Protecting-group-free synthesis of glycosyl 1-phosphates

Article abstract of DOI:10.1021/ol3019083

Glycosyl 1-phosphates enriched in the α-anomer are obtained without the use of protecting groups in two steps starting from the free hemiacetal. Condensation of free hemiacetals with toluenesulfonylhydrazide yields a range of glycosylsulfonohydrazide donors which can be oxidized using cupric chloride in the presence of phosphoric acid and the coordinating additive 2-methyl-2-oxazoline to give useful yields of the fully deprotected glycosyl 1-phosphates.

Full text of DOI:10.1021/ol3019083

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Protecting-Group-Free Synthesis
of Glycosyl 1ꢀPhosphates
Landon John G. Edgar, Somnath Dasgupta, and Mark Nitz*
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto,
Ontario, Canada M5S 3H6
mnitz@chem.utoronto.ca
Received July 10, 2012
ABSTRACT
Glycosyl 1-phosphates enriched in the R-anomer are obtained without the use of protecting groups in two steps starting from the free hemiacetal.
Condensation of free hemiacetals with toluenesulfonylhydrazide yields a range of glycosylsulfonohydrazide donors which can be oxidized using
cupric chloride in the presence of phosphoric acid and the coordinating additive 2-methyl-2-oxazoline to give useful yields of the fully deprotected
glycosyl 1-phosphates.
Glycosyl 1-phosphates are central intermediates in
carbohydrate metabolism and the biosynthesis of oligo-
saccharides.1 To access these useful intermediates a
variety of protocols, both chemical and enzymatic, have
been developed. Enzymatically, glycosyl 1-phosphates
are often prepared by glycosyl 1-phosphate kinases
although routes via phosphoglycomutases have also
been explored.2 Many monosaccharide-1-phosphate
kinases have been cloned, expressed, and characterized for
use in the scalable synthesis of glycosyl 1-phosphates.3,4
Corresponding sugar nucleotides may also be obtained
if a nucleotidyltransferase and inorganic pyrophospha-
tase are employed in addition to the glycosyl kinase in a
one-pot protocol.4 This methodology has been har-
nessed by recent work in glycorandomization which uses
non-native glycosyl 1-phosphates as substrates for nu-
cleotidyltransferase enzymes modified to have increased
substrate promiscuity via directed evolution.5 The resul-
tant non-native sugar nucleotides have been shown to be
effective glycosyl donors for glycosylation of a variety of
natural products.6
Although major advances have been made in the devel-
opment of enzymatic protocols, chemical approaches to
glycosyl 1-phosphates remain attractive as they are more
broad in their substrate scope.7 The majority of these
syntheses rely on hydroxyl protecting groups and as a
consequence are multistep procedures.7 For example, a
fully protected glycosyl donor such as a glycosyl trichlor-
oacetimidate, thioglycoside, glycosyl acetate, 1,2 orthoester,
glycosyl halide, pentenoyl glysoside, glycal, or oxosulfo-
nium glycoside can be used to glycosylate phosphate or a
phosphoester.8 Other methodologies couple unprotected
hemiacetals to protected electrophilic phosphorus(III)
sources such as chlorophosphates and phosphoramidates,
which require a separate oxidation step to generate
the corresponding phosphate.9 The resultant glycosyl
1-phosphate may then be converted to its corresponding
(1) Varki, A. Essentials of Glycobiology; Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory, 1999.
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r
10.1021/ol3019083
XXXX American Chemical Society

nucleotide sugar via exposure to a nucleoside phosphoro-
morpholidate, phosphoimidazolide, or another appro-
priate electrophilic phosphorus(V) source.7,10 Although
these methods can provide access to a desired depro-
tected glycosyl 1-phosphate, the number of steps re-
quired and difficult purifications limit their efficiency.
These synthetic issues culminate in limited commercial
availability and prohibitive prices for many glycosyl
1-phosphates.
Table 1. Acid-Catalyzed Condensation of p-Toluenesulfonyl-
hydrazide with Various Carbohydrates
Hanessian et al. have elegantly circumvented the use
of protecting groups in glycosyl 1-phosphate syn-
thesis by exploiting methoxypyridine (MOP) glycosyl
donors.11 Although the phosphorylation step in this
methodology is protecting-group-free, synthesis of each
MOP donor from free sugar requires a multistep pro-
tocol that involves protecting groups. We herein report
the first example of a two-step, protecting-group-free
protocol for the synthesis of glycosyl 1-phosphates from
hemiacetals.
We have previously reported the use of N0-glycosylto-
luenesulfonohydrazides (GSHs) as glycosyl donors for the
protecting-group-free synthesis of O-glycosides and glyco-
syl azides of N-acetyl-D-glucosamine (GlcNAc).12 These
GSH donors are easily prepared under mild conditions in
one step from free sugars and can be activated using a
source of electrophilic halogen such as N-bromosuccinimide
(NBS). Due to the robustness of O-glycosylation using
these donors, we speculated that they could be used to
synthesize glycosyl 1-phosphates, if anhydrous phosphoric
acid was used as the acceptor.
a Isolated yields.
A range of GSH donors were easily accessed via acid-
catalyzed condensation of free hemiacetals with p-tolue-
nesulfonyl hydrazide (Table 1). This protocol does not
require chromatographic purification; the donors were
isolated by precipitation in high purity and excellent yield.
Additionally, only β-anomers of GSH donors were formed
under these conditions, further simplifying isolation and
chacterization. The exclusive formation of the β-manno-
sylsulfonohydrazide (4) was confirmed by NOE experi-
ments which showed similar enhancements between H1,
H3, and H5.
Initial investigation into the phosphorylation of
GSH donor 1 employed the conditions that were successful
for O-glycosidations: NBS in DMF and anhydrous phos-
phoric acid as the acceptor.12 No conversion of donor 1 to
the corresponding glycosyl phosphate 7 was observed
under these conditions (Table 2). We hypothesized that
the poor nucleophilicity of triprotic phosphoric acid was
preventing formation of the glycosyl 1-phosphate. At-
tempts to improve the nucleophilicity of the phosphate
using less acidic conditions failed to improve the reaction
conversion (Table 2).
Next, Lewis acidic metal ion oxidants, which may
coordinate the phosphoric acid and activate the GSH
donor, were explored. Several transition metal salts were
screened as oxidizing agents of GSHs, by qualitatively
monitoring the reaction for gas evolution (N2) (Table 2).
Of the metal salts screened only cupric chloride (CuCl2)
was an effective oxidant. This is consistent with previous
reports of Cu(II)-mediated oxidation of anomeric
hydrazines.13
(8) (a) Plante, O. J.; Palmacci, E. R.; Andrade, R. B.; Seeberger, P. H.
J. Am. Chem. Soc. 2001, 123, 9545–9554. (b) Schmidt, R. R.; Wegmann,
B.; Jung, K. Liebigs Ann. Chem. 1991, 121–124. (c) Schmidt, R. R.;
Stumpp, M.; Michel, J. Tetrahedron Lett. 1982, 23, 405–408. (d) Veeneman,
G. H.; Broxterman, H. J. G.; van der Marel, G. A.; van Boom, J. H.
Tetrahedron Lett. 1991, 32, 6175–6178. (e) MacDonald, D. L. J. Org. Chem.
ꢀ
1962, 27, 1107–1109. (f) Ravida, A.; Liu, X.; Kovacs, L.; Seeberger, P. H.
Org. Lett. 2006, 8, 1815–1818. (g) Cori, C. F.; Colowick, S. P.; Cori,
G. T. J. Biol. Chem. 1937, 465–477. (h) Gokhale, U. B.; Hindsgaul, O.;
Palcic, M. M. Can. J. Chem. 1990, 68, 1063–1071. (i) Pale, P.; Whitesides,
G. M. J. Org. Chem. 1991, 56, 4547–4549. (j) Garcia, B. A.; Gin, D. Y.
Org. Lett. 2000, 2, 2135–2138.
(9) (a) Nunez, H. A.; O’Connor, J. V.; Rosevear, P. R.; Barker, R.
Can. J. Chem. 1981, 59, 2086–2095. (b) Westerduin, P.; Veeneman,
G. H.; Marugg, J. E.; van der Marel, G. A.; van Boom, J. H. Tetrahedron
Lett. 1986, 27, 1211–1214.
(10) (a) Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G.
J. Am. Chem. Soc. 1961, 83, 659–663. (b) Baisch, G.; Oehrlein, R. Bioorg.
Med. Chem. 1997, 5, 383–391.
(11) Hanessian, S.; Lu, P.; Ishida, H. J. Am. Chem. Soc. 1998, 120,
13296–13300.
Although the desired glycosyl phosphate 7 was formed
in the presence of CuCl2, the conversion was low and a
substantial amount of the hydrolysis product (GlcNAc)
was observed. Since all glycosylations were performed
(12) Gudmundsdottir, A. V.; Nitz, M. Org. Lett. 2008, 10, 3461–
3463.
(13) Hann, R. M.; Hudson, C. S. J. Am. Chem. Soc. 1934, 56, 957–
958.
B
Org. Lett., Vol. XX, No. XX, XXXX

Table 2. Optimization of Protecting-Group-Free Phosphorylationa
Table 3. Copper-Mediated Phosphorylation of Various
p-Toluenesulfonohydrazide Glycosyl Donorsa
oxidantb
additivec
7 (GlcNAc)d
NBS
none
0% (0%)
NBS
diisopropylethylamine (7.8 equiv)
none
0% (0%)
Cu(OTf)2
CuCl2
CuCl2
CuCl2
CuCl2
CuCl2
CuCl2
no reaction
22% (66%)
33% (66%)
66% (33%)
3% (26%)
0% (0%)
none
imidazole (4.1 equiv)
2-methyl-2-oxazoline (4.1 equiv)
2-methyl-2-oxazoline (1.0 equiv)
2-methyl-2-oxazoline (8.2 equiv)
2-methyl-2-oxazoline (4.1 equiv)e
91% (9%)
a Reaction time was 2 h except where otherwise indicated. b The
concentration of 1 in the reaction was held at 48 mM. c The number of
equivalents of additive is relative to compound 1. d Qualitative NMR
yield based on percent area of integration of all resonances in the
anomeric region of the spectrum (5.5ꢀ4.0 ppm). e Reaction time
was 18 h.
under anhydrous conditions the hydrolysis was thought to
arise from reaction with solvent. This process is presum-
ably facilitated by CuCl2, as little hydrolysis was observed
in the presence of NBS. In efforts to reduce the extent of
hydrolysis, various coordinating and noncoordinating
additives were investigated (Tables 2 and S1). Of the
additives screened, imidazole and 2-methyl-2-oxazoline
were the most promising, as they promoted GSH donor
oxidation by Cu(II). 2-Methyl-2-oxazoline proved to be
a superior additive, as less donor hydrolysis was ob-
served under reaction conditions. Optimization of the
number of equivalents of this additive produced inter-
esting results, with less than or more than 1 equiv per
Cu(II) ion promoting side-product formation. When 1
equiv of additive was included the desired R-glycosyl
1-phosphate was formed in a 2:1 ratio with hydrolyzed
donor, and no other carbohydrate related products
were observed. Increasing the duration of reaction
from 2 to 18 h led to glycosyl 1-phosphate 7 as the
major carbohydrate product. The time dependence
of the reaction suggests that an unstable intermediate
is formed, which is slowly converted to the glycosyl
1-phosphate.
The optimized reaction conditions proved versatile and,
when applied to donors 1ꢀ6, gave the desired R-glycosyl
1-phosphates asthe major product (Table 3). The mannose
GSH donor (4) provided exclusively the R-anomer of the
corresponding glycosyl phosphate 10. A particularly at-
tractive attribute of this protocol is its ability to provide
good yields of higher order glycosyl 1-phosphates, such as
disaccharide 12.
A general protocol was developed for the isolation of the
glycosyl 1-phosphate barium salts that does not require
chromatography. An initial precipitation of the crude
product from the reaction mixture with dichloromethane
a Minor noncarbohydrate impurities and trace amounts of corre-
sponding free hemiacetals were present in all isolated glycosyl 1-phos-
phates. Compounds 7, 10, 11, and 12 were further purified using
Sephadex A-25 anion exchange resin with NH4HCO3 as the eluent. This
polishing purification resulted in a 25ꢀ62% loss of yield relative to those
shown in the table.
removes the majority of noncarbohydrate impurities. The
resulting precipitate is dissolved in a solution of dry
DMF/ethanol and subsequently neutralized with barium
hydroxide to remove excess phosphate and cupric/cuprous
chloride via formation of water insoluble barium phos-
phate and copper hydroxide species respectively. The
barium salts of the phosphate sugars are water-soluble
and can be isolated after lyophilization of the aqueous
solution.14 The isolated yields reported in Table 3 reflect
the isolated product that contains small amounts of free
hemiacetal and minor impurities (see Supporting Infor-
mation (SI)). The barium salts of the phosphate sugars
can be converted into biologically useful ammonium
salts after a polishing purification step with anion ex-
change chromatography which yields analytically pure
samples (see SI).
The present study demonstrates that GSH donors
efficiently provide a wide variety of glycosyl 1-phos-
phates enriched in the R-anomer in yields comparable to
previous literature reports (Table 3). Investigations into
(14) Wood, T. J. Chromatogr. 1968, 35, 352–361.
Org. Lett., Vol. XX, No. XX, XXXX
C

bolstering the diastereoselectivity of this protocol and
the mechanism of the transformation are currently
underway.
Molecules and Polymers is gratefully acknowledged.
We thank Dr. T. Burrow, Dr. D. C. Burns, and Mr. D.
Pichugin for assistance in obtaining NMR spectral data.
Acknowledgment. Funding from the Natural Science &
Engineering Research Council of Canada, the Canadian
Foundation for Innovation (Project #19119), and the
Ontario Research Fund for funding of the Centre
for Spectroscopic Investigation of Complex Organic
Supporting Information Available. Full experimental
details and NMR spectra data are available. This material is
available free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
D
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