Photooxidation of alkaloids: Considerable quantum yield enhancement by rose bengal-sensitized singlet molecular oxygen generation

Article abstract of DOI:10.1111/j.1751-1097.2011.00994.x

The photooxidation of sanguinarine, coralyne and berberine was studied in oxygenated alkaline methanol solutions. Rose bengal as photosensitizer significantly accelerates the process, indicating the importance of singlet molecular oxygen in the reaction mechanism. The quantum yield of sensitized oxidation was found to increase significantly with pH and reaches 0.4 for berberine at pH 13.8. The direct oxidation of alkaloids is less efficient, the quantum yield does not exceed 0.01 even in oxygen-saturated solutions. The photoinduced electron ejection does not play a role in the oxidation. The uncharged pseudobase forms, which are present in alkaline medium, are oxidized much more easily than the alkaloid cations. The quantum yield of rose bengal-sensitized photooxidation of berberine and coralyne alkaloids increases significantly with pH. Singlet molecular oxygen plays an important role in the reaction mechanism.

Full text of DOI:10.1111/j.1751-1097.2011.00994.x

Photochemistry and Photobiology, 2011, 87: 1315–1320
Photooxidation of Alkaloids: Considerable Quantum Yield Enhancement
by Rose Bengal-sensitized Singlet Molecular Oxygen Generation
1
Helmut Gorner , Zsombor Miskolczy , Monika Megyesi and Laszlo Biczok*
2
2
2
¨
´
´
´
´
1Max-Planck-Institut fu¨r Bioanorganische Chemie, Mu¨lheim an der Ruhr, Germany
2Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary
Received 10 May 2011, accepted 18 August 2011, DOI: 10.1111/j.1751-1097.2011.00994.x
of a ketone with triethylamine or alcohol as photoinitiators
and a quantum yield of close to unity was achieved for
dihydroalkaloid formation (16).
ABSTRACT
The photooxidation of sanguinarine, coralyne and berberine was
studied in oxygenated alkaline methanol solutions. Rose bengal
as photosensitizer significantly accelerates the process, indicat-
ing the importance of singlet molecular oxygen in the reaction
mechanism. The quantum yield of sensitized oxidation was found
to increase significantly with pH and reaches 0.4 for berberine at
pH 13.8. The direct oxidation of alkaloids is less efficient, the
quantum yield does not exceed 0.01 even in oxygen-saturated
solutions. The photoinduced electron ejection does not play a role
in the oxidation. The uncharged pseudobase forms, which are
present in alkaline medium, are oxidized much more easily than
the alkaloid cations.
Knowledge of the oxidation processes of natural isoquin-
oline alkaloids is scarce. 8-Oxoberberine was synthesized by
oxidation of berberine (25). For sanguinarine in alkaline
media, a conversion of the alkanolamine form to oxysanguin-
arine was performed photochemically and the fluorescence
signal of sanguinarine in water at pH 11 is unstable due to an
oxidative photoreaction (21,24). The photooxidation followed
second-order kinetics and inclusion complex formation of
sanguinarine with cucurbit[7]uril significantly inhibits any
reaction (24). It was demonstrated that electron transfer to
O2(1Dg) plays an important role in the oxidation of indol
alkaloids (26). The reactions of O2(1Dg) with biologically active
heteroaromatics such as dihydropterins (27), purine nucleo-
tides (28), folic acid (29,30) and other oxidized pterins (31)
have also attracted considerable attention.
INTRODUCTION
Despite the large biological and pharmaceutical importance of
alkaloids, the mechanistic details of their photoinitiated
processes are still largely unknown. Alkaloids, such as berber-
ine, coralyne (1–17) and sanguinarine (18–24), are sensitive to
light and can induce adverse phototoxic reactions. The solvent
and molecular structure variations markedly affect the quan-
tum yield of singlet molecular oxygen, O2(1Dg) generation
(5–7,16). The quantum yields of triplet and O2(1Dg) formation
strongly diminish with increasing polarity of the environment
in the cases of berberine, palmitine and sanguinarine, but are
barely changed for coralyne (16). Binding to DNA signifi-
cantly enhanced the efficiency of the berberine-sensitized
formation of O2(1Dg) (11,12) and coralyne initiates easily the
photochemical DNA damage (9). The photoinduced processes
of berberine have been examined most intensively (5–7). The
transients produced upon exposure to UV light were identified
by laser flash photolysis (13,14,16), whereas the short-lived
intermediates of the reaction between berberine and strongly
oxidizing or reducing species were examined by pulse
radiolysis (17).
The main goal of the present work was to reveal the
reaction mechanism of the photooxidation of sanguinarine,
berberine and coralyne in alkaline media. To examine whether
O2(1Dg) plays an important role in the oxidation, rose bengal
was used as a sensitizer.
MATERIALS AND METHODS
Berberine (Sigma), coralyne (Acros) and sanguinarine (Fluka) as
chloride salts were used as received. The solvents (Merck) were of
the purest spectroscopic quality. The absorption spectra were
monitored on a spectrophotometer (HP, 8453). The molar absorp-
tion coefficients of coralyne and sanguinarine in (m)ethanol were
M
)1
taken as e420 = 1.5 · 105 and e327 = 3.1 · 104
cm)1
, respec-
tively. The initial rate of photoreactions was determined from the
changes in absorbance. The number of absorbed photons was
measured using the aberchrome 540 actinometer as reference (32).
The quantum yield Fox of photooxidation was calculated as a ratio
of these quantities. For the sensitized cases, 0.1, 0.2 or 1 cm cells
and the tail of the Hg-lamp spectrum above 500 nm were used (33).
At constant rose bengal concentration and the same irradiation
conditions, the initial rates of the alkaloid photooxidation were
compared to that of a reference reaction having a known quantum
yield. For this, the rose bengal-sensitized oxidation of 0.2 mM
5-hydroxy-1-naphthol in air-saturated methanol was used, whose
quantum yield is usen = 0.003 (34). Alternatively, the rose bengal-
sensitized oxidation of 1 mM 1-naphthol to 1,4-naphthoquinone in a
methanol and water 1:30 mixture at pH 10 was also used as a
reference with usen = 0.25 (33). On the basis of many experiments
The photoirradiation of various alkaloids in the presence of
triethylamine results in dihydroalkaloids, which were back-
converted on admission of oxygen (16). The rate of photore-
duction is significantly accelerated by the combined utilization
*Corresponding author email: biczok@chemres.hu (Laszlo Biczok)
´ ´ ´
ꢀ 2011 Wiley Periodicals, Inc.
Photochemistry and Photobiology ꢀ 2011 The American Society of Photobiology 0031-8655/11
with the same method for various compounds,
a conservative
1315

¨
1316 Helmut Gorner et al.
estimate of the experimental error of the quantum yield determina-
tions is
ments a Clark cell was used (33). An excimer laser (Lambda Physik,
EMG 200, pulse width of 20 ns and energy of <30 mJ) was used
for excitation at 308 nm (33).
30%. For photoinduced oxygen consumption measure-
A290
2
2
1
2
3
A
1
1
2.0
RESULTS AND DISCUSSION
Ground state of alkaloids
0
0
250
250
300
350
400
λ
300
350
(nm)
(nm) 400
λ
The absorption spectra of sanguinarine in a 9:1 mixture of
methanol and water change with pH in a manner that the orange
color disappears on going to higher pH and the maximum is
shifted from 320 to 290 nm. The equilibrium between the
iminium and alkanolamine forms is due to the introduction of
the OH) group at position 6, where HA-OH is the pseudobase
with the pKa = 7.14 value in water (24), see Scheme 1.
1.6
0
200
Time (s)
400
Figure 1. Plots of A290 as a function of the time of 313 nm irradiation
of sanguinarine in argon- (d), air- (D) and oxygen-saturated (s)
methanol–water (9:1, vol) at pH 8. Inset: absorption spectra in oxygen-
saturated solution at 0, 60 and 180 s irradiation at 313 nm, 1–3,
respectively.
HAþ þ OHꢀ Ð HA-OH
ð1Þ
Addition of saturated NaOH aqueous solution to berberine in
water leads to the corresponding pseudobase (35). For the
analogous methoxide adduct formation of berberine in metha-
nol, pKa = 15.4 has been reported (36). For the addition of
OH) to berberine in methanol–water mixtures, the pKa is
probably smaller. Note that the alkaloids are not stable in
aqueous solution at pH > 12, but coralyne and berberine are
stable in NaOH plus methanol for several minutes, a timeframe
during which our spectrophotometric and quantum yield mea-
surements were completed. The water-induced decomposition
at pH > 12 may be a ring opening. Reversible addition of OH)
followed by ring opening has been reported for coralyne (37).
anol–water (9:1, vol) mixtures at pH 8. A gradual absorbance
diminution around 290 nm is accompanied by the concomitant
rise of the absorption in the 355–400 nm range. Several
isosbestic points indicate that secondary photolysis does
not occur. The main photoproduct has been identified as
6-oxysanguinarine (21) (Scheme 2). A very slow reaction takes
place in argon-saturated solution, but a significant rate
enhancement was found in the presence of air and especially
under oxygen saturation. Table 1 summarizes the quantum
yields of photooxidation (Fox) derived from the initial slope of
the absorbance changes at 290 nm (A290) during photolysis. It
is seen that Fox remains below 0.01 under all conditions. The
primary step of photooxidation can be the formation of singlet
oxygen, O2(1Dg) or the solvated electron (esolv)). The former is
analogous to the sensitized oxidation of different related types
of heteroaromatics, e.g. dihydropterins (27), folic acid (30) and
alkaloid tertiary amines (26). In fact, photoinduced radical
Photoirradiation of sanguinarine
The inset to Fig. 1 shows the alteration of the absorption
spectra of sanguinarine upon irradiation at 313 nm in meth-
O
O
O
O
)
cation and esolv formation initiates the oxidation of phenols
(33). In the ionization reaction sequence 2–4, esolv) is converted
-
-
+OH
)
+
into the superoxide radical, O2Æ , which then reacts with the
N
N
−OH
CH3
CH3
O
O
HA-O· radical produced in the primary photoprocess.
H
O
O
OH
ꢁþ H
HA-OH þ hv ! HA-OHÆþ ! þesꢀolv ! HA-O
þ þ eꢀsolv
+
Sanguinarine (HA )
HA-OH
ð2Þ
O
O
ꢀ
ꢀ
ꢁ
esolv þ O2 ! O2
ð3Þ
ð4Þ
-
-
O
O
+OH
ꢀ
HA-O þ O2 ! A=O + HOꢀ2
ꢁ
ꢁ
−OH
+
N
N
CH3O
CH3O
OH
H
Table 1. Quantum yield Fox of direct photooxidation of alkaloids*
H
CH3O
CH3O
+
Berberine (HA )
HA-OH
Fox
Alkaloid
pH
Argon
Air
O2
OCH3
OCH3
OCH3
OCH3
Sanguinarine
5
8
12
12
8
<0.0001
0.003
0.0005
0.005
0.004
0.004
<0.001
0.010
-
-
CH3O
CH3O
+OH
CH3O
CH3O
N
+
N
Berberine
Coralyne
<0.0001
<0.0001
−OH
0.008
HO
CH3
CH3
12
+
HA-OH
Coralyne (HA )
Scheme 1. Cationic and pseudobase forms of the three alkaloids.
*In MeOH–H2O, 9:1, kirr = 313 nm.

Photochemistry and Photobiology, 2011, 87 1317
O
O
•
-
(4)
-
O
O
+ HO2
O2
+
O
N
O
N
O
•
CH3
O
O
CH3
O
H
•
A=O
HA-O
Scheme 2. Reaction 4 of sanguinarine.
is very small at pH 6–10 (Table 1). The most efficient
photooxidation is achieved at pH 12 (Fox = 0.01). Coralyne
is unstable above pH 12 due to the conversion to 6¢-
acetylpapaverine in a reversible process (37). The first step of
this reaction is addition of OH) at the 8th position (Scheme 1).
The more than one order of magnitude increase of Fox when
pH is changed from 8 to 12 implies the important role of the
OH) adduct in the photooxidation. At pH 8, coralyne is
present in the cationic form that is difficult to oxidize. The
extent of conversion from the cationic form to the adduct
increases with the OH) concentration. As the uncharged
species (coralyne-OH) is more prone to oxidation, a sub-
stantial rise of Fox is expected and indeed found at pH 12.
Transient absorption could not be detected by laser flash
photolysis in the 1–2 ls range because of the strong fluores-
cence of coralyne. No solvated electron absorption was found
after disappearance of this strong fluorescence signal (ca 1 ls).
Therefore, the first reaction step cannot be photoionization.
Berberine was also photooxidized upon exposure to 313 nm
light at pH 12 in oxygen-saturated 9:1 MeOH–H2O mixtures
and Fox = 0.004 was found (Table 1). To test the possibility
of photoionization, laser flash photolysis with 308 nm pulses
This reaction mechanism may be valid for the alkanolamine
form of sanguinarine (HA-OH), Scheme 2.
An alternative reaction mechanism is the O2(1Dg)-mediated
oxidation, as shown in Scheme 3. Here, O2(1Dg) is produced by
energy transfer from triplet sanguinarine and reacts with the
sanguinarine pseudobase (HA-OH in step 5), resulting in a
)
radical cation and O2Æ . The rate constant of this process (k5)
)1
is unknown, but for related cases k5 is (0.01–1) · 109
(33,34).
M
s)1
ꢀ
1
þ
ꢁ
þ HA-OHꢁ
O2ð DgÞ þ HA-OH ! O2
ð5Þ
ð6Þ
ð7Þ
ð8Þ
þ
þ
HA-OHꢁ ! þHA-Oꢁþ H
ꢀ
1
þ O2ð DgÞ ! A=O þ O2 þ Hþ
ꢁ
HA-Oꢁ
ꢀ
2O2 þ 2H2O ! H2O2 þ O2 þ 2OHꢀ
ꢁ
After deprotonation of the radical cation (HA-OH·+, step
6), a second electron transfer (step 7) can occur from the
HA-O· radical to O2(1Dg), which, after deprotonation, leads
eventually to the stable A=O keto-product. The formed
superoxide radicals (O2·)) undergo disproportionation in the
presence of water resulting in hydrogen peroxide (step 8). Since
was carried out. Despite the large molar absorption coefficient
)1
of the solvated electron (e700 = 1.5 · 104
M
cm)1), no sig-
nificant absorption was detected at 700 nm, indicating a
negligible yield of electron photoejection. For the structurally
similar palmatine, photoinduced O2·) formation has been
suggested (6). The quantum yield of singlet molecular oxygen
production is in the 0.39–0.42 range, irrespective of the solvent
polarity for coralyne, but low (<0.05) for berberine in polar
solvents (16). The low Fox values at pH 8–12 (Table 1) indicate
a too low reactivity (i.e. k5 is small) rather than a correlation
with the quantum yield of intersystem crossing (Fisc). If Fisc
controlled the yield of oxidation, Fox of coralyne would be
much larger than that of berberine, which is not the case
(Table 1).
)
no evidence was found for esolv formation by laser flash
photolysis and the sensitized O2(1Dg) production promotes the
photooxidation (vide infra), the reaction mechanism displayed
in Scheme 3 seems to be the correct one.
Irradiation of coralyne and berberine
Irradiation of coralyne at 313 nm in oxygen-saturated meth-
anol–water (9:1, vol) leads to significant absorption diminution
in oxygen-saturated solution above pH 10 (Fig. 2), but the
quantum yield of this photoreaction in the absence of oxygen
•
O
+
O
O
O
(7)
+1O2(Δ)
(5)
(6)
-H+
O
O
O
O
+1O2(Δ)
• -
•
-O2
-
-O2
CH3
OH
CH3
CH3
-H+
CH3
O
N
•
N
N
N
H
OH
H
OH
O
O
O
O
O
O
O
O
•
•
+
HA−OH
HA−OH
HA−O
A=O
Scheme 3. Reactions 5–7 of singlet molecular oxygen with sanguinarine.

¨
1318 Helmut Gorner et al.
Table 2. Quantum yield usen of rose-bengal-sensitized oxidation of
alkaloids*
1.5
1.5
1.5
usen
1
A
[Alkaloid]
(mM)
2
A300
1.0
1.0
Alkaloid
pH
Air
O2
3
0.5
0.5
Sanguinarine
Berberine
8–10
12.0
13.0
13.8
12.0
13.0
0.2
0.8
0.005†
0.015
0.007
0.02
0.2
0.4
0.012
0.1
0.
0.0
0
λ
(nm)
330000
350
400
400
350
0.3†
1.0
Coralyne
0.5
λ
(nm)
0.07†
*In MeOH–H2O, 99:1, using kirr = 500–550 nm. †Under argon:
usen < 0.002.
0
100
200
Time (s)
300
Figure 2. Plots of A300 as a function of the time of 313 nm irradiation
of coralyne in argon- (d) and oxygen-saturated (s) methanol–water
(9:1, vol) at pH 11. Inset: spectra in oxygen-saturated solution at 0, 90
and 300 s irradiation at 313 nm, 1–3, respectively.
1,5
1.5
1.5
A
1
2
Aobs
1,0
1.0
Sensitized irradiation of sanguinarine
0,
0.5
1.0
3
To confirm the essential role of O2(1Dg) in the photooxidation of
alkaloids, rose bengal was chosen as a sensitizer. Lambert and
Kochevar provided evidence for the very inefficient formation
of O2·) via electron transfer from triplet rose bengal to O2 and
concluded that this process has a negligible rate compared to the
energy transfer from triplet rose bengal to O2 producing O2(1Dg)
(38). When rose bengal was irradiated at 500–560 nm in the
presence of both oxygen and 0.1 mM sanguinarine in 99:1
methanol–water mixtures at pH 8–10, the spectral change
increased with the oxygen concentration, but the quantum
yields of the sensitized oxidation (usen) even in air- and oxygen-
saturated solutions remained small (Table 2). Figure 3 displays
the spectra together with the absorbance change at 280 and
380 nm as a function of the time of irradiation (inset). We
propose that the sensitized oxidation of the sanguinarine
alkanolamine form follows the mechanism shown in Scheme 4.
The reaction sequence involves the radical cation HA-OH·+,
the radical HA-O· and two molecules of O2(1Dg).
0
300
4
Time (s)
0
300
0.5
0.0
250
300
350
(nm)
400
λ
Figure 3. Absorption spectra of 0.2 mM sanguinarine in oxygen-
saturated methanol–water (99:1, vol) at pH 9 after 0, 30, 90 and
180 s irradiation of rose bengal at 500–560 nm, 1–4, respectively. Inset:
plots of A280
(
) and A380 (h) as a function of the irradiation time.
+O2
hν
1
3
*
Sen
Sen
Sen
*
Sen
-O2(1Δg)
+O2(1Δg)
Sensitized irradiation of berberine and coralyne
+O2(1Δg)
• -
•
+
HA-OH
HA-OH
A=O
When rose bengal is irradiated at 500–560 nm in oxygen-
saturated methanol–water (99:1, vol) at pH 11, A270, A345 and
A430 of berberine decrease strongly and isosbestics points at
290 and 310 nm appear. The latter indicates sensitized
conversion to 8-oxyberberine (A=O) without significant
formation of byproducts. Examples for spectral alteration
are shown in the insets of Figs. 4 and 5. The substantial
difference in the time dependences in the absence and presence
of O2 (Fig. 4) is convincing evidence for an oxygen-mediated
photoreaction. The spectral changes for relatively small
(0.1 mM) and large (0.8 mM) berberine concentrations are the
same. The usen values in oxygen-saturated solution are slightly
larger than under air at the same OH) concentration (Table 2),
but a marked difference is not to be expected. When the pH
was raised from 12 to 13.8, a considerable increase of usen was
observed reaching a value of 0.4. This effect probably indicates
that the uncharged pseudobase (Scheme 1), which is produced
in strongly alkaline medium in the ground state (35), can be
oxidized more efficiently by O2(1Dg) than the cationic berber-
•
-
−O2 -2H+
−O2
Scheme 4. Formation of singlet molecular oxygen and reaction with
the pseudobase forms of the three alkaloids.
ine. The latter form dominates at pH 12. Therefore, usen is
only 0.02 even in oxygen-saturated solution at pH 12. The
amount of the easier oxidizable pseudobase grows with
increasing OH) concentration. This effect accelerates the
reaction with O2(1Dg), resulting in a substantial rise of usen
with pH (Fig. 5). The decrease of usen above pH 13.8 probably
arises from the instability of the berberine pseudobase under
this condition. At pH 13.8, most berberine molecules are
converted to the pseudobase. Therefore, usen reaches a
maximum value of 0.4. As it can be seen in Fig. 5, usen is
very small below pH 11.5, when the solution contains
negligible amounts of the pseudobase. This indicates that
electron transfer to O2(1Dg) is necessary to induce sensitized

Photochemistry and Photobiology, 2011, 87 1319
alkaloid cation, thereby lessening the concentration of the
pseudobase form.
2.0
A345
1
2
2
CONCLUSIONS
1.5
1.0
A
2
3
4
The studied alkaloids are photochemically stable in neutral
aqueous solution in argon atmosphere, but undergo photoox-
idation in the presence of oxygen. Relatively efficient oxidation
occurs via the pseudobase form of the alkaloids. The suggested
primary photochemical reaction in the oxidation process is not
the electron ejection but the formation of O2(1Dg). The
sensitized O2(1Dg) generation using rose bengal significantly
accelerates the oxidation of coralyne and berberine. The
suggested reaction mechanism involves two electron transfer
steps to O2(1Dg). The substantial increase of the quantum yield
of photooxidation with pH is due to formation of the
pseudobase, which more readily accepts an electron from
O2(1Dg) than the cationic alkaloid.
1
1
0
0
300
300
400
400
λ (nm)
λ
(nm)
0
100
200
Time (s)
300
Figure 4. Plots of A345 as a function of the time of irradiation of rose
bengal at 500–560 nm in the presence of 0.8 mM berberine in argon-
(d) and oxygen-saturated (s) methanol–water (99:1 vol) at pH 11.
Inset: absorption spectra at 0, 30, 60 and 180 s, 1–4, respectively.
Acknowledgements—We thank Professor Wolfgang Lubitz for his
support and Mr. Leslie J. Currell for technical assistance. The authors
very much appreciate the support of this work by the Hungarian
Scientific Research Fund (OTKA, Grant K75015) and the bilateral
program between the Deutsche Forschungsgemeinschaft and the
Hungarian Academy of Sciences.
0.4
2
2
1
2
A
sen
1
1
0.2
3
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