Synthetic studies towards a new scaffold, spirobicycloimidazoline

Available online at www.sciencedirect.com

Carbohydrate Research 343 (2008) 1624–1635

Synthetic studies towards a new scaﬀold, spirobicycloimidazoline

Tatsuto Nakahara,^aNaoki Okamoto,^bKatsuhiko Suzuki^band Osamu Kanie^a,b,

*

^aTokyo Institute of Technology, Graduate School of Bioscience and Biotechnology,

4529 Nagatsuta-cho, Midori-ku, Yokohama 226-0018, Japan

^bMitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan

Received 17 March 2008; received in revised form 14 April 2008; accepted 17 April 2008

Available online 25 April 2008

Abstract—A new scaﬀold consisting of a carbocycle and a substituted imidazoline in an orthogonal arrangement was synthesized as

a potential speciﬁc inhibitor of glycosidases. The spirobicycloimidazoline, (5R,6R,7R,8R)-8-(hydroxymethyl)-2-phenyl-1,3-diaza-

spiro[4.4]non-1-ene-6,7-diol, was synthesized from methyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-a/b-^D-gluco-6-enopyranoside via

(1R,2S,3S,4R,5S)-3,4-bis(benzyloxy)-2-(4-methoxybenzyloxy)-5-vinyl-cyclopentanol. The ring contraction of the 6-enopyranoside

in the presence of zirconocene equivalent (‘Cp₂Zr’) reagent gave exclusively the corresponding cyclopentanol without cleavage of

the PMB protecting group. In the course of the study, a new a-mannosidase inhibitor, (1R,2R,3R,5R)-5-amino-3-hydroxy-

methyl-cyclopentane-1,2-diol, was also discovered.

Keywords: Glycosidase inhibitors; Spiro compounds; Imidazoline; Carbocyclic compounds

1. Introduction

result of a strong charge interaction between the ammo-

nium group and the carboxylate group in the active site

of glycosidase. A combinatorial chemistry approach is

expected to result in further improvement of activ-

ity.^14,15Cyclopentitols having a sugar-like carbocycle

are another class of inhibitors that deserve attention.

Mannostatin A, isolated from Streptoverticillium verti-

cillus, is a speciﬁc and potent inhibitor of Golgi a-man-

nosidase II.^16,17The structure of Mannostatin A does

not resemble the three-dimensional structure of a man-

nopyranoside, the substrate of the mannosidase, and

the inhibitory mechanism is thus of great interest.

Bicyclic cyclopentitol-related compounds have also

been studied as potential inhibitors of various glycosi-

dases. Trehazoline and allosamidin are known to be

good inhibitors of trehalase and chitinase, respec-

tively.^18,19Despite the accumulated information regard-

ing inhibitory activities, the prediction of enzyme

speciﬁcity in the inhibition by ﬁve-membered ring com-

pounds is, in general, diﬃcult because of the conforma-

tional freedom of these rings and the resulting

unpredictable three-dimensional placement of func-

tional groups.

The glycosidases, which are related to the control of var-

ious biological phenomena, are one of the most impor-

tant families of carbohydrate-related enzymes.^1,2

Inhibitors of glycosidases are useful tools when investi-

gating biochemical processes,^3–5in enzyme puriﬁcation

as aﬃnity ligands,^6,7and also as drug candidates.^8–11

Thus, many compounds, either isolated from natural

sources or chemically synthesized, have been reported

as inhibitors of glycosidases. Recent advances in three-

dimensional analysis based on the crystal structures of

glycosidases have also been used to design inhibitors

of these enzymes. A majority of compounds in the class

carry a cationic moiety in the structure that mimics the

oxocarbenium ion species in the transition state.

One well-known family of glycosidase inhibitors with

a ﬁve-membered ring is hydroxylated pyrrolidines, rep-

resented by DMDP and DAB-1 (Chart 1).^12,13It is be-

lieved that the powerful inhibitory activities are the

*

Corresponding author. E-mail: kokanee@mitils.jp

doi:10.1016/j.carres.2008.04.025

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

1625

OH

O

SMe

HO

H

HO

N

R

HO

NH₂

HO

OH

HO

HN

O

OH

N

OH

HO

OH

R = CH₂OH DMDP

OH

Mannostatin A

HO

R = H

DAB-1

Trehazolin

HO

O

NH

HN

OH

O

OH

N

O

NHAc

O

NH

Ph

HO

N

O

HO

O

NHAc

HO

OH

HO

OH

NMe₂

1

Hydantocidin

Allosamidin

Chart 1.

Hydantocidin, a naturally occurring spiroribofura-

nose derivative, was reported to show strong herbicidal

activity with no toxicity to microorganisms or ani-

mals.^20,21An analogue of hydantocidin, in which the

endocyclic oxygen of the sugar moiety has been replaced

by a methylene group, is known.^22–24This type of com-

pound, having a fused ring system in an orthogonal

arrangement, might provide valuable information and

hopefully serve as a source of potent glycosidase inhib-

itors. This consideration was based on fact that the nat-

ural product salacinol with a ‘semi-’ spiro system as the

result of intramolecular ion pairing, showed potent

inhibitory activity against a-glucosidase.²⁵However,

cancellation of charge in the molecule is considered to

be unfavourable for glycosidase inhibition. We thus de-

signed a new scaﬀold consisting of a carbocycle and a

substituted imidazoline, where the hydroxylated carbo-

cycle ring mimics the glycon moiety, the imidazoline

moiety provides hydrogen-bonding capability in the

catalytic site of the enzyme, and an aglycon moiety

can be incorporated at the amidino-group. We report

here the synthesis of spirobicycloimidazoline (1), which

is expected to become a compound in a novel class of

compounds. In the course of these studies a new a-man-

nosidase inhibitor, (1R,2R,3R,5R)-5-amino-3-hydroxy-

methyl-cyclopentane-1,2-diol, was also discovered.

the key steps were a ring contraction of an enopyrano-

side (i) to provide a cyclopentitol derivative (ii), subse-

quent conversion of ii into a ketone (iii) and ﬁnally

introduction of a C–C bond into iii leading eventually

to iv. Diamine iv is a precursor to imidazoline 1 (Scheme

1).

A suitably protected 6-enopyranoside (3), which was

to be subjected to the ring contraction, was synthesized

from ^D-glucose (Scheme 2). The 2-OH of compound 2,

which is transformed to the spiro-centre of 1, should

be distinct from other hydroxyl groups, so we chose p-

methoxybenzyl (PMB) ether as the protecting group.

Compound 2 was obtained in high yield by conventional

protection of the hydroxyl groups, and the spectral data

matched the reported data.^26,27Oxidation of the

hydroxymethyl group of 2 gave the corresponding

aldehyde, which was converted into 3 by a Wittig

reaction.

It had been reported that the ring contraction of 6-

enopyranoside was promoted by a zirconocene equiva-

lent (‘Cp₂Zr’) reagent.²⁸Although the reaction generally

required a Lewis acid to assist with elimination of the

aglycone group when methyl glycosides were used as

substrates, Ito et al. reported that this reaction some-

times proceeded in the absence of a Lewis acid.²⁹How-

ever, the details of the reaction conditions were not

revealed in the paper, and it was also anticipated that

in our case that the use of Lewis acid could prove trou-

blesome due to cleavage of the PMB group. After some

experimentation, we found suitable reaction conditions.

In the absence of a Lewis acid, the ring contraction of

the oleﬁn–Zr complex, which was generated in situ at

ꢀ78 °C, proceeded smoothly by rapidly increasing the

temperature. This suggests that multiple intermediates

may exist, although it does not indicate that all of them

become precursors of carbocyclic compound (4).

Although we could not determine the structure of these

species, compounds 3a and 3b were eﬃciently converted

into compound 4 accompanied by small amounts of

2. Results and discussion

2.1. Synthesis

Our target bicyclospiro compound, imidazoline (1), con-

sists of a ﬁve-membered carbocycle and an imidazoline.

In this study, we selected as a target backbone 5-amino-

3-hydroxymethyl-cyclopentane-1,2-diol, which might

provide new information when searching for and/or

designing new inhibitors of glycosidases. In the con-

struction of this scaﬀold we chose an approach in which

1626

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

OH

HO

PO

NH

2

O

OP'

PO

1

OMe

NH

2

OH

HO

OP

PO

OP

i

ii

iii

iv

P, P' = Protective group

Scheme 1.

6

OH

4

3

BnO

b)

a)

1

O

BnO

O

BnO

OH

D-Glucose

PMBO

OMe

e)

PMBO

OMe

OPMB

2

3

4

OR

OTr

TrO

BnO

c)

f)

S

BnO

OH

OPMB

O

N

OR

OPMB

5 R = H

6 R = Tr

8 R = PMB

9 R = H

d)

7

g)

Scheme 2. Reagents and conditions: (a) (1) (COCl)₂, Me₂SO, i-Pr₂NEt, CH₂Cl₂, ꢀ78 °C; (2) Ph₃PCH₃I, t-BuOK, toluene; 7:3, a/b, 64%; (b)

Cp₂ZrCl₂, n-BuLi, THF, ꢀ78 then 25 °C, 55% from 3-a, 50% from 3-b; (c) O₃then NaBH₄, MeOH, CH₂Cl₂, 76%; (d) TrCl, pyridine, 83%; (e)

(Im)₂C@S, DMAP, THF, 96%; (f) nBu₃SnH, AIBN, toluene, 94%; (g) DDQ, CH₂Cl₂, H₂O, 91%.

their respective diastereomers (65% yield, 85:12:3, 4:

1S,2S isomer:1S5R isomer). Under these conditions, it

was found that the PMB group was not at all aﬀected.

Compound 4 was converted into diol (5) by ozonoly-

sis and successive reduction using NaBH₄(76% yield).

After tritylation of the primary alcohol, the secondary

alcohol of compound 6 was deoxygenated by radical

reduction to aﬀord cyclopentitol (8). Compound 9,

obtained by cleavage of the PMB group in 8 (91%), is

the common precursor for the synthesis of amino-cyclo-

pentitols (13 and 19) (Scheme 3).

The azido group in compound 11 was introduced by

substitution of the corresponding mesylate (10, obtained

by mesylation of 9) with NaN₃in an S_N2 fashion (95%).

Deprotection of 11 was accomplished by hydrogenolysis

under acidic conditions to aﬀord 13, whose amino group

was protected as t-butyl carbamate (12, 86%) to facili-

tate puriﬁcation of some minor alkylation by-products.

Upon treatment with triﬂuoroacetic acid (TFA), com-

pound 12 was converted into known 13³⁰as a pure

TFA salt, in quantitative yield.

For the synthesis of 1, construction of a quaternary

carbon including the amino-functionality on the

cyclopentitol frame was required. This step was achieved

by introducing an azide via and a-chloroepoxide inter-

mediate (Scheme 4).^31,32Nucleophilic addition of

dichloromethyllithium to ketone (14) generated two iso-

meric branched-cyclopentitols (20-R and 20-S) in 67%

and 13% yield, respectively. An NOE was observed be-

tween the dichloromethyl protons and H-2, which sup-

ported an R conﬁguration of 20-R. The structure of

the isomeric 20-S was also supported by an NOE be-

tween H-5 and the dichloromethyl protons. The diaste-

reoselectivity of this reaction was similar to the

hydride reduction described for compound 15. The ma-

jor isomer (20-R) was converted into a-azido aldehyde

(21) with stereochemical inversion at the quaternary car-

bon (79%).

The stereochemistry of 21 was supported by the NOE

experiments of diamine (23), the synthesis of which fol-

lows. Introduction of the second nitrogen atom of the

imidazoline ring was achieved by reductive amination

of 21 using BnNH₂and NaBH₃CN (86%).³³Diamine

(23), obtained from 22 by hydrogenolysis under acidic

conditions, was easily puriﬁed by protection as an N-

Boc derivative, which was quantitatively converted back

into diamine (23) as the TFA salt. The NOE shown in

the structure suggested the relative stereochemistry of

23. Condensation of 23 with benzaldehyde using N-bro-

mosuccinimide (NBS) gave imidazoline (1; 33% after

Alternatively, the hydroxyl group in 9 was success-

fully epimerized by an oxidation–reduction procedure

via ketone (14) to aﬀord 15 (89%) together with 9

(10%). The diastereoselectivity of the reduction was con-

trolled by steric eﬀects, with the hydride attacking the

ketone in 14 from the less-hindered side. Compound

19 was obtained from 15 by way of azido compound

17 in the same manner as described for 13.

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

1627

OTr

OH

OTr

BnO

2

1

b)

HO

a)

c)

BnO

4

9

BnO

N

NHR

3

OMs

11

12 R = Boc

13 R = H

10

e)

d)

OH

OTr

HO

c)

3

BnO

b)

f)

1

NHR

N

O

OH

3

14

15 R = H

17

18 R = Boc

19 R = H

a)

d)

16 R = Ms

Scheme 3. Reagents and conditions: (a) MsCl, DMAP, pyridine; (b) NaN₃, DMF, 90% for 11, 92% for 17; (c) (1) H₂, Pd(OH)₂–C, HCl, MeOH,

THF; (2) (Boc)₂O, Et₃N, H₂O, MeOH, 86% for 12, 84% for 18; (d) TFA, CH₂Cl₂, quant.; (e) Dess–Martin periodinane, CH₂Cl₂, 91%; (f) NaBH₄,

MeOH, THF, 89%.

OTr

BnO

a)

b)

BnO

5

14

2

H

CHCl

2

Cl

OTr

HO

O

BnO

NOE

5.3%

H

20-R

OH

Cl HC

2

NOE

7.9%

20-

S

OH

OTr

HO

H

8

OTr

HO

1

d)

e)

c)

BnO

6

4

H

HO

H

N

NH

3'

2

N

NH

BnO

OHC

N

3

2

3

NOE

2.2%

NH

Ph

2

NHBn

H

HMBC

correlation

21

22

23

1

Scheme 4. Reagents and conditions: (a) i-Pr₂NH, n-BuLi, CH₂Cl₂, THF, 20-R 67%, 20-S 13%; (b) NaN₃, 15-crown-5, Me₂SO, 79%; (c) BnNH,

NaBH₃CN, AcOH, TFA, 86%; (d) (1) H₂, Pd(OH)₂–C, HCl, MeOH, THF; (2) (Boc)₂O, Et₃N, MeOH, H₂O; (3) TFA, CH₂Cl₂, 64%; (e) PhCHO,

NBS, Et₃N, MeOH, 33%.

puriﬁcation using HPLC) whose imidazoline structure

was supported by a correlation between C-2 and H-4

in the HMBC spectrum.³⁴

oryzae and a-mannosidase (EC 3.2.1.24) from jack bean.

It was observed that 13 and 19 had low activities against

a-glucosidase (IC₅₀= 0.16–0.18 mM), high activity

against a-mannosidase (13: IC₅₀= 1.55 lM, K_i=

0.85 lM; 19: IC₅₀= 17.5 lM, K_i= 16.0 lM), and no

activity against other glycosidases. Although 13 does

not have a cis-diol moiety, of which the presence has

been considered to be essential for a-mannosidase inhi-

bition, the inhibitory activity was found to be as power-

ful as swainsonine.³⁵This might suggest that the amino

group of 13 mimics a hydroxyl group, possibly 2-OH of

mannose. However, the observed inhibitory activity of

19 against a-mannosidase seems to be a contradiction

to this hypothesis because all functional groups are pre-

2.2. Glycosidase inhibition studies

The inhibitory activities of compounds 13, 19 and 1

against various glycosidases, namely, a/b-glucosidases,

b-N-acetylglucosaminidase, a-mannosidase and b-galac-

tosidase, were investigated. The sources of enzymes and

substrates are as follows: a-glucosidase (EC 3.2.1.20)

from yeast, b-glucosidase (EC 3.2.1.21) from almond,

b-N-acetylglucosaminidase (EC 3.2.1.52) from jack

beans, b-galactosidase (EC 3.2.1.23) from Aspergillus

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T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

sented in trans-orientation. Thus, detailed analysis and

further investigation are required to determine the inhib-

itory mechanism. Only low activities against a/b-gluco-

sidase (17% inhibition at 0.3 mM against a-glucosidase

and 17% inhibition at 0.5 mM against b-glucosidase)

were observed for imidazoline (1). We speculate that

the reasons for the lower activity of 1 compared with

13 and 19 are either the steric hindrance at an aglycon

site in the enzymes or a change of conformation of the

carbocycle resulting from the fused imidazoline.

vent peak (CDCl₃; 77.0 ppm, CD₃OD; 49.0 ppm or

C₆D₆; 128.0 ppm) as the internal standard.

3.2. Methyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-a/b-^D-

gluco-6-enopyranoside (3)

A solution of Me₂SO (0.73 mL, 10.3 mmol) in CH₂Cl₂

(4.40 mL) was added to a solution of (COCl)₂(2 M in

CH₂Cl₂, 2.57 mL, 5.14 mmol) under a nitrogen atmo-

sphere at ꢀ78 °C. To the mixture was added after

50 min a solution of 2 (1.27 g, 2.57 mmol, 3:1, a/b) in

CH₂Cl₂(30.0 mL), and then after stirring for 1 h a solu-

tion of N,N⁰-diisopropylethylamine (2.68 mL, 15.4

mmol) at ꢀ65 °C. The mixture was poured into H₂O,

and the organic layer was washed successively with 2%

aq HCl, H₂O and brine, dried over Na₂SO₄, concen-

trated in vacuo and co-evaporated with toluene three

times. To the toluene (8.40 mL) solution of the residue

(1.27 g) was added Ph₃P@CH₂(0.3 M in toluene,

17.1 mL, 51.4 mmol) over 0.5 h at ꢀ10 °C. After being

stirred for 2 h at rt, to the mixture was added large ex-

cess of acetone. The mixture was concentrated, and

the residue was puriﬁed by silica gel chromatography

(6:1 n-hexane–EtOAc) to aﬀord 3 (810 mg, 64%, 3:1,

2.3. Conclusion

A novel spirobicycloimidazoline expected to have potent

inhibitory activity against glucosidase was synthesized.

Although the compound was found to have only weak

inhibitory activity, it is hoped that the related com-

pounds may be more potent. However, these studies

did identify an amino-cyclopentitol that was a good

inhibitor of a-mannosidase. In the synthesis of the tar-

get, a key ring-contraction reaction in the presence of

zirconocene was achieved without using a Lewis acid,

which allowed us to use an acid labile protecting group

such as a PMB ether. Expansion of the choice of pro-

tecting groups is advantageous for the synthesis of addi-

tional cyclopentitol derivatives and analogues.

29

a/b):3a; R_f= 0.57 (2:1 n-hexane–EtOAc); ½aꢂ_Dꢀ11.6

(c 0.5, CHCl₃); ¹H NMR (CDCl₃): d 7.37–7.26 (m,

12H, aromatic), 6.86 (d, 2H, J = 8.6 Hz, p-OMe-Ph),

5.91 (ddd, 1H, J_5,6= 6.5 Hz, J_6,7-cis= 10.5 Hz,

J_6,7-trans= 17.2 Hz, H-6), 5.42 (d, 1H, H-7-cis), 5.26 (d,

1H, H-7-trans), 4.96 (d, 1H, J = 10.8 Hz, PhCH₂),

4.83 (d, 1H, J = 10.8 Hz, PhCH₂), 4.79 (d, 1H,

J = 10.7 Hz, PhCH₂), 4.75 (d, 1H, J = 11.8 Hz, PhCH₂),

4.62 (d, 2H, J = 11.3 Hz, PhCH₂), 4.55 (d, 1H,

J_1,2= 3.2 Hz, H-1), 4.09 (dd, 1H, J_4,5= 9.7 Hz, H-5),

3.98 (t, 1H, J_2,3= J_3,4= 9.3 Hz, H-3), 3.81 (s, 3H,

Ph–OCH₃), 3.52 (dd, 1H, H-2), 3.38 (s, 3H, OCH₃),

3.26 (t, 1H, H-4); ¹³C NMR (CDCl₃): d 138.6, 138.2

(aromatic C), 135.3 (C-6), 130.3–127.6 (aromatic C),

118.2 (C-7), 113.9 (aromatic C), 98.2 (C-1), 82.3 (C-4),

81.7 (C-3), 79.5 (C-2), 75.9, 75.2, 73.1 (PhCH₂ꢃ3),

55.3 (Ph–OCH₃), 55.2 (OCH₃). ESIMS m/z calcd for

[C₃₀H₃₄O₆+Na]⁺: 513.2248. Found: 513.2248: 3b;

R_f= 0.66 (2:1, n-hexane–EtOAc); mp 93.0–95.0 °C;

3. Experimental

3.1. General methods

Thin-layer chromatography was performed on Merck

Art. 5715, Kieselgel 60 F₂₅₄/0.25 mm thickness plates.

Visualization was accomplished with UV light and 1%

Ce(SO₄)₂–1.5% (NH₄)₆MoO₂₄ꢁ4H₂O–10% H₂SO₄solu-

tion or 0.5% ninhydrin n-BuOH solution followed by

heating. Silica gel column chromatography was con-

ducted on Kieselgel 60 (Merck Art. 7734) or Silica Gel

60 Spherical (Kanto Chemical). The HPLC was per-

formed with Waters-LCMS system (Column: Unizon

C-18; Mobile phase: 60% MeCN, 40% H₂O; Flow rate:

4.0 mL/min). Mass spectra were obtained on SHIMA-

DZU-LCQIT-TOF (Shimadzu) coupled with an electro-

spray interface. Melting points were measured with

Yanaco MP-S3 micro melting point apparatus. Optical

rotations were measured in a 1.0 dm tube with a Horiba

SEPA-200 polarimeter. ¹H NMR (500 MHz) spectra

were recorded with an AVANCE 500 spectrometer

(Bruker Biospin Inc.) in deuterated solvent using Me₄Si

(0.00 ppm) or the solvent peak (HDO; 4.65 ppm or

CD₃OH; 4.87 ppm) as the internal standard. ¹³C

NMR (125 MHz) spectra were recorded with an

AVANCE 500 spectrometer (Bruker Biospin Inc.) in

deuterated solvent using Me₄Si (0.00 ppm) or the sol-

23

1

½aꢂ_Dꢀ2.4 (c 0.8, CHCl₃); H NMR (CDCl₃): d 7.34–

7.26 (m, 12H, aromatic), 6.84 (d, 2H, J = 8.7 Hz, p-

MeO-Ph), 5.96 (ddd, 1H, J_5,6= 6.0 Hz, J_6,7-cis

=

10.6 Hz, J_6,7-trans= 17.3 Hz, H-6), 5.45 (d, 1H, H-7-

cis), 5.28 (d, 1H, H-7-trans), 4.89 (d, 1H, J = 10.9 Hz,

PhCH₂), 4.83 (d, 1H, J = 10.6 Hz, PhCH₂), 4.79 (d,

1H, J = 10.9 Hz, PhCH₂), 4.76 (d, 1H, J = 10.6 Hz,

PhCH₂), 4.64 (d, 1H, J = 10.6 Hz, PhCH₂), 4.61 (d,

1H, J = 10.6 Hz, PhCH₂), 4.34 (d, 1H, J_1,2= 7.8 Hz,

H-1), 3.79 (s, 3H, Ph–OCH₃), 3.77 (dd, 1H,

J_4,5= 9.5 Hz, H-5), 3.62 (t, 1H, J_2,3= J_3,4= 9.1 Hz,

H-3), 3.59 (s, 3H, OCH₃), 3.41 (dd, 1H, H-2), 3.30 (t,

1H, H-4); ¹³C NMR (CDCl₃): d 138.6, 138.0 (aromatic

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

1629

C), 134.6 (C-6), 130.6–127.6 (aromatic C), 117.9 (C-7),

113.8 (aromatic C), 104.5 (C-1), 84.2 (C-3), 82.0 (C-2),

82.0 (C-4), 75.8 (PhCH₂), 75.6 (C-5), 75.2, 74.5

(PhCH₂ꢃ 2), 57.2 (Ph–OCH₃), 55.3 (OCH₃). ESIMS

m/z calcd for [C₃₀H₃₄O₆+Na]^+:: 513.2248. Found:

513.2255.

(PhCH₂ꢃ 3), 55.3 (Ph–OCH₃), 50.9 (C-5). ESIMS m/z

calcd for [C₂₉H₃₂O₅+Na]⁺: 483.2142. Found:

483.2146. 1S,5R Isomer; R_f= 0.31 (3:1, n-hexane–

1

EtOAc); H NMR (CDCl₃): d 7.36–7.28 (m, 12H, aro-

matic), 6.88 (d, 2H, J = 8.6 Hz, p-MeO-Ph), 6.14 (ddd,

1H, J_6,5= 9.3 Hz, J_6,7-cis= 10.2 Hz, J_6,7-trans= 17.2 Hz,

H-6), 5.25 (br d, 1H, H-7-cis), 5.22 (br d, 1H, H-7-trans),

4.62, 4.52 (each d, 2H, J = 11.4 Hz, PhCH₂), 4.51 (s, 4H,

PhCH₂and p-MeO–PhCH₂), 4.16–4.11 (m, 2H, H-1, H-

3), 3.86 (dd, 1H, J_3,4= 2.7 Hz, J_4,5= 5.9 Hz, H-4), 3.83–

3.79 (m, 1H, H-2), 3.81 (s, 3H, Ph–OCH₃), 2.82 (ddd,

1H, J_1,5= 5.3 Hz, H-5), 2.63 (d, 1H, J = 8.6 Hz, OH);

¹³C NMR (CDCl₃): d 159.33, 137.93, 137.73, 133.16,

129.85, 129.61, 128.38, 128.34, 127.79, 127.77, 127.73,

127.71, 118.48, 113.82, 88.30 (C-3), 84.89 (C-2),

84.33 (C-4), 73.44 (C-1), 71.80, 71.61, 71.52, 55.25,

49.81 (C-5).

3.3. (1R,2S,3S,4R,5S)-3,4-Bis(benzyloxy)-2-(4-methoxy-

benzyloxy)-5-vinyl-cyclopentanol (4)

To a THF (20.0 mL) solution of bis(cyclopentadi-

enyl)zirconium dichloride (1.87 g, 6.39 mmol) was

added n-BuLi (2 M in n-hexane, 8.50 mL, 13.6 mmol)

at ꢀ78 °C. After being stirred for 1 h, to the solution

was added 3a (2.09 g, 4.26 mmol) in a cold (ꢀ78 °C)

THF (25.0 mL) solution over 20 min. After the mixture

was stirred for 15 min at ꢀ78 °C, the reaction tempera-

ture was quickly elevated to rt. The reaction was

stopped by addition of 5% aq NaOH at 0 °C, and insol-

uble materials were ﬁltered through a Celite pad. The

organic layer of ﬁltrate was washed with brine, followed

by extraction with CH₂Cl₂, dried over Na₂SO₄and con-

centrated in vacuo. The residue was puriﬁed by silica gel

chromatography (3:1 n-hexane–EtOAc) to aﬀord 4

(1.09 g, 55%) and its diastereomers (1S,2S isomer,

149 mg, 8% and 1S,5R isomer, 39 mg, 2%). The prod-

ucts were obtained from 3b by reacting in the same man-

ner as described for 3a:4; R_f= 0.31 (3:1 n-hexane–

3.4. (1R,2S,3S,4R,5S)-3,4-Bis(benzyloxy)-5-hydroxy-

methyl-2-(4-methoxybenzyloxy)-cyclopentanol (5)

Ozone was bubbled through a solution of 4 and 1S,2S

isomer (1.25 g, 2.71 mmol, 88:12, 4/1S,2S isomer) in

CH₂Cl₂–MeOH (1:1, 34.0 mL) at ꢀ78 °C until con-

sumption of the oleﬁn. To the solution was added

NaBH₄(1.03 g, 27.1 mmol), and the mixture was stirred

for 1 h at ꢀ78 °C and for 1 h at rt. The reaction mixture

was poured into satd NH₄Cl, then the products were ex-

tracted with EtOAc. The organic layer was washed suc-

cessively with H₂O and brine, dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by silica

gel chromatography (1:1 n-hexane–EtOAc) to aﬀord 5

(955 mg, 76%) and its 1S isomer (128 mg, 10%): 5;

R_f= 0.34 (2:3 n-hexane–EtOAc); mp 84.5–86.0 °C;

25

1

EtOAc); ½aꢂ_D+17.4 (c 1.3, CHCl₃); H NMR (CDCl₃):

d 7.36–7.26 (m, 12H, aromatic), 6.88 (d, 2H, J = 8.6 Hz,

p-MeO-Ph), 5.93 (ddd, 1H, J_5,6= 7.8 Hz, J_6,7-cis

=

10.3 Hz, J _6,7-trans= 17.4 Hz, H-6), 5.28 (d, 1H, H-7-

cis), 5.25 (d, 1H, H-7-trans), 4.62–4.52 (m, 6H, PhCH₂),

4.11 (ddd, 1H, J_1,2

=

2.9 Hz, J_1,5= 5.6 Hz,

25

1

J_1,OH= 4.3 Hz, H-1), 4.02 (dd, 1H, J_3,4= 5.1 Hz,

J_4,5= 8.1 Hz, H-4), 3.95 (t, 1H, J = 4.2 Hz, H-3), 3.80

(s, 3H, Ph–OCH₃), 3.79 (t, 1H, J = 3.5 Hz, H-2), 2.90

(ddd, 1H, J = 7.9 Hz, H-5), 1.75 (d, 1H, OH); ¹³C

NMR (CDCl₃): d 138.2, 138.0 (aromatic C), 134.6 (C-

6), 130.0–127.6 (aromatic C), 119.0 (C-7), 113.8 (aro-

matic C), 88.3 (C-3), 86.7 (C-2), 84.7 (C-4), 75.5 (C-1),

72.0, 71.9, 71.4 (PhCH₂ꢃ3), 55.3 (Ph–OCH₃), 50.9 (C-

5). ESIMS m/z calcd for [C₂₉H₃₂O₅+Na]⁺: 483.2142.

Found: 483.2148. 1S,2S Isomer; R_f= 0.31 (3:1 n-hex-

½aꢂ_D+22.9 (c 1.0, CHCl₃); H NMR (CDCl₃): d 7.36–

7.27 (m, 12H, aromatic), 6.88 (d, 2H, J = 8.6 Hz, p-

MeO-Ph), 4.67–4.58 (m, 6H, PhCH₂), 4.28 (td, 1H,

J_1,2= J_1,OH= 4.2 Hz, J_1,5= 6.8 Hz, H-1), 4.02 (dd,

1H, J_3,4= 5.8 Hz, J_4,5= 8.1 Hz, H-4), 3.93 (dd, 1H,

J_2,3= 5.2 Hz, H-3), 3.92 (ddd, 1H, H-5a), 3.86 (ddd,

0

1H, J_{5,5 b}= 5.9 Hz, J_{5 a,5 b}= 11.3 Hz, J_{5 b,OH}= 5.8 Hz,

H-5⁰b), 3.81 (s, 3H, Ph–OCH₃), 3.78 (t, 1H, J =

4.5 Hz, H-2), 2.59 (d, 1H, J = 4.5 Hz, OH), 2.30 (m,

1H, H-5), 2.17 (t, 1H, J = 5.6 Hz, OH); ¹³C NMR

(CDCl₃): d 138.2–127.7, 113.9 (aromatic C), 87.7 (C-

3), 87.6 (C-2), 81.7 (C-4), 75.7 (C-1), 72.2, 72.1, 71.7

(PhCH₂ꢃ 3), 61.2 (C-5⁰), 55.3 (Ph–OCH₃), 46.6 (C-5).

ESIMS m/z calcd for [C₂₈H₃₂O₆+Na]⁺: 487.2091.

Found: 487.2094. 1S Isomer R_f= 0.23 (2:3 n-hexane–

1

ane–EtOAc); H NMR (C₆D₆): d 7.34–7.09 (m, 12H,

aromatic), 6.76 (d, 2H, J = 8.6 Hz, p-MeO-Ph), 5.93

(ddd, 1H, J_5,6= 7.9 Hz, J_6,7-cis= 10.3 Hz, J _6,7-trans

=

17.1 Hz, H-6), 5.23 (d, 1H, H-7-trans), 5.07 (d, 1H,

H-7-cis), 4.61–4.30 (m, 6H, PhCH₂), 4.10 (t, 1H,

J = 3.6 Hz, H-3), 3.88 (ddd, 1H, J_1,2= 5.3 Hz,

J_1,5= 8.5 Hz, J_1,OH= 8.5 Hz, H-1), 3.75 (m, 2H, H-2,

H-4), 3.28 (s, 3H, Ph–OCH₃), 2.90 (q, 1H, J = 8.1 Hz,

H-5), 2.38 (d, 1H, OH); ¹³C NMR (CDCl₃): d 138.2,

138.0 (aromatic C), 134.6 (C-6), 130.0–127.6 (aromatic

C), 119.0 (C-7), 113.8 (aromatic C), 88.3 (C-3), 86.7

(C-2), 84.7 (C-4), 75.5 (C-1), 72.0, 71.9, 71.4

28

EtOAc); mp 101.0–103.5 °C; ½aꢂ_D+6.6 (c 0.5, CHCl₃);

¹H NMR (CDCl₃): d 7.38–7.28 (m, 12H, aromatic),

6.88 (d, 2H, J = 8.6 Hz, p-MeO-Ph), 4.68–4.51 (m, 6H,

PhCH₂), 4.00 (dd, 1H, J_2,3= 3.9 Hz, J_3,4= 4.1 Hz, H-

3), 3.93 (ddd, 1H, J_1,2= 7.8 Hz, J_1,5= 5.5 Hz, J_1,OH

=

7.9 Hz, H-1), 3.83–3.78 (m, 5H, H-2, H-5⁰a, Ph–

0

OCH₃), 3.74 (td, 1H, J = 5.4 Hz, J_{5 a,5 b}= 10.7 Hz,

1630

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

H-5⁰b), 3.67 (dd, 1H, J_4,5= 8.2 Hz, H-4), 2.61 (d, 1H,

J = 7.9 Hz, OH), 2.25 (m, 1H, H-5), 1.88 (t, 1H, J

5.3 Hz, OH). ¹³C NMR (CDCl₃): d 138.1–127.7, 113.9

(aromatic C), 86.6 (C-3), 82.6 (C-4), 81.6 (C-2), 72.1,

71.9, 71.7 (PhCH₂ꢃ 3), 71.6 (C-1), 63.1 (C-5⁰), 55.3

(Ph–OCH₃), 50.6 (C-5). ESIMS m/z calcd for

[C₂₈H₃₂O₆+Na]⁺: 487.2091. Found: 487.2089.

4.3 Hz, J_{5 a,5 b}= 9.1 Hz, H-5⁰a), 3.01 (t, 1H, J =

9.1 Hz, H-5⁰b), 2.38 (m, 1H, H-5); ¹³C NMR (CDCl₃):

d (OC@S), 143.4, 137.8–127.0, 113.9 (aromatic C),

88.9, 86.7 (CPh₃), 84.0, 83.9, 83.0, 72.1, 71.9, 71.6

(PhCH₂ꢃ 3), 59.8 (C-5⁰), 55.3 (Ph–OCH₃), 45.6 (C-5).

ESIMS m/z calcd for [C₅₁H₄₈N₂O₆S+Na]⁺: 839.3125.

Found: 839.3117.

0

3.5. (1R,2S,3S,4R,5S)-3,4-Bis(benzyloxy)-2-(4-methoxy-

benzyloxy)-5-trityloxymethyl-cyclopentanol (6)

3.7. (1S,2R,3S,5S)-2,3-Bis(benzyloxy)-5-(4-methoxybenz-

yloxy)-3-trityloxymethyl-cyclopentane (8)

To a pyridine (64.0 mL) solution of 5 (890 mg,

1.92 mmol) was added TrCl (695 mg, 2.50 mmol) and

the mixture was stirred for 30 h at 70 °C under a nitro-

gen atmosphere. After the mixture was concentrated in

vacuo, the residue was suspended in a mixed solvent

(2:1 n-hexane–EtOAc) and ﬁltered through a Celite

pad. The ﬁltrate was concentrated again, and the residue

was puriﬁed by silica gel chromatography (7:2 n-hex-

ane–EtOAc) to aﬀord 6 (1.13 g, 83%) as a syrup:

To a toluene (8.00 mL) solution of n-Bu₃SnH (0.61 mL,

2.25 mmol) was added a toluene (6.00 mL) solution of 7

(460 mg, 0.563 mmol) and azobisisobutyronitrile

(AIBN, 28 mg, 0.169 mmol) at reﬂux under a nitrogen

atmosphere. After being stirred for 1 h, the mixture

was concentrated in vacuo. The residue was puriﬁed

by silica gel chromatography (6:1 n-hexane–EtOAc) to

aﬀord 8 (365 mg, 94%) as a syrup: R_f= 0.66 (2:1 n-hex-

27

ane–EtOAc); ½aꢂ_D+16.3 (c 0.8, CHCl₃); ¹H NMR

28

R_f= 0.47 (2:1, n-hexane–EtOAc); ½aꢂ_D+8.8 (c 0.5,

(CDCl₃): d 7.43–7.17 (m, 27H, aromatic), 6.86 (d, 2H,

J = 8.6 Hz, p-MeO-Ph), 4.64–4.43 (m, 6H, PhCH₂),

3.94 (dd, 1H, J_1,5= 2.7 Hz, J_1,2= 3.6 Hz, H-1), 3.90

(m, 1H, H-5), 3.80 (m, 4H, H-2, Ph–OCH₃), 3.17 (dd,

1

CHCl₃); H NMR (CDCl₃): d 7.41–7.17 (m, 27H, aro-

matic), 6.86 (d, 2H, J = 8.7 Hz, p-MeO-Ph), 4.66–4.48

(m, 6H, PhCH₂), 4.25 (ddd, 1H, J_1,2= 2.9 Hz, J_1,5

6.0 Hz, J_1,OH= 3.0 Hz, H-1), 4.15 (dd, 1H, J_4,3

=

1H, J_{3,3 a}= 4.7 Hz, J_{3 a,3 b}= 9.2 Hz, H-3⁰a), 3.10 (t,

0

1H, J_{3,3 b}= 5.7 Hz, H-3⁰b), 2.41 (m, 1H, H-3), 1.93 (m,

0

6.2 Hz, J_4,5= 9.4 Hz, H-4), 3.93 (dd, 1H, J_2,3= 5.0 Hz,

H-3), 3.80 (dd, 1H, H-2), 3.79 (s, 3H, Ph–OCH₃), 3.45

2H, H-4a, H-4b); ¹³C NMR (CDCl₃): d 144.1, 138.5–

126.9, 113.7 (aromatic C), 90.0 (C-1), 86.3 (CPh₃), 84.8

(C-2), 80.9 (C-5), 72.1, 72.0, 70.6 (PhCH₂ꢃ 3), 63.8

(C-3⁰), 55.3 (Ph–OCH₃), 41.5 (C-3), 30.9 (C-4). ESIMS

m/z calcd for [C₄₇H₄₆O₅+Na]⁺: 713.3237. Found:

713.3236.

(dd, 1H, J_{5,5 a}= 6.1 Hz, J_{5 a,5 b}= 9.6 Hz, H-5⁰a), 3.39

0

(dd, 1H, J_{5,5 b}= 3.8 Hz, H-6⁰), 3.09 (d, 1H, OH), 2.38

0

(m, 1H, H-5); ¹³C NMR (CDCl₃): d 143.4, 138.4–

127.2, 113.8 (aromatic C), 88.6 (C-3), 87.4 (C-2), 87.3

(CPh₃), 82.0 (C-4), 74.9 (C-1), 72.2, 72.0, 71.3

(PhCH₂ꢃ3), 61.1 (C-5⁰), 55.3 (Ph–OCH₃), 45.6 (C-5).

ESIMS m/z calcd for [C₄₇H₄₆O₆+Na]⁺: 729.3187.

Found: 729.3179.

3.8. (1S,2R,3R,4R)-2,3-Bis(benzyloxy)-4-trityloxymeth-

yl-cyclopentanol (9)

3.6. (1R,2S,3S,4R,5S)-3,4-Bis(benzyloxy)-2-(4-methoxy-

benzyloxy)-5-trityloxymethyl-1-thiocarbonylimidazoyl-

oxy-cyclopentanol (7)

To a CH₂Cl₂(62.4 mL) solution of 8 (455 mg, 0.659

mmol) were added H₂O (3.50 mL) and 2,3-dichloro-

5,6-dicyano-p-benzoquinone (DDQ, 299 mg, 1.32

mmol) at 0 °C. After being stirred for 2 h at rt, the mix-

ture was ﬁltered through a Celite pad and poured into

satd NaHCO₃, then the product was extracted with

CH₂Cl₂. The organic layer was dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by silica

gel chromatography (3:1 n-hexane–EtOAc) to aﬀord 9

(343 mg, 91%) as a syrup: R_f= 0.36 (3:1 n-hexane–

The mixture of 6 (1.13 g, 1.60 mmol), 1,1⁰-thiocarbonyl

diimidazole (1.27 g, 6.39 mmol) and DMAP (78 mg,

0.64 mmol) in THF (6.00 mL) was stirred for 20 h at

80 °C. After the mixture was concentrated in vacuo,

the residue was puriﬁed by silica gel chromatography

(5:2 n-hexane–EtOAc) to aﬀord7 (1.25 g, 96%) as a syr-

25

27

1

up: R_f= 0.34 (2:1 n-hexane–EtOAc); ½aꢂ_D+66.3 (c 0.4,

EtOAc); ½aꢂ_D+25.3 (c 0.3, CHCl₃); H NMR (CDCl₃):

d 7.47–7.21 (m, 25H, aromatic), 4.57 (s, 2H, PhCH₂),

4.53 (s, 2H, PhCH₂), 4.14 (br s, 1H, H-1), 3.83 (m,

1

CHCl₃); H NMR (CDCl₃): d 7.97 (s, 1H, imidazole),

7.32–7.14 (m, 28H, aromatic), 6.94 (s, 1H, imidazole),

6.87 (d, 2H, J = 8.6 Hz, p-MeO-Ph), 5.99 (br d, 1H,

J = 4.5 Hz, H-1), 4.76 (d, 1H, J = 11.8 Hz, PhCH₂),

4.63 (d, 1H, J = 11.8 Hz, PhCH₂), 4.51 (d, 1H, J =

11.8 Hz, PhCH₂), 4.41 (d, 1H, J = 11.8 Hz, PhCH₂),

4.38 (s, 2H, PhCH₂), 3.99 (br s, 1H, H-3), 3.94 (br s,

1H, H-2), 3.82 (dd, 1H, J_3,4= 4.9 Hz, J_4,5= 8.9 Hz,

0

2H, H-2, H-3), 3.13 (ddd, 2H, J_{4,4 a}= 9.2 Hz,

J_{4,4 b}= 6.5 Hz, J_{4 a,4 b}= 15.8 Hz, H-4⁰a, H-4⁰b), 2.48

(m, 1H, H-4), 2.02 (d, 1H, J_1,OH= 6.1 Hz, OH), 1.92

(ddd, 1H, J = 4.6 Hz, J = 8.9 Hz, J_5a,5b= 13.7 Hz, H-

5a), 1.85 (ddd, 1H, J = 6.5 Hz, J = 7.7 Hz, H-5b); ¹³C

NMR (CDCl₃): d 144.1, 138.3, 128.8–127.0 (aromatic

C), 90.0 (C-2), 86.5 (CPh₃), 85.0 (C-3), 75.1 (C-1), 71.9

0

H-4), 3.79 (s, 3H, Ph–OCH₃), 3.35 (dd, 1H, J_{5,5 a}

=

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

1631

(PhCH₂ꢃ2), 64.9 (C-4⁰), 42.2 (C-4), 34.3 (C-5). ESIMS

3.11. (1R,2R,3R,5S)-5-t-Butyloxycarbonylamino-3-

hydroxymethyl-cyclopentane-1,2-diol (12)

m/z calcd for [C₃₉H₃₈O₄+Na]⁺: 593.2662. Found:

593.2662.

To a THF–MeOH (1:1, 1.16 mL) solution of 11

(20.7 mg, 34.8 lmol) were added Pd(OH)₂–C (ca.

10.0 mg) and 10% HCl (MeOH solution, 0.12 mL).

The mixture was stirred for 20 h under a hydrogen

atmosphere, ﬁltered through a Celite pad then concen-

trated in vacuo. To a MeOH–H₂O solution (1:1,

1.16 mL) of the residue were added Et₃N (10.0 lL,

69.5 lmol) and a MeOH (0.29 mL) solution of Boc₂O

(16.0 mg, 69.5 lmol) at 0 °C. After being stirred for

3 h at rt, the mixture was concentrated in vacuo. The

residue was puriﬁed by silica gel chromatography

(30:1, EtOAc–MeOH) to aﬀord 12 (7.4 mg, 86%) as a

3.9. (1S,2R,3R,4R)-2,3-Bis(benzyloxy)-4-trityloxymeth-

yl-cyclopentanoyl methanesulfonate (10)

To a pyridine (0.90 mL) solution of 9 (42.0 mg, lmol)

were added DMAP (3.0 mg, 22.1 lmol) and methanesul-

fonyl chloride (17.0 lL, 0.221 mmol) at 0 °C under a

nitrogen atmosphere. After being stirred for 20 h at rt,

the mixture was poured into 5% KHSO₄at 0 °C, then

the product was extracted with Et₂O. The organic layer

was washed with brine, dried over Na₂SO₄and concen-

trated in vacuo. The residue was puriﬁed by silica gel

chromatography (3:1 n-hexane–EtOAc) to aﬀord a cor-

responding mesylate 10 (45.3 mg, 95%) as a syrup;

25

syrup: R_f= 0.37 (10:1, EtOAc–MeOH); ½aꢂ_D+28.0 (c

1

0.2, MeOH); H NMR (CD₃OD): d 3.96 (m, 1H, H-5),

28

R_f= 0.44 (2:1 n-hexane–EtOAc); ½aꢂ_D+9.3 (c 0.3,

3.76 (dd, 1H, J_1,2= 4.9 Hz, J_1,5= 3.2 Hz, H-1), 3.66

1

0

CHCl₃); H NMR (CDCl₃): d 7.42–7.15 (m, 25H, aro-

(dd, 1H, J_2,3= 4.4 Hz, H-2), 3.60 (dd, 2H, J_{3,3 a}

=

0

matic), 5.04 (dt, 1H, J = 4.6 Hz, H-5), 4.68 (d, 1H,

J = 11.7 Hz, PhCH₂), 4.61 (d, 1H, J = 11.7 Hz, PhCH₂),

4.44 (d, 1H, J = 11.6 Hz, PhCH₂), 4.40 (d, 1H,

J = 11.6 Hz, PhCH₂), 4.10 (t, 1H, J_1,5= J_1,2= 4.6 Hz,

H-1), 3.82 (dd, 1H, J_2,3= 6.8 Hz, H-2), 3.20 (dd, 1H,

5.7 Hz, J_{3 a,3 b}= 10.6 Hz, H-3⁰a), 3.51 (dd, 1H,

J_{3,3 b}= 6.6 Hz, H-3⁰b), 2.11 (dt, 1H, J = 8.0 Hz,

0

J_4a,4b= 12.6 Hz, H-4a), 1.87 (m, 1H, H-3), 1.41 (s,

9H, t-Bu), 1.35 (dt, 1H, J = 9.4 Hz, H-4b); ¹³C NMR

(CD₃OD): d 157.9 (NH–CO), 80.1 (CMe₃), 80.0 (C-2),

78.7 (C-1), 65.1 (C-3⁰), 53.3 (C-5), 47.3 (C-3), 32.3 (C-

4), 28.7 (Me). ESIMS m/z calcd for [C₁₁H₂₁NO₅+Na]⁺:

270.1312. Found: 270.1308.

J_{3,3 a}= 4.6 Hz, H-3⁰a), 3.16 (dd, 1H, J_{3,3 b}= 5.3 Hz,

H-3⁰b), 2.98 (s, 3H, SO₂CH₃), 2.41 (m, 1H, H-3), 2.15

(m, 2H, H-4a, H-4b); ¹³C NMR (CDCl₃): d 143.4,

137.9–127.0 (aromatic C), 88.5 (C-1), 86.6 (CPh₃),

84.6 (C-2), 83.2 (C-5), 72.3, 72.2 (PhCH₂ꢃ 2), 63.5 (C-

3⁰), 41.9 (C-3), 38.5 (SO₂CH₃), 32.2 (C-4). ESIMS

m/z calcd for [C₄₀H₄₀O₆S+Na]⁺: 671.2438. Found:

671.2440.

0

3.12. (1R,2R,3R,5S)-5-Amino-3-hydroxymethyl-cyclo-

pentane-1,2-diol (13)

To a CH₂Cl₂(0.42 mL) solution of 12 (2.8 mg,

11.3 lmol) was added TFA (0.14 mL) at 0 °C. The mix-

ture was stirred for 1 h under a nitrogen atmosphere,

and concentrated in vacuo. The residue was dissolved

in H₂O then ﬁltered through Millex^Òﬁlter. The ﬁltrate

was concentrated in vacuo to aﬀord 13 (3.0 mg, quant.)

as a TFA salt: R_f= 0.39 (5:2:3, n-BuOH–AcOH–H₂O);

3.10. (1R,2R,3R,5S)-5-Azido-1,2-bis(benzyloxy)-3-trityl-

oxymethyl-cyclopentane (11)

To a DMF (0.87 mL) solution of this compound

(45.3 mg, 0.698 mmol) were added NaN₃(45.0 mg,

0.698 mmol), and the mixture was stirred for three days

at 80 °C. After cooling, the mixture was poured into ice

water then the product were extracted with a mixed sol-

vent (4:1, n-hexane–EtOAc). The organic layer was

washed with brine, dried over Na₂SO₄and concentrated

in vacuo. The residue was puriﬁed by silica gel chroma-

tography (10:1 n-hexane–EtOAc) to aﬀord 10 (39.5 mg,

23

1

½aꢂ_D+23.6 (c 0.6, MeOH); H NMR (D₂O): d 4.06 (t,

1H, J = 8.5 Hz, H-1), 3.80 (dd, 1H, J_1,2= 5.3 Hz,

J_1,5= 6.9 Hz, H-2), 3.69 (m, 1H, H-5), 3.68 (dd, 1H,

J_{3,3 a}= 5.0 Hz, J_{3 a,3 b}= 11.2 Hz, H-3⁰a), 3.59 (dd, 1H,

0

J_{3,3 b}= 6.5 Hz, H-3⁰b), 2.34 (td, 1H, J = 8.1 Hz, J_4a,4b

0

= 13.6 Hz, H-4a), 1.99 (m, 1H, H-3), 1.88 (ddd, 1H,

J_4b,5= 9.6 Hz, J_3,4b= 8.4 Hz, H-4b); ¹³C NMR

(CD₃OD): d 79.3 (C-2), 77.3 (C-1), 64.2 (C-3⁰), 53.0

(C-5), 47.3 (C-3), 30.5 (C-4). HRMS m/z calcd for

[C₆H₁₃NO₃+H]⁺: 148.0968. Found: 148.0966.

27

95%) as a syrup; R_f= 0.57 (3:1 n-hexane–EtOAc); ½aꢂ_D

1

ꢀ10.0 (c 0.5, CHCl₃); H NMR (CDCl₃): d 7.42–7.19

(m, 25H, aromatic), 4.58–4.47 (m, 4H, PhCH₂), 3.87

(t, 1H, J = 4.2 Hz, H-1), 3.82 (t, 1H, J = 4.3 Hz, H-2),

3.76 (ddd, 1H, H-5), 3.17 (m, 2H, H-3⁰a, H-3⁰b), 2.22

(m, 1H, H-4), 2.15 (ddd, 1H, H-4a), 1.72 (dt, 1H,

J = 7.8 Hz, J_4a,4b= 13.0 Hz, H-4b); ¹³C NMR (CDCl₃):

d 144.1–127.0 (aromatic C), 86.6 (CPh₃), 84.6 (C-1), 83.6

(C-2), 72.0, 71.9 (PhCH₂ꢃ2), 65.0 (C-3⁰), 60.6 (C-5),

42.2 (C-3), 29.6 (C-4). ESIMS m/z calcd for

[C₃₉H₃₇N₃O₃+Na]⁺: 618.2727. Found: 618.2729.

3.13. (2S,3R,4R)-2,3-Bis(benzyloxy)-4-trityloxymethyl-

cyclopentanone (14)

A mixture of 9 (209 mg, 0.366 mmol) and Dess–Martin

Periodinane (311 mg, 0.732 mmol) in CH₂Cl₂

(7.30 mL) was stirred for 20 h at rt. To the mixture

was added satd NaHCO₃and stirred for 5 min at

1632

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

0

0 °C. The organic layer was washed with brine, followed

by extraction with CH₂Cl₂, dried (Na₂SO₄) and concen-

trated in vacuo. The residue was puriﬁed by silica gel

chromatography (6:1 n-hexane–EtOAc) to aﬀord 14

(190 mg, 91%) as a syrup: R_f= 0.57 (3:1, n-hexane–

H-2), 3.19 (dd, 1H, J_{3,3 a}= 9.0 Hz, H-3⁰a), 3.18 (dd,

1H, J_{3,3 b}= 9.0 Hz, H-3⁰b), 2.89 (s, 3H, SO₂CH₃), 2.27

0

(ddd, 1H, H-4a), 2.26 (m, 1H, H-3), 1.94 (dt, 1H,

J = 4.9 Hz, J_4a,4b= 8.7 Hz, H-4b); ¹³C NMR (CDCl₃):

d 144.0, 138.2–127.0 (aromatic C), 86.6 (CPh₃), 83.9

(C-1), 83.3 (C-2), 79.4 (C-5), 72.4, 72.3 (PhCH₂ꢃ 2),

64.7 (C-3⁰), 40.9 (C-3), 38.7 (SO₂CH₃), 30.6 (C-4).

ESIMS calcd for [C₄₀H₄₀O₆S+Na]⁺: 671.2438. Found:

671.2432.

25

1

EtOAc); ½aꢂ_D+16.8 (c 0.6, CHCl₃); H NMR (CDCl₃):

d

7.41–7.14 (m, 25H, aromatic), 5.07 (d, 1H,

J = 11.4 Hz, PhCH₂), 4.74 (d, 1H, J = 11.5 Hz, PhCH₂),

4.71 (d, 1H, J = 11.5 Hz, PhCH₂), 4.47 (d, 1H,

J = 11.5 Hz, PhCH₂), 4.12 (t, 1H, J = 7.4 Hz, H-3),

4.06 (dd, J_2,3= 8.7 Hz, J_2,5= 1.6 Hz, H-2), 3.30 (d,

3.16. (1R,2R,3R,5R)-5-Azido-1,2-bis(benzyloxy)-3-trityl-

oxymethyl-cyclopentane (17)

2H, J = 3.9 Hz, H-4⁰a, H-4⁰b), 2.55 (ddd, 1H, J_4,5a

=

8.8 Hz, J_5a,5b= 19.1 Hz, H-5a), 2.37 (m, 1H, H-4),

2.43 (dd, 1H, J_4,5b= 10.8 Hz, H-5b); ¹³C NMR

(CDCl₃): d 213.1 (C-1),143.7, 137.9–127.0 (aromatic

C), 87.0 (C-2), 86.4 (CPh₃), 80.9 (C-3), 73.02, 72.98

(PhCH₂ꢃ 2), 61.2 (C-6), 38.2 (C-5), 37.7 (C-4). ESIMS

m/z calcd for [C₃₉H₃₆O₄+Na]⁺: 591.2506. Found:

591.2506.

Compound 17 was obtained from 16 by reacting in the

same manner as described for 11. Compound 17 was

puriﬁed by silica gel chromatography (8:1 n-hexane–

EtOAc) to aﬀord a syrup (quant.); R_f= 0.69 (3:1 n-hex-

28

ane–EtOAc); ½aꢂ_D+18.6 (c 0.5, CHCl₃); ¹H NMR

(CDCl₃): d 7.43–7.18 (m, 25H, aromatic), 4.66 (d, 1H,

J = 11.6 Hz,PhCH₂), 4.59 (d, 1H, J = 11.6 Hz, PhCH₂),

4.50 (d, 1H, J = 11.6 Hz, PhCH₂), 4.45 (d, 1H,

J = 11.6 Hz, PhCH₂), 4.10 (dd, 1H, J_1,5= 4.5 Hz,

J_1,2= 4.7 Hz, H-1), 3.82 (dd, 1H, J_2,3= 6.8 Hz, H-2),

3.14. (1R,2R,3R,4R)-2,3-Bis(benzyloxy)-4-trityloxy-

methyl-cyclopentanol (15)

3.18 (dd, 1H, J_{3,3 a}= 5.1 Hz, J_{3 a,3 b}= 9.3 Hz, H-3⁰a),

0

To a THF–MeOH (1:1, 2.80 mL) solution of 14

(81.0 mg, 0.142 mmol) was added NaBH₄(54.0 mg,

1.42 mmol) at ꢀ78 °C, then stirred for 1 h. After being

stirred for 1 h at 0 °C, the mixture was poured into satd

NH₄Cl. The organic layer was washed with brine, fol-

lowed by extraction with EtOAc, dried (Na₂SO₄), and

concentrated in vacuo. The residue was puriﬁed by silica

gel chromatography (5:1, n-hexane–EtOAc) to aﬀord 15

(73.0 mg, 89%) and 9 (8.0 mg, 10%): R_f= 0.49 (5:2, n-

3.16 (dd, 1H, J_{3,3 b}= 5.8 Hz, H-3⁰b), 2.33 (m, 1H,

0

H-3), 2.00 (dt, 1H, J = 7.2 Hz, J_4a,4b= 13.6 Hz, H-4a),

1.91 (ddd, 1H, J = 6.5 Hz, J = 8.6 Hz, H-4b); ¹³C

NMR (CDCl₃): d 144.0, 138.2–127.1 (aromatic C),

89.3 (C-2), 86.6 (CPh₃), 85.0 (C-1), 72.4, 72.2

(PhCH₂ꢃ 2), 63.9 (C-3⁰), 63.7 (C-5), 41.8 (C-3), 30.5

(C-4). ESIMS m/z calcd for [C₃₉H₃₇N₃O₃+Na]⁺:

618.2727. Found: 618.2732.

24

1

hexane–EtOAc); ½aꢂ_D+11.6 (c 0.5, CHCl₃); H NMR

(C₆D₆): d 7.58–7.00 (m, 25H, aromatic), 4.59 (s, 2H,

PhCH₂), 4.32 (d, 1H, J = 11.9 Hz, PhCH₂), 4.27 (d,

1H, J = 11.8 Hz, PhCH₂), 4.03 (m, 1H, H-1), 3.99 (t,

1H, J = 5.2 Hz, H-3), 3.68 (t, 1H, J = 4.8 Hz, H-2),

3.37 (dd, 1H, H-4⁰a), 3.13 (dd, 1H, H-4⁰b), 2.23 (m,

1H, H-4), 2.21 (d, 1H, J = 4.9 Hz, OH), 1.90 (ddd,

1H, H-5a), 1.68 (ddd, 1H, H-5b); ¹³C NMR (C₆D₆): d

144.9, 139.5–127.2 (aromatic C), 87.0 (CPh₃), 86.4 (C-

2), 84.7 (C-3), 72.0, 71.8 (PhCH₂ꢃ2), 70.6 (C-1), 66.2

(C-6), 42.3 (C-4), 33.3 (C-5). ESIMS m/z calcd for

[C₃₉H₃₈O₄+Na]⁺: 593.2662. Found: 593.2663.

3.17. (1R,2R,3R,5R)-5-t-Butyloxycarbonylamino-3-

hydroxymethyl-cyclopentane-1,2-diol (18)

Compound 18 was obtained from 17 by reacting in the

same manner as described for 12. Compound 18 was

puriﬁed by silica gel chromatography (30:1 EtOAc–

MeOH) to aﬀord a syrup (84%): R_f= 0.43 (10:1

25

1

EtOAc–MeOH); ½aꢂ_D+38.2 (c 0.2, MeOH); H NMR

(CD₃OD): d 3.63–3.51 (m, 4H, H-1, H-2, H-5, H-3⁰a),

3.47 (dd, 1H, J_{3,3 b}= 6.6 Hz, J_{3 a,3 b}= 10.6 Hz, H-3⁰b),

1.92 (m, 1H, H-3), 1.88 (dt, 1H, J = 7.0 Hz,

J_4a,4b= 13.0 Hz, H-4a), 1.61 (m, 1H, H-4b), 1.41 (s,

9H, t-Bu); ¹³C NMR (CD₃OD): d 83.3 (C-1 or 2), 80.0

(CMe₃), 78.6 (C-1 or 2), 64.5 (C-3⁰), 55.3 (C-5), 44.7

(C-3), 31.1 (C-4), 28.8, 28.5 (Me). ESIMS m/z calcd

for [C₁₁H₂₁NO₅+Na]⁺: 270.1312. Found: 270.1309.

0

3.15. (1R,2R,3R,4R)-2,3-Bis(benzyloxy)-4-trityloxy-

methyl-cyclopentanoyl methanesulfonate (16)

Compound 16 was obtained from 15 by reacting in the

same manner as described for 10. Corresponding mesy-

late 16 was puriﬁed by silica gel chromatography (7:2, n-

hexane–EtOAc) to aﬀord a syrup (92%); R_f= 0.29 (3:1,

3.18. (1R,2R,3R,5R)-5-Amino-3-hydroxymethyl-cyclo-

pentane-1,2-diol (19)

26

1

n-hexane–EtOAc); ½aꢂ_Dꢀ12.7 (c 0.4, CHCl₃); H NMR

(CDCl₃): d 7.44–7.20 (m, 25H, aromatic), 5.14 (ddd, 1H,

Compound 19 was quantitatively obtained from 18 by

reacting in the same manner as described for 13:

H-5), 4.67–4.51 (m, 4H, PhCH₂), 3.91 (dd, 1H, J_1,5

=

23

4.5 Hz, J_1,2= 4.7 Hz, H-1), 3.89 (dd, 1H, J_2,3= 6.8 Hz,

R_f= 0.40 (5:2:3 n-BuOH–AcOH–H₂O); ½aꢂ_D+47.6 (c

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

1633

0.4, MeOH); ¹H NMR (D₂O): d 3.88 (t, 1H, J = 8.5 Hz,

H-1), 3.67 (t, 1H, J = 8.5 Hz, H-2), 3.65 (dd, 1H,

m/z calcd for [C₄₀H₃₈Cl₂O₄+Na]⁺: 675.2039. Found:

675.2043.

J_{3,3 a}= 4.6 Hz, J _{3 a,3 b}= 11.2 Hz, H-3⁰a), 3.54 (dd, 1H,

0

J_{3,3 b}= 6.6 Hz, H-3⁰b), 3.37 (q, 1H, J=8.7 Hz, H-5),

0

3.20. (1S,2R,3R,4R)-1-Azido-2,3-bis(benzyloxy)-4-trityl-

oxymethyl-cyclopentane-carbaldehyde (21)

2.10 (m, 1H, H-3), 1.98 (ddd, 1H, H-4a), 1.88 (ddd,

1H, H-4b); ¹³C NMR (CD₃OD): d 81.3 (C-1), 77.8 (C-

2), 63.5 (C-3⁰), 55.0 (C-5), 44.6 (C-3), 28.1 (C-4). ESIMS

m/z calcd for [C₆H₁₃NO₃+H]⁺: 148.0968. Found:

148.0966.

To a Me₂SO (0.40 mL) solution of 20-R (22.0 mg,

33.7 lmol) were added 15-crown-5-ether (20.0 lL) and

NaN₃(22.0 mg, 0.337 mmol) and the mixture was stir-

red for two days at 80 °C under a nitrogen atmosphere.

To the mixture was added NaN₃(22.0 mg, 0.337 mmol)

again. After being stirred for 20 h, the mixture was

diluted with EtOAc, and poured into an ice water. The

organic layer was washed with brine, followed by

extraction with EtOAc, dried (Na₂SO₄) and concen-

trated in vacuo. The residue was puriﬁed by silica gel

chromatography (100:1, toluene–EtOAc) to aﬀord 21

(16.6 mg, 79%) as a syrup: R_f= 0.34 (40:1, toluene–

3.19. (1R,2S,3R,4R)-2,3-Bis(benzyloxy)-1-dichloro-

methyl-4-trityloxymethyl-cyclopentanol (20-R) and

(1S,2S,3R,4R)-2,3-bis(benzyloxy)-1-dichloromethyl-

4-trityloxymethyl-cyclopentanol (20-S)

To a THF (1.20 mL) solution of N,N⁰-diisopropylamine

(47.0 lL, 0.333 mmol) were added n-BuLi (1.6 M in n-

hexane, 0.21 mL) and the mixture was stirred for 1 h

at ꢀ78 °C under nitrogen atmosphere. To the mixture

were added THF (0.15 mL) solution of CH₂Cl₂

(0.214 mL, 3.33 mmol) and THF (0.30 mL) solution of

14 (47.4 mg, 83.4 lmol), successively. After being stirred

for 1 h, the mixture was poured into satd NH₄Cl. The

products were extracted with CH₂Cl₂then washed with

brine, dried (Na₂SO₄) and concentrated in vacuo. The

residue was puriﬁed by silica gel chromatography (8:1

n-hexane–EtOAc) to aﬀord 20-R (36.4 mg, 67%) and

20-S (7.0 mg, 13%): 20-R; R_f= 0.56 (7:2, n-hexane–

26

1

EtOAc); ½aꢂ_D+25.1 (c 0.5, CHCl₃); H NMR (CDCl₃):

d 9.65 (s, 1H, CHO), 7.42–7.13 (m, 25H, aromatic), 4.55

(d, 1H, J = 11.4 Hz, PhCH₂), 4.53 (d, 1H, J = 11.3 Hz,

PhCH₂), 4.48 (d, 1H, J = 11.5 Hz, PhCH₂), 4.42 (d,

1H, J = 11.4 Hz, PhCH₂), 4.04 (d, 1H, J = 5.7 Hz, H-

2), 4.01 (dd, 1H, J_3,2= 5.9 Hz, J_3,4= 7.8 Hz, H-3),

3.26 (m, 2H, H-4⁰a, H-4⁰b), 2.43 (m, 1H, H-4), 2.38

(dd, J_4,5a= 11.4 Hz, J_5a,5b= 13.1 Hz, H-5a), 1.95 (dd,

J_4,5b= 7.1 Hz, H-5b); ¹³C NMR (CDCl₃): d 197.1

(CHO), 143.9, 128.7–127.1 (aromatic C), 91.0 (C-2),

86.6 (CPh₃), 84.1 (C-3), 75.0 (C-1), 72.8, 72.7

(PhCH₂ꢃ 2), 62.5 (C-4⁰), 42.3 (C-4), 31.1 (C-5). ESIMS

m/z calcd for [C₄₀H₃₇N₃O₄+Na]⁺: 646.2676. Found:

646.2670.

24

1

EtOAc); ½aꢂ_D+10.5 (c 0.4, CHCl₃); H NMR (CDCl₃):

d 7.44–7.14 (m, 25H, aromatic), 5.56 (s, 1H, CHCl₂),

4.77 (d, 1H, J = 11.1 Hz, PhCH₂), 4.70 (d, 1H,

J = 11.1 Hz, PhCH₂), 4.49 (d, 1H, J = 11.4 Hz, PhCH₂),

4.45 (d, 1H, J = 11.4 Hz, PhCH₂), 4.15 (d, 1H,

J = 7.1 Hz, H-2), 3.95 (t, 1H, J = 7.5 Hz, H-3), 3.40 (s,

0

1H, OH), 3.31 (dd, 1H, J_{4,4 a}= 5.4 Hz, J_{4 a,4 b}= 9.3 Hz,

3.21. (1R,2R,3R,5R)-3-Azido-3-benzylaminomethyl-1,2-

bis(benzyloxy)-5-trityloxymethyl-cyclopentane (22)

H-4⁰a), 3.18 (dd, 1H, J_{4,4 b}= 5.8 Hz, H-4⁰b), 2.39 (dd,

0

J_4,5a= 9.5 Hz, J_5a,5b= 14.5 Hz, H-5a), 2.18 (m, 1H, H-

4), 1.91 (dd, J_4,5b= 8.0 Hz, H-5b); ¹³C NMR (CDCl₃):

d 144.0, 128.7–127.0 (aromatic C), 86.6 (CPh₃), 85.6

(C-3), 84.6 (C-2), 80.0 (C-1), 77.2 (CHCl₂), 73.7, 73.0

(PhCH₂ꢃ2), 63.9 (C-4⁰), 40.1 (C-4), 34.1 (C-5). ESIMS

m/z calcd for [C₄₀H₃₈Cl₂O₄+Na]⁺: 675.2039. Found:

To

a THF (1.10 mL) solution of 21 (23.5 mg,

37.7 lmol), BnNH₂(21.0 lL, 0.188 mmol) and AcOH

(22.0 lL, 0.377 mmol) was added NaBH₃CN (1 M in

THF solution, 0.113 mL) and the mixture was stirred

for 2 h at 0 °C under a nitrogen atmosphere. The mix-

ture was poured into 5% aq NaOH, and the product

was extracted with EtOAc. The organic layer was

washed with brine, dried (Na₂SO₄) and concentrated

in vacuo. The residue was puriﬁed by silica gel chroma-

tography (6:1 n-hexane–EtOAc) to aﬀord 22 (23.2 mg,

25

675.2044. 20-S; R_f= 0.43 (7:2, n-hexane–EtOAc); ½aꢂ_D

1

+28.9 (c 0.6, CHCl₃); H NMR (CDCl₃): d 7.42–7.13

(m, 25H, aromatic), 6.03 (s, 1H, CHCl₂), 4.58 (d, 1H,

J = 12.0 Hz, PhCH₂), 4.53 (d, 1H, J = 12.0 Hz, PhCH₂),

4.37 (d, 1H, J = 10.9 Hz, PhCH₂), 4.29 (d, 1H,

J = 10.9 Hz, PhCH₂), 3.85 (d, 1H, J = 3.5 Hz, H-3),

25

86%) as a syrup: R_f= 0.63 (7:2 n-hexane–EtOAc); ½aꢂ_D

3.72 (s, 1H, H-2), 3.20 (dd, 1H, J_{4,4 a}= 5.5 Hz,

+28.8 (c 0.2, CHCl₃); ¹H NMR (C₆D₆): d 7.54–7.00

(m, 30H, aromatic), 4.59 (d, 1H, J = 12.0 Hz, PhCH₂),

4.47 (d, 1H, J = 12.0 Hz, PhCH₂), 4.41 (d, 1H,

J = 11.5 Hz, PhCH₂), 4.35 (d, 1H, J = 11.5 Hz, PhCH₂),

4.02 (d, 1H, J = 4.1 Hz, H-2), 3.96 (dd, 1H,

J_1,2= 4.3 Hz, J_1,5= 6.8 Hz, H-1), 3.59 (s, 2H, NH–

0

J_{4 a,4 b}= 9.1 Hz, H-4⁰a), 3.07 (dd, 1H, J_{4,4 b}= 7.6 Hz,

H-4⁰b), 3.02 (s, 1H, OH), 2.74 (m, 1H, H-4), 2.15 (dd,

J_4,5a= 8.1 Hz, J_5a,5b= 13.4 Hz, H-5a), 1.67 (dd,

0

J_4,5b= 10.4 Hz, H-5b); ¹³C NMR (CDCl₃):

d

144.0, 128.7–127.0 (aromatic C), 87.9 (C-2), 86.6

(CPh₃), 85.7 (C-1), 84.6 (C-3), 76.4 (CHCl₂), 71.8, 71.6

(PhCH₂ꢃ 2), 65.0 (C-4⁰), 44.9 (C-4), 38.3 (C-5). ESIMS

0

CH₂Ph), 3.20 (dd, 1H, J_{5,5 a}= 5.4 Hz, J_{5 a,5 b}= 9.2 Hz,

H-5⁰a), 3.07 (dd, 1H, J_{5,5 b}= 5.2 Hz, H-5⁰b), 2.87

0

1634

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

(d, 1H, J = 12.3 Hz, CH₂NHBn), 2.75 (d, 1H, J =

12.3 Hz, CH₂NHBn), 2.52 (m, 1H, H-5), 1.98 (dd,

J_4a,5= 10.8 Hz, J_4a,4b= 13.7 Hz, H-4a), 1.87 (dd,

7.74–7.49 (m, 5H, aromatic), 3.96 (d, 1H, J = 9.1 Hz,

H-6), 3.95 (d, 1H, J = 11.7 Hz, H-4a), 3.66 (dd, 1H,

J_{8,8 a}= 4.8 Hz, J_{8 a,8 b}= 11.3 Hz, H-8⁰a), 3.59 (t, 1H,

0

J

_4b,5= 8.4 Hz, H-4b); ¹³C NMR (C₆D₆): d 144.6,

J = 8.9 Hz, H-7), 3.54 (dd, 1H, J_{8,8 b}= 6.6 Hz, H-8⁰b),

0

128.7–127.1 (aromatic C), 88.8 (C-2), 87.0 (CPh₃), 85.3

(C-1), 72.6, 72.3 (PhCH₂ꢃ2), 71.8 (C-3), 64.3 (C-5⁰),

54.6 (N–CH₂Ph), 53.6 (CH₂NHBn), 43.0 (C-5), 34.6

(C-4). ESIMS m/z calcd for [C₄₇H₄₆N₄O₃+H]⁺:

715.3643. Found: 715.3637.

3.45 (d, 1H, J = 11.6 Hz, H-4b), 2.25 (dd, 1H, J_8,9a=

11.2 Hz, J_9a,9b= 14.1 Hz, H-9a), 2.07 (m, 1H, H-8),

1.73 (dd, 2H, J_8,9b= 6.1 Hz, H-9b). HRMS m/z calcd

for [C₁₄H₁₈N₂O₃+H]⁺: 263.1390. Found: 263.1392.

3.24. Glycosidase assay

3.22. (1R,2R,3R,5R)-3-Amino-3-aminomethyl-5-

hydroxymethyl-cyclopentane-1,2-diol (23)

3.24.1. Materials. p-Nitrophenyl-a-^D-glucopyranoside

(PNP-a-Glc), p-nitrophenyl-b-^D-glucopyranoside (PNP-

To a THF–MeOH (1:1, 0.80 mL) solution of 22

(13.1 mg, 18.3 lmol) were added 20% Pd(OH)₂–C (ca.

10.0 mg) and 10% HCl–MeOH (65.0 lL). The mixture

was stirred for 20 h under a hydrogen atmosphere, ﬁl-

tered through a Celite pad then concentrated in vacuo.

To a H₂O solution (0.30 mL) of the residue were added

Et₃N (10.0 lL, 73.3 lmol) and a MeOH (0.30 mL) solu-

tion of Boc₂O (16.0 mg, 73.3 lmol) at 0 °C. After being

stirred for 4 h at rt, the mixture was concentrated in

vacuo. The residue was puriﬁed by silica gel chromato-

graphy (30:1 EtOAc–MeOH) to aﬀord a corresponding

Boc derivative (4.4 mg, 64%). To a CH₂Cl₂(0.36 mL)

solution of the Boc derivative (3.5 mg, 9.3 lmol) was

added TFA (0.12 mL) at 0 °C. The mixture was stirred

for 2 h under a nitrogen atmosphere, and concentrated

in vacuo. The residue was dissolved in H₂O then ﬁltered

through Millex^Òﬁlter. The ﬁltrate was concentrated in

vacuo to aﬀord 23 (3.8 mg, quant.) as a TFA salt:

R_f= 0.21 (5:2:3, n-BuOH–AcOH–H₂O); ¹H NMR

(D₂O): d 4.08 (d, 1H, J = 8.0 Hz, H-2), 3.80 (dd, 1H,

J_1,2= 8.1 Hz, J_1,5= 8.4 Hz, H-1), 3.65 (dd, 1H, H-5⁰a),

b-Glc),

p-nitrophenyl-N-acetyl-2-deoxy-b-^D-glucopy-

ranoside (PNP-b-GlcNAc), p-nitrophenyl-b-^D-galacto-

pyranoside (PNP-b-Gal) and p-nitrophenyl-a-^D-

mannopyranoside (PNP-a-Man) were from Sigma–Al-

drich Inc. (St. Louis, MO, USA). a-Glucosidase (EC

3.2.1.20) from yeast was purchased from Wako Pure

Chemical Industries Ltd (Osaka, Japan), b-Glucosidase

(EC 3.2.1.21) from almond, b-N-acetylglucosaminidase

(EC 3.2.1.52) from jack beans, b-galactosidase (EC

3.2.1.23) from Aspergillus oryzae and a-mannosidase

(EC 3.2.1.24) from jack bean were purchased from Sig-

ma–Aldrich Inc. (St. Louis, MO, USA). a-Glucosidase

was used in the phosphate buﬀer (25 mM, pH 7.0). b-

N-Acetylglucosaminidase was used in citrate buﬀer

(50 mM, pH 4.5). b-Glucosidase, a-mannosidase and

b-galactosidase were used in phosphate buﬀer (25 mM,

pH 5.8). Concentrations of enzymes were optimized

and arranged between 0.1 and 0.2 U/mL. PNP-glyco-

sides except PNP-a-Glc (0.5 mM) were used at

0.35 mM in solution.

3.56 (dd, 1H, J_{5,5 b}= 3.7 Hz, J_{5 a,5 b}= 11.5 Hz, H-5⁰b),

3.42 (d, 1H, J = 14.5 Hz, CH₂NH₂), 3.29 (d, 1H, J =

14.5 Hz, CH₂NH₂), 2.13 (m, 2H, H-4a, H-5), 1.78 (dd,

J_4b,5= 13.2 Hz, J_4a,4b= 18.7 Hz, H-4b); ¹³C NMR

(D₂O): d 81.7 (C-2), 76.0 (C-1), 61.2 (C-5⁰), 58.4 (C-3),

43.1 (CH₂NH₂), 41.4 (C-5), 32.3 (C-4). HRMS m/z calcd

for [C₇H₁₆N₂O₃+H]⁺: 177.1234. Found: 177.1238. NOE

(CH₂NH₂); 2.2% (H-3), 1.3% (H-5⁰).

0

3.24.2. Methods. Assays were performed on a BD Fal-

con microtest 96-well assay plate. After the mixture of

PNP-glycosides, various amounts of inhibitors (75–

500 lM) and various glycosidases in buﬀer solution

(90 lL) were incubated for 7 min at 25 °C, the reaction

was terminated by addition of 50 mM glycin buﬀer

(180 lL, pH 10.0). Concentrations of p-nitrophenol gen-

erated in the each well were detected by absorbance at

405 nm using Wallac 1420-ARVOsx multilabel counter

Version 2.0.

3.23. (5R,6R,7R,8R)-8-(Hydroxymethyl)-2-phenyl-1,3-

diazaspiro[4.4]non-1-ene-6,7-diol (1)

A MeOH (1.00 mL) solution of 23 (8.0 mg, 32 lmol),

Et₃N (22.0 lL), 0.161 mmol and benzaldehyde

(10.0 lL, 96.3 lmol) was stirred for 0.5 h at 0 °C. To

the mixture was added NBS (17.0 mg, 96.3 lmol). After

being stirred for 20 h at rt, the mixture was concentrated

in vacuo then ﬁltered through Dowex^Ò-1 ꢃ 8, followed

by eluting with H₂O. The product was puriﬁed by

HPLC to aﬀord 1 (2.8 mg, 33%) as a syrup: R_f= 0.34

(10:10:1, i-PrOH–H₂O–Et₃N); ¹H NMR (D₂O): d

Acknowledgements

This work was supported in part by the ‘Special Coordi-

nation Funds for Promoting Science and Technology’ of

the Japanese Ministry of Education, Culture, Sports,

Science and Technology (MEXT). We thank Shusaku

Daikoku for measuring the HRMS.

T. Nakahara et al. / Carbohydrate Research 343 (2008) 1624–1635

1635

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Article Doi

Synthetic studies towards a new scaffold, spirobicycloimidazoline

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