Synthesis and pH-dependent spectroscopic behavior of 2,4,6-trisubstituted pyridine derivatives

Article abstract of DOI:10.1002/jhet.1923

Seven 2,4,6-trisubstituted pyridine derivatives with N,N-diethylaniline substituents at the 4-position were synthesized, and their spectroscopic properties in the absence and presence of acid were studied. The spectral effects of protonation, molar absorptivities, pK_a values, and the structural origins of the observed spectral behavior were ascertained. The pyridine nitrogen was found to be more basic than the diethylamino nitrogen atom. Protonation of the pyridine ring nitrogen is associated with the appearance of a red-shifted intramolecular charge transfer peak in the UV-visible spectra. Favorable color indicating properties result from electron-donating substitution at the 2 and 6 positions of pyridine, which provide a greater absorptivity of the red-shifted peak associated with protonation of the pyridine nitrogen. These findings will assist in the design and optimization of these compounds for ion-indicating and pH-sensing applications.

Full text of DOI:10.1002/jhet.1923

Month 2014
Synthesis and pH-Dependent Spectroscopic Behavior of 2,4,6-
Trisubstituted Pyridine Derivatives
a
a
a
a
*
*
Gala Chapman, Isaac Solomon, Gabor Patonay, and Maged Henary
Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, Georgia 30302-4098
*
E-mail: gpatonay@gsu.edu; mhenary1@gsu.edu
Additional Supporting Information may be found in the online version of this article.
Received August 25, 2012
DOI 10.1002/jhet.1923
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
Seven 2,4,6-trisubstituted pyridine derivatives with N,N-diethylaniline substituents at the 4-position were
synthesized, and their spectroscopic properties in the absence and presence of acid were studied. The spectral
effects of protonation, molar absorptivities, pKa values, and the structural origins of the observed spectral
behavior were ascertained. The pyridine nitrogen was found to be more basic than the diethylamino nitrogen
atom. Protonation of the pyridine ring nitrogen is associated with the appearance of a red-shifted intramolec-
ular charge transfer peak in the UV-visible spectra. Favorable color indicating properties result from elec-
tron-donating substitution at the 2 and 6 positions of pyridine, which provide a greater absorptivity of the
red-shifted peak associated with protonation of the pyridine nitrogen. These findings will assist in the design
and optimization of these compounds for ion-indicating and pH-sensing applications.
J. Heterocyclic Chem., 00, 00 (2014).
INTRODUCTION
the result of a pronounced difference in dipole moment be-
tween the ground and excited states of the molecule because
of an intramolecular charge transfer (ICT) between the donor
and acceptor groups [6]. These charge transfer properties
give rise to a long wavelength ICT band in the absorption
spectrum because of the p ! p* transition [6].
Pyridine derivatives substituted at the 2, 4, and 6 positions
have a wide variety of applications, most of which are based
upon their photophysical properties. Applications of these
compounds include thermal recording materials [1],
photographic acid-mediated imaging media [2], photocur-
able compositions for stereolithography, tunable dye lasers
[3], and ion probes [4,5].
Pyridines conjugated to a heteroatom with electron-donating
resonance effects can be classified as a type of meropoly-
methine dye, which are of particular interest due to their
solvatochromic properties. Betaine dyes possessing a 2,4,6-
trisubstituted pyridinium moiety linked to a phenolate group
exhibit dramatic solvatochromic effects [6] and thermochro-
mic properties [7]; thus, these compounds have potential
applications in thermochromic media. These properties are
Substituents at the 2, 4, and 6 positions can act as donor
groups to the pyridine nitrogen because of their ortho or para
orientation; accordingly, they are potential modulators of the
spectroscopic properties of these compounds. When a donor
group is also nucleophilic, binding of cations can either occur
at the donor group or the acceptor pyridine group. To model
and evaluate the effects of substitution on the spectroscopic
properties of such ditopic compounds, spectral data can be
collected both for the compounds alone and in the presence
of a protonating agent or an appropriate metal ion. However,
by using an acid instead of a metal ion, the spectral behavior
© 2014 HeteroCorporation

G. Chapman, I. Solomon, G. Patonay, and M. Henary
Vol 000
corresponding to binding at each of the nucleophilic sites can
be modeled separately by simply changing the concentration
of a single acidic species rather than using different metals
with specific affinities for different nucleophilic sites.
Moreover, this approach circumvents the possibility of
partial conjugation at both of the nucleophilic sites by metal
ions with mixed affinities; hence, such protonation studies
provide a picture that is easier to interpret regarding the spec-
tral and physical changes in the unknown upon ion binding.
The primary purpose of our study was to synthesize dito-
pic 2,4,6-trisubstituted pyridine derivatives with para-N,
N-diethylaniline substitution at the 4 position and varying sub-
stitution at the 2 and 6 positions and to determine the effect of
varying substitution on the UV-visible spectroscopic behavior
in various solvents in the presence of various acids. To date,
limited data is available on the changes to the photophysical
characteristics associated with varying the substitution at the
2 and 6 positions of these compounds. Protonation studies
were carried out, and molar absorptivities of peaks
corresponding to the basic and singly protonated forms of
the compounds were determined. For selected compounds,
acid dissociation constants were determined spectroscopically,
and molar absorptivities of the nonprotonated and singly pro-
tonated forms were determined in different solvent systems
using different acids to determine the effects of solvent and
acid strength on the photophysical behavior. Finally, a corre-
lated NMR-absorption study was carried out on one of the
compounds to experimentally verify the order of protonation
of the basic nitrogen atoms. Similar NMR studies were also
carried out at higher and lower temperature to ensure that the
order of protonation is not temperature dependent. These stud-
ies to determine the effects of changing substitution at the 2
and 6 positions, and the effects of solvents and acid strength
will assist targeted design of these compounds for their myriad
of applications, particularly as ion probes and pH sensors.
shown in Equation 1. This simple synthetic route is
advantageous in that it requires the use of fewer starting
materials and less than half the reaction time of the more
commonly used three-step method [8]. Asymmetrically
substituted pyridines 6 and 7 were synthesized using a
three-step reaction [8] as outlined in Scheme 1. In
this scheme, a chalcone is synthesized as described in
the literature [9–13] and reacted with an N-
phenacylpyridinium salt to afford the asymmetrical
product. Compound 1 has been previously described in
the literature [14–16], but to the best of our knowledge,
compounds 2–7 are novel.
Protonation studies. Protonation studies were carried
out for all of the dyes using HCl and HClO4 in acetonitrile
(ACN). Absorption spectra from the studies using HCl
have not been included in this section to avoid redundancy,
as similar spectral behavior is observed in the presence of
both acids. Two well-resolved equilibria are present in
the protonation spectra, as indicated by two distinct sets
of isosbestic points. These equilibria are generalized in
Equation 2.
Because two distinct equilibria were present, the spectra
were split into two groupings to simplify interpretation and
avoid spectral crowding. The subsets of spectra belonging
to group (I) correspond to the first protonation equilibrium
(defined by pKa2), and include spectra of the base form of
the dye and of the dye in the presence of increasing concen-
trations of HClO4, up to the maximum observed absorptivity
for the red-shifted peak (typically 40–60 mM HClO4), at
which point the concentration of the singly protonated form
is presumed to be at a maximum. The subsets of spectra
belonging to group (II) correspond to the second protonation
equilibrium (defined by pKa1), and include spectra obtained
by increasing the concentration of acid past that
corresponding to the maximum observed absorptivity of
the red-shifted peak. As similar protonation behavior occurs
for all of the compounds, relevant peaks will be distinguished
by the conventions a (for the peak around 280nm), b (for the
ICT peak around 340 nm), and w (for the red-shifted proton-
ation ICT peak around 440 nm).
RESULTS AND DISCUSSION
Synthesis. Symmetrically substituted 2,4,6-trisubstituted
pyridines (1–5) were synthesized using a one-pot synthesis as
Equation 1. Synthetic route of symmetrical 2,4,6-trisubstituted pyridines.
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Synthesis and pH-Dependent Spectroscopic Behavior of
2,4,6-Trisubstituted Pyridine Derivatives
Scheme 1. Synthetic route of asymmetrical 2,4,6-trisubstituted pyridines.
N
R'
R
O
H
KOH
N
H2O
EtOH
N
N
O
R'
NH4AC
O
CH3CO2H
R
R
R'
6: R = H,
O
O
R' = OH
7: R = MeO, R' = H
N
Br
Acetone
Br
N
(I)
1
0.8
0.6
0.4
0.2
0
Equation 2. General diprotic acid–base equilibrium.
Absorption spectra from the protonation study of com-
pound 2 using HClO4 are provided in Figure 1. The pro-
tonation behavior exhibited by this derivative is
representative of the behavior exhibited by all of the aryl-
substituted dyes except compound 6, which will be
discussed in the following paragraphs.
280
330
380
430
480
530
580
(nm)
(II)
1
0.8
0.6
0.4
0.2
0
In the first equilibrium [group (I) spectra, characterized by
pKa2], upon adding increasing concentrations of acid, a
decrease in the a band (around 290 nm for compound 2)
and a decrease and blue shift in the b band (initially present
around 340 nm for compound 2) is observed, whereas the
red-shifted ICT w band (around 440 nm for compound 2)
appears and increases to a maximum. This shift has been
attributed to delocalization of the positive charge and
increased conjugation within the p system of the molecule
upon protonation [5], and the results in a visible color change
from colorless to yellow. In group (II) spectra, the a band
increases again; the b band undergoes a red-shift back to its
original wavelength, and the w band decreases in intensity
(all the way to zero in the presence of high enough concentra-
tions of acid). This behavior is associated with a disappear-
ance of the yellow color.
280
330
380
430
480
530
580
(nm)
Figure 1. Protonation study of compound 2 using HClO4, with labeled a,
b, and w bands. (I): 0–45 mM HClO4, (II): 45–4000 mM HClO4.
For alkyl substituted 5, the spectral behavior was simi-
lar, but one important difference between its spectra and
those of the aryl-substituted compounds was observed.
Absorption spectra from the protonation study of dye 5
using HClO4 are provided in Figure 2.
Pyridine is known to display an absorption band at 270 nm
because of the p ! p* excitation, and upon protonation, the
intensity of absorption of this band increases without any
appreciable spectral shift [17]. The lMAX of this absorption
band coincides closely the a band of the compounds. Thus,
it would seem that the increased absorptivity around
280 nm as acid concentrations are increased in the second
equilibrium is also most likely attributable to pyridine
protonation and the localization of charge associated with
protonation of the second basic site. It was this observation
that prompted the correlated NMR-absorption studies.
Similar spectral behavior was observed in the a and w
bands for both the aryl- and alkyl-substituted compounds.
However, for the aryl-substituted compounds 1–4 and 7,
the b band is present throughout both protonation equilib-
ria (spectral groupings (I) and (II)), whereas for alkyl
substituted 5, the b band decreases and disappears in the
first equilibrium as the w band increases and reaches its
maximum. As acid concentrations are increased beyond
the first equilibrium for derivative 5, the w band decreases
to zero, and the intensity of the a band increases, but there
is no reappearance of the b band. This behavior is
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G. Chapman, I. Solomon, G. Patonay, and M. Henary
Vol 000
(I)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
280
330
380
430
480
530
580
(nm)
(II)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
280
330
380
430
480
530
580
(nm)
Figure 3. Protonation study of compound 3 using HClO4, with labeled a,
b, and w bands. (I): 0–45 mM HClO4, (II): 45–4000 mM HClO4.
Figure 2. Protonation study of compound 5 using HClO4, with labeled a,
b, and w bands. (I): 0–45 mM HClO4, (II): 45–4000 mM HClO4.
(I)
0.5
0.4
0.3
0.2
0.1
0
reversible upon addition of base and is therefore not due to
dye oxidation or decomposition (as was initially thought
when the spectra were first observed). The continuous
presence of the b ICT band in all spectra for compounds
with aryl substituents at the 2 and 6 positions (as contrasted
with the disappearance of the b band in alkyl-substituted 5)
indicates that the 2 and 6 positions participate in the intra-
molecular charge transfer interaction in addition to the ani-
lino substituent at the 4 position, acting as “secondary
donors” of electron density to the pyridine nitrogen.
It is worth noting that most of the compounds display
greater absorptivity in the bathochromic w band relative
to the base form b band when protonated with HClO4;
the only exceptions are compounds 3 and 6 (spectra
provided in Figures 3 and 4, respectively). These two
compounds are expected to be the least basic, poorest
nucleophiles of the compounds studied, as derivative 3
possesses electron-withdrawing para-chlorophenyl substi-
tution, and derivative 6 possesses an ortho-hydroxyphenyl
moiety as its 2 position that is capable of intramolecular
hydrogen bonding with the pyridine nitrogen and compet-
ing with external protons.
260
310
360
410
460
510
560
(nm)
(II)
0.5
0.4
0.3
0.2
0.1
0
260
310
360
410
460
510
560
(nm)
Figure 4. Protonation study of compound 6 using HClO4, with labeled a,
b, and w bands. (I): 0–50 mM HClO4, (II): 50–4000 mM HClO4.
in spectral behavior is potentially attributable to the intra-
molecular hydrogen bonding previously posited.
For protonation studies carried out using HCl, nearly
identical spectral responses and patterns based on substitu-
tion were observed. The primary spectroscopic difference
observed was that when HCl was used, the maximum
absorptivity of the red-shifted w band was substantially
reduced for all compounds, as will be discussed further in
the “solvent and acid effects” section.
It is interesting to note that the spectra obtained from
compound 6 (Figure 4) behaves somewhat differently than
those obtained from all other aryl-substituted compounds.
Specifically, in the other aryl-substituted compounds (1–4
and 7), the a band is present around 290 nm, decreases in
group I spectra, and increases in group II spectra; whereas
for compound 6, the short wavelength absorption band
(denoted a) is present at around 260 nm and increases in
intensity in both group I and group II spectra (while
exhibiting a red-shift in group II spectra). This difference
Molar absorptivities of 2,4,6-trisubstituted pyridines.
Average molar absorptivities of the ICT peaks
corresponding to the singly protonated and nonprotonated
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Synthesis and pH-Dependent Spectroscopic Behavior of
2,4,6-Trisubstituted Pyridine Derivatives
forms of the dyes are provided in Table 1, along with their
standard deviations.
corresponding to the nonprotonated basic forms of
compounds 1–3 in different solvent systems relevant to this
study is provided in Table 2, along with standard deviations.
It is interesting to note that the base form peaks for the
compounds did not show any pronounced dependence of
absorptivity on solvent. There was a very slight solvatochro-
mic shift in wavelength observed for all of these dyes, with
ACN solutions resulting in the shortest wavelengths,
ethanol–water mixtures intermediate, and DMSO solutions
resulting in the longest wavelengths of maximum absorption.
A summary of the average molar absorptivities of the
red-shifted ICT peaks corresponding to the singly proton-
ated forms of compounds 1–3 in different solvent systems
relevant to this study is provided in Table 3, along with
standard deviations. Note that HClO4 was not used with
DMSO as a solvent because of the potential for explosive
oxidation reactions [18]. Note also that the 50% EtOH/
H2O (v/v) solvent system with I = 0.1M and HClO4 as a
protonating acid was chosen to replicate the conditions
used in the spectroscopic pKa determination.
The standard deviations of the molar absorptivities indi-
cated reasonably good reproducibility of the calculated
values, with the highest %RSD corresponding to the base
form of dye 3 (6.45%). Additionally, correlation coeffi-
cients indicated reasonably good linearity; the poorest R2
value calculated was 0.998.
The base forms of these compounds had wavelengths of
maximum absorption ranging from 287 to 352 nm, depend-
ing on substitution. Dyes with phenyl-based substitution at
the 2 and 6 positions had a comparatively narrow range of
lMAX between 343 and 359, despite variation of the phenyl
substitution. Dye 5, which possesses tert-butyl substituents
in place of the aryl groups possessed by all the other dyes
studied, exhibits a shorter lMAX (326 nm). This is to be
expected given that alkyl substituents at the 2 and 6 posi-
tions of the pyridine ring cannot contribute to the energy-
lowering conjugation that aryl substituents participate in.
]The spectral behavior discussed with respect to
protonation studies can also be observed in this data. The
red-shifted protonation peak absorptivity shows a strong
dependence on the substitution of the compounds, but the
basic ICT peak does not demonstrate any clear pattern of
dependence on substitution. In general, compounds with
electron-donating substituents (e.g. 2, 5, and 7) possess much
higher protonation peak absorptivities, whereas compounds
with electron-withdrawing or hydrogen-bonding substituents
(e.g. 3 and 6) possess substantially lower absorptivities.
Notably, this increase in absorptivity for compounds with
electron-donating substitution is observed both for
compounds with substituents that donate electrons either by
inductive effects (5) or by resonance effects (7).
As previously mentioned, when HClO4 is used as the
protonating acid, there is an increase in the observed
absorptivity of the red-shifted peak corresponding to the
Table 2
Average molar absorptivities of nonprotonated compounds 1–3 in aceto-
nitrile (ACN), DMSO, and 50% EtOH/H2O (v/v).
lMAX
(nm)
e(lMAX
)
Compound
Solvent
ACN
(Mꢀ1 cmꢀ1
)
1
347
354
350
344
349
347
352
362
358
(2.48 ꢁ 0.015) ꢂ 104
(3.24 ꢁ 0.020) ꢂ 104
(2.84 ꢁ 0.011) ꢂ 104
(3.48 ꢁ 0.091) ꢂ 104
(2.90 ꢁ 0.099) ꢂ 104
(2.82 ꢁ 0.095) ꢂ 104
(2.7 ꢁ 0.18) ꢂ 104
DMSO
50% EtOH/H2Oa
ACN
2
3
Solvent and acid effects. Another interesting observation
from these studies was that both the protonating acid and the
solvent choice strongly affects the absorptivity of the
bathochromic peak correlated to the singly protonated form
of the dye, but not that of the base form. A summary of
the average molar absorptivities of the ICT peaks
DMSO
50% EtOH/H2Oa
ACN
DMSO
(2.52 ꢁ 0.039) ꢂ 104
(2.6 ꢁ 0.20) ꢂ 104
50% EtOH/H2Oa
aI = 0.1M
Table 1
Wavelengths of maximum absorption (lMAX) and molar absorptivities (e) of protonated and nonprotonated 2,4,6-trisubstituted pyridines. Molar
absorptivities of protonated form given for bathochromic peak in [HCl] associated with maximum extent of bathochromic absorptivity observed.
Substituent (position)
Base form (B) in ACN
lMAX (nm)
e(lMAX) (Mꢀ1 cmꢀ1
Singly protonated form (HB+) in HCl/ACN (<0.1% H2O)
Compound
2
6
)
lMAX (nm)
e(lMAX) (Mꢀ1 cmꢀ1
(2.2 ꢁ 0.13) ꢂ 104
)
1
2
3
4
5
6
7
Ph
Ph
347
344
352
349
326
359
343
(2.48 ꢁ 0.015) ꢂ 104
(3.48 ꢁ 0.091) ꢂ 104
(2.7 ꢁ 0.18) ꢂ 104
(3.54 ꢁ 0.019) ꢂ 104
(3.0 ꢁ 0.20) ꢂ 104
(3.39 ꢁ 0.026) ꢂ 104
(3.58 ꢁ 0.070) ꢂ 104
450
448
456
456
425
447
448
p-Tolyl
p-ClPh
b-Np
p-Tolyl
p-ClPh
b-Np
(4.06 ꢁ 0.018) ꢂ 104
(8.7 ꢁ 0.41) ꢂ 103
(2.56 ꢁ 0.010) ꢂ 104
(4.12 ꢁ 0.050) ꢂ 104
(5.8 ꢁ 0.15) ꢂ 103
(3.77 ꢁ 0.076) ꢂ 104
tert-Butyl
o-OHPh
Ph
tert-Butyl
Ph
p-MeOPh
Ph, phenyl; b-Np, b-naphthalene; o-OHPh, o-hydroxyphenyl; p-MeOPh, p-methoxyphenyl; p-ClPh, p-chlorophenyl; ACN, acetonitrile.
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Vol 000
Table 3
Average molar absorptivities of singly protonated compounds 1–3 in acetonitrile (ACN), DMSO, and 50% EtOH/H2O (v/v), I = 0.1M.
Compound
Solvent
Acid
[Acid] (mM)
lMAX (nm)
e(lMAX) (Mꢀ1 cmꢀ1
)
1
ACN
ACN
HClO4
HCl
HCl
HClO4
HClO4
HCl
4.00E + 01
4.10E + 01
1.00E + 04
1.20E + 03
4.60E + 01
4.10E + 01
1.00E + 04
8.51E + 02
5.00E + 01
5.13E + 01
1.00E + 04
2.57E + 03
449
450
447
453
447
448
446
451
456
456
452
456
(2.99 ꢁ 0.066) ꢂ 104
(2.2 ꢁ 0.13) ꢂ 104
(7.56 ꢁ 0.097) ꢂ 103
(6.7 ꢁ 0.34) ꢂ 103
(4.8 ꢁ 0.12) ꢂ 104
(4.06 ꢁ 0.018) ꢂ 104
(1.23 ꢁ 0.010) ꢂ 104
(1.25 ꢁ 0.010) ꢂ 104
(1.90 ꢁ 0.064) ꢂ 104
(8.7 ꢁ 0.41) ꢂ 103
(1.33 ꢁ 0.010) ꢂ 103
(9.9 ꢁ 0.38) ꢂ 102
DMSO
50% EtOH/H2Oa
ACN
2
3
ACN
DMSO
HCl
50% EtOH/H2Oa
ACN
HClO4
HClO4
HCl
HCl
HClO4
ACN
DMSO
50% EtOH/H2Oa
aI = 0.1M
Spectroscopic pKa determination. The spectral results of
the pKa studies of compound 2 in 50% EtOH/H2O are
provided in Figure 5. For ease of interpretation and to avoid
crowded spectra, the spectra have again been grouped into
two plots, the first plot (I) depicting spectra corresponding
to the first protonation equilibrium (pKa2), and the second
plot (II) depicting spectra corresponding to the second
protonation equilibrium (pKa1). In both spectra, arrows
indicate the spectral changes associated with decreasing pH.
Both equilibria in both sets of spectra show clear
isosbestic behavior, reinforcing the assumption that the pKa
values are at least two units apart and validating the approach
to calculating pKa used in this section. Furthermore, the ab-
sorption of the red-shifted peak at 449 nm goes from zero
singly protonated form for all compounds relative to those
absorptivities measured using HCl as the protonating acid.
This behavior is apparent in the data provided in Table 3
for HCl and HClO4 in ACN. The reason for this difference
in absorptivity is presumably increased association between
the conjugate anion of the acid and the protonated com-
pounds in the case of weaker acids [5]. Hydrochloric acid
behaves as a weak acid in ACN (pKa 8.9) [19] and is not fully
dissociated, whereas perchloric acid behaves as a strong acid
and is known to be fully dissociated in ACN [20]. It follows
that the conjugate perchlorate anion would be less associated
with the protonated compounds than the chloride anion;
therefore as chloride is more likely to associate and assist
in charge stabilization, the extent of charge transfer within
the protonated compounds would be reduced [5].
The data provided in Table 3 also indicates that solvent
choices also substantially affected the absorptivities of the
red-shifted ICT peak correlated to the singly protonated form
of the dye. When the data for HCl in ACN is compared with
that obtained using HCl in DMSO, there is a substantial
decrease in the observed absorptivity for compounds 1
(66%), 2 (70%), and 3 (85%). Similarly, when the data for
HClO4 in ACN is compared with that obtained using HClO4
in 50% EtOH-H2O, there is a substantial decrease in the ob-
served absorptivity for compounds 1 (78%), 2 (74%), and 3
(95%). It can be generalized that the absorptivity seems to
decrease with increasing dielectric constant of the solvent or
solvent mixture. It should be noted that no substantial shift in
wavelength is observed in the protonation spectra upon chang-
ing solvents; however, the hypochromic effect observed in
more polar solvents does indicate solvatochromic properties,
as would be expected given the dipolar nature of the protonated
state [6]. The hypochromic effect observed may be due to
increased dipole-dipole interactions between the charged pro-
tonated form of the compounds and the polar solvent mole-
cules. These observations indicate that solvents must be
chosen carefully when metal-binding or protonation studies
are carried out.
(I)
0.6
0.5
0.4
0.3
0.2
0.1
0
280
330
380
430
480
530
(nm)
(II)
0.6
0.5
0.4
0.3
0.2
0.1
0
280
330
380
430
480
530
(nm)
Figure 5. Absorption spectra from pKa determination of compound 2
in 50% EtOH/H2O (25ꢃC, I = 0.100M except for pH < 1.00). (I) pH
6.18–3.20, (II) pH 3.20–0.92.
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Correlated NMR-absorption studies.
Provided in
to a maximum in the first equilibrium and decreases back to
zero in the second equilibrium, further validating the ap-
proach for calculating pKa. The absorption at 449 nm was
plotted as a function of pH; this plot is provided in Figure 6.
Provided in Table 4 are the experimentally determined
psKa values for the compounds in 50% EtOH/H2O. The
data in Table 4 indicates that when electron-donating
substituents (such as p-tolyl) are interchanged for
electron-withdrawing substituents (such as p-chlorophenyl)
at the 2 and 6 positions of pyridine, neither the psKa1
nor the psKa2 are decreased substantially (the decrease for
both in this case is approximately 0.4–0.5 units). Interest-
ingly, the psKa1 of “intermediate” phenyl-substituted 1
was actually observed to be below that of the more
electron-withdrawing chlorophenyl-substituted 3, whereas
the psKa2 is closer to that of electron-donating tolyl-substituted
2. This modulation of pKa by changing substitution more or
less follows the expected pattern (as electron-withdrawing
substituents are expected to make the dye less basic),
but the observed change in pKa is not as large as was
expected, especially given the large differences in
absorptivity of the protonation peak between those
compounds with electron-donating substitution and those
with electron-withdrawing substitution. One potential
explanation for this relatively minor change in pKa is stabili-
zation by the electron-donating diethylamino aryl group at
the 4-position of the pyridine ring.
Figure 7 is the structure of compound 1 with diagnostic
proton positions “a, b, c” (proximal and/or conjugated to
either of the basic nitrogen atoms) labeled. Provided in
Table 5 are the results of the correlated NMR-absorption
study at 25ꢃC, including the absorption at 450 nm (red-
shifted ICT peak corresponding to the singly protonated
form), chemical shifts of diagnostic positions in the
molecule, changes in chemical shift of diagnostic positions
in the molecule relative to the nonprotonated form of 1,
concentrations of acid in each sample, and calculated pH
(pcH). The pcH value was calculated assuming the activity
of the hydrogen ion is unity (pcH = ꢀlog[H+] = ꢀlog
[HClO4]). It should be noted that equating [H+] with
[HClO4] is valid given that perchloric acid is known to be
fully dissociated in ACN [14]. In Table 5, the first three
samples correspond to the first equilibrium, and the last
two correspond to the second; the fourth sample
corresponds to the maximum concentration of the singly
protonated species. Provided in Figure 8 is a graphic
representation of the data provided in Table 5, wherein
change in chemical shift for diagnostic positions a, b, and c
is plotted as a function of pcH. In this representation, the
line at pcH 3.26 represents the division between the first
and second protonation equilibria. Note that NMR studies
carried out at higher (37ꢃC) and lower (0ꢃC) temperatures
resulted in the same outcome.
It is readily apparent from Table 5 and Figure 8 that the
majority of the change in chemical shift for the protons in
proximity with and conjugated directly to the pyridine
nitrogen (positions b and c) occurs within the first equilib-
rium, whereas the chemical shift for position a (inductively
coupled and a to the aniline nitrogen) does not change
substantially until the second equilibrium. This provides
strong evidence supporting protonation of the pyridine ring
in the first equilibrium (pKa2) and protonation of the aniline
nitrogen in the second equilibrium (pKa1).
The finding that the pyridine nitrogen is protonated prior
to the aniline nitrogen supports the discussion of the spectro-
scopic behavior provided in the “protonation studies”
section. If pyridine is protonated first, the positive charge is
delocalized throughout the conjugated chromophore, as
illustrated in Figure 9. Delocalization of a positive charge
0.25
0.20
0.15
0.10
0.05
0.00
0
1
2
3
4
5
6
7
pH
Figure 6. Absorption of compound 2 at 449 nm as a function of pH from
pKa determination in 50% EtOH/H2O (25ꢃC, I = 0.100M except for
pH < 1.00).
a
N
Table 4
Calculated psKa values in 50% EtOH/H2O, I = 0.1M.
Substituents
Compound
(2 and 6)
psKa1
psKa2
b
N
1
2
3
Phenyl
p-Tolyl
p-Chlorophenyl
1.31 ꢁ 0.081
1.84 ꢁ 0.031
1.34 ꢁ 0.099
4.5 ꢁ 0.12
4.63 ꢁ 0.010
4.2 ꢁ 0.12
c
Figure 7. Structure of 1 with diagnostic positions a, b, and c labeled.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

G. Chapman, I. Solomon, G. Patonay, and M. Henary
Vol 000
Table 5
Chemical shifts (d) and changes in chemical shift relative to nonprotonated form of 1 (Δd) for diagnostic positions a, b, and c (Figure 7), absorption at
450 nm (A450), concentration of HClO4 in samples and calculated pH (pcH) from correlated NMR-absorption study at 25ꢃC.
Diagnostic chemical shifts (d) and change in chemical shift (Δd)
[H+] (mM)
pcH
A450
d a (ppm)
dΔ a (ppm)
d b (ppm)
dΔ b (ppm)
d c (ppm)
dΔ c (ppm)
0.000
0.100
0.300
0.550
1.000
5.000
N/A
4.00
3.52
3.26
3.00
2.30
0
3.4566
3.4691
3.4842
3.4873
3.6391
3.7115
0.0000
0.0125
0.0276
0.0307
0.1825
0.2549
8.2640
8.2173
8.0988
8.0233
8.0163
8.0159
0.0000
0.0468
0.1652
0.2407
0.2478
0.2481
7.4775
7.5255
7.5953
7.6720
7.7017
7.7231
0.0000
0.0480
0.1178
0.1945
0.2242
0.2456
0.585
2.136
3.254
0.685
0
N
N
N
H
N
H
Figure 9. Representative resonance structures illustrating charge delocal-
ization resulting from pyridine protonation.
Figure 8. Absolute value of change in chemical shift for diagnostic
positions a, b, and c (Figure 7) as a function of pcH with included division
between first and second equilibrium (dashed line labeled [HB+]MAX)and
marker indicating pcH at which all of 1 is in the doubly protonated form
corresponding color change (lMAX = 425–456nm, Table 1).
Preliminary studies on the effect of varying the substituent at
the 4 position indicate that the amino nitrogen of the diethy-
laminoaryl substituent is responsible for the magnitude of
this red shift (relative to alkyl- and alkoxy-substituted com-
pounds, data not shown). Upon further addition of acid to
these compounds, there is a reversal of the color change
and a corresponding hypsochromic shift, with the new peak
demonstrating a lMAX around 270–280 nm. The presence
of clear isosbestic points suggests that these two protonation
equilibria are distinct and well-resolved (i.e. pKa1 and pKa2
are separated by at least two pH units); results of pKa studies
confirm this finding. Correlated NMR-absorption studies
support the presumption that the pyridine nitrogen is in fact
the site of the first protonation (pKa2). Accordingly, the bath-
ochromic shift is likely attributable to resonance delocaliza-
tion of the positive charge throughout the chromophore.
Substitution with electron-donating groups was corre-
lated to an increase in the absorptivity of the red-shifted
protonation peak, which may be explained in terms of
charge transfer behavior; as the electron-donating nature
of the substituents increases, more extensive delocalization
of the positive charge throughout the structure is possible,
resulting in a greater absorptivity. Surprisingly, despite
the large differences in protonation peak absorptivity
observed upon varying substitution, the observed pKa
values are not strongly modulated by interchanging
(dashed line labeled [H2B2+
]MAX).
across a conjugated system is known to result in a red-shifted
absorption peak and higher absorptivity relative to that of an
uncharged system with the same extent of conjugation, as is
exemplified by other common classes of dyes such as the
cyanines [21]; this would readily explain the observed
bathochromic shift and increased absorptivity in spectra
associated with the first protonation equilibrium. Moreover,
this delocalization of charge is impossible if the aniline
nitrogen is protonated first. In the second equilibrium,
both nitrogen atoms become protonated, and resonance delo-
calization is no longer possible. The disappearance of the
red-shifted peak and the increase in blue-shifted peak absorp-
tivity in spectra corresponding to the second equilibrium is
likely explained by this “relocalization” of charges when
both nitrogen atoms are positively charged.
CONCLUSIONS
All compounds presented in this study were synthesized,
purified, and isolated in a good yield, then characterized by
1H-NMR and 13C-NMR. Protonation studies indicate that
each of these compounds displays a bathochromic shift of
approximately 100 nm upon addition of acid, with a marked
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2014
Synthesis and pH-Dependent Spectroscopic Behavior of
2,4,6-Trisubstituted Pyridine Derivatives
(CD3CN; 99.8% D) were obtained from Cambridge Isotope
Laboratories (Andover, MA) and stored under dry N2 (Airgas, Inc.,
Radnor, PA). All of the 2,4,6-trisubstituted pyridines were
synthesized in our lab. Purities were verified by 1H-NMR,
13C-NMR, and mp ranges. 1H-NMR spectra have been provided in
the Supporting Information, Figures S.1–S.7, and 13C-NMR
spectra provided in the Supporting Information, Figures S.8–S.14.
electron-withdrawing substituents for electron-donating
substituents at the 2 and 6 positions of the pyridine ring.
The solvent and the protonating acid also both have a
strong influence on the absorptivity of the bathochromic
protonation peak. Absorptivities of the protonation peak in
ACN were substantially higher than those found in either
DMSO or 50% EtOH/H2O when the same protonating acid
was used. Additionally, it was found that in ACN, HClO4
resulted in greater bathochromic peak absorptivity than
HCl, presumably because of increased association of the con-
jugate anion of weaker acids with the protonated compounds.
In this study, structural features of 2,4,6-trisubstituted
pyridines that affect the amplitude of spectroscopic response
to cations for applications as pH indicators and ion probes
have been determined and rationalized on the basis of known
spectroscopic phenomena. These findings should assist in the
future design of donor–acceptor pyridine ion probes with
optimal spectroscopic and color indicating responses for
applications as color indicators of ions.
General procedure for preparation of symmetrical 2,4,6-
trisubstituted pyridines.
Symmetrically substituted 2,4,6-
trisubstituted pyridines (1–5) were synthesized using a one-pot
synthesis as shown in Equation 1. In this one-pot synthesis,
1 equiv of 4-diethylaminobenzaldehyde (40mmol), 2 equiv of
variously substituted methyl ketones (80mmol), and 2 equiv of
ammonium acetate (80 mmol) were mixed together in a 250-mL
round-bottomed flask, followed by addition of a catalytic amount
of acetic acid. The reaction mixture was heated for 24h at 100ꢃC
to obtain the 2,4,6-trisubstituted pyridine product. TLC was
performed to monitor the extent of reaction, and upon completion,
200 mL of a saturated solution of sodium carbonate was added to
quench the reaction. The reaction mixture was extracted with
dichloromethane to isolate the product, and the organic layer was
washed three times with water. After the washing steps, the
organic layer was dried over anhydrous magnesium sulfate, which
was removed by filtration. The filtrate was concentrated under
vacuum and further purified by column chromatography on silica
gel using dichloromethane/hexanes as eluent. Yield: 58–76%.
EXPERIMENTAL
Instrumentation.
Absorption spectra were measured using
either a Perkin-Elmer Lambda 20 UV–vis-NIR Spectrophotometer
(Perkin-Elmer Incorporated, Waltham, MA) or a Cary 3G UV–vis
Spectrophotometer (Agilent Technologies Incorporated, Santa
Clara, CA). Disposable Fisherbrand polystyrene cuvettes with
pathlengths of 1.00 cm were obtained from Fisher Scientific
(Fairlawn, NJ), and quartz cuvettes of 1.000 cm pathlength were
obtained from Starna Cells, Inc. (Atascadero, CA). A quartz
cuvette of 0.200 cm pathlength (and a spacer to adjust the effective
sample compartment size) was obtained from NSG Precision Cells
(Farmingdale, NY) for the correlated absorption-NMR studies. A
ROSS pH electrode (operational pH range: 0–14; Orion Research,
Inc., Beverly, MA) was used for pH measurements and calibrated
using a three-point calibration method with pH 4.00, pH 7.00, and
pH 10.00 reference standard buffer solutions (EMD Millipore,
Billerica, MA). All calculations were carried out using Microsoft
Excel (Microsoft Corporation, Redmond, WA). RT (25ꢃC) and
higher temperature (37ꢃC) NMR studies were carried out on a
400 MHz Bruker Avance NMR spectrometer (Bruker BioSpin
Corporation, Billerica, MA). The lower temperature (0ꢃC) NMR
study was carried out on a 600 MHz Bruker Avance NMR
spectrometer. NMR spectra were processed using Topspin Software
(Bruker BioSpin Corporation, Billerica, MA). Standard 5 mm NMR
tubes were obtained from Wilmad-LabGlass (Vineland, NJ).
General synthesis of unsymmetrical 2,4,6-trisubstituted
pyridines.
Asymmetrically substituted pyridines 6 and 7 were
synthesized using a three-step synthesis. In the first step, a chalcone
was synthesized by reacting a substituted acetophenone (80 mmol)
with 4-diethylaminobenzaldehyde (80 mmol) in a 250-mL round-
bottomed flask containing 40 mL ethanol. The reaction was heated
for 10 min, and then 40 mL of a 55% potassium hydroxide solution
was added slowly with stirring. The reaction mixture was left for an
additional 24 h with continuous stirring. TLC was performed to
monitor the extent of reaction, and upon completion, the reaction
mixture was extracted with dichloromethane to isolate the product.
The organic layer was washed three times with water, and then
dried over anhydrous magnesium sulfate, which was removed by
filtration. The filtrate was then concentrated under vacuum to isolate
the desired chalcones. Yield: 76–83%.
In the second step, an N-phenacylpyridinium salt was synthe-
sized by combining 2-bromoacetophenone (80mmol) and anhy-
drous pyridine (160 mmol) in 200mL acetone in an Erlenmeyer
flask and stirring continuously for 48 h at RT, until a chunky, milky
precipitate formed. The resulting product was diluted with diethyl
ether (200mL) and then filtered. The filtrand was washed with
diethyl ether and dried under vacuum to furnish the desired product
as a colorless solid. Yield: 91%.
Chemicals and reagents. Perchloric acid (HClO4; 69.5%,
Lot No. 02829ER) was obtained from Aldrich Chemical Co.
(Milwaukee, WI). Hydrochloric acid (HCl; 12.309M) and
DMSO (≥99.8%, UV grade) were obtained from Sigma-Aldrich
(St. Louis, MO). Sodium chloride (NaCl; 99 + %) and ACN
(99.9% and 0.03% H2O) were obtained from Thermo Fisher
Scientific (Waltham, MA). ACN was stored over freshly dried
4 Å molecular sieves. Type 1 ultrapure water (18.2MΩ cm) was
obtained from a Barnstead Nanopure Water System (Thermo
Fisher Scientific). Koptec ethanol (EtOH; 200 proof; 99.5%
min) was obtained from VWR (Radnor, PA). Deuterated DMSO
(DMSO-d6; 99.9% D +0.05% V/V TMS) and deuterated ACN
In the third step, unsymmetrical 2,4,6-trisubstituted pyridine
derivatives were synthesized by combining the chalcone from step
1 (40mmol) and the N-phenacylpyridinium salt from step 2
(40 mmol) in a 500-mL round-bottomed flask in the presence of
glacial acetic acid (120 mL) and ammonium acetate (NH4Ac,
60g). The reaction was stirred under reflux for 24h. TLC was
performed to monitor the extent of reaction, and upon completion,
the reaction mixture was poured into 400mL of water and made
basic with sodium hydroxide. The reaction mixture was extracted
with dichloromethane to isolate the product, and the organic layer
was washed three times with water. After the washing steps, the
organic layer was dried over anhydrous magnesium sulfate, which
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

G. Chapman, I. Solomon, G. Patonay, and M. Henary
Vol 000
was removed by filtration. The filtrate was concentrated under vac-
uum to give a brown oil residue and further purified by column
chromatography on silica gel using dichloromethane/hexanes as
eluent. Yield: 52–57%.
4-[2-(2-Hydroxyphenyl)-6-phenylpyridin-4-yl]-N,N-diethylaniline
(6). Yellow solid, mp 140–142ꢃC, yield: 9.0 g (57%); TLC (70:30
CH2Cl2/Hexanes) Rf = 0.40; 1H-NMR (400 MHz, DMSO-d6)
d (ppm): 1.15 (t, J=7.0Hz, 6H), 3.44 (q, J=7.0Hz, 4H), 6.81
(d, J=8.9Hz, 2H), 6.94–6.98 (m, 2H), 7.34 (t, J= 7.8 Hz, 1H), 7.54
(t, J=7.2Hz, 1H), 7.60 (t, J= 7.6 Hz, 2H), 7.96 (d, J=8.9Hz, 2H),
8.08 (d, J= 7.6 Hz, 2H), 8.10 (s, 1H), 8.28 (d, J= 7.2 Hz, 1H), 8.32
(s, 1H); 13C-NMR (100 MHz, DMSO-d6) d (ppm): 12.5, 43.7,
111.4, 113.8, 115.2, 117.8, 118.8, 119.1, 122.5, 126.7, 127.5, 128.6,
129.2, 129.6, 131.3, 137.9, 148.6, 150.5, 154.1, 157.2, 159.4.
Thin layer chromatography. Normal phase TLC was carried
out periodically to determine the extent of reaction and the purity of
the compounds. The ideal mobile phase for both TLC and column
chromatography was determined by starting with a 50:50 mixture
of hexane and dichloromethane, then adjusting the polarity as
needed to achieve the desired separation of compounds.
4-[2-(4-Methoxyphenyl)-6-phenylpyridin-4-yl]-N,N-diethylaniline
Column chromatography.
Normal phase column
(7). White powdery crystals, mp 92–94ꢃC, yield: 8.5 g (52%); TLC
chromatography using a mixture of dichloromethane and hexanes
was carried out to purify pyridine derivatives (1–7). The column
was packed as a slurry using hexanes, and this solvent was eluted
prior to loading the crude product. The crude products were
dissolved in dichloromethane and loaded on the column, and a
mobile phase half the polarity of the TLC mobile phase was run
through the column; as elution slowed, the mobile phase polarity
was increased to that of the mobile phase used for TLC to obtain
optimal separation within the column.
4-(2,6-Diphenylpyridin-4-yl)-N,N-diethylaniline (1). Yellow-
orange solid, mp 96–97ꢃC, yield: 8.8 g (58%); TLC (70:30
CH2Cl2/Hexanes) Rf =0.43; 1H-NMR (400 MHz, DMSO-d6)
d (ppm): 1.15 (t, J= 7.0Hz, 6H), 3.43 (q, J= 7.0 Hz, 4H), 6.80
(d, J= 8.6 Hz, 2H), 7.47 (t, J= 7.1 Hz, 2H), 7.55 (t, J= 7.5 Hz, 4H),
7.90 (d, J = 8.6 Hz, 2H), 8.09 (s, 2H), 8.30 (d, J=7.5Hz, 4H); 13C-
NMR (100 MHz, DMSO-d6) d (ppm): 11.9, 43.2, 111.0, 114.3,
122.7, 126.3, 127.7, 128.2, 128.5, 138.6, 147.8, 148.9, 155.7.
4-[2,6-Bis(4-methylphenyl)pyridin-4-yl]-N,N-diethylaniline
(2). Light yellow powdery crystals, mp 133–134ꢃC, yield: 10.6 g
(65%); TLC (70:30 CH2Cl2/Hexanes) Rf =0.43; 1H-NMR
(400 MHz, DMSO-d6) d (ppm): 1.14 (t, J= 7.0 Hz, 6H), 2.39
(s, 6H), 3.42 (q, J= 7.0Hz, 4H), 6.79 (d, J =9.0Hz, 2H), 7.34
(d, J= 8.1Hz, 4H), 7.87 (d, J= 9.0 Hz, 2H), 8.02 (s, 2H), 8.19
(d, J=8.1Hz, 4H); 13C-NMR (100 MHz, DMSO-d6) d (ppm):
12.5, 20.9, 43.7, 111.5, 114.2, 123.4, 126.7, 128.2, 129.3, 136.5,
138.5, 148.2, 149.3, 156.1.
1
(70:30 CH2Cl2/Hexanes) Rf = 0.17; H-NMR (400 MHz, DMSO-d6)
d (ppm): 1.14 (t, J=7.0Hz, 6H), 3.42 (q, J= 7.0 Hz, 4H), 6.79
(d, J=9.0Hz, 2H), 7.09 (d, J=8.9Hz, 2H), 7.47 (t, J=7.3Hz, 1H),
7.54 (t, J= 7.7 Hz, 2H), 7.88 (d, J= 8.9 Hz, 2H), 8.02 (s, 2H), 8.25–
8.30 (m, 4H); 13C-NMR (100 MHz, DMSO-d6) d (ppm): 12.0, 43.2,
54.7, 111.0, 113.5, 113.6, 122.9, 126.3, 127.7, 128.1, 128.4, 131.1,
138.8, 147.7, 148.8, 155.4, 155.5, 159.6
Protonation studies and method of determining molar
absorptivities. For the protonation studies, working solutions
of all dyes were prepared containing 20 mM dye and varied
concentrations of acid (typically 20–200 mM) with negligible
(<0.1%) added water, and the absorption spectra were
measured. In ACN, studies were carried out using both HClO4
and HCl for all of the dyes. For derivatives 1–3, additional
protonation studies were carried out using HCl in DMSO and
HClO4 in 50% EtOH/H2O (v/v).
The absorption spectra of working solutions of varying dye con-
centrations were measured, and the absorption at a particular
wavelength of maximum absorption (lMAX) was determined. The
absorption of each sample at lMAX was plotted as a function of con-
centration; the linear regression equation was computed, and the
molar absorptivity (e) was taken as the slope, as per Beer’s law.
Absorptivities were determined in duplicate and averages, and per-
cent relative standard deviations were calculated. Molar absorptivi-
ties for the basic forms of all of the dyes were determined in ACN.
Absorptivities for the singly protonated forms of all compounds
were determined by adding a constant amount of HCl in ACN to
the solutions and taking the absorption at the bathochromic peak
lMAX corresponding to the singly protonated form of the
compound. Preliminary protonation studies using HCl were used
to determine the concentration of HCl resulting in the greatest
observed bathochromic peak absorptivity; this concentration of
HCl was held constant, whereas the dye concentration was
decreased to determine the protonated form molar absorptivity.
Final working solutions contained <0.1% additional added H2O
from the HCl. For clarity, representative spectra and plots of absorp-
tion versus concentration from the molar absorptivity determination
for the basic and singly protonated forms of one of the compounds
(7) are provided in the Supporting Information, Figures S.15–S.19,
along with a discussion of the procedures used to obtain the data.
4-[2,6-Bis(4-chlorophenyl)pyridin-4-yl]-N,N-diethylaniline
(3).
Light yellow powdery crystals, mp 174–176ꢃC, yield:
1
12.5g (70%); TLC (70:30 CH2Cl2/Hexanes) Rf = 0.73; H-NMR
(400 MHz, DMSO-d6) d (ppm): 1.14 (t, J = 7.0 Hz, 6H), 3.43
(q, J = 7.0Hz, 4H), 6.79 (d, J = 9.0Hz, 2H), 7.60 (d, J = 7.7Hz,
4H), 7.92 (d, J = 9.0 Hz, 2H), 8.14 (s, 2H), 8.35 (d, J = 6.8Hz,
4H); 13C-NMR (100 MHz, DMSO-d6) d (ppm): 12.5, 43.7, 111.4,
115.0, 122.9, 128.3, 128.6, 133.9, 137.8, 148.4, 149.7, 155.0.
4-[2,6-Bis(2-naphthyl)pyridin-4-yl]-N,N-diethylaniline (4). Finely
divided orange crystals, mp 196–198ꢃC, yield: 14.6g (76%); TLC
1
(70:30 CH2Cl2/Hexanes) Rf = 0.60; H-NMR (400 MHz, DMSO-
d6) d (ppm): 1.17 (t, J = 7.0Hz, 6H), 3.46 (q, J = 7.0 Hz, 4H),
6.85 (d, J = 9.0 Hz, 2H), 7.57–7.62 (m, 4H), 7.99–8.02 (m, 4H),
8.10–8.16 (m, 4H), 8.32 (s, 2H), 8.57 (dd, J = 8.6, 1.7 Hz, 2H),
8.93 (s, 2H); 13C-NMR (100 MHz, DMSO-d6) d (ppm): 12.5,
43.77, 111.5, 115.3, 123.2, 124.8, 126.1, 126.4, 126.7,
127.6,128.2, 128.3, 128.7, 133.2, 133.3, 136.6, 148.4, 149.5, 156.2.
4-[2,6-Bis(tert-butyl)-4-pyridinyl]-N,N-diethylaniline (5). White
powdery crystals, mp 95–97ꢃC, yield: 9.7 g (72%); TLC (70:30
CH2Cl2/Hexanes) Rf =0.10; 1H-NMR (400 MHz, DMSO-d6) d
(ppm): 1.12 (t, J= 6.7 Hz, 6H), 1.35 (s, 18H), 3.38 (q, J=6.7Hz,
4H), 6.75 (d, J= 8.5 Hz, 2H), 7.31 (s, 2H), 7.58 (d, J= 8.5 Hz, 2H);
13C-NMR (100 MHz, DMSO-d6) d (ppm): 12.4, 30.1, 37.3, 43.7,
111.6, 112.0, 124.7, 127.8, 147.8, 148.0, 167.1.
Molar absorptivities for the nonprotonated forms of com-
pounds 1–3 were also determined in DMSO and 50% EtOH/
H2O, and molar absorptivities of the singly protonated forms of
compounds 1–3 were determined using HCl as the protonating
acid for studies carried out in DMSO and using HClO4 as the pro-
tonating acid for studies carried out in 50% EtOH/H2O. All of the
aforementioned absorptivities were determined in duplicate by
linear regression, and the ideal concentrations of acid used to
determine absorptivities of the singly protonated forms were
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2014
Synthesis and pH-Dependent Spectroscopic Behavior of
2,4,6-Trisubstituted Pyridine Derivatives
determined from the protonation studies mentioned previously.
Molar absorptivities of the singly protonated forms of 1–3 using
HClO4 in ACN were also estimated by single point measurements
(rather than linear regression). These measurements were also
carried out at least twice. Linear regression determination of
absorptivity using perchloric acid in ACN was not possible when
the acid concentration was held constant, as the equilibrium was
perturbed too greatly when the concentration of dye was changed;
it is unknown why this behavior was only observed for HClO4
and not for HCl (although it may be related to the acid strength).
was 50% EtOH/H2O (v/v). It should be noted that ionic strengths
were adjusted to a constant value as preliminary studies carried
out on two of the compounds indicated that large changes in
ionic strength are capable of perturbing the protonation equilibrium
to some extent. Each solution was thoroughly mixed, and then part
of the solution was transferred into a cuvette for measurement
of the absorption spectrum. A pH electrode was inserted into the
remaining solution, and the pH was measured at the same time as
the absorption spectrum.
The psKa values of these compounds were determined plotting the
absorption at the red-shifted ICT wavelength corresponding to the
singly protonated species (Al, HB+) as a function of pH and solving
for the pKa. As the absorption at this wavelength is zero when no acid
is present (all dye is in the basic form) and goes to zero when suffi-
ciently high concentrations of acid are present (all dye is in the dou-
bly protonated form H2B2+), any absorption at this wavelength is
presumably proportional to the concentration of the singly proton-
ated species HB+ (CHB+) as per Beer’s law (Al, HB+ =ebCHB+). Plot-
ting absorption at lHB+ as a function of pH results in a curve with a
maximum absorption Al, HB+ MAX at the pH corresponding to the
maximum concentration of the singly protonated species.
Accordingly, both psKa values were determined by plotting absorp-
tion at l HB+ as a function of pH, determining Al, HB+ MAX, and solv-
ing for pH for Al, HB+ =½ Al, HB+ MAX at pH lower than the pH
corresponding to Al, HB+ MAX (to determine psKa1) and at pH higher
than the pH corresponding to Al, HB+ MAX (to determine psKa2).
Correlated NMR-absorption studies.
Derivative 1 was
selected for the correlated NMR-absorption studies because of
simplicity of spectral interpretation and the presumption that, given
its phenyl substitution, its behavior is representative of most of the
compounds studied. Solutions containing 0.5 mM of compound 1
were utilized to facilitate the measurement of both the absorption
and NMR spectra of each sample, and absorptivities were measured
using a reduced pathlength (0.2 cm) cuvette. Unfortunately, for
these samples, some of the measured absorption values were too
high for quantitative measurement (A > 2), but as no detector
saturation was observed, the measurements were still usable for
qualitative determination of the species present in a given sample.
An initial protonation study was carried out in which a 0.5-mM
solution of compound 1 was protonated with varying concentrations
of HClO4 in ACN, and the concentrations of acid corresponding to
the first and second protonation equilibria were determined. Using
this information, six samples were prepared, each containing
0.5 mM of compound 1 and varied concentrations of HClO4.
Concentrations of acid were chosen that corresponded to both the
first and second equilibria, including one sample containing no
added acid, one sample containing a concentration of acid
corresponding to the maximum concentration of the singly proton-
ated form of 1, one sample containing a concentration of acid
corresponding to all of compound 1 being in the doubly protonated
form, and the rest of the samples containing intermediate acid
concentrations in which two species are present. NMR and absorp-
tion spectra of each sample were acquired simultaneously at 25ꢃC
over the course of a single evening. Additional NMR studies were
carried out at higher (37ꢃC) and lower (0ꢃC) temperatures to ensure
that the observed behavior was not temperature dependent. These
higher and lower temperature studies were carried out without the
associated absorption measurements as no thermostatted UV-visible
spectrophotometers were available; however, the color changes and
intensities were noted visually.
Acknowledgments. This work is supported by the GSU Research
Initiation Grant and Georgia Research Alliance Grant to MH.
Additionally, the authors would like to acknowledge the Center
for Diagnostics and Therapeutics at Georgia State University for
their support. The funding agencies had no involvement in the
design, collection, analysis, or interpretation of data, the writing
of this report, or the decision to submit this article for
publication. The authors would also like to acknowledge Drs
Alex Spring, Sekar Chandrasekaran, and Markus Germann for
their assistance with the NMR spectrometers.
DISCLOSURE
This manuscript has been read and approved by all
authors. This article is unique, is not under consideration
by any other publication, and has not been published else-
where. The authors and peer reviewers of this article report
no conflicts of interest. The authors confirm that they have
permission to reproduce any copyrighted material.
Spectroscopic pKa determination. As the donor strength of
the substituents at the 2 and 6 positions was found to have a
marked effect on the molar absorptivities of the intermediate
singly protonated species, three dyes were chosen for further
study of this substitution effect. Compounds 1–3 were chosen on
the basis of a decreasing scale of electron-donating tendencies;
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