6
580
T. Meickle et al. / Bioorg. Med. Chem. 19 (2011) 6576–6580
lective HPLC was carried out using three separate conditions and
the retention times were compared to those of authentic stan-
Cancer cells were plated in 96-well plates (HCT-116, 10,000;
U2OS, 4000), and after 24 h were treated with various concentra-
tions of compounds 1, 2 (purity >95%) or solvent control (1% EtOH).
After 48 h of incubation, cell viability was measured using MTT
according to the manufacturer’s instructions (Promega). Assays
were run in quadruplicate and dose–response curves were gener-
ated using XLfit Excel (ID Business Solutions Ltd).
dards. Condition 1: 5% CH
min, Phenomenex Chirex 3126 (4.6 ꢁ 250 mm) column; elution
times (t , min) for standards: -alanine (6.2), -alanine (7.3),
-proline (9.2), and -proline (17.9). Condition 2: 15% CH CN in
O, 1.0 mL/min, Phenomenex Chirex 3126
, min) for standards:
-N-Me-phenylalanine (32.8). Condi-
CN in 2.0 mM CuSO in H O, 1.0 mL/min, Diacel
3 4 2
CN in 2.0 mM CuSO in H O, 1.0 mL/
R
L
D
L
D
3
2
4 2
.0 mM CuSO in H
(
4.6 ꢁ 250 mm) column; elution times (t
R
L-N-Me-phenylalanine (29.2),
D
Acknowledgments
tion 3: 15% CH
3
4
2
Chemical Laboratories, Ltd, Chiralpak MA (+) (4.6 ꢁ 50 mm) col-
umn; elution times (t , min) for standards: (2R,3S)-Hmpa (16.8),
2R,3R)-Hmpa (19.7), (2S,3R)-Hmpa (25.9), (2S,3S)-Hmpa (31.7).
The hydrolysate for 1 was analyzed by the above methods to reveal
the presence of -alanine (6.1 min), -proline (8.9 min), -N-Me-
This research was supported by the National Institutes of
Health, NIGMS Grant P41GM086210. We are grateful for the use
of the 600 MHz NMR spectrometer at Harbor Branch Oceano-
graphic Institute at Florida Atlantic University. We also thank the
Florida Atlantic University, Jupiter Campus, for use of their polar-
imeter and infrared spectrometer. HRMS analyses were performed
at the UCR Mass Spectrometry Facility, Department of Chemistry,
University of California at Riverside. This is contribution # 849 of
the Smithsonian Marine Station at Fort Pierce.
R
(
L
L
D
phenylalanine (32.5 min), and (2S,3S)-Hmpa (31.8 min). The
hydrolysate for 2 was analyzed by the above methods to reveal
the presence of
phenylalanine (32.0 min), and (2S,3S)-Hmpa (31.8 min). The pres-
ence of -N-Me-phenylalanine in both compounds was confirmed
by co-injection with the standard.
L-alanine (6.2 min), L-proline (8.9 min), D-N-Me-
D
Supplementary data
Supplementary data ( H, 13C, DQF COSY, HSQC, HMBC, and
1
3
.5. Analysis of 3-amino-2-methyloctanoic acid by advanced
Marfey’s method
3
NOESY NMR spectra in CD CN for compounds (1) and (2)) associ-
Compounds 1 and 2 were hydrolyzed (ꢃ0.8 mg each) in 6 N HCl
at 115 °C for 18 h in a sealed tube. The hydrolysates were dried un-
der air and then re-dissolved in 400
Then 100 L of each hydrolysate product was derivatized with
-FDLA and 100 L of each was derivatized with DL-FDLA using
standard procedures.
lL of 50:50 acetonitrile:water.
References
l
L
l
1. Liu, L.; Rein, K. S. Mar. Drugs 2010, 8, 1817.
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1
3,17
Standard samples of
L-valine, L-alanine,
L-proline, and N-Me-D-phenylalanine were also derivatized with
3.
Gademann, K.; Portmann, C. Curr. Org. Chem. 2008, 12, 326.
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-FDLA. The analysis was carried out by RP-HPLC using the same
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-FDLA derivatized Amoa standards were 25.7 min (2R,3S),
6.7 min (2S,3S), 45.6 min (2R,3R), and 54.1 min (2S,3R).12,14 The
retention time of the Amoa portion for 1 was 48.5 and 23.5 min
for -FDLA and -FDLA, respectively. The retention time of the
Amoa portion for 2 was 40.5 and 24.5 min for -FDLA and -FDLA,
respectively. The previously identified amino acids appeared at
2
2
002, 65, 996.
10. Kwan, J. C.; Rocca, J. R.; Abboud, K. A.; Paul, V. J.; Luesch, H. Org. Lett. 2008, 10,
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(2S,3S)-Amoa was inferred from (2R,3R)-Amoa derivatized with -FDLA.
7
L
D
1
L
D
2
2
1
0.1 min (L-Ala), 9.6 min (L-Pro), and 12.6 min (N-Me-D-Phe).
1
1
D
3
.6. Activity
D
1
1
1
5. Singh, S.; Kate, B. N.; Banerjee, U. C. Crit. Rev. Biotechnol. 2005, 25, 73.
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Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-
,5-diphenyl-tetrazolium bromide) (MTT) colorimetric assay.
2