Journal of Natural Products
Article
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Analysis of Enzyme Products by HPLC, NMR, and MS. An
Agilent HPLC series 1200 was used for analysis and isolation of the
enzyme products. A Multospher 120 RP-18 column (250 × 4 mm, 5
um C+S Chromatographie Service, Langerwehe, Germany) was used
for analysis at a flow rate of 1 mL/min, and a Multospher 120 RP18
column (250 × 10 mm, 5 μm) for isolation at a flow rate of 2.5 mL/
min. H2O (solvent A) and MeCN (solvent B), both containing 0.5%
TFA, were used as solvents. A linear gradient of 40−100% (v/v)
solvent B in 15 min was used for analysis of the enzymatic products.
The column was then washed with 100% solvent B for 5 min and
equilibrated with 40% solvent B for another 5 min. Detection was
carried out using a photodiode array detector. Solvents for isolation of
the enzyme products were H2O (solvent C) and MeCN (solvent D)
without acid. The enzyme products were isolated with a linear gradient
of 50−100% D in C in 25 min. After each run, the column was
equilibrated with 50% solvent D for 10 min. HPLC analysis of the (R)-
benzodiazepinedione reaction was carried out as described pre-
viously.21
for C20H18O5, 338.1154); H NMR (500 Hz, acetone-d6) δ 7.93 (2H,
d, J = 9.0 Hz, H-2′/H-6′), 7.02 (2H, d, J = 9.0 Hz, H-3′/H-5′), 6.62
(1H, s, H-3), 6.64 (1H, s, H-8), 5.27 (1H, tsept, J = 7.3, 1.4 Hz, H-2″),
3.35 (2H, d, J = 7.3 Hz, H-1″), 1.78 (3H, d, J = 1.0 Hz, H-5″), 1.65
(3H, d, J = 1.1 Hz, H-4″).
Compound 8b: tR = 10.52 min; UV (extracted from PDA)
(MeCN/H2O) λmax 215, 265 nm; MS m/z 338.1175 (calculated for
C20H18O5, 338.1154); H NMR (500 Hz, acetone-d6) δ 8.15 (1H, s,
1
H-2), 7.45 (2H, d, J = 6.5 Hz, H-2′/H-6′), 6.90 (2H, d, J = 8.7 Hz, H-
3′/H-5′), 6.51 (1H, s, H-8), 5.27 (1H, tsept, J = 7.2, 1.2 Hz, H-2″),
3.36 (2H, d, J = 7.2 Hz, H-1″), 1.78 (3H, d, J = 0.7 Hz, H-5″), 1.65
(3H, d, J = 1.2 Hz, H-4″).
Compound 8c: tR = 12.73 min; UV (extracted from PDA)
(MeCN/H2O) λmax 215, 266 nm; MS m/z 406.1768 (calculated for
1
C25H26O5, 406.1780); H NMR (500 Hz, acetone-d6) δ 8.10 (1H, s,
H-2), 7.32 (1H, d, J = 2.3 Hz, H-2′), 7.25 (1H, d, J = 8.3, 2.3 Hz, H-
6′), 6.88 (1H, d, J = 8.3 Hz, H-2′), 6.50 (1H, s, H-8), 5.36 (1H, m, H-
2‴), 5.26 (1H, m, H-2″), 3.35 (2H, d, J = 7.5 Hz, H-1‴), 3.33 (2H, d, J
= 6.0 Hz, H-1″), 1.76 (3H, d, J = 0.8 Hz, H-4‴), 1.71 (3H, d, J = 1.2
Hz, H-5″), 1.70 (3H, d, J = 1.2 Hz, H-5‴), 1.63 (3H, d, J = 1.1 Hz, H-
4″).
Compound 1b: tR = 10.02 min; UV (extracted from PDA)
(MeCN/H2O) λmax 230, 288 nm; MS m/z 340.1293 (calculated for
C20H20O5, 340.1311); 1H NMR (500 Hz, acetone-d6) δ 7.39 (2H, d, J
= 8.7 Hz, H-2′/H-6′), 6.89 (2H, d, J = 8.7 Hz, H-3′/H-5′), 6.03 (1H,
s, H-8), 5.43 (1H, dd, J = 12.9, 3.0 Hz, H-2), 5.23 (1H, tsept, J = 7.3,
1.5 Hz, H-2″), 3.17 (1H, dd, J = 17.1, 12.9 Hz, H-3), 3.24 (2H, d, J =
7.3 Hz, H-1″), 2.72 (1H, dd, J = 17.1, 3.0 Hz, H-3), 1.75 (3H, d, J =
0.8 Hz, H-5″), 1.64 (3H, d, J = 1.1 Hz, H-4″).
Compound 9b: tR = 13.55 min; UV (extracted from PDA)
(MeCN/H2O) λmax 215, 265 nm; MS m/z 352.1310 (calculated for
C21H20O5, 352.1311); H NMR (500 Hz, acetone-d6) δ 8.18 (1H, s,
H-2), 7.54 (2H, d, J = 8.8 Hz, H-2′/H-6′), 7.00 (2H, d, J = 8.8 Hz, H-
3′/H-5′), 6.51 (1H, s, H-8), 5.27 (1H, tsept, J = 7.2, 1.5 Hz, H-2″),
3.84 (3H, s, OCH3), 3.36 (2H, d, J = 7.2 Hz, H-1″), 1.78 (3H, d, J =
0.6 Hz, H-5″), 1.65 (3H, d, J = 1.1 Hz, H-4″).
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Compound 1c: tR = 9.52 min; UV (extracted from PDA) (MeCN/
H2O) λmax 230, 296 nm; MS m/z 340.1309 (calculated for C20H20O5,
1
340.1311); H NMR (500 Hz, acetone-d6) δ 7.29 (1H, d, J = 2.2 Hz,
H-2′), 7.21 (1H, dd, J = 8.3, 2.2 Hz, H-6′), 6.89 (1H, d, J = 8.3 Hz, H-
5′), 5.95 (1H, d, J = 2.2 Hz, H-6), 5.94 (1H, d, J = 2.2 Hz, H-8), 5.43
(1H, dd, J = 12.9, 3.0 Hz, H-2), 5.35 (1H, tsept, J = 7.4, 1.5 Hz, H-2″),
3.34 (2H, d, J = 7.4 Hz, H-1″), 3.18 (1H, dd, J = 17.1, 12.9 Hz, H-3),
2.71 (1H, dd, J = 17.1, 3.0 Hz, H-3), 1.71 (3H, d, J = 0.7 Hz, H-5″),
1.70 (3H, d, J = 1.4 Hz, H-4″).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
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S
Compound 2b: tR = 12.43 min; UV (extracted from PDA)
(MeCN/H2O) λmax 240, 277, 320 nm; MS m/z 308.1389 (calculated
NMR spectra; HPLC chromatograms of enzyme assays
with GPP; kinetic parameters (PDF)
1
for C20H20O3, 308.1412); H NMR (500 Hz, acetone-d6) δ 7.59 (1H,
s, H-5), 7.57 (2H, br d, J = 8.2 Hz, H-2′/H-6′), 7.44 (2H, t, J = 8.2 Hz,
H-3′/H-5′), 7.38 (1H, tt, J = 8.2, 1.4 Hz, H-4′), 6.49 (1H, s, H-8),
5.54 (1H, dd, J = 13.0, 3.0 Hz, H-2), 5.33 (1H, tsept, J = 7.4, 1.5 Hz,
H-2″), 3.28 (2H, d, J = 7.4 Hz, H-1″), 3.01 (1H, dd, J = 16.7, 13.0 Hz,
H-3), 2.72 (1H, dd, J = 16.7, 3.0 Hz, H-3), 1.73 (3H, d, J = 0.9 Hz, H-
5″), 1.71 (3H, d, J = 1.4 Hz, H-4″).
AUTHOR INFORMATION
Corresponding Author
28-22461/25365.
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Present Address
Compound 3c: tR = 8.99 min; UV (extracted from PDA) (MeCN/
H2O) λmax 230, 290 nm; MS m/z 356.1234 (calculated for C20H20O6,
‡Department of Chemical and Biomolecular Engineering,
University of California, Los Angeles, 420 Westwood Plaza,
Los Angeles, California 90095, United States.
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356.1260); H NMR (500 Hz, acetone-d6) δ 6.90 (1H, d, J = 2.0 Hz,
H-2′), 6.80 (1H, d, J = 2.0 Hz, H-6′), 5.95 (1H, d, J = 1.2 Hz, H-6),
5.94 (1H, d, J = 1.2 Hz, H-8), 5.37 (1H, dd, J = 12.9, 3.0 Hz, H-2),
5.35 (1H, tsept, J = 7.3, 1.5 Hz, H-2″), 3.35 (2H, d, J = 7.3 Hz, H-1″),
3.12 (1H, dd, J = 17.1, 12.9 Hz, H-3), 2.70 (1H, dd, J = 17.1, 3.0 Hz,
H-3), 1.71 (3H, d, J = 0.6 Hz, H-5″), 1.70 (3H, d, J = 1.1 Hz, H-4″).
Compound 5b: tR = 11.08 min; UV (extracted from PDA)
(MeCN/H2O) λmax 230, 290 nm; MS m/z 550.1839 (calculated for
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This work was financially supported in part by a grant from the
Deutsche Forschungsgemeinschaft (Li844/4-1 to S.-M.L.). K.Z.
is a recipient of a scholarship from China Scholarship Council
(201308440282). We thank N. Zitzer and S. Newel for taking
MS and NMR spectra, respectively.
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C30H30O10, 550.1900); H NMR (600 Hz, acetone-d6) Table 1.
Compound 6b: tR = 9.09 min; UV (extracted from PDA) (MeCN/
H2O) λmax 230, 290 nm; MS m/z 342.1438 (calculated for C20H22O5,
1
342.1467); H NMR (500 Hz, acetone-d6) δ 7.09 (2H, d, J = 8.5 Hz,
H-2′/H-6′), 6.74 (2H, d, J = 8.5 Hz, H-3′/H-5′), 6.07 (1H, s, H-8),
3.24 (2H, d, J = 7.2 Hz, H-1″), 5.22 (1H, tsept, J = 7.2, 1.1 Hz, H-2″),
3.33 (2H, t, J = 7.7 Hz, H-2), 2.88 (2H, t, J = 7.7 Hz, H-1), 1.74 (3H,
d, J = 0.7 Hz, H-5″), 1.62 (3H, d, J = 1.1 Hz, H-4″).
REFERENCES
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(1) Marais, J. P. J.; Deavours, B.; Dixon, R. A.; Ferreira, D. In The
Science of Flavonoids; Grotewold, E., Ed.; Springer Science + Business
Media, Inc: New York, 2006; Chapter 1, pp 1−46.
(2) Sandhar, H. K.; Kumar, B.; Prasher, S.; Tiwari, P.; Salhan, M.;
Sharma, P. Int. Pharm. Sci. 2011, 1, 25−41.
Compound 6c: tR = 8.70 min; UV (extracted from PDA) (MeCN/
H2O) λmax 230, 285 nm; MS m/z 342.1505 (calculated for C20H22O5,
1
342.1467); H NMR (500 Hz, acetone-d6) Table 2.
Compound 6d: tR = 11.59 min; UV (extracted from PDA)
(3) Agrawal, A. D. Int. J. Pharm. Sci. Nanotechnol. 2011, 4, 1394−
1398.
(MeCN/H2O) λmax 230, 290 nm; MS m/z 410.2071 (calculated for
1
C25H30O5, 410.2093); H NMR (500 Hz, acetone-d6) Table 2.
Compound 7b: tR = 11.55 min; UV (extracted from PDA)
(4) Botta, B.; Vitali, A.; Menendez, P.; Misiti, D.; Delle, M. G. Curr.
(MeCN/H2O) λmax 220, 277, 330 nm; MS m/z 338.1149 (calculated
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