Nitrone formation in phosphate buffer and aqueous solutions: Novel chemistry inspired by a natural product

Article abstract of DOI:10.1016/j.tetlet.2012.02.046

Nitrones are formed from the reaction of aspergillusol A (1) and a ketone/aldehyde in phosphate buffer and aqueous solutions with pH ranges of 6.8-8.6, resembling physiological conditions. The reaction of 1 with 1-substituted cyclohexanones gave the (1′S,2′R)-isomer, diastereoselectively.

Full text of DOI:10.1016/j.tetlet.2012.02.046

Tetrahedron Letters 53 (2012) 2129–2131
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Tetrahedron Letters
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Nitrone formation in phosphate buffer and aqueous solutions: novel
chemistry inspired by a natural product
Acharavadee Pansanit a, Nattha Ingavat a, Thammarat Aree b, Chulabhorn Mahidol a,c, Somsak
Ruchirawat a,c,d, Prasat Kittakoop a,c,d,
⇑
a Chulabhorn Graduate Institute, Chemical Biology Program, Vibhavadi-Rangsit Road, Laksi, Bangkok 10210, Thailand
b Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
c Chulabhorn Research Institute, Vibhavadi-Rangsit Road, Laksi, Bangkok 10210, Thailand
d Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitrones are formed from the reaction of aspergillusol A (1) and a ketone/aldehyde in phosphate buffer
and aqueous solutions with pH ranges of 6.8–8.6⁄, resembling physiological conditions. The reaction of 1
with 1-substituted cyclohexanones gave the (10S ,20R⁄)-isomer, diastereoselectively.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 January 2012
Revised 1 February 2012
Accepted 10 February 2012
Available online 17 February 2012
Keywords:
Nitrone
Reaction in water
Reaction in aqueous solution
Green chemistry
Natural products
Nature deploys wisely the molecular architecture of natural
products enabling them to perform functions essential for the
survival of living organisms under both threatened and normal
environments. Examples include antibiotics to combat harmful
microbes, hormones regulating vital functions, and pheromones
for sexual communication among insects. However, particular
functions or properties of natural products are sometimes unex-
pected and these unprecedented properties are utilized for research
and industrial benefits. For instance, cinchona alkaloids and proline
have significant impact in chemical syntheses as they are robust
catalysts for asymmetric synthesis, which is important in modern
chemistry and in chemical/pharmaceutical industries.1–3 In the
present Letter, we report novel chemistry inspired by a unique
property of the natural product, aspergillusol A (1) (also known as
JBIR-25), a metabolite of the fungus Aspergillus aculeatus4 and
Hyphomycetes sp.5 We found that cyclic nitrones (e.g., 2 and 3)
could be easily formed from the reaction of 1 and a ketone/aldehyde
under conditions that resemble physiological environments (pH
ranges of 6.8–8.6). Excellent diastereoselectivity was obtained from
the reaction of aspergillusol A (1) and 1-substituted cyclohexa-
nones, providing only the (10S⁄,20R⁄)-diastereoisomer.
(1). We tried to hydrolyze one of the ester bonds of 1 using a lipase
enzyme in phosphate buffer (0.1 M, pH 6.8). Due to the insolubility
of 1 in phosphate buffer, we added acetone (30%, v/v) to the reac-
tion mixture in order to increase the solubility of 1. We expected to
obtain erythritol and 4-hydroxyphenylpyruvic acid oxime, but in-
stead, we observed a quantitative yield of nitrone 2 and a tetraol
(erythritol). However, further examination revealed that the lipase
enzyme was not involved in the nitrone formation because a quan-
titative yield of nitrone 2 was also obtained without the addition of
lipase.
O
OH
O
OH
N
HO
HO
O
OH
N
O
N
2
1
2
2'
O
3'
3
1' O
O
4
N
OH
O
HO
O
O
S
Aspergillusol A (1) or JBIR-25
OH
3
We serendipitously discovered an anion-mediated nitrone for-
mation reaction while exploring the chemistry of aspergillusol A
We hypothesized that an anion might mediate this reaction and
consequently performed the reaction in various solutions using 2-
(methylthio)cyclohexanone as a ketone substrate (Table 1).
⇑
Corresponding author. Tel.: +66 86 975 5777; fax: +66 2 574 0622/1513.
E-mail address: prasat@cri.or.th (P. Kittakoop).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.046

2130
A. Pansanit et al. / Tetrahedron Letters 53 (2012) 2129–2131
Table 1
O
O
Yield of nitrone 3 from the reaction of 2-(methylthio)cyclohexanone and 1
O
R1
O
O
N
N
O
O
O
R2
HO
S
HO
R1
O
Aspergillusol A (1)
4, R1 = Me, R2 = Et
5, R1 = Me, R2 = Pr
6, R1 = Et, R2 = Et
7, R1 = Me, R2 =
+
N
S
R2
R3
O
HO
9, R1 = H, R2 = H, R3 = H
3
10, R1 = H, R2 = H, R3 = tert-butyl
11, R1 = H, R2 = H, R3 = Ph
12, R1 = Me, R2 = H, R3 = H
13, R1 = H, R2 = Me, R3 = H
14, R1 = H, R2 = H, R3 = Me
15, R1 = COOMe, R2 = H, R3 = H
16, R1 = H, R2 = COOMe, R3 = H
17, R1 = H, R2 = COOH, R3 = H
O
Entry
Buffer/solutiona
Conc (M)
pH
Yieldb (%)
O
21, R1 = H, R2 = Me
22, R1 = H, R2 = Et
1
2
3
4
5
6
H2O
—
—
0
Phosphate buffer
Sodium citrate
Sodium acetate
Sodium sulfate
Sodium nitrate
0.1
0.1
0.1
0.1
0.1
6.8
8.6
7.8
7.9
7.5
78
84
71
30
18
23, R1 = H, R2
=
O
O
a
30% EtOH was added to the reaction mixture in order to increase the solubility
N
O
of the ketone substrate.
O
HO
HO
b
The yield refers to 2 mol of 3 from 1 mol of 1.
8
O
N
O
O
HO
Product 3 was formed in the presence of 0.1 M sodium salts of
citrate (84%), acetate (71%), sulfate (30%), and nitrate (18%), while
3 was not obtained in H2O (Table 1). Interestingly, the pH ranges of
the solution/buffer were 6.8–8.6, which were relatively mild condi-
tions, resembling those of biological systems. These experiments
support the fact that anions play a role in nitrone formation from
1 and ketones. We further investigated the reaction between
aspergillusol A (1) and various ketones and aldehydes in phosphate
buffer (0.1 M, pH 6.8) and sodium citrate solution (0.1 M, pH 8.6);
in some cases, EtOH (30%, v/v) was added to increase the solubility
of non-polar ketones/aldehydes (Table 2).6 As expected, aliphatic
and cyclic ketones/aldehydes reacted with 1 in both phosphate
buffer and citrate solution with the yields ranging from 3% to
>99% (Table 2, Fig. 1). It should be noted that cyclohexanone and
its derivatives, except those with a bulky group (menthone), gave
higher yields (>70% in most cases) (Table 2). However, ketones/
19
O
O
N
X
O
O
N
3, X= S
O
18, X= O
HO
20
Figure 1. Nitrone products 3–23.
benzophenone, 1-(furan-2-yl)ethanone, benzaldehyde and its
derivatives, and carvone), as well as those with a bulky group at
position 2, (+)-methyl-(R)-3-(1-methyl-2-oxocyclohexyl) propio-
nate, did not react with 1. Interestingly, 3-oxo-1-cyclohexanecar-
boxylic acid did not react with 1 to give nitrone 17, while its
corresponding methyl ester reacted with 1 to give the nitrone 16
(Table 2).
aldehydes with
a conjugated double bond (acetophenone,
We next turned our attention to the issue of diastereoselectivi-
ty. We examined the stereochemistry of cyclic nitrone products
when using chiral cyclohexanone derivatives as substrates. Theo-
retically, when using 1-substituted cyclohexanone as a model sub-
strate, the nitrone products generated from this reaction should
exist as four possible isomers, consisti⁄ng of two pairs of diastereo-
mers designated as (10S⁄,20R⁄) and (10R ,20R⁄) (Fig. 2). The (10S⁄,20R⁄)
diastereomer contains two enantiomers, the (10S,20R)-isomer and
its mirror image (10R,20S), while the diastereomer (10R⁄,20R⁄) has
two enantiomers, the (10R,20R)-isomer and its mirror image
(10S,20S). Surprisingly, a single nitrone product was generated from
the reaction of 1 and chiral cyclohexanones, indicating the diaste-
reoselectivity obtained from this reaction. Fortunately, nitrone 3
obtained from the reaction of 2-(methylthio)cyclohexanone and
1 could be crystallized and subjected to single crystal X-ray analy-
sis,7 which revealed the structure as the (10S⁄,20R⁄)-isomer 3
( Fig. 3). In the solid state, 3 is stabilized by intermolecular
Table 2
Yield of nitrones from the reaction of 1 and various ketones or aldehydes in phosphate
buffer and sodium citrate solution
Ketone/aldehydea
Product Yield (%) PPb Yield (%) Ctc
Acetone
2-Butanone
Methyl propyl ketone
3-Pentanone
Methyl acetoacetate
Cyclopentanone
2
>99
49
38
50
25
12
90
72
75
64
77
93
69
>99
57
36
76
20
13
83
67
72
92
88
90
58
4
5
6
7
8
9
Cyclohexanone
4-tert-Butylcyclohexanone
4-Phenylcyclohexanone
2-Methylcyclohexanone
3-Methylcyclohexanone
4-Methylcyclohexanone
Methyl cyclohexanone-
2-carboxylate
10
11
12
13
14
15
Methyl cyclohexanone-
3-carboxylate
16
66
43
3-Oxo-1-cyclohexanecarboxylic acid 17
0
0
HO
2-(Methylthio)cyclohexanone
2-Methoxycyclohexanone
(+)-Menthone
3
75
92
3
84
80
8
10
>99
95
95
18
19
20
21
22
23
O
(-)-Menthone
3
O
O
Acetaldehyde
>99
94
94
N
O
N
R
1'
Propanaldehyde
O
R
1'
O
2-Methylbutylaldehyde
HO
2'
2'
a
30% EtOH was added to the reaction mixture, except in the cases of acetone and
acetaldehyde.
(1'S*,2'R*)-isomer
(1'R*,2'R*)-isomer
b
PP = phosphate buffer, 0.1 M, pH 6.8.
Ct = citrate solution, 0.1 M, pH 8.6.
Figure 2. Possible diastereomers generated from the reaction of
substituted cyclohexanone derivative.
1 and a 1-
c

A. Pansanit et al. / Tetrahedron Letters 53 (2012) 2129–2131
2131
logical molecules which has to be performed under physiological
conditions.
Acknowledgements
A.P. is a student under the Royal Golden Jubilee Ph.D. Program.
P.K. is supported by The Thailand Research Fund and the Center of
Excellence on Environmental Health and Toxicology, Science &
Technology Postgraduate Education and Research Development
Office (PERDO), Ministry of Education. T.A. is grateful to the Na-
tional Research University Project (FW657B) and the Thai Govern-
ment Stimulus Package 2 (TKK2555).
Supplementary data
Figure 3. ORTEP plot of the nitrone 3.
Supplementary data (experimental procedure for nitrone for-
mation, spectroscopic data, and 1H and 13C NMR spectra of new
compounds) associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2012.02.046.
hydrogen bonding, and H-20 of 3 is axial in the solid state. The X-
ray crystallographic analysis also revealed the inversion symme-
try-related enantiomer of 3, suggesting that th⁄is reaction had no
enantiomeric selectivity; therefore the (10S⁄,20R )-isomer of 3 was
a racemic mixture of the (10S,20R)-enantiomer and its mirror image
(10R,20S). At this stage, we conclude that the reaction ⁄of 1 and chiral
cyclohexanones ⁄gives diastereoselectively the (10S ,20R⁄)-isomer,
and not the (10R ,20R⁄)-isomer (Fig. 2).
References and notes
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Although several of the cyclic nitrones reported here have been
previously prepared, the conditions employed were not under
physiological conditions. For example, thermolysis of isonitroso
Meldrum’s acid,8–10 or in organic solvents (CH2Cl2 or CH3CN).11
Moreover, previous syntheses of the cyclic nitrones did not occur
with diastereoselectivity.8–10 The present study showed that nitro-
nes were formed diastereoselectively in water under physiological
conditions (pH ranges of 6.8–8.6). The reaction mechanism for this
nitrone formation is not known. However, we hypothesize that the
mechanism for the nitrone formation in water possibly involves
the nucleophilic addition of the oxime lone pair to the ketone car-
bonyl giving a zwitterion intermediate from which the oxime OH
proton is removed at mild basic pH (or removed by an anion) thus
giving an anion which attacks the ester carbonyl, leading to a 5-
exo-trig cyclization. We could not rule out a mechanism involving
the cycloaddition of a nitrosoketene intermediate and ketones,
which resembles the reaction mechanism of ketones and nitro-
soketene generated from the thermolysis of isonitroso Meldrum’s
acid.8–10,12
Although we do not know the precise role of aspergillusol A (1)
in biological systems and why the fungus A. aculeatus accumulates
large amounts of 1 in its cells,4 it is known that living cells produce
reactive aldehydes and ketones when they are under oxidative
stress,13 and that in humans, reactive aldehydes and ketones are
produced as a result of oxidative stress in several disease pro-
cesses.14 Since fungal cells contain several anions (including phos-
phate) and some ketones/aldehydes, they should have particular
mechanisms to control nitrone formation. We propose that fungi
possibly use aspergillusol A (1) to trap unwanted reactive alde-
hydes and ketones derived from oxidative stress which are harmful
to cells.
4. Ingavat, N.; Dobereiner, J.; Wiyakrutta, S.; Mahidol, C.; Ruchirawat, S.;
Kittakoop, P. J. Nat. Prod. 2009, 72, 2049–2052.
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6. Experimental details: (a) without the addition of EtOH and (b) with the addition
of EtOH). (a) To a solution of 1 (20.3 mg, 0.042 mmol) in acetone (0.75 mL) was
added 0.75 mL of phosphate buffer (0.1 M, pH 6.8) at room temperature. The
mixture was stirred at room temperature for 18 h, and then extracted with
EtOAc (3 ꢀ 3 mL). The EtOAc layers were combined and evaporated, yielding
nitrone 2 (20.0 mg). (b) Compound 1 (41.5 mg, 0.087 mmol) was dissolved in
1.0 mL of phosphate buffer (0.1 M, pH 6.8) and 0.45 mL of EtOH. To the solution
of
1 was added 2-methoxycyclohexanone (0.31 mmol, 3.7 equiv) at room
temperature. The mixture was stirred at room temperature for 18 h, and then
purified by reversed-phase HPLC, eluting with a gradient solvent system of
MeCN–H2O (35–100% MeCN), furnishing nitrone 18 (49.0 mg).
7. X-ray crystallographic analysis of 3: Crystals of 3 were obtained from the slow
solvent evaporation of ethanol without a solvent of crystallization in the
monoclinic space group P21/n (no. 14) with unit cell constants a = 12.5282(4) Å,
b = 7.1080(2) Å, c = 17.7905(5) Å, b = 96.202(1)°, V = 1574.98(8) Å3, Z = 4,
Dcalc = 1.355 g/cm3, MW = 321.38. A colorless block-like single crystal of 3
with dimensions 0.28 ꢀ 0.45 ꢀ 0.50 mm3 was mounted with epoxy glue on the
tip of a glass fiber. The X-ray diffraction experiment was performed at 298(2) K
using a Bruker X8 APEX II Kappa CCD area-detector diffractometer with Mo K
a
radiation (k = 0.71073 Å). A total of 8890 reflections were collected, integrated,
reduced by SAINT+, corrected for Lorentz, polarization and absorption effects,
and scaled by SADABS (Bruker Software Suite, APEX2, SAINT+ and SADABS.
Bruker AXS Inc., Madison, Wisconsin, USA, 2005) to yield 3935 unique
reflections (Rint = 0.022). The structure was solved by direct methods and
refined with full-matrix least squares on F2 using SHELXL-97 (Sheldrick, G.M.
Acta Cryst. 2008, A64, 112). Final R1(F2) = 0.039 and wR(F2) = 0.106 for 3020
reflections with F2 >2 (F2). Data have been deposited with the Cambridge
r
Crystallographic Data Centre (CCDC 821363). Copies of these data can be
obtained, free of charge, on application to the CCDC via www.ccdc.cam.ac.uk/
conts/retrieving.html (or 12 Union Road, Cambridge CB2 1EZ, UK, fax: +44 1223
336033, e-mail: deposit@ccdc.cam.ac.uk).
8. Katagiri, N.; Sato, H.; Kurimoto, A.; Okada, M.; Yamada, A.; Kaneko, C. J. Org.
Chem. 1994, 59, 8101–8106.
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10. Katagiri, N.; Kurimoto, A.; Yamada, A.; Sato, H.; Katsuhara, T.; Takagi, K.;
Kaneko, C. J. Chem. Soc., Chem. Commun. 1994, 281–282.
11. Flores, M. A.; Bode, J. W. Org. Lett. 2010, 12, 1924–1927.
12. Matsui, H.; Zückerman, E. J.; Katagiri, N.; Kaneko, C.; Ham, S.; Birney, D. M. J.
Phys. Chem. A 1997, 101, 3936–3941.
13. Grimsrud, P. A.; Xie, H.; Griffin, T. J.; Bernlohr, D. A. J. Biol. Chem. 2008, 283,
21837–21841.
In summary, we have demonstrated that nitrone formation oc-
curs in phosphate buffer and aqueous solutions that resemble
physiological conditions. The reaction of 1 with chiral cyclohexa-
nones provided good yields of nitrones and excellent diastereose-
lectivity. This reaction may have potential applications in click
chemistry, as well as in bioorthogonal chemistry for labelling bio-
14. Ellis, E. M. Pharmacol. Ther. 2007, 115, 13–24.