Determination of Key Hydrocarbon Autoxidation Products by Fluorescence

Article abstract of DOI:10.1021/acs.joc.6b01032

Hydroperoxides and carboxylic acids are key primary products that arise in the autoxidation of hydrocarbons. We have developed a simple approach to rapidly and simultaneously determine both types of products using hydroperoxide- and acid-sensitive moieties conjugated to nonpolar coumarin- and BODIPY-based fluorophores. The coumarin- and BODIPY-conjugated amine probes described here undergo 38- and 8-fold enhancement, respectively, upon protonation in a solvent system compatible with heavy hydrocarbons. The latter can be used directly with our previously described hydroperoxide-sensitive coumarin-conjugated phosphine probe to enable rapid quantification of both carboxylic acids and hydroperoxides in hydrocarbon samples. The utility of the approach is illustrated by the ready determination of the differing relative rates of hydroperoxide and acid formation with changes in hydrocarbon structure (e.g., n-hexadecane vs 1-hexadecene vs a lubricant base stock). The method offers significant versatility and automation compared with common but laborious titration approaches, and greatly improves screening efficiency and accuracy for the identification of novel radical-trapping antioxidants for high temperature applications. This application was demonstrated by the automated analysis of hydroperoxides and carboxylic acids (by microplate reader) in samples from 24 inhibited autoxidations of a lubricating oil, which were carried out on a parallel synthesizer at 160 °C in triplicate in a single day.

Full text of DOI:10.1021/acs.joc.6b01032

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Article
Determination of Key Hydrocarbon Autoxidation Products By Fluorescence
Ron Shah, and Derek A. Pratt
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01032 • Publication Date (Web): 06 Jul 2016
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Determination of Key Hydrocarbon Autoxidation Products By Fluorescence
Ron Shah & Derek A. Pratt*
Department of Chemistry and Biomolecular Sciences
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University of Ottawa, Ottawa, Canada K1N 6N5
dpratt@uottawa.ca
Abstract: Hydroperoxides and carboxylic acids are key primary products that arise in the
autoxidation of hydrocarbons. We have developed a simple approach to rapidly and
simultaneously determine both types of products using hydroperoxide- and acid-sensitive
moieties conjugated to non-polar coumarin- and BODIPY-based fluorophores. The
coumarin- and BODIPY-conjugated amine probes described here undergo 38- and 8-fold
enhancement, respectively, upon protonation in a solvent system compatible with heavy
hydrocarbons. The latter can be used directly with our previously described
hydroperoxide-sensitive coumarin-conjugated phosphine probe to enable rapid
quantification of both carboxylic acids and hydroperoxides in hydrocarbon samples. The
utility of the approach is illustrated by the ready determination of different relative rates
of hydroperoxide and acid formation with changes in hydrocarbon structure (e.g. n-
hexadecane vs. 1-hexadecene vs. a lubricant basestock). The method offers significant
versatility and automation compared to common, but laborious titration approaches and
greatly improves screening efficiency and accuracy for the identification of novel radical-
trapping antioxidants for high temperature applications. This application was
demonstrated by the automated analysis of hydroperoxides and carboxylic acids (by
microplate reader) in samples from 24 inhibited autoxidations of a lubricating oil, which
were carried out on a parallel synthesizer at 160°C in triplicate in a single day.
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Introduction
Autoxidation is the spontaneous radical-mediated process that converts hydrocarbons into
their corresponding hydroperoxides (Scheme 1). It is primarily responsible for the
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degradation of all hydrocarbon-based products such as plastics, rubbers and lubricants.
(1)
(2)
(3)
(4)
Initiation
RH
O2
R
Propogation
R
+
ROO
ROOH
ROO
ROO
+
RH
+
R
Termination
+
ROO
non-radical products
Scheme 1: Hydrocarbon autoxidation.
The sequence of reactions in Scheme 1 (in particular Eq. 2-3) constitutes a
generalized, but also greatly simplified, mechanism of hydrocarbon autoxidation. In fact,
the primary products of autoxidation are not simply monohydroperoxides and the
secondary products involve cleavage of carbon-carbon bonds. In fact, as first
demonstrated by Korcek and co-workers for the autoxidation of n-hexadecane,
ketohydroperoxides (of which the γ-isomer is predominant) are formed competitively
2
from very early on, and carboxylic acids and methyl ketones shortly thereafter. The
3
formation of each of these products can be rationalized by the mechanism in Scheme 2.
O
HO
O2
OOH OOH
O
O
OO OOH
OOH O
-HO
O
O
O O OH
+
HO
Scheme 2: Proposed mechanism of acid formation in n-alkane autoxidation.
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The accumulation of these autoxidation products (or subsequent decomposition
products thereof) can alter the properties of hydrocarbon-based products and/or materials
that come into contact with them. For instance, autoxidation of engine lubricants
increases their viscosity and the production of acids leads to corrosion of metallic engine
parts. As such, antioxidants are key additives to essentially all hydrocarbon-based
materials. Radical-trapping antioxidants (RTAs, A-H in Eq. 5), such as phenols and
aromatic amines, intervene directly in the radical chain-reaction depicted in Scheme 1 by
reducing the chain-carrying peroxyl radicals by H-atom transfer, thereby precluding
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4,5
chain-propagation.
(5)
A H
+
ROO
A
+
ROOH
In an attempt to speed up the identification and assessment of new chemical
entities as RTAs, we recently reported a strategy to monitor the production of
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hydroperoxides using fluorogenic phosphine 1. This probe enables rapid quantification
of hydroperoxides by measuring the pseudo-first order rate of fluorescence increase in a
microplate assay instead of more conventional methods, such as iodometric titration (i.e.
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ASTM method D3703-13) or lengthy HPLC or GC analyses. This method has proven
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-11
highly useful in quantifying hydroperoxides,
but ignores the acids that are produced.
In addition to their role in advancing corrosion, carboxylic acids can have a profound
impact on both the kinetics and mechanisms of the RTAs added to protect the substrate
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from autoxidation (e.g. diarylamines and hindered amine light stabilizers ). Thus, we
have extended our work on 1 (Scheme 3) to the development of an analogous strategy to
monitor the production of carboxylic acids in hydrocarbon autoxidations, which are
1
2
conventionally determined by acid-base titration (i.e. ASTM method D664-11a).
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p-Tol
ROOH
ROH
p-Tol p-Tol
P
P
p-Tol
O
-1 -1
k = 9.1 M s (MeOH)
Φƒ,ox/Φƒ,red = 10.2
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O
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Scheme 3: The reaction of fluorogenic coumarin-triarylphosphine 1 with hydroperoxides.
Although acid-sensitive fluorescent probes have been developed for the real-time
monitoring of intracellular pH by flow cytometry or fluorescence microscopy, they are
13-21
unsuitable for monitoring acid production in the current context for solubility reasons.
Moreover, the probes reported to date are relatively complex, and we envisioned simply
using the same trivially accessed 7-methoxycoumarin core as in hydroperoxide probe 1.
We surmised that a similar probe wherein the triarylphosphine moiety was replaced with
a basic trialkylamine unit would undergo fluorescence enhancement with increasing
acidity of the medium, similarly to the response of 1 to increasing hydroperoxide
concentration. The electron-rich trialkylamine is expected to quench the fluorescence of
2
2
the coumarin by photoinduced electron transfer (PeT); protonation would preclude PeT.
Results and Discussion
Coumarin 2 was easily prepared (see Experimental Section), and as expected, its
fluorescence was dramatically increased upon protonation (Figure 1B). Determination of
the fluorescence of 2 as a function of pH yielded a sigmoidal profile whose midpoint
!
!
indicates a ꢀꢀ of 7.9±0.1 (Figure 1B). The experiments were carried out in 4:1
!
MeOH:i-PrOH (v:v) to ensure a balance between solubility of hydrocarbon samples (vide
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infra), efficient quenching of coumarin fluorescence by PeT, and ionizability of 2. It
should be noted that this solvent mixture is characterized by an autoprotolysis constant of
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1
5.3 and associated neutrality at ꢀꢁ = 9.2. Under these conditions, the ratio of
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fluorescence quantum yields of 2 and its conjugate acid (2-H ) was determined to be 38
(Figure 1C).
Figure 1. (A) The relevant equilibrium of the coumarin-trialkylamine 2 and its conjugate
+
+
acid 2-H . (B) Excitation and emission spectra of 2 and 2-H . (C) Fluorescence of 2 (λex
=
315 nm; λem = 395 nm) as a function of pH in 4:1 MeOH:i-PrOH.
The foregoing suggested that carboxylic acid formation could be monitored in
autoxidation samples simply by determination of their fluorescence following addition of
2
4
2
and dissolution in the 4:1 MeOH:i-PrOH solvent system. Figure 2A reveals the linear
relationship between the fluorescence of 2 and added palmitic acid, a representative
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lipophilic carboxylic acid. Based on this standard, carboxylic acid formation in the initial
stages of an n-hexadecane autoxidation (carried out at 160°C in a stirred flow
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reactor)
were readily determined and yielded similar results to those obtained
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previously by (negative ion) electrospray mass spectrometric analysis (Figure 2B). The
previous results involved determination of each individual isomer of carboxylic acid
formed in the n-hexadecane autoxidations from the mass spectra, and then summing them
2
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in order to determine the total acid concentration.
It should be noted that 2 does not
react with hydroperoxides (see the Supporting Information for the results of the control
experiments).
Figure 2. (A) Fluorescence of 2 (λex = 315 nm; λem = 395 nm) determined as a function
of palmitic acid concentration in 4:1 MeOH:i-PrOH. (B) Acid formation in the initial
stages (<2% conversion) of an n-hexadecane autoxidation at 160˚C determined directly
by ESI-MS (￭) or indirectly by the fluorescence of 2 (●).
n-Hexadecane is a convenient model for heavy hydrocarbon autoxidation because
it produces the simplest possible product distribution. In fact, industrial and/or
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commercially relevant hydrocarbon-based products are usually mixtures of compounds
and/or constitutional isomers. Since the current method does not require knowledge of
the precise structure of each of the carboxylic acid products that are formed, acid
production can easily be determined in these complex mixtures. An example is shown in
Figure 3 where acid formation was determined in the initial stages of the autoxidation of
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a lubricant basestock (an American Petroleum Institute Group III base oil) at 160°C.
The hydroperoxides were determined in parallel (using 1), and are shown on the same
plot (Fig. 3B) alongside the corresponding data for an n-hexadecane autoxidation (Fig.
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A).
The data reveal that the base oil is more easily oxidized than n-hexadecane.
However, less acid is produced relative to hydroperoxide at these early stages of the
autoxidation. These results can be understood on the basis that the base oil contains
branched hydrocarbons and that H-atom abstraction from tertiary positions of
-3
hydrocarbons by peroxyl radicals is faster than from secondary positions (i.e. k = 7.0×10
-
4
-1 -1
and 1.5×10 M s for H-atom abstraction from the tertiary and secondary positions of 2-
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methylbutane, respectively, at 30°C) and this is the rate-determining propagation step
(Eq. 3) in the autoxidation. Moreover, the initially formed tertiary peroxyl radicals cannot
undergo the sequence of reactions to form carboxylic acids as in Scheme 2; only the
fraction of secondary peroxyl radicals that are formed – and that undergo intramolecular
H-atom abstraction from a secondary site in lieu of intermolecular propagation by
abstraction at a tertiary site – can contribute via this pathway. Of course, it must be
acknowledged that acids may arise from other mechanisms.
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Figure 3. Formation of hydroperoxides (￭) and acids (●) in the initial stages (<2%
conversion) of the autoxidation of either n-hexadecane (A) or a API Group III base oil
(
B) at 160°C or 1-hexadecene (C) at 120°C. Autoxidations of n-hexadecane and base oil
were initiated with tetralin hydroperoxide at 10 and 5 mM, respectively, while the 1-
hexadecene autoxidations were initiated with 10 mM dicumyl peroxide.
The results of corresponding autoxidations of 1-hexadecene, a model α-olefin, are
shown in Fig. 3C. These autoxidations were carried out at 120°C since 1-hexadecene is
an unsaturated hydrocarbon that oxidizes much more rapidly than saturated ones, such as
-1 -1
-1 -1
n-hexadecane (i.e. k
p
= 1 M s for 1-octene vs. k
p
= 0.0015 M s for n-octane at
3
0,31
3
0˚C).
Very little acid is produced relative to hydroperoxide in the early stages of
these autoxidations – presumably since intermolecular propagation is much faster than
intramolecular H-atom abstraction. The production of acid via the mechanism in Scheme
2 requires the intramolecular H-atom abstraction from an unactivated position to compete
with the intermolecular H-atom abstraction from an allylic position – which is unlikely.
Indeed, ESI-MS measurements of samples from the 1-hexadecene autoxidation (Fig. 4B)
reveal a very different profile of acids that are formed compared to that observed from
samples of the n-hexadecane autoxidation (cf. Fig. 4A), implying that the mechanism of
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acid formation in 1-hexadecene autoxidations is very different than in autoxidations of
linear saturated hydrocarbons. The two most predominant peaks in the mass spectra are
consistent with the molecular formulas of hexadeca-2-enoic acid and n-pentadecanoic
acid, which may derive from the expected allylic hydroperoxide arising in the
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autoxidation of 1-hexadecene as is shown in Scheme 4.
ꢀ
Figure 4. Representative negative ion electrospray mass spectra from the initial stages of
autoxidations of either n-hexadecane (A) or 1-hexadecene (B) at 160°C and 120 °C,
respectively. Samples (50 µL) were withdrawn 2400 or 4800 s into the autoxidation,
diluted with 4.95 mL of a 20% i-PrOH/80% MeOH solution containing 3,5-
dimethylbenzoic acid (10.5 µM) as an internal standard.
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Hock
fragmentation
OH
O
O
OH
R
R
R
R = C H
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1
3
-
H CO
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O
OH
OH
Scheme 4: Possible mechanisms for the formation hexadeca-2-enoic acid and n-
pentadecanoic acid in the autoxidation of 1-hexadecene.
6
Our motivation for the development of rapid means to quantify hydroperoxides –
and now acids – in hydrocarbon autoxidations was the ability to assess the efficacy of
RTAs. In Figure 5, we show the results of inhibited autoxidations of the same three
model hydrocarbons (n-hexadecane, the API Group III base oil and 1-hexadecene) in the
presence of diarylamines 3 and 4. Compound 3 is an alkylated diphenylamine
representative of catalytic diarylamine antioxidants found in a variety of hydrocarbon-
based products (e.g. lubricating oils and rubber) and compound 4 is representative of a
recently developed class of heterocyclic diarylamines that have been shown to better
9
,33-36
inhibit hydrocarbon autoxidation compared to industry standards.
H
H
N
N
N
C H
6
13
N
N
C H
C H
8 17
8
17
C H
6
13
3
4
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The increased reactivity of the heterocyclic diarylamine 4 relative to 3 is readily
observed in Figures 5A and B, where 4 was able to suppress acid formation to a greater
extent than 3 in each of n-hexadecane and the API Group III base oil, respectively. The
same trend is observed in the inhibition of 1-hexadecene autoxidation (Figure 5C), but
because acid formation lags considerably behind hydroperoxide formation in 1-
hexadecene (cf. Figure 4), resolution of the difference requires running the autoxidation
to much higher conversion.
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ꢀ
Figure 5. Formation of carboxylic acids (A-C) and hydroperoxides (D-F) in the
autoxidation of either n-hexadecane (A,D) or a API Group III base oil (B,E) at 160˚C or
1
-hexadecene (C,F) at 120˚C (￭) and corresponding data for autoxidations inhibited by
either 3 (●) or 4 (▲) at 40 µM in n-hexadecane and 1-hexadecene and 100 µM in base
oil. Autoxidations of n-hexadecane and base oil were initiated with tetralin hydroperoxide
at 10 and 5 mM, respectively, while the 1-hexadecene autoxidations were initiated with
1
0 mM dicumyl peroxide. Autoxidations were carried out in the presence of 2,4,6-tri-tert-
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1
1
1
1
1
2
2
2
2
2
2
2
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2
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6
butylpyridine (1 mM) in order to prevent deactivation of 4 by acid formed during the
autoxidation (see ref. 9).
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The corresponding hydroperoxide analyses of the inhibited autoxidation samples
using the fluorogenic phosphine 1 are shown in Figures 5D-F. Interestingly, the
heterocyclic diarylamine 4 appears to be more effective than the diphenylamine standard
3
at inhibiting hydroperoxide formation in the n-hexadecane autoxidation relative to the
base oil autoxidation, even though the profiles are similar when one considers acid
formation only. Since the base oil autoxidizes so rapidly, it is more difficult for the
antioxidants to compete with propagation (at this antioxidant loading). However, since
acid formation is relatively slower (cf. Figure 4), the differences in the antioxidant
efficacy are better resolved.ꢀ
In the foregoing experiments, hydroperoxides and acids were determined in
parallel. That is, duplicate samples were withdrawn from the hydrocarbon autoxidation,
added to separate wells of a microplate and solutions of either 1 or 2 were added to one or
the other and the fluorescence recorded. For operational expediency, and to limit the
amount of material that must be used for analysis, it would be desirable to carry out both
measurements on a single sample. However, since the fluorophores in 1 and 2 have
practically identical properties, this is not possible. Hence, we prepared the BODIPY-
1
3,37
substituted trialkylamine 5 (Figure 6) and examined its potential as an alternative to 2.
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ꢀ
Figure 6. (A) The relevant equilibrium of the BODIPY-trialkylamine 5 and its conjugate
+
+
acid 5-H . (B) Excitation and emission spectra of 5 and 5-H . (C) Fluorescence of 5 (λex
=
495; λem = 500) as a function of pH in 1:4 isopropanol/methanol. ꢀ
Although the fluorescence enhancement observed upon protonation was less than
!
!
for 2 (i.e., Φ5-H+/Φ
5
= 8 vs. Φ2-H+/Φ
2
= 38), it could be used interchangeably (i.e., ꢀꢀ =
!
8
.1±0.2 for 5 vs. 7.9±0.1 for 2 in in 4:1 MeOH:i-PrOH) and yielded essentially identical
results for the foregoing experiments (see Supporting Information for the data).
To illustrate how this dual-approach is amenable to rapid screening of antioxidant
efficacy, we carried out 24 inhibited autoxidations of base oil at 160˚C simultaneously in
a commercial parallel reactor system that was adapted in-house to enable continuous
oxygenation of all 24 reaction vessels. The reactions were supplemented with either of
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2
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2
2
2
2
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5
5
5
5
5
6
twelve different aromatic amines (6-17) or twelve different phenols (18-29). The
aromatic amines were assessed at lower loadings than the phenols (250 µM vs. 1 mM)
since they are known to trap multiple radicals at elevated temperature due to a catalytic
0
1
2
3
4
5
6
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8
9
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,38
mechanism wherein the substrate can be used to regenerate the amine. An aliquot from
each reaction vessel was taken one-hour post initiation and acid and hydroperoxide
concentrations were determined using probes 1 and 5 simultaneously using a microplate
reader. The entire experiment was completed in triplicate in a single day.
The data in Figure 7 indicate that, consistent with our previous (significantly
9
lower throughput) studies, the heterocyclic diarylamines 6-10 all outperform the
representative industrial standard dialkylated diphenylamine 11. Similarly reactive to the
heterocyclic diarylamines is phenoxazine (14), which was significantly better than its
sulfur analog phenothiazine (15). Interestingly, liproxstatin-1 (13), one of the most potent
3
9
inhibitors of cellular lipid peroxidation known, is also particularly effective at inhibiting
hydrocarbon autoxidations at high temperatures, while ferrostatin-1 (12), a similarly
4
0
potent antioxidant in cell culture, is noticeably less effective.
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H
N
NH
2
OH
H
N
N
HO
HO
6
12
18
24
N
N
N
O
O
OH
O
H
N
NH
H
N
HO
HO
7
8
9
13
14
15
19
20
21
25
26
27
Cl
N
N
H
NO2
N
N
N
10
11
12
13
14
15
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17
18
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H
N
H
N
HO
HO
N
N
O
O
OH
H
N
H
N
HO
N
N
N
S
H
N
H
N
N
HO
HO
HO
N
N
1
0
16
17
22
23
C
18
H
37 28
N
N
N
N
O
N
N
O
N
O
H
N
H
N
HO
11
29
O
Figure 7. Formation of carboxylic acids (black) and hydroperoxides (red) upon
autoxidation of a API Group III base oil for one hour at 160˚C (U = uninhibited) and
corresponding data for autoxidations inhibited by either 250 µM of aromatic amines 6-17
or 1 mM of phenols 18-29. Autoxidations were initiated with 5 mM tetralin
4
1
hydroperoxide and carried out in the presence of 2,4,6-tri-tert-butylpyridine (1 mM).
On the other hand, of the phenolic antioxidants we investigated, only pyrimidinols 22 and
23 suppressed autoxidation to a larger extent compared to the representative industrial
standard BHT, 18, which was similar to the di-tert-butylated catechol 24. Despite the
exciting reactivity of pyrimidinols as radical-trapping antioxidants at ambient
4
2,43
temperatures,
they have yet to be studied at elevated temperatures. This result, and
those obtained for phenoxazine and liproxstatin-1, underscore the value of this rapid
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2
2
2
2
2
2
2
2
2
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5
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5
5
5
5
5
5
5
6
assessment of high temperature RTA activity for further, more detailed, investigations of
promising compounds or classes of compound. Given that these analyses are generally
carried out in both industry and academic laboratories by laborious iodometric and acid-
base titrations, respectively, we anticipate that this will be a widely adopted screening
tool for the identification of useful compounds and unanticipated conceptually novel
chemistry.
0
1
2
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5
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8
9
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Experimental Section
General
Reagents were purchased from commercial suppliers and used without further
purification, unless otherwise indicated. Column chromatography was carried out using
1
13
flash silica gel (40-63 µm, 230-400 mesh). H and C NMR were recorded on a
spectrometer operating at 400 MHz and 100 MHz spectrometer, respectively, unless
specified otherwise. High-resolution mass spectra were obtained on a magnetic sector
electron impact mass spectrometer.
3-((Diethylamino)methyl)-7-methoxy-2H-chromen-2-one (2). To a solution of 3-
4
5
(
Bromomethyl)-7-methoxy-2H-chromen-2-one (0.53 g, 2.0 mmol), KI (0.033 g, 0.2
mmol) and K CO (0.4 g,3 mmol) in THF (20 mL) diethylamine (0.24 mL, 2.4 mmol)
2
3
was added dropwise. The reaction was stirred overnight at room temperature until
completion as determined by TLC. The mixture was added to a separatory funnel and
2 4
extracted using Et O and water. The organic layer was dried over MgSO , filtered and the
2
solvent was evaporated under vacuo. Column chromatography (25% Et O/Hexanes
1
eluent) afforded pure product. Yield: 0.45 g, 86%. Brown Oil. H NMR (400 MHz;
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aceton-d6): δ 7.90 (s, 1H), 7.59 (d, J = 8.5 Hz, 1H), 6.93-6.88 (m, 2H), 3.91 (s, 3H), 3.41
1
3
(
d, J = 1.3 Hz, 2H), 2.58 (q, J = 7.1 Hz, 4H), 1.05 (t, J = 7.1 Hz, 6H). C NMR (100
MHz; aceton-d6): δ 163.0, 161.6, 155.6, 139.8, 129.6, 124.9, 114.0, 112.9, 101.0, 56.2,
2.6, 48.2, 12.6. HRMS: m/z Calc: C15 : 261.1365 Found: 261.1369.
10
11
12
13
14
15
16
17
18
19
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23
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5
H19NO
3
7-Methoxy-3-methyl-2H-chromen-2-one. The precursor to 3-(bromomethyl)-7-methoxy-
4
5
2
H-chromen-2-one used in the synthesis of 2 was prepared using a previously reported
4
4
procedure with slight modifications. Ethyl 2-(triphenylphosphoranylidene)propanoate
(0.9 g, 6.0 mmol) and 2-hydroxy-4-methoxybenzaldehyde (2.1 g, 6.0 mmol) were stirred
in a sealed tube and heated to 200˚C overnight. The reaction was cooled and the crude
product was recrystallized from chloroform and hexanes. Yield: 1.05 g, 92%. Brown
1
needles. H-NMR (400 MHz; aceton-d6): δ 7.69 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 6.91-
6
.86 (m, 2H), 3.90 (s, 3H), 2.09 (s, 3H). NMR spectra were consistent with previously
4
4
reported data.
1
0-((Diethylamino)methyl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2',1'-
f][1,3,2]diazaborinin-4-ium-5-uide (5). To a solution of 10-(chloromethyl)-5,5-difluoro-
4
4
13
1,3,7,9-tetramethyl-5H-4λ ,5λ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine (0.1 g, 0.3
mmol), KI (0.005 g, 0.03 mmol) and K CO (0.062 g, 0.45 mmol) in THF (3 mL)
2
3
diethylamine (0.037 mL, 0.36 mmol) was added dropwise. The reaction was stirred
overnight at room temperature until completion as determined by TLC. The mixture was
added to a seperatory funnel and extracted using Et
2
O and water. The organic layer was
dried over MgSO , filtered and the solvent was evaporated under vaccuo. Column
4
2
chromatography (25% Et O/Hexanes eluent) afforded pure product. Yield: 0.042 g, 42%.
1
Orange solid. H NMR (400 MHz; aceton-d6): δ 6.45 (s, 1H), 6.17 (s, 1H), 3.83 (s, 2H),
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.66 (s, 3H), 2.58 (q, J = 7.1 Hz, 4H), 2.48 (s, 3H), 2.45 (s, 6H), 1.04 (t, J = 7.1 Hz, 6H).
C NMR (76 MHz; aceton-d6): δ 158.5, 154.0, 143.8, 142.4, 142.1, 132.7, 122.0, 120.8,
1
3
5
2 3
1.8, 48.7, 17.5, 17.3, 16.7, 14.4, 12.6. Calc: C18H26BF N : 333.2188 Found: 333.2189.
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0
General Procedure for Hydrocarbon Autoxidations
n-Hexadecane, 1-hexadecene or the base oil (100 mL) were thoroughly degassed with
argon and then heated to 160°C in a stirred flow reactor. Once the temperature stabilized,
inhibitor 3 or 4, base (2,4,6-tri-tert-butylpyridine, 1 mM final concentration) and an
appropriate amount of initiator (see text) were added to the solution and the flow of argon
2
was replaced with O . Aliquots (0.5 mL) were withdrawn every 5 minutes, and allowed
to cool to room temperature for analysis.
General Procedure for Determining Hydroperoxide Concentration
Four replicates (30 µL) of each sample were loaded into separate wells of a 96-well
microplate and the automated reagent dispenser of the microplate reader was used to
dilute each sample with tert-amyl alcohol (200 µL) and a solution of a fluorgenic
phosphine dye 1 (20 µL of a 250 µM stock solution in acetonitrile) immediately before
reading. The plate was stirred for 8 seconds, and allowed to rest for 2 seconds, and the
fluorescence of each well was measured every second for 60 seconds (excitation
wavelength = 340 nm; emission wavelength = 425 nm). The concentration of
hydroperoxide was determined from the rate of phosphine oxidation using the rate
constant for the reaction of the dye with secondary hydroperoxides in tert-amyl alcohol (k
-1 -1
=
1.2 M s ) assuming pseudo-first-order kinetics.
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General Procedure for Determining Acid Concentration
Four replicates (1.2 µL) of each sample were loaded into separate wells of a 96-well
microplate and the automated reagent dispenser of the microplate reader was used to
dilute each sample with 20% isopropyl alcohol in methanol (280 µL), and the plate was
stirred for 15 s. Afterward, a solution of fluorogenic amine 2 (20 µL of a 630 µM stock in
methanol) was added. The plate was stirred for 8 s, and the fluorescence of each well was
measured every second for 10 s (excitation wavelength = 315 nm; emission wavelength =
395 nm) and the acid concentration was determined from the average fluorescence
reading (RFU = 1771 [Acid] + 14065).
10
11
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General Procedure for Simultaneous Determination of Hydroperoxide and Acid
Concentration
Four replicates (5 µL) of each sample were loaded into separate wells of a 96-well
microplate and the automated reagent dispenser of the microplate reader was used to
dilute each sample with 20% isopropyl alcohol in methanol (280 µL), and the plate was
stirred for 15 s. Afterward, a solution containing fluorogenic phosphine dye 1 and
fluorogenic amine 5 (20 µL of a solution containing 250 µM of probe 1 and 630 µM of
probe 5 in acetonitrile) were added. The plate was stirred for 8 s, and the fluorescence of
each well was measured every five seconds for 60 s (excitation wavelength = 340 nm;
emission wavelength = 425 nm for probe 1 and excitation wavelength = 495 nm;
emission wavelength = 500 nm for probe 5). The acid concentration was determined from
the average fluorescence reading over 10 s (RFU = 352 [Acid] + 34925). The
concentration of hydroperoxide was determined from the rate of phosphine oxidation
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2
2
2
2
2
2
2
2
2
3
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
using the rate constant for the reaction of the dye with secondary hydroperoxides in 20%
-
1 -1
isopropyl alcohol in methanol (k = 5.1 M s ) assuming pseudo-first-order kinetics.
Screening of High Temperature Antioxidant Activity
0
1
2
3
4
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Base oil (3 mL) was added to test tubes in a 24-place parallel synthesizer, thoroughly
degassed with argon and then heated to 160°C. Once the temperature stabilized, an
appropriate amount of inhibitor 6-29 (see text), base (2,4,6-tri-tert-butylpyridine, 1 mM
final concentration) and 5 mM tetralin hydroperoxide were added to the test tubes and the
2
flow of argon was replaced with O . An aliquot (0.1 mL) was withdrawn 1-hour post
initiation, and allowed to cool to room temperature for analysis.
Supporting Information. Standardization data, results of autoxidations analysed using 5
and relevant NMR spectra. This material is available free of charge on the ACS
Publications website.
Acknowledgement. We acknowledge the support of the Natural Sciences and
Engineering Research Council (NSERC) of Canada and the Canada Foundation for
Innovation. DAP also acknowledges support from the University of Ottawa and the
Canada Research Chairs program, respectively, while RS acknowledges the support of
NSERC in the form of a Canada Graduate Scholarship.
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(23)
Fonrodona, G.; Ràfols, C.; Bosch, E.; Rosés, M. Anal. Chim. Acta 1996, 335,
2
91.
(24)
This assumes that carboxylic acids are the only acidic species formed in the
autoxidation, which will generally be true in the early stages of the reaction.
Igarashi, J.; Jensen, R. K.; Lusztyk, J.; Korcek, S.; Ingold, K. U. J. Am. Chem.
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We carried out ESI-MS in order to quantify acids produced during n-hexadecane
autoxidations since it was a facile approach compared to acid-base titrations.
This was enabled by the commercial availability of the linear carboxylic acid
standards. Deuterated standards are preferred, but were unavailable. Accurate
determination of acids beyond ca. 10 mM requires further sample dilutions due
to ion saturation (accounting for the deviation between the two data points at
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500 seconds in Figure 2B).
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API Group III base oil is highly refined, severely hydrocracked crude oil that
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Valgimigli, L.; Pratt, D. A. Acc. Chem. Res. 2015, 48, 966.
A similar BODIPY-based pH probe (see ref. 13) has been shown to undergo a
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4-fold fluorescence enhancement upon protonation by acids in 1:1 EtOH:H
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Autoxidations were carried out in the presence of 2,4,6-tri-tert-butylpyridine (1
mM) in order to prevent deactivation of 4 by acid formed during the autoxidation
(see ref. 9).
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