Arabinofuranose disaccharide analogs as inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.tet.2003.10.054

Several octyl 5-O-(α-D-arabinofuranosyl)-α-D-arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis (M.tb.), Mycobacterium avium complex (M

Full text of DOI:10.1016/j.tet.2003.10.054

Tetrahedron 59 (2003) 10239–10248
Arabinofuranose disaccharide analogs as inhibitors of
Mycobacterium tuberculosis
Ashish K. Pathak,a Vibha Pathak,a Manish Kulshrestha,a Darren Kinnaird,a William J. Suling,a
S. S. Gurcha,b Gurdyal S. Besrab and Robert C. Reynoldsa
,
*
aDrug Discovery Division, Southern Research Institute, PO Box 55305, Birmingham, AL 35255, USA
bSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
Received 10 September 2003; revised 16 October 2003; accepted 16 October 2003
Abstract—Several octyl 5-O-(a-D-arabinofuranosyl)-a-D-arabinofuranoside disaccharide analogs substituted at the 5-position of the non-
reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis (M.tb.), Mycobacterium avium complex (MAC)
as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory
activity in the cell free assay as well as against the whole microorganism in vitro.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
have increased treatment noncompliance leading to the
generation of drug resistant strains of M.tb.7
Tuberculosis (TB) is one of the primary killers worldwide,
especially in developing nations, in spite of the availability
of several potent antimycobacterial agents.1 Annually,
millions die worldwide due to this epidemic disease, and
the problem is amplified by the apparent synergism with
HIV. Furthermore, the appearance of multiple drug resistant
(MDR-TB) forms of TB throughout the world hinders its
control, and has raised the concern that this disease may
once again resurface as an incurable disease even in
developed nations.2 MDR-TB is not only fatal; it is very
expensive to treat and has become a significant economic
burden for developing countries. The development of
newer, faster acting and safer anti-tubercular agents as
well as more effective vaccines are urgently needed to fight
this devastating disease.3 In particular, there is a critical
awareness that new classes of drugs with mechanisms of
action that are unique from the presently used anti-TB
agents will be needed to treat drug resistant forms of the
disease.4 In fact, there are few treatment options for patients
infected with the highly intractable forms of MDR-TB.5 TB
chemotherapy relies largely on mycobacterial-specific
drugs inhibiting bacterial metabolism and the biosynthesis
of the cell envelope.6 Presently, the recommended therapy is
six months treatment with the combination of isoniazid
(INH), rifampin (RIF), pyrazinamide (PZA) and ethambutol
(EMB). The prolonged therapy times and drug side effects
The bacterial cell wall has been a robust target for
development of antimycobacterial drugs. For example, the
two clinical antitubercular drugs INH and EMB act
primarily through inhibition of cell wall biosynthesis,
specifically targeting mycolic acid and AG synthesis,
respectively.8,9 The mycobacterial cell wall possesses a
series of complex polysaccharides containing several
unique monosaccharides in well defined linkages.10 Pre-
dominantly, the sugars are mannopyranose (Manp),
rhamnopyranose (Rhap), galactofuranose (Galf) and
arabinofuranose (Araf) containing several different linkages
built into four basic superstructural motifs, lipoarabino-
mannan (LAM), arabinomannan (AM), arabinogalactan
(AG) and linker disaccharide.10 The arabinan portion
present in AM and AG is shown in Figure 1. These
important carbohydrate components are critical for the cell
wall integrity, and alteration at any site can lead to
disturbance in cell wall biosynthesis. On the basis of the
fact that Rhap, Galf and Araf are not found in mammalian
cells, derivatives of structurally relevant saccharides con-
taining these sugars might be acceptors/inhibitors that could
hamper mycobacterial cell wall biosynthesis.
Recently, we have reported the syntheses and anti-
mycobacterial activities of several 6,60-dideoxytrehalose
N,N0-dialkylamino- and 6,60-bis(sulfonamido) analogs that
might possibly interfere with mycolylation pathways in the
mycobacterial cell wall.11 With the goal of developing
novel and selective antitubercular agents with mechanisms
unique from current clinical agents such as EMB that target
Keywords:
Mycobacterium
tuberculosis;
glycosyltransferase;
arabinofuranose; inhibitors.
*
Corresponding author. Tel.: þ1-205-581-2454; fax: þ1-205-581-2877;
e-mail: reynolds@sri.org
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.054

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A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
Figure 1. Arabinan motif present in M. tb. cell wall polysaccharides.
the AG,9 we have also prepared several different disacchar-
ides containing Rhap, Galf and Araf that possess acceptor/
inhibitor capability.12–17 Specifically, octyl disaccharides
of Galf and Araf containing free hydroxyl groups, similar to
natural substrates, have shown acceptor capability with
inhibitor activity, albeit modest, in a cell free assay system.
Partially blocked disaccharides at the non-reducing end
have also shown modest inhibitory activity.12–15 Herein we
have synthesized a small number of disaccharides posses-
sing substitutions at the 5-position of the reducing sugar of
octyl Araf(a1!5)Araf disaccharide template (Fig. 2) with
the intention of probing the active, catalytic site-binding
region of the mycobacterial arabinosyltransferase(s). This
approach is based on the fact that enzymes that synthesize
and utilize Araf are essential to mycobacteria and the
premise that agents specifically designed against these
targets will result in highly selective and potent anti-
tubercular drugs.
2. Result and discussion
Utilization of the a(1!5)-linked Araf–Araf disaccharide
substrates by the appropriate mycobacterial arabinosyl-
transferase would involve the addition of an Araf unit at the
5-position of the non-reducing end of the disaccharide.
Herein, we have prepared 1-O-octyl-Araf(a1!5)Araf
disaccharides (12–25) possessing the general structure as
shown in Figure 2 in order to probe the effects of
modification in the catalytic site-binding region of a typical
substrate disaccharide.
2.1. Approach
In designing a synthetic route for the disaccharide
derivatives with the general structure depicted in Figure 2,
we endeavored to develop a reasonably adaptable and
efficient approach. We have previously synthesized the
disaccharide octyl 5-(a-D-arabinofuranosyl)-a-D-arabino-
furanose 114 (Fig. 3), and we attempted to introduce a
leaving group (tosyl or triflate) at the 5-position of the
reducing end (50-position) to afford structure 2. The amino
group could then be introduced via azide displacement and
reduction. Unfortunately, tosylation resulted in little or no
substitution, and under harsher conditions di- and tri-
substituted products were formed. As a result of the poor
results with tosylation of the disaccharide as an approach for
introduction of the amine function, we decided to
incorporate the incipient amine function as an azido group
into the glycosyl donor first. Hence, the building blocks 8
(the glycosyl donor possessing an azido group at the
Figure 2. General structure of octyl Araf(a 1!5)Araf disaccharide
derivatives.
Figure 3.

A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
10241
5-position) and 11 (the acceptor) were utilized to prepare
disaccharide 12.
(Fig. 3) possessing a free hydroxyl group at the 5-position,
available to undergo glycosylation.15 Utilizing similar
methodology, we prepared acceptor 11 starting from the
earlier synthesized octyl a-D-arabinofuranose 914 as shown
in Scheme 1. First, all the three hydroxyl groups present
were protected with a tert-butyldimethylsilyl (TBDMS)
functionality via the reaction with TBDMSCl in dimethyl-
formamide in the presence of imidazole at room tempera-
ture to afford 10 in 96% yield. The selective deblocking of
the TBDMS group at the 5-position was successfully
achieved in high yields using trifluoroacetic acid in water
(1:1) at 248C. After 4 h of reaction followed by column
chromatographic purification, acceptor 11 was obtained in
74% yield. The a-configuration at the anomeric center of 11
2.1.1. Synthesis of building blocks. We have extensively
used thiocresyl glycosides possessing ester-protecting
groups as glycosyl donors for 1,2-trans glycosylation in
both the galactofuranose and arabinofuranose disaccharide
series.13–15 Utilizing a similar approach we synthesized the
donor sugar 8. The synthesis of both 8 and 11 are outlined in
Scheme 1.
1
was assigned on the basis of the H NMR spectrum that
showed the anomeric proton at 4.79 ppm as a doublet with a
J1,2 coupling constant value of 1.4 Hz.
2.1.1.3. Glycosylation reaction and analog prep-
aration. As outlined in Scheme 2, 1,2-trans glycosylation
was carried out by the reaction between alcohol 11 and
glycosyl donor 8 in presence of N-iodosuccinimide and the
Lewis acid promoter tin(II) triflate in dry dichloromethane.
The reagents were added at 08C, and the reaction was
carried out at rt for 45 min. After column chromatographic
purification, pure disaccharide 12 was obtained in 69%
Scheme 1. Synthesis of building blocks 8 and 11. Reagents and conditions.
(a) TsCl, Py, 08C, 4 h, 49%; (b) NaN3, DMF, 508C, overnight, Ac2O, Py,
overnight, 69%; (c) TBDMSCl, imidazole, DMF, rt, 6 h, 96%; (d) TFA–
water (1:1), THF, 248C, 4 h, 74%.
1
yield. H NMR spectra confirmed the a-glycosylation as
H-10 was observed at 5.14 ppm as a singlet (J1 ,2 ¼0 Hz),
whereas the signal for H-1 was seen at 4.77 ppm with
J1,2¼1.9 Hz. Disaccharide 12 was deacetylated using 7N
NH3/MeOH at room temperature to afford partially blocked
disaccharide 13. The totally deblocked disaccharide 14
possessing an azido group at the 5-position of the non-
reducing end sugar was obtained by desilylating 13 with
tetraethylammonium fluoride in THF in excellent yield. The
1H NMR spectrum showed the H-1 proton as0 a doublet at
4.83 ppm with J1,2¼1.7 Hz, whereas the H-1 proton was
observed as a doublet at 4.95 ppm with J1,2¼1.6 Hz,
confirming the a-anomeric configuration in disaccharide
14. The azido group in disaccharide 14 was reduced to an
amino functionality by reduction with ammonium
formate over 10% Pd/C in methanol at room temperature.
Purification on a short Bio-Beadse SM-4 (20–50 mesh)
column using 5% methanol in water gave the amino
disaccharide 16 in 64% yield. Disaccharide analogs
19–22 (Scheme 3), on the other hand, were prepared via
intermediate disaccharide 15, synthesized from 13. The
disaccharide 13 was treated with TBDMSCl in the presence
of imidazole using DMF as the solvent at 508C to block both
the 2-OH and 3-OH at the reducing end,0which was utilized
further for derivatization at the 5 -position of this
disaccharide.
0
0
2.1.1.1. Building block 8. To access 8 we started with
1-thiocresyl-a-D-arabinofuranose 6 as reported,18 and in a
simple reaction sequence the 5-hydroxyl group was
selectively tosylated with tosyl chloride in pyridine at 08C
to afford 7 in 49% yield. In this reaction, unreacted starting
material 6 was recovered during purification by column
chromatography. The compound 7 was then heated over-
night at 508C with sodium azide in DMF and, after
concentration in vacuo, the crude material was dissolved
in dry pyridine and treated with acetic anhydride at room
temperature. After standard workup and purification by
column chromatography, pure donor 8 was obtained in 69%
yield. The 1H NMR and 13C NMR spectral data showed the
anomeric proton at 5.33 ppm as a doublet possessing
J1,2¼1.8 Hz, and the anomeric carbon appeared at
91.2 ppm respectively supporting the 1,2-trans configur-
ation as compared with sugar derivative 6 reported in
literatue.18
2.1.1.2. Building block 11. In a previous communication,
we described the synthesis of similar arabinofuranosyl
acceptors 3 and 4 used in the synthesis of disaccharide 1
(Fig. 3).14 During the synthesis of arabinofuranose dis-
accharides as photoaffinity probes, however, we have
demonstrated an effective route to prepare acceptor 5
Scheme 2. Glycosylation reaction. Reagents and conditions. (a) NIS, Sn(OTf)2, CH2Cl2, 08C–rt, 45 min, 69%; (b) 7N NH3/MeOH, MeOH, rt, 6 h, 74%;
(c) Et4NþF2, THF, rt, overnight, 96%; (d) TBDMSCl, imidazole, DMF, 508C, overnight, 79%; (e) HCO2NH4, 10% Pd/C, MeOH, rt, 4 h, 64%.

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A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
Scheme 3. 50-substututed Araf(a1!5)Araf disaccharides. Reagents and conditions.(a) C6H11CHO, 10% Pd/C, MeOH, rt, 1 h, 87%; (b) Et4NþF2, THF, rt,
overnight, 18: 96%, 23: 77%, 24: 91%, 25: 95%, 26: 88%; (c) HCO2NH4, 10% Pd/C, MeOH, rt, 2 h, RCOCl/RSO2Cl, N-methyl imidazole, MeOH, 08C,
8–12 h, 19: 86%, 20: 77%, 21: 72%, 22: 73%.
The disaccharide analog 17 was prepared by reductive
amination. Compound 13 was reacted with cyclohexane-
carboxyaldehyde in DMF over 10% Pd/C under an H2
atmosphere to afford analog 17 possessing dicyclohexyl
groups attached at the 50-amino group. The desilylation of
17 gave analog 18 in excellent yield. The 1H NMR and ESI-
MS supported the expected structure as the anomeric
protons H-1 and H-10 were observed at 4.76 ppm as a
doublet J1,2¼1.4 Hz and at 5.01 ppm as a singlet, respect-
ively.0 For the preparation of disaccharide analogs 19–22,
the 5 -azido group in 15 was reduced by reacting with
ammonium formate over 10% Pd/C in methanol. The
resulting amino analog was used in the ensuing coupling
reactions without further purification. In a typical reaction
sequence, the reduced disaccharide was reacted with
1.5 equiv. of the appropriate acid chloride or sulfonyl
chloride in methanol for 2 h. After the usual workup and
purification by column chromatography, pure analogs
19–22 were obtained in good overall yields. The fully
deblocked disaccharide analogs 23–26 were obtained by
desilylating with Et4NþF2 in dry THF followed by column
chromatographic purification.
All new compounds were characterized using 1H NMR and
ESI-MS spectral techniques. The NMR values of mono- and
di-saccharides were compared with the values reported in
the literature.14–19 Whenever needed, NOE, decoupling and
D2O exchange experiments were performed in order to
confirm NMR assignments.
2.2. Biological activity
2.2.1. In vitro activity. The disaccharide analogs were
screened in an in vitro bacterial growth inhibition assay
against M.tb. strain H37Ra.20 The results are tabulated in
Table 1. Compound0 18, possessing dicyclohexylamino
substitution at the 5 -position of the disaccharide, gave
good inhibition with an MIC of 8 mg/mL. This MIC
compares favorably with that of EMB (MIC 2–4 mg/mL),
the frontline antitubercular drug targeting the mycobacterial
arabinan. Interestingly, compound 18 also showed inhibitor
activity against M. avium complex (MAC) NJ211 with an
MIC of 16 mg/mL.
2.2.2. Cell free assay. The fully deblocked disaccharide
analogs were tested in the cell-free enzymatic arabinosyl-
transferase acceptor assay. Based on the previous use of
specific arabinose-based neoglycolipid acceptors14 com-
pounds 14, 16, 18, 23, 24–26 were synthesized and
compared as potential acceptors of [14C]Araf from DP-
[14C]A in the transferase assay.21 Assays performed in the
presence of membranes resulted in [14C]Araf incorporation
from DP-[14C]A only for the control a(1!5)-linked octyl
arabinofuranosyl disaccharide,14 and as predicted com-
pounds blocked at the accepting hydroxyl group on the non-
reducing end of the disaccharide did not show any
detectable acceptor activity. They did, however, have
promising inhibitory activity at a concentration of 3.6 mM
(with control acceptor at 0.4 mM). Further competition-
based experiments established that several compounds were
effective as inhibitors. Specific IC50 values were determined
and are shown in Table 1.
Table 1. Biological data on 50-substituted Araf(a1!5)Araf disaccharides
Compounds
In vitro
against
M. tb. MIC
in mg/mL
Cell Free
assay
IC50
in mM
14
15
.12.8
.12.8
.12.8
.12.8
8
.12.8
.12.8
.12.8
.12.8
.12.8
.12.8
.12.8
.12.8
2–4
2.93
–
–
–
1.56
–
–
–
–
2.49
2.25
3.55
1.82
–
16a
17
18
19
20
21
22
23
24
25
26
Ethambutol
3. Conclusion
a
In intial screening (3.6 mM with control acceptor at 0.4 mM), no
inhibition was observed hence no IC50 was measured.
Within this small set were included basic (amine), poorly

A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
10243
acidic (carboxamide), and relatively acidic (sulfonamide)
groups that may interact with acidic/basic residues within
the catalytic glycosyltransferase active site pocket. Com-
pound 18, possessing a sterically large dicyclohexylamino
substitution at the 50-position of the inhibitor disaccharide,
showed the most promising activity. It is also notable that
there are few reports22–24 showing that substitution of this
site within the catalytic pocket of glycosyltransferases is
tolerated and can lead to inhibitors. Our results verify earlier
data, and suggest that there may be significant steric
tolerance for substitution of the acceptor portion of these
compounds within the catalytic active site. Furthermore,
although it has been hypothesized that substitution at this
position with basic amino groups may be ideal to interact
with COOH functions in the catalytic active site,23 our data
with this particular transferase do not necessarily bear that
out. For example, the amino-substituted disaccharide 16
showed no inhibition in the initial transferase assay.
Although the dicyclohexylmethylamino derivative 18, a
basic substitution, was the most active with an IC50 of
1.56 mM, other analogs (e.g. 26—IC50 1.82 mM) that
contain carboxamides or sulfonamides are comparably
active. Hydrophobicity may play a more important role
for this particular glycosyltransferase in inhibition of these
types of disaccharides than specific interactions within the
catalytic active site. The present study will be useful for the
preparation of second-generation disaccharide analogs as
inhibitors of M.tb.that may better utilize the active site
cavity and take advantage of specific chemistry therein.
(4.47 g, 23.44 mmol) was added. The reaction mixture was
stirred overnight at 08C and 4 h at rt. The reaction mixture
was poured into an ice-water mixture and extracted with
CHCl3 (2£75 mL). The organic layer was dried over
Na2SO4 and concentrated to a syrup. Column chromato-
graphy using CHCl3/MeOH (9:1) as the eluant of the crude
product gave purified compound 7 (3.92 g, 49%) as a
1
colorless oil. MS-ESI (LiCl): m/z 417 [MþLi]þ. H NMR
(CDCl3): d 7.77 (2H, dd, J¼1.8, 6.6 Hz, Ar), 7.35–7.29
(4H, m, Ar), 7.09 (2H, d, J¼7.9 Hz), 5.20 (1H, d, J¼2.6 Hz,
H-1), 4.23 (2H, d, J¼3.1 Hz, H2-5), 4.13–4.10 (3H, m, H-2,
H-3, H-4), 2.42 (3H, s, CH3), 2.32 (3H, s, CH3). 13C NMR
(CDCl3): d 145.09, 137.69 (C), 132.24 (2£CH), 132.18 (C),
129.86, 129.68 (2£CH), 129.59 (C), 127.91 (2£CH), 91.14
(C-1), 81.21 (C-2), 79.54 (C-3), 76.46 (C-4), 68.62 (C-5),
21.57 (CH3), 21.04 (CH3).
4.1.2. 1,5-Dideoxy-2,3-di-O-acetyl-5-azido-1-thiocresyl-
(3.83 g,
a-D-arabinofuranoside (8). Compound
7
9.34 mmol) was dissolved in dry DMF (20 mL) and NaN3
(911 mg, 14.01 mmol) was added. The reaction mixture was
heated at 508C overnight, and was then concentrated in
vacuo to a syrup. The syrup was dissolved in dry pyridine
(15 mL) and Ac2O (1.98 mL, 21.00 mmol) was added. The
reaction mixture was stirred overnight at rt and poured into
an ice-water mixture. It was extracted with CHCl3
(2£75 mL), the organic layer was dried over Na2SO4 and
concentrated to a syrup. Column chromatography on silica
gel using CHCl3/MeOH (99:1) gave pure compound 8 as a
colorless oil (2.15 g, 69%). MS-ESI: m/z 388.21 [MþNa]þ.
1H NMR (CDCl3): d 7.42–7.39 (2H, m, Ar), 7.14 (2H, d,
J¼7.9 Hz), 5.33 (1H, d, J1,2¼1.8 Hz, H-1), 5.28 (1H, t,
J1,2¼J2,3¼1.8 Hz, H-2), 5.07–5.04 (1H, m, H-3), 4.46–4.42
(1H, m, H-4), 3.67 (1H, dd, J4,5a¼3.0 Hz, J5a,5b¼13.3 Hz,
H-5a), 3.49 (1H, dd, J4,5b¼4.9 Hz, J5a,5b¼13.3 Hz, H-5b),
2.34 (3H, s, CH3), 2.13, 2.12 (3H each, s, 2£OCH3). 13C
NMR (CDCl3): d 170.16, 169.67 (2£CvO), 138.12 (C),
132.67, 129.84 (4£CH), 129.48 (C), 91.20 (C-1), 81.65
(C-4), 81.48 (C-2), 77.80 (C-3), 51.12 (C-5), 21.11 (CH3),
20.72 (2£OCH3).
4. Experimental
4.1. Synthesis
All reactions were performed under a dry argon atmosphere
and reaction temperatures were measured externally.
Anhydrous solvents from Aldrich were used in the reactions
as such. Whenever necessary, compounds and starting
materials were dried by azeotropic removal of water with
toluene under reduced pressure. In the case of Et4NþF2,
however, this material was purchased from Aldrich as the
hydrated form and was used as such. Reactions were
monitored by thin-layer chromatography (TLC) on pre-
coated E. Merck silica gel (60F254) plates (0.25 mm) and
visualized using UV light (254 nm) and/or heating after
spray with (NH4)2SO4 solution (150 g ammonium sulfate,
30 mL H2SO4, 750 mL H2O). All solvents used for workup
and chromatography were reagent grade from Fisher
Scientific. Flash chromatography was carried out on Fischer
silica gel 60 (230–400 mesh). Melting points were
determined with a Mel-Temp II capillary melting points
apparatus and is uncorrected. 1H and 13C NMR spectra were
recorded on Nicolet NT 300NB instrument at 300 MHz and
75 MHz respectively. Coupling constants (J) are reported in
Hz and chemical shifts are in ppm (d) relative to residual
solvent peak or internal standard. ESI-MS was recorded on a
BioTof-2 time-of-flight mass spectrometer.
4.1.3. Octyl 2,3,5-tri-O-tert-butyldimethylsilyl-a-D-ara-
binofuranoside (10). Octyl a-D-arabinofuranoside 914
(2.00 g, 7.63 mmol) was dissolved in dry DMF (10 mL)
and TBDMSCl (4.01 g, 26.71 mmol) was added followed
by imidazole (1.56 g, 22.89 mmol). The reaction mixture
was stirred at rt for 6 h and was poured into an ice-water
mixture. Extraction with CHCl3 (3£50 mL) followed by
drying on Na2SO4 and concentration gave the crude product
as a syrup. Column chromatography on silica gel using
cyclohexane/EtOAc (96:4) gave pure compound 10 as a
colorless oil (4.43 g, 96%). MS-ESI: m/z 627.38 [MþNa]þ.
1H NMR (CDCl3): d 4.74 (1H, d, J1,2¼1.6 Hz, H-1), 4.00
(1H, dd, J1,2¼1.6 Hz, J2,3¼3.5 Hz, H-2), 3.96 (1H, dd,
J2,3¼3.5 Hz, J3,4¼5.7 Hz, H-3), 3.91–3.86 (1H, m, H-4),
3.77–3.63 (3H, m, H2-5, OCH2), 3.37–3.30 (1H, m,
OCH2), 1.56–1.51 (2H, m, CH2), 1.27 (10H, br s,
5£CH2), 0.90–0.86 (30H, m, 10£CH3), 0.082, 0.077,
0.072, 0.064 (s, 6£CH3).
4.1.1. 1-Deoxy-1-thiocresyl-5-tosyl-a-D-arabinofurano-
side (7). 1-Deoxy-1-thiocresyl-a-D-arabinofuranoside 618
(5.00 g, 19.53 mmol) was dissolved in dry pyridine
(20 mL), cooled to 08C under argon, and tosyl chloride
4.1.4. Octyl 2,3-di-O-tert-butyldimethylsilyl-a-D-ara-
binofuranoside (11). Compound 10 (4.00 g, 6.62 mmol)
was dissolved in dry THF (20 mL) and cooled to 248C. Ten

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A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
(10) mL of a TFA–water mixture (1:1) was added, and the
reaction mixture was stirred at 248C for 4 h. The reaction
mixture was then poured into an ice-water mixture and
extracted with CHCl3 (2£50 mL). The organic layer was
dried over Na2SO4 and concentrated to a syrup. Column
chromatography on silica gel using cyclohexane/EtOAc
(95:5) gave pure compound 11 as a colorless oil (2.65 g,
74%). MS-ESI: m/z 514.34 [MþNa]þ. 1H NMR (CDCl3): d
4.79 (1H, d, J1,2¼1.4 Hz, H-1), 4.06–3.94 (3H, m, H-2, H-3,
H-4), 3.82 (1H, ddd, J4,5a¼2.3 Hz, J5a,5-OH¼4.5 Hz,
J5a,5b¼12.1 Hz, H-5a), 3.73–3.60 (2H, m, H-5b, OCH2),
3.39–3.31 (1H, m, OCH2), 2.01 (1H, dd, J5a,5-OH¼4.5 Hz,
J5b,5-OH¼7.5 Hz, 5-OH), 1.59–1.52 (2H, m, CH2), 1.27
(10H, br s, 5£CH2), 0.91–0.87 (21H, m, 7£CH3), 0.102,
0.092, 0.087, 0.069 (each 3H, s, 4£CH3).
4.1.7. Octyl 5-O-(5-deoxy-5-azido-a-D-arabinofurano-
syl)-a-D-arabinofuranoside (14). Compound 13 (50 mg,
0.06 mmol) was dissolved in dry THF (4 mL) and Et4NþF2
(52 mg, 0.34 mmol) was added. The reaction mixture was
stirred overnight and concentrated to a syrup. Column
chromatography (CHCl3/MeOH, 8:1) gave compound 14 as
a colorless oil (23 mg, 96%). MS-ESI: m/z Found 442.2176
[MþNa]þ, calcd 442.2159 for C18H33N3O8. 1H NMR
0
0
0
(CD3OD): d 4.95 (1H, dd, J1 ,2 ¼1.6 Hz, H-1 ), 4.83 (1H,
0
0
0
0
d, J1,2¼1.7 Hz, H-1), 4.04 (1H, ddd, J4 ,5 a¼3.5 Hz, J4 ,5 b
¼
0
0
0
0
0
6.2 Hz, J3 ,4 ¼6.60Hz, H-4 ), 4.00 (1H, dd, J1 ,2 ¼1.6 Hz,
0
0
J2 ,3 ¼3.8 Hz, H-2 ), 4.03–3.99 (1H, m, H-4), 3.94 (1H, dd,
J1,2¼1.7 Hz, J2,3¼03.9 Hz, H-2), 3.87 (1H, dd, J2 ,3 ¼3.8 Hz,
J3 ,4 ¼6.6 Hz, H-3 ), 3.86–3.79 (2H, m, H-3, H-5a), 3.73–
0
0
0
0
3.65 (1H, m, OCH2), 3.65 (1H, dd, J4,5b¼3.6 Hz, J5a,5b
¼
0
0
0
0
11.1 Hz, H-5b), 3.50 (1H, dd, J4 ,5 a¼3.3 Hz, J5 a,5 b¼13.3
4.1.5. Octyl 5-O-(5-deoxy-5-azido-2,3-di-O-acetyl-a-D-
arabinofuranosyl)-2,3-di-O-tert-butyldimethylsilyl-a-D-
arabinofuranoside (12). Compound 11 (2.24 g,
Hz, H-50a), 3.44–3.36 (2H, m, OCH2), 3.37 (1H, dd,
J4 ,5 b¼6.2 Hz, J5 a,5 b¼13.3 Hz, H-50b), 1.62–1.53 (2H,
m, CH2), 1.30 (10H, br s, 5£CH2), 0.92–0.87 (3H, m,
CH3).
0
0
0
0
˚
4.57 mmol), activated powdered 4 A molecular sieves
(800 mg), and glycosylation donor 9 (2.00 g, 5.48 mmol)
in dry CH2Cl2 (25 mL) were cooled at 08C under an Argon
atmosphere. The mixture was stirred for 15 min, and NIS
(1.23 g, 5.48 mmol) followed by Sn(OTf)2 (287 mg,
0.69 mmol) were added to initiate coupling. The reaction
mixture was allowed to stir for 30 min at rt, and the reaction
was quenched by addition of Et3N (1.5 mL), diluted with
CH2Cl2 (50 mL) and filtered through a celite pad. The
filtrate was washed with 10% Na2S2O3 (2£15 mL),
followed by washing with saturated aqueous NaHCO3
(25 mL). The organic layer was dried over Na2SO4, the
solvent was removed in vacuo, and the residue was purified
by column chromatography (cyclohexane/EtOAc 3:1) to
give disaccharide 12 as a colorless oil (2.31 g, 69%). MS-
ESI: m/z 754.32 [MþNa]þ. 1H NMR (CDCl3): d 5.17 (1H,
4.1.8. Octyl 5-O-(5-deoxy-5-azido-2,3-di-O-tert-butyl-
dimethylsilyl-a-D-arabinofuranosyl)-2,3-di-O-tert-butyl-
dimethylsilyl-a-D-arabinofuranoside (15). Compound 13
(670 mg, 1.03 mmol) was dissolved in dry DMF (10 mL)
and TBDMSCl (390 mg, 2.60 mmol) was added followed
by imidazole (280 mg, 4.12 mmol). The reaction mixture
was stirred overnight at 508C and was then poured into an
ice-water mixture. Extraction with CHCl3 (3£15 mL)
followed by drying on Na2SO4 and concentration gave the
crude product as a syrup. Column chromatography on silica
gel using cyclohexane/EtOAc (95:5) gave pure compound
15 as a colorless oil (637 mg, 79%). MS-ESI: m/z Found
898.5594 [MþNa]þ, calcd 898.5618 for C42H89N3O8Si4. 1H
0
0
0
NMR (CDCl3): d 4.86 (1H, dd, J1 ,2 ¼1.8 Hz, H-1 ), 4.74
0
0
0
0
0
0
d, J2 ,3 ¼1.3 Hz, H-2 ), 5.14 (1H, s, H-1 ), 4.95 (1H, dd,
(1H, d, J1,2¼1.90Hz, H-1), 4.08 (1H, dd, J1 ,2 ¼1.8 Hz,
0
0
0
0
0
0
0
0
J2 ,3 ¼1.3 Hz, J3 ,4 ¼4.8 Hz, H-3 ), 4.77 (1H, d, J1,2¼1.9 Hz,
J2 ,3 ¼4.0 Hz, H-2 ), 4.05–3.99 (1H, m, H-4 ), 4.00 (1H, dd,
J1,2¼1.9 Hz, J2,3¼4.0 Hz, H-2), 3.98–3.90 (3H, m, H-3,
H-4, H-30), 3.79 (1H, dd, J4,5a¼4.5 Hz, J5a,5b¼11.0 Hz,
0
0
0
0
H-1), 4.22 (1H, 0 ddd, J4 ,5 a¼3.0 Hz, J3 ,4 ¼4.8 Hz,
3.7 Hz, H-2), 3.99–3.92 (2H, m, H-3, H-4), 3.80 (1H, dd,
0
0
J4 ,5 b¼4.9 Hz, H-4 ), 4.01 (1H, dd, J1,2¼1.9 Hz, J2,3
¼
H-5a), 3.72–3.65 (1H, m, OCH2), 3.56 (1H, dd, J4,5b
¼
0
0
J4,5a¼4.1 Hz, J5a,5b¼11.0 Hz, H-5a), 3.72–3.60 (3H, m,
3.1 Hz, J5a,5b¼11.0 Hz, H-5b), 3.48 (1H, dd, J4 ,5 a¼3.3 Hz,
H-5b, H-50a, OCH2), 3.44 (1H, dd, J4 ,5 b¼4.9 Hz, J5 a,5 b
¼
J5 a,5 b¼13.2 Hz, H-50a), 3.36–3.26 (2H, m, H-50b, OCH2),
1.60–1.50 (2H, m, CH2), 1.27 (10H, br s, 5£CH2), 0.91–
0.86 (39H, m, 13£CH3), 0.097, 0.093, 0.083, 0.077, 0.074,
0.064 (each s, 8£CH3).
0
0
0
0
0 0
13.2 Hz, H-50b), 3.37–3.30 (1H, m, OCH2), 4.77, 3.96 (each
3H, s, 2£OCH3), 2.09 (2H, m, CH2), 2.08 (10H, br s,
5£CH2), 1.69–1.27 (21H, m, 7£CH3), 0.897, 0.879, 0.857
(each s, 4£CH3).
4.1.9. Octyl 5-O-(5-deoxy-5-amino-a-D-arabinofurano-
syl)-a-D-arabinofuranoside (16). Compound 15 (30 mg,
0.07 mmol) was dissolved in MeOH (5 mL) and 10% Pd/C
(15 mg) was added followed by HCO2NH4 (18 mg,
0.29 mol). The reaction mixture was stirred 4 h at rt and
filtered through celite. The solvent was evaporated and the
resulting syrup was purified by passing the aqueous solution
(syrup in 5 mL) through a small column of Bio-Beadse
SM-4 (20–50 mesh) and eluting with H2O–MeOH (5%) to
yield compound 16 as a colorless oil (18 mg, 64%). MS-
ESI: m/z Found 416.2234 [MþNa]þ, calcd 416.2254 for
C18H35NO8. 1H NMR (MeOH-d4): d 4.98 (1H, s, H-10), 4.83
(1H, d, J1,2¼1.7 Hz, H-1), 4.06–3.98 (3H, m, H-4, H-20
H-40), 3.95 (1H, dd, J1,2¼1.7 Hz, J2,3¼3.7 Hz, H-2), 3.89–
3.80 (2H, m, H-3, H-5a), 3.76–72 (1H, m, H-30), 3.72–3.64
(2H, m, H-5b, OCH2), 3.44–3.36 (2H, m, OCH2), 3.12–
4.1.6. Octyl 5-O-(5-deoxy-5-azido-a-D-arabinofurano-
syl)-2,3-di-O-tert-butyldimethylsilyl-a-D-arabinofurano-
side (13). To a solution of compound 12 (2.00 g,
2.73 mmol) in dry methanol (20 mL) was added 7N NH3/
MeOH (50 mL). The reaction mixture was stirred at room
temperature for 6 h and concentrated in vacuo to a syrup.
Chromatography on silica gel using cyclohexane/EtOAc
(9:1) gave pure compound 13 as a colorless oil (1.31 g,
1
74%). MS-ESI: m/z 670.44 [MþNa]þ. H NMR (CDCl3,
D2O exchanged): d 5.08 (1H, s, H-10), 4.76 (1H, d, J1,2
¼
0
0
0
0
1.3 Hz, H-1), 4.17 (1H, ddd, J4 ,5 a¼2.8 Hz, J3 ,4 ¼3.7 Hz,
J4 ,5 b¼3.8 Hz, H-40), 4.08 (1H, br s, H-20), 4.03–3.97 (2H,
m, H-2, H-4), 3.87–3.78 (3H, m, H-3, H-5a, H-30), 3.67–3.59
(4H, m, H-5b, H2-50, OCH2), 3.53–3.28 (1H, m, OCH2),
1.60–1.50 (2H, m, CH2), 1.27 (10H, br s, 5£CH2), 0.90–0.86
(21H, m, 7£CH3), 0.093, 0.081, 0.058 (each s, 4£CH3).
0
0
3.06 (1H, m, H-50a), 2.94 (1H, dd, J4 ,5 b¼7.7 Hz,
0
0

A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
10245
J5 a,5 b¼13.1 Hz, H-50b), 1.62–1.53 (2H, m, CH2), 1.30
(10H, br s, 5£CH2), 0.92–0.87 (3H, m, CH3).
evaporated, and the resulting syrup was used as such for
further reaction. This crude amino disaccharide was
dissolved in 2 mL of dry dichloromethane and cooled to
08C. To it was added pivaloyl chloride (10 mL, 0.09 mmol),
N-methylimidazole (10 mL, 0.12 mmol), and the reaction
mixture was stirred for 8 h at 08C. The reaction mixture was
poured into an ice-water mixture and extracted with CHCl3
(2£10 mL). The organic layer was dried over Na2SO4 and
concentrated to syrup. Column chromatography using
cyclohexane/EtOAc (98:2) afforded purified 19 (55 mg,
96%) as an oil. MS-ESI: m/z Found 956.2284 [MþNa]þ,
0
0
4.1.10. Octyl 5-(5-deoxy-5-N-dicylohexyl-a-D-arabino-
furanosyl)-2,3-di-O-tert-butyldimethylsilyl-a-D-arabino-
furanoside (17). Compound 13 (50 mg, 0.07 mmol) was
dissolved in MeOH (3 mL) and 10% Pd/C (20 mg) was
added. Cyclohexane carboxyaldehyde (6 mL, 0.07 mmol)
was added, and the reaction mixture was stirred at rt under
H2 atmosphere for 1 h. Filtration through a celite pad and
concentration gave a viscous oil. Purification by column
chromatography on silica gel (cyclohexane/EtOAc, 3:1)
afforded 17 as a colorless oil (55 mg, 87%). MS-ESI: m/z
Found 814.6022 [MþH]þ, calcd 814.6042 for
1
calcd 956.6289 for C47H99NO9Si4. H NMR (CDCl3): d
5.95 (1H, t, J¼5.3 Hz, NH), 4.84 (1H, s, H-10), 4.74 (1H, d,
0
0
0
0
J1,2¼1.8 Hz, H-1), 4.08 (1H, dd, J1 ,2 ¼1.1 Hz, J2 ,3
¼
0
1
C44H87NO8Si2. H NMR (CDCl3): d 5.01 (1H, s, H-1 ),
4.76 (1H, d, J1,2¼1.4 Hz, H-1), 4.17 (1H, br s, H-40), 4.03–
3.97 (3H, m, H-2, H-4, H-20), 3.83–3.76 (3H, m, H-3, H-5a,
2.9 Hz, H-20), 4.01–3.95 (4H, m, H-2, H-3,0 H-4, H-40),
0
0
0
0
3.76 (1H, dd, J2 ,3 ¼2.9 Hz, J3 ,4 ¼6.6 Hz, H-3 ), 3.74 (1H,
dd, J4,5a¼3.7 Hz, J5a,5b¼11.3 Hz, H-5a), 3.73–3.64 (1H, m,
OCH2), 3.58–3.53 (1H, m, H-5b), 3.53–3.49 (2H, m, H2-50),
1.55 (1H, m, OCH2), 1.60–1.52 (2H, m, CH2), 1.30–1.25
(10H, m, 5£CH2), 1.20 (9H, s, 3£CH3), 0.89–0.87 (39H, m,
13£CH3), 0.093, 0.084, 0.076, 0.066 (each s, 8£CH3).
H-30), 3.68–3.58 (1H, m, OCH2), 3.59 (1H, dd, J4,5b
¼
3.2 Hz, J5a,5b¼10.4 Hz, H-5b), 3.35–3.28 (1H, m, OCH2),
2.71–2.60 (2H, m, H2-50), 2.44 (2H, dd, J¼7.7, 12.6 Hz,
NCH2), 2.16 (2H, dd, J¼5.2, 12.6 Hz, NCH2), 1.87–1.60
0
(4H, m, cyclohexyl CH2 s), 1.57–1.50 (10H, m, cyclo-
hexyl), 1.46–1.41 (2H, m, CH2), 1.37 (10H, br s, 5£CH2),
1.20–1.08 (8H, m, cyclohexyl), 0.90–0.86 (21H, m,
7£CH3), 0.09, 0.07, 0.06 (each s, 4£CH3). 13C NMR
(CDCl3): d 108.20 (C-1), 107.69 (C-10), 87.17 (C-40), 84.27
(C-2), 81.72 (C-4), 79.70, 79.66 (C-3, C-30), 78.00 (C-200),
67.67 (OCH2), 65.74 (C-5), 64.79 (2£CH2N), 58.25 (C-5 ),
35.70 (CH), 32.34, 32.04 (4£CH2), 31.80, 29.622, 29.35,
29.22 (4£CH2), 26.45, 26.17, 26.15, 26.05 (7£CH2), 25.81,
25.67 (6£CH3), 22.62 (CH2), 17.86, 17.79 (2£C), 14.06
(CH3), 24.28, 24.59, 24.70, 24.86 (4£CH3).
4.1.13. Octyl 5-(5-deoxy-5-phenylamido-2,3-di-O-tert-
butyldimethylsilyl-a-D-arabinofuranosyl)-2,3-di-O-tert-
butyldimethylsilyl-a-D-arabinofuranoside (20). Com-
pound 14 (60 mg, 0.07 mmol) was treated with HCO2NH4
(17 mg, 0.27 mmol) in MeOH (5 mL) over 10% Pd/C
(40 mg) followed by reaction with benzoyl chloride (10 mL,
0.09 mmol), N-methylimidazole (10 mL, 0.12 mmol) in
CH2Cl2 (4 mL) as described earlier for the preparation of
19. Purification by column chromatography (cyclohexane/
EtOAc, 98:2) yielded 20 (50 mg, 77%) as an oil. MS-ESI:
m/z Found 976.5971 [MþNa]þ, calcd 976.5976 for
C49H95NO9Si4. 1H NMR (CDCl3): d 8.07–8.05, 7.78–
7.75, 7.51–7.38 (each m, Ar), 6.44 (1H, t, J¼5.3 Hz, NH),
4.91 (1H, s, H-10), 4.73 (1H, d, J1,2¼1.9 Hz, H-1), 4.15–
4.1.11. Octyl 5-(5-deoxy-5-N-dicylohexyl-a-D-arabino-
furanosyl)-a-D-arabinofuranoside (18). Compound 17
(50 mg, 0.06 mmol) was treated with Et4NþF2 (28 mg,
0.18 mmol) in dry THF (3 mL) as described for the
preparation of 13. Purification by column chromatography
(CHCl3/MeOH, 9:1) yielded 18 (20 mg, 96%) as an oil. MS-
ESI: m/z Found 586.4305 [MþNa]þ, calcd 586.4313 for
4.11 (1H, m, H-40), 4.09 (1H, dd, J1 ,2 ¼0.9 Hz, J2 ,3
¼
0
0
0
0
2.6 Hz, H-20), 4.01 (1H, dd, J1,2¼1.9 Hz, J2,3¼4.2 Hz, H-2),
0
3.99–3.95 (2H, m, H-3, H-4), 3.87 (1H, dd, J2 ,3 ¼2.6 Hz,
0
00
0
0
J3 ,4 ¼5.7 Hz, H-3 ), 3.79–3.64 (3H, m, H-5a, H2-5 , OCH2),
3.50 (1H, dd, J4,5b¼3.2 Hz, J5a,5b¼11.2 Hz, H-5b), 3.34–
3.27 (1H, m, OCH2), 1.55–1.50 (2H, m, CH2), 1.27 (10H,
m, 5£CH2), 0.89–0.84 (39H, m, 13£CH3), 0.106, 0.084,
0.077, 0.072, 0.064, 0.052 (each s, 8£CH3).
0
1
C32H59NO8. H NMR (CDCl3): d 4.99 (2H, s, H-1, H-1 ),
4.20–4.14 (2H, m, H-4, H-40), 4.00–3.94 (4H, m, H-2, H-3,
H-30, H-5a), 3.88 (1H, br s, H-2), 3.76–3.67 (1H, m, OCH2),
3.69 (1H, dd, J4,5b¼3.3 Hz, J5a,5b¼10.0 Hz, H-5b), 3.47–
3.39 (1H, m, OCH2), 2.66 (2H, d, J¼3.5 Hz, H2-50), 2.39
(2H, dd, J¼7.2, 12.7 Hz, NCH2), 2.19 (2H, dd, J¼5.7,
12.7 Hz, NCH2), 1.83–1.63 (14H, m, cyclohexyl), 1.60–
1.54 (2H, m, CH2), 1.28 (10H, br s, 5£CH2), 1.20–1.11 (8H,
m, cyclohexyl), 0.90–0.86 (3H, m, CH3). 13C NMR
(CD3OD): d 109.52 (C-1, C-10), 83.72, 83.66, 83.47, 83.19
(C-2, C-4, C-20, C-30), 81.42 (C-40), 79.48 (C-3), 68.95
(OCH2), 68.17 (C-5), 64.44 (2£CH2N), 59.28 (C-50), 37.57
(2£CH), 33.08 (4£CH2), 33.02 (CH2), 30.72 (CH2), 30.51
(CH2), 30.43 (CH2), 28.07 (2£CH2), 27.33 (4£CH2), 27.29
(CH2), 23.72 (CH2), 14.43 (CH3).
4.1.14. Octyl 5-(5-deoxy-5-ethylsulfonamido-2,3-di-O-
tert-butyldimethylsilyl-a-D-arabinofuranosyl)-2,3-di-O-
tert-butyldimethylsilyl-a-D-arabinofuranoside
(21).
Compound 14 (60 mg, 0.07 mmol) was treated with
HCO2NH4 (17 mg, 0.27 mmol) in MeOH (5 mL) over
10% Pd/C (40 mg) followed by reaction with ethanesulfonyl
chloride (8 mL, 0.09 mmol), N-methylimidazole (10 mL,
0.12 mmol) in CH2Cl2 (4 mL) as described earlier for the
preparation of 19. Purification by column chromatography
(CHCl3/MeOH, 9:1) afforded 21 (50 mg, 77%) as an oil.
MS-ESI: m/z Found 964.5646 [MþNa]þ, calcd 964.5645
for C44H95NO10SSi4. 1H NMR (CDCl3): d 4.87 (1H, s,
H-10), 4.74 (1H, d, J1,2¼1.9 Hz, H-1), 4.59 (1H, t, J¼5.5 Hz,
4.1.12. Octyl 5-(5-deoxy-5-tert-butylamido-2,3-di-O-tert-
butyldimethylsilyl-a-D-arabinofuranosyl)-2,3-di-O-tert-
butyldimethylsilyl-a-D-arabinofuranoside (19). Com-
pound 14 (60 mg, 0.07 mmol) was dissolved in MeOH
(5 mL) and 10% Pd/C (40 mg) was added followed by
HCO2NH4 (17 mg, 0.27 mmol). The reaction mixture was
stirred 2 h at rt and filtered through celite. The solvent was
NH), 4.08–4.03 (1H, m, H-40), 4.07 (1H, dd, J1 ,2 ¼1.2 Hz,
0
0
0
H-2), 3.97–3.91 (2H, m, H-3, H-4), 3.90 (1H, dd, J2 ,3
0
0
J2 ,3 ¼2.7 Hz, H-2 ), 4.00 (1H, dd, J1,2¼1.9 Hz, J2,3¼3.5 Hz,
0
0
¼
0
0
0
2.7 Hz, J3 ,4 ¼5.5 Hz, H-3 ), 3.73 (1H, dd, J4,5a¼4.3 Hz,
J5a,5b¼11.2 Hz, H-5a), 3.72–3.64 (1H, m, OCH2), 3.57 (1H,

10246
A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
dd, J4,5b¼3.2 Hz, J5a,5b¼11.2 Hz, H-5b), 3.42–3.35 (1H, m,
H-50a), 3.35–3.27 (1H, m, OCH2), 3.24 (1H, dd, J4 ,5 b
J2,3¼3.7 Hz, J3,4¼5.7 Hz, H-3), 3.85–3.81 (2H, m, H-30,
H-50a), 3.71–3.63 (4H, m, H-50b, H2-5, OCH2), 3.42–3.35
(1H, m, OCH2), 1.59–1.54 (2H, m, CH2), 1.29 (10H, m,
5£CH2), 0.91–0.87 (3H, m, CH3).
0
0
¼
5.7 Hz, J5 a,5 b¼13.0 Hz, H-50b), 3.05(2H, dd, J¼7.4,14.7 Hz,
CH2), 1.56–1.50 (2H, m, CH2), 1.37 (3H, t, J¼7.4 Hz, CH3),
1.27 (10H, m, 5£CH2), 0.90–0.86 (39H, m, 13£CH3), 0.113,
0.105, 0.092, 0.083, 0.075, 0.071 (each s, 8£CH3).
0
0
4.1.18. Octyl 5-(5-deoxy-5-ethylsulfonamido-a-D-arab-
inofuranosyl)-a-D-arabinofuranoside (25). Compound 21
(40 mg, 0.04 mmol) treated with Et4NþF2 (38 mg,
0.35 mmol) in dry THF (4 mL) as described for the
preparation of 13. Purification by column chromatography
(CHCl3/MeOH, 9:1) yielded 25 (20 mg, 95%) as an oil. MS-
ESI: m/z Found 508.2192 [MþNa]þ, calcd 508.2186 for
4.1.15. Octyl 5-[5-deoxy-5-(p-methylphenyl)sulfona-
mido-2,3-di-O-tert-butyldimethylsilyl-a-D-arabinofura-
nosyl]-2,3-di-O-tert-butyldimethylsilyl-a-D-arabinofura-
noside (22). Compound 14 (60 mg, 0.07 mmol) was treated
with HCO2NH4 (17 mg, 0.27 mmol) in MeOH (5 mL) over
10% Pd/C (40 mg) followed by reaction with tosyl chloride
C20H39NO10S. 1H NMR (CD3OD): d 4.93 (1H, d,
0
0
0
(17 mg,
0.09 mmol),
N-methylimidazole
(10 mL,
J1 ,2 ¼1.2 Hz, H-1 ), 4.83 (1H, d, J1,2¼1.7 Hz, H-1), 4.02–
3.96 (2H, m, H-4, H-40), 4.00 (1H, dd, J1 ,2 ¼1.5 Hz,
0
0
0.12 mmol) in CH2Cl2 (4 mL) as described earlier for the
preparation of 19. Purification by column chromatography
(cyclohexane/EtOAc, 95:5) yielded 22 (50 mg, 73%) as an
oil. MS-ESI: m/z Found 1026.5787 [MþNa]þ, calcd
1026.5802 for C49H97NO10SSi4. 1H NMR (CDCl3): d
7.73, 7.30 (each 2H, d, J¼8.2 Hz, Ar), 4.78 (1H, s, H-10),
0
0
0
J2 ,3 ¼3.4 Hz, H-2 ), 3.94 (1H, dd, J1,2¼1.8 Hz, J2,3¼3.7 Hz,
H-2), 3.87 (1H, dd, J2,3¼3.7 Hz, J3,4¼6.4 Hz, H-3), 3.82
(1H, dd, J4,5a¼5.4 Hz, J5a,5b¼11.30Hz, H-5a), 3.78 (1H, dd,
0
0
0
0
J2 ,3 ¼3.4 Hz, J3 ,4 ¼5.8 Hz, H-3 ), 3.72–3.65 (1H, m,
OCH2), 3.64 (1H, dd, J4,5b¼3.5 Hz, J5a,5b¼11.3 Hz,
0
0
4.76 (1H, t, J¼5.7 Hz, NH), 4.72 (1H, d, J1,2¼1.8 Hz, H-1),
H-5b), 3.44–3.36 (1H, m, OCH2), 3.37 (1H, dd, J4 ,5 a
¼
¼
0
4.01 (1H, dd, J1 ,2 ¼1.3 Hz, J2 ,3 ¼3.0 Hz, H-2 ), 3.99 (1H,
3.7 Hz, J5 a,5 b¼13.7 Hz, H-50a), 3.23 (1H, dd, J4 ,5 b
0
0
0
0
0
0
0
0
6.6 Hz, J5 a,5 b¼13.7 Hz, H-50b), 3.13–3.05 (2H, m, CH2),
1.62–1.50 (2H, m, CH2), 1.35–1.26 (13H, m, 5£CH2,
CH3), 0.92–0.87 (3H, m, CH3).
0
0
dd, J1,2¼1.8 Hz, J2,3¼3.7 Hz, H-2), 3.96–3.90 (3H, m, H-03,
H-4, H-40), 3.84 (1H, dd, J2 ,3 ¼3.0 Hz, J3 ,4 ¼5.8 Hz, H-3 ),
3.71–3.64 (1H, m, OCH2), 3.65 (1H, dd, J4,5a¼3.4 Hz,
J5a,5b¼11.4 Hz, H-5a), 3.50 (1H, dd, J4,5b¼3.1 Hz,
J5a,5b¼11.4 Hz, H-5b), 3.34–3.27 (1H, m, OCH2), 3.20–
3.13 (1H, m, H-50a), 3.10–3.02 (1H, m, H-50b), 1.60–1.51
(2H, m, CH2), 1.27 (10H, br s, 5£CH2), 0.90–0.84 (39H, m,
13£CH3), 0.094, 0.082, 0.076, 0.067, 0.062, 0.045, 0.035,
0.023 (each 3H, m, CH3).
0
0
0
0
4.1.19. Octyl 5-[5-deoxy-5-(p-methylphenyl)sulfona-
mido-a-D-arabinofuranosyl]-a-D-arabinofuranoside
(26). Compound 22 (45 mg, 0.04 mmol) treated with
Et4NþF2 (41 mg, 0.27 mmol) in dry THF (4 mL) as
described for the preparation of 13. Purification by column
chromatography (CHCl3/MeOH, 8:1) afforded 26 (21 mg,
88%) as an oil. MS-ESI: m/z Found 570.2327 [MþNa]þ,
4.1.16. Octyl 5-(5-deoxy-5-tert-butylamido-a-D-arabino-
furanosyl)-a-D-arabinofuranoside (23). Compound 19
(45 mg, 0.05 mmol) was treated with Et4NþF2 (43 mg,
0.30 mmol) in dry THF (4 mL) as described for the
preparation of 13. Purification by column chromatography
(CHCl3/MeOH, 8:1) afforded 23 (21 mg, 91%) as an oil.
MS-ESI: m/z Found 500.2817 [MþNa]þ, calcd 500.2830
1
calcd 570.2343 for C25H41NO10S. H NMR (CD3OD): d
7.73 (2H, d, J¼8.2 Hz, Ar), 7.37 (1H, dd, J¼0.6, 8.2 Hz,
Ar), 4.85 (1H, s, H-10), 4.82 (1H, d, J1,2¼1.7 Hz, H-1),
3.99–3.88 (2H, m, H-4, H-40), 3.95 (1H, dd, J1 ,2 ¼1.4 Hz,
0
0
0
0
0
J2 ,3 ¼3.4 Hz, H-2 ), 3.93 (1H, dd, J1,2¼1.7 Hz, J2,3¼3.7 Hz,
H-2), 3.85 (1H, dd, J2,3¼3.7 Hz, J3,4¼6.6 Hz, H-3), 3.74
(1H, dd, J4,5a¼5.4 Hz, J5a,5b¼11.10Hz, H-5a), 3.73 (1H, dd,
0
1
for C23H43NO9. H NMR (CD3OD): d 4.94 (1H, s, H-1 ),
0
0
0
0
0
0
4.83 (1H, d, J1,2¼1.8 Hz, H-1), 4.06 (1H, ddd, J4 ,5 a
¼
J2 ,3 ¼3.4 Hz, J3 ,4 ¼5.4 Hz, H-3 ), 3.71–3.64 (1H, m,
J4 ,5 b¼4.8 Hz, J3 ,4 ¼5.3 Hz, H-40), 4.00 (1H, dd,
OCH2), 3.57 (1H, dd, J4,5b¼3.6 Hz, J5a,5b¼11.1 Hz,
0
0
0
0
0
0
0
0
0
0
0
J1 ,2 ¼1.2 Hz, J2 ,3 ¼2.8 Hz, H-2 ), 4.02–3.97 (1H, m,
H-4), 3.94 (1H, dd, J1,2¼1.8 Hz, J2,3¼3.7 Hz, H-2), 3.87
(1H, dd, J2,3¼3.7 Hz, J3,4¼6.5 Hz, H-3), 3.81 (1H, dd,
H-5b), 3.43–3.36 (1H, m, OCH2), 3.14 (1H, dd, J4 ,5 a
0
¼
¼
4.1 Hz, J5 a,5 b¼13.4 Hz, H-50a), 3.01 (1H, dd, J4 ,5 b
0
0
0
6.5 Hz, J5 a,5 b¼13.4 Hz, H-50b), 1.60–1.54 (2H, m, CH2),
0
0
0
0
J4,5a¼5.1 Hz, J5a,5b¼11.0 Hz, H-5a), 3.72 (1H, dd, J2 ,3
¼
1.29 (10H, m, 5£CH2), 0.91–0.87 (3H, m, CH3).
0
0
0
2.8 Hz, J3 ,4 ¼5.3 Hz, H-3 ), 3.69–3.62 (1H, m, OCH2), 3.63
(1H, dd, J4,5b¼3.7 Hz, J5a,5b¼11.0 Hz, H-5b), 3.44–3.36
(3H, m, H2-50, OCH2), 1.59–1.53 (2H, m, CH2), 1.30 (10H,
m, 5£CH2), 1.18 (9H, s, 3£CH3), 0.92–0.87 (3H, m, CH3).
4.2. Biological
4.2.1. In vitro assay. In vitro inhibition assays20 of the
arabinofuranosyl disaccharide analogs were performed on
Mycobacterium tuberculosis (H37Ra, ATCC 25177) and
Mycobacterium avium (NJ 211).
4.1.17. Octyl 5-(5-deoxy-5-phenylamido-a-D-arabino-
furanosyl)-a-D-arabinofuranoside (24). Compound 20
(50 mg, 0.05 mmol) was treated with Et4NþF2 (47 mg,
0.31 mmol) in dry THF (4 mL) as described for the
preparation of 13. Purification by column chromatography
(CHCl3/MeOH, 9:1) yielded 24 (20 mg, 77%) as an oil. MS-
ESI: m/z Found 520.2508 [MþNa]þ, calcd 520.2517 for
4.2.2. Arabinosyltransferase assay.21 Compounds 14, 16,
18, 23, 24–26 at a range of concentrations from 0.25 to
6.0 mM (which were stored as 100 mM ethanol stocks) and
DP[14C]A (20,000 cpm, 9 mM, 10 mL [stored in chloro-
form/methanol, 2:1]), were dried under a stream of argon in
a microcentrifuge tube (1.5 mL) and placed in a vacuum
desiccator for 15 min to remove any residual solvent. The
dried constituents of the assay were then resuspended in
8 mL of a 1% aqueous solution of Igepal. The remaining
1
C25H39NO9. H NMR (CD3OD): d 7.84–7.81, 7.55–7.49,
7.47–7.41 (each m, Ar), 4.99 (1H, s, H-10), 4.82 (1H, d,
J1,2¼1.7 Hz, H-1), 4.20–4.15 (1H, m, H-40), 4.03 (1H, dd,
0
0
0
0
0
J1 ,2 ¼1.2 Hz, J2 ,3 ¼2.9 Hz, H-2 ), 4.02–3.98 (1H, m, H-4),
3.94 (1H, dd, J1,2¼1.8 Hz, J2,3¼3.7 Hz, H-2), 3.88 (1H, dd,

A. K. Pathak et al. / Tetrahedron 59 (2003) 10239–10248
10247
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constituents of the arabinosyltransferase assay containing
50 mM MOPS (adjusted to pH 8.0 with KOH), 5 mM
b-mercaptoethanol, 10 mM MgCl2, 1 mM ATP, membranes
(250 mg) were added to a final reaction volume of 80 mL.
The reaction mixtures were then incubated at 378C for 1 h.
A CHCl3/CH3OH (1:1, 533 mL) solution was then added to
the incubation tubes and the entire contents centrifuged at
18,000g. The supernatant was recovered and dried under a
stream of argon and re-suspended in C2H5OH/H2O (1:1,
1 mL) and loaded onto a pre-equilibrated (C2H5OH/H2O
[1:1]) 1 mL Whatmann strong anion exchange (SAX)
cartridge which was washed with 3 mL of ethanol. The
eluate was dried and the resulting products partitioned
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(3 mL) and H2O (3 mL). The resulting organic phase was
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aqueous phase was again extracted twice with 3 ml of
n-butanol saturated water, the pooled extracts were back-
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The n-butanol-saturated water fraction was dried and
re-suspended in 200 mL of n-butanol. The total cpm of
radiolabeled material extractable into the n-butanol phase
was measured by scintillation counting using 10% of the
labeled material and 10 ml of EcoScintA (National
Diagnostics, Atlanta). The incorporation of [14C]Araf was
determined by subtracting counts present in control assays
(incubation of the reaction components in the absence of the
compounds). Another 10% of the labeled material was
subjected to thin-layer chromatography (TLC) in CHCl3/
CH3OH/NH4OH/H2O (65:25:0.5:3.6) on aluminium backed
Silica Gel 60 F254 plates (E. Merck, Darmstadt, Germany).
Autoradiograms were obtained by exposing TLCs to X-ray
film (Kodak X-Omat) for 3 days. Competition based
experiments were performed by mixing compounds
together at various concentrations, a(1!5)-linked octyl
arabinofuranosyl disaccharide13 at 0.4 mM with disacchar-
ide analogs at 0.5, 1.0, 2.0, 3.0 and 6.0 mM followed by
thin-layer chromatography/autoradiography as described
earlier to determine the extent of product formation and
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Acknowledgements
16. Pathak, A. K.; Pathak, V.; Suling, W. J.; Gurcha, S. S.;
Morehouse, C. B.; Besra, G. S.; Maddry, J. A.; Reynolds, R. C.
Bioorg. Med. Chem. 2002, 10, 923–928.
The authors are thankful to Dr J. M. Riordan for NMR
spectra and Mr M. D. Richardson for ESI-MS data. This
work was supported by NIH/NIAID grant R01AI45317.
GSB who is currently a Lister Institute-Jenner Research
Fellow acknowledges support from The Medical Research
Council (49343 and 49342). The M. avium strain (NJ211)
was kindly provided by Dr Leonid Heifets, National Jewish
Center for Immunology and Respiratory Diseases, Denver,
CO, USA.
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