Intermediates for incorporation of tetrahydroxypipecolic acid analogues of α- and β-D-mannopyranose into combinatorial libraries: Unexpected nanomolar-range hexosaminidase inhibitors. Synthesis of α- and β- homomannojirimycin

Article abstract of DOI:10.1016/S0957-4166(98)00365-6

Homoazasugars have the distinction as a class of natural products in that most of them have been synthesised before they were isolated. Syntheses of α-1 and β-homomannojirimycin 2 rely on the stereoselective and chemoselective sodium cyanoborohydride reduction of a [2.2.2] bicyclic imino lactone (6) to give a single [2.2.2] bicyclic amino-lactone (7). Methanolysis of 7 under basic conditions is accompanied by efficient epimerisation of the first formed α-amino-ester (8) to the more stable β-amino-ester (9) in which the 2,6-substitutents are equatorial. Both 7 and 9 are suitable intermediates for the incorporation of tetrahydroxypipecolic acid derivatives into combinatorial libraries containing α- and β-C-glycosyl analogues of aza-D-mannopyranose, respectively. Methylamides derived from 7 and 9 are shown to be specific and potent inhibitors of two β-N-acetylglucosaminidases but have no effect on an α-N-acetylgalactosaminidase. The synthesis of α- 14 and β-17 manno-pipecolic acids is also reported.

Full text of DOI:10.1016/S0957-4166(98)00365-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 3505–3516
Intermediates for incorporation of tetrahydroxypipecolic acid
analogues of α- and β-D-mannopyranose into combinatorial
libraries: unexpected nanomolar-range hexosaminidase inhibitors.
Synthesis of α- and β-homomannojirimycin
John P. Shilvock,a Robert J. Nash,b Janet D. Lloyd,b Ana L. Winters,b Naoki Asano c and
George W. J. Fleet a,∗
aDyson Perrins Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QY, UK
bInstitute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK
cFaculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-11, Japan
Received 3 September 1998; accepted 9 September 1998
Abstract
Homoazasugars have the distinction as a class of natural products in that most of them have been synthesised
before they were isolated. Syntheses of α-1 and β-homomannojirimycin 2 rely on the stereoselective and chemo-
selective sodium cyanoborohydridereduction of a [2.2.2] bicyclic imino lactone (6) to give a single [2.2.2] bicyclic
amino-lactone (7). Methanolysis of 7 under basic conditions is accompanied by efficient epimerisation of the first
formed α-amino-ester (8) to the more stable β-amino-ester (9) in which the 2,6-substitutents are equatorial. Both 7
and 9 are suitable intermediates for the incorporation of tetrahydroxypipecolic acid derivatives into combinatorial
libraries containing α- and β-C-glycosyl analogues of aza-D-mannopyranose, respectively. Methylamides derived
from 7 and 9 are shown to be specific and potent inhibitors of two β-N-acetylglucosaminidasesbut have no effect on
an α-N-acetylgalactosaminidase. The synthesis of α-14 and β-17 manno-pipecolic acids is also reported. © 1998
Elsevier Science Ltd. All rights reserved.
1. Introduction
Highly hydroxylated pipecolic acids provide an opportunity for the incorporation of a pyranose carbo-
hydrate motif into amide libraries to produce diverse and novel compounds for biological evaluation. The
natural1 distribution and biological properties2 of oxygenated pipecolic acids have attracted a number
of approaches to their synthesis.3 Modified pipecolic acids, as well as prolines, have the potential of
∗
Corresponding author.
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00365-6

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J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
inducing secondary structure in short sequences.4,5 Both bi- and mono-cyclic lactones — with relatively
reactive carbonyl groups — are flexible intermediates and allow the introduction of polyhydroxylated
proline analogues for such studies.6
This paper reports the synthesis of a bicyclic amino lactone (7) which may be used to introduce a
tetrahydroxypipecolic acid moiety (which corresponds to an α-C-mannopyranoside mimic) into amide
libraries; the formation of 7 relies on the efficient reduction of the bicyclic iminolactone by sodium
cyanoborohydride in acetic acid — conditions under which there are no nucleophiles present to cause
lactone ring opening. Ring opening of the reactive lactone 7 gives the ring opened methyl ester 8 which
undergoes an unanticipated but efficient epimerisation to 9 and should allow the generation of libraries
of β-C-mannopyranoside mimics. Reduction of 7 and 9 gives easy access to homoazasugars such as α-
1 and β-homomannojirimycin 2, while treatment with nucleophilic amines gives 3 and 4, respectively.
This paper also reports that 3 and 4 [where X=NHMe] are specific and potent inhibitors of some N-
acetylglucosaminidases; this result was completely unanticipated and augurs well for the likely value of
such libraries providing new leads for biological activity.
Naturally occurring7 and synthetic8 polyhydroxylated nitrogen heterocycles (azasugars) such as de-
oxynojirimycin have received much attention as selective inhibitors of glycosidases and glycosyltrans-
ferases. Homoazasugars with an additional anomeric hydroxymethyl such as α-HMJ 1 and β-HMJ
2, as well as some glycosides, have recently been shown to be in popular cultivated plants such as
Hyacinths9 and Aglaonema10 and are likely to be widespread in nature. For the most part, the synthesis11
of homoazasugars (2,6-imino-heptitols) has preceded their isolation as natural products; both 112,13 and
214,15 were synthesised prior to their isolation. Before the isolation of α-homonojirimycin (HNJ) as a
natural product,16 Liu17 had shown that an additional carbon substituent at the anomeric position of
DNJ might provide additional potency and specificity so that α-homonojirimycin (HNJ) and its 7-O-β-
glucopyranosyl derivative (MDL 25,637) might have significant advantages over deoxynojirimycin for
the treatment of late-onset diabetes. α-HNJ inhibited α-glucosidases and trehalase to a similar extent to
DNJ but had no inhibitory activity to other glycosidases, thus showing a greater selectivity than DNJ.
MDL 25,637 is an effective inhibitor of rat intestinal glucohydrolases in vitro and in vivo, and is more
effective against glycoprotein processing α-glucosidase II than α-glucosidase I;18 MDL 25,637 is also
a potent inhibitor of pig kidney trehalase.19 MDL 25,637 and the 5-α-galactoderivative of HNJ20 have
also been isolated as natural products and it is very probable that such compounds are defences for
plants. It is likely that these alkaloids reduce the nutritional value of plants to predators and additionally
interfere with other essential processes involving glycosidases and transferases. β-Homonojirimycin21,22
in contrast does not inhibit β-glucosidases, or indeed any other glycosidase, to any significant extent.10

J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
3507
The first synthesis12 of α-HMJ 1 involved a direct but rather inefficient reduction of the iminolactone 6.
This paper describes the efficient reduction of 6 by sodium cyanoborohydride in acetic acid to the bicyclic
aminolactone 7 which on further reduction gives access to α-HMJ 1 while reaction of 7 with methylamine
allows isolation of the α-amide 13. Reaction of 7 with methanol in the presence of sodium acetate as base
forms the α-methyl ester 8 first, which is efficiently epimerised to the β-methyl ester 9, which allows the
preparation of the β-amide 16. Thus C-aza-mannose moieties should be easily introduced into libraries
by the use of the intermediates 7 and 9. Surprisingly, screening of the amides 13 and 16 against a panel
of glycosidases showed that they are excellent and specific inhibitors of β-N-acetylglucosaminidases,
respectively; 7 and 9 may thus provide access to libraries of hexosaminidase inhibitors. The synthesis
and characterisation of α-14 and β-17 manno-pipecolic acids is also reported.
2. Synthesis
The original synthesis of α-homomannojirimycin 1 relied on reduction of the azido-ketone 5 by
phosphite with a concomitant aza-Wittig reaction, resulting in formation of a bicyclic imine lactone
6 (Scheme 1). Subsequent treatment of 6 with lithium aluminium hydride resulted in stereoselective
reduction of the imine and reductive ring opening of the lactone to give 10 in relatively low yield.
Chemoselective reduction of the imine functionality in 6 would allow the isolation of 7 which should
prove a suitable intermediate for incorporation of polyhydroxylated pipecolic acids into amide libraries.
The bicyclic lactone 7 contains the required piperidine ring with an unprotected nitrogen (capable of
elaboration by electrophilic attack) together with the carbonyl group of the lactone which is very sensitive
to nucleophilic ring opening.
All attempts at conversion of 6 to give 7 by hydrogenation were unsuccessful; it is necessary to use a
selective hydride reducing agent in a solvent which will not induce ring opening of the lactone. When
the bicyclic imine 6 was treated with sodium cyanoborohydride in acetic acid, an efficient and highly
chemo- and stereoselective reduction afforded the bicyclic amino-lactone 7 in 70% yield (Scheme 1).
The assignment of the configuration at C-6 in 7 was based on the observed four bond coupling between
H-4 and H-6 (4J4,6 1.4 Hz) in the 1H NMR spectrum, indicative of a W-system within the rigid bicyclic
framework (Fig. 1). None of the known13 C-6 epimer 7a was observable in the 1H NMR spectrum of the
crude reaction mixture; hydride delivery occurs solely from the least hindered face of the imine 6.
The relatively unstable lactone 7 is highly susceptible to nucleophilic attack. The lactone 7 with
sodium acetate in methanol gave initial ring opening to the α-amino-ester 8 accompanied by subsequent
epimerisation to the β-ester 9 in isolated yields of 63% and 20%, respectively. Treatment of 7 under more
basic conditions with sodium carbonate in methanol afforded the two esters 8 and 9 in yields of 13% and
59%. It is thus clear that, while this reaction needs to be carefully monitored, suitable conditions will
allow isolation of either 8 or 9 in good yield. The initial product 8 of ring opening of the lactone 7 has an
axial ester group, while 9 has a thermodynamically stable 2,6-diequatorially substituted piperidine ring
(Fig. 2). The relative ease of epimerisation of the ester 8 and the lack of fragmentation of the intermediate
anion are noteworthy.
Reduction of either the bicyclic lactone 7 or the α-ester 8 with Super-Hydride® in THF at −60°C
afforded 10 in yields of 64% and 67%, respectively. Subsequent deprotection of 10 with aqueous
hydrochloric acid afforded α-homomannojirimycin 1 in 79% yield. Similarly, reduction of β-ester 9 with
Super-Hydride® in THF at −60°C gave 11 in 69% yield which, on treatment with aqueous hydrochloric
acid, gave β-homomannojirimycin 2 in 82% yield. The correlation of 8 and 9 with the known natural
products 1 and 2 firmly establishes the configurations of the anomeric esters.

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J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
Scheme 1. (i) NaBH3CN, AcOH, (ii) NaOAc, MeOH, ∆, (iii) Na2CO3, MeOH, ∆, (iv) LiBHEt3, THF, −60°C, (v) HCl (aq)
Fig. 1.
The bicyclic amino-lactone 7 should provide a potential divergent intermediate for the generation
of libraries containing α-linked C-glycosyl azasugar mimics of D-mannopyranose for incorporation
into larger structures. Treatment of bicyclic amino-lactone 7 with methylamine in THF afforded the
methyl amide 12 in 93% yield (Scheme 2), and subsequent removal of the protecting groups using
methanolic hydrochloric acid afforded 13 in 86% yield. Since the lactone 7 is highly reactive towards
nucleophiles, this procedure should allow the incorporation of an α-manno-pipecolic acid moiety into

J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
3509
Fig. 2.
amide assemblies. The free manno-pipecolic acid 14 itself was obtained directly in 79% yield by the
treatment of 7 with aqueous trifluoroacetic acid.
Scheme 2. (i) MeNH2, THF, (ii) HCl, MeOH, (iii) TFA (aq)
Similarly, the β-ester 9 might be used to incorporate β-linked C-glycosyl azasugar mimics of D-
mannopyranose into larger structures. The use of the β-ester 9 has been investigated (Scheme 3) by
initial removal of the protecting groups using aqueous trifluoroacetic acid to give the ester 15; subsequent
treatment of 15 with an excess of methylamine in methanol afforded the methyl amide 16 in 65% yield
over the two steps. Although the methyl ester 15 is less reactive than the lactone 7 to nucleophilic attack,
treatment of 15 with primary amines should thus provide access to amide libraries containing a β-manno-
pipecolic acid fragment. α-manno-Pipecolic acid 17 was formed in 62% yield overall by hydrolysis of
ester 15 with aqueous sodium hydroxide and subsequent neutralisation with hydrochloric acid.
Scheme 3. (i) TFA (aq), (ii) MeNH2, THF, (iii) NaOH (aq), then HCl (aq)

3510
J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
3. Glycosidase inhibition
The most common natural occurring homoazasugar HNJ is a potent and fairly specific inhibitor of
various α-glucosidases (see earlier). α-HMJ 1 is a weak inhibitor of human liver α-mannosidases,
and has much the same potency and specificity towards human liver α-mannosidases as does DMJ,
although α-HMJ is more selective in regard to its lack of inhibition of other human glycosidases. DMJ
is usually a more potent inibitor of α-fucosidases than of α-mannosidases,23,24 and β-HMJ 2 is a potent
inhibitor of bovine epididymis α-L-fucosidase with a Ki of 0.45 µM. Neither 1 nor 2 inhibited either
α- or β-mannosidases from a number of other sources but were both good inhibitors of rat digestive
α-glucosidases.10
The amides 13 and 16 were screened against a range of glycosidases, including α-glucosidase
(brewer’s yeast and rice), α-fucosidase (human placenta and bovine kidney), β-glucosidase (Almond
emulsin), α-galactosidase (green coffee beans and Aspergillus niger), β-galactosidase (E. coli, A. niger
and bovine liver), α-mannosidase (jack bean) and naringinase (Penicillium decumbens) to see if there
was any significant recognition of either L-fucose or D-mannose. The α-amide (13) was not inhibitory to
any of the glycosidases at 650 µM; at the same concentration, the β-amide (16) was a weak inhibitor of
yeast α-glucosidase (IC50 220 µM) but not rice α-glucosidase, and also weakly inhibited α-galactosidase
from green coffee beans (IC50 90 µM). The pipecolic acid (17) was weakly inhibitory (40%) only to the
β-glucosidase at 690 µM.
However, both the α- 13 and the β-amide 16 showed strong competitive inhibition of two β-N-
acetylglucosaminidases. Against human placenta β-N-acetylglucosaminidase, using the p-nitrophenyl
glycoside as substrate, the Ki for the β-amide (16) is 39 µM and for the α-amide (13) is 0.01 µM. The
IC50 for inhibition of bovine liver β-N-acetylglucosaminidase for the two compounds were similar to the
IC50 for the human placenta enzyme, being 11 µM for 16 and 0.09 µM for 13. In contrast there was no
inhibition of α-N-acetylgalactosaminidase from chicken liver by either compound.
In summary, this paper reports the extension of the original synthetic methodology used to access
α-homomannojirimycin, via the synthesis of a key intermediate bicyclic amino-lactone bearing a D-
mannose configuration. This material was shown to be of use in the generation of α- and β-C-glycosyl
analogues of aza-D-mannopyranose. The unanticipated hexosaminidase inhibition by the amides 13 and
16 indicates that unexpected biological activities may arise from carbohydrate mimics which might be
generated by combinatorial library procedures. Such materials as the bicyclic lactone (7) and open chain
pipecolic acid ester (9) may provide valuable ‘core structures’ for incorporation into new classes of
inhibitors of carbohydrate processing enzymes. Further synthetic and modelling studies in attempts to
rationalise and optimise the hexosaminidase inhibition are in progress.25
4. Experimental
4.1. General
THF was distilled from sodium before use and hexane refers to petroleum ether boiling in the
range 60–80°C, distilled before use. All other solvents were used as supplied (AR or HPLC grade).

J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
3511
Super-Hydride® refers to lithium triethylborohydride. Other reagents were used as supplied. TLC was
performed on aluminium or plastic sheets coated with silica gel 60 F254, visualisation being effected
using 0.2% w/v cerium(IV) sulphate and 5% ammonium molybdate in 2 M sulphuric acid. Column
chromatography was performed on Sorbsil C 60 and ion-exchange chromatography was performed on
Amberlite IR-120 (H+ form). Melting points were recorded on a Kofler hot block and are uncorrected.
Optical rotations were recorded on a Perkin–Elmer 241 polarimeter with a path length of 1 dm, concen-
1
trations are quoted in g/100 ml. H NMR spectra were recorded on either a Bruker AM 500 or AMX
500 spectrometer (500 MHz) and 13C NMR spectra were recorded on a Bruker AC 200 spectrometer (50
MHz); chemical shifts (δ) are quoted in ppm and coupling constants (J) in hertz; residual signals from
solvents were used as internal reference and 13C NMR spectra in D2O were referenced to 1,4-dioxane
(δ 67.4). IR spectra were recorded on a Perkin–Elmer Paragon 1000 spectrophotometer using either thin
films on NaCl plates (film) or KBr discs (KBr). Low resolution mass spectra were recorded on either a VG
MASS LAB 20–250 using chemical ionisation (CI, NH3) or a VG Platform using atmospheric pressure
chemical ionisation (APCI). High resolution mass spectra were recorded on a VG Autospec spectrometer.
Elemental analysis was carried out by the microanalysis service of the Dyson Perrins Laboratory. The
bicyclic imine 6 was prepared as previously described.13
4.2. 7-O-tert-Butyldimethylsilyl-2,6-dideoxy-2,6-imino-3,4-O-isopropylidene-D-glycero-D-talo-
heptono-1,5-lactone 7
A solution of sodium cyanoborohydride (512 mg, 8.20 mmol) in acetic acid (10 ml) was treated with a
solution of the unstable bicyclic imine 6 (2.14 g, 6.27 mmol) in acetic acid (10 ml) at room temperature.
TLC (30% ethyl acetate:hexane) indicated the complete conversion of the starting material (Rf 0.5) to a
major product (Rf 0.3). Toluene (60 ml) was added to the solution and the mixture concentrated in vacuo.
The residue was subjected to flash chromatography on silica (30% ethyl acetate:hexane) to afford bicyclic
25
amino-lactone 7 as a colourless oil (1.50 g, 70%). [α]D −1.6 (c 1.91; CHCl3); δH (500 MHz; CDCl3)
0.07 (6H, s, Si(CH3)2), 0.90 (9H, s, SiC(CH3)3), 1.38, 1.63 (6H, 2s, C(CH3)2), 3.48 (1H, dddd, J4,6=1.4,
0
0
J
5,6=2.2, J6,7=6.5, J6,7 =7.9, H-6), 3.78 (1H, d, J2,3=2.9, H-2), 3.79 (1H, dd, J6,7=6.5, J7,7 =9.7, H-7),
0
0
0
4.04 (1H, dd, J6,7 =7.9, J7,7 =9.7, H-7 ), 4.42 (1H, dd, J2,3=2.9, J3,4=8.2, H-3), 4.55 (1H, ddd, J3,4=8.2,
J
4,5=4.5, J4,6=1.4, H-4), 4.81 (1H, dd, J5,6=2.2, J4,5=4.5, H-5); δC (50 MHz; CDCl3) −5.4 (q, Si(CH3)2),
18.2 (s, SiC(CH3)3), 23.9, 24.8 (2q, C(CH3)2), 25.8 (q, SiC(CH3)3), 54.5, 57.8 (2d, C-2, C-6), 63.5 (t,
C-7), 71.9, 73.0, 74.4 (3d, C-3, C-4, C-5), 113.6 (s, C(CH3)2), 170.8 (s, C-1); νmax (film)/cm−1 3352
(br, NH), 1773 (s, C_O); m/z (CI, NH3) 344 (MH+, 100%); HRMS m/z (CI+). Found 344.1896 (MH+),
C16H30NO6Si requires 344.1893.
4.3. Methyl 7-O-tert-butyldimethylsilyl-2,6-dideoxy-2,6-imino-3,4-O-isopropylidene-D-glycero-D-talo-
heptonate 8 and methyl 7-O-tert-butyldimethylsilyl-2,6-dideoxy-2,6-imino-3,4-O-isopropylidene-D-
glycero-D-galacto-heptonate 9
4.3.1. Method 1
A solution of bicyclic amino-lactone (7) (450 mg, 1.31 mmol) and sodium acetate (119 mg, 1.46
mmol) in methanol (10 ml) was stirred at reflux for 6 h. TLC (50% ethyl acetate:hexane) indicated the
complete conversion of the starting material (Rf 0.8) into two major products (Rf 0.5 and 0.2) and the
reaction mixture was concentrated in vacuo. The residue was subjected to flash chromatography on silica
(gradient elution: 25–60% ethyl acetate:hexane) to afford ester 8, first eluted, as a colourless oil (310 mg,
25
63%). [α]D −35.8 (c 2.27; CHCl3); δH (500 MHz; CDCl3) 0.09, 0.11 (6H, 2s, Si(CH3)2), 0.91 (9H,

3512
J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
0
s, SiC(CH3)3), 1.37, 1.51 (6H, 2s, C(CH3)2), 2.50 (1H, ddd, J5,6=10.0, J6,7=5.0, J6,7 =4.3, H-6), 3.64
0
(1H, dd, J4,5=7.2, J5,6=10.0, H-5), 3.78 (1H, dd, J6,7=5.0, J7,7 =9.9, H-7), 3.80 (3H, s, CO2CH3), 3.86
0
0
0
(1H, d, J2,3=3.0, H-2), 3.95 (1H, dd, J6,7 =4.3, J7,7 =9.9, H-7 ), 4.02 (1H, dd, J3,4=5.2, J4,5=7.2, H-4),
4.43 (1H, dd, J3,4=5.2, J2,3=3.0, H-3); δC (50 MHz; CDCl3) −5.5 (q, Si(CH3)2), 18.1 (s, SiC(CH3)3),
25.7 (q, SiC(CH3)3), 26.3, 28.1 (2q, C(CH3)2), 52.4 (q, O(CH3), 55.5, 56.9 (2d, C-2, C-6), 63.4 (t, C-
7), 72.0, 73.8, 79.4 (3d, C-3, C-4, C-5), 109.2, (s, C(CH3)2), 171.8 (s, C-1); νmax (film)/cm−1 3458
(br, NH, OH), 1737 (s, C_O); m/z (APCI+) 376 (MH+, 100%); HRMS m/z (CI+). Found 376.2154
(MH+), C17H34NO6Si requires 376.2155, and ester 9, second eluted, as a white solid (102 mg, 20%).
25
M.p. 97–99°C with phase change at 81–83°C (diethyl ether:hexane); [α]D −34.3 (c 0.47; CHCl3);
δH (500 MHz; CDCl3) 0.08 (6H, s, Si(CH3)2), 0.91 (9H, s, SiC(CH3)3), 1.41, 1.55 (6H, 2s, C(CH3)2),
0
2.65 (1H, ddd, J5,6=9.6, J6,7=6.2, J6,7 =4.6, H-6), 3.26 (1H, dd, J4,5=7.2, J5,6=9.6, H-5), 3.74 (1H, dd,
0
0
0
0
J
J
6,7=6.2, J7,7 =9.8, H-7), 3.78 (3H, s, CO2CH3), 3.82 (1H, dd, J6,7 =4.6, J7,7 =9.8, H-7 ), 4.02 (1H, d,
2,3=2.6, H-2), 4.06 (1H, dd, J3,4=5.5, J4,5=7.2, H-4), 4.56 (1H, dd, J3,4=5.5, J2,3=2.6, H-3); δC (50 MHz;
CDCl3) −5.5, −4.9 (2q, Si(CH3)2), 18.1 (s, SiC(CH3)3), 25.8 (q, SiC(CH3)3), 26.5, 28.1 (2q, C(CH3)2),
52.3 (q, CO2CH3), 57.4, 58.0 (2d, C-2, C-6), 63.4 (t, C-7), 71.8, 76.4, 81.0 (3d, C-3, C-4, C-5), 109.9 (s,
C(CH3)2), 170.1 (s, C-1); νmax (film)/cm−1 3459 (br, NH, OH), 1755 (s, C_O); m/z (APCI+) 376 (MH+,
100%); HRMS m/z (CI+). Found 376.2150 (MH+), C17H34NO6Si requires 376.2155.
4.3.2. Method 2
A solution of bicyclic amino-lactone 7 (500 mg, 1.46 mmol) and sodium carbonate (159 mg, 1.46
mmol) in methanol (10 ml) was stirred at reflux for 20 min. TLC (50% ethyl acetate:hexane) indicated
the complete conversion of the starting material (Rf 0.8) into two major products (Rf 0.5 and 0.2) and the
reaction mixture was concentrated in vacuo. The residue was subjected to flash chromatography on silica
(gradient elution: 25–60% ethyl acetate:hexane) to afford ester 8, first eluted, as a colourless oil (71 mg,
13%) and ester 9, second eluted, as a white solid (323 mg, 59%), both materials possessing identical data
to that listed in Method 1.
4.4. Methyl 7-O-tert-butyldimethylsilyl-2,6-dideoxy-2,6-imino-3,4-O-isopropylidene-D-glycero-
D-galacto-heptonate 9
A solution of ester 8 (100 mg, 0.30 mmol) and sodium carbonate (10 mg, 94 mmol) in methanol
(3 ml) was stirred at 70°C for 3 h. TLC (ethyl acetate) indicated the presence of starting material (Rf
0.8) and a major product (Rf 0.5). The solvent was removed in vacuo and the residue subjected to flash
chromatography on silica (70% ethyl acetate:hexane) to afford the starting material 8, first eluted, as
a colourless oil (21 mg, 21%) and ester 9, second eluted, as a white solid (52 mg, 52%), the material
identical to that listed above.
4.5. 1-O-tert-Butyldimethylsilyl-2,6-dideoxy-2,6-imino-4,5-O-isopropylidene-D-glycero-D-manno-
heptitol 10
4.5.1. Method 1
Super-Hydride® (1.0 M solution in THF, 0.64 ml, 0.64 mmol) was added to a stirred solution of
bicyclic amino-lactone 7 (100 mg, 0.29 mmol) in THF (3 ml) at −20°C and the reaction mixture allowed
to warm to room temperature over 30 min. TLC (60% ethyl acetate:hexane) indicated the complete
conversion of the starting material (Rf 0.9) to a major product (Rf 0.2) and saturated ammonium chloride
solution (5 drops) was added. The mixture was concentrated in vacuo and the residue subjected to flash

J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
3513
chromatography on silica (ethyl acetate) to afford 10 as a white solid (65 mg, 64%). M.p. 163–165°C,
23
20
lit.13 165–166°C; [α]D −26.8 (c 0.37; CHCl3), lit.13 [α]D −28.5 (c 1.0; CHCl3); δH (500 MHz;
CDCl3) 0.09 (6H, 2s, Si(CH3)2), 0.91 (9H, s, SiC(CH3)3), 1.37, 1.52 (6H, 2s, C(CH3)2), 2.60 (1H, m,
0
H-2), 3.30 (1H, ddd, J2,3=9.9, J1,2=3.1, J1 ,2=5.0, H-2), 3.49 (1H, dd, H-3), 3.63–3.66 (2H, m), 3.73 (1H,
dd, J=7.4, J=9.4), 3.95–4.02 (3H, m).
4.5.2. Method 2
Super-Hydride® (1.0 M solution in THF, 0.36 ml, 0.36 mmol) was added to a stirred solution of ester
8 (50 mg, 0.13 mmol) in THF (2 ml) at −30°C and the reaction mixture allowed to warm to room
temperature over 30 min. TLC (ethyl acetate) indicated the complete conversion of the starting material
(Rf 0.8) to a major product (Rf 0.2) and saturated ammonium chloride solution (5 drops) was added. The
mixture was concentrated in vacuo and the residue subjected to flash chromatography on silica (ethyl
acetate) to afford 10 as a white solid (31 mg, 67%). Material identical to that described in Method 1.
4.6. 7-O-tert-Butyldimethylsilyl-2,6-dideoxy-2,6-imino-3,4-O-isopropylidene-D-glycero-D-galacto-
heptitol 11
Super-Hydride® (1.0 M solution in THF, 1.1 ml, 1.1 mmol) was added to a stirred solution of ester 9
in THF (5 ml) at −60°C and the reaction mixture allowed to warm to room temperature over 30 min.
TLC (ethyl acetate) indicated the complete conversion of starting material (Rf 0.5) to a major product
(Rf 0.3) and saturated ammonium chloride solution (5 drops) was added. The mixture was concentrated
in vacuo and the residue subjected to flash chromatography on silica (ethyl acetate) to afford 11 as an
21
amorphous solid (95 mg, 69%). [α]D −55.9 (c 0.89; CHCl3), lit. [α]D −6.5 (c 0.80; CHCl3); δH (500
MHz; CDCl3) 0.09, 0.10 (6H, 2s, Si(CH3)2), 0.91 (9H, s, SiC(CH3)3), 1.38, 1.53 (6H, 2s, C(CH3)2),
0
0
2.50 (1H, ddd, J5,6=10.1, J6,7=4.5, J6,7 =5.8, H-6), 3.09 (1H, ddd, J2,3=2.8, J1,2=5.0, J1 ,2=4.1, H-2),
0
3.63 (1H, dd, J4,5=7.4, J5,6=10.1, H-5), 3.74 (1H, dd, J06,7=5.0, J7,7 =9.9, H-7), 3.81 (1H, dd, J1,2=4.5,
0
0
0
0
0
0
J1,1 =11.3, H-1), 3.86 (1H, dd, J1 ,2=5.8, J1,1 =11.3, H-1 ), 3.94 (1H, dd, J6,7 =4.1, J7,7 =9.9, H-7 ), 4.00
(1H, dd, J3,4=5.2, J4,5=7.4, H-4), 4.18 (1H, dd, J3,4=5.2, J2,3=2.8, H-3)
4.7. 2,6-Dideoxy-2,6-imino-D-glycero-D-manno-heptitol 1 (α-homomannojirimycin)
A solution of 10 (50 mg, 0.14 mmol) in hydrochloric acid (2.0 M, 5 ml) was stirred at room temperature
for 2 h. TLC (20% methanol:ethyl acetate) indicated the complete conversion of the starting material (Rf
0.8) to a single product (Rf 0.2). The solution was concentrated in vacuo and the residue subjected to
ion-exchange chromatography (Amberlite IR-120, H+ form: eluting with 1.0 M ammonium hydroxide)
to afford 1 as a hygroscopic solid (22 mg, 79%). [α]D +6.8 (c 0.65; H2O), lit.13 [α]D +7.45 (c 0.55;
H2O); δH (500 MHz; D2O) 2.75 (1H, m, H-2), 3.14 (1H, m, H-6), 3.59 (6H, m), 3.96 (1H, m, H-5).
24
20
4.8. 2,6-Dideoxy-2,6-imino-D-glycero-D-galacto-heptitol 2 (β-homomannojirimycin)
A solution of 11 (70 mg, 0.20 mmol) in hydrochloric acid (2.0 M, 5 ml) was stirred at room temperature
for 2 h. TLC (20% methanol:ethyl acetate) indicated the complete conversion of the starting material (Rf
0.8) to a single product (Rf 0.2). The solution was concentrated in vacuo and the residue subjected to
ion-exchange chromatography (Amberlite IR-120, H+ form: eluting with 1.0 M ammonium hydroxide)
to afford 2 as a hygroscopic solid (32 mg, 82%). [α]D −5.1 (c 0.75; MeOH), lit.14 [α]D −4.3 (c
24
20
0
1.3; MeOH); δH (500 MHz; CD3OD) 2.50 (1H, ddd, J5,6=9.8, J6,7=3.0, J6,7 =6.0, H-6), 2.76, (1H, m,

3514
J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
J2,3=1.3, H-2), 3.36 (1H, dd, J4,5=9.4, J3,4=3.1, H-4), 3.53 (1H, dd, J4,5=9.4, J5,6=9.8, H-5), 3.65 (2H,
0
m, H-1, H-10), 3.67 (1H, J7,7 =11.0, J6,7 =6.0, H-7 ), 3.84 (1H, J7,7 =11.0, J6,7=3.0, H-7), 3.87 (1H, dd,
0
0
0
J
2,3=1.3, J3,4=3.1, H-3).
4.9. Methyl 7-O-tert-butyldimethylsilyl-2,6-dideoxy-2,6-imino-3,4-O-isopropylidene-D-glycero-D-talo-
heptonamide 12
Methylamine (33% w/w solution in industrial methylated spirit, 0.2 ml, 1.75 mmol) was added to a
solution bicyclic amino-lactone 7 (200 mg, 0.58 mmol) in dry THF (3 ml) and the mixture stirred at room
temperature for 2 h. TLC (ethyl acetate) indicated the complete conversion of the starting material (Rf 0.9)
to a single product (Rf 0.5) and the reaction mixture was concentrated in vacuo. The residue was subjected
to flash chromatography on silica (80% ethyl acetate/hexane) to afford methyl amide 12 as a colourless
23
foam (203 mg, 93%). [α]D −47.0 (c 1.53; CHCl3); δH (500 MHz; CDCl3) 0.09 (6H, s, Si(CH3)2), 0.91
0
(9H, s, SiC(CH3)3), 1.42, 1.52 (6H, 2s, C(CH3)2), 2.35 (1H, ddd, J5,6=10.0, J6,7=3.4, J6,7 =3.2, H-6),
2.35 (1H, br m, OH/NH), 2.84 (3H, d, J=5.0, CONHCH3), 3.64 (1H, dd, J4,5=7.8, J5,6=10.0, H-5), 3.69
0
0
0
0
(1H, dd, J6,7=3.4, J7,7 =10.0, H-7), 3.84 (1H, m, H-2), 3.93 (1H, dd, J6,7 =4.3, J7,7 =9.9, H-7 ), 3.95 (1H,
m, H-4), 4.93 (1H, dd, J3,4=5.3, J2,3=1.9, H-3), 7.37 (1H, m, CONHCH3); δC (50 MHz; CDCl3) −5.5
(q, Si(CH3)2), 18.2 (s, SiC(CH3)3), 25.8 (q, SiC(CH3)3), 26.0 (q, CONHCH3), 26.4, 28.2 (2q, C(CH3)2),
55.0, 56.9 (2d, C-2, C-6), 61.9 (t, C-7), 69.8, 73.9, 79.4 (3d, C-3, C-4, C-5), 108.5, (s, C(CH3)2), 170.6
(s, C-1); νmax (KBr)/cm−1 3353 (br, NH, OH), 1665 (s, amide I), 1530 (m, amide II); m/z (APCI+) 375
(MH+, 100%). Found C: 54.52, H: 9.40, N: 7.39, C17H34N2O5Si requires C: 54.51, H: 9.15, N: 7.48%.
4.10. Methyl 2,6-dideoxy-2,6-imino-D-glycero-D-talo-heptonamide 13
A solution of methyl amide 12 (160 mg, 0.43 mmol) in 3% methanolic hydrochloric acid was stirred
at room temperature for 24 h. The solution was concentrated in vacuo (co-evaporation with methanol,
3×10 ml) and the residue purified by ion-exchange chromatography (Amberlite IR-120, H+ form: eluting
22
with 1.0 M ammonium hydroxide) to afford 13 as a hygroscopic foam (81 mg, 86%). [α]D −10.0 (c
0
0.66; H2O, pH 9); δH (500 MHz; D2O, pH 9) 2.61 (1H, ddd, J5,6=9.5, J6,7=3.1, J6,7 =5.6, H-6), 2.84
(3H, s, CONHCH3), 3.45 (1H, dd, J4,5=9.5, J3,4=3.0, H-04), 3.56 (1H, dd, J4,5=J5,6=9.5, H-5), 3.67 (1H,
0
0
0
d, J2,3=2.9, H-2), 3.71 (1H, dd, J6,7 =5.7, J7,7 =11.6, H-7 ), 3.79 (1H, dd, J6,7=3.1, J7,7 =11.6, H-7), 4.40
(1H, dd, J3,4=J2,3=2.9, H-3); δC (50 MHz; D2O, pH 9) 26.4 (q, CONHCH3), 58.2, 61.7 (2d, C-2, C-6),
61.0 (t, C-7), 68.5, 70.4, 73.0 (3d, C-3, C-4, C-5), 173.5 (s, C-1); νmax (KBr)/cm−1 3400 (br, NH, OH),
1654 (s, amide I), 1535 (m, amide II); m/z (APCI+) 221 (MH+, 100%); HRMS m/z (CI+). Found 221.1131
(MH+), C8H17N2O5 requires 221.1137.
4.11. 2,6-Dideoxy-2,6-imino-D-glycero-D-talo-heptonic acid 14
A solution of bicyclic amino-lactone 7 (150 mg, 0.44 mmol) in 50% aqueous trifluoroacetic acid (4
ml) was stirred at room temperature for 18 h. The solvent was removed in vacuo and the residue purified
by ion-exchange chromatography (Amberlite IR-120, H+ form: eluting with 1.0 M aqueous pyridine)
22
to afford pipecolic acid 14 as a hygroscopic foam (71 mg, 79%). [α]D +9.8 (c 0.54; H2O); δH (500
0
MHz; D2O) 3.30 (1H, ddd, J5,6=10.6, J6,7=3.1, J6,7 =6.2, H-6), 3.43 (1H, dd,0J4,5=9.6, J3,4=2.9, H-4),
0
0
3.70 (1H, dd, J4,5=9.6, J5,6=10.6, H-5), 3.73 (1H, dd, J6,7 =6.2, J7,7 =12.4, H-7 ), 3.86 (1H, dd, J6,7=3.1,
0
J7,7 =12.4, H-7), 3.89 (1H, d, J2,3=2.9, H-2), 4.40 (1H, dd, J3,4=3.0, J2,3=2.9, H-3); δC (50 MHz; D2O,
pH 9) 58.2, 62.3 (2d, C-2, C-6), 58.9 (t, C-7), 66.0, 69.5, 71.4 (3d, C-3, C-4, C-5), 170.7 (s, C-1); νmax

J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
3515
(KBr)/cm−1 3400 (br, NH, OH), 1625 (s, C_O); m/z (−ve electrospray) 106 (M−H+, 100%); HRMS m/z
(CI+). Found 208.0827 (MH+), C7H14NO6 requires 208.0821.
4.12. Methyl 2,6-dideoxy-2,6-imino-D-glycero-D-galacto-heptonamide 16
A solution of ester 9 (50 mg, 0.13 mmol) in 50% aqueous trifluoroacetic acid (2 ml) was stirred at
room temperature for 18 h. The solvent was removed in vacuo by co-evaporation with water (×3) and
the residue dissolved in methanol (2 ml). The solution was treated with methylamine (33% w/w solution
in industrial methylated spirit, 1 ml, 8.6 mmol) and stirred for 5 h. The solvent was removed in vacuo
and the residue purified by ion-exchange chromatography (Amberlite IR-120, H+ form: eluting with 1.0
M ammonium hydroxide) to afford methyl amide 16 as a hygroscopic foam (19 mg, 65%). [α]D22 +16.4
00
(c 0.72; H2O, pH 9); δH (500 MHz; D2O, pH 9) 2.56 (1H, ddd, J5,6=9.5, J6,7=3.1, J6,7 =5.1, H-6), 2.76
(3H, s, CONHCH3), 3.50 (1H, m, H-2), 3.55 (1H, dd, J4,5=9.7, J5,6=9.5, H-5), 3.60 (1H, dd, J4,5=9.7
0
0 0 0
3,4=2.9, H-4), 3.74 (1H, dd, J6,7=5.2, J7,7 =11.7, H-7), 3.79 (1H, dd, J6,7 =3.1, J7,7 =11.7, H-7 ), 4.18
J
(1H, dd, J3,4=2.9, J2,3=1.6, H-3); δC (50 MHz; D2O, pH 9) 26.5 (q, CONHCH3), 60.2 (d, C-6), 61.5 (d
and t, C-2, C-7), 68.6, 71.2, 75.4 (3d, C-3, C-4, C-5), 173.5 (s, C-1); νmax (KBr)/cm−1 3410 (br, NH,
OH), 1648 (s, amide I), 1560 (m, amide II); m/z (APCI+) 221 (MH+, 100%); HRMS m/z (CI+). Found
221.1138 (MH+), C8H17N2O5 requires 221.1137.
4.13. 2,6-Dideoxy-2,6-imino-D-glycero-D-galacto-heptonic acid 17
A solution of ester 9 (50 mg, 0.13 mmol) in 50% aqueous trifluoroacetic acid (2 ml) was stirred at
room temperature for 18 h. The solvent was removed in vacuo by co-evaporation with water (×3) and the
residue redissolved in water (2 ml) and treated with sodium hydroxide solution (0.5 M, 0.5 ml) and left
standing for 2 h. The solution was taken to pH 8 by the addition of 2 M hydrochloric acid and the solvent
removed in vacuo. The residue was purified by ion-exchange chromatography (Amberlite IR-120, H+
form: eluting with 1.0 M aqueous pyridine) to afford pipecolic acid (17) as a hygroscopic foam (17 mg,
22
0
62%). [α]D +18.7 (c 0.63; H2O); δH (500 MHz; D2O) 3.15 (1H, ddd, J5,6=10.2, J6,7=3.2, J6,7 =6.6,
0
H-6), 3.73 (1H, dd, J4,5=9.6, J3,4=3.0, H-4), 3.086 (2H, m, H-2, H-5), 3.89 (1H, dd, J6,7=3.2, J7,7 =12.6,
0
0
H-7), 4.02 (1H, dd, J6,7 =6.2, J7,7 =12.4, H-7 ), 4.45 (1H, dd, J3,4=3.0, J2,3=1.6, H-3); δC (50 MHz;
D2O, pH 9) 59.0, 62.4 (2d, C-2, C-6), 59.9 (t, C-7), 66.5, 69.3, 73.9 (3d, C-3, C-4, C-5), 171.8 (s, C-1);
ν
max (KBr)/cm−1 3400 (br, NH, OH), 1625 (s, C_O); m/z (APCI+) 208 (MH+, 100%); HRMS m/z (CI+).
Found 208.0825 (MH+), C7H14NO6 requires 208.0821.
4.14. Enzyme assays
Activity against a range of commercially available glycosidases (Sigma) was assayed at a microtitre
scale at the pH optimum for each enzyme; the range of enzymes used is listed in the discussion. The
incubation mixture was 20 µl of enzyme solution (approx. 0.1 unit per ml), 20 µl of inhibitor solution
and 100 µl of 5 mM of the appropriate p-nitrophenyl-glycopyranoside substrate made up in 50 mM
phosphate citrate buffer. Enzyme and inhibitor were pre-incubated for 15 min at 30°C (those extracted
from animal tissues which were evaluated at 37°C) before starting the reaction by addition of the
substrate. The reaction was quenched after a period of 10 min by the addition of 160 µl glycine solution
(0.4 M, pH 10.4) and absorbance measured at 405 nm. Ki values were determined for inhibitors of β-
N-acetylglucosaminidase (human placenta) with substrate concentrations ranging from 0.18 to 1.8 mM
using Lineweaver–Burk analysis.

3516
J. P. Shilvock et al. / Tetrahedron: Asymmetry 9 (1998) 3505–3516
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