Pennelliiside D, a New Acyl Glucose from Solanum pennellii and Chemical Synthesis of Pennelliisides

Acyl glucoses are a group of specialized metabolites produced by Solanaceae. Solanum pennellii, a wild-type tomato plant, produces acyl glucoses in its hair-like epidermal structures known as trichomes. These compounds have been found to be herbicides, microbial growth inhibitors, or allelopathic compounds. However, there are a few reports regarding isolation and investigation of biological activities of acyl glucoses in its pure form due to the difficulty of isolation. Here, we report a new acyl glucose, pennelliiside D, isolated and identified from S. pennellii. Its structure was determined by 1D NMR and 2D NMR, together with FD-MS analysis. To clarify the absolute configuration of the acyl moiety of 2-methylbutyryl in the natural compound, two possible isomers were synthesized starting from β-D-glucose pentaacetate. By comparing the spectroscopic data of natural and synthesized compounds of isomers, the structure of pennelliiside D was confirmed to be 3,4-O-diisobutyryl-2-O-((S)-2-methylbutyryl)-D-glucose. Pennelliiside D and its constituent fatty acid moiety, (S)-2-methylbutanoic acid, did not show root growth-inhibitory activity. Additionally, in this study, chemical synthesis pathways toward pennelliisides A and B were adapted to give 1,6-O-dibenzylpennelliisides A and B.


Introduction
Plants are considered a rich source of natural products that possess diverse structures and corresponding biological activities, such as antiherbivory, antimicrobial, and antioxidant activities [1]. Acyl sugars (sugar esters), nonvolatile secondary metabolites, are specialized natural products produced in the hair-like epidermal structures, known as trichomes, of many Solanaceae families, such as Solanum [1], Nicotina [2], Datura [3], and Petunia [4]. The backbone of acyl sugars basically consists of either a glucose or sucrose moiety attached to one or more straight or branched-chain fatty acids via O-acylation [1].
Although some studies have been conducted on the biosynthesis of acyl sugars [17], their full discovery remains unclear because of the availability of vastly diverse acyl sugars [15]. This implies the potential to present considerably diverse acyl glucoses in S. pennellii as well. However, there are few reports regarding isolation and investigation of their biological activities in their pure form due to the difficulty of isolation [13,17]. pennellii as well. However, there are few reports regarding isolation and investigation of their biological activities in their pure form due to the difficulty of isolation [13,17]. That is because of α and β anomerization at the C-1 position of the glucose moiety. We recently found that α and β anomerization can be successfully control by benzylation of hydroxyl groups present at the glucose moiety. Using this strategy, three compounds, 2,3,4-O-triisobutyryl-D-glucose, 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-D-glucose, and 3-O-decanoyl-2,4-O-diisobutyryl-D-glucose, namely, pennelliisides A-C, were reported [14].
As a part of our ongoing research, another new analogue of acyl glucose was identified from S. pennellii. To determine the absolute configuration of its fatty acid moiety, total synthesis was carried out. Additionally, in this report, chemical synthesis of previously reported 1,6-O-dibenzyl penneliisides A and B are presented. Root-growth inhibitory activity of the newly identified compound and its synthesized compound was also investigated.

Isolation and Identification of Pennelliiside D (1)
The aerial parts of 80-day-old S. pennellii (1.7 kg) were dipped in EtOH for 30 s, and an extract of epicuticular lipophilic wax was obtained by evaporating the organic solvent under reduced pressure. The extract was partitioned between EtOAc and sat. NaHCO3. The extract obtained from the EtOAc layer was roughly purified using silica gel column chromatography to give acyl glucoses, followed by benzylation with 2,4,6-tris(benzyloxy)-1,3,5-triazine (TriBOT) to hinder α and β anomerization as previously reported [14,18,19]. The obtained benzylated derivatives of acyl glucoses were purified using silica gel column chromatography and HPLC to give dibenzyl pennelliiside D (2, 19 mg, Figure 1B). Compound 2 was obtained as a colorless oil. The molecular formula and molecular weight were found to be C33H44O9 and m/z 584.2992 [M] Figure S3, Supplementary Materials) also indicated that the glucose moiety exhibited a β anomeric structure.  (Table 1, and Figure S2, Supplementary Materials). The presence of glucopyranose was further confirmed by comparing COSY correlations between the signals at H-1/H-2, H-2/H-3, H-3/H-4, and H-4/H-5 ( Figure 2A and Figure S4, Supplementary Materials) together with their corresponding coupling constants (Table 1). Meanwhile, NOESY interactions observed due to the cross-peaks of H-2/H-4 and H-1/H-3/H-5 ( Figure 2B and Figure S7, Supplementary Materials), and the signal observed at δ C 100.3 in 13 C NMR (Table 1 and Figure S3, Supplementary Materials) also indicated that the glucose moiety exhibited a β anomeric structure.     Figure S6, Supplementary Materials) were identified as methylene protons corresponding to benzylidene attached to the C-1 and C-6 positions. Moreover, the presence of two isobutyryl ester moieties was determined according to the 1 H NMR and 13 C NMR spectra and COSY and HMBC correlations (Table 1 and Figures S2-S6, Supplementary Materials), and these moieties were attached to C-3 and C-4 positions in the glucose moiety ( Figure 2). Similarly, the 2-methylbutyryl fatty acid moiety attached to C-2 was revealed based on COSY and HMBC correlations, as shown in Figure 2. Therefore, the detailed analysis of 2D NMR data clarified the structure of 2 to be 1, Figure 1).
To afford 1, compound 2 was subjected to debenzylation with palladium black under a hydrogen gas atmosphere (Scheme 1). Compound 1 was obtained as a colorless oil, and the molecular formula and molecular weight were found to be C 19 Table 2. Although 1 H NMR, 13 C NMR, COSY, HSQC, and HMBC (Figures S9-S14, Supplementary Materials) data were complex due to the interference of α and β anomers, assignment of H and C corresponding to the α and β anomers of 1 were done partially. Assignments of α and β anomers are shown in Figure S12 in the Supplementary Materials. Based on the NMR data, the chemical structure of 1 was determined to be 3,4-O-diisobutyryl-2-O-(2-methylbutyryl)-D-glucose ( Figure 1A), although the absolute configuration of the 2-methylbutyryl fatty acid moiety was still unclear [1,13,17]. HSQC, and HMBC (Figures S9-S14, Supplementary Materials) data were complex due to the interference of α and β anomers, assignment of H and C corresponding to the α and β anomers of 1 were done partially. Assignments of α and β anomers are shown in Figure  S12 in the Supplementary Materials. Based on the NMR data, the chemical structure of 1 was determined to be 3,4-O-diisobutyryl-2-O-(2-methylbutyryl)-D-glucose ( Figure 1A), although the absolute configuration of the 2-methylbutyryl fatty acid moiety was still unclear [1,13,17].
Removal of benzyl ether. (12), were synthesized to determine the absolute configuration of the fatty acid moiety, 2-methylbutyryl, attached to C-2, although the naturally available ester of 2-methylbutyryl in other natural sources is mostly in the (S) configuration [20,21].
Then, we compared the 1 H NMR and 13 C NMR data of natural and synthesized compounds (S/R) ( Table 1 and Table S1, Supplementary Materials). Synthesized (S) isomer of dibenzyl pennelliiside D (2) had good accordance with natural dibenzyl pennelliiside D (2). In the 1 H NMR, the differences between synthesized (S/R) with the natural compound were found in the resonances around δ 1.65 and δ 1.32 as shown in Figure 3. Furthermore, a significant difference was shown when comparing specific rotation values with 12, while natural 2 and synthesized 2 showed almost the same value. The specific rotation values measured for natural and synthesized 2, and 12 were [α] 25 D = −10.5, −10.7, and −21.3 (c 0.6, MeOH), respectively. Based on the above observations, we concluded that the absolute configuration of the 2-methylbutyryl fatty acid moiety in natural 2 was (S) and confirmed its structure, as shown in Figure 1B. Then, we compared the 1 H NMR and 13 C NMR data of natural and synthesized compounds (S/R) (Tables 1 and S1, Supplementary Materials). Synthesized (S) isomer of dibenzyl pennelliiside D (2) had good accordance with natural dibenzyl pennelliiside D (2). In the 1 H NMR, the differences between synthesized (S/R) with the natural compound were found in the resonances around δ 1.65 and δ 1.32 as shown in Figure 3. Furthermore, a significant difference was shown when comparing specific rotation values with 12, while natural 2 and synthesized 2 showed almost the same value. The specific rotation values measured for natural and synthesized 2, and 12 were [α] 25 D = −10.5, −10.7, and −21.3 (c 0.6, MeOH), respectively. Based on the above observations, we concluded that the absolute configuration of the 2-methylbutyryl fatty acid moiety in natural 2 was (S) and confirmed its structure, as shown in Figure 1B.   Then, we compared the 1 H NMR and 13 C NMR data of natural and synthesized compounds (S/R) (Tables 1 and S1, Supplementary Materials). Synthesized (S) isomer of dibenzyl pennelliiside D (2) had good accordance with natural dibenzyl pennelliiside D (2). In the 1 H NMR, the differences between synthesized (S/R) with the natural compound were found in the resonances around δ 1.65 and δ 1.32 as shown in Figure 3. Furthermore, a significant difference was shown when comparing specific rotation values with 12, while natural 2 and synthesized 2 showed almost the same value. The specific rotation values measured for natural and synthesized 2, and 12 were [α] 25 D = −10.5, −10.7, and −21.3 (c 0.6, MeOH), respectively. Based on the above observations, we concluded that the absolute configuration of the 2-methylbutyryl fatty acid moiety in natural 2 was (S) and confirmed its structure, as shown in Figure 1B.   By debenzylation of synthesized 2 with palladium black under a hydrogen gas atmosphere (Scheme 1), synthesized 1 (5.9 mg) was obtained as a colorless oil. The molecular formula and molecular weight were similar to those of the natural 1, which were C 19 Table 3. 1 H NMR, 13 C NMR, COSY, HSQC, and HMBC data are shown in Figures S52-S59 in the Supplementary Materials. Similar to natural 1, partial assignment of H and C corresponding to the α and β anomers of D-glucose for synthesized 1 is shown in Figure S55 in the Supplementary Materials. Comparison of 1 H NMR and 13 C NMR spectra of natural and synthesized 1 also showed similar data (Tables 2 and 3, and   Previously, it has been reported that the acyl sucrose showed root growth-inhibitory effect on velvetleaf [23]. Therefore, root growth-inhibitory activity against natural and synthesized 1 and its constituent fatty acid, (S)-2-methylbutanoic acid, was assessed. Arabidopsis thaliana seeds and 10 µM, 50 µM, and 100 µM concentrations of compounds were used in this experiment. As the control, A. thaliana seeds were germinated in the MS medium without adding any compound. The data revealed that neither compound showed root growth-inhibitory activity at any tested concentration (Figure 4), which might support that acyl glucose contains longer chain carbon fatty acids shows root growth-inhibitory effect.

Synthesis of Dibenzyl Pennelliisides A and B
Using the same strategy of synthesis of 2, synthesis of dibenzyl pennelliisides A and B (17a, b) were conducted using 6 as the starting material (Scheme 3). Isobutyryl chloride and 8-methylnonanoic acid were used to obtain dibenzyl pennelliisides A and B (17a, b). In order to synthesize dibenzyl pennelliiside A, 6 was reacted with isobutyryl chloride to yield 13 that has two isobutyryl fatty acid moieties. Next, deprotection was carried out followed by another reaction with isobutyryl chloride to give the desired compound, dibenzyl pennelliiside A (17a). Using the same starting compound, the synthesis of dibenzyl pennelliiside B was commenced with a condensation reaction with 8-methylnonanoic acid to esterify the fatty acid moiety selectively to C-3. Then, 14 was reacted with isobutyryl chloride followed by deprotection and another esterification with isobutyryl chloride to yield dibenzyl pennelliiside B (17b). The chemical structures of all compounds were characterized using 1 H NMR, 13 C NMR, 2D NMR, and FD-MS ( Figures S76-S102, Supplementary Materials). It has been already proven that the removal of benzyl groups can be accomplished as Scheme 1 to obtain pennelliisides A and B. Using the same synthesis pathway, it is possible to synthesize other acyl glucoses.

Synthesis of Dibenzyl Pennelliisides A and B
Using the same strategy of synthesis of 2, synthesis of dibenzyl pennelliisides A and B (17a, b) were conducted using 6 as the starting material (Scheme 3). Isobutyryl chloride and 8-methylnonanoic acid were used to obtain dibenzyl pennelliisides A and B (17a, b). In order to synthesize dibenzyl pennelliiside A, 6 was reacted with isobutyryl chloride to yield 13 that has two isobutyryl fatty acid moieties. Next, deprotection was carried out followed by another reaction with isobutyryl chloride to give the desired compound, dibenzyl pennelliiside A (17a). Using the same starting compound, the synthesis of dibenzyl pennelliiside B was commenced with a condensation reaction with 8-methylnonanoic acid to esterify the fatty acid moiety selectively to C-3. Then, 14 was reacted with isobutyryl chloride followed by deprotection and another esterification with isobutyryl chloride to yield dibenzyl pennelliiside B (17b). The chemical structures of all compounds were characterized using 1 H NMR, 13

General Experimental Procedures
Optical rotations were obtained with a JASCO P-2200 polarimeter. NMR spectra were recorded in C6D6, CD3OD and CDCl3 using a JNM-EX 270 FT-NMR spectrometer (JEOL, 1

Plant Material
Seeds of S. pennellii were obtained from the National Bioresource Project (NBRP, Tsukuba). The plants were grown under 16 h of light and 8 h of dark for 80 days at 25 • C in an artificial weather room at the Faculty of Agriculture, Hokkaido University, Hokkaido, Japan.