Biosynthetic studies on the α-glucosidase inhibitor acarbose: The chemical synthesis of dTDP-4-amino-4,6-dideoxy-α-D-glucose

Article abstract of DOI:10.1016/S0008-6215(01)00323-8

To study the biosynthesis of the pseudotetrasaccharide acarbose, dTDP-4-amino-4,6-dideoxy-α-D-glucose (3) was prepared from galactose in 16 steps. After initial protecting-group manipulations, the 6-position of galactose was deoxygenated by hydride displa

Full text of DOI:10.1016/S0008-6215(01)00323-8

Carbohydrate Research 337 (2002) 297–304
www.elsevier.com/locate/carres
Biosynthetic studies on the a-glucosidase inhibitor acarbose: the
chemical synthesis of dTDP-4-amino-4,6-dideoxy-a- -glucose
D
Simeon G. Bowers, Taifo Mahmud, Heinz G. Floss*
Department of Chemistry, Uni6ersity of Washington, Box 351700, Seattle, WA 98195-1700, USA
Received 12 October 2001; accepted 29 November 2001
Abstract
To study the biosynthesis of the pseudotetrasaccharide acarbose, dTDP-4-amino-4,6-dideoxy-a-
D
-glucose (3) was prepared from
galactose in 16 steps. After initial protecting-group manipulations, the 6-position of galactose was deoxygenated by hydride
displacement of a tosylate. Similarly a tosyl group at the 4-position was displaced upon reaction with sodium azide. Conversion
of the resulting glycoside to a glycosyl phosphate was accomplished by reaction of a glycosyl trichloroacetimidate with dibenzyl
phosphate. Subsequent removal of the benzyl protecting groups and reduction of the azide by hydrogenation and coupling with
an activated nucleoside phosphate gave dTDP-4-amino-4,6-dideoxy-a-
D-glucose. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Acarbose biosynthesis; Aminodeoxy sugar
1. Introduction
cyclitol moiety is derived from the pentose phosphate
pathway.2 In particular, through feeding experiments
with various isotopically labeled cyclitols to Acti-
noplanes sp. SN223/29, it was found that only 2-epi-5-
epi-[6-2H2]valiolone (2) was incorporated into acarbose,
giving 15% specific incorporation.3 2-epi-5-epi-Vali-
olone in turn is formed from sedoheptulose 7-phos-
phate in a reaction catalyzed by a cyclase encoded by
the acbC gene of the acarbose biosynthetic gene cluster
of Actinoplanes, a homolog of dehydroquinate syn-
thases.4 Interestingly, neither the C-2 epimer (which has
the same stereochemistry as acarbose) nor the C-5
epimer of 2, nor their dehydrated forms, 2-epi-va-
lienone and valienone, were incorporated.3 The biosyn-
thesis of the deoxyhexose unit involves the conversion
Acarbose (1, Fig. 1) a pseudotetrasaccharide isolated
from the fermentation broth of Actinoplanes sp., is an
inhibitor of a-glucosidase and a clinically useful drug
for the treatment of type II insulin-independent dia-
betes.1 Structurally acarbose consists of an unsaturated
cyclitol linked via a nitrogen bridge to a deoxyhexose,
which is in turn linked to maltose. Previous studies on
the biosynthesis of acarbose have demonstrated that the
of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose
by an intramolecular hydride transfer5 catalyzed by
dTDP-glucose 4,6-dehydratase, which is encoded by the
acbB gene.4 It is proposed that this keto-sugar is subse-
quently converted to dTDP-4-amino-4,6-dideoxy-D-glu-
cose (3) by the action of a transaminase. The maltose
moiety of 1 is derived directly from maltose in the
fermentation medium.2,6
Fig. 1. Structure of acarbose.
Two possible pathways for the biosynthesis of the
pseudodisaccharide portion of acarbose are outlined in
Scheme 1. The first proposes that condensation of the
* Corresponding author. Tel.: +1-206-5430310; fax: +1-
206-5438318.
E-mail address: floss@chem.washington.edu (H.G. Floss).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00323-8

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S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
Scheme 1. Proposed pathways for the biosynthesis of acarbose.
keto-cyclitol 2 with amino sugar 3 gives the intermedi-
ate imine 4. Subsequent epimerization at C-2 and dehy-
dration between C-5 and C-6 followed by reduction of
the imine to an amine, which all would have to occur on
one enzyme to explain the absence of free intermediates,
would afford the pseudodisaccharide nucleotide 5.3
an alternative hypothetical pathway involves the
reduction of keto-sugar 2 to a 1-epi-valiolol derivative,
followed by activation of the C-1 hydroxyl group
as a phosphate and subsequent nucleophilic dis-
placement by the nitrogen of amino sugar 3 to give
pseudodisaccharide 5. Such displacements are prece-
dented in the biosynthesis of aristeromycin and ne-
planocin A.7 In this pathway, the dehydration between
C-5 and C-6 and the epimerization at C-2 of the cyclitol
could occur either before or after the coupling with the
amino sugar.
In order to determine whether either of the two
proposed routes is operating in acarbose formation and
to identify the actual biosynthetic pathway, it was
necessary to have amino sugar 3 available as a substrate
in the search for an enzyme coupling it to the cyclitol
moiety and as a reference sample to aid in the determi-
nation whether the compound is generated enzymati-
cally in acarbose-producing Actinoplanes sp. In this
paper we describe the chemical synthesis of compound
3. An alternative synthesis of 3 has also recently been
reported by Thorson and co-workers.8
2. Results and discussion
The chemical synthesis of amino sugar 3 started from
fully protected galactoside 6 which is readily available
from D
-galactose in three steps.9 The hydroxyl groups at
the 2- and 3-positions were protected as benzyl ethers as
follows (Scheme 2). Zemple´n deacetylation of 6 was
followed by protection of the 4,6-diol as a benzylidene
acetal upon treatment with benzaldehyde dimethyl acetal
and CSA to afford 7 in 79% yield. Subsequent benzyla-
tion of the 2- and 3-positions of 7 gave fully protected
8, which was converted to diol 9 upon acid hydrolysis of
the benzylidene acetal. Deoxygenation of the 6-position
was accomplished by hydride displacement of a p-tolu-
enesulfonyl leaving group. Chemoselective tosylation of
the primary hydroxyl group of 9 was achieved using a
slight excess of p-toluenesulfonyl chloride in combina-
tion with prolonged reaction times to afford tosylate 10
in 84% yield. Under these conditions no tosylation of the
secondary hydroxyl was observed. Subsequent displace-
ment of the primary tosylate with LiAlH4 in THF
afforded 6-deoxy derivative 11 in 83% yield.

S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
299
Scheme 2. Reagents and conditions: (i) NaOCH3 (0.2 equiv), CH3OH, 18 h; (ii) benzaldehyde dimethyl acetal (1.2 equiv), CSA,
CH3CN, 5 h, 79% (two steps); (iii) BnBr (3 equiv), NaH (4 equiv), DMF, 5 h, 89%; (iv) HOAc–water, 50 °C, 18 h, 93%; (v) TsCl
(1.5 equiv), pyridine, 4 days, 84%; (vi) LiAlH4 (2.2 equiv), THF, reflux, 18 h, 83%; (vii) TsCl (5 equiv), DMAP (0.5 equiv),
pyridine, 50 °C, 18 h, 78%; (viii) NaN3 (5 equiv), DMF, 70 °C, 3 days, 80%.
In a similar fashion nitrogen was introduced at the
4-position by displacement of a leaving group with
azide. Since the tosylation of the 6-position required
extended reaction times, it was expected that the sec-
ondary hydroxyl group of 11 would be much less
reactive and possibly inert. Fortunately, when 11 re-
acted with a large excess of tosyl chloride and DMAP
in pyridine at elevated temperatures, complete conver-
sion to tosylate 12 was observed. Displacement of the
tosyl group with sodium azide in DMF at 70 °C for 3
sugar hemi acetal, which is subsequently oxidized to the
glycosyl phosphate.13–15 The latter method was at-
tempted first. The 2-(trimethylsilyl)ethyl anomeric pro-
tecting group was removed from fully protected
glycoside 13 upon treatment with TFA in CH2Cl2 to
give hemi acetal 14 in 89% yield (Scheme 3).16 After
treating 14 with the trivalent phosphitylating reagent,
dibenzyl N,N-diisopropylphosphoramidite, and 1H-tet-
razole, followed by addition of MCPBA and purifica-
tion, the desired phosphate could not be isolated. After
the failure of this approach, an alternative method for
the preparation of the glycosyl phosphate was at-
tempted using a glycosyl trichloroacetimidate as a gly-
cosyl donor.11,12 Hemi acetal 14 was treated with
trichloroacetonitrile and potassium carbonate in
CH2Cl2 and after purification, trichloroacetimidate 15
was obtained in 74% yield (1:5 a/b). Subsequent reac-
tion of 15 with dibenzyl phosphate in CH2Cl2 afforded
1
days afforded azide 13 in 80% yield. Furthermore, H
NMR spectroscopy indicated that the azido group of
compound 13 was equatorial.
With compound 13 at hand, it remained to introduce
an anomeric phosphate which would be coupled with
an activated TMP derivative to give the final compound
3. Various procedures exist for the preparation of gly-
cosyl phosphates, including the reaction of dibenzyl
phosphate with glycosyl donors such as thiogly-
cosides,10 glycosyl trichloroacetimidates,11,12 and glyco-
syl bromides.13 Alternatively it is possible to prepare an
intermediate glycosyl phosphite from the corresponding
1
protected glycosyl phosphate 16 in 82% yield. H NMR
analysis of purified 16 indicated a mixture of anomers
with an anomeric ratio of 5:1 a/b. All attempts to
anomerize the mixture to the thermodynamically more
Scheme 3. Reagents and conditions: (i) 2:1 TFA–CH2Cl2 (v/v), 1 h, 89%; (ii) CCl3CN (10 equiv), K2CO3 (5 equiv), CH2Cl2, 12
h, 74%; (iii) (BnO)2P(O)OH (1.3 equiv), CH2Cl2, 1 h, 82%; (iv) H2, Pd–C, 1:1 dioxane–water (v/v), 18 h; (v) 4-morpholine-N,N%-
dicyclohexylcarboxamidinium thymidine 5%-monophosphomorpholidate (18) (1.2 equiv), 1H-tetrazole (3 equiv), pyridine, 5 days.

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S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
stable a anomer using p-toluenesulfonic acid11 were
unsuccessful and this material was used in the next step.
Removal of the protecting groups and conversion of
the azide to an amine was accomplished in one step via
hydrogenation. Thus, compound 16 was stirred under a
hydrogen atmosphere in the presence of Pd–C for 18 h,
and the reaction mixture was maintained at pH 6 by the
addition of 1 M NaOH. The product was purified on a
Dowex 1X8 anion-exchange column (Cl−) which was
eluted with an NaCl gradient. The resulting product
was further purified on a BioGel P2 column (45×2 cm)
to give 17 as a mixture of anomers (5:1 a/b) in 87%
yield.
The final step involved the coupling of glycosyl phos-
phate 17 with the activated nucleoside phosphate,
thymidine 5%-monophosphomorpholidate (18) (Scheme
3) using Wong’s modified Moffat procedure.17 A solu-
tion of 17, 4-morpholine-N,N%-dicyclohexylcarboxami-
dinium thymidine 5%-monophosphomorpholidate and
1H-tetrazole in pyridine was stirred for 14 days.17
Purification of the resulting products using Dowex 1X8
and size-exclusion chromatography on BioGel P2 resin
gave 3 in 36% yield.
2 - (Trimethylsilyl)ethyl 4,6 - O - benzylidene - i -
actopyranoside (7).—2-(Trimethylsilyl)ethyl 2,3,4,6-
tetra-O-acetyl-b- -galactopyranoside (6) (41.9 g, 93.4
D-gal-
D
mmol) was dissolved in MeOH and NaOCH3 (1 g, 18.7
mmol) was added. The reaction mixture was stirred for
18 h after which TLC (eluant 3:2 hexane–EtOAc)
indicated complete conversion to a lower-running
product. The solution was neutralized by the addition
of Dowex 50 (H+), filtered and the filtrate concentrated
under vacuum. The resulting oily residue was dissolved
in CH3CN (200 mL) and benzaldehyde dimethyl acetal
(16.7 mL, 111.2 mmol) was added. Camphorsulfonic
acid was added until the reaction mixture reached pH 3
and the resulting solution was stirred for 5 h, after
which Et3N (3 mL) was added dropwise and the sol-
vents were removed under vacuum. The residue was
redissolved in CH2Cl2 (200 mL) and washed with satd
aq NaHCO3 (3×200 mL), brine (2×200 mL), dried
(MgSO4), filtered, and the filtrate was concentrated
under vacuum. The resulting residue was purified by
column chromatography (eluant 9:1–1:1 hexane–
EtOAc). Concentration of the appropriate fractions
afforded 7 (26.9 g, 72.9 mmol, 79% over two steps) as
a white foam; Rf 0.50 (EtOAc); [h]2D3 −43.6° (c 1,
CHCl3); 1H NMR (500 MHz, CDCl3) lH 7.53–7.32 (m,
5 H, ArH), 5.49 (s, 1 H, PhCH), 4.32 (dd, 1 H, J5,6a
1.2, J6a,6b 12.4 Hz, H-6a), 4.28 (d, 1 H, J1,2 7.4 Hz,
H-1), 4.11 (d, 1 H, J3,4 3.7 Hz, H-4), 4.10–4.04 (m, 2 H,
H-6b, OCHHCH2Si), 3.76 (dd, 1 H, J1,2 7.4, J2,3 9.9
Hz, H-2), 3.68 (dd, 1 H, J3,4 3.7, J2,3 9.9 Hz, H-3),
3.66–3.01 (m, 1 H, OCHHCH2Si), 3.42 (bs, 1 H, H-5),
1.16–0.90 (m, 2 H, OCH2CH2Si), 0.00 (s, 9 H,
(CH3)3Si); 13C NMR (125 MHz, CDCl3) lC 137.4
(ArCq), 128.9, 128.0, 126.3 (ArC), 102.1, 101.2 (C-1,
CHPh), 75.3, 72.5, 71.5, 66.5 (C-2, C-3, C-4, C-5), 69.1,
67.2 (C-6, OCH2CH2Si), 18.2 (OCH2CH2Si), −1.3
(Si(CH3)3). Electrospray-MS: m/z 391.2 [M+Na]+.
Electrospray-HRMS: m/z 391.1544 [M+Na]+, Calcd
for C18H28NaO6Si: 391.1553.
Currently, compound 3 is being utilized as a sub-
strate for putative transaminases and transferases,
which have been expressed from candidate genes in the
acarbose biosynthetic gene cluster, in an attempt to
identify the role, if any, of each enzyme in the forma-
tion of acarbose. The results will be reported separately
in due course.
3. Experimental
General methods.—The 1H and 13C NMR spectra
were recorded on a Bruker AF-300 or AM-500 NMR
spectrometer with MACNMR 5.5 PCI as the instrument
controller and data processor. Low-resolution electro-
spray mass spectra were recorded on a Bruker–Esquire
ion-trap mass spectrometer with electrospray, APCI,
and nanospray ionization sources. A Fison VG Quattro
II electrospray ionization-mass spectrometer was used
to measure the high-resolution mass spectroscopy. Op-
tical rotations were measured on a Jasco DIP-370 digi-
tal polarimeter. All reactions were carried out under an
atmosphere of dry argon in oven dried glassware unless
noted otherwise. Reactions were monitored by TLC
(Silica Gel 60 F254, E. Merck) with detection by UV
light and by charring with H2SO4–MeOH solution. All
chemicals were purchased from Aldrich or Sigma and
used without further purification unless otherwise
noted. Column chromatography was performed on
230–400 mesh silica gel (Aldrich). Dowex 50 and
Dowex 1 were from Aldrich. BioGel P-2 was purchased
from Bio-Rad.
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-4,6-di-O-ben-
zylidene-i-
lyl)ethyl
D
-galactopyranoside
(8).—2-(Trimethylsi-
-galactopyranoside
4,6-di-O-benzylidene-b-
D
(7) (25 g, 67.9 mmol) was dissolved in DMF (300 mL),
and the solution was cooled to 0 °C. Sodium hydride
(10.86 g, 271.6 mmol of 60% suspension) was added
and the resulting suspension was stirred for 15 min.
Benzyl bromide (24.2 mL, 203.6 mmol) was added
dropwise and the reaction mixture was stirred for 5 h.
Methanol (2 mL) was added and the resulting solution
was extracted with ether (3×300 mL). The ether ex-
tracts were combined and washed with brine (2×700
mL), dried (MgSO4), filtered, and the filtrate was con-
centrated under vacuum. The resulting residue was
purified by column chromatography (eluant 19:1–4:1
hexane–EtOAc). Concentration of the appropriate
fractions afforded 8 (33.2 g, 60.5 mmol, 89%) as a white

S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
301
foam; Rf 0.44 (7:3 hexane–EtOAc); [h]2D3 18.2° (c 1,
CHCl3); 1H NMR (500 MHz, CDCl3) lH 7.71–7.32 (m,
15 H, ArH), 5.51 (s, 1 H, PhC(H)O2), 5.03, 4.92 (AB,
2 H, CH2Ph), 4.90–4.84 (m, 2 H, CH2Ph), 4.50 (d, 1 H,
J1,2 7.4 Hz, H-1), 4.38 (d, J6a,6b 12.4 Hz, H-6a), 4.21–
4.18 (m, 2 H, H-4, OCHHCH2Si), 4.10 (d, 1 H, J6a,6b
12.4 Hz, H-6b), 3.92 (dd, 1 H, J1,2 7.4, J2,3 9.3 Hz, H-2),
3.75–3.65 (m, 2 H, H-3, OCHHCH2Si), 3.35 (s, 1 H,
H-5), 1.20–1.14 (m, 2 H, OCH2CH2Si), 0.05 (s, 9 H,
(CH3)3Si); 13C NMR (75 MHz, CDCl3) lC 138.9, 138.4,
137.9, 128.7, 128.1, 127.9, 127.6, 127.4, 126.4, (ArC),
103.1, 101.0 (C-1, CHPh), 79.1, 78.5, 75.0, 73.6, 71.6,
69.1, 67.2, 66.1, 18.4, −1.3. Electrospray-MS: m/z
571.2 [M+Na]+. Electrospray-HRMS: m/z 571.2474
[M+Na]+, Calcd for C32H40NaO6Si: 571.2492.
under vacuum. The resulting residue was purified by
column chromatography (eluant 9:1–7:3 hexane–
EtOAc). Concentration of the appropriate fractions
afforded 10 (22.4 g, 36.4 mmol, 84%) as a clear oil; Rf
0.41 (7:3 hexane–EtOAc); [h]2D3 −5.3° (c 1.0, CHCl3);
1H NMR (300 MHz, CDCl3) lH 7.72 (d, 2 H, J 8.3 Hz,
ArH), 7.32–7.20 (m, 12 H, ArH), 4.85 (1/2 AB, 1 H,
JAB 11.4 Hz, CHHPh), 4.66–4.58 (m, 3 H, CHHPh,
CH2Ph), 4.26 (d, 1 H, J1,2 7.3 Hz, H-1), 4.21 (dd, 1 H,
J5,6a 5.7, J6a,6b 10.4 Hz, H-6a), 4.12 (dd, 1 H, J5,6b 6.8,
J6a,6b 10.4 Hz, H-6b), 3.96–3.86 (m,
1
H,
OCHHCH2Si), 3.85 (d, 1 H, J3,4 3.1 Hz, H-4), 3.59 (dd,
1 H, J5,6a 5.7, J5,6b 6.8 Hz, H-5), 3.55–3.45 (m, 2 H,
H-2, OCHHCH2Si), 3.40 (dd, 1 H, J2,3 9.3, J3,4 3.1 Hz,
H-3), 2.35 (s, 3 H, Ar-CH3), 0.99 (dd, 2 H, J 8.8, 8.3
Hz, OCH2CH2Si), 0.01 (s, 9 H, (CH3)3Si); 13C NMR
(75 MHz, CDCl3) lC 145.7, 139.4, 138.4, 133.4 (4×
ArCq), 130.7–128.4 (ArC), 103.8 (C-1), 80.9, 79.5, 72.4,
67.0 (C-2, C-3, C-4, C-5), 7.9, 73.3, 69.2, 68.3 (C-6,
2×CH2Ph, OCH2CH2Si), 22.4 (Ar-CH3), 19.2
(OCH2CH2Si), −0.6 (Si(CH3)3). Electrospray-MS: m/z
637.0 [M+Na]+. Electrospray-HRMS: m/z 637.2263
[M+Na]+, Calcd for C32H42NaO8SSi: 637.2267.
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-i-
pyranoside (9).—2-(Trimethylsilyl)ethyl 4,6-di-O-ben-
zylidene-2,3-di-O-benzyl-b- -galactopyranoside (8)
D-galacto-
D
(30.1 g, 54.8 mmol) was dissolved in 4:1 AcOH–water
(300 mL) and heated to 50 °C. The reaction mixture
was stirred for 18 h. The mixture was extracted with
CH2Cl2 (5×100 mL) and the organic layers were com-
bined and washed with satd aq NaHCO3 (6×300 mL),
brine (2×300 mL), dried (MgSO4), filtered, and the
filtrate was concentrated under vacuum. The resulting
residue was purified by column chromatography (eluant
4:1–1:1 hexane–EtOAc). Concentration of the appro-
priate fractions afforded 9 (23.6 g, 51.2 mmol, 93%) as
a white solid; Rf 0.26 (1:1 hexane–EtOAc); [h]2D3 1.3° (c
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-6-deoxy-i-
galactopyranoside (11).—A solution of 2-(trimethyl-
silyl)ethyl 2,3-di-O-benzyl-6-O-p-tolylsulfonyl-b-
D-
D
-
galactopyranoside (10) (16.3 g, 26.5 mmol) in THF (200
mL) was cooled to −10 °C, and LiAlH4 (2.21 g, 58.2
mmol) was added. The reaction mixture was stirred for
10 min and subsequently heated to reflux for 18 h. The
resulting suspension was cooled to −10 °C and
quenched by the dropwise addition of EtOAc (50 mL),
CH3OH (50 mL) and satd aq potassium sodium tar-
trate (50 mL). The mixture was extracted with ether
(3×100 mL), the organic layers were combined,
washed with satd aq potassium sodium tartrate (2×
300 mL), brine (2×300 mL), dried (MgSO4), filtered,
and the filtrate was concentrated under vacuum. The
resulting residue was purified by column chromatogra-
phy (eluant 4:1 hexane–EtOAc). Concentration of the
appropriate fractions afforded 11 (9.73 g, 21.9 mmol,
83%) as a clear oil; Rf 0.47 (7:3 hexane–EtOAc); [h]D23
6.4° (c 1, CHCl3); 1H NMR (300 MHz, CDCl3) lH
7.46–7.09 (m, 10 H, ArH), 4.86 (1/2 AB, 1 H, JAB 10.9
Hz, CHHPh), 4.26 (d, 1 H, J1,2 7.8 Hz, H-1), 4.68–4.64
(m, 3 H, CHHPh, CH2Ph), 4.04–3.92 (m, 1 H,
CHHCH2Si), 3.66 (d, 1 H, J3,4 3.1 Hz, H-4), 3.58–3.37
(m, 4 H, H-2, H-3, H-5, CHHCH2Si), 1.27 (d, 3 H, J5,6
6.2 Hz, H-6), 0.97 (dd, 2 H, J 8.8, 8.3 Hz, CH2CH2Si),
0.01 (s, 9 H, (CH3)3Si); 13C NMR (75 MHz, CDCl3) lC
139.6, 138.8 (ArCq), 129.2–128.3 (ArC), 103.9, (C-1),
81.7, 79.7, 70.7, 70.3 (C-2, C-3, C-4, C-5), 75.9, 73.1,
69.9 (2×CH2Ph, OCH2CH2Si), 19.3 (OCH2CH2Si),
17.2 (C-6), −0.6 (Si(CH3)3). Electrospray-MS: m/z
467.24 [M+Na]+. Electrospray-HRMS: m/z 467.2223
[M+Na]+, Calcd for C25H36NaO5Si: 467.2230.
1
0.44, CHCl3); H NMR (300 MHz, CDCl3) lH 7.50–
7.12 (m, 10 H, ArH), 4.87, 4.69 (AB, 2 H, JAB 10.9 Hz,
CH2Ph), 4.68–4.61 (m, 2 H, CH2Ph), 4.32 (d, 1 H, J1,2
7.8 Hz, H-1), 4.03–3.95, 3.88–3.71, (2×m, 4 H, H-4,
H-6a, H-6b, OCHHCH2Si), 3.68–3.50 (m, 2 H, H-2,
OCHHCH2Si) 3.42 (dd, 1 H, J2,3 9.3, J3,4 3.1 Hz, H-3),
3.62 (dd, 1 H, J5,6a 5.2, J5,6b 6.2 Hz, H-5), 0.95 (dd, 2 H,
J 8.8, 8.3 Hz, OCH2CH2Si); 0.01 (s, 9 H, (CH3)3Si); 13C
NMR (75 MHz, CDCl3) lC 139.5, 138.7 (ArCq), 129.2–
128.4 (ArC), 104.2 (C-1), 81.3, 79.9, 74.8, 67.8 (C-2,
C-3, C-4, C-5), 75.9, 73.1, 68.3, 62.6 (C-6, 2×CH2Ph,
OCH2CH2Si), 19.3 (OCH2CH2Si), −0.5 (Si(CH3)3).
Electrospray-MS: m/z 483.2 [M+Na]+. Electrospray-
HRMS: m/z 483.2181 [M+Na]+, Calcd for
C25H36NaO6Si: 483.2179.
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-6-O-(p-tolyl-
sulfonyl)-i-
lyl)ethyl 2,3-di-O-benzyl-b-
D
-galactopyranoside (10).—2-(Trimethylsi-
-galactopyranoside (9) (20
D
g, 43.4 mmol) was dissolved in pyridine (200 mL) and
p-toluenesulfonyl chloride (12.4 g, 65.1 mmol) was
added. The reaction mixture was stirred for 4 days at rt
after which TLC indicated complete conversion to a
product of higher Rf. The solution was concentrated to
50 mL and subsequently diluted with CH2Cl2 (200 mL).
The resulting solution was washed with satd aq
NaHCO3 (5×300 mL), brine (2×300 mL), dried
(MgSO4), filtered, and the filtrate was concentrated

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S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-6-deoxy-4-O-
(p-tolylsulfonyl)-i- -galactopyranoside (12).—2-(Tri-
methylsilyl)ethyl 2,3-di-O-benzyl-6-deoxy-b- -galacto-
MHz, CDCl3) lC 138.3, 137.8 (ArCq), 128.3–127.6
D
(ArC), 102.8 (C-1), 82.6, 82.3, 70.3, 67.7 (C-2, C-3, C-4,
C-5), 75.5, 74.7, 67.5 (2×CH2Ph, OCH2CH2Si), 18.5
(OCH2CH2Si), 18.4 (C-6), −1.4 (Si(CH3)3). Electro-
spray-MS: m/z 492.2 [M+Na]+. Electrospray-HRMS:
m/z 492.2280 [M+Na]+, Calcd for C25H35N3NaO4Si:
492.2295.
D
pyranoside (11) (9 g, 20.2 mmol), was dissolved in
pyridine (200 mL) and p-toluenesulfonyl chloride (19.3
g, 101 mmol), and DMAP (1.24 g, 10.1 mmol) were
added. The mixture was stirred for 18 h at 50 °C after
which TLC indicated complete conversion to a product
of higher Rf. The reaction mixture was concentrated to
50 mL and diluted with CH2Cl2 (300 mL). The resulting
solution was washed with satd aq NaHCO3 (5×300
mL), brine (2×300 mL), dried (MgSO4), filtered, and
the filtrate was concentrated under vacuum. The result-
ing residue was purified by column chromatography
(eluant 4:1 hexane–EtOAc). Concentration of the ap-
propriate fractions afforded 12 (9.48 g, 15.8 mmol,
78%) as a white solid; Rf 0.62 (7:3 hexane–EtOAc);
4-Azido-2,3-di-O-benzyl-4,6-dideoxy-
(14).—2-(Trimethylsilyl)ethyl 4-azido-2,3-di-O-benzyl-
4,6-dideoxy-b- -glucopyranoside (13) (2 g, 4.26 mmol)
D-glucopyranose
D
was dissolved in CH2Cl2 (15 mL), and TFA (30 mL)
was added dropwise over 1 h. The reaction mixture was
stirred for a further 2 h and subsequently diluted with
EtOAc (10 mL) and toluene (20 mL). The solution was
concentrated to 20 mL and toluene (30 mL) was added.
This solution was evaporated to dryness and the residue
taken up in toluene (3×20 mL) and evaporated. The
resulting residue was purified by column chromatogra-
phy (eluant 7:3–1:1 hexane–EtOAc). Concentration of
the appropriate fractions afforded 14 (1.38 g, 3.73
mmol, 89%, a:b 6:4) as a white solid; Rf 0.32 (7:3
hexane–EtOAc); 1H NMR (300 MHz, CDCl3) lH
7.37–7.06 (m, 10 H, ArH), 5.05 (d, 0.6 H, J1a,2 3.1 Hz,
H-1a), 4.88–4.59 (m, 4 H, 2×CH2Ph), 4.57 (d, 0.4 H,
J1b,2 7.3 Hz, H-1b), 3.80–3.69 (m, 1.2 H, H-3a, H-5a),
3.45 (dd, 0.6 H, J1a,2 3.3, J2,3 9.5 Hz, H-2a), 3.43–3.38
(m, 0.4 H, H-3b), 3.30 (dd, 0.4 H, J1b,2 7.3, J2,3 9.3 Hz,
H-2b), 3.19 (dd, 0.4 H, J4,5b 9.9, J5b,6b 5.7 Hz, H-5b),
3.15–2.98 (m, 1 H, H-4a, H-4b), 1.28 (d, 1.2 H, J5b,6b
5.7 Hz, H-6b), 1.20 (d, 1.8 H, J5a,6a 6.2 Hz, H-6a); 13C
NMR (125 MHz, CDCl3) lC 138.7, 138.5, 138.4, 138.2
(ArCq), 129.2–128.6 (ArC), 97.9, 91.8 (C-1), 83.9, 83.4,
80.8, 80.2, 71.4, 68.6, 68.3, 66.9 (C-2, C-3, C-4, C-5),
76.5, 76.4, 75.6, 73.9 (CH2Ph), 19.3, 19.2 (C-6). Electro-
spray-MS: m/z 392.1 [M+Na]+. Electrospray-HRMS:
m/z 392.1598 [M+Na]+, Calcd for C20H23N3NaO4:
392.1586.
1
[h]2D3 71.6° (c 1, CHCl3); H NMR (300 MHz, CDCl3)
lH 7.60 (d, 2 H, ArH), 7.40–7.15 (m, 10 H, ArH), 7.05
(d, 2 H, ArH), 4.93 (d, 1 H, J3,4 2.1 Hz, H-4), 4.78, 4.60
(2 H, AB, J 11.4 Hz, CH2Ph), 4.68, 4.48 (2 H, AB, J
11.9 Hz, CH2Ph), 4.24 (d, 1 H, J1,2 7.3 Hz, H-1),
3.96–3.85 (m, 1 H, OCHHCH2Si), 3.55 (q, 1 H, J 6.2
Hz, H-5), 3.48–3.36 (m, 3 H, H-2, H-3, OCHHCH2Si),
2.28 (s, 3 H, ArCH3), 1.22 (d, 3 H, J5,6 6.2 Hz, H-6),
0.94 (dd, 2 H, J 8.3, 8.8 Hz, OCH2CH2Si), −0.01 (s, 9
H, (CH3)3Si); 13C NMR (75 MHz, CDCl3) lC 144.8,
139.4, 138.6, 135.5 (ArCq), 130.0–128.3 (ArC), 103.9
(C-1), 79.8, 79.6, 79.1, 69.7 (C-2, C-3, C-4, C-5), 75.8,
73.2, 68.4 (2×CH2Ph, OCH2CH2Si), 22.3, 17.9 (C-6,
ArCH3), 19.3 (OCH2CH2Si), −0.6 (Si(CH3)3). Electro-
spray-MS: m/z 621.2 [M+Na]+. Electrospray-HRMS:
m/z 621.2304 [M+Na]+, Calcd for C32H42NaO7SSi:
621.2318.
2-(Trimethylsilyl)ethyl 4-azido-2,3-di-O-benzyl-4,6-
dideoxy-i-
ethyl 2,3-di-O-benzyl-6-deoxy-4-O-(p-tolylsulfonyl)-b-
-galactopyranoside (12) (9 g, 15.0 mmol) was dis-
D
-glucopyranoside (13).—2-(Trimethylsilyl)-
D
4-Azido-2,3-di-O-benzyl-4,6-dideoxy-i-
nosyl trichloroacetimidate (15).—4-Azido-2,3-di-O-ben-
zyl-4,6-dideoxy- -glucopyranose (14) (1.3 g, 3.52
D-glucopyra-
solved in DMF (150 mL) and NaN3 (4.89 g, 75.2
mmol) was added. The reaction mixture was stirred at
70 °C for 3 days after which the solution was cooled to
rt and extracted with ether (3×100 mL). The combined
ether layers were washed with water (3×200 mL),
dried (MgSO4), filtered, and the filtrate was concen-
trated under vacuum. The resulting residue was purified
by column chromatography (eluant 19:1–4:1 hexane–
EtOAc). Concentration of the appropriate fractions
afforded 13 (5.62 g, 12.0 mmol, 80%) as a clear oil; Rf
D
mmol), was dissolved in CH2Cl2 (20 mL), and
trichloroacetonitrile (3.53 mL, 35.2 mmol) was added
followed by anhyd K2CO3 (2.43 g, 17.6 mmol). The
reaction mixture was stirred for 12 h after which TLC
(eluant 7:3 hexane–EtOAc) indicated that the reaction
was complete. The suspension was filtered through a
plug of Celite, and the filtrate was concentrated under
vacuum. The resulting residue was purified by column
chromatography (eluant 9:1–7:3 hexane–EtOAc, plus
0.5% Et3N). Concentration of the appropriate fractions
afforded 15 (1.33 g, 2.59 mmol, 74%) as a clear oil; Rf
0.58 (7:3 hexane–EtOAc); 1H NMR (300 MHz, CDCl3)
lH 8.75 (s, 1 H, NH), 7.45–7.30 (m, 10 H, ArH), 5.79
(d, 1 H, J1,2 7.9 Hz, H-1), 5.03–4.67 (m, 4 H, 2×
CH2Ph), 3.75 (dd, 1 H, J1,2 7.9, J2,3 8.8 Hz, H-2), 3.64
(dd, 1 H, J2,3 8.8, J3,4 9.3 Hz, H-3), 3.45 (dq, 1 H, J4,5
1
0.65 (9:1 hexane–EtOAc); [h]2D3 87.7° (c 1, CHCl3); H
NMR (300 MHz, CDCl3) lH 7.23–7.10 (m, 10 H,
ArH), 4.90, 4.83, 4.68, 4.65 (2×AB, 4 H, 4×
CHHPh), 4.27 (d, 1 H, J1,2 7.8 Hz, H-1), 3.93–3.82 and
3.56–3.43 (2×m, 2 H, OCHHCH2Si), 3.41–3.28 (m, 2
H, H-2, H-3), 3.17–3.00 (m, 2 H, H-4, H-5), 1.26 (d, 3
H, J5,6 6.2 Hz, H-6), 0.96 (dd, 2 H, J 8.3, 8.8 Hz,
OCH2CH2Si), 0.01 (s, 9 H, (CH3)3Si); 13C NMR (75

S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
303
(C-6). Electrospray-MS: negative ion m/z 242.2 [M−
H]-, positive ion m/z 266.0 [M+Na]+. Electrospray-
HRMS: m/z 266.0403 [M+Na]+, Calcd for
C6H14NaNO7: 266.0406.
9.8, J5.6 6.2 Hz, H-5), 3.26 (dd, 1 H, J3,4 9.3, J4,5 9.8 Hz,
H-4), 1.41 (d, 3 H, J5,6 6.2 Hz, H-6); 13C NMR (75
MHz, CDCl3) lC 161.1 (C(CCl3)ꢀNH), 137.7, 137.6
(ArCq), 128.4–127.6 (ArC), 97.9 (C-1), 90.8
(C(CCl3)ꢀNH), 82.7, 81.0, 71.5, 67.3 (C-2, C-3, C-4,
C-5), 75.7, 74.9 (CH2Ph), 18.4 (C-6). Electrospray-MS:
m/z 535.1 [M+Na]+. Electrospray-HRMS: m/z
535.0683 [M+Na]+, Calcd for C22H23Cl3NaN4O4:
535.0683.
Thymidine
5%-diphospho-4-amino-4,6-dideoxy-h-
D-
glucopyranose (3).—4-Amino-4,6-dideoxy-a-
D
-glucopy-
ranosyl phosphate (17) (33 mg, 136 mmol) and 4-
morpholine-N,N%-dicyclohexylcarboxamidinium thymi-
dine 5%-monophosphomorpholidate (186 mg, 272 mmol)
were dissolved in pyridine (1.5 mL) and the resulting
solution was concentrated to dryness. This process was
repeated twice and the residue was dried at rt under
vacuum for 48 h. To the dry residue was added pyridine
(400 mL) and 1H-tetrazole (29 mg, 408 mmol), and the
solution was stirred at rt for 14 days. Water (1 mL) was
added and the solution was concentrated under vac-
uum. The resulting residue was purified by size-exclu-
sion chromatography on Bio-Gel P2 resin, eluting with
250 mM NH4HCO3 to give 3 (26.5 mg, 48.4 mmol, 36%)
as a white solid; Rf 0.39 (2:1 iPrOH–1 M NH4HCO3);
1H NMR (300 MHz, CDCl3) lH 7.50 (s, 1 H, H-6%%),
6.11 (t, 1 H, J 6.7 Hz, H-1%), 5.35 (dd, 1 H, J1,P 6.8, J1,2
3.1 Hz, H-1), 4.42–4.38 (m, 1 H, H-3%), 4.00–3.87 (m, 3
H, H-4%, H-5a%, H-5b%), 3.80 (m, 1 H, H-5), 3.58 (dd, 1
H, J2,3 9.3, J3,4 9.9 Hz, H-3), 3.40–3.31 (m, 1 H, H-2),
2.61 (dd, 1 H, J3,4=J4,5 9.9 Hz, H-4), 2.19–2.09 (m, 2
H, H-2a%, H-2b%), 1.70 (s, 3 H, H-5%%-CH3), 1.09 (d, 3 H,
J5,6 5.7 Hz, H-6). Electrospray-MS: m/z 570.1 [M+
Na]+. Electrospray-HRMS: m/z 570.0845 [M+Na]+,
Calcd for C16H27N3NaO14P2: 570.0866.
4-Azido-2,3-di-O-benzyl-4,6-dideoxy-h-
nosyl dibenzyl phosphate (16).—4-Azido-2,3-di-O-ben-
zyl-4,6-dideoxy-b- -glucopyranosyl trichloroacetimi-
D-glucopyra-
D
date (15) (1.2 g, 2.34 mmol) was dissolved in CH2Cl2
and dibenzyl phosphate (0.84 g, 3.04 mmol) was added.
The reaction mixture was stirred for 1 h, after which
TLC (eluant 7:3 hexane–EtOAc) indicated complete
conversion to a product of lower Rf. The resulting
solution was concentrated under vacuum, and the
residue was purified by column chromatography (eluant
4:1 hexane–EtOAc). Concentration of the appropriate
fractions afforded 16 (1.21 g, 1.92 mmol, 82%) as a
1
clear oil; Rf 0.24 (7:3 hexane–EtOAc); H NMR (500
MHz, CDCl3) lH 7.51–7.25 (m, 20 H, ArH), 5.92 (dd,
1 H, J1,2 3.1, J1,P 6.8 Hz, H-1), 5.18–5.09 (m, 4 H,
CH2Ph), 4.98, 4.84 and 4.82, 4.72 (2×AB, 4 H, 2×
CH2Ph), 3.80, (t, 1 H, J 9.9 Hz, H-3), 3.73 (dq, 1 H, J4,5
9.9, J5,6 6.2 Hz, H-5), 3.66 (dt, 1 H, J 9.9 Hz, 3.1, H-2),
3.18 (t, 1 H, J 9.9 Hz, H-4), 1.26 (d, 3 H, J5,6 6.2 Hz,
H-6). Electrospray-MS: m/z 652.2 [M+Na]+. Electro-
spray-HRMS: m/z 652.2167 [M+Na]+, Calcd for
C34H36N3NaO7P: 652.2189.
4-Amino-4,6-dideoxy-h-
(17).—4-Azido-2,3-di-O-benzyl-4,6-dideoxy-a-
D
-glucopyranosyl phosphate
-gluco-
D
Acknowledgements
pyranosyl dibenzyl phosphate (16) (206 mg, 327 mmol)
was dissolved in 1:1 1,4-dioxane–water (6 mL), and
Pd–C (100 mg, 50% wet) was added. The reaction
mixture was stirred vigorously under an atmosphere of
hydrogen for 18 h while maintaining pH 6 by the
addition of 1 N NaOH. The resulting suspension was
filtered through a plug of Celite and concentrated under
vacuum. The residue was purified on a Dowex 1X8
anion-exchange column (Cl−, 7×1.5 cm) and eluted
with a NaCl gradient (0–700 mmol, 80 mL). The
appropriate fractions were combined and lyophilized to
dryness. The resulting white solid was redissolved in a
minimum volume of water and purified on a Bio-Gel
P2 column (45×2 cm) which was eluted with water to
give 17 (69 mg, 284 mmol, 87%) as a white solid; Rf 0.13
(2:1 iPrOH–1 M NH4HCO3); 1H NMR (500 MHz,
CDCl3) lH 5.22 (dd, 1 H, J1,2 3.6, J1,P 6.7 Hz, H-1),
3.98 (dq, 1 H, J4,5 10.4, J5,6 6.2 Hz, H-5), 3.64 (dd, 1 H,
This work was supported by a grant from Bayer AG,
Germany, and by research grant GM 60662 from the
National Institutes of Health. Acquisition of the Bruker
Esquire ion trap mass spectrometer was supported by
the National Science Foundation through grant No.
9807748.
References
1. Truscheit, E.; Frommer, W.; Junge, B.; Mu¨ller, L.;
Schmidt, D.; Wingender, W. Angew. Chem., Int. Ed.
Engl. 1981, 20, 744–761.
2. Degwert, U.; Hu¨lst, R.; Pape, H.; Herrold, R. E.; Beale,
J. M.; Keller, P. J.; Lee, J. P.; Floss, H. G. J. Antibiot.
1987, 40, 855–861.
3. Mahmud, T.; Tornus, I.; Egelkrout, E.; Wolf, E.; Uy, C.;
Floss, H. G.; Lee, S. J. Am. Chem. Soc. 1999, 121,
6973–6983.
4. Stratmann, A.; Mahmud, T.; Lee, S.-S.; Distler, J.; Floss,
H. G.; Piepersberg, W. J. Biol. Chem. 1999, 274, 10889–
10896.
J
2,3=J3,4 9.9 Hz, H-3), 3.40–3.32 (m, 1 H, H-2), 2.75
(dd, 1 H, J3,4 9.9, J4,5 10.4 Hz, H-4), 1.09 (d, 3 H, J5,6
6.2 Hz, H-6); 13C NMR (125 MHz, CDCl3) lC 97.9
(C-1), 72.4, 69.2, 65.9, 57.7 (C-2, C-3, C-4, C-5), 17.7

304
S.G. Bowers et al. / Carbohydrate Research 337 (2002) 297–304
5. Snipes, C. E.; Brillinger, G.-U.; Sellers, L.; Mascaro, L.;
Floss, H. G. J. Biol. Chem. 1997, 252, 8113–8117.
6. Lee, S.; Sauerbrei, B.; Niggemann, J.; Egelkrout, E. J.
Antibiot. 1997, 50, 954–960.
11. Hoch, M.; Heinz, E.; Schmidt, R. R. Carbohydr. Res.
1989, 191, 21–28.
12. Schmidt, R. R.; Stumpp, M.; Michel, J. Tetrahedron Lett.
1982, 23, 405–408.
7. Hill, J. M.; Jenkins, G. N.; Rush, C. P.; Turner, N. J.;
Willets, A. J.; Buss, A. D.; Dawson, M. J.; Rudd, B. A.
M. J. Am. Chem. Soc. 1995, 117, 5391–5392.
8. Jiang, J.; Biggins, J. B.; Thorson, J. S. Angew. Chem., Int.
Ed. Engl. 2001, 40, 1502–1505.
9. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnus-
son, G. J. Org. Chem. 1988, 53, 5629–5647.
10. Veeneman, G. H.; Broxterman, H. J. G.; van der Marel,
G. A.; van Boom, J. H. Tetrahedron Lett. 1991, 32,
6175–6179.
13. Ichikawa, Y.; Sim, M. M.; Wong, C.-H. J. Org. Chem.
1992, 57, 2943–2946.
14. Yu, K.-L.; Fraser-Reid, B. Tetrahedron Lett. 1988, 29,
979–982.
15. Bannwarth, W.; Trzeciak, A. Hel6. Chim. Acta 1987, 70,
175–186.
16. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnus-
son, G. J. Org. Chem. 1988, 53, 5629–5647.
17. Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62,
2144–2147.