ISSN 10619348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 13, pp. 1130–1135. © Pleiades Publishing, Ltd., 2013. Original Russian Text © V.M. Babaev, R.Z. Musin, M.G. Korochkina, 2012, published in Massspektrometria, 2012, Vol. 9, No. 3, pp. 175–180.

ARTICLES

Investigation of Diterpenoid Isosteviol Ammonium Derivatives by MatrixAssisted Laser Desorption/Ionization Mass Spectrometry V. M. Babaeva, b, R. Z. Musina, and M. G. Korochkinaa a

Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, ul. Arbuzova 8, Kazan, 420088 Russia b Kazan National Research Technological University, ul. Karla Marksa 68, Kazan, 420015 Russia email: [email protected]

Abstract—Matrixassisted laser desorption/ionization (MALDI) mass spectra of diterpenoid isosteviol ammonium derivatives have been analyzed. The compounds were synthesized as antibacterial agents. It has been found that peaks of salt cations were detected in positive ion MALDI mass spectra of monoquaternary derivatives. The fragmentation of bisquaternary salts depended on the distance between the ammonium cen ters. Clusters containing dication and acidic matrix anion have been observed for compounds with two quat ernized nitrogen atoms connected by a nonanomethylene or a longer spacer. Similar clusters did not form for compounds containing pentamethylene or a shorter spacer between two quaternized nitrogen atoms. Keywords: mass spectrometry, matrixassisted laser desorption/ionization, isosteviol, ammonium com pounds DOI: 10.1134/S1061934813130029

INTRODUCTION Mass spectrometry is one of widely used methods for the identification and structural investigation of various naturallyoccurring compounds and their syn thetic derivatives [1–5], including terpenoids. Cur rently various methods of ionization are used for their analysis; however, matrixassisted laser desorp tion/ionization (MALDI) is of special interest as an efficient method of soft ionization. It is particularly important for thermolabile compounds. The implementation of this method is complicated because the number of variables in MALDI is signifi cantly higher than those in other methods. The yield of ions is affected by the procedure of sample preparation and application (matrix nature, layer thickness, and target surface), the ratio sample : matrix, laser energy, and the presence of impurities and additives [6, 7]. Therefore, the analysis of new compounds is an inde pendent problem [8] and often requires the develop ment of special approaches. The study of derivatives of naturallyoccurring compounds bearing charged centers by MALDI is of current interest, because the quaternization of natural metabolites, usually possessing complex structures, can substantially simplify the mass spectrometric anal ysis of their neutral precursors [2]. It should be noted that the analysis of compounds with the salt structure by this method has certain specific features and is not a simple task [9]. Moreover, bisquaternary ammo

nium compounds are attractive objects as models for the development of mass spectrometric approaches to studying multicharged molecules [10]. Thus, some general regularities of the analysis of MALDI data for such compounds were obtained in detailed mass spec trometric investigations of the organic bisammonium decamethoxine salt by secondary ion mass spectrome try, electrospray ionization mass spectrometry, and MALDI [9, 10]. In this work we recorded for the first time and com prehensively studied MALDI mass spectra of a series of mono (compounds 1 and 2) and bis (com pounds 3–7) ammonium derivatives of diterpenoid isosteviol (Fig. 1). Isosteviol (16oxoentbeyerane 19oic acid) is a diterpenoid isolated from the avail able food sweetener SWETA, a mixture of enzymati cally treated glycosides of the plant Stevia rebaudiana Bertoni, using acid hydrolysis. Isosteviol demonstrates antihypertensive and antihypotensive activity [11– 13], antituberculosis activity [14], and inhibits the transport of monosaccharides and oxidative phospho rylation [15–17]. Isosteviol derivatives with one onium nitrogen atom are active in relation to St. aureus, and their activity enhances with increasing hydrophobicity of the substituent at the onium nitro gen atom. Bisammonium derivatives of isosteviol studied in this work demonstrate high antimicrobial activity, depending on the length of the polymethylene chain between the nitrogen atoms [18]. As a whole, the

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1131

Fig. 1. Structures of the studied (1, 2) monoquaternary and (3–7) bisquaternary ammonium derivatives of diterpenoid isoste viol.

Mass spectra were acquired on a UltraFlex III TOF/TOF timeofflight mass spectrometer (Bruker Daltonik, Bremen, Germany) in the linear and reflec tron mode. The measurements were performed in the range m/z 200–5000. The data were processed using the FlexAnalysis 3.0 software (Bruker Daltonik). Sample preparation. The samples were dissolved in methanol to the concentration 10–2 mg/mL. 2,5 Dihydroxybenzoic acid (DHB), αcyano4hydroxy cinnamic acid (HCCA), 3hydroxypicolinic acid (HPA), and pnitroaniline (pNA) were used as matri ces. The solutions of matrices in methanol were pre pared in concentrations 3–5 mg/mL. The samples were applied by the drieddroplet method. A solution of matrix (0.3 μL) was applied onto a Anchor Chip tar get (Bruker Daltonik) using a dispenser. After solvent evaporation 0.3 μL of a sample solution were applied onto the target. JOURNAL OF ANALYTICAL CHEMISTRY

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596 594

100

Cat+

The studied compounds were obtained by the reac tion of an excess of isosteviol amino derivative with 1,ωdibromoalkanes with prolonged heating in aceto nitrile according to the method similar to the one described in the work [18]. Their structures were con firmed by NMR, mass spectrometry, and elemental analysis.

Intense peaks of cations (Cat+) at m/z 538 Br)/540 (81Br) for compound 1 and m/z 594 79Br)/596 (81Br) for compound 2 (Fig. 2) were ( observed in the MALDI mass spectra of ammonium derivatives of diterpenoid isosteviol 1 and 2 because of the dissociation of salts. The fact of the dissociation of ammonium compounds agrees with the published data [19]. The presence of peaks at m/z 458 and 514 for salts 1 and 2, respectively, is, probably, due to the elim ination of an HBr molecule from the corresponding cation because of fragmentation occurring under MALDI conditions. The mass spectrum of bisquaternary ammonium salt 5 is presented in Fig. 3. The main peak in the figure at m/z 1058 corresponds to the dication cluster with a (79

80

[Cat – HBr]+

EXPERIMENTAL

RESULTS AND DISCUSSION

Relative intensity, %

antimicrobial activity of the bisquaternary derivatives of isosteviol exceeds the activity of the corresponding monoquaternary derivatives.

60 40

514

20 345

550

0 300 350 400 450 500 550 600 650 700 m/z Fig. 2. MALDI mass spectrum of compound 2 acquired with DHB as matrix. No. 13

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Fig. 3. MALDI mass spectrum of compound 5 acquired with DHB as matrix.

1043

60 40 20 500

700

900

1100

984

The results of the preformed studies suggest that the cluster ion [Dicat · Br]+ decomposes by path ways (a–c) indicated in Scheme 1. Direction a of the decomposition of cluster [Dicat · Br]+ is associated with the abstraction of a neutral CH3Br molecule from the cluster ion and the forma tion of the ion [Dicat ⋅ Br – CH3Br]+. Similarly to pathway a, pathway b associated with the abstraction of an RBr molecule from the cluster is also possible. Pathway c is characteristic for bisammonium salts [9, 21]; it consists of the break of the spacer bond C⎯N with the addition of a bromine atom and the ejection of a neutral amine molecule RN(CH3)2. Similar path ways are observed in the decomposition of the cluster ion [Dicat + DHB – H]+ as well. The fragmentation of ions [Dicat ⋅ Br]+ and [Dicat + DHB – H]+, also gave ions at m/z 345 of the composition R+.

Relative intensity, %

[Dicat + HPA – H]+

80

For several ions of bisammonium salt 5 post source decay (PSD) mass spectra were recorded. In Figs. 5 and 6 mass spectra of the fragmentation of [Dicat + DHB – H]+ (m/z 1058) and [Dicat ⋅ Br]+ (m/z 984/986) ions are presented. It should be noted that the peaks of fragment ions are also observed in the mass spectra obtained in the linear mode with increas ing power of laser radiation.

100

890

[Dicat – CH3]+

Relative intensity, %

100

0 300

[Dicat ⋅ Br]+

deprotonated molecule of the matrix [Dicat + DHB – H]+. The formation of this ion was supposed and observed in the work [9] in the study of decamethox ine; the authors paid special attention to its origin. Probably, bisquaternary ammonium compounds interact with the carboxylate anion of the molecule of dihydroxybenzoic acid. The interaction of the quater nary ammonium group with the carbonyl group C=O, quite strong as it follows from the studies of the prop erties of hydrogen bond of the R3N+–C–H⋅⋅⋅O=C type [20], is possible as well. The formation of cluster ions is typical not only for DHB. Thus, the use of 3hydroxypicolinic acid as a matrix led to the forma tion of an intense peak of the cluster ion [Dicat + HPA – H]+ (m/z 1043) (Fig. 4). In the case of αcyano4hydroxycinnamic acid, a peak of cluster ion [Dicat + HCCA – H]+ (m/z 1092) was observed as well. Because of the possibility of the cation–πinter action of quaternary ammonium groups with the aro matic ring we studied this salt using pnitroaniline as a matrix. It was found that the cluster of the dication with a molecule of pnitroaniline bearing an aromatic ring does not form. This is, probably, because of the basic nature of pnitroaniline preventing its action as a counterion for the dication of the bisquaternary salt.

The cluster of the dication with the counterion is the main characteristic ion by which the identification of individual bisquaternary salts can be performed.

1136

1300

80 60 40

345

20 0 300

400

560

514 571

500

600

m/z Fig. 4. MALDI mass spectrum of compound 5 acquired with 3HPA as matrix.

668

700 m/z

890 [Dicat – CH3]+

0 300 400 500 600 700 800 900 1000 1100 m/z

[Dicat + DHB – RN(CH3)2 – H]+

984

[Dicat – R]+

890

20

The regularities found are in agreement with the general behavior of bisammonium compounds under MALDI conditions described by Pokrovsky with co authors in [9]; in particular, in our case no peaks of the doubly charged ion Dicat2+ and ion [Dicat – H]+ were observed.

[Dicat – RN(CH3)2]+

60 40

[Dicat + DHB – H]+

[Dicat – CH3]+

Relative intensity, %

80

[Dicat ⋅ Br]+

1058

100

[Dicat – RN(CH3)2(CH2)9N(CH3)2]+

1132

800

900 1000

Fig. 5. PSD mass spectrum of ion [Dicat + DHB – H]+ (m/z 1058) of compound 5.

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R

+

N

(CH2)n

+

N

b –RBr

CH3

CH3 R

+

N

(CH2)n

CH3

R Br–

[Dicat · Br]+ a –CH3Br

N

R

R

+

N CH3

CH3

[Dicat · Br – CH3Br]+

c

–RN(CH3)2

N

R

+

N

O R=

CH3

CH3 (CH2)n

1133

(CH2)n

Br

[Dicat · Br – RBr]+

O H2C CH2

O

CH3

CH3

[Dicat · Br – RN(CH3)2]+

n = 3, 5, 9, 10, 12

Scheme 1. The main fragmentation pathways of the ion [Dicat ⋅ Br]+.

500

600

700 m/z

984

800

900 1000

Fig. 6. PSD mass spectrum of ion [Dicat ⋅ Br]+ (m/z 984/986) of compound 5. JOURNAL OF ANALYTICAL CHEMISTRY

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100 80 60 40 20 404 520 0 300 500

[Dicat + DHB – H]+

[Dicat – Br]+

476

[Dicat – R – 2CH3 + 3]+

The revealed regularities in the behavior of bis quaternary salts of diterpenoid isosteviol under MALDI conditions ensure accurate and reliable anal ysis at the stage of their synthesis and the judgment about the reaction path. The MALDI mass spectra of the reaction mixtures formed in the interaction of the

Relative intensity, %

514

atoms of dication of the studied compounds. The authors of [10, 21] found a strong dependence of the degree of fragmentation on the distance between the two charges r1–2. Ions for which r1–2 > 1 nm are rela tively stable. Thus, for bisammonium salts 3 and 4, the Coulomb repulsion between the two positive charges is strong, which apparently leads to the frag mentation of the dication and presence of intense peaks of the corresponding products in the mass spec tra. Unlike compounds 3 and 4, the dication of salts 5–7 is stable, which is revealed in the mass spec tra by the presence of peaks of clusters with counteri ons and/or a matrix molecule.

890

594

[Dicat – CH3]+

560

[Dicat⋅Br – RN(CH3)2]+

Relative intensity, %

100 80 345 60 40 20 0 300 400

[Dicat – RN(CH3)2]+ [Dicat – R]+

[Dicat – RN(CH3)2(CH2)9N(CH3)2]+

The MALDI mass spectra of compounds 6 and 7 are similar to the mass spectrum of compound 5. Figure 7 presents a mass spectrum of bisammo nium salt 4. It should be noted that the mass spectra of isosteviol bisquaternary derivatives of 3 and 4 radi cally differ from the mass spectra of bisquaternary ammonium salts 5–7. The intense peaks for salts 3 and 4 are peaks of ions at m/z 448 and 476, respectively. The formation of these ions is an interesting experi mental fact and requires additional studies. The peaks corresponding to clusters of the dication with a matrix molecule either possess low intensity or are not observed at all. The difference between the MALDI mass spectra of diterpenoid isosteviol bisammonium derivatives 3 and 4 and 5–7 is undoubtedly due to with different lengths of the spacer between the two charged nitrogen

1002

700

900 m/z

1100

1300

Fig. 7. MALDI mass spectrum of compound 4 acquired with DHB as matrix. No. 13

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CONCLUSIONS [Dicat + DHB – H]+

[Cat]+

Fig. 8. MALDI mass spectrum of a reaction mixture in the synthesis of target compound 4 (compound 1 is present as an impurity) acquired with DHB as matrix.

isosteviol amino derivative with 1,5dibromopentane and 1,9dibromononane, respectively, (collected after the completeness of the reaction) are presented in Figs. 8 and 9. As can be seen in Figs. 8 and 9, the MALDI mass spectra of the reaction mixtures formed upon the interaction of two initial compounds contain charac teristic peaks of two new compounds: mono (by product) and bisquaternary (target product) isoste viol derivatives. The characteristic peaks for com pound 1 are m/z 458, 538/540; for compound 4, m/z 476, 520, 1002; for compound 2, m/z 514, 594/596; and for compound 5 m/z 532, 559, 668, 890, 984/986, 1058.

20

0 300

345 390

400

[Cat]+

532 559

514

500

1058

594

[Dicat – R]+

[Dicat – RN(CH3)2]+ [Dicat – R – 2CH3 + 3]+

40

[Dicat – RN(CH3)2(CH2)9 + H]+

60

[Dicat – RN(CH3)2(CH2)9N(CH3)2]+

Relative intensity, %

80

This work was supported by the Ministry of Educa tion and Science of the Russian Federation (state con tract no. 16.552.11.7008), Program of the Department of Chemistry and Materials Science of the Russian

[Dicat + DHB – RN(CH3)2 – H]+

550

100

ACKNOWLEDGMENTS

[Dicat ⋅ Br]+

Relative intensity, %

100 80 476 60 40 494 458 520 20 390404 1002 0 300 400 500 600 700 800 900 1000 1100 m/z

Matrixassisted laser desorption/ionization mass spectrometry was successfully used to study diterpe noid isosteviol ammonium derivatives. It was demon strated that, for monoquaternary ammonium deriva tives under MALDI conditions, peak of the corre sponding salt cation is registered in the detection of positive ions. It was found that the nature of processes of charac teristic ion the formation in the fragmentation of iso steviol bisammonium derivatives under MALDI con ditions depends on the length of the polymethylene chain linking two quaternary nitrogen atoms: —for the compounds with quaternary nitrogen atoms separated by nine and more methylene groups, the formation of cluster ions of the salt dication with a deprotonated matrix molecule and/or a counterion is typical; —for compounds with quaternary nitrogen atoms separated by five and less methylene groups cluster ions practically do not form. Thus, MALDI mass spectrometry is an efficient instrument for the analysis of quaternary salts of diter penoid isosteviol, ensuring the study of reaction mix tures for their synthesis and also the identification of the target and byproducts.

890

984

[Dicat + DHB – H]+

538

[Dicat – CH3]+

[Dicat – R – 2CH3 + 3]+

1134

1220

668

600

700

800 m/z

900

1000

1100

1200

1300

Fig. 9. MALDI mass spectrum of a reaction mixture in the synthesis of target compound 5 (compound 2 is present as an impurity) acquired with DHB as matrix. JOURNAL OF ANALYTICAL CHEMISTRY

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Translated by I. Duchovni

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