Isolation and Structure Elucidation of Novel Mycosporine-like Amino Acids from the Two Intertidal Red Macroalgae Bostrychia scorpioides and Catenella caespitosa

This study presents a phytochemical survey of two common intertidal red algal species, Bostrychia scorpioides and Catenella caespitosa, regarding their MAA (mycosporine-like amino acid) composition, which are known as biogenic sunscreen compounds. Six novel MAAs from Bostrychia scorpioides named bostrychines and two novel MAAs from Catenella caespitosa named catenellines were isolated using a protocol which included silica gel column chromatography, flash chromatography on reversed phase material and semipreparative HPLC (High-Performance Liquid Chromatography). The structure of the novel MAAs was elucidated using NMR (Nuclear Magnetic Resonance) and HR-MS (High-Resolution Mass Spectrometry), and their absolute configuration was confirmed by ECD (Electronic Circular Dichroism). All isolated MAAs possess a cyclohexenimine scaffold, and the metabolites from B. scorpioides are related to the known MAAs bostrychines A-F, which contain glutamine, glutamic acid and/or threonine in their side chains. The new MAAs from C. caespitosa contain taurine, an amino sulfonic acid that is also present in another MAA isolated from this species, namely, catenelline. Previous and new data confirm that intertidal red algae are chemically rich in MAAs, which explains their high tolerance against biologically harmful ultraviolet radiation.


Introduction
Bostrychia scorpioides (Hudson) Montagne ex Kützing and Catenella caespitosa (Withering) L. M. Irvine are two of the most common red macroalgae growing in the intertidal zone of estuarine to marine coasts in Europe [1].
Bostrychia scorpioides is the only species out of 40 accepted taxa of the genus Bostrychia abundant on European coasts [2,3].It is an Atlantic estuarine and marine species living as epiphyte on salt marsh plants such as Halimione portulacoides in the upper intertidal from the British Isles to Morocco.Other Bostrychia species mainly occur as epiphytes on mangrove root systems in warm-temperate to tropical regions [4].These algae tolerate long periods of desiccation during the tidal cycle due to their high-shore position [5], and they survive several weeks exposed to air by adjusting the intracellular solute content, resulting in partial or complete turgor regulation.This is achieved by changing internal concentrations of the main ions K + , Na + and Cl − and adopting in the organic osmolyte content such as polyols (sorbitol and dulcitol), amino acids or quaternary ammonium Mar.Drugs 2023, 21, 543 2 of 15 compounds [2].Additionally, B. scorpioides produces a set of unique mycosporine-like amino acids (MAAs), bostrychines A to F (approximately 2-10 mg/g of dry weight in total) [6,7], compounds with established photoprotective but questionable osmoprotective properties [8].Because of its high number of representatives of this compound class (eight to twelve MAAs), it stands out among other red algae of the same biogeographic region [9] or other Bostrychia species from all over the world.B. scorpioides has also been reported to produce a high number of the carotenoids violaxanthin, zeaxanthin and β-carotene (in total, 0.65 mg/g DW) and a significantly increased concentration of phycoerythrin and chlorophyll a in summer, as compared to in winter, possibly due to the higher exposure to light radiation and desiccation stress [9].
Only five species of the genus Catenella have been accepted taxonomically [3].One of them, C. caespitosa, has a wide biogeographic distribution in Europe and America, and it grows on sheltered shady rocks or sediment between high tide levels and supralittoral zones [1,10].Catenelline, a sulfonic acid-bearing MAA, has been isolated as the main MAA from the former species Catenella repens (synonym of C. caespitosa) with an amount ranging from 0.3-1.8mg/g of dry weight, but at least one additional but unknown MAA is reported to be present in lower amounts [10].
The main focus of the present study was to complete the phytochemical profile of two intertidal red macroalgae found in Europe, B. scorpioides and C. caespitosa.Previous studies indicated that both produce MAAs, which had not been identified [6,10].Thus, this study focused on the isolation of yet-unexplored UV-sunscreen molecules.
MAAs are commercially attractive compounds since they are biocompatible, biodegradable and have no toxic properties [23,24].Some commercial formulations such as Helioguard ® 365 and Helionori ® contain them as active ingredients [25].They show a promising future for applications in the pharmaceutical and cosmetic industries as natural sunscreens and anti-photoaging molecules, being in demand because of the rapid increase in skin damage in humans due to UV radiation [26] and the harmful effects of synthetic sunscreens on human health and the environment [14,23].Unfortunately, the utilization of their biological sources for the mass production of these economically important molecules has been hindered because of the incomplete understanding of their effect on human skin and their low abundance in the producing organisms [13,17].Algaculture, biotechnology or semisynthetic approaches could be alternatives for overcoming the supply issue [17,27].

Isolation
The combined aqueous and methanolic extracts of B. scorpioides and C. caespitosa were first fractionated on a silica gel column in order to remove non-polar constituents such as chlorophylls in the early eluting fractions and sugars in the later, more polar fractions so as to obtain MAA-enriched fractions (details in Section 4.2).This initial step was followed by separations using reversed phase material on flash chromatography and semipreparative HPLC.The purification procedure resulted in the isolation of six novel compounds from B. scorpioides and two novel compounds from C. caespitosa.

Structure Elucidation
Characteristic NMR (Nuclear Magnetic Resonance) shifts of compound 1 (Tables 1 and 2) indicated the presence of a cyclohexenimine-type MAA scaffold.The side chain was identified as glutamine based on a COSY (Correlation Spectroscopy) chain correlation of H-9/H-11/H-12 and two carbonyl groups at δ C 179.5 and 181.1 and by comparison with the literature values [6].Its position was confirmed by long-range correlations visible in the HMBC (Heteronuclear Multiple Bond Correlation) spectrum (H-9 at δ H 4.21 to C-3 at δ C 161.7).Specifically, a methine group at δ H 4.21 (H-9) showed a correlation in the COSY spectrum with the protons of the methylene at δ H 2.18 and δ H 2.27 (H-11) and an HMBC correlation with the carbonyl group at δ C 179.5 (C-10).Furthermore, the protons of the methylene at C-12 (δ H 2.45) showed a correlation in the COSY spectrum with the protons of the methylene at position 11 (δ H 2.18 and 2.27) and an HMBC correlation with the carbonyl group at δ C 181.1 (C-13).However, the 1 H-NMR spectrum revealed extra signals, two doublets of doublets of a methylene at δ H 3.44/3.51(H-1 ), a multiplet of an oxygenated methine at δ H 4.03 (H-2 ) and a doublet of a methyl-group at δ H 1.24 (J = 6.4 Hz, H-3 ), which showed a correlation in the COSY spectrum (H-1 /H-2 /H-3 ).These signals, in addition to the HMBC connectivities (H-3 /C-2 and C-1 , H-1 /C-2 and C-3 ), indicated the presence of a threamine moiety.The HMBC correlation of the same proton (H-1 ) to carbon C-1 at δ C 163.5 confirmed that threamine is attached to position C-1.The two amino acid residues of the side chains of this MAA have already been observed individually in other MAAs; glutamine has been found in MAAs from B. scorpioides-for example, in bostrychines A and B [6], while threamine is a constituent of the MAAs aplysiapalythine A and bostrychine E [6,28].The advanced Marfey's method, an established LC-MS (Liquid Chromatography-Mass Spectrometry)-based procedure for determining the absolute configuration, has confirmed the presence of L-glutamic acid and R-threamine as constituent of other MAAs in this species [7].In order to determine the absolute stereochemistry of C-5, geometrical optimization followed by ECD (Electronic Circular Dichroism) calculation at the m062x/6−31 + g(d,p)/smd//wb97xd/6−31 + g(d,p) level in H 2 O resulted in an ECD spectrum showing a high similarity to the experimentally obtained spectrum.The absolute configuration was established as 5R,1 S,3 R, and therefore, compound 1 (Figure 1 The COSY spectrum revealed an extra coupling network, including the protons of three methylene units H-9/H-10/H-11.Additionally, HMBC correlations of H-10 (δ H 1.96) and H-11 (δ H 2.39) to a carbonyl group at δ C 180.7 (C-12) revealed the presence of 4-aminobutanamide as a side chain (Figure 2).An HMBC correlation of proton H-9 (δ H 3.51) to carbon C-1 at δ C 163.4 indicated at which position the 4aminobutanamide moiety is attached to the cyclohexenimine scaffold.The advanced Marfey's method of bostrychines B, D and F has shown the presence of L-threonine as a constituent of the final product of the reaction, indicating that this amino acid residue in the MAA has an L-configuration [7].The determination of the absolute stereochemistry of compound 2 by geometrical optimization followed by ECD calculation at the m062x/6−31 + g(d,p)/smd//wb97xd/6−31 + g(d,p) level in H 2 O resulted in an ECD spectrum showing an excellent match with the experimentally obtained spectrum, therefore establishing the absolute configuration as 5R,1 S,3 R. Compound 2 was finally identified as a new MAA, (2S,3R)-2-(((R,1E,3E)-3-((4-amino-4-oxobutyl)-l4-azaneylidene)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohexan-2-ylium-1-ylidene)-l4-azaneyl)-3-hydroxybutanoate, for which we propose the trivial name bostrychine H. Compound 2 was assigned the molecular formula C16H27N3O7, as established by a positive ion (for [M + H] + , m/z = 374.1913;calculated for C16H28N3O7, 374.1927) in the HR-MS (High-Resolution-Mass Spectrometry) spectrum.Characteristic NMR chemical shifts indicated the presence of a cyclohexenimine-type MAA.The side chain was identified as threonine based on a COSY chain correlation of H-1′/H-3′/H-4′ and a carbonyl group at δC 178.3 and by comparison with the literature values.Its position was confirmed by longrange correlations visible in the HMBC spectrum (H-1′ at δH 4.05 to C-3 at δC 162.2).The COSY spectrum revealed an extra coupling network, including the protons of three methylene units H-9/H-10/H-11.Additionally, HMBC correlations of H-10 (δH 1.96) and H-11 (δH 2.39) to a carbonyl group at δC 180.7 (C-12) revealed the presence of 4-aminobutanamide as a side chain (Figure 2).An HMBC correlation of proton H-9 (δH 3.51) to carbon C-1 at δC 163.4 indicated at which position the 4-aminobutanamide moiety is attached to the cyclohexenimine scaffold.The advanced Marfey's method of bostrychines B, D and F has shown the presence of ʟ-threonine as a constituent of the final product of the reaction, indicating that this amino acid residue in the MAA has an ʟ-configuration [7].The determination of the absolute stereochemistry of compound 2 by geometrical optimization followed by ECD calculation at the m062x/6−31 + g(d,p)/smd//wb97xd/6−31 + g(d,p) level in H2O resulted in an ECD spectrum showing an excellent match with the experimentally obtained spectrum, therefore establishing the absolute configuration as 5R,1′S,3′R.Compound 2 was finally identified as a new MAA, (2S,3R)-2-(((R,1E,3E)-3-((4-amino-4-oxobutyl)-l4-azaneylidene)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohexan-2-ylium-1-ylidene)-l4-azaneyl)-3-hydroxybutanoate, for which we propose the trivial name bostrychine H. Compounds 3 and 4 had similar retention times in the HPLC chromatogram (Figure 3), and they were obtained as a mixture.The integration of the signals in the 1 H-NMR spectra showed that the relative ratio of the two compounds was 0.7:1 (compound 3: compound 4).The side chain of both compounds at position 3 was identified as glutamine based on COSY chain correlations of H-9/H-11/H-12 and characteristic NMR values which were in accordance with the literature values.The second side chain of compound 3 contained a cis conformation double bond, which was indicated by characteristic 1 H-NMR (H-1 , δ H 6.39 and H-2 , δ H 5.42) and 13 C-NMR (C-1 , δ C 124.7 and C-2 , δ C 120.3) values and the coupling constants of H-1 (J = 8.0/1.2Hz).Moreover, a methyl group at δ H 1.78 (H-3 ) showed a correlation in the COSY spectrum with the protons of H-2 at δ H 5.42 and an HMBC correlation with the carbons of the double bond at δ C 120.3 and 124.7 (C-2 and C-1 ), revealing the presence of a propene side group.Its position was confirmed by long-range correlations visible in the HMBC spectrum (H-1 at δ H 6.39 to C-1 at δ C 158.0).The NMR signals of compound 4 were highly similar to those of compound 3.However, protons H-1 and H-2 were deshielded (H-1 , δ H 6.57 and H-2 , δ H 5.77), and the coupling constants of H-1 were higher (J = 13.6/2.0Hz), indicating that the conformation of the double bond was trans.Thus, compounds 3 and Compounds 3 and 4 had similar retention times in the HPLC chromatogram (Figure 3), and they were obtained as a mixture.The integration of the signals in the 1 H-NMR spectra showed that the relative ratio of the two compounds was 0.     When analyzing the C. caespitosa extract by HPLC, compound 7 eluted after catenelline (Figure 4), and characteristic NMR shifts indicated the presence of a cyclohexenimine-type MAA, too.The side chain was identified as 2-aminoethane-1-sulfonic acid based on a COSY chain correlation of H-9/H-10 and by comparison with the literature values [9].Its position was evidenced by long-range correlations visible in the HMBC spectrum (H-9 at δH 3.87 to C-3 at δC 163.3), and relevant connectivities are indicated by arrows in Figure 2. Specifically, a methylene group at δH 3.87 (H-9) showed a correlation in the COSY spectrum with the protons of the methylene at δH 3.23 (H-10), as well as a HMBC correlation with the carbon at δC 42.1 (C-10).2-aminoethane-1-sulfonate has already been found as a moiety in catenelline.The latter was first described by Hartmann et al. in 2015 [12].A comparison of the experimental and calculated spectrum at the m062x/6−31 + g(d,p)/smd//wb97xd/6−31 + g(d,p) level in the H2O of compound 7 (Figure 5) indicated the S chirality of C-5.Compound 7 was finally identified as a new MAA, (S,Z)- The characteristic NMR shifts (Tables 1 and 2) and 2D-NMR data of compound 8 indicated the same substructure as that of compound 7.However, the 1 H-NMR spectrum showed two additional signals, two triplets at δH 3.60 (J 5.4 Hz, H-11) and δH 3.77 (J = 5.4 Hz, H-12).The protons of these two methylene groups showed a correlation in the COSY spectrum, and in the HMBC, the protons of H-11 showed a correlation with the carbon at δC 63.1 (C-12), revealing the presence of a 2-aminoethan-1-ol moiety.Furthermore, the HMBC correlation of the same proton (H-11) to carbon C-3 at δC 163.3 indicated that this side chain is attached to position C-3.The purity of this compound was approximately 70%; thus, the optical rotation value and the molar absorption coefficient (ε) were not determined.The ECD spectrum of compound 8 was highly similar to that of compound

Compound 4
Pale yellow amorphous powder; UV λmax = 360 nm; 1 H and 13 C NMR data (600/151 MHz; D2O), Tables 1 and 2   The characteristic NMR shifts (Tables 1 and 2) and 2D-NMR data of compound 8 indicated the same substructure as that of compound 7.However, the 1 H-NMR spectrum showed two additional signals, two triplets at δ H 3.60 (J 5.4 Hz, H-11) and δ H 3.77 (J = 5.4 Hz, H-12).The protons of these two methylene groups showed a correlation in the COSY spectrum, and in the HMBC, the protons of H-11 showed a correlation with the carbon at δ C 63.1 (C-12), revealing the presence of a 2-aminoethan-1-ol moiety.Furthermore, the HMBC correlation of the same proton (H-11) to carbon C-3 at δ C 163.3 indicated that this side chain is attached to position C-3.The purity of this compound was approximately 70%; thus, the optical rotation value and the molar absorption coefficient (ε) were not determined.The ECD spectrum of compound 8 was highly similar to that of compound  1 and 2 1 and 2 1 and 2

Discussion
Mycosporine-like amino acids are compounds with photoprotective, antioxidant [22], anti-inflammatory [29], wound-healing [30] and immunomodulatory [31] properties.The producing organisms are mainly various micro-and macroalgae, and the prevailing hypothesis is that they synthesize MAAs because they protect against the UV damages of sun irradiation and thermal stress [32].They might also participate in the osmotic equilibrium, especially in photosymbiotic partnerships [33].The osmotic function of MAAs in marine organisms, however, has been questioned because the intracellular concentrations are generally too low for a major contribution to the osmotic potential [34].MAAs also have other physiological functions; they serve as antioxidants [32], ROS-scavengers [35,36], reproductive regulators [37] and an intracellular nitrogen reservoir [17], while some studies suggest that MAAs, especially in cyanobacteria, play a negligible role in the protection against UV radiation and might be involved in the formation of extracellular matrix and cell-cell interactions [38][39][40].
Recently, MAAs have attracted increasing attention, and there are a large number of patents in international databases for several products and methods with MAAs [22,29].They offer a great potential for the development of novel UV sunscreens because of their direct and indirect protective properties.Therefore, the investigation of unexplored MAAproducing organisms and their unambiguous structure elucidation increase the understanding on chemical diversity [29].It is noteworthy to mention that most of the MAAs utilized in these patents provide minimal protection in the more damaging UVB range.Additionally, the MAA concentration in the algal extract is often very low when compared to the concentration of other photoprotective ingredients in most sunscreen products [22,41]; thus, the identification of organisms producing higher yields of these metabolites or the preparation of semisynthetic analogues is very important.
In the present study, MAAs of two intertidal red macroalgae, B. scorpioides and C. caespitose, were investigated.The presence of these MAAs had been described in previous studies, although their isolation and structural assignment were not feasible previously [6,9,10].After the usage of a slightly different isolation protocol in this study, six novel MAAs were obtained from B. scorpioides and two novel compounds were obtained from C. caespitosa using chromatographic techniques.Their structures were elucidated, and their stereochemistry was revealed after the combination of NMR and ECD experiments.
The newly isolated MAAs from B. scorpioides are related to the reported bostrychines A-F [6] since they contain the amino acid residues threonine, threamine, glutamine and glutamic acid in their side chains.Compounds 3-6 (bostrychines I to L) additionally contain (E)-and (Z)-prop-1-en-1-amine, which is also a substituent of the known MAAs usujirene [42] and palythene [43] and another MAA tentatively identified as dehydroxylusujirene [44].It is notable that the latter three MAAs, along with the newly isolated bostrychines I-L, all containing an extra double bond in their side chain, have a λ max of 357-360 nm in water.This is uncommon since the majority of cyclohexenimine-type MAAs have an λ max of 325-337 nm in the same solvent, while those with a cyclohexenone scaffold show a maximum absorption of around 310-324 nm in water [22,33,45].Regarding the MAAs isolated from C. caespitosa, both compounds contained taurine in their side chain, which is also reported to be a moiety of catenelline, isolated from the same species previously [10].Additionally, there is a high similarity of catenelline B and mycosporinetaurine isolated from the sea anemone Anthopleura elegantissima, with the difference being that the first MAA possesses the cyclohexenimine scaffold (λ max : 320 nm in water), whereas the second one possesses the cyclohexenone scaffold (λ max : 309 nm) [46].
Bostrychia scorpioides is an ecologically fascinating red alga, as it grows as an epiphyte on the basal stems of saltmarsh vegetation.Consequently, it is immersed by seawater only during extreme high tides and hence exhibits a rather atmophytic lifestyle due to long periods of exposure to air, leading to enhanced desiccation and osmotic and radiation stress [6].These stressors can be well compensated by various ecophysiological and biochemical traits.Most important is the capability to synthesize and accumulate protective organic compounds like the polyols sorbitol and dulcitol [2].In addition, B. scorpioides is regularly confronted with high solar insolation including ultraviolet radiation (UVR).UVR affects various biological functions in living organisms, and extensive exposure can lead to significant stress and deleterious effects at the molecular and cellular level [47][48][49].Adaptive mechanisms against enhanced UVR typically include in many micro-and macroalgae photoprotective MAAs [50].Most algae investigated so far typically contain a small set of MAAs (one to five) [9,51], while B. scorpioides exhibited at least twelve chemically different MAAs, named in a previous publication and in the present publication as bostrychines A to L [6,7].The biosynthesis of each of these MAAs requires individual enzymatic steps [8,52], and hence, the question arises of why this red alga invests so much metabolic energy for the formation of such an array of chemically similar compounds.As we can only speculate at this stage, it might be possible that bostrychines A to L all exhibit slightly different functions as anti-stress compounds, i.e., some act as UV-sunscreens and others as antioxidants.
Catenella caespitosa also grows as an intertidal species mainly exposed to the atmosphere.Although it can occur as epiphyte on saltmarsh plants, too, this species preferentially grows in the upper littoral zone on exposed rocky shores.For salinity acclimation, C. caespitosa uses as an organic osmolyte floridoside, and for UVR protection, it uses the previously identified MAA catenelline [10].In the present study, two additional and chemically related MAAs (catenelline B and C) were identified, which might also be involved in the protection against UVR and oxygen radicals.

Biological Material
The algae investigated in this study were collected in 2018 next to Roscoff, Brittany, France and morphologically identified by some of the authors (Maria Orfanoudaki, Anja Hartmann and Markus Ganzera), together with Ulf Karsten, from the University of Rostock, Germany, using their taxonomic expert knowledge in conjunction with standard identification keys [3,53].Voucher samples were deposited at the Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Austria (B.scorpioides) and at the University of Rostock, Germany (C. caespitosa).

Extraction and Isolation
The combined aqueous and methanolic extract of B. scorpioides was first fractionated on a silica gel column, followed by further purification using flash chromatography on reversed phase material and semipreparative HPLC.The procedure resulted in the isolation of six compounds.The same extraction and isolation protocol was used for the isolation and identification of MAAs from C. caespitosa, with the addition of column chromatography on Sephadex LH-20 as the last purification step.
B. scorpioides (approximately 350 g) was extracted three times in an ultrasonic bath (Bandelin Sonorex 35 KHz, Berlin, Germany) for 15 min successively with methanol and water (100%).Afterwards, the combined and dried extract (50 g) was fractionated on a silica gel column using EtOAc and methanol (from 10:0 to 0:10) as the eluent, resulting in 15 subfractions, in order to remove non-polar constituents such as chlorophylls in the early eluting fractions and sugars in the polar fractions.The HPLC analysis of the fractions indicated that fractions 9-12 contained MAAs; therefore, they were combined and used for further purification.This collective fraction (approximately 15 g) was separated with reversed phase flash chromatography using water-methanol (10:0-0:10) to give ten subfractions.Subfraction 6 (5 g) was subjected to semipreparative HPLC (H 2 O-MeOH) to yield compounds 1 (2 mg, 0.0006% yield) and 2 (2 mg, 0.0006% yield).Subfraction 7 (4 g) was also separated by semipreparative HPLC (H 2 O-MeOH) to give a mixture of compounds 3 and 4 (9.2 mg, 0.003% yield) and a mixture of compounds 5 and 6 (0.9 mg, 0.0003% yield).
7:1 (compound 3: compound 4).The side chain of both compounds at position 3 was identified as glutamine based on COSY chain correlations of H-9/H-11/H-12 and characteristic NMR values which were in accordance with the literature values.The second side chain of compound 3 contained a cis conformation double bond, which was indicated by characteristic 1 H-NMR (H-1′, δH 6.39 and H-2′, δH 5.42) and 13 C-NMR (C-1′, δC 124.7 and C-2′, δC 120.3) values and the coupling constants of H-1′ (J = 8.0/1.2Hz).Moreover, a methyl group at δH 1.78 (H-3′) showed a correlation in the COSY spectrum with the protons of H-2′ at δH 5.42 and an HMBC correlation with the carbons of the double bond at δC 120.3 and 124.7 (C-2′ and C-1′), revealing the presence of a propene side group.Its position was confirmed by longrange correlations visible in the HMBC spectrum (H-1′ at δH 6.39 to C-1 at δC 158.0).The NMR signals of compound 4 were highly similar to those of compound 3.However, protons H-1′ and H-2′ were deshielded (H-1′, δH 6.57 and H-2′, δH 5.77), and the coupling

Figure 5 .
Figure 5. Electronic Circular Dichroism spectra (calculated: red; calculated mirror image: blue) of compounds 1, 2 (Bostrychia scorpioides) and 7, 8 (Catenella caespitosa) compared to their experimentally recorded spectra (black curves) in H2O.Δε is the molar ellipticity of the compounds, "Calc." is the abbreviation for "calculated for" and mirror displays the opposite spectra corresponding to the respective enantiomer of each compound.

Figure 5 .
Figure 5. Electronic Circular Dichroism spectra (calculated: red; calculated mirror image: blue) of compounds 1, 2 (Bostrychia scorpioides) and 7, 8 (Catenella caespitosa) compared to their experimentally recorded spectra (black curves) in H 2 O. ∆ε is the molar ellipticity of the compounds, "Calc." is the abbreviation for "calculated for" and mirror displays the opposite spectra corresponding to the respective enantiomer of each compound.

Table 1 .
1H NMR (Nuclear Magnetic Resonance) data of compounds