Protonation-deprotonation equilibria in tetrapyrroles Part 4: Mono- and diprotonations of deutero-, hemato-, meso-, and protoporphyrin IX dimethyl esters in methanolic hydrochloric acid

Article abstract of DOI:10.1142/S1088424614500126

The N-protonations in the deutero, hemato, meso and protoporphyrin IX dimethyl esters (DME) were investigated by spectrophotometric titrations using HCl as the acid and methanol as the solvent. Two spectroscopically different protonated species were observed for each porphyrin DME in addition to the neutral form. These were assigned to the N-protonated monocation and dication. There were no difficulties encountered in observing the monocation formation in the HCl-MeOH system. Very sharp isosbestic points were characteristic of each protonation stage. The pK³ values for the porphyrins in the above order were 3.23, 4.70, 2.93 and 3.37; the pK⁴ values were 2.48, 2.62, 2.41 and 2.64, respectively. For all porphyrins studied, no further spectral changes were observed after the dication was completely formed. This was interpreted as indicating that the formation of more highly protonated species is not possible in fullydelocalized porphyrins possessing the 18 π-electron [18]diazaannulene delocalization pathway. When the titration was performed on the free dicarboxylic acid porphyrins, aggregation obscured the first protonation step and no clear monocation spectrum could be distinguished. However, also in that case the titration ended up to a UVvis spectrum typical of the dication and the effect of aggregation on the pK₄ values was negligible. The UVvis spectrometric parameters are given for the neutral forms and for the protonated species of the porphyrin DMEs. The results are discussed in terms of the NH tautomerization connected to the π-electron delocalization pathway (aromaticity), which tends to hinder outofplane distortions in the porphyrin plane, and in terms of solvation and counterion stabilization of the protonated forms.

Full text of DOI:10.1142/S1088424614500126

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2014; 18: 385–395
DOI: 10.1142/S1088424614500126
Published at http://www.worldscinet.com/jpp/
Protonation-deprotonation equilibria in tetrapyrroles.
Part 4. Mono- and diprotonations of deutero-, hemato-,
meso-, and protoporphyrin IX dimethyl esters in methanolic
hydrochloric acid
Paavo H. Hynninen*◊
Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E),
FI-00014 Helsinki, Finland
Received 10 December 2013
Accepted 24 February 2014
ABSTRACT: The N-protonations in the deutero-, hemato-, meso- and protoporphyrin IX dimethyl
esters (DME) were investigated by spectrophotometric titrations using HCl as the acid and methanol as
the solvent. Two spectroscopically different protonated species were observed for each porphyrin DME
in addition to the neutral form. These were assigned to the N-protonated monocation and dication. There
were no difficulties encountered in observing the monocation formation in the HCl–MeOH system. Very
sharp isosbestic points were characteristic of each protonation stage. The pK3 values for the porphyrins
in the above order were 3.23, 4.70, 2.93 and 3.37; the pK4 values were 2.48, 2.62, 2.41 and 2.64,
respectively. For all porphyrins studied, no further spectral changes were observed after the dication
was completely formed. This was interpreted as indicating that the formation of more highly protonated
species is not possible in fully-delocalized porphyrins possessing the 18 p-electron [18]diazaannulene
delocalization pathway. When the titration was performed on the free dicarboxylic acid porphyrins,
aggregation obscured the first protonation step and no clear monocation spectrum could be distinguished.
However, also in that case the titration ended up to a UV-vis spectrum typical of the dication and the
effect of aggregation on the pK4 values was negligible. The UV-vis spectrometric parameters are given
for the neutral forms and for the protonated species of the porphyrin DMEs. The results are discussed
in terms of the NH tautomerization connected to the p-electron delocalization pathway (aromaticity),
which tends to hinder out-of-plane distortions in the porphyrin plane, and in terms of solvation and
counter-ion stabilization of the protonated forms.
KEYWORDS: porphyrin, acid-baseproperties, π-electrondelocalization, conformation, NHtautomerism.
hydrochloric acid (MeOH–HCl), the first protonation
INTRODUCTION
produced relatively easily the H+N22 monocation of
In this series, Parts 1 and 2 described the acid-base
the phorbin (pK3 = 4.14) but the second protonation,
behavior of phytyl/methyl pyropheophorbide a, a
phorbin possessing the isocyclic ring E. This sub-ring
was found to rigidify the macrocyclic ring, thereby
hindering conformational alterations demanded by NH
yielding the H+N22, H+N24 dication, was a difficult
reaction (pK4 ≈ 0) [1, 2]. Part 3 of this series focused
on the acid-base properties of chlorin e6 trimethyl ester
(TME) and the 71-acetal of rhodin g7 trimethyl ester in
tautomerization and N-protonations. In methanolic
the MeOH–HCl system [3]. This investigation resulted
in the following pKa values: pK3 = 4.63 and pK4 = 0.62
for chlorin e6 TME and pK3 = 4.40 and pK4 = 0.60 for
◊SPP full member in good standing
the acetal of rhodin g7 TME. It was concluded that the
low pK4 values originated in the steric hindrance caused
*Correspondenceto:PaavoH.Hynninen,email:paavo.hynninen@
helsinki.fi, tel: +358 9-19159180, fax: +358 9-19159138
Copyright © 2014 World Scientific Publishing Company

386
P. H. HYNNINEN
71
R3
3
C
5
7
R3
4
6
2
1
21
8
9
R8
B
A
R8
N
H
N
21
22
N
N
N
10
20
24
23
N
Ha Hb
11
12
19
N
H
C
16
D
N
121
181
14
18
13
131
132
17
15
171
172
CO CH
2
133
CO2CH3
173
3
CO2CH3
H3CO2C
1 Deuteroporphyrin IX DME: R3 = R8 = H
R3
R3
2 Hematoporphyrin IX DME: R3 = R8 = CH(OH)CH3
3 Mesoporphyrin IX DME: R3 = R8 = CH2CH3
4 Protoporphyrin IX DME: R3 = R8 = CH=CH2
R8
R8
N
N
N
N
N
N
N
Ha
Hb
Hb
Ha
Fig. 1. Structures of deuteroporphyrin IX DME (1),
hematoporphyrin IX DME (2), mesoporphyrin IX DME (3) and
protoporphyrin IX DME (4)
N
H3CO2C
CO2CH3
H3CO2C
CO2CH3
by the C15-methoxycarbonylmethyl substituent and
the interruption of the 18 p-electron [18]diazaannulene
delocalizationpathwayinthereducedringDofthechlorin.
In order to see how the electronic delocalization pathway,
which is intimately connected to NH tautomerization,
influences the pKa values of fully-delocalized porphyrins
(porphyrins proper) in the MeOH–HCl system, we shall
deal in this paper with the acid-base properties of the
dimethyl esters (DME) of deutero-(1), hemato-(2), meso-
(3) and protoporphyrin IX (4) (Fig. 1).
B
A
Scheme 1. The tautomeric NH exchange in the solutions of
3,8-substituted deuteroporphyrin IX dimethyl esters (DME).
A and B represent trans-NH tautomers and C is an example of
cis-NH tautomers (cf. the cis-NH tautomers in Scheme 2). The
conversion from A to B must go via C. The thick black line shows
the 18 p-electrons [18]diazaannulene delocalization pathway
These are all fully delocalized (aromatic)
b-substituted porphyrins that tend to maintain planarity
in the macrocycle by resonance (delocalization) energy,
reviewed by George [4]. Fully-delocalized porphyrins
with various b-substituents undergo NH tautomerization
values reported by us [14] for the NH tautomerization of
chlorin e6 TME (DG1‡ = 10.8 kcal.mol-1) and the 71-acetal
of rhodin g7 TME (DG1‡ = 10.6 kcal.mol-1).
The acid-base properties of porphyrins 1–4 have
inspired an appreciable amount of interest in almost all
fields of chemistry and not least in biochemistry and
clinical chemistry [15–23]. Protoporphyrin IX is one
of the most important functional porphyrins, because it
occurs as the organic constituent of the hemes (= iron-
complexes of protoporphyrin IX or its derivatives). The
hemes in turn occur as co-enzymes or prosthetic groups
in the hemoproteins, which function in oxygen-transfer,
electron-transfer and oxygen activation. In addition,
protoporphyrin IX can be used in photodynamic therapy
(PDT) by applying its precursor, 5-aminolevulinic acid
(d-ALA), topically or systemically in the PDT of skin
cancers (e.g. basal cell carcinoma and squamous cell
carcinoma) [24–26].
For PDT, the first legalized porphyrins (in Canada
1994) are the so-called hematoporphyrin derivative
(HpD) and its partially purified form (DHE) [27–
29]. The HpD has been found to contain hemato-,
monovinylhydroxyethyldeutero- and protoporphyrin
monomers as well as small amounts of their dimers and
oligomers, where the porphyrin units are covalently
1
(Scheme 1) fast at the room temperature on the H
NMR time-scale [5–11]. In Scheme 1, the aromatic 18
p-electron [18]diazaannulene delocalization pathway
is shown with thick lines for the more stable trans-NH
tautomers A and B with the NH groups in opposite sub-
rings, as well as for one example of an unstable cis-NH
tautomer C with the NH groups in adjacent sub-rings. For
the application of the 18 p-electron [18]diazaannulene
delocalization pathway to metal-free porphyrinoids, the
reader is advised to read the review by Lash [12].
There are several possible cis-NH tautomers as is
shown subsequently in Scheme 2. Though the cis-NH
tautomers are unstable intermediates in the tautomerism,
they are obligatory for the NH exchange process. The free
activation energy (DG‡) for the tautomeric NH exchange
process of fully-delocalized porphyrins has been deter-
mined by several authors [6, 8, 13]. For instance, for
deuteroporphyrin IX DME and its dideuteriated form,
DG‡ is 10.3 kcal.mol-1 (H2 form) and 11.6 kcal.mol-1 (D2
form)[6]. Interestingly, these values are close to the DG1‡
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 386–395

PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
387
linked with ether, ester or carbon–carbon bonds. DHE
nitrogen atoms. Consequently, while varying the structure
of the tetrapyrrole, the other relevant parameters, such
as the temperature, concentration of tetrapyrrole,
solvent, acid and counterion, should be optimal. On the
other hand, the requirement of systematisation sets up
insurmountable difficulties for the selection of solvent,
as the structurally different tetrapyrroles exhibit a great
variation in their solubility properties.
This paper describes the protonation titration results
on the dimethyl ester (DME) of deuteroporphyrin IX
and its 3,8-substituted derivatives in the methanolic
hydrochloric acid system. To see the effects of the
maximally delocalized p-system on the protonation
behavior of the fully-delocalized porphyrins, we selected
the experimental conditions, in addition to the acid,
solvent and counter-ion, closely similar to the conditions
used by us in the previous protonation studies [1, 3], i.e.
the concentration of the derivative was ca. 10-5 M and the
temperature was 20°C. To ascertain the reversibility of the
protonations and to eliminate the possible side-reaction
effects, we regularly recorded the UV-vis spectrum of the
titrated sample after neutralization with aqueous NaOAc
and extraction of the pigments into Et2O.
is obtained from HpD by removing the monomers.
The mechanism of the selective accumulation of some
unrecognized HpD/DHE component(s) into cancerous
tissues is unknown, but among the various hypotheses,
it has been proposed [30–33] that the protonated
cationic porphyrins would behave in this regard more
selectively than the neutral forms. It is also noteworthy
that the N-protonated cationic porphyrin would have
photophysical properties different from those of the
neutral form of the same porphyrin.
In spite of the enormous functional importance
of the b-substituted porphyrins, the knowledge of
their acid-base properties is still incomplete (see the
reviews of Refs. [15–22]). A good knowledge of these
properties would be of fundamental significance when
trying to understand better the other long-standing
major problems in porphyrin chemistry such as the
p-electron delocalization pathway (aromaticity), the
electronic configurations of the central N-atoms (sp2
vs. sp3 hybridization), NH tautomerism as well as the
metallation and demetallation mechanisms. These are all
intimately associated with the protonation-deprotonation
equilibria at the central N-atoms of porphyrins.
Further, the knowledge of the acid-base properties of
porphyrins is important in the clinical laboratory [23].
b-Substituted porphyrins such as proto-, copro- and
uroporphyrins occur at elevated levels in blood, urine and
fecesinvariousporphyrias,whichareabnormalconditions
of porphyrin metabolism [23]. For the diagnosis of these
diseases, the porphyrins are usually extracted from
urine or feces with an acidified organic solvent and then
quantified spectrophotometrically, fluorometrically or by
HPLC. Considering that the neutral forms, monocations
and dications of the porphyrins have very different
absorption and fluorescence properties [15, 16], it is
obvious that considerable systematic errors may interfere
with the determinations, if not the right amounts of the
right solvents and acids are used in the extractions and
measurements. Hence, to be able to select the optimum
conditions for the determinations, precise knowledge of
the acid-base properties of porphyrins is needed.
The inner N-protonation behavior of a cyclic
tetrapyrrole is determined by a delicate interplay between
several factors, e.g. the steric factors determined by the
substitution pattern and size of the substituents, p-electron
delocalization pathway, which is intimately associated
with NH-tautomerism, van der Waals repulsion and
electrostatic repulsion between the positively charged
nitrogen atoms, as well as solvate and ion-pair formation
of the protonated species. Aggregation and some
other reaction possibilities, may considerably further
complicate the situation [1]. Owing to the complexity
of the acid-base behavior of tetrapyrroles, it is important
to seek a systematic approach, if one wishes to learn
something about how the structural differences of various
tetrapyrroles are reflected in the basicities of the sub-ring
In this presentation, the acid dissociation constants
(Ka) are denoted as in Part 1 [1]. The numbering system
and Fischer trivial names accepted by IUPAC-IUB
[34] are used for the porphyrins studied (Fig. 1, 1–4).
The notations for the assignments of absorption bands
to electronic transitions are performed according to
the Fischer–Stern Notation and the Platt–Gouterman
Notation [35].
RESULTS AND DISCUSSION
The results from the protonation titrations on the DMEs
of deutero-, hemato-, meso- and protoporphyrin IX in the
HCl–MeOH system are illustrated in Figs 2–5. These
figures clearly show that the four porphyrin derivatives
behave very similarly when the HCl concentration is
increased in MeOH. Each porphyrin derivative can be
seen to give two clearly distinguishable sets of spectra
with several sharp isosbestic points. The interpretation
of the results is straightforward. The first set of spectra
represents the conversion of the neutral form of porphyrin
to its monocation and the second set the conversion of the
monocation into the dication.
The spectral changes occurring in the monocation
formation are closely comparable for the four porphyrin
derivatives. Inspection of, for instance Fig. 2, shows
that the typical changes are as follows. Band I (Qy, 0-0) is
shifted to the blue by ca. 20 nm with a slight reduction
in its intensity. Band II (Qy, 0-1) also experiences a blue
shift by ca. 10 nm, but its intensity is clearly increased.
The location and intensity of band III (Qx, 0-0) remain
approximately constant. Interesting is the behavior of
band IV (Qx, 0-1). It seems to disappear completely as
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 387–395

388
P. H. HYNNINEN
Fig. 2. Spectrophotometric titration spectra for the protonations
of deuteroporphyrin IX DME (1) in the HCl–MeOH system.
Upper spectra: conversion of the neutral form to the monocation.
Lower spectra: conversion of the monocation to the dication
Fig. 3. Spectrophotometric titration spectra for the protonations
of hematoporphyrin IX DME (2) in the HCl–MeOH system.
Upper spectra: conversion of the neutral form to the monocation.
Lower spectra: conversion of the monocation to the dication
was observed also for pyropheophytin a [1] and the
chlorophyll-related chlorins [3]. The Soret band (S/B) is
shifted to the blue by ca. 5 nm and its intensity is slightly
decreased for deuteroporphyrin IX DME but increased
for the other three porphyrin derivatives.
addition of proton to the vinyl group yielding the 31- or
81-carbocation, which then reacts with the methanol
nucleophile.
After reaching the terminal dication spectrum, dry
gaseous HCl was conducted into the solution. The
spectra remained approximately constant; only a slight
reduction in the Soret band intensity of protoporphyrin
IX DME could be observed on prolonged HCl bubbling.
The UV-vis spectra of the porphyrins recovered from the
titrated solutions were practically identical with those of
the original porphyrins.
On the whole, our results are very similar to those
obtained previously by Corwin et al. [36] when titrating
mesoporphyrin IX DME in the acetic acid (60%)–
acetone (40%) system with HCl (the legends of Figs
2 and 3 in Ref. [36] should be interchanged). Our
results show that no special conditions are necessary
for observing the monocation formation, in contrast to
the earlier belief. Using the simple HCl–MeOH system
and a very accurate titration technique [1, 3], we have
been able to observe the monocation formation for every
tetrapyrrole derivative studied by us this far. Evidently,
The spectral changes in the second protonation step,
representing the formation of the dications, are also
similar for the four porphyrins. Bands I and II are shifted
further to the blue by ca. 10 nm with some elevation in
intensities. Now band III seems to vanish completely or
perhaps bands II and III merge to form a new band at ca.
550 nm. The behavior of the Soret band is noteworthy. It
experiences a red shift by ca. 15 nm and a huge increase
in intensity. The increase in the Soret band intensity
is clearly less for protoporphyrin IX DME (Fig. 5) as
compared with the other three porphyrin DMEs. Also the
isosbestic points at ca. 400 and 570 nm can be seen to
disintegrate slightly in Fig. 5, whereas in Figs 2–4, all
isosbestic points are very sharp. These results for the
protoporphyrin DME can be interpreted as indicating that,
to a slight extent, some side-reactions occur in addition
to the N-protonations. One probable side-reaction is the
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 388–395

PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
389
Fig. 4. Spectrophotometric titration spectra for the protonations
Fig. 5. Spectrophotometric titration spectra for the protonations
of protoporphyrin IX DME (4) in the HCl–MeOH system.
Upper spectra: conversion of the neutral form to the monocation.
Lower spectra: conversion of the monocation to the dication
of mesoporphyrin IX DME (3) in the HCl–MeOH system.
Upper spectra: conversion of the neutral form to the monocation.
Lower spectra: conversion of the monocation to the dication
the remarkable suitability of MeOH to stabilize the
porphyrin monocation is due to the very high ability of
MeOH to solvate cations [37, 38]. The lmax and e values
for the spectrometric parameters of the neutral form,
monocation and dication of each porphyrin 1–4, are listed
in Table 1. When the solvent effects are taken into account,
these values compare surprisingly well with those given by
Falk (see Tables 28–32 on pp 232–239 in reference 15).
No lmax and e values can be found in Falk’s book [15]
for the molecular species of the four porphyrin DMEs in
methanol and the values for the monocation and dication
of hematoporphyrin IX (DME) are missing as well.
The pK3 and pK4 values obtained from the titrations
above are listed in Table 2. Comparison of the pKa values
in Table 2 with those reported in the literature for the same
dicarboxylic porphyrins or their DMEs shows that our pK3
values for deutero-, meso- and protoporphyrin IX DMEs
are 1.5–3.0 pKa units lower than the pK3 values given by
Phillips and co-workers [17–20] and Caughey et al. [35]
for the same porphyrins in 2.5% sodium dodecylsulphate.
The pK4 values of Table 2 are approximately equal for the
four porphyrin derivatives, and are higher by 0.5–1.0 pKa
units than the values reported by Dempsey et al. [19]. The
distinctly higher pK3 value of 4.70 for hematoporphyrin
IX DME in methanol is consistent with the values of 4.7
and 5.0 reported by Brault and co-workers [30, 39] for
the free porphyrin in aqueous solutions.
The pK3 values of Table 2 seem to afford a reasonable
explanation to the long-standing debate about the exist-
ence of a porphyrin monocation as a separate species
[1]. The conspicuously higher pK3 and pK3-pK4 values
for the hematoporphyrin DME probably cannot solely
be attributed to the higher basicity of its pyridine-type
nitrogen atoms. Another relevant factor contributing
to the higher pK3 value presumably results from the
higher solvation degree of the hematoporphyrin DME
in methanol and the formation of a more stable ion-pair
between the solvated porphyrin monocation and the
solvated Cl- counter-ion, leading to the stabilization of
the monocation. Compared with the hematoporphyrin
DME, the other porphyrin DMEs cannot be solvated to a
similar extent by methanol, because they possess 3- and
8-substituents that cannot from hydrogen bonds with
methanol. Thus, the exceptionally low pK3 value of 2.93
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 389–395

390
P. H. HYNNINEN
Table 1. Spectrometric parameters for the neutral and protonated molecular species of dicarboxylic porphyrin DMEs in
methanol at 20°C
Porphyrin
Neutral form Neutral form Monocation
Monocation
Dication
max, nm
Dication
lmax, nm
e, mM-1.cm-1
lmax, nm
e, mM-1.cm-1
l
e,· mM-1.cm-1
Deuteroporphyrin IX DME
617
593
565
526
494
392.5
3.49
0.10
598
—
3.04
—
590
4.41
—
—
5.54
556
526
—
10.97
8.27
—
548
13.6
—
7.20
—
12.87
169.3
—
—
387.5
160.2
402
314.2
Hematoporphyrin IX DME
620
595
567
531
498
396.5
—
4.22
0.15
6.75
9.38
14.76
189.8
—
602
—
3.94
—
591
572 sh
550
—
5.38
5.85
17.19
—
561
531
—
13.65
10.39
—
—
—
390
294
192.1
13.23
403
—
393.9
—
Mesoporphyrin IX DME
Protoporphyrin IX DME
617
566
4.22
6.54
600.5
559
529
—
3.68
12.18
9.20
590
548
—
4.96
14.98
—
529
9.30
496
12.33
160.9
—
—
—
393.5
388
181.9
402
379.0
627.5
574
4.89
6.27
609
565
535
—
3.66
12.17
8.37
599
556
—
5.62
15.18
—
537
9.91
502.5
13.54
163.0
—
—
—
399
396.5
185.0
408.5
270.8
Table 2. pKa-values for the protonated molecular species of free
dicarboxylic porphyrins and their DMEs in methanol at 20°C
line would lead to the conclusion that, the less solvated
the porphyrin monocation in the solvent used, the lower
is the pK3 value and the less stable the ion-pair formed
between the porphyrin monocation and the counter-ion;
solvents, such as CHCl3 or CH2Cl2, with a low solvation
capability for ions, were frequently used in the earlier
protonation studies of porphyrins. Hence, obviously we
are approaching a situation where the pK3 and pK4 values
are nearly identical. This situation implies that we cannot
see the formation of the monocation at all, because it is a
short-lived transient species, which is rapidly converted
to the neutral form and the dication (disproportionation).
However, it should be noted that the failure in observing
the monocation in the earlier protonation studies cannot
be ascribed solely to the selection of a wrong solvent,
because the stability of the ion-pair between the porphyrin
monocation and counter-ion also depends on the nature
of the counter-ion. We shall examine in further detail the
role of the counter-ion in the ion-pair stabilization when
dealing with the results from the protonation studies in
acetic acid.
Porphyrin
Derivative
Free
pK3
pK4
pK3-pK4
Deuterop. IX
—
—
—
0.75
—
Dimethyl ester +3.23 +2.48
Free +2.65
Dimethyl ester +4.70 +2.62
Free +2.68
Dimethyl ester +2.93 +2.41
Free +2.37
Dimethyl ester +3.37 +2.64
a Obscured by aggregation.
Hematop. IX
Mesop. IX
Protop. IX
a
2.08
—
a
0.52
—
a
0.73
for the mesoporphyrin IX DME would be explained by
its low solvation degree with methanol, leading to a less
stable ion-pair between the porphyrin monocation and
the Cl- counter-ion. Continuation of thinking on this
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 390–395

PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
391
cations and the negatively charged anions to form an ion-
pair between the porphyrin monocation and the counter-
ion. It is noteworthy that there exist variably solvated
ion-pairs: (1) a solvated contact (intimate) ion-pair,
(2) a solvent-shared ion-pair, (3) a solvent-separated ion-
pair and (4) unpaired solvated ions of a 1:1 ionophore in
solution; (see Figs. 2–14 in reference 38). In the present
case, the counter-ion is AcO-, where the negative charge
is delocalized equally between the two carboxylate
oxygen atoms. Such a counter-ion is less effective in
stabilizing the porphyrin monocation than the smaller Cl-
anion, in which the negative charge is concentrated on
one atom. In AcOH, the mesoporphyrin monocation can
be expected to be the least solvated of the four porphyrin
monocations. Thus, the reduced solvation degree and
insufficientcounter-ionstabilizationmakethemonocation
of mesoporphyrin DME a short-lived transient species,
whose presence cannot be clearly seen in the UV-vis
absorption spectrum. The AcO- is comparable with ClO4-
as a counter-ion. Karaman and Bruice [40] concluded
that the localized negatively charged counter-ions, such
as Cl-, stabilize the protonated porphyrin by forming
strong “tight-contact pairs” with the porphyrin cation,
whereas delocalized negatively charged counter-ions,
-
such as ClO4 , form weak ion-pairs and hence destabilize
the protonated porphyrin by increasing its acidity.
However, they found no evidence for the accumulation of
the monocation of the tetraphenylporphyrin, H3TPP+(X-),
in chloroform or nitrobenzene.
In part, the diverse results from the earlier protonation
studies on porphyrins [1] can be ascribed to their high
Fig. 6. UV-vis spectra of the dicarboxylic porphyrin DMEs
aggregation tendency. Titrations were usually carried
out on free (unesterified) dicarboxylic porphyrins in
relatively nonpolar solvents such as CHCl3 or CH2Cl2,
in which the free porphyrins are only slightly soluble.
To see the possible effects of aggregation in detail, we
performed titrations on the free dicarboxylic deutero-,
meso-, proto- and hematoporphyrin IX in the HCl–MeOH
system. The results were mutually quite similar for all
these porphyrins and, hence, only the results obtained for
free mesoporphyrin IX are shown in Fig. 7.
The UV-vis spectra in Fig. 7 show clearly that the
aggregation obscures the formation of the monocation
almost entirely. Spectrum no. 1 is the initial spectrum
measured from the solution of mesoporphyrin in neat
MeOH. When small increments of HCl are added to
the solution, the intensity of the Soret band is markedly
decreased and, in the case of spectrum no. 5, there is
very little left of its original intensity. The position of the
Soret band is shifted to the red by only 2–3 nm. The low-
intensity bands IV–I in the range 480–650 nm are shifted
to the red by 5–8 nm and their base level is lifted. These
aggregation effects on porphyrin spectra are comparable
with those described earlier [41, 42]. The first sign of
the monocation formation can be seen in the spectrum
no. 6 of Fig. 7. A new Soret band starts rising at 390 nm
and there is a small hump at 600 nm, indicating the
(1)–(4) in glacial acetic acid
To learn more about the roles played by the solvent
and counter-ion in the protonation equilibria, we
dissolved each porphyrin IX DME in glacial acetic acid
and measured the UV-vis spectra (Fig. 6). Comparison
of the spectra in Fig. 6 with those in Figs 2–5 indicates
that the proto-, hemato- and deuteroporphyrin IX DMEs
occur in AcOH as mixtures of the neutral forms and the
monocations (ratio roughly 1:1), whereas the spectrum
for mesoporphyrin DME in AcOH represents a mixture
of the dication and monocation, the dication being the
predominant species. The spectra in Fig. 6 for the DMEs
of proto-, deutero- and mesoporphyrins in AcOH are
quite consistent with those reported by Caughey et al.
[35]. Addition of dry HCl to the AcOH solutions resulted
in dication spectra, which were identical with those in
Figs 2–5.
An interesting feature in the results of Fig. 6 is that the
mesoporphyrin DME is the most extensively protonated
porphyrin in AcOH, although it has the lowest pK3 and
pK4 values in the HCl–MeOH system. The explanation
to this peculiarity can be derived straightforwardly from
a consideration of the solvent effects on the solvation
ability of the solvent to stabilize the positively charged
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 391–395

392
P. H. HYNNINEN
energies, because the induction effect (electron-density
withdrawing or donating effect) is of about the same
order of magnitude for the various b-substituents of
the sub-rings; (note that precisely taken, the porphyrins
1–4 are unsymmetric). In addition, Scheme 2 shows as
obligatory intermediates the participation of 8 cis-NH
tautomers, which have the NH groups in adjacent
sub-rings. Though aromatic, the cis-NH tautomers
are unstable because of the van der Waals repulsion
between the too closely spaced central NH hydrogen
atoms. Hence, the presence of a cis-NH tautomer in the
tautomerizing solution of a fully-delocalized porphyrin
has never been observed.
Comparison of Scheme 2 for chlorins in reference
[3] with Scheme 2 in this paper for fully-delocalized
porphyrins shows a clear difference between the two
schemes. In the latter case, the 18 p-electron [18]-
diazaannulene delocalization pathway can be applied
to every NH tautomer, whereas in Scheme 2 for
chlorins the full delocalization is interrupted in the
reduced ring D, because the delocalization of the lone-
pair electrons of the ring D nitrogen-atom consumes
energy. Thus, the freedom of tautomeric NH exchange
is clearly greater for fully-delocalized porphyrins than
for chlorins.
In the protonations, the protons occupy the lone-
pair electrons of the N-atoms in the pyrrolenine-type
sub-rings of a trans-NH tautomer. Consequently, in the
monoprotonation, the proton can occupy either N22
or N24 of the type A trans-NH tautomer or either N21
or N23 of the B type trans-NH tautomer (Scheme 1).
Correspondingly, in the diprotonation, the proton can
occupy both N22 and N24 of the A type trans-NH
tautomer or N21 and N23 of the B type trans-NH
tautomer. Owing to the approximately similar inductive
effects of the b-substituents in porphyrins 1–4, the A
and B type trans-NH tautomers of the neutral form, are
expected to give similar UV-vis spectra. This argument
is likely to hold also for the monocation or the dication
of the A and B type trans-NH tautomers. However, in the
case of more unsymmetric porphyrins, it is possible that
the A and B type trans-NH tautomers of the monocation
or the dication have different energies, originating from
differences in substituent effects, whose nature may be
steric and/or inductive. That situation is then likely to
lead to different populations of the protonated tautomeric
species. This possibility is interesting considering the
results from the investigations of Crossley and co-workers
on the NH tautomerism of differently substituted, fully-
delocalized porphyrins by 1H NMR in the slow exchange
range [7–11].
Fig. 7. Spectrophotometric titration spectra for the protonations
of the unesterified mesoporphyrin IX in the HCl–MeOH
system. Upper spectra: the effect of aggregation on the UV-vis
spectrum of the neutral form of mesoporphyrin IX. Lower
spectra: conversion of the aggregated mesoporphyrin IX to its
monomeric dication
formation of the monocation. The Soret band intensity
of the monocation reaches its maximum in the case of
spectrum no. 10, which also shows the rising Soret band
of the dication at 415 nm. The terminal spectrum reached
(no. 12) is typical of the mesoporphyrin dication (Fig. 3).
No well-defined isosbestic points can be distinguished
in this case. Owing to the aggregation effects, no pK3
value could be estimated from the spectra. These results
show clearly that the DMEs of dicarboxylic porphyrins
should always be used in the titrations instead of the free
dicarboxylic acids.
To understand in further detail the effects of the
central N-protonations on the structure, conformation
and chemical reactivity of porphyrins 1–4, let us
consider first all possible reactions in the tautomeric
NH exchange process of the neutral forms of the
porphyrins (Scheme 2). Inspection of Scheme 2
indicates that there are 4 different trans-NH tautomers,
which can be estimated to have approximately equal
In the dication (diacid), the increased number of
hydrogen atoms leads to a significant congestion in the
central hole of the porphyrin macrocycle. This situation
is described in terms of steric hindrance, steric strain,
van der Waals repulsion and electrostatic repulsion. The
overcrowding is relieved by conformational alterations,
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 392–395

PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
393
A
N
N
N
N
Ha
Hb
N
N
N
N
N
N
N
N
Ha
Hb
HaHb
D'
C
N
N
N
N
N
N
N
N
Ha
Hb
HaHb
N
N
N
N
N
N
Ha
Hb
Hb
Ha
B'
B
N
N
N
N
N
N
N
N
N
Hb
Ha
HbHa
N
N
N
N
N
N
N
N
Hb
Ha
HbHa
N
N
N
N
Hb
Ha
N
D
C'
A'
Scheme 2. The total tautomeric NH exchange process between the aromatic trans- and cis-NH tautomers of the unsubstituted free-
base porphyrins. The thick black line shows the 18 p-electrons [18]diazaannulene delocalization pathway
which appear as out-of-plane distortions in the macro-
cycle of the dication [43]. Stone and Fleischer [43]
mention three possible causes for the pronounced tilting
EXPERIMENTAL
Preparation of porphyrins and their dimethyl esters
ofthesub-ringsofcrystallinemeso-tetraphenylporphyrin
and meso-tetrapyridylporphyrin dications: “(1) the
steric hindrance that would result if all four inner
hydrogen atoms were coplanar, (2) the electrostatic
repulsion of the inner nitrogen atoms, assuming each
of them to have a charge of +½ and (3) general packing
effects.” In addition, they are of the opinion that “causes
1 and 2 apply to the molecule in solution as well as in
the solid state, while cause 3 is applicable only to the
solid state.” Senge and Kalisch [44] and Senge [45] have
carried out a thorough comparative crystallographic
analysis of the conformation of porphyrin dications
with various substituent types. The results from this
analysis reveal appreciable differences in the degree
of nonplanarity and distortion modes. Varying the
type, location and number of substituents, porphyrins,
possessing macrocycles with a ruffled, saddle, wave or
dome distortion mode, could be prepared and analyzed
by crystallography.
Protohemin IX was isolated from bovine blood
according to the method of Labbe and Nishida [46].
Deutero-, meso- and protoporphyrins IX (1, 3, 4) were
prepared from crystalline protohemin IX and purified
by multiplicative liquid–liquid partition as described in
reference 47. Hematoporphyrin IX (2) was purchased
from Koch-Light Co. and further purified by multiplicative
liquid–liquid partition [47]. The dimethyl esters of the
porphyrins were obtained by permitting each dicarboxylic
porphyrin to stand in dry methanol saturated with dry HCl
gas. Methanol (Absolute, ACS, “Baker analysed reagent”
from J. T. Baker) was dried with 3 Å molecular sieves.
Spectrophotometric protonation titrations
A Cary model 219 UV-vis spectrophotometer was used
for the measurement of the electronic absorption spectra
(UV-vis). Quartz cuvettes provided with Teflon-stoppers
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 393–395

394
P. H. HYNNINEN
Table 3. Experimental details for the protonation titration of hematoporphyrin IX DME, c = 9.48 × 10-6 M
Spectrum no.
Volume of
HCl–MeOH
added, mL
cHCl in
MeOH, M
Total amount of
HCl added, mM
Total volume
of solution in
cuvette, mL
cH+, M
-log cH+
pKa
1
2
0.0
4.0
0.00
0.01
0.01
0.01
0.01
0.01
0.10
0.10
1.00
1.00
1.00
1.00
1.00
1.00
5.00
5.00
5.00
0.00
0.04
3000
3004
3006
3008
3010
3014
3016
3026
3028
3030
3032
3037
3042
3052
3057
3087
3187
—
—
—
1.332 × 10-5
1.996 × 10-5
2.660 × 10-5
3.322 × 10-5
4.645 × 10-5
1.127 × 10-4
4.428 × 10-4
1.103 × 10-3
1.762 × 10-3
2.421 × 10-3
4.063 × 10-3
5.700 × 10-3
8.958 × 10-3
1.222 × 10-2
6.069 × 10-2
2.157 × 10-1
4.876
4.700
4.575
4.479
4.333
3.948
3.354
2.957
2.754
2.616
2.391
2.244
2.048
1.913
1.217
0.666
—
3
2.0
0.06
4.70 = pK3
4
2.0
0.08
—
5
2.0
0.10
—
6
4.0
0.14
—
7
2.0
0.34
—
8
10.0
2.0
1.34
—
9
3.34
—
10
11
12
13
14
15
16
17a
2.0
5.34
—
2.0
7.34
2.62 = pK4
5.0
12.34
17.34
27.34
37.34
187.34
687.34
—
—
—
—
—
—
5.0
10.0
2.0
30.0
100.0
a After spectrum no. 17, the titration was continued by conducting dry HCl gas into the solution in the cuvette on an ice-bath
for a period of 30 s for each successive spectrum. Virtually no changes in the absorption spectrum were observed.
and having a light path of 1.0 cm and a volume of 3 mL
were employed in the measurements. Spectra were taken
after sufficiently small additions of HCl–MeOH to a
ca. 10-5 M solution of each porphyrin derivative in dry
methanol. The titration technique was similar to that
described in Part 1 [1]. The experimental details of the
titration for hematoporphyrin IX DME are shown in
Table 3 as an example.
Hence, the pK3-pK4 values are: 0.75, 2.08, 0.52 and
0.73, respectively.
3. Even in the case of mesoporphyrin IX dimethyl ester,
where the pK3-pK4 value is as low as 0.52, the forma-
tion of the monocation could be clearly observed as a
separate species. This result is in disagreement with
the conclusions from some earlier investigations that
the monocation is too unstable to be observable (for
references, see reference [1]). However, it should be
noted that the earlier acid-base studies on porphyrins
were carried out in relatively nonpolar solvents, e.g.
CHCl3 and CH2Cl2, which have inadequate solvation
capability for cations and anions.
4. The use of unesterified porphyrins instead of dies-
ters may cause interference of aggregation with the
N-protonations. This was shown by the protonation
titration experiment performed on unesterified meso-
porphyrin IX using the HCl–MeOH system. In this
experiment, the free porphyrin remained in aggre-
gated state at low HCl concentrations and no sign
of the monocation formation was observed. When
the HCl concentration was increased to a certain
level, the porphyrin aggregate was dissolved and the
monomeric porphyrin dication appeared as could be
judged from the measured UV-vis spectrum.
CONCLUSIONS
1. The spectrophotometric protonation titrations with
HCl as titrant and MeOH as solvent verified that each
of the fully-delocalized porphyrins, such as deu-
tero-, hemato- meso- or protoporphyrin IX dimethyl
ester, is monoprotonated to form the monocation
and diprotonated to form the dication. In these cat-
ion species, the protons occupy the N-atoms of the
pyrrolenine type sub-rings. There are no difficulties
in observing the monocation formation in the HCl–
MeOH system. This is explained by the good solva-
tion properties of MeOH for both cations and anions
which enables electrostatic stabilization between the
solvated monocation and the solvated chloride anion.
2. The following pKa values were obtained for the por-
phyrins in the above order: pK3 = 3.23, 4.70, 2.93 and
3.37; pK4 = 2.48, 2.62, 2.41 and 2.64, respectively.
5. The porphyrins studied in this paper were regarded
as 18 p-electrons [18]diazaannulenes. Comparison
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 394–395

PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
395
of the total tautomeric NH exchange process of chlo-
23. Labbe RF and Lamon JM. In Textbook of Clinical
Chemistry, Tietz NW (Ed.). Saunders: Philadelphia,
1986; pp 1589–1614.
24. Kennedy JC, Pottier RH and Pross DC. J. Photo-
chem. Photobiol. B Biol. 1990; 6: 143–148.
25. Kennedy JC and Pottier RH. J. Photochem. Photo-
biol. B Biol. 1992; 14: 275–292.
rins with that of fully-delocalized porphyrins indi-
cates that there is more freedom for NH exchange
in the porphyrins. Apparently, this might explain,
besides the differences between the pKa values of the
porphyrins and chlorins, the faster NH-exchange rate
of the porphyrins as compared with that of chlorins.
26. Peng Q, Berg K, Moan J, Kongshaug M and Nesland
JM. Photochem. Photobiol. 1997; 65: 235–251.
27. Photodynamic Therapy, Gomer CJ (Ed.). Photo-
chem. Photobiol. 1987; 46(5): 561–952.
28. Pandey RK and Zheng G. In The Porphyrin Hand-
book, Vol. 6, Kadish KM, Smith KM and Guilard R
(Eds.). Academic Press: San Diego, San Francisco,
NewYork, Boston, London, Sydney, Toronto, 1999;
pp 157–260.
REFERENCES
1. Hynninen PH. J. Chem. Soc. Perkin Trans. 2 1991;
669–678.
2. Hynninen PH and Mesilaakso M. J. Porphyrins
Phthalocyanines 2012; 16: 39–46.
3. Hynninen PH. J. Porphyrins Phthalocyanines 2012;
16: 1167–1176.
29. Nyman ES and Hynninen PH. J. Photochem. Photo-
biol. B Biol. 2004; 73: 1–28.
30. Brault D and Vever-Bizet C. J. Chem. Soc. Faraday
Trans. 1992; 88: 1519–1524.
31. Taima H, Okubo A, Yoshioka N and Inoue H.
Tetrahedron Lett. 2005; 46: 4161–4164.
32. Pandey RK, Smith NW, Shiau F-Y, Dougherty TJ
and Smith KM. J. Chem. Soc. Chem. Commun.
1991; 1637–1638.
33. Kessel D, Woodburn K, Henderson BW and Chang
CK. Photochem. Photobiol. 1995; 62: 875–881.
34. International Union of Pure and Applied Chemistry
(IUPAC) and International Union of Biochemistry
(IUB). Nomenclature of Tetrapyrroles, Moss GB
(Ed.). Pure Appl. Chem. 1987; 59: 779–832.
35. Caughey WS, Fujimoto WY and Johnson BP.
Biochemistry 1966; 5: 3830–3843.
4. George P. Chem. Rev. 1975; 75: 85–111.
5. Storm CB and TekluY. J. Am. Chem. Soc. 1972; 94:
1745–1747.
6. Storm CB, Teklu Y and Sokoloski EA. Ann. New
York Acad. Sci. 1973; 206: 631–640.
7. Crossley MJ, Harding MM and Sternhell S. J. Am.
Chem. Soc. 1986; 108: 3608–3613.
8. Crossley MJ, Field LD, Harding MM and Sternhell
S. J. Am. Chem. Soc. 1987; 109: 2335–2341.
9. Crossley MJ, Harding MM and Sternhell S. J. Org.
Chem. 1988; 53: 1132–1137.
10. Crossley MJ, Harding MM and Sternhell S. J. Org.
Chem. 1992; 57: 1833–1837.
11. Crossley MJ, Harding MM and Sternhell S. J. Am.
Chem. Soc. 1992; 114: 3266–3272.
12. Lash TD. J. Porphyrins Phthalocyanines 2011; 15:
1093–1115.
36. Corwin AH, Chivvis AB, Poor RB, Whitten DG and
Baker EW. J. Am. Chem. Soc. 1968; 90: 6577–6583.
37. Sarmini K and Kenndler E. J. Biochem. Biophys.
Methods 1999; 38: 123–137.
38. Reichardt C. Solvents and Solvent Effects in Organic
Chemistry, VCH Verlagsgesellschaft mbH: D-6940
Weinheim, 1988; pp 27–50.
13. Irving CS and Lapidot A. J. Chem. Soc. Chem.
Comm. 1977; 184–186.
14. Helaja J, Stapelbroek-Möllmann M, Kilpeläinen I and
Hynninen PH. J. Org. Chem. 2000; 65: 3700–3707.
15. Falk JE. Porphyrins and Metalloporphyrins, Elsevier:
Amsterdam, London and NewYork, 1964; pp 26–29,
236–239.
39. Brault D, Vever-Bizet C and Le Doan T. Biochim.
Biophys. Acta 1986; 857: 238–250.
40. Karaman R and Bruice TC. Inorg. Chem. 1992; 31:
2455–2459.
16. Smith KM. In Porphyrins and Metalloporphyrins,
Smith KM (Ed.). Elsevier:Amsterdam, 1975; pp 3–28.
17. Phillips JN. Rev. Pure Appl. Chem. (Australia)
1960; 10: 35–60.
41. WhiteWI. In The Porphyrins, Dolphin D (Ed.).Vol. 5;
Academic Press: New York, 1978; pp 303–339.
42. Karns GA, Gallagher WA and Elliot WB. Bioorg.
Chem. 1979; 8: 69–81.
43. Stone A and Fleischer EB. J. Am. Chem. Soc. 1967;
90: 2735–2748.
44. Senge MO and Kalisch WW. Z. Naturforsch. 1999;
54b: 943–959.
45. Senge MO. Z. Naturforsch. 2000; 55b: 336–344.
46. Labbe RF and Nishida G. Biochim. Biophys. Acta
1957; 26: 437.
18. Phillips JN. In Comprehensive Biochemistry, Vol. 9,
Florkin M and Stotz EH (Eds.). Elsevier:Amsterdam,
1963; pp 34–72.
19. Dempsey B, Love MB and Phillips JN. In Hematin
Enzymes, Falk J, Lemberg R and Morton RK (Eds.).
Pergamon Press: London, 1961; pp 29–40.
20. Falk JE and Phillips JN. In ChelatingAgents and Metal
Chelates, Dwyer FP and Mellor DP (Eds.). Academic
Press: NewYork and London, 1964; pp 441–490.
21. Hambright P. Coord. Chem. Rev. 1971; 6: 247–268.
22. Hambright P. In Porphyrins and Metalloporphy-
rins, Smith KM (Ed.). Elsevier: Amsterdam, 1975;
pp 233–278.
47. Hynninen PH and Ellfolk N. Acta Chem. Scand.
1973; 27: 1795–1806.
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 395–395