6
Nisler et al.
J ¼ 5.6 Hz, OH) 5.21 (1H, t, J ¼ 6.8 Hz, CH¼), 7.82 (4H, m, ArH). The crude product
ꢀ
(
with m.p. 86-90 C) was used in the next reaction.
(Z)-4-Hydroxy-3-methyl-2-but-2-enylamine hemioxalate (4a)
ꢀ
Compound 9 (15.2 g, 65 mmol) was dissolved in 80% ethanol (80 mL, 50 C), then cooled,
hydrate (Caution! Corrosive! 3.35 mL, 80 mmol) was added and the mixture was stirred at
ꢀ
r. t. overnight before being heated to 60 C (2 h). The reaction mixture was cooled to r. t.,
H SO (35 ml, 2 M) was added and the mixture was stirred for 30 min. After cooling in a
2
4
refrigerator, the phthalhydrazide deposit was filtered off. The filtrate was neutralized using
NH OH. The resulting brownish oil was treated with methanol saturated with NH OH
4
4
(20 ml), then mixed, chloroform (20 ml) was added and the solution was placed in a
refrigerator overnight. Precipitated (NH ) SO was filtered off and the filtrate was washed
4
2
4
2
6
x with absolute ethanol. A brown dense oil (7.3 g) was obtained. Oxalic acid (8.2 g,
5 mmol) was dissolved in water (80 mL) and slowly added to the crude product. The hem-
ioxalate was immediately formed and, after being placed in a refrigerator for a few hours,
the crystallized hemioxalate was filtered off. Additional hemioxalate was obtained by con-
centration of the solvent volume to about half. The resulting crystals were washed with
ethanol and diethyl ether and dried at r. t. The hemioxalate (6.7 g, 45 mmol) was isolated
and TLC analysis (chloroform-methanol-conc. NH OH, 6:4:0.4, v:v:v) showed one single
4
spot. A sample of the product was re-crystallized from EtOH-water (10:1), m.p. 182-
ꢀ ꢀ
82.5 C. Tolman found m.p. 168–170 C (dec.). H NMR (D O): d 1.84 (3H, dt,
2
8
1
1
J¼ 1.1 Hz, 0.75 Hz, CH C¼); 3.68 (2H, dq, J¼ 7.5 Hz, 1.1 Hz, CH N); 4.15 (2H, d,
3
2
J¼ 0.75 Hz, CH OH); 5.46 (1H, ttq, J ¼ 7.5Hz, 1.1Hz, 0.75 Hz, CH¼).
2
cis-Zeatin
Hemioxalate (4a, 2.92 g, 20 mmol) obtained from the previous step was treated in n-
propanol (80 mL) with 6-chloropurine (2.8 g, 18.1 mmol) and triethylamine (6.8 mL,
ꢀ
4
9 mmol). The mixture was heated under argon to 98 C for 6 h. After placing the mix-
ture overnight in a refrigerator, yellow crystals were filtered off, then washed with n-
propanol (2 x 15 mL) and diethyl ether (10 mL). After drying in a desiccator, 3.2 g of
crude product was isolated. The product was purified by heating with n-propanol
(
30 mL) to give a yield of 2.9 g (66%, based on the starting hemioxalate) of yellowish
ꢀ
ꢀ
ꢀ
cis-zeatin, m.p. 213-214 C (m.p. in ref.5,206–208 C; in ref.9,212–214 C; in
ꢀ
1
ref.8,214–215 C). H-NMR (DMSO-d ): d 1.70 (3H, bs, ¼C-CH ); 4.04 (2H, d,
6
3
J ¼ 5.2 Hz, CH OH); 4.13 (2H, m, NCH ); 4.76 (1H, t, J ¼ 5.2 Hz, OH), 5.35 (1H, t,
2
2
J ¼ 6.8 Hz, ¼CH-), 7.56 (1H, bs, NH), 8.06 and 8.16 (1H each, s, purine protons).
HPLC: 99.5% of cis-zeatin, containing 0.15% of trans-zeatin.
Acknowledgments
This work was supported by project grants of the Ministry of Education, Youth and
Sports CZ (LO1204) and the Grant Agency of the Czech Republic (15-22322S).
References
1
. S. Gajdo ꢀs ov ꢁa , L. Sp ꢁı chal, M. Kam ꢁı nek, K. Hoyerov ꢁa , O. Nov ꢁa k, P. I. Dobrev, P. Galuszka,
P. Kl ꢁı ma, A. Gaudinov ꢁa , E. Zizkov ꢁa , J. Hanus, M. Dan ꢀc ꢁa k, B. Tr ꢁa vn ꢁı ꢀc ek, B. Pesek, M.
Krupicka, R. Vankov ꢁa , M. Strnad, and V. Motyka, J. Exp. Bot., 62, 2827 (2011).