Synthesis of lipid-linked arabinofuranose donors for glycosyltransferases

Article abstract of DOI:10.1021/jo302507p

Mycobacteria and corynebacteria use decaprenylphosphoryl-β-D- arabinofuranose (DPA) as a critical cell wall building block. Arabinofuranosyltransferases that process this substrate to mediate cell wall assembly have served as drug targets, but little is known about the substrate specificity of any of these enzymes. To probe substrate recognition of DPA, we developed a general and efficient synthetic route to β-D-arabinofuranosyl phosphodiesters. In this approach, the key glycosyl phosphodiester bond-forming reaction proceeds with high β-selectivity. In addition to its stereoselectivity, our route provides the means to readily access a variety of different lipid analogues, including aliphatic and polyprenyl substrates.

Full text of DOI:10.1021/jo302507p

Article
pubs.acs.org/joc
Synthesis of Lipid-Linked Arabinofuranose Donors for
Glycosyltransferases
Matthew B. Kraft,† Mario A. Martinez Farias,† and Laura L. Kiessling*,†,‡
‡
†Department of Chemistry and Department of Biochemistry, University of WisconsinMadison, Madison, Wisconsin 53706,
United States
S
* Supporting Information
ABSTRACT: Mycobacteria and corynebacteria use decap-
renylphosphoryl-β-D-arabinofuranose (DPA) as a critical cell
wall building block. Arabinofuranosyltransferases that process
this substrate to mediate cell wall assembly have served as drug
targets, but little is known about the substrate specificity of any
of these enzymes. To probe substrate recognition of DPA, we
developed a general and efficient synthetic route to β-D-
arabinofuranosyl phosphodiesters. In this approach, the key
glycosyl phosphodiester bond-forming reaction proceeds with
high β-selectivity. In addition to its stereoselectivity, our route
provides the means to readily access a variety of different lipid analogues, including aliphatic and polyprenyl substrates.
INTRODUCTION
■
Mycobacteria and corynebacteria have a robust cell wall that is
crucial for their survival and pathogenesis. In the case of
Mycobacterium tuberculosis, the enzymes responsible for cell wall
biosynthesis have been identified as promising targets for the
development of new drugs to treat tuberculosis.1 A significant
proportion of the mycobacterial cell envelope is composed of
arabinofuranose (Araf), which is found in the form of a large
branched heteropolysaccharide known as the arabinogalactan
(AG, Figure 1a) as well as the lipoarabinomannan (LAM).1−3
The assembly of the arabinan polysaccharide is catalyzed by a
series of arabinofuranosyltransferases, which utilize the lipid-
linked donor substrate, decaprenylphosphoryl-β-D-arabinofur-
anose (DPA, Figure 1b), to add single Araf residues to a
growing arabinan polysaccharide.4 Some of these arabinofur-
anosyltransferases are targets of the first line antitubercular
agent ethambutol.5 Despite their known potential value as
therapeutic targets, little is known about the mechanism of
substrate preferences of any of these enzymes.
Figure 1. (a) Schematic depiction of a portion of the mycobacterial
cell wall showing the galactofuranose (red) and arabinofuranose
(yellow) polysaccharide components and (b) decaprenylphosphoryl-β-
D-arabinofuranose (DPA) is the building block used to incorporate
arabinofuranose residues.
Paramount to the study of glycan biosynthesis is the ability to
readily obtain renewable quantities of glycosyl donor and
acceptor substrates or functional analogues thereof.6,7 DPA can
be accessed by total synthesis; however, the required
decaprenol precursor is costly and only available in small
quantities.5,8 Additionally, the long decaprenyl lipid of DPA
confers poor aqueous solubility upon the glycosyl donor,
thereby complicating the study of arabinofuranosyltransferases.
A more readily available, shorter lipid analogue of DPA could
facilitate a wide range of mechanistic studies. A recent report
provides impetus to generate DPA analogues, as proteolipo-
somes containing the mycobacterial arabinofuranosyltransferase
AftC could use a (Z,Z)-farnesyl lipid analogue to transfer a
single Araf residue to a synthetic acceptor.9 To explore
arabinose incorporation into the cell wall, we sought to prepare
various analogues of DPA with both saturated and unsaturated
lipid substituents. Using the known synthetic methods, we were
unable to generate a wide range of β-D-arabinofuranosyl
Special Issue: Howard Zimmerman Memorial Issue
Received: November 15, 2012
Published: February 1, 2013
© 2013 American Chemical Society
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Figure 2. Synthetic approaches to DPA and analogues.
phosphodiesters. Some methods have restrictions on the type
of lipid that can be appended, and many approaches give rise to
mixtures of glycosylphosphate derivatives that are difficult to
separate. Because only β-glycosyl phosphodiesters are candidate
substrates for the arabinofuranosyltransferases, the most
valuable synthetic method would be one that generates the β-
anomer selectively. Here we describe a route to β-D-
arabinofuranosyl phosphodiesters that provides both saturated
and unsaturated products with high stereoselectivity for the β-
anomer.
phosphate salt 4 and a lipid alcohol (Figure 1c). Of the
known coupling agents for linking alcohols and phosphoryl
groups to form phosphodiesters (e.g., carbodiimides, sulfonyl
chlorides, Mitsunobu conditions, etc.),13 we were attracted to
the method of Cramer and co-workers, which uses trichlor-
oacetonitrile to activate the phosphoryl group.14−17 Applying
this approach to our targets involves generating iminophos-
phate intermediate 5, which can undergo nucleophilic attack by
an alcohol. When we attempted to carry out this reaction on a
TBS-protected substrate, we found the phosphorylated Araf
derivative 4 to be quite unstable. We traced this instability to
the lability of its silyl groups, which underwent cleavage even
during storage at −20 °C. When phosphodiester coupling was
attempted using freshly prepared 4 and dodecanol, only the
minor α-anomer underwent coupling whereas the desired β-
anomer afforded the corresponding C1,C2 cyclic phosphate 7.18
Although it was not noted, others appear to have encountered
this problem previously.19 The production of this undesired
product is a consequence of the deleterious removal of the C2
tert-butyldimethylsilyl (TBS) ether and subsequent intra-
molecular cyclization via the electrophilic iminophosphate
intermediate. Thus, the unwanted desilylation not only
decreased the yield of our target compound but also precluded
the production of the requisite β-anomer.
To obtain our desired DPA analogues, a protecting group
strategy for Araf was needed that circumvented the propensity
of the TBS ether at the 2-position to undergo cleavage. It has
previously been shown that nonparticipating protecting groups
such as benzyl groups provide high selectivity for the β-anomer
during phosphorylation of the corresponding protected α-
glycosyl bromide intermediate.20 Still, the conditions needed to
remove the benzyl protecting groups would interfere with
accessing substrates that possess alkene-containing lipids.
Alternatively, ester protecting groups are compatible with
installing a wide variety of lipids but offer poor selectivity in the
glycosylation reaction due to competing neighboring group
RESULTS AND DISCUSSION
■
To date, there have been two different approaches to the
synthesis of DPA and shorter polyprenyl analogues. One
approach developed by Lee and co-workers uses a phosphor-
amidite coupling method to join 2,3,5-silyl protected Araf (1)
with a corresponding polyprenol (Figure 2a).5,10 After
oxidation of the intermediate phosphite, the desired phospho-
diester is formed as a 5:1 mixture of α- and β-anomers (2 and
3). This approach is advantageous in that any number of DPA
analogues, both saturated and unsaturated, can be prepared by
simply changing the alcohol coupling partner. A disadvantage,
however, is that the desired β-anomer is the minor component
of the product mixture. Another approach developed by Liav
and Brennan relies on first installing a phosphoryl group at the
C1 position of arabinose. In a subsequent step, the resulting
Araf phosphate (4) is coupled to a lipid-linked trichloroaceti-
midate intermediate (Figure 2b).8,9,11,12 This approach favors
formation of the desired β-anomer (2:1−4:1 β/α), but it is
applicable only to activated lipid acetimidates (i.e., allylic or
benzylic) and not aliphatic substrates.13 Furthermore, this
approach requires an extra synthetic step to convert the lipid
moiety into the corresponding trichloroacetimidate derivative.
To access both saturated and unsaturated substrates, we
envisioned using an alternative strategy in which the
phosphodiester moiety is generated directly from mono-
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Scheme 1. Synthesis of β-D-Arabinofuranosyl Phosphodiesters 12−18
participation.20−22 For the synthesis of DPA derivatives, silyl
groups offer an ideal combination of selectivity and functional
group tolerance. We therefore reasoned that a potential
solution to the observed instability of the TBS protecting
group would be to use a more hindered tert-butyldiphenylsilyl
(BPS) protecting group.
mechanism, one might anticipate that the ratio of glycosyl
bromide anomers would be reflected in the ratio of
phosphorylated products.20 NMR analysis of the crude glycosyl
bromides, however, revealed that the identity of the silyl group
has little influence on the ratio of glycosyl bromides; both
substrates had a preference for the α-configuration (92:8 α/β
for TBS and >95:5 α/β for BPS).25,26 More flexibility in the
TBS-protected substrate may allow the reaction to proceed
through not only the SN2 reaction but also a competing SN1-
like pathway involving an oxocarbenium intermediate.27 The
increased steric demands in the BPS-protected substrate may
make it difficult for this species to adopt a conformation that
can accommodate an oxocarbenium intermediate. Whatever the
origins of the selectivity, this phosphorylation reaction is a
remarkable case in which the identity of the silyl group has a
large effect on selectivity in a furanose ring system. It should be
noted that bulky silicon protecting groups have been exploited
in pyranose systems to control ring conformation and anomeric
selectivity in glycosylation reactions.28−30 Moreover, Bols and
co-workers demonstrated that conformational preferences in
pyranose glycosyl donors enforced by bulky silicon protecting
groups can dramatically enhance their reactivity in glycosylation
reactions.31−34 No such effects had been reported in furanose
sugars. We postulate that the dramatic influence of the sterically
demanding silyl substituents that we observed may be exploited
to produce other furanosides with high stereoselectivity.
With the C1 phosphoryl group installed in the desired β-
configuration, we focused on forming the requisite phospho-
diester. First, the benzyl substituents on the phosphotriester
were removed by hydrogenolysis to provide the intermediate
monophosphate. As expected, this entity was stable; we found
no evidence of the silyl group lability that plagued our reactions
when TBS was used as a protecting group. We could promote
phosphodiester formation using a variety of alcohols (poly-
prenyl, saturated n-alkanes, and naphthyl) by heating the
alcohol and protected Araf monophosphate in pyridine at 55
°C in the presence of trichloroacetonitrile. The crude
phosphodiesters were then treated with ammonium fluoride
in a 15% solution of aqueous ammonium hydroxide in
methanol to afford the corresponding β-D-arabinofuranosyl
phosphodiesters 12−18 in 62−79% yield over two steps.
On the surface, the use of the BPS rather than TBS group
seemed to be a straightforward perturbation, but there were
uncertainties. The first was whether this exchange would indeed
allow us to generate the β-linked Araf derivatives (vide infra).
Perhaps the most pressing unknown, however, was whether the
steric demands of this protecting group would prevent
generating the target glycosyl donor. For example, a recent
attempt by Dureau and co-workers to persilylate galactose with
BPSCl led only to a mixture of partially silylated products.23
Fortunately, when D-arabinose was treated with BPSCl and
imidazole, the desired tetra-BPS-protected Araf intermediate 8
was obtained in 70% yield. This product was obtained as a 10:1
mixture of anomers favoring the α-configuration (Scheme 1).
Selective removal of the anomeric BPS group was accomplished
by treating 7 with excess trifluoroacetic acid for 5 min, followed
by carefully pouring the reaction mixture into a solution of
ammonium hydroxide in methanol at −15 °C. The desired
product, 2,3,5-(BPS)-Araf (9), was obtained as a 3:1 mixture of
α- and β-anomers. Acetylation of 9 resulted in glycosyl acetate
10, which was formed as a 9:1 ratio of anomers, again favoring
the α-anomer.
The C1 phosphate moiety was installed in two steps: first,
conversion of acetate 10 into the corresponding glycosyl
bromide intermediate was effected using bromotrimethylsilane,
and second, displacement of the anomeric bromide substituent
was carried out with dibenzyl phosphate serving as a
nucleophile to afford 11. This two-step sequence was highly
selective for the desired β-anomer (>10:1 β/α).24 This
selectivity is notable. When the same reaction sequence was
performed on the analogous TBS-protected intermediate, the
process was much less selective (a 2:1−4:1 mixture of β- and α-
anomers). When BPS protection was employed, therefore, not
only did the desired β-linked phosphotriester predominate, but
it was obtained in high yield.
We considered the origin of the higher selectivity obtained
with the substrate bearing BPS relative to that possessing TBS
protecting groups. Since the glycosylation reaction of the
phosphodiester is anticipated to proceed through an SN2
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warmed to rt. The mixture was partitioned between 1:1 CH2Cl2/water
(50 mL). The phases were separated, and the aqueous phase was
extracted with CH2Cl2 (3 × 30 mL). The combined organic phase was
dried over Na2SO4, filtered, and concentrated under reduced pressure.
Purification was accomplished by flash column chromatography on
silica gel, eluting with 4% EtOAc/hexanes. The product-containing
fractions were combined and concentrated under reduced pressure to
provide 9 (681 mg, 69%) as a clear, highly viscous oil: Rf = 0.31 (10%
EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) δ 7.63−7.12 (m,
30H), 5.28 (dd, J = 12.6, 3.4 Hz, 0.25H, H-1β), 5.10 (d, J = 12.1,
0.75H, H-1α), 4.56 (dd, J = 6.9, 6.9 Hz, 0.75H, H-4α), 4.26 (dd, J =
1.7, 1.7 Hz, 0.25H, H-2β), 4.22 (s, 0.75H, H-2α), 4.05 (s, 1H, H-
3α,β), 3.97 (dd, J = 5.8, 5.8 Hz, 0.25H, H-4β), 3.72 (dd, J = 10.2, 6.7
Hz, 0.75H, H-5α), 3.68 (d, J = 12.1 Hz, 0.75H, OHα), 3.57−3.49 (m,
0.75H, H-5′α), 3.39 (dd, J = 10.5, 6.1 Hz, 0.25H, H-5β), 3.29 (dd, J =
10.5, 5.7 Hz, 0.25H, H-5′β), 0.98 (s, 2.3H, BPS-β), 0.96 (s, 6.7H, BPS-
α), 0.95 (s, 6.7H, BPS-α), 0.92 (s, 2.3H, BPS-β), 0.88 (s, 2.3H, BPS-
β), 0.80 (s, 6.7H, BPS-α); 13C NMR (101 MHz, CDCl3) δ 136.0,
135.9, 135.9, 135.8, 135.8, 135.7, 135.7, 135.0, 133.8, 133.5, 133.5,
133.4, 133.2, 133.0, 132.9, 132.58, 132.5, 132.5, 132.4, 132.2, 130.2,
130.2, 130.1, 130.0, 129.9, 129.9, 129.9, 129.8, 129.8, 129.7, 128.1,
128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 104.0, 98.7, 88.8, 85.5,
81.2, 79.4, 78.7, 78.1, 65.2, 65.0, 27.1, 27.0, 27.0, 26.9, 26.8, 19.3, 19.3,
19.3, 19.2, 19.1; HRMS (ESI-TOF+) calcd for C53H68NO5Si3 (M +
CONCLUSION
■
The synthetic sequence described here can provide β-D-
arabinofuranosyl phosphodiesters in 8 synthetic steps with an
overall yield of 13−16%. This approach is not only highly
selective for the β-anomer, but it also offers flexibility.
Essentially any derivative can be prepared from the
corresponding alcohol and Araf monophosphate salt. We
envision that this route will facilitate the study of the
mycobacterial arabinofuranosyltransferases as it efficiently and
reliably provides access to shorter and more aqueous soluble
analogues of the endogenous Araf donor, DPA.
EXPERIMENTAL SECTION
■
General Experimental Procedures, Materials, and Instru-
mentation. Solvents were purified according to the guidelines in
Purification of Common Laboratory Chemicals.35 All reactions were run
under nitrogen atmosphere unless otherwise stated. Analytical thin-
layer chromatography (TLC) was carried out on E. Merck
(Darmstadt) TLC plates precoated with silica gel 60 F254 (250 μm
layer thickness). Analyte visualization was accomplished using a UV
lamp and by charring with p-anisaldehyde solution. Flash column
chromatography was performed with Silicycle flash silica gel (40−63
μm, 60 Å pore size) using reagent-grade hexanes and ACS grade ethyl
acetate (EtOAc) or methanol and CH2Cl2. 1H and 13C nuclear
magnetic resonance (NMR) spectra were recorded on a 300 MHz
+
NH4 ) 882.4400, found 882.4431.
1-O-Acetyl-2,3,5-O-tert-butyldiphenylsilyl-β-D-arabinofura-
nose (10). To a stirred solution of 9 (0.64 g, 0.74 mmol) in pyridine
(7.4 mL) were added 4-(dimethylamino)pyridine (9 mg, 0.07 mmol)
and acetic anhydride (0.21 mL, 2.2 mmol). The reaction mixture was
stirred at rt for 3.5 h and was then quenched by the addition of
saturated aqueous NaHCO3 solution (10 mL). The mixture was
partitioned between CH2Cl2 (15 mL) and water (15 mL), the phases
were separated, and the aqueous phase was extracted with CH2Cl2 (3
× 10 mL). The combined organic phase was dried over Na2SO4,
filtered, and concentrated under reduced pressure. Purification was
accomplished by flash column chromatography on a 2.5 × 19 cm silica
gel column, eluting with 4% EtOAc/hexanes, collecting 13 × 100 mm
test tube fractions. The product containing fractions (19−29) were
combined and concentrated under reduced pressure to provide 10
(473 mg, 70%) as a clear, highly viscous oil: Rf = 0.30 (6% EtOAc/
spectrometer (acquired at 300 MHz for 1H NMR and 75 MHz for 13
C
NMR), a 400 MHz spectrometer (acquired at 400 MHz for 1H NMR
and 101 MHz for 13C NMR), or a 500 MHz spectrometer (acquired at
500 MHz for 1H NMR and 126 MHz for 13C NMR). Chemical shifts
are reported relative to tetramethylsilane or residual solvent peaks in
parts per million (CHCl3: 1H, 7.27, 13C, 77.23; MeOH: 1H, 3.31, 13C,
49.15). Peak multiplicity is reported as singlet (s), doublet (d), doublet
of doublets (dd), doublets of doublets of doublets (ddd), triplet (t),
doublet of triplets (dt), etc. High-resolution mass spectra (HRMS)
were obtained on an electrospray ionization-time of flight (ESI-TOF)
mass spectrometer.
1,2,3,5-Tetra-O-tert-butyldiphenylsilyl-D-arabinofuranose
(8). A mixture of D-(−)-arabinose (200 mg, 1.33 mmol) and imidazole
(633 mg, 9.31 mmol) were azeotropically dried by evaporation with
toluene (3 × 3 mL). The mixture was taken up in DMF (9 mL), and
tert-butyldiphenylsilyl chloride (2.1 mL, 8.0 mmol) was added. The
reaction mixture was stirred for 48 h at 70 °C and was then quenched
by the addition of diethanolamine (0.38 mL, 4.0 mmol). Stirring was
continued for 2 h, and the reaction mixture was then poured into a
stirring mixture of 15% EtOAc/hexanes (25 mL) and water (25 mL).
The phases were separated, and the organic phase was washed with
water (3 × 25 mL) and brine (30 mL). The organic phase was dried
over Na2SO4, filtered, and concentrated under reduced pressure.
Purification was accomplished by flash column chromatography on
silica gel, eluting with 4% EtOAc/hexanes. The product containing
fractions were combined and concentrated under reduced pressure to
provide 8 (1.03g, 70%) as a white foam: Rf = 0.53 (10% EtOAc/
hexanes); 1H NMR (400 MHz, CDCl3) δ 7.71−7.49 (m, 16H), 7.41−
7.07 (m, 34H), 5.23 (s, 1H), 4.51 (ddd, J = 7.4, 5.2, 2.3 Hz, 1H), 4.29
(s, 1H), 4.10 (d, J = 2.3 Hz, 1H), 3.53 (dd, J = 10.4, 7.2 Hz, 1H), 3.41
(dd, J = 10.5, 5.2 Hz, 1H), 1.00 (s, 9H) 0.97−0.95 (m, 18H), 0.70 (s,
9H); 13C NMR (101 MHz, CDCl3) δ 136.1, 136.1, 136.1, 136.0,
135.8, 135.8, 135.7, 134.2, 133.8, 133.7, 133.5, 133.4, 132.9, 129.8,
129.8, 129.7, 129.7, 129.6, 129.5, 129.2, 128.4, 127.8, 127.8, 127.8,
127.8, 127.7, 127.7, 127.6, 127.5, 125.5, 103.9, 88.9, 84.8, 81.2, 65.5,
27.1, 27.1, 27.0, 26.8, 19.4, 19.4, 19.4, 19.1; HRMS (ESI-TOF+) calcd
1
hexanes); H NMR (400 MHz, CDCl3) δ 7.62−7.13 (m, 30H), 5.97
(s, 1H), 4.49 (ddd, J = 7.6, 6.2, 1.3 Hz, 1H), 4.33 (s, 1H), 4.16 (d, J =
0.9 Hz, 1H), 3.66 (dd, J = 10.3, 6.4 Hz, 1H), 3.49 (dd, J = 10.3, 7.3 Hz,
1H), 1.94 (s, 3H), 0.97 (s, 9H), 0.96 (s, 9H), 0.83 (s, 9H); 13C NMR
(101 MHz, CDCl3) δ 169.9, 135.9, 135.9, 135.9, 135.8, 135.7, 133.7,
133.5, 133.4, 133.3, 132.7, 132.6, 130.1, 130.0, 129.9, 129.9, 129.8,
129.7, 127.9, 127.9, 127.8, 127.8, 102.6, 90.5, 82.4, 79.1, 64.8, 27.0,
26.9, 21.3, 19.4, 19.4, 19.1; HRMS (ESI-TOF+) calcd for
+
C55H70NO6Si3 (M + NH4 ) 924.4506, found 924.4528.
Dibenzyl (2,3,5-O-tert-Butyldiphenylsilyl-β-D-arabinofurano-
syl)-1-phosphate (11). To a stirred solution of 10 (0.46 g, 0.50
mmol) in CH2Cl2 (5 mL) was added trimethylsilyl bromide (133 μL,
1.01 mmol). The reaction mixture was stirred at rt for 6 h and was
then concentrated under reduced pressure. The crude glycosyl
bromide was azeotropically dried with toluene (3 × 2 mL) and then
used immediately.
To a stirred solution of azeotropically dried dibenzyl phosphate
(281 mg, 1.01 mmol) in toluene (3 mL) were added powdered 4 Å
molecular sieves (400 mg) and triethylamine (182 μL, 1.31 mmol).
The mixture was cooled to 0 °C, and a solution of the aforementioned
glycosyl bromide in toluene (1 mL) was added slowly via cannula. The
transfer was completed by rinsing twice with toluene (2 × 0.5 mL).
Stirring was continued at 0 °C for 1 h and then at rt overnight. The
reaction mixture was filtered through a pad of sand and Celite, rinsing
with EtOAc, and the solvent was removed under reduced pressure.
Purification was accomplished by flash column chromatography on a
2.5 × 19 cm silica gel column, eluting with 15% EtOAc/hexanes,
collecting 13 × 100 mm test tube fractions. The product containing
fractions (10−29) were combined and concentrated under reduced
pressure to provide 11 (356 mg, 63% over two steps) as a clear viscous
+
for C69H86NO5Si4 (M + NH4 ) 1120.5578, found 1120.5588.
2,3,5-Tri-O-tert-butyldiphenylsilyl-D-arabinofuranose (9). To
a stirred solution of 8 (1.27g, 1.15 mmol) in CH2Cl2 (11.5 mL) was
added trifluoroacetic acid (2.90 mL, 37.9 mmol). The reaction mixture
was stirred at rt for 5 min and was then slowly poured into a 15%
solution of concentrated ammonium hydroxide in MeOH (38 mL) at
−15 °C. The mixture was stirred at −15 °C for 30 min and was then
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1
oil: Rf = 0.26 (15% EtOAc/hexanes); H NMR (300 MHz, CDCl3) δ
3.63 (dd, J = 12.2, 5.8 Hz, 1H), 2.17−2.01 (m, 4H), 1.74 (s, 3H), 1.67
(s, 3H), 1.61 (s, 3H); 13C NMR (101 MHz, CD3OD) δ 141.0, 132.9,
125.2, 123.5(d), 99.2(d), 85.1, 79.5(d), 75.3, 64.1, 63.5(d), 33.2, 27.9,
26.1, 23.8, 17.9; HRMS (ESI-TOF−) calcd for C15H26O8P (M−)
365.1370, found 365.1373.
7.60−7.41 (m, 13H), 7.40−7.10 (m, 27H), 5.78 (dd, J = 5.0, 3.0 Hz,
1H), 4.98 (dd, J = 11.9, 6.9 Hz, 1H), 4.87 (dd, J = 11.9, 7.7 Hz, 1H),
4.77 (d, J = 6.5 Hz, 2H), 4.32 (s, 1H), 4.24 (td, J = 7.0, 1.5 Hz, 1H),
4.12 (dd, J = 2.4, 2.4 Hz, 1H), 3.72 (dd, J = 10.4, 7.2 Hz, 1H), 3.61
(dd, J = 10.4, 6.8 Hz, 1H), 0.92 (s, 9H), 0.92 (s, 9H), 0.87 (s, 9H); 13C
NMR (101 MHz, CDCl3) δ 136.2, 136.1, 136.1, 136.0, 136.0, 135.9,
135.9, 135.7, 133.7, 133.5, 133.2, 132.8, 132.8, 130.0, 129.9, 129.9,
129.8, 129.7, 128.5, 128.5, 128.4, 128.3, 128.0, 127.9, 127.9, 127.9,
127.9, 127.8, 127.7, 101.1(d), 87.0, 78.3, 78.2(d), 69.1(d), 69.0(d),
64.7, 27.1, 27.0, 26.9, 19.5, 19.3; HRMS (ESI-TOF+) calcd for
((R)-Citronellyl)phosphoryl-β-D-arabinofuranose (14). Pre-
pared in the same manner as described for compound 12 from
intermediate monophosphate salt S3 (30 mg, 0.026 mmol) and (R)-
citronellol (21 μL, 0.11 mmol). Compound 14 (6.2 mg, 62%) was
generated as a clear colorless oil: Rf = 0.24 (4% H2O/25% MeOH/
1
71% CH2Cl2); H NMR (400 MHz, CD3OD) δ 5.47 (t, J = 4.6 Hz,
+
C67H81NO8Si3 (M + NH4 ) 1142.5003, found 1142.5027.
1H), 5.14−5.08 (m, 1H), 4.08 (dd, J = 8.1, 6.9 Hz, 1H), 4.00−3.89
(m, 3H), 3.81−3.69 (m, 2H), 3.62 (dd, J = 12.1, 5.8 Hz, 1H), 2.09−
1.91 (m, 2H), 1.75−1.63 (m, 1H), 1.67 (s, 3H), 1.61(s, 3H), 1.48−
1.33 (m, 3H), 1.22−1.11 (m, 1H), 0.92 (d, J = 6.5 Hz, 3H); 13C NMR
(101 MHz, CD3OD) δ 132.0, 126.1, 99.2(d), 85.1, 79.5(d), 75.3,
65.3(d), 64.2, 39.0(d), 38.5, 30.5, 26.6, 26.0, 19.9, 17.9, 17.8. HRMS
(ESI-TOF−) calcd for C15H28O8P (M−) 367.1527, found 367.1534.
n-Octylphosphoryl-β-D-arabinofuranose (15). Prepared in the
same manner as described for compound 12 from intermediate
monophosphate salt S3 (30 mg, 0.026 mmol) and n-octanol (18 μL,
0.11 mmol). Compound 15 (7.3 mg, 78%) was generated as a clear
colorless oil: Rf = 0.27 (4% H2O/25% MeOH/71% CH2Cl2); 1H
NMR (400 MHz, CD3OD) δ 5.47 (t, J = 4.5 Hz, 1H), 4.07 (dd, J =
8.3, 6.9 Hz, 1H), 3.99−3.93 (m, 1H), 3.89 (q, J = 6.4 Hz, 2H), 3.82−
3.69 (m, 2H), 3.62 (dd, J = 12.1, 5.7 Hz, 1H), 1.63 (p, J = 6.8 Hz, 2H),
1.47−1.21 (m, 10H), 0.90 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz,
CD3OD) δ 99.2(d), 85.1, 79.5(d), 75.3, 67.0(d), 64.2, 33.2, 32.0(d),
30.6, 27.1, 27.0, 23.9, 14.6; HRMS (ESI-TOF−) calcd for C13H26O8P
(M−) 341.1370, found 341.1365.
Bis-triethylammonium (2,3,5-O-tert-Butyldiphenylsilyl-β-D-
arabinofuranosyl)-1-phosphate (S3). To a stirred solution of 11
(259 mg, 0.230 mmol) in 10% EtOH/EtOAc (8.2 mL) were added
triethylamine (0.8 mL, 5.75 mmol) and 10% palladium on carbon (17
mg, 0.016 mmol). The reaction vessel was purged with N2 and then
equipped with a H2-filled balloon. The reaction mixture was stirred at
rt overnight and was then filtered through a plug of sand and Celite
with 10% EtOH/EtOAc. The solvent was removed under reduced
pressure to provide monophosphoryl salt S3 (259 mg, 98%) as a white
powder: Rf = 0.5 (15% MeOH/CH2Cl2); 1H NMR (500 MHz,
CDCl3) δ 7.75−7.67 (m, 2H), 7.56 (m, 4H), 7.53−7.48 (m, 2H),
7.46−7.40 (m, 2H), 7.40−7.21 (m, 14H), 7.15 (m, 4H), 7.07 (t, J =
7.5 Hz, 2H), 5.86 (dd, J = 7.1, 3.0 Hz, 1H), 4.22 (s, 1H), 4.10 (brt, J =
2.4 Hz, 1H), 4.05 (t, J = 7.2 Hz, 1H), 3.70 (dd, J = 10.4, 7.6 Hz, 1H),
3.47 (m, 1H), 2.97 (q, J = 7.3 Hz, 12H), 1.25 (t, J = 7.3 Hz, 18H), 0.93
(s, 9H), 0.92 (s, 9H), 0.85 (s, 9H); 13C NMR (126 MHz, CDCl3) δ
136.7, 136.0, 135.8, 135.2, 135.7, 134.1, 133.7, 133.7, 133.6, 133.0,
132.7, 129.7, 129.7, 129.6, 129.4, 127.8, 127.7, 127.7, 127.6, 99.8(d),
85.3, 78.9, 78.5(d), 65.2, 45.6, 27.2, 27.0, 27.0, 19.6, 19.3, 19.2, 8.7.
+
HRMS (ESI-TOF+) calcd for C53H69NO8PSi3 (M + NH4 ) 962.4064,
(8-Phenyloctyl)phosphoryl-β-D-arabinofuranose (16). Pre-
pared in the same manner as described for compound 12 from
intermediate monophosphate salt S3 (30 mg, 0.026 mmol) and 8-
phenyl-1-octanol (24 mg, 0.11 mmol). Compound 16 (7.4 mg, 65%)
was generated as a clear colorless oil: Rf = 0.30 (4% H2O/25%
found 962.4080.
(Z,Z)-Farnesylphosphoryl-β-D-arabinofuranose (12).9 A mix-
ture of monophosphate salt S3 (86 mg, 0.075 mmol) and (Z,Z)-
farnesol36 (67 mg, 0.30 mmol) were azeotropically dried with
anhydrous pyridine (3 × 0.5 mL). The mixture was taken up in
pyridine (1 mL), and trichloroacetonitrile (75 μL, 0.75 mmol) was
added. The reaction mixture was stirred at 55 °C for 12 h. The solvent
was removed under reduced pressure, and the crude phosphodiester
was carried onto deprotection without purification.
1
MeOH/71% CH2Cl2); H NMR (400 MHz, CD3OD) δ 7.24 (t, J =
7.6 Hz, 2H), 7.19−7.08 (m, 3H), 5.47 (t, J = 4.6 Hz, 1H), 4.15−4.03
(m, 1H), 3.99−3.92 (m, 1H), 3.92−3.81 (m, 2H), 3.80−3.70 (m, 2H),
3.62 (dd, J = 12.1, 5.8 Hz, 1H), 2.59 (t, J = 7.7 Hz, 2H), 1.67−1.56 (m,
4H), 1.49−1.20 (m, 8H); 13C NMR (101 MHz, CD3OD) δ 144.1,
129.5, 129.4, 126.7, 99.2(d), 85.1, 78.9, 75.3, 67.0(d), 64.2, 37.1, 32.9,
31.9(d), 30.75, 30.6, 30.4, 27.0; HRMS (ESI-TOF−) calcd for
C19H30O8P (M−) 417.1683, found 417.1693.
n-Dodecylphosphoryl-β-D-arabinofuranose (17). Prepared in
the same manner as described for 12 from intermediate mono-
phosphate salt S3 (89 mg, 0.078 mmol) and n-dodecanol (70 μL, 0.31
mmol). Compound 17 (23 mg, 71%) was generated as a colorless wax:
Rf = 0.30 (4% H2O/30% MeOH/66% CH2Cl2); 1H NMR (300 MHz,
CD3OD) δ 5.47 (t, J = 4.6 Hz, 1H), 4.12−4.03 (m, 1H), 3.97 (ddd, J =
8.1, 4.3, 2.2 Hz, 1H), 3.90 (q, J = 6.5 Hz, 2H), 3.82−3.69 (m, 2H),
3.68−3.58 (m, 1H), 1.69−1.55(m, 2H), 1.48−1.17 (m, 18H), 0.90 (t,
J = 7.1 Hz, 3H); 13C NMR (75 MHz, CD3OD) δ 99.2(d), 85.08,
79.4(d), 75.33, 67.0(d), 64.2, 33.2, 32.0, 31.9, 30.9, 30.9, 30.9, 30.7,
30.6, 27.0, 23.9, 14.6, 9.3; HRMS (ESI-TOF−) calcd for C17H34O8P
(M−) 397.1996, found 397.1995.
(2-Naphthalenemethyl)phosphoryl-β-D-arabinofuranose
(18). Prepared in the same manner as described for compound 12
from intermediate monophosphate salt S3 (30 mg, 0.026 mmol) and
2-naphthalenemethanol (18 mg, 0.11 mmol). Compound 18 (6.6 mg,
65%) was generated as a clear colorless oil: Rf = 0.24 (4% H2O/25%
MeOH/71% CH2Cl2); 1H NMR (400 MHz, CD3OD) δ 7.90 (s, 1H),
7.88−7.79 (m, 3H), 7.55 (dd, J = 8.5, 1.8 Hz, 1H), 7.50−7.41 (m,
2H), 5.58 (t, J = 4.7 Hz, 1H), 5.13 (d, J = 5.9 Hz, 2H), 4.11 (t, J = 7.6
Hz, 1H), 4.03−3.97 (m, 1H), 3.85−3.69 (m, 2H), 3.64 (dd, J = 12.1,
5.8 Hz, 1H); 13C NMR (101 MHz, CD3OD) δ 137.4, 137.3, 134.9,
134.6, 129.1, 129.1, 128.8, 127.18, 127.0, 126.8, 99.4(d), 85.2, 79.5,
75.3, 68.7(d), 64.3; HRMS (ESI-TOF−) calcd for C16H18O8P (M−)
369.0744, found 369.0743.
To a solution of the aforementioned BPS-protected phosphodiester
in a 15% solution of concd ammonium hydroxide in methanol (1.5
mL) was added ammonium fluoride (83 mg, 2.2 mmol). The reaction
mixture was stirred at 55 °C for 10 h. After the mixture was cooled to
rt, CH2Cl2 (2 mL) was added, and the precipitate that had formed was
removed by filtration through Celite. The solvent was removed under
reduced pressure, and purification was accomplished by flash column
chromatography on a 1 × 6 cm silica gel column. The column was
eluted with 10% MeOH/CH2Cl2 (fractions 1−10) then 1% H2O/20%
MeOH/CH2Cl2 (fractions 11−60) while collecting 1.5 mL test tube
fractions. The product-containing fractions (14−65) were combined
and concentrated under reduced pressure to provide 12 (27 mg, 79%)
as a clear colorless oil: Rf = 0.27 (4% H2O/25% MeOH/71% CH2Cl2);
1H NMR (400 MHz, CD3OD) δ 5.48 (t, J = 4.5 Hz, 1H), 5.45−5.38
(m, 1H), 5.19−5.07 (m, 2H), 4.43 (t, J = 6.3 Hz, 2H), 4.13−4.04 (m,
1H), 3.90−3.93 (m, 1H), 3.82−3.69 (m, 2H), 3.63 (dd, J = 12.2, 5.7
Hz, 1H), 2.17−1.98 (m, 8H), 1.74 (s, 3H), 1.68 (s, 6H), 1.61 (s, 3H);
13C NMR (101 MHz, CD3OD) δ 140.9, 136.7, 132.5, 125.9, 125.5,
123.5(d), 99.2(d), 85.1, 79.5(d), 75.3, 64.2, 63.4(d), 33.4, 33.1, 27.8,
27.7, 26.1, 23.9, 23.8, 17.9; HRMS (ESI-TOF−) calcd for C20H34O8P
(M−) 433.1996, found 433.1991.
(Z)-Nerylphosphoryl-β-D-arabinofuranose (13). Prepared in
the same manner as described for compound 12 from intermediate
monophosphate salt S3 (30 mg, 0.026 mmol) and nerol (20 μL, 0.11
mmol). Compound 13 (7.6 mg, 76%) was generated as a clear
colorless oil: Rf = 0.26 (4% H2O/25% MeOH/71% CH2Cl2); 1H
NMR (400 MHz, CD3OD) δ 5.48 (t, J = 4.6 Hz, 1H), 5.41 (t, J = 6.9
Hz, 1H), 5.15−5.09 (m, 1H), 4.49−4.38 (m, 2H), 4.08 (dd, J = 8.0,
7.0 Hz, 1H), 3.96 (ddd, J = 8.1, 4.3, 2.1 Hz, 1H), 3.80−3.69 (m, 2H),
2132
dx.doi.org/10.1021/jo302507p | J. Org. Chem. 2013, 78, 2128−2133

The Journal of Organic Chemistry
Article
of DPA. While reaction times are shorter using microwave-assisted
conditions, our analysis of the authors’ NMR data suggests that mainly
the α-anomer was formed. This is consistent with our own attempt to
form the phosphodiester from intermediate 4: Shih, H.-W.; Chen, K.-
T.; Cheng, W.-C. Tetrahedron Lett. 2012, 53, 243−246.
(20) Maryanoff, B. E.; Reitz, A. B.; Tutwiler, G. F.; Benkovic, S. J.;
Benkovic, P. A.; Pilkis, S. J. J. Am. Chem. Soc. 1984, 106, 7851−7853.
(21) Maryanoff, B. E.; Reitz, A. B.; Nortey, S. O. Tetrahedron 1988,
44, 3093−3106.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: kiessling@chem.wisc.edu.
Notes
■
(22) Smellie, I. A.; Bhakta, S.; Sim, E.; Fairbanks, A. J. Org. Biomol.
Chem. 2007, 5, 2257−2266.
̀
(23) Dureau, R.; Legentil, L.; Daniellou, R.; Ferrieres, V. J. Org.
Chem. 2011, 77, 1301−1307.
The authors declare no competing financial interest.
(24) It should be noted that after prolonged storage (>6 months) at
−20 °C compound 11 equilibrated to the α-anomer. It is therefore
recommended that this intermediate is carried forward to avoid
erosion of the anomeric stereochemistry.
(25) Gillard, J. W.; Israel, M. Tetrahedron Lett. 1981, 22, 513−516.
(26) See the Supporting Information for 1H and 13C NMR spectra of
the glycosyl bromides.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health (R01-AI063596). The UWMadison Chemistry NMR
facility is supported by the NSF (CHE-9208463 and CHE-
9629688) and NIH (1 s10 RRO 8389). M.B.K. was supported
by an NIH postdoctoral fellowship (F32 GM100729). M.A.M.
was supported by an NIH predoctoral fellowship (F31
GM101953).
́
(27) Bohe, L.; Crich, D. C. R. Chim. 2011, 14, 3−16.
(28) Shuto, S.; Terauchi, M.; Yahiro, Y.; Abe, H.; Ichikawa, S.;
Matsuda, A. Tetrahedron Lett. 2000, 41, 4151−4155.
(29) Ichikawa, S.; Shuto, S.; Matsuda, A. J. Am. Chem. Soc. 1999, 121,
10270−10280.
DEDICATION
■
This paper is dedicated to the memory of our colleague
Howard Zimmerman, who inspired us with his relentless
pursuit of mechanistic understanding.
(30) Hosoya, T.; Ohashi, Y.; Matsumoto, T.; Suzuki, K. Tetrahedron
Lett. 1996, 37, 663−666.
(31) Heuckendorff, M.; Pedersen, C. M.; Bols, M. J. Org. Chem. 2012,
77, 5559−5568.
(32) Jensen, H. H.; Pedersen, C. M.; Bols, M. Chem.Eur. J. 2007,
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