Unveiling the Absolute Configuration of Diarylheptanoid Hirsutanonol: Going Beyond Optical Rotations

Abstract

Our chemical studies on Renealmia petasites led to the isolation of (+)-hirsutanonol, a diarylheptanoid that presents several inconsistencies in the literature regarding its absolute configuration (AC). In this paper, a detailed investigation of optical rotation (OR) and electronic circular dichroism (ECD) chiroptical properties supported by quantum chemical calculations was carried out to unequivocally determine its AC. In addition, our analysis indicated the source of conflicting AC results for hirsutanonol and its derivatives came from the direct comparison of experimental OR and ECD data obtained for structurally related molecules with different chromophoric systems. As an alternative, ECD spectroscopy combined with density functional theory (DFT) calculations is proposed as a more reliable and sensitive technique for the stereochemical investigation of diarylheptanoids.

Keywords:
stereochemistry; natural products; electronic circular dichroism; optical rotation; molecular modeling; absolute configuration

Introduction

Natural products continue to play a significant role in drug discovery and development processes. These compounds are widely known to present a vast structural diversity, intrinsically endowed with both architectural and stereochemical complexity.¹ The assignment of absolute configuration (AC) is a crucial step in the structural characterization of natural products, especially for those subjected to biological assays. However, the configurational assignment of natural products remains a challenging task in current research, being considered a major bottleneck for natural product chemistry.² Therefore, this topic has a long history of significant importance in the field of molecular stereochemistry.³,⁴ Because of this preeminent attention to chiral secondary metabolites, improved techniques have been developed over the years to solve this problem.⁵

Currently, the main techniques available for AC determination are stereocontrolled organic synthesis, X-ray crystallography, nuclear magnetic resonance (NMR) methods, and chiroptical spectroscopy. However, each of the methods has certain limitations when applied to natural products. Stereocontrolled organic synthesis, although it was one of the first methodologies successfully applied for this purpose, is an expensive, laborious, and time-consuming technique, intrinsically dependent on the correct AC of both starting materials and products.³ X-ray crystallography is the golden standard for stereochemical studies and provides resolution at the atomic level. Although presents unquestionable importance, it requires a single and well-defined crystal, restricting its application to substances in the solid state, which can often be difficult to achieve in the case of natural products due to their waxy and amorphous nature and therefore, may preclude it use entirely.⁶,⁷ NMR spectroscopy is one of the main techniques applied for structural elucidation of natural products, however, it is intrinsically achiral and has no sensitivity to the differentiation of enantiomers in an isotropic medium, as it is normally used. To that end, it requires the use of chiral auxiliaries, chiral solvents, and related methods.⁸,⁹ On the other hand, chiroptical spectroscopy has emerged in the last decades as the most widely used method for stereochemical assignment as it is non-destructive, naturally sensitive to chirality, and investigate different aspects of optical activity.¹⁰ Chiroptical techniques include OR (optical rotation), ORD (optical rotatory dispersion) and ECD (electronic circular dichroism), all of then based on electronic transitions, and VCD (vibrational circular dichroism), and ROA (Raman optical activity), based on vibrational transitions.² The modern applications of chiroptical methods are based on simultaneous evaluation of the experimental spectra with reference to those predicted using reliable quantum chemical methods. The availability of time-dependent density functional theory (TD-DFT) for reliable predictions of the ECD spectra has offered one of the most established approaches for AC assignments with a high level of confidence for compounds with different kinds of structural frameworks, including natural products. However, even the modern chiroptical spectroscopy methodologies present limitations. ECD, for example, requires that the molecule to be evaluated presented ultraviolet-visible (UV-Vis) chromophores. While VCD, on the other hand, as presents smaller intensities, required larger quantities of samples to obtain a good quality spectrum. On the theoretical point of view, sometimes obtaining an accurate simulation of chiroptical spectra can be challenging, especially for large molecules with high conformational freedom, and theoretical predictions may not always perfectly match experimental observations in such cases.² However, without any doubt, even with the limitations inherent to each method, chiroptical spectroscopy stands out and its application has updated and broadened research activity in the AC assignments of natural products to the point that nowadays such studies are routinely found in the high-impact literature.

A common practice within the natural products community is known as empirical correlations, which involves the AC assignment of natural products by direct comparisons of the OR and/or ECD experimental data with those described for analogous molecules, which, despite being structurally related, present different chromophores. Although successful in some cases, such empirical correlations are prone to constant breakdowns and exceptions for two main reasons. First, the fact that the OR signal often presents a strong dependence on the solvent used. Second, similar molecules containing the same absolute stereochemistry have been reported with oppositely signed OR values.³,¹¹-¹⁶ Another cumbersome factor is that molecules commonly used as standards for AC assignment by empirical correlations sometimes do not have their AC unambiguously assigned in the first place. It is commonly found in old literature the application of circular dichroism (CD) semiempirical rules to interpret ECD experimental data and therefore establish their AC. There are several known CD semiempirical rules, such as the octant rule, benzene sectoral rule, and ellipticity rules, among others, frequently applied mainly in antique reports.¹⁷ This type of vicious cycle can easily lead to error propagation. As a result, an increasing number of absolute configuration reassignments have been reported in the literature¹¹,¹³,¹⁸,¹⁹ in the last decade.

Diarylheptanoids are an important class of natural compounds characterized by 1,7-diphenylheptane skeleton, and called curcumins. These natural products are commonly found in the genera Curcuma, Zingiber, Alpinia, Alnus, Betula, Myrica and Renealmia, and have also been applied extensively in traditional and folk medicine.²⁰-²³ Although several diarylheptanoid derivatives have been described for decades, determining their AC remains a challenge due to the combination of trace amounts of isolated natural products and the presence of chiral centers in a flexible hydrocarbon chain in linear diarylheptanoids. While recent studies²¹ have successfully assigned the AC of some diarylheptanoid glucoside derivatives, most studies on the isolation of diarylheptanoid derivatives have relied entirely on empirical methods, using OR and/or ECD data comparisons with similar molecules reported in early literature.

This is precisely the case with hirsutanonol and its derivatives. It has been isolated for decades from various plants, such as Alnus japonica, Alpinia officinarum, Amomum muricarpum, Alnus hirsuta, among others, and has demonstrated significant biological activities, including antineoplastic, anti-inflammatory, antioxidant, antiestrogenic, hepatoprotective, antileishmanial, and neuroprotective agents.²¹,²⁴ More significantly, diarylheptanoids like hirsutanonol and its derivatives yielded a remarkable non-toxicity even when administered at elevated dosages, underscoring their immense potential as therapeutic agents.²¹

Although hirsutanonol and its derivatives have only a single chiral center in their structure, as far as we know, all stereochemical analyzes were made based on empirical correlations, through comparison with similar molecules present in early literature.

It was known by Kuroyanagi et al.²⁵ that the potent antioxidant effect of diarylheptanoids is directly related to some structural features, mainly the catechol structure and the chiral center at C-5, which sometimes be linked to different types of sugar moieties. Therefore, determining the AC of C-5 in hirsutanonol and its derivatives is crucial to evaluating their biological activities. Since our phytochemical studies on Renealmia petasites Gagnep. led to the isolation of (+)-hirsutanonol (Figure 1), this work takes this opportunity to reassess the previously reported AC assignment based on empirical OR and ECD analyzes. Furthermore, it conducts a detailed investigation of the ECD chiroptical properties of (+)-hirsutanonol, supported by quantum chemical calculations, to unequivocally determine its AC.

Figure 1
Chemical structure of (+)-hirsutanonol.

When searching the literature regarding the AC analysis of the previously reported hirsutanonol, to our bewilderment, several inconsistencies were identified which may have direct implications for other diarylheptanoids and related derivatives. Therefore, besides reporting the AC assignment of (+)-hirsutanonol with a high level of confidence, this paper aims to highlight the risks associated with empirically based OR and ECD stereochemical assignments for this class of compounds. As an alternative, ECD spectroscopy combined with DFT calculations is proposed as a more reliable and sensitive technique for the stereochemical investigation of diarylheptanoids.

Experimental

Materials and reagents

All solvents used for extract preparation and thin layer chromatography (TLC) analyzes were of analytical grade (Neon Comercial Reagentes Analíticos Ltda, Suzano, Brazil), whereas solvents used for high-performance liquid chromatography (HPLC) and high-speed countercurrent chromatography (HSCCC) were of HPLC grade, purchased from Merck (Darmstadt, Germany). Ultrapure water used in mobile phase preparation was obtained from a Purelab purification system (ELGA, High Wycombe, UK).

Plant material

Rhizomes of R. petasites Gagnep. were collected in September 2021 in the municipality of Guarapari (20°36’08.7” S, 40°30’32.6” W), Espírito Santo, Brazil. The species was identified by Dr Solange Schneider. A voucher specimen (UVVES-2737) has been deposited in the herbarium of Universidade de Vila Velha, Brazil. The genetic heritage access activity was registered under number A65446E in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen).

Extraction and HSCCC separation

The rhizome (107.75 g) was successively extracted with 1.45 L of n-hexane and 0.6 L of absolute ethanol, yielding 4.0 and 3.6 g, respectively.

Approximately 340.0 mg of the ethanolic extract was subjected to HSCCC under isocratic elution in reverse mode using the n-hexane:ethyl acetate:ethanol:water biphasic solvent system (4:6:4:6, v/v),²⁶,²⁷ with a rotation speed of 880 rpm, a flow rate of 2.0 mL min^-1, and a temperature of 30 °C. The stationary phase retention (Sf) was 68%. Fractions of 4.0 mL per test tube were collected.

General experimental procedures

TLC was performed using SiliaPlate TLC plates (silica F254, SiliCycle, Quebec, Canada). The mobile phase consisted of chloroform:methanol (9:1, v/v).

HSCCC was performed using an AECS Quattro QuikPrep system (Gloucestershire, UK) equipped with two polytetrafluoroethylene multilayer coils (4 × 112 mL) with a 10 mL sample loop, 2.1 mm internal diameter, and adjustable speed (0-1000 rpm). The solvent was pumped using an ECP 2010 pump (ECOM®, Prague, Czech Republic), and fractions were collected using a Gilson^® FC203B fraction collector (Wisconsin, USA).

The liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using a Nexera X2 HPLC system (Shimadzu, Kyoto, Japan); column ODS Hypersil C18, 150 × 2.1 mm, 3 μm particle size (Thermo Fisher Scientific, Waltham, USA); oven temperature of 30 °C; flow rate of 0.35 mL min^-1; injection volume of 5 μL using the Nexera X2 autosampler (SIL-30AC, Shimadzu). The mobile phase was a gradient system composed of (A) 0.1% formic acid in water, (B) methanol, varying the mobile phase B for 0.0 min to 10%, 0.5 min to 15%, 10.0 min to 100%, 15.5 min to 10%, 16.0 min to 10%, 19.0 min to stop. The mass analyzes was performed using a Bruker Maxis Impact (Q-TOF) high resolution mass spectrometer with an internal calibration of 100 µM sodium formate in water:acetonitrile (1:1); data-dependent acquisition mode (DDA/AutoMS), with isolation/fragmentation of 5 precursors per cycle; scan range of 50-1500 m/z; acquisition rate of 3.0 Hz; electrospray ionization source; negative polarity; instrument settings were: nebulizer pressure of 3.0 bar; dry gas flow of 9.0 L min^-1; dry gas temperature of 200 °C; capillary voltage of 3500 V; end plate offset of –800 V; quadrupole low mass of 100 m/z; fragmentation energy of 15 and 30 eV (combined). The samples (1.0 mg) were dissolved in 500 μL of methanol:water (1:1, v/v)

The NMR spectra of the isolated compounds were acquired using an NMR Bruker Avance III 400 MHz spectrometer (Karlsruhe, Germany). Tetramethylsilane (TMS) was used as an internal reference. The compound was dissolved in 0.6 mL of deuterated methanol for analysis at 25 °C. Chemical shifts were reported in parts per million (ppm) relative to the applied frequency, and coupling constants (J) were expressed in hertz (Hz).

The optical rotation was obtained in a YK-P100 digital automatic polarimeter (Yuke Jiahang, Shangai, China) in acetonitrile solution (1.0 mg mL^-1) at 20 °C in 3 accumulations. The experimental ECD spectra were recorded with a Jasco J-1100 spectrometer (Jasco, Tokyo, Japan) in the 200-400 nm region using the following parameters: bandwidth 1 nm; response 1 s; scanning speed 100 nm min^-1; 3 accumulations; room temperature (25 °C); sample in acetonitrile solution; 0.1 cm cell path length; concentration 0.1 mg mL^-1. The obtained CD spectra were smoothed in Origin 8 software.²⁸

Theoretical methods

For the simulation of ECD spectra, randomized conformational searches were performed for all the possible stereoisomers using the Monte Carlo algorithm with a Merck molecular force field (MMFF) in Spartan’14 software.²⁹ All the conformers within a relative free energy window of 10 kcal mol^-1 were selected for geometric optimization calculations in the gas phase, employing the B3LYP/6-31G(d) level of theory. Vibrational frequency calculations were performed at the same level of theory to confirm that the stationary points correspond to minima on the potential energy surface. Subsequently, conformers within a relative energy window of 3 kcal mol^-1 were selected. For the ECD simulations, the TD-DFT level of theory was applied: CAM-B3LYP/TZVP, employing a polarizable continuous model with integral equation formalism (IEF-PCM) to implicitly simulate acetonitrile (ACN) as the solvent. The final ECD spectra were generated based on Boltzmann statistics of the selected conformers and plotted using Origin 8 software.²⁸ All quantum-mechanical calculations were performed using the Gaussian 16 software package.³⁰

Results and Discussion

Upon reviewing the literature, conflicting results regarding the AC of C-5 in hirsutanonol were found. Tung et al.,²⁴ who isolated it from the bark of Alnus japonica, determined the AC of C-5 as R based on the positive sign of the OR and the CD spectrum (both obtained in MeOH). This conception was achieved mainly by the positive Cotton effect associated with the carbonyl η-π* transition at ٣٠٠ nm of (+)-hirsutanonol, in comparison with those of muricarpone A, isolated from Amomum muricarpum.³¹ This is where the inconsistencies began. Although muricarpone A, which was also assigned as R and yielded a similar positive Cotton effect associated with the carbonyl η-π* transition (however, at ٣٥٠ instead of ٣٠٠ nm), the sign of the reported OR was negative (also obtained in MeOH), contradicting the assignment as R for (+)-hirsutanonol. It is important to highlight that the source of these misleading results regarding the AC assignment at C-5 may come from the fact that muricarpone A is the methoxylated form of hirsutanonol at C-5.²⁵ Although they are very similar compounds, they present different chromophores, which may lead to misassignment. As mentioned before, several studies²,¹¹,¹⁵,¹⁸,³²,³³ are showing the dangers of assigning the AC entirely based on comparisons of spectroscopy data with similar compounds, in which small changes in the chromophore drastically alter both the OR and Cotton effects. In addition, it has already been demonstrated in the literature that hirsutanonol derivatives present OR signs that are strongly dependent on the solvents used to measure the rotations,³⁴ a factor that can also easily lead to stereochemical assignment errors if the analysis is carried out based solely on comparisons of OR values.

In turn, Lee et al.³⁵ also isolated (–)-hirsutanonol from the leaves of Alnus hirsuta Turcz. During the structural elucidation process, the authors determined the AC of (–)-hirsutanonol as S by comparing the negative signal obtained (in Me₂CO) from OR with the data reported for oregonin, a hirsutanonol derivative in which C-5 is directly linked to a xylose moiety.³⁶ Once again, this generates a conflicting AC result when compared with the data reported by Giang et al.,³¹ which also obtained a negative OR sign and established the absolute configuration for their (–)-hirsutanonol derivative as R.

These conflicting data on OR values and AC assignments for these compounds make clear the dangers associated with basing all stereochemical analysis solely on empirical comparisons. The use of different solvents to obtain the OR and the comparison of OR data from similar compounds can easily lead to opposite OR signals and induce stereochemical assignment errors, therefore, should be avoided.

In addition, when investigating the assignment made by Giang et al.³¹ as R for muricarpone A, the authors also compared OR and CD data with those of a series of analogous compounds isolated by Itokawa et al.³⁷ from Alpinia officinarum Hance. In this paper, Itokawa et al.³⁷ proposed that a relationship can be obtained between the chirality of the hydroxyl group chromophore and the sign of Cotton effect and OR values. According to the authors, derivatives with an S configuration on C-5 yielded a positive OR value and negative Cotton effect at 300 nm, whereas derivatives with an R configuration on C-5 yielded a negative OR value and a positive Cotton effect. As far as we know, this paper was the base standard for AC determination of several diarylheptanoid compounds in literature, including the ones mentioned above. However, as this paper was published in 1985, Itokawa et al.³⁷ based their analyzes on the octant rule, an antique semiempirical method of analysis that predicts the sign of the Cotton effect based on the spatial arrangement of the carbonyl group dividing the three-dimensional space into sectors, proposed by Moffitt et al.³⁸ in the early sixties. As stated by the authors:³⁷ “i.e., the hydroxyl group situated in the lower site makes a negative contribution to the Cotton effect, whereas the hydroxyl group situated in the upper site leads to a positive Cotton effect. In this way, it seems that the hydroxyl group determines the sign of the Cotton effect and the absolute stereochemistry.” Although this antique method has been successful in many cases, it is extremely error-prone and there are several reports of reassignment.²,¹⁷ Perform the stereochemistry analysis based on this semiempirical method is especially dangerous for this class of linear diarylheptanoids derivatives, due to the presence of a flexible polyhydric main chain, which produces high conformational freedom where the position of the hydroxyl group at C-5 can vary considerably between the possible conformers.

As mentioned before, this closes the vicious cycle, where the AC assignment analyzes have been entirely based on a comparison between spectroscopic data of similar compounds, and the first report, used as standards for the AC assignment back in 1985, does not have its own AC unambiguously assigned in the first place, and was carried out based on an antique method that is easily subject to errors. This clearly justifies the propagation of the elevated number of inconsistencies found in the literature, such as those discussed above. Therefore, it is evident that there is an urge to determine the AC of C-5 of hirsutanonol with a high level of confidence and to definitively clarify the previously reported inconsistencies.

Hence, in an intent to break this cycle of unresolved reports regarding the AC assignment, for our isolated (+)-hirsutanonol, we applied the modern and robust combination of ECD and quantum chemical calculations, which has emerged as a powerful and reliable tool for both conformational and configurational analysis of natural product.

(+)-Hirsutanonol (29.7 mg) was isolated from the ethanolic extract of Renealmia petasites rhizomes (340.0 mg) by HSCCC in test tubes 17 to 21 (see Figure S1, presented in the Supplementary Information (SI) section). The Q-TOF mass analysis showed the deprotonated molecule m/z 345.1341 [M – H]^– (calcd. for C₁₉H₂₁O₆: 345.1344) and deduced the molecular formula of the isolated compound as C₁₉H₂₂O₆ (Figure S2, SI section). The ¹H NMR spectrum shows two tri-substituted aromatic rings, each exhibiting an ABX system (Figures S3-S6, SI section). The protons H-6’/H-6” couple in ortho (J 8.0 Hz) with the protons H-5’/H-5” and couple in meta (J 2.5 Hz) with the protons H-2’/H-2”. The proton at position 5 showed a chemical shift (δ_H ca. 4.0 ppm) characteristic of a secondary alcohol. The ¹H-¹H COSY (homonuclear correlation spectroscopy) spectrum shows the couplings of the protons in the aliphatic chain in sequence: H-4, H-5, H-6, and H-7 (Figure S7, SI section). The coupling of protons H-1 and H-2 is not observed due to the signal overlap at ca. 2,70 ppm. The ¹³C APT NMR (attached proton test nuclear magnetic ressonance) spectrum shows a characteristic carbonyl chemical shift at 212.0 ppm (C-3) and a secondary alcohol chemical shift at 68.3 ppm (C-5) (Figure S8, SI section). In the heteronuclear single quantum coherence (HSQC) spectrum, the coupling between H-5 and C-5 can be highlighted as important for determining the position of the hydroxyl group in the linear chain. Additionally, signal overlaps of protons H-1 and H-2 (ca. 2.70 ppm) are observed, which are not present in their respective carbon signals (30.1 and 46.4 ppm) (Figure S9, SI section). The heteronuclear multiple bond correlation (HMBC) spectrum shows the coupling (2,3 J_CH) of the carbon at position 5 (δ_C 68.3 ppm) with protons at positions 4 (δ_H 2.64-2.41 ppm) and 6 (δ_H 1.70-1.60 ppm) confirming the hydroxyl group at position 5 (Figure S10, SI section). The carbonyl group at position 3 was confirmed by the carbon (δ_C 212.0 ppm) couplings with the proton at positions 2 (δ_H 2.72-2.68 ppm) and 4 (δ_H 2.64-2.41 ppm). These data shown in Table S1, presented in the SI section, are consistent with the literature on hirsutanonol.³⁹,⁴⁰

Although the good practices for circular dichroism analysis dictate that stereochemical determination should not be performed based only on a single region of the spectrum²,⁴¹ as done by Itokawa et al.,³⁷ a superficial analysis based only on the 300 nm region of the CD spectrum of the isolated compound was initially performed (see Figure 2) to evaluate the correlation of OR values and the signal of Cotton effect as proposed by Itokawa et al.³⁷ in 1985.

Figure 2
Circular dichroism spectrum at 270-320 nm obtained for (+)-hirsutanonol.

The positive OR value obtained and the small negative Cotton effect at 300 nm are compatible with the S configuration at C-5 according to the correlation proposed by Itokawa et al.³⁷ in 1985. However, it contradicts the proposal made by Tung et al.,²⁴ in 2010, which isolated the (+)-hirsutanonol with the same positive OR values but proposed the C-5 as R based on the positive sign of the Cotton effect. On the other hand, it also contradicts the proposal made by Lee et al.,³⁵ which although also assigned the C-5 as S by (–)-hirsutanonol, yielded the opposite negative OR values.

As previously described in the literature,²¹ the analysis of chiroptical properties of diarylheptanoids is commonly complex due to the presence of many factors that can influence the signs of the Cotton effects, mainly the presence of the flexible polyhydric main chains in linear diarylheptanoids. Therefore, under the circumstances shown above, intending to unveil the stereochemistry of (+)-hirsutanonol and eliminate inconsistencies, the best approach in this case to determine the stereochemistry with a high level of confidence is to perform the comparison of the full wavelength CD spectra of the isolated compound with the corresponding simulated spectra for (+)-hirsutanonol with S configuration (according to the proposal made by Itokawa et al.)³⁷ as shown in Figure 3.

Figure 3
Experimental and calculated ECD spectra of (+)-hirsutanonol.

In a deeper analysis of Figure 3, the observed ECD spectrum in acetonitrile solution yielded two negatives (ca. 215-225 nm) and a weak positive π-π* (ca. 250 nm) Cotton effects with a slight negative η-π* (ca. 300 nm) Cotton effect.

In turn, for the simulated spectra, range-separated hybrid functional CAM-B3LYP was used in combination with the TZVP basis sets. This theoretical level was selected due to its better performance in ECD calculations.⁴¹ The simulated spectra in Figure 3, obtained by the final Boltzmann weighted theoretical ECD spectra for all conformers of (+)-hirsutanonol with S configuration (Figure S11, SI section), yielded all the expected Cotton effect patterns, except for the weak negative Cotton effect at 300 nm. However, when evaluating the contribution of individual conformers, opposite signs at this weak band at 300 nm were observed. While some conformers presented a weak negative band, others presented a weak positive one. This signal inversion originates due to the high conformational freedom of the flexible polyhydric main chain, where the position of the hydroxyl group at C-5 can vary considerably between the possible conformers, directly influencing the signal of the η-π* Cotton effect at ca. 300 nm.

Therefore, this comprehensive analysis of the simulated CD spectra clearly shows the risks of basing all stereochemical analysis of molecules of biological interest like (+)-hirsutanonol solely on the octant rule, since the signal of this single band can be affected by small conformational changes. Finally, the good match between simulated and experimental full-length CD spectra clearly confirms the stereochemistry of (+)-hirsutanonol as S.

Conclusions

In this paper, it was demonstrated several inconsistencies in the literature regarding the AC of hirsutanonol and its derivatives. The dangers of trying to determine their stereochemistry based only on the comparison of OR values with similar compounds found in the literature were also highlighted. In addition, the generalized application of the octant rule for this class of secondary metabolites is strongly discouraged without quantum chemical calculations, once it was shown that the sign Cotton effect at 300 nm is strongly dependent on conformation and inversion of the signs of η-π* transition Cotton effects can occur between different conformers. Therefore, in conclusion, for reliable assignment of the absolute configuration of structurally related diarylheptanoids derivatives, the use of full-length ECD spectroscopy, especially in combination with the theoretical spectrum, is recommended.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

Data Availability Statement

All data are available in the text.

Acknowledgments

The authors would like to thank the Brazilian agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES, finance code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant No. E-26/210-313/2022 and E-26/200.241/2023) for the financial support and fellowships. Research developed with the support of the Núcleo Avançado de Computação de Alto Desempenho (NACAD) - COPPE, and Centro de Espectrometria de Massas de Biomoléculas (CEMBio), Universidade Federal do Rio de Janeiro (UFRJ).

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Edited by

• Editor handled this article:
  Hector Henrique F. Koolen (Associate)

Publication Dates

• Publication in this collection
  14 July 2025
• Date of issue
  2025

History

• Received
  07 Apr 2025
• Accepted
  03 June 2025