Scalable syntheses of both enantiomers of DNJNAc and DGJNAc from glucuronolactone: The effect of N-alkylation on hexosaminidase inhibition

Article abstract of DOI:10.1002/chem.201200110

The efficient scalable syntheses of 2-acetamido-1,2-dideoxy-D-galacto- nojirimycin (DGJNAc) and 2-acetamido-1,2-dideoxy-D-gluco-nojirimycin (DNJNAc) from D-glucuronolactone, as well as of their enantiomers from L-glucuronolactone, are reported. The evaluation of both enantiomers of DNJNAc and DGJNAc, along with their N-alkyl derivatives, as glycosidase inhibitors showed that DGJNAc and its N-alkyl derivatives were all inhibitors of α-GalNAcase but that none of the epimeric DNJNAc derivatives inhibited this enzyme. In contrast, both DGJNAc and DNJNAc, as well as their alkyl derivatives, were potent inhibitors of β-GlcNAcases and β-GalNAcases. Neither of the L-enantiomers showed any significant inhibition of any of the enzymes tested. Correlation of the in vitro inhibition with the cellular data, by using a free oligosaccharide analysis of the lysosomal enzyme inhibition, revealed the following structure-property relationship: hydrophobic side-chains preferentially promoted the intracellular access of iminosugars to those inhibitors with more-hydrophilic side-chain characteristics. Getting the NAc of it: Scalable syntheses of DGJNAc and DNJNAc from D-glucuronolactone are reported. DGJNAc and its N-alkyl derivatives were inhibitors of α-GalNAcase and both DGJNAc and DNJNAc were potent inhibitors of β-GlcNAcases and β-GalNAcases. Copyright

Full text of DOI:10.1002/chem.201200110

FULL PAPER
DOI: 10.1002/chem.201200110
Scalable Syntheses of Both Enantiomers of DNJNAc and DGJNAc from
Glucuronolactone: The Effect of N-Alkylation on Hexosaminidase Inhibition
Andreas F. G. Glawar,[a, b] Daniel Best,[b] Benjamin J. Ayers,[b] Saori Miyauchi,[c]
Shinpei Nakagawa,[c] Matilde Aguilar-Moncayo,[d] Josꢀ M. Garcꢁa Fernꢂndez,[e]
Carmen Ortiz Mellet,[d] Elizabeth V. Crabtree,[a, b] Terry D. Butters,[a] Francis X. Wilson,[f]
Atsushi Kato,[c] and George W. J. Fleet*[a, b]
Abstract: The efficient scalable synthe-
ses of 2-acetamido-1,2-dideoxy-d-galac-
to-nojirimycin (DGJNAc) and 2-acet-
amido-1,2-dideoxy-d-gluco-nojirimycin
(DNJNAc) from d-glucuronolactone,
as well as of their enantiomers from l-
glucuronolactone, are reported. The
evaluation of both enantiomers of
DNJNAc and DGJNAc, along with
their N-alkyl derivatives, as glycosidase
inhibitors showed that DGJNAc and
its N-alkyl derivatives were all inhibi-
tors of a-GalNAcase but that none of
the epimeric DNJNAc derivatives in-
hibited this enzyme. In contrast, both
DGJNAc and DNJNAc, as well as
their alkyl derivatives, were potent in-
hibitors of b-GlcNAcases and b-Gal-
NAcases. Neither of the l-enantiomers
showed any significant inhibition of
any of the enzymes tested. Correlation
of the in vitro inhibition with the cellu-
lar data, by using a free oligosaccharide
analysis of the lysosomal enzyme inhib-
ition, revealed the following structure–
property relationship: hydrophobic
side-chains preferentially promoted the
intracellular access of iminosugars to
those inhibitors with more-hydrophilic
side-chain characteristics.
Keywords:
cell penetration
iminosugars · oligosaccharides
carbohydrates
·
·
·
inhibitors
Introduction
Although there are many naturally occurring hydroxylated
piperidines that are related to sugars in which the oxygen
atom of the pyranose ring has been replaced by a nitrogen
atom,[1] neither DGJNAc (2-acetamido-1,2-dideoxy-d-galac-
to-nojirimycin, 1D) nor DNJNAc (2-acetamido-1,2-dideoxy-
d-gluco-nojirimycin, 2D), which are analogues of GalNAc
and GlcNAc, respectively, have yet been isolated as natural
products (Figure 1). Nagstatin (3),[2] which is isolated from
the fermentation broth of Streptomyces amakusaensis
MG846-fF3,[3] and pochonicine (4),[4] which is isolated from
Pochonia suchlasporia var. suchlasporia TAMA 87, are
potent inhibitors of b-hexosaminidases but show no inhibi-
tion of a-GalNAcases. Although synthetic piperidines are
common,[5] LABNAc (2-acetamido-1,4-imino-1,2,4-trideoxy-
[a] A. F. G. Glawar,+ Dr. E. V. Crabtree, Dr. T. D. Butters,
Prof. G. W. J. Fleet
Oxford Glycobiology Institute, University of Oxford
South Parks Road, Oxford, OX1 3QU (UK)
Fax : (+44)1865285002
E-mail: george.fleet@chem.ox.ac.uk
[b] A. F. G. Glawar,+ Dr. D. Best,+ B. J. Ayers, Dr. E. V. Crabtree,
Prof. G. W. J. Fleet
Chemistry Research Laboratory, Department of Chemistry
University of Oxford Mansfield Road, Oxford, OX1 3TA (UK)
[c] S. Miyauchi, S. Nakagawa, Prof. A. Kato
Department of Hospital Pharmacy, University of Toyama
2630 Sugitani, Toyama 930-0194 (Japan)
[d] M. Aguilar-Moncayo, Prof. C. Ortiz Mellet
Departamento de Quꢀmica Orgꢁnica, Facultad de Quꢀmica
Universidad de Sevilla, Profesor Garcꢀa Gonzꢁlez 1
41012 Sevilla (Spain)
[e] Prof. J. M. Garcꢀa Fernꢁndez
Instituto de Investigaciones Quꢀmicas
CSIC-Universidad de Sevilla
Amꢂrico Vespucio 49, Isla de la Cartuja
41092 Sevilla (Spain)
[f] Dr. F. X. Wilson
Summit PLC, 91, Milton Park
Abingdon, Oxon OX14 4RY (UK)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200110.
Figure 1. Hexosaminidase inhibitors.
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l-arabinitol, 5) is a rare example of a pyrrolidine hexosami-
nidase inhibitor[6] that has promise as a chaperone in Sandh-
off[7] and Tay–Sachs diseases.[8] The interest in selective in-
hibitors of hexosaminidases is due to their potential use in
the chemotherapy of a wide range of diseases,[9] including
O-GlcNAcase inhibition[10] and cancer metastasis.[11]
DGJNAc is rare example of a potent inhibitor of a-GalNA-
cases.[12] Specific a-GalNAcase inhibition is of interest in the
studies of Schindler–Kanzaki disease,[13] the treatment of
cancer by the protection of macrophage-activating factor,[14]
and the modification of a number of pathogens by the inhib-
ition of endo-GalNAcases.[15] DNJNAc[16] and its N-alkyl de-
rivatives[17] are highly potent and specific inhibitors of b-hex-
osaminidases but show no significant inhibition of a-GalNA-
cases. Herein, we report scalable syntheses of DGJNAc
(1D) and DNJNAc (2D) from d-glucuronolactone (6D), as
well as of their enantiomers (1L and 2L, respectively) from
l-glucuronolactone (6L), which is readily available from d-
glucoheptonolactone.[18] A comparison of the inhibition pro-
files of both enantiomers of compounds 1 and 2, together
with a number of N-alkyl derivatives, is reported; the alkyla-
tion of DGJNAc (1D) decreases the inhibition of a-GalNA-
cases but not of b-hexosaminidases. The cellular lysosomal
inhibition of b-hexosaminidases by compounds 1D, 1Da–
1Dc, and 1De by using protein-derived carbohydrate analy-
sis was performed, thereby revealing enhanced cellular pen-
etration of hydrophobic side-chain derivatives.
Results and Discussion
Synthesis: d-Glucuronolactone (6D) has been used for the
efficient synthesis of many iminosugars and amino acids,
wherein a nitrogen moiety is introduced at the C5 position
and then joined to the C1 atom to form the nitrogen center
in the piperidine ring.[19] For the preparation of both
DGJNAc (1D) and DNJNAc (2D) from d-glucuronolac-
tone (6D, Scheme 1): i) an azide group was introduced with
inversion of the configuration at the C5 position as a precur-
sor to the exocyclic acetamido group. Sequential reduction
of the lactone at the C6 position and protection of the C6
primary alcohol gave silyl ether 7L as a divergent intermedi-
ate. For the synthesis of DGJNAc (1D): ii) inversion of the
configuration at the C3 position was necessary. iii) Protec-
tion as the benzyl ether and subsequent change in the pro-
tecting groups gave diol 8L. iv) Activation of diol 8L al-
lowed double nucleophilic displacement of the leaving
groups on the C2 and C6 positions by benzylamine to form
the bicyclic piperidine (9L). v) Subsequent functional-group
manipulation gave DGJNAc (1D). Performing analogous
transformations (iii), (iv), and (v) without inversion of the
C3 OH group in compound 7L afforded access to DNJNAc
(2D). The enantiomers (1L and 2L, respectively) were pre-
pared in an identical manner from l-glucuronolactone (6L);
l-enantiomers of many iminosugars have surprising biologi-
cal activities compared to their d-natural analogues.[8,20]
Scheme 1. (i) Introduction of a N atom with inversion of the stereochem-
istry at the C5 position and reduction of the C6 atom; (ii) inversion of
the stereochemistry at the C3 position; (iii) protection of the oxygen
atom at the C3 position; (iv) ring-closure between the C2 and C6 posi-
tions; (v) functional-group manipulation.
The synthesis of the divergent silyl ether in the d-series
(7L) is shown in Scheme 2. d-Glucuronolactone (6D) was
condensed with acetone in the presence of concentrated sul-
furic acid to give the known acetonide (12D) in 90% yield.
Esterification of the free alcohol group at the C5 position in
compound 12D with trifluoromethanesulfonic (triflic) anhy-
dride in CH2Cl2 in the presence of pyridine gave the corre-
sponding triflate (13D), which, on treatment with sodium
azide in DMF, afforded azide 14L with inversion of the con-
figuration at the C5 position (92% yield). Compound 14L
was very sensitive to base, such that direct reduction with
sodium borohydride gave a very low yield of the required
diol (16L). A two-step procedure was much more efficient:
initial reduction of lactone 14L with diisobutylaluminum hy-
dride (DIBAL-H) in CH2Cl2 gave lactol 15L (84% yield),
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Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
Scheme 2. (i) Me2CO, conc. H2SO4, 90%; (ii) (CF3SO2)2O, CH2Cl2, pyri-
dine; (iii) NaN3, DMF, 92% over two steps; (iv) DIBAL-H, CH2Cl2,
84%; (v) NaBH4, MeOH, 90%; (vi) tBuMe2SiCl, pyridine, 88%.
which, on treatment with sodium borohydride in MeOH,
provided compound 16L in 90% yield. Selective protection
of the primary alcohol in compound 16L with tert-butyldi-
methylsilyl chloride in pyridine afforded silyl ether 7L
(88% yield). Over 30 g of this key intermediate was pre-
pared in one batch and 55% overall yield from d-glucurono-
lactone. The corresponding enantiomer (7D) was also pre-
pared in an identical manner starting from l-glucuronolac-
tone (6L).
The synthesis of DGJNAc (1D) required the inversion of
the configuration of the alcohol at the C3 position in com-
pound 7L. Oxidation of compound 7L with pyridinium
chlorochromate (PCC) in CH2Cl2 in the presence of molecu-
lar sieves gave ketone 17L (81% yield), which, on treatment
with sodium borohydride in aqueous EtOH, underwent
a highly diastereoselective reduction to give the more-hin-
dered alcohol (18L) in 91% yield (Scheme 3). Treatment of
compound 18L with benzyl bromide and sodium hydride in
DMF afforded the completely protected azide (19L, 89%).
Treatment of compound 19L with HCl in MeOH caused the
concomitant removal of the silyl ether and the acetonide
protecting groups to form an anomeric mixture of methyl
furanosides 8L (93% yield; a/b ratio: 7:1), which contained
unprotected alcohol functionalities at the C2 and C6 posi-
tions. Esterification of both OH groups in compounds 8L
with triflic anhydride in CH2Cl2 in the presence of pyridine
gave stable ditriflate 20L. Reaction of ditriflate 20L with
benzylamine in THF proceeded in a stepwise manner (as in-
ferred by monitoring the reaction by TLC) that probably in-
volved initial nucleophilic substitution of the more reactive
primary triflate group. Only the major a-anomer underwent
subsequent displacement of the secondary triflate group at
the C2 position to afford bicyclic piperidine 9La (69% from
8L), whilst the minor anomer remained unreacted.
Scheme 3. (i) PCC, CH2Cl2, molecular sieves, 81%; (ii) NaBH4, EtOH,
H2O, 91%; (iii) PhCH2Br, NaH, DMF, 89%; (iv) AcCl, MeOH, 93%;
(v) (CF3SO2)2O, CH2Cl2, pyridine; (vi) PhCH2NH2, THF, 69% from 8L;
(vii) Et2O·BF3, Ac2O, 97%; (viii) DIBAL-H, CH2Cl2; then NaBH4,
MeOH; then Ac2O, pyridine, 72% from 21L; (ix) Zn, CuSO4 (aq), THF/
AcOH/Ac2O 3:2:1, 91%; (x) MeONa, MeOH; then H2, Pd/C (10%),
HCl, 1,4-dioxane, H2O, 99%; (xi) HCHO, H2, Pd/C (10%), 1,4-dioxane,
H2O, 100%; (xii) MeCHO, H2, Pd/C (10%), EtOH, 100%; (xiii)
MeCH2CH2CHO, NaBH3CN, AcOH, EtOH, 100%; (xiv) PhCHO,
NaBH3CN, AcOH, EtOH, 68%; (xv) HOCH2CHO, H2, Pd/C (10%), 1,4-
dioxane, H2O, 95%.
by leading to a mixture of the acetylated hemiacetals (21L,
97% yield); the first equivalent of the Lewis acid was
needed to complex the amine, thereby allowing subsequent
catalytic acetolysis of the glycosidic bond. A two-step reduc-
tion of compound 21L by sequential treatment with
DIBAL-H and sodium borohydride in MeOH gave the cor-
responding diol, which was transformed into diacetate 22D
by conventional acetylation with acetic anhydride in pyri-
dine (72% yield). Reduction of azide 22D with zinc in
a mixture of THF/acetic-acid/acetic-anhydride in the pres-
ence of aqueous copper(II) sulfate[21] produced di-O-acety-
lated acetamide 23D in 91% yield. Selective removal of the
acetate esters in compound 23D with sodium methoxide in
MeOH, followed by hydrogenolysis of the benzyl protecting
groups in the presence of 10% palladium/HCl, gave the
target iminosugar DGJNAc (1D) in 99% yield, which corre-
sponded to an overall yield of 27% from silyl ether 7L. This
procedure was suitable for the synthesis of DGJNAc (1D)
All attempts to hydrolyze methyl furanoside 9La by acid
treatment were unsuccessful, instead leading to complex
mixtures. Alternatively, acetolysis of compound 9La could
be achieved by conducting the reaction with over one equiv-
alent of boron trifluoride etherate in acetic anhydride, there-
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G. W. J. Fleet et al.
on a multigram scale in 15% yield from d-glucuronolactone
(6D); the enantiomer l-DGJNAc (1L) was made by a simi-
lar sequence from l-glucuronolactone (6L, Scheme 3).
A series of N-alkylated derivatives of compound 1D was
prepared to determine the effect on hexosaminidase inhibi-
tion. Reductive amination of compound 1D by hydrogena-
tion with formaldehyde and glycoaldehyde in 1,4-dioxane/
H2O (1:1) in the presence of palladium gave N-methyl-
DGJNAc (1Da) and N-(2-hydroxyethyl)-DGJNAc (1De) in
yields of 100% and 95%, respectively. Hydrogenation of
compound 1D in EtOH in the presence of ethanal and pal-
ladium formed N-ethyl-DGJNAc (1Db) in 100% yield.
Treatment of compound 1D in EtOH with butyraldehyde
and benzaldehyde in the presence of sodium cyanoborohy-
dride gave the N-butyl- (1Dc) and N-benzyl analogues
ACHTUNGTRENNUNG
first treated with benzyl bromide and sodium hydride in
DMF to give the benzyl ether derivative (24L, 100%),
which, on treatment with HCl in MeOH, afforded an easily
separable mixture of approximately equal amounts of fura-
noside anomers 10Lb (48%) and 10La (43%, Scheme 4).
Treatment of anomers 10La or 10Lb with triflic anhy-
dride in CH2Cl2 in the presence of pyridine gave ditriflate
32, which was found to be much less stable than its C3
epimer (20L). The benzyl ether at the C3 position, which
was in a cis disposition with respect to the primary triflate at
the C6 position in compound 32, spontaneously began to
form 3,6-anhydro derivative 33 (Scheme 5); subsequent
treatment with benzylamine in the next step gave mono-
amine 34, which was not readily separable from the required
bicycle (11L). All attempts to generate the 2,6-di-O-trifyl
ester and react it in situ with benzylamine for the isolation
of compound 11L in good yield were unsuccessful. Replac-
ing the triflyl groups by methanesulfonyl (mesyl) groups led
to a stable disulfonate ester; however, following displace-
ment at the C6 position with benzylamine, the compound
was insufficiently reactive at the C2 position to allow ring
closure. Therefore, it was necessary to introduce a less-reac-
tive leaving group at the C6 position to suppress the forma-
tion of a 3,6-anhydro derivative whilst keeping a reactive tri-
flate at the C2 position to facilitate ring closure.
Scheme 4. (i) PhCH2Br, NaH, DMF, 100%; (ii) AcCl, MeOH, 48% 10Lb,
43% 10La; (iii) MeSO2Cl, 2,4,6-collidine, CH2Cl2, 86%; (iv) TsCl, 2,4,6-
collidine, CH2Cl2, 74%; Ts=4-toluenesulfonyl; (v) (CF3SO2)2O, CH2Cl2,
pyridine; (vi) PhCH2NH2, THF, 70% from 25Lb, 68% from 27La;
(vii) Et2O·BF3, Ac2O, 75% from 11Lb, 71% from 11La; (viii) DIBAL-H,
CH2Cl2, pyridine; then NaBH4, MeOH; then Ac2O, pyridine, 78%;
(ix) Zn, CuSO4 (aq), THF/AcOH/Ac2O 3:2:1, 79%; (x) MeONa, MeOH;
then H2, Pd/C (10%), HCl, 1,4-dioxane, H2O, 94%; (xi) HCHO, H2, Pd/C
(10%), H2O, 94%; (xii) MeCH2CH2CHO, H2, Pd/C (10%), 1,4-dioxane,
H2O, 96%.
In the case of the b-anomer (10Lb), reaction with mesyl
chloride in CH2Cl2 in the presence of 2,4,6-collidine allowed
selective esterification of the primary alcohol group to pro-
duce the corresponding monomesylate (25Lb, 86% yield;
Scheme 4). Subsequent activation of the secondary alcohol
in compound 25Lb by reaction with triflic anhydride formed
mixed disulfonate 26Lb, which, on treatment with benzyla-
mine, gave the bicyclic piperidine (11Lb) in 70% yield. The
a-anomer (10La) did not undergo selective mesylation
under the above conditions. However, the reaction of com-
pound 10La with p-toluenesulfonyl (tosyl) chloride afforded
the primary tosylate (27La) in satisfactory yield (74%).
Esterification of compound 27La with triflic anhydride
formed the triflate (28La), which underwent double nucleo-
philic displacement with benzylamine to give the fully pro-
Scheme 5. Unwanted formation of THF.
tected piperidine (11La, 68%). Acetolysis of bicyclic
amines 11La and 11Lb with boron trifluoride etherate in
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Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
acetic anhydride led to the same epimeric mixture of the di-
acetates (29L) in 71% and 75% yield, respectively. A two-
step reduction of compound 29L by sequential treatment
with DIBAL-H and sodium borohydride in MeOH, fol-
lowed by acetylation with acetic anhydride in pyridine, gave
the diacetate (30D) in 78% yield. Reductive acetylation of
the azide group in compound 30D with zinc/copper-sulfate
in THF/acetic-acid/acetic-anhydride provided amide 31D
(79% yield), which, on deprotection by conventional cata-
lytic transesterification and hydrogenolysis, formed
DNJNAc (2D) in 94% yield. Enantiomer l-DNJNAc (2L)
was prepared in a similar sequence from l-glucuronolactone
(6L). Reductive alkylation of compound 2D by hydrogena-
tion with formaldehyde in water and butyraldehyde in aque-
ous 1,4-dioxane gave N-methyl-DNJNAc (2 Da, 94%) and
N-butyl-DNJNAc (2Dc, 96%), respectively.
DNJ, Zavesca), which is structurally related to N-butyl-
DGJNAc (1Dc), has been approved for the treatment of
the lysosomal-storage disorder Type 1 Gaucher disease by
substrate-reduction therapy. The N-hydroxyethyl derivative
of DNJ (Glyset, Miglitol), which is analogous to compound
1De, is used for the treatment of non-insulin-dependent dia-
betes mellitus.
Comparison of DGJNAc (1D) versus DNJNAc (2D):
Table 1 shows the inhibitory data of the enantiomers of
DGJNAc (1) and DNJNAc (2); the l-enantiomers of d-imi-
nosugar competitive glycosidase inhibitors are often non-
competitive inhibitors of the same enzyme.[25] The syntheses
reported herein from the enantiomers of glucuronolactone
ensured the enantiomeric purity of inhibitors 1 and 2. Nei-
ther of the l-enantiomers (1L and 2L) significantly inhibit-
ed any of the glycosidases at the highest concentration
tested; l-DGJNAc (1L) showed very weak inhibition
(IC50 =830 mm) of human placenta b-GlcNAcase. In contrast,
both compounds 1D and 2D were good inhibitors of b-
GlcNAcases, with the exception of enzyme from Aspergillus
oryzae. Therefore, b-GlcNAcases were inhibited by both
epimers at the C4 position with either gluco- or galacto-ste-
reochemistry; the same was true for b-GalNAcase inhibition
against the enzyme that was derived from HL60 cell lysate.
These results suggested that the stereochemistry at the C4
OH group was not strictly distinguished by b-GalNAcases
and b-GlcNAcases.
Biological evaluation: Both enantiomers of DGJNAc and
DNJNAc, as well as their N-alkyl derivatives, were tested in
vitro for their glycosidase-inhibiting potential against the
following enzymes: b-N-acetyl-glucosaminidase (b-GlcNA-
case), b-N-acetyl-galactosaminidase (b-GalNAcase), and a-
N-acetyl-galactosaminidase (a-GalNAcase; Tables 1–3); the
influence of DGJNAc (1D) and its derivatives 1 Da, 1Db,
1Dc, and 1De on lysosomal glycosidase inhibition was in-
vestigated by using a free oligosaccharide (FOS) analysis in
HL60 cells (Figure 2, Table 4). This set of glycosidases has
been implicated in a range of pathological disorders. In
cancer patients that carry pancreatic adenocarcinoma[22] or
gliomas,[23] b-HexNAcase activity is elevated in the blood in
comparison to healthy individuals. a-GalNAcase activity
was identified as a biomarker for patients that suffer from
melanoma.[14a] Furthermore, inhibitors of these enzymes
could be employed to treat lysosomal-storage disorders,
such as Tay–Sachs, Sandhoff, and Schindler–Kanzaki diseas-
es, by improving mutant-enzyme activity through a chaper-
one-mediated pathway.[24] N-Butyl deoxynorjirimycin (NBu-
DGJNAc (1D) was a potent inhibitor of a-GalNAcase
with an IC50 value of 0.32 mm against chicken liver and a Ki
value of 0.17 mm against Charonia lampas.[12] None of the
other three NH iminosugars (compounds 1L, 2D, or 2L)
significantly inhibited a-GalNAcase from chicken liver
(Table 1).
N-Alkyl derivatives of DNJNAc (2D): The N-alkylated de-
rivatives of DNJNAc 2D (2Da and 2Dc) showed similar in-
Table 1. Concentrations of the iminosugars that give 50% inhibition of various glycosidases.
IC50 [mm]
Enzyme
DGJNAc (1D)
l-DGJNAc (1L)
DNJNAc (2D)
l-DNJNAc (2L)
b-N-acetyl-glucosaminidase
human placenta
bovine kidney
Aspergillus oryzae
HL60
8.3
4.2
830
7.0
7.4
891
9.8
2.9
NI (31.2)
NI (24.4)
NI (0.7)
NI (25.9)
NI (28.8)
NI (46.8)
NI (0.8)
NI (31.3)
NI (46.3)
NI[a] (8.8)[b]
7.1
1.8
jack beans
a-N-acetyl-galactosaminidase
chicken liver
0.32
NI (22.6)
NI (5.0)
NI (36.9)
b-N-acetyl-galactosaminidase
Aspergillus oryzae
HL60
NI (9.6)
23
NI (0)
NI (12.9)
998
17
NI (0)
NI (14.8)
[a] NI=No inhibition (less than 50% inhibition at 1000 mm). [b] Inhibition (%) at 1000 mm are given in parentheses.
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G. W. J. Fleet et al.
Figure 2. HL60 cells were homogenized and the FOS was extracted as de-
scribed in the Experimental Section. After labeling with 2-AA, the FOS
were separated by using HPLC. a) Control cells; b) cells that were treat-
ed with DGJNAc (1D, 500 mm). The peaks are numbered and their corre-
sponding structures are given in Table 4.
Figure 3. Ratio of the integration of peak 5 to the control peak 7 is
shown for the derivatives of compound 1D (50 mm). Because all of the
derivatives have very comparable in vitro inhibition, the variation can be
attributed to the differences in cell- and organelle penetration. To test
for significance, a student t-test was performed (99% confidence inter-
val).
hibition of b-GlcNAcase or b-GalNAcase to that of the NH
parent compound (Table 2). In general, N-alkylation of com-
pound 2D had little effect on enzyme inhibition, in contrast
to the observed increased inhibition for the l-lysine-linked
dansyl derivatives of DNJNAc that were generated by Stein-
er et al.;[17b] however, inhibition of the Aspergillus oryzae
enzyme was significantly increased by alkylation, notably for
N-methyl-DNJNAc (2Da). None of the derivatives of com-
pound 2D inhibited a-GalNAcase.
seemed to be less-well-tolerated by the same enzyme. This
trend was reflected in the Ki values against the enzyme that
was derived from Charonia lampas, with the exception of
the N-methyl derivative.
Free oligosaccharide (FOS) analysis[26] was performed on
HL60 cells after treatment for 24 h with the N-alkyl deriva-
tives of DGJNAc (1 Da, 1Db, 1Dc, and 1De), which gave
additional free glycans (FOS) in the HPLC profiles that
were derived from HL60 homogenates (Figure 2b) in com-
parison to the untreated control reactions (Figure 2a). Sub-
sequent treatment of these FOSs with jack bean b-GlcNA-
case identified the newly produced glycans as non-reducing
terminal GlcNAc-containing species; their identity was veri-
fied by comparison with the retention times in glucose units
N-Alkyl derivatives of DGJNAc (1D): All of the derivatives
of DGJNAc (1D) were good inhibitors of b-GlcNAcases
(except for Aspergillus oryzae, Table 3), although N-benzyl-
DGJNAc (1Dd) was significantly weaker. N-Ethyl- (1Db),
N-benzyl- (1Dd), and N-hydroxyethyl-DGJNAc (1De) were
all good inhibitors of a-GalNAcase that was derived from
chicken liver. However, the methyl and butyl groups
Table 2. Concentrations of DNJNAc and its N-alkyl derivatives that give 50% inhibition of various glycosidases.
IC50 [mm]
Enzyme
DNJNAc (2D)
N-Me-DNJNAc (2Da)
N-Bu-DNJNAc (2Dc)
b-N-acetyl-glucosaminidase
human placenta
bovine kidney
Aspergillus oryzae
HL60
7.0
7.4
891
9.8
2.9
18
12
225
22
17
10
8.1
478
16
jack beans
24
a-N-acetyl-galactosaminidase
chicken liver
NI[a] (5.0)[b]
NI (0.8)
NI (9.6)
b-N-acetyl-galactosaminidase
Aspergillus oryzae
HL60
998
17
167
74
381
62
[a] NI=No inhibition (less than 50% inhibition at 1000 mm). [b] Inhibition (%) at 1000 mm are given in parentheses.
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Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
Table 3. Concentrations of DGJNAc and its N-alkyl derivatives that give 50% inhibition of various glycosidases.
IC50 [mm]
Enzyme
DGJNAc (1D)
N-Me-DGJNAc
(1Da)
N-Et-DGJNAc
(1Db)
N-Bu-DGJNAc
(1Dc)
N-Bn-DGJNAc
(1Dd)
N-(2-Hydroxyethyl)-
DGJNAc (1De)
b-N-acetyl-gluco-
saminidase
human placenta
bovine kidney
Aspergillus
oryzae
8.3
4.2
5.7
2.1
NI (22.1)
7.3
3.1
NI (26.5)
2.7
1.2
NI (22.6)
45
40
NI (11.4)
8.2
3.7
NI (46.9)
NI[a] (8.8)[b]
HL60
jack beans
7.1
1.8
5.4
1.5
7.9
3.4
1.7
2.7
52
22
8.3
1.9
a-N-acetyl-galac-
tosaminidase
chicken liver
Charonia lampas
b-N-acetyl-galac-
tosaminidase
Aspergillus
oryzae
0.32
A
107
5.6
166
11
[14]
6.3
ACHTUNGTNER[NUNG 3.9]
[0.17][c]
A
A
A
NI (9.6)
23
NI (29.4)
26
NI (26.3)
44
NI (29.8)
14
NI (5.8)
187
926
35
HL60
[a] NI=No inhibition (less than 50% inhibition at 1000 mm). [b] Inhibition (%) at 1000 mm are given in parentheses. [c] Ki values are given in square brackets.
(GUs) reported by Boomkamp et al. (Table 4).[26] A struc-
ture of the glycans (peaks 8, 11, and 13) was proposed based
on their retention times and the b-GlcNAcase-digestion re-
sults. The appearance of non-reducing terminal GlcNAc-
containing glycans was dose-dependent for all of the inhibi-
tors tested over the range 500 mm, 100 mm, and 50 mm; more-
over, their appearance was due to inhibition of lysosomal b-
HexNAcase, thus leading to incomplete degradation of the
glycans.
DNJ, shows a volume of distribution (VD) of 1.04–
1.31 LkgÀ1 (calculated for an 80 kg individual),[27] whilst Mi-
glitol (Glyset), which is the N-hydroxyethyl derivative of
DNJ, has a value of VD =0.18 LkgÀ1, which is 5.7–7.3-fold
lower.[28] Therefore, whilst Miglustat is able to penetrate tis-
sues beyond the bloodstream, Miglitol is primarily restricted
to the extracellular fluid. Previous results have also demon-
strated that the N-alk(en)yl derivatives (C4, C9, C18) of DNJ
are taken up by cells in less than 1 min.[29]
Quantitative
(GlcNAc2Man3GlcNAc1)
comparison
with
of
control
glycan 5
glycan 7
In the design of a potential therapeutic agent, a polar N-
alkyl group, such as N-hydroxyethyl 1De, on the ring nitro-
gen atom could restrict the iminosugar to the extracellular
space, whilst a hydrophobic side-chain derivative, such as N-
butyl, would assist in the cell-membrane-penetration of the
inhibitor. Therefore, the correlation of the in vitro data with
those in cells could be used to estimate how well the inhibi-
tor is able to reach an intracellular therapeutic target. If the
target is secreted into extracellular space, this method could
be used to estimate the side-effects of intracellular enzyme
inhibition. The iminosugar derivatives presented herein all
showed this inhibition to some degree, albeit only at very
non-physiological high concentrations of 50 mm (the maxi-
mum plasma concentration of Miglustat in a multiple dose
regime is 12.7 mm; the maximum plasma concentration of
Miglitol in a 100 mg oral dose is 8.4 mm).[30,31]
(Man5GlcNAc1) allowed an estimation of the degree of lyso-
somal b-GlcNAcase inhibition. At a concentration of 50 mm,
the levels of inhibition that were displayed by the deriva-
tives showed strong differences depending on the side-chain
that was present on the inhibitor. Increasing the length of
the alkyl side-chain in comparison to parent compound 1D
led to stronger inhibition in this cell-based assay, whilst the
hydroxyethyl variant showed lower levels of inhibition
(Figure 3). With the in vitro inhibition activity against b-
GlcNAcase of all of the inhibitors being of comparable
strength (HL60 data, Table 3), the apparent differences be-
tween the inhibition of lysosomal b-GlcNAcase in the cells
could be attributed to differential penetration of the cells or
organelles by the inhibitors. Accordingly, the N-butyl deriva-
tive (1Dc) was approximately 5-fold more-effective at inhib-
iting lysosomal b-GlcNAcase than the N-hydroxyethyl deriv-
ative (1De). This result correlated with the bio-distribution
of two iminosugar inhibitors of DNJ that are already used
clinically. Miglustat (Zavesca), the N-butyl derivative of
Chem. Eur. J. 2012, 00, 0 – 0
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G. W. J. Fleet et al.
Table 4. Structures, number of glucose units (GUs), and percentage proportion of isolated FOS peaks from the control experiment and 500 mm
DGJNAc-treated HL60 cells.[a]
ID
1
Untreated GUs
–
Treated GUs
3.77
Structure name
Structure
In control cells [%]
–
In treated cells [%]
GlcNAc1Man2GlcNAc1
2.45
1.62
A
2
–
3.88
GlcNAc2Man2GlcNAc1
–
(Æ0.21)
3
4
4.13
4.95
4.16
4.99
Man3GlcNAc1
Man4GlcNAc1
3.44
G
1.66
5.20
(Æ0.88)
14.37
5
6
–
–
5.28
5.65
GlcNAc2Man3GlcNAc1
GlcNAc3Man3GlcNAc1
–
–
17.98
15.08
G
6A
7
5.62
5.87
–
Man5GlcNAc1
Man5GlcNAc1
4.22
G
–
5.91
38.76
16.81
N
8
–
6.16
GlcNAc4Man3GlcNAc1
–
2.92
N
9
–
6.49
6.67
6.96
7.37
GlcNAc4Man3GlcNAc1
Glc1Man5GlcNAc1
–
10.00
7.37
1.35
3.24
N
10
11
12
6.62
14.92
(Æ0.13)
–
GlcNAc5Man3GlcNAc1
Glc2Man5GlcNAc1
–
7.31
6.17
(Æ0.35)
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Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
Table 4. (Continued)
FULL PAPER
ID
Untreated GUs
Treated GUs
Structure name
Structure
In control cells [%]
In treated cells [%]
13
–
7.73
GlcNAc6Man3GlcNAc1
–
4.51
N
14
15
16
7.99
8.47
8.79
8.04
8.42
8.87
Man7GlcNAc2
Man8GlcNAc2
Man8GlcNAc2
2.25
1.25
5.39
G
0.88
1.03
3.88
G
17
9.46
9.55
Man9GlcNAc2
9.24
(Æ0.54)
4.05
N
[a] The peak IDs correspond to the labels in Figure 2. The mean values were each taken from three independent measurements (ÆS.D.). The species
were assigned in reference to the GU units in ref. [26]. The labels are as follows:
were used as supplied (analytical or HPLC grade). The reactions that
were performed under an atmosphere of argon or hydrogen were main-
Conclusion
tained by an inflated balloon. All water sensitive reactions were per-
formed under an inert atmosphere of argon/nitrogen unless otherwise
stated. All solutions are saturated unless otherwise stated. Thin layer
chromatography (TLC) analysis was performed on aluminum sheets
(Merck) that were coated with 60 F254 silica. The sheets were either vi-
This paper describes efficient scalable syntheses of both
enantiomers of DGJNAc (1) and DNJNAc (2) from glucur-
onolactone, thereby allowing easy access to this class of hex-
osaminidase inhibitor. DGJNAc (1D) and its N-alkyl deriv-
sualized by using a dip of 0.2% w/v cerium(IV) sulfate and 5% ammoni-
atives (1 Da–1De) were all inhibitors of a-GalNAcase but
none of the epimeric DNJNAc (2) derivatives inhibited the
same enzyme. In contrast, both DGJNAc (1D) and
DNJNAc (2D), as well as their alkyl derivatives, were
potent inhibitors of b-hexosaminidases. Neither of their
enantiomers (1L and 2L, respectively) showed any signifi-
cant inhibition against this enzyme. The correlation of the in
vitro inhibition data against b-GlcNAcase with the cellular
lysosomal inhibition data of this enzyme by using protein-
derived carbohydrate analysis revealed a side-chain-depen-
dent variation in cell- and organelle penetration. Hydropho-
bic side-chains of increasing length promote cellular uptake
whilst hydrophilic variants restrict access of the inhibitor to
intracellular organelles.
um molybdate in 2m sulfuric acid or potassium permanganate (0.5% in
1m NaOH). Flash chromatography was either performed on Sorbsil C60
40/60 silica or Merck grade 9385, 230–400 mesh, 60 ꢅ. Where chromato-
graphic techniques that involve triethylamine-containing mobile phases
were employed, silica was doped with triethylamine before use. Melting
points were recorded on a Kofler hot block and are uncorrected. Optical
rotations were recorded on a Perkin–Elmer 241 polarimeter with a path
length of 1 dm. Concentrations are quoted in g100 mLÀ1. IR spectra were
recorded on a Bruker Tensor 27 FTIR spectrophotometer by using thin
films on either NaCl or Ge plates. Only the characteristic peaks are
quoted. NMR spectra were record on a Bruker AMX500 (1H: 500 MHz
and 13C: 125.7 MHz) or on
a
Bruker AV400 spectrometer (1H:
400.2 MHz and 13C: 100.6 MHz) in the stated deuterated solvent. Chemi-
cal shifts (d) are quoted in ppm and coupling constants (J) are given in
Hz. Residual signals from the solvents were used as an internal reference.
MeCN or MeOH were used as an internal reference when D2O was used
as the solvent. MS (ESI) was recorded on a Micromass LCT spectrome-
ter. HRMS (ESI) was performed on a Bruker MicroTof (resolution=
10000 FWHM). Assays for the inhibition of glycosidases were performed
according to literature procedures.[32]
Experimental Section
Synthesis of divergent protected silyl ether 7
1,2-O-Isopropylidene-a-d-glucurono-3,6-lactone (12D): d-Glucurono-3,6-
lactone (6D, 30.0 g, 170 mmol) was dissolved in acetone (250 mL) and
All commercial reagents were used as supplied. Dry pyridine and DMF
were purchased from Aldrich in Sure-Sealꢄ bottles. All other solvents
Chem. Eur. J. 2012, 00, 0 – 0
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G. W. J. Fleet et al.
concentrated sulfuric acid (8.3 mL). The mixture was stirred at RT for
4 h, after which time TLC analysis (EtOAc) indicated the complete con-
sumption of the starting material (Rf =0.40, streaks to baseline) and the
formation of a major product (Rf =0.88). The reaction mixture was neu-
tralized by the addition of solid sodium bicarbonate, filtered, and concen-
trated in vacuo. The crude residue was recrystallized (toluene) to afford
acetonide 12D as a white crystalline solid (33.0 g, 90%). M.p. 118–1208C
(toluene) [lit. 120.5–121.58C[19c] (toluene)]; [a]D25 =+59.2 (c 2.00, CHCl3)
[lit. [a]2D0 =+52.5[19c] (c 1.95, CHCl3)]; 1H NMR (CDCl3, 400 MHz): d=
and the reaction mixture was stirred vigorously at RT for 18 h. The or-
ganic phase was collected and the aqueous layer was extracted with
CH2Cl2 (8ꢆ300 mL). The organic fractions were combined, dried
(MgSO4), filtered, and concentrated in vacuo to afford crude lactol 15L
as a pale-yellow crystalline solid (18.6 g, 84%) in a 2:3 (A/B) ratio of the
anomers. This crude product was reacted on without further purification.
M.p. 53–558C; [a]D25 =À21.4 (c 1.10, CHCl3); 1H NMR (CDCl3,
A
400 MHz): d=1.33 (s, 3H; CH3B), 1.35 (s, 3H; CH3A), 1.50 (s, 6H; CH3
and CH3B), 3.45 (br s, 1H; OH6B), 4.08 (s, 1H; H5A), 4.08 (d, J
ACHTUNGTRENNUNG
4.1 Hz, 1H; H5B), 4.64 (d, J(2,1)=3.8 Hz, 1H; H2B), 4.72 (d, J
ACTHNUGTRENNUGN ACHTUNGTRENNUNG
1.36, 1.53 (s, 2ꢆ3H; CACTHNGUTRENUN(GN CH3)2), 3.02 (s, 1H; OH5), 4.53 (d, JACHTGNUTRENNUNG
4.4 Hz, 1H; H5), 4.83 (d, J
N
ACHTUNGTRENNUNG
2.7 Hz, 1H; H3), 4.95 (dd,
J
U
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
6.00 ppm (d, J
26.5, 26.9 (C(CH3)2), 70.6 (C5), 78.1 (C4), 81.3 (C3), 82.9 (C2), 106.6
(C1), 113.6 (CACHTUNGTRENNUNG(CH3)2), 173.7 ppm (C6); IR (thin film): n˜ =1790 (s; C=O),
ACHTUNGTRENNUNG
(1,2)=3.4 Hz, 1H; H1); 13C NMR (CDCl3, 100 MHz): d=
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
U
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
3441 cmÀ1 (s; OH); LRMS (ESI+): m/z (%): 239 (54) [M+Na]+, 271 (30)
[M+MeOH+Na]+, 455 cmÀ1 (100) [2M+Na]+; HRMS (ESI+): m/z calcd
for C9H12NaO6: 239.0526 [M+Na]+; found: 239.0526.
AHCTUNGTRENNUNG
106.5 (C1B), 106.8 (C1A), 112.7 (C(CH3)2B), 113.0 ppm (C(CH3)2A); IR
ACTHNUTRGENNUG CAHTUNGTRENNUGN
(thin film): n˜ =2113 (s, N3), 3425 cmÀ1 (br s; OH); LRMS (ESI+): m/z
(%): 298 (100) [M+MeOH+Na]+, 573 (83) [2M+2MeOH+Na]+, 589
(48) [2M+2MeOH+K]+; HRMS (ESI+): m/z calcd for C10H17N3NaO6:
298.1010 [M+MeOH+Na]+; found: 298.1010.
For enantiomer 12L: M.p. 117–1198C (toluene); [a]2D5 =À62.3 (c 2.00,
CHCl3).
5-Azido-5-deoxy-1,2-O-isopropylidene-b-l-idurono-3,6-lactone (14L): Tri-
fluoromethanesulfonic anhydride (39.1 mL, 232 mmol) was added drop-
wise to a solution of lactone 12D (38.65 g, 178.8 mmol) in CH2Cl2
(400 mL) and pyridine (43.4 mL, 536 mmol) at À408C. The reaction mix-
ture was stirred for 1 h at between À358C and À258C. After this time,
TLC analysis (cyclohexane/EtOAc, 1:1) indicated the complete consump-
tion of the starting material (Rf =0.32) and the formation of a major
product (Rf =0.80). The reaction mixture was diluted with CH2Cl2
(150 mL), washed with 2m HCl (3ꢆ600 mL), and the organic phase was
dried (MgSO4), filtered, and concentrated in vacuo to afford crude tri-
flate 13D. M.p. 160–1628C [lit. 1608C[19c] (EtOH)]; [a]D25 =+55.1 (c 1.01,
CHCl3) [lit. [a]D20 =+49.2[19c] (c 1.28, CHCl3)]; 1H NMR (CDCl3,
For enantiomer 15D: M.p. 57–598C; [a]2D5 =+27.4 (c 1.05, CHCl3).
5-Azido-5-deoxy-1,2-O-isopropylidene-b-l-idofuranose (16L): Sodium
borohydride (1.16 g, 30.6 mmol) was added slowly to a solution of lactol
15L (18.6 g, 76.5 mmol) in MeOH (200 mL) at À308C. The reaction mix-
ture was stirred for 90 min, with an internal temperature maintained be-
tween À208C and À108C; after which time, TLC analysis (cyclohexane/
EtOAc, 2:1) indicated the consumption of the starting material (major
Rf =0.40, minor Rf =0.39) and the formation of a major product (Rf =
0.15). The reaction was neutralized with glacial acetic acid and concen-
trated in vacuo. The crude residue was dissolved in EtOAc (600 mL),
washed with a saturated solution of sodium bicarbonate (600 mL), and
the aqueous phase was extracted with EtOAc (2ꢆ800 mL). The organic
fractions were combined, dried (MgSO4), filtered, concentrated in vacuo,
and purified by column chromatography on silica gel (cyclohexane/
EtOAc, 2:1 to 1:9) to afford diol 16L as a white crystalline solid (16.9 g,
90%); M.p. 122–1248C [lit. 120–1228C];[12] [a]D25 =À75.4 (c 1.00, CHCl3)
[lit. [a]2D5 =À69.6[12] (c 0.94, CHCl3)]; 1H NMR ([D6]acetone, 400 MHz):
400 MHz): d=1.37, 1.54 (s, 2ꢆ3H; C
1H; H2), 4.94 (d, J(3,4)=2.7 Hz, 1H; H3), 5.08 (dd, J
(4,5)=4.3 Hz, 1H; H4), 5.42 (d, J(5,4)=4.3 Hz, 1H; H5), 6.06 ppm (d, J-
ACHTUNGTRENNUNG(1,2)=3.6 Hz, 1H; H1).
A
ACHTUNGTREN(NUNG 2,1)=3.6 Hz,
G
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
Sodium azide (15.20 g, 232.4 mmol) was added in one portion to a solu-
tion of crude triflate 13D in DMF (400 mL) at À308C. After 90 min,
TLC analysis (cyclohexane/EtOAc, 2:1) indicated the complete consump-
tion of the starting material (Rf =0.58) and formation of a major product
(Rf =0.72). The reaction mixture was partitioned between EtOAc
(600 mL) and a water/sat. brine (4:1) solution (300 mL), separated, and
the organic phase was washed with water/sat. brine (4:1, 2ꢆ300 mL),
dried (MgSO4), filtered, and concentrated in vacuo. The crude residue
was purified by column chromatography on silica gel (cyclohexane/
EtOAc, 5:1) to afford the azide (14L) as a white crystalline solid (39.7 g,
92%). M.p. 110–1128C [lit. 114–1168C];[19c] [a]D25 =+240.1 (c 1.00, CHCl3)
d=1.27, 1.41 (s, 2ꢆ3H; C
5.8 Hz, J(6,6’)=10.6 Hz, 1H; H6), 3.70–3.75 (1H, m; H5), 3.78 (ddd, J-
(6’,5)=3.4 Hz, J(6’,OH6)=5.1 Hz, J(6’,6)=10.6 Hz, 1H; H6’), 4.16–4.18
(m, 2H; H3 and H4), 4.27 (app t, J(OH6,6)=J(OH6,6’)=5.5 Hz, 1H;
OH6), 4.52 (d, J(OH3,3)=4.8 Hz, 1H; OH3), 4.54 (d, J(2,1)=3.8 Hz,
1H; H2), 5.91 ppm (d, J
(1,2)=3.8 Hz, 1H; H1); 13C NMR ([D6]acetone,
100 MHz): d=26.8, 27.4 (C(CH3)2), 62.8 (C6), 65.0 (C5), 75.5, 81.9 (C3,
C4), 87.0 (C2), 105.9 (C1), 112.3 ppm (C(CH3)2); IR (thin film): n˜ =2108
ACHTUGNTRENNU(NG CH3)2), 3.66 (app dt, JAHCNUTRTGEG(NNUN 6,5)=JACHTUNGTRENNUNG(6,OH6)=
AHCTUNGTRENNUNG
A
E
ACHTUNGTRENNUNG
T
ACHTUNGTRENNUNG
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
(s; N3), 3335 cmÀ1 (br s; OH); LRMS (ESI+): m/z (%): 268 (93)
[M+Na]+, 513 cmÀ1 (100) [2M+Na]+; (ESIÀ): 244 (25) [MÀH]À, 280
(100) [M+35Cl]À, 282 (70) [M+37Cl]À, 489 (55) [2MÀH]À; HRMS
(ESI+): m/z calcd for C9H15N3NaO5: 268.0904 [M+Na]+; found:
268.0903.
[lit. [a]2D0 =+243[19c] (c 1.1, CHCl3)]; H NMR (CDCl3, 400 MHz): d=1.35,
1
1.51 (s, 2ꢆ3H; C
H4), 4.84 (d, J(2,1)=3.8 Hz, 1H; H2), 4.95 (d, J
5.94 ppm (d, J
(1,2)=3.8 Hz, 1H; H1); 13C NMR (CDCl3, 100 MHz): d=
26.5, 27.0 (C(CH3)2), 61.2 (C5), 81.1 (C4), 81.8 (C2), 84.9 (C3), 106.2
(C1), 113.4 (C(CH3)2), 170.7 ppm (C6); IR (thin film): n˜ =1792 (s; C=O),
2124 cmÀ1 (s; N3); LRMS (ESI+): m/z (%): 264 (25) [M+Na]+, 296 (100)
R
ACHTUNGTREN(NGNU 4,3)=3.1 Hz, 1H;
T
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
For enantiomer 16D: M.p. 119–1208C; [a]2D5 =+63.1 (c 1.15, MeOH).
ACHTUNGTRENNUNG
5-Azido-6-O-tert-butyldimethylsilyl-5-deoxy-1,2-O-isopropylidene-b-l-ido-
furanose (7L): tert-Butyldimethylsilyl chloride (19.5 g, 129 mmol) was
added to a solution of diol 16L (21.1 g, 86.0 mmol) in pyridine (210 mL).
The reaction mixture was stirred at RT for 18 h, after which time TLC
analysis (cyclohexane/EtOAc, 2:1) indicated the complete consumption
of the starting material (Rf =0.15) and the formation of a major product
(Rf =0.60). The reaction was quenched by the addition of MeOH (4 mL)
and concentrated in vacuo. The crude residue was dissolved in EtOAc
(750 mL), washed with 2m HCl (3ꢆ450 mL), dried (MgSO4), filtered,
concentrated in vacuo, and purified by column chromatography on silica
gel (cyclohexane/EtOAc, 20:1 to 3:1) to afford the silyl ether 7L as a col-
orless oil (27.2 g, 88%); [a]2D5 =À13.9 (c 1.40, CHCl3); 1H NMR (CDCl3,
[M+MeOH+Na]+,
328
(15)
[M+2MeOH+Na]+,
569
(85)
[2M+2MeOH+Na]+; (ESIÀ): 276 (100) [M+35Cl]À, 278 (40) [M+37Cl]À;
HRMS
(ESI+):
m/z
calcd
for
C10H15N3NaO6:
296.0853
[M+MeOH+Na]+; found: 296.0855.
For enantiomer 14D: M.p. 111–1138C; [a]2D5 =À213.5 (c 0.96, CHCl3).
5-Azido-5-deoxy-1,2-O-isopropylidene-b-l-ido-hexodialdo-1,4:3,6-difura-
nose (15L): Diisobutylaluminum hydride (1.5m in toluene, 91.0 mL,
137 mmol) was added dropwise to a solution of azide 14L (21.9 g,
90.8 mmol) in CH2Cl2 (200 mL) under an argon atmosphere at À788C.
The reaction mixture was stirred at this temperature for 90 min. After
this time, TLC analysis (cyclohexane/EtOAc, 2:1) indicated the complete
consumption of the starting material (Rf =0.72) and formation of two
400 MHz): d=0.13 (s, 6H; Si
(s, 2ꢆ3H; C(CH3)2), 3.22 (d, J
(m, 1H; H5), 3.77–3.82 (m, 2H; H6 and H6’), 4.12 (dd, JAHCTUNGTRENNUNG
G
ACHTUGNTRENN(UNG CH3)3), 1.32, 1.50
products (major Rf =0.40, minor Rf =0.39).
sodium potassium tartrate (500 mL) and CH2Cl2 (600 mL) were added
A
saturated solution of
E
ACHTUNGTRENNUNG
&
10
&
www.chemeurj.org
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
A
E
E
5-Azido-3-O-benzyl-6-O-tert-butyldimethylsilyl-5-deoxy-1,2-O-isopropyli-
dene-b-l-talofuranose (19L): Sodium hydride (60% min. oil suspension,
3.74 g, 93.4 mmol) was added to a solution of alcohol 18L (22.4 g,
62.2 mmol) and benzyl bromide (11.1 mL, 93.4 mmol) in DMF (200 mL)
at 08C. The reaction mixture was allowed to warm to RT and stirred for
1 h. TLC analysis (cyclohexane/EtOAc, 2:1) indicated the complete con-
sumption of the starting material (Rf =0.58) and the formation of a major
product (Rf =0.70). The reaction mixture was quenched with MeOH
(2 mL) at 08C, diluted with EtOAc (500 mL), washed with water/sat.
brine (1:1) solution (3ꢆ200 mL), dried (MgSO4), filtered, and concentrat-
ed in vacuo. The crude residue was purified by column chromatography
on silica gel (cyclohexane/EtOAc, 50:1 to 20:1) to afford the benzyl ether
(19L) as a colorless oil (24.9 g, 89%); [a]2D5 =+110.0 (c 0.90, CHCl3);
4.55 (d, JACHTUNGTRENNUNG(2,1)=3.5 Hz, 1H; H2), 5.97 ppm (d, JACHTUGNTRENNNUG
13C NMR (CDCl3, 100 MHz): d=À5.7, À5.7 (Si
N
ACHTUNGTRENNUNG
25.7 (SiCACHTUNGTRENNUNG(CH3)3), 26.2, 26.8 (CAHCTUNGTERN(NUGN CH3)2), 61.9 (C5), 63.3 (C6), 75.2 (C3),
82.4 (C4), 85.0 (C2), 104.5 (C1), 111.7 ppm (CACTHNURGTNEG(UN CH3)2); IR (thin film): n˜ =
2099 (s; N3), 3464 cmÀ1 (br s; OH); LRMS (ESI+): m/z (%): 382 (100)
[M+Na]+, 741 (98) [2M+Na]+; (ESIÀ): 358 (76) [MÀH]À, 394 (100)
[M+35Cl]À, 396 (74) [M+37Cl]À, 404 (80) [M+HCOO]À, 717 (98)
[2MÀH]À; HRMS (ESI+): m/z calcd for C15H29N3NaO5Si: 382.1769
[M+Na]+; found: 382.1769.
For enantiomer 7D: [a]2D5 =+15.1 (c 1.2, CHCl3).
Synthesis of DGJNAc and derivatives
5-Azido-6-O-tert-butyldimethylsilyl-5-deoxy-1,2-O-isopropylidene-b-l-
lyxo-hexofuranos-3-ulose (17L): Pyridinium chlorochromate (110.1 g,
510.7 mmol) was added to a solution of alcohol 7L (30.6 g, 85.1 mmol) in
CH2Cl2 (450 mL) over 3 ꢅ molecular sieves. The reaction mixture was
stirred under a nitrogen atmosphere at RT for 18 h, after which time
TLC analysis (cyclohexane/EtOAc, 2:1) indicated almost complete con-
sumption of the starting material (Rf =0.60) and the formation of a major
product (Rf =0.30 streaking to 0.80). The reaction mixture was filtered
through Celite and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (cyclohexane/EtOAc, 5:1 to 3:1) to
afford the ketone (17L) as a colorless oil (24.6 g, 81%); [a]2D5 =+130.8 (c
1.09, CHCl3); 1H NMR (CDCl3, 400 MHz): d=0.10, 0.11 (s, 2ꢆ3H; Si-
1H NMR (CDCl3, 400 MHz): d=0.09, 0.10 (s, 2ꢆ3H; Si
9H; SiC(CH3)3), 1.36, 1.58 (s, 2ꢆ3H; C(CH3)2), 3.58 (ddd, J
2.0 Hz, J(5,6)=4.8 Hz, J(5,6’)=8.9 Hz, 1H; H5), 3.85 (dd, J(6,5)=4.4 Hz,
(6,6’)=10.6 Hz, 1H; H6), 3.88 (dd, J(3,2)=5.1 Hz, J(3,4)=8.9 Hz, 1H;
H3), 3.90 (dd, J(6’,5)=8.9 Hz, J(6’,6)=10.6 Hz, 1H; H6’), 4.04 (dd, J-
(4,5)=2.1 Hz, (4,3)=8.9 Hz, 1H; H4), 4.56 (app t, (2,1)=J(2,3)=
4.1 Hz, 1H; H2), 4.57 (d, Jgem =12.1 Hz, 1H; OCH2Ph), 4.80 (d, Jgem
ACHTUNGTRENNUNG
G
E
ACHTUNGTRENNUNG
R
U
ACHTUNGTRENNUNG
J
G
E
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
A
J
N
J
G
ACHTUNGTRENNUNG
=
12.1 Hz, 1H; OCH2Ph), 5.72 (d, JACHTNUTRGNE(NUG 1,2)=3.4 Hz, 1H; H1), 7.34–7.40 ppm
(5H, m; ArH); 13C NMR (CDCl3, 100 MHz): d=À5.5, À5.5 (Si
ACHTUNGTRENNUNG(CH3)2),
18.1 (SiCACTHGNUTREN(UNNG CH3)3), 25.8 (SiCAHCNUTRTEGNGN(UN CH3)3), 26.5, 26.8 (CACHTNGUTREN(NUGN CH3)2), 62.1 (C5), 64.4
(C6), 72.3 (OCH2Ph), 76.9 (C2), 76.9 (C4), 77.8 (C3), 104.3 (C1), 113.1
(CACTHUNGRTNEUNG(CH3)2), 128.0, 128.2, 128.5 (ArCH), 137.3 ppm (ArCC); IR (thin
A
R
ACHTUGNTRNEN(UNG CH3)2), 3.80
film): n˜ =2098 cmÀ1 (s; N3); LRMS (ESI+): m/z (%): 472 (97) [M+Na]+,
921 (100) [2M+Na]+; HRMS (ESI+): m/z calcd for C22H35N3NaO5Si:
472.2238 [M+Na]+; found: 472.2239.
A
R
ACHTUNGTRENNUNG
J
N
N
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
For enantiomer 19D: [a]2D5 =À95.2 (c 0.59, CHCl3).
A
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
Methyl-5-azido-3-O-benzyl-5-deoxy-l-talofuranoside (8L): A solution of
benzyl ether 19L (19.2 g, 42.7 mmol) in a mixture of MeOH (200 mL)
and acetyl chloride (20 mL) was stirred at 558C for 2 h. TLC analysis (cy-
clohexane/EtOAc, 2:1) indicated the complete consumption of the start-
ing material (Rf =0.72) and the formation of a major product (Rf =0.20).
The reaction was neutralized by the addition of solid sodium bicarbonate,
filtered, and concentrated in vacuo. The crude residue was purified by
column chromatography on silica gel (cyclohexane/EtOAc, 9:1 to 2:3) to
afford the methyl talofuranoside (8L) as a pale-yellow oil (12.3 g, 93%)
A
E
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
208.9 ppm (C3); IR (thin film): n˜ =1777 (s; C=O), 2111 cmÀ1 (s; N3);
LRMS (ESI+): m/z (%): 380 (100) [M+Na]+, 412 (95)
[M+MeOH+Na]+, 737 (15) [2M+Na]+; (ESIÀ): 424 (100)
[M+MeOH+35Cl]À, 426 (40) [M+MeOH+37Cl]À; HRMS (ESI+): m/z
calcd for C16H31N3NaO6Si: 412.1874 [M+MeOH+Na]+; found: 412.1873.
For enantiomer 17D: [a]2D5 =À140.1 (c 1.11, CHCl3).
1
as a 7:1 ratio of the anomers; H NMR (CDCl3, 400 MHz, major anomer
5-Azido-6-O-tert-butyldimethylsilyl-5-deoxy-1,2-O-isopropylidene-b-l-ta-
lofuranose (18L): A solution of sodium borohydride (2.59 g, 68.4 mmol)
in water (50 mL) was added dropwise to a solution of ketone 17L
(24.6 g, 68.4 mmol) in EtOH (200 mL) at 08C. The reaction mixture was
allowed to warm to RT and stirred for 90 min. After this time, TLC anal-
ysis (cyclohexane/EtOAc, 2:1) indicated the complete consumption of
the starting material (Rf =0.30 streaking to 0.80) and the formation of
a major product (Rf =0.59). The reaction was neutralized with glacial
acetic acid, concentrated in vacuo, dissolved in EtOAc (400 mL), and
washed with saturated solutions of sodium bicarbonate (2ꢆ400 mL) and
brine (400 mL). The organic phase was dried (MgSO4), filtered, concen-
trated in vacuo, and purified by column chromatography on silica gel (cy-
clohexane/EtOAc, 50:1 to 3:1) to afford alcohol 18L as a colorless oil
(22.4 g, 91%); [a]D25 =+65.3 (c 1.30, CHCl3); 1H NMR (CDCl3,
only): 2.19 (dd, J
(d, J(OH2,2)=2.6 Hz, 1H; OH2), 3.39 (s, 3H; OCH3), 3.43 (td, J
4.3 Hz, J(5,4)=J(5,6)=6.1 Hz, 1H; H5), 3.71 (ddd, J(6,OH6)=5.2 Hz, J-
(6,5)=6.1 Hz, J(6,6’)=12.0 Hz, 1H; H6), 3.77 (ddd, J(6’,5)=4.3 Hz, J-
(6’,OH6)=7.6 Hz, (6’,6)=12.0 Hz, 1H; H6’), 4.04 (dd, (2,OH2)=
2.6 Hz, J(2,3)=4.3 Hz, 1H; H2), 4.13 (dd, J(4,5)=6.1 Hz, J(4,3)=7.5 Hz,
1H; H4), 4.20 (dd, J(3,2)=4.3 Hz, J(3,4)=7.5 Hz, 1H; H3), 4.55 (d,
gem =11.4 Hz, 1H; OCH2Ph), 4.62 (d, Jgem =11.4 Hz, 1H; OCH2Ph), 4.89
ACHUTGTNREN(UNG OH6,6)=5.2 Hz, JACTHUNGTRENNNUG
G
ACHTUNGTRENNUNG
E
R
ACHTUNGTRENNUNG
N
G
ACHTUNGTRENNUNG
E
J
A
JACHTUNGTRENNUNG
E
E
ACHTUNGTRENNUNG
C
ACHTUNGTRENNUNG
J
(s, 1H; H1), 7.32–7.42 ppm (m, 5H; ArH); 13C NMR (CDCl3, 100 MHz,
major anomer only): d=55.6 (OCH3), 62.5 (C6), 65.4 (C5), 72.5 (C2),
73.1 (OCH2Ph), 79.6 (C3), 81.0 (C4), 108.6 (C1), 128.2, 128.7, 128.7, 128.8
(ArCH), 136.4 ppm (ArCC); IR (thin film): n˜ =2116 (s; N3), 3427 cmÀ1
(br s; OH); LRMS (ESI+): m/z (%): 332 (100) [M+Na]+, 641 (41)
[2M+Na]+; HRMS (ESI+): m/z calcd for C14H19N3NaO5: 332.1217
[M+Na]+; found: 332.1217.
400 MHz): d=0.11 (s, 6H; Si
(s, 2ꢆ3H; C(CH3)2), 2.58 (d, J
(5,4)=3.4 Hz, J(5,6)=5.5 Hz, J
3.4 Hz, (4,3)=8.5 Hz, 1H; H4), 3.87 (dd,
10.6 Hz, 1H; H6), 3.91 (dd, J(6’,5)=7.9 Hz, J(6’,6)=10.6 Hz, 1H; H6’),
4.06 (ddd, J(3,2)=5.1 Hz, J(3,4)=8.9 Hz, J(3,OH3)=9.9 Hz, 1H; H3),
4.59 (dd, J(2,1)=3.8 Hz, J (1,2)=
(CH3)2),
A
ACHTUGNTRENN(UNG CH3)3), 1.37, 1.56
A
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
Methyl-5-azido-2-N,3-O-dibenzyl-2,5,6-trideoxy-2,6-imino-l-galactofura-
noside (9La): Trifluoromethanesulfonic anhydride (10.6 mL, 62.8 mmol)
and pyridine (10.2 mL, 126 mmol) were added to a solution of diol 8L
(7.2 g, 23 mmol) in CH2Cl2 (75 mL) at À408C under an argon atmos-
phere. After 1 h, with the internal temperature rising to À208C, TLC
analysis (cyclohexane/EtOAc, 2:1) indicated the complete consumption
of the starting material (Rf =0.19) and the formation of two products
(major Rf =0.77, minor Rf =0.65), which were thought to be a mixture of
anomeric ditriflates. The reaction mixture was diluted with CH2Cl2
(100 mL), washed with 2m HCl (3ꢆ60 mL), dried (MgSO4), filtered, and
concentrated in vacuo. The crude ditriflate (20L) was dissolved in THF
(75 mL), cooled to À308C under an argon atmosphere, and benzylamine
(7.6 mL, 70 mmol) was added dropwise. The reaction mixture was al-
lowed to warm to RT and stirred for 2 h, after which time TLC analysis
J
A
J
A
JACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHUTGTNREN(UNG 2,3)=5.1 Hz, 1H; H2), 5.82 ppm (dd, JACHTUNGTRENNUNG
3.8 Hz, 1H; H1); 13C NMR (CDCl3, 100 MHz): d=À5.5, À5.5 (Si
ACHTUNGTRENNUNG
18.1 (SiCACHTUNGTRENNUNG(CH3)3), 25.7 (SiCACHTUTGNENRNGU(CH3)3), 26.5, 26.5 (CACTUHNGTRENNUGN
(C6), 72.2 (C3), 78.4 (C2), 79.3 (C4), 104.1 (C1), 112.9 ppm (CACHTUNGTRENNUNG
IR (thin film): n˜ =2099 (s; N3), 3473 cmÀ1 (br s; OH); LRMS (ESI+): m/
z (%): 382 (100) [M+Na]+, 741 (70) [2M+Na]+; (ESIÀ): 358 (100)
[MÀH]À, 394 (48) [M+35Cl]À, 396 (15) [M+37Cl]À, 717 (64) [2MÀH]À;
HRMS (ESI+): m/z calcd for C15H29N3NaO5Si: 382.1769 [M+Na]+;found:
382.1768.
For enantiomer 18D: [a]2D5 =À70.7 (c 1.08, CHCl3).
Chem. Eur. J. 2012, 00, 0 – 0
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
11
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ÞÞ
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G. W. J. Fleet et al.
(cyclohexane/EtOAc/triethylamine, 79:20:1) indicated the consumption
of both ditriflates and the formation of the two anomeric monosubstitut-
ed triflates (major Rf =0.42, minor Rf =0.30). The reaction was heated to
608C for 16 h. TLC analysis indicated the complete consumption of the
major component (Rf =0.42), the formation of a new major product (Rf =
0.67), and the persistence of the minor component (Rf =0.30). The reac-
tion mixture was concentrated in vacuo and purified by column chroma-
tography on silica gel (cyclohexane/EtOAc/triethylamine, 99:0:1 to
96:3:1) to afford the bicyclic compound (9La) as a pale-yellow oil and
a single anomer (6.12 g, 69%). [a]2D5 =+16.2 (c 1.1, CHCl3); 1H NMR
78.0 mmol) was added dropwise to a solution of mixed acetal 21L
(7.54 g, 15.6 mmol) in CH2Cl2 (75 mL) at À788C. After 45 min, TLC
analysis (cyclohexane/EtOAc/triethylamine, 59:40:1) indicated the con-
sumption of the starting material (Rf =0.70, 0.73) and the appearance of
three new spots (Rf =0.20, 0.60, and 0.80). The reaction mixture was
quenched and diluted with EtOAc (400 mL) and allowed to warm to RT.
A saturated solution of sodium potassium tartrate (400 mL) was added
and the biphasic mixture was stirred vigorously for 90 min. The organic
layer was collected and the aqueous layer was extracted with EtOAc (3ꢆ
300 mL). The organic fractions were combined, dried (MgSO4), filtered,
and concentrated in vacuo. TLC analysis (cyclohexane/EtOAc/triethyla-
mine, 59:40:1) indicated the disappearance of the spot at Rf =0.80 during
the work-up procedure. The crude residue was dissolved in MeOH
(100 mL) and sodium borohydride (590 mg, 15.6 mmol) was added at
08C. The reaction mixture was allowed to warm to RT and stirred for
3 h, after which time, TLC analysis (cyclohexane/EtOAc/triethylamine,
59:40:1) indicated the formation of one major product (Rf =0.25) with
the disappearance of all previous spots. The reaction mixture was concen-
trated in vacuo, co-evaporated with MeOH (3ꢆ50 mL), and dissolved in
pyridine (75 mL) and acetic anhydride (75 mL). The reaction mixture
was stirred at RT for 16 h, after which time, TLC analysis (cyclohexane/
EtOAc/triethylamine, 59:40:1) indicated the consumption of the starting
material (Rf =0.25) and the formation of a major product (Rf =0.65). The
reaction mixture was concentrated in vacuo and purified by column chro-
matography on silica gel (cyclohexane/EtOAc/triethylamine, 97:2:1 to
89:10:1) to afford the diacetyl piperidine (22D) as a light-yellow oil
(5.08 g, 72% over 3 steps). [a]2D5 =+65.7 (c 0.60, CHCl3); 1H NMR
(CDCl3, 400 MHz): d=2.74 (d, J
H2), 3.52 (dd, J(6’,5)=4.1 Hz, J
OCH3), 3.87 (app t, J(5,4)=J(5,6’)=4.4 Hz, 1H; H5), 3.90 (d, Jgem
13.8 Hz, 1H; NCH2Ph), 4.01 (d, Jgem =13.8 Hz, 1H; NCH2Ph), 4.11 (d, J-
(3,2)=1.0 Hz, 1H; H3), 4.30 (d, J(4,5)=5.1 Hz, 1H; H4), 4.52 (d, Jgem
11.8 Hz, 1H; OCH2Ph), 4.58 (d, Jgem =11.8 Hz, 1H; OCH2Ph), 5.32 (d, J-
(1,2)=2.7 Hz, 1H; H1), 7.26–7.37 ppm (m, 10H; ArH); 13C NMR
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
R
=
A
R
=
ACHTUNGTRENNUNG
(CDCl3, 100 MHz): d=48.6 (C6), 57.6 (OCH3), 59.7 (C5), 59.9
(NCH2Ph), 61.4 (C2), 71.0 (OCH2Ph), 78.7 (C4), 81.5 (C3), 109.7 (C1),
127.0, 127.7, 127.8, 128.3, 128.4 (ArCH), 137.7, 139.1 ppm (ArCC); IR
(thin film): n˜ =2109 cmÀ1 (s; N3); LRMS (ESI+): m/z (%): 381 (100)
[M+H]+, 403 (80) [M+Na]+; HRMS (ESI+): m/z calcd for
C21H24N4NaO3: 381.1921 [M+H]+; found: 381.1920.
For enantiomer 9Da: [a]2D5 =À21.4 (c 1.20, CHCl3).
4-O-Acetyl-5-azido-2-N,3-O-dibenzyl-2,5,6-trideoxy-2,6-imino-l-galactose
acetyl methyl acetal (21L): Boron trifluoride diethyl etherate (5.1 mL,
40 mmol) was added dropwise to a solution of bicyclic acetal 9La (6.12 g,
16.1 mmol) in acetic anhydride (60 mL) at À308C. The reaction was al-
lowed to warm to RT and stirred for 2.5 h, after which time TLC analysis
(cyclohexane/EtOAc/triethylamine, 69:30:1) indicated the complete con-
sumption of the starting material (Rf =0.72) and the formation of major
products (Rf =0.47, 0.56). The reaction mixture was concentrated in
vacuo and the residue was dissolved in EtOAc (200 mL), washed with
a saturated solution of sodium bicarbonate (3ꢆ100 mL), dried (MgSO4),
filtered, and concentrated in vacuo. The crude residue was purified by
column chromatography on silica gel (cyclohexane/EtOAc/triethylamine,
97:2:1 to 79:20:1) to afford the mixed-acetal piperidine (21L) as a pale-
yellow oil (7.54 g, 97%), in a 3:2 (A/B) mixture of diastereoisomers.
1H NMR (CDCl3, 400 MHz): d=2.05 (s, 3H; COCH3B), 2.10 (s, 3H;
COCH3A), 2.13 (s, 3H; COCH3B), 2.14 (s, 3H; COCH3A), 2.38 (dd, J-
(CDCl3, 400 MHz): d=2.04, 2.14 (s, 2ꢆ3H; COCH3), 2.26 (dd, J
8.9 Hz, J(1,1’)=12.6 Hz, 1H; H1), 2.95–3.00 (m, 1H; H5), 3.02 (dd, J-
(1’,2)=4.4 Hz, J(1’,1)=12.7 Hz, 1H; H1’), 3.64 (d, Jgem =14.1 Hz, 1H;
NCH2Ph), 3.88 (d, Jgem =14.1 Hz, 1H; NCH2Ph), 3.96 (app td, J(2,1’)=
4.1 Hz, J(2,1)=J(2,3)=8.5 Hz, 1H; H2), 4.04 (app t, J(4,3)=J(4,5)=
2.7 Hz, 1H; H4), 4.23 (dd, J(6,5)=6.1 Hz, J(6,6’)=11.6 Hz, 1H; H6),
4.55 (dd, (6’,5)=6.1 Hz, (6’,6)=11.6 Hz, 1H; H6’), 4.57 (d, Jgem
11.4 Hz, 1H; OCH2Ph), 4.75 (d, Jgem =11.4 Hz, 1H; OCH2Ph), 4.90 (dd,
(3,4)=2.7 Hz, J(3,2)=8.5 Hz, 1H; H3), 7.26–7.40 ppm (m, 10H; ArH);
ACHTUNGTRENNUNG(1,2)=
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
J
A
J
A
=
J
N
ACHTUNGTRENNUNG
13C NMR (CDCl3, 100 MHz): d=21.0, 21.0 (COCH3), 51.4 (C1), 56.5
(C2), 56.9 (NCH2Ph), 60.7 (C5), 61.9 (C6), 74.3 (OCH2Ph), 74.8 (C4),
75.1 (C3), 127.3, 127.8, 127.9, 128.4, 128.5, 128.7 (ArCH), 137.8, 138.0
(ArCC), 170.1, 170.6 ppm (COCH3); IR (thin film): n˜ =1740 (s; C=O),
2108 cmÀ1 (s; N3); LRMS (ESI+): m/z (%): 475 (100) [M+Na]+; HRMS
(ESI+): m/z calcd for C24H28N4NaO5: 475.1952 [M+Na]+; found:
475.1950.
A
R
ACHTUNGTREN(NGNU 6,5)=10.8 Hz, J-
A
R
ACHTUNGTRENNUNG
For enantiomer 22L: [a]2D5 =À75.6 (c 0.87, CHCl3).
A
ACHTUNGTRENNUNG
A
E
ACHTUNGTRENNUNG
2-Acetamido-3,6-di-O-acetyl-1-N,4-O-dibenzyl-1,2,5-trideoxy-1,5-imino-d-
galactitol (23D): Powdered zinc (9.0 g, 130 mmol) was added to a solution
of azide 22D (3.0 g, 6.6 mmol) in THF/glacial-acetic-acid/acetic-anhy-
dride (3:2:1, 72 mL) and the mixture was stirred vigorously before the
dropwise addition of a saturated copper(II) sulfate solution (9 mL) to ini-
tiate the reaction. After 30 min, TLC analysis (cyclohexane/EtOAc/trie-
thylamine, 49:50:1) indicated the complete consumption of the starting
material (Rf =0.78) and the formation of a major product (Rf =0.15). The
reaction mixture was filtered (GF/A glass microfiber), concentrated in
vacuo, and the crude residue was purified by column chromatography on
silica gel (cyclohexane/EtOAc/triethylamine, 94:5:1 to 29:70:1) to afford
the acetamide (23D) as a white crystalline solid (2.83 g, 91%). M.p. 112–
1148C; [a]2D5 =+20.3 (c 1.02, acetone); 1H NMR (CDCl3, 500 MHz): d=
1.87 (s, 3H; NHCOCH3), 2.05, 2.11 (s, 2ꢆ3H; COCH3), 2.20 (dd, J-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
J
G
U
ACHTUNGTRENNUNG
G
A
ACHTUNGTRENNUNG
G
R
ACHTUNGTRENNUNG
J
=
N
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
A
JACHTUNGTRENNUNG
7.43 ppm (m, 20H; ArHA and ArHB); 13C NMR (CDCl3, 100 MHz): d=
20.9, 21.0 (COCH3B), 20.0, 21.1 (COCH3A), 51.6 (C6B), 51.9 (C6A), 53.9
(NCH2PhB), 53.9 (C5B), 54.4 (C5A), 54.7 (NCH2PhA), 56.4 (OCH3A), 56.9
(OCH3B), 63.2 (C2A), 64.2 (C2B), 74.9 (OCH2PhA), 75.2 (OCH2PhB), 76.0
(C3A), 76.8 (C3B), 77.2 (C4B), 77.6 (C4A), 95.1 (C1A, C1B), 126.7, 127.0,
127.1, 127.4, 127.5, 127.7, 127.8, 128.3, 128.4, 128.5, 128.6 (ArCHA and
ArCHB), 138.0, 138.2, 139.3, 139.6 (ArCCA and ArCCB), 170.2, 170.2,
A
ACHUTGTNRNEUG(N 1,1’)=12.6 Hz, 1H; H1), 3.04 (dd, JAHCTUNGTRNEN(UGN 1’,2)=3.1 Hz, J-
AHCTUNGTRENNUNG
J
AHCTUNGTRENNUNG
170.6, 171.0 ppm (COCH3 and COCH3B); IR (thin film): n˜ =1743 (s; C=
A
JACHTUNGTRENNUNG
O), 2106 cmÀ1 (s; N3); LRMS (ESI+): m/z (%): 423, (95) [MÀOAc]+,
483 (98) [M+H]+, 505 (100) [M+Na]+, 521 (83) [M+K]+, 955 (20)
[2M+H]+, 987 (95) [2M+Na]+; HRMS (ESI+): m/z calcd for
C25H30N4NaO6: 505.2058 [M+Na]+; found: 505.2058.
A
ACHTUNGTRENNUNG
JACHTUNGTRENNUNG
NHCOCH3), 7.25–7.38 ppm (m, 10H; ArH); 13C NMR ([D6]acetone,
126 MHz): d=21.0, 21.0 (COCH3), 22.9 (NHCOCH3), 46.6 (C2), 50.1
(C1), 58.2 (NCH2Ph), 61.5 (C5), 61.6 (C6), 72.5 (C3), 73.4 (OCH2Ph),
75.5 (C4), 127.6, 128.1, 128.2, 128.9, 128.9, 129.3 (ArCH), 139.4, 140.1
3,6-Di-O-acetyl-2-azido-1-N,4-O-dibenzyl-1,2,5-trideoxy-1,5-imino-d-ga-
lactitol (22D): Diisobutylaluminium hydride (1.5m in toluene, 52.0 mL,
&
12
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
(ArCC), 169.6, 170.2, 170.7 ppm (COCH3; NHCOCH3); IR (thin film):
n˜ =1541 (br s; amide II), 1655 (s; amide I), 1739 (s; C=O), 3296 cmÀ1
(br s; OH); LRMS (ESI+): m/z (%): 469 (100) [M+H]+, 491 (55)
[M+Na]+; (ESIÀ): 503 (55) [M+35Cl]À, 505 (15) [M+37Cl]À, 513 (100)
[M+HCOO]À; HRMS (ESI+): m/z calcd for C26H33N2O6: 491.2153
[M+H]+; found: 491.2151.
(m, 2H; H2 and H4); 13C NMR (D2O, 126 MHz): d=22.6 (NHCOCH3),
41.5 (NCH3), 47.8 (C2), 58.4 (C1), 61.3 (C6), 65.9, 70.0, 73.1 (C3, C4 and
C5), 174.9 ppm (NHCOCH3); IR (thin film): n˜ =1561 (s; amide II), 1639
(s; amide I), 3289 cmÀ1 (br s; OH); LRMS (ESI+): m/z (%): 459 (100)
[2M+Na]+; HRMS (ESI+): m/z calcd for C9H18N2NaO4: 241.1159
[M+Na]+; found: 241.1158.
For enantiomer 23L: M.p. 113–1158C; [a]2D5 =À28.6 (c 1.06, CHCl3).
2-Acetamido-1,2,5-trideoxy-1-N-ethyl-1,5-imino-d-galactitol,
(N-ethyl-
DGJNAc, 1Db): Ethanal (0.01 mL, 0.2 mmol) and palladium (10% on
carbon, 8 mg) were added to a solution of DGJNAc (1D, 20.9 mg,
0.10 mmol) in EtOH (1 mL). The vessel was degassed, charged with hy-
drogen, and the reaction mixture was stirred at RT for 4.5 h, after which
LRMS indicated the absence of the starting material. The reaction mix-
ture was filtered (GF/A glass microfiber) and concentrated in vacuo. The
residue was loaded onto a short column of Dowex (50W-X8, H+) and the
resin was washed with water. The product was liberated with aqueous
ammonia (2m) and the ammoniacal fractions were concentrated in vacuo
to afford N-ethyl-DGJNAc (1Db) as a colorless oil (24 mg, quant.);
[a]2D5 =+18.1 (c 1.00, MeOH); 1H NMR (D2O, 400 MHz): d=1.02 (t, J=
2-Acetamido-1,2,5-trideoxy-1,5-imino-d-galactitol
(DGJNAc,
1D):
Sodium methoxide (10 mg, cat.) was added to a solution of the protected
piperidine (23D, 2.83 g, 6.03 mmol) in MeOH (40 mL) at RT and the
mixture was stirred for 16 h. TLC analysis (EtOAc/triethylamine, 99:1)
indicated the complete consumption of the starting material (Rf =0.50)
and the formation of a major product (Rf =0.14 streaking to 0.0). The re-
action mixture was neutralized with glacial acetic acid and concentrated
in vacuo. The crude residue was dissolved in water/2m HCl/1,4-dioxane
(10:2:1, 78 mL) and palladium (10% on carbon, 600 mg) was added. The
vessel was degassed, charged with hydrogen, and the reaction mixture
was stirred at RT for 24 h. LRMS and 1H NMR indicated that a single
product had been formed, with no trace of any benzylated intermediates
observed. The reaction mixture was filtered (GF/A glass microfiber) and
concentrated in vacuo to afford crude DGJNAc (1D) as its hydrochlo-
ride salt.
6.9 Hz, 3H; NCH2CH3), 2.01 (s, 3H; NHCOCH3), 2.23 (app t, J
ACHTUNGTRENNUNG(1,1’)=J-
A
=
13.6 Hz, 1H; NCHH’CH3), 2.85 (dd, J=6.9 Hz,
NCHH’CH3), 2.98 (dd, J(1’,2)=4.0 Hz, J
(br d, J=10.3 Hz, 1H; H3), 3.80 (dd, J
1H; H6), 3.87 (dd, J(6’,5)=3.9 Hz, J(6’,6)=11.4 Hz, 1H; H6’), 4.04–
J
gem =13.6 Hz, 1H;
A
ACHTUNGTRENNUNG
Selected data for DGJNAc·HCl (1D): 1H NMR (D2O, 400 MHz): d=
A
ACHTUNGTRENNUNG
1.96 (s, 3H; NHCOCH3), 2.82 (app t, J
3.34 (br s, 1H; H5), 3.41 (dd, J(1’,2)=5.0 Hz, J
3.72 (dd, J(3,4)=2.6 Hz, J(3,2)=10.7 Hz, 1H; H3), 3.75 (dd, J
7.5 Hz, J(6,6’)=12.3 Hz, 1H; H6), 3.82 (dd, J(6’,5)=5.5 Hz, J(6’,6)=
12.3 Hz, 1H; H6’), 4.11 (dd, J(4,5)=1.0 Hz, J(4,3)=2.6 Hz, 1H; H4),
4.26 ppm (ddd, (2,1’)=5.1 Hz, (2,3)=11.0 Hz, (2,1)=11.9 Hz, 1H;
A
ACHTUNGERTN(NUNG 1,2)=12.5 Hz, 1H; H1),
A
ACHTUNGTRENNUNG
4.20 ppm (m, 2H; H2 and H4); 13C NMR (D2O, 100 MHz): d=8.6
(NCH2CH3), 22.7 (NHCOCH3), 46.6 (NCH2CH3), 47.8 (C2), 53.3 (C1),
60.8 (C6), 62.8 (C5), 70.0 (C4), 73.0 (C3), 175.0 ppm (NHCOCH3); IR
(thin film): n˜ =1560 (s; amide II), 1637 (s; amide I), 3317 cmÀ1 (br s;
OH); LRMS (ESI+): m/z (%): 487 (100) [2M+Na]+; HRMS (ESI+): m/
z calcd for C10H21N2O4: 233.1496 [M+H]+; found: 233.1495.
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
J
A
J
A
JACHTUNGTRENNUNG
H2); LRMS (ESI+): m/z (%): 205 (100) [M+H]+, 409 (35) [2M+H]+;
(ESIÀ): 203 (100) [MÀH]À.
2-Acetamido-1-N-butyl-1,2,5-trideoxy-1,5-imino-d-galactitol,
(N-butyl-
The crude salt of compound 1D was loaded onto a short column of
Dowex (50W-X8, H+) and the resin was washed with water until neutral
fractions were obtained. The product was liberated with aqueous ammo-
nia (2m) and the ammoniacal fractions were concentrated in vacuo. The
residue was co-evaporated with EtOH (3ꢆ50 mL) to give DGJNAc (1D)
as a pale-yellow crystalline solid (1.22 g, 99%). M.p. 153–1568C (EtOH);
[a]2D5 =+49.3 (c 0.65, H2O); 1H NMR (D2O, 400 MHz): d=1.88 (s, 3H;
DGJNAc, 1Dc): Butyraldehyde (0.01 mL, 0.1 mmol) and sodium cyano-
borohydride (6 mg, 0.1 mmol) were added to a solution of DGJNAc (1D,
22.4 mg, 0.110 mmol) in EtOH/acetic-acid (200:1, 10 mL). The mixture
was stirred at RT for 2.5 h after which LRMS indicated the absence of
the starting material. The reaction mixture was concentrated in vacuo
and the crude residue was purified by cation-exchange chromatography
(Bond Elut SCX 1 cc cartridge, residue loaded in 50 mm HCl and eluted
with 3.5% ammonium hydroxide in MeOH/water 9:1) and reverse-phase
chromatography (SepPak C18 1 cc cartridge, residue loaded in water and
eluted with MeOH) to afford N-butyl-DGJNAc (1Dc) as a white crystal-
line solid (32 mg, quant.). M.p. 180–1848C; [a]2D5 =+ 14.4 (c 0.62 in
MeOH); 1H NMR (D2O, 400 MHz): d=0.89 (t, J=7.3 Hz, 3H;
NCH2CH2CH2CH3), 1.20–1.32 (m, 2H; NCH2CH2CH2CH3), 1.35–1.52
(m, 2H; NCH2CH2CH2CH3), 2.01 (s, 3H; NHCOCH3), 2.18 (app t, J-
NHCOCH3), 2.24 (app t, J
(5,6)=J(5,6’)=6.5 Hz, 1H; H5), 2.96 (dd,
13.1 Hz, 1H; H1’), 3.45–3.53 (m, 3H; H3, H6, and H6’), 3.83 (app td, J-
(2,1’)=4.9 Hz, J(2,1)=J(2,3)=10.9 Hz, 1H; H2), 3.89 ppm (s, 1H; H4);
ACHUTGTNRNEUG(N 1,1’)=JACHTGNUTRENNUNG
J
G
E
J
N
JACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
13C NMR (D2O, 100 MHz): d=22.6 (NHCOCH3), 47.7 (C1), 49.1 (C2),
59.3 (C5), 61.8 (C6), 68.9 (C4), 73.2 (C3), 175.1 ppm (NHCOCH3); IR
(thin film, Ge): n˜ =1563 (s; amide II), 1638 (s; amide I), 3285 cmÀ1 (br s;
OH/NH); LRMS (ESI+): m/z (%): 205 (100) [M+H]+, 409 (30)
[2M+H]+; (ESIÀ): 203 (100) [MÀH]À; HRMS (ESI+): m/z calcd for
C8H17N2O4: 205.1183 [M+H]+; found: 205.1184; elemental analysis calcd
(%) for C8H16N2O4: C 47.05, H 7.90, N 13.72; found: C 46.88, H 7.86,
N 13.55.
A
ACHTUNGTRENNUNG
J
J
N
JACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
For enantiomer 1L: M.p. 152–1568C (EtOH); [a]2D5 =À46.6 (c 0.73,
H2O).2-Acetamido-1,2,5-trideoxy-1,5-imino-1-N-methyl-d-galactitol, (N-
methyl-DGJNAc, 1Da): Formaldehyde (37% in water, 0.03 mL,
0.3 mmol) and palladium (10% on carbon, 6 mg) were added to a solution
of DGJNAc (1D, 30 mg, 0.15 mmol) in 1,4-dioxane/water (1:1, 2 mL).
The vessel was degassed, charged with hydrogen, and the reaction mix-
ture was stirred at RT for 18 h. The reaction mixture was filtered (GF/A
glass microfiber) and concentrated in vacuo. The residue was loaded
onto a short column of Dowex (50W-X8, H+) and the resin washed with
water until neutral fractions were obtained. The product was liberated
with aqueous ammonia (2m) and the ammoniacal fractions were concen-
trated in vacuo to afford N-methyl-DGJNAc (1Da) as a brown, crystal-
line solid (35 mg, quant.). M.p. 203–2068C; [a]2D2 =+23.6 (c 0.65, H2O);
1H NMR (D2O, 400 MHz): d=1.88 (s, 3H; NHCOCH3), 2.04 (app t, J-
G
G
ACHTUNGTRENNUNG
H4), 4.07–4.13 ppm (m, 1H; H2); 13C NMR (D2O, 126 MHz): d=13.8
(NCH2CH2CH2CH3), 20.7 (NCH2CH2CH2CH3), 22.7 (NHCOCH3), 25.9
(NCH2CH2CH2CH3), 48.0 (C2), 52.4 (NCH2CH2CH2CH3), 54.4 (C1), 61.0
(C6), 63.1 (C5), 70.1 (C4), 73.4 (C3), 175.0 ppm (NHCOCH3); IR (thin
film, Ge): n˜ =1559 (s; amide II), 1644 (s; amide I), 3286 cmÀ1 (br s; OH);
LRMS (ESI+): m/z (%): 261 (100) [M+H]+; HRMS (ESI+): m/z calcd
for C12H25N2O4: 261.1809 [M+H]+; found: 260.1809.
Data for the HCl salt: 1H NMR (D2O, 400 MHz): d=0.93 (t, J=7.4 Hz,
3H; NCH2CH2CH2CH3), 1.31–1.45 (m, 2H; NCH2CH2CH2CH3), 1.58–
1.81 (m, 2H; NCH2CH2CH2CH3), 2.03 (s, 3H; NHCOCH3), 3.01 (app t,
J
G
(1,1’)=11.9 Hz,
1H;
3.16–3.38
(m,
2H;
ACHTUNGTRENNUNG(3,4)=
AHCTUNGTRENNUNG
N
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
J
A
R
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
Chem. Eur. J. 2012, 00, 0 – 0
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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ÞÞ
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G. W. J. Fleet et al.
(NCH2CH2CH2CH3), 46.0 (C2), 52.1 (C1), 53.8 (NCH2CH2CH2CH3), 59.8
(C6), 64.5 (C5), 70.1 (C4), 70.6 (C3), 175.3 ppm (NHCOCH3).
(s, 2ꢆ3H; Si
(CH3)2), 3.53 (dd, J
(m, 2H; H5 and H6’), 3.89 (d, J
3.0 Hz, J(4,5)=8.3 Hz, 1H; H4), 4.44 (d, Jgem =11.9 Hz, 1H; OCH2Ph),
G
ACHTUNGTREN(NNUG CH3)3), 1.33, 1.49 (s, 2ꢆ3H; C-
E
E
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
2-Acetamido-1-N-benzyl-1,2,5-trideoxy-1,5-imino-d-galactitol, (N-benzyl-
DGJNAc, 1Dd): Benzaldehyde (0.01 mL, 0.1 mmol) and sodium cyano-
borohydride (6 mg, 0.1 mmol) were added to a solution of DGJNAc (1D,
20 mg, 0.10 mmol) in EtOH/acetic-acid (200:1, 10 mL). The reaction was
stirred at RT for 3 h, after which time LRMS indicated the absence of
the starting material. The reaction mixture was concentrated in vacuo
and the crude residue was purified by cation-exchange chromatography
(Bond Elut SCX 1 cc cartridge, residue loaded in 50 mm HCl and eluted
with 3.5% ammonium hydroxide in MeOH/water 9:1). A large propor-
tion of the product was eluted during the wash fractions, which were con-
centrated and purified by column chromatography on Dowex (50W-X8,
H+, washed with water). The product was liberated with aqueous ammo-
nia (2m) and the ammoniacal fractions were concentrated in vacuo. The
products of both columns were combined to afford N-benzyl-DGJNAc
(1Dd) as a colorless oil (20 mg, 68%); [a]2D5 =+ 17.6 (c 0.25, MeOH);
1H NMR (D2O, 400 MHz): d=1.95 (s, 3H; NHCOCH3), 2.30 (app t, J-
AHCTUNGTRENNUNG
4.67 (d, JACHTNURTGNEUNG(2,1)=3.8 Hz, 1H; H2), 4.71 (d, Jgem =11.9 Hz, 1H; OCH2Ph),
5.97 (d,
JACHTNGUTERN(UNG 1,2)=3.8 Hz, 1H; H1), 7.30–7.39 ppm (m, 5H; ArH);
13C NMR (CDCl3, 100 MHz): d=À5.7, À5.6 (Si
ACHTUNTGRNENGU(CH3)2), 18.1 (SiCACHTUNGTRENNUNG(CH3)3),
25.7 (SiCACHTUNRTGENNUG(CH3)3), 26.4, 26.7 (CACHTUGNTERN(NUGN CH3)2), 61.9 (C5), 63.1 (C6), 71.5
(OCH2Ph), 78.8 (C4), 81.3 (C3), 81.8 (C2), 104.7 (C1), 111.9 (CACHTUNGTRENNUNG(CH3)2),
128.0, 128.2, 128.6 (ArCH), 136.8 ppm (ArCC); IR (thin film): n˜ =2100
(s; N3); LRMS (ESI+): m/z (%): 472 (67) [M+Na]+, 551 (56), 916 (93)
[2M+NH4]+, 921 (100) [2M+Na]+; HRMS (ESI+): m/z calcd for
C22H35N3NaO5Si: 472.2238 [M+Na]+; found: 472.2235.
For enantiomer 24D: [a]2D5 =+30.1 (c 1.21, CHCl3).
Methyl 5-azido-3-O-benzyl-5-deoxy-l-idofuranosides (10L): A solution of
benzyl ether 24L (7.36 g, 16.4 mmol) in a mixture of MeOH (50 mL) and
acetyl chloride (2.5 mL) was stirred at 508C for 1 h. TLC analysis (cyclo-
hexane/EtOAc, 3:1) indicated the complete consumption of the starting
material (Rf =0.60) and the formation of two products (Rf =0.00; tolu-
ene/acetone (4:1): Rf =0.25, 0.35). The reaction mixture was neutralized
by the addition of solid sodium bicarbonate, filtered, and concentrated in
vacuo. The crude residue was purified by column chromatography on
silica gel (toluene/acetone, 49:1 to 3:1) to afford anomeric methyl furano-
side 10Lb as a white crystalline solid (Rf =0.35, 2.42 g, 48%) and 10La
as a colorless oil (Rf =0.25, 2.19 g, 43%).
A
R
ACHTUNGTRNE(NUNG 1’,2)=
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
J
(D2O, 126 MHz): d=22.5 (NHCOCH3), 46.5 (C2), 52.7 (C1), 56.7
(NCH2Ph), 60.5 (C6), 64.6 (C5), 70.2 (C4), 72.3 (C3), 128.9, 129.4, 131.3,
131.8 (ArCH), 136.8 (ArCC), 175.0 ppm (NHCOCH3); IR (thin film,
Ge): n˜ =1558 (s; amide II), 1635 (s; amide I), 3385 cmÀ1 (br s; OH);
LRMS (ESI+): m/z (%): 295 (100) [M+H]+, 317 (75) [M+Na]+; HRMS
(ESI+): m/z calcd for C15H23N2O4: 295.1652 [M+H]+; found: 295.1648.
Data for compound 10Lb: M.p. 46–508C; [a]2D5 =+79.1 (c 1.39, CHCl3);
1H NMR (CDCl3, 400 MHz): d=2.04 (t, J
N
ACHTUNGTREN(NGNU OH6,6’)=6.3 Hz,
1H; OH6), 2.85 (d, J
3.57 (app dt, J
(ddd, J
ACHTUNGTRENNUNG
G
R
ACHTUNGTRENNUNG
G
N
ACHTUNGTRENNUNG
2-Acetamido-1,2,5-trideoxy-1-N-hydroxyethyl-1,5-imino-d-galactitol, (N-
hydroxyethyl-DGJNAc, 1De): Glycoaldehyde dimer (180 mg, 1.50 mmol)
and palladium (10% on carbon, 12 mg) were added to a solution of
DGJNAc (1D, 30 mg, 0.15 mmol) in 1,4-dioxane/water (1:1, 4 mL). The
vessel was degassed, charged with hydrogen, and the reaction mixture
was stirred at RT for 16 h. The reaction mixture was filtered (GF/A glass
microfiber) and concentrated in vacuo. The residue was loaded onto
a short column of Dowex (50W-X8, H+) and the resin was washed with
water. The product was liberated with aqueous ammonia (2m) and the
ammoniacal fractions were concentrated in vacuo to afford N-hydrox-
yethyl-DGJNAc (1De) as an off-white, viscous gum (35 mg, 95%);
A
R
ACHTUNGTRENNUNG
A
J
E
J
N
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
=
AHCTUNGTRENNUNG
(m, 5H; ArH); 13C NMR (CDCl3, 100 MHz): d=55.8 (OCH3), 62.4 (C6),
62.9 (C5), 71.7 (OCH2Ph), 76.4 (C2), 77.9 (C4), 83.1 (C3), 101.6 (C1),
127.9, 128.0, 129.5 (ArCH), 137.2 ppm (ArCC); IR (thin film): n˜ =2105
(s; N3), 3424 (br s; OH); LRMS (ESI+): m/z (%): 332 (91) [M+Na]+,
348 (78) [M+K]+, 641 (100) [2M+Na]+; HRMS (ESI+): m/z calcd for
C14H19N3NaO5: 332.1217 [M+Na]+; found: 332.1215.
1
[a]2D5 =+14.2 (c 0.96, MeOH); H NMR (D2O, 400 MHz): d=2.00 (s, 3H;
For enantiomer 10Db: M.p. 43–448C; [a]2D5 =À73.2 (c 1.32, CHCl3).
NHCOCH3), 2.21 (app t, J
(m, 2H; H5 and NCHH’CH2OH), 2.85–2.96 (m, 1H; NCHH’CH2OH),
3.02 (dd, J(1’,2)=4.5 Hz, J(1’,1)=11.8 Hz, 1H; H1’), 3.50 (dd, J=2.8 Hz,
J=10.5 Hz, 1H; H3), 3.68 (app dt, J=5.7 Hz, gem =11.8 Hz, 2H;
NCH2CH2OH), 3.79 (dd, J(6,5)=5.9 Hz, J(6,6’)=11.5 Hz, 1H; H6), 3.85
(dd, J(6’,5)=5.1 Hz, J(6’,6)=11.5 Hz, 1H; H6’), 3.96–4.12 ppm (m, 2H;
ACHUTGTNRENNUG(1,1’)=JACHTUNGTRNE(NUGN 1,2)=11.4 Hz, 1H; H1), 2.52–2.71
Data for compound 10La: [a]2D5 =À41.4 (c 0.92, CHCl3); 1H NMR
A
ACHTUNGTRENNUNG
(CDCl3, 400 MHz): d=2.20 (t,
JACTHUNGTRENNU(G OH6,6)=JACHTNUGTREN(NUNG OH6,6’)=6.5 Hz, 1H;
J
OH6), 2.51 (d, J
(app dt, J(6,5)=J
(6’,5)=3.5 Hz, J(6’,OH6)=6.1 Hz, J
(5,6’)=3.8 Hz, J(5,6)=5.8 Hz, J(5,4)=8.1 Hz, 1H; H5), 3.94 (dd, J-
(3,4)=5.6 Hz, 1H; H3), 4.33 (app dt, J(2,1)=J(2,3)=
(2,OH2)=4.0 Hz, 1H; H2), 4.35 (dd, J(4,3)=5.6 Hz, J(4,5)=
8.3 Hz, 1H; H4), 4.49 (d, Jgem =11.9 Hz, 1H; OCH2Ph), 4.73 (d, Jgem
11.9 Hz, 1H; OCH2Ph), 4.86 (d, J(1,2)=1.5 Hz, 1H; H1), 7.30–7.39 ppm
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
J
J
U
U
ACHTUNGTRENNUNG
H2 and H4); 13C NMR (D2O, 100 MHz): d=22.6 (NHCOCH3), 48.2
(C2), 53.5 (NCH2CH2OH), 54.8 (C1), 58.7 (NCH2CH2OH), 61.4 (C6),
64.1 (C5), 70.6 (C4), 73.2 (C3), 175.0 ppm (NHCOCH3); IR (thin film,
Ge): n˜ =1561 (s; amide II), 1638 (s; amide I), 3318 cmÀ1 (br s; OH);
LRMS (ESI+): m/z (%): 249 (27) [M+H]+, 271 (66) [M+Na]+, 519 (100)
[2M+Na]+; HRMS (ESI+): m/z calcd for C10H20N2NaO5: 271.1264
[M+Na]+; found: 271.1260.
R
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
=
AHCTUNGTRENNUNG
(m, 5H; ArH); 13C NMR (CDCl3, 100 MHz): d=56.2 (OCH3), 62.2 (C6),
64.2 (C5), 72.1 (OCH2Ph), 78.3 (C2), 80.7 (C4), 82.3 (C3), 109.6 (C1),
128.1, 128.1, 129.6 (ArCH), 137.0 ppm (ArCC); IR (thin film): n˜ =2106
(s; N3), 3417 (br s; OH); LRMS (ESI+): m/z (%): 332 (74) [M+Na]+,
348 (57), [M+K]+, 641 (100) [2M+Na]+; HRMS (ESI+): m/z calcd for
C14H19N3NaO5: 332.1217 [M+Na]+; found: 332.1214.
Synthesis of DNJNAc and derivatives
5-Azido-3-O-benzyl-6-O-tert-butyldimethylsilyl-5-deoxy-1,2-O-isopropyli-
dene-b-l-idofuranose (24L): Sodium hydride (60% min. oil suspension,
1.00 g, 25.0 mmol) was added to
a solution of alcohol 7L (5.90 g,
For enantiomer 10Da: [a]2D5 =+33.6 (c 1.03, CHCl3).
16.4 mmol) and benzyl bromide (3.0 mL, 25 mmol) in DMF (50 mL) at
08C. The reaction mixture was allowed to warm to RT and stirred for
1 h. TLC analysis (cyclohexane/EtOAc, 3:1) indicated the complete con-
sumption of the starting material (Rf =0.30) and the formation of a single
product (Rf =0.60). The reaction mixture was diluted with EtOAc
(150 mL), washed with a water/sat. brine solution (1:1, 3ꢆ150 mL), dried
(MgSO4), filtered, and concentrated in vacuo. The crude residue was pu-
rified by column chromatography on silica gel (cyclohexane/EtOAc, 49:1
to 9:1) to afford benzyl ether 24L as a colorless oil (7.36 g, 100%).
[a]2D5 =À39.4 (c 1.78, CHCl3); 1H NMR (CDCl3, 400 MHz): d=0.01, 0.03
Methyl-5-azido-3-O-benzyl-5-deoxy-6-O-methanesulfonyl-b-l-idofurano-
side (25Lb): Methanesulfonyl chloride (0.29 mL, 3.8 mmol) was added
dropwise to a solution of diol 10Lb (982 mg, 3.17 mmol) and pyridine
(0.61 mL, 7.6 mmol) in CH2Cl2 (9 mL) at 08C. After 3 h, TLC analysis
(toluene/acetone, 4:1) indicated the complete consumption of the starting
material (Rf =0.35) and the formation of a major product (Rf =0.45) and
a
minor product (Rf =0.50). The reaction mixture was diluted with
CH2Cl2 (15 mL), washed with 2m HCl (3ꢆ20 mL), dried (MgSO4), fil-
tered, and concentrated in vacuo. The crude residue was purified by
&
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
column chromatography on silica gel (CH2Cl2/MeOH, 60:1) to afford the
2105 (s; N3), 3443 (br s; OH); LRMS (ESI+): m/z (%): 486 (100)
[M+Na]+; HRMS (ESI+): m/z calcd for C21H25N3NaO7S: 486.1305
[M+Na]+; found: 486.1303.
primary mesylate (25Lb) as a colorless oil (582 mg, 86%); [a]2D5 =+72.6
(c 0.97, CHCl3); 1H NMR (CDCl3, 400 MHz): d=2.74 (d, J
ACHTUNGTRENNUNG
For enantiomer 27Da: [a]2D5 =+11.0 (c 1.15, CHCl3).
G
E
N
ACHTUNGTRENNUNG
Methyl-5-azido-2-N,3-O-dibenzyl-2,5,6-trideoxy-2,6-imino-a-l-gulofurano-
side (11La): Trifluoromethanesulfonic anhydride (0.20 mL, 1.2 mmol)
and pyridine (0.20 mL, 2.5 mmol) were added to a solution of tosylate
27La (483 mg, 1.04 mmol) in CH2Cl2 (5 mL) at À408C. After 1 h, with
the internal temperature rising to À308C, TLC analysis (cyclohexane/
EtOAc, 2:1) indicated the complete consumption of the starting material
(Rf =0.15) and the formation of a single product (Rf =0.55). The reaction
mixture was diluted with CH2Cl2 (15 mL), washed with 2m HCl (3ꢆ
20 mL), dried (MgSO4), filtered, and concentrated in vacuo. The crude
triflate (28La) was dissolved in benzylamine (2.5 mL) and the mixture
was stirred at 1008C for 18 h. TLC analysis (cyclohexane/EtOAc,
80:19:1) indicated the complete consumption of the triflate (Rf =0.35)
and the formation of a major product (Rf =0.60). The reaction mixture
was preadsorbed onto silica gel and purified by column chromatography
on silica gel (cyclohexane/EtOAc/triethylamine, 99:0:1 to 94:5:1) to
afford the bicyclic piperidine (11La) as a pale-yellow oil (267 mg, 68%).
[a]2D5 =+39.2 (c 1.05, CHCl3); 1H NMR (CDCl3, 400 MHz): d=3.00 (d, J-
J
G
J
E
JACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
G
R
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
1H; H1), 7.30–7.40 ppm (m, 5H; ArH); 13C NMR (CDCl3, 100 MHz):
d=37.5 (SO2CH3), 55.9 (OCH3), 60.0 (C5), 68.5 (C6), 71.9 (OCH2Ph),
76.6 (C2), 76.7 (C4), 82.8 (C3), 101.7 (C1), 128.1, 128.6 (ArCH),
137.1 ppm (ArCC); IR (thin film): n˜ =2110 (s; N3), 3518 (br s; OH);
LRMS (ESI+): m/z (%): 410 (100) [M+Na]+; HRMS (ESI+): m/z calcd
for C15H21N3NaO7S: 410.0992 [M+Na]+; found: 410.0991.
For enantiomer 25Db: [a]2D5 =À65.5 (c 0.87, CHCl3)
Methyl-5-azido-2-N-benzyl-3-O-benzyl-2,6-imino-2,5,6-trideoxy-b-l-gulo-
furanoside (11Lb): Trifluoromethanesulfonic anhydride (0.69 mL,
4.2 mmol) and pyridine (0.68 mL, 8.4 mmol) were added to a solution of
mesylate 25Lb (1.28 g, 3.38 mmol) in CH2Cl2 (14 mL) at À408C. After
1 h, with the internal temperature rising to À308C, TLC analysis
(CH2Cl2/MeOH, 30:1) indicated the complete consumption of the start-
ing material (Rf =0.45) and the formation of a single product (Rf =0.65).
The reaction mixture was diluted with CH2Cl2 (25 mL), washed with 2m
HCl (3ꢆ20 mL), dried (MgSO4), filtered, and concentrated in vacuo. The
crude triflate (26Lb) was dissolved in benzylamine (5.8 mL) and stirred
at 1008C for 18 h. TLC analysis (cyclohexane/EtOAc/triethylamine,
66:33:1) indicated the complete consumption of the triflate (Rf =0.30)
and the formation of a major product (Rf =0.70). The reaction mixture
was pre-adsorbed onto silica gel and purified by column chromatography
on silica gel (cyclohexane/EtOAc/triethylamine, 99:0:1 to 94:5:1) to
afford the bicyclic piperidine (11Lb) as a pale-yellow crystalline solid
(1.09 g, 70%). M.p. 50–548C; [a]2D5 =+120.3 (c 0.46, CHCl3); 1H NMR
A
R
ACHTUNGTREN(NUNG 2,3)=3.7 Hz, 1H; H2),
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
J
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ArH); 13C NMR (CDCl3, 100 MHz): d=49.0 (C6), 56.4 (C5), 56.8
(OCH3), 57.6 (C2), 60.1 (NCH2Ph), 71.5 (OCH2Ph), 74.9 (C4), 75.1 (C3),
107.3 (C1), 127.1, 127.6, 127.7, 128.2, 128.3, 129.1 (ArCH), 137.7,
138.4 ppm (ArCC); IR (thin film): n˜ =2114 (s; N3); LRMS (ESI+): m/z
(%): 381 (97) [M+H]+, 403 (100) [M+Na]+; HRMS (ESI+): m/z calcd
for C21H25N4O3: 381.1921 [M+H]+; found: 381.1921.
(CDCl3, 400 MHz): d=3.02 (dd, J
H6), 3.06 (d, J(6’,6)=13.1 Hz, 1H; H6’), 3.18 (d, J
H2), 3.35 (s, 3H; OCH3), 3.49–3.52 (m, 1H; H5), 3.73 (s, 2H; NCH2Ph),
4.16 (app t, J(3,2)=J(3,4)=4.6 Hz, 1H; H3), 4.26 (t, J(4,3)=J(4,5)=
4.6 Hz, 1H; H4), 4.51 (d, Jgem =11.9 Hz, 1H; OCH2Ph), 4.63 (d, Jgem
11.9 Hz, 1H; OCH2Ph), 4.94 (s, 1H; H1), 7.28–7.47 ppm (m, 10H; ArH);
13C NMR (CDCl3, 100 MHz): d=49.4 (C6), 55.4 (OCH3), 56.3 (C5), 60.1
(NCH2Ph), 61.5 (C2), 70.7 (OCH2Ph), 72.2 (C4), 73.1 (C3), 101.4 (C1),
127.5, 127.6, 127.7, 128.3, 128.4, 129.1 (ArCH), 137.5, 137.9 ppm (ArCC);
IR (thin film): n˜ =2114 (s; N3); LRMS (ESI+): m/z (%): 381 (75)
[M+H]+, 403 (77) [M+Na]+, 783 (100) [M+Na]+; HRMS (ESI+): m/z
calcd for C21H24N4NaO3: 403.1741 [M+Na]+; found: 403.1737.
(6,5)=4.3 Hz, J
E
For enantiomer 11Da: [a]2D5 =À40.0 (c 1.58, CHCl3).
G
ACHTUNGTRENNUNG
4-O-Acetyl-5-azido-2-N,3-O-dibenzyl-2,5,6-trideoxy-2,6-imino-l-gulose
acetyl methyl acetal (29L): Boron trifluoride diethyl etherate (0.82 mL,
6.6 mmol) was added dropwise to a solution of bicyclic acetal 11Lb
(797 mg, 2.09 mmol) in acetic anhydride (8 mL) at 08C. The reaction mix-
ture was allowed to warm to RT and stirred for 36 h, after which time
TLC analysis (cyclohexane/EtOAc/triethylamine, 80:19:1) indicated the
complete consumption of the starting material (Rf =0.60) and the forma-
tion of a major product (Rf =0.50). The reaction mixture was concentrat-
ed in vacuo and the residue was dissolved in EtOAc (40 mL), washed
N
G
N
ACHTUNGTRENNUNG
=
with
a saturated solution of sodium bicarbonate (3ꢆ20 mL), dried
(MgSO4), filtered, and concentrated in vacuo. The crude residue was pu-
rified by column chromatography on silica gel (cyclohexane/EtOAc/trie-
thylamine, 99:0:1 to 90:9:1) to afford the mixed-acetal piperidine (29L)
as a pale-yellow oil (951 mg, 94%), in a 2:1 (A/B) ratio of diastereoiso-
mers.
For enantiomer 11Db: M.p. 48–508C; [a]2D5 =À109.2 (c 0.67, CHCl3).
Methyl-5-azido-3-O-benzyl-5-deoxy-6-O-p-toluenesulfonyl-a-l-idofurano-
side (27La): p-Toluenesulfonyl chloride (450 mg, 2.36 mmol) was added
to
a solution of diol 10La (358 mg, 1.16 mmol) and 2,4,6-collidine
Similar treatment of the epimer (11La, 96 mg, 0.25 mmol, Rf =0.55 in cy-
clohexane/EtOAc/triethylamine, 80:19:1) with boron trifluoride etherate
for 3 h afforded compound 29L as a pale-yellow oil (86 mg, 71%), as
a 2:1 (A/B) ratio of diastereoisomers.
(0.60 mL, 4.5 mmol) in CH2Cl2 (5 mL) at RT. After 24 h, TLC analysis
(toluene/acetone, 4:1) indicated a trace of starting material (Rf =0.25), as
well as the formation of a major product (Rf =0.50) and a minor product
(Rf =0.75). The reaction mixture was diluted with CH2Cl2 (10 mL),
washed with 2m HCl (3ꢆ10 mL), dried (MgSO4), filtered, and concen-
trated in vacuo. The crude residue was purified by flash chromatography
(toluene/acetone, 99:1 to 19:1) to afford the primary tosylate (27La) as
a colorless oil (397 mg, 74%). [a]2D5 =À7.0 (c 1.32, CHCl3); 1H NMR
(CDCl3, 400 MHz): d=2.07 (br s, 1H; OH2), 2.44 (s, 3H; SO2C6H4CH3),
Data for compound 29L: 1H NMR (CDCl3, 400 MHz): d=2.06 (s, 3H;
COCH3B), 2.07, 2.09 (s, 2ꢆ3H; COCH3A), 2.14 (s, 3H; COCH3B), 2.16–
2.23 (m, 2H; H6A and H6B), 2.82–2.85 (m, 2H; H2A and H2B), 2.96 (dd,
J
N
(6’,6)=12.4 Hz, 1H; H6’A), 2.96 (dd, J
ACHUTGTNRENNUG ACHUTNTGREN(NUGN 6’,5)=4.5 Hz, J-
AHCTUNGTRENNUNG
3.40 (s, 3H; OCH3), 3.90–3.95 (m, 2H; H3 and H5), 4.05 (dd, J
5.8 Hz, J(6,6’)=10.4 Hz, 1H; H6), 4.09 (dd, J(6’,5)=3.8 Hz, J(6’,6)=
10.4 Hz, 1H; H6’), 4.28–4.29 (m, 1H; H2), 4.30 (dd, J=6.1 Hz, J=
7.6 Hz, 1H; H4), 4.46 (d, Jgem =11.6 Hz, 1H; OCH2Ph), 4.67 (d, Jgem
11.6 Hz, 1H; OCH2Ph), 4.81 (d, J
ACHTUNGTRNE(NUNG 6,5)=
G
E
ACHTUNGTRENNUNG
R
ACHTUNGTRENNUNG
G
E
=
=
AHCTUNGTRENGN(UN 1,2)=1.5 Hz, 1H; H1), 7.30–7.39 (m,
J
7H; ArH), 7.74–7.77 ppm (m, 2H; ArH); 13C NMR (CDCl3, 100 MHz):
d=21.7 (SO2C6H4CH3), 56.1 (OCH3), 60.8 (C5), 68.9 (C6), 72.3
(OCH2Ph), 78.3 (C2), 79.7 (C4), 82.5 (C3), 109.6 (C1), 128.0, 128.1, 128.2,
128.6, 129.9 (ArCH), 132.5, 137.0, 145.1 ppm (ArCC); IR (thin film): n˜ =
J
J
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Chem. Eur. J. 2012, 00, 0 – 0
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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G. W. J. Fleet et al.
(COCH3B), 52.0 (C6A), 52.4 (C6B), 57.0 (NCH2PhA), 57.2 (OCH3B), 57.6
(OCH3A), 58.3 (NCH2PhB), 58.7 (C5A), 58.9 (C5B), 66.2 (C2A), 66.8
(C2B), 74.2 (OCH2PhA), 74.3 (OCH2PhB), 76.6, 76.8, 76.9, 76.9 (C3A, C3B,
C4A, C4B), 96.9 (C1A), 97.2 (C1B), 127.1, 127.2, 127.5, 127.7, 127.7, 127.7,
128.0, 128.1, 128.4, 128.4, 128.6, 128.9 (ArCH), 137.9, 138.7, 138.7, 139.0
(ArCC), 170.0, 170.2, 170.2, 171.0 ppm (COCH3); IR (thin film): n˜ =1746
(s, C=O), 2104 (s, N3); LRMS (ESI+): m/z (%): 483 (97) [M+H]+, 505
(100) [M+Na]+, 987 (92) [2M+Na]+; HRMS (ESI+): m/z calcd for
C25H31N4O6: 483.2238 [M+H]+; found: 483.2242.
1H; NCH2Ph), 3.72 (app t, J
dq, J(2,1’)=4.5 Hz, J(2,1)=J(2,3)=J
gem =13.6 Hz, 1H; NCH2Ph), 4.35 (dd, J
1H; H6), 4.64 (dd, J(6’,5)=2.8 Hz, J(6’,6)=12.6 Hz, 1H; H6), 4.65 (d,
gem =11.1 Hz, 1H; OCH2Ph), 4.72 (d, Jgem =11.1 Hz, 1H; OCH2Ph), 4.90
(dd, J(3,4)=8.6 Hz, J(3,2)=9.6 Hz, 1H; H3), 6.77 (d, J(NH,2)=9.3 Hz,
G
ACHTUNGTNER(NUNG 4,5)=8.8 Hz, 1H; H4), 4.05 (app
E
E
G
ACHTUNGTRENNUNG
J
A
ACHTUNGTRENNUNG
U
ACHTUNGTRENNUNG
J
G
E
ACHTUNGTRENNUNG
1H; NHCOCH3), 7.21–7.37 ppm (m, 10H; ArH); 13C NMR
([D6]acetone), 100 MHz): d=20.8, 21.0 (COCH3), 22.9 (NHCOCH3),
49.0 (C2), 54.6 (C1), 57.3 (NCH2Ph), 60.6 (C6), 64.7 (C5), 75.2
(OCH2Ph), 77.7 (C3), 77.9 (C4), 127.8, 128.4, 128.7, 128.1, 128.1, 128.5
(ArCH), 139.3, 140.0 (ArCC), 169.5, 170.9, 170.9 ppm (COCH3;
NHCOCH3); IR (thin film, Ge): n˜ =1546 (s; amide II), 1660 (s; amide I),
1739 (s; C=O); LRMS (ESI+): m/z (%): 469 (100) [M+H]+, 491 (64)
[M+Na]+; HRMS (ESI+): m/z calcd for C26H33N2O6: 469.2333 [M+H]+;
found: 469.2334.
3,6-Di-O-acetyl-2-azido-1-N,4-O-dibenzyl-1,2,5-trideoxy-1,5-imino-d-glu-
citol (30D): Diisobutylaluminium hydride (16.8 mL, 16.8 mmol) was
added dropwise to a solution of mixed acetal 29L (1.6 g, 3.3 mmol) in
CH2Cl2 (13 mL) at À788C. After 40 min, TLC analysis (cyclohexane/
EtOAc/triethylamine, 79:20:1) indicated the complete consumption of
the starting material (Rf =0.40) and the formation of a major product
(Rf =0.25). The reaction mixture was quenched and diluted with EtOAc
(88 mL) and allowed to warm to RT. A saturated solution of sodium po-
tassium tartrate (88 mL) was added and the biphasic mixture was stirred
vigorously for 1 h. The organic layer was collected and the aqueous layer
was extracted with EtOAc (3ꢆ20 mL). The organic fractions were com-
bined, dried (MgSO4), filtered, and concentrated in vacuo. The crude res-
idue was dissolved in MeOH (18 mL) and sodium borohydride (125 mg,
3.30 mmol) was added at À108C. After 2 h, TLC analysis (cyclohexane/
EtOAc/triethylamine, 59:40:1) indicated the complete consumption of
the intermediate (Rf =0.55) and the formation of a major product (Rf =
0.25). The reaction mixture was neutralized with glacial acetic acid, con-
centrated in vacuo, and dissolved in pyridine (9 mL) and acetic anhydride
(9 mL). The reaction was stirred at RT for 16 h, after which time TLC
analysis (cyclohexane/EtOAc/triethylamine, 59:40:1) indicated the com-
plete consumption of the starting material (Rf =0.25) and the formation
of a major product (Rf =0.40). The reaction mixture was concentrated in
vacuo and purified by column chromatography on silica gel (cyclohex-
ane/EtOAc/triethylamine, 96:3:1 to 84:15:1) to afford the diacetyl piperi-
dine (30D) as a white crystalline solid (1.315 g, 88% over 3 steps). M.p.
89–918C; [a]2D2 =+3.3 (c 1.20, CHCl3); 1H NMR (CDCl3, 400 MHz): d=
For enantiomer 31L: M.p. 144–1488C; [a]2D5 =À2.6 (c 1.18, acetone).
2-Acetamido-1,2,5-trideoxy-1,5-imino-d-glucitol (DNJNAc, 2D). Sodium
methoxide (60 mg, cat.) was added to a solution of the protected piperi-
dine (31D, 1.11 g, 2.63 mmol) in MeOH (25 mL) at RT and the mixture
was stirred for 16 h. TLC analysis (EtOAc/triethylamine, 99:1) indicated
the complete consumption of the starting material (Rf =0.70) and the for-
mation of a major product (Rf =0.25). The reaction mixture was neutral-
ized with solid CO2, concentrated in vacuo, and purified by column chro-
matography on silica gel (CH2Cl2/MeOH, 20:1 to 9:1). The crude residue
was dissolved in water/2m HCl/1,4-dioxane (13:3:4, 35 mL) and palladi-
um (10% on carbon, 408 mg) was added. The vessel was degassed,
charged with hydrogen, and the reaction mixture was stirred at RT for
22 h. LRMS and 1H NMR spectroscopy indicated that a single product
had been formed, with no trace of benzylated intermediates observed.
The reaction mixture was filtered through Celite and concentrated in
vacuo to afford crude DNJNAc (2D) as its hydrochloride salt.
Selected data for DNJNAc·HCl (2D): 1H NMR (D2O, 400 MHz): d=
2.03 (s, 3H; NHCOCH3), 2.99 (app t, 1H, J
3.23 (ddd, J(5,6’)=3.2 Hz, J(5,6)=5.0 Hz, J(5,4)=10.1 Hz, 1H; H5), 3.50
(dd, J(1’,2)=5.0 Hz, J(1’,1)=12.6 Hz, 1H; H1’), 3.63 (app t, J(3,2)=J-
(3,4)=9.6 Hz, 1H; H3), 3.67 (dd, J(4,3)=9.1 Hz, J(4,5)=10.2 Hz, 1H;
H4), 3.90 (dd, J(6,5)=5.1 Hz, J(6,6’)=12.8 Hz, 1H; H6), 3.96 (dd, J-
(6’,5)=3.2 Hz, (6’,6)=12.8 Hz, 1H; H6’), 3.72 ppm (ddd, (2,1’)=
5.0 Hz, J(2,3)=10.1 Hz, J(2,1)=12.3 Hz, 1H; H2).
ACHUTNGTRENNUG(1,2)=JACHTUGNTREN(NUGN 1,1’)=12.5 Hz; H1),
A
R
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
2.06 (app t, J
COCH3), 2.55 (app dt, J
2.98 (dd, (1’,2)=4.8 Hz,
13.3 Hz, 1H; NCH2Ph), 3.47 (app dt, J
10.4 Hz, 1H; H2), 3.66 (app t, J(4,3)=J(4,5)=9.4 Hz, 1H; H4), 4.09 (d,
gem =13.3 Hz, 1H; NCH2Ph), 4.31 (dd, J(6,5)=2.7 Hz, J(6,6’)=12.6 Hz,
1H; H6), 4.59 (d, Jgem =10.9 Hz, 1H; OCH2Ph), 4.64 (dd, J(6’,5)=2.4 Hz,
(6’,6)=12.8 Hz, 1H; H6’), 4.66 (d, Jgem =10.9 Hz, 1H; OCH2Ph), 5.08
(app t, J(3,2)=J(3,4)=9.6 Hz, 1H; H3), 7.26–7.38 ppm (m, 10H; ArH);
ACHTUNGTRENNUNG(1,1’)=JACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
J
A
J
A
=
A
J
A
JACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
The crude salt of compound 2D was loaded onto a short column of
Dowex (50W-X8, H+) and the resin washed with water until neutral frac-
tions were obtained. The product was liberated with aqueous ammonia
(2m) and the ammoniacal fractions were concentrated in vacuo to afford
DNJNAc (2D) as a white crystalline solid (510 mg, 95%). M.p. 224–
2268C [lit. 227–2288C];[16c] [a]D25 =+14.6 (c 0.86, H2O) [lit. [a]2D0 =+16.4[16c]
(c 1.00, H2O)]; 1H NMR (D2O, 400 MHz): d=2.00 (s, 3H; NHCOCH3),
J
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
JACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
13C NMR (CDCl3, 100 MHz): d=20.9, 20.9 (COCH3), 53.6 (C1), 56.5
(NCH2Ph), 59.3 (C2), 59.9 (C6), 63.9 (C5), 75.0 (OCH2Ph), 76.9 (C4),
77.4 (C3), 127.4, 128.0, 128.0, 128.5, 128.5, 128.7 (ArCH), 137.3, 137.6
(ArCC), 170.0, 170.6 ppm (COCH3); IR (thin film): n˜ =1743 (s, C=O),
2104 (s, N3); LRMS (ESI+): m/z (%): 453 (94) [M+H]+, 475 (100)
[M+Na]+, 927 (99) [2M+Na]+; HRMS (ESI+): m/z calcd for
C24H28N4NaO5: 475.1952 [M+H]+; found: 475.1954.
2.43 (dd, J
3.0 Hz, J(5,6)=6.1 Hz, J
(1’,1)=12.6 Hz, 1H; H1’), 3.30 (app t, J
3.39 (dd, J(4,3)=9.1 Hz, J(4,5)=10.0 Hz, 1H; H4), 3.66 (dd, J
(6,6’)=11.6 Hz, 1H; H6), 3.72 (ddd, J(2,1’)=4.9 Hz, J
(2,1)=11.4 Hz, 1H; H2), 3.82 ppm (dd, (6’,5)=2.9 Hz, J-
A
R
ACHTUNGTRENNUNG(5,6’)=
A
R
ACHTUNGTRENNUNG
J
N
N
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
6.1 Hz, J
N
N
ACHTUNGTRENNUNG
For enantiomer 30L: M.p. 90–928C; [a]2D5 =À4.3 (c 2.2, CHCl3).
10.2 Hz,
J
G
JACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(6’,6)=11.6 Hz, 1H; H6’); 13C NMR (D2O, 100 MHz): d=22.6
2-Acetamido-3,6-di-O-acetyl-1-N,4-O-dibenzyl-1,2,5-trideoxy-1,5-imino-d-
glucitol (31D): Powdered zinc (3.8 g, 58 mmol) was added to a solution
of azide 30D (1.31 g, 2.91 mmol) in THF/glacial-acetic-acid/acetic-anhy-
dride (3:2:1, 35 mL) and the mixture was stirred vigorously before the
dropwise addition of a saturated solution of copper(II) sulfate (9.2 mL)
to initiate the reaction. After 20 min, TLC analysis (cyclohexane/EtOAc/
triethylamine, 79:20:1) indicated the complete consumption of the start-
ing material (Rf =0.50) and the formation of a major product (Rf =0.10).
The reaction mixture was filtered through Celite, concentrated in vacuo,
and the crude residue was purified by column chromatography on silica
gel (CH2Cl2/MeOH, 40:1) to afford the acetamide (31D) as a white crys-
talline solid (1.11 g, 81%). M.p. 149–1518C; [a]2D5 =+0.9 (c 0.92, acetone);
1H NMR ([D6]acetone), 400 MHz): d=1.74 (s, 3H; NHCOCH3), 1.98,
(NHCOCH3), 47.5 (C1), 52.8 (C2), 61.0 (C5), 61.8 (C6), 72.5 (C3), 76.4
(C4), 174.9 ppm (NHCOCH3); IR (thin film, Ge): n˜ =1561 (s, amide II),
1638 (s, amide I), 3286 (br s; OH/NH); LRMS (ESI+): m/z (%): 205
(100) [M+H]+, 227 (90) [M+Na]+, 431 (91) [2M+Na]+; HRMS (ESI+):
m/z calcd for C8H17N2O4: 205.1183 [M+H]+; found: 205.1183.
For enantiomer 2L: M.p. 2208C (dec.); [a]2D5 =À18.8 (c 0.57, H2O).
2-Acetamido-1,2,5-trideoxy-1,5-imino-1-N-methyl-d-glucitol,
(N-methyl-
DNJNAc, 2Da): Formaldehyde (37% in water, 0.10 mL) and palladium
(10% on carbon, 5 mg) were added to a solution of DNJNAc (2D,
11.5 mg, 56.9 mmol) in water (1 mL). The vessel was degassed, charged
with hydrogen, and the reaction mixture was stirred at RT for 24 h. The
reaction mixture was filtered (GF/A glass microfiber) and concentrated
in vacuo. The residue was loaded onto a short column of Dowex (50W-
X8, H+) and the resin washed with water until neutral fractions were ob-
tained. The product was liberated with aqueous ammonia (2m) and the
2.06 (s, 2ꢆ3H; COCH3), 2.14 (dd, J
H1), 2.63 (app dt, J(5,6)=J(5,6’)=3.0 Hz, J
(dd, J(1’,2)=4.5 Hz, J(1’,1)=11.6 Hz, 1H; H1’), 3.34 (d, Jgem =13.6 Hz,
A
ACHTUNGTREN(NUGN 1,1’)=11.6 Hz, 1H;
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
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ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Scalable Syntheses of Enantiomers of DNJNAc & DGJNAc
FULL PAPER
ammoniacal fractions were concentrated in vacuo to afford N-methyl-
DNJNAc (2Da) as a white crystalline solid (11.6 mg, 94%). M.p. 2488C
(dec.); [a]2D5 =+20.3 (c 0.53, H2O); 1H NMR (D2O, 400 MHz): d=1.99
the time course of the reaction was confirmed by using a series of incuba-
tion times.
Materials: Enzyme a-N-acetyl-d-galactosaminidase (Charonia lampas)
was purified from the natural sources (Oxford Glycobiology Institute) in
50 mm citrate-phosphate buffer at pH 5 that contained 1 mgmLÀ1 bovine
serum albumin (BSA) and 0.02% NaN3. Substrate: p-nitrophenyl-2-acet-
amido-2-deoxy-a-d-galactopyranoside (Koch-Light Ltd.). Method: Solu-
tions of the enzyme (5 mL) and the inhibitor (5 mL) were added separate-
ly to a 96-well microtitre plate that was pre-incubated at 378C for 5 min
before starting the reaction by addition of the substrate solution (40 mL).
After a further 40 min of incubation at 378C, the reaction was quenched
by the addition of 0.5m aq. Na2CO3 (200 mL). Absorbance at 405 nm was
measured immediately by using a microtitre plate reader (Molecular De-
vices UVmax kinetic microplate reader and SOFTmax 2.35 software).
(app dt, J
NHCOCH3), 2.17 (t, J
NCH3), 2.89 (dd, J(1’,2)=4.8 Hz, J
(3,4)=9.3 Hz, J(3,2)=10.1 Hz, 1H; H3), 3.46 (app t, J
9.6 Hz, 1H; H4), 3.78–3.85 (m, 2H; H2 and H6), 3.82 ppm (dd, J
2.5 Hz, J
(6’,6)=12.9 Hz, 1H; H6’); 13C NMR (D2O, 100 MHz): d=22.6
A
N
ACHTUNGTRNE(NUNG 5,4)=9.9 Hz, 1H; H5), 2.01 (s, 3H;
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
(NHCOCH3), 41.3 (NCH3), 50.5 (C2), 58.1 (C1), 58.1 (C6), 68.5 (C5),
70.9 (C4), 76.3 (C3), 174.9 ppm (NHCOCH3); IR (thin film, Ge): n˜ =
1565 (s; amide II), 1635 (s; amide I), 3300 (br s; OH); LRMS (ESI+): m/
z (%): 219 (70) [M+H]+, 459 (100) [2M+Na]+; HRMS (ESI+): m/z
calcd for C9H19N2O4: 219.1339 [M+H]+; found: 219.1340.
2-Acetamido-1-N-butyl-1,2,5-trideoxy-1,5-imino-d-glucitol,
(N-Butyl-
Data analysis
DNJNAc, 2Dc): Butyraldehyde (0.10 mL, 1.1 mmol) and palladium
(10% on carbon, 5 mg) were added to a solution of DNJNAc (2D,
11.0 mg, 53.9 mmol) in water/1,4-dioxane (1:1, 2 mL). The vessel was de-
gassed, charged with hydrogen, and the reaction mixture was stirred for
24 h. The reaction mixture was filtered (GF/A glass microfiber) and con-
centrated in vacuo. The residue was loaded onto a short column of
Dowex (50W-X8, H+) and the resin washed with water until neutral frac-
tions were obtained. The product was liberated with 25% aqueous am-
monia/MeOH (3:17) and the ammoniacal fractions were concentrated in
vacuo to afford N-butyl-DNJNAc (2Dc) as a white crystalline solid
(13.4 mg, 96%). M.p. 192–1968C; [a]2D5 =+13.3 (c 0.56, H2O); 1H NMR
(D2O, 400 MHz): d=0.89 (t, J=7.3 Hz, 3H; NCH2CH2CH2CH3), 1.27
(sext, J=7.3 Hz, 2H; NCH2CH2CH2CH3), 1.40–1.48 (m, 2H;
Ki values: Data sets were analyzed on Prism 4.0a by using a Lineweaver–
Burk plot (1/rate versus 1/[substrate concentration]) for 4–7 different
substrate concentrations that were selected from a suitable range (4, 2, 1,
0.75, 0.5, 0.2, 0.1 mm) and 3–5 inhibitor concentrations. The slopes were
plotted against the inhibitor concentration and the Ki value was obtained
from the intercept on the x axis.
Free oligosaccharide assay
Materials: Tissue culture media and supplies were purchased from PAA
(Pashing, Austria); sodium cyanoborohydride, bicinchoninic acid/copper(-
II) sulfate reagent, sodium acetate trihydrate, and anthranilic acid (2-
AA) were purchased from Sigma–Aldrich (Dorset, UK); water was
Milli-QTM grade; MeCN (HPLC grade) was purchased from Merk
(Darmstadt, Germany); boric acid was purchased from BDH; MeOH
was purchased from VWR (Lutterworth, Leicestershire, UK); ion-ex-
change resins AG50-X12 and AG4-X4 were purchased from Biorad
(Hemel Hampstead, Hertfordshire, UK), Spe-ed amide 2 was purchased
from Applied Separations (Allentown, Pennsylvania, U.S.); an Amicon
Ultra centrifugal filter was purchased from Millipore (Croxley, Watford,
UK).
NCH2CH2CH2CH3), 2.01 (s, 3H; NHCOCH3), 2.25 (app t, J
(1,2)=11.6 Hz, 1H; H1), 2.25 (app dt, J(5,6)=J(5,6’)=2.5 Hz, J
9.6 Hz, 1H; H5), 2.55–2.62 (m, 1H; NCHH’CH2CH2CH3), 2.71–2.79 (m,
1H; NCHH’CH2CH2CH3), 2.98 (dd, (1’,2)=4.5 Hz, (1’,1)=11.9 Hz,
1H; H1’), 3.32 (dd, J(3,4)=9.3 Hz, J(3,2)=10.1 Hz, 1H; H3), 3.45 (app t,
(4,3)=J(4,5)=9.3 Hz, 1H; H4), 3.79 (app dt, J(2,1’)=4.5 Hz, J(2,1)=J-
(2,3)=10.9 Hz, 1H; H2), 3.84 (dd, J(6,5)=2.8 Hz, J(6,6’)=12.6 Hz, 1H;
H6), 3.92 ppm (dd, J(6’,5)=2.3 Hz, J
(6’,6)=12.6 Hz, 1H; H6’); 13C NMR
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
J
N
JACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
J
G
E
N
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
Cell culture HL60: The human acute amyloid leukemia cell-line HL60
(ATCC) was cultured in RPMI 1640 medium that contained 10% fetal
calf serum, 100 unitsmLÀ1 of penicillin, and 100 mgmLÀ1 streptomycin
(PAA) at 378C and 5% CO2. Cell viability was checked by using Trypan
Blue (Sigma Aldrich).
(D2O, 100 MHz): d=13.8 (NCH2CH2CH2CH3), 20.8 (NCH2CH2CH2CH3),
22.6 (NHCOCH3), 25.9 (NCH2CH2CH2CH3), 50.6 (C2), 52.2
(NCH2CH2CH2CH3), 53.9 (C1), 58.2 (C6), 65.7 (C5), 71.2 (C4), 76.4 (C3),
174.9 ppm (NHCOCH3); IR (thin film, Ge): n˜ =1562 (s; amide II), 1640
(s; amide I), 3296 (br s; OH); LRMS (ESI+): m/z (%): 261 (94)
[M+H]+, 331 (74) [M+MeOH+K]+, 543 (100) [2M+Na]+; HRMS
(ESI+): m/z calcd for C12H25N2O4: 261.1809 [M+H]+; found: 261.1816.
Inhibition treatment of HL60 cells and FOS isolation: Having been propa-
gated at a higher density, HL60 cells were seeded in a 6 well plate at
a concentration of 5ꢆ105 cellsmLÀ1 and treated with a fresh medium
(2 mL) that contained the derivatives of DGJNAc (1D) at a concentra-
tion of 500 mm, 100 mm, and 50 mm for 24 h. NB-DNJ (500 mm) and un-
treated wells were used as controls. Following incubation, the cells were
centrifuged at 350 g, 108C, for 7 min and the pellet was washed three
times with PBS. The washed cell pellets were stored at À208C until use.
Following thawing at RT, the samples were resuspended in water
(650 mL) and Dounce homogenized. An aliquot (50 mL) was removed
and treated with 0.25m NaOH solution for 16 h prior to determination of
the protein concentration in comparison to a standard by using bicincho-
ninic acid/copper(II) sulfate reagent (Sigma Aldrich). For desalting and
deproteination, the homogenate (500 mL) was subjected to a mixed-bed
ion-exchange column [AG50-X12 (0.2 mL), (H+, 100–200 mesh), AG4-X4
(0.4 mL), (OHÀ, 100–200 mesh)], which had been preequilibrated with
water (5ꢆ1 mL). The homogenate was added to the column and eluted
with water (4ꢆ1 mL). The eluent that contained the free oligosaccharides
(FOS) was collected and dried by lyophilization.
Biological experiments
In vitro enzyme inhibition, IC50 determination: The b-N-acetyl-glucosami-
nidases (from human placenta, bovine kidney, Aspergillus oryzae, and
jack beans), a-N-acetyl-galactosaminidase (from chicken liver), b-N-
acetyl-galactosaminidase (from Aspergillus oryzae), and p-nitrophenyl
glycosides were purchased from Sigma–Aldrich. The cell lysate of the
human acute amyloid leukemia cell-line HL60 (RBRC), which was cul-
tured in RPMI 1640 medium that contained 10% fetal calf serum,
100 unitsmLÀ1 of penicillin, and 100 mgmLÀ1 streptomycin (Invitrogen) at
378C under 5% CO2, was used as the source of b-N-acetyl-glucosamini-
dase and b-N-acetyl-galactosaminidase. The glycosidase activities were
determined by using an appropriate p-nitrophenyl glycoside as the sub-
strate at the optimum pH value of each enzyme. The reaction mixture
(1 mL) contained 2 mm of the substrate and the appropriate amount of
enzyme. The reaction was stopped by adding of 400 mm Na2CO3 (2 mL).
The released p-nitrophenol was measured spectrometrically at 400 nm.
a-N-Acetyl-d-galactosaminidase assay, Ki determination: Before use, the
enzyme solutions were thawed at RT then put on ice. The solutions were
stored at À208C. The inhibitors were dissolved in deionized H2O to
a stock concentration of 50 mm. These solutions were stored at RT. The
enzyme substrate solutions were 4 mm in 50 mm citrate/citric acid buffer,
pH 5.0. These solutions were stored at 48C. Assays were carried out in
triplicate/duplicate by using H2O in place of the inhibitor as a positive
control; replacing both the enzyme and inhibitor with H2O was used as
a blank to determine the background of the experiment. Linearity over
Carbohydrate fluorescent labeling: The lyophilized samples were resus-
pended in water (300 mL), transferred to an Eppendorf tube, and evapo-
rated to dryness by using a Thermo Savant (SPD121P) SpeedVac system.
As previously described,[33] anthranilic acid (2-AA) was dissolved at
a concentration of 30 mgmLÀ1 in a MeOH solution that contained 4%
sodium acetate trihydrate (w/v) and 2% boric acid (w/v). To this mixture
was added sodium cyanoborohydride to
a final concentration of
45 mgmLÀ1 (labeling buffer). The dried samples were dissolved in water
Chem. Eur. J. 2012, 00, 0 – 0
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G. W. J. Fleet et al.
(30 mL) and 2-AA labeling buffer (80 mL) was added, followed by incuba-
tion at 808C for 1 h.
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(97:3, 1 mL) was added to the labeled FOS samples and the samples
were vortexed and added to a spe-ed amide-2 column that had been pre-
equilibrated as follows: MeCN (1 mL), water (2 mL), MeCN (2 mL). The
eluent was discarded and the column was washed with MeCN/water
(95:5, 2 mL) and discarded. The purified 2-AA-labeled FOS was eluted
by gravity by using water (2ꢆ 0.75 mL). The samples were stored at
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Carbohydrate analysis by normal-phase HPLC: As previously described,
the chromatography system that was used consisted of a Water Alliance
2695 separations module and an in-line Waters 474 fluorescence detector
that was set at Exl 360 nm and Eml 425 nm.[34] The gain for the detector
was set to 1000 and the emission bandwidth was set to 40 nm. Chroma-
tography was performed at 308C. Solvent A was MeCN; solvent B was
water; solvent C consisted of ammonium hydroxide (800 mm) that was ti-
trated to pH 3.85 with acetic acid in water and was prepared by using
a standard 5 m ammonium hydroxide solution (Sigma Aldrich). Data col-
lection and -processing were performed by using Waters Empower soft-
ware. Glucose units were derived by comparison to a 2-AA-labeled glu-
cose oligomer ladder (which was obtained by the partial digestion of dex-
tran) as an external standard. A sample/MeCN mixture (1:1, 50 mL) was
injected for each run. Separation was performed on a 4.6ꢆ250 mm TSK
gel-Amide 80 column (5 mm; Anachem, Luton, UK) and the following
gradient conditions were used for the analysis of the FOS samples:
time=0 min (t=0), 71.6% A, 25.9% B, 2.5% C (0.8 mLminÀ1); t=6,
71.6% A, 25.9% B, 2.5% C (0.8 mLminÀ1); t=45, 46.2% A, 51.3% B,
2.5% C (0.8 mLminÀ1); t=46, 35% A, 62.5% B, 2.5% C (0.8 mLminÀ1);
t=48, 35% A, 62.5% B, 2.5% C (0.8 mLminÀ1); t=49, 71.6% A, 25.9%
B, 2.5%
C C
(0.8 mLminÀ1); t=51, 71.6% A, 25.9% B, 2.5%
(1.2 mLminÀ1); t=64, 71.6% A, 25.9% B, 2.5% C (1.2 mLminÀ1); t=65,
71.6% A, 25.9% B, 2.5% C (0.8 mLminÀ1).
Enzyme (jack bean b-HexNAcase) digest: Glycosidase digest using jack
bean b-HexNAcase (10 mL of 6 UmLÀ1 in 50 mm citrate buffer (pH 5.0)
that contained 1 mgmLÀ1 BSA and 0.02% sodium azide, purified in
house) was performed on a representative sample (100 mL) of the com-
plete FOS population from 500 mm DGJNAc-treated HL60 cells. After
16 h incubation at 378C, the enzyme digest was diluted with water
(90 mL). An Amicon Ultra centrifugal filter (Millipore, 10000 MWCO)
was pretreated with water (150 mL) and used to remove proteins follow-
ing centrifugation at 13000 g for 15 min at 48C. The filter was washed
with water (100 mL) and the combined eluate was evaporated to dryness
before being reconstituted in MeCN/water (1:1, 100 mL) for HPLC analy-
sis.
Statistical analysis: Experiments were performed in triplicate. To test for
significance, Prism 4.0a software was used to perform a student t-test by
using a 99% confidence interval.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research (C)
(23590127) from the Japanese Society for the Promotion of Science
(JSPS), by the Spanish Ministerio de Ciencia e Innovaciꢇn (contract no.:
SAF2010-15670), and by the Fundaciꢇn Ramꢇn Areces. A.G. was sup-
ported by the Medicinal Chemistry for Cancer Graduate Programme
from Cancer Research UK (CRUK). B.A. was supported by the Newton
Abraham Research Fund.
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Received: January 11, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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G. W. J. Fleet et al.
Enzyme Inhibitors
A. F. G. Glawar, D. Best, B. J. Ayers,
S. Miyauchi, S. Nakagawa,
M. Aguilar-Moncayo,
J. M. Garcꢀa Fernꢁndez,
C. Ortiz Mellet, E. V. Crabtree,
T. D. Butters, F. X. Wilson, A. Kato,
G. W. J. Fleet* ................. &&&& — &&&&
Getting the NAc of it: Scalable synthe-
ses of DGJNAc and DNJNAc from d-
glucuronolactone are reported.
were inhibitors of a-GalNAcase and
both DGJNAc and DNJNAc were
potent inhibitors of b-GlcNAcases and
b-GalNAcases.
Scalable Syntheses of Both Enantio-
mers of DNJNAc and DGJNAc from
Glucuronolactone: The Effect of N-
Alkylation on Hexosaminidase Inhibi-
tion
DGJNAc and its N-alkyl derivatives
&
20
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www.chemeurj.org
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!