α- and β-Homogalactonojirimycins (α- and β-Homogalactostatins): Synthesis and further biological evaluation

Article abstract of DOI:10.1016/S0968-0896(00)00343-6

The homoiminosugars α- and β-homogalactonojirimycins were prepared from a common intermediate, tetra-O-benzyl-D-galacto-heptenitol 6, by way of highly stereoselective reaction sequences involving, as the key steps, an internal amidomercuration (α-epimer) and a double reductive amination (β-epimer). α-Homogalactonojirimycin retains a large part of the potent activity of the parent galactonojirimycin and 1-deoxygalactonojirimycin as an inhibitor of α-galactosidases. However, by contrast with the parent iminosugars, it does not inhibit β-galactosidases, with the exception of the Jack beans enzyme. β-Homogalactonojirimycin is a weak α-galactosidase inhibitor and is completely devoid of activity towards β-galactosidases. Thus, a marked selectivity toward one family of enzymes has been achieved by the addition of an α-CH₂OH group in the structure of the parent iminosugars.

Full text of DOI:10.1016/S0968-0896(00)00343-6

Bioorganic & Medicinal Chemistry 9 (2001) 1269±1278
ꢀ- and ꢁ-Homogalactonojirimycins
(ꢀ- and ꢁ-Homogalactostatins):
Synthesis and Further Biological Evaluation
Olivier R. Martin,a,* Oscar M. Saavedra,a Fang Xie,a Li Liu,a Sylviane Picasso,b
Pierre Vogel,b Haruhisa Kizuc and Naoki Asanoc
aDepartment of Chemistry, State University of New York, Binghamton, NY 13902-6016, USA
b
Â
Institut de Chimie Organique, Universite de Lausanne, BCH, 1015 Lausanne, Switzerland
cFaculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-11, Japan
Received 26 October 2000; accepted 21 December 2000
AbstractÐThe homoiminosugars a- and b-homogalactonojirimycins were prepared from a common intermediate, tetra-O-benzyl-
d-galacto-heptenitol 6, by way of highly stereoselective reaction sequences involving, as the key steps, an internal amidomercuration
(a-epimer) and a double reductive amination (b-epimer). a-Homogalactonojirimycin retains a large part of the potent activity of the
parent galactonojirimycin and 1-deoxygalactonojirimycin as an inhibitor of a-galactosidases. However, by contrast with the parent
iminosugars, it does not inhibit b-galactosidases, with the exception of the Jack beans enzyme. b-Homogalactonojirimycin is a weak
a-galactosidase inhibitor and is completely devoid of activity towards b-galactosidases. Thus, a marked selectivity toward one
family of enzymes has been achieved by the addition of an a-CH2OH group in the structure of the parent iminosugars. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
As a result of their remarkable biological activities, pri-
marily as glycosidase inhibitors, iminosugars have been
the object of an intense research e€ort during the past
decade.1 Potential therapeutic applications, for example
for the treatment of lysosomal storage diseases (Fabry2
and Gaucher3 diseases) or of viral infections,4 are now
starting to emerge. These useful properties have prompted
the search for more ecient and/or more selective com-
pounds and iminosugar derivatives of great structural
diversity have been prepared5,6 or isolated from natural
sources.1b One modi®cation of the archetypal 1-deoxy-
nojirimycin structure that has received particular atten-
tion is the addition of a CH2OH group at the pseudoa-
nomeric carbon such as, for example, in a- and b-
homonojirimycin 17 and 2.8,9
Such compounds are truly homoiminosugars since they
result formally from the insertion of a CH2 group into
the anomeric CÀO bond of the labile 5-amino-5-deoxy-
hexoses, and constitute also the simplest examples of
iminosugar C-glycosides. Both 1 and 2 are natural
products which, interestingly, have been synthesised7À9
before their discovery in Nature.10,11 While 1 is a potent
inhibitor of several a-glucosidases,10,12 2 is a very weak
b-glucosidase inhibitor.9,12 Homoiminosugars in the
d-gluco, d-manno, l-fuco, and d-allo13 series as well as
N-alkylated derivatives have been the object of extensive
studies.12,13
*Corresponding author at new address: I.C.O.A., Faculte des Sciences,
BP 6759, 45067 Orleans cedex 2, France. Tel.: +33-2-38-49-45-81; fax:
+33-2-38-41-72-81; e-mail: olivier.martin@univ-orleans.fr
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00343-6

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O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
We have reported in a preliminary communication14 the
®rst synthesis of the galacto epimer of 1, namely a-
homogalactonojirimycin 3 (a-homogalactostatin). We
have also recently15 completed the synthesis of the b-epimer
4, b-homogalactonojirimycin (b-homogalactostatin), by
way of an internal double reductive amination, process
that we had developed for the synthesis of b-homo-
nojirimycin 2.9 We wish to report in this article the full
details of our syntheses of 3 and 4 as well as to provide
new data on the activities of 3 and 4 as glycosidase
inhibitors. The synthesis of 3 and 4 by a di€erent
approach, as well as their activity toward selected
galactosidases, have been reported recently by Fleet and
co-workers.16
derivative 9 could be obtained from 6 by a double
inversion process under Mitsunobu's conditions, by way
of the l-altro heptenitol 8. It is noteworthy that the two
displacement reactions proceeded without complica-
tions: similar reactions of the d-gluco epimer of 6 are
indeed accompanied by rearrangements and lead to
useless mixtures of several products.20
Exchange of the protecting group of the amine function
provided the benzyloxycarbonyl derivative 11 which
was submitted to internal amidomercuration using
mercury(II) tri¯uoroacetate.21 This reaction led to a labile
organomercurial which could be isolated as bromo-
mercury derivative 11a in 76% yield after ligand
exchange with KBr and ¯ash chromatography. This
product was then treated with iodine to achieve iodo-
demercuration; remarkably, the reaction gave directly
the cyclic carbamate 12 in 89% yield. Isolation of the
organomercurial intermediate was not necessary: treat-
ment of 11 with the mercury(II) salt followed by in situ
treatment of the resulting intermediate with iodine gave
12 in 71% isolated yield. This very useful reaction pro-
vided thus directly the oxygen-functionalized methylene
group as a result of the participation of the vicinal benzyl
carbamate. In addition, the cyclisation was highly ste-
reoselective and no pseudo-b-epimer could be detected.
Results and Discussion
Synthesis
Our synthesis of 3 was achieved in 10 steps from a d-
galactose derivative (5) by way of a chain±extension±
amination±cyclisation sequence (Scheme 1). This
approach was designed on the basis of the expectation
that the cyclisation of a d-galacto-con®gured amino-
heptenitol such as 11 by internal amidomercuration
would proceed with a very high degree of stereo-
selectivity7a and provide the desired pseudo-a-epimer
(i.e., an iminoheptitol with the d-glycero-l-galacto con-
®guration).
Wittig methylenation of tetra-O-benzyl-d-galactopyr-
anose 5 by Sinay's procedure17 as previously described18
gave the corresponding heptenitol 6 in high yield. The
substitution of the 6-OH group in 6 by an amino group
using an oxidation±reductive amination sequence or the
reduction of an oxime gave preponderantly the undesired
l-altro-epimer.19 However, the d-galacto amino heptenitol
The NMR parameters of cyclic carbamate 12 indicated
that the six-membered ring adopts a conformation dif-
ferent from the usual 4C1(D) form of d-galactopyranose
Scheme 1.

O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
1271
derivatives. The 3JH,H coupling constants provide evidence
that the conformation is near an inverted 1C4 chair
conformation, a consequence of the strain imposed by
the fused ®ve-membered ring.
galacto con®guration and adopts a chair conformation
with four equatorial substituents and a single axial sub-
stituent. As expected, both reduction steps were highly
stereoselective; their selectivity was not a€ected by the
presence of the incipient axial substituent in the inter-
mediate cyclic iminium ions derived from 16 and
ammonia.23
Deprotection of 12 in two steps a€orded a-homo-
galactonojirimycin 3 in 15% overall yield from 5. The
3
ring JH,H coupling constants and in particular the
magnitude of J4,5 (9.8 Hz) indicated that 3 adopts in
solution a chair conformation similar to that of the
parent d-galactopyranosides.
Cleavage of the t-butyldimethylsilyl ether of 17 gave 18, a
partially protected b-homogalactonojirimycin derivative
particularly useful for the synthesis of further glycomi-
metics containing an aza-galactose unit. Final debenzy-
lation of 18 using iodotrimethylsilane or by catalytic
hydrogenation a€orded `b-homogalactonojirimycin' 4,
thus completing a seven step synthesis from 5 (ꢀ20%
overall yield).
The epimeric b-homogalactonojirimycin 4 was reached
in six steps from the same heptenitol 6 (Scheme 2). The
synthetic plan consisted in forming the piperidine ring
of 4 by way of a double reductive amination process;22
according to our previous studies in the nojirimycin (d-
gluco) series,9 we had predicted that the double reductive
amination of an l-arabino-2,6-heptodiulose derivative
such as 16 would a€ord predominantly if not exclusively a
homoiminosugar having the l-glycero-l-galacto (pseudo-
b-d-galacto) con®guration. The diulose 16 was obtained
in three steps from 6: dihydroxylation of the double
bond with catalytic OsO4 gave the l-glycero-l-galacto
heptitol derivative 14 in high yield and with a high
degree of stereoselectivity (6S con®guration, d.e.
90%); a sample of diastereomerically pure 14 could be
obtained by crystallization from MeOH and its con®g-
uration was unambiguously established by further che-
mical transformation (see below). Selective protection of
the primary alcohol function of 14 as a t-butyldi-
methylsilyl ether gave the diol 15 which was oxidized to
the corresponding diketone under Swern conditions.
The crude diketone was submitted to reductive amina-
tion conditions (ammonium formate and NaBH3CN):
this reaction gave a single piperidine derivative, 17, in
44% combined yield from 15. As shown clearly by its
NMR parameters, compound 17 has a pseudo-b-d-
The con®guration of 14 was determined as follows
(Scheme 3). Protection of the vicinal diol function of 14
by formation of an isopropylidene acetal a€orded com-
pound 19. The isolated alcohol function of 19 was then
oxidized to give a fully protected 2-heptulose derivative
(20). The l-manno con®guration of this heptulose was
established on the basis of the NMR parameters of the
pyranoside 21 formed upon cleavage of the isopropyl-
idene group from 20 using iodine in MeOH. This reaction
gave also a small amount of the 2,7-anhydro-l-manno-
heptulose derivative 22. The major stereoisomer (14)
resulting from the dihydroxylation of 6 is thus the one
predicted from Kishi's empirical rule.24
Enzymatic assays
Compounds 3 and 4 have been submitted to a screening
of their activity as inhibitors toward a wide range of
glycosidases. The most signi®cant results, namely their
action on galactosidases, are gathered in Table 1 and
compared with the reported activity of the parent
Scheme 2.

1272
O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
Scheme 3.
Table 1. Inhibition of a- and b-galactosidases by galactonojirimycin, 1-deoxygalactonojirimycin and their homo analogues 3 and 4. IC50 and Ki
values are in mM
Iminosugars
Enzymes:
IC50
Ki
IC50
Ki
IC50
Ki
IC50
Ki
a-Gal, Co€ee beans
a-Gal, Rat epididymis
a-Gal, Human a-Gal.A
a-Gal, E. Coli
b-Gal, E.Coli
b-Gal, Bovine liver
b-Gal, A. niger
Ð
Ð
Ð
0.0007a
Ð
0.003b
Ð
0.0016a
Ð
0.022
4.5
0.21
68
0.015
nd
0.17
1.4
2.4
nd
nd
0.107
Ð
0.004c
Ð
Ð
Ð
Ð
0.17a
0.045a
Ð
0.24a
12.5a
NI
17% inh. at 1 mM
0.17d
616d
NI
NI
NI
NI
NI
NI
NI
Ð
Ð
Ð
74% inh. at 1 mMe
b-Gal, Jack beans
0.494d
Ð
4.7
0.86
aRef 25.
bRef 30.
cRef 26.
dRef 28.
eRef 27. nd=not determined. NI=no inhibition.
galactonojirimycin (5-amino-5-deoxy-d-galactose)25,28 and
1-deoxygalactonojirimycin.26,27 In addition, a-homo-
galactonojirimycin 3 is an inhibitor of chicken liver a-N-
acetylgalactosaminidase (IC50=10 mM, Ki=1.7 mM),
and the b-epimer 4 is a weaker inhibitor of the same
enzyme (IC50=220 mM, Ki=78 mM). It was found that 3
and 4 did not inhibit the following enzymes:
cated above, with the exception of the rat epididymis
galactosidase and of the human a-galactosidase A. As
expected from its altered shape and of the non-basic
character of its nitrogen atom, no activity was found for
this compound (1 mM concentration), save a very weak
e€ect on the co€ee beans a-galactosidase (58% inhibition
at 1 mM concentration).
Bovine epididymis
a-l-fucosidase
Yeast and rice
a-glucosidases
Bakers' yeast isomaltase
Caldocellum sacch.
b-glucosidase
Jack beans and almonds
The homogalactonojirimycins 3 and 4 are extremely spe-
ci®c for enzymes that hydrolyse galactosides: by contrast
with galactonojirimycin25 and its 1-deoxy derivative,29
which retain some activity toward certain glucosidases,
3 and 4 are not inhibitors of any of the enzymes that act
on non-galacto substrates.
a-mannosidases
Snail b-mannosidase
A. niger amyloglucosidase A. niger b-xylosidase
Rhizopus amyloglucosidase Jack beans
b-N-acetylglucosaminidase
Furthermore, a-homoiminosugar 3 remains a potent
inhibitor of a-galactosidases, with Ki values for compe-
titive inhibition one order of magnitude larger than those
for 1-deoxygalactonojirimycin. Interestingly, compound
3 is a good inhibitor of human lysosomal galactosidase
A (a-Gal A): this enzyme is responsible for one of the
Almonds b-glucosidase
Bovine epididymis A & B
b-N-acetylglucosaminidase
The cyclic carbamate derivative of 3, compound 13, was
also evaluated as an inhibitor of all the enzymes indi-

O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
1273
steps involved in the degradation of glycosphingolipids
in the lysosome (hydrolysis of the terminal galactose
unit from a ceramide trihexoside) and a de®ciency in
this enzyme is the cause of Fabry's disease.2 We have
shown recently that low concentrations of a-Gal A
inhibitors could be used to restore the mutant enzyme's
activity in Fabry's patients and that compound 3 was
one of the most active agents.30 These studies provide
the basis of a method of treatment of this lysosomal storage
disease (`chemical chaperone therapy'). By contrast with
both galactonojirimycin and its 1-deoxy derivative, a-
homogalactonojirimycin is completely devoid of activity
toward b-galactosidases, with the exception of the Jack
beans enzyme. It is particularly signi®cant to note that a
high degree of selectivity was achieved between two
galactosidases of the same microorganism: while the parent
iminosugar and iminohexitol inhibit both the a- and the
b-galactosidases of E. coli, the presence of the a-
CH2OH group in the homo analogue 3 abolished the
activity toward the b-glycosidase, a result that emphasizes
the usefulness of such a structural change.
inhibitor devoid of all activity toward b-galactosidases.
These results demonstrate that it is possible to modulate
the selectivity of iminosugars in the 1-deoxynojirimycin
series by the addition of a hydroxymethyl substituent in
the a-orientation.
Experimental
Optical rotations were measured with an automatic
polarimeter for solutions in a 0.1 dm cell at 22 Æ 3 ꢁC.
1H and 13C NMR spectra were recorded at 360 and
90 MHz, or at 400 and 100 MHz, respectively, on a
Bruker AM-360 instrument or on a JEOL JNM-GX
400 spectrometer, using chloroform-d as the solvent
unless otherwise indicated and TMS as the internal
reference (dTMS=0.00 ppm). TSP (sodium 3-trimethyl-
silylpropionate) was used as the reference for spectra
recorded in D2O (dTSP=0.00 ppm). For 13C NMR
spectra in CDCl3, the signal of the solvent (d=77.00)
was used as the reference. Chemical shifts (in ppm) and
coupling constants (in Hz) were obtained from ®rst-
order analysis of the spectra. A minus sign (À) is used to
indicate negative 13C NMR-signals in the DEPT mode
(yy=135ꢁ).
b-Homogalactonojirimycin 4 is a generally weaker inhi-
bitor than 3. Compound 4 still retains some activity
towards the co€ee beans a-galactosidase. However, as had
been noted by Fleet and co-workers,16b compound 4 is
devoid of inhibitory activity towards all the b-galactosi-
dases investigated. Thus, if an increased selectivity towards
a-galactosidases is observed for the a-homoiminosugar 3, a
similar trend does not exist for the b-epimer: the presence of
the b-CH2OH group is detrimental to the biological activity
of this compound. This behaviour parallels that of a- and
b-homonojirimycins 1 and 2 towards a- and b-glucosidases.
Analytical TLC was performed on glass plates pre-
coated with silica gel 60 F-254 as the adsorbent (layer
thickness: 0.25 mm). The developed plates were air-dried,
exposed to UV light for inspection, sprayed with a
solution of ammonium phosphomolybdate, and heated
to 120±140 ꢁC. Flash chromatography was performed
using silica gel 60 (230±400 mesh). The following solvent
systems were used: A, hexane/ethyl acetate 1:1; B, 1:3;
C, 1:4; D, 1:5; E, 1:6; F, 1:9.
The detrimental e€ect of the b-CH2OH group may be
justi®ed by steric e€ects. However, as it is now well
documented for b-glucosidases,31 the lack of a heteroatom
at the pseudo-anomeric center may be an important
factor as well: the fact that galactonojirimycin (i.e. 5-
amino-5-deoxy-d-galactose) is generally a potent inhi-
bitor of mammalian b-galactosidases,28 much more
potent than its 1-deoxy derivative, may indeed be
explained by the fact that the amino-sugar can exist
with a b-hydroxy group and thus interact favorably
with a lateral proton donor.31
Separations by HPLC were achieved using a preparative
HPLC system equipped with a gradient programmer, a
variable wavelength UV detector, and a 21.2Â250 mm
preparative column of silica gel 60 (10 mm).
Solvents were evaporated under reduced pressure and
below 40 ꢁC. Catalytic hydrogenations and hydrogen-
olysis under pressure (P>1 atm) were performed in a
stainless steel benchtop pressure reactor equipped with a
magnetic stirring drive and a pressure and temperature
controller (maximum pressure: 2000 psig).
Conclusion
The synthesis of a- and b-homogalactonojirimycins 3
and 4 has been achieved in nine and six steps respec-
tively from a common precursor, d-galacto-heptenitol
derivative 6. Both compounds were obtained by way of
highly stereoselective processes which provided control
of the pseudoanomeric con®guration, namely an internal
amido-mercuration for the a-epimer, and a double
reductive amination of a 2,6-heptodiulose for the b-epi-
mer.
3,4,5,7-Tetra-O-benzyl-1,2-dideoxy-D-galacto-hept-1-enitol
(6). To a suspension of methyltriphenylphosphonium
bromide (7.10 g, 19.9 mmol) in dry toluene (120 mL) was
added dropwise a 1.6 M solution of butyllithium in
hexane (12 mL, 19.2 mmol) at 0 ꢁC under N2 and the
reaction mixture was stirred for 2 h at room temperature. A
suspension of 2,3,4,6-tetra-O-benzyl-a-d-galactopyranose
14 (3.58 g, 6.62 mmol) in dry toluene (30 mL) was added
in one portion and the reaction mixture was stirred for
48 h at the same temperature. The reaction was quenched
with acetone (20 mL), the mixture was diluted with ether
and extracted with water (100 mL). The aqueous phase
was extracted with ether (2Â100 mL), the organic layers
Compound 3 is a potent a-galactosidase inhibitor; it is
much more selective for a-galactoside-hydrolysing
enzymes than the parent galactonojirimycin and 1-
deoxygalactonojirimycin. The b-epimer 4 is a weaker

1274
O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
were combined, dried (Na2SO4) and concentrated. The
residue was puri®ed by ¯ash chromatography (solvent
C) to a€ord 6 (3.0 g, 84%) as a syrup: [a]1D8 À10.8 (c 1.2,
138.28, 138.30 and 138.42 (4ArC). Anal. calcd for
C35H38O5: C, 78.04; H, 7.11. Found: C, 78.08; H, 7.15.
1
CHCl3); H NMR: d 3.04 (d, 1H, J6,OH=5.2 Hz, HO-
3,4,5,7-Tetra-O-benzyl-1,2,6-trideoxy-6-phthalimido-D-
galacto-hept-1-enitol (9). To a solution of heptenitol 8
(730 mg, 1.36 mmol), triphenylphosphine (1.07 g,
4.08 mmol) and phthalimide (400 mg, 4.71 mmol) in dry
THF (80 mL) was added dropwise DEAD (0.64 mL,
4.06 mmol) at room temperature under N2. After having
been stirred overnight, the reaction mixture was con-
centrated and the residue was triturated with ether
(5 mL). The precipitate was removed by ®ltration and
the ®ltrate concentrated under reduced pressure. The
residue was puri®ed by ¯ash chromatography (solvent
D) to a€ord 9 (720 mg, 79%) as a syrup: [a]1D8 +5.8 (c
1.55, CHCl3); 1H NMR: d 3.74 (dd, 1H, J4,3=6.6,
C6), 3.49 (dd, 1H, J7A,6=6.5, J7A,7B=9.4 Hz, H-7A),
3.54 (dd, 1H, J7B,6=6.2 Hz, H-7B), 3.8 (dd, 1H,
J5,4=5.9, J5,6=1.5 Hz, H-5), 3.83 (dd, 1H, J4,3=4.0 Hz,
H-4), 4.09 (dd, 1H, J3,2=7.9, J3,1A=0.4, J3,1B=0.8 Hz,
H-3), 4.13 (m, 1H, H-6), 4.34±4.77 (m, 8H,
OCHAHBPh), 5.32 (ddd, 1H, J1A,1B=1.6, J1A,2
4
=
10.3 Hz, H-1A), 5.35 (ddd, 1H, J1B,2=17.4 Hz, H-1B),
5.89 (ddd, 1H, H-2), 7.16±7.38 (m, 20H, 4 C6H5); 13C
NMR: d 69.57 (C-6), 70.21, 71.11, 73.00 (2C) and 75.06
[all (À), C-7 and 4 OCH2Ph], 76.61, 80.68 and 82.06 (C-
3,4,5), 118.94 [(À), C-1], 127.4±128.2 (ArCH), 135.66 (C-
2), 137.98, 138.08, 138.13 and 138.18 (4 ArC). Anal. calcd
for C35H38O5: C, 78.04; H, 7.11. Found: C, 77.72; H, 7.11.
J4,5=2.8 Hz, H-4), 3.77 (dd, 1H, J7A,6=5.1, J7A,7B
=
10.0 Hz, H-7A), 4.03 (br t, 1H, J7B,6=9.4 Hz, H-7B),
4.06 (br dd, 1H, J3,2=7.6, J3,1A=1.0, J3,1B=0.5 Hz, H-
3), 4.21 (dd, 1H, J5,6=8.3 Hz, H-5), 4.26±4.84 (m, 8H, 4
OCHAHBPh), 4.92 (ddd, 1H, H-6), 5.37 (ddd, 1H,
J1A,1B=1.7, J1A,2=17.4 Hz, H-1A), 5.38 (ddd, 1H,
J1B,2=10.3 Hz, H-1B), 5.92 (ddd, 1H, H-2), 6.97±7.72
(m, 24H, 4 C6H5, PhthN); 13C NMR: d 52.25 (C-6),
67.07, 70.71, 72.37, 73.15 and 74.64 [all (À), C-7, 4
OCH2Ph], 77.12, 81.45, 83.05 (C-3, 4, 5), 119.41 [(À), C-
1)], 122.91, 127.2±128.2 and 133.46 (ArCH), 135.48 (C-
2), 132.08 and 138.02±138.5 (5ArC), 168.64 (CˆO).
Anal. calcd for C43H41NO6: C, 77.34; H, 6.19. N, 2.10.
Found: C, 77.26; H, 6.20; N, 2.08.
3,4,5,7-Tetra-O-benzyl-1,2-dideoxy-6-O-(4-nitrobenzoyl)-
L-altro-hept-1-enitol (7). To a cold (0 ꢁC), stirred solution
of 6 (130 mg, 0.24 mmol), triphenylphosphine (184 mg,
0.72 mmol) and 4-nitrobenzoic acid (160 mg, 0.96 mmol)
in dry THF (20 mL) was added dropwise DEAD
(0.15 mL, 0.95mmol) under N2. After having been stirred
overnight, the reaction mixture was concentrated under
reduced pressure. The residue was puri®ed by ¯ash
chromatography (solvent F) to a€ord 7 (130 mg, 78%)
1
as a syrup. H NMR: d 3.74 (t, 1H, J4,3=J4,5=5.3 Hz,
H-4), 3.80 (m, 1H, J7A,6=3.5, J7A,7B=11 Hz, H-7A),
3.90 (m, 1H, J7B,6=6.5 Hz, H-7B), 4.07 (dd, 1H,
J5,6=3.5 Hz, H-5), 4.12 (dd, 1H, J3,2=7.6 Hz, H-3),
4.22±4.81 (m, 8H, 4 OCHAHBPh), 5.32 (dd, 1H, J1A,1B
1.5, J1A,2=10.4 Hz, H-1A), 5.38 (dd, 1H, J2,1B
=
=
3,4,5,7-Tetra-O-benzyl-6-[(benzyloxycarbonyl)amino]-1,2,6-
trideoxy-D-galacto-hept-1-enitol (11). Compound
9
17.6 Hz, H-1B), 5.80 (dt, 1H, H-6), 5.92 (ddd, 1H, H-2),
7.18±7.53 (m, 20H, 4 C6H5), 7.86±8.24 (m, 4H, C6H4);
13C NMR: d 68.71, 70.54, 72.91, 73.17 and 74.49 [all
(À), C-7 and 4 OCH2Ph], 74.68, 78.77, 80.74 and 81.69
(C-3,4,5 and 6), 119.14 [(À), C-1)], 123.44, 127.47±
128.33 and 130.70 (ArCH), 135.58 (C-2), 135.79, 137.97,
138.07 and 138.26 (4 ArC), 163.80 (CˆO).
(830 mg, 1.24 mmol) was heated in MeOH (80 mL) in
the presence of hydrazine hydrate (3 mL) at a Tꢂ70 ꢁC
for 1 h. The reaction mixture was concentrated under
reduced pressure. The residue was immediately dis-
solved in THF (100 mL) containing anhydrous K2CO3
(15 g) and the slurry was stirred under N2 at 0 ꢁC for
5 min. A solution of benzyl chloroformate (0.8 mL) in
THF (5 mL) was added at 0 ꢁC and the mixture was
stirred for 1 h at the same temperature. Water (30 mL)
was then added and the mixture was extracted with
ether (2Â50 mL). The organic phases were combined,
dried (Na2SO4), and concentrated. The residue was
puri®ed by ¯ash chromatography (solvent E) to a€ord
compound 11 (700 mg, 84%) as a syrup; [a]2D3 +1.7 (c
3,4,5,7-Tetra-O-benzyl-1,2-dideoxy-L-altro-hept-1-enitol
(8). To a solution of p-nitrobenzoate 7 (110 mg,
0.16 mmol) in MeOH (3 mL) was added a solution of
MeONa (5 mg) in MeOH (0.8 mL). The reaction mix-
ture was stirred for 5 h, and then neutralised with
Amberlite1 IR-120 (H+) ion exchange resin. The resin
was removed by ®ltration, washed with MeOH and water,
and the ®ltrate was concentrated. The residue was puri®ed
by ¯ash chromatography (solvent D) to give 8 (70 mg,
1
1.15, CHCl3); H NMR: d 3.45 (br t, 1H, J7A,6=8.4,
J7A,7B=9.3 Hz, H-7A), 3.55 (dd, 1H, J7B,6=5.8 Hz, H-
7B), 3.59 (dd, 1H, J4,3=3.7, J4,5=7.5 Hz, H-4), 4.02 (dd,
1H, J5,6=1.1 Hz, H-5), 4.11 (dd, 1H, J3,2=7.8 Hz, H-3),
4.33 (dddd, 1H, J6,NH=8.3 Hz, H-6), 4.30±4.65 (m, 8H,
4 OCHAHBPh), 5.04 (d, 1H, JA,B=12.1 Hz, Cbz-HA),
5.07 (d, 1H, Cbz-HB), 5.31 (br d, 1H, J1A,1B=0,
J1A,2=10.3 Hz, H-1A), 5.31 (d, 1H, NH), 5.37 (br d, 1H,
J1B,2=17.4 Hz, H-1B), 5.96 (ddd, 1H, H-2), 7.07±7.37
(m, 25H, 5 C6H5); 13C NMR: d 50.30 (C-6), 66.56,
69.59, 70.19, 72.79, 73.47 and 74.96 [all (À), C-7, 5
OCH2Ph], 75.89, 80.50, 81.42 (C-3, 4, 5), 118.68 [(À), C-
1)], 127.40±128.36 (ArCH), 136.07 (C-2), 138.03 (2C)
and 138.34 (2C) (ArC), 156.02 (CˆO). Anal. calcd for
C43H45NO6: C, 76.87; H, 6.75. N, 2.08. Found: C,
76.52; H, 6.76; N, 2.08.
77%) as a syrup: [a]D18 À10.8 (c 1.2, CHCl3); H NMR: d
1
2.82 (d, 1H, JHO,6=4.0 Hz, HO-C6), 3.61 (dd, 1H,
J7A,6=6.3, J7A,7B=9.9 Hz, H-7A), 3.67 (dd, 1H,
J7B,6=3.0 Hz, H-7B), 3.78 (dd, 1H, J5,4=4.0, J5,6=
6.2 Hz, H-5), 3.8 (dd, 1H, J4,3=5.4 Hz, H-4), 4.14 (m,
1H, H-6), 4.15 (br dd, 1H, J3,2=7.8, J3,1A=0.6,
J3,1B=0.9 Hz, H-3), 4.37±4.81 (s and 3 AB, 8H, 4
OCHAHBPh), 5.31 (ddd, 1H, J1A,1B=1.7, J1A,2=10.3 Hz,
H-1A), 5.34 (ddd, 1H, J1B,2=17.3 Hz, H-1B), 5.9 (ddd,
1H, H-2), 7.16±7.35 (m, 20H, 4 C6H5); 13C NMR: d 70.52
[(À) C-7], 70.90 (C-6), 71.35, 72.74, 73.22 and 74.60 [all
(À), 4 OCH2Ph], 79.43, 81.46, 82.51 (C-3, 4, 5), 118.77
[(À), C-1], 127.39±128.24 (ArCH), 135.83 (C-2), 138.14,

O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
1275
1,3,4,5-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-D-glycero-
L-galacto-heptitol O(7), N-cyclic carbamate (12). A
solution of compound 11 (200 mg, 0.31 mmol) and mer-
curic tri¯uoroacetate (250 mg, 0.62 mmol) in dry THF
(15 mL) was stirred for 48 h at room temperature in the
dark and under N2. Solid NaHCO3 (ꢀ200 mg) was then
added and the suspension stirred for 2 h. To the cold
(0 ꢁC) reaction mixture was added dropwise a solution
of I2 (500mg) in THF (30 mL) until persistence of the
brown color. The mixture was stirred for 45min (0±
25ꢁC). An aqueous solution of Na2S2O3 [10% (w/v),
10 mL] was then added. The mixture was extracted with
ether (2Â40 mL). The organic phases were combined,
dried (Na2SO4), ®ltered over Celite and concentrated.
The residue was puri®ed by ¯ash chromatography (sol-
vent C then A) to give iminoheptitol 12 (127 mg, 71%)
as a syrup: [a]1D9 +32.4 (c 3.3, CHCl3); 1H NMR: d 3.32
(dd, 1H, J5,6=2.4, J4,5=3.9 Hz, H-5), 3.82 (br dd, 1H,
an ion-exchange column (IR-120 (H+) resin). The resin
was washed with water and the product was then eluted
with 10% aqueous NH3. The eluted fractions were con-
centrated, thus a€ording iminoheptitol 3 (10 mg, 81%).
A very pure sample was obtained by chromatography
on Dowex 1X2 (OHÀ) ion exchange resin (elution with
H2O), followed by chromatography on CG-50 (NH+4 )
ion exchange resin (elution with water) (yield of pur-
i®cation: ꢀ60%). [a]D +86.3 (c 0.15, H2O) [lit.16b [a]D
+72.0 (c 0.54, H2O)]; 1H NMR (D2O, 400 MHz): d 3.05
(m, 1H, H-2), 3.30 (m, 1H, H-6), 3.67 (dd, 1H,
J1A,2=7.5, J1A,1B=11.0 Hz, H-1A), 3.70 (dd, 1H, J3,4
=
3.0, J4,5=9.8 Hz, H-4), 3.72 (dd, 1H, J1B,2=5.8 Hz, H-1B),
3.78±3.85 (m, 2H, H-7A,7B), 4.00 (dd, 1H, J2,3=2.6 Hz,
H-3), 4.04 (dd, 1H, J5,6=5.9 Hz, H-5); 13C NMR (D2O,
100 MHz): good agreement with lit. data.16b HR-
FABMS (matrix: glycerol), calcd for C7H16NO5
([M+H]+): m/z 194.1029; found: 194.1028.
J3,4=2.7 Hz, H-4), 3.87 (dd, 1H, J1A,2=3.0, J1A,1B
=
11.3 Hz, H-1A), 3.94 (dd, 1H, J2,3=6.8 Hz, H-3), 3.95
(ddd, 1H, J6,7A=4.0, J6,7B=8.0 Hz, H-6), 4.01 (dd, 1H,
J1B,2=9.9 Hz, H-1B), 4.08 (dd, 1H, J7A,7B=8.6 Hz, H-
7A), 4.15 (br t, 1H, H-7B), 4.30±4.72 (m, 8H, 4
OCH2Ph), 4.56 (br ddd, 1H, H-2), 7.10±7.38 (m, 20H, 4
C6H5); 13C NMR: d 49.61 and 51.63 (C-2,6), 62.82 and
65.59 (C-1,7), 71.67, 72.33, 72.38 and 73.84 [all (À), 4
OCH2Ph], 73.35, 74.00, 75.38 (C-3, 4,5), 127.50±128.60
(ArCH), 137.13, 137.86, 138.04 and 138.48 (4ArC),
157.69 (CˆO). Anal. calcd for C36H37NO6: C, 74.59; H,
6.43; N, 2.42. Found: C, 74.67; H, 6.44; N, 2.37.
1,3,4,5-Tetra-O-benzyl-L-glycero-L-galacto-heptitol (14).
To a solution of heptenitol 6 (1.88 g, 4.49 mmol) and
N-methylmorpholine N-oxide (900 mg, 7.68 mmol) in a
mixture of acetone (36 mL) and water (9 mL) was added
a 2.5% (w/v) solution of OsO4 in tBuOH (1.8 mL) and
the reaction mixture was stirred for 14 h at room tem-
perature. A 5% (w/v) solution of NaHSO3 in water
(20 mL) was then added, the resulting mixture was passed
through a layer of Florisil and the Florisil layer was
washed with EtOAc (100 mL). The eluted phase was
extracted with N H2SO4 (50 mL), water (50 mL) and
brine (50 mL), dried (Na2SO4), and concentrated to
a€ord a homogeneous mixture of l-glycero-l-galacto
and d-glycero-l-galacto epimers [ꢀ20:1(NMR), 1.96 g,
98%]. The major epimer 14 was obtained as a solid by
crystallization from MeOH: mp 101±102 ꢁC; [a]D À16 (c
1.0, CHCl3); 1H NMR: d 3.49 (dd, 1H, J1A,1B=9.4,
J1A,2=6.5 Hz, H-1A), 3.55 (dd, 1H, J1B,2=5.9 Hz, H-
1B), 3.61 (dd, 1H, J7A,6=5.1, J7A,7B=11.5 Hz, H-7A),
3.68 (dd, 1H, J7B,6=5.1 Hz, H-7B), 3.74 (dd, 1H,
J5,4=4.0, J5,6=7.2 Hz, H-5), 3.89 (ddd, 1H, H-6), 3.92
(dd, 1H, J3,2=2.1, J3,4=5.3 Hz, H-3), 3.94 (dd, 1H, H-
2,6-Dideoxy-2,6-imino-D-glycero-L-galacto-heptitol O(7),
N-cyclic carbamate (13). To a solution of compound 12
(100 mg, 0.17 mmol) in a mixture of AcOH (10 mL) and
EtOH (40 mL), Pd/C (10% Pd, 100 mg) was added and
the reaction mixture was stirred overnight under H2 (90
psi, 50 ꢁC) in a Parr hydrogenation apparatus. The catalyst
was removed by ®ltration and the solution concentrated
under reduced pressure to give compound 13 (28 mg,
75%) as a solid: mp 210 ꢁC (dec.); [a]1D8 À36.1 (c 0.36,
1
H2O); IR nmax (®lm) 1728 (CˆO). H NMR (D2O): d
3.78 (dd, 1H, J1A,1B=12.9, J1A,2=1.3 Hz, H-1A), 3.80
(dd, 1H, J5,6=2.0, J4,5=5.8 Hz, H-5), 3.91 (t, 1H, J3,4=
4), 4.13 (ddd, 1H, H-2), 4.41±4.73 (m, 8H, 4
OCHAHBPh), 7.20±7.32 (m, 20H, 4 C6H5); 13C NMR: d
63.62 [(À) C-7], 69.76 (C-2), 71.07 [(À) C-1], 71.66 (C-6),
73.30, 73.40, 74.09 and 74.13 [all (À), 4 OCH2Ph], 78.15
(C-3), 78.51 (C-5), 79.61 (C-4), 127.7±128.4 (ArCH),
137.73, 137.78, 137.85 and 137.90 (4 ArC). Anal. calcd
for C35H40O7: C, 73.40; H, 7.04. Found C, 73.23; H,
7.08.
J4,5=5.8 Hz, H-4), 3.95 (dd, 1H, J1B,2=8.4 Hz, H-1B),
3.99 (ddd, 1H, J2,3=2.9 Hz, H-2), 4.15 (dd, 1H, H-3),
4.24 (ddd, 1H, J6,7A=4.2, J6,7B=9.0 Hz, H-6), 4.31 (dd,
1H, H-7A), 4.41 (t, 1H, J7A,7B=9.0 Hz, H-1B); 13C NMR
(D2O; internal reference CH3OH d 49.60): d 50.47 and
56.28 (C-2,6), 58.43 and 64.88 [(À), C-1,7], 65.03, 69.79,
71.34 (C-3, 4, 5), 160.65 (CˆO). Anal. calcd for
C8H13NO6: C, 43.84; H, 5.98; N, 6.39. Found: C, 43.60;
H, 6.05; N, 6.17.
1,3,4,5-Tetra-O-benzyl-7-O-(t-butyldimethylsilyl)-L-glycero-
L-galacto-heptitol (15). To a solution of the mixture of
epimeric triols obtained in the preceding reaction
(1.54 g, 2.7 mmol) in anhydrous CH2Cl2 (10 mL) was
added successively DMAP (80 mg), Et3N (0.56 mL) and
t-butyldimethylsilyl chloride (450 mg, 3.0 mmol). The
mixture was stirred for 1 h at room temperature. The
solvent was removed under reduced pressure and the
residue puri®ed by ¯ash chromatography (solvent D) to
a€ord a mixture of silylated epimers [1.54 g, 82%; ratio
ꢀ20:1 (NMR)]. Data for the major isomer (15): 1H
NMR: d 0.04 and 0.05 [2s, 6H, Si(CH3)2], 0.89 [s, 9H,
SiC(CH3)3], 3.52 (dd, 1 H, J1A,1B=9.4, J1A,2=6.5 Hz,
2,6-Dideoxy-2,6-imino-D-glycero-L-galacto-heptitol (ꢀ-
homogalactonojirimycin) (3). To a solution of 13
(14 mg, 0.064 mmol) in MeOH (1 mL) was added 50%
aqueous KOH (5 mL) and the mixture was stirred
overnight at room temperature. Thereafter the solution
was heated to 60 ꢁC for 2 h. The pH of the cooled reac-
tion mixture was then reduced to ꢀ9 using MeOH-
washed IR-120(H+) ion-exchange resin. The resin was
removed by ®ltration and the volume of the ®ltrate was
reduced to about 1 mL. The solution was loaded onto

1276
O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
H-1A), 3.58 (dd, 1H, J1B,2=6.3 Hz, H-1B), 3.73 (dd,
1H, J7A,6=5.2, J7A,7B=10.2 Hz, H-7A), 3.79 (dd, 1H,
J5,4=3.5, J5,6=7.7 Hz, H-5), 3.8 (dd, 1H, J7B,6=3.6 Hz,
H-7B), 3.92 (dd, 1H, J3,2=3.5, J3,4=6.4 Hz, H-3), 3.93
(ddd, 1H, H-6), 4.10 (dd, 1H, H-4), 4.20 (ddd, 1H, H-2),
4.46±4.78 (m, 8H, 4 OCHAHBPh), 7.21±7.36 (m, 20H, 4
C6H5); 13C NMR: d À5.41 [2C, Si(CH3)2], 18.20
[SiC(CH3)3], 25.85 [SiC(CH3)3], 64.03 [(À) C-7], 69.67
(C-2), 71.19 (C-6), 71.39 [(À) C-1], 72.84, 73.21, 73.42
and 74.59 [all (À), 4 OCH2Ph], 77.65 (C-3), 78.13 (C-5),
79.52 (C-4), 127.5±128.1 (ArCH), 138.08 (2C), 138.25
and 138.44 (4 ArC). Anal. (mixture of epimers). Calcd for
C41H54O7Si: C, 71.69; H, 7.92. Found: C, 71.82; H, 8.01.
3.99 (dd, 1H, H-5), 4.05 (dd, 1H, H-3), 4.42±5.05 (m,
8H, 4 OCHAHBPh), 7.23±7.40 (m, 20H, 4 C6H5); 13C
NMR: d À5.65, À5.71 [Si(CH3)2], 14.16 [SiC(CH3)3],
25.27 [SiC(CH3)3], 57.01 (C-2), 60.52 (C-6), 61.95 [(À)
C-7], 69.85 [(À) C-1], 71.90, 73.08, 73.78 and 75.05 [all
(À), 4 OCH2Ph], 74.01 (C-3), 76.56 (C-5), 85.92 (C-4),
126.9±128.1 (ArCH), 137.90, 138.45, 138.70 and 138.96
(4 ArC). Anal. calcd for C41H53NO5Si: C, 73.72; H,
8.00; N, 2.10. Found: C, 73.61; H, 8.03; N, 2.15.
1,3,4,5-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-L-glycero-
L-galacto-heptitol (18). A solution of imino heptitol 17
(242 mg, 0.36 mmol) in a mixture of THF (2 mL), water
(2 mL), and CH3COOH (6 mL) was stirred overnight at
55 ꢁC. The solution was then co-evaporated under
reduced pressure with MeOH (2Â50mL) and then toluene
(2Â50 mL). The residue was puri®ed by ¯ash chromato-
graphy (solvent A for elution of impurities, then 100%
EtOAc) to give pure 18 (157 mg, 78%): solid; mp 81±
3,4,5,7-Tetra-O-benzyl-1-O-(t-butyldimethylsilyl)-L-arabino-
2,6-heptodiulose (16). To a solution of oxalyl chloride
(0.89 mL, 10.2 mmol) in anhydrous CH2Cl2 (25 mL) was
added dropwise
a solution of DMSO (0.79 mL,
11.1 mmol) in CH2Cl2 (5 mL) at À78 ꢁC over a period of
3 min; the mixture was further stirred for 10 min at the
same temperature. A solution of diol 15 (1.3 g,
1.89 mmol) in anhydrous CH2Cl2 (10 mL) was added
dropwise while the temperature of the mixture was
maintained below À60 ꢁC. The mixture was stirred for
1 h at À78 ꢁC. Et3N (7.1 mL) was then added dropwise,
the mixture was stirred for 15 min at À78 ꢁC and then
allowed to warm-up to room temperature. The solids
were removed by ®ltration through a membrane
(Magna-R, Nylon, 0.22 micron), the ®ltrate was con-
centrated, co-evaporated once with toluene (15 mL),
and dried under high vacuum to give crude 16 (1.5 g).
This product was used in the next step without pur-
i®cation. A sample of crude 16 was rapidly processed
[extraction with cold N HCl and brine] for analysis by
NMR. 13C NMR: d À5.89, À5.95 [Si(CH3)2], 18.20
[SiC(CH3)3], 25.35 [SiC(CH3)3], 68.13 [(À) C-1], 72.31,
72.77, 73.60, 73.81 and 73.96 [all (À), C-7 and 4
OCH2Ph], 79.97, 80.59 and 81.41 (C-3, 4, 5), 127.5±
128.1 (ArCH), 136.33, 136.52, 136.74 and 136.99 (4
ArC), 206.48 and 207.27 (C-2, 6).
82 ꢁC; [a]D À11.8 (c 1.1, CHCl3); H NMR: d 2.2 (br s,
1
1H, HO-C7), 2.61 (ddd, 1H, J=9.1, 5.4, 3.3 Hz, H-6),
2.83 (ddd, 1H, J2,1A=8.8, J2,1B=6.2, J2,3 ꢀ0.0 Hz, H-2),
3.35 (dd, 1H, J1A,1B=8.8 Hz, H-1A), 3.47 (dd, 1H, H-
1B), 3.52±3.57 (m, 2H, H-4, 5), 3.73±3.78 (m, 2H, H-
7A,7B), 4.0 (br s, 1H, H-3), 4.36±4.96 (m, 8H, 4
OCHAHBPh), 7.17±7.37 (m, 20H, 4 C6H5); 13C NMR: d
57.32 (C-2), 60.85 (C-6), 62.36 [(À) C-7], 69.82 [(À) C-1],
72.11, 73.27, 74.23 and 75.04 [all (À), 4 OCH2Ph], 74.11
(C-3), 77.12 (C-5), 85.84 (C-4), 127.4±128.3 (ArCH),
137.80, 138.41 (2C), and 138.76 (ArC). Anal. calcd for
C35H39NO5: C, 75.92; H, 7.10; N, 2.53. Found: C,
75.84; H, 7.05; N, 2.48.
2,6-Dideoxy-2,6-imino-L-glycero-L-galacto-heptitol (ꢁ-
Homogalactonojirimycin 4). To a solution of imino hep-
titol 18 (127.6 mg, 0.23 mmol) in CH2Cl2 (4 mL) was
added, dropwise at 0 ꢁC, iodotrimethylsilane (0.25 mL).
The reaction mixture was allowed to warm up to room
temperature and stirred for 12 h. Water (25 mL) and
CH2Cl2 (25 mL) were added, the organic phase was
separated, and the aqueous phase was extracted with
CH2Cl2 (25 mL). The volume of the aqueous phase was
then reduced to 2 mL by evaporation under reduced
pressure. The solution was loaded onto a column of
Dowex 1x2-200 (OHÀ) ion-exchange resin. The product
was eluted with water (40 mL) and the fractions con-
taining 4 were combined and lyophilized to a€ord 4 as a
1,3,4,5-Tetra-O-benzyl-7-O-(t-butyldimethylsilyl)-2,6-di-
deoxy-2,6-imino-L-glycero-L-galacto-heptitol (17). To a
stirred solution of crude 16 (1.5 g) in MeOH (25 mL)
was added HCO2NH4 (227 mg, 3.6 mmol), powdered
molecular sieves (3A, 200 mg), and then, NaBH3CN
(380 mg, 6.1 mmol) in one portion. The reaction mixture
was stirred for 1 h at room temperature. The solids were
removed by ®ltration through Celite, the Celite bed was
washed with MeOH (100 mL), the ®ltrate and washings
were combined and concentrated under reduced pressure.
The residue was dissolved in CH2Cl2 (50 mL), the solu-
tion washed with water (30 mL), saturated aqueous
NaHCO3 (30 mL) and brine (20 mL), dried (Na2SO4),
and concentrated. The residue was puri®ed by ¯ash
chromatography (solvent C), thus a€ording pure 17
1
glass (34.1 mg, 68%); H NMR (CD3OD): good agree-
ment with lit. data.16b 13C NMR (CD3OD): d 60.69 (C-
2), 62.78 (C-6), 63.21 and 63.74 [both (À), C-1, 7], 70.86
(C-3), 71.10 (C-5), 77.69 (C-4). HR-FABMS (matrix: 3-
nitrobenzyl alcohol), calcd for C7H16NO5 ([M+H]+):
m/z 194.1029; found: 194.1022. Note: the debenzylation
can also be performed by catalytic hydrogenation
[Pd(OH)2 on carbon, EtOH containing HCl, 1 atm H2,
12 h at room temperature].
1
(577 mg, 44%): syrup, [a]D À29.1 (c 1.03, CHCl3); H
NMR: d 0.05 [s, 6H, Si(CH3)2], 0.86 [s, 9H, SiC(CH3)3],
2.60 (ddd, 1H, J6,5=9.6, J6,7A=2.2, J6,7B=3.5 Hz, H-6),
2.88 (ddd, 1H, J2,1A=8.4, J2,1B=5.8, J2,3=0 Hz, H-2),
3.39 (dd, 1H, J1A,1B=8.6 Hz, H-1A), 3.52 (dd, 1H, H-
1B), 3.58 (dd, 1H, J4,3=2.4, J4,5=9.5 Hz, H-4), 3.76
(dd, 1H, J7A,7B=9.7 Hz, H-7A), 3.90 (dd, 1H, H-7B),
1,3,4,5-Tetra-O-benzyl-6,7-O-isopropylidene-L-glycero-L-
galacto-heptitol (19). To a suspension of 14 (192 mg,
0.335 mmol) in 2,2-dimethoxypropane (5 mL) was
added p-TsOH (10 mg, 0.06 mmol). The mixture became
homogeneous after 2 min and TLC analysis indicated
that the reaction was complete. The solution was diluted

O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
1277
with CH2Cl2 (10 mL), washed with saturated aqueous
NaHCO3 (10 mL), dried (Na2SO4) and concentrated.
The crude product was puri®ed by ¯ash chromato-
graphy (solvent D) to give 19 (187.2 mg, 91%) as a
syrup; [a]D À19.8 (c 1.1, CHCl3); H NMR: d 1.29 and
74.92 and 75.13 [all (À), 4 OCH2Ph], 72.94, 74.72, 75.23,
81.29 (C-3-6), 101.12 (C-2), 127.4±128.4 (ArCH), 137.0,
138.4, 138.6 and 138.8 (4 ArC). Anal. calcd for
C36H40O7: C, 73.95; H, 6.89. Found: C, 73.85; H, 6.93.
1
1.38 [2s, 2Â3H, C(CH3)2], 2.92 (d, 1H, J=5.8 Hz, HO-
C2), 3.45 (dd, 1H, J=6.5, 9.3 Hz) and 3.56 (dd, 1H,
J=6.4, 9.3 Hz) (H-1A, 1B), 3.89 (m, 3H, H-3, 4, 5), 3.95
(d, 2H, J=6.7 Hz, 2H-7), 4.15 (tt, Jꢀ5.8 Hz, H-2), 4.26
(tt, J=6.6, 5.4, 6.4 Hz, H-6), 4.42±4.80 (m, 8H, 4
OCHAHBPh), 7.20±7.40 (m, 20 H, 4 C6H5); 13C NMR:
d 24.96 and 26.46 [C(CH3)2)], 66.39 [(À) C-7], 69.54 (C-
2), 71.28 [(À) C-1], 73.22, 73.46, 73.53 and 74.64 [all (À),
4 OCH2Ph], 76.37, 71.17, 79.12 and 80.21 (C-3, 4, 5, 6),
108.52 [C(CH3)2)], 127.6±128.3 (ArCH), 137.99 (2C),
138.09 and 138.35 (4 ArC). Anal. calcd for C38H44O7:
C, 74.49; H, 7.24. Found: C, 74.47; H, 7.28.
(22): 1H NMR (CD3OD):
d
3.44 (d, 1H,
J1A,1B=10.7 Hz, H-1A), 3.65 (dd, 1H, J5,6=1.5,
J5,4=1.6 Hz, H-5), 3.72 (dd, 1H, J7A,6=5.9,
J7A,7B=7.1 Hz, H-7A), 3.82 (ddd, 1H, J4,3=5.3,
J4,6=1.4 Hz, H-4), 3.84 (d, 1H, H-3), 3.92 (d, 1H,
H-1B), 4.16 (dd, 1H, J7B,6=0.9 Hz, H-7B), 4.61±4.65
(m, 2H, H-6 and 0.5 OCHAHBPh), 4.34±4.58 (m, 7H,
3.5 OCHAHBPh), 7.21±7.42 (m, 20H, 4 C6H5); 13C
NMR (CD3OD): d 66.04 [(À) C-7], 70.91 [(À) C-1],
72.48, 72.94, 72.16 and 74.85 [all (À), 4 OCH2Ph],
75.26 (C-3), 76.24 (C-4), 77.17 (C-6), 78.09 (C-5),
108.52 (C-2), 127.9±129.8 (ArCH), 139.38, 139.48,
139.53 and 139.54 (4 ArC).
1,3,4,5-Tetra-O-benzyl-6,7-O-isopropylidene-L-manno-2-
heptulose (20). To a solution of heptitol 19 (116.7 mg,
0.19 mmol) in CH2Cl2 (3 mL) containing molecular
sieves (3 A, 200 mg), PCC (150 mg, 0.69 mmol) was
added in one portion. After having been stirred for 4 h
at room temperature, the reaction mixture was poured
into a column containing ether and ¯ash grade silica gel
(f=1 cm, 10 cm silica gel, 20 cm Et2O). The column was
rapidly eluted and the silicagel bed washed with Et2O
(100 mL). The resulting clear solution was concentrated,
thus giving homogeneous 19 (110 mg, 95%): syrup; IR
Enzymatic assays
Human a-Gal A was expressed from Sf-9 insect cells
infected with a recombinant baculovirus encoding normal
a-Gal A gene and puri®ed to homogeneity by Con-
canavalin A-Sepharose and Mono-Q (Pharmacia LKB
Biotechnology).33 Other glycosidases were commercially
available (Sigma). Assays were performed as previously
described.32,33 The following typical conditions were
used: enzyme (0.01±0.03 U/mL) in a bu€er at the
appropriate pH (see below), 5 mM substrate (p-nitro-
phenyl glycopyranoside), inhibitor, for a ®nal volume
of 0.1 mL. Enzyme and inhibitor were preincubated
for 5 min at room temperature in the bu€er, and the
reaction started by addition of the substrate. After
20 min at 37 ꢁC, the reaction was stopped by the addi-
tion of 0.2M sodium borate bu€er (pH 9.8, 0.25 mL).
Concentration of the p-nitrophenolate ion released was
measured at 410 nm.
1
nmax (®lm) 1732.1 cmÀ1 (CˆO); H NMR: d 1.29 and
1.38 [2s, 2Â3H, C(CH3)2], 3.87 (dd, 1H, J=6.7, 8.1 Hz,
H-7A), 3.88 and 3.99 (2t, 2H, Jꢀ5.6 Hz, H-4, 5), 3.93
(dd, 1H, J=6.5, 8.1 Hz, H-7B), 4.19 (d, 1H, J1A,1B
=
17.9 Hz, H-1A), 4.24 (dt, 1H, J=6.5, 5.6, 5.6 Hz, H-6),
4.29 (d, 1H, J3,4=5.6 Hz, H-3), 4.3 (d, 1H, H-1B), 4.36±
4.64 (m, 8H, 4 OCHAHBPh), 7.21±7.32 (m, 20H, 4
C6H5); 13C NMR: d 24.98, 26.43 [C(CH3)2)], 66.34 [(À)
C-7], 72,47, 73.12, 74.37, 74.46 and 74.61 [all (À) C-1, 4
OCH2Ph], 75.94, 79.08, 80.89 and 81.13 (C-3-6), 108.58
[C(CH3)2], 127.6±128.4 (ArCH), 136.89, 137.34, 137.61
and 138.04 (4 ArC), 207.22 (C-2).
For each enzyme, the following bu€ers and pH were
used [bu€er A=0.1M Na phosphate/citrate; B=0.1M
Na citrate; C=0.1M K phosphate; D=0.1M Na citrate/
0.1M Zn(OAc)2]: bovine epididymis a-l-fucosidase (B, pH
6.5); E. Coli a-galactosidase (C, pH 7); b-galactosidases
from E. Coli (C, pH 7.3), bovine liver (A, pH 7.3), A.
niger (B, pH 4), Jack beans (B, 3.5); a-glucosidases
from yeast (A, pH 6.8), rice (B, pH 4), Baker's yeast
(isomaltase)(A, pH 6.8); amyloglucosidases from A.
niger (B, pH 5), Rhizopus mold (B, pH 5); b-glucosidases
from almonds (B, pH 4.5), caldocellum saccharolyticum
(B, pH 5); a-mannosidases from jack beans (D, pH 5),
almonds (B, pH 4.5); b-mannosidase from helix pomatia
(B, pH 4); b-xylosidase from A. niger (B, pH 5); a-N-
acetylgalactosaminidases from chicken liver (B, pH 4);
b-N-acetylglucosaminidase from jack beans (B, pH 5),
form bovine epididymis A and B (B, pH 4); human
lysosomal a-galactosidase A (B, pH 4.5).33
Methyl 1,3,4,5-tetra-O-benzyl-L-manno-2-heptulopyrano-
side (21) and 2,7-Anhydro-1,3,4,5-tetra-O-benzyl-L-manno-
2-heptulose (22). Compound 20 (110 mg, 0.18 mmol)
was dissolved in a 1% (w/v) solution of I2 in MeOH
(5 mL) and the reaction mixture was stirred overnight at
room temperature. The solution was then concentrated,
the residue dissolved in CH2Cl2 (20 mL), the solution
washed with 5% (w/v) aqueous Na2S2O3, dried
(Na2SO4) and concentrated, to a€ord a crude mixture
of 21 and 22 (97.4 mg). Flash chromatography (solvent
B) of the mixture a€orded pure 21 (50.5 mg, 50%) as
well as a sample of 22 (20 mg, 20%).
1
(21): H NMR: d 3.17 (s, 3H, OCH3), 3.44 (d, 1 H,
J1A,1B=10.1 Hz, H-1A), 3.55 (ddd, 1 H, J6,5=9.7,
J6,7A=4.6, J6,7B=3.0 Hz, H-6), 3.63 (d, 1H, H-1B), 3.70
(dd, 1 H, J7A,7B=11.7 Hz, H-7A), 3.78 (dd, 1H, H-7B),
3.95 (dd, 1H, J5,4=9.7 Hz, H-5), 4.08 (d, J3,4=2.8 Hz,
H-3), 4.12 (dd, H-4), 4.40±4.94 (m, 8H, 4 OCHAHBPh),
7.23±7.36 (m, 20 H, 4 C6H5); 13C NMR: d 47.84
(OCH3), 62.37 [(À) C-7], 65.28 [(À) C-1], 72.12, 73.45,
Acknowledgements
Support of this research by a grant from the National
Insitutes of Health (grant DK 35766) is gratefully

1278
O. R. Martin et al. / Bioorg. Med. Chem. 9 (2001) 1269±1278
acknowledged. We also thank Laureen Connell for
technical assistance.
13. Martin, O. R.; Compain, P.; Kizu, H.; Asano, N. Bioorg.
Med. Chem. Lett. 1999, 9, 3171.
14. Martin, O. R.; Xie, F.; Liu, L. Tetrahedron Lett. 1995, 36,
4027.
15. Connell, L.; Saavedra, O. M.; Martin, O. R. Abstr. Pap.
213th Meeting Am. Chem. Soc., San Francisco, April 1997,
Abstract CHED-235.
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