Diketopiperazines as Cross-Communication Quorum-Sensing Signals between Cronobacter sakazakii and Bacillus cereus.

Herein, we reveal a second quorum-sensing system produced by Cronobacter sakazakii. A cyclo(l-Pro-l-Leu) diketopiperazine, detected in pure and mixed cultures of C. sakazakii and Bacillus cereus explains the coexistence of both in the same industrial environments. The molecule was identified by gas chromatography-mass spectrometry (GC-MS), 1H, and 13C NMR, including 2D NMR (correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear single quantum correlation), and the absolute configuration was compared with that of four synthetic standards produced by solid phase peptide synthesis using a chiral column on a GC-flame ionization detection. This article provides a new method to determine the absolute configuration of cyclo(Pro-Leu) diketopiperazine replacing the joint use of 1H NMR and Marfey's method.

Introduction

Cronobacter sakazakii, a Gram-negative, yellow-pigmented, rod-shaped, and facultatively anaerobic bacterium, is an opportunistic pathogen that is life-threatening for newborns and elderly adults.[1] This microorganism is naturally found in the environment and is present in dry foods such as powdered milk, spices, teas, and starches, surviving desiccated conditions. This bacterium can adhere to surfaces, be soaked in an exopolysaccharide matrix (biofilm), and be protected from neighboring threats.[2] The biofilm formation is controlled by low-molecular-weight molecules in a process of intercommunication called quorum-sensing (QS), and it is known that Gram-negative bacteria are likely to communicate by producing acyl-homoserine lactones (AHL).[3]C. sakazakii is known to produce 3-oxo-hexanoyl-HSL, 3-oxo-octanoyl-HSL,[4] (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL, (S)-N-tetradecanoyl-HSL,[5] and short chain acyl-HSL[6] for QS. The exopolysaccharide matrix can shelter other microorganisms like Bacillus cereus, which is a Gram-positive bacterium and also an infecting agent that causes intestinal and nonintestinal infections in humans; it is commonly associated with food poisoning,[7] causing acute diarrheal disease, which is related to the production and secretion of a variety of hemolysins, phospholipases, and toxins.[7] This spore-forming bacterium withstands low temperatures, making the control of its contamination and biofilm formation on plastics, glasses, wool, and inox steel difficult, which impacts public health and the economy.[8,9] Some Bacillus sp. have been investigated for secondary metabolites, revealing the presence of cyclic dipeptides, called 2,5-diketopiperazines (DKPs), used as antifungal and antibacterial agents. Bacillus cereus, from an entomopathogenic nematode, produces cyclo(l-Pro–l-Arg) with antibacterial and antitumor actions,[10] and B. cereus subsp. thuringiensis produces an antifungal cyclo(l-Pro–d-Leu).[11] The four isomers occur in nature, however, their optical rotation data are highly unreliable, thus preventing a rapid absolute configuration determination.[12−14]

Araujo and co-workers[5] studied C. sakazakii and B. cereus in a co-culture and detected C. sakazakii’s acyl-homoserine lactone depletion by B. cereus enzymes (acyl-HSL lactonase and acyl-homoserine acylase). However, this depletion did not inhibit the biofilm formation or C. sakazakii growth, suggesting the presence of alternative signaling molecules for the C. sakazakii and B. cereus QS system. Therefore, the aim of this work is to reveal the cross-talking signal system between C. sakazakii and B. cereus.

Results and Discussion

The absence of biofilm inhibition in co-cultures of C. sakazakii and B. cereus, as previously reported, was intriguing. Thus, the presence of a secondary communication exclusive to C. sakazakii and a cross-communication system between both species, was the focus of the present investigation. Consequently, C. sakazakii and B. cereus were cultivated individually, and co-cultivated, to evaluate the second mechanism of intra- and inter-species chemical communication. The cell-free supernatants were extracted with ethyl acetate, and a common group of DKPs was effectively detected that was completely unaccounted for in our previous investigation. Isolation of the polar fractions was achieved using a silica column eluted with ethyl acetate; methanol allowed the identification of the major DKP as the cyclic dipeptide cyclo(Pro–Leu) (Figure ). The retention time and fragmentation pattern were consistent with data found in the literature.[15,16]

Figure 1

Structure of cyclo(Pro–Leu).

NMR data confirmed the structure of the DKP and showed the 1H and 13C shifts for cyclo(Pro–Leu) isolated from B. cereus, C. sakazakii, and the culture of both bacteria (see Supporting Information). 2,5-DKPs are QS molecules commonly found in Gram-positive bacteria and are not usually present in Gram-negative bacteria such as C. sakazakii, where the QS is based on AHL. However, previous studies have shown that DKPs can modulate regulatory proteins for gene expression, substituting AHLs in Gram-negative bacteria’s communication.[17,18] From this point of view, this work shows that despite the B. cereus enzymes destroying C. sakazakii’s AHLs, both B. cereus and C. sakazakii share a common signal for communication, the cyclo(Pro–Leu). Following this discovery, a few pending questions remained unanswered. The most important question was the absolute configuration of the cyclo(Pro–Leu) and whether this was important for the intercrossing communication. The first attempts to determine the absolute configuration employing specific optical rotation were complicated by the broad range of values of specific optical rotations reported in the literature (Table ). An additional drawback was the lack of a rapid chiral chromatographic method to check the enantiopurity of the samples.

Table 1

Optical Rotation Data for Cyclo(Pro–Leu) Isomers

DKPs	[α]D (deg)	solvent	references
cyclo(l-Pro–l-Leu)	–109	EtOH	(12)
cyclo(l-Pro–l-Leu)	–124	EtOH	(12)
cyclo(l-Pro–l-Leu)	–143	EtOH	(19)
cyclo(l-Pro–l-Leu)	–142	EtOH	(20)
cyclo(l-Pro–l-Leu)	–88	EtOH	(21)
cyclo(l-Pro–l-Leu)	–133	EtOH	(22)
cyclo(l-Pro–l-Leu)	–156	NaOH in MeOH/H2O 1:1	(23)
cyclo(l-Pro–l-Leu)	–89	MeOH	(24)
cyclo(l-Pro–l-Leu)	–105.8	MeOH	(25)
cyclo(l-Pro–l-Leu)	–91.3	H2O	(26)
cyclo(l-Pro–d-Leu)	–78	EtOH	(27)
cyclo(l-Pro–d-Leu)	–78.3	MeOH	(28)
cyclo(l-Pro–d-Leu)	–91.2	NaOH in MeOH/H2O 1:1	(23)
cyclo(l-Pro–d-Leu)	–38	MeOH	(13)
cyclo(d-Pro–l-Leu)	+35	EtOH	(29)
cyclo(d-Pro–l-Leu)	+34	CHCl3	(14)
cyclo(d-Pro–d-Leu)	+152	EtOH	(12)

The optical rotation ([α]D) values were mainly dependent on the absolute configuration of proline in proline-based DKPs. Negative [α]D values were observed for l-proline DKPs, and positive [α]D for d-proline DKPs. Additionally, the magnitude was variable, but l-Pro–l-Leu showed higher negative values and l-Pro–d-Leu showed lower negative values of [α]D. The same result was observed for d-proline derivatives, where d-Pro–d-Leu presented higher positive values and d-Pro–l-Leu presented lower positive values.[30]

Comparing the optical rotation of the standards and natural samples, we became aware that different amounts of stereoisomers were present in the mixtures. The B. cereus cyclo(Pro–Leu) had an [α]D of −28, and C. sakazakii cyclo(Pro–Leu) had an [α]D of −21, but the mixed culture had an [α]D of −27. The results were clear but not conclusive. Other procedures were needed to determine the absolute configuration.

Many literature reports[11,24,31,32] have utilized Marfey’s method for enantiomeric identification, which consists of hydrolysis and derivatization using a chiral compound (1-fluoro-2,4-dinitrophenyl-5-l-alanine amide), and high-performance liquid chromatography elution in a silica gel column.[33] This method has been very effective in revealing the absolute configurations of the amino acids, which are the 2,5-diketopiperazine components. However, the method depends on hydrolysis and derivatization of the DKPs, which is a time-demanding procedure. The use of gas chromatography (GC) is expected to be less demanding but requires the selection of a column with effective enantio- and diastereodiscrimination. Thus, to investigate the enantiodiscrimination of cyclo(Pro–Leu) by the available chiral columns, we either had to have racemic standards or pure samples of the four isomers, that is, cyclo(d-Pro–d-Leu), cyclo(l-Pro–l-Leu), cyclo(l-Pro–d-Leu), and cyclo(d-Pro–l-Leu).

Optimization of the chiral gas chromatographical method was undertaken with the four stereoisomeric standards, which are not commercially available, and were obtained by solid phase synthesis. Remarkably, these molecules are able to epimerize, and this phenomenon involves a change of configuration at just one of several chiral centers present in the molecule (Scheme ). As observed by Adamczeski et al.,[30] epimerization occurred preferentially at H6 with DKPs containing proline in experiments where d-prolyl–l-norvaline went from +91 to +81 in alkaline solution (0.01 M NaOH in 1:1 v/v H2O/MeOH) in 24 h. The decreasing positive [α]D values indicated the formation of l-prolyl–l-norvaline, which is negative. Epimerization occurs preferentially at positions 6 and 9, and it was observed that standard cyclo(l-Pro–l-Leu) epimerization was larger (21%), followed by that of cyclo(l-Pro–d-Leu) (18%), and that of cyclo(d-Pro–l-Leu) and cyclo(d-Pro–d-Leu) (both 11%). The cis enantiomers (cyclo(l-Pro–l-Leu) and cyclo(d-Pro–d-Leu)) were harder than the trans enantiomers (cyclo(l-Pro–d-Leu) and cyclo(d-Pro–l-Leu)) to separate, leading to a long chromatographic run (85 min). Despite the epimerization of the synthetic standards, it was possible to determine the retention times of each stereoisomer based on the major peak. The retention times were 64.197 min for cyclo(d-Pro–d-Leu), 64.474 min for cyclo(l-Pro–l-Leu), 69.592 min for cyclo(l-Pro–d-Leu), and 70.834 min for cyclo(d-Pro–l-Leu) (Table ).

Scheme 1

Stereoisomers of Cyclo(Pro–Leu)

Adapted from ref (34).

Table 2

Retention Times, Relative Quantification and Optical Rotations of Cyclo(Pro–Leu) Synthetic Standards

			cyclo(Pro–Leu)
standard	retention time (min)a,b	relative retention time (RRT)a,c	(l–l) (%)	(l–d) (%)	(d–l) (%)	(d–d) (%)	[α]D (deg)b
cyclo(l-Pro–l-Leu)	64.47	1.064	79	1	0	0	–25
cyclo(l-Pro–d-Leu)	69.59	0.982	7	90	2	9	–6
cyclo(d-Pro–l-Leu)	70.83	0.970	14	4	82	2	+19
cyclo(d-Pro–d-Leu)	64.19	1.069	0	5	16	89	+28

Retention time and index of the major peak for synthetic standards.

Measured from partial epimerized standards.

Relative to C23H48 peak.

For more reliable and reproducible standardizations we used a RRT, which overcomes the uncertainty of the identity of the analyzed substance.[35] Thus, the four isomers of cyclo(Pro–Leu) were injected with a tricosane standard (C23H48), and the calculation of RRT was performed, giving rise to the data in Table .

With this chromatographical separation, we could monitor the absolute configuration of the cyclo(Pro–Leu) produced by these microorganisms. C. sakazakii produced higher amounts of cyclo(l-Pro–l-Leu) compared to cyclo(d-Pro–d-Leu) and cyclo(d-Pro–l-Leu), whereas B. cereus produced cyclo(l-Pro–l-Leu) and cyclo(d-Pro–l-Leu). The mixed culture showed that both cultures produced the same molecules, which are depicted in Table . The presence of the same enantiomers in different bacterial cultures can be attributed to the natural use of d-amino acids by microorganisms, or to epimerization. The epimerization percentages of C. sakazakii and the co-culture were similar to those of the cyclo(l-Pro–l-Leu) standard (14 and 17%), indicating a possible epimerization of the C. sakazakii cyclo(Pro–Leu) and not a natural occurrence.

Table 3

Relative Quantification and Optical Rotation of Natural Cyclo(Pro–Leu)

	cyclo
culture	l-Pro–l-Leu (%)	l-Pro–d-Leu (%)	d-Pro–l-Leu (%)	d-Pro–d-Leu (%)	[α]D (deg)
B. cereus	95	0	5	0	–28
C. sakazakii	86	0	12	2	–21
co-culture	83	0	12	5	–27

We observed that (1) the major stereoisomer in the bacterial extracts was cyclo(l-Pro–l-Leu), and that (2) there was similar epimerization for the standards and the natural products. Therefore, the cross-talking signal responsible for a second communication system between C. sakazakii and B. cereus is cyclo(l-Pro–l-Leu).

Other DKPs

The presence of cyclo(Pro–Leu) in the cultures dominated over other polar molecules. However, other DKPs occurred in the extracts (Figure ), that is, cyclo(Val–Val) and cyclo(Pro–Leu) in the B. cereus culture, and cyclo(Val–Val), cyclo(Pro–Leu), cyclo(Leu–Leu), cyclo(Phe–Val), cyclo(Phe–Leu), and cyclo(Phe–Pro) in the C. sakazakii culture and co-culture. The fragmentation patterns are in the Supporting Information.

Figure 2

Secondary DKPs found in the extract of C. sakazakii and B. cereus.

DKPs are able to modulate communication (QS) in microorganism cultures. DKPs and short chain AHLs bind to the same regulatory sites allowing the co-sharing of an ecological niche by different species of microorganisms.[17]

Conclusions

DKPs are important chemical signals in the communication between C. sakazakii and B. cereus. Acyl-HSL is one of the C. sakazakii communication signals that is easily destroyed by the action of other microorganisms (i.e., B. cereus). In contrast, cyclo(l-Pro–l-Leu) produced as a second mechanism of communication is not destroyed by Bacillus sp. enzymes and is a common signal between C. sakazakii and B. cereus. Finally, this explains why they coexist in the same industrial environment. The absolute configuration cyclo(l-Pro–l-Leu) was determined by chiral GC, which is a new tool for the absolute configuration of the four cyclo(Pro–Leu) isomers as an alternative to the joint application of NMR and Marfey’s method. Further studies will evaluate the biological activity of these compounds.

Experimental Section

Unless stated otherwise, the reactions were conducted under a N2 atmosphere using reagent-grade solvents. All commercially available reagents were purchased from Sigma-Aldrich Co., Germany and were used without further purification.

Culture Conditions

B. cereus and C. sakazakii were cultivated at 30 °C in nutrient agar (NA) (“Lab-Lemco” Powder 0.1%, yeast extract 0.2%, peptone 0.5%, NaCl 0.5%, agar 1.5%, and initial pH 7.4) and grown at 30 °C at 200 rpm in a Luria–Bertani growth medium (tryptone 1%, yeast extract 0.5%, NaCl 1%, and initial pH 7.0) for 24 h.

2,5-Diketopiperazine Extraction

C. sakazakii’s 2,5-Diketopiperazine (ES-DKP)

C. sakazakii CCT4821 was inoculated to LB medium (1 L) and was incubated for 24 h at 30 °C. The cells were removed by centrifugation at 3500 rpm for 30 min at 4 °C. The supernatant was extracted with ethyl acetate (3 × 300 mL), and the solvent was evaporated under reduced pressure (Büchi Rotavapor R-200). The residue (80 mg) was submitted to column chromatography on silica gel and eluted with a hexane/ethyl acetate gradient. The fractions were monitored by thin layer chromatography (TLC) and GC–MS. Fractions displaying characteristic DKP fragmentation [cyclo(Val–Val):m/z 156, 113, 72; cyclo(Leu–Leu):m/z 170, 86; cyclo(Pro–Leu):m/z 154, 86, 70; cyclo(Phe–Val):m/z 246, 127, 91; cyclo(Phe–Leu):m/z 204, 169, 141, 91; cyclo(Phe–Pro):m/z 153, 125, 91, 70] and adequate purity were pooled, and the solvent was evaporated. The major molecule found was cyclo(Pro–Leu), the only product completely isolated, which yielded 1.2 mg from the C. sakazakii culture.

The same procedure was repeated for B. cereus CCT4060 and the co-culture of B. cereus and C. sakazakii, yielding 90 and 70 mg of brute extract and 0.8 and 3.1 mg of the pure DKP fraction, respectively.

Enantiomer Standards Synthesis

The production of synthetic diketopiperazine cyclo(l-Pro–l-Leu), cyclo(l-Pro–d-Leu), cyclo(d-Pro–l-Leu), and cyclo(d-Pro–d-Leu) was carried out by solid phase peptide synthesis (SPPS) with the Fmoc (9-fluorenylmethyloxycarbonyl) strategy. A total of 100 mg of Wang resin (Sigma-Aldrich) already coupled with l- or d-leucine-Fmoc-OH (0.7 mmol/gresin) was submitted to a 3 mL dichloromethane (DCM) bath for 20 min for activation. Next, 3 mL of 4-methylpiperidine was poured into the reaction flask and stirred for 20 min, twice. After vacuum filtration, the resin was washed with 3 mL of methanol and 3 mL of DCM, three times each. The solvents were removed with vacuum filtration.

l- or d-Proline-Fmoc-OH (Sigma-Aldrich) was added to the system in 3:1 mmol equivalency in relation to l-leucine-Fmoc. 1-Hydroxybenzotriazole hydrate (Sigma-Aldrich) and N,N′-diisopropylcarbodiimide (DIC), dissolved in 3 mL of dimethylformamide, were added to the system in 6:1 proportions relative to the quantity of amino acid coupled to the resin (mmol). The system was stirred for 4 h at room temperature, and afterwards, it was vacuum filtered. The procedure of deprotection of proline was repeated to assure the availability of the amino terminal for spontaneous cyclization.

After vacuum filtration to remove the reactants, 3 mL of the cleavage cocktail (95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% distilled water) was added to the system and stirred for 4 h at room temperature. Filtration and evaporation (Büchi Rotavapor R-200) produced a colorless oil (24 mg) that was dissolved in methanol and analyzed by GC–MS.

Cyclo(l-Pro–l-Leu)

(3S, 8aS)-3-isobutylhexahydropyrrolo[1,2-α]pyrazine-1,4-dione (24 mg)

TLC (hexane/ethyl acetate/methanol, stain solution: phosphomolybdic acid (10% m/v in ethanol)) Rf 0.25.

[α] −25 (c = 0.1 g/100 mL, 20 °C, MeOH);

H NMR (400.18 MHz, CD3OD, δCD 3.31 ppm): δ 4.45 (1H, dd, J = 9.7 and 5.5 Hz, H-6), 4.31 (1H, dd, J = 8.6 and 6.3 Hz, H-9), 3.42 (1H, m, H-3a), 3.34 (1H, m, H-3b), 2.45 (1H, m, H-5b), 2.15 (1H, m, H-5a), 2.05 (2H, m, H-4), 1.73 (1H, m, H-11), 1.65 (2H, m, H-10), 0.98 (3H, d, J = 6.3 Hz, H-12), 0.94 (3H, d, J = 6.2 Hz, H-13).

C NMR (100.63 MHz, CD3OD, δCD 49.15 ppm): δ 175.5 (C, C-7), 170.0 (C, C-1), 61.1 (CH, C-6), 52.6 (CH, C-9), 47.6 (CH2, C-3), 41.4 (CH2, C-10), 31.1 (CH2, C-5), 26.2 (CH, C-11), 25.0 (CH2, C-4), 23.5 (CH3, C-12), 21.8 (CH3, C-13).

EI/MS (70 eV)m/z (relative intensity): 210 (M•+), 154 (99); 70 (47), 86 (13), 125 (9), 155 (8), 124 (7), 69 (6), 55 (5), 68 (5), 96(4).

Cyclo(l-Pro–d-Leu)

(3R, 8aS)-3-isobutylhexahydropyrrolo[1,2-α]pyrazine-1,4-dione (20 mg)

TLC (hexane/ethyl acetate/methanol, stain solution: phosphomolybdic acid (10% m/v in ethanol)) Rf 0.25.

[α] −6 (c = 0.1 g/100 mL, 20 °C, MeOH);

H NMR (400.18 MHz, CD3OD, δCD 3.31 ppm): δ 4.45 (1H, m, H-6), 4.29 (1H, m, H-9), 3.38 (2H, m, H-3a and -3b), 2.47 (1H, m, H-5b), 2.03 (3H, m, H-5a and -4), 1.68 (3H, m, H-10 and -11), 0.98 (3H, d, J = 6.0 Hz, H-12), 0.93 (3H, d, J = 6.1 Hz, H-13).

C NMR (100.63 MHz, CD3OD, δCD 49.15 ppm): δ 175.6 (C, C-7), 169.9 (C, C-1), 61.3 (CH, C-6), 52.5 (CH, C-9), 47.5 (CH2, C-3), 41.6 (CH2, C-10), 31.4 (CH2, C-5), 26.4 (CH, C-11), 25.2 (CH2, C-4), 23.5 (CH3, C-12), 21.8 (CH3, C-13).

EI/MS (70 eV)m/z (relative intensity): 210 (M•+), 154 (99); 70 (51), 125 (17), 124 (12), 86 (10), 155 (8), 69 (6), 68 (6), 96 (5), 98 (5).

Cyclo(d-Pro–l-Leu)

(3S, 8aR)-3-isobutylhexahydropyrrolo[1,2-α]pyrazine-1,4-dione (30 mg)

TLC (hexane/ethyl acetate/methanol, stain solution: phosphomolybdic acid (10% m/v in ethanol) Rf 0.25.

[α] + 19 (c = 0.1 g/100 mL, 20 °C, MeOH);

H NMR (400.18 MHz, CD3OD, δCD 3.31 ppm): δ 4.45 (1H, m, H-6), 4.30 (1H, dd, J = 8.8 and 7.1 Hz, H-9), 3.38 (2H, m, H-3), 2.45 (1H, m, H-5b), 2.03 (3H, m, H-4 and -5a), 1.68 (3H, m, H-10 and -11), 0.98 (3H, d, J = 6.1 Hz, H-12), 0.93 (3H, d, J = 6.2 Hz, H-13).

C NMR (100.63 MHz, CD3OD, δCD 49.15 ppm): δ 175.6 (C, C-7), 169.9 (C, C-1), 61.3 (CH, C-6), 52.5 (CH, C-9), 47.5 (CH2, C-3), 41.6 (CH2, C-10), 31.4 (CH2, C-5), 26.4 (CH, C-11), 25.2 (CH2, C-4), 23.5 (CH3, C-12), 21.8 (CH3, C-13).

EI/MS (70 eV)m/z (relative intensity): 210 (M•+), 154 (99); 70 (53), 98 (26), 125 (18), 124 (12), 86 (11), 155 (8), 68 (6), 96 (6), 55 (6).

Cyclo(d-Pro–d-Leu)

(3R, 8aR)-3-isobutylhexahydropyrrolo[1,2-α]pyrazine-1,4-dione (17 mg)

TLC (hexane/ethyl acetate/methanol, stain solution: phosphomolybdic acid (10% m/v in ethanol)) Rf 0.28.

[α] + 28 (c = 0.1 g/100 mL, 20 °C, MeOH);

H NMR (400.18 MHz, CD3OD, δCD 3.31 ppm): δ 4.45 (1H, dd, J = 9.7 and 5.4 Hz, H-6), 4.31 (1H, dd, J = 8.6 and 6.4 Hz, H-9), 3.42 (1H, m, H-3a), 3.35 (1H, m, H-3b), 2.45 (1H, m, H-5b), 2.15 (1H, m, H-5a), 2.04 (2H, m, H-4), 1.68 (3H, m, H-10 and -11), 0.98 (3H, d, J = 6,3 Hz, H-12), 0.94 (3H, d, J = 6.1 Hz, H-13).

C NMR (100.63 MHz, CD3OD, δCD 49.15 ppm): δ 175.5 (C, C-7), 170.0 (C, C-1), 61.1 (CH, C-6), 52.6 (CH, C-9), 47.6 (CH2, C-3), 41.4 (CH2, C-10), 31.1 (CH2, C-5), 26.2 (CH, C-11), 25.0 (CH2, C-4), 23.5 (CH3, C-12), 21.8 (CH3, C-13).

EI/MS (70 eV)m/z (relative intensity): 210 (M•+), 154 (99); 70 (43), 86 (14), 155 (9), 125 (8), 124 (8), 69 (6), 68 (6), 55 (5), 139 (5).

Identification and Structure Elucidation

GC–Mass Spectrometry (GC–MS)

GC–MS was performed with an Agilent 6890 gas chromatograph (Santa Clara, CA) coupled with a Hewlett-Packard 5973 mass spectrometer, equipped with a capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness) coated with silica in splitless mode. The injector temperature was 270 °C, and the carrier gas (He) flow was 1 mL/min. One microliter samples (1 mg/mL) in ethyl acetate were injected into the GC–MS. The oven temperature program was as follows: initial temperature of 150 °C for 3 min; increasing the temperature from 150 to 275 °C at 25 °C/min; and maintaining the temperature of 275 °C for 10 min. The MS conditions included an ionization energy of 0.7 kV. Analysis was conducted in full scan mode (m/z 40–400). Spectral interpretation was aided by the National Institute of Standards and Technology (NIST) 05 spectral library stored in the GC–MS controller unit.

1H NMR and 13C NMR

1H NMR spectra were recorded on Bruker spectrometers (400 MHz). Chemical shifts are reported in parts per million (ppm) relative to the residual solvent peaks (CD3OD δ 3.30 and 4.84 ppm). 1H NMR coupling constants (J) are reported in hertz (Hz), and multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m (multiplet), brs (broad singlet), and dd (doublet of doublet). 13C NMR spectra were recorded at 100 MHz, and all chemical shift values are reported in ppm, using the signal at δ 49.15 ppm as the internal reference.

Absolute Configuration of Compounds

A chiral Lipodex-E fused silica capillary column (octakis-(3-O-butyryl-2,6-di-O-n-pentyl)-γ-cyclodextrin) (28 m length, 0.25 mm I.D., film thickness of 12 μm) was installed on an Agilent GC-FID Model 6850. The run started at 120 °C for 5 min and then it increased 1 °C/min to 180 °C and stood for 20 min. The four enantiomeric standards, as well as the samples of each culture and co-culture, were injected at 1 mg/mL. The enantiomers were also injected separately to determine the retention time for comparison with those of the natural ones to determine their absolute configurations. RRT was obtained by co-injecting tricosane (C23H48, RT 68.649) with the standards and calculated using eq .

Optical Rotation

Optical rotations were measured at 20 °C on a PerkinElmer 341 polarimeter at 589 nm using PerkinElmer 10-mm cuvettes. The concentrations (c) are expressed in g/100 mL.