2
704
M. Matuszewski, E. Sochacka / Bioorg. Med. Chem. Lett. 24 (2014) 2703–2706
6
6
9
6
O
published for ct A and t A molecules. Additionally, Ac
ct A was
O
OH
OH
3
O
N
O
N
1
13
further characterized by H NMR (D
and IR spectra (Figs. S10–S15), and its purity was confirmed by
HPLC (Fig. S16). The minor product (yield 12%) was identified on
the basis of H NMR spectrum in DMSO-d
3
ct A additionally acetylated at the b-hydroxyl function of
the threonine residue (abbreviated as Ac ct A ). This compound
was the only product when the dehydration reaction was
performed in neat acetic anhydride (Supplementary data, p. 24).
2
O), C NMR, FAB MS, UV,
HN
N
N
H
N
N
N
OH
H
N
N
N
N
HO
HO
1
(Fig. S17) as a derivative
O
O
6
6
of Ac
HO OH
HO
OH
6
Ac
6
6
3
t A
ct A
6
6
Figure 1. Structures of t A and ct A.
6
It is noteworthy that in the case of synthesis of Ac
3
ct A, the
formation of the threonine cyclic ester under acetic anhydride
treatment in methylene chloride appeared to be much more
convenient than the procedure employing soluble carbodiimide
0
0
0
6
2
,3 ,5 -tri-O-acetyl-adenosine N -carbamate with an
L
-threonine
residue (details are given in Supplementary data). The structure
of Ac t A was unambiguously confirmed by H NMR, C NMR,
3
6
1
13
(
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)),21 as the
FAB MS, UV, IR spectra and HPLC data (Supplementary data,
Figs. S1–S8) and obtained results conform with earlier published
latter methodology provides difficult to separate urea side-products
(
unpublished data).
To study the susceptibility of the oxazolone type Ac
1
6–19
6
t6A cyclic
data.
3
Dehydration of Ac t A by the use of 5-fold molar excess
3
1
of acetic anhydride (a convenient procedure for formation of amino
ester to hydrolysis under different conditions H NMR technique
was used, as the spectra (in D
Ac
2
0
6
acid oxazolones ) furnished the cyclic form of threonine residue.
The reaction was performed at room temperature in an 85/15 (v/
v) mixture of anhydrous methylene chloride and dimethylformam-
ide (Scheme 1).
2
O) of cyclic Ac
3
ct A and opened
6
3
t A are sufficiently different (Fig. 2 and Fig. S24). The signals
of heterobase protons H8 and H2 are diagnostic for quantification
of the product formation.
6
After 3.5 h, TLC analysis (CHCl
complete disappearance of the nucleoside substrate and formation
of two products, the major one with R = 0.40 and the minor with
= 0.56, which were isolated and subjected to H NMR analysis
in anhydrous DMSO-d . The spectrum of the major product (iso-
lated in 82% yield) clearly confirmed the structure of the desired
3
/MeOH 9:1, v/v) revealed almost
Cyclic Ac
3
ct A was subjected to hydrolysis in a 0.2 M PBS/D
2
O
buffer (pD 7.4). The reactions were performed at 23 and 37 °C in
NMR tubes, and the time course of the process was monitored
f
1
R
f
(Fig. 3, panel Ia and Ib, respectively, and Fig. S26 and Fig. S27).
6
The hydrolyses carried out at 23 and 37 °C required ca. 230 and
7
0 h, respectively, to reach the mid-point. The reactions followed
6
Ac
3
ct A nucleoside (Fig. S9). The signal for the diagnostic H11 atom
the kinetics of a pseudo-first order process (Fig. S28 and Fig. S29)
ꢁ
4
ꢁ1
(
NH) at d 8.74 ppm is located upfield to that at 9.85 ppm for the
with the rate constant values of 66.7 ꢀ 10 min
and
6
ꢁ4
ꢁ1
same proton in opened Ac
3 6
t A (compare the spectra in DMSO-d
2
.17 ꢀ 10 min , respectively.
Our next studies confirmed that the cyclic form easily hydro-
lyzes under the conditions of the conventional isolation of modi-
on Fig. S1 and Fig. S9), and the observed relation is the same as that
1
0
6
fied tRNA nucleosides.
3
Treatment of Ac ct A with 0.1 M
O 13 OH
O
ammonium acetate (pH 5.3) at 45 °C for 2 h, followed by 1 M
ammonium bicarbonate (pH 8.2) at 37 °C for additional 2 h, gave
almost exclusively the open form (Fig. 3, panel IIa). Most likely
the reaction mixture contained the carboxylic acid and amide
15
14
O
HN 10
N
O
N
1
1
14 15
N
12
N
N
N
OAc
N
N
OH
H
H
N
N
OH
N
N
N
N
N
AcO
AcO
(
CH
3
CO)
2
O
AcO
AcO
AcO
6
1
3
derivatives of Ac t A, as in the H NMR region of the open nucleo-
O
O
O
CH
2
Cl
2
RT
/DMF
side the signals of H2 and H8 protons were splitted. The presence
of the amide is possible since treatment of the cyclic nucleoside
OAc
OAc
AcO
OAc
6
Ac ct6A (82%)
6 Ac
Ac
3
t A
Ac ct A (12%)
only with 1 M ammonium bicarbonate (pH 8.2) afforded the amide
3
3
6
derivative of Ac
3
t A in good yield (Supplementary data, p. 26,
0
0
0
Scheme 1. Synthesis of the cyclic form of 2 ,3 ,5 -tri-O-acetyl-threonylcarbamoyl-
Figs. S18–S23 and Fig. S25). The new methodology for isolation
6
3
adenosine (Ac ct A).
Figure 2. Comparison of 1H NMR spectra in D
O of Ac
t6A (an upper panel) and Ac
ct A (a lower panel); the protons H2 and H8 diagnostic for Ac
6
ct A stability studies are
6
2
3
3
3
marked with ovals.