Design and synthesis of 2-acetamidomethyl derivatives of isofagomine as potential inhibitors of human lysosomal β-hexosaminidases

Article abstract of DOI:10.1016/j.bmc.2003.12.040

As part of a program towards the development of specific inhibitors of human lysosomal β-hexosaminidase for use as chemical chaperones in therapy of G_M2 gangliosidosis related diseases, the synthesis of 2-acetamidomethyl derivatives of isofagom

Full text of DOI:10.1016/j.bmc.2003.12.040

Bioorganic & Medicinal Chemistry 12 (2004) 891–902
Design and synthesis of 2-acetamidomethyl derivatives
of isofagomine as potential inhibitors of human lysosomal
ꢀ-hexosaminidases
Richard J. B. H. N. van den Berg,a Wilma Donker-Koopman,b Jacques H. van Boom,a
Hans M. F. G. Aertsb and Daan Noortc,*
aLeiden Institute of Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502,
Leiden NL-2300 RA, The Netherlands
bDepartment of Biochemistry, Academic Medical Centre, University of Amsterdam, PO Box 22700,
Amsterdam NL-1100 DE, The Netherlands
cDivision of Chemical & Biological Protection, TNO Prins Maurits Laboratory, PO Box 45,
Rijswijk NL-2280 AA, The Netherlands
Received 22 September 2003; accepted 22 December 2003
Abstract—As part of a program towards the development of specific inhibitors of human lysosomal b-hexosaminidase for use as
chemical chaperones in therapy of GM2 gangliosidosis related diseases, the synthesis of 2-acetamidomethyl derivatives of iso-
fagomine has been undertaken. Key event in this synthesis is the conversion of a C-2 substituted gluconolactone derivative into the
corresponding lactam, followed by reduction to the corresponding amine. The 1-N-imino-2 acetamidomethyl derivative 5 proved to
be a rather selective inhibitor with a Ki of 2.4 mM for homogenate of human spleen lysosomal b-hexosaminidase.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
ciency of lysosomal b-hexosaminidase (E.C 3.2.1.52)
that cleaves terminal b-N-acetyl hexosamine residues.
In eukaryotic cells the catabolism of oligosaccharides,
glycolipids, glycoproteins, and glycosaminoglycans is
predominantly carried out in the lysosome by soluble
hydrolases, e.g., glycosidases. Precursors of these lyso-
somal enzymes are synthesized on ribosomes in
the endoplasmic reticulum (ER). During transport via the
ER and the Golgi compartment, these precursors are
further processed (e.g., by proteolysis, glycosylation and
phosphorylation) to their near mature form as present
in the lysosomes.1 Genetic defects may cause incorrect
folding of a lysosomal enzyme, resulting in the retention
of the enzyme in the ER, and premature degradation.2,3
The resulting low activity of the lysosomal enzyme causes
accumulation of corresponding substrates within the
lysosomes, which may result in a specific pathology.4 A
specific type of lysosomal storage disease is GM2 gang-
liosidosis that results in the degeneration of the central
nervous system. GM2 gangliosidosis is caused by a defi-
A potential therapy towards GM2 gangliosidosis might
be derived from the recently postulated ‘chemical
chaperone’ approach for Fabry disease,5 a related lyso-
somal storage disorder which is caused by a deficiency
of a-galactosidase A (a-Gal A). The new therapeutic
intervention is based on administrating competitive
inhibitors, so-called ‘chemical chaperones’, at sub-
inhibitory intracellular concentrations. These inhibitors
should help mutant enzyme precursors to fold properly,
avoiding their excessive pre-lysosomal degradation.
Intralysosomally, a-Gal A is not effectively reduced in
activity due to sub-inhibitory concentrations of the
competitive inhibitors. Thus, 1-deoxygalactonojirimycin,
a competitive inhibitor of a-Gal A, increased the intra-
cellular a-Gal A activity by 14-fold in Fabry lympho-
blasts.5b To explore whether the concept of ‘chemical
chaperone’ is also applicable to GM2 ganglioside related
diseases, such as Tay-Sachs and Sandhoff disease,
specific inhibitors of human lysosomal b-hexosaminidase
are needed.
* Corresponding author. Tel.: +31-15-2843497; fax: +31-15-2843963;
e-mail: noortd@pml.tno.nl
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.040

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R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
An important class of reversible competitive inhibitors
of glycosidases consists of polyhydroxylated piperidines
(iminosugars) which are widespread found in plants and
micro-organisms. In the ongoing process of unraveling
the mechanism of glycosidases it has been established
for b-glycosidases that a positive charge is generated at
the anomeric position rather than at the position of the
ring oxygen.6 Pioneering work of Bols7 and Ichikawa8
on the syntheses of iminosugars in which the anomeric
carbon atom was replaced by a nitrogen atom, resulted
in various powerful and selective inhibitors of b-glyco-
sidases. For example, isofagomine 1 (Fig. 1) is a very
potent inhibitor of b-glucosidase (Ki=0.11 mM, sweet
almond), but only a poor inhibitor of a-glucosidase
(yeast, 780 times less strong).9 A discrete class of glyco-
sidase inhibitors, the gem-diamine 1-N-iminosugars,
based on natural Siastatin B,10 proved to be powerful
although less selective inhibitors of glycosidases. For
example, gluco-type 1-N-iminosugar 211 was as active
towards a-(IC50=0.19 mM, yeast) as b-glucosidase
(IC50=0.42 mM, almond). Interestingly, Schuster12
showed that introduction of a 2-hydroxymethylene
substituent at the C-2 position of 1, leading to homo-
isofagomine 3, had no dramatic effect on the inhibitory
activity towards b-glucosidase (Ki=6.6 mM, sweet
almond). In contrast, for a-glucosidase (yeast) the inhibi-
tory effect was absent. Moreover, Takaoka et al. repor-
ted13 that 2-acetamidomethyl derivatives 4 of 2,5-
dihydroxymethyl-3,4-dihydroxypyrolidine were potent
inhibitors of N-acetylglucosaminidase. In view of the
above-mentioned findings we reasoned that the 1-N-
imino-2-acetamidomethyl isofagomine derivative 5,
which contains a 2-acetamidomethyl moiety as a stable
mimic of the N-acetyl function, might be a potent inhi-
bitor of human lysosomal b-hexosaminidase. In this
paper the synthesis of 5 is described. In addition, since it
has been shown that various sugar lactams are potent
glycosidase inhibitors,6c the closely related sugar lactam
6 was synthesized. Although the lactam function is
orientated more or less reversely as compared to ‘nor-
mal’ sugar lactams, this compound has a highly polar-
ized bond as well as a half-chair conformation and can
therefore be regarded as a transition state analogue for
the hydrolysis of a glycosidic linkage. The inhibitory
effect on human lysosomal b-hexosaminidase is also
reported.
2. Results and discussion
The retro-synthetic analysis of compounds 5 and 6 is
outlined in Scheme 1. It was envisaged that the 1-N-
imino moiety of 5 and the lactam function of 6 should
be introduced through conversion of a lactone, carrying
a protected 2-hydroxymethyl moiety at C-2 (8), into the
corresponding lactam 7. Reduction of the latter will give
the precursor of amine 5. Virtual rotation of the
required lactone synthon reveals that it should be readily
accessible from Cerny epoxide 9.14
The assembly of the 2-substituted gluco-d-lactone deri-
vative commences with the introduction of a benzyloxy-
methyl moiety at C-2 of Cerny epoxide 9 via the
reaction sequence depicted in Scheme 2. Regio- and
stereoselective opening of the oxirane functionality of 9
with vinylmagnesium bromide in refluxing tetrahydro-
furan led to the exclusive isolation of alkene 10 in an
excellent yield of 91%. Subsequent oxidation of the
double bond of 10 with sodium periodate in the presence
of osmium tetroxide15 gave the labile aldehyde, which
was directly converted into hydroxymethyl derivative 11
by reduction with sodium borohydride in a mixture of
tetrahydrofuran and isopropyl alcohol (64% over 2
steps). The hydroxyl groups of 11 were masked with a
benzyl group to give compound 12 in a yield of 90%.
Surprisingly, cleavage of the 1,6-anhydro bond of 12
under the conditions reported by Jespersen et al.7b for
opening the 1,6-anhydro bond in 11, led to an intractable
mixture of products. When slightly modified conditions
were used (dioxane/1 M H2SO4, 2/1, v/v, 72 h under
reflux), lactol 14 was isolated in a disappointing yield of
Figure 1.

R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
893
Scheme 1. Retro-synthetic analysis of compounds 5 and 6.
Scheme 2. Conditions: (i) CH2=CHMgBr, THF, 91%; (ii) (a) OsO4, NaIO4, dioxane, H2O; (b) NaBH4, isopropanol, THF, 64%; (iii) BnBr, NaH,
DMF, 90%; (iv) TFA, Ac2O, 78%; (v) NaOMe, MeOH, 96%; (vi) TBDMSCl, imidazole, DMF, 82%; (vii) DMSO, Ac2O, 92%; (viii) NH3/MeOH,
53%; (ix) DMSO, Ac2O, 81%; (x) NaCNBH3, HCO2H, CH3CN, 63%.

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R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
32%. In contrast, cleavage of the 1,6-anhydro bond
of 12 was readily accomplished using trifluoroacetic acid
in acetic anhydride,16 affording diacetate 13 as a mix-
ture of anomers (a/b=3:1). Zemplen deacetylation of 13
gave lactol 14 in an overall yield of 75%. The primary
hydroxyl function of 14 was protected with a tert-butyl-
dimethylsilyl group to afford silyl-ether 15. Oxidation of 15
under Albright–Goldman conditions17 provided lactone
16 in a yield of 75% over the two steps. Introduction of
the endocyclic imino function was accomplished
according to the method originally described by the
groups of Vasella18 and Pandit,19 and more recently by
our groups.20 Thus, reaction of 16 with methanolic
ammonia resulted in the isolation of hydroxy-amide 17
in a moderate yield of 53%, together with the C-2 epimer
of 17 (15%). The C-2 stereoisomer of 17 is presumably
formed by base-promoted racemization. The gluco-
amino function in 23 with acetyl chloride gave acetam-
ido derivative 24 in 67% yield. The identity of 24 was
unambiguously corroborated by mass spectrometry,
and 1H- and 13C NMR spectroscopy. Unmasking of
compound 24 by hydrogenation over palladium on carbon
in dimethyl formamide afforded the requisite lactam 6
which was evaluated as an inhibitor of various hexos-
aminidases (vide infra).
The synthesis of isofagomine derivative 5 was continued
with the reduction of the lactam function of compound
19 as is depicted in Scheme 3. Thus, treatment of lactam
19 with borane in tetrahydrofuran25 provided the
desired iminosugar 25 in reasonable yield (59%). Alter-
natively, reduction of 19 with borane-methyl sulfide
complex26 gave 25 in 61% yield together with a con-
siderable amount of starting material 19 (32%). The
configuration and conformation of 25 was ascertained
by NMR spectroscopy. The di-axial proton coupling
constants of the ring sugar moiety (J1ax,2=11.6 Hz,
J2,3=10.6 Hz, J3,4=9.0 Hz and J4,5=9.6 Hz) are char-
1
configuration of 17 was irrefutably ascertained by H
NMR spectroscopy. The large coupling constants
between H2 and H3, and H4 and H5 (J2,3=6.8 Hz and
J4,5=7.6 Hz) and the small coupling constant between
H3 and H4 (J3,4=3.0 Hz) are in full accordance with
the proposed identity of compound 17.21 Dimethyl
sulfoxide-acetic anhydride mediated oxidation17 of 17
resulted in a diastereomeric mixture of 18 in good yield
(81%). The 5-R-isomer of 18 could be separated from
the 5-S-isomer of 18 by silica gel column chromato-
graphy (ratio R:S=78:22), enabling the firm establish-
ment of their respective chemical structures. Coupling
constants of the 5-R-isomer of 18 (J2,3=9.6 Hz and
J3,4=9.1 Hz) indicate a 4H3 conformation; coupling
constants of the 5-S-isomer of 18 (J2,3=5.0 Hz and
4
acteristic for the gluco-configuration and the C1 con-
formation.7b Subsequently the endocyclic nitrogen atom
of compound 25 was protected with a benzyl group,
giving imino-glucitol 26 in 49% yield. TBAF-mediated
desilylation of 26 afforded 27 in quantitative yield.
Surprisingly, reaction of 27 with mesyl chloride did not
afford the O-methanesulfonyl derivative. Instead the
corresponding chloride 28 was isolated in almost quan-
titative yield (97%). Compound 28 could be smoothly
converted by treatment with sodium azide into the
desired azido derivative 29 in a yield of 91%. The high
reactivity of the chloro-derivative 28 towards nucleo-
philic substitution suggests that the endocyclic nitrogen
atom might participate in the displacement reaction, by
forming an aziridinium intermediate. Reduction of the
azido function in 29 was accomplished with triphenyl-
phosphine in aqueous tetrahydrofuran24 to give the
amine 30 in 71% yield. Subsequent acylation with acetyl
chloride furnished imino-glucitol 31 in 85% yield.
Deprotection of 31 by catalytic hydrogenation over
palladium on carbon in acidic aqueous ethanol
afforded the desired homoisofagomine derivative 5 in
good yield.
3
J3,4=6.3 Hz) suggest a H4 conformation which is in
accordance with published data for analogous com-
pounds.22 Reduction of the diastereomeric mixture of
18 with sodium cyanoborohydride under acidic condi-
tions afforded a mixture of the d-gluco isomer (19;
63%) and the l-ido isomer (17%), which were separated
by silica gel column chromatography. When the R- and
S-isomers of 18 were reduced separately under identical
conditions, diastereomeric 19 was obtained in the same
ratio of isomers and yield as obtained after reduction of
the diastereomeric mixture of 18. This indicates that in
both cases reduction proceeds via the same intermediate
N-acyliminium ion. Evidently, hydride attack occurs
preferentially from the Si-face, affording the d-gluco
derivative as the major product.23 The stereochemistry of
19 was firmly established by NMR spectroscopy, the di-
axial coupling constants of the ring protons (J2,3=9.6 Hz,
J3,4=9.1 Hz, J4,5=9.0 Hz), together with NOE’s observed
between H2 and H4 and between H3 and H5, clearly
show the d-gluco-configuration and a 4C1 conformation
of 19. In addition to its use for the preparation of iso-
fagomine derivative 5, it was used for the preparation of
lactam 6 (Scheme 3). Desilylation of 19 with TBAFgave
the hydroxy-amide 20. Subsequent reaction of 20 with
methanesulfonyl chloride in pyridine afforded methyl-
sulfonyl derivative 21. Treatment of 21 with sodium
azide in DMFat elevated temperature gave the azido
derivative 22 in a yield of 80% over the three steps.
Reduction of the azido function in 22 under the agency
of triphenylphospine in aqueous tetrahydrofuran24 gave
the amino compound 23. Subsequent acetylation of the
It is interesting to note that compound 25 could also be
converted into the known12 b-glycosidase inhibitor
homoisofagomine 32 in two steps (see Scheme 3). The
originally reported synthesis of 32 was based on a
chemical-enzymatic approach and afforded 32 as C-5
epimeric mixture in a ratio of 1:1.2 (5-S:5-R). In the first
step, the TBDMS group of 25 was removed by treat-
ment with TBAF. Subsequent catalytic hydrogenation
over palladium on carbon in aqueous ethanol containing
hydrochloric acid afforded 32 in an overall yield of
70%.
The inhibitory potency of iminosugar 5 and lactam 6
was tested towards homogenate of human spleen lyso-
somal b-hexosaminidase. For comparison, the earlier
reported 2-acetamido-2-deoxy-d-glucono-d-lactam20,22 (33)
and 2-acetamido-2-deoxy-d-glucono-deoxynojirimycin20,22
(34) were evaluated for their inhibitory potency. The

R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
895
Scheme 3. Conditions: (i) TBAF, THF, 91%; (ii) MsCl, pyridine, 99%; (iii) NaN3, DMF, 89%; (iv) triphenylphosphine, H2O, THF, 25%; (v) AcCl,
TEA, DCM, 67%; (vi) DMF, 10% Pd/C, H2, 69%; (vii) BH3Me2S, DCM, 61%; (viii) BnBr, TEA, CH3CN, 49%; (ix) TBAF, THF, 100%; (x) MsCl,
TEA, DCM, 97%; (xi) NaN3, DMF, 91%; (xii) triphenylphosphine, H2O, THF, 71%; (xiii) AcCl, TEA, DCM, 85%; (xiv) HCl, EtOH, 10% Pd/C,
H2, 94% (xv) (a) TBAF, THF (b) HCl, EtOH, H2O, 10% Pd/C, H2, 70%.
results show that 2-acetamidomethyl-isofagomine (5) is
a relatively strong inhibitor of human lysosomal b-hexo-
saminidase. Furthermore, the high inhibitory effect of 33
towards human lysosomal b-hexosaminidase (see
Table 1) is striking. Apparently, the half chair con-
formation of 33 contributes strongly to the inhibi-
tory activity of this compound. This is in full agreement
with a recent study on conformation and inhibitory
potency of various d-glycono-d-lactams.6c In order to
determine the specificity of the inhibitors, two other b-
N-acetylglucosaminidases were included in the enzy-
matic assays, that is, b-hexosaminidase of Aspergillus
niger and purified human chitotriosidase, an endo-N-
acetylglucosaminidase.27 No inhibition of human chito-
triosidase was observed. The most powerful inhibitors (5
and lactam 33) were only poor inhibitors (Ki 612 and
34.5 mM, respectively). These results indicate that these
compounds are fairly selective inhibitors of human
lysosomal b-hexosaminidases. Further studies are
required to determine whether these iminosugars are
actually useful as enhancers of human lysosomal b-hex-
osaminidase activity in cultured cell lines from patients
suffering from Tay-Sachs or Sandhoff disease.
3. Experimental
3.1. General methods and materials
Column chromatography was performed on Silica gel
60 (220–440 mesh ASTM, Fluka). TLC analysis was
performed with silica gel TLC plates (Fluka) with
detection by UV absorption (254 nm) where applicable
and charring with 20% H2SO4 in MeOH or ammonium
molybdate (25 g/L) and ceric ammonium sulfate (10 g/L)
in 20% H2SO4. H NMR and 13C NMR were recorded
1
with a Varian VXR-400S (399.9/100.6 MHz). 1H and
13C chemical shifts are given in ppm (d) relative to tetra-
methylsilane (d=0.00), DMSO-d5 (d=2.525), DMSO-d6
(d=39.6) and CDCl3 (d=77.10) as internal standard.
The purity of the compounds was established by 1H
NMR spectroscopy: >95% in all cases. Mass spectra
were recorded with a VG Quattro II triple quadropole
mass spectrometer (Fisons Instruments, Altrincham,
UK). High resolution mass spectra and tandem MS
spectra were recorded on a Q-TOFmass spectrometer
(Micromass, Manchester, UK). The Q-TOFinstrument
was calibrated; electrospray MS and electrospray MS-MS
spectra were recorded with resolution 8000 (Full Width

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R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
a
Table 1. Apparent IC50 values and Ki values of compounds 5, 6, 3320,22 and 3420,22 for human lysosomal b-hexosaminidase and corresponding
isoenzymes
Compd
Enzyme
Homogenate of human spleen
lysosomal b-hexosaminidase
5 mM
Ki=2.4 mM
20%b
N.D.c
2.5 mM
16 mM
0.85d
0.87d
Human spleen lysosomal
b-hexosaminidase A
Pi=5.0
Human spleen lysosomal
b-hexosaminidase B
Pi=7.2
4.5 mM
Ki=2.8 mM
5.0 mM
Ki=3.1 mM
20 mM
Ki=16 mM
3.0 mM
Ki=1.55 mM
N.D.c
2.5 mM
Ki=1.8 mM
16 mM
Ki=16 mM
2.33d
a b-Hexosaminidase activities were determined with 4-methylumbelliferyl-N-acetyl-b-d-glucosaminide as substrate.
b Inhibition at a concentration of 1200 mM.
c Not determined.
d KM value (mM) of the specific enzyme for used substrate, that is, 4-methylumbelliferyl-N-acetyl-b-d-glucosaminide.
Half Mass). Positive ion electrospray MS spectra were
recorded at a cone voltage of 15 V. Argon gas pressure
was 10ꢀ4 mBar. Product ion spectra (MS-MS) were
recorded at a collision energy between 15 and 28 eV.
Prior to reactions that require anhydrous conditions,
traces of water were removed by coevaporation with dry
toluene. These reactions were conducted under dry
argon atmosphere. Hydrogenations were executed at
atmospheric pressure under an atmosphere of hydrogen
gas maintained by an inflated balloon.
water (70 mL, 5:2). Neat NaIO4 (1.58 g, 7.4 mmol) was
added and the mixture was stirred for 1 h, subsequently
diluted with water (50 mL) and extracted with ethyl
acetate (5ꢂ50 mL). The combined organic layers were
dried (MgSO4), filtered and concentrated. The residue
was dissolved in a mixture of THF/isopropyl alcohol
(50 mL, 9:1). The solution was cooled (0 ꢁC) and
NaBH4 (0.14 g, 3.7 mmol) was added. After 1 h, the
reaction mixture was poured into aqueous NH4Cl (2 M,
50 mL) and extracted with ethyl acetate (5ꢂ50 mL). The
organic layer was dried (MgSO4), filtered and con-
centrated. Column chromatography of the residue over
silica gel with DCM/MeOH (100:0!95:5) gave 11 as a
colorless oil. Yield 0.57 g (64%). TLC (silica gel,
MeOH/DCM, 6:94) Rf=0.54. 1H NMR (CDCl3):
d=1.92 (m, 1H, H-2), 2.55 (br. s, 1H, OH), 2.94 (br. s,
1H, OH), 3.41 (m, 1H, H-4), 3.68 (dd, 1H, H-6a,
J6a,6b=7.4 Hz, J5,6a=5.6 Hz), 3.71–3.83 (m, 3H, H2-20,
H-3), 4.04 (dd, 1H, H-6b, J6a,6b=7.4 Hz, J5,6b=0.8 Hz),
4.56 (d, 1H, H-5), 4.64 (AB, 2H, CH2 Bn), 5.53 (s, 1H,
H-1), 7.25–7.36 (m, 5H, CH-arom Bn). 13C{1H}-NMR
(CDCl3): d=48.95 (C-2), 62.19 (C-20), 65.62 (C-6), 69.04
(C-3), 71.78 (CH2 Bn), 74.95 (C-5), 79.61 (C-4), 102.06
(C-1), 127.84-128.91 (CH-arom Bn), 137.78 (Cq Bn).
3.1.1. 1,6-Anhydro-4-O-benzyl-2-deoxy-2-C-vinyl-ꢀ-D-
glucose (10). Vinylmagnesium bromide in THF(1.0 M,
100 mL, 100 mmol) was added to 9 (3.20 g, 13.7 mmol)
in THF(70 mL). The mixture was refluxed for 4 h,
cooled to 0 ꢁC and poured into aqueous NH4Cl (2 M,
100 mL). The aqueous layer was extracted with ethyl
acetate (3ꢂ100 mL). The combined organic layers were
dried (MgSO4), filtered and concentrated. Column
chromatography of the residue over silica gel with hex-
ane/ethyl acetate (4:1!7:3) gave 10 as a colorless oil.
Yield 3.28 g (91%). TLC (silica gel, ethyl acetate/hex-
1
ane, 1:1) Rf=0.40. H NMR (CDCl3): d=2.42 (m, 1H,
H-2), 2.81 (d, 1H, OH-3, J=5.4 Hz), 3.41 (m, 1H, H-4),
3.68 (dd, 1H, H-6a, J6a,6b=7.4 Hz, J5,6a=5.5 Hz), 3.76
(br. s, 1H, H-3), 4.05 (dd, 1H, H-6b, J6a,6b=7.4 Hz,
J5,6b=0.8 Hz), 4.57–4.68 (m, 3H, H-5, CH2 Bn), 5.16
(m, 2H, H2-30), 5.39 (s, 1H, H-1), 5.96 (m, 1H, H-20),
7.29–7.36 (m, 5H, CH-arom Bn). 3C{1H}-NMR
(CDCl3): d=51.99 (C-2), 65.64 (C-6), 70.72 (C-3), 71.49
(CH2 Bn), 74.87 (C-5), 79.06 (C-4), 103.71 (C-1), 117.79
(C-30), 127.69; 127.84; 128.51 (CH-arom Bn), 135.74 (C-
20), 137.42 (Cq Bn).
3.1.3. 1,6-Anhydro-3,4-di-O-benzyl-2-C-benzyloxymethyl-
2-deoxy-ꢀ-D-glucose (12). Sodium hydride (55%, 0.85
g, 19.5 mmol) was added to a cooled (0 ꢁC) and stirred
solution of 11 (0.95 g, 3.55 mmol) in anhydrous DMF
(50 mL). Benzyl bromide (0.84 mL, 7.1 mmol) was
added to the mixture. After 4 h, MeOH (2 mL) was
added and the reaction mixture was diluted with ethyl
acetate (100 mL). The organic layer was washed with
water, dried (MgSO4), filtered, and concentrated. Col-
umn chromatography of the residue over silica gel with
DCM/MeOH (100:0!99:1) gave 12 as a yellow oil.
Yield 1.42 g (90%). TLC (silica gel, MeOH/DCM,
3.1.2. 1,6-Anhydro-4- O-benzyl-2-deoxy-2-C-hydroxy-
methyl-ꢀ-D-glucose (11). Osmium tetroxide (0.1 M in
dioxane/water, 5:2, 0.4 mL, 0.04 mmol) was added to a
stirred mixture of 10 (0.88 g, 3.35 mmol) in dioxane/
1
0.5:99.5) Rf=0.48. H NMR (CDCl3): d=2.23 (t, 1H,

R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
897
H-2), 3.36 (br.s, 1H, H-4), 3.48 (dd, 1H, H-20a), 3.51 (m,
1H, H-3), 3.61 (dd, 1H, H-20b), 3.74 (t, 1H, H-6), 4.11
(dd, 1H, H-60), 4.41–4.54 (m, 7H, H-5, 3ꢂCH2 Bn), 5.53
(d, 1H, H-1), 7.24–7.34 (m, 15H, CH-arom Bn).
13C{1H}-NMR (CDCl3): d=43.65 (C-2), 64.73 (C-6),
69.13 (C-20), 70.80, 71.22, 73.16 (3ꢂCH2 Bn), 73.30 (C-
3), 74.38 (C-5), 76.78 (C-4), 101.08 (C-1), 127.60-128.49
(CH-arom Bn), 137.85, 138.16, 138.24 (3ꢂCq Bn).
(C-5 (a,b)), 75.57–73.35 (CH2 Bn), 77.44, 79.20 (C-3
(a,b)), 79.44, 79.79 (C-4 (a,b)), 93.70, 95.68 (C-1 (a,b)),
128.57–127.69 (CH-arom Bn), 137.48–138.40 (Cq Bn).
3.1.6. 3,4-Di- O-benzyl-2-C-benzyloxymethyl-6-O-tert-
butyldimethylsilyl-2-deoxy-ꢁ,ꢀ-D-glucose (15). To a stir-
red solution of 14 (2.21 g, 4.76 mmol) in DMF(47 mL)
were added imidazole (0.68 g, 10.0 mmol) and tert-
butyldimethylchlorosilane (0.97 g, 6.43 mmol). After 6
h, the reaction mixture was diluted with HCl (1M, 100
mL). The aqueous layer was separated and extracted
with ethyl acetate (3ꢂ100 mL). The combined organic
layers were washed with aqueous NaHCO3 (1%, 100
mL), water (100 mL), dried (MgSO4), filtered and con-
centrated. Column chromatography of the residue over
silica gel with hexane/ethyl acetate (80:20!70:30) gave
15 as a colorless oil. Yield 2.28 g (82%). TLC (silica gel,
ethyl acetate/hexane, 1:1) Rf=0.88. 13C{1H}-NMR
(CDCl3): d=ꢀ5.26, ꢀ4.87 (Si(CH3)2), 14.20, 18.45 (Cq
tBu (a,b)), 25.21 (CH3 tBu (a,b)), 46.77, 49.86 (C-2
(a,b)), 62.32, 62.56 (C-6 (a,b)), 65.99, 67.17 (C-20 (a,b)),
72.33, 72,79 (C-5 (a,b)), 73.38–75.39 (CH2 Bn), 77.31,
77.36 (C-3 (a,b)), 79.32, 79.71 (C-4 (a,b)), 94.06, 95.67
(C-1 (a,b)).
3.1.4. 1,6-Di-O-acetyl-3,4-di-O-benzyl-2-C-benzyloxy-
methyl-2-deoxy-ꢁ,ꢀ-D-glucose (13). TFA (2.70 mL, 35.3
mmol) was added to a cooled (0 ꢁC) and stirred solution
of 12 (2.98 g, 6.39 mmol) in acetic anhydride (90 mL,
0.95 mol). After 45 min, the mixture was concentrated
and the residue was coevaporated with toluene (3ꢂ50
mL). Column chromatography of the residue over silica
gel with hexane/ethyl acetate (80:20!50:50) gave 13 as
a colorless oil. Yield 2.37 g (78%) TLC (silica gel, ethyl
acetate/hexane, 1:2) Rf=0.51. 1H NMR [a-anomer]
(CDCl3): d=1.92 (s, 3H, CH3 acetyl), 2.00 (s, 3H, CH3
acetyl), 2.31 (m, 1H, H-2), 3.38 (t, 1H, H-20a), 3.61 (dd,
1H, H-4, J3,4=8.8 Hz, J4,5=10.0 Hz), 3.65 (m, 1H, H-
20b), 3.81 (dd, 1H, H-3, J2,3=11.2 Hz, J3,4=8.8 Hz),
3.91 (dt, H-5, J4,5=10.0 Hz, J5,6a=J5,6b=3.2 Hz), 4.27
(d, 2H, H2-6), 4.29–4.88 (3ꢂAB, 6H, 3ꢂCH2 Bn), 6.34
(d, 1H, H-1, J1,2=3.3 Hz), 7.23–7.32 (m, 15H, CH-
arom Bn). 13C{1H}-NMR [a-anomer] (CDCl3):
d=20.64, 20.69 (2ꢂCH3 acetyl), 45.56 (C-2), 62.77 (C-
6), 66.37 (C-20), 71.31 (C-5), 73.00, 74.97, 74.99 (3ꢂCH2
Bn), 78.15 (C-3), 78.70 (C-4), 91.67 (C-1), 127.51–128.41
(CH-arom Bn), 137.55, 137.85, 137.95 (3ꢂCq Bn),
168.76, 170.43 (2ꢂC¼O). 1H NMR [b-anomer]
(CDCl3): d=1.88 (m, 1H, H-2), 1.96 (s, 3H, CH3 ace-
tyl), 1.99 (s, 3H, CH3 acetyl), 3.41 (m, 1H, H-20a), 3.55
(t, 1H, H-4, J3,4=8.8 Hz, J4,5=9.7 Hz), 3.63 (m, 1H, H-
5) 3.67 (m, 1H, H-20b), 3.95 (dd, 1H, H-3, J2,3=10.8 Hz,
J3,4=10.8 Hz), 4.27 (d, 2H, H2-6), 4.29–4.88 (m, 6H,
3ꢂCH2 Bn), 5.81 (d, H-1, J1,2=9.2 Hz), 7.23–7.32 (m,
15H, CH-arom Bn). 13C{1H}-NMR [b-anomer]
(CDCl3): d=20.69, 20.71 (2ꢂCH3 acetyl), 47.71 (C-2),
62.90 (C-6), 63.65 (C-20), 73.53 (C-5), 72.92, 74.66, 75.16
(3ꢂCH2 Bn), 78.75 (C-4), 78.98 (C-3), 91.97 (C-1),
127.51-128.41 (CH-arom Bn), 137.66, 137.82, 138.18
(3ꢂCq Bn), 168.76, 170.43 (2ꢂC¼O acetyl).
3.1.7. 3,4-Di- O-benzyl-2-C-benzyloxymethyl-6-O-tert-
butyldimethylsilyl-2-deoxy-L-gluco-ꢂ-lactone (16). Acetic
anhydride (3.0 mL) was added to a solution of 15 (0.44
g, 0.76 mmol) in DMSO (5.0 mL). The mixture was
stirred for 16 h, after which it was diluted with diethyl
ether (100 mL), washed thoroughly with aqueous
NaHCO3 (10%, 3ꢂ10 mL) and brine (30 mL). The
organic layer was dried (Na2SO4) and concentrated.
Column chromatography of the residue over silica gel
with hexane/ethyl acetate (100:0!85:15) gave 16 as a
colorless oil. Yield 0.40 g (92%). TLC (silica gel, ethyl
acetate/hexane, 1:5) Rf=0.84. 1H NMR (CDCl3):
d=0.08 (d, 6H, 2ꢂCH3, TBDMS), 0.89 (t, 9H, 3ꢂCH3,
tBu), 2.64 (m, 1H, H-2), 3.69 (dd, 1H, H-20a), 3.89 (d,
2H, H2-6), 3.98 (t, 1H, H-4), 4.00 (dd, 1H, H-20b), 4.09
(t, 1H, H-3), 4.14 (m, 1H, H-5), 4.37–4.89 (3ꢂAB, 6H,
3ꢂCH2 Bn), 7.23–7.35 (m, 15H, CH-arom Bn). 13C{1H}-
NMR (CDCl3): d=ꢀ5.37, ꢀ5.13 (2ꢂCH3 TBDMS),
18.31 (Cq tBu), 25.93 (CH3 tBu), 48.79 (C-2), 61.43 (C-6),
66.67 (C-20), 73.41, 74.81, 74.85 (3 CH2 Bn), 76.78 (C-3/
C-4), 79.27 (C-5), 127.74–128.62 (CH-arom Bn), 137.99,
138.02, 138.06 (3ꢂCq Bn), 170.45 (C¼O lactone).
3.1.5. 3,4-Di-O-benzyl-2-C-benzyloxymethyl-2-deoxy-ꢁ,ꢀ-
D-glucose (14). Sodium methoxide (100 mg, 1.85 mmol)
was added to a solution of 13 (2.73, 4.97 mmol) in
MeOH (100 mL). After 45 min, the reaction mixture
was neutralized with Dowex 50 Wꢂ8 (H+), filtered and
concentrated. Yield 2.21 g of a colorless oil (96%). TLC
(silica gel, ethyl acetate/hexane, 1:1) Rf=0.52. 1H NMR
[a-anomer] (CDCl3): d=2.06 (m, 1H, H-2), 3.57 (m, 1H,
H-4), 3.65 (m, 2H, H2-20), 3.85 (m, 2H, H2-6), 3.91 (dd,
1H, H-3), 3.98 (m, 1H, H-5), 4.30–4.90 (3ꢂAB, 6H,
3ꢂCH2 Bn), 5.37 (d, 1H, H-1), 7.15–7.40 (m, 15H, CH-
3.1.8. 3,4-Di- O-benzyl-2-C-benzyloxymethyl-6-O-tert-
butyldimethylsilyl-2-deoxy-D-gluco-amide (17). Lactone
16 (1.52 g, 2.63 mmol) was dissolved in methanolic
ammonia (4 M, 50 mL). After stirring for 2 h, the mix-
ture was concentrated and traces of ammonia were
removed by coevaporation with toluene (1ꢂ10 mL).
Column chromatography of the residue over silica gel
with hexane/ethyl acetate (70:30!50:50) gave 17 as a
colorless oil. Yield 0.83 g (53%). TLC (silica gel, ethyl
acetate/hexane, 1:1) Rf=0.41 and Rf=0.28. The com-
pound with Rf=0.41 turned out to be the desired gluco-
1
arom Bn). H NMR [b-anomer] (CDCl3): d=1.72 (m,
1H, H-2), 3.39 (m, 1H, H-5), 3.57 (m, 1H, H-4), 3.78
(dd, 1H, H-3), 3.69 (m, 2H, H2-20), 3.85 (m, 2H, H2-6),
4.30–4.90 (3ꢂAB, 6H, 3ꢂCH2 Bn), 4.88 (d, 1H, H-1),
7.15–7.40 (m, 15H, CH-arom Bn). 13C{1H}-NMR [a,b-
anomer] (CDCl3): d=46.76, 49.72 (C-2 (a,b)), 61.94,
62.01 (C-6 (a,b)), 65.78, 67.18 (C-20 (a,b)), 71.68, 76.78
1
derivative. H NMR (CDCl3): d=0.05 (d, 6H, 2ꢂCH3,
TBDMS), 0.90 (s, 9H, tBu), 2.75 (br. s, 1H, OH-5), 3.63
0
0
(m, 1H, H-2, J2,3=6.8 Hz, J2,2 a=5.4 Hz, J2,2 b=6.5
Hz), 3.62 (dd, 1H, H-4, J3,4=3.0 Hz, J4,5=7.6 Hz), 3.69

898
R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
(dd, 1H, H-6a, J5,6a=5.4 Hz, J6a,6b=10.2 Hz), 3.77 (dd,
1H, H-6b, J5,6b=3.6 Hz, J6a,6b=10.2 Hz), 3.87 (m, 3H,
H2-20, H-5, J4,5=8.2 Hz, J5,6a=5.4 Hz, J5,6b=3.6 Hz),
4.37 (dd, 1H, H-3, J2,3=6.8 Hz, J3,4=3.1 Hz), 4.35–4.72
(3ꢂAB, 6H, 3ꢂCH2 Bn), 5.36 (s, 1H, NH), 6.42 (s, 1H,
NH), 7.24–7.36 (m, 15H, CH-arom Bn). 13C{1H}-NMR
(CDCl3): d=ꢀ5.3 (Si(CH3)2), 18.33 (Cq tBu), 25.98
(CH3 tBu), 47.71 (C-2), 64.14 (C-6), 68.35 (C-20), 71.46
(C-5), 73.49, 73.92, 74.32 (3ꢂCH2 Bn), 77.58 (C-3),
79.21 (C-4), 127.73–128.53 (CH-arom Bn), 137.81, 138.14,
138.36 (3ꢂCq Bn), 175.24 (C¼O amide). ES–MS; m/z:
594.42, [M+H]+; mono-isotopic MW calculated for
C34H47N1O6Si1=593.32.
3.1.10. 3,4-Di-O-benzyl-2-C-benzyloxymethyl-6-O-tert-
butyldimethylsilyl-2-deoxy-D-gluco-ꢂ-lactam (19). NaC-
NBH3 (0.078 g, 1.24 mmol) was added to mixture of 18
(0.74 g, 1.24 mmol; mixture of isomers) in acetonitrile
(13 mL) and formic acid (1.40 mL). The reaction mix-
ture was refluxed for 2 h, cooled to 0 ꢁC, and quenched
with aqueous HCl (0.1 N, 12.4 mL). After 15 min, the
solution was poured into a mixture of ethyl acetate and
aqueous NaHCO3 (10%, 200 mL, 1:1). The aqueous
layer was separated and extracted with ethyl acetate
(3ꢂ50 mL). The combined organic layers were washed
with brine (50 mL). The organic phase was dried
(MgSO4) and concentrated. Column chromatography
of the residue over silica gel with hexane/ethyl acetate
(80:20!55:45) gave 19 as a colorless oil. Yield R-isomer
0.45 g (63%), yield S-isomer 0.12 g (17%). 1H NMR [R-
isomer] (CDCl3): d=0.02 (d, 6H, 2ꢂCH3 Si(CH3)2),
0.86 (s, 9H, CH3 tBu), 2.44 (m, 1H, H-2, J2,3=9.6 Hz,
3.1.9. 5-(R/S)-3,4-Di-O-benzyl-2-C-benzyloxymethyl-6-
O-tert-butyldimethylsilyl-5-dehydro-2-deoxy-5-hydroxy-
D-gluco-ꢂ-lactam (18). Acetic anhydride (6.0 mL) was
added to a solution of 17 (0.91 g, 1.54 mmol) in DMSO
(10 mL). The mixture was stirred for 16 h, after which it
was diluted with diethyl ether (100 mL), washed thor-
oughly with aqueous NaHCO3 (10%, 3ꢂ20 mL) and
brine (50 mL). The organic layer was dried (Na2SO4)
and concentrated. Column chromatography of the resi-
due over silica gel with hexane/ethyl acetate
(80:20!40:60) gave 18 as a colorless oil. Yield 0.74 g
(81%), mixture of isomers (S-configuration 78%, R-
configuration 22%). TLC (silica gel, ethyl acetate/hex-
0
0
J2,2 a=5.4 Hz, J2,2 b=2.8 Hz), 3.29 (dd, 1H, H-6a,
J6a,6b=9.8 Hz), 3.39 (m, 1H, H-5, J4,5=8.9 Hz,
J5,6a=8.4 Hz, J5,6b=3.0 Hz), 3.48 (t, 1H, H-4,
J3,4=J4,5=9.1 Hz), 3.71 (dd, 1H, H-20a), 3.75 (dd, 1H,
H-6b), 4.09 (dd, 1H, H-20b), 4.11 (t, 1H, H-3), 4.38–4.94
(3ꢂAB, 6H, 3ꢂCH2 Bn), 6.06 (s, 1H, NH), 7.21–7.35
(m, 15H, CH-arom Bn). 13C{1H}-NMR [R-isomer]
(CDCl3): d=ꢀ5.39 (SiCH3), ꢀ5.35 (SiCH3), 18.26 (Cq
tBu), 25.91 (CH3 tBu), 48.56 (C-2), 55.69 (C-5), 64.14
(C-6), 65.79 (C-20), 73.33, 74.89, 75.10 (3ꢂCH2 Bn),
77.71 (C-3), 78.68 (C-4), 127.63-128.64 (CH-arom Bn),
137.82, 138.34, 138.21 (3ꢂCq Bn), 170.23 (C¼O amide).
ES–MS; m/z: 576.34, [M+H]+; mono-isotopic MW
calculated for C34H45N1O5Si1=575.31. 1H NMR [S-
isomer] (CDCl3): d=0.02 (d, 6H, 2ꢂCH3 Si(CH3)2),
0.88 (s, 9H, CH3 tBu), 2.78 (m, 1H, H-2), 3.62–3.76 (m,
4H, H-4, H-5, H2-6), 3.85 (m, 2H, H2-2), 4.16 (dd, 1H,
H-3), 4.35–4.79 (3ꢂAB, 6H, 3ꢂCH2 Bn), 6.01 (s, 1H,
NH), 7.20–7.32 (m, 15H, CH-arom Bn). 13C{1H}-NMR
[S-isomer] (CDCl3): d=ꢀ5.39 (SiCH3), ꢀ5.34 (SiCH3),
18.30 (Cq tBu), 25.95 (CH3 tBu), 47.24 (C-2), 53.69 (C-
5), 63.69 (C-6), 68.53 (C-20), 72.15, 72.43, 73.07 (3ꢂCH2
Bn), 71.89 (C-3), 74.78 (C-4), 127.63–128.72 (CH-arom
Bn), 137.50, 138.14, 138.34 (3ꢂCq Bn), 170.25 (C¼O
amide). ES–MS; m/z: 576.34, [M+H]+; HR-MS 575.314;
mono-isotopic MW calculated for C34H45N1O5Si1=
575.3067. Tandem MS data: 468.27 (MH+ꢀbenzyl
alcohol), 444.22 (MH+ꢀtert-butyldimethylsilyl alco-
hol), 91.06 (benzyl cation).
1
ane, 1:1) Rf=0.73 and 0.37. H NMR [S-configuration]
(CDCl3): d=0.03 (d, 6H, 2ꢂCH3, Si(CH3)2), 0.87 (s,
0
9H, tBu), 2.51 (m, 1H, H-2, J2,3=8.7 Hz, J2,2 a=2.9 Hz,
0
J2,2 b=5.8 Hz), 3.27 (s, 1H, OH-5), 3.28 (d, 1H, H-6a,
J6a,6b=10.2 Hz), 3.52 (d, 1H, H-6b, J6a,6b=10.2 Hz),
3.65 (dd, 1H, H-20a, J2 a,2 b=9.0 Hz, J2,2 a=2.9 Hz),
0
0
0
3.72 (d, 1H, H-4, J3,4=9.6 Hz), 4.07 (dd, 1H, H-20b,
0
0
J2 a2 b=9.0 Hz), 4.39 (d, 1H, CH Bn, J=12.1 Hz), 4.42
(dd, 1H, H-3, J2,3=8.7 Hz, J3,4=9.6 Hz), 4.56 (d, 1H,
CH Bn, J=12.1 Hz), 4.57 (d, 1H, CH Bn, J=11.0 Hz),
4.68 (d, 1H, CH Bn, J=11.4 Hz), 4.82 (d, 1H, CH Bn,
J=11.0 Hz), 4.97 (d, 1H, CH Bn, J=11.4 Hz), 6.32 (s,
1H, NH amide), 7.22–7.38 (m, 15H, CH-arom Bn).
13C{1H}-NMR [S-configuration] (CDCl3): d=ꢀ5.39
(SiCH3), ꢀ5.31 (SiCH3), 18.31 (Cq tBu), 25.92 (CH3
tBu), 49.47 (C-2), 66.57 (C-20), 67.06 (C-6), 73.36, 75.04,
75.24 (3ꢂCH2 Bn), 75.11 (C-3), 79.45 (C-4), 81.99 (C-5),
127.73-128.70 (CH-arom Bn), 137.54, 138.04, 138.22
(3ꢂCq Bn), 170.57 (C¼O amide). ES–MS; m/z:
592.28, [M+H]+; monoisotopic Mw calculated for
C34H45N1O6Si1=591.30. 1H NMR [R-configuration]
(CDCl3): d=0.06 (d, 6H, Si(CH3)2), 0.90 (s, 9H, tBu),
3.1.11. 3,4-Di-O-benzyl-2-C-benzyloxymethyl-2-deoxy-D-
gluco-ꢂ-lactam (20). TBAF(1 M in THF, 1.0 mL) was
added to a cooled (0 ꢁC) solution of 19 (0.30 g, 0.52
mmol) in THF(8 mL). After 30 min, the reaction was
quenched with aqueous sodium acetate (2 M, 1.5 mL).
Ethyl acetate (20 mL) was added and the organic layer
was washed with water (10 mL), dried (MgSO4), and
concentrated. Column chromatography of the residue
over silica gel with MeOH/DCM (0:100!6:94) gave 20
as a colorless oil. Yield 0.22 g (91%). TLC (silica gel,
0
0
2.83 (m, 1H, H-2, J2,3=5.0 Hz, J2,2 a=4.2 Hz, J2,2 b=6.9
Hz), 3.74 (dd, 2H, H2-6, J6a,6b=9.7 Hz), 3.82 (m, 2H, H-
0
20a, H-4, J3,4=6.7 Hz, J2 a,2 b=9.0 Hz, J2,2 a=4.2 Hz),
0
0
3.87 (dd, 1H, H-20b, J2 a,2 b=9.0 Hz, J2,2 b=6.9 Hz), 4.16
(dd, 1H, H-3, J2,3=5.1 Hz, J3,4=6.3 Hz), 4.24 (s, 1H,
OH-5), 4.45–4.67 (3ꢂAB, 6H, 3ꢂCH2 Bn), 6.28 (s, 1H,
NH), 7.20–7.33 (m, 15H, CH-arom Bn). 13C{1H}-NMR
[R-configuration] (CDCl3): d-5.33 (SiCH3),-5.29 (SiCH3),
18.46 (Cq tBu), 25.99 (CH3 tBu), 47.31 (C-2), 67.17 (C-
20), 68.01 (C-6), 73.19, 73.31, 73.85 (3ꢂCH2 Bn), 73.39
(C-3), 78.53 (C-4), 83.97 (C-5), 127.70–128.59 (CH-
arom Bn), 137.48, 137.51, 138.30 (3ꢂCq Bn), 168.94
(C¼O amide). ES–MS; m/z: 592.28, [M+H]+; mono-
isotopic MW calculated for C34H45N1O6Si1= 591.30.
0
0
0
1
ethyl acetate/hexane, 4:1) Rf=0.20. H NMR (CDCl3):
0
d=2.44 (m, 1H, H-2, J2,3=9.3 Hz, J2,2 a=2.8 Hz,
0
J2,2 b=5.6 Hz), 3.21 (br. s, 1H, OH-6), 3.38 (m, 1H, H-
5, J4,5=9.0 Hz, J5,6a=3.0 Hz, J5,6b=5.8 Hz), 3.56 (dd,
1H, H-6a, J5,6a=5.9 Hz, J6a,6b=11.6 Hz), 3.61 (t, 1H,

R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
899
H-4, J3,4=9.3 Hz, J4,5=9.1 Hz), 3.67 (dd, 1H, H-20b,
3.1.14. 6-Amino-3,4-di-O-benzyl-2-C-benzyloxymethyl-2,6-
dideoxy-D-gluco-ꢂ-lactam (23). Water (0.5 mL) and tri-
phenylphosphine (72 mg, 0.27 mmol) were added to a
solution of 22 (84 mg, 0.18 mmol) in THF(5 mL). After
refluxing for 6 h, the reaction mixture was concentrated.
Column chromatography of the residue over silica gel
with CHCl3/MeOH/acetic acid/H2O, (94:6:1.75:0.75!
88:12:3.5:1.5) gave 23 as a colorless oil. Yield 21 mg
(25%). TLC (silica gel, CHCl3/MeOH/acetic acid/H2O,
88:12: 3.5:1.5) Rf=0.38. 1H NMR (CDCl3): d=2.50 (m,
0
0
J2 a,2 b=8.9 Hz), 3.79 (dd, 1H, H-6b, J5,6b=2.8 Hz,
0
J6a,6b=11.6 Hz), 4.01 (dd, 1H, H-20a, J2,2 a=2.8 Hz,
0
0
J2 a,2 b=8.9 Hz), 4.08 (t, 1H, H-3, J2,3=J3,4=9.4 Hz),
4.34–4.91 (3 AB, 6H, 3 CH2 Bn), 7.19–7.36 (m, 15H,
CH-arom Bn). 13C{1H}-NMR (CDCl3): d=48.70 (C-2),
56.26 (C-5), 62.19 (C-6), 65.94 (C-20), 73.28, 74.93, (CH2
Bn), 77.46 (C-3), 78.18 (C-4), 127.69–128.63 (CH-arom
Bn), 137.88, 138.21, 138.23 (3ꢂCq Bn), 171.99 (C¼O
amide). ES–MS; m/z: 461.23, [M+H]+; mono-isotopic
MW calculated for C28H31N1O5=461.22.
0
0
1H, H-2, J2,3=9.0 Hz, J2,2 a=2.9 Hz, J2,2 b=3.1 Hz),
2.75 (br. s, 1H, H-6a), 3.13 (br. s, 1H, H-6b), 3.40 (br. t,
1H, H-5), 3.57 (t, 1H, H-4, J3,4=J4,5=8.7 Hz), 3.70 (dd,
3.1.12. 3,4-Di-O-benzyl-2-C-benzyloxymethyl-2-deoxy-6-
O-mesyl-D-gluco-ꢂ-lactam (21). Mesyl chloride (8.0 mL,
0.10 mmol) was added to a cooled (0 ꢁC) solution of 20
(26 mg, 56.4 mmol) in pyridine (5 mL). After 18 h,
methanol (1 mL) was added to the mixture and subse-
quently concentrated. The residue was dissolved in die-
thyl ether, the organic layer washed with water (3ꢂ10
mL), brine (10 mL), dried (Na2SO4), and concentrated.
Column chromatography of the residue over silica gel
with DCM/MeOH (100:0!96:4) gave 21 as a colorless
oil. Yield 30 mg (99%). TLC (silica gel, hexane/ethyl
1H, H-20a, J2,2 a=2.9 Hz, J2 a,2 b=8.9 Hz), 4.02 (dd, 1H,
0
0
0
H-20b, J2 a,2 b=8.9 Hz, J2,2 b=3.1 Hz), 4.08 (t, 1H, H-3,
J2,3=9.1 Hz, J3,4=8.9 Hz), 4.34–4.91 (3ꢂAB, 6H,
3ꢂCH2 Bn), 7.19–7.36 (m, 15H, CH-arom Bn), 7.55 (s,
1H, NH lactam). 13C{1H}-NMR (CDCl3): d=42.17
(C-6), 48.43 (C-2), 54.88 (C-5), 65.99 (C-20), 73.28,
74.65, 74.76 (3ꢂCH2 Bn), 77.46 (C-3), 78.50 (C-4),
127.67–128.66 (CH-arom Bn), 137.76, 138.15, 138.28
(3ꢂCq Bn), 171.76 (C¼O lactam). ES–MS; m/z: 461.17,
[M+H]+; HR-MS: 460.239; mono-isotopic MW
calculated for C28H32N2O4=460.2362. Tandem MS data:
m/z 353.18 (MH+ꢀbenzyl alcohol), 245.12 (MH+ꢀ
2ꢂbenzyl alcohol).
0
0
0
1
acetate 1:4) Rf=0.32. H NMR (CDCl3): d=2.50 (m,
1H, H-2, J2,3=8.9 Hz), 2.96 (s, 3H, CH3 Ms), 3.63 (m,
2H, H-4, H-5), 3.71 (dd, 1H, H-20a, J2.2 a=3.0 Hz,
0
J2 a,2 b=8.9 Hz), 4.03 (dd, 1H, H-20b, J2,2 b=3.3 Hz,
0
0
0
0
0
J2 a,2 b=8.9 Hz), 4.13 (m, 2H, H-3, H-6a), 4.29 (dd, 1H,
H-6b, J5,6b=2.4 Hz, J6a,6b=10.4 Hz), 4.37–4.93 (3ꢂAB,
6H, 3ꢂCH2 Bn), 7.21–7.37 (m, 15H, CH-arom Bn).
13C{1H}-NMR (CDCl3): d=37.55 (CH3 Ms), 48.41 (C-
2), 53.74 (C-5), 66.02 (C-20), 68.20 (C-6), 73.33, 74.75
(CH2 Bn), 76.90 (C-3, C-4), 127.74-128.79 (CH-arom
Bn), 137.38, 137.94, 138.16 (3ꢂCq Bn), 170.81 (C¼O
lactam). ES–MS; m/z: 540.18, [M+H]+; mono-isotopic
MW calculated for C29H33N1O7S1=539.19.
3.1.15. 6-Acetamido-3,4-di-O-benzyl-2-C-benzyloxymethyl-
2,6-dideoxy-D-gluco-ꢂ-lactam (24). Acetyl chloride (1.6
mL, 23 mmol) and TEA (6.4 mL, 46 mmol) were added to
a cooled (0 ꢁC) solution of 23 (5.3 mg, 11.5 mmol) in
DCM (1 mL). After 15 min, the reaction mixture was
concentrated. Column chromatography of the residue
over silica gel with DCM/MeOH (100:0!94:6) gave 24
as a colorless oil. Yield 3.9 mg (67%). TLC (silica gel,
DCM/MeOH, 95:5) Rf=0.56. 1H NMR (CDCl3):
d=1.78 (s, 3H, CH3 acetamido), 2.47 (m, 1H, H-2,
0
0
3.1.13. 6-Azido-3,4-di-O-benzyl-2-C-benzyloxymethyl-2,6-
dideoxy-D-gluco-ꢂ-lactam (22). Sodium azide (0.13 g,
2.0 mmol) was added to a solution of 21 (0.36 g, 0.67
mmol) in DMF(10 mL) and stirred for 4 h at 70 ꢁC.
The mixture was concentrated, and the residue was dis-
solved in DCM (50 mL). The organic layer was washed
with water (20 mL), dried (MgSO4), and concentrated.
Column chromatography of the residue over silica gel
with DCM/MeOH (100:0!97:3) gave 22 as a colorless
oil. Yield 0.29 g (89%). TLC (silica gel, hexane/ethyl
J2,3=8.8 Hz, J2,2 a=3.0 Hz, J2,2 b= 3.1 Hz), 3.11 (m,
1H, H-6a, J6a,6b=14.3 Hz, J5,6a=4.8 Hz, JNH,6a=6.1
Hz), 3.36 (m, 1H, H-5, J4,5=9.1 Hz, J5,6a=4.3 Hz,
J5,6b=3.9 Hz), 3.43 (t, 1H, H-4, J3,4=8.8 Hz, J4,5=9.1
Hz), 3.55 (m, 1H, H-6b, J6a,6b=14.3 Hz, J5,6b=3.4 Hz,
JNH,6b=6.7 Hz), 3.66 (dd, 1H, H-20a, J2 a,2 b=8.9 Hz,
0
0
J2,2 a=3.0 Hz), 4.01 (dd, 1H, H-20b, J2 a,2 b=8.9 Hz,
0
0
0
0
J2,2 b=3.2 Hz), 4.08 (t, 1H, H-3, J2,3= 8.8 Hz, J3,4=8.7
Hz), 4.35–4.92 (3ꢂAB, 6H, 3ꢂCH2 Bn), 5.13 (br. t, 1H,
NH amide, JNH,6a=JNH,6b=6.5 Hz), 6.63 (s, 1H, NH
lactam), 7.22–7.44 (m, 15H, CH-arom Bn). 13C{1H}-
NMR (CDCl3): d=23.03 (CH3 acetamido) 39.94 (C-6),
49.08 (C-2), 54.88 (C-5), 66.50 (C-20), 73.33, 74.13, 74.78
(3ꢂCH2 Bn), 77.04 (C-3), 78.28 (C-4), 127.74–129.15
(CH-arom Bn), 137.83, 138.11, 138.22 (3ꢂCq Bn),
170.99, 171.76 (C¼O amide, lactam). ES–MS; m/z:
503.26, [M+H]+; mono-isotopic MW calculated for
C30H34N2O5=502.25.
1
acetate, 1:4) Rf=0.76. H NMR (CDCl3): d=2.48 (m,
0
0
1H, H-2, J2,3=9.4 Hz, J2,2 a=2.8 Hz, J2,2 b=6.6 Hz),
3.31 (dd, 1H, H-6a, J5,6a=5.4 Hz, J6a,6b=12.5 Hz), 3.41
(m, 1H, H-5, J4,5=8.3 Hz, J5,6a=5.2 Hz, J5,6b=3.0 Hz),
3.58 (dd, 1H, H-6b, J5,6b=3.0 Hz, J6a,6b=12.5 Hz), 3.63
(t, 1H, H-4, J4,5=8.9 Hz, J3,4=9.2 Hz), 3.70 (dd, 1H,
H-20a, J2,2 a=2.9 Hz, J2 a,2 b=8.9 Hz), 4.08 (m, 2H, H-
0
0
0
20b, H-3, J2 a,2 b=8.8 Hz, J2,2 b=6.5 Hz, J2,3=9.3 Hz,
J3,4=9.1 Hz), 4.35–4.93 (3ꢂAB, 6H, 3ꢂCH2 Bn), 7.18–
7.36 (m, 15H, CH-arom Bn), 7.53 (s, 1H, NH lactam).
13C{1H}-NMR (CDCl3): d=48.46 (C-2), 51.88 (C-6),
53.96 (C-5), 65.79 (C-20), 73.19, 74.74, 74.81 (3ꢂCH2
Bn), 77.24 (C-3), 78.11 (C-4), 127.56-128.52 (CH-arom
Bn), 137.68, 138.07, 138.19 (3ꢂCq Bn), 171.47 (C¼O
lactam). ES–MS; m/z: 487.18, [M+H]+; mono-isotopic
MW calculated for C28H30N4O4=486.22.
0
0
0
3.1.16. 6-Acetamido-2,6-dideoxy-2-C-hydroxymethyl-D-
gluco-ꢂ-lactam (6). Pd/C (10%, 5 mg) was added to a
solution of 24 (2.5 mg, 5.0 mmol) in DMF(0.5 mL).
Hydrogen was passed through the stirred mixture for 16
h. The reaction mixture was filtered and concentrated.
1
Yield 0.8 mg of a colorless oil (69%). H NMR (D2O):
d=2.09 (s, 3H, CH3 acetamido), 2.46 (m, 1H, H-2,

900
R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
0
0
J2,3=10.1 Hz, J2,2 a=J2,2 b=2.8 Hz), 3.46 (m, 1H, H-5,
J4,5=9.4 Hz, J5,6a=3.4 Hz, J5,6b=5.0 Hz), 3.57 (dd, 1H,
H-6a, J5,6a= 3.4 Hz, J6a,6b=14.5 Hz), 3.62 (t, 1H, H-4,
J3,4=9.7 Hz, J4,5=9.4 Hz), 3.63 (dd, 1H, H-6b,
J5,6b=5.0 Hz, J6a,6b=14.5 Hz), 3.95 (t, 1H, H-3,
J2,3=10.0 Hz, J3,4= 9.9 Hz) 3.96 (dd, 1H, H-20a,
H-5, J4,5=9.1 Hz, J5,6a=3.6 Hz, J5,6b=2.1 Hz), 2.83 (dd,
1H, H-1eq, J1ax,1eq=11.7 Hz, J1eq,2=3.7 Hz), 3.25 (d,
1H, NCHa Bn, JHa,Hb=13.6 Hz), 3.41 (dd, 1H, H-20a,
0
J2 a,2 b=9.3 Hz, J2,2 a=2.8 Hz), 3.54 (m, 2H, H-3, H-
0
0
20b, J2,3=10.4 Hz, J3,4=8.5 Hz, J2 a,2 b=9.1 Hz, J2,2 b
0
0
0
=
5.9 Hz), 3.67 (t, 1H, H-4, J3,4=8.8 Hz, J4,5=8.9 Hz),
3.98 (dd, 1H, H-6a, J6a,6b=11.3 Hz, J5,6a=3.6 Hz), 4.07
(dd, 1H, H-6b, J6a,6b=11.3 Hz, J5,6b=2.1 Hz), 4.24 (d,
1H, NCHb Bn, JHa,Hb=13.6 Hz), 4.30–4.94 (3ꢂAB, 6H,
3ꢂCH2 Bn), 7.18–7.38 (m, 20H, CH-arom Bn). 13C{1H}-
NMR (CDCl3): d=ꢀ5.31 (SiCH3), 18.25 (Cq tBu),
26.02 (CH3 tBu), 41.43 (C-2), 53.28 (C-1), 56.99 (NCH2
Bn), 60.28 (C-6), 67.77 (C-5), 69.13 (C-20), 72.94, 74.79,
74.81 (3ꢂCH2 Bn), 80.59 (C-4), 82.83 (C-3), 127.48-128.86
(CH-arom Bn), 138.60, 138.90, 138.99, 139.97 (4ꢂCq Bn).
ES–MS; m/z: 652.4, [M+H]+; HR-MS: 651.365; mono-
isotopic MW calculated for C41H53 N1O4Si1=651.3744.
Tandem MS data: m/z 544.32 (MH+- benzyl alcohol),
520.29 (MH+- tert-butyldimethylsilyl alcohol).
J2 a,2 b=11.4 Hz, J2,2 a=2.8 Hz), 4.16 (dd, 1H, H-20b,
0
0
0
J2 a,2 b=11.4 Hz, J2,2 b=2.8 Hz). 13C{1H}-NMR (D2O):
d=22.67 (CH3 acetyl) 40.76 (C-6), 49.89 (C-2), 55.54
(C-5), 58.23 (C-20), 69.03 (C-4), 70.75 (C-3). ES–MS; m/
z: 233.1, [M+H]+; HR-MS 232.104. mono-isotopic
MW calculated for C9H16N2O5=232.1059. Tandem MS
data: 215.10 (MH+- H2O), 197.10 (MH+-2ꢂH2O),
179.07 (MH+- 3ꢂH2O).
0
0
0
3.1.17. 3,4-Di-O-benzyl-2-C-benzyloxymethyl-6-O-tert-
butyldimethylsilyl - 1,2,5 -trideoxy -1,5 -imino - D -glucitol
(25). BH3Me2S complex (2 M, 0.65 mL, 1.3 mmol) was
added to a cooled (0 ꢁC) solution of 19 (94 mg, 0.16
mmol) in DCM (5 mL). The reaction mixture was stir-
red for 16 h at ambient temperature. EtOH (4 mL) was
added and the mixture was concentrated. Column
chromatography of the residue over silica gel with hex-
ane/ethyl acetate (80:20!60:40) gave 25 as a colorless
oil. Yield 56 mg (61%). TLC (silica gel, ethyl acetate/
3.1.19. N-Benzyl-3,4-di-O-benzyl-2-C-benzyloxymethyl-
1,2,5-trideoxy-1,5-imino-D-glucitol (27). TBAF(1M in
THF, 300 mL) was added to a cooled (0 ꢁC) solution of
26 (28 mg, 43 mmol) in THF(250 mL). After 6 h, the
mixture was quenched with aqueous sodium acetate
(2 M, 500 mL). The water layer was separated and
extracted with DCM (3ꢂ2 mL). The combined organic
layers were dried (MgSO4) and concentrated. Column
chromatography of the residue over silica gel with ethyl
acetate/hexane, (0:100!40:60) gave 27 as a colorless oil.
Yield 23 mg (100%). TLC (silica gel, ethyl acetate/hex-
1
hexane, 1:4) Rf=0.14. H NMR (CDCl3): d=0.04 (d,
6H, 2ꢂCH3, TBDMS), 0.89 (t, 9H, 3ꢂCH3, tBu), 1.64
(br. s, 1H, NH), 1.83 (m, 1H, H-2), 2.60 (m, H-5,
J4,5=9.7 Hz, J5,6a=4.8 Hz, J5,6b=2.8 Hz), 2.64 (dd, 1H,
H-1ax, J1ax,1eq=12.8 Hz, J1ax,2=11.6 Hz), 3.16
(dd, 1H, H-1eq, J1ax,1eq=13.0 Hz, J1eq,2=4.2 Hz),
3.40 (t, 1H, H-4, J3,4=9.0 Hz, J4,5=9.5 Hz), 3.52
(dd, 1H, H-3, J2,3=10.6 Hz, J3,4=9.0 Hz), 3.57 (m, 2H,
H2-20, J2 a,2 b=9.1 Hz, J2,2 a=3.1 Hz, J2,2 b=5.3 Hz),
3.74 (dd, 1H, H-6a, J6a,6b=9.8 Hz, J5,6a=4.7 Hz),
3.78 (dd, 1H, H-6b, J6a,6b=9.7 Hz, J5,6b=2.7 Hz),
4.41–4.95 (3ꢂAB, 6H, 3ꢂCH2 Bn), 7.24–7.35 (m, 15H,
CH-arom Bn). 13C{1H}-NMR (CDCl3): d=ꢀ5.35
(SiCH3), ꢀ5.29 (SiCH3), 18.32 (Cq tBu), 26.01
(CH3 tBu), 45.62 (C-2), 47.66 (C-1), 61.82 (C-5),
63.12 (C-6), 68.98 (C-20), 73.17, 75.06, 75.31 (3ꢂCH2
Bn), 82.13 (C-4), 82.92 (C-3), 127.61–128.54 (CH-
arom Bn), 138.57, 138.76, 138.80 (3ꢂCq Bn). ES–
MS; m/z: 562.35, [M+H]+; mono-isotopic MW calcu-
lated for C34H47N1O4Si1=561.33.
1
ane, 1:4) Rf=0.25. H NMR (CDCl3): d=1.91 (m, 1H,
H-2), 2.23 (t, 1H, H-1ax, J1ax,1eq=11.7 Hz), 2.32 (br. s,
1H, OH-6), 2.36 (m, 1H, H-5, J4,5=9.3 Hz, J5,6a=1.5
Hz, J5,6b=2.7 Hz), 2.97 (dd, 1H, H-1eq, J1ax,1eq=11.8
Hz, J1eq,2=3.8 Hz), 3.28 (d, 1H, NCHa Bn,
0
0
0
0
JHa,Hb=13.7 Hz), 3.44 (dd, 1H, H-20a, J2 a,2 b=9.3 Hz,
0
0
J2,2 a=2.8 Hz), 3.52 (m, 2H, H-3, H-20b, J2,3= 10.5 Hz,
0
0
0
0
J3,4=9.1 Hz, J2 a,2 b=9.2 Hz, J2,2 b=5.7 Hz), 3.72 (t,
1H, H-4, J3,4=J4,5=9.3 Hz), 3.88 (dd, 1H, H-6a,
J6a,6b=11.8 Hz, J5,6a=1.5 Hz), 4.01 (dd, 1H, H-6b,
J6a,6b=11.8 Hz, J5,6b=3.0 Hz), 4.08 (d, 1H, NCHb Bn,
JHa,Hb=13.5 Hz), 4.34–4.98 (3ꢂAB, 6H, 3ꢂCH2 Bn),
7.19–7.39 (m, 20H, CH-arom Bn). 13C{1H}-NMR
(CDCl3): d=41.23 (C-2), 54.02 (C-1), 57.08 (NCH2 Bn),
58.02 (C-6), 66.66 (C-5), 68.77 (C-20), 73.03, 74.86, 75.44
(3ꢂCH2 Bn), 80.34 (C-4), 82.28 (C-3), 127.31–128.85
(CH-arom Bn), 138.19, 138.44, 138.59, 138.69 (4ꢂCq
Bn). ES–MS; m/z: 538.3, [M+H]+; mono-isotopic MW
calculated for C35H39N1O4=537.29.
3.1.18. N-Benzyl-3,4-di-O-benzyl-2-C-benzyloxymethyl-
6-O-tert-butyldimethylsilyl-1,2,5-trideoxy-1,5-imino-D-
glucitol (26). TEA (10 mL, 72 mmol) and benzyl bro-
mide (3.2 mL, 27 mmol) were added to a solution of 25
(10.2 mg, 18.1 mmol) in acetonitrile (250 mL). After 16 h,
the mixture was diluted with DCM (2 mL) and washed
with aqueous NaHCO3 (1%, 2 mL). The aqueous phase
was extracted with DCM (3ꢂ2 mL). The combined
organic layers were dried (MgSO4) and concentrated.
Column chromatography of the residue over silica gel
with hexane/ethyl acetate (100:0!70:30) gave 26 as a
colorless oil. Yield 5.8 mg (49%). TLC (silica gel, ethyl
acetate/hexane, 4:1) Rf=0.70. 1H NMR (CDCl3):
d=0.01 (d, 6H, 2ꢂCH3, TBDMS), 0.89 (s, 9H, 3ꢂCH3,
tBu), 1.94 (m, 1H, H-2, J1ax,2=11.3 Hz, J1eq,2=3.7 Hz,
3.1.20. N-benzyl-3,4-di-O-benzyl-2-C-benzyloxymethyl-6-
chloro-1,2,5,6-tetradeoxy-1,5-imino-D-glucitol (28). Mesyl
chloride (5 mL, 64 mmol) and TEA (18 mL, 129 mmol)
were added to a cooled (0 ꢁC) solution of 27 (11.6 mg,
21.5 mmol) in DCM (1 mL). After 16 h, methanol (50 mL)
was added and the reaction mixture was concentrated.
Column chromatography of the residue over silica gel
with hexane/ethyl acetate (100:0!70:30) gave 28 as a
colorless oil. Yield 11.6 mg (97%). TLC (silica gel, hex-
ane/ethyl acetate, 4:1) Rf=0.71. 1H NMR (CDCl3):
d=1.93 (m, 1H, H-2), 2.15 (t, 1H, H-1ax, J1ax,1eq=11.6
Hz, J1ax,2=11.4 Hz), 2.52 (m, 1H, H-5, J4,5=8.9 Hz,
0
0
J2,3=10.4 Hz, J2,2 a=2.8 Hz, J2,2 b=5.9 Hz), 2.09 (t, 1H,
H-1ax, J1ax,1eq=11.6 Hz, J1ax,2=11.3 Hz), 2.38 (m, 1H,

R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
901
J5,6a=3.0 Hz, J5,6b=1.8 Hz), 2.88 (dd, 1H, H-1eq,
J1ax,1eq=11.8 Hz, J1eq,2=3.9 Hz), 3.16 (d, 1H, NCHa
Hz), 3.69 (t, 1H, H-4, J3,4=9.1 Hz, J4,5=9.3 Hz), 4.05
(d, 1H, NCHb Bn, JHa,Hb=13.7 Hz), 4.33–4.97 (3ꢂAB,
6H, 3ꢂCH2 Bn), 7.18–7.40 (m, 20H, CH-arom Bn).
13C{1H}-NMR (CDCl3): d=38.39 (C-6), 41.25 (C-2),
53.92 (C-1), 56.48 (NCH2 Bn), 66.99 (C-5), 68.96 (C-20),
72.98, 74.83, 75.02 (3ꢂCH2 Bn), 79.78 (C-4), 82.96 (C-3),
127.03-129.02 (CH-arom Bn), 138.53, 138.66, 138.84,
139.02 (4ꢂCq Bn). ES–MS; m/z: 537.3, [M+H]+; mono-
isotopic MW calculated for C35H40N2O3=536.30.
Bn, JHa,Hb=13.0 Hz), 3.40 (dd, 1H, H-20a, J2 a,2 b=9.4
0
0
Hz, J2,2 a=2.7 Hz), 3.54 (m, 2H, H-3, H-20b, J2,3=10.1
0
0
0
0
Hz, J3,4=8.9 Hz, J2 a,2 b=9.4 Hz, J2,2 b=5.5 Hz), 3.77
(t, 1H, H-4, J3,4=J4,5=8.8 Hz), 3.97 (dd, 1H, H-6a,
J6a,6b=12.3 Hz, J5,6a=3.2 Hz), 4.10 (dd, 1H, H-6b,
J6a,6b=12.3 Hz, J5,6b=1.8 Hz), 4.11 (d, 1H, NCHb Bn,
JHa,Hb=13.0 Hz), 4.31–4.98 (3ꢂAB, 6H, 3ꢂCH2 Bn),
7.19–7.39 (m, 20H, CH-arom Bn). 13C{1H}-NMR
(CDCl3): d=41.38 (C-2), 42.16 (C-6), 52.83 (C-1), 56.62
(NCH2 Bn), 65.40 (C-5), 68.80 (C-20), 72.95, 74.73,
75.28 (3ꢂCH2 Bn), 80.44 (C-4), 82.24 (C-3), 127.16–
129.20 (CH-arom Bn), 138.48, 138.50, 138.58, 138.74
(4ꢂCq Bn). ES–MS; m/z: 556.3, [M+H]+; mono-iso-
topic MW calculated for C35H38Cl1N1O3= 555.25.
3.1.23. 6-Acetamido-N-benzyl-3,4-di-O-benzyl-2-C-benzyl-
oxymethyl-1,2,5,6-tetradeoxy-1,5-imino-D-glucitol (31).
TEA (5 mL, 36 mmol) and acetyl chloride (1.5 mL, 21
mmol) were added to a cooled (0 ꢁC) solution of 30 (2.5
mg, 4.7 mmol) in DCM (0.5 mL). After 15 min, MeOH
(100 mL) was added and the reaction mixture was con-
centrated. Column chromatography of the residue over
silica gel with DCM/EtOH (100:0!95:5) gave 31 as a
colorless oil. Yield 2.4 mg (85%). TLC (silica gel,
3.1.21. 6-Azido-N-benzyl-3,4-di- O-benzyl-2-C-benzyl-
oxymethyl-1,2,5,6-tetradeoxy-1,5-imino-D-glucitol (29).
Sodium azide (7.0 mg, 107 mmol) was added to a solu-
tion of 28 (5.8 mg, 10.4 mmol) in DMF(250 mL) and the
1
MeOH/DCM, 5:95) Rf=0.42. H NMR (CDCl3): d=
1.93 (br. s, 4H, H-2, CH3 acetamido), 2.18 (t, 1H, H-1ax,
J1ax,1eq=J1ax,2=11.7 Hz), 2.56 (m, 1H, H-5, J4,5=9.0
Hz, J5,6a=4.1 Hz, J5,6b=2.0 Hz), 2.95 (dd, 1H, H-1eq,
J1ax,1eq=12.0 Hz, J1eq,2=3.7 Hz), 3.26 (d, 1H, NCHa
Bn, JHa,Hb=13.8 Hz), 3.37–3.56 (m, 5H, H2-20, H-3, H-4,
H-6a), 3.98 (d, 1H, NCHb Bn, JHa,Hb=13.9 Hz), 4.01
ꢁ
mixture was stirred for 1 h at 100 C. The mixture was
concentrated. Column chromatography of the residue
over silica gel with hexane/ethyl acetate (100:0!70:30)
gave 29 as a colorless oil. Yield 5.4 mg (91%). TLC
(silica gel, hexane/ethyl acetate, 4:1) Rf=0.65. 1H NMR
(CDCl3): d=1.95 (m, 1H, H-2), 2.12 (t, 1H, H-1ax,
J1ax,1eq=11.6 Hz, J1ax,2=11.4 Hz), 2.52 (m, 1H, H-5,
J4,5=9.0 Hz, J5,6a=5.9 Hz, J5,6b=3.0 Hz), 2.89 (dd, 1H,
H-1eq, J1ax,1eq=11.8 Hz, J1eq,2=3.8 Hz), 3.25 (d, 1H,
NCHa Bn, JHa,Hb=13.4 Hz), 3.41 (dd, 1H, H-20a,
(m, 1H, H-6b, J6a,6b=14.3 Hz, J5,6b=2.0 Hz, J6b,NH
=
7.1 Hz ), 4.33–4.95 (3ꢂAB, 6H, 3ꢂCH2 Bn), 7.21–7.37
(m, 20H, CH-arom Bn). 13C{1H}-NMR (CDCl3):
d=23.34 (CH3 acetamido), 36.70 (C-6), 41.41 (C-2),
54.02 (C-1), 56.86 (NCH2 Bn), 63.91 (C-5), 68.72 (C-20),
73.06, 75.01, 75.54 (3ꢂCH2 Bn), 80.74 (C-4), 82.35 (C-
3), 127.34-128.99 (CH-arom Bn), 138.13, 138.26, 138.40,
138.55 (4ꢂCq Bn), 170.20 (C¼O acetamido). ES–MS;
m/z: 579.3, [M+H]+; HR-MS 578.299 mono-isotopic
MW calculated for C37H42N2O4=578.3145. Tandem
MS data: 471.26 (MH+-benzyl alcohol), 412.22 (MH+-
benzyl alcohol–CH3C(O)NH2).
0
0
0
J2 a,2 b=9.3 Hz, J2,2 a=2.8 Hz), 3.51 (dd, 1H, H-3,
J2,3=10.3 Hz, J3,4=8.8 Hz), 3.55 (dd, 1H, H-20b,
0
0
0
J2 a,2 b=9.4 Hz, J2,2 b=5.5 Hz), 3.61 (dd, 1H, H-6a,
J6a,6b=12.6 Hz, J5,6a=2.9 Hz), 3.65 (t, 1H, H-4,
J3,4=J4,5=8.8 Hz), 3.87 (dd, 1H, H-6b, J6a,6b=12.6 Hz,
J5,6b=2.9 Hz), 4.11 (d, 1H, NCHb Bn, JHa,Hb=13.4 Hz),
4.32–4.98 (3ꢂAB, 6H, 3ꢂCH2 Bn), 7.18–7.37 (m, 20H,
CH-arom Bn). 13C{1H}-NMR (CDCl3): d=41.20 (C-2),
48.45 (C-6), 53.49 (C-1), 57.48 (NCH2 Bn), 65.37 (C-5),
68.81 (C-20), 72.98, 74.71, 75.13 (3ꢂCH2 Bn), 80.79 (C-4),
82.35 (C-3), 127.19–128.97 (CH-arom Bn), 138.29, 138.50,
138.68 (Cq Bn). ES–MS; m/z: 563.3, [M+H]+; mono-
isotopic MW calculated for C35H38N4O3=562.29.
3.1.24. 6-Acetamido-2-C-hydroxymethyl-1,2,5,6-tetrade-
oxy-1,5-imino-D-glucitol (5). Compound 31 (1.8 mg, 3.1
mmol) was treated as described for the preparation of
1
compound 6. Yield 0.7 mg of a colorless oil (94%). H
NMR (D2O): d=2.07 (m, 1H, H-2), 2.12 (s, 3H, CH3
acetamido), 3.05 (t, 1H, H-1ax, J1ax,1eq=J1ax,2=13.0
Hz), 3.29 (m, 1H, H-5, J4,5=10.2 Hz, J5,6a=6.6 Hz,
J5,6b=3.2 Hz), 3.57 (t, 1H, H-4, J3,4=8.8 Hz, J4,5=9.1
Hz), 3.58 (dd, 1H, H-1eq, J1ax,1eq=13.0 Hz, J1eq,2=4.3
Hz), 3.62 (dd, 1H, H-3, J2,3=9.9 Hz, J3,4=9.0 Hz), 3.64
(dd, 1H, H-6a, J6a,6b=15.3 Hz, J5,6a=6.6 Hz), 3.80 (dd,
3.1.22. 6-Amino-N-benzyl-3,4-di-O-benzyl-2-C-benzyloxy-
methyl-1,2,5,6-tetradeoxy-1,5-imino-D-glucitol (30). Tri-
phenylphosphine (5.0 mg, 19 mmol) and water (10 mL) were
added to a solution of 29 (5.4 mg, 9.5 mmol) in THF(0.5
mL). The reaction mixture was stirred for 2 h at 60 ꢁC,
after which the mixture was concentrated. Column
chromatography of the residue over silica gel with CHCl3/
MeOH (100:0!90:10) gave 30 as a colorless oil. Yield 3.6
mg (71%). TLC (silica gel, DCM/MeOH, 95:5) Rf=0.32.
1H NMR (CDCl3): d=1.48 (s, 3H, NH3), 1.93 (m, 1H, H-
2), 2.15 (t, 1H, H-1ax, J1ax,1eq= J1ax,2=11.7 Hz), 2.30 (m,
1H, H-5, J4,5=9.3 Hz, J5,6a=2.5 Hz, J5,6b=3.3 Hz), 2.91
(dd, 1H, H-1eq, J1ax,1eq=11.9 Hz, J1eq,2=3.9 Hz), 3.03
(dd, 1H, H-6a, J6a,6b=14.0 Hz, J5,6a=2.5 Hz), 3.17 (dd,
1H, H-6b, J6a,6b=13.8 Hz, J5,6b=3.6 Hz), 3.20 (d, 1H,
NCHa Bn, JHa,Hb=13.6 Hz), 3.43 (dd, 1H, H-20a,
1H, H-20a, J2 a,2 b=11.7 Hz, J2,2 a=6.0 Hz), 3.85 (dd,
1H, H-6b, J6a,6b=15.3 Hz, J5,6b=3.1 Hz), 3.89 (dd, 1H,
0
0
0
H-20b, J2 a,2 b=11.7 Hz, J2,2 b=3.3 Hz). 13C{1H}-NMR
(D2O): d=22.71 (CH3 acetamido), 38.82 (C-6), 41.57
(C-2), 45.65 (C-1), 59.88 (C-20), 60.34 (C-5), 71.34 (C-4),
71.87 (C-3). ES–MS; m/z: 219.2, [M+H]+; HR-MS
218.119; mono-isotopic MW calculated for C9H18N2O4=
218.1267. Tandem MS data: m/z 201.13 (MH+ꢀH2O),
183.11 (MH+ꢀ2ꢂH2O), 165.11 (MH+ꢀ3ꢂH2O), 142.08
(MH+ꢀH2O–CH3C(O)NH2).
0
0
0
J2 a,2 b=9.3 Hz, J2,2 a=2.7 Hz), 3.53 (m, 2H, H-20b, H-3,
3.1.25. 1,2,5-Trideoxy-2-C-hydroxymethyl-1,5-imino-D-
glucitol (32). Compound 25 (3.3 mg, 5.8 mmol) was
0
0
0
0
0
0
J2 a,2 b=9.3 Hz, J2,2 b=9.4 Hz, J2,3=10.7 Hz, J3,4=9.3

902
R. J. B. H. N. van den Berg et al. / Bioorg. Med. Chem. 12 (2004) 891–902
treated with TBAFas described for the preparation of
compound 20. Subsequently, the residue was dissolved
in aqueous EtOH (90%, 200 mL) and hydrochloric acid
(1N, 5 mL) and Pd/C (10%, 5 mg) were added. Hydrogen
was passed through the stirred mixture for 1 h. The reac-
tion mixture was filtered and concentrated. Yield 1.2 mg
2. Bychkova, V. E.; Ptitsyn, O. B. FEBS Lett. 1995, 359, 6.
3. Kuznetsov, G.; Nigam, S. K. N. Engl. J. Med. 1998, 339,
1688.
4. Winchester, B.; Vellodi, A.; Young, E. Biochem. Soc.
Trans. 2000, 28, 150.
5. (a) Suzuki, Y.; Nanba, E.; Ohno, K.; Matsuda, J.; Ogawa,
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Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O. R.;
Fan, J. Q. Eur. J. Biochem. 2000, 267, 4179. (c) Asano, N.
J. Enzym. Inhib. 2000, 15, 215. (d) Asano, N.; Nash, R. J.;
Molyneux, R. J.; Fleet, G. W. J. Tetrahedron Asymm.
2000, 11, 1645. (e) Fan, J. Q.; Ishii, S.; Asano, N.; Suzuki,
Y. Nat. Med. 1999, 5, 112.
6. (a) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y.
J. Am. Chem. Soc. 1998, 120, 3007. (b) Zechel, D. L.;
Wither, S. G. Acc. Chem. Res. 2000, 33, 11. (c) Nishi-
mura, Y.; Adachi, H.; Satoh, T.; Shitara, E.; Nakamura,
H.; Kojima, F.; Tacheuchi, T. J. Org. Chem. 2000, 65,
4871.
7. (a) Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup,
T.; Lundt, I.; Bols, M. Angew. Chem., Int. Ed. Engl 1994,
33, 1778. (b) Jespersen, T. M.; Bols, M.; Dong, W.; Sierks,
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3, 161. (b) Ichikawa, M.; Igarashi, Y.; Ichikawa, Y. Tet-
rahedron Lett. 1995, 36, 1767. (c) Ichikawa, Y.; Igarashi,
Y. Tetrahedron Lett. 1995, 36, 4585.
9. (a) Dong, W.; Jespersen, T.; Bols, M.; Skrydstrup, T.;
Sierks, M. R. Biochemistry 1996, 35, 2788. (b) Ichikawa,
Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J. Am. Chem.
Soc. 1998, 120, 3007.
1
of a colorless oil (70%). H NMR (D2O): d=2.07 (m,
1H, H-2), 3.01 (t, 1H, H-1ax, J1ax,1eq=J1ax,2=13.0 Hz),
3.24 (m, 1H, H-5, J4,5=9.8 Hz, J5,6a=5.7 Hz, J5,6b=3.0
Hz), 3.60 (dd, 1H, H-1eq, J1ax,1eq=12.9 Hz, J1eq,2=4.2
Hz), 3.63 (t, 1H, H-3, J2,3=10.3 Hz, J3,4=9.5 Hz), 3.68
(t, 1H, H-4, J3,4=9.5 Hz, J4,5=9.9 Hz), 3.81 (dd, 1H,
H-20a, J2 a,2 b=11.8 Hz, J2,2 a=6.2 Hz), 3.91 (dd, 1H,
0
0
0
H-20b, J2 a,2 b=11.7 Hz, J2,2 b=2.9 Hz), 3.97 (dd, 1H,
H-6a, J6a,6b=12.6 Hz, J5,6b=5.6 Hz), 4.03 (dd, 1H, H-
6b, J6a,6b=12.6 Hz, J5,6b=3.0 Hz). ES–MS; m/z:
178.11, [M+H]+; HR-MS: 177.097 mono-isotopic MW
calculated for C7H15N1O4=177.1001. Tandem MS
data: m/z 160.10 (MH+ꢀH2O), 142.08 (MH+ꢀ2ꢂH2O),
124.08 (MH+ꢀ3ꢂH2O).
0
0
0
3.1.26. Purification of homogenate of human spleen lyso-
somal ꢀ-hexosaminidase and isolation of isoenzymes.
Spleen from a control subject was homogenized in 50
mM potassium phosphate buffer (pH 6.5) and centrifuged
for 1 h at 60,000g. The supernatant was collected and
used to measure total b-hexosaminidase activity.
Preparative isoelectric focussing of the supernatant
fraction in a granulated flat bed gel was used to purify
b-hexosaminidase A (Pi 5.0) and b-hexosaminidase B
(Pi 7.5). Immunoprecipitation with specific antisera
revealed that the b-hexosaminidase A and B fractions
contained no other b-hexosaminidases.
10. Umezawa, H.; Aoyagi, T.; Komiyama, T.; Morishima,
H.; Hamada, M.; Takeuchi, T. J. Antibiotics 1974, 27, 963.
11. Shitara, E.; Nishimura, Y.; Kojima, F.; Takeuchi, T.
Bioorg. Med. Chem. 1999, 7, 1241.
12. Schuster, M. Bioorg. Med. Chem. Lett. 1999, 9, 615.
13. Takaoka, Y.; Kajimoto, T.; Wong, C.-H. J. Org. Chem.
1993, 58, 4809.
14. Trnka, T.; Cerny, M. Collection Czechoslov. Chem. Com-
mun. 1971, 36, 2216.
3.2. Enzyme assays
15. Schroder, M. Chem. Rev. 1980, 80, 187.
¨
IC50 values were determined by variation of inhibitory
concentrations in the substrate mixtures. b-Hexo-
saminidase activities were determined with the
fluorogenic substrate 4-methylumbelliferyl-N-acetyl-b-
d-glucosaminide. Substrate mixtures contained 1.58
mM substrate in 75 mM McIlvain buffer (50 mM citric
acid/100 mM sodium phosphate), pH=4.0. The assays
were performed at 37 ꢁC and stopped by the addition of
excess glycine-NaOH (0.3 M, pH=10.6). Liberated
4-methylumbelliferone was fluorometrically measured
using excitation and emission wavelengths of 366 and
445 nm, respectively.27
16. Paulsen, H.; Lockhoff, O. Chem. Ber. 1981, 114, 3102.
17. Albright, J. D.; Goldman, L. J. Am. Chem. Soc. 1967, 89,
2416.
18. Hoos, R.; Naughton, A. B.; Vasella, A. Helv. Chim. Acta
1993, 76, 1802.
19. Overkleeft, H. S.; Van Wiltenburg, J.; Pandit, U. K. Tet-
rahedron Lett. 1993, 34, 2527.
20. Van den Berg, R. J. B. H.N.; Noort, D.; Milder-Ena-
chace, E. S.; Van der Marel, G. A.; Van Boom, J. H.;
Benschop, H. P. Eur. J. Org. Chem. 1999, 2593.
21. Ermert, P.; Vasella, A. Helv. Chim. Acta 1991, 74, 2043.
22. Granier, T.; Vasella, A. Helv. Chim. Acta 1998, 81, 865.
23. Overkleeft, H. S.; Van Wiltenburg, J.; Pandit, U. K. Tet-
rahedron 1994, 50, 4215.
24. Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett.
1983, 24, 763.
References and notes
25. Brown, H. C.; Heim, P. J. Org. Chem. 1973, 38, 912.
26. Hembre, E. J.; Pearson, W. H. Tetrahedron 1997, 53, 11021.
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J. M. J. Clin. Invest. 1994, 93, 1288.
1. Braulke, T. in Biology of the Lysosome, Subcellular Bio-
chemistry, Vol. 27 Lloyd, J.B.; Mason, R.W. Eds., Plenum
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