A Biomimetic Approach to Premyrsinane-Type Diterpenoids: Exploring Microbial Transformation to Enhance Their Chemical Diversity

Premyrsinane-type diterpenoids have been considered to originate from the cyclization of a suitable 5,6- or 6,17-epoxylathyrane precursor. Their biological activities have not been sufficiently explored to date, so the development of synthetic or microbial approaches for the preparation of new derivatives would be desirable. Epoxyboetirane A (4) is an 6,17-epoxylathyrane isolated from Euphorbia boetica in a large enough amount to be used in semi-synthesis. Transannular cyclization of 4 mediated by Cp2TiIIICl afforded premyrsinane 5 in good yield as an only diasteroisomer. To enhance the structural diversity of premyrsinanes so their potential use in neurodegenerative disorders could be explored, compound 5 was biotransformed by Mucor circinelloides NRRL3631 to give rise to hydroxylated derivatives at non-activated carbons (6–7), all of which were reported here for the first time. The structures and absolute configurations of all compounds were determined through extensive NMR and HRESIMS spectroscopic studies.


Introduction
The Euphorbia genus is the largest of the Euphorbiaceae family; it contains more than 2000 species, and has a widespread distribution and a high chemical diversity [1][2][3][4][5].A great number of polycyclic and macrocyclic diterpenoids with a broad range of therapeutically relevant biological activities have been isolated from Euphorbia species including jatrophane [6], tiglianes [7], ingenanes [8], and lathyranes [9].
Premyrsinanes-type diterpenoids have been isolated from approximately 20 Euphorbia species and Jatropha jurcus [10].They are characterized by a 5/7/6/3-tetracyclic carbon framework with trans-oriented A/B and B/C ring junctions.As described in several reports, they often exhibit three characteristic oxymethyne protons at C-3, C-5, and C-7 positions and one oxygenated methylene group at C-17 [10,11].
From a biogenetic point of view, premyrsinanes could be derived from a lathyrane precursor by intramolecular cyclization between the C-12 and C-6 positions.However, their biogenetic relationships still remain unclear.For instance, 5,6-and 6-17-epoxylathyranes could be considered biosynthetic precursors of premyrsinanes [12,13].Appendino et al. proposed a biogenetic relationship between lathyranes and premyrsinanes based on the cooccurrence of these diterpenoids with a similar functionalization pattern (Scheme 1).They Plants 2024, 13, 842 2 of 11 suggested that the conversion of a suitable 6,17-epoxylathyrane would lead to premyrsinane derivative through a transannular cyclization but so far it has not been achieved using synthetic approaches [14].Recently, Xiao et al. performed the chemical conversion, catalyzed by Fe(acac) 3 , of Euphorbia factor L 3 , a ∆ 6(17),12 lathyradiene (1), into premyrsinane skeleton compounds.The reaction involved an intramolecular Michael addition via free radicals to produce a mixture of diasteroisomeric premyrsinanes 2a-b [15,16] (Scheme 2).
Plants 2024, 13, x FOR PEER REVIEW 2 of 12 these diterpenoids with a similar functionalization pattern (Scheme 1).They suggested that the conversion of a suitable 6,17-epoxylathyrane would lead to premyrsinane derivative through a transannular cyclization but so far it has not been achieved using synthetic approaches [14].Recently, Xiao et al. performed the chemical conversion, catalyzed by Fe(acac)3, of Euphorbia factor L3, a Δ 6(17),12 lathyradiene (1), into premyrsinane skeleton compounds.The reaction involved an intramolecular Michael addition via free radicals to produce a mixture of diasteroisomeric premyrsinanes 2a-b [15,16] (Scheme 2).
Studies of the biological activities of premyrsinanes are scarce in the literature; this is likely because of the reduced amount of the isolated compounds [10].Nevertheless, several studies have reported that premyrsinanes possess promising anticancer [17][18][19], multidrug resistance reversal [20], and anti-inflammatory activities, inhibiting the lipopolysaccharide-induced NO production [21].Furthermore, their potential biosynthetic precursors, euphoboetirane A (3) and epoxyboetirane A (4), have demonstrated a promising activity as promoters of neural stem cells (NSC) proliferation [22].For this reason, it is necessary to develop synthetic or microbial methodologies to obtain new premyrsinane derivatives in order to explore its biological activity and to study structure-activity relationships.
Cp2Ti III Cl has emerged as a useful reagent in organic chemistry because of its versatility to generate single-electron-transfer reactions [23][24][25][26][27].This complex is easily prepared from commercially available Cp2Ti IV Cl2 by reduction with metals such as Zn [28] or Mn [29].The reaction of Cp2Ti III Cl with oxiranes produces its homolytic cleavage to afford a β-titanoxyl radical [30].This radical can also react with activated olefins to form carboncarbon bonds [31].Consequently, we postulate that the titanium-catalyzed cyclization of 6,17-epoxylathyranes could be a powerful tool for the preparation of premyrsinane derivatives.
On the other hand, microbial transformations have advantages over conventional organic synthesis due to their high stereo-and regioselectivity under mild reaction conditions [32][33][34][35].In the literature, there are few examples of microbial transformations of lathyrane-type compounds involving regioselective hydroxylations, glycosylations, deoxygenations, and cyclopropane ring openings [36][37][38].Recently, we reported the microbial transformation of euphoboetirane A (3) by Streptomyces puniceus BC-5GB.11;this resulted in regioselective oxidation of non-activated carbons, isomerization of the Δ 12 double bond, and cyclopropane rearrangements [39].To the best of our knowledge, this strategy has not been used for the functionalization of non-activated positions of premyrsinane-type diterpenoids.
Studies of the biological activities of premyrsinanes are scarce in the literature; this is likely because of the reduced amount of the isolated compounds [10].Nevertheless, several studies have reported that premyrsinanes possess promising anticancer [17][18][19], multidrug resistance reversal [20], and anti-inflammatory activities, inhibiting the lipopolysaccharide-induced NO production [21].Furthermore, their potential biosynthetic precursors, euphoboetirane A (3) and epoxyboetirane A (4), have demonstrated a promising activity as promoters of neural stem cells (NSC) proliferation [22].For this reason, it is necessary to develop synthetic or microbial methodologies to obtain new premyrsinane derivatives in order to explore its biological activity and to study structure-activity relationships.
Cp2Ti III Cl has emerged as a useful reagent in organic chemistry because of its versatility to generate single-electron-transfer reactions [23][24][25][26][27].This complex is easily prepared from commercially available Cp2Ti IV Cl2 by reduction with metals such as Zn [28] or Mn [29].The reaction of Cp2Ti III Cl with oxiranes produces its homolytic cleavage to afford a β-titanoxyl radical [30].This radical can also react with activated olefins to form carboncarbon bonds [31].Consequently, we postulate that the titanium-catalyzed cyclization of 6,17-epoxylathyranes could be a powerful tool for the preparation of premyrsinane derivatives.
On the other hand, microbial transformations have advantages over conventional organic synthesis due to their high stereo-and regioselectivity under mild reaction conditions [32][33][34][35].In the literature, there are few examples of microbial transformations of lathyrane-type compounds involving regioselective hydroxylations, glycosylations, deoxygenations, and cyclopropane ring openings [36][37][38].Recently, we reported the microbial transformation of euphoboetirane A (3) by Streptomyces puniceus BC-5GB.11;this resulted in regioselective oxidation of non-activated carbons, isomerization of the Δ 12 double bond, and cyclopropane rearrangements [39].To the best of our knowledge, this strategy has not been used for the functionalization of non-activated positions of premyrsinane-type diterpenoids.Studies of the biological activities of premyrsinanes are scarce in the literature; this is likely because of the reduced amount of the isolated compounds [10].Nevertheless, several studies have reported that premyrsinanes possess promising anticancer [17][18][19], multidrug resistance reversal [20], and anti-inflammatory activities, inhibiting the lipopolysaccharideinduced NO production [21].Furthermore, their potential biosynthetic precursors, euphoboetirane A (3) and epoxyboetirane A (4), have demonstrated a promising activity as promoters of neural stem cells (NSC) proliferation [22].For this reason, it is necessary to develop synthetic or microbial methodologies to obtain new premyrsinane derivatives in order to explore its biological activity and to study structure-activity relationships.
Cp 2 Ti III Cl has emerged as a useful reagent in organic chemistry because of its versatility to generate single-electron-transfer reactions [23][24][25][26][27].This complex is easily prepared from commercially available Cp 2 Ti IV Cl 2 by reduction with metals such as Zn [28] or Mn [29].The reaction of Cp 2 Ti III Cl with oxiranes produces its homolytic cleavage to afford a βtitanoxyl radical [30].This radical can also react with activated olefins to form carbon-carbon bonds [31].Consequently, we postulate that the titanium-catalyzed cyclization of 6,17epoxylathyranes could be a powerful tool for the preparation of premyrsinane derivatives.
On the other hand, microbial transformations have advantages over conventional organic synthesis due to their high stereo-and regioselectivity under mild reaction conditions [32][33][34][35].In the literature, there are few examples of microbial transformations of lathyrane-type compounds involving regioselective hydroxylations, glycosylations, deoxygenations, and cyclopropane ring openings [36][37][38].Recently, we reported the microbial transformation of euphoboetirane A (3) by Streptomyces puniceus BC-5GB.11;this resulted in regioselective oxidation of non-activated carbons, isomerization of the ∆ 12 double bond, and cyclopropane rearrangements [39].To the best of our knowledge, this strategy has not been used for the functionalization of non-activated positions of premyrsinane-type diterpenoids.
Therefore, as a part of our ongoing research on compounds with diterpene scaffold for the treatment of neurodegenerative disorders, we describe here an unprecedented titanium-catalyzed cyclization of a 6,17-epoxylathyrane isolated from Euphorbia boetica, epoxyboetirane A (4) [22,40], to premyrsinane 5. Furthermore, we also describe the bio-Plants 2024, 13, 842 3 of 11 transformation of compound 5 by Mucor circinelloides NRRL3631 in order to increase the structural diversity of premyrsinane-type diterpenoids.
Encouraged by the postulated biogenetic relationship between 6,17-epoxylathyranes and premyrsinanes [14], we explored a transannular cyclization of 4 catalyzed by Cp 2 Ti III Cl.This reagent was generated by reduction of catalytic quantities of Cp 2 Ti IV Cl 2 (0.2 equiv.)with excess of Mn powder (8 equiv.) in deoxygenated and dry THF in the presence of 2,4,6-collidine and TMSCl [45].After 15 min, the mixture turned to the characteristic lime green color of Ti(III) solutions.Gratifyingly, premyrsinane 5 was isolated in good yield after 2 h as a result of a 6-endo-trig intramolecular cyclization.Interestingly, the reaction occurred with high regio-and diastereoselectivity (Scheme 3).Contrary to the Fe-mediated cyclization of Euphorbia factor L 3 [21], premyrsinane 5 was obtained at room temperature as only one diastereoisomer, with the same stereochemistry at C-6, C-12, and C-13 than most naturally occurring premyrsinane-type diterpenoids [10].Our newly developed protocol would represent the first report of a biomimetic cyclization of a 6,17-epoxylathyrane into premyrsinane derivative.
Therefore, as a part of our ongoing research on compounds with diterpene scaffold for the treatment of neurodegenerative disorders, we describe here an unprecedented titanium-catalyzed cyclization of a 6,17-epoxylathyrane isolated from Euphorbia boetica, epoxyboetirane A (4) [22,40], to premyrsinane 5. Furthermore, we also describe the biotransformation of compound 5 by Mucor circinelloides NRRL3631 in order to increase the structural diversity of premyrsinane-type diterpenoids.
Encouraged by the postulated biogenetic relationship between 6,17-epoxylathyranes and premyrsinanes [14], we explored a transannular cyclization of 4 catalyzed by Cp2Ti III Cl.This reagent was generated by reduction of catalytic quantities of Cp2Ti IV Cl2 (0.2 equiv.)with excess of Mn powder (8 equiv.) in deoxygenated and dry THF in the presence of 2,4,6-collidine and TMSCl [45].After 15 min, the mixture turned to the characteristic lime green color of Ti(III) solutions.Gratifyingly, premyrsinane 5 was isolated in good yield after 2 h as a result of a 6-endo-trig intramolecular cyclization.Interestingly, the reaction occurred with high regio-and diastereoselectivity (Scheme 3).Contrary to the Femediated cyclization of Euphorbia factor L3 [21], premyrsinane 5 was obtained at room temperature as only one diastereoisomer, with the same stereochemistry at C-6, C-12, and C-13 than most naturally occurring premyrsinane-type diterpenoids [10].Our newly developed protocol would represent the first report of a biomimetic cyclization of a 6,17epoxylathyrane into premyrsinane derivative.Compared with the starting material (compound 4), the 12,13-double bond and 13vinylic methyl group characteristic signals were absent in the NMR spectra of compound 5 (Figures S3-S9) [40].Furthermore, resonances which could be attributed to a hydroxymethyl group (δH 3.84 (d) and 3.68 (d); δC 63.2) and a methyl attached to a methyne (δH 1.10 (d); δC 19.7; H3-20) were present in the NMR spectra of 5, which could be located at C-6 and C-13, respectively, on a premyrsinane skeleton (Table 1).Analysis of the 1 H- Compared with the starting material (compound 4), the 12,13-double bond and 13vinylic methyl group characteristic signals were absent in the NMR spectra of compound 5 (Figures S3-S9) [40].Furthermore, resonances which could be attributed to a hydroxymethyl group (δ H 3.84 (d) and 3.68 (d); δ C 63.2) and a methyl attached to a methyne (δ H 1.10 (d); δ C 19.7; H 3 -20) were present in the NMR spectra of 5, which could be located at C-6 and C-13, respectively, on a premyrsinane skeleton (Table 1).Analysis of the 1 H-1 H COSY spectrum (Figure S5) revealed two spin systems: H 2 -1/H-2/H 3 -16/H-3/H-4/H-5 and H 3 -20/H-13/H-12/H-11/H-9/H 2 -8/H 2 -7.Moreover, HMBC correlations (Figure S7) from H 2 -17 to C-6 and C-12, H 3 -20 to C-12, C-13 and C-14, and H-5 to C-6 and C-12 were observed, consistent with a 5/7/6/3-fused tetracyclic ring skeleton for 5, characteristic of premyrsinane derivatives.1D and 2D NOESY correlations (Figures S8 and S9) between H-2/H-3/H-4/H 2 -17/H-8α/H-9/H 3 -18/H-11/H-13 and H 2 -17/H-13 indicated that they all are α-oriented, while correlations between H-5/H-12/H 3 -19 and H-5/H-7β supported their β-orientation (Figure 1).consistent with a 5/7/6/3-fused tetracyclic ring skeleton for 5, characteristic of premyrsinane derivatives.1D and 2D NOESY correlations (Figures S8 and S9) between H-2/H-3/H-4/H2-17/H-8α/H-9/H3-18/H-11/H-13 and H2-17/H-13 indicated that they all are αoriented, while correlations between H-5/H-12/H3-19 and H-5/H-7β supported their β-orientation (Figure 1).Based on the above-mentioned NOESY correlations and the absolute configuration described above for epoxyboetirane A (4), the structure of 5 was determined to be (2S,3S,4R,5R,6R,9S,11S,12S,13S,15R)-3,5,15-triacetoxy-7,13-dideoxipremyrsinol.Based on the above-mentioned NOESY correlations and the absolute configuration described above for epoxyboetirane A (4), the structure of 5 was determined to be (2S,3S,4R,5R,6R,9S,11S,12S,13S,15R)-3,5,15-triacetoxy-7,13-dideoxipremyrsinol.In accordance with the mechanism admitted for catalytic cyclization mediated by Cp 2 Ti III Cl, a catalytic cycle is proposed in Scheme 4 to rationalize the formation of 5 [45].The homolytic opening of the epoxide leads to a β-titanoxyl radical (I), which is trapped by the electron deficient 12,13 double bond in 4 [46].The control of the observed diastereoselectivity in the cyclization step could be explained on the basis of the 14d20d preferred conformation for 4 (Figure S1).This conformation would be consistent with a Ti-carbonyl coordination [47], favoring an approach of the radical to the double bond leading to a transfused ring.Then, the carbon-centered radical resulting from the cyclization step is trapped by a second molecule of Cp 2 Ti III Cl to produce the alkyltitanium species III.Ti-carbonyl coordination would be again essential to explain the β-orientation of the methyl group at C-20.Finally, the 2,4,6-trimethylsilylpyridinium chloride obtained by mixing 2,4,6-collidine and TMSCl is capable of regenerating Cp 2 Ti IV Cl 2 and finally yields compound 4. In accordance with the mechanism admitted for catalytic cyclization mediated by Cp2Ti III Cl, a catalytic cycle is proposed in Scheme 4 to rationalize the formation of 5 [45].The homolytic opening of the epoxide leads to a β-titanoxyl radical (I), which is trapped by the electron deficient 12,13 double bond in 4 [46].The control of the observed diastereoselectivity in the cyclization step could be explained on the basis of the 14d20d preferred conformation for 4 (Figure S1).This conformation would be consistent with a Ti-carbonyl coordination [47], favoring an approach of the radical to the double bond leading to a trans-fused ring.Then, the carbon-centered radical resulting from the cyclization step is trapped by a second molecule of Cp2Ti III Cl to produce the alkyltitanium species III.Ticarbonyl coordination would be again essential to explain the β-orientation of the methyl group at C-20.Finally, the 2,4,6-trimethylsilylpyridinium chloride obtained by mixing 2,4,6-collidine and TMSCl is capable of regenerating Cp2Ti IV Cl2 and finally yields compound 4.  In order to enrich the chemical diversity of premyrsinane-type diterpenoids, compound 5 was subjected to biotransformation.Initial screenings were performed to determine the optimal microorganism and culture conditions.Based on previous experiments of euphoboetirane A (3) with about 20 microorganisms (fungi and bacteria), Mucor circinelloides NRRL3631 was selected for its ability to biotransform macrocyclic diterpenes [48].A scaled-up biotransformation of 5 involved feeding of a 3-day-old culture of M. circinelloides with 5 and incubation for further 5 days.Two closely related premyrsinane derivatives were obtained: compounds 6 (29% conversion) and 7 (45% conversion), as a result of the regioselective hydroxylation at non-activated positions (Scheme 5).
On the other hand, the 1 H NMR spectrum of compound 7 (Figure S21) showed a double of triplet signal at δH 4.37, which was correlated in the gHSQC spectrum (Figure S24) to an oxygenated methine carbon at δC 64.9 (Table 1).These data suggested the presence of a hydroxyl group, which was located at C-8 based on HMBC correlations (Figures 2 and S24

Plant Material
E. boetica was collected in March 2020 from El Pinar del Hierro (Chiclana de la Frontera), Cádiz, Spain, under the authorization of the competent national authorities (reference numbers ESNC64 and 201999901092011).

Microorganism
M. circinelloides NRRL3631 was kindly provided by Prof. Cerdá-Olmedo of the University of Sevilla (Spain).Conidia of this strain were preserved in 80% glycerol at −40 • C.

Extraction and Isolation of Euphoboetirane A (3) and Epoxyboetirane A (4)
The aerial parts of fresh E. boetica plant (3.0 kg) were frozen with liquid nitrogen, ground into powder, and subsequently extracted with MeOH (2.5 L × 3) at room temperature for 24 h.After solvent removal, the crude extract was suspended in water (1 L) and extracted with hexane (1.5 L × 3).The obtained crude extract (33.3 g) was subjected to column chromatography on silica gel, using an increasing gradient of EtOAc in n-hexane (10-100%) as the mobile phase.Fractions containing lathyranes were further purified by column chromatography, employing an increasing gradient of CH 2 Cl 2 in acetone (0-2%) to give 2.0 g of euphoboetirane A (3) and 345.0 mg of epoxyboetirane A (4) [38].

Preparation of Epoxyboetirane A (4) by Epoxidation of Euphoboetirane A (3)
A solution of euphoboetirane A (3) (43.1 mg, 0.1 mmol) in CH 2 Cl 2 (4 mL) was treated with m-chloroperbenzoic acid (21.0 mg, 0.1 mmol, 77% w/w) and stirred at room temperature for 7 h.Then, a solution of saturated NaHCO 3 was added and the mixture was extracted with CH 2 Cl 2 (×3).The organic phase was washed with H 2 O and dried over anhydrous Na 2 SO 4 .Subsequently, the solvent was evaporated using a rotary evaporator, and the resulting reaction mixture was subjected to column chromatography to give 12.3 mg of euphoboetiranne A (3) and 28.3 mg of epoxyboetirane A (4) (63% yield, 89% conversion).The reaction was repeated several times, with progressively longer reaction times, in an attempt to enhance conversion.In all cases, unreacted starting material was observed not improving yield and conversion stated above.

Extraction, Isolation, and Characterization of Biotransformation Products
The culture broth and mycelium were separated by vacuum filtration using a 200 µm pore size Nytal filter.The culture medium was saturated with NaCl and then extracted with EtOAc (×3).The organic layer was dried over anhydrous Na 2 SO 4 and the solvent was removed using a rotary evaporated, yielding 106.0 mg of a crude extract.The resulting residue containing the biotransformation products was purified by column chromatography on silica gel, using an increasing gradient of EtOAc in n-hexane as mobile phase.Fractions collected were further purified by semipreparative and/or analytical HPLC to afford compounds 5 (21.6 mg), 6 (14.1 mg, 29% conversion), and 7 (22.0mg, 45% conversion).