PROTONATION-DEPROTONATION EQUILIBRIA IN TETRAPYRROLES 1175
prominent conformational alteration. On the contrary, the
pK4-values of these chlorins (chlorin e6 TME, pK4 = 0.62
CONCLUSION
The protonation titrations on chlorin e6 TME and
rhodin g7 TME acetal in the HCl-MeOH system afforded
clearly interpretable results suggesting the formation
of the N22-protonated monocation and the N22, N24-
protonated dication in this system. The use of HCl-MeOH
to investigate the acid-base properties of these chlorin
derivatives clearly has several advantages. Firstly, the
chlorinstaysmonomericinitstriesterforminHCl-MeOH,
thus preventing the possible interference by aggregation
of the chlorin. This aspect is important, because the
aggregation is a general property of fully-conjugated
porphyrins and chlorins (17,18-dihydroporphyrins) in
water and nonpolar solvents and has seriously limited
the acid-base studies of these tetrapyrroles. Secondly,
besides aggregation, other side-reaction options, such as
additions to the C3-vinyl group, isomerization converting
the chlorin to the corresponding chloroporphyrin (cf.
Fischer’s HI isomerization [53]) and electrophilic
substitution at C20 (C20-chlorination) [35, 54, 55], can
be avoided or minimized. As to the addition of HCl to the
vinyl group, it should be noted that MeOH saturated with
HCl has long been used to convert quantitatively free
chlorin e6 to its trimethyl ester [56]. This shows that the
methanol-solvated chloride anion is a weak nucleophile
that cannot attack the carbocation position of the H3C-
HC+-group, which is formed from the vinyl as a result of
protonation. It is also possible that the methanol-solvate
layer surrounding H+, might be capable of preventing the
proton from approaching the p-orbital of the vinyl group
closely enough to produce the H3C-HC+-group in the HCl-
MeOH system. The isomerization involves the apparent
transfer of the two H-atoms from the reduced sub-ring
D to the vinyl group, which is converted to an ethyl
group. Carbocation rearrangement has been proposed
[57] to be a likely mechanism for this isomerization.
No isomerization yielding the chloroporphyrin e6
derivative nor electrophilic substitution affording the
20-chlorochlorin e6 derivative has been observed by
us under the dry and non-oxidizing conditions of the
HCl-MeOH system. The side-reactions were firmly
excluded by the recovery procedures consisting of the
neutralization of each chlorin sample with NaOAc after
the protonation titration, extraction of the chlorin with
Et2O and recording of the UV-vis spectrum from the Et2O
extract. The UV-vis spectrum of the recovered chlorin
sample was found practically identical with the initial
spectrum of the neutral form of the chlorin derivative.
The several clear isosbestic points, observed between
the two sets of UV-vis spectra for each protonation step of
thechlorinderivatives,supporttheconclusionthatonlytwo
molecular forms of the chlorin were in equilibrium with
one another at each protonation step. The determined pK3-
values of chlorin e6 TME (pK3 = 4.63) and rhodin g7 TME
acetal (pK3 = 4.40) indicate that the first N-protonation is a
relatively facile reaction, which probably does not involve
and rhodin g7 TME acetal, pK4 = 0.60) manifest of the
difficulty of the second N-protonation. This difficulty
may be attributed to the hindrance of conformational
alterations, which are demanded by the steric crowdedness
in the center and among the carboxylate ester side-chains
of the diprotonated chlorin.
The relatively high level of the pK3-values of the
chlorin derivatives is particularly interesting from
the viewpoint of possible applications, e.g. to PDT. The
interstitial fluid of cancerous tissues has frequently been
found to show significantly lower pH-values than 7.4 of
the normal tissue [7, 10, 13]. The lower interstitial pH has
been observed to increase the selective uptake of chlorin
e6 derivatives by cancer cells and in this fashion improve
the efficacy of PDT treatments [7, 10, 13]. Further, there
is evidence that the cationic photosensitizer molecules
are taken up more selectively by cancer cells [11, 58, 59].
However, it has rarely been realized in the connection
of PDT investigations that the cationic charge can be
introduced by N-protonation to the center of a porphyrin
or chlorin photosensitizer.
REFERENCES
1. Fischer H and SternA. Die Chemie des Pyrrols, Vol.
II/2, Akademische Verlagsgesellschaft: Leipzig,
1940; (reprinted by Johnson Reprint Corporation:
New York, 1968).
2. Woodward RB, Ayer WA, Beaton JM, Bickelhaupt
F, Bonnet R, Buchschacher P, Closs GL, Dutler H,
Hannah J, Hauck FP, Ito S, Langemann A, Le Goff
E, Leimgruber W, Lwowski W, Sauer J, Valenta Z
and Volz H. Tetrahedron 1990; 46: 7599–7659.
3. Inhoffen HH, Buchler JW and Jäger P. Fortschr.
Chem. Org. Naturst. 1968; 26: 284–355.
4. Stapelbroek-Möllmann ME and Hynninen PH.
Japan — Northern Baltic Symposium on Synthetic
Chemistry, June 25–26, 1996, Helsinki, Finland.
5. Stapelbroek-Möllmann ME. Studien zur Synthese
enantiomerenreiner Chlorophylle a und b. Doctoral
Thesis. University of Helsinki and University of
Bremen: Helsinki/Bremen, 1997; 108–129.
6. Minaeva LI, Kabachnik MM, Ponomarev GV,
Morozova JV and Beletskaya IP. Synthesis 2010;
14: 2451–2455.
7. Vermathen M, Marzorati M, Vermathen P and
Bigler P. Langmuir 2010; 26: 11085–11094.
8. Ol’shevskaya VA, Nikitina RG, Savchenko AN,
Malshakova MV, Vinogradov AM, Golovina GV,
Belykh DV, Kutchin AV, Kaplan MA, Kalinin VN,
Kuzmin VA and Shtil AA. Bioorg. Med. Chem.
2009; 17: 1297–1306.
9. Chin WWL, Heng PWS and Olivo M. BMC
Pharmacology 2007; 7: 15.
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J. Porphyrins Phthalocyanines 2012; 16: 1175–1176