Studies of nitrile oxide cycloadditions, and the phenolic oxidative coupling of vanillin aldoxime by Geobacillus sp. DDS012 from Italian rye grass silage

Article abstract of DOI:10.1039/b716915a

During studies directed towards the discovery of nitrile hydrolysing enzymes from thermophiles, vanillin aldoxime was incubated with the thermophilic organism, Geobacillus sp. DDS012 isolated from Italian rye grass (Lolium multiflorum) silage. The predominant product was a dihydro-dimer, which could only be characterised by LC-MS. This was initially imagined to be the product of cycloaddition of vanillin aldoxime with the corresponding nitrile oxide, but preparation of the supposed adduct and model studies excluded this possibility. The rate constant for the second order dimerisation of 4-O-acetyl vanillin nitrile oxide was measured (1.21 × 10^-4 M^-1 s ^-1, 0.413 M, 25 °C) and the ¹³C-NMR signal for the nitrile oxide carbon was observed (δ_C 34.4, br. t ¹J¹³C,¹⁴N circa 50 Hz). Treatment of vanillin aldoxime with potassium persulfate and iron sulfate gave material with the same LC-MS properties as the natural product, which is therefore identified as 5,5′-dehydro-di-(vanillin aldoxime) 1d formed by phenolic oxidative coupling. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b716915a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Studies of nitrile oxide cycloadditions, and the phenolic oxidative coupling of
vanillin aldoxime by Geobacillus sp. DDS012 from Italian rye grass silage†‡
b
David R. Kelly,*a Simon C. Baker,§ David S. King,a Deepa S. de Silva,b Gwyn Lordb and Jason P. Taylorb
Received 2nd November 2007, Accepted 11th December 2007
First published as an Advance Article on the web 21st January 2008
DOI: 10.1039/b716915a
During studies directed towards the discovery of nitrile hydrolysing enzymes from thermophiles,
vanillin aldoxime was incubated with the thermophilic organism, Geobacillus sp. DDS012 isolated from
Italian rye grass (Lolium multiflorum) silage. The predominant product was a dihydro-dimer, which
could only be characterised by LC-MS. This was initially imagined to be the product of cycloaddition
of vanillin aldoxime with the corresponding nitrile oxide, but preparation of the supposed adduct and
model studies excluded this possibility. The rate constant for the second order dimerisation of
4-O-acetyl vanillin nitrile oxide was measured (1.21 × 10−4 M−1 s−1, 0.413 M, 25 ◦C) and the 13C-NMR
signal for the nitrile oxide carbon was observed (dC 34.4, br. t 1J13C,14N circa 50 Hz). Treatment of
vanillin aldoxime with potassium persulfate and iron sulfate gave material with the same LC-MS
properties as the natural product, which is therefore identified as 5,5ꢀ-dehydro-di-(vanillin aldoxime) 1d
formed by phenolic oxidative coupling.
of the product from spent reagent is expensive. Consequently
there has been much interest in developing enzymatic methods
Introduction
for nitrile hydrolysis, which proceed under mild conditions.3 The
most notable success is the Nitto process for the hydrolysis of
acrylonitrile to acrylamide by R. rhodochrous J1 whole cells, which
contain a nitrile hydratase that can constitute up to 50% of the
soluble protein in the cell. The productivity is greater than 7 Kg
of acrylamide per g of dry cells and production is currently circa
30 000 tons per annum.4
There are two classes of enzymes, which hydrolyse nitriles 2.¶
Nitrilases (EC 3.5.5.1) hydrolyse nitriles directly to carboxylic
acids 4 without forming free amide intermediates, whereas nitrile
hydratases (EC 4.2.1.84) give the amide 3. Almost invariably, the
amide 3 so formed is hydrolysed to the carboxylic acid 4 by a
co-expressed amidase (EC 3.5.1.4).1 In microbiological studies
prospecting for nitrile hydrolysis activity, nitrile hydratases are
found far more commonly than nitrilases. This appears to be a
consequence of limited genetic capability, because in a survey of
150 sequenced bacterial genomes, only ten were found to harbour
nitrilase genes.2
Screening of more than six hundred biotope specific environ-
mental DNA libraries yielded 137 characterised nitrilases. The ac-
tivity of a few has been optimised for synthetic applications by site-
directed saturation mutagenesis, but it remains to be demonstrated
how many will be useful synthetically.5 Screening of environmental
samples for nitrile hydrolysis activity with representative nitriles is
a powerful selection strategy, which avoids the necessity to develop
expensive high speed screening techniques. Unfortunately, nitriles
are almost invariably toxic to whole cells reducing the chances
of inducing nitrile hydrolysis activity by feeding experiments. We
have employed the strategy of feeding aldoximes 1, which undergo
in situ dehydration to nitriles, catalysed by aldoxime dehydratase.6
Our intention is to reduce the nitrile concentration below the toxic
threshold7 and moreover aldoximes are generally more soluble
than nitriles in aqueous media. A survey of 975 micro-organisms
has shown that aldoxime dehydratase activity is almost invariably
linked to nitrile hydrolysis activity.8 We have adopted vanillin
aldoxime 1b as the standard substrate for screening for nitrilases,
because the hydroxyl and methoxy groups confer extra solubility,
and at least thus far, aromatic nitriles have been found to be the
preferred substrates for most nitrilases.1
The hydrolysis of nitriles to amides and carboxylic acids requires
vigorous conditions with strong acids or bases and separation
aThe Tatem Laboratories, School of Chemistry, Cardiff University, Cardiff,
CF10 3AT, UK. E-mail: KellyDR@Cardiff.ac.uk
bSchool of Biological and Chemical Sciences, Birkbeck College, University
of London, Malet Street, London, WC1E 7 HX, UK
† The HTML version of this article has been enhanced with colour images.
‡ Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b716915a
Using enzymes from thermophilic species in biocatalysis confers
considerable benefits.9,10 The solubility of hydrophobic substrates
and rates of diffusion increase as temperature increases and
because mesophilic species cannot tolerate high temperatures,
it is easier to prevent contamination in whole cell biotransfor-
mations. Most thermophilic organisms have been isolated from
§ Current address: School of Life Sciences, Oxford Brookes University,
Headington Campus, Gipsy Lane, Oxford OX3 0BP, UK
¶ Compounds are numbered using a reverse Markush system. If no
alphabetical character is present, the carbon chain is undefined and hence
the discussion refers to the generalised chemistry of the functional group
indicated.
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If silage is exposed to air21 then yeast, fungi and bacteria cause
aerobic decomposition.22 If this occurs in bulk silage, (e.g. due to
leaks in the container), particularly if it too dry (25–45% water)
the temperature rises excessively. 70 ◦C is considered to be an
indication of danger, 82 ◦C the onset of charring and 93 ◦C* will
cause severe losses of material and fire.
We have investigated aerobically decomposing silage as a source
of thermophilic micro-organisms with nitrile hydrolysis activity.23
Surprisingly, silage has rarely been investigated for thermophilic
organisms. The first thermophile was isolated by Miquel from the
waters of the Seine in 1879, and it was demonstrated that the
self heating of hay was due to microorganisms in 1907.24 Silage
was recommended as a good source of thermophiles in 1919,25
and a review in 1921 reported four previous studies in which
thermophilic bacteria growing at 60 ◦C or above were isolated from
silage or hay,26 but subsequently the area has been largely fallow.
Nearly fifty years later, thermophilic “Bacillus caldotenax” and “B.
caldovelox” strains were isolated from silage and used as a source
of novel bacteriophages.27,28 Around the same time Thermoactino-
myces candidus (synonym T. vulgaris) a thermophilic actinomycete
geothermally heated environments in exotic locations, such as hot
springs and deep sea vents, but many of these are now covered
by strict licenses for bioprospecting. However under favourable
conditions, comparable temperatures are generated by the aerobic
decomposition of plant materials in more mundane processes such
as composting and ensilaging.
◦
During composting a succession of microorganisms11 partici-
pate in decomposition as the temperature rises to a maximum of
65–82 ◦C. At the lower end of this range (65–69 ◦C), Geobacillus
(previous synonym Bacillus) stearothermophilus strains (which are
obligate thermophiles) frequently predominate,12,13 and above this
(TOpt 50 C), was first isolated from an air-conditioner duct, but
was subsequently found in moldy silage dust. The proteolytic
antigens from this species29 and others30 are probably one of
the causes of hypersensitivity pneumonitis (e.g. farmers’ lung,
humidifier fever). More recently, Enterococcus faecalis K-4, was
isolated from grass silage in Thailand. Essentially identical cell
◦
temperature (70–75 C) Thermus thermophilus strains (e.g. HB8,
◦
facultative thermophiles) and several thermophilic Bacillus sp.12,14
are found in high numbers.15 Although many other species have
identified,16 there is currently much debate over the virtues of
different methods (e.g. culture, 16S rDNA, chemical markers etc.)
for determining decomposition-significant microbial populations,
rather than those which are amenable to easy identification.17
Composting differs from ensilaging by being predominantly
aerobic, consequently much of the plant material is lost as
carbon dioxide. It is also strongly affected by scale; as the weight
of biomass increases, anoxic regions develop at the bottom of
the compost pile, which results in the emergence of different
microbial populations. Composts also tend to be composed of
heterogeneous material at different stages of decomposition. Even
in the laboratory, where dog food is the preferred substrate,
composting is subject to considerable variation in microbial
populations and rates of degradation.18 In contrast, silage is
generally composed of a single plant species, which is harvested
at a specific stage of maturity and immediately ensilaged, with
controlled water content.
growth occurred in the range 30–45 C, but was greatly reduced
at 50 ◦C and maximal amounts of◦a bacteriocin (an antibacterial
peptide) were produced at 43–45 C.31 By the usual definitions,
both of these species are on the cusp of the temperature range
between mesophiles (10–47 ◦C) and thermophiles (40–68 ◦C).32
Results and discussion
1. Microbiology and biotransformation
Italian rye grass silage samples were incubated on four different
media (Thermus 878; Difco nutrient broth, Castenholz, Luria-
Bertani) at 70 ◦C in an orbital shaker for 24 hours. Aliquots
from enrichment cultures were serially diluted and streaked on
Thermus 878 agar plates to give 156 colonies with dissimilar colony
morphology, which were isolated as pure cultures. Of these, 17
cultures were selected for further investigation on the basis of
colony morphological diversity. All the species were Gram positive
or Gram variable rods, except for one which was a Gram variable
coccid (DDS016) and all produced acid from glucose, fructose
and mannose as assessed by reduction in pH. Carbohydrate
assimilation profiles (API 50 CHB, bioMe´rieux, France) and 16S
rDNA analysis indicated that all the species could be putatively
assigned to the genus Geobacillus (formerly Bacillus group 5).33
All were obligate thermophiles (65 ◦C), except for one (DDS011)
Silage is manufactured from crops such as grass, maize, oats,
barley, peas, corn or alfalfa (water content 55–70%), which
are compressed (in a silo, clamp or bags) to exclude atmo-
spheric oxygen and allowed to undergo anaerobic fermentation
by indigenous, epiphytic microorganisms at 20–35 ◦C. These
are frequently augmented in modern agricultural practice, by
inocula of Lactobacillus, Pediococcus and Enterococcus sp.19,20 to
improve reliability and reproducibility. Initially a burst of aerobic
decomposition occurs, but when the oxygen in the spaces between
the crop is exhausted, anaerobic fermentation commences. In the
early stages, sugars are converted to acetic, propionic or butyric
acids and in the later stages to lactic acid. The pH drops to around
4, which prevents further degradation, and allows silage to be
stored for feeding to cud-chewing animals such as cattle and sheep.
◦
which also grew at 37 C. Sixteen of the isolates were capable of
growth as measured by increase in turbidity (OD 600 nm, 24 h)
on ammonium chloride or vanillin aldoxime 1b as sole sources of
nitrogen, and one (DDS014) was only capable of slow growth on
ammonium chloride. For all the other isolates, vanillin aldoxime
* These seemingly overly precise values are a consequence of conversion
from Fahrenheit to Celsius; 180 ◦F = 82 ◦C, 200 ◦F = 93 ◦C.
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1b as sole source of nitrogen caused a significant increase in growth
yield (p < 0.001) compared to the control (succinic acid), but 35–
50% less than ammonium chloride (p < 0.01).
the rate of transformation of 5 mM vanillin aldoxime 1b, was
0.35 mM h−1 mg−1 of dry cells, but thereafter sharply declined to
24 hours and was complete at 48 hours. After 60 hours, no traces
of any compounds were detected by HPLC-UV (254 nm). At 24 h,
vanillin aldoxime 1b (5 mM) was converted by isolate DDS012
to a mixture containing traces of vanillin nitrile 2b, vanillamide
3b, vanillinic acid 4b and vanillin 5b, plus a new component 1d
with a HPLC retention time which did not match any of the
standards or conceivable impurities. The vanillin derivatives 2b–5b
were identified by HPLC-QTOF-MS (ESI+) by comparison with
commercial samples except for vallinamide 3b.
There is only one reported synthesis of vallinamide 3b via a
five step sequence; in which vanillin 5b was converted to the
aldoxime 1b, O-acetylated and dehydrated to give the 4-O-acetyl
nitrile 2c. Cleavage of the O-acetyl group gave vanillin nitrile 2b
and electrophilic addition of butanol in hydrogen chloride gave
the butyl amidoester hydrochloride salt 6b, which was finally
pyrolysed at 170 ◦C to give vanillamide 3b in a reported 95%
yield.37 We repeated this procedure, but found that addition to
the nitrile was difficult to achieve in good yield and that the
pyrolysis gave no more than a trace of impure product. The
obvious synthesis by treating vanillic acid chloride with ammonia
is reported to fail,37 plausibly due to 1,6-elimination of HCl to
give a 1,4-quinoketene. We found that a convenient procedure
was the DCC mediated coupling of vanillic acid 4b with N-
hydroxysuccinimide in DMF. The adduct was soluble whereas
most of the DCU precipitated and was removed by filtration. The
concentrated solution was then added to concentrated ammonia
from which vanillamide 3b precipitated and was recrystallised to
high purity from ethanol in 69% yield.
The predominant component 1d gave peaks in the mass
spectrum at m/z 333 (M + H), 355 (M + Na). Analysis of the
accurate mass measurement of the ion at m/z 333 gave seven
possible formulae ( 10 ppm), from which the best fit and most
plausible corresponded to (2 × vanillin aldoxime − 2H), i.e. a
dehydro-dimer. Thefunctionalgroups attachedtothevanillinring,
could yield many compounds fitting this formula, from reactions
such as cycloaddition, peroxide formation or phenolic oxidative
coupling. Given that we were seeking new nitrile hydrolysis
activity and we were aware of the well precedented dehydration
of aldoximes to nitriles by micro-organisms,6,8 we reasoned that
the aldoxime 1b would undergo dehydration to the nitrile 2b and
oxidation to the nitrile oxide 8b. Nitrile oxides are well known
to undergo cyclodimerisation, which in the current case would
give the cycloadducts 9b–11b, which in turn could be reduced to
the dehydro-dimers 12b–16b. However under abiotic conditions
the rate of cyclodimerisation of nitrile oxides is slow and hence
the biotic equivalent would require high local concentrations or
catalysis of a bimolecular reaction of two highly reactive species.
An apparently better alternative was the cycloaddition of nitrile
oxide 8b to the aldoxime 1b to give dehydro-dimer cycloadducts
12b–16b directly. Such cycloadditions are unknown in Nature and
rare in abiotic synthetic chemistry, but this proposal was more
plausible, because it required the reaction of the highly reactive
nitrile oxide 8b with the more stable aldoxime 1b, which at least in
the early stages of the process would be present in high excess.
Aromatic nitrile oxides 8 most commonly cyclodimerise to
give 3,4-diaryl-1,2,5-oxadiazol-2-oxides 9, but 3,5-diaryl-1,2,4-
oxadiazol-4-oxides 10 are occasionally found as byproducts.
The ability of each of the seventeen isolates to transform vanillin
aldoxime 1b, was monitored by HPLC-UV at 254 nm. Traces of
hydrolysis products were formed (see below), but the predominant
product was a new, unknown compound (later shown to be the
dehydro-dimer 1d) that did not match any of the standards. The
degree of transformation was quantified from the UV response of
the dehydro-dimer 1d, relative to that of the initial concentration
of vanillin aldoxime 1b (5 mM). At 24 hours, conversion of vanillin
aldoxime to the unknown was a minimum of 11% (DDS017) with
the remainder in the range 21 to 54%. At 48 hours, the degree of
conversion was evenly distributed in the range 49 to 96% for all
seventeen isolates. Even DDS014 which was completely incapable
of growth on vanillin aldoxime 1b alone, nevertheless was capable
of forming the dehydro-dimer 1d, albeit at the lower end of the
range observed (24 h, 25%; 48 h 56%). We were not able to repeat
the HPLC-UV measurements after the dehydro-dimer 1d was
correctly identified. Moreover, the UV spectra of the dehydro-
dimer 1d in methanol showed a disproportionately increased
adsorption at 230–240 nm as the concentration increased, however
the maximum which was in the range 260–270 nm (e circa 25 000)
was about twice that of vanillin aldoxime 1b (270 nm, 12 920).
Consequently, the percentage conversions reported above are a
rough but fair measure of the conversion of vanillin aldoxime 1b.
Isolate DDS012 had average activity at 24 hours (31%), but
had the highest activity at 48 hours and was selected for further
investigation. When vanillin aldoxime 1b was incubated with heat-
killed DDS012 cells no transformation occurred. DDS012 grew
on vanillin aldoxime 1b as sole source of nitrogen in the range
1–10 mM, with an optimum of 5 mM (0.0312 doubling h−1). Slow
growth occurred in the control which lacked vanillin aldoxime
(0.0125 doubling h−1) but 25 or 50 mM vanillin aldoxime
inhibited virtually all growth (0.002 doubling h−1). The general
bacteriological properties of DDS012 are fairly typical of the
group as a whole; it is a Gram positive, small curved rod, with no
spores and an obligate thermophile/facultative hyperthermophile.
The best species match for carbohydrate assimilation profile (API
50 CHB) was Bacillus firmus (ID% 94.4, T-index 0.61) and by
16S rDNA, Geobacillus thermodenitrificans OHT-1 (97% sequence
similarity, Genbank Accession number EF426762). However the
type stain of G. thermodenitrificans is able to produce acids from all
the sugars tested, but DDS012 was unable to utilise the pentoses:
arabinose, ribose, and xylose, the disaccharides: cellobiose and
lactose, or galactose and inositol.
As the name of the genus implies, Geobacillus is frequently found
in soil samples. G. thermodenitrificans34 is closely related to G.
stearothermophilus which is more commonly isolated from warm
composts and comparable niches. The strain G. thermodenitrificans
OHT-1 has been isolated from horse manure compost under
◦
aerobic conditions at 60 C35 and G. thermodenitrificans CBG-
A1◦, was selected on the basis of L-arabinose isomerase activity at
60 C from Korean compost.36
2. Analysis of biotransformation products
The biotransformation of vanillin aldoxime 1b by DDS012, as
described above was reinvestigated in more detail. Up to 7.5 h
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C; 125.6, 2-C) were reassuringly close to that for 3,6-di(2-pyridyl)-
1,4,2,5-dioxadiazine (dC 160.63, 1-C)52 and more relevantly for
3,6-di(4-tolyl)-1,4,2,5-dioxadiazine (dC 162.8, 1-C), although only
this shift was reported with no supporting data.53
Whereas in the presence of nucleophiles (e.g. pyridine38,39 or
trimethylamine40), addition, cycloaddition, elimination gives 3,6-
diaryl-[1,4,2,5]dioxadiazines 11 or tricyclic derivatives.41 To fur-
ther increase the chemical diversity; dimerisation of benzonitrile
oxide 8a in the presence of excess boron trifluoride gives 3,6-
diphenyl-[1,4,2,5]dioxadiazines 11a, but with only half an equiv-
alent of BF3; 3,5-diaryl-1,2,4-oxadiazol-4-oxides 10a,42,43 whereas
with boron trifluoride etherate, both of these products are formed
plus the isocyanate.44
As far as we are aware there has only been one report of the
cycloaddition of a nitrile oxide to oximes. Benzonitrile oxide 8a
underwent addition to two aldoximes and two ketoximes catalysed
by boron trifluoride etherate to give 4-hydroxy-4,5-dihydro-1,2,4-
oxadiazoles 12,45 although clearly there are other possibilities 13–
16. Of these latter four ring systems, the sole isolated example is 3-
chloro-3,4-diphenyl-3H-furazan-2-ol46 (cf. 15), although another
example may be a minor component of a tautomeric mixture.47
Most of this work was reported before the advent of high
field NMR and some structures for the adducts have been
controversial.48,49 Faced with this melange of possible structures
and a paucity of reliable NMR data, we took prudent refuge on
the safer ground of initial model studies.
To give some assurance that we were truly preparing the nitrile
oxide-aldoxime adduct, a split experiment was devised. Treatment
of oximyl chloride 7a with aqueous sodium bicarbonate as before,
gave a diethyl ether solution of nitrile oxide 8a which was divided
into two. One portion was allowed to stand at room temperature
and gave 3,4-diphenyl-1,2,5-oxadiazol-2-oxide 9a as the sole
1
product after 20 h by H-NMR spectrometry and was isolated
in 58% yield. The other portion was added to (Z)-benzaldoxime
1a but otherwise treated in an identical way. The crude reaction
mixture containing the nitrile oxide–aldoxime adduct 12a, dimer
9a and other products was separated by column chromatogra-
phy. Crystallisation yielded 3,5-diphenyl[1,2,4]oxadiazol-4-ol 12a
(32%), which was unstable in deuterated chloroform. However the
adduct was perfectly stable in deuterated benzene and the NMR
spectra were fully assignable. The NMR chemical shifts for 5-C/H
(dH 6.08, dC 101.7, C6D6) were poorly predicted by chemical shift
increment models (dH 5.64, dC 84.1, CDCl3) even allowing for the
solvent difference. The key signal validating the assignment was
a
13C–1H–3J-correlation signal between 5-H and 3-C (dC 159.2;
C6D6). The mass spectrum (EI+) showed no molecular ion, but
a strong M–H2O peak, consistent with in situ dehydration to 3,3-
diphenyl-1,2,4-oxadiazole 17a.54
3. Cycloaddition of phenyl derivatives
Chlorination of (Z)-benzaldoxime 1a50 with N-chlorosuccimide
in THF yielded the oximyl chloride 7a (82% yield), which could
be stored in the freezer for several weeks without decomposition.
Treatment of the oximyl chloride 7a with aqueous sodium bi-
carbonate gave 3,4-diphenyl-1,2,5-oxadiazol-2-oxide 9a, whereas
treatment with triethylamine and pyridine gave 3,6-diphenyl-
[1,4,2,5]-dioxazine 11a as reported previously.38 The 13C-NMR
data for 3,4-diphenyl-1,2,5-oxadiazol-2-oxide 9a in DMSO-d6
was identical to partial data reported previously (dC 114.5, 3-C;
dC 156.6, 4-C).51 Virtually nothing could be deduced from the
1H-NMR spectrum, because of overlap, however in deuterated
chloroform, virtually all the signals could be assigned. The key
NMR data for 3,6-diphenyl-[1,4,2,5]-dioxazine 11a (dC 162.7, 1-
4. Cycloaddition of vanillin derivatives
4-O-Acetyl vanillyl nitrile oxide 8c proved to be very difficult to
handle. After considerable experimentation we found that addition
of the oximyl chloride 7c to a vigorously stirred sodium carbonate
solution, rapid work-up and dissolution in deuterated benzene
allowed us to obtain NMR spectra of the nitrile oxide 8c which
slowly dimerised to the dimer 9c at ambient temperature. In the
first 12 h we were able to detect the 13C-NMR signal for the
nitrile oxide carbon at dC 34.4, which appeared as a very broad
triplet with peak heights in the ratio 1 : 1.6 : 1 and a coupling
of circa 50 Hz (1J13C,14N). Coupling to 14N should yield three
equally intense, but broad lines due to 14N nuclear quadrupole
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relaxation, however the extreme line broadening causes the height
of the central line to be increased by overlap with the outer
lines. Our results are comparable to those of reported for 15N-
total of 380.07 h (15.8 days) the reaction mixture consisted almost
entirely of dimer 9c, plus traces of unidentified impurities (circa
2%) and nitrile oxide 8c. Ignoring the impurities, the ratio of
nitrile oxide 8c to dimer 9c was 2.8 : 97.2, which corresponds
to 5.86 mM nitrile oxide 8c. Remarkably, the predicted value
from the rate equation was 5.93 mM an error of just 1.2%. In
the sole prior report of the kinetics of dimerisation of nitrile
oxides, rate constants were measured for benzonitrile oxide and
four monosubstituted derivatives, Values ranged from 2.37 (p-
methoxy) through 6.78 (p-chloro) to 8.53 (m-chloro) × 10−3 M−1 s−1
at 40 ◦C in carbon tetrachloride, which is appreciably higher than
the value we measured (0.121 × 10−3 M−1 s−1 at circa 25 ◦C),
even allowing for the temperature difference. At 25 ◦C the rate
constant for the dimerisation of p-chlorobenzonitrile oxide in
carbon tetrachloride was 1.81 × 10−3 M−1 s−1 but in chloroform
only 0.177 × 10−3 M−1 s−1. Hence dimerisation rates in carbon
tetrachloride are up to ten times faster than in other solvents.60
No data was reported for benzene or even a comparable solvent,
so no sensible comparison can be made between the two sets of
data. However by using the data for p-chlorobenzonitrile oxide in
chloroform at 25 ◦C and at 40 ◦C in carbon tetrachloride, the rate
constant for the dimerisation of 4-O-acetyl-vanillin nitrile oxide
in carbon tetrachloride at 40 ◦C is estimated to be circa 4.6 ×
10−3 M−1 s−1 which is in satisfactory agreement with the prior
data. The structure of the dimer 9c was determined from NMR
spectra and by reduction with neat trimethylphosphite at reflux to
yield the symmetrical product; 3,4-diaryl-1,2,5-oxadiazole 18c.
The cycloaddition of 4-O-acetyl vanillyl nitrile oxide 8c with 4-
O-acetylvanillin aldoxime 1c proved to be very difficult and despite
testing a range of reaction conditions, only barely acceptable
results were finally achieved. Initially, 4-O-acetyl vanillyl oximyl
chloride 7c was treated with saturated sodium carbonate in diethyl
ether. Workup gave a solution of nitrile oxide 8c which was
added to a 1 : 1 mixture of 4-O-acetyl vanillin aldoxime 1c and
boron trifluoride etherate according to the literature method.45
An aliquot of the nitrile oxide solution showed a 75 : 25 mixture
2,4,6-trimethylbenzo- (dC 34.9,55 d, 1J-13C,15N 77.5
0.5 Hz,
in CD2Cl256), 2,4,6-trinitrobenzo- (dC 28.9357), aceto- (dC 35.6,
◦
br t, −51 C58) tert-butylaceto- (dC 42.2, br t, > 50 Hz59) and
triphenylmethylaceto-(dC 38.4, br59) nitrile oxides in CDCl3. The
chemical shift of the “nitrile oxide” carbon is best rationalised as
the iminyl carbanion canonical form 8c–II rather than the nitrile
oxide 8c–I.
1
of nitrile oxide 8c to dimer 9c by H-NMR. This measurement
indicates that the solution contained essentially pure nitrile oxide
with little or no dimer, because the time taken to prepare and run
the NMR spectrum, allows time for dimerisation. After two hours
the reaction consisted of a 50 : 50 mixture of nitrile oxide 8c
and the aldoxime 1c, which became a 23 : 77 mixture of dimer
9c and aldoxime 1c after 60 hours. In a control reaction a 1 : 1
mixture of 4-O-acetyl vanillin aldoxime 1c and boron trifluoride
etherate in diethyl ether was stirred for 48 hours and the aldoxime
1c was recovered in quantitative yield. Reverting to the conditions
used for the phenyl nitrile oxide 8a and benzaldoxime 1a were
equally unrewarding. The oximyl chloride 7c was treated with
sodium carbonate in THF and the nitrile oxide solution added
ortho-Disubstituted nitrile oxides are frequently sufficiently
stable that they can be isolated and analysed, however the case
in hand lacks this steric barrier which tempted us to determine the
rate constant for dimerisation. The method of preparation does
not allow an accurate measure of the start time, consequently this
was estimated and used as an additional data point. Fortuitously,
the single O-methyl group of the nitrile oxide was easily distin-
1
guishable from the two of the dimer by H-NMR and there was
no overlap with other signals, consequently integration of these
signals enabled a relatively accurate measure of the composition
of the reaction mixture. Six data points were measured over a
molar composition range; 84 : 16 to 15 : 85 for nitrile oxide 8c :
dimer 9c over 60.75 h, which were analysed by plotting 1/[nitrile
oxide] vs. time, with the assumption of second order dimerisation
kinetics (Fig. 1). The rate constant from linear regression was
0.434 M−1 h−1 or 1.21 × 10−4 M−1 s−1 (R2 = 0.976) for a solution
which was initially 0.413 M (95% C6D6, 5% THF wt/wt). The
initial half life was calculated to be 5.6 h, and the curve fit to
the data indicated a value around 4.8 h (for 66 : 33, 8c : 9c). In
the initial stages the conversion is very rapid and the effect of
the poorly estimated starting time has maximum effect. After a
1
to 1.5 equivalents of aldoxime 1c in dichloromethane. H-NMR
monitoring of aliquots showed a 21 : 79 mixture of nitrile oxide 8c :
aldoxime 1c after 2.5 hours and a 13 : 87 ratio after 23.5 hours and
after workup a 7 : 93 ratio. Curiously, no dimer 9c was observed at
any stage in this reaction. In an attempt to mimic the conditions of
the successful dimerisation of nitrile oxide 8c, essentially the same
reaction was conducted in deuterated benzene. After 2 hours a 1H-
NMR spectrum of an aliquot showed a 9 : 13 : 78 ratio of nitrile
oxide 8c : dimer 9c : aldoxime 1c, which became an 18 : 82 ratio of
dimer 9c : aldoxime 1c after 17 hours. Preparation of scrupulously
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Fig. 1 Nitrile oxide and dimer conversion, % units.
dry nitrile oxide from ethereal solutions, whilst working quickly to
avoid dimerisation is difficult. Consequently, we decided to prepare
the nitrile oxide 8c and react it with the aldoxime 1c in chloroform,
also obtained a second set of NMR data in DMSO-d6 to make
absolutely certain that the structure was rigorously assigned.
When this material was analysed by HPLC-QTOF-MS (ESI+),
the retention time was identical to that of the natural product,
but the mass spectrum was quite different. The natural product
1d showed a high abundance precursor ion (M + H, m/z 333.1)
plus a series of product ions of diminishing abundance down to
m/z 212.1, due to small losses (H2O, CH3OH etc.), whereas the
supposed natural product, showed an almost negligible precursor
ion, two high abundance product ions at m/z 168.1 (plausibly
protonated vanillin aldoxime 1b + H+) and 135.1 and some weaker
ions in between (Fig. 2).
1
which is easier to dry. The crude H-NMR revealed a new down
field singlet and column chromatography gave the desired adduct
12c in 10% yield. The connectivity of the heterocyclic ring was
established from NMR spectra in which C-1, dC 101.1 was attached
3
to a hydrogen at dH 6.07 with a J-13C–1H coupling to a carbon
at dC 159.2 (C-11). Deacetylation with sodium tert-butoxide in
methanol gave the imagined natural product 12b in a miserable
28% yield after column chromatography. The NMR spectroscopic
data for C-1/H-1 and C-9 (C6D6 dH 6.23; dC 101.7; 159.6) were
essentially identical to that of the acetylated precursor and we
5. Phenolic oxidative coupling
At this stage we reassessed the mass spectrum data for the natural
dehydro-dimer and the circumstantial evidence surrounding its
formation. Firstly, the mass spectrum of the heterocyclic dehydro-
dimer 12b clearly showed cleavage of the heterocyclic ring yet the
mass spectrum of the natural product 1d showed only progressive
losses of small fragments, consistent with a single carbon chain.
Secondly the natural dehydro-dimer was only formed with vanillin
aldoxime 1b and no analogous compound was formed from
2,3-dimethoxy- or 2,4-dimethoxybenzaldoxime indicating that
the free phenol substituent of vanillin aldoxime was crucial for
dimerisation formation. Finally, formation of the dimer was
always attended by formation of a yellow pigment, with an
apparently high e-value, because the colour was strong even at high
dilution. Again this could not be isolated, but the yellow colour
was reminiscent of quinones formed by an oxidative process.
Taken together, this admittedly tenuous evidence suggested that
the dehydro-dimer was the product of phenolic oxidative coupling.
Phenol oxidative coupling of vanillin 5c is a well known
process and indeed dehydro-divanillin 5d is a commercial product.
It was first prepared in 1916 using potassium persulfate and
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Fig. 2 HPLC-QTOF-MS/MS (ESI+) spectra of vanillin aldoxime dehydro-dimer 1d (top) and the imagined-natural product, 3,5-di(4-hydroxy-
3-methoxyphenyl)-5H-[1,2,4]oxadiazol-4-ol 12b (bottom).
catalytic ferrous sulfate in almost quantitative yield (97%61),
but repetition of this procedure has been less fruitful (22%62
(23%). Formation of the dialdoxime 1d was extremely slow, using
hydroxylamine and sodium acetate. Nevertheless after 7 days at
50 ◦C, 40% conversion was determined by 1H-NMR and the
dehydro-dimer 1d was isolated (33% yield). Alternatively treatment
of vanillin aldoxime 1b with potassium persulfate and catalytic
ferrous sulfate gave a good yield of crude product 1d (71%), which
was hugely eroded by washing/crystallisation to give material of
high purity (19%). The dehydodimer 1d produced by each route
was identical and identical to the natural product by HPLC-MS,
on the basis of retention time, accurate mass measurement and
mass spectral fragmentation patterns.
&
86%63). Other procedures have used ferric chloride·hexahydrate
(57%64), TEMPO derived oxoammonium salts (85%65) or [(diace-
toxy)iodo]benzene (56%66). The kinetics of octacyanotungstate(V)
ion mediated oxidation in alkaline solution (80%) are first order
in vanillin anion and [W(CN)8]3−.67 It has also been prepared by
the Ullman coupling of 5-bromovanillin with copper powder in
anhydrous DMF, but no yield was reported.68 Biotransformation
based methods are discussed below.
For our own satisfaction we performed the phenolic oxidative
coupling of vanillin with; potassium persulfate and iron sulfate61
and achieved a moderate yield of dehydrodivanillin 5d (47%,
identical to commercial material), plus recovered vanillin 1d
One of the reasons that we initially discounted phenolic
oxidative coupling as a route to a dehydro-dimer 1d was that
we were unable to find an example of this reaction for aryl
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oximes in the literature. It appeared reasonable that the aldoxime
would be become oxidised either to a nitro-compound or some
intermediate oxidation state. However careful reading revealed
that Erdtman had reported that the dehydro-dimer 1d could be
prepared by potassium persulfate and iron sulfate coupling of
vanillin aldoxime 1b. The product was not isolated (and hence
does not appear in the databases) but was immediately acetylated
and dehydrated to the acetylated nitrile 4,4ꢀ-di-O-acetyl-2d.69 This
appears to be the only reported case of phenolic oxidative coupling
of an aryl oxime. However in recent syntheses of spiroisoxazole
sponge metabolites, homobenzylic aldoximes have been used to
capture phenolic radicals and thereby yield spiro-1,2-isoxazolinyl-
monoquinones.70
and Enterococcusfaecium77 and dehydro-divanillinic acid 4d to 5-
carboxyvanillate by the soil bacterium Pseudomonas paucimobilis
SYK-6.78,79
16S rDNA data indicated that isolate DDS012 was related to
Geobacillus thermodenitrificans OHT-1. The closest related species
for which full genetic data is available is G. stearothermophilus.
This was originally reported to express a novel peroxidase from
the perA gene,80 but after sequencing errors in the gene were
corrected, it is now termed Catalase I.81 This is a member of
Class I of the superfamily of heme containing plant, fungal
and bacterial peroxidases, which consists of bacterial catalases
with broad spectrum peroxidase activity. Despite high catalase
activity, they have minimal sequence homology with typical heme
containing monofunctional catalases, but have high sequence
homology to plant ascorbate peroxidase and fungal cytochrome
c peroxidase.82 Catalase I has the interesting property that upon
heating to 70 ◦C for ten minutes it undergoes a conformation
change to a form with higher catalase activity, which only reverts
to the lower temperature form after denaturation, refolding and
reconstitution with heme.83 Clearly, Catalase I is an ideal candidate
for catalysing the oxidative dimerisation of vanillin aldoxime
1b at high temperatures. Perforce the benefit of this process to
the organism is open to many interpretations, but we imagine
that conversion to the highly insoluble dehydro-dimer 1d may
constitute detoxification by sequestration. Moreover consumption
of peroxide is beneficial to all aerobic organisms, but particularly
for thermophiles.
Acknowledgements
We gratefully acknowledge funding of this work by the BBSRC
(grant E17088) and the EPSRC (grant GR/S 25456/01 for a
500 Mhz NMR spectrometer) and for an allocation of time
at the EPSRC National Mass Spectrometry Service Centre at
Swansea University. We thank Dr Jane Nicklin and Professor
Christopher J. Knowles for helpful advice and Robert Jenkins
and Jenny Smale for technical assistance.
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