COMMUNICATIONS
enzymatic activities are present within the extract to mediate
the entire biosynthesis. Incubation of a synthetic sample of 5’-
FDA (5), the initial product of enzymatic fluorination, with
the cell-free extract resulted in the accumulation of 3 (and 10),
thus confirming the role of 5 as a biosynthetic intermediate of
GC-MS analyses were performed on an Agilent 6890 gas chromatograph
connected to an Agilent 5973 mass-selective detector. The methods used
for analysis were similar to those previously described.[ Fluoroacetate was
analyzed by GC-MS in the scan mode after preparation of its p-phenyl-
8]
[8]
phenacyl derivative. MS (EI þ ): m/z (%): 272 (9.5), 182 (13.7), 181 (100),
152 (65.9). 4-Fluorothreonine (2) was analyzed as its per(trimethylsily-
lated) derivative (obtained by treatment with MSTFA) by GC-MS
operated in the scan mode. MS (EI þ ): m/z (%): 236 (53.4), 218 (100),
1
and 2. An overview of this biotransformation in shown in
Scheme 3. The accumulation of 3 in this case appears to have
promoted the biosynthesis of 2 as well as 1. A transient signal
1
47 (15.6), 128 (14.2), 100 (11.2).
1
9
: White powder; m.p. 159±1608C; H NMR (300 MHz, CDCl
), 1.64 (s, 3H; CH
), 4.52 (dm, 1H; 4’-H), 4.62 (ddd, J ¼
46.7, 14.5, 10.4 Hz, 1H; 5’-H /5’-H ), 4.63 (ddd, J ¼ 46.7, 13.5, 10.4 Hz, 1H;
3
, 258C): d ¼
1
9
(
d ¼ ꢀ227 ppm) was also observed in the F NMR spectrum
1.40 (s, 3H; CH
3
3
in this experiment, which is presumably a metabolic inter-
mediate between 5 and 3. This compound has not yet been
identified.
a
b
5’-H
.37 (dm, J(2’H,3’H) ¼ 6.3 Hz, 1H; 2’-H), 5.68 (br s, 2H; NH
J(1’H,2’H) ¼ 1.93 Hz, 1H; 1’-H), 7.93 (s, 1H; 2-H/8-H), 8.36 ppm (s, 1H; 2-
a
/5’-H
b
), 5.10 (dd, J(3’H,2’H) ¼ 6.3 Hz, J(3’H,4’H) ¼ 3.8 Hz, 1H; 3’-H),
5
2
), 6.19 (d,
1
3
H/8-H); C NMR (75 MHz, CDCl
(
8
3
, 258C): d ¼ 25.7 (CH
3 3
), 27.5 (CH ), 81.1
d, J(C3’, F) ¼ 6.8 Hz; C3’), 83.3 (d, J(C5’,F) ¼ 171.8 Hz; C5’), 84.9 (C’2),
OH
5.9 (d, J(C4’,F) ¼ 19.5 Hz; C4’), 91.3 (C’1), 115.0, 120.5, 139.7 (C2/C8),
19
N
N
149.8, 153.7 (C2/C8), 156.0 ppm; F NMR (282.2 MHz, CDCl
, 258C): d ¼
):
3
ꢀ
228.86 ppm (dt, J(F,4 ’H) ¼ 23.6 Hz, J(F, 5 ’H) ¼ 46.7 Hz); MS (CI, CH
4
N
N
þ
m/z (%): 310 (100) [M þH], 290 (10) [ꢀHF], 251 (8), 207 (5), 175 (7), 75
F
O
þ
(
3
40); HRMS (CI): calcd for C13
H
17FN
5
O
3
[MþH ]: 310.1315, found:
10.1321.
HO
OH
: White powder, m.p 203±2048C [lit.[12] 205±2068C]; H NMR (300 MHz,
1
5
6
[
3
1
D
6
]DMSO, 258C): d ¼ 4.11 (dm, J(F,4 ’H) ¼ 23.8, 1H; 4’-H), 4.26 (m, 1H;
’-H), 4.60 (m, 1H; 2’-H), 4.64 (dm, J(F, 5 ’CH ), 5.46 (br s,
) ¼ 47.8, 2H; CH
H; OH), 5.65 (br s, 1H; OH), 5.94 (d, J(1’H,2’H) ¼ 4.86 Hz, 1H; 1’-H),
), 8.16 (s, 1H; 2-H/8-H), 8.27 ppm (s, 1H; 2-H/8-H);
C NMR (75 MHz, [D
]DMSO, 258C); d ¼ 69.7 (d, J(C3’,F) ¼ 6.0 Hz), 73.4
OH
2
2
deaminase
10
F
7.33 (br s, 2H; NH
2
1
3
6
alcohol
dehydrogenase
(
1
C’2), 82.6 (d, J(C4’,F) ¼ 18.0 Hz), 83.3 (d, J(C5’,F) ¼ 171.8 Hz), 88.0 (C1’),
NH2
O
19
19.4, 139.7, (C2/C8), 149.7, 153.1 (C2/C8), 156.4 ppm;
F NMR
N
N
N
O
(282.2 MHz, [D
J(F,5 ’H) ¼ 47.8 Hz); MS (ES): m/z (%): 269.98 (100) [M ].
6
]DMSO, 258C); d ¼ ꢀ230.0 ppm (dt, J(F,4 ’H) ¼ 23.8 Hz,
þ
F
N
O
1
O
unknown steps
F
H
Received: August 5, 2002 [Z19887]
F 3
HO
OH
OH
5
CO2
[
1] D. B. Harper, D. O©Hagan, Nat. Prod. Rep. 1994, 123 ± 133.
non-enzymatic
[2] M. Sanada, T. Miyano, S. Iwadare, J.M. Williamson, B. H. Arison, J.L.
F
NH3
Smith, A. W. Douglas, J.M. Liesch, E. Inamine, J. Antibiot. 1984, 39,
2
2
59 ± 265.
[
3] K. A. Reid, J. T. G. Hamilton, R. D. Bowden, D. O’Hagan, L.
Dasaradhi, M. R. Amin, D. B. Harper, Microbiology 1995, 141,
tris(hydroxymethyl)-aminoethane-
fluoroacetaldehyde-complex
1
385 ± 1393.
Scheme 3. Schematic representation of the bioconversions observed after
incubating 5’-FDA with the S. cattleya cell-free extract.
[
4] D. O©Hagan, C. Schaffrath, S. Cobb, J. T. G. Hamilton, C. D. Murphy,
Nature 2002, 416, 279 ± 279.
[
5] S. J. Moss, C. D. Murphy, J. T. G. Hamilton, W. C. McRoberts, D.
O©Hagan, C. Schaffrath, D. B. Harper, Chem. Commun. 2000, 2281 ±
Experimental Section
2
282.
Streptomyces cattleya NRRL 8057 was grown in 500-mL flasks in a medium
of the composition previously described. After 6 days of incubation, the
[6] C. D. Murphy, S. J. Moss, D. O©Hagan, Appl. Environ. Microbiol. 2001,
67, 4919 ± 4921.
[
3]
cells were harvested and washed with Tris buffer (50 mm, pH 7.8) and
[7] C. D. Murphy, D. O©Hagan, C. Schaffrath, Angew. Chem. 2001, 113,
4611 ± 4613; Angew. Chem. Int. Ed. 2001, 40, 4479 ± 4481.
[8] J. T. G. Hamilton, C. D. Murphy, M. R. Amin, D. O©Hagan, D. B.
Harper, J. Chem. Soc. Perkin Trans. 1 1998, 759 ± 767.
[9] Z. Wang, F. A. Quiocho, Biochemistry 1998, 37, 8314 ± 8324.
[10] D. Gani, A. W. Johnson, J. Chem. Soc. Perkin Trans. 1 1982, 1197 ±
1204.
ꢀ
1
resuspended in the same buffer (0.1 g wet-cell weight mL ). Ultrasonica-
tion and centrifugation gave a supernatant that was used directly as the cell-
free extract. Biotransformations were initiated by supplementation of
SAM (0.4 mm) and NaF (10 mm) or by the addition of 5’-FDA (10 mm) to
the cell-free extract, and the reactions were incubated at 288C for up to
1
9
2
0 h. For real-time analysis by F NMR spectroscopy, the sample was
1
9
prepared in the same manner but D
2
O was added (100 mL). F NMR
[11] S. J. Moss, PhD thesis, University of Durham, 1999.
[12] H. M. Kissman, M. J. Weiss, J. Am Chem. Soc. 1958, 80, 5559 ± 5564.
spectra were recorded on a Varian Inova 500-MHz NMR spectrometer.
Angew. Chem. Int. Ed. 2002, 41, No. 20
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0044-8249/02/4120-3915 $ 20.00+.50/0
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