H_2b), 2.56 (d, J_OH,3= 2.5 Hz, 1H, OH), 1.92

(m, 1H, L-camphor), 1.86 (m, 1H, L-camphor), 1.75 (m, 1H,

L-camphor), 1.72 (m, 1H, L-camphor), 1.46 (d, J = 13.0 Hz, 1H,

L-camphor), 1.39 (m, 1H, L-camphor), 1.22 (m, 1H, L-camphor),

1.07 (s, 3H, CH₃^L-camphor), 0.873 (s, 3H, CH₃L-camphor),

0.868 (s, 3H, CH₃^L-camphor). ¹³C-NMR (CDCl₃, 125 MHz): d

138.42, 138.33, 138.00, 137.11 (ArC), 129.20, 128.44, 128.36,

128.30, 128.19, 128.08, 127.85, 127.82, 127.71, 127.59, 127.52,

126.46 (ArCH), 118.10 (C_acetalicL-camphor), 102.03 (CH_benzylic

benzylidene), 95.97 (C_1b), 81.95 (C_4b), 80.65 (C_4a), 80.59 (C_5a),

78.12 (C_6a), 76.85 (C_3a), 75.99 (C_1a), 75.20, 74.71 (CH_2benzylic),

73.94 (C_2a), 72.61 (CH_2benzylic), 68.76 (C_6b), 68.58 (C_3b), 63.09

(C_2b), 62.18 (C_5b), 51.60, 47.96 (C L-camphor), 45.15 (CH

L-camphor), 44.81, 29.87, 26.97 (CH₂L-camphor), 20.60, 20.36,

9.71 (CH₃L-camphor). FAB-MS: m/z 860 M⁺, 882 (M + Na)⁺.

Anal. calcd. for C₅₀H₅₇O₁₀N₃: C 69.83%; H 6.68%; N 4.89%;

found C 69.59%; H 6.90%; N 5.03%.

2-Azido-3-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-a-D-

glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trime-

thyl-[2,2,1]-bicyclohept-2-ylidene)-D-myo-inositol 57. To

a

solution of 56 (350 mg, 0.406 mmol) and imidazol (88 mg,

1.293 mmol) in dry DMF (5 mL) under an argon atmosphere,

a solution of TBDMSCl (73 mg, 0.484 mmol) in dry DMF

(3 mL) was added at −15 ^◦C. The solution was stirred for

2 h, diluted with AcOEt (50 mL) and washed with 10% HCl

solution (50 mL). The aqueous layer was extracted with AcOEt

(2 × 25 mL) and the combined organic layers were washed

with sat. NaCl solution (3 × 100 mL), dried over MgSO₄

and concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography (hexane–AcOEt 19 : 1) to yield 380 mg (96%)

of 57 as a white foam. [a]²_D⁰= +37.5 (c = 1.7, CHCl₃). ¹H-NMR

(CDCl₃, 500 MHz): d 7.44 − 7.26 (m, 20H, ArH), 5.55 (d,

J_1,2= 3.5 Hz, 1H, H_1b), 4.88 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.86 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.81 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.76 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.72 (d, J =

11.0 Hz, 2H, 2 × CH_benzylic), 4.70 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.69 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.28 (dd, J_2,1= 3.5 Hz,

J_2,3= 6.0 Hz, 1H, H_2a), 4.01 (t, J_1,2= J_1,6= 7.0 Hz, 1H, H_1a),

3.96 (dd, J_6,1= 7.0 Hz, J_6,5= 10.0 Hz, 1H, H_6a), 3.88 (br dt,

2-Azido-4,6-O-benzylidene-2-deoxy-3-O-dibenzylphosphate-a-

D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-tri-

methyl-[2,2,1]-bicyclohept-2-ylidene)-D-myo-inositol 60. To a

solution of 59 (300 mg, 0.349 mmol) and 1H-tetrazol (61 mg,

0.871 mmol) in dry CH₂Cl₂(7 mL) under an argon atmosphere,

N,N-diisopropyl dibenzyl phosphoramidite (230 ll,

0.699 mmol) was added. The solution was stirred for 30 min

and 70% MCPBA (172 mg, 0.698 mmol) was added at 0 ^◦C.

After 10 min the mixture was diluted with AcOEt (50 mL) and

washed with sat. NaHCO₃(2 × 50 mL) and sat. NaCl (3 ×

50 mL) solutions. The organic layer was dried over MgSO₄,

concentrated in vacuo and the residue was puriﬁed by double

ﬂash chromatography (hexane–AcOEt 2 : 1 and hexane–AcOEt

9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1) to yield 375 mg

(96%) of 60 as a white foam. [a]²_D⁰= +55.6 (c = 1.1, CHCl₃).

¹H-NMR (CDCl₃, 500 MHz): d 7.38 − 7.18 (m, 25H, ArH),

7.14 (m, 3H, ArH), 7.04 (m, 2H, ArH), 5.70 (d, J = 4.0 Hz,

J_5,6= J_5,6= 4.0 Hz, J_5,4= 9.5 Hz, 1H, H_5b), 3.82 (t, J_3,2= J_3,4=

ꢀ

9.5 Hz, 1H, H_3b), 3.81 (t, J_4,3= J_4,5= 8.5 Hz, 1H, H_4a), 3.78 (dd,

J_3,2= 4.0 Hz, J_3,4= 8.0 Hz, 1H, H_3a), 3.74 (td, J_4,OH= 3.0 Hz,

J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.71 (dd, J_6,5= 3.5 Hz, J_6,6=

ꢀ

11.0 Hz, 1H, H_6b), 3.60 (dd, J_{6 ,5}

H_6b

ꢀ

= 5.0 Hz, J_{6 ,6}= 11.0 Hz, 1H,

ꢀ

), 3.42 (dd, J_5,4= 7.5 Hz, J_5,6= 9.5 Hz, 1H, H_5a), 3.25 (dd,

J_2,1= 3.5 Hz, J_2,3= 10.5 Hz, 1H, H_2b), 2.35 (d, J_OH,4= 3.0 Hz,

1H, OH), 1.88 (m, 2H, L-camphor), 1.75 (m, 1H, L-camphor),

1.72 (m, 1H, L-camphor), 1.46 (d, J = 13.0 Hz, 1H, L-camphor),

1.38 (m, 1H, L-camphor), 1.22 (m, 1H, L-camphor), 1.07 (s, 3H,

CH₃^L-camphor), 0.87 (s, 3H, CH₃L-camphor), 0.86 (s, 12H,

CH₃L-camphor + (CH₃)C TBDMS), 0.02 (CH₃TBDMS),

0.01 (CH₃TBDMS). ¹³C-NMR (CDCl₃, 125 MHz): d 138.56,

138.34, 138.26, 138.20 (ArC), 128.51, 128.36, 128.35, 128.31,

128.25, 128.23, 127.88, 127.834, 127.825, 127.71, 127.66, 127.56

(ArCH), 118.04 (C_acetalicL-camphor), 95.30 (C_1b), 80.80 (C_5a),

80.65 (C_4a), 79.10 (C_3b), 77.66 (C_6a), 77.20 (C_3a), 76.19 (C_1a),

75.38, 74.92, 74.86 (CH_2benzylic), 73.90 (C_2a), 72.84 (C_4b), 72.53

(CH_2benzylic), 70.47 (C_5b), 63.56 (C_6b), 62.73 (C_2b), 51.59, 47.94

(C L-camphor), 45.13 (CH L-camphor), 44.89, 29.82, 26.98

(CH₂L-camphor), 25.94 ((CH₃)₃C TBDMS), 20.62, 20.37 (CH₃

L-camphor), 18.34 ((CH₃)₃C TBDMS), 9.78 (CH₃L-camphor),

−5.22 (2 × CH₃TBDMS). FAB-MS: m/z 975 M⁺, 998 (M +

Na)⁺. Anal. calcd. for C₅₆H₇₃O₁₀SiN₃: C 68.89%; H 7.54%; N

4.30%; found C 68.62%; H 7.56%; N 4.07%.

1H, H_1b), 5.45 (s, 1H, CH_benzylicbenzylidene), 5.08 (dd, J₁

=

7.0 Hz, J₂= 11.5 Hz, 1H, CH_benzylic), 4.99 (dd, J₁= 8.0 Hz,

J₂= 11.5 Hz, 1H, CH_benzylic), 4.97 (q, J = 9.5 Hz, 1H, H_3b), 4.95

(dd, J₁= 7.0 Hz, J₂= 12.0 Hz, 1H, CH_benzylic), 4.85 (dd, J₁=

8.0 Hz, J₂= 12.0 Hz, 1H, CH_benzylic), 4.76 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.751 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.749 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.71 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.68 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.67 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.30 (dd broad, J₁= 3.5 Hz, J₂= 5.5 Hz, 1H, H_2a),

4.21 (td, J₁= 5.0 Hz, J₂= 10.0 Hz, 1H, H_5b), 4.14 (dd, J₁=

5.0 Hz, J₂= 10.5 Hz, 1H, H_6b), 4.06 (m, 2H, H_6a+ H_1a), 3.83

(m, 2H, H_4a+ H_3a), 3.68 (t, J = 9.5 Hz, 1H, H_4b), 3.63 (t, J =

2-Azido-4,6-O-benzylidene-2-deoxy-a-D-glucopyranosyl-(1→

6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-bicyclo-

hept-2-ylidene)-D-myo-inositol 59. To a solution of 58 (445 mg,

0.454 mmol) in a mixture CH₂Cl₂–H₂O 9 : 1 (9 mL), DDQ

(155 mg, 0.683 mmol) was added. The mixture was stirred for

1 h, diluted with AcOEt (50 mL) and washed with sat. NaHCO₃

(2 × 50 mL) and sat. NaCl (3 × 50 mL) solutions. The organic

layer was dried over MgSO₄, concentrated in vacuo and the

residue was puriﬁed by ﬂash chromatography (hexane–AcOEt

9 : 1, 4 : 1) to yield 352 mg (90%) of 59 as a white foam.

10.5 Hz, 1H, H_6b

ꢀ

), 3.47 (m, 1H, H_5a), 3.32 (dd, J₁= 4.0 Hz,

J₂= 10.5 Hz, 1H, H_2b), 1.92 (m, 1H, L-camphor), 1.86 (m, 1H,

L-camphor), 1.76 (m, 1H, L-camphor), 1.72 (m, 1H, L-camphor),

1.46 (d, J = 13.0 Hz, 1H, L-camphor), 1.40 (m, 1H, L-camphor),

1.22 (m, 1H, L-camphor), 1.07 (s, 3H, CH₃L-camphor), 0.88 (s,

3H, CH₃^L-camphor), 0.86 (s, 3H, CH₃L-camphor). ³¹P-NMR

(CDCl₃, 202 MHz): d −2.52. ¹³C-NMR (CDCl₃, 125 MHz): d

138.35, 138.31, 137.86, 136.96 (ArC), 135.93 (d, J_C,P= 7.5 Hz,

ArC phosphate), 135.93 (d, J_C,P= 7.8 Hz, ArC phosphate),

129.07, 128.41, 128.38, 128.35, 128.34, 128.27, 128.21, 128.16,

128.09, 128.04, 127.82, 127.80, 127.69, 127.63, 127.54, 127.48,

126.56 (ArCH), 118.08 (C_acetalL-camphor), 102.05 (CH_benzylic

benzylidene), 96.76 (C_1b), 80.55 (C_4a), 80.38 (C_5a), 80.29 (d,

J_C,P= 2.9 Hz, C_4b), 78.74 (C_6a), 76.55 (C_3a), 75.81 (C_1a), 75.07

(CH_2benzylic), 74.82 (d, J_C,P= 6.4 Hz, C_3b), 74.41 (CH_2benzylic),

1

[a]²_D⁰= +56.3 (c = 1.2, CHCl₃). H-NMR (CDCl₃, 500 MHz):

d 7.38 − 7.23 (m, 17H, ArH), 7.18 (m, 3H, ArH), 5.63 (d,

J_1,2= 4.0 Hz, 1H, H_1b), 5.94 (s, 1H, CH_benzylicbenzylidene),

4.78 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.77 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.75 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.72 (d, J =

7 7 4

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

73.91 (C_2a), 72.72 (CH_2benzylic), 69.23 (d, J_C,P= 5.4 Hz, CH_2benzylic

phosphate), 69.20 (d, J_C,P= 5.4 Hz, CH_2benzylicphosphate), 68.64

(C_6b), 62.49 (d, J_C,P= 3.1 Hz, C_2b), 62.45 (C_5b), 51.57, 47.95 (C

L-camphor), 45.16 (CH L-camphor), 44.69, 29.92, 26.97 (CH₂

L-camphor), 20.59, 20.34, 9.76 (CH₃L-camphor). FAB-MS: m/z

1119 M⁺, 1142 (M + Na)⁺. Anal. calcd. for C₆₄H₇₀O₁₃N₃P:

C 68.62%; H 6.30%; N 3.75%; found C 68.40%; H 6.10%; N

3.56%.

(C_5b), 71.56 (C_1a), 70.89 (C_3a), 68.95 (C_4b), 59.90 (C_6b), 54.27

(C_2b). HRMS m/z calcd. for C₁₂H₂₄O₁₃NPNa⁺: 444.0876; found

444.0885 (M + Na)⁺.

2-Azido-2-deoxy-3,6-di-O-benzyl-4-O-dibenzylphosphate-a-D-

glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trime-

thyl-[2,2,1]-bicyclohept-2-yliden)-D-myo-inositol 62. To

a

solution of 53 (200 mg, 0.210 mmol) and 1H-tetrazol

(37 mg, 0.528 mmol) in dry CH₂Cl₂(4.2 mL) under an

argon atmosphere, N,N-diisopropyl dibenzyl phosphoramidite

(138 ll, 0.420 mmol) was added. The solution was stirred for

30 min and 70% MCPBA (129 mg, 0.525 mmol) was added at

0 ^◦C. After 10 min the mixture was diluted with AcOEt (50 mL)

and washed with sat. NaHCO₃(2 × 50 mL) and sat. NaCl (3 ×

50 mL) solutions. The organic layer was dried over MgSO₄,

concentrated in vacuo and the residue was puriﬁed by double

ﬂash chromatography (hexane–AcOEt 2 : 1 and hexane–AcOEt

9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1) to yield 242 mg

(95%) of 62 as a white foam. [a]²_D⁰= +48.4 (c = 1.9, CHCl₃).

¹H-NMR (CDCl₃, 500 MHz): d 7.44 (m, 2H, ArH), 7.37 − 7.19

(m, 28H, ArH), 7.14 (m, 5H, ArH), 5.60 (d, J_1,2= 3.5 Hz, 1H,

H_1b), 4.89 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.89 − 4.79 (m, 5H,

4 × CH_benzylicphosphate + CH_benzylic), 4.77 (br d, J = 11.0 Hz,

2H, 2 × CH_benzylic), 4.74 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.676

(q, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 4.674 (d, J = 11.5 Hz, 1H,

CH_benzylic), 4.65 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.62 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.43 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.34 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.28 (br dd, J_2,3= 3.0 Hz,

2-Azido-2-deoxy-3-O-dibenzylphosphate-a-D-glucopyranosyl-

(1→6)-3,4,5-tri-O-benzyl-D-myo-inositol 61. To a solution of

60 (300 mg, 0.268 mmol) in CH₂Cl₂(13.4 mL), H₂O (483 ll,

26.811 mmol) and TFA (2.06 mL, 26.83 mmol) were added

and the resulting mixture was stirred for 2 h. The mixture was

diluted with AcOEt (100 mL) and washed with sat. NaHCO₃

(2 × 100 mL) and sat. NaCl (3 × 100 mL) solutions. The organic

layer was dried over MgSO₄, concentrated in vacuo and the

residue was puriﬁed by ﬂash chromatography (hexane–AcOEt

1 : 1, 1 : 4, AcOEt 100%) to yield 197 mg (82%) of 61 as

1

a colorless syrup. [a]_D²⁰= +49.8 (c = 1.3, CHCl₃). H-NMR

(CDCl₃, 500 MHz): d 7.38 − 7.22 (m, 25H, ArH), 5.47 (d, J =

3.5 Hz, 1H, H_1b), 5.16 − 5.03 (m, 4H, 4 × CH_benzylicphosphate),

5.01 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.92 (d, J = 10.5 Hz, 1H,

CH_benzylic), 4.80 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.73 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.70 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.62 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.60 (ddd, J_3,P= 6.5 Hz,

J_3,4= 8.5 Hz, J_3,2= 10.0 Hz, 1H, H_3b), 4.32 (d, J_OH,4= 3.0 Hz,

1H, OH_4b), 4.15 (br t, J_2,1= J_2,3= 2.5 Hz, 1H, H_2a), 3.98 (t,

J_4,3= J_4,5= 9.5 Hz, 1H, H_4a), 3.95 (t, J_6,5= J_6,1= 9.5 Hz, 1H,

J_2,1= 6.0 Hz, 2H, H_2a), 4.16 (br dt, J_5,6= J_5,6

ꢀ

= 2.0 Hz, J_5,4

=

H_6a), 3.80 (dt, J_5,6= J_5,6

ꢀ

= 3.0 Hz, J_5,4= 10.0 Hz, 1H, H_5b),

10.5 Hz, 1H, H_5b), 4.02 (m, 2H, H_6a+ H_1a), 3.97 (t, J_3,2= J_3,4

3.69 (td, J_4,OH= 3.0 Hz, J_4,3= J_4,5= 9.0 Hz, H_4b), 3.61 (ddd,

J_1,2= 2.5 Hz, J_1,OH= 5.5 Hz, J_1,6= 8.5 Hz, 1H, H_1a), 3.48 (dd,

J_3,2= 2.5 Hz, J_3,4= 9.5 Hz, 1H, H_3a), 3.44 (d, J_OH,1= 5.5 Hz,

9.5 Hz, 1H, H_3b), 3.80 (m, 2H, H_4a+ H_3a), 3.49 (dd, J_6,5

2.5 Hz, J_6,6

ꢀ

= 11.0 Hz, 1H, H_6b), 3.44 (dd, J_{6 ,5}

ꢀ

= 1.5 Hz, J_{6 ,6}

ꢀ

11.0 Hz, 1H, H_6b

ꢀ

), 3.43 (br dd, J_5,4= 4.0 Hz, J_5,6= 9.5 Hz,

1H, OH_1a), 3.41 (dt, J_6,OH= J_6,5= 3.5 Hz, J_6,6

ꢀ

= 12.0 Hz,

1H, H_5a), 3.39 (dd, J_2,1= 3.5 Hz, J_2,3= 10.5 Hz, 1H, H_2b),

1.89 (m, 2H, L-camphor), 1.75 (m, 1H, L-camphor), 1.71 (m,

1H, L-camphor), 1.46 (d, J = 13.0 Hz, 1H, L-camphor), 1.37

(m, 1H, L-camphor), 1.21 (m, 1H, L-camphor), 1.06 (s, 3H, CH₃

L-camphor), 0.87 (s, 3H, CH₃L-camphor), 0.82 (s, 3H, CH₃

L-camphor). ³¹P-NMR (CDCl₃, 202 MHz): d − 2.96. ¹³C-NMR

(CDCl₃, 125 MHz): d 138.31 (ArC), 138.28 (2 × ArC), 137.94,

137.73 (ArC), 135.88 (d, J_C,P= 7.6 Hz, ArC phosphate), 135.85

(d, J_C,P= 7.1 Hz, ArC phosphate), 128.41, 128.37, 128.32,

128.27, 128.25, 128.21, 128.17, 128.12, 127.95, 127.82, 127.77,

127.73, 127.67, 127.60, 127.57, 127.26 (ArCH), 118.09 (C_acetalic

L-camphor), 95.42 (C_1b), 80.67 (C_4a), 80.51 (C_5a), 78.04 (C_6a),

78.00 (d, J_3,P= 2.5 Hz, C_3b), 76.75 (C_3a), 76.03 (C_1a), 75.47 (d,

J_4,P= 7.3 Hz, C_4b), 74.90, 74.56, 74.00 (CH_2benzylic), 73.80 (C_2a),

73.04, 72.57 (CH_2benzylic), 69.45 (d, J_5,P= 4.9 Hz, C_5b), 69.26 (d,

J_C,P= 4.9 Hz, CH_2benzylicphosphate), 69.24 (d, J_C,P= 4.8 Hz,

CH_2benzylicphosphate), 67.50 (C_6b), 62.59 (C_2b), 51.54, 47.92 (C

L-camphor), 45.09 (CH L-camphor), 44.69, 29.83, 26.94 (CH₂

L-camphor), 20.57, 20.34, 9.68 (CH₃L-camphor). FAB-MS: m/z

1234 (M + Na)⁺. Anal. calcd. for C₇₁H₇₈O₁₃N₃P: C 70.34%; H

6.49%; N 3.47%; found C 70.49%; H 6.81%; N 3.27%.

1H, H_6b), 3.38 (dd, J_2,1= 4.0 Hz, J_2,3= 10.0 Hz, H_2b), 3.36 (t,

J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.34 (dt, J_{6 ,OH}= J_{6 ,5}= 4.0 Hz,

ꢀ

J_{6 ,6}

J_OH,6

ꢀ

= 12.0 Hz, 1H, H_6b

ꢀ

), 2.62 (s, 1H, OH_2a), 1.51 (br t, J_OH,6

=

ꢀ

= 4.0 Hz, 1H, OH_6b). ³¹P-NMR (CDCl₃, 202 MHz): d

−0.41. ¹³C-NMR (CDCl₃, 125 MHz): d 138.34, 138.12, 137.60

(ArC), 135.32 (d, J_C,P= 6.8 Hz, ArC phosphate), 135.24 (d,

J_C,P= 7.1 Hz, ArC phosphate), 128.67, 128.60, 128.58, 128.54,

128.39, 128.35, 128.03, 128.00, 127.96, 127.93, 127.83, 127.64,

127.61, 127.48 (ArCH), 98.00 (C_1b), 81.59 (C_4a), 80.67 (C_5a),

80.61 (d, J_3,P= 5.5 Hz, C_3b), 79.84 (C_6a), 79.57 (C_3a), 75.81,

75.39, 72.87 (CH_2benzylic), 72.72 (C_1a), 71.42 (C_5b), 70.12 (d, J_C,P

=

5.8 Hz, CH_2benzylicphosphate), 70.05 (d, J_C,P= 5.8 Hz, CH_2benzylic

phosphate), 69.70 (C_4b), 69.56 (C_2a), 62.59 (d, J_2,P= 6.8 Hz,

C_2b), 61.19 (C_6b). FAB-MS: m/z 920 (M + Na)⁺. Anal. calcd.

for C₄₇H₅₂O₁₃N₃P·H₂O: C 61.63%; H 5.94%; N 4.59%; found C

61.81%; H 6.11%; N 4.40%.

2-Ammonio-2-deoxy-3-O-phosphate-a-D-glucopyranosyl-(1→

6)-D-myo-inositol 8. A suspension of 61 (120 mg, 0.134 mmol)

and 10% Pd on charcoal (285 mg, 0.268 mmol) in a mixture

of MeOH–H₂O 9 : 1 (13 mL) under a hydrogen atmosphere

was stirred for 5 h. The mixture was ﬁltered through a pad of

celite and the ﬁlter cake washed with H₂O. The solution was

concentrated in vacuo and lyophilised to yield 56 mg (100%) of

8 as a white solid. [a]_D²⁰= +55.0 (c = 0.5, H₂O). NMR data at

pH = 5.6: ¹H-NMR (D₂O, 500 MHz): d 5.38 (d, J_1,2= 3.5 Hz,

2-Azido-3,6-di-O-benzyl-4-O-dibenzylphosphate-2-deoxy-a-D-

glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-D-myo-inositol

63.

To a solution of 62 (180 mg, 0.149 mmol) in CH₂Cl₂(7.4 mL),

H₂O (268 ll, 14.876 mmol) and TFA (1.15 mL, 14.977 mmol)

were added and the resulting mixture was stirred for 2 h. The

mixture was diluted with AcOEt (50 mL) and washed with sat.

NaHCO₃(2 × 50 mL) and sat. NaCl (3 × 50 mL) solutions.

The organic layer was dried over MgSO₄, concentrated in

vacuo and the residue was puriﬁed by ﬂash chromatography

(hexane–AcOEt 2 : 1, 1 : 1, 1 : 4) to yield 133 mg (83%) of

1H, H_1b), 4.34 (q, J_3,2= J_3,4= 9.0 Hz, 1H, H_3b), 4.07 (dt, J_5,6

=

J_5,6

3.0 Hz, 1H, H_2a), 3.82 (dd, J_6,5= 3.5 Hz, J_6,6

H_6b), 3.77 (dd, J_{6 ,5}= 12.5 Hz, 1H, H_6b

= 2.5 Hz, J_{6 ,6}

(dd, J_1,2= 3.0 Hz, J_1,6= 10.0 Hz, 1H, H_1a), 3.69 (t, J_6,1= J_6,5

10.0 Hz, 1H, H_6a), 3.65 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.60

ꢀ

= 3.0 Hz, J_5,4= 10.0 Hz, 1H, H_5b), 3.99 (t, J_2,1= J_2,3

ꢀ

= 12.5 Hz, 1H,

ꢀ

), 3.74

=

1

63 as a white syrup. [a]_D²⁰= +40.9 (c = 1.8, CHCl₃). H-NMR

(t, J_4,3= J_4,5= 10.0 Hz, 1H, H_4a), 3.50 (dd, J_3,2= 3.0 Hz, J_3,4

=

(CDCl₃, 500 MHz): d 7.43 (m, 2H, ArH), 7.35 − 7.10 (m, 33H,

ArH), 5.44 (d, J_1,2= 4.0 Hz, 1H, H_1b), 5.00 (d, J = 11.0 Hz,

1H, CH_benzylic), 4.94 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.91 (d,

J = 11.0 Hz, 1H, CH_benzylic), 4.88 − 4.75 (m, 6H, 4 × CH_benzylic

phosphate + 2 × CH_benzylic), 4.73 (d, J = 11.5 Hz, 1H, CH_benzylic),

10.0 Hz, 1H, H_3a), 3.40 (t, J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.40

(dd, J_2,1= 4.0 Hz, J_2,3= 10.0 Hz, 1H, H_2b). ³¹P-NMR (D₂O,

202 MHz): d 2.54. ¹³C-NMR (D₂O, 125 MHz): d 96.61 (C_1b),

80.52 (C_6a), 73.08 (br s, C_3b), 72.53 (C_4a+ C_5a), 72.41 (C_2a), 71.66

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 7 5

View Article Online

4.70 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.67 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.61 (q, J_4,P= J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 4.27 (d,

J = 11.5 Hz, 1H, CH_benzylic), 4.23 (d, J = 11.5 Hz, 1H, CH_benzylic),

(C_3a), 69.39 (C_3b), 68.82 (C_4b), 63.08 (d, J_6,P= 4.9 Hz, C_6b), 54.44

(C_2b). HRMS m/z calcd. for C₁₂H₂₅O₁₃NP⁺: 422.1056; found

422.1063 (M + H)⁺.

4.17 (br t, J = 3.0 Hz, 1H, H_2a), 4.04 (br dt, J_5,6= J_5,6= 2.0 Hz,

ꢀ

6-O-tert-Butyldiphenylsilyl-2,3,4-tri-O-benzyl-a-D-mannopy-

ranosyl-(1→4)-2-azido-2-deoxy-3,6-di-O-benzyl-a-D-glucopyra-

nosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-

bicycle-hept-2-yliden)-D-myo-inositol 74. To a solution of 68

(567 mg, 0.680 mmol) and 53 (540 mg, 0.567 mmol) in dry

J_5,4= 9.5 Hz, 1H, H_5b), 3.99 (t, J_4,3= J_4,5= 9.0 Hz, 1H, H_4a),

3.98 (t, J_6,5= J_6,6

ꢀ

= 9.5 Hz, 1H, H_6a), 3.97 (t, J_3,2= J_3,4

=

9.5 Hz, 1H, H_3b), 3.63 (ddd, J_1,2= 3.0 Hz, J_1,OH= 5.0 Hz, J_1,6

9.0 Hz, 1H, H_1a), 3.52 (d, J_OH,1= 5.0 Hz, 1H, OH_eq.), 3.50 (dd,

J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H, H_2b), 3.48 (dd, J_3,2= 3.0 Hz,

J_3,4= 9.5 Hz, 1H, H_3a), 3.39 (t, J_5,4= J_5,6= 9.5 Hz, 1H, H_5a),

˚

Et₂O (11.3 mL) with 4 A molecular sieves under an argon

atmosphere, TMSOTf (5 ll, 0.028 mmol) at −40 ^◦C was added.

The solution was stirred for 30 min, neutralised with Et₃N

and concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography (hexane–AcOEt 19 : 1, 14 : 1, 9 : 1) to yield

856 mg (93%) of 74 as a white foam. [a]²_D⁰= +41.4 (c = 1.8,

CHCl₃). ¹H-NMR (CDCl₃, 500 MHz): d 7.71 (dd, J_A= 1.5 Hz,

3.34 (dd, J_6,5= 3.0 Hz, J_6,6= 11.0 Hz, 1H, H_6b), 3.21 (dd,

ꢀ

J_{6 ,5}

ꢀ

= 1.5 Hz, J_{6 ,6}

ꢀ

= 11.0 Hz, 1H, H_6b

ꢀ

), 2.52 (s, 1H, OH_ax.).

³¹P-NMR (CDCl₃, 202 MHz): d − 2.94. ¹³C-NMR (CDCl₃,

125 MHz): d 138.17, 138.08, 137.96, 137.53, 137.33 (ArC),

135.61 (d, J_C,P= 6.9 Hz, ArC phosphate), 135.55 (d, J_C,P

=

6.6 Hz, ArC phosphate), 128.41, 128.35, 128.32, 128.27, 128.22,

128.16, 128.13, 128.01, 127.87, 127.83, 127.80, 127.75, 127.66,

127.55, 127.48, 127.31, 127.21, 127.17 (ArCH), 97.96 (C_1b),

J_B= 8.0 Hz, 2H, ArH TBDPS), 7.64 (dd, J_A= 1.0 Hz, J_B

=

8.0 Hz, 2H, ArH TBDPS), 7.40 − 7.15 (m, 41H, ArH), 7.13 −

7.06 (m, 4H, ArH), 6.96 (br t, J = 7.5 Hz, 1H, ArH TBDPS),

5.60 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.28 (d, J_1,2= 1.5 Hz, 1H,

H_1c), 4.97 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.89 (d, J = 11.0 Hz,

1H, CH_benzylic), 4.724 (br d, J = 12.5 Hz, 2H, 2 × CH_benzylic),

4.717 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.69 (s, 2H, CH_2benzylic),

4.67 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.60 (d, J = 10.5 Hz, 1H,

CH_benzylic), 4.57 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.54 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.44 (d, J = 12.5 Hz, 1H, CH_benzylic),

4.43 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.35 (d, J = 12.0 Hz, 1H,

81.50 (C_4a), 80.55 (C_5a), 79.65 (C_6a), 79.56 (C_3a), 78.65 (d, J_3,P

=

2.9 Hz, C_3b), 75.77 (CH_2benzylic), 75.34 (d, J_4,P= 7.0 Hz, C_4b),

75.12, 74.55, 72.89 (CH_2benzylic), 72.73 (C_1a), 72.47 (CH_2benzylic),

69.85 (d, J_5,P= 4.3 Hz, C_5b), 69.36 (C_2a), 69.25 (d, J_C,P= 5.1 Hz,

2 × CH_2benzylicphosphate), 67.17 (C_6b), 63.36 (C_2b). FAB-MS:

m/z 1100 (M + Na)⁺. Anal. calcd. for C₆₁H₆₄O₁₃N₃P: C 67.96%;

H 5.98%; N 3.90%; found C 67.86%; H 6.27%; N 3.64%.

2-Ammonio-2-deoxy-4-O-phosphate-a-D-glucopyranosyl-(1→

6)-D-myo-inositol 34. A suspension of 63 (80 mg, 0.074 mmol)

and 10% Pd on charcoal (157 mg, 0.148 mmol) in a mixture

of MeOH–H₂O 9 : 1 (7.4 mL) under a hydrogen atmosphere

was stirred for 5 h. The mixture was ﬁltered through a pad of

celite and the ﬁlter cake washed with H₂O. The solution was

concentrated in vacuo and lyophilised to yield 31 mg (100%) of

34 as a white solid. [a]_D²⁰= +59.4 (c = 0.2, H₂O). NMR data at

pH = 5.5: ¹H-NMR (D₂O, 500 MHz): d 5.40 (d, J_1,2= 3.5 Hz,

CH_benzylic), 4.32 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4c), 4.28 (t, J_2,1

=

J_2,3= 3.5 Hz, 1H, H_2a), 4.27 (d, J = 12.5 Hz, 1H, CH_benzylic),

4.17 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.02 (m, 2H, H_6a+ H_1a),

4.01 (m, 1H, H_5b), 3.92 (dd, J_6,5= 3.0 Hz, J_6,6= 11.0 Hz, 1H,

ꢀ

H_6c), 3.86 (t, J_4,3= J_4,5= 9.0 Hz, 1H, H_4b), 3.831 (t, J_3,2= J_3,4

=

9.5 Hz, 1H, H_3b), 3.827 (dd, J_3,2= 3.0 Hz, J_3,4= 9.5 Hz, 1H,

H_3c), 3.77 (m, 2H, H_4a+ H_3a), 3.74 (t, J_2,1= J_2,3= 2.5 Hz, 1H,

H_2c), 3.70 (dd, J_{6 ,5}

ꢀ

= 1.0 Hz, J_{6 ,6}

ꢀ

= 11.0 Hz, 1H, H_6c

ꢀ

), 3.59

1H, H_1b), 4.10 (br dt, J_5,6= J_5,6

ꢀ

= 3.0 Hz, J_5,4= 10.0 Hz, 1H,

(br d, J_5,4= 9.5 Hz, 1H, H_5c), 3.48 (dd, J_6,5= 3.0 Hz, J_6,6

11.0 Hz, 1H, H_6b), 3.44 (dd, J_{6 ,5}= 11.0 Hz, 1H,

= 1.5 Hz, J_{6 ,6}

H_6b

), 3.43 (m, 1H, H_5a), 3.34 (dd, J_2,1= 3.5 Hz, J_2,3= 9.5 Hz,

ꢀ

=

H_5b), 4.07 (dd, J_3,4= 9.0 Hz, J_3,2= 10.5 Hz, 1H, H_3b), 3.99 (br t,

J_2,1= J_2,3= 2.5 Hz, 1H, H_2a), 3.98 (q, J_4,P= J_4,3= J_4,5= 9.0 Hz,

ꢀ

1H, H_4b), 3.88 (dd, J_6,5= 3.5 Hz, J_6,6

ꢀ

= 13.0 Hz, 1H, H_6b), 3.73

1H, H_2b), 1.89 (m, 2H, L-camphor), 1.74 (m, 1H, L-camphor),

1.71 (m, 1H, L-camphor), 1.46 (d, J = 12.5 Hz, 1H, L-camphor),

1.38 (m, 1H, L-camphor), 1.21 (m, 1H, L-camphor), 1.06 (s,

3H, CH₃^L-camphor), 1.01 (s, 9H, (CH₃)₃C TBDPS), 0.87 (s,

3H, CH₃^L-camphor), 0.82 (s, 3H, CH₃L-camphor). ¹³C-NMR

(CDCl₃, 125 MHz): d 139.12, 138.72, 138.61, 138.40, 138.33,

138.21, 138.13, 137.86 (ArC), 135.92, 135.62 (ArCH TBDPS),

133.92, 133.17 (ArC TBDPS), 129.45 (ArCH TBDPS), 128.46,

128.34, 128.27, 128.25, 128.20, 128.10, 128.04, 127.87, 127.81,

127.68, 127.61, 127.56, 127.48, 127.45, 127.31, 127.21, 127.11,

127.07, 127.05 (ArCH), 118.09 (C_acetalL-camphor), 100.78

(C_1c), 95.41 (C_1b), 80.85 (C_5a), 80.58 (C_4a), 79.89 (C_3c), 79.67

(C_3b), 77.94 (C_6a), 77.18 (C_4b), 76.91 (C_3a), 76.41 (C_2c), 76.08

(C_1a), 75.01, 74.90, 74.72 (CH_2benzylic), 74.23 (C_4c), 73.87 (C_2a),

73.69 (C_5c+ CH_2benzylic), 72.99, 72.56, 72.27, 72.18 (CH_2benzylic),

70.10 (C_5b), 68.37 (C_6b), 62.98 (C_2b), 62.72 (C_6c), 51.60, 47.96

(C L-camphor), 45.12 (CH L-camphor), 44.77, 29.83, 26.97

(CH₂L-camphor), 26.78 ((CH₃)₃C TBDPS), 20.59, 20.38 (CH₃

L-camphor), 19.31 ((CH₃)₃C TBDPS), 9.67 (CH₃L-camphor).

(dd, J_{6 ,5}

ꢀ

= 1.5 Hz, J_{6 ,6}

ꢀ

= 13.0 Hz, 1H, H_6b

ꢀ

), 3.71 (dd, J_1,2

=

3.0 Hz, J_1,6= 9.5 Hz, 1H, H_1a), 3.69 (t, J_6,1= J_6,5= 10.0 Hz,

1H, H_6a), 3.61 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4a), 3.49 (dd,

J_3,2= 3.0 Hz, J_3,4= 10.5 Hz, 1H, H_3a), 3.37 (dd, J_2,1= 4.0 Hz,

J_2,3= 10.0 Hz, 1H, H_2b), 3.36 (t, J_5,4= J_5,6= 9.0 Hz, 1H, H_5a).

³¹P-NMR (D₂O, 202 MHz): d 0.71. ¹³C-NMR (D₂O, 125 MHz):

d 96.20 (C_1b), 80.44 (C_6a), 72.68 (br s, C_4b), 72.57 (C_5a), 72.52

(C_4a), 72.38 (C_2a), 71.61 (C_1a), 71.33 (br d, J_5,P= 3.9 Hz, C_5b),

70.89 (C_3a), 69.15 (C_3b), 59.79 (C_6b), 54.34 (C_2b). HRMS m/z

calcd. for C₁₂H₂₄O₁₃NPNa⁺: 444.0876; found 444.0843 (M +

Na)⁺.

2-Ammonio-2-deoxy-6-O-phosphate-a-D-glucopyranosyl-(1→

6)-D-myo-inositol 9. From 63: A suspension of 63 (30 mg,

0.033 mmol) and 10% Pd on charcoal (70 mg, 0.066 mmol) in

a mixture MeOH–H₂O–AcOH_glacial9 : 1 : 0.1 (3.3 mL) under

a hydrogen atmosphere was stirred for 24 h. The mixture was

ﬁltered through a pad of celite and the ﬁlter cake washed with

H₂O. The solution was concentrated in vacuo and lyophilised to

yield 14 mg (100%) of 9 as white solid. From 61: The suspension

was stirred for 48 h in this case, using the same procedure.

[a]²_D⁰= +55.0 (c = 0.2, H₂O). NMR data at pH = 6.3: ¹H-NMR

(D₂O, 500 MHz): d 5.39 (d, J_1,2= 3.5 Hz, 1H, H_1b), 4.14 (br d,

¹³C-NMR-undecoupled (CDCl₃, 125 MHz): d 100.78 (d, J_C,H

170.0 Hz, C_1c), 95.41 (d, J_C,H= 173.5 Hz, C_1b). FAB-MS: m/z

1645 (M + Na)⁺. Anal. calcd. for C₁₀₀H₁₁₁O₁₅N₃Si: C 74.00%; H

6.89%; N 2.59%; found C 73.75%; H 6.55%; N 2.68%.

=

J_5,4= 10.0 Hz, 1H, H_5b), 4.11 (m, 1H, H_6b), 3.99 (br t, J_2,1

=

2,3,4-Tri-O-benzyl-a-D-mannopyranosyl-(1→4)-2-azido-3,6-

di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-ben-

zyl-D-myo-inositol 75. A solution of 74 (690 mg, 0.425 mmol)

in a mixture TFA–H₂O 9 : 1 (8.5 mL) was stirred for 30 min, con-

centrated in vacuo and coevaporated with AcOEt (2 × 10 mL)

and Et₃N (5 mL). To a solution of the above residue in dry

MeOH (8.5 mL), MeONa (1.0 M solution in MeOH, 43 ll)

was added. The solution was stirred for 10 min, neutralized

with Amberlite IR-120, ﬁltered and concentrated in vacuo. The

J_2,3= 2.5 Hz, 1H, H_2a), 3.98 (m, 1H, H_6b

ꢀ

), 3.90 (dd, J_3,4

9.5 Hz, J_3,2= 10.5 Hz, 1H, H_3b), 3.70 (m, 2H, H_1a+ H_6a), 3.63

(t, J_4,3= J_4,5= 9.0 Hz, 1H, H_4b), 3.62 (t, J_4,3= J_4,5= 9.5 Hz,

1H, H_4a), 3.49 (dd, J_3,2= 3.0 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 3.36

(dd, J_2,1= 4.0 Hz, J_2,3= 10.5 Hz, 1H, H_2b), 3.35 (t, J_5,4= J_5,6

=

9.5 Hz, 1H, H_5a). ³¹P-NMR (D₂O, 202 MHz): d 0.86. ¹³C-NMR

(D₂O, 125 MHz): d 96.47 (C_1b), 80.62 (C_6a), 72.62 (C_5a), 72.57

(C_4a), 72.36 (C_2a), 71.59 (C_1a), 71.40 (d, J_5,P= 7.6 Hz, C_5b), 70.89

7 7 6

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

residue was puriﬁed by ﬂash chromatography (hexane–AcOEt

4 : 1, 2 : 1, 1 : 1) to yield 478 mg (90%) of 75 as a colourless syrup.

[a]²_D⁰= +48.8 (c = 1.8, CHCl₃). ¹H-NMR (CDCl₃, 500 MHz): d

7.37 − 7.12 (m, 40H, ArH), 5.44 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.22

(d, J_1,2= 2.0 Hz, 1H, H_1c), 4.98 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.90 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.88 (d, J = 11.5 Hz, 1H,

CH_benzylic), 4.87 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.76 (d, J =

10.5 Hz, 1H, CH_benzylic), 4.74 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.71 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.70 (d, J = 11.5 Hz, 1H,

CH_benzylic), 4.67 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.59 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.57 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.50 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.39 (d, J = 12.0 Hz, 1H,

CH_benzylic), 4.37 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.29 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.183 (d, J = 12.0 Hz, 1H, CH_benzylic),

inositol 76. To a solution of 74 (290 mg, 0.179 mmol) in dry

THF (3.6 mL) under an argon atmosphere, TBAF (1.0 M

solution in THF, 1.8 mL) was added. The solution was stirred

for 48 h, diluted with AcOEt (50 mL) and washed with sat.

NaHCO₃solution (50 mL). The aqueous layer was extracted

with AcOEt (2 × 25 mL) and the combined organic layers

were washed with sat. NaCl solution (3 × 100 mL), dried over

MgSO₄and concentrated in vacuo. The residue was puriﬁed

by ﬂash chromatography (hexane–AcOEt 9 : 1, 6 : 1, 4 : 1)

to yield 233 mg (94%) of 76 as a white foam. [a]²_D⁰= +46.6

1

(c = 1.7, CHCl₃). H-NMR (CDCl₃, 500 MHz): d 7.38 − 7.19

(m, 37H, ArH), 7.14 − 7.06 (m, 3H, ArH), 5.61 (d, J_1,2

=

3.5 Hz, 1H, H_1b), 5.25 (d, J_1,2= 2.0 Hz, 1H, H_1c), 4.90 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.87 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.76 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.74 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.72 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.70 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.69 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.63 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.60 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.58 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.54 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.51 (s, 2H, CH_2benzylic), 4.44 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.34 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.29 (br dd, J_2,1= 3.0 Hz, J_2,3= 6.0 Hz, 1H, H_2a), 4.14 (d,

4.176 (t, J_2,1= J_2,3= 3.0 Hz, 1H, H_2a), 4.00 (t, J_4,3= J_4,5

=

9.5 Hz, 1H, H_4a), 3.99 (t, J_6,1= J_6,5= 9.5 Hz, 1H, H_6a), 3.88 (m,

2H, H_5b+ H_4b), 3.85 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4c), 3.83 (m,

1H, H_3b), 3.79 (dd, J_3,2= 2.5 Hz, J_3,4= 9.0 Hz, 1H, H_3c), 3.69 (t,

J_2,1= J_2,3= 2.5 Hz, 1H, H_2c), 3.64 (br dt, J_1,2= J_1,OH= 3.0 Hz,

J_1,6= 9.5 Hz, 1H, H_1a), 3.58 (br d, J_6,5= J_{6 ,5}= 3.0 Hz, 2H, H_6c

ꢀ

+ H_6c

ꢀ

), 3.512 (br dt, J_5,6= J_5,6

ꢀ

= 3.5 Hz, J_5,4= 10.0 Hz, 1H,

H_5c), 3.506 (br d, J_OH,1= 3.0 Hz, 1H, OH_eq.), 3.49 (dd, J_3,2

3.0 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 3.46 (dd, J_2,1= 3.5 Hz, J_2,3

=

J = 12.0 Hz, 1H, CH_benzylic), 4.05 (br dt, J_5,6= J_5,6= 2.5 Hz,

ꢀ

9.5 Hz, 1H, H_2b), 3.41 (br d, J_6,6

ꢀ

= 11.5 Hz, 1H, H_6b), 3.40 (t,

J_5,4= 10.0 Hz, 1H, H_5b), 4.03 (m, 2H, H_6a+ H_1a), 3.92 (t,

J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.87 (t, J_4,3= J_4,5= 9.0 Hz,

1H, H_4c), 3.84 (dd, J_3,4= 9.0 Hz, J_3,2= 10.0 Hz, 1H, H_3b),

3.80 (m, 2H, H_4a+ H_3a), 3.77 (dd, J_3,2= 2.5 Hz, J_3,4= 9.0 Hz,

1H, H_3c), 3.66 (t, J_2,1= J_2,3= 2.5 Hz, 1H, H_2c), 3.64 (m, 2H,

J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.19 (br d, J_{6 ,6}

ꢀ

= 11.5 Hz, 1H,

H_6b

ꢀ

), 2.54 (br s, 1H, OH_ax.), 2.11 (br s, 1H, OH_6c). ¹³C-NMR

(CDCl₃, 125 MHz): d 138.47, 138.42 (ArC), 138.35 (3 × ArC),

138.17, 137.66, 137.58 (ArC), 128.57, 128.50, 128.44, 128.34,

128.23, 128.15, 128.04, 128.01, 127.95, 127.93, 127.89, 127.78,

127.67, 127.65, 127.59, 127.53, 127.48, 127.44, 127.37, 127.25,

126.94, 126.89 (ArCH), 100.53 (C_1c), 98.48 (C_1b), 81.59 (C_4a),

80.92 (C_5a), 80.73 (C_3b), 80.40 (C_6a), 79.79 (C_3a), 79.25 (C_3c),

76.66 (C_4b), 76.35 (C_2c), 75.91, 75.03 (CH_2benzylic), 74.84 (CH_2benzylic

+ C_4c), 74.58, 73.58 (CH_2benzylic), 73.34 (C_5c), 72.79 (C_1a), 72.76,

72.54, 72.15 (CH_2benzylic), 71.17 (C_5b), 69.54 (C_2a), 68.03 (C_6b),

64.50 (C_2b), 62.29 (C_6c). FAB-MS: m/z 1272 (M + Na)⁺. Anal.

calcd. for C₇₄H₇₉O₁₅N₃·H₂O: C 70.07%; H 6.44%; N 3.31%;

found C 70.25%; H 6.41%; N 3.34%.

H_6c+ H_6c

3.60 (m, 1H, H_5c), 3.47 (dd, J_{6 ,5}

H_6b

), 3.44 (m, 1H, H_5a), 3.34 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz,

ꢀ

), 3.61 (dd, J_6,5= 2.5 Hz, J_6,6

ꢀ

= 11.5 Hz, 1H, H_6b),

ꢀ

= 1.5 Hz, J_{6 ,6}= 11.5 Hz, 1H,

ꢀ

1H, H_2b), 2.23 (br s, 1H, OH), 1.91 (m, 2H, L-camphor), 1.75

(m, 1H, L-camphor), 1.72 (m, 1H, L-camphor), 1.47 (d, J =

13.0 Hz, 1H, L-camphor), 1.40 (m, 1H, L-camphor), 1.22 (m,

1H, L-camphor), 1.07 (s, 3H, CH₃L-camphor), 0.88 (s, 3H, CH₃

L-camphor), 0.87 (s, 3H, CH₃L-camphor). ¹³C-NMR (CDCl₃,

125 MHz): d 138.59, 138.47, 138.44, 138.38, 138.33, 138.29,

138.12, 137.85 (ArC), 128.52, 128.46, 128.36, 128.32, 128.30,

128.27, 128.12, 127.89, 127.84, 127.80, 127.73, 127.71, 127.67,

127.61, 127.57, 127.54, 127.45, 127.31, 127.22, 127.00 (ArCH),

118.13 (C_acetalL-camphor), 100.73 (C_1c), 95.44 (C_1b), 80.76 (C_5a),

80.56 (C_4a), 80.05 (C_3b), 79.30 (C_3c), 78.18 (C_6a), 76.88 (C_3a),

76.84 (C_4b), 76.59 (C_2c), 76.09 (C_1a), 74.95 (CH_2benzylic), 74.91

(C_4c), 74.76, 74.73 (CH_2benzylic), 74.04 (CH_2benzylic), 73.90 (C_2a),

73.59 (CH_2benzylic), 73.37 (C_5c), 72.59, 72.51, 72.24 (CH_2benzylic),

70.36 (C_5b), 68.03 (C_6b), 63.27 (C_2b), 62.41 (C_6c), 51.62, 47.98 (C

L-camphor), 45.14 (CH L-camphor), 44.78, 29.88, 26.98 (CH₂

L-camphor), 20.60, 20.38, 9.78 (CH₃L-camphor). FAB-MS: m/z

1406 (M + Na)⁺. Anal. calcd. for C₈₄H₉₃O₁₅N₃: C 72.86%; H

6.77%; N 3.04%; found C 72.65%; H 6.67%; N 2.92%.

a-D-Mannopyranosyl-(1→4)-2-ammonio-2-deoxy-a-D-glucopy-

ranosyl-(1→6)-D-myo-inositol 10. A suspension of 75 (115 mg,

0.092 mmol) and 10% Pd on charcoal (196 mg, 0.184 mmol)

in a mixture MeOH–H₂O 9 : 1 (9.2 mL) under a hydrogen

atmosphere was stirred for 12 h. The mixture was ﬁltered

through a pad of celite and the ﬁlter cake washed with H₂O. The

solution was concentrated in vacuo, loaded on a small column

of Amberlite IRA-408 (Cl⁻), eluted with H₂O and lyophilised

to yield 50 mg (100%) of 10 as a white solid. [a]²_D⁰= +75.0 (c =

0.1, H₂O). NMR data at pH = 6.8: ¹H-NMR (D₂O, 500 MHz):

d 5.37 (d, J_1,2= 3.0 Hz, 1H, H_1b), 5.25 (d, J_1,2= 1.5 Hz, 1H,

H_1c), 4.12 (dt, J_5,6= J_5,6= 2.5 Hz, J_5,4= 10.0 Hz, 1H, H_5b),

ꢀ

4.04 (t, J_3,2= J_3,4= 10.0 Hz, 1H, H_3b), 4.03 (dd, J_2,1= 2.0 Hz,

J_2,3= 3.0 Hz, 1H, H_2c), 3.99 (t, J_2,1= J_2,3= 2.5 Hz, 1H, H_2a),

6-O-Dibenzylphosphate-2,3,4-tri-O-benzyl-a-D-mannopyranos-

yl-(1→4)-2-azido-2-deoxy-3,6-O-dibenzyl-a-D-glucopyranosyl-

(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-bicyc-

3.86 (dd, J_6,5= 1.0 Hz, J_6,6

ꢀ

= 12.0 Hz, 1H, H_6c), 3.84 (dd, J_6,5

=

3.5 Hz, J_6,6

ꢀ

= 12.5 Hz, 1H, H_6b), 3.81 (dd, J_{6 ,5}

ꢀ

= 2.5 Hz, J_{6 ,6}

ꢀ

lehept-2-yliden)-D-myo-inositol 77. To

a solution of 76

H_3c), 3.75 (t, J_4,3^ꢀ= J_4,5= 9.5 Hz, 1H, H_4b), 3.73 (dd, J_{6 ,5}

ꢀ

=

12.5 Hz, 1H, H_6b), 3.78 (dd, J_3,2= 3.0 Hz, J_3,4= 9.0 Hz, 1H,

(190 mg, 0.137 mmol) and 1H-tetrazol (24 mg, 0.343 mmol)

in dry CH₂Cl₂(2.7 mL) under an argon atmosphere, N,N-

diisopropyl dibenzyl phosphoramidite (90 ll, 0.274 mmol) was

added. The solution was stirred for 30 min and 70% MCPBA

(68 mg, 0.276 mmol) was added at 0 ^◦C. After 10 min the

mixture was diluted with AcOEt (50 mL) and washed with sat.

NaHCO₃(2 × 50 mL) and sat. NaCl (3 × 50 mL) solution.

The organic layer was dried over MgSO₄and concentrated in

vacuo. The residue was puriﬁed by double ﬂash chromatography

(hexane–AcOEt 2 : 1 and hexane–AcOEt 9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 :

1, 4 : 1, 3 : 1, 2 : 1) to yield 214 mg (95%) of 77 as a white foam.

[a]²_D⁰= +36.8 (c = 0.5, CHCl₃). ¹H-NMR (CDCl₃, 500 MHz): d

7.37 − 7.12 (m, 47H, ArH), 7.10 (m, 2H, ArH), 7.03 (m, 1H,

ArH), 5.61 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.23 (d, J_1,2= 2.0 Hz,

1H, H_1c), 4.98 − 4.85 (m, 4H, 4 × CH_benzylicphosphate), 4.88

5.5 Hz, J_{6 ,6}

ꢀ

= 12.0 Hz, 1H, H_6c

ꢀ

), 3.71 (m, 2H, H_6a+ H_1a), 3.672

(dd, J_4,3= 8.0 Hz, J_4,5= 11.5 Hz, 1H, H_4c), 3.670 (m, 1H, H_5c),

3.61 (t, J_4,3= J_4,5= 10.0 Hz, 1H, H_4a), 3.49 (dd, J_3,2= 3.0 Hz,

J_3,4= 10.0 Hz, 1H, H_3a), 3.35 (m, 1H, H_5a), 3.29 (br d, J_2,3

=

9.5 Hz, 1H, H_2b). ¹³C-NMR (D₂O, 125 MHz): d 100.10 (C_1c),

96.11 (C_1b), 79.90 (C_6a), 75.37 (C_4b), 73.34 (C_5c), 72.24 (C_5a),

72.13 (C_4a), 71.98 (C_2a), 71.19 (C_1a), 70.47 (C_3a+ C_5b), 69.90

(C_3c), 69.80 (C_2c+ C_3b), 66.07 (C_4c), 60.44 (C_6c), 59.51 (C_6b),

54.13 (C_2b). HRMS m/z calcd. for C₁₈H₃₃O₁₅NNa⁺: 526.1738;

found 526.1748 (M + Na)⁺.

2,3,4-Tri-O-benzyl-a-D-mannopyranosyl-(1→4)-2-azido-2-de-

oxy-3,6-di-O-benzyl-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-ben-

zyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-bicyclehept-2-yliden)-D-myo-

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 7 7

View Article Online

(d, J = 10.5 Hz, 1H, CH_benzylic), 4.87 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.74 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.73 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.71 (s, 2H, CH_2benzylic), 4.68 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.61 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.60 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.54 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.51 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.49 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.40 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.38 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.292 (d, J = 12.0 Hz, 1H,

1H, H_3a), 3.42 (dd, J_2,1= 3.5 Hz, J_2,3= 9.5 Hz, 1H, H_2b), 3.39

(t, J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.38 (dd, J_6,5= 3.0 Hz, J_6,6=

ꢀ

11.0 Hz, 1H, H_6b), 3.28 (dd, J_{6 ,5}

H_6b

ꢀ

= 1.5 Hz, J_{6 ,6}= 11.0 Hz, 1H,

ꢀ

), 2.53 (br s, 1H, OH_ax.). ³¹P-NMR (CDCl₃, 202 MHz): d −

1.80. ¹³C-NMR (CDCl₃, 125 MHz): d 138.48, (ArC), 138.39

(2 × ArC), 138.34, 138.28, 138.18, 137.68, 137.54 (ArC), 135.93

(d, J_C,P= 7.5 Hz, ArC phosphate), 135.91 (d, J_C,P= 7.4 Hz,

ArC phosphate), 128.56, 128.48, 128.44, 128.41, 128.34, 128.33,

128.30, 128.27, 128.24, 128.20, 128.10, 128.03, 127.92, 127.85,

127.78, 127.75, 127.62, 127.59, 127.50, 127.47, 127.36, 127.28,

127.15, 127.00, 126.79 (ArCH), 100.42 (C_1c), 98.25 (C_1b), 81.55

(C_4a), 80.98 (C_5a), 80.53 (C_3b), 80.08 (C_6a), 79.75 (C_3a), 79.58

(C_3c), 77.20 (C_4b), 75.88 (CH_2benzylic+ C_2c), 75.04, 75.00, 74.36

CH_benzylic), 4.286 (m, 1H, H_2a), 4.21 (ddd, J_6,5= 3.5 Hz, J_6,P

=

5.5 Hz, J_6,6

ꢀ

= 11.0 Hz, 1H, H_6c), 4.17 (ddd, J_{6 ,5}

ꢀ

= 1.5 Hz, J_{6 ,P}

ꢀ

5.0 Hz, J_{6 ,6}

ꢀ

= 11.0 Hz, 1H, H_6c

ꢀ

), 4.13 (d, J = 12.0 Hz, 1H,

CH_benzylic), 4.03 (m, 3H, H_6a+ H_1a+ H_5b), 4.00 (t, J_4,3= J_4,5

=

9.5 Hz, 1H, H_4c), 3.86 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.82 (t,

J_3,2= J_3,4= 10.0 Hz, 1H, H_3b), 3.79 (m, 2H, H_4a+ H_3a), 3.77 (dd,

J_3,2= 3.0 Hz, J_3,4= 9.5 Hz, 1H, H_3c), 3.67 (t, J_2,1= J_2,3= 2.5 Hz,

(CH_2benzylic), 73.85 (C_4c), 73.22 (CH_2benzylic), 72.76 (CH_2benzylic

+

C_1a), 72.39, 72.01 (CH_2benzylic), 71.75 (d, J_5,P= 7.9 Hz, C_5c), 70.89

(C_5b), 69.59 (C_2a), 69.15 (d, J_C,P= 5.6 Hz, CH_2benzylicphosphate),

69.07 (d, J_C,P= 5.4 Hz, CH_2benzylicphosphate), 68.43 (C_6b), 66.29

(d, J_6,P= 5.5 Hz, C_6c), 64.38 (C_2b). FAB-MS: m/z 1533 (M +

Na)⁺.

1H, H_2c), 3.66 (br d, J = 9.5 Hz, 1H, H_5c), 3.50 (m, 2H, H_6b

+

H_6b

ꢀ

), 3.43 (m, 1H, H_5a), 3.31 (dd, J_2,1= 3.5 Hz, J_2,3= 9.5 Hz,

1H, H_2b), 1.90 (m, 2H, L-camphor), 1.75 (m, 1H, L-camphor),

1.72 (m, 1H, L-camphor), 1.46 (d, J = 13.0 Hz, 1H, L-camphor),

1.39 (m, 1H, L-camphor), 1.22 (m, 1H, L-camphor), 1.07 (s, 3H,

CH₃^L-camphor), 0.88 (s, 3H, CH₃L-camphor), 0.85 (s, 3H, CH₃

L-camphor). ³¹P-NMR (CDCl₃, 202 MHz): d − 1.80. ¹³C-NMR

(CDCl₃, 125 MHz): d 138.60 (ArC), 138.38 (3 × ArC), 138.34,

138.33, 138.18, 137.79 (ArC), 135.96 (d, J_C,P= 7.8 Hz, ArC

phosphate), 135.93 (d, J_C,P= 7.4 Hz, ArC phosphate), 128.49,

128.45, 128.43, 128.40, 128.36, 128.30, 128.28, 128.25, 128.23,

128.09, 127.89, 127.87, 127.82, 127.80, 127.71, 127.68, 127.65,

127.62, 127.60, 127.54, 127.52, 127.32, 127.25, 127.15, 126.91

(ArCH), 118.13 (C_acetalL-camphor), 100.60 (C_1c), 95.42 (C_1b),

80.83 (C_5a), 80.61 (C_4a), 79.80 (C_3b), 79.66 (C_3c), 77.98 (C_6a),

77.16 (C_4b), 76.88 (C_3a), 76.09 (C_1a), 76.04 (C_2c), 74.97, 74.92,

74.74 (CH_2benzylic), 73.91 (C_4c), 73.88 (C_2a), 73.75, 73.14, 72.58,

72.35, 72.07 (CH_2benzylic), 71.74 (d, J_5,P= 7.9 Hz, C_5c), 70.07

(C_5b), 69.17 (d, J_C,P= 5.8 Hz, CH_2benzylicphosphate), 69.07 (d,

6-O-Phosphate-a-D-mannopyranosyl-(1→4)-2-ammonio-2-de-

oxy-a-D-glucopyranosyl-(1→6)-D-myo-inositol 17.

A

sus-

pension of 78 (90 mg, 0.060 mmol) and 10% Pd on charcoal

(128 mg, 0.120 mmol) in a mixture of MeOH–H₂O 9 : 1 (6 mL)

under hydrogen atmosphere was stirred for 6 h. The mixture was

ﬁltered through a pad of celite and the ﬁlter cake washed with

H₂O. The solution was concentrated in vacuo and lyophilised to

yield 35 mg (100%) of 17 as a white solid. [a]²_D⁰= +107.9 (c =

0.2, H₂O). NMR data at pH = 6.5: ¹H-NMR (D₂O, 500 MHz):

d 5.40 (d, J_1,2= 4.0 Hz, 1H, H_1b), 5.25 (d, J_1,2= 1.5 Hz, 1H,

H_1c), 4.17 (dt, J_5,6= J_5,6= 2.5 Hz, J_5,4= 10.0 Hz, 1H, H_5b), 4.11

ꢀ

(dd, J_3,4= 9.5 Hz, J_3,2= 10.5 Hz, 1H, H_3b), 4.08 (br dd, J_6,5

5.0 Hz, J_6,6), 4.01 (dd,

= 10.0 Hz, 1H, H_6c), 4.02 (m, 1H, H_6c

J_2,1= 2.0 Hz, J_2,3= 3.0 Hz, 1H, H_2c), 3.99 (br s, 1H, H_2a), 3.84

(dd, J_6,5= 3.5 Hz, J_6,6= 12.0 Hz, 1H, H_6b), 3.82 (m, 1H, H_6b),

3.80 (m, 1H, H_5c), 3.79 (m, 1H, H_3c), 3.78 (m, 1H, H_4b), 3.75

=

ꢀ

J_C,P= 5.5 Hz, CH_2benzylicphosphate), 68.37 (C_6b), 66.37 (d, J_6,P

=

5.3 Hz, C_6c), 63.08 (C_2b), 51.62, 47.98 (C L-camphor), 45.14 (CH

L-camphor), 44.78, 29.86, 26.99 (CH₂L-camphor), 20.60, 20.39,

9.72 (CH₃L-camphor). FAB-MS: m/z 1667 (M + Na)⁺.

(dd, J_A= 9.5 Hz, J_B= 12.0 Hz, 1H, H_4c), 3.70 (m, 2H, H_6a

+

=

H_1a), 3.61 (t, J_4,3= J_4,5= 10.0 Hz, 1H, H_4a), 3.49 (dd, J_3,2

2.5 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 3.351 (t, J_5,4= J_5,6= 9.0 Hz,

1H, H_5a), 3.345 (dd, J_2,1= 3.5 Hz, J_2,3= 10.5 Hz, 1H, H_2b).

³¹P-NMR (D₂O, 202 MHz): d 0.80. ¹³C-NMR (D₂O, 125 MHz):

d 100.58 (C_1c), 95.79 (C_1b), 79.89 (C_6a), 75.33 (C_4b), 72.38 (d,

J_5,P= 7.6 Hz, C_5c), 72.20 (C_5a), 72.14 (C_4a), 71.97 (C_2a), 71.19

(C_1a), 70.47 (C_3a), 70.20 (C_5b), 69.84 (C_2c), 69.73 (C_3c), 69.21

(C_3b), 65.79 (C_4c), 63.54 (C_6c), 59.48 (C_6b), 54.11 (C_2b). HRMS

m/z calcd. for C₁₈H₃₄O₁₈NPNa⁺: 606.1401; found 606.1411

(M + Na)⁺.

6-Dibenzylphosphate-2,3,4-tri-O-benzyl-a-D-mannopyranosyl-

(1→4)-2-azido-2-deoxy-3,6-O-dibenzyl-a-D-glucopyranosyl-(1→

6)-3,4,5-tri-O-benzyl-D-myo-inositol 78. To a solution of 77

(170 mg, 0.103 mmol) in CH₂Cl₂(5.2 mL), H₂O (186 ll, 10.325

mmol) and TFA (791 ll, 10.302 mmol) were added and the

resulting mixture was stirred for 2 h. The mixture was diluted

with AcOEt (50 mL) and washed with sat. NaHCO₃(2 ×

50 mL) and sat. NaCl (3 × 50 mL). The organic layer was

dried over MgSO₄and concentrated in vacuo. The residue was

puriﬁed by ﬂash chromatography (hexane–AcOEt 4 : 1, 2 : 1,

6-O-tert-Butyldiphenylsilyl-2,3,4-tri-O-benzyl-a-D-galactopy-

ranosyl-(1→4)-2-azido-2-deoxy-3,6-di-O-benzyl-a-D-glucopyra-

nosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-

bicyclehept-2-yliden)-D-myo-inositol 79. To a solution of 53

1 : 1) to yield 143 mg (92%) of 78 as a colourless syrup. [a]_D²⁰

=

+34.3 (c = 1.3, CHCl₃). ¹H-NMR (CDCl₃, 500 MHz): d 7.36 −

7.07 (m, 50H, ArH), 5.46 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.20

(d, J_1,2= 2.0 Hz, 1H, H_1c), 4.96 − 4.84 (m, 4H, 4 × CH_benzylic

phosphate), 4.88 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.87 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.86 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.74 (br d, J = 12.0 Hz, 2H, 2 × CH_benzylic), 4.73 (d, J = 11.5 Hz,

1H, CH_benzylic), 4.70 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.61 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.58 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.53 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.45 (d, J = 12.0 Hz, 1H,

CH_benzylic), 4.33 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.30 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.25 (d, J = 11.5 Hz, 1H, CH_benzylic),

˚

(540 mg, 0.567 mmol) with 4 A molecular sieves in dry Et₂O

(5.6 mL) under an argon atmosphere, TMSOTf (10 ll, 0.055

mmol) was added at 0 ^◦C. A solution of 73 (945 mg, 1.134

mmol) in dry Et₂O (5.6 mL) was slowly added (1 drop per

sec) and the mixture was stirred for 1 h after the addition was

completed. The mixture was neutralised, concentrated in vacuo

and puriﬁed by ﬂash chromatography (hexane–AcOEt 24 : 1,

19 : 1, 9 : 1) to yield 653 mg (71%) of 79 as a white foam. [a]_D²⁰

=

+59.0 (c = 0.4, CHCl₃). ¹H-NMR (CDCl₃, 500 MHz): d 7.59 −

7.55 (m, 5H, ArH), 7.38 − 7.12 (m, 42H, ArH), 7.11 − 7.06

4.17 (ddd, J_6,5= 3.5 Hz, J_6,P= 6.0 Hz, J_6,6

4.16 (t, J_2,1= J_2,3= 2.5 Hz, 1H, H_2a), 4.14 (d, J = 12.0 Hz,

1H, CH_benzylic), 4.10 (ddd, J_{6 ,5}= 6.0 Hz, J_{6 ,6}

= 2.0 Hz, J_{6 ,P}

11.5 Hz, 1H, H_6c

), 4.00 (t, J_6,1= J_6,5= 9.5 Hz, 1H, H_6a), 3.99 (t,

J_4,3= J_4,5= 9.5 Hz, 1H, H_4c), 3.97 (t, J_4,3= J_4,5= 9.0 Hz, 1H,

H_4a), 3.90 (m, 1H, H_5b), 3.82 (m, 2H, H_4b+ H_3b), 3.78 (dd, J_3,2

ꢀ

= 11.5 Hz, 1H, H_6c),

(m, 3H, ArH), 5.59 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.44 (d, J_1,2=

3.5 Hz, 1H, H_1c), 4.96 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.93 (d,

J = 11.5 Hz, 1H, CH_benzylic), 4.84 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.74 (d, J = 11.5 Hz, 2H, 2 × CH_benzylic), 4.71 (d, J = 11.0 Hz,

1H, CH_benzylic), 4.69 (d, J = 11.0 Hz, 2H, 2 × CH_benzylic), 4.66 (d,

J = 12.0 Hz, 1H, CH_benzylic), 4.64 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.613 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.610 (d, J = 12.0 Hz,

1H, CH_benzylic), 4.56 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.49 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.33 (d, J = 12.5 Hz, 1H, CH_benzylic),

ꢀ

=

ꢀ

=

3.0 Hz, J_3,4= 9.5 Hz, 1H, H_3c), 3.69 (t, J_2,1= J_2,3= 2.5 Hz,

1H, H_2c), 3.63 (m, 1H, H_1a), 3.59 (br d, J_5,4= 9.5 Hz, 1H, H_5c),

3.479 (br s, 1H, OH_eq.), 3.477 (dd, J_3,2= 3.0 Hz, J_3,4= 9.5 Hz,

7 7 8

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

4.28 (br dd, J_2,1= 3.5 Hz, J_2,3= 6.0 Hz, 1H, H_2a), 4.24 (d, J =

12.5 Hz, 1H, CH_benzylic), 4.11 (br dt, J_5,6= J_5,6= 2.5 Hz, J_5,4

9.5 Hz, 1H, H_5b), 4.04 (t, J_6,1= J_6,5= 8.0 Hz, 1H, H_6a), 4.03 (t,

128.24, 127.99, 127.93, 127.89, 127.74, 127.61, 127.55, 127.50,

127.36, 127.22, 127.15, 126.88 (ArCH), 99.16 (C_1b), 97.81 (C_1c),

82.17 (C_6a), 81.70 (C_4a), 81.07 (C_3b), 81.05 (C_5a), 79.75 (C_3a),

79.20 (C_3c), 75.90 (CH_2benzylic), 75.71 (C_2c), 74.49 (C_4c), 74.43,

74.34, 74.14, 74.01, 73.74 (CH_2benzylic), 73.17 (C_4b), 72.86, 72.67

(CH_2benzylic), 72.37 (C_1a), 71.68 (C_5b), 71.49 (C_5c), 69.26 (C_2a),

68.66 (C_6b), 64.98 (C_2b), 62.26 (C_6c). FAB-MS: m/z 1249 M⁺,

1275 (M + Na)⁺. Anal. calcd. for C₇₄H₇₉O₁₅N₃: C 71.08%; H

6.37%; N 3.36%; found C 71.06%; H 6.30%; N 2.96%.

ꢀ

=

J_3,2= J_3,4= 9.5 Hz, 1H, H_3b), 4.014 (dd, J_1,2= 3.0 Hz, J_1,6

=

7.0 Hz, 1H, H_1a), 4.006 (br s, 1H, H_4c), 3.98 (dd, J_2,1= 4.0 Hz,

J_2,3= 10.5 Hz, 1H, H_2c), 3.97 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b),

3.86 (br t, J_5,6= J_5,6= 7.0 Hz, 1H, H_5c), 3.82 (dd, J_3,4= 2.5 Hz,

ꢀ

J_3,2= 10.5 Hz, 1H, H_3c), 3.80 (m, 2H, H_3a+ H_4a), 3.72 (dd,

J_6,5= 8.0 Hz, J_6,6

ꢀ

= 10.0 Hz, 1H, H_6c), 3.67 (dd, J_{6 ,5}

ꢀ

= 6.0 Hz,

= 11.5 Hz,

J_{6 ,6}

ꢀ

= 10.0 Hz, 1H, H_6c

ꢀ

), 3.59 (dd, J_6,5= 4.0 Hz, J_6,6

ꢀ

a-D-Galactopyranosyl-(1→4)-2-ammonio-2-deoxy-a-D-gluco-

1H, H_6b), 3.46 (br t, J_5,4= J_5,6= 9.0 Hz, 1H, H_5a), 3.45 (dd,

pyranosyl-(1→6)-D-myo-inositol 13.

A suspension of 80

J_{6 ,5}

ꢀ

= 2.0 Hz, J_{6 ,6}

ꢀ

= 11.5 Hz, 1H, H_6b), 3.30 (dd, J_2,1= 3.5 Hz,

ꢀ

(100 mg, 0.080 mmol) and 10% Pd on charcoal (170 mg, 0.160

mmol) in a mixture MeOH–H₂O 9 : 1 (8 mL) under a hydrogen

atmosphere was stirred for 24 h. The mixture was ﬁltered

through a pad of celite and the ﬁlter cake washed with H₂O. The

solution was concentrated in vacuo, loaded on a small column

of Amberlite IRA-408 (Cl⁻), eluted with H₂O and lyophilised

to yield 43 mg (100%) of 13 as a white solid. [a]²_D⁰= +88.7 (c =

0.4, H₂O). NMR data at pH = 5.8: ¹H-NMR (D₂O, 500 MHz):

d 5.42 (d, J_1,2= 4.0 Hz, 1H, H_1c), 5.41 (d, J_1,2= 3.5 Hz, 1H,

H_1b), 4.18 (dd, J_3,4= 9.0 Hz, J_3,2= 10.5 Hz, 1H, H_3b), 4.16 (br

J_2,3= 10.0 Hz, 1H, H_2b), 1.91 (m, 2H, L-camphor), 1.75 (m, 1H,

L-camphor), 1.72 (m, 1H, L-camphor), 1.46 (d, J = 13.0 Hz, 1H,

L-camphor), 1.37 (m, 1H, L-camphor), 1.21 (m, 1H, L-camphor),

1.07 (s, 3H, CH₃^L-camphor), 1.05 (s, 9H, (CH₃)₃C TBDPS),

0.88 (s, 3H, CH₃^L-camphor), 0.84 (s, 3H, CH₃L-camphor).

¹³C-NMR (CDCl₃, 125 MHz): d 138.78, 138.73, 138.60 (ArC),

138.47 (2 × ArC), 138.43 (ArC), 138.36 (2 × ArC), 135.59,

135.55 (ArCH TBDPS), 133.21, 133.06 (ArC TBDPS), 129.69,

129.67, 128.35, 128.31, 128.27, 128.20, 128.12, 128.06, 127.97,

127.84, 127.74, 127.70, 127.63, 127.53, 127.44, 127.42, 127.38,

127.21, 127.16 (ArCH), 118.04 (C_acetalL-camphor), 99.12 (C_1c),

95.21 (C_1b), 80.76 (C_4a), 80.69 (C_5a), 79.67 (C_3b), 78.97 (C_3c),

77.87 (C_6a), 77.20 (C_3a), 76.39 (C_4b), 76.06 (C_2c), 76.02 (C_1a),

75.09 (CH_2benzylic), 75.00 (C_4c), 74.80, 74.70 (CH_2benzylic), 73.89

(C_2a), 73.70, 73.21, 72.80, 72.75, 72.61 (CH_2benzylic), 71.37 (C_5c),

70.25 (C_5b), 68.99 (C_6b), 63.08 (C_2b), 62.43 (C_6c), 51.60, 47.96

(C L-camphor), 45.13 (CH L-camphor), 44.77, 29.79 (CH₂

L-camphor), 26.98 ((CH₃)₃C TBDPS), 26.90 (CH₂L-camphor),

20.62, 20.39 (CH₃L-camphor), 19.16 ((CH₃)₃C TBDPS), 9.74

(CH₃L-camphor). FAB-MS: m/z 1645 (M + Na)⁺.

dt, J_5,6= J_5,6

H_2a+ H_5c), 3.97 (br d, J_4,3= 3.0 Hz, 1H, H_4c), 3.88 (dd, J_6,5

3.5 Hz, J_6,6

= 12.0 Hz, 1H, H_6b), 3.86 (dd, J_2,1= 4.0 Hz, J_2,3

ꢀ

= 3.0 Hz, J_5,4= 10.0 Hz, 1H, H_5b), 3.99 (m, 2H,

=

ꢀ

10.0 Hz, 1H, H_2c), 3.82 (dd, J_3,2= 3.0 Hz, J_3,4= 10.5 Hz, 1H,

H_3c), 3.80 (dd, J_6,5= 2.5 Hz, J_6,6

(t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.71 (m, 4H, H_1a+ H_6a

H_6c+ H_6c= 10.0 Hz, 1H, H_4a), 3.49 (dd,

), 3.62 (t, J_4,3= J_4,5

ꢀ

= 12.0 Hz, 1H, H_6b

ꢀ

), 3.77

ꢀ

+

ꢀ

J_3,2= 3.0 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 3.36 (m, 1H, H_5a), 3.34

(dd, J_2,1= 4.0 Hz, J_2,3= 10.5 Hz, 1H, H_2b). ¹³C-NMR (D₂O,

125 MHz): d 99.26 (C_1c), 95.86 (C_1b), 79.86 (C_6a), 75.58 (C_4b),

72.23 (C_5a), 72.12 (C_4a), 71.99 (C_2a), 71.35 (C_5c), 71.23 (C_1a),

70.51 (C_3a), 70.25 (C_5b), 69.77 (C_3b), 68.84 (C_3c), 68.74 (C_4c),

68.09 (C_2c), 60.75 (C_6c), 59.46 (C_6b), 53.89 (C_2b). HRMS: m/z

calcd. for C₁₈H₃₃O₁₅NNa⁺: 526.1738; found 526.1748 (M +

Na)⁺.

2,3,4-Tri-O-benzyl-a-D-galactopyranosyl-(1→4)-2-azido-2-de-

oxy-3,6-di-O-benzyl-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-ben-

zyl-D-myo-inositol 80. To a solution of 79 (270 mg, 0.166

mmol) in CH₂Cl₂(8.3 mL), H₂O (0.3 mL, 16.7 mmol) and TFA

(1.3 mL, 16.9 mmol) were added and the resulting mixture was

stirred for 2 h. The mixture was diluted with AcOEt (50 mL)

and washed with sat. NaHCO₃(2 × 50 mL) and sat. NaCl (3 ×

50 mL) solutions. The organic layer was dried over MgSO₄,

concentrated in vacuo and the residue was puriﬁed by ﬂash

chromatography (hexane–AcOEt 4 : 1, 2 : 1, 1 : 1) to yield

191 mg (92%) of 80 as a colourless syrup. [a]²_D⁰= +44.0 (c =

2,3,4-Tri-O-benzyl-a-D-galactopyranosyl-(1→4)-2-azido-2-de-

oxy-3,6-di-O-benzyl-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-ben-

zyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-bicyclehept-2-yliden)-D-myo-

inositol 81. To a solution of 79 (256 mg, 0.158 mmol) in dry

THF (3.2 mL) under an argon atmosphere, TBAF (1.0 M

solution in THF, 468 ll) was added. The solution was stirred

for 4 h, diluted with AcOEt (50 mL) and washed with sat.

NaCl solution (50 mL). The aqueous layer was extracted with

AcOEt (2 × 25 mL) and the combined organic layers were

washed with sat. NaCl solution (3 × 100 mL), dried over

MgSO₄and concentrated in vacuo. The residue was puriﬁed by

ﬂash chromatography (hexane–AcOEt 9 : 1, 6 : 1, 4 : 1) to yield

210 mg (96%) of 81 as colourless syrup. [a]²_D⁰= +68.7 (c = 1.4,

CHCl₃). ¹H-NMR (CDCl₃, 500 MHz): d 7.37 − 7.08 (m, 40H,

ArH), 5.61 (d, 2 × J_1,2= 4.0 Hz, 2H, H_1b+ H_1c), 4.88 (d, J =

11.5 Hz, 2H, 2 × CH_benzylic), 4.81 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.75 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.73 (d, J = 11.5 Hz, 2H,

2 × CH_benzylic), 4.71 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.684 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.676 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.63 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.62 (d, J = 10.5 Hz, 1H,

CH_benzylic), 4.61 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.58 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.54 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.50 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.49 (d, J = 11.5 Hz, 1H,

CH_benzylic), 4.29 (br dd, J_2,3= 3.5 Hz, J_2,1= 5.5 Hz, 1H, H_2a),

4.09 (m, 1H, H_5b), 4.08 (m, 1H, H_4b), 4.04 (m, 2H, H_1a+ H_6a),

4.03 (m, 1H, H_3b), 3.98 (dd, J_2,1= 4.0 Hz, J_2,3= 10.0 Hz, 1H,

H_2c), 3.80 (m, 2H, H_3a+ H_4a), 3.77 (br d, J_4,3= 2.5 Hz, 1H, H_4c),

1

1.1, CHCl₃). H-NMR (CDCl₃, 500 MHz): d 7.38 − 7.14 (m,

40H, ArH), 5.58 (d, J_1,2= 4.0 Hz, 1H, H_1c), 5.32 (d, J_1,2

=

3.5 Hz, 1H, H_1b), 4.97 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.94

(d, J = 11.5 Hz, 1H, CH_benzylic), 4.89 (d, J = 10.5 Hz, 1H,

CH_benzylic), 4.88 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.80 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.730 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.734 (s, 2H, CH_2benzylic), 4.72 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.70 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.66 (d, J = 12.0 Hz, 1H,

CH_benzylic), 4.63 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.58 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.56 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.32 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.20 (br s, 1H, H_2a), 4.18

(d, J = 12.0 Hz, 1H, CH_benzylic), 4.05 (br t, J_4,3= J_4,5= 9.0 Hz,

1H, H_4b), 4.02 (m, 1H, H_4a), 4.01 (m, 1H, H_2c), 4.00 (m, 1H,

H_3b), 3.99 (m, 1H, H_5b), 3.96 (d, J_OH,1= 3.5 Hz, 1H, OH_eq.),

3.95 (t, J_6,5= J_6,1= 9.5 Hz, 1H, H_6a), 3.77 (br s, 1H, H_4c), 3.76

(dd, J_3,2= 3.0 Hz, J_3,4= 10.0 Hz, 1H, H_3c), 3.60 (dt, J_1,2

=

3.0 Hz, J_1,6= 9.5 Hz, 1H, H_1a), 3.57 (br t, J_5,6= J_5,6= 6.0 Hz,

ꢀ

1H, H_5c), 3.56 (dd, J_6,5= 2.5 Hz, J_6,6= 12.0 Hz, 1H, H_6b),

ꢀ

3.52 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H, H_2b), 3.50 (m, 1H,

H_6c), 3.49 (dd, J_3,2= 2.5 Hz, J_3,4= 9.0 Hz, 1H, H_3a), 3.41 (t,

J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.33 (ddd, J_{6 ,OH}

ꢀ

= 5.0 Hz, J_{6 ,5}

ꢀ

=

3.74 (dd, J_3,4= 2.5 Hz, J_3,2= 10.5 Hz, 1H, H_3c), 3.71 (dd, J_6,5

2.5 Hz, J_6,6

= 12.0 Hz, 1H, H_6b), 3.56 (m, 1H, H_6c), 3.55 (m, 1H,

H_5c), 3.52 (br d, J_6,6), 3.47 (m, 1H, H_5a), 3.36

= 12.0 Hz, 1H, H_6b

=

7.5 Hz, J_{6 ,6}

J_{6 ,6}

J_OH,6

ꢀ

= 12.5 Hz, 1H, H_6c

ꢀ

), 3.23 (br dd, J_{6 ,5}

ꢀ

= 1.5 Hz,

ꢀ

= 12.0 Hz, 1H, H_6b

ꢀ

), 2.53 (s, 1H, OH_ax.), 1.71 (br t, J_OH,6

=

ꢀ

= 4.5 Hz, 1H, OH_6c). ¹³C-NMR (CDCl₃, 125 MHz): d

(dd, J_2,1= 3.5 Hz, J_2,3= 9.5 Hz, 1H, H_2b), 3.35 (dt, J_6,5= J_6,OH=

ꢀ

=^ꢀ7.5 Hz, 1H, OH_6c), 1.75 (m, 1H,

138.81 (ArC), 138.38 (2 × ArC), 138.16 (2 × ArC), 138.09,

137.74, 137.71 (ArC), 128.54, 128.45, 128.43, 128.32, 128.26,

7.5 Hz, J_6,6

(br dd, J_OH,6= 4.0 Hz, J_OH,6

= 13.5 Hz, 1H, H_6c), 1.92 (m, 2H, L-camphor), 1.91

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 7 9

View Article Online

L-camphor), 1.72 (m, 1H, L-camphor), 1.47 (d, J = 13.0 Hz, 1H,

L-camphor), 1.39 (m, 1H, L-camphor), 1.22 (m, 1H, L-camphor),

1.07 (s, 3H, CH₃^L-camphor), 0.87 (s, 6H, 2 × CH₃L-camphor).

¹³C-NMR (CDCl₃, 125 MHz): d 138.53 (ArC), 138.50 (2 ×

ArC), 138.40, 138.34, 138.25, 138.21, 138.17 (ArC), 128.45,

128.41, 128.39, 128.35, 128.31, 128.28, 128.26, 128.08, 127.88,

127.85, 127.80, 127.69, 127.59, 127.56, 127.51, 127.36, 127.33,

127.28, 127.26, 127.09 (ArCH), 118.06 (C_acetalL-camphor), 98.10

(C_1c), 95.42 (C_1b), 80.79 (C_5a), 80.53 (C_4a), 80.16 (C_3b), 79.16

(C_3c), 78.33 (C_6a), 76.89 (C_3a), 76.03 (C_1a), 75.87 (C_2c), 74.72

(C_4c+ CH_2benzylic), 74.62, 74.31 (CH_2benzylic), 73.96 (C_4b), 73.87

(C_2a), 73.69, 73.44, 73.40, 72.86, 72.60 (CH_2benzylic), 71.47 (C_5c),

70.73 (C_5b), 68.57 (C_6b), 63.14 (C_2b), 62.53 (C_6c), 51.61, 47.96 (C

L-camphor), 45.14 (CH L-camphor), 44.73, 29.89, 26.99 (CH₂

L-camphor), 20.60, 20.38, 9.83 (CH₃L-camphor). FAB-MS: m/z

1406 (M + Na)⁺. Anal. calcd. for C₈₄H₉₃O₁₅N₃: C 72.86%; H

6.77%; N 3.04%; found C 72.46%; H 7.00%; N 3.16%.

CH_2benzylicphosphate), 68.91 (C_6b), 65.79 (d, J_6,P= 5.1 Hz, C_6c),

63.00 (C_2b), 51.59, 47.95 (C L-camphor), 45.14 (CH L-camphor),

44.71, 29.87, 26.99 (CH₂L-camphor), 20.60, 20.39, 9.73 (CH₃

L-camphor). FAB-MS: m/z 1667 (M + Na)⁺. Anal. calcd. for

C₉₈H₁₀₆O₁₈N₃P·H₂O: C 70.78%; H 6.55%; N 2.53%; found C

70.85%; H 6.80%; N 2.55%.

6-O-Dibenzylphosphate-2,3,4-tri-O-benzyl-a-D-galactopyran-

osyl-(1→4)-2-azido-2-deoxy-3,6-di-O-benzyl-a-D-glucopyranos-

yl-(1→6)-3,4,5-tri-O-benzyl-D-myo-inositol 83. To a solution

of 82 (100 mg, 0.061 mmol) in CH₂Cl₂(3 mL), TFA (468 ll,

6.095 mmol) and H₂O (110 ll, 6.106 mmol) were added and the

resulting mixture was stirred for 2 h. The mixture was diluted

with AcOEt (25 mL) and washed with sat. NaHCO₃(2 × 25 mL)

and sat. NaCl (3 × 25 mL) solutions. The organic layer was

dried over MgSO₄and concentrated in vacuo. The residue was

puriﬁed by ﬂash chromatography (hexane–AcOEt 4 : 1, 2 : 1,

1 : 1) to yield 83 mg (90%) of 83 as white foam. [a]²_D⁰= +45.9

1

(c = 2.3, CHCl₃). H-NMR (CDCl₃, 500 MHz): d 7.37 − 7.10

6-O-Dibenzylphosphate-2,3,4-tri-O-benzyl-a-D-galactopyran-

osyl-(1→4)-2-azido-2-deoxy-3,6-O-di-benzyl-a-D-glucopyranosyl-

(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl-[2,2,1]-bicyc-

(m, 50H, ArH), 5.45 (d, J_1,2= 3.5 Hz, 1H, H_1c), 5.27 (d, J_1,2

=

4.0 Hz, 1H, H_1b), 4.96 − 4.91 (m, 5H, 4 × CH_benzylicphosphate

+ CH_benzylic), 4.91 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.89 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.85 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.81 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.72 (s, 2H, CH_2benzylic),

4.70 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.67 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.66 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.58 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.55 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.53 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.50 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.20 (s, 2H, CH_2benzylic), 4.19 (br t, J_2,1= J_2,3= 3.0 Hz,

lehept-2-yliden)-D-myo-inositol 82. To

a solution of 81

(105 mg, 0.076 mmol) and 1H-tetrazol (13 mg, 0.186 mmol)

in dry CH₂Cl₂(1.5 mL) under an argon atmosphere, N,N-

diisopropyl dibenzyl phosphoramidite (50 ll, 0.152 mmol) was

added. The solution was stirred for 30 min and 70% MCPBA

(18 mg, 0.150 mmol) was added at 0 ^◦C. After 10 min the

mixture was diluted with AcOEt (50 mL) and washed with sat.

NaHCO₃(2 × 50 mL) and sat. NaCl (3 × 50 mL) solutions.

The organic layer was dried over MgSO₄, concentrated in vacuo

and the residue was puriﬁed by double ﬂash chromatography

(hexane–AcOEt 2 : 1 and hexane–AcOEt 9 : 1, 8 : 1, 7 : 1, 6 :

1, 5 : 1, 4 : 1, 3 : 1, 2 : 1) to yield 120 mg (96%) of 82 as a

white foam. [a]²_D⁰= +68.3 (c = 1.2, CHCl₃). ¹H-NMR (CDCl₃,

500 MHz): d 7.37 − 7.08 (m, 50H, ArH), 5.58 (d, J_1,2= 3.5 Hz,

1H, H_1b), 5.54 (d, J_1,2= 3.5 Hz, 1H, H_1c), 4.99 − 4.84 (m, 4H,

4 × CH_benzylicphosphate), 4.87 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.86 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.81 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.73 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.702 (br d,

J = 11.5 Hz, 2H, 2 × CH_benzylic), 4.700 (d, J = 12.0 Hz, 1H,

CH_benzylic), 4.67 (d, J = 12.5 Hz, 1H, CH_benzylic), 4.64 (d, J =

12.0 Hz, 1H, CH_benzylic), 4.58 (d, J = 10.5 Hz, 1H, CH_benzylic),

4.57 (s, 2H, CH_2benzylic), 4.51 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.49 (d, J = 12.5 Hz, 1H, CH_benzylic), 4.48 (d, J = 12.0 Hz, 1H,

CH_benzylic), 3.39 (d, J = 12.5 Hz, 1H, CH_benzylic), 4.28 (br dd,

1H, H_2a), 4.02 (br dd, J_6,5= 6.5 Hz, J_6,6= 10.0 Hz, 1H, H_6c),

ꢀ

4.00 (m, 2H, H_4a+ H_5b), 3.97 (m, 1H, OH_ax.), 3.96 (m, 3H,

H_3b+ H_4b+ H_2c), 3.94 (t, J_6,1= J_6,5= 10.0 Hz, 1H, H_6a), 3.93

(br dd, J_{6 ,5}

ꢀ

= 5.5 Hz, J_{6 ,6}

ꢀ

= 10.0 Hz, 1H, H_6c

ꢀ

), 3.81 (br t, J_5,6

=

J_5,6

2.5 Hz, J_3,2= 10.5 Hz, 1H, H_3c), 3.57 (dt, J_1,2= 3.5 Hz, J_1,6

9.5 Hz, 1H, H_1a), 3.56 (dd, J_6,5= 3.0 Hz, J_6,6

ꢀ

= 6.5 Hz, 1H, H_5c), 3.72 (br s, 1H, H_4c), 3.71 (dd, J_3,4

ꢀ

= 10.5 Hz, 1H,

H_6b), 3.47 (dd, J_3,2= 3.0 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 3.40 (t,

J_5,4= J_5,6= 9.5 Hz, 1H, H_5a), 3.39 (dd, J_2,1= 3.0 Hz, J_2,3

=

9.5 Hz, 1H, H_2b), 3.28 (dd, J_{6 ,5}

ꢀ

= 2.0 Hz, J_{6 ,6}= 10.5 Hz, 1H,

ꢀ

H_6b

ꢀ

), 2.51 (s, 1H, OH_eq.). ³¹P-NMR (CDCl₃, 202 MHz): d −

1.72. ¹³C-NMR (CDCl₃, 125 MHz): d 138.77, 138.49, 138.37,

138.29, 138.20, 138.13, 137.81, 137.77 (ArC), 135.73 (d, J_C,P

=

6.6 Hz, 2 × ArC phosphate), 128.57, 128.52, 128.36, 128.29,

128.26, 128.23, 128.21, 128.16, 128.13, 127.94, 127.91, 127.87,

127.83, 127.69, 127.65, 127.55, 127.53, 127.48, 127.35, 127.31,

127.28, 127.25, 127.20 (ArCH), 99.00 (C_1b), 97.98 (C_1c), 82.17

(C_6a), 81.69 (C_4a), 81.14 (C_5a), 80.73 (C_3b), 79.67 (C_3a), 78.69

J_2,3= 2.5 Hz, J_2,1= 5.5 Hz, 1H, H_2a), 4.12 (br dt, J_5,6= J_5,6=

ꢀ

3.0 Hz, J_5,4= 9.5 Hz, 1H, H_5b), 4.03 (m, 2H, H_6a+ H_6c), 4.02

(C_3c), 75.82 (CH_2benzylic), 75.54 (C_2c), 74.58 (2 × CH_2benzylic

+

(m, 1H, H_1a), 4.01 (m, 1H, H_3b), 4.00 (m, 1H, H_4b), 3.99 (m,

C_4b), 74.16 (CH_2benzylic), 74.08 (C_4c), 73.77, 73.08, 72.80, 72.60

(CH_2benzylic), 72.20 (C_1a), 71.32 (C_5b), 69.69 (d, J_5,P= 9.8 Hz, C_5c),

69.27 (d, J_C,P= 5.4 Hz, 2 × CH_2benzylicphosphate), 69.20 (C_2a),

69.02 (C_6b), 65.91 (d, J_6,P= 5.3 Hz, C_6c), 64.82 (C_2b). FAB-MS:

m/z 1533 (M + Na)⁺. Anal. calcd. for C₈₈H₉₂O₁₈N₃P·2H₂O:

C 68.34%; H 6.26%; N 2.72%; found C 68.47%; H 6.14%; N

2.66%.

1H, H_6c

(br t, J_5,6= J_5,6

3.77 (m, 1H, H_4c), 3.72 (dd, J_3,4= 2.5 Hz, J_3,2= 10.0 Hz, 1H,

H_3c), 3.64 (dd, J_6,5= 3.5 Hz, J_6,6= 11.0 Hz, 1H, H_6b), 3.51 (br

dd, J_{6 ,5}= 11.0 Hz, 1H, H_6b), 3.46 (m, 1H, H_5a),

= 2.5 Hz, J_{6 ,6}

ꢀ

), 3.95 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H, H_2c), 3.82

ꢀ

= 6.5 Hz, 1H, H_5c), 3.78 (m, 2H, H_4a+ H_3a),

ꢀ

3.27 (dd, J_2,1= 3.5 Hz, J_2,3= 9.5 Hz, 1H, H_2b), 1.91 (m, 2H,

L-camphor), 1.75 (m, 1H, L-camphor), 1.72 (m, 1H, L-camphor),

1.46 (d, J = 13.0 Hz, 1H, L-camphor), 1.39 (m, 1H, L-camphor),

1.22 (m, 1H, L-camphor), 1.07 (s, 3H, CH₃L-camphor), 0.88 (s,

3H, CH₃^L-camphor), 0.85 (s, 3H, CH₃L-camphor). ³¹P-NMR

(CDCl₃, 202 MHz): d − 1.79. ¹³C-NMR (CDCl₃, 125 MHz):

d 138.50 (ArC), 138.46 (2 × ArC), 138.42 (2 × ArC), 138.37

(2 × ArC), 138.21 (ArC), 135.76 (d, J_C,P= 6.8 Hz, 2 × ArC

phosphate), 128.56, 128.48, 128.40, 128.34, 128.25, 128.20,

128.12, 128.07, 128.02, 127.89, 127.86, 127.82, 127.80, 127.69,

127.66, 127.61, 127.59, 127.53, 127.49, 127.42, 127.38, 127.32,

127.28, 127.21, 127.17 (ArCH), 118.02 (C_acetalL-camphor), 98.20

(C_1c), 95.42 (C_1b), 80.70 (C_5a), 80.62 (C_4a), 79.96 (C_3b), 78.69

(C_3c), 78.16 (C_6a), 76.83 (C_3a), 76.05 (C_1a), 75.66 (C_2c), 74.75,

74.64 (CH_2benzylic), 74.60 (C_4b), 74.57 (CH_2benzylic), 74.17 (C_4c),

73.85 (C_2a), 73.37, 73.28, 72.94, 72.74, 72.57 (CH_2benzylic), 70.10

(C_5b), 69.49 (d, J_5,P= 10.0 Hz, C_5c), 69.26 (d, J_C,P= 5.4 Hz, 2 ×

6-O-Phosphate-a-D-galactopyranosyl-(1→4)-2-ammonio-2-de-

oxy-a-D-glucopyranosyl-(1→6)-D-myo-inositol 35.

A

sus-

pension of 83 (60 mg, 0.040 mmol) and 10% Pd on charcoal

(85 mg, 0.080 mmol) in a mixture of MeOH–H₂O 9 : 1

(4 mL) under a hydrogen atmosphere was stirred for 6 h. The

mixture was ﬁltered through a pad of celite and the ﬁlter cake

washed with H₂O. The solution was concentrated in vacuo and

lyophilised to yield 23 mg (100%) of 35 as a white solid. [a]_D²⁰

=

+65.0 (c = 0.2, H₂O). NMR data at pH = 5.8: ¹H-NMR (D₂O,

500 MHz): d 5.42 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.40 (br d, J_1,2

=

2.0 Hz, 1H, H_1c), 4.25 (dd, J_3,4= 9.5 Hz, J_3,2= 10.5 Hz, 1H,

H_3b), 4.23 (dt, J_5,6= J_5,6= 3.0 Hz, J_5,4= 10.0 Hz, 1H, H_5b),

ꢀ

H_4c), 4.00 (br t, J_2,1= J_2,3= ^ꢀ2.5 Hz, 1H, H_2a), 3.95 (m, 2H,

4.15 (br dd, J_5,6= 5.5 Hz, J_5,6= 7.0 Hz, H_5c), 4.04 (br s, 1H,

H_6c+ H_6c

ꢀ

), 3.90 (dd, J_6,5= 3.5 Hz, J_6,6= 12.5 Hz, 1H, H_6b),

ꢀ

7 8 0

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

3.85 (m, 2H, H_2c+ H_3c), 3.84 (dd, J_{6 ,5}

1H, H_6b

ꢀ

= 2.5 Hz, J_{6 ,6}

ꢀ

= 12.5 Hz,

a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-tri-

ꢀ

), 3.78 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.72 (m, 2H,

methyl-[2,2,1]-bicyclehept-2-yliden)-D-myo-inositol

86. To

H_1a+ H_6a), 3.62 (t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4a), 3.50 (dd, J_3,2

=

a solution of 85 (140 mg, 0.108 mmol) and 1H-tetrazol

(38 mg, 0.542 mmol) in dry CH₂Cl₂(2.2 mL) under an

argon atmosphere, N,N-diisopropyl dibenzyl phosphoramidite

(142 ll, 0.432 mmol) was added. The solution was stirred for

30 min and 70% MCPBA (18 mg, 0.150 mmol) was added at

0 ^◦C. After 10 min the mixture was diluted with AcOEt (50 mL)

and washed with sat. NaHCO₃(2 × 50 mL) and sat. NaCl (3 ×

50 mL) solutions. The organic layer was dried over MgSO₄,

concentrated in vacuo and the residue was puriﬁed by double

ﬂash chromatography (hexane–AcOEt 1 : 1 and hexane–AcOEt

9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1) to yield 184 mg

(94%) of 86 as a white foam. [a]²_D⁰= +57.0 (c = 1.0, CHCl₃).

¹H-NMR (CDCl₃, 500 MHz): d 7.36 − 7.18 (m, 46H, ArH),

7.16 − 7.07 (m, 9H, ArH), 5.51 (d, J_1,2= 3.5 Hz, 1H, H_1b), 5.43

(d, J_1,2= 4.0 Hz, 1H, H_1c), 5.03 − 4.90 (m, 8H, 8 × CH_benzylic

phosphate), 4.84 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.83 (d, J =

11.0 Hz, 1H, CH_benzylic), 4.76 (d, J = 11.5 Hz, 1H, CH_benzylic),

4.72 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.70 (d, J = 11.5 Hz, 1H,

CH_benzylic), 4.68 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.65 (d, J =

12.5 Hz, 1H, CH_benzylic), 4.61 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.60 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.58 (s, 2H, CH_2benzylic),

4.49 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.48 (d, J = 10.5 Hz,

1H, CH_benzylic), 4.44 (d, J = 12.0 Hz, 1H, CH_benzylic), 4.26 (br

dd, J_2,3= 3.5 Hz, J_2,1= 6.5 Hz, 1H, H_2a), 4.23 (m, 1H, H_6b),

3.0 Hz, J_3,4= 10.0 Hz, 1H, H_3a), 3.37 (t, J_5,4= J_5,6= 9.5 Hz,

1H, H_5a), 3.36 (dd, J_2,1= 3.5 Hz, J_2,3= 10.5 Hz, 1H, H_2b).

³¹P-NMR (D₂O, 202 MHz): d 3.19. ¹³C-NMR (D₂O, 125 MHz):

d 98.57 (C_1c), 95.76 (C_1b), 79.90 (C_6a), 75.64 (C_4b), 72.22 (C_5a),

72.13 (C_4a), 71.99 (C_2a), 71.22 (C_1a), 70.49 (C_3a), 70.11 (d, J_5,P

=

7.6 Hz, C_5c), 69.91 (C_5b), 69.14 (C_3b), 68.64 (C_3c), 68.41 (C_4c),

68.02 (C_2c), 63.64 (br s, C_6c), 59.48 (C_6b), 53.98 (C_2b). HRMS

m/z calcd. for C₁₈H₃₄O₁₈NPNa⁺: 606.1411 (M + Na)⁺.

2,3,4-Tri-O-benzyl-a-D-galactopyranosyl-(1→4)-2-azido-3-O-

benzyl-2-deoxy-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-

O-(L-1,7,7-trimethyl-[2,2,1]-bicyclehept-2-yliden)-D-myo-inositol

˚

85. To a solution of 57 (330 mg, 0.338 mmol) with 4 A

molecular sieves in dry Et₂O (3.4 mL) under an argon

atmosphere, TMSOTf (6 ll, 0.033 mmol) was added at 0 ^◦C. A

solution of 73 (845 mg, 1.014 mmol) in dry Et₂O (5.1 mL) was

slowly added (0.5 drop per sec) and the mixture was stirred

for 30 min after the addition was completed. The mixture was

neutralised with Et₃N, diluted with AcOEt (10 mL), ﬁltered

and concentrated in vacuo. To a solution of the above residue in

dry THF (6.8 mL) under an argon atmosphere, TBAF (1.0 M

solution in THF, 5.1 mL) was added and the resulting mixture

was stirred for 6 h. The mixture was diluted with AcOEt (50 mL)

and washed with sat. NaHCO₃solution (50 mL). The aqueous

layer was extracted with AcOEt (2 × 25 mL) and the

combined organic layers were washed with sat. NaCl solution

(3 × 100 mL), dried over MgSO₄and concentrated in vacuo.

The residue was puriﬁed by double ﬂash chromatography

(hexane–AcOEt 6 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1 and toluene–AcOEt

2 : 1) to yield 143 mg (45%) of 85 as a white foam. TLC

(toluene–AcOEt 2 : 1) R_f= 0.64; [a]²_D⁰= +66.1 (c = 1.0, CHCl₃).

¹H-NMR (CDCl₃, 500 MHz): d 7.38 − 7.21 (m, 27H, ArH),

7.21 − 7.11 (m, 8H, ArH), 5.74 (d, J_1,2= 4.0 Hz, 1H, H_1c), 5.56

(d, J_1,2= 4.0 Hz, 1H, H_1b), 4.88 (d, J = 12.0 Hz, 1H, CH_benzylic),

4.84 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.77 (d, J = 11.0 Hz,

1H, CH_benzylic), 4.751 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.748 (d,

J = 11.0 Hz, 1H, CH_benzylic), 4.74 (br s, 2H, CH_{2 benzylic}), 4.71

(d, J = 11.5 Hz, 1H, CH_benzylic), 4.68 (s ancho, 2H, CH_{2 benzylic}),

4.67 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.63 (d, J = 11.0 Hz, 1H,

CH_benzylic), 4.59 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.49 (d, J =

4.16 (m, 1H, H_6b

7.5 Hz, J_6,5= 9.5 Hz, 1H, H_6a), 4.01 (m, 1H, H_6c

H_1a), 3.98 (t, J_3,2= J_3,4= 10.0 Hz, 1H, H_3b), 3.94 (dd, J_2,1

ꢀ

), 4.14 (m, 2H, H_5b+ H_6c), 4.05 (dd, J_6,1

=

ꢀ

), 4.00 (m, 1H,

=

3.5 Hz, J_2,3= 10.0 Hz, 1H, H_2c), 3.88 (t, J_4,3= J_4,5= 9.0 Hz,

1H, H_4b), 3.80 (m, 2H, H_4c+ H_5c), 3.77 (m, 1H, H_3a), 3.75 (m,

2H, H_4a+ H_3c), 3.43 (dd, J_5,6= 6.5 Hz, J_5,4= 9.5 Hz, 1H, H_5a),

3.08 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H, H_2b), 1.90 (m, 1H,

L-camphor), 1.86 (m, 1H, L-camphor), 1.74 (m, 1H, L-camphor),

1.68 (m, 1H, L-camphor), 1.45 (d, J = 12.5 Hz, 1H, L-camphor),

1.33 (m, 1H, L-camphor), 1.20 (m, 1H, L-camphor), 1.05 (s,

3H, CH₃^L-camphor), 0.89 (s, 3H, CH₃L-camphor), 0.82 (s,

3H, CH₃^L-camphor). ³¹P-NMR (CDCl₃, 202 MHz): d − 1.45,

−1.77. ¹³C-NMR (CDCl₃, 125 MHz): d 138.48 (ArC), 138.44

(2 × ArC), 138.39, 138.31, 138.29, 138.16 (ArC), 136.04 (d,

J_C,P= 6.9 Hz, ArC phosphate), 136.01 (d, J_C,P= 6.9 Hz, ArC

phosphate), 135.95 (d, J_C,P= 6.9 Hz, ArC phosphate), 135.90 (d,

J_C,P= 6.9 Hz, ArC phosphate), 128.55, 128.51, 128.50, 128.46,

128.45, 128.39, 128.37, 128.33, 128.25, 128.22, 128.09, 128.07,

127.99, 127.93, 127.89, 127.84, 127.82, 127.77, 127.71, 127.69,

127.62, 127.55, 127.54, 127.48, 127.44, 127.36, 127.32, 127.21,

127.19 (ArCH), 118.07 (C_acetalL-camphor), 98.31 (C_1c), 95.54

(C_1b), 80.81 (C_4a), 80.46 (C_5a), 79.42 (C_3b), 78.53 (C_3c), 78.42

(C_6a), 76.54 (C_3a), 76.05 (C_1a), 75.62 (C_2c), 74.65 (CH_2benzylic),

74.42 (C_4b+ CH_2benzylic), 74.21 (C_4c), 73.79 (C_2a), 73.36, 73.33,

72.70, 72.60 (CH_2benzylic), 69.81 (d, J_5,P= 10.5 Hz, C_5c), 69.66

(d, J_5,P= 7.0 Hz, C_5b), 69.27 (d, J_C,P= 5.5 Hz, CH_2benzylic

phosphate), 69.21 (d, J_C,P= 5.4 Hz, CH_2benzylicphosphate), 69.06

(d, J_C,P= 5.5 Hz, CH_2benzylicphosphate), 68.94 (d, J_C,P= 5.1 Hz,

CH_2benzylicphosphate), 66.68 (d, J_6,P= 6.3 Hz, C_6b), 65.74 (d,

J_6,P= 6.4 Hz, C_6c), 62.79 (C_2b), 51.54, 47.95 (C L-camphor),

45.12 (CH L-camphor), 44.65, 29.92, 26.97 (CH₂L-camphor),

20.57, 20.36, 9.84 (CH₃L-camphor). FAB-MS: m/z 1837 (M +

Na)⁺. Anal. calcd. for C₁₀₅H₁₁₃O₂₁N₃P₂: C 69.48%; H 6.28%; N

2.32%; found C 69.74%; H 6.57%; N 2.08%.

11.5 Hz, 1H, CH_benzylic), 4.30 (br s, 1H, H_2a), 4.07 (t, J_4,3= J_4,5

=

9.0 Hz, 1H, H_4b), 4.04 (m, 2H, H_1a+ H_6a), 4.035 (m, 1H, H_3b),

4.02 (m, 1H, H_2c), 3.99 (br d, J_5,4= 10.5 Hz, 1H, H_5b), 3.86

(br s, 1H, H_4c), 3.82 (m, 2H, H_4a+ H_3a), 3.81 (m, 1H, H_3c), 3.79

(m, 1H, H_6c), 3.67 (br dd, J_5,6= 2.5 Hz, J_5,6= 8.0 Hz, 1H,

H_5c), 3.60 (br s, 2H, H_6b+ H_6b), 3.47 (m, 1H, H_5a), 3.45 (br dd,

J_{6 ,5}= 2.5 Hz, J_{6 ,6}= 12.0 Hz, 1H, H_6c), 3.36 (dd, J_2,1= 4.0 Hz,

ꢀ

J_2,3= 10.0 Hz, 1H, H_2b), 2.53 (br s, 1H, OH_6c), 2.37 (br s, 1H,

OH_6b), 1.89 (m, 2H, L-camphor), 1.75 (m, 1H, L-camphor), 1.71

(m, 1H, L-camphor), 1.46 (d, J = 12.5 Hz, 1H, L-camphor),

1.39 (m, 1H, L-camphor), 1.22 (m, 1H, L-camphor), 1.07 (s, 3H,

CH₃^L-camphor), 0.87 (s, 6H, 2 × CH₃L-camphor). ¹³C-NMR

(CDCl₃, 125 MHz): d 138.43, 138.35 (ArC), 138.30 (3 × ArC),

138.10, 138.06 (ArC), 128.43, 128.40, 128.35, 128.33, 128.27,

128.09, 127.92, 127.86, 127.76, 127.69, 127.64, 127.46, 127.43,

127.32, 127.27, 126.81 (ArCH), 118.05 (C_acetalL-camphor), 98.10

(C_1c), 95.89 (C_1b), 80.72 (C_5a), 80.53 (C_4a), 80.40 (C_3b), 79.26

(C_3c), 78.67 (C_6a), 76.59 (C_3a), 75.95 (C_1a), 75.63 (C_2c), 74.69

(C_4c), 74.45, 74.39, 74.25 (CH_2benzylic), 73.80 (C_2a), 73.63, 73.38,

72.87, 72.66 (CH_2benzylic), 72.40 (C_5c), 72.12 (C_4b), 70.46 (C_5b),

63.43 (C_2b), 63.00 (C_6c), 60.78 (C_6b), 51.56, 47.95 (C L-camphor),

45.14 (CH L-camphor), 44.55, 29.93, 26.96 (CH₂L-camphor),

20.59, 20.33, 9.87 (CH₃L-camphor). FAB-MS: m/z 1316 (M +

Na)⁺. Anal. calcd. for C₇₇H₈₇O₁₅N₃·H₂O: C 70.46%; H 6.84%;

N 3.20%; found C 70.43%; H 6.85%; N 2.93%.

6-O-Dibenzylphosphate-2,3,4-tri-O-benzyl-a-D-galactopyran-

osyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-6-O-dibenzylphosphate-

a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-D-myo-inositol 87.

To a solution of 86 (140 mg, 0.077 mmol) in CH₂Cl₂(3.8 mL),

TFA (590 ll, 7.684 mmol) and H₂O (139 ll, 7.716 mmol)

were added and the resulting mixture was stirred for 2 h. The

mixture was diluted with AcOEt (50 mL) and washed with sat.

NaHCO₃(2 × 50 mL) and sat. NaCl (3 × 50 mL) solutions.

The organic layer was dried over MgSO₄, concentrated in

6-O-Dibenzylphosphate-2,3,4-tri-O-benzyl-a-D-galactopyran-

osyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-6-O-dibenzylphosphate-

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 8 1

View Article Online

vacuo and the residue was puriﬁed by ﬂash chromatography

(hexane–AcOEt 2 : 1, 1 : 1, 1 : 2) to yield 106 mg (82%) of 87

as a colourless syrup. [a]²_D⁰= +29.0 (c = 0.5, CHCl₃). ¹H-NMR

(CDCl₃, 500 MHz): d 7.35 − 7.15 (m, 54H, ArH), 7.11 (m, 1H,

ArH), 5.38 (d, J_1,2= 3.5 Hz, 1H, H_1c), 5.32 (d, J_1,2= 3.5 Hz,

1H, H_1b), 4.99 − 4.87 (m, 8H, 8 × CH_benzylicphosphate), 4.89

(br d, J = 11.0 Hz, 2H, 2 × CH_benzylic), 4.86 (d, J = 10.5 Hz,

1H, CH_benzylic), 4.84 (d, J = 10.5 Hz, 1H, CH_benzylic), 4.78 (d, J =

11.5 Hz, 1H, CH_benzylic), 4.71 (s, 2H, CH_2benzylic), 4.69 (d, J =

10.5 Hz, 1H, CH_benzylic), 4.65 (d, J = 11.0 Hz, 1H, CH_benzylic),

4.63 (d, J = 11.5 Hz, 1H, CH_benzylic), 4.62 (br s, 2H, CH_2benzylic),

4.50 (d, J = 11.0 Hz, 1H, CH_benzylic), 4.49 (d, J = 12.0 Hz, 1H,

CH_benzylic), 4.16 (t, J_2,1= J_2,3= 2.5 Hz, 1H, H_2a), 4.12 (m, 1H,

was added in small portions. 2,4-Pentadione (38 mL, 0.37 mol)

was then added dropwise to the stirred suspension and after

2.5 h of stirring at 5 ^◦C, no starting material was detected by

TLC-analysis. The solids were removed by ﬁltration through

a pad of celite, the ﬁltrate was concentrated in vacuo and the

residue was puriﬁed by ﬂash chromatography (hexane–AcOEt 8 :

1, 2 : 1,1 : 1) to obtain the amine 89 (2.04 g, 5.58 mmol, 82%)

as a fair yellow solid next to a small amount of hydrazine as

the autocoupling product (460 mg, 0.61 mmol). TLC (hexane–

1

AcOEt 4 : 1) R_f= 0.36. H-NMR (300 MHz, CDCl₃): d 7.50

(d, 6H, J_ortho/meta= 7.7Hz, ortho aromat. Trit), 7.29 (m, 6H, meta

aromat. Trit), 7.22 (m, 3H, para aromat. Trit.), 7.15 (d, 2H,

J_ortho/meta= 8.3Hz, meta aromat.), 6.66 (d, 2H, J_ortho/meta= 8.3Hz,

ortho aromat.), 4.03 (s, 2H, CH₂OTr), 3.62 (br s, 2H, NH₂).

¹³C-NMR (75 MHz, CDCl₃): d 196.52, 144.39, 137.98, 136.98,

129.09, 128.30, 128.07, 127.49, 125.03, 97.95, 87.47, 65.63, 29.59,

20.32. FAB-MS: m/z 388 (M + Na⁺).

H_6c), 4.08 (m, 1H, H_6b), 4.04 (m, 1H, H_6b

H_6c

), 3.96 (dd, J_2,1= 3.5 Hz, J_2,3= 9.5 Hz, 1H, H_2c), 3.95

(t, J_6,1= J_6,5= J_4,3= J_4,5= 9.5 Hz, 2H, H_6a+ H_4a), 3.93 (dd,

ꢀ

), 4.01 (m, 2H, H_5b+

ꢀ

J_3,4= 9.0 Hz, J_3,2= 10.0 Hz, 1H, H_3b), 3.823 (t, J_4,3= J_4,5

=

9.5 Hz, 1H, H_4b), 3.818 (br s, 1H, H_4c), 3.80 (m, 1H, H_5c), 3.77

(dd, J_3,2= 2.5 Hz, J_3,4= 10.0 Hz, 1H, H_3c), 3.67 (br s, 1H,

OH_eq.), 3.56 (br dd, J_1,2= 3.0 Hz, J_1,6= 9.5 Hz, 1H, H_1a), 3.44

1-Glutarylamino-4-triphenylmethyloxymethylbenzene 90. To

a solution of 89 (2.5 g, 6.8 mmol) in pyridine (90 mL), glutaric

anhydride (931 mg, 8.16 mmol) was added and the reaction

mixture was stirred at 45 ^◦C for 2 h, when TLC-analysis indicated

the complete consumption of the starting material. The reaction

crude was extracted with water–ethyl acetate, dried over MgSO₄

and after concentration in vacuo the residue was puriﬁed by ﬂash

chromatography (CH₂Cl₂–MeOH 15 : 1) to obtain 90 (3.01 g,

6.46 mmol, 95%) as a white solid. TLC (CH₂Cl₂–MeOH 20 :

1) R_f= 0.55. ¹H-NMR (300 MHz, DMSO): d 4.76 (s, 1H,

NH), 7.59 − 7.24 (m, 19H, PhH), 4.00 (s, 2H, OCH₂Ph), 3.37

(br s, 1H, COOH), 2.35 (t, 2H, J = 7.2Hz, CH₂), 2.27 (t, 2H,

J = 7.2Hz, CH₂), 1.81 (q, 2H, J = 7.1Hz, CH₂). ¹³C-NMR

(75 MHz, DMSO): d 171.60, 144.65, 139.30, 133.78, 129.07,

128.86, 128.21, 127.96, 119.92, 87.27, 65.82, 41.14, 36.28, 33.92,

21.39. FAB-MS: m/z 502 (M + Na⁺).

(dd, J_3,2= 2.5 Hz, J_3,4= 9.5 Hz, 1H, H_3a), 3.37 (t, J_5,4= J_5,6

=

9.5 Hz, 1H, H_5a), 3.15 (dd, J_2,1= 3.5 Hz, J_2,3= 10.0 Hz, 1H,

H_2b), 2.62 (br s, 1H, OH_ax.). ³¹P-NMR (CDCl₃, 202 MHz): d −

1.63, −1.67. ¹³C-NMR (CDCl₃, 125 MHz): d 138.55, 138.47

(ArC), 138.35 (2 × ArC), 138.23, 137.85, 137.74 (ArC), 135.96

(d, J_C,P= 6.5 Hz, ArC phosphate), 135.91 (d, J_C,P= 6.5 Hz, ArC

phosphate), 135.83 (d, J_C,P= 6.8 Hz, ArC phosphate), 135.79 (d,

J_C,P= 6.2 Hz, ArC phosphate), 128.51, 128.47, 128.44, 128.34,

128.31, 128.25, 128.22, 128.16, 128.06, 127.95, 127.93, 127.90,

127.87, 127.81, 127.77, 127.73, 127.57, 127.53, 127.50, 127.45,

127.32, 127.25, 127.22 (ArCH), 98.20 (C_1c), 97.74 (C_1b), 81.54

(C_4a), 80.92 (C_5a), 79.99 (C_6a), 79.92 (C_3b), 79.63 (C_3a), 78.44

(C_3c), 75.72 (CH_2benzylic), 75.56 (C_2c), 74.85, 74.62 (CH_2benzylic),

74.54 (C_4b), 74.15 (C_4c), 73.96, 73.57, 72.72 (CH_2benzylic), 72.66

(C_1a), 72.57 (CH_2benzylic), 70.28 (d, J_5,P= 6.5 Hz, C_5b), 69.93 (d,

J_5,P= 9.8 Hz, C_5c), 69.63 (C_2a), 69.30 (d, J_C,P= 6.1 Hz, CH_2benzylic

phosphate), 69.24 (d, J_C,P= 5.6 Hz, CH_2benzylicphosphate), 69.08

(d, J_C,P= 4.8 Hz, CH_2benzylicphosphate), 69.05 (d, J_C,P= 4.5 Hz,

CH_2benzylicphosphate), 66.72 (d, J_6,P= 5.3 Hz, C_6b), 65.75 (d,

J_6,P= 5.0 Hz, C_6c), 63.87 (C_2b). FAB-MS: m/z 1703 (M + Na)⁺.

Attachment of linker 90 to PS-NH₂resin (resin 91). Prior

to use, the PS-NH₂resin (substitution: 1.23 mmol g⁻¹, Cal-

biochem/Novabiochem AG, La¨ufelﬁngen, Switzerland; 423 mg,

1.23 mmol g⁻¹loading, 0.52 mmol) was washed 3 times with

diisopropylamine (5% solution in CH₂Cl₂, 5 mL for each cycle)

and with CH₂Cl₂(3 × 5 mL) in a specially designed reactor

equipped with a sintered glass ﬁlter. After drying the resin

under a high vacuum for two hours, the resin was swollen in

CH₂Cl₂(3 mL) and the linker 90 (250 mg, 0.52 mmol) was

added as a solution in DMF (3 mL). After addition of 1-

hydroxybenzotriazol (703 mg, 5.2 mmol) and DIC (162 lL,

1.1 mmol) the resin was shaken on an IKA-vibramax-shaker

for three days. The resin was washed subsequently with water

(3 × 5 mL), MeOH (4 × 5 mL), CH₂Cl₂(4 × 5 mL) and THF (3 ×

5 mL) and dried in vacuo until weight remained constant. Weight

gain of the resin (+245 mg after reaction, 98% transformation)

and check for free amine groups by the test of Kaiser,⁶³indicated

quantitative completion of the reaction. Remaining free amine

groups were capped by treatment with Ac₂O–pyr (2 : 1, 5 mL)

for 2 h at rt, followed by washing of the resin 91 with water (3 ×

5 mL), MeOH (3 × 5 mL), CH₂Cl₂(3 × 5 mL) and THF (2 ×

5 mL) and drying under a high vacuum until weight remained

constant (weight of resin after linker attachment: 668 mg, 99%

of attachment).

6-O-Phosphate-a-D-galactopyranosyl-(1→4)-2-ammonio-2-de-

oxy-6-O-phosphate-a-D-glucopyranosyl-(1→6)-D-myo-inositol 36.

A suspension of 87 (80 mg, 0.048 mmol) and 10% Pd on

charcoal (102 mg, 0.096 mmol) in a mixture of MeOH–H₂O 9 :

1 (4 mL) under a hydrogen atmosphere was stirred for 12 h. The

mixture was ﬁltered through a pad of celite and the ﬁlter cake

washed with H₂O. The solution was concentrated in vacuo and

lyophilised to yield 32 mg (100%) of 36 as a white solid. [a]_D²⁰

=

+134.0 (c = 0.2, H₂O). NMR data at pH = 5.9: ¹H-NMR (D₂O,

500 MHz): d 5.45 (d, J_1,2= 4.0 Hz, 1H, H_1c), 5.40 (d, J_1,2

=

3.0 Hz, 1H, H_1b), 4.40 (br d, J_5,4= 10.0 Hz, 1H, H_5b), 4.30 (t,

J_3,2= J_3,4= 10.0 Hz, 1H, H_3b), 4.19 (br t, J_5,6= J_5,6= 6.5 Hz,

1H, H_5c), 4.11 (br s, 2H, H_6b+ H_6b

), 4.03 (br d, J_4,3= 3.0 Hz,

1H, H_4c), 4.00 (br s, 1H, H_2a), 3.96 (br t, J_{6+6 ,5}= 6.0 Hz,

= J_{6+6 ,P}

2H, H_6c+ H_6c

ꢀ

), 3.88 (dd, J_3,4= 3.0 Hz, J_3,2= 10.5 Hz, 1H,

H_3c), 3.823 (dd, J_2,1= 3.5 Hz, J_2,3= 10.5 Hz, 1H, H_2c), 3.816

(t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4b), 3.72 (m, 2H, H_1a+ H_6a), 3.63

(t, J_4,3= J_4,5= 9.5 Hz, 1H, H_4a), 3.50 (dd, J_3,2= 3.0 Hz, J_3,4

=

10.0 Hz, 1H, H_3a), 3.39 (dd, J_2,1= 3.5 Hz, J_2,3= 10.5 Hz, 1H,

H_2b), 3.37 (m, 1H, H_5a). ³¹P-NMR (D₂O, 202 MHz): d 0.80,

−0.06. ¹³C-NMR (D₂O, 125 MHz): d 97.25 (C_1c), 95.78 (C_1b),

80.34 (C_6a), 74.71 (C_4b), 72.26 (C_5a), 72.10 (C_4a), 71.96 (C_2a),

71.12 (C_1a), 70.46 (C_3a), 70.08 (d, J_5,P= 7.4 Hz, C_5c), 68.76 (d,

J_5,P= 7.5 Hz, C_5b), 68.72 (C_3b), 68.55 (C_3c), 68.47 (C_4c), 67.94

(C_2c), 63.77 (C_6c), 63.26 (C_6b), 54.06 (C_2b).

Detritylation of resin 91 (resin 92). Linker-equipped resin

91 (668 mg, 0.51 mmol) was treated with a TFA-solution in

sec-butanol (1 mL TFA in 2 mL sec-butanol) and shaken for

30 min. After removal of the liquid phase by ﬁltering through

the incorporated sintered glass and washing of the resin with

CH₂Cl₂(4 × 5 mL), the detritylation procedure was repeated

three times until TLC-analysis of the liquid phase indicated

absence of any UV-active compound. Washing of the resin with

CH₂Cl₂(4 × 5 mL) and drying under a high vacuum afforded the

resin 92 (548 mg, 0.49 mmol, 97%), as determined by weight loss

(−120 mg).

1-Amino-4-triphenylmethyloxymethylbenzene 89.

A solu-

tion of 1-nitro-4-triphenylmethyl oxymethylbenzene⁶¹(2.7 g,

6.8 mmol) in THF (250 mL) under an argon atmosphere was

◦

cooled to 0 C and freshly prepared “Zn–Cu-couple”⁶²(7.2 g)

7 8 2

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

Preparation of PS-benzylaminolinker-TCA resin (resin 93,

0.72 mmol g⁻¹). To resin 92 bearing free benzylaminotype-

linker (548 mg, 0.89 mmol g⁻¹loading) swollen in CH₂Cl₂

(5 mL), trichloroacetonitrile (2.3 mL, 23 mmol) was added and

2.63 (m, 1H, CH₂Lev), 2.60 − 2.55 (m, 1H, CH₂Lev), 2.37 −

2.30 (m, 1H, CH₂Lev), 2.26 − 2.20 (m, 1H, CH₂Lev), 2.13 (s,

3H, CH₃Lev), 1.91 − 1.82 (m, 2H, L-camphor), 1.74 − 1.72

(m, 1H, L-camphor), 1.69 − 1.67 (m, 1H, L-camphor), 1.45 (d,

J = 12.9 Hz, 1H, L-camphor), 1.41 − 1.35 (m, 1H, L-camphor),

1.22 − 1.17 (m, 1H, L-camphor), 1.05 (s, 3H, CH₃L-camphor),

0.86 (s, 3H, CH₃^L-camphor), 0.85 (s, 3H, CH₃L-camphor).

¹³C-NMR (125 MHz, CDCl₃): d 205.98, 173.14, 138.35, 138.31,

138.29, 138.13, 137.83, 128.50, 128.46, 128.43, 128.40, 128.34,

128.17, 128.07, 127.96, 127.94, 127.91, 127.89, 127.86, 127.82,

127.76, 127.72, 127.68, 127.55, 127.53, 127.44, 127.42, 119.69,

95.33, 80.82, 80.69, 80.65, 80.42, 79.58, 77.09, 76.96, 76.91,

76.82, 76.70, 76.07, 74.98, 74.96, 74.78, 74.74, 74.67, 74.62,

73.89, 73.03, 72.63, 71.14, 71.12, 69.73, 69.61, 62.99, 62.95,

60.55, 53.69, 51.60, 48.78, 47.99, 45.16, 45.06, 37.68, 29.76,

29.71, 29.52, 27.71, 26.99, 26.92, 20.62, 20.57, 20.48, 20.38,

11.52, 9.79. FAB-MS: m/z 982 (M + Na⁺).

◦

the resin was shaken for 10 min at rt. After cooling to 0 C, a

solution of DBU (154 lL in 1 mL CH₂Cl₂)_◦was added over a

period of 5 min. The resin was shaken at 0 C for 40 min, the

liquid phase removed by pressure-assisted ﬁltering through the

sintered glass, and after subsequent washing of the resin with

CH₂Cl₂(4 × 5 mL) and THF (4 × 5 mL) the resin was dried

under a high vacuum. Formation of the trichloroacetimidate was

monitored by IR, following the disappearance of the hydroxy

stretching band at ∼3500 cm⁻¹of the original polymer with the

−1

=

appearance of a strong C N stretching band at 1664 cm . The

yield was determined by weight gain (+64 mg, 0.44 mmol, 90%

transformation) after drying under a high vacuum (weight of

resin 612 mg, 0.44 mmol, 0.72 mmol g⁻¹).

2-Azido-3-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-4-O-

levulinoyl-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-

(L-1,7,7-trimethyl[2.2.1]bicyclohept-6-ylidine)-D-myo-inositol 94.

To a solution of 57 (635 mg, 0.65 mmol) and DCC (670 mg, 3.25

mmol) in CH₂Cl₂(5 mL) levulinic acid (668 lL, 6.5 mmol) was

added at rt. After a few seconds precipitation of the formed urea

was observed. DMAP (8 mg, 0.07 mmol) dissolved in CH₂Cl₂

(100 lL) was added by syringe and the reaction stirred at rt

for 1.5 h. The mixture was diluted with CH₂Cl₂(20 mL) and

ﬁltered through a pad of celite to remove insoluble urea. The

solvent was removed under reduced pressure and the obtained

residue puriﬁed by column chromatography (hexane–AcOEt 8 :

1) to furnish 94 (670 mg, 0.62 mmol, 96%) as a colorless oil.

Loading of high-loading resin 93 with acceptor 95 (resin 96,

0.71 mmol g⁻¹) and cleavage. Resin 93 (111 mg, 0.08 mmol)

weighed into a 5 mL SP-reactor was suspended in cyclohexane

(1.5 mL) and shaken for 10 min under an atmosphere of inert

gas. The acceptor 95 (168 mg, 0.19 mmol, 2.4 eq.) as a solution

in CH₂Cl₂(1.5 mL) was added via syringe and the components

mixed thoroughly by shaking for 5 min. BF₃·Et₂O (200 lL of

a 0.2 M solution in CH₂Cl₂) was added at rt and shaking of

the resin continued for 2 h, followed by capping with MeOH

(1 mL) and further shaking for 15 min. After separation of

the liquid components, the resin was washed with MeOH (3 ×

4 mL) and CH₂Cl₂(5 × 4 mL) and dried under a high vacuum

overnight. Loading with 95 was determined by cleaving the

alcohol from a resin aliquot (27 mg) with DDQ in CH₂Cl₂–H₂O

9 : 1 and weighing the returned alcohol 95 (17 mg, 0.019 mmol,

0.71 mmol g⁻¹). MALDI-TOF MS m/z 985 (M + Na + 2H)⁺,

calcd. for C₅₅H₆₅N₃O₁₂: 960.

TLC (hexane–AcOEt 3 : 1) R_f= 0.48; [a]²⁰= +39.7^◦(c = 0.55,

D

CHCl₃). ¹H-NMR (500 MHz, CDCl₃): d 7.39 − 7.22 (m, 20H,

PhH), 5.66 (d, 1H, J_1,2= 3.4Hz, H_1b), 5.15 − 5.07 (m, 1H, H_4b),

4.86 − 4.68 (m, 6H, CH₂Ph), 4.59 − 4.55 (m, 2H, CH₂Ph),

4.15 − 4.13 (m, 1H, H_2a), 4.02 − 3.95 (m, 4H, H_5b, H_1a, H_6a,

H_4a), 3.63 (dd, 1H, J_2,3= 2.98 Hz, J_3,4= 8.0 Hz, H_3a), 3.52 (m,

Delevulination of resin 96 (resin 97). Resin 96 (90 mg,

0.71 mmol g⁻¹, 0.064 mmol) was swollen in CH₂Cl₂(2 mL) and

shaken for 10 min at rt. Hydrazine-acetate (18 mg, 0.2 mmol) as

a solution in MeOH (400 lL) was added and the resin shaken at

rt for 18 h. After washing with CH₂Cl₂–MeOH 9 : 1 (4 × 5 mL),

CH₂Cl₂(4 × 5 mL) and THF (4 × 5 mL) the resin was dried in

vacuo. A resin sample (6 mg) was treated with DDQ (1.5 mg) in

CH₂Cl₂–H₂O (20 : 1, 250 lL) for 1 h, and the liquid phase used

as reference for TLC-analysis (hexane–AcOEt 2 : 1; R_f= 0.39)

and MALDI-TOF-monitoring of the reaction. MALDI-TOF

MS m/z 887 (M + Na + 2H)⁺, calcd. for C₅₀H₅₉N₃O₁₀: 862.

1H, H_6b), 3.43 − 3.39 (m, 1H, H_5a), 3.37 − 3.31 (m, 2H, H_6b

ꢀ

,

H_2b), 2.64 − 2.52 (m, 2H, CH₂Lev), 2.40 (m, 1H, L-camphor),

2.38 − 2.26 (m, 2H, CH₂Lev), 2.11 (s, 3H, CH₃Lev), 1.69 −

1.65 (m, 3H, L-camphor), 1.58 − 1.55 (m, 3H, L-camphor), 0.96

t

(s, 3H, CH₃, L-camphor), 0.84 (s, 9H, Bu TBDMS), 0.81 (s,

3H, CH₃^L-camphor), 0.76 (s, 3H, CH₃L-camphor), −0.03 (s,

3H, CH₃TBDMS), −0.04 (s, 3H, CH₃TBDMS). ¹³C-NMR

(125 MHz, CDCl₃): d 128.50, 128.40, 128.36, 128.33, 128.28,

128.27, 128.18, 128.14, 128.02, 127.97, 127.92, 127.90, 127.80,

127.71, 127.60, 119.39, 94.69, 80.61, 78.07, 76.22, 74.19, 73.08,

70.12, 62.86, 53.58, 48.83, 47.99, 45.16, 37.77. FAB-MS: m/z

1096 (M + Na⁺).

Glycosylation of liberated acceptor 97 with glycosyl donor 68

and cleavage from resin. Resin-bound acceptor 97 (84 mg, 0.06

mmol) was washed with freshly distilled THF (3 × 3 mL) to

remove traces of water and dried in vacuo. Donor 68 (123 mg,

0.147 mmol) was divided into three aliquots and each aliquot

dissolved in CH₂Cl₂(2 mL). The resin was swollen with donor-

solution (0.049 mol in 2 mL CH₂Cl₂) by shaking for 10 min at rt.

After cooling the reactor to 0 ^◦C, 200 lL of a freshly prepared

TMSOTf solution (0.05 M in CH₂Cl₂) was added, the mixture

shaken for 1 h at rt and then quenched with triethylamine

(100 lL). After removal of the liquid phase by ﬁltration, the

resin was washed with MeOH (3 × 3 mL), THF (3 × 3 mL)

and CH₂Cl₂(3 × 3 mL). After drying the resin in vacuo, the

glycosylation was repeated twice with the remaining two donor-

aliquots. After repeated washing and drying of the resin to

weight constancy the crude yield of the transformation was

calculated to be 13% as suggested by the weight gain (+5 mg)

observed after performing the three glycosylation cycles. A

sample of the resin (7 mg) was subjected to the oxidative cleavage

conditions with DDQ (1 mg) in CH₂Cl₂–H₂O 20 : 1 (0.5 mL)

for 4 h. TLC and MALDI-TOF analysis showed the presence

of unreacted acceptor 56 besides a small portion of the attached

donor employed in the glycosylation. The formation of the

2-Azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-a-D-glucopyranos-

yl-(1→6)-3,4,5-tri-O-benzyl-1,2-O-(L-1,7,7-trimethyl[2.2.1]bicyc-

lohept-6-ylidine)-D-myo-inositol 95. Pseudodisaccharide 94

(670 mg, 0.686 mmol) was dissolved in THF (50 mL) under

an argon atmosphere placed in a sealed PE-ﬂask, cooled to

−20 ^◦C and treated with AcOH (14 mL) and HF–pyridine

complex (8 mL). The solution was allowed to warm up to rt

and left stirring for 4 h . After dilution with Et₂O the reaction

mixture was neutralized with sat. NaHCO₃solution and the

organic layer washed with sat. NaHCO₃solution and brine,

dried over MgSO₄and the solvent removed in vacuo. The

residue was puriﬁed by ﬂash chromatography (hexane–AcOEt

2 : 1) to furnish 95 as a colourless oil (598 mg, 0.68 mmol, 99%).

1

TLC (hexane–AcOEt 3 : 1) R_f= 0.17. H-NMR (500 MHz,

CDCl₃): d 7.38 − 7.21 (m, 20H, PhH), 5.59 (d, 1H, J_1,2

=

3.5Hz, H_1b), 4.93 (t, 1H, J_3,4= J_4,5= 10.1 Hz, H_4b), 4.83 − 4.62

(m, 8H, CH₂Ph), 4.27 (dd, 1H, J_2,3= 3.9 Hz, J_2,1= 5.6 Hz,

H_2a), 4.19 − 4.00 (m, 2H, H_1a+ H_6a), 3.95 − 3.91 (m, 2H,

H_3b+ H_5b), 3.83 − 3.77 (m, 2H, H_4a+ H_3a), 3.49 − 3.45 (m, 1H,

H_6b), 3.41 (t, 1H, J_4,5= J_5,6= 9.4 Hz, H_5a), 3.37 (dd, 1H, J_1,2

=

3.5 Hz, J_2,3= 10.2Hz, H_2b), 3.35 − 3.21 (m, 1H, H_6b), 2.68 −

ꢀ

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 8 3

View Article Online

desired trisaccharide was not observed. MALDI-TOF MS m/z

of donor was monitored by TLC-analysis of the liquid phase.

The resin was washed extensively with CH₂Cl₂–MeOH 9 : 1

(5 × 4 mL) and CH₂Cl₂(5 × 4 mL) and dried under a high

vacuum until the weight remained constant. Weight gain of the

resin suggested a transformation of 50% for the ﬁrst coupling.

Cleavage of the product from a resin sample by treatment

with DDQ in CH₂Cl₂–H₂O (20 : 1) and subsequent analysis

by TLC and MALDI-TOF demonstrated the appearance of

a two new products with the consistant weight of the desired

trisaccharide 102, next to unreacted disaccharidic acceptor 56.

The appearance of two isomeric forms were attributed to the

4^ꢀ-OH and 6^ꢀ-OH-glycosylated products due to the 2 : 1 mixture

obtained when attaching the diol 56 to the linker. The reaction

was repeated 4 times with donor charges ranging from 3.5 to

5 eq., resulting in a ﬁnal transformation of 60% as suggested

by the observed weight gain and the acceptor:product ratio on

TLC-sheet and in the mass-spectra. TLC (hexane–AcOEt 3 : 1)

R_f= 0.52 of major product 102; (hexane–AcOEt 3 : 1) R_f= 0.17

acceptor 56. MALDI-TOF MS m/z 885 (M + Na⁺), 901 (M +

K⁺) unreacted acceptor 56; 1371 (M + Na⁺), 1385 (M + K⁺)

trisaccharide 102.

887 (M + Na + 2H)⁺calcd. for C₅₀H₅₉N₃O₁₀: 862.

Preparation of low-loading PS-benzylaminolinker-TCA resin

(93, 0.20 mmol/g). Prior to use the PS-NH₂resin (1 g,

1.23 mmol, substitution: 1.23 mmol g⁻¹, Calbiochem/Novabio-

chem AG, La¨ufelﬁngen, Switzerland) was washed 3 times with

diisopropylamine (5% solution in CH₂Cl₂, 5 mL for each cycle)

and CH₂Cl₂(3 × 5 mL) in a specially designed reactor equipped

with a sintered glass ﬁlter. After drying the resin under a high

vacuum for 2 h, the resin was swollen in DCM (3 mL) and

a substechiometric amount of linker 90 (96 mg, 0.2 mmol)

was added as a solution in DMF (6 mL). After addition of

1-hydroxybenzotriazol (306 mg, 2 mmol) and DIC (66 lL,

0.2 mmol), the resin was shaken on an IKA-vibramax-shaker

for three days. The resin was washed subsequently with water

(3 × 6 mL), MeOH (4 × 6 mL), CH₂Cl₂(4 × 6 mL) and THF

(3 × 6 mL) and dried in vacuo. Free amine groups were capped

by treatment with Ac₂O–pyr (2 : 1 5 mL) for 2 h at rt, followed by

washing of the resin with water (3 × 5 mL), MeOH (3 × 5 mL),

CH₂Cl₂(3 × 5 mL) and THF (2 × 5 mL) and drying under a high

vacuum until weight remained constant. Weight gain of the resin

(+1.32 g after reaction) and check for free amine groups by the

Kaiser-test,⁶³indicated quantitative completion of the reaction.

Acidic cleavage of the trityl ether and formation of the benzylic

trichloroacetimidate 93 (loading 0.20 mmol g⁻¹) was performed

following the procedure for the high-loading resin 93.

Resin-bound acceptor 99. ArgoGel-Wang-Cl (loading:

0.43 mmol g⁻¹from Argonaut Technologies, San Carlos, CA,

USA; 274 mg, 118 lmol) and sodium hydride (8.5 mg, 0.35

mmol) were weighed into a ﬂask and purged with argon. A

solution of diol 56 (406 mg, 0.47 mmol) in dry THF (2.5–3 mL)

was added and the reaction was stirred at rt. After 16 h., the resin

was successively washed with MeOH (1 × 4 mL), THF (1 ×

4 mL) and CH₂Cl₂(3 × 4 mL). These organic layers were washed

with sat. NH₄Cl solution (25 mL) and water (25 mL), dried over

MgSO₄and concentrated in vacuo to recover the excess of diol 56.

The resin was washed further with H₂O (1 × 4 mL), sat. NH₄Cl

solution (1 × 4 mL), H₂O (5 × 4 mL), DMF (2 × 4 mL), THF

(2 × 4 mL), CH₂Cl₂(2 × 4 mL) and ﬁnally dried under a high

vacuum overnight. According to the amount of product isolated

from the resin, the new loading value was around 0.15 mmol g⁻¹.

Gel-phase¹³C-NMR(75MHz, CDCl₃):d 159.30, 138.89, 138.73,

138.61, 138.34, 130.47, 130.27, 129.73, 129.33, 128.93, 128.80,

128.61, 128.25, 128.15, 128.05, 118.45, 115.21, 114.85, 95.96

(C₁), 81.15, 81.03, 80.16, 79.60, 78.53, 76.50, 75.70, 75.61, 75.41,

75.26, 75.19, 74.29, 73.44, 72.94, 71.76, 71.25, 70.15, 70.08,

69.97, 69.54, 68.41, 63.78, 63.11, 52.01, 48.37, 46.70, 45.52,

45.27, 43.28, 30.26, 27.42, 21.08, 20.86, 11.97, 10.27, 10.16.

Selective attachment of diol 56 to low-loading PS-benzylamino-

linker-TCA resin 93 (resin 98, 0.14 mmol g⁻¹). PS-resin 93

(914 mg, 0.2 mmol g⁻¹, 0.182 mmol) was washed prior to

use with freshly distilled THF (4 × 5 mL) and dried under a

vacuum to remove traces of moisture. The resin was suspended

in cyclohexane (6 mL) and the diol 56 (486 mg, 0.543 mmol) was

added as a solution in CH₂Cl₂(6 mL). Shaking was continued at

rt for 10 min to achieve a thorough mixture of the components,

the reaction then cooled to −15 ^◦C and BF₃·OEt₂(45 lL) was

added by syringe. After shaking at −15 ^◦C for 1.5 h, MeOH was

added to cap unreacted linker sites and shaking was continued

for 10 min. The liquid phase was separated by ﬁltration and the

resin was washed subsequently with MeOH (3 × 10 mL) and

CH₂Cl₂(4 × 8 mL). Weight gain after drying indicated the yield

of attachment (109 mg, 0.126 mmol, 70%) resulting in a loading

of 0.14 mmol g⁻¹based on the diol attached. MALDI-TOF MS

m/z 885 (M + Na⁺), 901 (M + K⁺), calcd. for C₅₀H₅₉N₃O₁₀: 862.

2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2-azido-3-

O-benzyl-2-deoxy-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-

D-myo-inositol 104. Resin-bound acceptor 99 (123 mg,

18 lmol) was swollen in a solution of donor 103⁶¹(58 mg, 0.12

mmol) in dry CH₂Cl₂(1.5 mL), shaken for 30 min and cooled at

−20 ^◦C. TMSOTf (100 lL of a 0.06 M solution in dry CH₂Cl₂,

6 lmol) was added and the reaction mixture was shaken at

−20 ^◦C. After 4 h., the resin was washed with CH₂Cl₂(5 ×

4 mL), H₂O (2 × 4 mL), DMF (2 × 4 mL), THF (2 × 4 mL) and

CH₂Cl₂(2 × 4 mL). The glycosylation (103 (30 mg, 0.06 mmol),

TMSOTf (75 lL of a 0.06 M solution in dry CH₂Cl₂, 4.5 lmol))

and washing were repeated and the resin was dried under a

vacuum for 12 h. The reaction was monitored by removing

several mg of resin and cleaving from resin using TFA–CH₂Cl₂–

H₂O, followed by TLC analysis and MALDI-TOF mass

spectrometry. Resin-bound trisaccharide (110 mg, 16 lmol) was

swollen in CH₂Cl₂(2 mL) and shaken for 5 min. TFA (146 lL,

1.89 mmol) and water (34 lL, 1.89 mmol) were added and the

reaction mixture was shaken for 6 h. at rt. The solution and

CH₂Cl₂washings (4 × 4 mL) were collected by ﬁltration. This

organic layer was washed with sat. NaHCO₃solution (15 mL)

and H₂O (15 mL), dried (MgSO₄) and concentrated in vacuo.

The puriﬁcation of the residue was carried out by ﬂash column

chromatography (toluene–acetone 2 : 1) to give 104 (12 mg,

Determination of the selectivity of attachment of 56 to low-

loading resin 93. The resin 93 was swollen in CH₂Cl₂(5 mL)

and pyridine (2.5 mL) and treated with benzoyl chloride

(325 lL) for 20 h to determine the selectivity of the attach-

ment reaction. Cleavage of the benzoylated compound mixture

was performed by swelling an aliquot of the resin (383 mg,

0.054 mmol) in CH₂Cl₂–H₂O (20 : 1, 4 mL) and shaking

vigorously after addition of DDQ (21 mg, 0.093 mmol) for 2 h.

The cleavage was repeated once under the same conditions and

the ﬁltered liquid fractions combined, washed with sat. NaHCO₃

solution, dried over MgSO₄and puriﬁed through a short plug

of silica gel (hexane–AcOEt 3 : 1) to afford the benzoylated

pseudodisaccharide (15.1 mg, 0.016 mmol, 30%) as a mixture

1

of regioisomers. The selectivity was determined by H-NMR

spectroscopy to be 2 : 1 for the ratio of 4-OBz versus 6-OBz in

the glucosamine-moiety. ¹H-NMR (500 MHz, CDCl₃) selected

data: anomeric signals of major fraction d 5.71 (d, 1H, J_1,2

3.6 Hz, H_1b, 4-Benzoyl), anomeric signals minor fraction 5.53

=

(d, 1H, J_1,2= 3.4 Hz, H_1b, 6-benzoyl).

Glycosylation of resin-bound acceptor 98 with donor 100 (resin-

bound trisaccharide 101). Resin 98 (265 mg, 0.053 mmol) was

directly swollen in a CH₂Cl₂solution (2 mL) of donor 100⁶⁴

(216 mg, 0.29 mmol, 5.5 eq.), shaken for 10 min at rt and then

cooled to 0 ^◦C. TMSOTf solution (200 lL of a 0.46 M solution)

was added by syringe and the resin shaken at rt. for 1 h. Reaction

71%). TLC: (toluene–acetone 2 : 1) R_f= 0.21. [a]²⁰= +18.3^◦

D

(c = 0.42, CHCl₃). ¹H NMR (300 MHz, CDCl₃): d 7.43 − 7.15

7 8 4

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

View Article Online

107: TLC (toluene–acetone 2 : 1) R_f= 0.25. [a]²_D⁰= +33.4^◦(c =

(m, 20H, PhH), 5.30 (dd, 1H, J_3,4= J_4,5= 3.0 Hz, H_4c), 5.25

1

(d, 1H, J_1,2= 3.9 Hz, H_1b), 5.17 (dd, 1H, J_1,2= 8.1 Hz, J_2,3

=

1.25, CHCl₃). H-NMR (500 MHz, CDCl₃): d 7.32 − 7.21 (m,

10.5 Hz, H_2c), 5.10 − 4.97 (2d, 2H, J = 10.5 Hz, CH₂Ph), 4.96

(dd, 1H, J_3,4= 3.3 Hz, J_2,3= 10.5 Hz, H_3c), 4.90 (1d, 1H, J =

10.2 Hz, CH₂Ph), 4.77 − 4.65 (m, 6H, H_1c+ CH₂Ph), 4.22

(br s, 1H, H_2a), 4.14 (d, 1H, J = 3.5 Hz, OH), 4.01 (dd, 1H,

J_3,4= J_4,5= 9.5 Hz, H_4a), 3.97 − 3.88 (m, 4H), 3.85 − 3.74

(m, 3H), 3.61 (m, 1H, H_1a), 3.54 − 3.46 (m, 2H, H_2b, H_3a),

3.42 − 3.36 (m, 3H), 2.56 (br s, 1H, OH), 2.12, 1.96, 1.96,

1.89 (4s, 12H, OCOCH₃). ¹³C-NMR (125 MHz, CDCl₃): d

20H, Ph), 5.37 (d, 1H, J_1,2= 3.5 Hz, H_1b), 5.05 − 4.63 (m, 8H,

CH₂Ph), 4.15 (br s, 1H, H_2a), 3.98 (dd, 1H, J_3,4= J_4,5= 9.5 Hz,

H_4a), 3.94 (dd, 1H, J_5,6= J_1,6= 9.5 Hz, H_6a), 3.76 (dd, 1H, J_2,3

=

J_3,4= 9.5 Hz, H_3b), 3.70 (m, 2H), 3.62 − 3.57 (m, 2H, H_1a, H_4b),

3.46 (dd, 1H, J_2,3= 3.0 Hz, J_3,4= 10.0 Hz, H_3a), 3.40 − 3.29

(m, 4H), 2.72 (s, 1H, OH), 2.32 (br s, 1H, OH). Anal. calcd. for

C₄₀H₄₅N₃O₁₀·1/4H₂O: C, 65.60%; H, 6.26%; N, 5.74%; found:

C, 65.70%; H, 6.62%; N, 5.45%.

108: TLC (toluene–acetone 6 : 1) R_f= 0.30. [a]²⁰= +32.8^◦

=

170.14, 170.02, 169.39, 157.47 (C O), 138.60 − 126.54 (Ph),

D

(c = 0.25, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): d 7.36 − 7.12

(m, 40H, Ph), 5.27 (d, 1H, J_1,2= 3.6 Hz, H_1b), 4.96 − 4.59 (m,

14H, CH₂Ph, H_1c), 4.49 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.39 (2d,

2H, J = 12.0 Hz, CH₂Ph), 4.15 (br s, 1H, H_2a), 3.97 (dd, 1H,

J_3,4= J_4,5= 9.5 Hz, H_4a), 3.94 − 3.90 (m, 2H, H_6a+ H_2c), 3.87 −

3.82 (m, 2H, H_6b, OH), 3.79 (dd, 1H, J_2,3= J_3,4= 9.5 Hz, H_3b),

3.74 (dd, 1H, J_3,4= 2.5 Hz, J_2,3= 10.0 Hz, H_3c), 3.70 − 3.65 (m,

2H, H_4b, H_5c), 3.59 − 3.55 (m, 3H, H_1a+ H_5b+ OH), 3.45 (dd,

100.72 and 99.21 (C_1band C_1c), 82.35, 81.67, 80.75, 79.84,

78.65, 76.24, 75.86, 75.03, 74.22, 72.64, 72.37, 71.74, 71.07,

70.64, 69.72, 69.18, 66.84, 64.42, 60.42, 59.89, 20.61 and 20.53

(OCOCH₃). MALDI-TOF-MS: m/z 1080 (MNa⁺). Anal.

calcd. for C₅₄H₆₃N₃O₁₉·1/2H₂O: C, 60.78%; H, 6.05%; N,

3.94%; found: C, 60.75%; H, 6.47%; N, 3.72%.

2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl-(1→4)-2-azido-3-

O-benzyl-2-deoxy-a-D-glucopyranosyl)-(1→6)-3,4,5-tri-O-benzyl-

D-myo-inositol 106. Resin-bound acceptor 99 (157 mg,

24 lmol) was swollen in a solution of donor 105⁶⁵(113 mg,

0.16 mmol) in dry CH₂Cl₂(1.5 mL), shaken for 30 min and

cooled at −20 ^◦C. TMSOTf (100 lL of a 0.08 M solution

in dry CH₂Cl₂, 8 lmol) was added and the reaction mixture

was shaken at −20 ^◦C. After 4 h, the resin was washed with

CH₂Cl₂(5 × 4 mL), H₂O (2 × 4 mL), DMF (2 × 4 mL), THF

(2 × 4 mL) and CH₂Cl₂(2 × 4 mL). The glycosylation (105

(70 mg, 0.10 mmol), TMSOTf (50 lL of a 0.10 M solution

in dry CH₂Cl₂, 5 lmol)) and washing were repeated and the

resin was dried under a vacuum for 12 h. The reaction was

monitored by removing several mg of resin and cleaving from

resin using TFA–CH₂Cl₂–H₂O, followed by TLC analysis and

MALDI-TOF mass spectrometry.

1H, J_2,3= 2.5 Hz, J_3,4= 9.5 Hz, H_3a), 3.39 − 3.34 (m, 3H, H_6c

+

H_2b+ H_5a), 3.29 (dd, 1H, J_6,6

ꢀ

= 9.5 Hz, J_5,6

ꢀ

= 6.0 Hz, H_6c

ꢀ

), 3.14

(dd, 1H, J_5,6

ꢀ

= 5.0 Hz, J_6,6

ꢀ

= 10.5 Hz, H_6b

ꢀ

), 2.47 (s, 1H, OH_2a).

¹³C-NMR (125 MHz, CDCl₃): d 138.47 − 127.19 (Ph), 98.67

and 98.48 (C_1band C_1c), 81.74, 80.98, 80.07, 79.82, 79.04, 76.11,

75.87, 75.32, 74.93, 74.70, 74.59, 73.52, 73.37, 73.08, 72.74,

72.68, 70.70, 69.81, 69.44, 69.28, 68.33, 63.85. FAB-MS: m/z

(MNa⁺) calcd for C₇₄H₇₉O₁₅N₃Na 1272.5409; found 1272.5386.

109: TLC (toluene–acetone 6 : 1) R_f= 0.23. [a]²⁰= +16.5^◦

D

(c = 0.17, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): d 7.38 − 7.08

(m, 40H, Ph), 5.28 (d, 1H, J_1,2= 4.0 Hz, H_1b), 4.95 − 4.54 (m,

14H, CH₂Ph), 4.39 (2d, 2H, J = 11.5 Hz, CH₂Ph), 4.15 (m, 2H,

H_2a+ H_1c), 3.95 (dd, 1H, J_3,4= J_4,5= 9.5 Hz, H_4a), 3.89 (dd, 1H,

J_1,6= J_5,6= 9.5 Hz, H_6a), 3.84 − 3.77 (m, 2H), 3.76 − 3.69 (m,

4H), 3.58 − 3–31 (m, 9H), 2.58 (br s, 1H, OH), 2.54 (br s, 1H,

OH_2a). ¹³C-NMR (125 MHz, CDCl₃): d 138.22 − 127.34 (Ph),

103.56 (C_1c), 98.76 (C_1b), 82.26, 81.70, 80.91, 80.70, 80.10, 79.81,

78.80, 75.84, 75.06, 75.03, 74.68, 73.52, 73.41, 72.88, 72.76,

72.67, 71.84, 70.72, 69.39, 68.50, 67.69, 63.90. FAB-MS: m/z

(MNa⁺) calcd. for C₇₄H₇₉O₁₅N₃Na 1272.5409, found 1272.5381.

Resin-bound trisaccharide (157 mg, 24 lmol) was swollen in

CH₂Cl₂(2 mL) and shaken for 5 min. TFA (229 lL, 2.97 mmol)

and water (53 lL, 2.97 mmol) were added and the reaction

mixture was shaken for 6 h. at room temperature. The solution

and CH₂Cl₂washings (4 × 4 mL) were collected by ﬁltration.

This organic layer was washed with sat. NaHCO₃solution

(15 mL) and H₂O (15 mL), dried (MgSO₄) and concentrated

in vacuo. The puriﬁcation of the residue was carried out by ﬂash

chromatography (toluene–acetone 8 : 1 → 4 : 1 → 2 : 1 ) to

give 106 (12 mg, 40%). TLC: (toluene–acetone 2 : 1) R_f= 0.41.

[a]²_D⁰= +43.1^◦(c = 0.58, CHCl₃). ¹H-NMR (500 MHz, CDCl₃): d

7.38 − 7.11 (m, 40H, PhH), 5.61 (d, 1H, J_1,2= 4.0 Hz, H_1c), 5.34

(d, 1H, J_1,2= 3.5 Hz, H_1b), 4.99 − 4.47 (m, 15H, CH₂Ph), 4.38

(d, 1H, J = 12.0 Hz, CH₂Ph), 4.17 (br s, 1H, H_2a), 4.05 − 3.97

(m, 4H, H_4a+ H_3b+ H_4b+ H_2c), 3.94 (dd, 1H, J_1,6= J_5,6= 9.5 Hz,

Acknowledgements

We thank the Spanish Ministry of Science and Technology

(Grant BQU 2002-03734) and Rodaris Pharmaceuticals for

ﬁnancial support. N.-C. Reichardt thanks the European Union

for a fellowship (Grant HRN-CT-2000-00001/Glycotrain).

References

1 (a) For reviews see:D. R. Jones and I. Varela-Nieto, Int. J. Biochem.

Cell Biol., 1998, 30, 313–326; (b) M. C. Field, Glycobiology, 1997, 7,

161–168; (c) P. Stralfors, BioEssays, 1997, 19, 327–335.

2 J. M. Mato, K. L. Kelly, A. Abler and L. Jarret, J. Biol. Chem., 1987,

262, 2131–2137.

3 J. Larner, L. C. Huang, C. F. Schwarz, A. S. Oswald, T. Y. Shen, M.

Kinter, G. Z. Tang and K. Zeller, Biochem. Biophys. Res. Commun.,

1988, 151, 1416–1426.

4 J. Larner, J. D. Price, D. Heimark, L. Smith, G. Rule, T. Piccariello,

M. C. Fonteles, C. Pontes, D. Vale and L. Huang, J. Med. Chem.,

2003, 46, 3283–3291.

5 G. Romero, L. Lutrell, A. Mogol, K. Zeller, E. Hewlett and J. Larner,

Science, 1988, 240, 509–511.

H_6a), 3.86 (m, 2H, H_4c+ H_5b), 3.81 − 3.75 (m, 3H, H_3c+ H_5c

OH_1a), 3.58 (m, 1H, H_1a), 3.52 − 3.45 (m, 4H, H_3a+ H_2b+ H_6b

+

H_6c), 3.38 (dd, 1H, J_4,5= J_5,6= 9.5 Hz, H_5a), 3.33 (dd, 1H, J_6,6

ꢀ

=

12.5 Hz, J_5,6

9.5 Hz, H_6c

ꢀ

= 5.0 Hz, H_6b

ꢀ

), 3.26 (dd, 1H, J_5,6

ꢀ

= 4.5 Hz, J_6,6

ꢀ

=

ꢀ

), 2.76 (m, 1H, OH_6b), 2.54 (br s, 1H, OH_2a). ¹³C-

NMR (125 MHz, CDCl₃): d 138.64 − 126.96 (Ph), 99.14 (C_1b),

98.34 (C_1c), 81.66, 81.48, 81.03, 79.85, 79.22, 75.88, 75.40, 74.66,

74.56, 74.37, 74.09, 73.86, 73.54, 72.71, 71.73, 70.85, 69.38,

69.27, 64.87, 60.30. Anal. calcd. for C₇₄H₇₉N₃O₁₅·1/2H₂O: C,

70.57%; H, 6.40%; N, 3.34%; found: C, 70.46%; H, 6.60%; N,

3.09%.

6 M. Mart´ın-Lomas, N. Khiar, S. Garc´ıa, J.-L. Koessler, P. M. Nieto

and T. W. Rademacher, Chem. Eur. J., 2000, 6, 3608–3621.

7 (a) For reviews see:P. T. Englund, Annu. Rev. Biochem., 1993, 62, 121–

138; (b) M. J. McConville and M. A. J. Ferguson, Biochem. J., 1993,

294, 305–324; (c) V. L. Stevens, Biochem. J., 1995, 310, 361–370.

8 R. Plourde, M. D’Alarcao and A. R. Saltiel, J. Org. Chem., 1992, 37,

2606–2610.

9 A. Zapata, Y. Leo´n, J. M. Mato, I. Varela-Nieto, S. Penade´s and M.

Mart´ın-Lomas, Carbohydr. Res., 1994, 264, 21–31.

10 H. Dietrich, J. F. Espinosa, J. L. Chiara, J. Jime´nez-Barbero, Y. Leo´n,

I. Varela-Nieto, F. H. Cano, C. Foces-Foces and M. Mart´ın-Lomas,

Chem. Eur. J., 1999, 5, 320–336.

Tetraol 2-azido-3-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1→

6)-3,4,5-tri-O-benzyl-D-myo-inositol 107 and 6-O regioisomers

2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl-(1→6)-2-azido-3-O-

benzyl-2-deoxy-a-D-glucopyranosyl-(1→6)-3,4,5-tri-O-benzyl-D-

myo-inositol 108 and 2,3,4,6-tetra-O-benzyl-b-D-galactopy-

ranosyl-(1→6)-2-azido-3-O-benzyl-2-deoxy-a-D-glucopyranosyl-

(1→6)-3,4,5-tri-O-benzyl-D-myo-inositol 109. These com-

pounds were also isolated from the reaction mixture due

to incompleted coupling and formation of 4-O resin-bound

acceptors during the immobilization of diol 56, respectively.

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

7 8 5

View Article Online

11 W. Frick, A. Bauer, J. Bauer, S. Wied and G. Mu¨ller, Biochemistry,

1998, 37, 13421–13436.

12 J. Larner, G. Romero, A. S. Kennington, K. Lilley, E. Kilgour, C.

Zhang, D. Heimark, C. Gamez, D. B. Houston, L. C. Huang, in The

Biology of Signal Transduction, ed. Y. Nishizuka, Raven Press, New

York, 1990, pp. 290–294.

37 Sybyl^ꢀR6.6, Tripos Inc., 1699 South Hanley Rd., St Louis, Missouri,

63144, USA. All molecules were minimised using Tripos Force Field.

Structure optimization was carried out for 500 steps using TNG

method until gradient was less than 0.01. Charges were assigned

by MOPAC program within Sybyl, using AM1 without geometry

optimization.

13 P. Cohen, Nature Rev. Drug Discovery, 2002, 1, 309–315.

14 (a) For reviews see:S. S. Taylor, D. R. Knighton, J. Zheng, L. F.

Ten Eyk and J. M. Sowadski, Annu. Rev. Cell. Biol., 1992, 8, 429–

462; (b) S. S. Taylor, J. A. Buechler and W. Yonemoto, Annu. Rev.

Biochem., 1990, 59, 971–1005.

15 S. K. Hanks, A. M. Quinn and T. Hunter, Science, 1988, 241, 42–52.

16 C. Breitenlechner, M. Gassel, H. Hidaka, V. Kinzel, R. Huber, R. A.

Engh and D. Bossemeyer, Structure, 2003, 11, 1595–1607.

17 B. Shoichet, S. L. McGovern, B. Wei and J. J. Irwin, Curr. Opin.

Chem. Biol., 2002, 6, 439–446.

38 Program GRID 18, Molecular Discovery Limited, West Way House,

Elms Parade, Oxford, UK. Calculation was carried out using

1ATP.pdb as a model for the active site after removal of every

˚

substructure but the enzyme. A grid of 61, 76 and 56 A was built

˚

with a 1.0 A. spacing. A phosphate anion probe was used. A picture

of the most favourable regions is shown at supporting information.

39 N.-C. Reichardt and M. Mart´ın-Lomas, Angew. Chem., Int. Ed.,

2003, 42, 4674–4677.

40 B. La Ferla, L. Lay, M. Guerrini, L. Poletti, L. Panza and G. Russo,

Tetrahedron, 1999, 55, 9867–9888.

18 D. R. Knighton, J. H. Zheng, L. F. Ten Eyck, V. Ashford, N.-H.

Xuong, S. S. Taylor and J. M. Sowadski, Science, 1991, 253, 407–

414.

19 D. R. Knighton, J. H. Zheng, L. F. Ten Eyck, V. Ashford, N.-H.

Xuong, S. S. Taylor and J. M. Sowadski, Science, 1991, 253, 414–

420.

20 D. Bossemeyer, R. A. Engh, V. Kinzel, H. Postingl and R. Huber,

EMBO J., 1993, 12, 849–859.

21 R. Karlson, J. Zhen, N.-H. Xuong, S. S. Taylor and J. M. Sowadski,

Acta Crytallogr., Sect. D: Biol. Crstallogr., 1993, 49, 381–388.

22 D. R. Knighton, S. M. Bell, J. Zheng, L. F. Ten Eyck, F. Xuong,

S. S. Taylor and J. M. Sowadski, Acta Crystallogr., Sect. D: Biol.

Crytallogr., 1993, 49, 362–365.

23 J. Zheng, D. R. Knighton, L. F. Ten Eyck, R. Karlsson, N.-H. Xuong,

S. S. Taylor and J. M. Sowadski, Biochemistry, 1993, 32, 2154–2161.

24 J. Zheng, D. R. Knighton, N.-H. Xuong, S. S. Taylor, J. M. Sowadski

and L. F. Ten Eyck, Protein Sci., 1993, 2, 1559–1573.

25 J. Madhusudan, E. A. Trafney, N.-H. Xuong, J. A. Adams, L. F. Ten

Eyck, S. S. Taylor and J. M. Sowadski, Protein Sci., 1994, 3, 176–183.

26 R. A. Engh, A. Girod, V. Kinzel, R. Huber and D. Bossemeyer,

J. Biol. Chem., 1996, 271, 26157–26164.

27 J. M. Sowadski, C. A. Ellis and J. Madhusudan, J. Bioenerg.

Biomembr., 1996, 28, 7–12.

28 N. Narajana, S. Cox, S. Shaltiel, S. S. Taylor and N.-H. Xuong,

Biochemistry, 1997, 36, 4438–4448.

41 C. Murakata and T. Ogawa, Carbohydr. Res., 1992, 234, 75–91.

42 A. Toepfer and R. R. Schmidt, J. Carbohydr. Chem., 1993, 12, 809–

822.

43 M. M. Palme and A. Vasella, Helv. Chim. Acta, 1995, 78, 959–969.

44 J.-L. de Paz, R. Ojeda, N.-C. Reichardt and M. Mart´ın-Lomas,

Eur. J. Org. Chem., 2003, 3308–3324.

¨

45 J. Lindberg, L. Ohberg, P. J. Garegg and P. Konradsson, Tetrahedron,

2002, 58, 1387–1398.

46 P. J. Garegg, H. Hultberg and S. Wallin, Carbohydr. Res., 1982, 108,

97–101.

47 M. Ek, P. J. Garegg, H. Hultberg and S. Oscarson, J. Carbohydr.

Chem., 1983, 2, 305–311.

48 M. Oikawa, W. C. Liu, Y. Nakai, S. Koshida, K. Fukase and S.

Kusumoto, Synlett, 1996, 1179–1180.

49 A. Liptak, J. Imre, J. Harangi and P. Nanasi, Tetrahedron, 1982, 38,

3721–3727.

50 L. Jiang and T. H. Chan, Tetrahedron Lett., 1998, 39, 355–358.

51 Y. Oikawa, T. Yoshioka and O. Yomenitsu, Tetrahedron Lett., 1982,

23, 885–888.

52 K. Y. Yu and B. Fraser-Reid, Tetrahedron Lett., 1988, 29, 979–982.

53 J. Xue, N. Shao and Z. Guo, J. Org. Chem., 2003, 68, 4020–4029.

54 K. Fukase, Y. Nakai, K. Egusa, J. A. Porco and S. Kusumoto, Synlett,

1999, 7, 1074–1078.

55 M. MacCoss and D. J. Kameron, Carbohydr. Res., 1978, 60, 206–

209.

29 N. Narajana, S. Cox, N.-H. Xuong, L. F. Ten Eyck and S. S. Taylor,

Structure, 1997, 5, 921–935.

30 L. Prade, R. A. Engh, A. Girod, V. Kinzel, R. Huber and D.

Bossemeyer, Structure, 1997, 5, 1637–1637.

31 L. M. Toledo and N. B. Lydon, Structure, 1997, 5, 1551–1556.

32 P. M. Taxler, Expert Opin. Ther. Pat., 1997, 7, 571–588.

33 D. S. Lawrence and J. K. Niu, Pharmacol. Ther., 1988, 77, 81–114.

34 M. Mart´ın-Lomas, P. M. Nieto, N. Khiar, S. Garc´ıa, M. Flores-

Mosquera, E. Poirot, J. Angulo and J. L. Mun˜oz, Tetrahedron:

Asymmetry, 2000, 11, 37–51.

35 AutoDock 3.0.G, M. Morris, D. S. Goodsell, R. S. Halliday, R.

Huey, W. E. Hart, R. K. Belew and A. J. Olson, J. Comp. Chem.,

1998, 19, 1639–1662. For docking calculation details see supporting

information.

56 S. Hanessian and F. Xie, Tetrahedron Lett., 1998, 39, 733–736.

57 J.-H. van Boom and P. M. J. Burgers, Tetrahedron Lett., 1976, 52,

4875–4878.

58 H. Shimizu, Y. Ito, O. Kanie and T. Ogawa, Biorg. Med. Chem. Lett.,

1996, 5, 2841–2846.

59 X. Wu, M. Grathwohl and R. R. Scmidt, Angew. Chem., Int. Ed.,

2002, 41, 4489–4493.

60 L. Yan and D. Khane, Synlett, 1995, 523–524.

61 M. F. Hawthorne and D. J. Cram, J. Am. Chem. Soc., 1954, 76,

3451–3453.

62 K. Fukase, S. Hase, T. Ikenaka and S. Kusumoto, Bull. Chem. Soc.

Jpn., 1992, 65, 436–445.

63 E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Anal.

Biochem., 1970, 34, 595.

36 N. Narayana, T. C. Diller, K. Koide, M. E. Bunnage, K. C. Nicolaou,

L. L. Brunton, N.-H. Xuong, L. F. Ten Eyck and S. S. Taylor,

Biochemistry, 1999, 38, 2367–2376.

64 N.-C. Reichardt, PhD Thesis, University of Seville, 2004.

65 R. R. Schmidt and W. Kinzy, Adv. Carbohydr. Chem. Biochem., 1994,

50, 21–123.

7 8 6

O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 6 4 – 7 8 6

Article Doi

Design and synthesis of inositolphosphoglycan putative insulin mediators

DOI: 10.1039/b418041k

Source and publish data:

Authors:

Article abstract of DOI:10.1039/b418041k

Full text of DOI:10.1039/b418041k

Products guided by the article

R&D Labs maybe for 850428-42-1

Relevant to this article

Hot Product