Letter pubs.acs.org/OrgLett
Bile Salt-like Dienones Having a Novel Skeleton or a Rare Substitution Pattern Function as Chemical Cues in Adult Sea Lamprey Ke Li,† Anne M. Scott,† Cory O. Brant,† Skye D. Fissette,† Joseph J. Riedy,† Thomas R. Hoye,‡ and Weiming Li*,† †
Department of Fisheries and Wildlife, Michigan State University, Room 13 Natural Resources Building, 480 Wilson Road, East Lansing, Michigan 48824, United States ‡ Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States S Supporting Information *
ABSTRACT: Two novel sulfated bile salt-like dienones, featuring either a unique, rearranged side chain or a rare cis-11,12-diol on the steroidal C-ring, herein named petromyzene A (1) and B (2), respectively, were isolated from water conditioned with spawning male sea lamprey (Petromyzon marinus; a jawless vertebrate animal). The structures of these natural products were elucidated by mass spectrometry and NMR spectroscopy. Petromyzenes A and B exhibited high olfactory potency for adult sea lamprey and strong behavioral attraction for spawning females.
B
ile salts are biosynthesized from cholesterol by a sequence of enzymatic modifications in vertebrate animals.1 The intermediates and the end products involved in the biosynthetic pathways show common structural featuresa steroidal tetracyclic core (rings A−D) with a 5-carbon (α) or 8-carbon (α,ε-dilhexyl) upper side chain to form a skeleton containing 24 or 27 carbon atoms in total. Structural variants of bile salts, differing in the oxidation patterns throughout the carbon skeleton and typically containing one or more ionizable functional group, have different functional roles in a variety of organisms.2 Bile salt derivatives are known to be pheromones in the sea lamprey (Petromyzon marinus),3 whose invasion represents a major ecological disaster in the Laurentian Great Lakes. This pheromone system offers the potential for a selective and environmentally benign approach for control of the sea lamprey. We have previously reported five sulfated bile alcohol derivativesnamely, 3-ketopetromyzonol (3kPZS, 3),3 3,12diketopetromyzene (DkPES, 4),4 and petromyzones A-C5in an extensive investigation of pheromones emitted by spawning male sea lamprey. All of these reported compounds conform to the basic skeleton of bile salts. Here we report (i) a bile salt-like dienone with a novel carbon skeleton as well as (ii) a bile salt derivative with a rare hydroxylation pattern (Figure 1). Petromyzene A (1) contains an unprecedented arrangement of the carbon atoms in its side chain. Petromyzene B (2) has a rare6 cis-dihydroxylation feature at C11/C12 in its steroidal Cring. We describe the isolation and structure elucidation of these new compounds as well as the olfactory and behavioral responses that they elicit in adult sea lamprey. © 2017 American Chemical Society
Figure 1. Chemical structures of petromyzene A (1) and B (2), 3kPZS (3), and DkPES (4).
Petromyzene B (2) was obtained as a yellowish oil. The molecular formula was judged to be C27H42O7S from consideration of the HRESIMS (negative ion at m/z 509.2592 [M − H]−) in conjunction with the 13C NMR (namely, the carbon count) data. The constitution of 2 was determined by 1D- and 2D-NMR analysis (Figure 2a and Table 1). The 1H, 13C, and HSQC NMR spectra (Supporting Information) of 2 in methanol-d4 showed signals for two, 3proton singlets representing the methyl groups C-18 and C-19 and three doublets for the methyl groups C-21, C-26, and C-27. Received: June 23, 2017 Published: August 17, 2017 4444
DOI: 10.1021/acs.orglett.7b01921 Org. Lett. 2017, 19, 4444−4447
Letter
Organic Letters Table 1. NMR Data of 1 and 2c petromyzene A (1) no. 1α
1.71 ddd (13.8, 13.6, 5.0)
1β 2α 2β
1.96 m 2.64 ddd (5.5, 14.4, 18.0) 2.39 dddd (18.1, 5.0, 2.1, 1.0)
3 4 5 6 7 8β 9α 10 11α 11β 12α 12β 13 14α 15α 15β
Figure 2. a) 1H−1H COSY (bold bonds) and HMBC (base-to-head of arrow: proton-to-carbon) correlations and b) NOESY correlations in petromyzene B (2).
These methyl group signals and the predicted molecular formula indicated a classic 27-carbon bile salt skeleton. Among the HMBC correlations for 2 was a long-range communication between the methyl protons for Me-19 (δH 1.27) to a downfield qC (δC 167.9). The COSY spectrum showed correlation between a pair of CH doublets at δH 6.16 and 6.21 (J = 9.8 Hz), and the latter also showed an HMBC correlation to the qC at δC 167.9 (Figure 2a). These relationships were highly reminiscent of the conjugated Δ4(5),Δ6(7)-dienone present in DkPES (4).4 The chemical shifts of all 5 of carbons C-3−C-7 matched well with those of 4.4,7 The protons of the C-18 methyl group correlated with a carbon resonance at δC 79.0 (Figure 2), which established the location of a hydroxyl group at C-12. The crucial correlation between H11 to H-12 in the 1H−1H COSY spectrum identified the additional hydroxylation to reside at C-11. The H-11 carbinol proton (δH 4.01) was a doublet of doublets with couplings to two vicinal protons of 10.9 and 3.1 Hz, respectively. This identified that proton as having an axial orientation that, in turn, indicated that the C-11 and C-12 hydroxyl groups had a cis relative configuration, each with an α orientation. C-11 hydroxylation has rarely been observed in natural bile acids and salts;8 C-3, C-7, and/or C-12 are the common positions for hydroxylation on the steroidal rings, as is seen, for example, in major bile acids such as taurine, glycine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid.9 To our knowledge, cis-dihydroxylation at C-11 and C-12 of a steroidal natural product has been reported only twice.6 A CH resonance at δH 4.13 was attached to a carbon having a chemical shift of δC 85.9 (HSQC). This is a characteristic set of chemical shifts for a sulfated secondary alcohol,10 which accounted then for all heteroatoms present in 2. Long-range correlation from two methyl doublet signals having similar chemical shifts (δH 0.94 and 0.95) to the carbon at δC 85.9 suggested that the sulfate was substituted on C-24. Compound 2 has a side chain identical with that in petromyzonamine disulfate (PADS), previously isolated from the conditioned larval water of the same species.10 The relative configuration of 2 was supported by NOESY (Figure 2b) and coupling constant analysis.11 The NOESY
δH, mult (J in Hz)
5.71 s
16α 16β 17α
6.25 d (1.9) 6.25 d (1.9) 2.74 br dd (11.4, 11.4) 1.58 m absent 2.13 dd (13.3, 4.5) 2.71 dd (13.5, 13.5) absent absent absent 1.58 m 2.00 ma 1.63 dddd (11.5, 11.5, 11.5, 5.1)a 1.57 ma 1.94 ma 2.30 ddd (9.5, 9.5, 9.5)
18 19 20 21 22a 22b 23a 23b 24a
1.22 s 1.24 s absent 2.28 s 2.70 ddd (11.5, 9.4, 3.1) absent 1.98 m 1.98 m 3.95 ddd (10.3, 6.7, 5.1)
24b 25 26 27
3.81 ddd (10.3, 8.4, 5.9) absent absent absent
petromyzene B (2) δC 34.62
δH, mult (J in Hz) b
34.56b
202.0 124.6 165.6 129.5 141.1 38.3 53.4 37.8 38.3 214.5 58.9 55.1 23.8
26.4 45.6 12.5 16.1 215.5 29.3 52.6 31.3 67.3
absent absent absent
2.73 ddd (14.2, 5.3, 2.2) 1.97 m 2.33 ma 2.64 ddd (5.3, 14.6, 18.1)a 5.65 s 6.16 dd (2.7, 9.8) 6.21 dd (1.9, 9.7) 2.30 m 1.64 t (10.7, 10.7) absent absent 4.01 dd (3.1, 10.9) absent 3.84 d (3.1) absent 1.80 m 1.29 ma 1.81 ma 1.43 ma 1.99 ma 1.93 ddd (9.6, 9.5, 9.5) 0.84 s 1.27 s 1.46 m 1.03 d (6.6) 1.27 ma 1.54 ma 1.54 ma 1.72 ma 4.13 ddd (4.8, 4.8, 6.9) absent 2.07 m 0.95 d (6.9) 0.94 d (6.9)
δC 37.2
35.1
203.1 124.5 167.9 129.0 143.1 37.7 50.4 38.8 70.9 79.0 48.4 45.9 24.5
28.8 48.7 12.5 17.3 37.1 18.1 32.2 27.9 85.9
32.1 18.3 18.6
The assignments for this pair of α and β (or a and b) methylene protons may be interchanged. bThe assignments of these two carbon resonances may be interchanged. cIn methanol-d4, δ value in ppm, J values in Hz. a
correlations observed from H-18 to H-8β, H-11β, and H-21 as well as from H-14α to H-9α indicated the relative configuration for each ring junction to be trans. The coupling constant for the proton on position 12 is 3.1 Hz, in good agreement with a known cis-11,12 dihydroxylated analog.6b The correlations from H-11β to H-8β, H-19, and H-18 indicated that all of these protons were axially oriented, confirming that the hydroxyl at C-11 had an equatorial orientation. Known structures of natural products having the same side-chain constitution as that of 2 all have the 24-R configuration.10−12 The chemical shifts of H-24 and C-24 are nearly identical to those present in these compounds. However, it is also known that the epimer having the 24-S configuration has a nearly identical set of shifts as well.10 Nevertheless, the chemical shifts of the C-26 and C-27 4445
DOI: 10.1021/acs.orglett.7b01921 Org. Lett. 2017, 19, 4444−4447
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Organic Letters
model compound.7 An HMBC interaction between the C-18 methyl protons to a saturated carbonyl carbon (δC 214.5) supported the location of one of the two remaining keto-groups at C-12. The 1H chemical shift of the third methyl singlet along with its strong HMBC correlation with the third carbonyl carbon resonance (δC 215.5) indicated the presence of a methyl ketone. This led us to postulate two possible arrangements for the side chain constitution. These are shown in Figures 3c and 3d. Although a few steroidal sulfates are known that bear the OSO3− substituent at C-21,13 as is present in the substructure in Figure 3c, the absence of a COSY interaction between the methylene protons bearing the sulfated oxygen and an adjacent methine proton provided the first clear evidence that petromyzene A (1) did not possess such an arrangement. Instead, a very clear COSY correlation with each of a pair of diastereotopic methylene protons led us to propose the highly unusual arrangement shown in Figure 3d. This connectivity was then confirmed by the indicated, full, and self-consistent set of 2- and 3-bond HMBC correlations indicated in that substructure. Additional strong evidence for the RCH2CH2OSO3− substructure was seen in the resonances for each of the diastereotopic CH2OSO3− protons (H-24a and H24b, Table 1). Each was a ddd with one geminal (10.3 Hz) and two vicinal (ranging from 5.1−8.4 Hz) coupling constants. In the NOESY spectrum of 1 (Figure 3b14), correlations were observed between both Me-19 and Me-18 with H-8β, indicating that all three were on the β-face. Similarly, correlations between both of H-9α and H-17α with H-14α established that all were on the α-face. Thus, the structure of 1 was assigned as 17(20 → 22)abeo-3,12,20-triketo-4,6-cholandiene-24-sulfate and trivially named petromyzene A. Petromyzenes A (1) and B (2) were both potent odorants that stimulated the adult sea lamprey olfactory epithelium in a concentration dependent fashion, as measured in electroolfactogram (EOG) assays (Figure 4). The threshold of
methyl proton and carbon resonances are uniquely distinctive for the two epimeric compounds,10 and the shifts exhibited by 2 clearly support the assignment of the 24-R configuration. Thus, the structure of 2 was assigned as 3-keto-11S,12R-dihydroxy4,6-cholestadiene-24R-sulfate. Petromyzene A (1) was obtained as a yellowish oil. We emphasize at the outset that we have deduced that this compound contains an unprecedented carbon-connectivity within its side chain. The evidence for this assignment is detailed below. The molecular formula of 1 was established as C24H32O7S from its HRESIMS (m/z 463.1813, [M − H]−) in conjunction with the carbon count indicated by the 13C NMR data. This indicated nine degrees of unsaturation. The constitution of 1 was deduced to be a bile alcohol with a sulfate half ester (−OSO3Na) through analysis of 1D- (Table 1) and 2D-NMR (Figures 3a and 3b) data. The connectivity of
Figure 3. a) 1H−1H COSY (bold bonds) and HMBC (base-to-head of arrow: proton-to-carbon) correlations in the A−D tetracyclic substructure of 1, b) NOESY correlations in that tetracycle, and c,d) two possible connectivities for the side chain (R in 1-tetracycle).
Figure 4. Semilogarithmic plot of electro-olfactogram (EOG) concentration response curves shows that 1 and 2 (petromyzenes A and B, respectively) are stimulatory to the adult sea lamprey olfactory epithelium and have low detection thresholds. The numbers on the right of the figure correspond to each compound. Data are presented as the mean normalized EOG amplitude (n = 7). Vertical bars represent one standard error of the mean. Insert: Expanded view of responses showing response threshold concentrations.
most of the tetracyclic steroidal backbone was established through the interpretation of the 1H−1H COSY and HMBC correlations (spectra provided in the Supporting Information). The 13C NMR spectrum showed characteristic resonances for a 4,6-dien-3-one (carbonyl carbon at C-3 and four olefinic carbons for CH-4, qC-5, CH-6, and CH-7).7 This suggested that 1 shared a tetracyclic structure common to that deduced earlier to be present in 2 and DkPES (4).4 Notably, the 1H NMR spectrum of 1 contained three singlets representing methyl groups. This is unusual for a steroidal compound. One of these was shown to be the C-19 methyl resonance by HMBC correlation of H-19 to C-5. Another was assigned to be the C18 methyl resonance by comparison with a related steroid
detection, the lowest concentration that elicited an olfactory response greater than the blank water control, for 1 was less than 10−13 M and for 2 was 10−11 M (paired t test, p < 0.05). Petromyzenes A (1) and B (2) also attracted all spawningphase females tested in two-choice behavioral assays (Supporting Information). Of the 7 females assayed for each odorant, all 7 spent more time in the channel with compound 1 or 4446
DOI: 10.1021/acs.orglett.7b01921 Org. Lett. 2017, 19, 4444−4447
Letter
Organic Letters Notes
compound 2 compared to the adjacent channel treated with the vehicle methanol. Females were attracted to 1 at 10−12 M (mean index of preference ± S.E., 0.442 ± 0.108, n = 7) and to 2 at 10−12 M (mean index of preference ± S.E.; 0.422 ± 0.089, n = 7; Wilcoxon signed-rank test, p < 0.05; Figure 5). These levels of olfactory potencies and behavioral activities suggest strongly that 1 and 2 are male pheromones.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The staff of U.S. Geological Survey Hammond Bay Biological Station who provided research facilities is acknowledged. The U.S. Fish and Wildlife Service and Fisheries and Oceans Canada collected the sea lamprey. This work was supported by grants from the Great Lakes Fishery Commission to W.L., K.L., and C.B. T.H. was supported by National Institute of General Medical Sciences of the NIH (R01 GM108762). We thank Dr. Daniel Holmes for a helpful discussion.
■ Figure 5. Spawning phase female sea lamprey were attracted to petromyzenes A and B (compounds 1 and 2, respectively) in a twochoice maze. The time the lamprey spent in each channel of the maze before and after odorant exposure was used to calculate an index of preference (see eq 2 in the Supporting Information) to assess its behavioral response to the odorant. A positive value of the index of preference indicates attraction. Data are presented as the mean (n = 7). Error bars represent the standard error of the mean. An asterisk indicates a significant index of preference (α = 0.05) as evaluated using a Wilcoxon signed-rank test.
In conclusion, two novel bile salts have been identified from the washing of sexually mature male sea lamprey. Among natural steroids, compound 1 possesses unprecedented connectivity within its side chain as shown below.15 Compound 2 contains a rarely occurring oxygenation pattern. Compounds 1 and 2 represent new additions to the structure diversity of the steroid family. Notably, the isolation of 1 and 2 from spawningphase males and the behavior modification by 1 and 2 of spawning-phase females indicate that these two novel molecules play an essential role in sea lamprey reproduction.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01921. 1 H, 13C NMR, DFQ-COSY, HSQC, HMBC, MS, and HR-MS spectra of petromyzenes A (1) and B (2) (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*Phone: +1-517-432-6705. E-mail:
[email protected]. ORCID
Ke Li: 0000-0003-4869-5393 Thomas R. Hoye: 0000-0001-9318-1477 4447
DOI: 10.1021/acs.orglett.7b01921 Org. Lett. 2017, 19, 4444−4447
