2,3,6-Trideoxy sugar nucleotides: Synthesis and stability

Article abstract of DOI:10.1016/j.tetlet.2011.08.132

The synthesis and characterization of highly challenging 2,3,6-trideoxy sugar nucleotides were described for the first time. The study of their hydrolysis kinetics in aqueous buffers provided insight into their application as glycosyl donors.

Full text of DOI:10.1016/j.tetlet.2011.08.132

Tetrahedron Letters 52 (2011) 5799–5801
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
2,3,6-Trideoxy sugar nucleotides: synthesis and stability
Mingxuan Wu a,c, Qingqing Meng a, Min Ge b, Linquan Bai c, Huchen Zhou a,
⇑
a School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
b College of Biotechnololy and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
c School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2011
Revised 16 August 2011
Accepted 23 August 2011
Available online 31 August 2011
The synthesis and characterization of highly challenging 2,3,6-trideoxy sugar nucleotides were described
for the first time. The study of their hydrolysis kinetics in aqueous buffers provided insight into their
application as glycosyl donors.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Sugar nucleotides
2,3,6-Trideoxy
Synthesis
Stability
Introduction
tible to hydrolysis.10,11 The stability of sugar nucleotides decreases
with the decreasing number of hydroxyls on sugar ring.12 In the
Glycosyltransferases play key roles in important cellular pro-
cesses such as cell wall biosynthesis in bacterial pathogens and sig-
nal transduction, and carcinogenesis in human.1–3 Glycosylated
natural products produced by microorganisms, such as erythromy-
cin and vancomycin, represent an indispensable source of biomedi-
cally useful molecules.1–3 The biosynthesis of these
glycoconjugates is mediated by glycosylation reactions catalyzed
by specific glycosyltransferases which require activated sugar do-
nors in the form of sugar nucleotides as substrates.2–4 A variety
of sugars appended to natural product scaffolds are of crucial
importance in tuning the therapeutic properties of these complex
molecules.1–3 Interestingly, it was found that glycosyltransferases
are rather promiscuous with respect to sugar nucleotide sub-
strates, which can be utilized to produce diversified compounds.5
Unfortunately, this effort, as well as the study of glycoconjugate
biosynthesis, were hindered by the limited availability of required
unusual sugar nucleotides.
past, although the synthesis of 2-deoxy sugar nucleotides13 and a
stable isostere with C-glycosidic phosphonate linker14 have been
reported using both chemical and enzymatic methods, the synthe-
sis of 2,3,6-trideoxy sugar nucleotides has never been explored de-
spite of their existence in a large number of natural products
including L-rhodinose in urdamycin B and L-amicetose in tetrocar-
cin A (Fig. 1).15,16 Although they have significant value in the elu-
CH3
O
O
OH
H3C
O
HO
O
OH
O
H3C
O
H3C
O
O
1
HO
HO
OCH3
NH
Sugar nucleotides present a synthetic challenge due to a num-
ber of complications, including susceptibility to hydrolytic cleav-
age, low solubility in organic solvents, and the presence of polar
and charged groups.6–9 The deoxy-sugar nucleotides are especially
difficult to synthesize because the removal of electron-withdraw-
ing hydroxyls makes the positively charged oxocarbenium, which
is the intermediate of hydrolytic cleavage, more stable, thus the su-
gar nucleotides and their synthetic intermediates are more suscep-
O
OH
O
O
O
O
O
NO2
O
H
O
O
O
OH
O
HO
O
O
H
OH
AcO
O
2
CHO
⇑
Corresponding author. Tel.: +86 21 3420 6721; fax: +86 21 3420 4744.
E-mail address: hczhou@sjtu.edu.cn (H. Zhou).
Figure 1. Examples of natural products containing 2,3,6-trideoxy sugars. Urdamy-
cin B (1) has -rhodinose and tetrocarcin A (2) has -amicetose.
L
L
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.08.132

5800
M. Wu et al. / Tetrahedron Letters 52 (2011) 5799–5801
O
N
cidation of biosynthetic mechanisms and discovery of new medic-
inal molecules, the knowledge of the synthetic feasibility and the
stability of 2,3,6-trideoxy sugar nucleotides were nonexistent.
Here we undertook the synthesis of NDP-L-rhodinose as repre-
sentatives of 2,3,6-trideoxy sugar nucleotides and the study of
their stability in aqueous buffers. The 4-OH-, as well as the
NH
O
O
O
P
O
P
O
P
O
P
H3O+
O
O
O
O
O
H
O
O
U
O
OH OH
OH OH
O
OH
H
OH OH
13dUDP
4-OCH3- and 4-OAc-NDP-
L-rhodinose (13b–d) were successfully
Transition State
synthesized and characterized, while the 4-OTBS-
L-rhodinose
monophosphate (12a) was too labile to get through the final pyro-
phosphate bond formation (Scheme 1). This is the first time the
synthesis and stability study of 2,3,6-trideoxy sugar nucleotides
were reported. It was found that the NDP was the primary hydro-
lytic cleavage product (Scheme 2) and the rate of cleavage is
determined by a through-bond inductive effect as well as a
through-space electronic effect. And the kinetics study of the
hydrolysis provided a basis for the selection of enzymatic reaction
conditions and guidelines for the synthesis and storage of 2,3,
6-trideoxy sugar nucleotides.
O
-UDP
H2O
O
OH
OH
O
H
Oxocarbenium
Scheme 2. Hydrolytic reaction of 2,3,6-trideoxy sugar nucleotide.
Results and discussion
Firstly, the 4-OTBS-, 4-OCH3-, and 4-OAc-
L
-rhodinose (10a–c)
OH
OBn
OBn
O
were synthesized from (S)-ethyl lactate (3) using methods adapted
from Schlessinger et al. (Scheme 1).17 After protection with benzyl
group, ester 4 was reduced to aldehyde 5 with DIBALH. The chela-
tion controlled stereoselective addition with tri-n-butylallylstann-
ane in the presence of MgBr2–Et2O gave olefin 6, which in turn was
converted to its TBS, methyl, or acetyl derivatives 7a–c under con-
ditions of TBSCl, CH3I/NaH, or Ac2O/pyridine. Hydroboration of ole-
fin 7a–c with 9-BBN followed by oxidation with H2O2–NaOH (7a
and b) or H2O2–NaOAc (7c) gave the corresponding alcohols 8a–
c. After oxidation with PCC, the resulting aldehydes 9a–c were con-
BnBr, NaH
DMF
DIBALH
CH2Cl2
OEt
H
OEt
-78 o
C
O
O
3
(55%)
4
5
OBn
n-Bu3Sn
Et2O-MgBr2, CH2Cl2
-10 to -78 o
C
OR
6: R = H (83% over 2 steps)
7a: R = TBS (95%)
7b: R = CH3 (93%)
CH3I
NaH
TBSCl
Ac2O
Pyridine
verted to 4-OTBS-, 4-OCH3-, and 4-OAc-L-rhodinose (10a–c) under
7c: R = Ac (90%)
Pd/C catalyzed hydrogenation. It was necessary to use anhydrous
methanol as the solvent to suppress the hydrolysis of acetyl group
on 10c.
OBn
1. 9-BBN, THF
2. H2O2, NaOH
or H2O2, NaOAc
OBn
O
OH
H
The glycosyl monophosphates 12a–c were obtained via the gly-
cosyl chloride intermediates 11a–c using the method adapted from
Kahne et al. (Scheme 1).7 The stability of the glycosyl chlorides de-
creases when the electron-withdrawing ability of the sugar ring
substituents decreases due to the stabilization of the oxocarbeni-
um intermediate.10,11 Indeed, chloride 11c with an electron-with-
drawing 4-OAc substituent was easily generated at 0 °C, while
chloride 11a with an electron-donating 4-OTBS substituent was
only successfully prepared under temperatures as low as À80 °C.
Consistently, chloride 11b with a moderate electron-donating 4-
OCH3 was prepared at À50 °C. Decomposition of 11a and b was ob-
served when temperature was raised to 0 °C. Subsequent phos-
phorylation with tetrabutylammonium dihydrogen phosphate
(Bu4NH2PO4) in the presence of DIPEA in dichloromethane gave
monophosphates 12a–c. In this case, the steric effect instead of
PCC
OR
OR
CH2Cl2
8a: R = TBS (83%)
8b: R = CH3 (82%)
8c: R = Ac (86%)
9a: R = TBS (77%)
9b: R = CH3 (77%)
9c: R = Ac (70%)
OH
10a: R = TBS (94%)
10b: R = CH3 (92%)
10c: R = Ac (90%)
H2, Pd/C
O
MeOH
OR
O
P
Cl
(COCl)2
DMF
DIPEA
O
OH
O
n-Bu4NH2PO4
O
OH
CH2Cl2
OR1
CH2Cl2
OR1
β/α
11a: R1 = TBS, -80 oC
11b: R1 = CH3, -50 o
C
12a: R1 = TBS(-80 oC, 35%, 1:2)
12b: R1 = CH3 (-50 oC, 40%, 3:2)
electronic effect determined the b/
tuted chlorides, 11a with the bulkiest 4-OTBS substitution gave the
lowest b/ ratio of 1:2 presumably because the steric presence of
a ratio. Among the three substi-
11c: R1 = Ac, 0 o
C
12c: R1 = Ac
(0 oC, 48%, 1:1)
O
a
R2
OTBS hindered the SN2 trajectory thus disfavoring the formation
of the corresponding b-anomer. At the same time, the 4-OCH3
substituted chloride 11b which is the least sterically hindered gave
NH
NMP morpholidate
N
O
O
O
P
tetrazole
O
O
P
O
O
O
pyridine
the highest b/a ratio of 3:2. Phosphates 12b and c were success-
OH OH
OR1
OH R3
fully converted to their UDP and TDP derivatives using the method
developed by Wong et al.18 Since the b-anomer is the bioactive
configuration in most cases, the isolation and characterization of
pure b-anomer were emphasized on purification. Acetylated sugar
13bUDP: R1 = CH3, R2 = H, R3 = OH (8%)
13bTDP: R1 = CH3, R2 = CH3, R3 = H (8%)
13cUDP: R1 = Ac, R2 = H, R3 = OH (34%)
13dUDP: R1 = H, R2 = H, R3 = OH (20%)
13cTDP: R1 = Ac, R2 = CH3, R3 = H (33%)
13dTDP: R1 = H, R2 = CH3, R3 = H (14%)
NaOMe
MeOH
nucleotides 13cUDP and 13cTDP were converted to NDP-L-rhodinos-
es (13dUDP, 13dTDP) by removal of acetyl group under NaOMe/
MeOH conditions. Thus, six 2,3,6-tridexoy sugar nucleotides
13bUDP, 13bTDP, 13cUDP, 13cTDP, 13dUDP, and 13dTDP were success-
fully synthesized. Although they are susceptible to hydrolysis in
NaOMe
MeOH
Scheme 1. Synthesis of NDP-L-rhodinose.

M. Wu et al. / Tetrahedron Letters 52 (2011) 5799–5801
5801
pH P 7.0 and their half life (t1/2) increased 30- to 130-fold when
temperature was lowered from 40 to 5 °C. The 4-OAcÀ substituted
13cUDP is stabilized by the electron-withdrawing acetyl group. It is
intriguing to observe the unusually fast hydrolysis of 4-OH-L-rho-
dinose nucleotide 13dUDP that has a half life of 15 min at pH 7.0
and 30 °C (Fig. S3). It can be rationalized by the through-space elec-
tronic effect of the axial 4-OH that donates electron density to the
positively charged ring oxocarbenium,21 thus stabilizing the transi-
tion state (Scheme 2).
In summary, using
L-rhodinose as an example, the highly
interesting but elusive 2,3,6-trideoxy sugar nucleotides were syn-
thesized and characterized for the first time. The stability of its
4-OH, 4-OAc, and 4-OCH3 derivatives in aqueous buffers was inves-
tigated and their stability increased with the elevation of pH and
decreased when temperature was raised. The 4-OH derivative
showed significant instability in aqueous buffer presumably due
to the through space participation of the 4-OH electrons in
stabilizing the oxocarbenium intermediate.
Figure 2. Kinetics of the hydrolysis of 13cUDP
a- and b-anomers at different
Acknowledgments
concentrations (0.08–10 mM, pH 8.0, T = 30 °C) proved first order reaction (R >0.999
in all cases). k = 4.99 Â 10À5 sÀ1 and t1/2 = 232 min kb = 1.14 Â 10À5 sÀ1 and t1/
a
We acknowledge National Science Foundation of China
(20702031), Ministry of Science and Technology of China
(2009CB918404), E-Institutes of Shanghai Universities (EISU)
Chemical Biology Division, and National Comprehensive Technol-
ogy Platforms for Innovative Drug R&D (2009ZX09301-007) for
financial support of this work.
2 = 1011 min.
aqueous solution, they can be stored at À20 °C without decompo-
sition over a few months as lyophilized powder.
Considering the susceptibility of 2,3,6-trideoxy sugar nucleo-
tides toward hydrolytic cleavage, appropriate selection of the
buffer conditions is critical for carrying out successful enzymatic
reactions. Thus, we took UDP-L-rhodinose derivatives 13bUDP (4-
Supplementary data
OCH3), 13cUDP (4-OAc), and 13dUDP (4-OH) as representatives to
study their stability in aqueous buffers. First the hydrolysis reac-
tion was demonstrated to be first order by the linearity of the
kinetic curve in Figure 2 (Fig. S1) and the product was found by
HPLC and 31P NMR to be UDP instead of UMP (Scheme 2, Fig. S2),
which is due to the absence of neighboring group participation
from the 2-position.19,20 The stability decreases in the order of
Supplementary data associated (experimental procedures, char-
acterization of new compounds, and kinetics experiments) with
this Letter can be found, in the online version, at doi:10.1016/
j.tetlet.2011.08.132.
References and notes
13cUDP-b > 13cUDP-a > 13bUDP-b >> 13dUDP-b (Fig. 3). The b-anomer
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100
5000
0
0
5
6
7
8
9
10 11 12
5
15
25
35
45
Temp (oC)
pH
Figure 3. (Left) Effect of pH on the half life (t1/2) of sugar nucleotides (30 °C); (right)
Effect of temperature on the half life (t1/2) of sugar nucleotides (pH 7.0). 13bUDP-b
(s); 13cUDP-b (j); 13cUDP-a (N); 13dUDP-b (Â).