Mass Spectra Analyses of Amides and Amide Dimers of Steviol, Isosteviol, and Steviolbioside

1 Department of Microbiology and Immunology, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, 11031 Taipei, Taiwan 2 Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, 250 Wu-Shing Street, Taipei 11031, Taiwan 3 Department of Chemistry, Tamkang University, 151 Yingzhuan Road, Danshui District, 25137 New Taipei City, Taiwan 4 Departments of Biochemistry and Chemistry, University of Washington, 4311 11th Avenue NE, Seattle, WA 98105-4068, USA 5 Department of Medicinal Chemistry, College of Pharmacy, Taipei Medical University, 250 Wu-Shing Street, Taipei 11031, Taiwan


Introduction
1.1.Fragmentation Pattern of Steviolbioside Amide Derivatives.The mass spectrometry of these safe and sweet compounds such as steviol, isosteviol, and steviolbioside and their amides and amide dimer derivatives are an interesting current subject.Some reports [1] indicated that more than 50 of kaurane derivatives had been reviewed and presented in terms of specific activities which are antiparasitic, antimicrobial, antifertility, anti-inflammatory, and steroidogenesis.Bruno et al. [2] indicated the semisynthetic of ent-kauranes and the ester form displayed the antifeedant activity on insects.Compadre et al. [3] reported that the mass spectra analysis of this diterpenoid and its analogs revealed differences in stereochemistry, and Hussain et al. [4] used chemical ionization mass spectra to examine steviol and its aglycone.There were many reports to mention the toxicities [5], metabolism [6], bioactivities [7], microbial transformations [8], anti-HIV effects [9], genotoxicity [10], anti-inflammatory effects [11], and synthesis [12][13][14] of steviol, stevioside, isosteviol, and their derivatives dimers [15].In this paper, we report our works on steviol derivatives, steviolbioside, and their synthetic compounds which were examined by a number of mass spectrometric techniques including electron impact (EI), fast atom bombardment (FAB), and electrospray with tandem mass spectrometry LC/MS/MS ESI.

Experimental
For the synthetic purpose, steviol, isosteviol, and steviolbioside were prepared from stevioside, which was as obtained parts from plant extracts in China and purchased from Kyowa Foods Co. (Japan) as commercial food additives, via hydrolysis and purification by chromatography.The IR and NMR spectra were identified with those of the authentic sample [8,12].

Test Compounds
As shown in Scheme 3, sample of compounds 4, 5, 6, and 7 was prepared by previous method.

Results and Discussion
A series of substituted steviolbioside amides and amide dimers obtained from the synthesis of title compounds have been investigated by FAB-MS in our laboratory.Some steviolbioside derivative molecules exhibited molecular ion of low intensity and showed the main fragmentation corresponded to the loss of their side chains, such as the sugar adjacent to the nitrogen atom.Steviolbioside 3 has a free carboxylic acid at the carbon-4 position of skeleton.In order to facilitate the recognition of these compounds, we produced its indolyl amide 4p, steary amide 4q, and oleyl amide 4r in good yields (81%-86%).In the same way, the steviolbioside amide dimers 5a-5g, 6, and 7 were prepared by using various alkyldiamines (n = 2, 4, 5, 7, 8, 10, and 12) (Scheme 3).Among these diamine derivatives, aromatic diamines did not seem to react with bulkyl steviolbioside even in their ami-nation reactions.We did not obtain the aromatic amides from this work that might be explained by the weak basicity of the aniline derivatives; the pKa values of the anilines or rather anilinium ions were around 4.8∼5.4.In this work, the elec-tron donor effects seemed to enhance the resultant amination reactions, and we observed that these glycoside sugars did not perturb amide reactions or amide dimer couplings.
In the case of the 2-indolylethyl steviolbioside amide 4p, the fragmentation pathways proposed for the molecule ion [M+Na], [C 42 H 58 N 2 O 12 Na] + are shown in Figure 1 and Scheme 2. The main fragmentation pathway can be International Journal of Spectroscopy described by two major events.(1) α-Cleavage at the C-C bond resulted in the loss of ethylindolyl, and β-cleavage resulted in the loss of methylindolyl moieties producing the formation of molecules and the simultaneous rearrangement of the resulting fragment-formed ion generation at m/z 664.5 and m/z 347.1.(2) The main peaks at m/z 459.4,621.5 and m/z 603.4 were, respectively, found to eliminate 2 glucose, 1 glucose, and 1 o-glucose molecules or indolyl from the parent peaks (Figure 1).In the FAB mass spectra of compound 4q, we observed that the main peak was formed by the molecule, [C 50 H 87 NO 12 ] + at m/z 893 and fragment species at m/z 731 and 556.This could have been due to the lost of 1 and 2 glucose molecules.In its LC/MS/MS spectra, we observed an intense peak at m/z 916.6, which was found to be characteristic of the N-stearyl steviolbioside amide 4q, with [M+Na] ± and the elimination of 1 glucose specie at m/z 754.3.Further loss of the second glucose was occurred mainly through C-C bond β-cleavage with the loss of the branching part of C 16 H 33 in the stearyl amide at m/z 365.1.Followed by elimination of water at position C-13, the fragment ion was formed at m/z 347.1 (Table 2).In compound 4r, we observed that the base peak was the 4r molecule [C 50 H 85 NO 12 ] ± at m/z 891 in the FAB mass spectra.With the further loss of 1, and 2 glucoses molecules, two product ions at m/z 729 and m/z 567 were produced, respectively.In its LC/MS/MS spectra we observed the molecule peak with Na at m/z 914.5 and some relative intensities of fragment ion peaks at m/z 858.4 (loss of a butyl species of oleyl), at m/z 752.5 (loss of 1 glucose from the m/z 774.6 fragment ion), and at m/z 612.6 (loss of 2 glucoses molecules and Na from the molecule peak).We also observed a small fragment species at m/z 365.2; this could have been due to the loss of 2 glucose molecules and the C-C bond β-cleavaged to amine nitrogen with loss of the C 16 H 31 moiety from the olelyl group followed by loss of water (at m/z 347.2).An interesting fragment was observed at m/z 207.0 that was assumed to occur through γ-cleavage from the side chain of olelyl.In its ES − fragmentation pattern we observed some intense fragment species at m/z 864.4 and m/z 836.4; and it was assumed that they were the result of the loss of ethyl and butyl molecules of side chains of oleyl.via α-cleavage of the side chains occurred.In the LC/MS/MS spectra of compound 6, we observed an intense peak at m/z 683.5 that was the characteristic of steviol amide dimer (n = 2) molecule [M+Na] ± .Its base peak at m/z 600.5 was probably formed by the loss of two products [CH 2 CO] ± and [CH 2 CCH 2 ] ± , which were produced by the opening of the C/D ring of steviolbioside.We also observed in the daughter spectrum that the molecular peak was at 661.4 and loss of 1 H 2 O molecule was at m/z 643.5.In the parent peak at m/z 273.2 and the base peak there was the loss of a water molecule at m/z 255.2.In compound 7, we checked the FAB + mass spectrum at m/z 661 as a base peak [M+H] + and the parent peak at m/z 273 and its LC/MS/MS spectra of the isosteviol amide dimer (n = 2).It seemed to be the same fragmentation pattern as compound 6.
[M+Na] ± peak was at m/z 683.6 and there was a loss of Na at m/z 661.6, followed by losing of [CH 2 CO] ± at the D ring to form the fragment species at m/z 641.6 and to produce an intense second-generation ion at m/z 451.4.The C-N bond was then cleaved to form the peaks at m/z 344.3 and 273.2.It probably was formed by the cleavage of the C-C bond resulting in the formation of species "d" (Scheme 1) at m/z 121.2 and 151.0 followed by the loss of Na to form the base peak at m/z 128.8.(Scheme 1).
In conclusion, due to the observed fragmentation pattern pathways, cleavage formed and their generated peaks further encourage us to check the synthetic products in biological modification that will be useful in future studies on natural products.

Scheme 1 :
Scheme 1: Proposed fragmentation pathway leading to the generation of fragment peaks from steviolbioside-intermediate (linkage with benzotriazolyl).

2 Scheme 2 :
Scheme 2: Proposed fragmentation pathway leading to the generation of Fragment Peaks from steviolbioside amide 4p.

Table 1 :
Main ions observed in FAB mass spectra of compounds 5, 6, and 7.