Synthesis of regio- and stereospecifically C-deuterated derivatives of glycosidase inhibitors 1-deoxymannonojirimycin and 2,5-dideoxy-2,5-imino-D-mannitol by intramolecular reductive amination employing deuterium gas

Article abstract of DOI:10.1016/S0008-6215(98)00159-1

6-Azido-1,3,4-tri-O-benzyl-6-deoxy-d-fructofuranose can be easily obtained in two steps from the known 6,6'-diazido-6,6'-dideoxysucrose, which is available in two steps from sucrose. Cyclisation of this material by controlled hydrogenation with ²

Full text of DOI:10.1016/S0008-6215(98)00159-1

Carbohydrate Research 309 (1998) 367±374
Note
Synthesis of regio- and stereospeci®cally
C-deuterated derivatives of glycosidase
inhibitors 1-deoxymannonojirimycin and
2,5-dideoxy-2,5-imino-d-mannitol by
intramolecular reductive amination
employing deuterium gas1
Martin H. Fechter, Gunther Gradnig, Andreas Berger, Christian Mirtl,
Werner Schmid, Arnold E. Stutz *
Institut fur Organische Chemie der Technischen Universitat Graz, Stremayrgasse 16, A-8010 Graz,
È
È
Austria
Received 7 May 1998; accepted 16 June 1998
Abstract
6-Azido-1,3,4-tri-O-benzyl-6-deoxy-d-fructofuranose can be easily obtained in two steps from
the known 6,60-diazido-6,60-dideoxysucrose, which is available in two steps from sucrose. Cycli-
2
sation of this material by controlled hydrogenation with H2 over Raney nickel and concomitant
intramolecular reductive amination gave 3,4,6-tri-O-benzyl-1,5-dideoxy-1,5-imino-d-(5-2H)man-
nitol, a partially protected derivative of 1-deoxymannonojirimycin. Conventional deprotection
furnished 1-deoxy-(5-2H)mannonojirimycin. Reduction and intramolecular reductive amination
of free 5-azido-5-deoxy-d-fructopyranose gave 2,5-dideoxy-2,5-imino-d-(5-2H)mannitol in one
step. Likewise, 2,5-dideoxy-2,5-imino-d-(5-2H)glucitol was obtained from 5-azido-5-deoxy-l-sor-
bopyranose. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: C-Deuteration; Deuterium gas; Glycosidase inhibitor
Over the past decade, sugars with nitrogen
instead of oxygen in the ring (sometimes referred to
as aza-sugars) have emerged as the most important
class of reversible glycosidase inhibitors [1]. Spurred
by biochemical and medicinal research, glycosi-
dases involved in the modi®cation of glycoproteins
and glycolipids have become of eminent interest
due to their crucial roles in a wide variety of bio-
logical processes. In context with a programme
aiming for the structure elucidation of glycosidase
active sites, as well as approaches to non-invasive
in vivo inhibition studies, we have resorted to
NMR spectroscopic investigations of interactions
1
Dedicated to Professor Herfried Griengl on the occa-
sion of his 60th birthday.
* Corresponding author. Fax: +43-316-873-8740; e-mail:
stuetz@orgc.tu-graz.ac.at
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00159-1

368
M.H. Fechter et al./Carbohydrate Research 309 (1998) 367±374
of selected glycosidases with suitable iminoalditols
as model systems. For the purpose of these experi-
ments, we have recently synthesised several potent
glycosidase inhibitors substituted with stable iso-
topes such as 19F and 13C. This improves detection
under the assay conditions employed. A number of
19F-substituted derivatives of the powerful glycosi-
dase inhibitors 1-deoxynojirimycin (1), 2-acet-
amido-1,2-dideoxynojirimycin (2) and 2,5-dideoxy-
2,5-imino-d-mannitol (3) such as, for example, 1,2-
[2] and 1,6- [3] dideoxy¯uoronojirimycins (4 and
5), 1,2, 5-trideoxy-2-¯uoroacetamido-1,5-imino-
d-glucitol (6) [4], as well as 1,2,5-trideoxy-1-¯uoro-
2,5-imino-d-mannitol (7) [2], have been prepared.
For 13C NMR applications, 13C-substituted com-
pounds such as 1-deoxy-(1-13C)nojirimycin [5] have
been synthesised. In a related line of research
aiming for compounds exhibiting simpli®ed cou-
pling patterns as well as derivatives substituted
with two or more di€erent stable isotopes (13C,
15N) in the same molecule, selected regio- and stereo-
speci®cally C-deuterated inhibitors have now
become available.
elucidation of the biosynthesis of compound 1 [16].
A common disadvantage of these approaches is the
comparably expensive deuterating step that is
required at an early or intermediate step of the
synthesis. Additional synthetic transformations
and subsequent deprotection reactions lower the
yields of the desired ®nal products. Furthermore,
depending on the chemical environment of the
deuterium substituent and the reaction conditions,
isotopic scrambling, as well as partial exchange of
the introduced isotope, may occur [7,17,18]. Con-
sequently, we decided to combine the advantage of
the comparably moderate price of deuterium gas
[18,19] with the possibility to introduce the desired
labeling as late as possible in the synthesis. Indeed
this can be achieved in the last step of the pre-
paration, which is conventionally [20] the forma-
tion of the iminoalditol ring by intramolecular
reductive amination of an azidodeoxyaldose or
-ketose.
In order to avoid large losses of deuterium gas
through manipulations of the reaction vessel, the
glass apparatus depicted in Fig. 1 was employed.
This allowed purging of the reaction vessel with
nitrogen, replacing N2 with deuterium gas and,
upon work-up, the storage of excess D2 (albeit
diluted with nitrogen) in a storage ¯ask for con-
venient recycling of up to 70% of the deuterium.
Preliminary experiments had shown that with short
Compounds substituted with deuterium atoms
have conventionally been prepared by reduction of
suitable functional groups with complex metal
hydrides [6±9]. In the case of aldehydes and
ketones, mono-C-deuterio derivatives of the corre-
sponding alcohols can be prepared. On the other
hand, gem-dideuterated compounds are available
from carboxylic esters as demonstrated in the pre-
paration of 1,1,6-trideuterated 1-deoxynojirimycin
by Leontein and co-workers [10]. Other approaches
in the carbohydrate ®eld have taken advantage of a
deuterioboration protocol [11] or hot D2O in the
presence of Raney Ni [12] as well as the nucleo-
philic displacement of halides [13,14] or sulfonates
[14,15] employing LiAlD4. Another example in this
class of compounds was given in context with the
Fig. 1. Deuteration apparatus with D2 recycling option.

M.H. Fechter et al./Carbohydrate Research 309 (1998) 367±374
369
reaction times water was sucient to pump the
gases between the reaction vessel and the storage
¯asks despite some hydrogen/deuterium exchange
with the gas phase.
Prolonged deuteration procedures resulted in
reduced deuterium content of the products.
Moisture, as well as chemically bound water on
the catalyst, were found to have negative e€ects on
the yields of deuterium substitution. Under opti-
mised conditions, palladium-on-charcoal (10%)
was pre-deuterated in methanol-d1 for 24 h. Dry
next. As could be expected from published data
[22,23], catalytic intramolecular reductive amina-
tion of 5-azido-5-deoxy-d-fructopyranose (14) in
methanol-d1 employing palladium-on-activated-
charcoal (10%) gave mono-C-deuterated 2,5-di-
deoxy-2,5-imino-d-mannitol (15) in approximately
80% isolated yield (Scheme 2). As previously
observed with the non-deuterated counterpart, this
compound was formed practically stereo-
speci®cally over its C-2 epimer, which could not be
detected to have been formed under the conditions
employed. Applying a reaction time of 20 min, the
yield of deuterium introduction was found to range
between 90 and 95%, as determined by integration
over the 1H NMR spectrum of this product (Fig. 2,
spectrum A). After a reaction time of 120 min, this
value dropped to approximately 80%. Likewise,
employing 5-azido-5-deoxy-l-sorbose (16) [23],
azidodeoxyhexose was added in
a
minimal
amount of the same solvent, and hydrogenation
was performed under ambient pressure. Reaction
times of 20 min were found to be sucient for the
complete conversion of the starting materials.
Starting materials were prepared according to pre-
viously established procedures. Taking advantage
of the known regioselectivity of the reaction [21], in
initial model experiments applying the established
[4] ®ve-step approach from sucrose but employing
C-5-deuterated
2,5-dideoxy-2,5-imino-d-glucitol
(17) was identi®ed as the sole product of the reac-
tion. In this case, yields of deuterium incorporation
were found to be around 95% (Fig. 3, spectrum A)
with isolated yields of compound 17 of 75 to 80%.
As expected, N/O-perdeuterated product 17
showed a molecule ion of 169 mass units (Fig. 4).
All fragments were found to contain one mass unit
more than the corresponding N/O-perdeuterated,
unsubstituted parent compound (Fig. 5). Sym-
metrical compound 15 exhibited a very similar
fragmentation pattern.
In conclusion, catalytic reduction of cyclic aldi-
mines with deuterium gas was, for the ®rst time,
conveniently applied to the synthesis of selected
iminoalditols. By optimisation of the reaction con-
ditions as well as the selection of suitable starting
materials, satisfying deuterium substitution could
be achieved. Deuterium incorporation was found
to proceed with perfect regio- and practically com-
plete stereoselectivity. However, the need for
2
Raney nickel and H2 instead of hydrogen gas in
the hydrogenation step of 6-azido-1,3,4-tri-O-ben-
zyl-6-deoxy-d-fructofuranose (8), a 10:1 mixture of
3,4,6-tri-O-benzyl-1,5-dideoxy-1,5-imino-d-(5-2H)-
mannitol (9) and the corresponding l-gulo epimer
(10) were obtained in nearly 80% isolated yield
from compound 8. Separation could be achieved
after protection of the ring nitrogen as the N-benzyl-
oxycarbonyl derivatives 11 and 12. Intermediate 11
was smoothly deprotected under standard condi-
tions to yield 1-deoxy-(5-2H)mannonojirimycin (13)
in 60% overall yield (Scheme 1). From the integral
1
over the H NMR spectrum of this product incor-
poration of deuterium at C-5 was found to be in
the range of around 80%. In accordance with the
aim to introduce deuterium as late in the sequence
as possible, C-deuteration by catalytic reduction of
selected unprotected sugar derivatives was probed
Scheme 1.

370
M.H. Fechter et al./Carbohydrate Research 309 (1998) 367±374
Scheme 2.
Fig. 2. 1H NMR Spectrum of compound 15 (A) and non-deuterated 2,5-dideoxy-2,5-imino-d-mannitol (B) recorded at 300 MHz.
methanol-d1 as the reaction medium as well as the
pre-deuteration of the catalyst remain a dis-
advantage of this method. Nonetheless, de®nite
advantages of this approach are the introduction of
deuterium in the last step with compounds such as
15 and 17, together with the high deuterium con-
tent found in the a€orded products.
1. Experimental
General methods.ÐMelting points of crystalline
intermediates were recorded on a Tottoli apparatus
and are uncorrected. Optical rotations were mea-
sured on a Perkin±Elmer 341 Digital Polarimeter
with a path length of 10 cm. NMR spectra were

M.H. Fechter et al./Carbohydrate Research 309 (1998) 367±374
371
Fig. 3. 1H NMR Spectrum of compound 17 (A) and nondeuterated 2,5-dideoxy-2,5-imino-d-glucitol (B) recorded at 300 MHz.
Fig. 4. Mass spectra of N/O-perdeuterated compound 17.

372
M.H. Fechter et al./Carbohydrate Research 309 (1998) 367±374
Fig. 5. Mass spectra of N/O-perdeuterated 2,5-dideoxy-2,5-imino-d-glucitol.
recorded at 200 and 300 MHz (1H), and at 50.29
and 75.47 MHz (13C), in D2O. EI (70 eV) DI/MS
data were recorded on a Shimadzu Pro®le mass
spectrometer. TLC was performed on precoated
aluminum sheets (E. Merck 5554). For column
chromatography Silica Gel 60 (E. Merck) or ion-
exchange resin (Amberlite CG 50) was used. Deu-
terium (99.8%) was purchased from Cambridge
Isotope Laboratories (DLM-408). Raney Ni
(Fluka) was pre-deuterated by stirring in D2O for
18 h, after which time the solvent was removed by
®ltration. The catalyst was then rinsed with a
minimal amount of methanol-d1 and immediately
used in the respective hydrogenation experiment.
General procedure for the reaction of free 5-
azido-5-deoxyketoses.ÐTypically, a suspension of
Pd/C (10%, 300 mg) in methanol-d1 (10 mL) was
pre-deuterated twice at ambient pressure for 12 h
after which time the solvent was decanted. A 2%
solution of the respective free 5-azidodeoxyketose
(100 mg, 0.49 mmol) in methanol-d1 was added,
and the mixture was stirred under an atmosphere
of deuterium gas for 20 min. After removal of the
catalyst by ®ltration, the solvent was removed
under reduced pressure (with larger scale reactions,
the deuterated solvent can be recycled), and the
residue was chromatographed on silica gel
employing a mixture of 100:100:1 CHCl3±MeOH±
concd aq ammonia as the solvent system.
3,4,6-Tri-O-benzyl-1,5-dideoxy-1,5-imino-d-(5-2H)-
mannitol (9) and -l-(5-2H)gulitol (10).ÐTo
a
suspension of predeuterated Raney nickel (2 g)
(Caution: pyrophoric), a 2% solution of com-
pound 8 (300 mg, 0.63 mmol) in dry methanol-d1
was added, and the mixture was stirred under an
atmosphere of deuterium at ambient temperature
for 18 h. After ®ltration the solvent was removed
under reduced pressure to give an inseparable
mixture of partially protected iminoalditols 9
and 10, which was immediately used in the next
step.
3,4,6-Tri-O-benzyl-N-benzyloxycarbonyl-1,5-di-
deoxy-1,5-imino-d-(5-2H)mannitol (11).ÐTo a 5%
solution (5:1 dichloromethane±methanol) of the
crude mixture of intermediates 9 and 10 obtained
from the previous reaction, solid sodium bicarbo-
nate (160 mg, 1.9 mmol, 3 equiv), as well as benzy-
loxycarbonyl chloride (0.1 mL, 1.2 equiv), were
added, and the mixture was stirred at ambient
temperature for 30 min. Dichloromethane (100 mL)
was added, and the mixture was washed with
water, the aqueous layer was extracted once with

M.H. Fechter et al./Carbohydrate Research 309 (1998) 367±374
373
dichloromethane (100 mL), and the combined
organic layers were dried (sodium sulfate).
Removal of the solvent under reduced pressure
and chromatography of the residue gave com-
pound 11 (250 mg, 70%) as a colourless syrup.
This was identical by TLC with the non-deuter-
ated analogue [4] and was directly employed in the
subsequent deprotection step. Despite our best
e€orts, spectra of this compound did not resolve
due to the e€ects of pyramidal inversion at the
ring nitrogen as well as the presence of rotamers
as was previously reported ([4] and refs. cited
therein).
3.38 (dd, 1 H, H-2); 13C NMR (D2O): 79.9, 78.2 (C-
3, C-4), 65.4 (t, JC,D 21.5 Hz, C-5), 63.0 (C-1), 61.8,
61.0 (C-1, C-6).
Acknowledgements
This work was kindly supported by the Austrian
Fonds zur Forderung der Wissenschaftlichen For-
schung, Vienna, Projects 10067 and 10805 CHE.
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removed by ®ltration and the ®ltrate was con-
centrated under reduced pressure. The product was
puri®ed by ion-pair chromatography on Amberlite
CG 50 (200:1 water±concd aqueous ammonia) to
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