Protonation-deprotonation equilibria in tetrapyrroles Part 2:

Article abstract of DOI:10.1142/S1088424611004348

The two-dimensional ¹H, ¹³C and ¹H, ¹⁵N heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR techniques were applied to investigate the formation of N-protonated c

Full text of DOI:10.1142/S1088424611004348

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 39–46
DOI: 10.1142/S1088424611004348
Published at http://www.worldscinet.com/jpp/
Protonation-deprotonation equilibria in tetrapyrroles. Part 2.
Mono- and diprotonation of methyl pyropheophorbide a in
1
13
methanolic hydrochloric acid as verified by the H, C HSQC
1
15
and H, N HMBC NMR experiments
a◊
b
Paavo H. Hynninen* and Markku Mesilaakso
a
Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, A. I. Virtasen Aukio 1, P.O. Box
5, FI-00014, Finland
Finnish Institute for Verification of the Chemical Weapons Convention, University of Helsinki, A. I. Virtasen Aukio 1,
5
b
P.O. Box 55, FI-00014, Finland
Received 10 May 2011
Accepted 19 June 2011
1
13
1
15
ABSTRACT: The two-dimensional H, C and H, N heteronuclear single quantum coherence (HSQC)
and heteronuclear multiple bond correlation (HMBC) NMR techniques were applied to investigate the
formation of N-protonated cationic species of methyl pyropheophorbide a in methanolic hydrochloric
1
13
1
15
acid (CD OH-HCl). The H, C HSQC and H, N HMBC NMR spectra, recorded at the temperature
3
+
of 278 K, verified that the CD OH-HCl solution with [H] = 0.021 M, contained the N22-protonated
monocations of methyl pyropheophorbide a, whereas the CD OH-HCl solution with [H] = 5.0 M
3
+
3
contained the N22, N24-protonated dications of the phorbin. No further protonations to form the trication
or tetracation were observed. Consequently, the two middle electronic spectra, which were previously
tentatively interpreted as representing the dication and trication, were now attributed to conformational
alterations originating from steric hindrance between the central hydrogens and changes in counter-ion
and solvation interactions and/or conformational alterations intimately connected to NH-tautomerism.
These possibilities to explain the previously observed middle electronic spectra are briefly discussed.
KEYWORDS: porphyrin, chlorin, acid-base properties, p-electron delocalization, conformation,
NH-tautomerism, nuclear magnetic resonance.
NH-tautomerism [10, 12, 13–22], isomerization [23, 24],
INTRODUCTION
substitution pattern [25, 26] and stereochemistry [25, 26]
of a cyclic tetrapyrrole. Furthermore, knowledge of the
foregoing aspects may also be of immense importance
when trying to elucidate the reaction-center mechanism
of natural photosynthesis [27–29], disorders of porphyrin
metabolism [30] and the mechanism of photodynamic
therapy (PDT) [31–36].
The acid-base properties belong to fundamental
aspects in the chemistry of tetrapyrroles, such as
porphyrins proper, chlorins (17,18-dihydroporphyrins)
and bacteriochlorins (7,8,17,18-tetrahydroporphyrins).
The examination of these properties is expected to provide
information on, e.g., the mechanisms of metalation
and demetalation [1–7], p-electron delocalization
pathway (aromaticity) [8–10], resonance energy [11],
The reviews by Phillips [5, 6] and by Falk and Phillips
[2] are still the best sources of information on the
acid-base properties of cyclic tetrapyrroles. Following
the commonly used notation, the neutral form of
a porphyrin or chlorin with two H-atoms at positions

SPP full member in good standing
2
1 and 23 (Scheme 1) is called “free-base porphyrin”
*Correspondence to: Paavo H. Hynninen, email: paavo.hynninen
@helsinki.fi, tel: +358 9-19150358, fax: +358 9-19150466
or “free-base chlorin” (PH ). This can be deprotonated
2
Copyright © 2012 World Scientific Publishing Company

4
0
P. H. HYNNINEN AND M. MESILAAKSO
Methanol has rarely been used for the acid-base
studies of porphyrins, even though its pK value
a
(ca. -2, the exact value is unknown) is quite close
to that of water (pK = -1.84) [38]. Methanol
a
also has a high solvation power for many kinds
of organic molecules owing to its high hydrogen
bonding capability. In addition, it is noteworthy
that the pK value of the pyridinium ion (pK =
a
a
5
.2) has been found to be precisely the same in
methanol and water [39].
An ester-exchange method, that has long
been used to convert chlorophyll a (1) or
pheophytin a (3) to methyl pheophorbide a (5) and
pyrochlorophyll a (2) or pyropheophytin a (4) to
methyl pyropheophorbide a (6) (Fig. 1), involves
standing of the phytyl ester in dry methanol
containing sulfuric acid (5%, v/v) or saturated with
hydrochloric acid, liberated from sodium chloride
and dried in a standard gas generation apparatus.
The procedure results in a beautiful dark-blue
solution containing N-protonated form(s) of 5 or 6.
Interestingly, it should be established which
of the four central nitrogen atoms of 5 or 6 are
protonated in these solutions.
Scheme 1. Theoretically possible deprotonation-protonation equilibria
in an unsubstituted chlorin
Part 1 of this series [37] described the
spectrophotometric titrations of pyropheophytin a (4) in
methanolic hydrochloric acid. In addition to the neutral
form, four spectrally different protonated species were
observed with clear isosbestic points in the sets of spectra
whentheHClconcentrationwasgraduallyincreasedinthe
methanol solution of 4. As the most likely interpretation,
-
to the corresponding monoanion (PH ) and dianion
2
-
(
P ). K and K denote the acid ionization constants of
1 2
these deprotonations. The acid ionization constants of
the four theoretically possible N-protonation steps have
been denoted by Phillips with symbols K , K , K and K .
3
4
5
6
These acid-base equilibria are illustrated in Scheme 1 for
an unsubstituted chlorin.
Of the six theoretically predictable ionic species, only
-
2-
the monoanion (PH ), the dianion (P ), the monocation
(
+
2+
PH ), and the dication (PH ) have been reported to
3
4
be experimentally observable with certainty for fully
conjugated porphyrins (for references, see Ref. 37). The
existence of the porphyrin monocation as a separate
species has aroused a long dispute and thus far there is no
conclusive evidence for the formation of the porphyrin
3
+
4+
trication (PH5 ) or tetracation (PH6 ). In the case of
chlorins, the reported information of N-deprotonated
or N-protonated ionic species is even meager and more
controversial. Apparently, though the studies of the
central deprotonation-protonation equilibria in chlorins
and bacteriochlorins are extremely challenging, mainly
due to the functions of these compounds in Nature,
such studies are extremely demanding to carry out
experimentally and the results are difficult to interpret
unambiguously. Owing to the general low solubility and
high aggregation tendency of porphyrins, chlorins and
bacteriochlorins in water, various organic solvents have
to be used in the investigations of the acid-base properties
of these compounds. Regarding the acid ionization
constants of the tetrapyrroles, this brings about an enigma,
2
Fig. 1. Structures of 13 (R)-chlorophyll a (1), pyrochlorophyll
2
a (2), 13 (R)-pheophytin a (3), pyropheophytin a (4), methyl
3 (R)-pheophorbide a (5), methyl pyropheophorbide a (6);
pyro = 13 -demethoxycarbonyl; the semisystematic numbering
system and nomenclature of IUPAC-IUB [40] is used in this
paper
2
1
2
which is hard to solve: How is the K value determined
a
in water related to that determined in an organic solvent?
Copyright © 2012 World Scientific Publishing Company
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PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
41
the four different spectra were assigned to arise from the
[37]. The results from the protonation studies by NMR
described in this paper led us to reinterpret the nature of
the higher N-protonated species of 6.
N-protonated mono-, di-, tri- and tetracationic species.
Nevertheless, this interpretation was intended to be only
tentative as appears from the quotation [37]: “However,
this evidence cannot be considered conclusive, and
much more experimental work is necessary for the
final clarification of the protonation behavior of
various tetrapyrroles.” Alternative reaction possibilities,
including C-protonation to the vinyl group or a methine
RESULTS AND DISCUSSION
1
15
Figure 2 shows the H, N HMBC spectrum of methyl
pyropheophorbide a (6) in the CD OH-HCl solution
3
+
with [H] = 0.021 M. The correlation signals, arising
from 10-CH, 5-CH and 20-CH methine (meso) protons
and protonated N atoms, HN21, H N22 and HN23,
are localized at the upper part of Fig. 2 and have
1
bridge, O-protonation to the 13 -oxo function followed by
ketalization, electrophilic substitution at a methine bridge
and aggregation, were considered but found as less likely
options. The possible involvement of conformational
alterations and NH-tautomerism in the spectral changes
was also considered but found difficult to assess because
relatively little was known about those matters in the case
of chlorins at the time of the appearance of the first part
15
+
15
N
chemical shifts at 107, 115 and 111 ppm, respectively.
+
The correlation signals of H N22 and HN23 overlap
each other almost completely as can be more clearly
seen from Fig. 3, showing the expanded upper part of
1
15
the H, N HMBC spectrum in Fig. 2. At the lower part
[
37]. In addition, at that time, an NMR instrument with a
15
of Fig. 2, there is one correlation signal with d( N) at
sufficiently high magnetic field was not available for us.
During the past 25 years, the NMR instruments
and two-dimensional (2D) methods have experienced
an enormous development, allowing for instance
2
92 ppm. This correlation signal obviously arised from
the unprotonated N24 atom. These assignments indicate
1
15
that the H, N HMBC spectrum in Fig. 2 clearly belongs
+
to the H N22 monocation and not to the dication or
1
13
1
15
measurements of 2D heteronuclear H, C and H,
N
trication. The origin of the low-intensity satellite signals
in Fig. 2 remained unclear.
HSQC and HMBC NMR spectra (HSQC = heteronuclear
single quantum coherence, HMBC = heteronuclear
multiple bond correlation) at the natural abundances
A prerequisite for the above assignments was that
the assignment order of the methine (meso) proton
signals had to be determined first. This was performed
1
3
15
of C and N [41–50]. This development and the
availability of a 500 MHz NMR instrument enabled us
to focus meanwhile on investigations of NH-tautomerism
in natural chlorins [21, 22]. Subsequently, it also became
possible for us to revisit the problem concerning the
protonation of pyropheophytin a (4, Fig. 1) or methyl
pyropheophorbide a (6) in methanolic hydrochloric acid
1
13
by recording the H, C HSQC spectrum (Fig. 4), which
straightforwardly indicated that the correct order is the
following: 10-CH (d = 10.32), 5-CH (d = 9.93) and
H
H
2
0-CH (dH = 9.37). This order is in agreement with
our previous investigation [51] for the monocation of
pyropheophytin a (4, Fig. 1). Our previous investigation
also showed that the proton derived from
trifluoroacetic acid (TFA) resided at the N22 of
subring B.
1
15
The H, N HMBC spectrum (Fig. 5),
+
recorded from the NMR sample with [H] = 5.0
M, indicated that a higher protonated species
of phorbin 6 was formed. In this spectrum, the
1
5
N chemical shifts of N21, N23 and N22 were
localized at 103, 105 and 108 ppm, respectively.
These values are quite close to those obtained
1
5
for the monocation. However, the N chemical
1
5
shift of N24, d( N24) = 136 ppm, is distinctly
different from the corresponding value of the
1
5
monocation, d( N24) = 292 ppm. These results
1
15
verify that the H, N HMBC spectrum in
+
+
Fig. 5 represents the H N22, H N24-dication
of methyl pyropheophorbide a (6).
1
15
The H, N HSQC spectrum at 278 K of
the dicationic solution of 6 exhibited two
cross-signals (Fig. 6): the first cross-signal was
1
15
localized at d( H) = 1.00 ppm, d( N) = 115 ppm
1
15
and the second one at d( H) = 0.10 ppm, d( N)
118 ppm. These cross-signals can be assigned
to the H-N21 and H-N23 groups, respectively.
1
15
Fig. 2. H, N HMBC spectrum at 278 K of the monocation of methyl
=
+
pyropheophorbide a (6) in the CD OH-HCl solution with [H] = 0.021 M
3
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4
2
P. H. HYNNINEN AND M. MESILAAKSO
when the HCl concentration is increased gradually
up to saturation. From the results above follows
that the pK values of the different protonation steps
must be reestimated. The pK value, belonging
3
to the formation of the monocation in the first
protonation step, remains the same as reported
before, pK = +4.14. However, because the results
3
above clearly indicate that, from the higher
protonations, only the formation of the dication in
the second protonation step is possible, the new
pK value must replace the old pK value (pK =
4
6
6
-0.34 ± 0.04) and be around zero. This conclusion
is in agreement with the titration results for chlorin
1
e trimethyl ester (pK = 0.62) and the 7 -acetal
6
4
of rhodin g trimethyl ester (pK = 0.60) (P.H.
7
4
Hynninen, Protonation-deprotonation equilibria
in tetrapyrroles. Part 3, in preparation).
The previous paper also included the results
from the spectrophotometric titration of pyro-
pheophytin a (4) in the AcOH-HCl system, where
only two sets of electronic spectra were observed
[37]. The first set of spectra with several sharp
isosbestic points (see Fig. 10 in Ref. 37) was
interpreted as representing the conversion of the
neutral form of 4 (Fig. 1) to its monocation and
the second set of spectra (see Fig. 11 in Ref. 37),
the conversion of the monocation to the tetracation
of 4. In addition, the end spectrum of the titration
was virtually identical with that from the titration
in the MeOH-HCl system. On the basis of the
NMR results of this paper, it seems clear that the
second set of spectra (see Fig. 11 in Ref. 37) must
be reinterpreted as representing the conversion of
the monocation to the dication.
1
15
Fig. 3. Expanded upper part of the H, N HMBC spectrum in
Fig. 2; the expanded partial spectrum shows clearly that the N22 and
N23 cross-signals overlap almost completely, whereas the N21 and
N22 cross-signals appear as virtually separate signals
Previously, alternative reactions to the four
N-protonations were sought. These included
C-protonation to the vinyl group or the 20-methine
1
bridge(mesoposition), O-protonationtothe13 -oxo
function followed by ketalization, electrophilic
substitution (chlorination) to the 20-methine
bridge and aggregation. Furthermore, the
possible involvement of changes in conformation,
NH-tautomerism, counter-ion binding and
solvation were briefly discussed but could not
be treated more profoundly. Nowadays, these
phenomena are well-known for the crystalline
dications (diacids) of many variously substituted
fully-conjugated porphyrins and porphyrin
isomers [52–55], but comparable information is
still missing for the dications of chlorins. For the
1
13
Fig. 4. H, C HSQC spectrum at 278 K of the monocation of methyl
+
pyropheophorbide a (6) in the CD OH-HCl solution with [H] = 0.021 M
3
dications of fully-conjugated porphyrins, the degrees and
modes of distortion from planarity of the macrocycle to
relieve conformational strain in it (saddle conformation,
ruffled conformation) have been thoroughly investigated.
For some dications of the porphyrins, an interesting
new possibility to relieve conformational strain in the
core of the macrocycle has been mentioned by Senge
Continued conduction of the HCl gas into the dicationic
solution resulted in no tricationic or tetracationic species.
The foregoing results verify conclusively that only two
+
different N-protonated species, the H N22 monocation
+
+
and the H N22, H N24 dication, are formed in the
methanolic solution of methyl pyropheophorbide a (6)
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PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
43
pyropheophorbide a (6), it now seems likely
that those spectra represent different conformers
of the monocation of 4 or 6, because different
conformers of a molecule with p-electrons
may produce different electronic spectra. As
major reasons for conformational changes in
the monocation we see, besides moderate steric
hindrance (crowding) in the center, changes in
the solvation and counter-ion binding of the
monocationicspecies,whentheHClconcentration
in the solution was gradually increased and the
long, nonpolar phytyl chain was replaced with a
methyl group in the ester exchange.
Furthermore, it is noteworthy that confor-
mational changes are intimately connected to
NH tautomerism in a cyclic tetrapyrrole [56,
57]. All possible aromatic and less aromatic
NH tautomers of several natural chlorins were
studied by the Helsinki Chlorophyll Group
(
under the supervision of P.H.H.) using the two-
1
13
dimensional (2D), heteronuclear H, C and
1
15
H, N HSQC and HMBC NMR techniques, as
1
15
Fig. 5. H, N HMBC spectrum at 278 K of the dication of methyl
well as variable-temperature NMR [21, 22].
The structures of the cis and trans
NH-tautomers and the equilibria between
them are shown in Scheme 2. The variable-
temperature NMR investigations provided
+
pyropheophorbide a (6) in the CD OH-HCl solution with [H] = 5.0 M
3
‡
the activation free-energies (DG ) for
the NH-tautomerization of six different
demetallated chlorophyll derivatives, one
of which was methyl pyropheophorbide a
‡
(
6). The second activation free-energy, DG ,
2
describing the over-all tautomeric exchange
A  A′ in Scheme 2), was found to attain
(
‡
-1
the highest value, DG2 > 18.0 kcal.mol ,
for phorbin 6. This high energy barrier was
attributed to the macrocycle rigidifying effect
of the isocyclic ring E in the phorbin. The
rigidifying effect was considered capable
of hindering conformational distortions
in the macrocycle, demanded by the
NH-tautomeric process. This conclusion is in
line with the observation that the two lines,
representing the temperature dependence of
1
5
the d( NH) values for the H-N21 and H-N23
groups of phorbin 6, remained far away
from each other in the temperature range
of 210–330 K (see Fig. 4 in Ref. 22).
1
15
Fig. 6. H, N HSQC spectrum at 278 K of the dication of methyl
+
pyropheophorbide a (6) in the CD OH-HCl solution with [H] = 5.0 M
3
Consequently, the over-all NH-tautomerization seems
unlikely in chlorins possessing the isocyclic ring E.
However, the tautomeric conversion between an aromatic
trans NH-tautomer (A/A′, in Scheme 2) and an aromatic
cis NH-tautomer (C/C′, D/D′ in Scheme 2), might be
feasible also for derivative 6. Such a partial tautomeric
reaction would imply changes in the protonation sites, as
well as changes in the counter-ion binding and solvation
of the cations formed by N-protonations.
[
53, 54]: that is the pyramidalization of the N–H units by
2
3
sp → sp rehybridization, which removes the inner
hydrogen atoms farther away from each other, thus relieving
the strain. Such a rehybridization might also be possible in
the core of chlorin dications in the solution state.
When seeking a reasonable explanation for the two
middle electronic spectra, formerly observed in the
protonation of pyropheophytin a (4, Fig. 1) or methyl
Copyright © 2012 World Scientific Publishing Company
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4
4
P. H. HYNNINEN AND M. MESILAAKSO
Scheme 2. The total tautomeric NH exchange process between fully aromatic (thick black line) and less aromatic trans and cis
NH-tautomers; adapted from Ref. 22
The solvation phenomena of molecules in solution
are an interesting but a theoretically demanding subject
still today. Applying density functional theory (DFT)
EXPERIMENTAL
NMR spectra
and the implicit solvation method (dielectric polarizable
continuum model, DPCM) or alternatively the hybrid
solvation method (cluster-continuum model, CCM),
For the preparation of two NMR samples of methyl
pyropheophorbide a (6, Fig. 1), an adequate volume of
CD OH (methanol-d , Aldrich, 99 atom% D) solution,
Mehta and Datta [58] were able to calculate the pK3
3
3
value for the monocation of pyropheophytin a (4) in
methanol. The DFT-DPCM calculation resulted in
saturated with HCl, i.e. [HCl] = 5.0 M, was first prepared
by conducting dry, O -less HCl gas (for preparation, see
2
pK = 6.12 whereas the CCM gave pK = 4.70. The latter
3
3
Ref. 37) through 3.0 mL of CD OH in a 5 mL volumetric
3
value, obtained by the cluster-continuum model, is in
a better agreement with the experimentally observed
flask, on an ice bath. After the conduction, the flask was
closed air-tight with a Teflon stopper. On the basis of
Table 1 in Ref. 37, it was calculated that 3.0 mL of the
value, pK = 4.14 [37]. The authors explain this by the
3
presence of a strong intermolecular hydrogen bonding
interaction between the solute and the solvent which is
taken into account by the hybrid solvation model, CCM.
The authors predict that “consideration of a large number
of solvent molecules would further improve the result”.
In addition, they are of the opinion that “a pure quantum
mechanical treatment on a complex of a large number of
atoms is still not computationally feasible, and then one
would have to resort to a mixed quantum mechanical-
molecular mechanical treatment” [58].
CD OH solution with [HCl] = 5.0 M would be required
3
to produce 0.70 mL of CD OH-HCl solution with
3
+
[
H] = 0.021 M, assuming complete ionization of HCl. At
this [H] , complete formation of the previously postulated
+
dication was expected. Hence, a 3.0 mL volume of the
CD OH solution with [HCl] = 5.0 M was added with a
3
Carlsberg-type micropipette into a test tube, containing
0
.70 mL of CD OH solution of methyl pyropheophorbide
3
a(6, 10mg, 0.0182mmol)toresultinthefirstNMRsample,
transferred with a Pasteur pipette into a 5 mm NMR tube,
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PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES
45
sealed air-tight. The second NMR sample was prepared
5. Phillips JN. Rev. Pure Appl. Chem. (Australia)
1960; 10: 35–60.
6. Phillips JN. In Comprehensive Biochemistry, Vol. 9,
Florkin M and Stotz EH. (Eds.) Elsevier: Amster-
dam, 1963; pp 34–72.
7. Dempsey B, Love MB and Phillips JN. In Hematin
Enzymes, Falk J, Lemberg R and Morton RK. (Eds.)
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8. Krygowski TM, Cyranski MK, Czarnocki Z, Häfe-
linger G and Katritzky AR. Tetrahedron 2000; 56:
1783–1796.
by dissolving in another test tube 10 mg (0.0182 mmol) of
+
phorbin 6 in 0.70 mL of CD OH-HCl solution with [H] =
3
5
.0 M. The solution was transferred with a Pasteur pipette
into a second 5 mm NMR tube, sealed air-tight.
1
15
The two-dimensional H, N HSQC and HMBC
spectra were recorded at 278.0 K (4.85 °C) on a Bruker
1
13
DRX 500 spectrometer and the H, C HSQC spectrum
at 278.0 K on a Varian Unity 500 spectrometer, using a
non-spinning sample tube, an inverse broad-band probe-
1
head with z-gradients and the appropriate inverse ( H
detected) pulse sequence provided by the spectrometer
library. The acquisition/processing parameters were as
9. Balaban AT, Oniciu DC and Katritzky AR. Chem.
Rev. 2004; 104: 2777–2812.
10. Stepien M and Latos-Grazynski L. Top. Heterocycl.
Chem. 2009; 19: 83–153.
11. George P. Chem. Rev. 1975; 75: 85–111.
12. Claramunt RM, Elguero J and Katritzky AR. Adv.
Heterocycl. Chem. 2000; 77: 1–50.
1
13
follows: n( H) = 500.13 MHz, n( C) = 125.03 MHz
1
5
1
13
and n( N) = 50.68 MHz. The H and C chemical shifts
were referenced to internal Me S (tetramethylsilane), d =
.0 ppm, and the N chemical shifts to DMF (dimethyl
formamide), d( N) = 103.8 ppm [21, 22].
4
1
5
0
1
5
Chlorophyll a (1). Chlorophyll a (1, Fig. 1) was
isolated from fresh or frozen leaves of red clover
13. Crossley MJ, Harding HH and Sternhell S. J. Am.
Chem. Soc. 1986; 108: 3608–3613.
(
[
Trifolium pratense) by the method described previously
14. Crossley MJ, Field LD, Harding HH and Sternhell S.
J. Am. Chem. Soc. 1987; 109: 2335–2341.
15. Braun J, Schlabach M, Wehrle B, Köcher M, Vogel
E and Limbach HH. J. Am. Chem. Soc. 1994; 116:
6593–6604.
16. Schlabach M, Wehrle B, Rumpel H, Braun J,
Scherer G and Limbach HH. Ber. Bunsen-ges. Phys.
Chem. 1992; 96: 821–833.
59], adapted to large-scale preparation.
2
Pyrochlorophyll a [13 -(demethoxycarbonyl)chlor-
ophyll a] (2). Chlorophyll a was converted quantitatively
into pyrochlorophyll a (2), when its deaerated pyridine
solution was heated overnight at 100 °C in a sealed
tube [60].
2
Pyropheophytin a [13 -(demethoxycarbonyl)pheo-
phytin a] (4). Removal of the magnesium ion from 2 with
17. Garcia-Ortega H, Crusats J, Feliz M and Ribo JM.
J. Org. Chem. 2002; 67: 4170–4176.
1
3
1
2.5% (w/w) HCl afforded pyropheophytin a (4). The C
NMR spectrum of 4 was identical with that previously
18. Storm CB and TekluY. J. Am. Chem. Soc. 1972; 94:
1745–1746.
19. Bonnett R, Djelal BD, Hawkes GE, Haycock P
and Pont F. J. Chem. Soc., Perkin Trans. 2 1994;
1839–1843.
reported [51, 61].
2
Methyl pyropheophorbide a [13 -(demethoxycar-
bonyl)pheophorbide a methyl ester] (6). The phytyl ester
4
was converted to the methyl ester 6 by ester exchange in
dried MeOH containing 5% (v/v) sulfuric acid (note that a
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