Are Isoursenol and γ-Amyrin Rare Triterpenes in Nature or Simply Overlooked by Usual Analytical Methods?

Article abstract of DOI:10.1021/acs.orglett.5b01851

Among pentacyclic triterpenes commonly found in plants, γ-amyrin and isoursenol are seldom reported and considered rare in nature. It was hypothesized that these triterpenes are instead routinely overlooked due to inadequate spectral characterization. γ-Amyrin was prepared by HCOOH isomerization of α-amyrin, and isoursenol was isolated from products of a heterologously expressed oxidosqualene cyclase. With precise NMR and GC-MS data, a metabolomics strategy was used to identify isoursenol and γ-amyrin in a wide range of plants.

Full text of DOI:10.1021/acs.orglett.5b01851

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Letter
pubs.acs.org/OrgLett
Are Isoursenol and γ‑Amyrin Rare Triterpenes in Nature or Simply
Overlooked by Usual Analytical Methods?
Hui Shan,† William K. Wilson,† Dorianne A. Castillo,‡ and Seiichi P. T. Matsuda*,†,‡
‡
†Biosciences Department and Department of Chemistry, Rice University, Houston, Texas 77005, United States
S
* Supporting Information
ABSTRACT: Among pentacyclic triterpenes commonly found in
plants, γ-amyrin and isoursenol are seldom reported and considered
rare in nature. It was hypothesized that these triterpenes are instead
routinely overlooked due to inadequate spectral characterization. γ-
Amyrin was prepared by HCOOH isomerization of α-amyrin, and
isoursenol was isolated from products of a heterologously expressed
oxidosqualene cyclase. With precise NMR and GC-MS data, a
metabolomics strategy was used to identify isoursenol and γ-amyrin
in a wide range of plants.
riterpenes and their triterpenoid derivatives are widely
distributed in plants as specialized metabolites of medicinal,
proved to be more accurate than all experimental NMR data ever
reported for 13.13 We attempted to obtain enough material for
2D NMR characterization by repeatedly purifying the silica-gel
fraction by reversed-phase HPLC. However, the near coelution
of δ-amyrin (2) and 13 by HPLC gave at best an ∼20:1 ratio of
2:13 (Figure 2B). Considering Bauer’s similarly futile efforts,11
we discontinued our efforts to isolate 13 from tomato wax.
Seeking an alternative route to 13, we envisioned synthesis
from α-amyrin (14) via the Δ11,13(18) diene5 or acid
isomerization.14 Given the high cost of 14, we explored its
isomerization to 13 on a low milligram scale. Treatment of 14
with triflic acid14a (50 mM in CDCl3 at 23 °C) gave only 3% 13 in
a 63:11:7:7:6:3:3 mixture of 14, 2, 18-epi-β-amyrin14a (20),
canarenes15 (21), unidentified triterpenes, 1, and 13 (Figure S4).
As in other work,14a our TfOH isomerizations equilibrated most
of the triterpenes in Figure 3A. CF3COOH gave at best a
50:30:10:4 mixture of 14, canarenes, triterpene epimers, and 13.
No reaction occurred in AcOH at 90−95 °C for 20 h (Figure S5).
We then tried isomerization in formic acid. Relative to acetic
acid, HCOOH is more acidic by 1 pKa unit, has a 10-fold higher
dielectric constant, and can add reversibly to olefinic bonds. In a
series of HCOOH isomerizations of 14 ranging from 170 h at 40
°C to 4 h at 93 °C (Figure S6), the product consistently
contained ∼4% 13 and ∼2% total 1−3 (from the 2% impurity of
3 in 14). The amount of recovered 14 dropped with rising
temperature from 93% to 40% due to formation of canarenes15
(21) via dehydration to the C3 cation, A-ring contraction, and
backbone rearrangement. The results indicate that HCOOH
isomerization of 14 is limited to the ursane energy trough
(magenta bars in Figure 3A), with 13 as the only isomer formed.
Based on these exploratory findings, 14 (20 mg) was
isomerized in supersaturated HCOOH solution at 65 °C to a
94:6 mother-liquor mixture of 14 and 13 as formate esters 14f
T
agricultural, and ecological importance.1 More than 100
triterpene skeletons are constructed by oxidosqualene cyclases
(OSCs).2 Much of this structural diversity arises in plants from
OSCs that make a mixture of oleanane (1−7) and ursane (8−18)
derivatives. These pentacyclic triterpenes are generally well
documented (Graphical Abstract3), with detailed spectral
characterization (Figure 1).
Two notable exceptions are γ-amyrin4 (urs-13(18)-en-3β-ol,
13) and isoursenol (15). Prior to 1970, 135 and 156 were isolated
and characterized by melting point and optical rotation but not
by NMR or MS. Only limited spectral data for identifying 13 and
15 were reported in later references, which include a recent
erroneous structure elucidation of 13 (see Figure S1).7 Largely
due to this lack of data, we8 and others9,10 have overlooked 15 in
biological samples.
We had often anticipated the presence of 13 and 15 in
metabolomics studies and as OSC products but had no NMR or
GC-MS guidance to identify them in triterpene mixtures. Now
serendipity has led us to 13 and 15. Here we describe their
preparation, spectral characterization, and broad distribution
among higher plants.
Recently we encountered a GC-MS study of 13 in Stefan
Bauer’s 2002 Ph.D. dissertation.9,11 Bauer tentatively proposed
that tomato wax contains 13 on the basis of MS fragmentation
and GC retention time patterns. This extensive discussion of 13
was not indexed by Chemical Abstracts, nor was 13 mentioned in
Bauer’s subsequent papers on tomato wax lipids.12
We repeated Bauer’s9 extraction and purification of tomato
wax triterpenes and found a chromatographic fraction that
showed a small GC-MS peak corresponding to Bauer’s data for
13 (Figure 2A). Unable to locate any useful NMR data for 13, we
1
estimated H and 13C NMR chemical shifts from quantum
1
mechanical calculations. Nine H−13C signal pairs from an
HSQC spectrum of crude hexane extracts of tomato wax
matched well with the predicted values (Table S12), which
Received: June 26, 2015
© XXXX American Chemical Society
A
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Letter
Figure 1. Mechanistic pathways to oleanane and ursane triterpenes. The extent of NMR and GC-MS characterization in the literature is summarized for
each triterpene skeleton, with emphasis on NMR chemical shift precision suitable for mixture analysis (see Tables S2−S4 for details). Ursanes 10−12
and 18 are not well-established substances. Glutinol and other intermediates between 6 and 7 and 17 and 18 are omitted due to space limitations.
Figure 3. (A) Predicted free energies (ΔG, 25 °C) relative to 20 from
B3PW91/6-311G(2d,p)//B3LYP/6-31G* calculations. Atom number-
ing is given in Figure 1. Product ratios are governed thermodynamically
by energies of neutral species. Cation migration occurs readily via 1,2-
shifts in TfOH but not in HCOOH. Thus, TfOH gives a wider
isomerization range than HCOOH, whose largely neutral mechanism
Figure 2. GC-MS total ion chromatograms (TMS ethers) from efforts
to isolate 13 from tomato wax by (A) flash chromatography on silica gel
and (B) C18 HPLC. (C) Isolation of 13 by HCOOH isomerization of
14, followed by C18 HPLC. (D, E) Subtle differences in MS ion
cannot bridge the ursane and oleanane energy troughs. (B) Structures of
lupeol (its C20 cation being the precursor of 1−18) and isomerization
intensities9 for m/z 189, 190, 205, and 218 distinguish pure 13 and 2 as
byproducts: 18-epi-β-amyrin and representative canarene isomers.
TMS ethers but are usually obscured by overlapping impurities.
removed 14 and most other impurities, and analytical HPLC
and 13f. Partial HCOOH evaporation enriched the mother
liquor to a 3:1 ratio of 14f:13f, and precipitated 14f was recycled
for further isomerization. After saponification, preparative HPLC
mostly removed 2 to give ∼1 mg of 13 in 93% purity (Figures 2C
and S7F). NMR data at the precision required for mixture
analysis16 are given for 13 and acetate 13a (Tables 1, S7, S8, S10,
B
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Letter
and S11). Consistency of the NMR data with calculated values
(Table S12) confirmed that the olefin isomerization in HCOOH
was not accompanied by epimerization.
Table 1. 1H and 13C NMR Data for Methyl Signals of 13 and
a
15
γ-amyrin (13)
δH, mult (J)
isoursenol (15)
δH, mult (J)
atom
δC
δC
23
24
25
26
27
28
29
30
28.07
15.48
16.33
17.83
21.86
28.32
23.15
20.48
0.989, s
28.00
15.47
15.20
26.29
19.39
37.05
27.48
22.46
0.975, s
0.771, s
0.799, s
0.868, s
0.915, s
0.922, s
1.058, s
1.121, s
0.931, s
1.102, s
0.934, s
1.086, d (7.6)
0.843, d (7.0)
1.021, d (6.9)
0.977, d (6.6)
a
Conditions: ∼2 mM in CDCl3, 25 °C, 800 MHz; 13C chemical shifts
1
( 0.01 ppm) were referenced to CDCl3 (77.00 ppm); H chemical
shifts ( 0.001 ppm) were referenced to SiMe4; coarse J couplings are
given in Hz. See Tables S7−S9 for full NMR data and fine couplings.
Like γ-amyrin, isoursenol (15) is an established substance. Its
structure was determined in 1961−65 by synthesis from 14,6a,b
acidic rearrangement back to 14,6a and a photooxidative reverse
rearrangement.6b In 1966, 15 was isolated from tree leaves of an
asterid and rigorously identified by isomerization to 14 and
careful comparison with reported6a values for mp and [α]D. More
recent references cite this early work but provide little useful
spectral data.17
Figure 4. Detection of 13 and 15 in HSQC spectra. (A) Upfield methyl
region of crude hexane extracts of tomato wax. (B and C) Expansions
showing peaks for 13 and 15. (D and E) Similar HSQC expansions show
13 and 15 in the triterpene fraction of PEN6 products.
While investigating the enzymatic products of an Arabidopsis
thaliana OSC (PEN6), we observed in a triterpene mixture a
downfield olefinic NMR signal with the distinctive coupling
pattern of taraxerol (dd, 8 and 3 Hz) but at a different chemical
shift. Sensing that this unknown was a taraxerol analogue, we
obtained quantum mechanical estimates of NMR chemical shifts
for the postulated ursane derivative 15. As with γ-amyrin, the
NMR predictions confirmed the structure by their matches to the
observed HSQC peaks.
After chromatographic purification, 15 and its acetate 15a
were characterized by GC-MS and NMR (Tables 1 and S9−
S12), which matched known 13C chemical shifts17a and EI-MS
fragmentation17c for 15a. We also reproduced the reported6a,c
acid-catalyzed isomerization of 15 to 14 by using dilute TfOH. As
in our γ-amyrin experience, a lack of useful spectral data had
caused 15 to be overlooked in tomato wax,9 PEN6 products,10
and our own plant analyses.8
With precise NMR and GC-MS data now available for 13 and
15, we looked for their presence in a variety of plants by using
established methods for analyzing complex mixtures.16a First we
revisited our existing spectra and found 13 also in PEN6 products
and 15 also in tomato wax. The strongest evidence was from
analysis of HSQC results (Figures 4 and S8 and Tables S6 and
S15).
Figure 5. Observation of 15 and 13 in diverse plant species. Relative
amounts of 15 and 13 are indicated by the symbol sizes; symbols in
boxes denote previous isolations of 15. In this simplified AGP III
phylogeny, branch lengths have no significance. Figure S45 shows a
more detailed phylogeny, with genus and species names.
Among ∼25 additional plants we studied,18 15 was found in 18
plants and 13 was in 4 of these plants (Figure 5). Our results raise
the number of plants proven to make 15 from 3 to 23 and for 13
from 0 to 6. Figure 5 illustrates the broad taxonomic distribution
of 15 and 13. Most plants containing 14 also make 15 as a minor
OSC byproduct. Our less frequent detection of 13 is attributable
to steric hindrance of cation deprotonation (Figure S47) and to a
lack of the spectral markers that allowed detection of 15 at trace
levels.
We identified 13, 15, and other triterpenes in complex
mixtures by comparing observed HSQC chemical shifts, GC
retention times, and MS fragmentation against spectral data-
bases. An Excel macro searched thousands of HSQC peaks
against the precise data in Table 1 to give an objective appraisal of
evidence for 13 and 15.
Midpathway OSC byproducts 4−6 and 15−17 normally
represent <1% of total triterpenes. However, kale contained
nearly 1 mg/kg of 15 (3:2:1 ratio of 14, 3, and 15), suggesting a
C
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J. Chem. Soc. 1955, 2610−2615. (c) Yagishita, K. Bull. Agric. Chem. Soc.
Jpn. 1959, 23, 217−222.
possible defense function in cruciferous vegetables. We detected
13 and 15 in many other common plant foods. Some traditional
diets might contain up to 1 mg/day of 13 and 15 among an
abundance of biologically active triterpenoids having medicinal
and health benefits.
Regarding the title question, we have shown that 13 and 15 are
widespread in nature but routinely overlooked by usual analytical
methods. How many more secondary metabolites are similarly
overlooked in plants and microorganisms? A metabolomics
approach can reveal a vast complexity of hydrophobic substances.
Plant metabolic profiling, still in its infancy, might benefit from
our GC-MS and HSQC strategy for mixture analysis,16a which
can definitively identify and quantify numerous nonpolar
metabolites at 1 μg levels.
(6) (a) Laird, W.; Spring, F. S.; Stevenson, R. J. Chem. Soc. 1961, 2638−
2642. (b) Agata, I.; Corey, E. J.; Hortmann, A. G.; Klein, J.; Proskow, S.;
Ursprung, J. J. J. Org. Chem. 1965, 30, 1698−1710. (c) Chivers, H.;
Corbett, R. E.; Mitchell, R. E. M. J. Chem. Soc. C 1966, 1814−1816.
(7) Hua, J.; Luo, S.-h.; Li, S.-h.; Hua, Y. Tianran Chanwu Yanjiu Yu
Kaifa 2012, 24, 761−763, 831.
(8) Shan, H.; Wilson, W. K.; Phillips, D. R.; Bartel, B.; Matsuda, S. P. T.
Org. Lett. 2008, 10, 1897−1900.
(9) Bauer, S. Ph.D. dissertation, Westfalischen Wilhelms-Universitat:
̈
̈
Munster, 2002.
̈
(10) Shibuya, M.; Xiang, T.; Katsube, Y.; Otsuka, M.; Zhang, H.;
Ebizuka, Y. J. Am. Chem. Soc. 2007, 129, 1450−1455.
(11) Our past familiarity with Bauer’s work involved targeted searches
for specific GC-MS data. The description of 13 was found during an
unhurried exploration of the entire dissertation. Bauer’s lack of an
authentic standard or definitive literature data precluded unambiguous
identification of 13. His evidence for 13 included parallels between
oleanane and ursane GC retention times. However, the retention
patterns did not extend well to HPLC, and this finding raised misleading
doubts about the identification of 13. His diligent efforts could not
separate 13 from 2 by HPLC (MeOH or MeCN in water) or AgNO3-
TLC, and separation on Ag+-HPLC was slight.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.5b01851.
Experimental procedures, plant analyses, molecular
modeling, GC-MS and NMR figures (PDF)
Excel macro to analyze HSQC data for the presence of
specific compounds (ZIP)
(12) (a) Bauer, S.; Schulte, E.; Thier, H.-P. Eur. Food Res. Technol.
2004, 219, 487−491. (b) Bauer, S.; Schulte, E.; Thier, H.-P. Eur. Food
Res. Technol. 2005, 220, 5−10.
(13) (a) Misra, T. N.; Singh, R. S.; Upadhyay, J.; Srivastava, R. J. Nat.
Prod. 1984, 47, 368−372. (b) Reference 7. The impressive performance
of DFT calculations is consistent with the typical accuracy of predicted
and reported NMR chemical shifts (each roughly 0.5 ppm for 13C):
(c) Plainchont, B.; Nuzillard, J.-M. Magn. Reson. Chem. 2013, 51, 54−59.
(14) Triterpene isomerization in TfOH: (a) Surendra, K.; Corey, E. J. J.
Am. Chem. Soc. 2009, 131, 13928−13929. Isomerization with BF3-
etherate or H2SO4: (b) Ageta, H.; Shiojima, K.; Arai, Y. Chem. Pharm.
Bull. 1987, 35, 2705−2716. HCOOH isomerization is less common:
Reference 4b.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: matsuda@rice.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(15) (a) Fayez, M. B. E.; Grigor, J.; Spring, F. S.; Stevenson, R. J. Chem.
Soc. 1955, 3378−3383. (b) Kamdem, R. S. T.; Wafo, P.; Yousuf, S.; Ali,
Z.; Adhikari, A.; Rasheed, S.; Khan, I. A.; Ngadjui, B. T.; Fun, H.-K.;
Choudhary, M. I. Org. Lett. 2011, 13, 5492−5495.
The Robert A. Welch Foundation (C-1323) supported this work.
We thank Kaylah Dunbar and Matias I. Kinzurik (Rice
University) for carrying out early experiments. NMR instru-
mentation was supported by the W. M. Keck Foundation
(University of Houston) and the John S. Dunn Sr. Gulf Coast
Consortium for Magnetic Resonance (Rice University).
Quantum mechanical calculations were supported by the NSF
under Grants OCI-0959097 and EIA-0216467.
(16) NMR precision of 0.001 and 0.01 for 1H and 13C (broadly
reproducible for nonpolar, nonacidic, nonbasic substances) has long
been essential for our spectral analyses: (a) Castillo, D. A.; Kolesnikova,
M. D.; Matsuda, S. P. T. J. Am. Chem. Soc. 2013, 135, 5885−5894.
(b) Herrera, J. B.; Bartel, B.; Wilson, W. K.; Matsuda, S. P. T.
Phytochemistry 1998, 49, 1905−1911. (c) Wilson, W. K.; Sumpter, R.
M.; Warren, J. J.; Rogers, P. S.; Ruan, B.; Schroepfer, G. J., Jr. J. Lipid Res.
1996, 37, 1529−1555.
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D
DOI: 10.1021/acs.orglett.5b01851
Org. Lett. XXXX, XXX, XXX−XXX