Ismael Leon

Total assignment of 13C and 1H spectra of three isomeric triterpenol derivatives by 2D NMR: an investigation of the potential utility of 1H chemical shifts in structural investigations of complex natural products

It is shown that a recently proposed indirect heteronuclear shift-correlated pulse sequence, XCORFE, can be used to unambiguously assign the 13C spectra of three isomeric (C30H50O) triterpenols: taraxasterol (1a), pseudo-taraxasterol (2) and lupeol (3). This sequence gives excellent resolution combined with sensitivity far in excess of that given by 13C-13C connectivity experiments. Direct heteronuclear shift-correlated spectra are used to totally assign 1H spectra for 1a, 2, 3 and taraxasteryl acetate (1b). 1H chemical shifts are mainly sensitive to local environment and often show values which are characteristic of a particular environment. Knowledge of 1H chemical shifts and splitting patterns also places useful constraints on assignment of 13C chemical shifts for 13C1Hn units. It is strongly recommended that natural products chemists routinely use 2D NMR to assign 1H chemical shifts of complex organic derivatives in order to build up a data bank of 1H spectral data.

Investigation of the Advantages and Limitations of Forward Linear Prediction for Processing 2D Data Sets

Although the potential advantages of f1 forward linear prediction for the processing of multi‐dimensional NMR spectra are well established, this method is surprisingly little used for 2D spectra used for organic structure determination. A detailed investigation of the advantages and limitations of f1 forward linear prediction for this purpose is reported. This is a reliable technique which is particularly useful for 1H‐detected 13C–1H shift correlation spectra, allowing up to 16‐fold linear prediction of the 13C axis of HSQC spectra. In general, the use of linear prediction allows one to obtain comparable 2D spectra in one quarter of the time or double the sensitivity in a comparable time relative to similar spectra without linear prediction. The one exception is the absolute value COSY spectrum, where linear prediction beyond a factor of two gives poor results. Linear prediction is generally superior to zero filling as a time‐saving technique, although the difference between the two approaches disappears as the f1 data point resolution approaches the natural linewidth. By contrast, f2 forward linear prediction is not recommended.

Isolation and identification by 2D NMR of two new complex saponins from Michrosechium helleri

Two complex saponins, amole F and G, were characterized and their spectra were assigned using only 1D and 2D 1H and NMR methods. Amole F and G have seven and six monosaccharides, respectively, linked to the triterpene aglycone bayogenin. In addition to standard 2D methods, a series of TOCSY spectra with different mixing times and a high‐resolution coupled HSQC spectrum were particularly useful for assigning the monosaccharide units. It is concluded that saponins of this complexity are approaching the limit of structural complexity that can be solved by NMR alone, although the limit might be pushed further by access to ultra‐high field NMR spectrometers.

Chemical constituents and cytotoxic activity of Smallanthus maculatus

Smallanthus maculatus (Cav) H. Rob. (Asteraceae, nomenclatural synonym: Polymnia maculata Cav. Icon. [1, 2]) is used by the Highlands Mayas of Chiapas, Mexico, for the treatment of gastrointestinal diseases [3]. To our knowledge, no phytochemical investigation has been reported on S. maculatus; however, from P. maculata has been isolated ent-kaur-16-en19-oic acid, kauradiene-9,16-dioic acid, polymatin A, polymatin B, polymatin C [4], maculatin [5], melampodin D, and 9-desacetoxymelcanthin F [6]; consequently, these are chemical constituents of S. maculatus in reality. In our hands, the acetone extract from the aerial parts of S. maculatus was cytotoxically active (ED50 = 17.0 μg/mL against HCT-15 COLADCAR and ED50 = 18.4 μg/mL against OVCAR-5). Their bioguided analysis yield two active fractions: F-4 (ED50 = 17.4 μg/mL against HCT-15 COLADCAR, ED50 = 15.5 μg/mL against UISO-SQC-1 and ED50 = 19.5 μg/mL against OVCAR-5) and F-5 (ED50 = 7.2 μg/mL against HCT-15 COLADCAR, ED50 = 16.2 μg/mL against KB, ED50 = 4.0 μg/mL against UISO-SQC-1 and ED50 = 3.0 μg/mL against OVCAR-5), both composed of the mixture of ent-kaur-16-en-19-ioc acid and ent-kaur-15-en19ioc acid [7], 15α-angeloyloxy-ent-kaur-16-en-19-oic acid [7], 12α-hydroxy-ent-kaur-16-en-19-oic acid [8], and ursolic acid (1, [9, 10]) in different proportions. The cytotoxic analysis of these natural products showed that 1 is a unique cytotoxic compound from this plant (ED50 = 3.7 μg/mL against HCT-15 COLADCAR, ED50 = 3.4 μg/mL against UISO-SQC-1, and ED50 = 3.6 μg/mL against OVCAR-5). Compound 1 has been previously isolated from several species and its antitumor activity has been documented in mouse and human cell lines [10] as well as in vivo induced mice skin cancer [11]. The aerial parts of S. maculatus were collected in Rancho Merced Bason, Huixtan, Chiapas, Mexico and were identified by M. C. Abigail Aguilar. A voucher specimen (number 12700) was deposited at the Instituto Mexicano del Seguro Social Herbarium (IMSSH, Mexico City). The air-dried aerial parts (1.3 kg) were powdered and extracted by maceration at room temperature with acetone (10 L × 3) and concentrated to dryness in vacuum to obtain 52.6 g of a syrup residue, which was adsorbed on 53 g of silica gel and chromatographed on open CC over silica gel 60 (400 g), using a gradient of n-hexane:acetone as eluent. The composition of the fractions obtained (400 mL each) was monitored by TLC, visualizing the compounds by spraying with a 1% solution of CeSO4⋅NH3 in H2SO4 2N. The chromatographically identical fractions were combined, yielding six mixtures of compounds: F-1 [3.2 g, n-hexane 100%], F-2 [6.1 g, n-hexane-acetone (95:05)], F-3 [1.4 g, n-hexane-acetone (95:05)], F-4 [4.8 g, n-hexane-acetone (90:10)], F-5 [3.2 g, n-hexane-acetone (80:20)], and F-6 [2.0 g, n-hexane-acetone (60:40)]. The natural products were isolated by means of successive chromatographic process, F-1: caryophyllene β-oxide, F-2: triacontanol and stigmasterol, F-3: stigmasterol, ent-kaur-16-en-19-oic acid and ent-kaur-15-en-19-oic acid, F-4: ent-kaur-16-en19-oic acid, ent-kaur-15-en-19-oic acid, 15α-angeloyloxy-ent-kaur-16-en-19-oic acid, 12α-hydroxy-ent-kauren-19-oic acid, and ursolic acid (1), F-5: 12α-hydroxy-ent-kauren-19-oic acid and 1 and F-6: β-sitosteryl β-D-glucopyranoside, stigmasteryl β-D-glucopyranoside and sucrose. All these compounds were identified by comparison of their IR, H, and C NMR data with those previously reported. The cytotoxic activity of the extract, fractions, and pure compounds was accomplished according to the previously described procedure [12].