Total synthesis of a reported fluorometabolite from Streptomyces sp. TC1 indicates an incorrect assignment. the isolated compound did not contain fluorine

Article abstract of DOI:10.1021/np500260z

3,5-Di-tert-butyl-4-fluorophenylpropionic acid (1) was recently reported as a natural product from Streptomyces sp. TC1. This was a notable disclosure because fluorinated natural products are exceedingly rare, and in this case it suggested that the bacterium had the capacity to mediate an enzymatic aryl fluorination reaction. However, a synthesis of the putative metabolite 1 demonstrates that the spectroscopic data are inconsistent with the proposed structure. There is no evidence that the isolated compound contained a fluorine atom.

Full text of DOI:10.1021/np500260z

Communication
pubs.acs.org/jnp
Total Synthesis of a Reported Fluorometabolite from Streptomyces
sp. TC1 Indicates an Incorrect Assignment. The Isolated Compound
Did Not Contain Fluorine
Mohammed Salah Ayoup,†,‡ David B. Cordes,† Alexandra M. Z. Slawin,† and David O’Hagan*,†
†School of Chemistry, University of St Andrews, North Haugh, St Andrews, KY16 9ST, U.K.
‡Department of Chemistry, Faculty of Science, Alexandria University, P.B 426, Ibrahimia, Egypt
S
* Supporting Information
ABSTRACT: 3,5-Di-tert-butyl-4-fluorophenylpropionic acid (1) was recently reported as a natural
product from Streptomyces sp. TC1. This was a notable disclosure because fluorinated natural
products are exceedingly rare, and in this case it suggested that the bacterium had the capacity to
mediate an enzymatic aryl fluorination reaction. However, a synthesis of the putative metabolite 1
demonstrates that the spectroscopic data are inconsistent with the proposed structure. There is no
evidence that the isolated compound contained a fluorine atom.
i-tert-butylfluorophenylpropionic acid (1) was recently
reported in this journal as a novel fluorometabolite,
D
isolated from the microorganism Streptomyces sp. TC1.1 The
isolation immediately attracted attention2 because the identi-
fication of fluorine-containing metabolites is extremely rare.3,4
Also this would be a particularly intriguing compound, as it
suggests that the microorganism has an enzyme that can
accomplish an aryl fluorination, a class of enzyme reaction
without precedent. Such an enzyme would have exciting
biotechnological potential in view of the large-scale utility of
aryl fluorides in the fine chemicals industry. However, the 19F
NMR signal in the Supporting Information (SI)1 is extremely
broad (4000 Hz) and is not consistent with a soluble low
molecular weight organo-fluorine compound, which would be
expected to have a sharp, highly resolved signal. Additionally
the 1H and 13C NMR spectra do not appear to have any proton
to fluorine (4JHF) or carbon to fluorine (1JCF) coupling
constants. None are visible or reported. Thus, there is no
evidence from the submitted NMR data that the compound
actually contains a fluorine atom. The structure of 1 is claimed
based primarily on X-ray structure analysis. However, it is well
known that it is difficult for a crystallographer to distinguish
hydroxyl from fluorine, as these substituents have a similar
number of electrons and the hydrogen of the OH is not easily
resolvable. On the face of it, the X-ray structure could actually
be that of phenol 2. Thus, we decided to prepare compound 1
by synthesis to compare its spectroscopic properties with the
reported data of the compound isolated from Streptomyces sp.
TC1.
a
Scheme 1. Synthesis of 1
a
Reagents and conditions: (i) AlCl3, t-BuCl; (ii) NBS, (PhCO)2O2,
CCl4; (iii) NaH, THF, CH2(COOEt)2; (iv) (a) LiOH, THF, H2O;
(b) HCl; (c) aq H2SO4, reflux.
RESULTS AND DISCUSSION
■
alkylated (5) products was achieved in a ratio of 2:5 as
The synthesis route to 1 is shown in Scheme 1. 4-
Fluorotoluene (3) was treated with 2-chloro-2-methylpropane
and aluminum trichloride, following a Friedel-Craft’s protocol
described for a similar reaction with toluene.5 A complete
conversion of fluorotoluene (3) to the mono- (4) and di-
determined by 19F NMR. These products could be separated by
Received: March 21, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Communication
fractional distillation, and the desired disubstituted product 5
was isolated in 35% yield. Compound 5 was a crystalline solid,
and X-ray analysis supported its structure as shown in Figure
conducted in both +ve and −ve ion modes and gave
characteristic ions of [M + Na]+ = 303.1 amu and [M − H]−
= 279.1 amu. We have also carried out a FAB mass
spectrometry analysis, to compare to the FAB spectrum of
the compound isolated in the original paper. This gave as
expected [M + H]+ = 281.1 amu for 1; however the original
FAB reported a signal at 280.18 amu in positive ion mode,
which suggests a molecular ion of 279.1 amu. This does not fit
with the organofluorine compound 1, or with phenol 2, which
would be expected to report a FAB [M + H]+ of 279.1.
It is clear that the NMR data for synthetic 1 do not match
the compound reported from Streptomyces sp. TC1.1 The very
broad peak observed in the originally reported 19F NMR (δF
−168.01 ppm) most probably arises from the Teflon lining in
the NMR probe, with can give rise to a featureless and broad
baseline signal over a long acquisition and contrasts with the
very sharp signal (δF −110.1 ppm) for the synthetic sample of
1. This may have led to the conclusion that the X-ray structure
contained fluorine; however OH and F can be difficult to
distinguish by X-ray analysis. The isolate was shown to have
antioxidant activity.1 We tentatively suggest that the compound
that was isolated, or contaminated the isolation procedure, was
the phenolpropionic acid 2 and that there was a misinter-
pretation of the X-ray, NMR,10 and possibly mass spec data.
This structural motif is found widely in commercial
antioxidants. For example the methyl ester of 2, that is
compound 8, is a relatively well-known industrial antioxidant
(called “stabiliff”) used in fragrance mixtures and perfumes,11
and Irganox 1010 (9)12 is used in the plastics industry to
protect products against thermo-oxidative degradation.
1
4
1a. In the H NMR spectrum of 5 there was a very clear JHF
Figure 1. X-ray structures of (a) Friedel−Crafts alkylation product 5
(CCDC 992852) and (b) a synthetic sample of 1 (CCDC 992853).
coupling constant (7.6 Hz) between the fluorine and the 2/6
aryl protons. Bromination of 5 was efficiently achieved by
treatment with elemental bromine (Br2) and benzoylperoxide
in CCl4 as the solvent5 to generate benzyl bromide 6 in 78%
yield. Benzyl bromide 6 was then treated with diethyl malonate
and sodium hydride,6 to give alkylated malonate 7, followed by
treatment in aqueous base to mediate a hydrolysis.7 Carboxylic
acid 1 was then prepared by acid (aq H2SO4)-catalyzed
decarboxylation of the intermediate malonic acid.
The 1H, 19F, and 13C NMR spectra all provide signatures for
the presence of fluorine. A summary of coupling constants is
4
shown in Figure 2. Most obviously the JHF coupling (7.2 Hz)
Figure 3. Representative industrial antioxidants (stabiliff, 8, and
Irganox 1010, 9) carrying the 3,5-di-tert-butyl-4-fluorophenolpropio-
nate motif.
Figure 2. Significant JFH and JCF coupling constants determined by
NMR analysis of synthetic 1. Coupling constants to carbon around the
ring identify the presence of fluorine. These are not reported or
obvious in the data presented for the compound isolated from
Streptomyces sp. TC1.1
EXPERIMENTAL SECTION
■
General Experimental Procedures. All chemicals were pur-
chased from Sigma-Aldrich, Alfa Aesar, Fisher Scientific, and
Fluorochem. All reactions were carried out in oven-dried glassware
under an argon atmosphere using a double-vacuum manifold with the
inert gas passing through a bed of silica gel and molecular sieves.
Anhydrous CH2Cl2 and THF were obtained from an MBraun MB
SPS-800 solvent purification system, where the solvent was dried by
passage through activated filter columns and dispensed under an
atmosphere of argon. Petroleum ether refers to the fraction with a
boiling point between 40 and 60 °C. All chemicals were used as
supplied. All NMR spectra were recorded using Bruker Avance III 500
observed here between fluorine and the 2/6 aryl hydrogens is
not apparent in the reported data for structure 1 in the isolation
paper.1 Also there are obvious fluorine to carbon couplings to
all of the aryl ring carbons in the data for synthetic 1. A notable
2h
observation is a through-hydrogen
J
CF
coupling of 3.8 Hz
between the fluorine atom and the methyl hydrogens of the
tert-butyl groups. Carbon−fluorine scalar couplings can be
transmitted through short noncovalent hydrogen to fluorine
contacts.8 The shortest contact distance between the fluorine
atom and the closest tert-butyl methyl hydrogens is 2.25 Å
(from the X-ray structure), a distance significantly shorter than
1
and Bruker Avance 300 or 500 spectrometers. H NMR spectra were
recorded at either 300 or 500 MHz. 13C NMR spectra were recorded
using the DEPTQ or UDEFT pulse sequence and broadband proton
decoupling at either 75, 100, or 125 MHz. 19F NMR spectra were
recorded at 282, 376, or 470 MHz. All chemical shifts, δ, are stated in
the van der Waal contact distance (2.47 Å).9 Thus, the
2h
1
observed
J
coupling is consistent with this short contact.
units of parts per million (ppm), relative to a standard. For H NMR
CF
Electrospray ionization mass spectrometry (EIMS) of 1 was
and 13C NMR the reference point is TMS (δH and δC: 0.00 ppm). For
B
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Journal of Natural Products
Communication
19F NMR the reference point is CCl3F (δF: 0.00 ppm). Melting points
were determined using a Griffin MPA350 or an Electrothermal 9100
melting point apparatus and are uncorrected. High- and low-resolution
mass spectra were obtained by electospray ionization (ESI) using
either an LTQ Orbitrap XL spectrometer or a Waters Micromass LCT
spectrometer in positive or negative mode.
(d, 3JCF = 6.4 Hz, C-2), 35.8 (C, CH2COOH), 34.5 (C, PhCH2), 30.6
(C(CH3)3), 30.1 (d, 2hJCF = 3.8 Hz, 6 × CH3); 19F NMR (CDCl3, 282
MHz) δ −110.1 (s, 1F); HR-EIMS (−ve ion mode) m/z 279.1763
(calcd for C17H24FO2, 279.1760), which corresponds to [M − H]−;
(+ve ion mode) m/z 303.1727 (calcd for C17H25FNaO2, 303.1736),
which corresponds to [M + Na]+; FAB m/z 281.1, which corresponds
to [M + H]+.
3,5-Di-tert-butyl-4-fluorotoluene, 5. 2-Chloro-2-methylpropane
(8.4 g, 91 mmol) was added in two portions over a period 30 min
to a rapidly stirred solution of AlCl3 (1.5 g, 11.3 mmol) in p-
fluorotoluene (5.0 g, 45.4 mmol) at 0 °C for 6 h. The reaction was
continued for a further 6 h at room temperature. Water was added, the
product was extracted into CH2Cl2, then the solvent was removed
under reduced pressure, and the product was purified by Vigreux
distillation. The product 5 crystallized on standing (3.5 g, 35%). Mp
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Analytical data including H, 13C, and 19F NMR spectra and
selected mass spectra for compounds 5, 6, 7, and 1 are
illustrated. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
82−83 °C; H NMR (CDCl3, 300 MHz) δ 6.88 (2H, dd, JHF = 7.23,
0.5 Hz, H-2, H-6), 2.30 (3H, s, CH3), 1.30(18H, d, JHF = 1.1 Hz, 6
1
CH3); 13C NMR (CDCl3, 125 MHz) δ 159.6 (d, JCF = 249.4 Hz, C-
AUTHOR INFORMATION
Corresponding Author
*Tel: +44-1334-467176. Fax: +44-1334463808. E-mail: do1@
st-andrews.ac.uk.
■
2
4
4), 137.3 (d, JCF = 14 Hz, C-3), 131.7 (d, JCF = 3.4 Hz, C-1), 125.6
3
2h
(d, JCF = 6 Hz, C-2), 34.4 (CH3), 30.1 (d,
J
= 4 Hz, 6 × CH3),
CF
21.3 (C, C(CH3)3); 19F NMR (CDCl3, 282 MHz) δ −112.2 (s, 1F);
HR-CIMS, m/z 222.1779 (calcd for C15H23F, 222.1784).
Notes
3,5-Di-tert-butyl-4-fluoro(bromomethyl)benzene, 6. A solution of
3,5-di-tert-butyl-4-fluorotoluene (5; 2 g, 9.0 mmol), N-bromosuccini-
mide (1.6 g, 9.0 mmol), and benzoylperoxide (0.05 g, 0.2 mmol) in
CCl4 (120 mL) was heated under reflux for 3 h. The solution was
cooled, filtered, and concentrated under reduced pressure, and the
product was purified by flash column chromatography (petrol) to
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
One of us (M.A.) is grateful for a scholarship from the Egyptian
Government under the Channel Scheme and to Prof. L. Fathy
at the University of Alexandria, Department of Chemistry,
Egypt.
1
afford a colorless oil, 5 (2.1 g, 78%). H NMR (CDCl3, 300 MHz) δ
7.23 (2H, d, 4JHF = 7.0, H-2, H-6), 4.52 (2H, s, CH2Br), 1.42 (18H, d,
JHF = 1.1 Hz, 6 × CH3); 13C NMR (CDCl3, 125 MHz) δ 162.3 (d, 1JCF
= 250 Hz, C-4), 138.4 (d, 2JCF = 14 Hz, C-3), 132.4 (d, 4JCF = 3.6 Hz,
C-1), 126.5 (d, 3JCF = 7.3 Hz, C-2), 41.9 (CH2Br), 35.4 (C, C(CH3)3),
REFERENCES
■
4
30.4 (d, JCF = 4 Hz, 6 × CH3); 19F NMR (CDCl3, 282 MHz) δ
(1) Jaivel, N.; Uvarani, C.; Rajesh, R.; Velmurugan, D.; Marimuthu, P.
J. Nat. Prod. 2014, 77, 2−8.
−106.7 (s, 1F); HR-EIMS, m/z 221.1700 (calcd for C15H22F,
221.1706), which corresponds to [M − Br]+.
(2) Hill, R. A.; Sutherland, A. Nat. Prod. Rep. 2014, 31, 414−418.
(3) (a) O’Hagan, D.; Harper, D. B. J. Fluorine Chem. 1999, 100, 125−
131. (b) Harper, D. B.; O’ Hagan, D. Nat. Prod. Rep. 1994, 11, 123−
134.
(4) O’Hagan, D.; Perry, R.; Lock, J. M.; Meyer, J. J. M.; Dasaradhi, L.;
Hamilton, J. T. G.; Harper, D. B. Phytochemistry 1993, 33, 1043−1046.
(5) Plater, M. J.; Aiken, S.; Bourhill, G. Tetrahedron 2002, 58, 2405−
2413.
Diethyl (3,5-Di-tert-butyl-4-fluorobenzyl)malonate, 7. Diethyl
malonate (1.38 g, 8.6 mmol) was added dropwise to a stirred
suspension of sodium hydride (60% dispersion in mineral oil, 0.21 g,
8.6 mmol) in THF (10 mL) at 0 °C. A solution of 3,5-di-tert-butyl-4-
fluoro(bromomethyl)benzene (6; 2 g, 6.66 mmol) in anhydrous THF
(10 mL) was added after 30 min over a period of 10 min, followed by
stirring for 2 h at room temperature. The reaction mixture was then
diluted with H2O (20 mL), and the product extracted into EtOAc,
washed, and concentrated under reduced pressure. The residue was
then purified by flash column chromatography (petrol/Et2O, 9:1) to
(6) Chavan, S. P.; Katod, S. H. Tetrahedron: Asymmetry 2012, 23,
1410−1415.
(7) Musso, D. L.; Cochran, F. R.; Kelly, J. L.; McLean, E.; Selph, J. L.;
Rigdon, G. C.; Orr, G. F.; Davis, R. G.; Cooper, B. R.; Styles, V. L.;
Thompson, J. B.; Hall, W. R. J. Med. Chem. 2003, 46, 399−408.
(8) (a) Giuffredi, G. T.; Gouverneur, V.; Bernet, B. Angew. Chem., Int.
Ed. 2013, 52, 10524−10528. (b) Mele, A.; Vergani, B.; Viani, F.;
Meille, S. V.; Farina, A.; Bravo, P. Eur. J. Org. Chem. 1999, 187−196.
(c) Mele, A.; Salani, G.; Viani, F.; Bravo, P. Magn. Reson. Chem. 1997,
35, 168−174. (d) Gribble, G. W.; Kelly, W. J. Tetrahedron Lett. 1985,
26, 3779−3782.
1
afford diester 7 as a viscous oil (1.5 g, 60%). H NMR (CDCl3, 300
MHz) δ 6.90 (2H, d, 4JHF = 7.1 Hz, H-2, H-6), 4.10 (4H, q, J = 7 Hz,
2CH2CH3), 3.54 (1H, t, J = 8 Hz, CH), 3.07 (2H, d, J = 8 Hz,
PhCH2), 1.28 (18H, d, JHF = 1.1 Hz, 6 × CH3), 1.15 (6H, q, J = 7 Hz,
2CH3); 13C NMR (CDCl3, 125 MHz) δ 169.0 (2 × COOEt), 160.3
(d, 1JCF = 250 Hz, C-4), 137.3 (d, 2JCF = 14 Hz, C-3), 131.9 (d, 4JCF
=
3
3.6 Hz, C-1), 125.5 (d, JCF = 6.5 Hz, C-2), 61.5 (2 × OCH2), 54.1
(CH), 34.6 (2 × C(CH3)3), 34.5 (PhCH2), 30.1 (d, 4JCF = 3.6 Hz, 6 ×
CH3), 14.1 (2 × CH2CH3); 19F NMR (CDCl3, 282 MHz) δ −110.3
(s, 1F); HR-CIMS m/z 380.2368 (calcd for C22H33FO4, 380.2363).
3-(3,5-Di-tert-butyl-4-fluoropheny)propanoic acid, 1. A solution
of LiOH/H2O (30 mg 0.7 mmol) in H2O (2 mL) was added to a
stirred solution of diethyl (3,5-di-tert-butyl-4-fluorobenzyl)malonate
(7; 100 mg 0.27 mmol) in THF (2 mL), and stirring was continued
for 2 h. The reaction was then neutralized with cHCl to pH = 2, and
the product extracted into EtOAc. The organic extracts were
concentrated under reduced pressure. The product was heated
under reflux in aqueous sulfuric acid for 6 h. On cooling, the product
crystallized and was purified by flash chromatography (EtOAc/petrol
ether, 2:1) to afford 1 (60 mg, 81%) as colorless needles. Mp 165−166
°C; 1H NMR (CDCl3, 300 MHz) δ 6.92 (2H, d, J = 7.20 Hz, H-2, H-
6), 2.85 (2H, t, J = 7.2 Hz, PhCH2), 2.60 (2H, t, J = 7.2 Hz,
(9) Bondi, A. J. Phys. Chem. 1964, 68, 441−451.
(10) Bebbington, D.; Monck, N. J. T.; Gaur, S.; Palmer, A. M.;
Benwell, K.; Harvey, V.; Malcolm, C. S.; Porter, R. H. P. J. Med. Chem.
2000, 43, 2779−2782.
(11) Marteau, C.; Nardello-Rataj, V.; Favier, D.; Aubry, J.-M. Flavour
Fragr. J. 2013, 28, 30−38.
(12) Beer, S.; Teasdale, I.; Brueggemann, O. Eur. Polym. J. 2013, 49,
4257−4264.
5
CH2COOH), 1.30 (18H, d, JHF = 1.1 Hz, 6 × CH3); 13C NMR
1
(CDCl3, 125 MHz) δ 170.2 (COOH), 160.1 (d, JCF = 250.6 Hz, C-
4), 137.4 (d, 2JCF = 14 Hz, C-3), 134.1 (d, 4JCF = 3.50 Hz, C-1), 124.8
C
dx.doi.org/10.1021/np500260z | J. Nat. Prod. XXXX, XXX, XXX−XXX