Analytica Chimica Acta 429 (2001) 151170

Automatic identication of terpenoid skeletons through 13C nuclear magnetic resonance data disfunctionalization
Marcelo J.P. Ferreira a , Antnio J.C. Brant a , Gilberto V. Rodrigues b , Vicente P. Emerenciano a,
a

Instituto de Qu mica, Universidade de So Paulo, Caixa Postal 26077, 05513-970 So Paulo, Brazil b Departamento de Qu mica, ICEx, Universidade Federal de Minas Gerais, 30161-000 Belo Horizonte, Brazil Received 11 July 2000; accepted 25 October 2000

Abstract The proposal of this paper is to present a procedure that utilizes 13 C NMR for terpenoid skeletons identication. By this reason, a novel program named REGRAS was developed for the specialist system SISTEMAT. This program carries out an analysis of the 13 C NMR data from a given compound and, from ranges of chemical shifts, recognizes the chemical functions existing on specic positions of carbon skeletons. At the end of this procedure, the program matches the types of carbon atoms obtained against a database, displaying as analysis results the likely skeletons of the questioned substance. The program REGRAS was tested on skeleton elucidation of 35 compounds from the most varied classes of terpenoids, exhibiting excellent results in skeleton prevision precesses. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Terpenoids; Natural products; 13 C NMR; Skeletons elucidation; Expert system

1. Introduction The nuclear magnetic resonance (NMR) techniques for 1 H or 13 C and their bidimensional variant are currently the most powerful existing techniques referring to furnishment of data about an organic substance. These techniques are relevant when one treats structure elucidation of new natural products, due to the great diversity and the structural complexity found within these classes of substances [13]. Structural determination of natural products is realized by experienced spectroscopists who, in their
Corresponding author. Fax: +55-11-38155579. E-mail address: vdpemere@quim.iq.usp.br (V.P. Emerenciano).

majority, by analyzing a set of spectral data, track usually the reasoning logic as follows: 1. identication of the natural product class, i.e. if the questioned substance is a terpenoid, an alkaloid, a avonoid among others; 2. identication of the substance subclass, for example, if it is a terpenoid, if this subclass belongs to monoterpenes, sesquiterpenes, diterpenes, triterpenes or steroids; 3. determination of the substance carbon skeleton; 4. functionalization of the substance skeleton, that is, determination of the organic functions and their respective positions on the carbon skeletons; 5. determination of the relative stereochemistry of the molecule chiral centers.

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 2 6 8 - X

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The steps described in a and b are simple stages of the process, for the conditions and the solvents utilized in the extraction process lead to restrict the problem [4]. The fundamental stage of the procedure previously described is the determination of the substance carbon skeleton, since this forms the basic unit to which the substance belongs. However, the skeleton elucidation is not an easy task towards a great diversity of carbonic rearrangements which determined groups of substances can exhibit, for example, in sesquiterpenes or diterpenes, where there are some hundreds of possible skeletons [1]. In order to help the user to resolve these problems, numerous expert systems were developed and tested for automatic identication of substructures [512]. Throughout out last decade, we have developed an expert system SISTEMAT [1315], whose main purpose is to support this user in processes of structural determination of natural products. Thus, in the developed system was implanted the carbon skeleton denition, so that the system by itself, during a research, can nd the skeleton of the analyzed substance. At that rate, some classes of substance, e.g. monoterpenes, sesquiterpenes, diterpenes and triterpenes [1620] were successfully analyzed, so that most diverse natural product researchers could have one more available tool for making easier the processes of structural determination of new compounds from these classes.

In order to quicken carbonic skeleton determination of a substance in the specialist system SISTEMAT, one has elaborated a program termed REGRAS, which carries out the analysis of 13 C NMR data through automatic recognition of certain functional groups and further count of types of carbon atoms (methyl, methylene, methine and quaternary carbons) present in a substance. This process is denominated disfunctionalization, and it is worthwhile to point out here that this type of analysis is often mentally realized by experimented spectroscopists to obtain likely candidate skeletons. The advantage of the development and utilization of this program is its speed when compared with the specialists one, in addition to its possible usage by unskilled people at dened classes of substances. The objective of this paper is to introduce the program REGRAS, developed for the expert system SISTEMAT, its performance methodology and the results achieved for unprecedented substances recently published in the literature.

2. The program REGRAS 2.1. Methodology At this rst stage of the project SISTEMAT, we have directed our studies to a natural product class

Fig. 1. Example of calculation of carbon atom types present in a skeleton.

M.J.P. Ferreira et al. / Analytica Chimica Acta 429 (2001) 151170 Table 1 Chemical shift ranges utilized for disfunctionalization of the NMR data Chemical function C=O CHO RCOOR C=C Initial multiplicity 1 2 1 1 2 3 1 2 3 1 2 Final multiplicity 3 4 4 2 3 4 2 3 4 3 4

153

13 C

Chemical shift ranges 190.0250.0 190.0250.0 165.5190.0 113.0165.5 104.0167.0 100.0167.0 61.0100.0 54.090.0 54.090.0 100.0113.0 90.0104.0

COH

C(OR)2 CH(OR)2

that possesses a great variety of structures as well as a great skeleton diversity: the terpenoids. By this reason, we have collected from the literature thousands of substances bearing the most varied types of skeletons [12,1621]. So, from these skeletons, we calculated for each one the number of kinds of carbons present, i.e. the whole number of carbons encompassing quaternary, methine, methylene and methyl carbons, as outlined in Fig. 1. These data were obtained for all the skeletons observed in the bibliographic review. From 13 C NMR data of thousands of led substances, we have created 13 C NMR chemical shifts ranges usually noted for a given functional group. These chemical shifts ranges are shown in Table 1, together with the initial multiplicity they exhibit in a 13 C NMR spectrum, and the nal multiplicity they must present after data disfunctionalization. This information together with the types of carbon extant on each skeleton were recorded in a database allied to the program REGRAS, which is written in PASCAL languague. In Fig. 2 is presented a ow chart of the program. This program is available at the ftp site address ftp://143.107.53.186/PUB and runs in PC/DOS environment. 2.2. Program performance To typify operation and utilization of the program REGRAS, we have selected from the literature the

Fig. 2. Flowchart of the REGRAS program.

steroid whose skeleton is a withanolide, isolated from Ajuga parviora [22], shown in Fig. 3. After database creation with 800 types of skeletons gathered in the literature and their respective carbon atom types, as well as another database of dened chemical shift ranges represented in Table 1, we initiated data from 13 C NMR entry, that is, chemical shifts and multiplicities of the substance in Fig. 3. After entry of spectral data, we started the research on skeletons by the program REGRAS, which carries out the disfunctionalization of 13 C NMR data through the ranges displayed in Table 1, for further comparison with the data generated around the skeleton types observed in the literature. The program REGRAS, at the beginning, shows a screen (Table 2) with the chemical shift corresponding to the carbon that will be disfunctionalized and the range wherein the latter is tted with the respective multiplicity; it also shows the extant function before

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Fig. 3. Example of a withanolide employed for testing the program REGRAS.

the process disfunctionalization and the sort of carbon which will be generated after nishing the procedure. In Table 2, this step corresponds to disfunctionalizing C=O into CH2 . At the end of the screen, the program still exhibits the occurrence number of each multiplicity in the original spectrum and the total number of carbons. This process is repeated until the chemical shifts which are found in the ranges shown in Table 1 are completely analyzed. After the whole analysis of 13 C NMR data, the program displays the occurrence number of

each multiplicity in the disfunctionalized spectrum (Table 3). After realizing the research in the database, the program shows the probability of the substance belonging to a determined skeleton; in this case, the program showed withanolide skeleton with a 100.0% probability. Through this result, one can verify that the program REGRAS furnished correctly the skeleton of the substance exhibited in Fig. 3. Another example that can be given is the complex case of structural determination of limonoids. Limonoids are highly oxidized and degraded tetra-

Table 2 Initial screen of the REGRAS programa Disfunctionalization Multiplicity 1 2 3 4 No. of carbons
a

Table 3 Final screen of the REGRAS programa Disfunctionalization Occurrence 10 6 8 4 28 Multiplicity 1 2 3 4 No. of carbons
a

Occurrence 2 8 12 6 28

Chemical shift: 190 210.5 250; multiplicity: 1; disfunctionalizing C=O into CH2 .

Chemical shift: 54 58.1 250; multiplicity: 3; disfunctionalizing COH into CH3 .

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Fig. 4. Some limonoid skeletons.

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Fig. 4. (Continued).

cyclic triterpenes, which constitute true challenges on process of their structural determination, due to their high oxidation degree and skeleton diversity [23]. In Fig. 4 are shown the most common types of limonoid skeletons. To test the efciency of REGRAS program on this class of structures, we choose from the literature the Elegantin A limonoid (Fig. 5), isolated from Trichilia elegans ssp. elegans [24], and put its 13 C NMR data into the program. After data disfunctionalization process, the program REGRAS proposed the likely skeleton of the substance, the limonoid of type IX, according to Fig. 4, which represents the correct skeleton of the referred compound.

3. Results and discussion In order to evaluate the program REGRAS, we selected randomly from the literature ve unprecedented substances pertaining to each terpenoid class (totalizing 35 substances) and from their 13 C NMR data tests were realized. The structures of the utilized substances for testing the program REGRAS are exhibited in Fig. 6. The results obtained with this program are in Table 4, where one can nd the number of the substance corresponding to the structure in Fig. 6, 13 C NMR data, the actual skeleton of the substance and the skeleton proposed by the program, and the respective references. The structures of skeletons suggested by

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Fig. 5. Limonoid used to test the program REGRAS.

the program REGRAS are shown in Fig. 7. It is worth noting here to mention that the chemical shifts concerning the substituents are not exhibited in Table 4, once these are previously excluded by the program MACRONO, that identies the kinds of substituents as well as the signals belonging to the same. This program has widely been tested and evaluated in other papers [25,26]. Tests of 35 samples were realized by the program REGRAS, where, from the results displayed in Table 4, we veried that in all the cases the program proposed the right skeleton of the substance, so that in 62.9% of the cases, this was the unique proposed skeleton, and in the remaining cases the right skeleton was proposed together with other skeletons, being the major proposal number observed in the substance of test XXVII, for which were proposed as a total seven probable skeletons. One can verify that in the cases in which the program REGRAS has proposed other skeletons besides the correct one, only the types of carbon atoms present in the substance skeleton are not enough to dene only a skeleton proposal, however, it is important to point

out that at this point of the analysis, we have a meaningful reduction of the likely skeletons. For example, if we analyze the substance XXVII for which the program has shown seven skeleton proposals, we can note that there has been an extremely signicant reduction of proposals, for there are 180 likely skeleton types for triterpenes [20], therefore, resulting a clear reduction of analysis time. This kind of analysis will furtherly be integrated with the system PICKUP [16], which allows search and obtaining of characteristic 13 C NMR chemical shift ranges of the skeletons, in order that, from the proposals generated by the program REGRAS, there is a consultation, what reduces even more the skeleton probability offered by the programs. This methodology adapted for analysis of terpenoids should be revised for analysis of aromatic compounds, for instance, avonoids, in that the 13 C NMR chemical shift ranges accepted for disfunctionalization in program REGRAS do not allow the treatment of two organic functions present on the same carbon atom, what is an often observed case in aromatic compounds.

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Fig. 6. Substances utilized to test the program REGRAS.

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Fig. 6. (Continued).

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Fig. 6. (Continued).

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Fig. 6. (Continued).

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Fig. 7. Skeletons proposed by the program REGRAS.

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Fig. 7. (Continued).

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Fig. 7. (Continued).

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Fig. 7. (Continued).

4. Conclusions From the results obtained with the program REGRAS, one can conclude that this is introduced as a valuable tool for the process of terpenoid skeleton determination, in that it reduces considerably the number of skeleton proposals for a determined substance. This program in next future, if coupled with the characteristic 13 C NMR ranges already obtained for several terpenoids [1619], could be utilized as a restriction

module for the structure generator that is being developed for the expert system SISTEMAT, i.e. instead of the generator working with all the skeletons of a class of substance to start the process of generation of likely structures, it will have to initiate the process by utilizing only the skeletons previously proposed by the program REGRAS. The immediate consequence of the utilization of this program on to the structure generator will be the reduction of the computational time and the number of displayed structural propos-

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als, what avoids the combinatorial explosion problem observed in other specialist systems developed up to now [6266].

Acknowledgements This work was supported by grants from the Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP) and by the Conselho Nacional de Pesquisa (CNPq).

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