1372 Journal of Natural Products, 2009, Vol. 72, No. 8
Klausmeyer et al.
d4, 500 MHz) δ 7.15 (2H, s, H-12 and H-16), 4.39 (1H, dd, J ) 5.0,
1.5 Hz, H-3), 4.10 (1H, ddd, J ) 10.3, 7.0, 2.0 Hz, H-6), 3.56 (1H, m,
H-9a), 3.40 (1H, m, H-9b), 3.10 (1H, dd, J ) 14.0, 5.0 Hz, H-10a),
2.97 (1H, dd, J ) 14.0, 5.0 Hz, H-10b), 2.17 (1H, m, H-7a), 1.87 (2H,
m, H-8a and H-8b), 1.40 (1H, m, H-7b); 13C NMR (CD3OD, 125 MHz)
δ 170.8 (CO, C-5), 166.6 (CO, C-2), 151.2 (COH, C-14), 130.6 (CH,
C-12), 130.6 (CH, C-16), 128.2 (C, C-11), 123.3 (CCl, C-13), 123.3
(C, C-15), 59.8 (CH, C-6), 57.3 (CH, C-3), 45.7 (CH2, C-9), 36.3 (CH2,
C-10), 29.2 (CH2, C-7), 22.5 (CH2, C-8); LRESIMS (cone voltage )
70 V) m/z 327.0 [M - H]- (85), 174.0 [M - C7H10N2O2]- (100), 153.1
[M - C7H5Cl2O]- (78); HRESIMS m/z [M - H]- 327.0287 (calcd for
C14H13Cl2N2O3, 327.0303).
Chlorination of Tyrosine. Chlorine gas was generated by the
addition of 0.5 mL of concentrated HCl to 1.2 mL of household bleach
in a septum-plugged test tube. The headspace above the mixture was
sampled using a 20 mL syringe and injected into 2 mL of 10 mg/mL
methanolic D/L-Tyr or D-Tyr (Sigma-Aldrich) solution. The addition
of the chlorine gas and the subsequent reaction rapidly enabled
solublization of the solid into CH3OH. The high yield of the product
precluded the need for purification prior to derivatization with FDAA.
3,5-Dichlorotyrosine (3). 1H NMR (CD3OD, 400 MHz): δ 7.21 (2H,
s, H-3 and H-5), 4.29 (1H, dd, J ) 7.6, 6.4 Hz, H-R), 3.17 (1H, dd, J
) 14.8, 6.4 Hz, H-ꢀ), 3.03 (1H, dd, J ) 14.8, 7.6 Hz, H-ꢀ).
the maximum number of cells migrating to 100 ng/mL of CCL2 was
set to 100%, and reductions in migration were calculated relative to
the number of cells migrating to 100 ng/mL CCL2. The mean and
standard error of the mean for each treatment (N ) 6) are given.
Acknowledgment. This project has been funded in whole or in part
with federal funds from the National Cancer Institute, National Institutes
of Health, under contract NO1-CO-12400. The content of this publica-
tion does not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by the U.S.
Government. This research was supported [in part] by the Develop-
mental Therapeutics Program in the Division of Cancer Treatment and
Diagnosis of the National Cancer Institute. We gratefully acknowledge
R. Collins for obtaining the Leptoxyphium spores, J. Klose for NMR
experiments, T. Meragelman for informative NMR discussions, R. Akee
for assistance with amino acid derivatization, J. Britt for robotics
support, J. J. Oppenheim for critical evaluation, and E. Hudson for
technical support.
Supporting Information Available: This material is available free
Acid Hydrolysis and Determination of the Configuration of 1. 6
Compound 1 (25 µg) was dissolved in MeOH (100 µL) and transferred
to a hydrolysis tube. The MeOH was removed under vacuum, and the
tube was filled with 6 N HCl. After degassing, the solution was heated
in an oil bath at 105 °C for 18 h, at which time the hydrolysis reaction
was stopped. HCl was evaporated under a nitrogen stream, and the
hydrolysis products were rinsed twice with MeOH followed by
overnight drying under vacuum.
The hydrolysate of 1 and the amino acid standards D-Pro, D/L-Pro
(Sigma-Aldrich), 3,5-dichloro-D-Tyr, and 3,5-dichloro-D/L-Tyr were
dissolved in MeOH/H2O (4:1, at 1 mg/mL) and transferred (25 µL) to
separate HPLC autosampler vials. All traces of solvent were removed
under vacuum. To each sample was added 15 µL of 6% TEA and 7.5
µL of FDAA (Marfey’s reagent, Pierce). The samples were heated at
40 °C for 1 h and allowed to cool prior to C18 HPLC analysis with
CH3CN/5% aq HOAc (1:9 to 1:1).
References and Notes
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CCL2-Induced Monocyte Migration Bioassay. Human monocytes
were used to study the inhibition of CCL2-induced cell migration as
previously described.16,17 Fungal extracts and fractions were diluted
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response; therefore monocyte migration toward 100 ng/mL CCL2 was
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was determined using the normalization protocols described in Graph
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media control (also labeled BM) was subtracted from all replicates,
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