Isolation and structure identification of new cytokinins from Gleditsia caspia

Phytochemical investigation of the n -BuOH- soluble fraction of the alcoholic extract of the fruits of Gleditsia caspia (Leguminosae) resulted in the isolation and identification of three new cytokinin derivatives identified as 6-N -(3-methylbut-2-enylamino)-2-hydroxy-9- β -D- glucopyranosyl purine, 6-N -( cis -4-hydroxy-3-methylbut-2-enylamino)-2-hydroxy-9- β -D- glucopyranosyl purine, and 6-N -(3-methylbut-2-enylamino)-2-hydroxy-9-[ β - D-apiofuranosyl-(1'' → 6')- β -D-glucopyranosyl] purine. The structures of the isolated compounds were established by 1D-( 1 H, 13 C-and DEPT) and 2D-(COSY, HMQC and HMBC) NMR analysis.


Introduction
Cytokinins are plant hormones which regulate plant cell proliferation. 1 Most natural cytokinins are N-6-substituted adenine derivatives generated as degradation products during depurination of DNA. 2 Since their discovery, a substantial number of biochemical and genetic studies have focused on elucidating the diverse roles of cytokinins in plant growth and, in particular, watertransport and water-stress conditions. 3Cytokinins are also involved in other essential processes such as the sink/source relationship, vascular development, chloroplast differentiation, apical dominance, and senescence.It seems that these effects result from molecular interactions with other plant hormones and environmental signals. 4Studies using structure-activity relationships have revealed the importance of the isoprenoid side chain for cytokinin activity. 5Although the effects of cytokinins in plants are well known, the mechanism of their action is still not fully understood. 6,7Moreover, N-6-substituted adenine derivatives show pharmacological activity in humans, have found application in molecular medicine. 2Gleditsia caspia (Leguminosae) is a perennial shrub that is found in temperate and sub-tropical North and South America, tropical Africa, and Central and Eastern Asia.In traditional Chinese medicine the genus Gleditsia is used in the treatment of apoplexy, as an expectorant, and for a pesticide. 80][11] We report here the isolation and structural characterization of three new cytokinins isolated for the first time from the natural source.

Results and Discussion
The fruits of Gleditsia caspia were extracted with ethanol and then the dried ethanolic extract was suspended in water and partitioned successively with petroleum, EtOAc, and n-BuOH.The n-BuOH fraction was passed through a polyamide column.The water eluate was chromatographed over reversed-phase silica gel to yield four fractions [A-D].Fraction A was repeatedly chromatographed over Sephadex LH-20 and silica gel to yield three cytokinins 1-3.
Compound 1, was obtained as a white amorphous powder and gave on TLC yellowish-brown color with vanillin/H 2 SO 4 reagent at Rf 0.78 with CHCl 3 -MeOH-H 2 O (61-32-7) as developing solvent It displayed a [M+H] + ion at m/z 382.1742 and a [M+Na] + ion at m/z 404.1546 in the positive ion HR-FAB-MS spectrum, consistent with the molecular formula C 16 H 23 N 5 O 6 .Its UV spectrum showed absorption bands at 210 and 270 nm which are indicative of N-6-substituted adenine derivatives. 12The IR spectrum showed absorption peaks at 3440, 1640 cm -1 , indicating the presence of hydroxyl and conjugated C=N groups, respectively.The presence of a hydroxyadenine skeleton was indicated from the 1 H-NMR spectrum by the characteristic aromatic proton singlet at δ 8.03 and from the 13 C-and DEPT-NMR by the presence of five sp 2 carbons with δ 102.90, 143.47, 153.58, 153.95 and 155.40, corresponding to C-4, -8, -6, -5 and -2, respectively, and from the presence of five nitrogen atoms as indicated from the FAB-MS.The isopentenyl side chain was indicated by an olefinic proton at δ 5.21 (1H, br, H-12), geminal methylene protons at δ 4.48 (2H, br, H 2 -11) and two sharp singlets at δ 1.73 and 1.58 due to two methyl protons; H 3 -14 and H 3 -15, respectively, along with one methylene carbon at δ 39.95 (C-11), one methine carbon at δ 120.61 (C-12), one quaternary carbon at δ 135.34 (C-13) and two methyl carbons at 18.54 (C-14) and 25.94 (C-15), as shown in the 13 C-and 13 C-DEPT NMR spectra (Table 1).The 1 H-NMR spectrum also showed broad signals at δ 4.82 (1H), 5.14 (1H), 5.41 (1H), 5.71 (2H) and 7.09 (1H) corresponding to -OH and -NH protons.For the assignment of the two methyl carbons in compound 1, the more deshielded signal was assigned to the one which is trans-to the amino group. 13,14 The splitting pattern was masked by the water signal.
The glucose moiety was identified from the one anomeric proton and carbon resonances at δ H 5.39 (H-1'), δ C 86.85 (C-1') of glucose.The identity of the sugar moiety was determined by a combination of COSY, HMQC and HMBC experiments (Table 1).The β-anomeric configuration of the glucose moiety was evident from the J-H1, H2 value of 8.6 Hz. 15 The attachment of the glucose moiety to the N-6-position of the hydroxyl adenine skeleton was confirmed by the longrange correlations between H-1' (δ 5.39) of glucose and the methine carbon at δ 143.47 (C-8) and the quaternary carbon at δ 153.95 (C-4), in the HMBC spectrum of 1. Therefore the structure of 1 was established to be 6-N-(3-methylbut-2-enylamino)-2-hydroxy-9-β-D-glucopyranosyl purine.
Compound 2, was also obtained as a white amorphous powder, and gave on TLC yellowishbrown color with vanillin/H 2 SO 4 reagent at Rf 0.56 with CHCl 3 -MeOH-H 2 O (61-32-7) as developing solvent.It has an [M+H] + ion at m/z= 398.1694 and [M+Na] + ion at m/z 420.1515 in the HR FAB-MS, 16 mass units more than that of 1, suggesting the molecular formula of 2 to be C 16 H 23 N 5 O 7 .The UV spectrum showed absorption bands at 215 and 266 nm, and the IR spectrum showed absorption bands for hydroxyl group at 3445 and for conjugated C=N groups at 1640 cm -1 .The NMR data of 2 were superimposable with those of 1, verifying a close structural relationship between 2 and 1.The difference between 2 and 1 was the presence of hydroxyl isopentenyl side chain in 2 versus the iso-pentenyl side chain in 1.This was established from the 1 H-NMR of 2 which indicates the presence of signals for two geminal methylene protons at δ4.55 (2H, br, H 2 -11) and δ4.07 (2H, brs, H 2 -14), a methine proton at δ5.23 (1H, br, H-12) and one methyl group, δ 1.64 (3H, s, H 3 -15). 13C-and 13 2).
Compound 3, was obtained as a white amorphous powder with molecular formula C 21 H 31 N 5 O 10 as indicated from the HR-FAB-MS ions at m/z 514.2169 [M+H] + and 536.1988 [M+Na] + .Combined with the evidence from monosaccharide composition analysis shown in the Experimental Section, this suggested that in compound 3 an apiose moiety (132 mass units) was present in addition to the skeleton present in 1.It gave on TLC yellowish-brown color with vanillin/H 2 SO 4 reagent at Rf 0.31 with CHCl 3 -MeOH-H 2 O (61-32-7) as developing solvent.Its UV and IR spectra showed absorption bands similar to 1 and 2. The 1 H-NMR of 3 was very similar to that of 1, but had extra signals for an additional sugar moiety indicated by the signals of two anomeric protons, the usual one at δ 5.42 (1H, d, J = 8.4 Hz, H-1' of glucose) and a second at δ 4.82 (1H, d, J = 3.0 Hz, H-1'' of apiose).The 13 C-NMR spectrum of 3 also showed a signal arising from an extra anomeric carbon at δ 109.65 (Table 3).The β-configuration on C-1 anomeric orientation of apiose was confirmed by comparing the 1 H-and 13 C-NMR of 3 with those of α-D-δ C 104.5 and β-D-apiofuranosides δ C 111.5, respectively. 18,19 he signal of the glucosidic carbon, C-6' in 3 showed a typical glycosidation shift downfield by 6.6 ppm when compared to the unsubstituted C-6' of the glucose moiety in 1, supporting the attachment of the apiose moiety to the glucosyl unit at C-6'.This was confirmed by HMBC cross-peaks between the anomeric proton of apiose and C-6' of glucose, and between H 2 -6' of glucose and the anomeric carbon of apiose.Furthermore, the HMBC spectrum of 3 showed correlations between the anomeric proton of the glucose and C-4 and C-8 of the hydroxyadenine skeleton, confirming the attachment of the disaccharide moiety to the N-6-position of the aglycon.

Experimental Section
General.Melting points were obtained on a Stuart SMP3 apparatus.UV spectra were determined with a Hitachi 340 spectrophotometer, IR spectra from a Nicolet 205 FT-IR spectrometer connected to a Hewlett-Packard Color Pro.Plotter.The 1 H-and 13 C-NMR measurements were obtained with a Jeol JNM ECA 500 spectrometer operating at 500 MHz (for 1 H-) and 125 MHz (for 13 C-) in DMSO-d 6 solution, and chemical shifts are expressed in δ (ppm) with reference to TMS, and coupling constants (J) in Hertz.The 13 C-multiplicities were determined by the DEPT pulse sequence (135 o ).COSY, HMBC and HMQC-NMR experiments were carried out using a Jeol JNM ECA-500 high field spectrometer.HR-FAB MS were taken on a VGZAB-HF reversed geometry mass spectrometer (BE configuration, where "B" is a magnetic sector and "E" is an electrostatic analyzer).Polyamide (ICN Biomedicals), and silica gel (Si gel 60, Merck), were used for open column chromatography.Flash column liquid chromatography was performed using J.T. Baker glassware with 40 µm Si gel (Baker) and Sepralyte C 18 (40 µm) as stationary phases.TLC was performed on precoated silica gel 60 F 254 (Merck) plates.Developed chromatograms were visualized by spraying with 1% vanillin-H 2 SO 4 , followed by heating at 100 ºC for 5 min or spraying the developed plates with 2% ninhydrin in acetone.
* The splitting pattern was masked by the water signal.