Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Discovery of Populusone, a Skeletal Stimulator of Umbilical Cord Mesenchymal Stem Cells from Populus euphratica Exudates Kai-Xin Liu,†,∥ Yan-Xia Zhu,‡,∥ Yong-Ming Yan,† Yue Zeng,‡ Ya-Bin Jiao,† Fu-Ying Qin,† Jia-Wang Liu,† Yuan-Yuan Zhang,‡ and Yong-Xian Cheng*,† †
School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, P.R. China Shenzhen Key Laboratory for Anti-Ageing and Regenerative Medicine, Department of Medical Cell Biology & Genetics, Shenzhen University Health Science Center, Shenzhen 518060, P.R. China
‡
Org. Lett. Downloaded from pubs.acs.org by WEBSTER UNIV on 02/27/19. For personal use only.
S Supporting Information *
ABSTRACT: Populusone (1), a cembrane-type macrocyclic trinorditerpenoid, was isolated from the exudates of Populus euphratica and shown to have an unprecedented carbon skeleton, The structure was identified using spectroscopic methods and X-ray crystallography. A possible pathway for the biosynthesis of 1 was proposed. Populusone (10 μM) was found to promote proliferation and differentiation of umbilical cord derived mesenchymal stem cells into keratinocyte like cells.
C
utaneous injuries such as burns and chronic wounds require subjecting patients to long-term treatment protocols. Although various strategies, including those relying on growth factor and gene delivery, have been developed to promote wound healing, cell therapies exemplified by stem cell-based approaches have been shown to be viable for enhancing healing, particularly in the case of nonhealing wounds.1 Because of their self-renewal, multipotency, and low immunogenicity, along with their availability, mesenchymal stem cells (MSCs) have high potential for use in cell therapy. Because MSCs are isolated from umbilical cord stem cells (UC-MSCs) by using a noninvasive procedure, their use does not suffer from ethical problems. In addition, they have a higher expansion capacity compared to that of BM-MSCs,2 which makes them potentially superior to MSCs from other sources for use in drug screening.3,4 Therefore, an approach involving UC-MSC stimulation could be beneficial for treatment of cutaneous injuries. Populus euphratica (Salicaceae), a tree commonly called desert poplar, grows in saline and arid environments. Under biotic (bites) and abiotic stress (saline, drought), the tree produces resins (tears of poplar) as a protective response. The resin from P. euphratica has been a traditional medicine in China since ancient times.5 It is used for the treatment of throat, tuberculosedadenitis, and duodenal ulcer swelling and pain. Despite its medicinal importance, the resin and the leaves of P. euphratica have not been subjected to detailed chemical investigation.5 In an effort to uncover substances that have wound-healing properties,6 we recently initiated a study of P. euphratica resin. As described below, this investigation resulted in the isolation and characterization of populusone (1), which has an unprecedented, snail-shaped trinorditerpenoid skeleton (Figure 1). In addition, the effort has revealed the UC-MSC promotion property of 1. Populusone (1)7 was isolated as colorless crystals (MeOH) and shown to have the molecular formula C17H26O2 (5 degrees © XXXX American Chemical Society
Figure 1. Structure of 1.
of unsaturation) by using HRESIMS (m/z 263.2008 [M + H]+, calcd for 263.2006), 13C NMR, and DEPT methods. The 1 H NMR spectrum of 1 (Table 1) contains resonances for three methyls [δH 1.26 (s, H-17); δH 1.15 (s, H-15); δH 1.00 (d, J = 6.5 Hz, H-16)] and one olefinic proton [δH 5.82 (s, HTable 1. 1H (600 MHz) and 13C NMR (150 MHz) Data of 1 in CDCl3 (δ in ppm, J in Hz) no.
δH
δC
no.
1
Ha: 2.84 ddd (14.6, 12.7, 2.2) Hb: 2.25 br dd (14.6, 6.5) Ha: 3.06 ddd (18.2, 12.7, 2.2) Hb: 2.45 ddd (18.2, 6.5, 2.2)
26.9
9
2.42 m
37.4
10
Ha: 1.92 m Hb: 1.18 m
19.9
11
Ha: 1.69 m Hb: 1.22 m
33.1
2
3 4 5 6 7 8
2.65 m Ha: 1.81 m Hb: 1.13 m Ha: 1.54 m Hb: 1.12 m 1.08 m
41.6
216.0 47.0 35.4 21.6 42.0 75.9
12 13 14 15 16 17
δH
5.82 s 1.15 s 1.00 d (6.5) 1.26 s
δC
71.3 129.8 144.8 24.0 17.5 24.6
Received: January 31, 2019
A
DOI: 10.1021/acs.orglett.9b00423 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters 5)]. The 13C NMR and DEPT spectra of 1 show that its structure contains three methyls, seven methylenes, two methines (one olefinic and one aliphatic), four non-protonated carbons (one ketone, one sp2-carbon, and two oxygenated sp3carbons. These data indicate that 1 might be a norditerpenoidal derivative. The structure of 1 was assigned mainly by using 2D NMR data (Figure 2). The 1H−1H COSY spectrum of 1
assign the relative configurations of chiral centers in the macrocyclic system present in 1 owing to its flexibility. To confirm the structure of 1 and determine both relative and absolute configurations at its stereogenic centers, singlecrystal X-ray diffraction analysis was carried out using CuKα radiation. The results (Figure 3) enable assignment of the
Figure 3. Plot of X-ray crystallographic data for populsone.
absolute configuration at C-4 and confirm the spectroscopically derived configuration assignments at the other stereogenic centers. Collectively, the absolute stereochemistry of populusone was determined to be (4S,8R,9S,12R). It is evident that 1 is a trinorditerpenoid that represents the first example of a cembrane-type diterpenoid containing a fused 10/6 carbon skeleton. Populusone belongs to the cembrane diterpenoid family, natural products that are commonly present in marine organisms such as corals.8 However, thus far, only a few cembrane diterpenoids have been isolated from terrestrial plants despite the fact that they were first discovered in tobacco and Pinus plants.9−13 The unprecedented skeleton of populusone led us to envisage possible routes by which it could be biosynthesized. Geranylgeranyl pyrophosphate (GGPP) is the typical precursor of macrocyclic diterpenoids. Thus, a possible pathway for generation of 1 could begin with GGPP and could involve the intermediate of the oxidized cembrane i (Scheme 1), which could be the precursor of intermediate ii
Figure 2. Key 1H−1H COSY, HMBC, and ROESY correlations of populsone.
shows correlations of H2-1/H2-2, H3-16/H-4/H2-5/H2-6/H27, and H-9/H2-10/H2-11, suggesting the existence of three spin systems. The HMBC spectrum of this substance shows correlations of H3-16, H-4, H-5, H-1, H-2/C-3 (δC 216.0), indicating that C-2 is connected to C-4 via a ketone carbon. Similarly, HMBC correlations of H3-15, H-7/C-8 (δC 75.9), C9, H3-15/C-7 indicate that C-7 is attached to C-9 through an oxygenated sp3-carbon (C-8), and a methyl is connected to C8. Further HMBC correlations of H-1, H-2, H-9, H-10/C-14 (δC 144.8) enable assignment of a linkage between C-9 and C14, indicating that 1 contains a 10-membered ring with one exocyclic double bond between C-13 and C-14. Inspection of the remaining HMBC cross peaks associated with H-13, H-11, and H3-17/C-12 show that C-11 is linked with C-13 via C-12 (δC 71.3) and a methyl group is present at C-12. These findings establish that a six-membered ring fused to a 10membered ring via C-9−C-14 exists in the structure of this substance. Moreover, the chemical shifts of C-8 and C-12 indicate that they are bonded to oxygen, which along with consideration of the one remaining degree of unsaturation show that these carbons are connected via an oxygen. As can be seen by viewing the correlations displayed in Figure 2, the relative configurations at two bridged-head carbons (C-9 and C-12) are easily assigned owing to the rigidity of the two six-membered rings. In addition, ROESY correlations of H3-15/Ha-10, and Ha-11 clearly indicate the orientation of the C-8 CH3. Correlations of H-4, H-9/Ha-2 and H3-16/Hb-2 observed in the ROESY spectrum show that these protons are oriented vicinally, which establishes the relative configuration of C-4. However, it is challenging to
Scheme 1. Possible Pathway for Biosynthesis of Populusone
B
DOI: 10.1021/acs.orglett.9b00423 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
cell differentiation.16 We observed that the keratinocytes relative protein cytokeratin-19, P63, and involucrin are expressed in UC-MSCs after coculturing with HaCat (Figures 6A−C). Furthermore, 1 was found to promote the expression
formed by oxidation-promoted loss of the isopropyl moiety. Base-promoted enolate iii formation and transannular substitution at the epoxide carbon would then lead to formation of the fused 6/10 bicyclic intermediate iv. Subsequent reduction followed by dehydration would form intermediate vi, which upon acid promoted ether formation would produce populusone. Because UC-MSCs have high proliferation and differentiation potentials, they are ideal for tissue regeneration.14 The activity of populsone against UC-MSC proliferation and differentiation was evaluated using nontoxic concentrations and methods described in a previous study.6 We found that cocultivation with this substance for 1 week promotes proliferation of UC-MSCs (Figure 4). To assess the effect of
Figure 4. Growth curves of UC-MSC in the presence and absence of populsone.
1 on differentiation of UC-MSCs, multiple specific markers for keratinocytes (keratins, involucrin, P63, and integrin-β1) were selected and evaluated using real time PCR, immunocytofluorescence, as well as Western blotting. After coculturing with HaCat and 1 for 2 weeks, the morphology of UC-MSCs changed from fibroblast-like to epithelioid cells (Figure 5). It is known that the most representative cytokeratins of basal epidermal cells, CK5, CK14, integrin-β1, P63, and cytokeratin 19, are markers for epithelial stem cells and that cytokeratin-10 is considered to be an early marker of epidermal cell differentiation.15 During differentiation, keratinocytes express more CK18, and involucrin marks the terminal of epidermal
Figure 6. Specific gene and protein expression of keratinocytes after coculturing with populsone. *p < 0.05 versus coculturing group: (A) examination of cytokeratin 19 expression by immunocytofluorescence; (B) examination of P63 expression using immunocytofluorescence; (C) expression of keratinocyte specific proteins detected using Western blot; (D) keratinocyte specific protein change analyzed by semiquantitative method; (E) keratinocyte specific gene expression. UC-MSC: UC-MSCs only (control group). Coculture: UC-MSCs cocultured with HaCat without populsone. Co + 1: 10 μM populsone was added when cocultured. HaCat: HaCat cells served as a positive control.
of cytokeratin-19, P63, and involucrin in keratinocyte-like cells (KLCs) and indicated that the expression levels of P63 and involucrin are 10 times higher than that of the coculturing group only (Figure 6D). The values are superior to those reported in the literature, which range from 10% to 60% transdifferentiation.17,6 All of these genes were significantly upregulated in cells cocultured with 1 compared to that of the coculture group only. Overall, the results demonstrate that populusone not only promotes phenotypic changes but also induces genotypic changes in UC-MSCs. This finding shows that this substance has the capacity to promote transdifferentiation of UC-MSCs into KLCs and possibly the formation of a stratified epidermis-like structure.
Figure 5. Morphological changes of UC-MSC after coculturing with HaCat and populsone. UC-MSC: UC-MSCs only. Coculture: UCMSCs cocultured with HaCat without populsone. Co + 1: 10 μM populsone was added when cocultured. HaCat: HaCat cells served as a positive control. C
DOI: 10.1021/acs.orglett.9b00423 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
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(11) Luo, P.; Yin, Z. Y.; Sun, Z. J.; Chen, H.; Li, C. J.; Zhou, H. H.; Gu, Q.; Xu, J. Fitoterapia 2018, 129, 162−166. (12) Hanson, J. R. Nat. Prod. Rep. 1987, 4, 399−413. (13) Cretton, S.; Saraux, N.; Monteillier, A.; Righi, D.; Marcourt, L.; Genta-Jouve, G.; Wolfender, J. L.; Cuendet, M.; Christen, P. Phytochemistry 2018, 154, 39−46. (14) Gopinath, M.; Diliddo, R.; Marotta, F.; Murugesan, R.; Banerjee, A.; Sriramulu, S.; Jothimani, G.; Subramaniam, V. D.; Narasimhan, S.; Priya, K. S.; Sun, X. F.; Pathak, S. Int. J. Hematol. Oncol. Stem Cell Res. 2018, 12, 153−165. (15) Jensen, K. B.; Jensen, O. N.; Ravn, P.; Clark, B. F.; Kristensen, P. Mol. Cell. Proteomics 2003, 2, 61−69. (16) Watt, F. M. J. Invest. Dermatol. 1983, 81, 100−103. (17) Stocum, D. L. Science 2002, 298, 1901−1903.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00423. NMR spectra and HRESIMS, separation procedures, Xray data, and biological evaluation (PDF) Accession Codes
CCDC 1894400 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yan-Xia Zhu: 0000-0003-3247-3593 Yong-Xian Cheng: 0000-0002-1343-0806 Author Contributions ∥
K.X.L. and Y.-X.Z. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge financial support from the National Science Fund for Distinguished Young Scholars (81525026), the National Key Research and Development Program of China (2017YFA0503900), the Medical Scientific Research Foundation from Guangdong Province (A2018555), and Shenzhen Science and Technology Innovation Commission grants (JCYJ20170818100526471). We are also indebted to the Instrumental Analysis Center of Shenzhen University (Xili Campus) for NMR data measurement.
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REFERENCES
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DOI: 10.1021/acs.orglett.9b00423 Org. Lett. XXXX, XXX, XXX−XXX
