Sucrose esters associated with glandular trichomes of wild lycopersicon species

Phytochemistry, Vol. 29, No. 7, pp. 2115-2118, Printed in Great Britain.

SUCROSE

1990.

0031-9422/90

ESTERS ASSOCIATED WITH GLANDULAR WILD LYCOPERSZCOIV SPECIES RUSSELL

$3.00+0.00

Pergamon Press plc

TRICHOMES

OF

R. KING, LARRY A. CALHOUN,* RUDRA P. SINGH and ALAIN BOUCHER

Research Station, Research Branch, Agriculture Canada, Fredericton, New Brunswick, Canada, E3B 427; *Department Chemistry, University of New Brunswick, Fredericton, New Brunswick, Canada, E3B 6E2

of

(Received in reoisedfirm 15 November 1989) Key Word Index-Lycopersicon

sp.; Solanaceae; tomato tricho?es; sucrose esters; structural determination.

Abstract-Complexes of 2,3,4,1’-tetra-O- and 2,3,4-tri-0-acylated sucrose esters were identified as the major nonvolatile constituents in the exudate from glandular trichomes of Lycopersicon hirsutum and L. hirsutum f. sp. glabratum, respectively. The principal tetra-0-acylated ester was resolved by reversed phase HPLC and characterized as 2,4,1’-tri-O-(3-methylbutyryl)-3-O-(2-methylbutyryl)sucrose. A single 2,3,1’-tri-O-acylated sucrose ester was predominant in the exudate from the glandular trichomes of L. peruuianum var. glandulosum.

INTRODUCTION In common with many members of the Solanaceae, the foliage of some Lycopersicon species is provided with a

vesture of glandular trichomes that freely secrete mucilaginous substances. These sticky exudates may help entrap or otherwise deter potential predators [l, 21. Investigations regarding similar secretions in other members of the Solanaceae, e.g. Datura, [3], Nicotiania [4,5], Petunia [6] and Solarium [7-l l] revealed that complexes of either glucose or sucrose esters (sometimes both) constituted a majority of the nonvolatiles. This paper details the findings of related analyses involving L. hirsutum, L. hirsutum f. sp. glabratum and L. peruvianum var. glandulosum.

RESULTS AND DISCUSSION

Preliminary analysis of the chloroform soluble extracts of foliage from accessions of L. hirsutum, L. hirsutum f. sp. glabratum and L. peruvianum var. glandulosum indicated that sucrose esters constituted a major portion of the nonvolatiles and the amounts varied with the density of the secreting glandular trichomes. These results were in accordance with previous findings involving other genera and were taken to indicate that the secretory glandular trichomes were the source of the sucrose esters. Sequential fractionation of the exudate from the L. hirsutum by preparative TLC on silica gel isolated two complexes of sucrose esters hereafter designated as F, (72.3%) and F, (27.7%) (traces of glucose esters later determined to be derived from some of the sucrose esters were also found). Subsequent ‘HNMR studies [8,10] of the F, and F, esters indicated that they were composed of 2,3,4,62,3,4,1’- or 2,3,4,6’-tetra-O- and 2,3,4-tri-O-acylated sucrose esters, respectively. Fractionation of the F, complex by RP Cis TLC yielded three distinct zones hereafter designated F,,, (63.8%), F,., (22.5%) and F,,, (13.7%) in order of decreasing R, value (Fig. 1). GC of the F,,, isolates as

their acetyl derivatives indicated the presence of one major and several minor constituents. Similar manipulations of the F,,, and F,,, fractions indicated that they both contained a number of unresolved compounds. To determine acyl group composition, portions of all three fractions were transesterified with sodium ethoxide. Comparative capillary GC-MS (EI) of the ethyl esters confirmed the presence of substantial quantities of 2methylbutyryl and 3-methylbutyryl groups in all fractions. However, the major distinguishing feature of the fractions was that F,,, also contained large quantities of iso-undecanoyl substttuents while F,, 3 had substantial numbers of dodecanoyl groups. Good resolution of individual compounds in the three fractions by repetitive RP C,, TLC or HPLC was successful only for the F, i fraction. In this instance the major component 1 was isolated greater than 90% pure as measured by GC of its tetraacetate derivative. GC-MS (EI) of this tetraacetate derivative yielded a high mass ion mfz 457 corresponding to a tetra-0-acylated glucopyranosyl unit containing three methylbutyryl and one acetyl group. Also prominent in the mass spectrum were fragment ions (m/z 373, 253 and 211) that could be ascribed to degraded elements of a tetraacylated fructofuranosyl residue that contained acetates at C-3’, C-4’, C-6 and a methylbutyryl group in the C-l’ position [12]. A ‘H NMR homonuclear shift correlation experiment of 1 exhibited discrete downfield signals for H-l, H-2, H-3, H4 and (based on mass spectral analysis) H-l’, respectively (Table 1). A corresponding 13C NMR indicated the presence of one 2-methylbutyryl and three 3-methylbutyryl substituents. In order to complete the structural assignment the compound was subjected to mild hydrolysis conditions (methanolic ammonium hydroxide at room temperature). Interestingly, these conditions catalysed initial migration of the 4-O- substituent to the 6-0position thus yielding a 2,3,6,1’-tetra-O-acylated derivative (2) (see ref. [ 111 for a similar migration in a glucose derivative). The transformation product proved amenable to purification by crystallization and its evident 2115

2116

R. R.

KING et al.

Trichome exuate

Fz 2,3,4-tri-o-

F1 1’2,3,4-tetra-O-

r---Pi

fi t.‘l,Z

FI,l

(C,

(Cs + Cl I)

(Cr + Cl L) (Cs + Cl 2)

CC,)

t2,2

FZ,l

FlJ

OH-

+Crz) OH-

1’,2,3,6-tetra-O-

2,6-di-O(Cs

acyls)

1’,2,6-tri-O-

(CS acyls) Fig.

/

1. Analysis of L. hirsutum

OR4

Table H OH

1

2

3

sucrose esters

R’ =

Ra =

Rs =

RZ =

MeCH2tMe)CHC,

RI =

R4 =

R2 =

MeCH2(Me)CHC,

RI =

~4 =

~5 =

R2 =

R”=

l,

Rs =

(Me)lCHCH2C;I

R’ =

H

1 (Me)aCHCH,CR

R3 =

1

1.

‘H NMR

data (6) of sucrose esters

2

3

4

1

5.86

5.71

5.56

5.57

2

4.71

4.78

4.73

4.90

3

5.55

5.36

4.08

5.36

4

4.90

3.53

3.47

3.46 4.14

5

4.17

4.12

4.00

6

3.60

4.40

4.58, 4.25

3.92, 3.74

1’

4.05

4.10

4.05

4.04

3’

4.15

4.12

4.14

4.21

4’

4.28

4.30

4.33

4.40

5’

3.65

3.68

3.78

3.82

6

3.84, 3.78

3.89, 3.70

3.88, 3.65

3.82

H

8 (M~)~CHCH~C-

homogeneity was utilized in a ‘HNMR homonuclear shift correlation experiment to confirm the l’-O- position of the acyl substituent on the fructofuranosyl moiety, that is, a clearly resolved two proton downfield singlet displaying no evidence of vicinal proton coupling attributable to the H- 1’ protons. Subsequent partial hydrolysis of the transformation product yielded the 2,6,1’-tri-0-(3methylbutyryl)-sucrose (3) analogue. Consequently, the structure of the original majority component from fraction F,, 1is confirmed as 2,4,1’-tri-0-(3-methylbutyryl)-30-(2-methylbutyryl)-sucrose (1).

When sucrose esters in the F,, 2 and F,, 3 fractions were subjected to mild hydrolysis conditions there apparently was also an initial migration of C-4 substituents to the C6 positions. A ‘HNMR of the secondary products of hydrolysis from both fractions indicated that they consisted of 2,6,1’-tri-0-acylated sucrose esters. Subsequent transesterification of these 2,6,1’-tri-0-acylated compounds indicated that they were devoid of any long chain acyl groups. From these observations it was concluded that the original sucrose esters in the F,, z and F,, 3 fractions contained iso-undecanoyl and dodecanoyl substituents, respectively, in the C-3 positions. Fractionation of the F, complex on RP C,, TLC plates yielded two distinct zones designated as F,, , (65.3%) and F,,, (34.7%) in order of decreasing R, value (Fig. 1). Capillary GC-MS (EI) of the F,, 1isolates as their

Sucrose esters from Lycopersicon

acetyl derivatives indicated the presence of several compounds, but similar manipulations of the F,,, fraction demonstrated the existence of two mainly unresolved components. Transesterification of both fractions with sodium ethoxide and comparative capillary GC-MS (EI) of the resultant ethyl esters confirmed the presence of 2methylbutyryl, 3-methylbutyryl and iso-undecanoyl groups in the F,,, fraction while 2-methylbutyryl, 3methylbutyryl and dodecanoyl groups were predominant in the F,,, fraction. When the sucrose esters in the F, r and Fz.z fractions were subjected to mild hydrolysis conditions initial migration of C-4 substituents to the C-6 positions was again observed. These 2,3,6-tri-0-acyl sucrose transformation products still comprised two distinct fractions containing chromatographically unresolvable components. However, further hydrolysis yielded common 2,6-di-0-acylated sucrose esters that were devoid of any long chain acyl groups. From these observations it was concluded that the F,, 1 and F,,, fractions contained iso-undecanoyl and dodecanoyl substituents, respectively, in the C-3 positions. The preceding observations suggest a biosynthetic route proceeding through 2,3,4-tri-0-acylation of the glucose moiety prior to 1’-O-acylation of the fructose portion of sucrose in the glandular trichomes of L. hirsutum. It was also apparent from these investigations that long chain (C,, or Crz) substituents were situated preferentially in the C-3 position and that their presence tended to inhibit further substitution at the C-l’ position. Similar investigations of the trichome exudate from L. hirsutum f. sp. glabratum indicated that the sucrose ester isolates consisted predominantly of the 2,3,4-tri-Oacylated forms found in the L. hirsutum. When trichome exudate from L. peruvianum var. glandulosum species was processed the yield of sucrose ester material was extremely small and resolution by normal and reverse phase TLC resulted in the isolation of just one sucrose ester (>90% purity by GC of the acetylated derivative). A ‘H NMR homonuclear shift correlation experiment of this isolate (4) yielded data that was indicative of a 2,3,1’-tri-0-acylated sucrose ester. This represents another new substitution pattern for sucrose esters associated with glandular trichomes in the Solanaceae. A corresponding 13CNMR of the compound indicated the presence of one acetyl, one 2-methylbutyryl and one dodecanoyl substituent. Mass spectrometry (EI) of the compound yielded a high mass ion of m/z 387 corresponding to a glucopyranosyl unit containing one acetyl and one dodecanoyl group. Also prominent in the mass spectrum were fragment ions, m/z 247 and 211, that could be ascribed to degraded elements of a fructofuranosy1 residue that contained a methylbutyryl group in the C-l’ position. Due to lack of material partial hydrolysis studies that may have pinpointed the relative placings of the two different substituents, acetyl and dodecanoyl groups, on the glucose moiety were not undertaken and analysis of the mass spectrum was inconclusive. However, based on spatial considerations and in accordance with the results from L. hirsutum and L. hirsutum f. sp. glabratum sucrose ester make-up, the C-3 position is the most likely position for the dodecanoyl group. In contrast to sucrose esters previously characterized in trichome exudates, L. hirsutum is the first example to incorporate two different sucrose ester substitution patterns in the same plant, i.e. 2,3,4-tri-O-acyl and 2,3,4,1’tetra-O-acyl. More notably, L. hirsutum and L. per-

2117

uvianum var. glandulosum sucrose esters are also the first examples reported wherein sucrose moieties contain substituents in the C-l’ position. Of special interest regarding these findings are in L. hirsutum where all the atoms responsible for the perceived sweetness of sucrose, viz. hydroxyl groups on C-l’, C-2 and C-4 [13], have been esterified. These distinctive features could conceivably make them the most bitter tasting sucrose esters yet identified in glandular trichomes of the Solanaceae. It is of interest to speculate whether such seemingly coincidental features may, in fact, have some specific antipredator significance. EXPERIMENTAL

Plant material. Plants were grown in a greenhouse from seeds obtained from C. M. Rick (College of Agricultural and Environmental Services, University of California, Davis, California, USA) A total of 10 seedlings were examined (with a binocular microscope) for each species. Analytical methods. Mp: uncorr. IR: CHCI,. NMR spectra unless otherwise stated were recorded in CDCI, at 200 MHz for ‘H and 50 MHz for 13C. Chemical shifts were measured downfield from int. TMS and further details of the general procedures

for NMR and MS determinations are outlined in a previous paper [8]. Capillary GC was performed using on-column inj. and a 30 m x 0.25 nm id fused silica column with a 0.25 pm film of DB5. HPLC determinations were performed on a RP Crs column (10 pm particle size, 4.6 mm x 25 cm) in conjunction with a RI detector and MeOH-H,O as mobile phase. Isolation of carbohydrate esters. Composite samples of fr. collected foliage from mature plants (preliminary studies indicated that plant or leaf age did not alter the ester types present) were extracted with CHCl, (5 ml per g) by soaking for 5 min. CHCI, was removed in uacuo and the residue taken up in a min. vol. of Me&O, cooled to 0” and vacuum filtered through Whatman no. 1 filter paper to remove co-extracted plant waxes. After removal of Me&O in uacuo, a sample of the residue was

subjected to TLC (silica gel, CHCl,-MeOH, 9: 1)with detection by charring after spraying with 5% H,SO, in EtOH. For preliminary detection charred areas in the R, range 0.3 do.6 were usually indicative of the presence of carbohydrate esters. Positive and indeterminate samples were treated with Ac,O and pyridine (2: 1) with stirring at room temp. overnight. The reaction mixt. could be sampled directly (or quenched in NaHCO, soln, prior to extraction of the acetylated esters with an organic solvent) for comparative capillary GC analysis. Transesterification of carbohydrate esters. Portions of the carbohydrate esters (5-10 mg) were dissolved in dry EtOH (2 ml) and treated for 10 min. at room temp. with 0.1 M NaOEt (0.5 ml). The reaction mixt. was deionized with Amberlite IR-120 (H+) resin and the Et esters analysed by GC-MS (EI) as described previously [S]. Separation and identijication of sucrose esters from L. hirsutum (PI U1353) and L. hirsutum 1: sp. glabratum (PI LA1223). Isolation of the sucrose esters from foliage (610 g) of L. hirsutum on 0.25 mm silica gel TLC plates yielded F, (R/ 0.47, 613 mg) and F, (R,0.29, 235 mg), respectively. Chromatography of the F, fr. (575 mg) on 0.2 mm RP C,, TLC plates (Me&G-H,G, 7:3) yielded F,, 1 (R, 0.45, 294 mg), F,,, (R, 0.19, 104 mg) and F,,,(R,0.14,63 mg). HPLC(MeOH-H,O, 7:3)of F,., afforded 2,4,1’-tri-O-(3-methylbutyryl)-3-O-(2-methylbutyryl) sucrose (1) as a viscous semi-solid with v,,, 3520 and 1735 cm-‘. Pertinent ‘H NMR, and MS data are given in the text. i3C NMR signals at 611.41 (4). 15.99 (4). 26.55 (t). 40.58 (d) and 177.30 (s) corresponded with C-4, C-3’, C-3, C-2 and C-l, respectively, of the one

2118

R. R. KING et al.

2-methylbutyryl group; signals at 622.32 (6 q), 25.27 (d), 25.39 (d), 25.65 (d), 42.92 (t), 43.111 (t), 43.24 (t), 172.02 (s), 172.22 (s) and 172.26 (s) corresponded to C-4, C-4’, C-3, C-2 and C-l of the three 3-methylbutyryl groups; signals at 659.52 (t),61.39 (t).63.74 (t), 68.50(d), 68.63 (d) 70.86(d), 71.78 (d), 72.22 (d), 78.31 (d), 80.82 (d), 88.60 (d) and 103.38 (s) corresponded to the sucrose moiety. A portion of compound 1 (116 mg) was dissolved in 5% NH,OH in MeOH (15 ml) and stirred at room temp. until TLC studies (silica gel, CHCI,-MeOH, 9: 1) indicated that a majority of the starting material had been transformed (ca 30 min). Solvents were then removed in U~CUOat room temp. The residue was fractionated on 0.25 rnrb silica gel TLC developed in CHCl,-MeOH, 9: 1). The zone at R, 0.48 yielded 2,6,1’-tri-O-(3methylbutyryl)-3-0-(2-methylbutyryl) sucrose (2) as crystals (from hexane-Et,O) with mp 120-122”; Y,,, 3520 and 1735 cm-‘. A FAB positive MS exhibited a high mass peak at m/z 701.3 [M+Na]+ corresponding to a molecular formula C,,H,,O,,. ‘HNMR data is given in the text and ‘%NMR signals at b 11.41 (q), 15.98 (q), 26.67 (t), 40.60 (d) and 177.11 (s) corresponded to C-4, C-3’, C-3, C-2 and C-l, respectively, of the one Zmethylbutyryl group; signals at 622.36 (6q), 25.64 (3d), 43.01 (t), 43.15 (t), 43.18 (t). 172.35 (s), 173.61 (s) and 173.94 (s) corresponded to C-4, C-4’, C-3, C-2 and C-l of the three 3methylbutyryl groups; signals at 659.75 (t),62.12 (t), 63.45 (t),69.15 (d),70.15(d),71.53(d),71.69(6),72.91 (d),78.28(d),81.23(d),89.33 (d) and 103.39 (s) corresponded to the sucrose moiety. Further hydrolysis of compound 2 yielded 2,6,1’-tri-0-(3-methylbutyryl) sucrose (3) (R, 0.67 on 0.2 mm silica gel, CHCl,-MeOH, 17:3) which was characterized by its ‘H NMR (Table 1) and GC-MS @I). Partial hydrolysis of portions of F,,, and F,,, frs with NH,OH-MeOH as described for F,, , rapidly yielded mixts of 2,6,1’-tri-0-acylated sucrose esters. In both instances a compound identical by HPLC and GC (of the acetylated derivative) to compound 3 constituted the major portion. Chromatography of F, fr. (200mg) by RPC,, TLC (Me&O-H,O, 7: 3) yielded F,, , (R, 0.35, 98 mg) and F,,, (R, 0.28, 53 mg). None of the major components in either of these two frs could be satisfactorily resolved by HPLC or TLC. Partial hydrolysis of portions of frs F,,, and F,., with NH,OH-MeOH as outhned for F,. , readily yielded similar mixts (by GC of the acylated derivatives) of 2,6-di-0-acylated sucrose esters. ‘H NMR of the major component had 6 5.46 (d, IH, J = 3.5, H-l), 4.75 (dd, lH, 5=3.5, 10.2, H-2), 4.27 (m, 2H, H-6) and transesterification studies indicated that it contained only methylbutyryl groups. Isolation of the sucrose esters from foliage of L. hirsutum f. sp. glubratum (263 g) on 0.25 silica gel TLC yielded 212 mg of material with R, 0.29. The isolates were demonstrated by chromatographic and spectrometric means to be similar to the sucrose esters present in the F, fr. of L. hirsutum. Separation and identijication of sucrose esters from L. peruvianum var. glandulosum (PI LA1292). Isolation of the sucrose

esters from foliage (110 g) of L. peruvianurn var. glundulosum on silica gel TLC yielded a major fr. (R, 0.3 I, 13 mg) and traces of a minor fr. (R, 0.45,0.5 mg). Further resolution of the major fr. on RP C, s TLC (Me,CO-H,O, 7: 3) yielded one major product (R, 0.43,7 mg) as a viscous semi-solid with Y,,, 3520 and 1735 cm- ‘. Pertinent ‘H NMR and MS data are given in the text. 13C NMR signals at 620.73 (q) and 170.39 (s) corresponded to C-2 and C-l, respectively, of the acetate group; signals at 6 11.57 (q), 16.46 (4). 26.68 (t), 40.89 (d) and 175.04 (s) corresponded to C-4, C-3’, C-3, C-2 and C-l, respectively, of the 2-methylbutyryl group; signals at 614.12 (q), 22.68 (t), 31.9 (t), 29.03 (t). 29.24 (t), 29.32 (t),29.44 (2t), 29.60 (t), 24.98 (t), 34.38 (t) and 173.0 (s) corresponded to C-12 through C-l of the dodecanoyl group; signals at 659.40 (f), 61.37 (t), 62.63 (t), 69.85 (d), 70.05 (d), 72.07 (d), 72.37 (d), 74.88 (d), 78.80 (d), 81.61 (d), 89.50 (d) and 102.51 (s) corresponded to the sucrose moiety. Acknowledgements-We thank Jean Embleton (Agriculture Canada, Frederiction, New Brunswick) for HPLC, See Hua Tan (New Brunswick Research and Productivity Council, Frederiction, New Brunswick) for GC-MS and Pierre Lafontaine (Plant Research Institute, Agriculture Canada, Ottawa, Ontario) for FABMS.

REFERENCES 1. McKinney K. B. (1938) J. &on. Enromol.31, 638. 2. Dimock, M. B. and Kennedy, G. G. (1983) Entom.Exp. Appl. 33, 263. 3. King, R. R. and Calhoun, L. A. (1988) Phyrochemistry 27, 3761. 4. Severson, R. F., Arrendale, R. F., Chortyk, 0. T., Green, C. R., Thomas, F. A., Stewart, J. L. and Johnson, A. W. (1985) J. Agric. Food Chem. 33, 870. 5. Wahlberg, I., Walsh, E. B., Forsblom, I., Oscarson, S., Enzell, C. R., Ryhage, R. and Isaksson, R. (1986) Actu Chem. Stand. B 40, 724. 6. King, R. R., Singh R. P. and Boucher, A. (1987) Am. Potato J. 64, 529. 7. King, R. R., Pelletier, Y., Singh, R. P. and Calhoun, L. A. (1986) J. Chem. Sot. Chem. Commun. 1078. 8. King, R. R., Singh, R. P. and Calhoun, L. A. (1987) Carbohydr. Res. 166, 113. 9. Burke, B. A., Goldsby, G. and Mudd, J. B. (1987) Phytochemistry 26, 2567. 10. King, R. R., Singh, R. P. and Calhoun, L. A. (1988) Carbohydr. Rex 173, 235. 11. King, R. R., Calhoun, L. A. and Singh, R. P. (1988) Phytochemistry 21, 3765. 12. Clode, D. M., Laurie, W. A., McHale, D, and Sheridan, J. B. (1985) Carbohydr. Res. 139, 147. 13. Hough, L. and Emsley (1986) New Scienrist No. 1513, 48.