Lipophilic sugar nucleotide synthesis by structure-based design of nucleotidylyltransferase substrates

Article abstract of DOI:10.1039/b716955h

Structure-based design of alkyl sugar-1-phosphates provides an efficient nucleotidylyltransferase-catalyzed synthesis of a series of new lipophilic sugar nucleotides possessing long or branched alkyl chains, thereby demonstrating the utility of nucleotidy

Full text of DOI:10.1039/b716955h

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Lipophilic sugar nucleotide synthesis by structure-based design of
nucleotidylyltransferase substrates†
Malcolm P. Huestis,a Gaia A. Aish,a Joseph P. M. Hui,b Evelyn C. Soob,c and David L. Jakeman*a,c
Received 2nd November 2007, Accepted 22nd November 2007
First published as an Advance Article on the web 11th December 2007
DOI: 10.1039/b716955h
Structure-based design of alkyl sugar-1-phosphates provides an efficient nucleotidylyltransferase-c
atalyzed synthesis of a series of new lipophilic sugar nucleotides possessing long or branched alkyl
chains, thereby demonstrating the utility of nucleotidylyltransferases to catalyze the synthesis of sugar
nucleotides with potential applications in lipopolysaccharide and lipoglycopeptide biosynthesis.
Sugar nucleotides play pivotal roles in glycobiology studies as
substrates for glycosyltransferases and other sugar nucleotide
processing enzymes involved in controlling a myriad of biolog-
ical processes.1–4 One key step in bacterial growth includes the
processing of sugar nucleotides into the O-antigen5 and lipid A6
components of gram negative bacterial cells. Inhibition of the
enzymes responsible for lipopolysaccharide biosynthesis offers
a potential route for new antibiotic therapy distinct to current
clinical antibiotics.7 Some natural products contain lipophilic
sugars, and in the case of teicoplanin, a lipoglycopeptide, the
N-decanoyl glucosamine component may explain the improved
activity relative to vancomycin, a glycopeptide, towards type B
strains of vancomycin-resistant enterococci.8 The glycosyltrans-
ferase responsible for attaching glucosamine is also able to transfer
N-decanoyl glucosamine,9 indicating that attachment of lipophilic
sugars onto natural products using glycosyltransferases is feasible.
Directed evolution has been used to significantly broaden the
substrate specificity of glycosyltransferases and may improve
catalytic efficiencies with lipophilic sugar nucleotides.10,11 The
synthetic approaches towards the prerequisite sugar nucleotides
that are involved in lipopolysaccharide or natural product biosyn-
thesis, or structurally related enzyme inhibitor synthesis, remain
challenging due to a variety of reasons. Some difficulties arise
from the lipophilic nature of the sugar nucleotides, the need to
develop more efficient phosphate–phosphate coupling procedures
to supersede classical morpholidate-mediated methodology12,13 or
the challenges associated with control of anomeric stereocontrol
observed in general carbohydrate synthesis.14–17
yields and lack of anomeric stereochemical control required
using this enzymatic approach to sugar nucleotide synthesis, the
substrate specificities of several homologous nucleotidylyltrans-
ferases have been explored with respect to both substrates.19–25
The enzymes studied from primary metabolic pathways accept
a variety of primarily a-D-sugar-1-phosphate analogues and a
variety of nucleoside triphosphates. Site-specific mutagenesis has
also been used to alter the sugar-1-phosphate specificity26,27 and
the base specificity28 of the nucleoside triphosphate to provide
enzymatic access to a variety of sugar nucleotides. Recently, three
homologous wild-type thymidylyltransferases (RmlA, Cps2L
and RmlA3) from different gram positive organisms (Strepto-
coccus mutans, Streptococcus pneumoniae and Aneurinibacillus
thermoaerophilus, respectively)29 were described to have broad
substrate specificity with a variety of nucleoside triphosphates,
pyranose-1-phosphates30 and furanose-1-phosphates.31 Herein, we
provide insight into a new aspect of the substrate specificity of
these recombinant wild-type enzymes. Based on an analysis of the
crystal structure of a homologous nucleotidylyltransferase, RmlA
from Pseudomonas aeruginosa, bound to the product a-D-glucose
thymidine diphosphate (Fig. 1),32 we observed a pocket adjacent to
One enzymatic approach to sugar nucleotide synthesis requires
nucleotidylyltransferases to efficiently couple sugar-1-phosphates
and nucleoside triphosphates (Scheme 1a).18 As a result of high
aCollege of Pharmacy, Burbidge Building, Dalhousie University, 5968 College
St., Halifax, Nova Scotia, Canada B3H 3J5. E-mail: david.jakeman@
dal.ca; Fax: 1 902 494 1396; Tel: 1 902 494 7159
bMS Metabolomics Group, NRC-Institute for Marine Biosciences, 1411
Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1. E-mail: evelyn.
soo@nrc-cnrc.gc.ca
cDepartment of Chemistry, Dalhousie University, Halifax, Nova Scotia,
Canada B3H 4J3
† Electronic supplementary information (ESI) available: Experimental
procedures and NMR data for compounds 2b–h to 8a–h, NMR spectra
for compounds 6a–h and 8a–h. Details describing enzymatic assays,
HPLC and ESI-MS/MS data for sugar nucleotides (94 pages). See DOI:
10.1039/b716955h
Fig. 1 A cut-away view of the structure of RmlA from Pseudomonas
aeruginosa (pdb: 1G1L) showing dTDP-a-D-glucose (ball-and-stick repre-
sentation) bound to the solvent accessible enzyme surface. Only residues
forming a pocket surrounding the glucose moiety are shown for clarity.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 477–484 | 477
©

View Article Online
Scheme 1 (a) Physiological reaction catalyzed by a-D-glucopyranosyl-1-phosphate thymidylyltransferases, and (b) synthesis of 3-O-alkyl-a-D-
glucopyranosyl phosphates: (a) RBr, NaH, DMF, 17–76%; (b) (i) Amberlite 120 H+, 5 : 1 MeOH–H2O, 60 ◦C; (ii) Ac2O, py, 32–100%; (c) (i) hydrazine
acetate, DMF; (ii) ClPO(OPh)2, DMAP, CH2Cl2, 23–61%; (d) (i) H2, PtO2, 1 : 1 EtOAc–EtOH; (ii) 2 : 2 : 1 MeOH–H2O–NEt3, 50 ◦C; (iii) Amberlite
120 H+, NH4OH, 42–100%.
the enzyme active site and anticipated binding of substrates with
chemical functionality extending from the C3-OH of the glucose
moiety into this pocket. A similar pocket was also observed in the
crystal structure of the enzyme with the inhibitor b-L-rhamnose
thymidine diphosphate (Fig. 2).
This pocket is directed away from the centre of catalysis where
the new phosphoryl bond is formed and would, therefore, still
enable formation of a productive ternary enzyme complex.32 These
results would provide the first support for nucleotidylyltrans-
ferases to affect the synthesis of lipophilic sugar nucleotides of
relevance to lipopolysaccharide or lipoglycopeptide biosynthesis.
The synthetic strategy employed in the chemical preparation
of the 3-O-alkyl-a-D-glucopyranosyl phosphates is outlined in
Scheme 1b. 1,2:5,6-Di-O-isopropylidene-a-D-glucofuranose (1)
was treated with sodium hydride and an appropriate alkyl bromide
to furnish the alkylated furanoses.33 Good yields were obtained
for all products except the branched derivatives 2g and 2h
where the steric bulk likely decreased reactivity. Acid hydrolysis
of the diacetonides followed by acetylation of the 3-O-alkyl
glucopyranoses afforded 4a–h,33 with the exception of compound
4a, which was acetylated directly from commercially available 3-
O-methyl-D-glucopyranose. The beta anomer was isolated in all
cases except for that of compound 4a. Selective deacetylation at
the anomeric centre was accomplished using hydrazine acetate,34
and after column chromatography, access to the fully protected 3-
O-alkyl-a-D-glucopyranosyl-1-phosphates (6a–h) was provided by
stereoselective phosphorylation using diphenyl chlorophosphate.35
Deprotection of the phenyl phosphate esters was effected using
platinum(IV) oxide-mediated catalytic hydrogenolysis,35 followed
by deacetylation in 2 : 2 : 1 MeOH–H2O–NEt3 resulting in the
triethylammonium salt of the target sugar-1-phosphates (7a–h).36
After cation ion exchange chromatography on Amberlite 120 H+
resin, the ammonium salts (8a–h) were isolated by titration to
pH 9–10 with ammonium hydroxide and lyophilized to remove
any residual ammonium acetate. The 1JC1,H1 coupling constants for
8a–8h were in the range 171–174 Hz, confirming the configuration
at the anomeric centre as alpha.37
Fig. 2 A cut-away view of the structure of RmlA from Pseudomonas
aeruginosa (pdb: 1G3L) showing dTDP-b-L-rhamnose (ball-and-stick
representation) bound to the solvent accessible enzyme surface. Only
residues forming a pocket surrounding the rhamnose moiety are shown
for clarity.
Enzyme assays were performed as described previously30 and
results are presented in Fig. 3 for Cps2L (for RmlA3 see ESI†).
478 | Org. Biomol. Chem., 2008, 6, 477–484
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
An alternative representation of the crystal structure (Fig. 4)
indicated to us that the observed pocket described in Fig. 1 is
formed primarily by the side-chains of the bc sheet and the a8
helix. Thus, the lipophilic functionality of the dodecyl- (8e) and
branched analogues (8g, 8h), may fit between the side-chains of
these two secondary structural elements, since the size of the
pocket is likely too small to accommodate these larger substrates
without a significant steric clash. The acceptance of the alkyl
sugar-1-phosphates by the nucleotidylyltransferases is potentially
aided by an ordered Bi–Bi catalytic reaction mechanism where
glucose-1-phosphate binds after dTTP, as has been observed for
homologous enzymes.26,39 Substrate binding and protein engineer-
ing studies are underway to substantiate these hypotheses.
Fig. 3 Nucleotidylyltransferase-catalyzed conversion of sugar-1-phos-
phates (8a–h) to (a) dTDP-sugars (9a–h) and (b) UDP-sugars (10a–h) by
Cps2L. Products 9c and 10a co-eluted with dTTP and UDP, respectively.
Product formation was confirmed by ESI-MS/MS (ESI†).38
Both Cps2L and RmlA3 afforded quantitative conversion of all
eight synthesized sugar-1-phosphates to the corresponding dTDP-
sugars over 48 h with the exception of 8f. In general, the longer
alkyl chain derivatives were converted to product more slowly than
the shorter chain derivatives. The two branched chain analogues
(8g, 8h) were also converted readily, indicating that within the
extended substrate binding pocket it is possible to accommodate
more bulky substrate analogues. The significant surfactant-like
properties and the limited solubility, in addition to the longer
alkyl chain, may be contributing factors to explain why 8f was
a particularly poor substrate. In otherwise identical reaction
conditions, neither enzyme was able to match the quantitative
conversion observed with dTTP when using UTP as the nucleoside
triphosphate substrate. RmlA3 obtained the highest conversion
with UTP and 8b (30% over the 48 h). This is in contrast to the high
level of activity of these enzymes with various commercial sugar-
1-phosphates,30 but consistent with the catalysis observed with
furanosyl-1-phosphates.31 We observed no GDP-sugar formation
on incubation of 8a–8h with GTP. The second ionization constant
of the phosphate (pKa2) may influence the ability of the phosphate
to act as a nucleophile in the enzymatic reaction. We observed
an increase of + 0.6 units in the pKa2 over the series 8b, 8d,
8g to 8e, consistent with an inductively donating effect of the
alkyl substituent upon the sugar-1-phosphate. The decrease in the
acidity of the phosphate as indicated by an increase in pKa2 does
not appear to significantly affect catalysis.
Fig. 4 RmlA from Pseudomonas aeruginosa (pdb: 1G1L) as a cartoon
representation showing dTDP-a-D-glucose (ball-and-stick representation)
with the glucose C3 hydroxyl pointing between the side-chains of the bc
sheet and the a8 helix.
In conclusion, structure-based substrate design of a series of
alkyl sugar-1-phosphates has demonstrated a remarkably diverse
new activity for thymidylyltransferases. This is the first report of
the enzymatic preparation of lipophilic sugar nucleotides. Thus,
these catalysts will likely be of significant importance in providing
access to more elaborate sugar nucleotides for glycobiology studies
involving lipopolysaccharide or lipoglycopeptide biosynthesis.
Experimental
General methods
Dichloromethane and THF were dried over alumina (Innovative
˚
Technology) and stored over 3 A molecular sieves. All other
reagents and solvents were purchased and used without fur-
ther purification. All reactions were monitored by thin layer
chromatography (TLC) using Silicycle precoated silica gel plates
(250 lm thickness). The TLC plates were visualized with a potas-
sium permanganate solution (3 g potassium permanganate, 20 g
potassium carbonate, 5 mL 5% aqueous sodium hydroxide, 300 mL
distilled water), a phenol solution (10 g phenol, 5 mL H2SO4,
95 mL ethanol), or ultraviolet light (k = 254 nm). Unless otherwise
specified, flash chromatographic purification was performed on a
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 477–484 | 479
©

View Article Online
Biotage SP1 HPFC. 1H, 13C and 31P NMR spectra were obtained
on a Bruker AVANCE-500 NMR Spectrometer operating at
frequencies of 500.13 MHz, 125.76 MHz, and 202.45 MHz,
respectively. Assignments are based on COSY and HSQC 2D
H2O (20 mL) and extracted with dichloromethane (2 × 20 mL).
The combined organic layers were washed with brine (10 mL)
and dried (Na2SO4), filtered and concentrated. The major product
was purified by flash chromatography to forge the selectively
deacetylated pyranose (5a–h). Under a nitrogen atmosphere, a
solution of the selectively deacetylated product (5a–h) (1.49 mmol)
and 4-dimethylaminopyridine (438 mg, 3.58 mmol) in anhydrous
dichloromethane (8 mL) was stirred for 15 min at rt. The flask was
cooled to −10 ◦C and charged with diphenyl chlorophosphate
(464 lL, 2.24 mmol). After 1.5 h, the mixture was diluted with
H2O (20 mL) and extracted with dichloromethane (2 × 15 mL).
The combined organic extracts were washed with brine (10 mL)
and dried (Na2SO4), filtered and concentrated. Purification by
flash chromatography afforded the title compounds (6a–g).
1
NMR experiments. For chloroform-d, H NMR chemical shifts
are reported as d in units of parts per million (ppm) downfield
from tetramethylsilane (d 0.0) and 13C NMR chemical shifts are
reported as d in units of parts per million (ppm) relative to the
residual solvent signal of chloroform-d (d 77.16). For methanol-
d4, 1H and 13C NMR chemical shifts are reported as d in units of
parts per million (ppm) relative to the residual solvent signal of
methanol-d4 (d 3.31 and d 49.00, respectively). 31P NMR chemical
shifts are reported as d in units of parts per million (ppm) relative
to 85% H3PO4. Enzymatic reactions were monitored by HPLC
performed on a Hewlett Packard Series 1050 instrument with
an Agilent Zorbax 5 lm Rx-C18 column (150 mm × 4.6 mm).
Compounds with a nucleotide base chromophore were monitored
using a UV detector (k = 254 nm). The linear gradient used was 90 :
10 A–B to 40 : 60 A–B over 8.0 min, followed by a plateau at 40 : 60
A–B from 8.0 to 10.0 min at 1.0 mL min−1 where A is an aqueous
buffer containing 12 mM NBu4Br, 10 mM KH2PO4, and 5% HPLC
grade CH3CN (pH 4.9) and B is HPLC grade CH3CN except for
assays containing compound 8f, whereby the gradient was run to
80% CH3CN instead of 60%. High resolution mass spectra were
recorded on a micrOTOF instrument (Bruker Dalton) running
in negative ion mode (ESI). Low resolution mass spectra were
recorded on an LCQDuo ion trap instrument (Thermo Finnigan)
running in positive ion mode (ESI).
An Agilent 1100 LC system was coupled to an Applied
Biosystems-MDS SCIEX hybrid triple quadrupole linear ion trap
(4000QTRAP) mass spectrometer equipped with a Turbo V source
for electrospray ionization for the ESI-MS/MS experiments. The
sample was analyzed by flow-injection analysis in 75 : 25 (v/v)
acetonitrile–de-ionized water using a flow-rate of 200 lL min−1.
Precursor ion scanning of m/z 323 ([UMP − H]−) and m/z
383 ([TDP − H]−) were used initially to selectively detect for
uridine- and thymidine-linked sugar nucleotides, respectively, in
the sample. ESI-MS/MS analysis in the enhanced product ion
(EPI) mode was then performed to confirm the presence of
the expected synthetic sugar nucleotide. The mass spectrometer
settings for precursor ion scanning were: ionspray voltage 4.5 kV;
mass range Q1 m/z 300–800; scan time 5 sec, Q1 and Q3 set
to unit resolution. For the ESI-MS/MS experiments: ionspray
voltage 4.5 kV; Q3 m/z 100–800; scan speed 1000 amu per sec;
trap fill time was set to dynamic; Q1 and Q3 set to unit resolution.
All acquisitions were made in the negative ion mode.
Diphenyl 2,4,6-tri-O-acetyl-3-O-methyl-a-D-glucopyranosyl-1-
phosphate (6a). Compound 5a (RF = 0.28 (hexanes–EtOAc, 60 :
40)), a colorless liquid, was provided by silica gel chromatography
(hexanes–EtOAc, 60 : 40) (556 mg, 72%) and immediately
carried onto the subsequent phosphorylation. Chromatographic
purification (hexanes–EtOAc, 72 : 28) furnished compound 6a as
a colorless liquid (552 mg, 67%); RF = 0.36 (hexanes–EtOAc, 67 :
33); 1H NMR (CDCl3) d 7.38–7.20 (m, 10H, Ph × 2), 6.06 (dd, 1H,
1JC,H = 180 Hz, J1,P = 6.5 Hz, J1,2 = 3.3 Hz, H-1), 5.07 (dd, 1H,
J4,5 = 10.2 Hz, J3,4 = 9.6 Hz, H-4), 4.90 (ddd, 1H, J2,3 = 10.0 Hz,
J1,2 = 3.1 Hz, 4J1,P = 3.1 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz,
J5,6a = 4.3 Hz, H-6a), 4.02 (ddd, 1H, J4,5 = 10.4 Hz, J5,6a = 4.2 Hz,
J5,6b = 2.1 Hz, H-5), 3.90 (dd, 1H, J6a,6b = 12.2 Hz, J5,6b = 2.2 Hz,
H-6b), 3.73 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 9.7 Hz, H-3), 3.45 (s, 3H,
CH3), 2.10, 2.00, 1.94 (s, 9H, C(O)CH3 × 3); 13C NMR (CDCl3)
d 170.8, 170.0, 169.4 (C(O)CH3 × 3), 150.6–120.1 (12C, Ph × 2),
2
3
95.6 (d, J1,P = 6.3 Hz, C-1), 77.9 (C-3), 71.9 (d, J1,P = 7.1 Hz,
C-2), 70.3 (C-5), 68.7 (C-4), 61.5 (C-6), 60.6 (CH3), 20.9, 20.8, 20.7
(C(O)CH3 × 3); 31P NMR (CDCl3) d −13.98 (s, 1P, P-1); LRMS
m/z calcd for C25H29O12P [M + Na]+: 575.1. Found 575.0.
Diphenyl
2,4,6-tri-O-acetyl-3-O-butyl-a-D-glucopyranosyl-1-
phosphate (6b). Compound 5b (RF = 0.43 (hexanes–EtOAc, 67 :
33)), a colorless liquid, was provided by silica gel chromatography
(hexanes–EtOAc, 72 : 28) (690 mg, 79%) and immediately
carried onto the subsequent phosphorylation. Chromatographic
purification (hexanes–EtOAc, 78 : 22) furnished compound 6b as
a colorless liquid (602 mg, 68%); RF = 0.26 (hexanes–EtOAc, 75 :
1
25); H NMR (CDCl3) d 7.38–7.20 (m, 10H, Ph × 2), 6.06 (dd,
1
1H, JC,H = 183 Hz, J1,P = 6.6 Hz, J1,2 = 3.3 Hz, H-1), 5.08 (dd,
1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4), 4.90 (ddd, 1H, J2,3
10.0 Hz, J1,2 = 3.2 Hz, 4J1,P = 3.2 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b
=
=
pKa values were measured in the following way: using an IQ
Scientific Instruments IQ150 fitted with an ISFET probe, a 0.01 M
solution of the diammonium salt was adjusted to pH 10 with 0.2 M
NaOH. Titration was done with 5 lL aliquots of 0.2 M HCl until
pH 2, and the pKa values were determined by plotting in GraFit
5.0.4 (Erithacus Software Limited).
12.5 Hz, J5,6a = 4.2 Hz, H-6a), 4.01 (ddd, 1H, J4,5 = 10.3 Hz,
J5,6a = 4.1 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.5 Hz,
J5,6b = 2.2 Hz, H-6b), 3.79 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.8 Hz,
H-3), 3.56 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 × 3),
1.46 (m, 2H, OCH2CH2), 1.30 (m, 2H, OCH2CH2CH2), 0.89 (t,
3H, J = 7.5 Hz, (CH2)3CH3); 13C NMR (CDCl3) d 170.7, 169.9,
General procedure for selective deacetylation and phosphorylation
(5a–h and 6a–h, respectively). To a stirring solution of the
acetylated glucopyranoside (4a–h) (2.41 mmol) in anhydrous
DMF (10 mL) was added hydrazine acetate (0.33 g, 3.62 mmol)
and the solution was subsequently heated to 60 ◦C for 15 min.
The mixture was then stirred for 1 h at rt before being diluted with
169.3 (C(O)CH3 × 3), 150.5–120.1 (12C, Ph × 2), 95.7 (d, 2J1,P
=
6.0 Hz, C-1), 76.5 (C-3), 73.0 (OCH2), 72.1 (d, 3J1,P = 7.1 Hz, C-2),
70.3 (C-5), 68.9 (C-4), 61.5 (C-6), 32.3 (OCH2CH2), 20.8, 20.7,
20.6 (C(O)CH3 × 3), 19.1 (OCH2CH2CH2), 13.9 ((CH2)3CH3);
31P NMR (CDCl3) d −14.00 (s, 1P, P-1); LRMS m/z calcd for
C28H35O12P [M + Na]+: 617.2. Found 617.2.
480 | Org. Biomol. Chem., 2008, 6, 477–484
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
Diphenyl 2,4,6-tri-O-acetyl-3-O-hexyl-a-D-glucopyranosyl-1-
phosphate (6c). Diethyl ether was used instead of dichloro-
methane as an extraction solvent en route to compound 5c (RF =
0.30 (hexanes–EtOAc, 70 : 30)), a colorless liquid, which was
provided by silica gel chromatography (hexanes–EtOAc, 63 : 37)
(772 mg, 82%) and immediately carried onto the subsequent
phosphorylation. Diethyl ether was again used instead of
dichloromethane as an extraction solvent for compound 6c,
a colorless liquid, which was isolated by chromatographic
purification (hexanes–EtOAc, 75 : 25) (538 mg, 58%); RF = 0.29
the phosphorylation, diethyl ether was again used instead of
dichloromethane as an extraction solvent, which was isolated by
chromatographic purification (hexanes–EtOAc, 80 : 20) to furnish
compound 6e as a colorless liquid (463 mg, 44%); RF = 0.38
1
(hexanes–EtOAc, 75 : 25); H NMR (CDCl3) d 7.38–7.20 (m,
10H, Ph × 2), 6.05 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.6 Hz, J1,2
=
3.4 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.3 Hz, J3,4 = 9.7 Hz, H-4),
4.90 (ddd, 1H, J2,3 = 10.0 Hz, J1,2 = 3.1 Hz, 4J1,P = 3.1 Hz, H-2),
2
4.14 (dd, 1H, J6a,6b = 12.5 Hz, J5,6a = 4.2 Hz, H-6a), 4.01 (ddd,
1H, J4,5 = 10.4 Hz, J5,6a = 4.1 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd,
1
(hexanes–EtOAc, 75 : 25); H NMR (CDCl3) d 7.38–7.20 (m,
1H, J6a,6b = 12.5 Hz, J5,6b = 2.1 Hz, H-6b), 3.79 (dd, 1H, J2,3 =
10H, Ph × 2), 6.06 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.5 Hz, J1,2
=
9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.55 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s,
9H, C(O)CH3 × 3), 1.47 (m, 2H, OCH2CH2), 1.33–1.19 (m, 18H,
OCH2CH2(CH2)9CH3), 0.88 (t, 3H, J = 6.7 Hz, O(CH2)11CH3);
13C NMR (CDCl3) d 170.8, 169.9, 169.3 (C(O)CH3 × 3), 150.6–
3.4 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4),
4
4.90 (ddd, 1H, J2,3 = 9.9 Hz, J1,2 = 3.0 Hz, J1,P = 3.0 Hz, H-2),
2
4.14 (dd, 1H, J6a,6b = 12.5 Hz, J5,6a = 4.2 Hz, H-6a), 4.01 (ddd,
2
1H, J4,5 = 10.4 Hz, J5,6a = 4.1 Hz, J5,6b = 2.0 Hz, H-5), 3.89 (dd,
120.1 (12C, Ph × 2), 95.7 (d, J1,P = 6.1 Hz, C-1), 76.6 (C-3),
3
1H, J6a,6b = 12.6 Hz, J5,6b = 2.1 Hz, H-6b), 3.79 (dd, 1H, J2,3
=
73.5 (OCH2), 72.1 (d, J1,P = 7.3 Hz, C-2), 70.4 (C-5), 68.9 (C-
9.7 Hz, J3,4 = 9.7 Hz, H-3), 3.55 (m, 2H, OCH2), 2.08, 2.00, 1.92
(s, 9H, C(O)CH3 × 3), 1.47 (m, 2H, OCH2CH2), 1.28 (m, 6H,
OCH2CH2(CH2)3CH3), 0.88 (t, 3H, J = 6.8 Hz, O(CH2)5CH3);
13C NMR (CDCl3) d 170.8, 169.9, 169.3 (C(O)CH3 × 3), 150.5–
120.1 (12C, Ph × 2), 95.7 (d, 2J1,P = 6.0 Hz, C-1), 76.5 (C-3), 73.4
(OCH2), 72.1 (d, 3J1,P = 7.3 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5
(C-6), 31.7, 25.8, 22.7 (OCH2CH2(CH2)3CH3), 30.3 (OCH2CH2),
20.9, 20.8, 20.6 (C(O)CH3 × 3), 14.2 (O(CH2)5CH3); 31P NMR
(CDCl3) d −14.00 (s, 1P, P-1); LRMS m/z calcd for C30H39O12P
[M + Na]+: 645.2. Found 645.1.
4), 61.5 (C-6), 32.1, 29.8, 29.8, 29.8, 29.8, 29.6, 29.5, 26.1, 22.8
(OCH2CH2(CH2)9CH3), 30.4 (OCH2CH2(CH2)9CH3), 20.9, 20.8,
20.7 (C(O)CH3 × 3), 14.2 (O(CH2)11CH3); 31P NMR (CDCl3) d
−14.00 (s, 1P, P-1); LRMS m/z calcd for C36H51O12P [M + Na]+:
729.3. Found 729.1.
Diphenyl 2,4,6-tri-O-acetyl-3-O-hexadecyl-a-D-glucopyranosyl-
1-phosphate (6f). Chloroform was used instead of dichloro-
methane as an extraction solvent en route to compound 5f (RF =
0.21 (hexanes–EtOAc, 67 : 33)), a white solid, which was provided
by silica gel chromatography (hexanes–EtOAc, 67 : 33) (524 mg,
41%) and immediately carried onto the subsequent phosphory-
lation. Chromatographic purification (hexanes–EtOAc, 80 : 20)
furnished compound 6f as a colorless liquid (625 mg, 55%); RF =
0.25 (hexanes–EtOAc, 80 : 20); 1H NMR (CDCl3) d 7.39–7.18 (m,
Diphenyl
2,4,6-tri-O-acetyl-3-O-octyl-a-D-glucopyranosyl-1-
phosphate (6d). Diethyl ether was used instead of dichloro-
methane as an extraction solvent en route to compound 5d
(RF = 0.27 (hexanes–EtOAc, 67 : 33)), a colorless liquid which
was provided by silica gel chromatography (hexanes–EtOAc,
63 : 37) (625 mg, 62%) and immediately carried onto the
subsequent phosphorylation. Chromatographic purification
(hexanes–EtOAc, 81 : 19) furnished compound 6d as a colorless
10H, Ph × 2), 6.06 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.6 Hz, J1,2
=
3.3 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4),
4.90 (ddd, 1H, J2,3 = 10.0 Hz, J1,2 = 3.0 Hz, 4J1,P = 3.0 Hz, H-2),
2
4.14 (dd, 1H, J6a,6b = 12.6 Hz, J5,6a = 4.3 Hz, H-6a), 4.01 (ddd,
1
liquid (717 mg, 74%); RF = 0.45 (hexanes–EtOAc, 67 : 33); H
1H, J4,5 = 10.3 Hz, J5,6a = 3.9 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd,
NMR (CDCl3) d 7.38–7.20 (m, 10H, Ph × 2), 6.06 (dd, 1H,
1JC,H = 181 Hz, J1,P = 6.6 Hz, J1,2 = 3.4 Hz, H-1), 5.08 (dd, 1H,
J4,5 = 10.2 Hz, J3,4 = 10.2 Hz, H-4), 4.90 (ddd, 1H, J2,3 = 10.1 Hz,
J1,2 = 3.2 Hz, 4J1,P = 3.2 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz,
J5,6a = 4.2 Hz, H-6a), 4.01 (ddd, 1H, J4,5 = 10.2 Hz, J5,6a = 3.9 Hz,
J5,6b = 2.0 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.6 Hz, J5,6b = 2.1 Hz,
H-6b), 3.78 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.55 (m,
2H, OCH2), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 × 3), 1.47 (m, 2H,
OCH2CH2), 1.34–1.19 (m, 10H, OCH2CH2(CH2)5CH3), 0.88 (t,
3H, J = 7.1 Hz, (CH2)7CH3); 13C NMR (CDCl3) d 170.8, 169.9,
1H, J6a,6b = 12.5 Hz, J5,6b = 2.2 Hz, H-6b), 3.79 (dd, 1H, J2,3 =
9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.54 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s,
9H, C(O)CH3 × 3), 1.47 (m, 2H, OCH2CH2), 1.33–1.19 (m, 26H,
OCH2CH2(CH2)13CH3), 0.88 (t, 3H, J = 7.1 Hz, O(CH2)15CH3);
13C NMR (CDCl3) d 170.8, 169.9, 169.3 (C(O)CH3 × 3), 150.6–
120.1 (12C, Ph × 2), 95.7 (d, 2J1,P = 6.2 Hz, C-1), 76.5 (C-3), 73.4
(OCH2), 72.1 (d, 3J1,P = 7.2 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5
(C-6), 32.0, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5,
26.1, 22.8 (OCH2CH2(CH2)13CH3), 30.3 (OCH2CH2(CH2)13CH3),
20.9, 20.7, 20.6 (C(O)CH3 × 3), 14.2 (O(CH2)15CH3); 31P NMR
(CDCl3) d −14.00 (s, 1P, P-1); LRMS m/z calcd for C40H59O12P
[M + Na]+: 785.4. Found 785.4.
169.3 (C(O)CH3 × 3), 150.6–120.1 (12C, Ph × 2), 95.7 (d, 2J1,P
=
3
6.1 Hz, C-1), 76.5 (C-3), 73.5 (OCH2), 72.1 (d, J1,P = 7.2 Hz,
C-2), 70.4 (C-5), 68.9 (C-4), 61.5 (C-6), 32.0, 29.5, 29.4, 26.1, 22.8
(OCH2CH2(CH2)5CH3), 30.3 (OCH2CH2(CH2)5CH3), 20.9, 20.8,
20.6 (C(O)CH3 × 3), 14.2 ((CH2)7CH3); 31P NMR (CDCl3) d
−14.00 (s, 1P, P-1); LRMS m/z calcd for C32H43O12P [M + Na]+:
673.2. Found 673.3.
Diphenyl
2,4,6-tri-O-acetyl-3-O-(2-methylpropyl)-a-D-gluco-
pyranosyl-1-phosphate (6g). Compound 5g (RF = 0.23 (hexanes–
EtOAc, 67 : 33)), a colorless liquid, was provided by silica gel
chromatography (hexanes–EtOAc, 60 : 40) (341 mg, 39%)
and immediately carried onto the subsequent phosphorylation.
Chromatographic purification (hexanes–EtOAc, 71 : 29) furnished
compound 6g as a colorless liquid (585 mg, 66%); RF = 0.36
Diphenyl 2,4,6-tri-O-acetyl-3-O-dodecyl-a-D-glucopyranosyl-1-
phosphate (6e). Diethyl ether was used instead of dichloro-
methane as an extraction solvent en route to compound 5e (RF =
0.29 (hexanes–EtOAc, 67 : 33)), a colorless liquid, which was
not purified by silica gel chromatography (675 mg, 59%). For
1
(hexanes–EtOAc, 66 : 33); H NMR (CDCl3) d 7.39–7.18 (m,
10H, Ph × 2), 6.05 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.5 Hz, J1,2
=
3.2 Hz, H-1), 5.10 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4),
Org. Biomol. Chem., 2008, 6, 477–484 | 481
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
4
1
4.92 (ddd, 1H, J2,3 = 9.9 Hz, J1,2 = 3.0 Hz, J1,P = 3.0 Hz, H-2),
(dd, 1H, JC,H = 176 Hz, J1,P = 7.2 Hz, J1,2 = 3.0 Hz, H-1), 3.80
(ddd, 1H, J4,5 = 9.6 Hz, J5,6b = 4.7 Hz, J5,6a = 2.0 Hz, H-5), 3.75
2
4.14 (dd, 1H, J6a,6b = 12.6 Hz, J5,6a = 4.2 Hz, H-6a), 4.01 (ddd,
2
1H, J4,5 = 10.4 Hz, J5,6a = 4.2 Hz, J5,6b = 2.3 Hz, H-5), 3.90 (dd,
(dd, 1H, J6a,6b = 12.2 Hz, J5,6a = 2.0 Hz, H-6a), 3.65 (dd, 1H,
1H, J6a,6b = 12.5 Hz, J5,6b = 2.2 Hz, H-6b), 3.78 (dd, 1H, J2,3
=
J6a,6b = 12.4 Hz, J5,6b = 4.9 Hz, H-6b), 3.52 (s, 3H, CH3), 3.47 (m,
9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.37 (dd, 1H, J = 6.2 Hz, 2J = 8.4 Hz,
1H, H-2), 3.44 (m, 1H, H-3), 3.39 (m, 1H, H-4); 13C NMR (D2O) d
2
OCH2a), 3.28 (dd, 1H, J = 6.4 Hz, J = 8.8 Hz, OCH2b), 2.08,
94.2 (d, 2J1,P = 5.7 Hz, C-1), 82.8 (C-3), 72.3 (C-5), 71.4 (d, 3J1,P
=
2.00, 1.92 (s, 9H, C(O)CH3 × 3), 1.75 (nonet, 1H, J = 6.6 Hz,
OCH2CH), 0.84 (d, 6H, J = 6.6 Hz, OCH2CH(CH3)2); 13C
NMR (CDCl3) d 170.8, 170.0, 169.3 (C(O)CH3 × 3), 150.6–120.2
7.5 Hz, C-2), 69.0 (C-4), 60.5 (C-6), 60.0 (CH3); 31P NMR (D2O)
d 0.77 (s, 1P, P-1); HRMS m/z calcd for C7H13O9P2− [M + H]−:
273.0386. Found 273.0390.
2
(12C, Ph × 2), 95.8 (d, J1,P = 6.1 Hz, C-1), 80.3 (OCH2), 76.7
3-O-Butyl-a-D-glucopyranosyl phosphate diammonium salt (8b).
Compound 8b was obtained as a colorless foam (63 mg, 47%);
3
(C-3), 72.1 (d, J1,P = 7.2 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5
(C-6), 29.0 (OCH2CH), 20.9, 20.8, 20.6 (C(O)CH3 × 3), 19.2,
19.2 (OCH2CH(CH3)2); 31P NMR (CDCl3) d −14.00 (s, 1P, P-1);
LRMS m/z calcd for C28H35O12P [M + Na]+: 617.2. Found 617.2.
1H NMR (D2O) d 5.33 (dd, 1H, JC,H = 172 Hz, J1,P = 7.4 Hz,
1
J1,2 = 3.4 Hz, H-1), 3.79 (ddd, 1H, J4,5 = 10.1 Hz, J5,6b = 5.0 Hz,
2
J5,6a = 2.2 Hz, H-5), 3.75 (dd, 1H, J6a,6b = 12.5 Hz, J5,6a
=
2.2 Hz, H-6a), 3.72 (t, 2H, J = 6.7 Hz, OCH2), 3.63 (dd, 1H,
Diphenyl
2,4,6-tri-O-acetyl-3-O-(2-ethylbutyl)-a-D-gluco-
0.23
J6a,6b = 12.5 Hz, J5,6b = 5.2 Hz, H-6b), 3.50 (dd, 1H, J2,3
9.5 Hz, J3,4 = 9.2 Hz, H-3), 3.42 (ddd, 1H, J2,3 = 9.6 Hz, J1,2
=
=
=
pyranosyl-1-phosphate (6h). Compound 5h (RF
=
(hexanes–EtOAc, 67 : 33)), a white solid, was provided by silica
gel chromatography (hexanes–EtOAc, 61 : 39) (612 mg, 65%)
and immediately carried onto the subsequent phosphorylation.
Chromatographic purification (hexanes–EtOAc, 78 : 22) furnished
compound 6h as a colorless liquid (724 mg, 78%); RF = 0.24
(hexanes–EtOAc, 78 : 22); 1H NMR (CDCl3) d 7.39–7.17 (m, 10H,
Ph × 2), 6.05 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.6 Hz, J1,2 = 3.4 Hz,
H-1), 5.09 (dd, 1H, J4,5 = 10.2 Hz, J3,4 = 9.5 Hz, H-4), 4.91 (ddd,
4
3.3 Hz, J2,P = 2.0 Hz, H-2), 3.33 (dd, 1H, J3,4 = 9.6 Hz, J4,5
9.6 Hz, H-4), 1.49 (p, 2H, J = 7.0 Hz, OCH2CH2), 1.26 (sextet,
2H, J = 7.5 Hz, O(CH2)2CH2CH3), 0.79 (t, 3H, J = 7.8 Hz,
O(CH2)3CH3); 13C NMR (D2O) d 94.1 (d, J1,P = 5.5 Hz, C-1),
2
81.5 (C-3), 73.1 (OCH2), 72.3 (C-5), 71.8 (d, 3J1,P = 7.4 Hz, C-2),
69.3 (C-4), 60.7 (C-6), 31.5 (OCH2CH2), 18.5 (OCH2CH2CH2),
13.1 (O(CH2)3CH3); 31P NMR (D2O) d 1.32 (s, 1P, P-1); HRMS
m/z calcd for C10H19O9P2− [M + H]−: 315.0856. Found 315.0856.
4
1H, J2,3 = 10.0 Hz, J1,2 = 3.2 Hz, J1,P = 3.2 Hz, H-2), 4.13 (dd,
1H, 2J6a,6b = 12.6 Hz, J5,6a = 4.4 Hz, H-6a), 4.01 (ddd, 1H, J4,5
10.4 Hz, J5,6a = 4.2 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd, 1H, J6a,6b
=
=
=
3-O-Hexyl-a-D-glucopyranosyl phosphate diammonium salt (8c).
Compound 8c was obtained as a colorless foam (110 mg, 76%);
12.5 Hz, J5,6b = 2.2 Hz, H-6b), 3.78 (dd, 1H, J2,3 = 9.7 Hz, J3,4
1H NMR (D2O) d 5.34 (dd, 1H, JC,H = 173 Hz, J1,P = 6.9 Hz,
1
2
9.7 Hz, H-3), 3.52 (dd, 1H, J = 4.6 Hz, J = 9.0 Hz, OCH2a),
J1,2 = 3.0 Hz, H-1), 3.78 (m, 1H, H-5), 3.74 (m, 1H, H-6a), 3.70
2
3.42 (dd, 1H, J = 4.4 Hz, J = 8.8 Hz, OCH2b), 2.08, 2.00, 1.92
(t, 2H, J = 6.6 Hz, OCH2), 3.64 (dd, 1H, J6a,6b = 12.0 Hz, J5,6b
=
(s, 9H, C(O)CH3 × 3), 1.28 (m, 5H, OCH2CH(CH2CH3)2), 0.83
(t, 6H, J = 7.0 Hz, OCH2CH(CH3)2); 13C NMR (CDCl3) d 170.8,
4.5 Hz, H-6b), 3.49 (dd, 1H, J2,3 = 9.3 Hz, J3,4 = 9.3 Hz, H-3), 3.44
(ddd, 1H, J2,3 = 9.7 Hz, H-2), 3.35 (dd, 1H, J3,4 = 9.5 Hz, J4,5
=
170.0, 169.3 (C(O)CH3 × 3), 150.5–120.2 (12C, Ph × 2), 95.7 (d,
9.5 Hz, H-4), 1.50 (p, 2H, J = 7.0 Hz, OCH2CH2), 1.22 (m, 6H,
2J1,P = 6.1 Hz, C-1), 76.6 (C-3), 75.4 (OCH2), 72.3 (d, J1,P
=
3
OCH2CH2(CH2)3CH3), 0.76 (m, 3H, O(CH2)5CH3); 13C NMR
7.3 Hz, C-2), 70.4 (C-5), 69.0 (C-4), 61.5 (C-6), 42.0 (OCH2CH),
23.2, 23.2 (OCH2CH(CH2CH3)2), 20.9, 20.8, 20.6 (C(O)CH3 ×
3), 11.3, 11.3 (OCH2CH(CH2CH3)2); 31P NMR (CDCl3) d −13.99
(s, 1P, P-1); LRMS m/z calcd for C30H39O12P [M + Na]+: 645.2.
Found 645.2.
2
(D2O) d 94.4 (d, J1,P = 5.7 Hz, C-1), 81.4 (C-3), 73.4 (OCH2),
3
72.4 (C-5), 71.6 (d, J1,P = 7.7 Hz, C-2), 69.1 (C-4), 60.6 (C-6),
30.9, 24.8, 22.0 (OCH2CH2(CH2)3CH3), 29.2 (OCH2CH2), 13.4
(O(CH2)5CH3); 31P NMR (D2O) d 0.41 (s, 1P, P-1); HRMS m/z
calcd for C12H23O9P2− [M + H]−: 343.1169. Found 343.1176.
General procedure for deprotection and ion exchange (7a–h and
8a–h, respectively). A solution of phosphorylated pyranoside
(0.384 mmol) and platinium(IV) oxide (0.242 mmol) in 1 : 1 EtOH–
EtOAc (4 mL) was shaken under H2 in a Parr apparatus at 54
PSI for 2 h. The reaction mixture was filtered through Celite and
concentrated. The residue was ta◦ken up in 2 : 2 : 1 MeOH–H2O–
NEt3 (10 mL) and stirred at 50 C for 36 h. Again, the mixture
was concentrated and the residue was partitioned between H2O
(10 mL) and EtOAc (10 mL). The aqueous layer containing the
triethyl ammonium salt (7a–h) was passed through Amberlite IR-
120 PLUS(H) ion exchange resin. The resulting acidic aqueous
fraction was immediately adjusted to pH 8 with NH4OH 0.1 M,
concentrated to 5 mL, and lyophilized. Titration with NH4OH
0.1 M and lyophilization was repeated twice more to remove
NH4OAc and isolate the ammonium salt (8a–h).
3-O-Octyl-a-D-glucopyranosyl phosphate diammonium salt (8d).
Diethyl ether was used instead of ethyl acetate as an extraction
solvent en route to compound 8d, a colorless foam (66 mg, 42%);
1
1H NMR (D2O) d 5.36 (dd, 1H, JC,H = 173 Hz, J1,P = 7.2 Hz,
J1,2 = 3.2 Hz, H-1), 3.78 (ddd, 1H, J4,5 = 9.8 Hz, J5,6b = 5.0 Hz,
J5,6a = 2.1 Hz, H-5), 3.75 (m, 1H, H-6a), 3.72 (t, 2H, J = 7.0 Hz,
OCH2), 3.66 (dd, 1H, J6a,6b = 12.3 Hz, J5,6b = 4.9 Hz, H-6b), 3.50
(m, 1H, H-3), 3.45 (m, 1H, H-2), 3.37 (dd, 1H, J3,4 = 9.4 Hz, J4,5
=
9.4 Hz, H-4), 1.50 (p, 2H, J = 7.2 Hz, OCH2CH2), 1.30–1.12 (m,
10H, OCH2CH2(CH2)5CH3), 0.77 (t, 3H, J = 7.0 Hz, (CH2)7CH3);
2
13C NMR (D2O) d 94.6 (d, J1,P = 5.9 Hz, C-1), 81.4 (C-3), 73.5
(OCH2), 72.5 (C-5), 71.5 (d, 3J1,P = 7.7 Hz, C-2), 69.1 (C-4), 60.5
(C-6), 31.1, 28.5, 28.4, 25.1, 22.0 (OCH2CH2(CH2)5CH3), 29.3
(OCH2CH2), 13.4 (O(CH2)7CH3); 31P NMR (D2O) d 0.12 (s, 1P, P-
1); HRMS m/z calcd for C14H28O9P2− [M + H]−: 371.1482. Found
371.1490.
3-O-Methyl-a-D-glucopyranosyl phosphate diammonium salt
(8a)40. Deacetylation was performed at rt and compound 8a was
isolated as a colorless foam (50 mg, 42%); 1H NMR (D2O) d 5.35
3-O-Dodecyl-a-D-glucopyranosyl phosphate diammonium salt
(8e). Deacetylation was performed at 70 ◦C instead of 50 ◦C
482 | Org. Biomol. Chem., 2008, 6, 477–484
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
and diethyl ether was used instead of ethyl acetate as an extraction
solvent. Triethylamine (1 mL) was added before rotary evaporation
to suppress the surfactant-like behavior of the salt. After the
third NH4OAc sublimation, the residue was dissolved in water
and filtered through a short pad of Bio-Rad Chelex 100. The
solution was then passed through Amberlite IR-120 PLUS(H) ion
exchange resin, titrated with NH4OH 0.1 M, and lyophilized to
HRMS m/z calcd for C10H19O9P2− [M + H]−: 315.0856. Found
315.0869.
3-O-(2-Ethylbutyl)-a-D-glucopyranosyl phosphate diammonium
salt (8h). Compound 8h was obtained as a colorless foam
1
1
(145 mg, 76%); H NMR (D2O) d 5.32 (dd, 1H, JC,H = 172 Hz,
J1,P = 4.6 Hz, J1,2 = 3.0 Hz, H-1), 3.79 (m, 1H, H-5), 3.75
2
(d, 1H, J6a,6b = 12.6 Hz, H-6a), 3.63 (m, 1H, H-6b), 3.61
forge compound 8e as a colorless foam (103 mg, 58%); 1H NMR
(m, 2H, OCH2), 3.49 (dd, 1H, J2,3 = 9.2 Hz, J3,4 = 9.2 Hz,
1
(MeOD) d 5.48 (dd, 1H, JC,H = 173 Hz, J1,P = 6.9 Hz, J1,2
=
H-3), 3.42 (d, 1H, J2,3 = 9.6 Hz, H-2), 3.33 (dd, 1H, J3,4
=
3.3 Hz, H-1), 3.85 (ddd, 1H, J4,5 = 9.8 Hz, J5,6b = 5.7 Hz, J5,6a
=
9.2 Hz, J4,5 = 9.2 Hz, H-4), 1.39 (m, 1H, OCH2CH(CH2CH3)2),
1.25 (m, 4H, OCH2CH(CH2CH3)2), 0.76 (t, 6H, J = 6.7 Hz,
2.1 Hz, H-5), 3.81 (m, 2H, OCH2), 3.80 (m, 1H, H-6a), 3.63 (dd,
1H, J6a,6b = 11.6 Hz, J5,6b = 5.5 Hz, H-6b), 3.47 (dd, 1H, J2,3
=
OCH2CH(CH2CH3)2); 13C NMR (D2O) d 94.1 (d, 2J1,P = 5.6 Hz,
9.0 Hz, J3,4 = 9.0 Hz, H-3), 3.42 (ddd, 1H, J2,3 = 9.6 Hz, J1,2
=
3
C-1), 81.7 (C-3), 75.7 (OCH2), 72.3 (C-5), 71.8 (d, J1,P
=
3.0 Hz, 4J1,P = 3.0 Hz, H-2), 3.32 (m, 1H, H-4), 1.62 (p, 2H, J =
7.3 Hz, OCH2CH2), 1.41–1.23 (m, 18H, OCH2CH2(CH2)9CH3),
0.90 (t, 3H, J = 7.0 Hz, (CH2)11CH3); 13C NMR (MeOD) d 96.6
7.3 Hz, C-2), 69.3 (C-4), 60.7 (C-6), 41.0 (OCH2CH), 22.5,
22.4 (OCH2CH(CH2CH3)2), 10.2, 10.2 (OCH2CH(CH2CH3)2); 31P
NMR (D2O) d 1.62 (s, 1P, P-1); HRMS m/z calcd for C12H23O9P2−
[M + H]−: 343.1169. Found 343.1159.
2
(d, J1,P = 6.2 Hz, C-1), 83.6 (C-3), 74.6 (OCH2), 74.5 (C-5),
3
74.0 (d, J1,P = 7.5 Hz, C-2), 71.4 (C-4), 62.8 (C-6), 33.1, 30.8,
30.8, 30.8, 30.8, 30.7, 30.5, 27.1, 23.7 (OCH2CH2(CH2)9CH3), 31.4
(OCH2CH2), 14.4 (O(CH2)11CH3); 31P NMR (MeOD) d −1.86 (s,
1P, P-1); HRMS m/z calcd for C18H35O9P2− [M + H]−: 427.2108.
Found 427.2115.
General procedure for nucleotidylyltransferase-catalyzed syn-
thesis of sugar nucleotides (9a–h, 10a–h). Enzymatic reactions
were performed according to the method of Timmons (MgCl2,
1.0 mM NTP, 2.0 mM S1P, 0.5EU inorganic pyrophosphatase,
2EU nucleotidylyltransferase in Tris-HCl buffer-total 50 lL)30
except that MgCl2 was used at a final concentration of 5.5 mM
and incubations were performed at 41 ◦C. Sugar nucleotides were
characterized by ESI-MS/MS (ESI†).
3-O-Hexadecyl-a-D-glucopyranosyl phosphate diammonium salt
(8f). Deacetylation was performed at 70 ◦C instead of 50 ◦C
and diethyl ether was used instead of ethyl acetate as an extraction
solvent. Triethylamine (1 mL) was added before rotary evaporation
to suppress the surfactant-like behavior of the salt. After the
third NH4OAc sublimation, the residue was dissolved in water
and filtered through a short pad of Bio-Rad Chelex 100. The
solution was then passed through Amberlite IR-120 PLUS(H) ion
exchange resin, titrated with NH4OH 0.1 M, and lyophilized to
forge compound 8f as a colorless foam (179 mg, 90%). Coupling
constants are omitted from the 1H NMR spectrum since the
compound’s insolubility in a variety of NMR solvents led to
Notes and references
1 R. S. Narayan and M. S. VanNieuwenhze, Eur. J. Org. Chem., 2007,
1399–1414.
2 A. V. Nikolaev, I. V. Botvinko and A. J. Ross, Carbohydr. Res., 2007,
342, 297–344.
3 A. L. Lovering, L. H. de Castro, D. Lim and N. C. Strynadka, Science,
2007, 315, 1402–1405.
4 D. Kapitonov and R. K. Yu, Glycobiology, 1999, 9, 961–978.
5 C. Whitfield, Annu. Rev. Biochem., 2006, 75, 39–68.
6 A. H. Williams, R. M. Immormino, D. T. Gewirth and C. R. Raetz,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 10877–10882.
7 C. R. Raetz and C. Whitfield, Annu. Rev. Biochem., 2002, 71, 635–700.
8 R. G. Kruger, W. Lu, M. Oberthur, J. Tao, D. Kahne and C. T. Walsh,
Chem. Biol., 2005, 12, 131–140.
significant line-broadening; 1H NMR (D2O) d 5.44 (d, 1H, 1JC,H
=
174 Hz, H-1), 3.86 (m, 1H, H-5), 3.86 (m, 1H, H-6a), 3.74 (m,
2H, OCH2), 3.69 (m, 1H, H-6b), 3.56 (m, 1H, H-2), 3.52 (m,
1H, H-3), 3.39 (m, 1H, H-4), 1.58 (m, 2H, OCH2CH2), 1.42–1.15
(m, 26H, OCH2CH2(CH2)13CH3), 0.84 (m, 3H, (CH2)15CH3); 13
C
NMR (D2O) d 94.9 (d, 2J1,P = 5.1 Hz, C-1), 81.0 (C-3), 72.7 (C-5),
72.2 (OCH2), 71.1 (d, 3J1,P = 7.3 Hz, C-2), 69.4 (C-4), 61.0 (C-6),
32.1, 30.3, 30.3, 30.3, 30.3, 30.3, 30.3, 30.3, 30.1, 30.1, 30.0, 29.7,
26.2, 22.7 (OCH2(CH2)14CH3), 13.8 (O(CH2)15CH3); 31P NMR
(D2O) d −0.57 (s, 1P, P-1); HRMS m/z calcd for C22H43O9P2−
[M + H]−: 483.2734. Found 483.2708.
9 A. R. Howard-Jones, R. G. Kruger, W. Lu, J. Tao, C. Leimkuhler, D.
Kahne and C. T. Walsh, J. Am. Chem. Soc., 2007, 129, 10082–10083.
10 A. Aharoni, K. Thieme, C. P. Chiu, S. Buchini, L. L. Lairson, H. Chen,
N. C. Strynadka, W. W. Wakarchuk and S. G. Withers, Nat. Methods,
2006, 3, 609–614.
11 G. J. Williams, C. Zhang and J. S. Thorson, Nat. Chem. Biol., 2007, 3,
657–662.
12 J. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc., 1961, 39, 649–659.
13 Z. B. Zhao, L. Hong and H. W. Liu, J. Am. Chem. Soc., 2005, 127,
7692–7693.
14 M. Arlt and O. Hindsgaul, J. Org. Chem., 1995, 60, 14–15.
15 T. Uchiyama and O. Hindsgaul, J. Carbohydr. Chem., 1998, 17, 1181–
1190.
3-O-(2-Methylpropyl)-a-D-glucopyranosyl phosphate diammo-
nium salt (8g). Compound 8g was obtained as a colorless foam
1
1
(69 mg, 51%); H NMR (D2O) d 5.29 (dd, 1H, JC,H = 171 Hz,
J1,P = 7.3 Hz, J1,2 = 3.3 Hz, H-1), 3.76 (ddd, 1H, J4,5 = 10.0 Hz,
J5,6b = 4.8 Hz, J5,6a = 1.8 Hz, H-5), 3.72 (dd, 1H, 2J6a,6b = 12.3 Hz,
J5,6a = 1.9 Hz, H-6a), 3.60 (dd, 1H, J6a,6b = 12.1 Hz, J5,6b = 5.2 Hz,
H-6b), 3.47 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.4 Hz, H-3), 3.46 (d, 2H,
J = 6.1 Hz, OCH2), 3.41 (m, 1H, H-2), 3.31 (dd, 1H, J3,4 = 9.5 Hz,
J4,5 = 9.5 Hz, H-4), 1.74 (nonet, 1H, J = 6.5 Hz, OCH2CH), 0.81,
0.81 (d, 3H, J = 6.7 Hz, OCH2CH(CH3)2); 13C NMR (D2O) d 94.0
(d, 2J1,P = 5.6 Hz, C-1), 81.7 (C-3), 80.1 (OCH2), 72.2 (C-5), 71.8
16 C. Ernst and W. Klaffke, J. Org. Chem., 2003, 68, 5780–5783.
17 S. C. Timmons and D. L. Jakeman, Org. Lett., 2007, 9, 1227–1230.
18 J. Q. Jiang, J. B. Biggins and J. S. Thorson, J. Am. Chem. Soc., 2000,
122, 6803–6804.
19 J. Q. Jiang, C. Albermann and J. S. Thorson, ChemBioChem, 2003, 4,
443–446.
20 J. Bae, K. H. Kim, D. Kim, Y. Choi, J. S. Kim, S. Koh, S. I. Hong and
D. S. Lee, ChemBioChem, 2005, 6, 1963–1966.
21 R. M. Mizanur, C. J. Zea and N. L. Pohl, J. Am. Chem. Soc., 2004, 126,
15993–15998.
3
(d, J1,P = 7.2 Hz, C-2), 69.3 (C-4), 60.7 (C-6), 28.2 (OCH2CH),
22 K. S. Ko, C. J. Zea and N. L. Pohl, J. Am. Chem. Soc., 2004, 126,
18.6, 18.6 (OCH2CH(CH3)2); 31P NMR (D2O) d 1.76 (s, 1P, P-1);
13188–13189.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 477–484 | 483
©

View Article Online
23 Z. Zhang, M. Tsujimura, J. Akutsu, M. Sasaki, H. Tajima and Y.
Kawarabayasi, J. Biol. Chem., 2005, 280, 9698–9705.
24 J. M. Murrell, W. Liu and B. Shen, J. Nat. Prod., 2004, 67, 206–213.
25 J. C. Errey, B. Mukhopadhyay, K. P. R. Kartha and R. A. Field, Chem.
Commun., 2004, 23, 2706–2707.
26 W. A. Barton, J. Lesniak, J. B. Biggins, P. D. Jeffrey, J. Q. Jiang, K. R.
Rajashankar, J. S. Thorson and D. B. Nikolov, Nat. Struct. Biol., 2001,
8, 545–551.
27 W. A. Barton, J. B. Biggins, J. Jiang, J. S. Thorson and D. B. Nikolov,
Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 13397–13402.
28 R. Moretti and J. S. Thorson, J. Biol. Chem., 2007, 282, 16942–16947.
29 M. Graninger, B. Kneidinger, K. Bruno, A. Scheberl and P. Messner,
Appl. Environ. Microbiol., 2002, 68, 3708–3715.
32 W. Blankenfeldt, M. Asuncion, J. S. Lam and J. H. Naismith, EMBO J.,
2000, 19, 6652–6663.
33 W. Tjarks, A. K. M. Anisuzzaman, L. Liu, A. H. Soloway, R. F.
Barth, D. J. Perkins and D. M. Adams, J. Med. Chem., 1992, 35, 1628–
1633.
34 G. Excoffier, D. Gagnare and J. P. Utille, Carbohydr. Res., 1975, 39,
368–373.
35 Z. Dinev, A. Z. Wardak, R. T. Brownlee and S. J. Williams, Carbohydr.
Res., 2006, 341, 1743–1747.
36 S. Sabesan and S. Neira, Carbohydr. Res., 1992, 223, 169–185.
37 J. Duus, C. H. Gotfredsen and K. Bock, Chem. Rev., 2000, 100, 4589–
4614.
38 D. J. McNally, J. P. Hui, A. J. Aubry, K. K. Mui, P. Guerry, J. R. Brisson,
S. M. Logan and E. C. Soo, J. Biol. Chem., 2006, 281, 18489–18498.
39 A. C. Lamerz, T. Haselhorst, A. K. Bergfeld, M. von Itzstein and R.
Gerardy-Schahn, J. Biol. Chem., 2006, 281, 16314–16322.
40 J. Jiang, C. Albermann and J. Thorson, ChemBioChem, 2003, 4, 443–
446.
30 S. C. Timmons, R. H. Mosher, S. A. Knowles and D. L. Jakeman, Org.
Lett., 2007, 9, 857–860.
31 S. C. Timmons, J. P. M. Hui, J. Pearson, P. Peltier, R. Daniellou, C.
Cugier-Chauvin, E. C. Soo, R. T. Syvitski, V. Ferrieres and D. L.
Jakeman, Org. Lett., 2007, DOI: 10.1021/ol7023949.
484 | Org. Biomol. Chem., 2008, 6, 477–484
This journal is
The Royal Society of Chemistry 2008
©