First synthesis of enantio-uracil dinucleotide, comparison of physicochemical properties of their enantiomers, and separation by chiral column chromatography

Article abstract of DOI:10.1016/j.tetlet.2003.09.142

Enantio-uracil dinucleotide 5, which consists of two L-uridylic acids and one pyrophosphate, was synthesized for the first time in our laboratory. Benzolyated L-uridine was prepared by a stereoselective glycosylation of silylated uracil with L-1-O-acetyl-2,3,5-tri-O-benzoylribose (L-ABR 7). After deprotection, L-uridine 9 was converted to P¹,P⁴-di(L- uridine 5′-) tetraphosphate tetrasodium salt (L-UP₄U 5) by treatment of L-UMP morpholidate 10c with triethylammonium pyrophosphate (TEA-PPi 11b). Spectral data of synthesized L-UP₄U 5 are given in the references. All spectral data were identical with those of UP₄U 3 except the specific rotation, which showed a positive value compared to UP₄U 3 having a negative value. Furthermore, the separation by chiral column chromatography was investigated.

Full text of DOI:10.1016/j.tetlet.2003.09.142

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 8605–8607
First synthesis of enantio-uracil dinucleotide, comparison of
physicochemical properties of their enantiomers, and separation
by chiral column chromatography
Takanori Miyashita, Shinji Sakata and Hiroyuki Hayakawa*
Chemistry & Pharmacology Laboratory, Biochemicals Division, Yamasa Corporation, 10-1, Araoicho 2-Chome, Choshi,
Chiba-Ken 288-0056, Japan
Received 22 July 2003; revised 29 August 2003; accepted 12 September 2003
Abstract—Enantio-uracil dinucleotide 5, which consists of two
first time in our laboratory. Benzolyated -uridine was prepared by a stereoselective glycosylation of silylated uracil with
-1-O-acetyl-2,3,5-tri-O-benzoylribose ( -ABR 7). After deprotection, -uridine 5%-)
-uridine 9 was converted to P1,P4-di(
tetraphosphate tetrasodium salt ( -UP4U 5) by treatment of -UMP morpholidate 10c with triethylammonium pyrophosphate
(TEA-PPi 11b). Spectral data of synthesized -UP4U 5 are given in the references. All spectral data were identical with those of
L-uridylic acids and one pyrophosphate, was synthesized for the
L
L
L
L
L
L
L
L
UP4U 3 except the specific rotation, which showed a positive value compared to UP4U 3 having a negative value. Furthermore,
the separation by chiral column chromatography was investigated.
© 2003 Elsevier Ltd. All rights reserved.
Purine and pyrimidine (P2) receptors are attractive
drug targets.1 We are interested in dinucleotide deriva-
tives because they have both P2 receptor agonist and
antagonist activities. Among them, we also have
focused on the preparation of uracil dinucleotides 1–4
(Fig. 1) and elucidated their structures including physico-
chemical properties.2
nucleotides are also considered as attractive targets for
the exploration of new probes of P2Y ligands. How-
ever, there are no reports on the synthesis and analysis
of these dinucleotides.
As a part of our program to study both chirality and
physicochemical properties of these compounds, we
planned to make -UP4U 5 (Fig. 2). The present report
L
P1,P4-Di(uridine 5%-) tetraphosphate (3, uracil dinucleo-
tide, UP4U) has been developed as a drug for the
treatment of Dry Eyes disease and chronic obstructive
pulmonary disease.3
However, it is known that these dinucleotides are
unstable to enzymatic degradation. Additionally, new
methods for the analysis of stereoisomers in relation to
impurities were required.
Figure 1.
On the contrary, it is considered that enantio dinucleo-
tides will be probably stable to enzymatic degradation.
Furthermore, both enantio dinucleotides and meso di-
Keywords: enantio-uracil dinucleotide; uracil dinucleotide; L-uridylic
acids; TEA-PPi; specific rotation; separation; HPLC; chiral column
chromatography; stereoisomers.
* Corresponding author. Tel.: 81-479-22-0095; fax: 81-479-22-9821;
e-mail: hayakawa@yamasa.com
Figure 2.
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.09.142

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T. Miyashita et al. / Tetrahedron Letters 44 (2003) 8605–8607
L
-UP4U 5 was purified twice by chromatography, first
on an anion-exchange resin, and next on charcoal. The
appropriate fractions containing -UP4U 5, after
adjusting pH with NaOH, were concentrated to crystal-
lize -UP4U 5 (Scheme 2).
describes the first synthesis of
L
-UP4U 5 bearing the
L
-ribose moiety, comparison of physicochemical prop-
L
erties in enantiomers, and then separation of enan-
tiomers by chiral column chromatography.
L
Synthesis of L-UP4U 5
After recrystallization from aqueous ethanol,
L
-UP4U 5
was obtained in 0.4% overall yield, HPLC (99.4%).6,7
Preparation of
10b): -1-O-Acetyl-2,3,5-tri-O-benzoylribose (
was made by the modified method used for the prepara-
tion of peracylated sugars from -ribose 6, and then it
was derived to protected -uridine 8 by stereoselective
glycosylation of silylated uracil with
-ABR 7.4
L
-ABR 7 and
L
-uridylic acid (
L-UMP
L
L
-ABR 7)
Elucidation of the structure of
of
-UP4U 5 was confirmed by H, 13C, 31P NMR MS,
UV, IR, elemental analysis (EA), and specific rotation.
The spectral data of -UP4U 5 are summarized in Ref.
L-UP4U 5: The structure
1
L
L
L
L
L
6, and other data such as mp and EA are noted in Ref.
7.
After deprotection of
with sodium methoxide (NaOMe) to give
this compound was converted to the corresponding
L
-2,3,5-tri-O-benzoyluridine 8
L
-uridine 9,
All spectral data were identical with those of UP4U 3
except the specific rotation, which showed +8.5° (c 1,
water, 20°C) compared to that of UP4U (−9.5°). The
L
-uridylic acid 10 by the improved method5 used for the
preparation of uridine 5%-monophosphophoric acid
melting point of
temperature (228°C).
L
-UP4U 5 showed a slightly higher
(UMP) (Scheme 1).
Preparation of
monophosphoric acid, triethylammonium salt (
2TEA 10a) in deionized water was acidified to uridine-
5%-monophosphoric acid ( -UMP, free, 10b) by passing
through a cation-exchange resin (H+ form).
-UMP,
free was converted to its morpholidate 10c with mor-
pholine and dicyclohexyl carbodiimide (DCC).
L
-UMP morpholidate 10c:
L
-Uridine 5%-
Separation of enantiomers (
HPLC
L-UP4U 5 and UP4U 3) by
L
-UMP,
L
Initially, we tried to distinguish several enantiomers
(nucleosides or 5%-mononucleotides, which were compo-
nents of the desired dinucleotides) by HPLC through
these studies. However, we could not separate enan-
L
tiomers such as UMP and
uridine) by using even chiral column chromatography.
Interestingly, enantiomer ( -UP4U 5) was separated
from -derivative 3 by the same technique. Mixtures of
UP4U 3 and -UP4U 5 were analyzed by HPLC using
L-UMP (uridine and L-
Preparation of TEA-PPi 11b: In a similar manner,
pyrophosphoric acid, tetrasodium salt (PPi, 4Na, 11a)
was acidified by passing through a cation-exchange
resin, then neutralized with triethylamine to give a
triethylammonium salt (TEA-PPi 11b).
L
D
L
YMC chiral b-CD BR column (see operating condi-
tion). Figure 3 shows the HPLC profile of a mixture of
Dehydrated -UMP morpholidate 10c was treated with
L
L
-UP4U 5 and UP4U 3 with the ratio of 1:1. -UP4U 5
L
TEA-PPi 11b in pyridine to afford a crude mixture
was eluted at 18.49 min of retention time about 2 min
containing -UP4U 5.
L
faster than UP4U 3.
Scheme 1.
Scheme 2.

T. Miyashita et al. / Tetrahedron Letters 44 (2003) 8605–8607
8607
new probes of P2Y ligands as agonists and/or
antagonists.
Further investigations are in progress in our laboratory.
Acknowledgements
The authors are thankful to Ms. Tamiko Nemoto for
her technical assistance.
Figure 3. Operating condition. Detector: spectrophotometer
(wavelength: 262 nm). Column: A stainless steel column (4.6
mm I.D.×25 cm L) (5 mm, CHIRAL b-CD BR, YMC).
Column temperature: room temperature. Mobile phase: mix-
ture of K-P (potassium phosphate 80 mM) and TBAS (tetra-
butylammonium hydrogen sulfate 20 mM) in water (pH 6.7).
Flow rate: 0.5 mL/min.
References
1. Jacobson, K. A.; Jarvis, M. F.; Williams, M. J. Med.
Chem. 2002, 45, 4057–4093.
2. (a) Pendergast, W.; Yerxa, B. R.; Douglass, J. G., III;
Shaver, S. R.; Dougherty, R. W.; Redick, C. C.; Sims, I.
F.; Rideout, J. L. Bioorg. Med. Chem. Lett. 2001, 11,
157–160; (b) Pat. No. JP324768; (c) Unpublished results in
our laboratory.
These results suggest the possibility that chiral HPLC
will become a useful technique to elucidate the
stereoisomers of these dinucleotides. Therefore, we can
identify a large number of impurities and the desired
products related to these dinucleotides.
3. Pat. No. WO9905155, Pat. No.WO9834593.
4. Sivets, G. G.; Klennitskaya, T. V.; Zhernosek, E. V.;
Miklailopulo, I. A. Synthesis 2002, 253–259.
5. Yoshikawa, M.; Kato, T.; Takenishi, T. Bull. Chem. Soc.
Jpn. 1969, 42, 3505–3508.
Stability of L-UP4U 5 to enzymatic degradation
6. Spectral data: IR (KBr) 3380 cm−1, 1690 cm−1, 1250 cm−1
,
Enantiomers were treated with two enzymes. UP4U 3
was completely degraded when exposed to 5%-
phosphodiesterase8 and subsequent acid phosphatase in
aqueous ammonium acetate (37°C, pH 5.0). On the
1140 cm−1, 1113 cm−1, 890 cm−1; FAB+MS 879 (M+H)+;
1H NMR (D2O) lppm 7.97 (2H, d, H-6), 6.00 (2H, d, H-1%),
5.98 (2H, d, H-5), 4.43 (2H, m, H-3%), 4.39 (2H, m, H-2%),
4.29 (2H, m, H-4%), 4.25 (2H, m, H2-5%); 13C NMR (D2O)
lppm 169.3 (C-4), 154.9 (C-2), 144.5 (C-6), 105.6 (C-5), 91.0
(C-1%), 86.4 (C-4%), 76.6 (C-2%), 72.6 (C-3%), 67.9 (C-5%); 31P
NMR (D2O) lppm −10.6 (P-1), −22.2 (P-2); UV (0.01N
HCl) umax 262 nm. [h]D20=+8.5° (c 1, water, 20°C).
7. Other data. Anal. calcd for (C18H22N4Na4O23P4·3.5H2O):
C, 22.97; H, 3.11; N, 5.95. Found: C, 22.96; H, 3.13; N,
5.92; mp 228°C (dec.).
contrary,
reactions.
L
-UP4U 5 was stable to these enzymatic
In summary, we have prepared enantio-uracil dinucleo-
tide 5 for the first time. All spectral data were identical
with those of UP4U 3 except the specific rotation. The
melting point of the enantiomer showed slightly higher
temperature. The chiral column chromatography was
effective to separate these enantiomers. Enantio-uracil
dinucleotide 5 is resistant to enzymatic degradation.
8. Fujimoto, M.; Fujiyama, K.; Midorikawa, Y.; Fujishima,
T.; Kuninaka, A.; Yoshino, H. Agric. Biol. Chem. 1977,
41, 737–744.
According to these results,
L-dinucleotides might be