CureCuma–cationic curcuminoids with improved properties and enhanced antimicrobial photodynamic activity

Article abstract of DOI:10.1016/j.ejmech.2017.09.072

The naturally occurring photosensitizer curcumin has excellent biocompatibility, but its antimicrobial photodynamic efficacy is limited by (i) weak adherence to Gram(-) bacteria cell walls, (ii) low (photo-)stability and (iii) limited solubility in water.

Full text of DOI:10.1016/j.ejmech.2017.09.072

Accepted Manuscript
CureCuma–cationic curcuminoids with improved properties and enhanced
antimicrobial photodynamic activity
Andreas Spaeth, Andre Graeler, Tim Maisch, Kristjan Plaetzer
PII:
S0223-5234(17)30787-0
10.1016/j.ejmech.2017.09.072
EJMECH 9787
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2017
Revised Date: 20 June 2017
Accepted Date: 29 September 2017
Please cite this article as: A. Spaeth, A. Graeler, T. Maisch, K. Plaetzer, CureCuma–cationic
curcuminoids with improved properties and enhanced antimicrobial photodynamic activity, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.072.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
CureCuma – Cationic curcuminoids with improved
properties and enhanced antimicrobial photodynamic
activity
1
*
2
3
2
Andreas Spaeth , Andre Graeler , Tim Maisch and Kristjan Plaetzer
1
2
Department of Organic Chemistry, University of Regensburg, Regensburg, Germany
Laboratory of Photodynamic Inactivation of Microorganisms, Department Cell Biology and
Physiology, University of Salzburg, Salzburg, Austria
3
Department of Dermatology, University Medical Centre Regensburg, Regensburg, Germany
*
Corresponding author:
Andreas Späth, PhD
Department of Organic Chemistry
University of Regensburg
Universitätsstraße 31, 93040 Regensburg, Germany
e-mail: andreas.spaeth@chemie.uni-regensburg.de
Tel.: +49 941 943 4087

ACCEPTED MANUSCRIPT
Keywords
antimicrobial
photodynamic
curcumin
diferoylmethane
cationic photosensitizer
Escherichia coli

ACCEPTED MANUSCRIPT
Abstract
The naturally occurring photosensitizer curcumin has excellent biocompatibility, but its
antimicrobial photodynamic efficacy is limited by (i) weak adherence to Gram(-) cell walls,
(ii) low (photo-)stability and (iii) limited solubility in water.
In this study novel curcuminoids bearing cationic substituents were prepared by different
synthetic routes. The derivatives exhibit excellent water solubility, improved photostability
and low aggregation.
All novel curcuminoids showed antibacterial photodynamic effects (>3 log10 reduction of
CFU) against Escherichia coli and Staphylococcus aureus upon blue light illumination. In
contrast to natural curcumin, effective photokilling of E. coli was possible without the
addition of permeabilizing agents. Ten micromolar of the most active compound (8) achieved
a 7 log decrease of E. coli after light activation with a fluence of 33.8 J/cm², whereas S.
1
0
aureus was inactivated by more than 4 log at a fluence of 5.3 J/cm². Overall the reduction in
1
0
bacterial count was at least 100-fold more effective with these new curcuminoids in
comparison to natural curcumin.

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Introduction
Humans are constantly exposed to pathogenic microorganisms and in most situations the
immune system of healthy persons can handle bacterial attacks efficiently. In the past
bacterial infections were easily cured by administration of antibiotics, but it is estimated that
due to increasing antimicrobial resistance 10 million lives a year and a cumulative 100 trillion
USD of economic output are at risk by 2050.[1] The extensive use and also misuse of
antibiotics in human and veterinary medicine poses a huge selection pressure towards
resistance.[2] It facilitates the fast spread of genes encoding antibiotic resistance within a
bacteria population, providing a competitive advantage of such mutated resistant strains. As a
consequence resistance to antibiotics is dramatically increasing. The development of new
antibiotics is not able to keep up with the evolutionary pace. Only two new classes of
antibiotics (oxazolidinone, cyclic lipopeptide) have been marketed in the past 15 years, which
are active against Gram(+) bacteria, such as MRSA (methicillin-resistant Staphylococcus
aureus). For Gram(-) bacteria the situation is even more critical.[3]
The WHO addresses antibiotic resistance as one of the most urgent priorities for public health
and stated that this “a problem so serious that it threatens the achievements of modern
medicine”[4] and the CDC report of 2013 calls the development of antibioitic resistance “one
of our most serious health threats “.[5] In the recent UN General assembly it was stated that
antibiotic resistant bacteria will render high-quality universal healthcare coverage more
difficult if not impossible and undermine sustainable food production.[6]
This situation not only demands new antibiotics urgently, but also development of alternative
disinfection approaches, which do not initiate resistance. In addition, adjuvants to
conventional antibiotics, like drug efflux pump inhibitors,[7] can be used to inhibit resistance
or to potentiate the activity of antibiotics.[8] Alternative therapy or precaution treatments

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include cold atmospheric pressure plasma,[9] monoclonal antibodies,[10] and phage
treatment.[11]
Photodynamic inactivation of bacteria (PDI) could serve both, the medical and the food
sector: a per se harmless photoactive compound, the photosensitizer (PS), is administered and
subsequently excited by visible light of appropriate wavelengths to generate reactive oxygen
species (ROS). Photocatalytical ROS formation occurs via effective intersystem crossing and
in consequence charge transfer to generate radicals (Type I photochemical reaction) or energy
transfer to produce singlet oxygen (Type II photochemical reaction). The short-lived ROS
immediately attack cellular structures and biomolecules in close proximity. The irreversible
damage to the pathogens is caused by multi-target processes, thus avoiding rapid development
of resistances of the microorganisms.[12] PDI has proven highly efficient against fungal, viral
or bacterial infections.[13] In case that application of such light-activated PS are designated
for food decontamination, decolonization of livestock or treatment of infection, the
photosensitizer molecule should feature high biocompatibility, a negligible toxicity profile,
accessibility for a reasonable price, and good biodegradability yielding only non-toxic
fragments. In this regard, natural molecules, like curcumin /curcuma, with photosensing
abilities should be considered.
Curcuma, obtained from the dried and grounded rhizome of Curcuma longa and Curcuma
domestica, has been used for centuries as spice, in medicine, cosmetics and in fabric
dying[14] and is attributed to have anti-inflammatory and digestive properties.[15, 16]
The main ingredient of the plant extract is curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-
1
,6-heptadiene-3,5-dione), a diferuloylmethane with bright orange color. Intensive research
over the last decades showed its high therapeutic and pharmacological potential[17, 18]:
Several studies confirm anti-inflammatory[19], anti-rheumatic,[20, 21] anti-tumor[22, 23] and
anti-carcinogenic[24] properties. Recently, the substance was discussed as therapeutic for
Alzheimer´s disease.[25] These properties are mainly attributed to the antioxidant effects [17,

ACCEPTED MANUSCRIPT
2
6] based on the elimination of free radicals [27, 28] and thereby reducing oxidative stress in
the cell.[29, 30] There is also evidence that curcuminoids have antibacterial properties
without illumination[31], but in presence of blue light curcumin shows clear phototoxic
characteristics.[32]
Hydrogen bonding and charge delocalisation are important features of the molecule[33] for
interactions with the outer bacterial cell wall,[34] and photochemical processes.[15]
Nevertheless, its antimicrobial photodynamic efficacy is limited due to the weak adherence to
Gram(-) bacteria,[12, 35, 36] but also due to low photostability and limited solubility of the
natural substance. Limited photostability of a photosensitizer is not necessarily a major
drawback of a photosensitizer, as it prevents long or pronounced photosensitivity after
systemic administration. On the contrary, for disinfection high photostability provides a long-
lasting antimicrobial effect.[37]
For the application of curcuminoids in clinical therapy and food preservation their
photostability should be improved when compared to the natural compound, but not result in
an extensive long-term stability. Derivatives should also feature lower anti-oxidative potential
than curcumin for not counteracting photodynamic efficacy, but this property should not be
entirely lost to maintain the health benefits of the natural molecule. Anti-oxidative properties
of curcumin were mainly attributed to phenolic OH groups.[38, 39] Blocking these groups
may result in lower pH sensitivity and as the central unit is not affected, the non-toxic nature
of the chromophore should be maintained.
Addition of cationic charges has already been proven to enhance photodynamic effectivity
against Gram(-) bacteria in other photosensitizer molecules like methylene blue.[40] In this
study alkyl chains bearing cationic charged moieties are attached to the phenolic hydroxyl
groups to the parent compound. Both the number of charges and the lipophilicity were altered
in these derivatives, thereby enhancing the photodynamic inactivation efficacy.[40].

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Therefore here we examine the impact of our approach of adding cationic charges to
curcumin on photodynamic efficacy against two Gram(+) and Gram(-) bacterial model
species (S. aureus and E. coli).

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Material and methods
Characterization of prepared compounds
Thin layer chromatography (TLC) analyses were performed on silica gel 60 F-254 with 0.2
mm layer thickness and detection via UV light at 254 nm / 366 nm or through staining with
ninhydrin in ethanol. Flash column chromatography was performed on Merck silica gel (Si 60
4
0-63 µm) either manually or on a Biotage® solera™ flash purification system. Column
chromatography was performed on silica gel (70–230 mesh) from Merck.
Melting points were measured on a SRS melting point apparatus (MPA100 Opti Melt) and are
uncorrected.
1
13
NMR spectra were recorded on Bruker Avance 300 ( H 300.13 MHz, C 75.47 MHz, T =
1
13
3
00 K) or Bruker Avance 400 ( H 400.13 MHz, C 100.61 MHz, T = 300 K) instruments.
The chemical shifts are reported in δ[ppm] relative to external standards (solvent residual
peak). The spectra were analyzed by first order, the coupling constants J are given in Hertz
[
Hz]. Characterization of the signals: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, bs = broad singlet, psq = pseudo quintet, dd = double doublet, dt = doublet of
triplets, ddd = double double doublet. Integration is determined as the relative number of
1
3
atoms. Assignment of signals in C-spectra was determined with DEPT technique (pulse
angle: 135 °) and given as (+) for CH or CH, (–) for CH and (C ) for quaternary C . Error of
3
2
q
q
1
13
reported values: chemical shift 0.01 ppm ( H NMR) and 0.1 ppm ( C NMR), coupling
constant J 0.1 Hz. The solvents used for the measurements are reported for each spectrum.
IR spectra were recorded with a Bio-Rad FT-IR-FTS 155 spectrometer. Absorption spectra
were recorded on a “Cary BIO 50” UV/VIS/NIR spectrometer from Varian. All
measurements were performed in 1 cm quartz cuvettes (Hellma) and UV-grade solvents
(Baker or Merck) at 25 °C.

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Mass spectra were recorded on Finnigan MAT95 (EI-, CI- and FAB-MS), Agilent Q-TOF
6
540 UHD (ESI-MS, APCI-MS) or Thermo Quest Finnigan TSQ 7000 (ES-MS, APCI-MS)
spectrometer. Xenon serves as the ionization gas for FAB.
Synthesis and purification of the compounds
Commercial reagents and starting materials were purchased from Acros Organics, TCI
Europe, Fluka, Merck or Sigma-Aldrich and used without further purification. The dry
solvents tetrahydrofurane and dichloromethane were purchased from Sigma-Aldrich (puriss.,
absolute) or Roth (RotiDry Sept), respectively. They were stored over molecular sieves under
nitrogen and were used as received. Curcumin (95 % purity) was purchased from ABCR and
was purified by recrystallization from ethanol.
The PS presented in this study are water-soluble dyes based on the curcumin structure (1).
H
O
O
O
O
O
R =
R
R
R
R
OH
1)
(
O
+
+
+
+
NH3
NH3
NH3
NH3
O
O
O
O
O
(
2)
(3)
(4)
(5)
+
+
NH3
NH3
O
O
O
+
O
O
O
NH3
+
+
+
O
NH3
NH3
NH3
O
O
O
+
(
6)
(7)
(8)
(9)
NH3
Figure 1: The parent compound in its tautomeric forms (1) and curcumin derivatives
investigated in this study (2 – 9); all compounds were prepared as chloride salts

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The starting materials for the synthesis are prepared by the substitution of
hydroxybenzaldehydes with an alkyl bromide or tosylate in N,N-dimethylformamide (DMF)
at elevated temperature:
O
R1
R3
O
R1
R3
R2
R2
X
H
H
OH
R1 = R2 = R3 = H
(
(
(
(
(
(
10) R1 = R2 = R3 = H
(17)
(18)
(19)
(20)
(21)
(22)
X = OCH CH NHBoc
2
2
R1 = R3 = H, R2 = Me
X = OCH CH NHBoc
11) R1 = R3 = H, R2 = Me
12) R1 = R3 = H, R2 = OMe
13) R2 = R3 = H, R1 = OMe
14) R1 = R3 = H, R2 = OBz
12) R1 = R3 = H, R2 = OMe
2
2
a
R1 = R3 = H, R2 = OMe
X = OCH CH NHBoc
2
2
R2 = R3 = H, R1 = OMe
X = OCH CH NHBoc
2
2
R1 = R3 = H, R2 = OBz
b
c
X = OCH CH CH NHBoc
2
2
2
R1 = R3 = H, R2 = OMe
X = (OCH CH ) NHBoc
2
2 2
R1 = R3 = H, R2 = X =
OCH CH NHBoc
(
(
15) R1 = R3 = H, R2 = OH
16) R1 = H, R2 = R3 = OH
(23)
(24)
2
2
a
R1 = H, R2 = R3 = X =
OCH CH NHBoc
2
2
Scheme 1: Synthesis of the substituted aldehydes; conditions: a) BocHNCH CH Br, DMF,
2
2
K CO , KI, 80°C, overnight, 61 - 87 %; b) TsOCH CH OCH CH NHBoc, DMF, K CO , KI,
2
3
2
2
2
2
2
3
8
0°C, overnight, 72 %; c) BocHNCH CH Br, DMF, K CO , KI, 80°C, overnight, 84 %;
2 2 2 3

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Synthesis of the protected curcumin derivatives was performed analog to the common
approach via aldehyde/acetylacetone in presence of boric anhydride[41] (Scheme 2) and,
starting directly from curcumin using the Mitsonubo reaction [42] (Scheme 3).
Purification of the deprotected derivatives was achieved by precipitation with diethylether and
re-precipitation from dichloromethane with diethylether, until the UV/Vis spectra showed
constant absorption.
O
R1
R3
R1
R3
O
O
R1
R3
R2
X
R2
X
R2
X
H
R1 = R2 = R3 =
X =
X =
X =
+
+
(
(
(
(
(
(
17)
18)
19)
H
H
H
H
H
H
H
H
H
H
OCH
OCH
OCH
OCH
OCH
2
CH
CH
CH
CH
CH
2
NHBoc
NHBoc
NHBoc
NHBoc
(25) OCH
2
CH
2
NHBoc
NHBoc
NHBoc
NHBoc
(2) OCH
(3) OCH
(4) OCH
(5) OCH
(6) OCH
2
CH
CH
CH
CH
CH
2
NH
NH
NH
NH
3
Me
(26) OCH
(27) OCH
(28) OCH
(29) OCH
CH
CH
CH
CH
2
2
2
2
2
2
2
3
+
+
OMe
H
a
b
2
2
2
2
2
3
3
20) OMe
2
2
2
2
2
2
2
2
2
2
2
2
+
21)
22)
H
H
OBz
OMe
CH
2
NHBoc
CH
2
NHBoc
CH
2
NH
3
+
(OCH
CH
)
NHBoc
(30) (OCH
CH
)
NHBoc
(7) (OCH
CH
)
NH
2
2
2
2
2
2
2
2
2
3
R1 = R3 =
H
R2 = X =
OCH CH NHBoc
R2 = X =
OCH CH NHBoc
R2 = X =
OCH CH NH
3
+
(
23)
24)
2
2
a
a
(31)
(32)
2
2
b
b
(8)
(9)
2
2
R1 =
H
R2 = R3 = X =
OCH CH NHBoc
R2 = R3 = X =
OCH CH NHBoc
R2 = R3 = X =
+
(
2
2
2
2
OCH CH NH
2 2 3

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Scheme 2: Synthesis and deprotection of the substituted curcumins (25–32) and their
deprotection; conditions: a) Ethyl acetate, H BO , B(OBu) , BuNH , 80°C, 5 h, 41 – 87 %; b)
3
3
3
2
TFA, DCM, RT, 5 h, quant.; then ion exchanger Amberlite IRA-958, water, MeCN
O
O
O
O
O
O
O
O
O
O
O
O
OH
BocHN
NHBoc
HO
n
n
(
(
27) n= 0 81%
30) n= 1 76%
(
1)
Scheme 3: Substitution of curcumin by Mitsonubo reaction; conditions: THF, DEAD, PPh3,
RT, overnight
The purity of all synthesized compounds was determined by NMR spectroscopic methods
1
(
Bruker Avance 300, DMSO-d6) and HPLC-MS confirming a purity of > 97%. H-NMR
spectra of selected compounds and the synthesis procedure for 17 – 24 can be found in the
supporting information.
General procedure 1: Substitution of curcumin by Mitsonubo reaction
The reaction was conducted under subdued light. Curcumin (0.36 g, 1 mmol),
triphenylphosphine (1.04 g, 4 mmol) and the boc-protected aminoalcohol (3 mmol) were
dissolved in dry THF (4 mL). DEAD (0.7 g, 4 mmol, 40% in toluene) diluted with dry THF (6
mL) was added dropwise over the course of 20 mins at 2-5 °C. The reaction mixture was
stirred for additional 30 mins in the ice bath, then 4 h at room temperature.
Ethyl acetate (40 mL) was added, the organic layer was washed with water (20 mL) trice,
separated and dried over MgSO . All volatiles were removed at reduced pressure and the
4
residue was purified by column chromatography with silica gel and acetone / petroleum ether
as the eluent (dryload with acetone). The product was dissolved in the minimum amount of

ACCEPTED MANUSCRIPT
ethyl acetate and precipitated by slow addition of the tenfold volume of petroleum ether. The
orange powder was filtered off and washed with petroleum ether.
Compound (27)
2
-(N-tert-butoxycarbonyl-amino)ethanol (483 mg, 3 mmol) was reacted to yield an orange
solid (465 mg, 71% yield). The product was purified with acetone / petroleum ether 1:3 ‰ 1:2
as the eluent.
Compound (30)
2
-(2-(N-tert-butoxycarbonyl-amino)ethoxy)ethanol (715 mg, 3 mmol) was reacted to give an
orange solid (386 mg, 52% yield). The product was purified with acetone / petroleum ether
:2 ‰ 2:3 as the eluent.
1
General procedure 2
Acetylacetone (0.1 g, 1 mmol) and B O (0.07 g, 1 mmol) are stirred in dried ethyl acetate (2
2
3
mL) for 30 mins at 50°C. The substituted aldehyde (20 mmol) in ethyl acetate (3 mL) is
added, followed by tri-n-butyl borate (0.7 g, 3 mmol) and stirring is continued for 30 mins at
5
0°C. n-Butylamine (0.1 mL in 1 mL ethyl acetate) was added dropwise over 5 mins. After 5
h of stirring at 50°C the mixture is allowed to stand overnight at room temperature.
Ethyl acetate (30 mL) is added followed by 60 mL of diluted acetic acid (50 %). For
hydrolysis of the borium complex the mixture is stirred for 12 h at room temperature avoiding
illumination. The solvents are evaporated at reduced pressure. The residue is extracted with
trice with ethyl acetate (30 mL). The combined organic extracts are washed with water (50
mL), dried over MgSO and evaporated. The residue was purified by column chromatography
4
with silica gel and acetone / petroleum ether 1:3 ‰ 2:3 as the eluent (dryload with acetone), if

ACCEPTED MANUSCRIPT
not stated otherwise. The product was dissolved in the minimum amount of ethyl acetate and
precipitated by slow addition of the tenfold volume of petroleum ether. The orange powder
was filtered off and washed with petroleum ether.
[
2-(4-{7-[4-(2-tert-Butoxycarbonylamino-ethoxy)-phenyl]-3,5-dioxo-hepta-1,6-dienyl}-
phenoxy)-ethyl]-carbamic acid tert-butyl ester (25)
Compound 17 (530 mg, 2 mmol) was reacted according to general procedure 2. The product
is a yellow solid (363 mg, 61% yield).
1
MP (not corr.) = 136-138°C; - H NMR (300 MHz, CDCl ), δ = 7.61 (d, J = 15.8 Hz, 2H),
3
7
.50 (d, J = 8.8 Hz, 4H), 6.89 (d, J = 8.7 Hz, 4H), 6.50 (d, J = 15.8 Hz, 2H), 5.02 (s, 2H), 4.05
(
t, J = 5.1 Hz, 4H), 3.60 – 3.48 (m, 4H), 1.45 (s, 18H); - MS (ESI, CH Cl /MeOH + 10 mmol
2
2
+
+
NH OAc): 595.3 (100%, MH ), 539.2 (18%, MH -C H ); - HRMS (PI-LSIMS FAB,
4
4
9
+
+
glycerine): calc. for (M+H ) : 595.3014, found: 595.3020; - Molecular Weight = 594.71
g/mol; - Molecular Formula = C H N O
3
3
42
2
8
[
2-(4-{7-[4-(2-tert-Butoxycarbonylamino-ethoxy)-3-methoxy-phenyl]-3,5-dioxo-hepta-1,6-
dienyl}-2-methoxy-phenoxy)-ethyl]-carbamic acid tert-butyl ester (27)
Using compound 19 (590 mg, 2 mmol) in general procedure 2 the product is isolated as an
orange solid (590 mg, 61% yield).
1
MP (not corr.) = 149-151°C; - H NMR (300 MHz, CDCl ), δ = 7.58 (d, J = 15.7 Hz, 2H),
3
7
.14 – 6.99 (m, 4H), 6.87 (d, J = 8.3 Hz, 2H), 6.48 (d, J = 15.8 Hz, 2H), 5.16 (s, 2H), 4.09 (t,
J = 4.9 Hz, 4H), 3.90 (s, 6H), 3.56 (m, 4H), 1.44 (s, 18H); - MS (ESI, CH Cl /MeOH + 10
2
2
+
+
mmol NH OAc): 655.3 (100%, MH ), 555.3 (26%, MH -boc); - HRMS (PI-LSIMS FAB,
4
+
+
glycerine): calc. for (M+H ) : 655.3239, found: 655.3241; - Molecular Weight = 654.76
g/mol; - Molecular Formula = C H N O
3
5
46
2
10

ACCEPTED MANUSCRIPT
[
2-(4-{7-[4-(2-tert-Butoxycarbonylamino-ethoxy)-2-methoxy-phenyl]-3,5-dioxo-hepta-1,6-
dienyl}-3-methoxy-phenoxy)-ethyl]-carbamic acid tert-butyl ester (28)
Compound 20 (530 mg, 2 mmol) was reacted according to general procedure 2 to give an
orange-yellow solid (393 mg, 60% yield).
1
MP (not corr.) = 143-145°C; - H NMR (300 MHz, CDCl ), δ = 7.89 (d, J = 16.0 Hz, 2H),
3
7
.47 (d, J = 8.5 Hz, 2H), 6.62 (d, J = 16.0 Hz, 2H), 6.52 – 6.39 (m, 4H), 4.99 (s, 2H), 4.06 (t,
J = 5.1 Hz, 4H), 3.88 (s, 6H), 3.55 (m, 4H), 1.45 (s, 18H); - MS (ESI, CH Cl /MeOH + 10
2
2
+
mmol NH OAc): 655.3 (100%, MH ); - HRMS (PI-LSIMS FAB, glycerine): calc. for
4
+
+
(
M+H ) : 655.3225, found: 655.3231; - Molecular Weight = 654.76 g/mol; - Molecular
Formula = C H N O
3
5
46
2
10
[
2-(4-{7-[4-(2-tert-Butoxycarbonylamino-ethoxy)-3-methyl-phenyl]-3,5-dioxo-hepta-1,6-
dienyl}-2-methyl-phenoxy)-ethyl]-carbamic acid tert-butyl ester (26)
Using compound 18 (559 mg, 2 mmol) in general procedure 2 the product is isolated as an
orange solid (442 mg, 71% yield).
1
MP (not corr.) = 139-141°C; - H NMR (300 MHz, CDCl ), δ = 7.59 (d, J = 15.8 Hz, 2H),
3
7
4
+
.36 (d, J = 10.8 Hz, 4H), 6.80 (d, J = 8.3 Hz, 2H), 6.49 (d, J = 15.8 Hz, 2H), 4.94 (s, 2H),
.06 (t, J = 5.0 Hz, 4H), 3.58 (m, 4H), 2.25 (s, 6H), 1.46 (s, 18H); - MS (ESI, CH Cl /MeOH
2
2
+
+
10 mmol NH OAc): 623.3 (100%, MH ), 567.3 (21%, MH -C H ); - HRMS (PI-LSIMS
4
4
9
+
+
FAB, glycerine): calc. for (M+H ) : 623.3332, found: 623.3336; - Molecular Weight =
22.77 g/mol; - Molecular Formula = C H N O
6
3
5
46
2
8

ACCEPTED MANUSCRIPT
[
2-(4-{7-[4-(2-tert-Butoxycarbonylamino-ethoxy)-3-benzyloxy-phenyl]-3,5-dioxo-hepta-1,6-
dienyl}-2-benzyloxy-phenoxy)-ethyl]-carbamic acid tert-butyl ester (29)
Compound 21 (738 mg, 2 mmol) was reacted according to general procedure 2 to yield an
orange solid (5183 mg, 62% yield).
1
MP (not corr.) = 138-140°C; - H NMR (300 MHz, CDCl ), δ = 7.55 (d, J = 15.8 Hz, 2H),
3
7
5
1
.45 – 7.31 (m, 10H), 7.14 – 7.04 (m, 4H), 6.86 (d, J = 8.3 Hz, 2H), 6.46 (d, J = 15.8 Hz, 2H),
.39 (s, 2H), 5.22 (s, 4H), 4.14 (t, J = 5.9 Hz, 4H), 3.39 (m, 4H), 2.09 – 2.00 (m, 4H), 1.41 (s,
+
8H); - MS (ESI, CH Cl /MeOH + 10 mmol NH OAc): 857.4 (100%, MNa ), 835.4 (76%,
2
2
4
+
+
+ +
MH ), 735.4 (53%, MH -boc); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+H ) :
8
35.4178, found: 835.4179; - Molecular Weight = 835.02 g/mol; - Molecular Formula =
C H N O
4
9
58
2
10
(
2-{2-[4-(7-{4-[2-(2-tert-Butoxycarbonylamino-ethoxy)-ethoxy]-3-methoxy-phenyl}-3,5-
dioxo-hepta-1,6-dienyl)-2-methoxy-phenoxy]-ethoxy}-ethyl)-carbamic acid tert-butyl ester
30)
(
Reaction of compound 22 (530 mg, 2 mmol) by general procedure 2 yields an orange yellow
solid as the product (349 mg, 47% yield). The eluent for purification by column
chromatography was acetone / petroleum ether 1:2 ‰ 1:1.
1
MP (not corr.) = 105-107°C; - H NMR (300 MHz, CDCl ), δ = 7.59 (d, J = 15.8 Hz, 2H),
3
7
4
.18 – 7.03 (m, 4H), 6.90 (d, J = 8.3 Hz, 2H), 6.49 (d, J = 15.8 Hz, 2H), 5.07 (s, 2H), 4.24 –
.15 (m, 4H), 3.91 (s, 6H), 3.88 – 3.83 (m, 4H), 3.61 (t, J = 5.1 Hz, 4H), 3.33 (m, 4H), 1.43
+
(
s, 18H); - MS (ESI, CH Cl /MeOH + 10 mmol NH OAc): 671.3 (51%, MNa ), 655.3 (86%,
2
2
4
+
+
+ +
MH ), 555.3 (100%, MH -boc); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+H ) :

ACCEPTED MANUSCRIPT
7
43.3750, found: 743.3752; - Molecular Weight = 742.87 g/mol; - Molecular Formula =
C H N O
12
39
54
2
{
2-[4-{7-[3,4-Bis-(2-tert-butoxycarbonylamino-ethoxy)-phenyl]-3,5-dioxo-hepta-1,6-dienyl}-
-(2-tert-butoxycarbonylamino-ethoxy)-phenoxy]-ethyl}-carbamic acid tert-butyl ester (31)
2
Compound 23 (850 mg, 2 mmol) was reacted according to general procedure 2. The product
is an orange-red solid (374 mg, 41% yield). The eluent for purification by column
chromatography was acetone / petroleum ether 1:3 ‰ 1:2.
1
MP (not corr.) = 95-97°C; - H NMR (300 MHz, CDCl ), δ = 7.57 (d, J = 15.8 Hz, 2H), 7.19
3
–
7.12 (m, 4H), 6.98 – 6.86 (m, 2H), 6.50 (d, J = 15.8 Hz, 2H), 5.28 (s, 4H), 4.10 (t, J = 4.4
Hz, 8H), 3.55 (m, 8H), 1.46 (s, 18H), 1.45 (s, 18H); - MS (ESI, CH Cl /MeOH + 10 mmol
2
2
+
+ 2+
NH OAc): 913.5 (100%, MH ), 457.3 (7%, (M+2H ) ); - HRMS (PI-LSIMS FAB,
4
+
+
glycerine): calc. for (M+H ) : 913.4810, found: 913.4812; - Molecular Weight = 913.08
g/mol; - Molecular Formula = C H N O
4
7
68
4
14
{
2-[4-{7-[3,4,5-Tris-(2-tert-butoxycarbonylamino-ethoxy)-phenyl]-3,5-dioxo-hepta-1,6-
dienyl}-2,6-bis-(2-tert-butoxycarbonylamino-ethoxy)-phenoxy]-ethyl}-carbamic acid tert-
butyl ester (32)
Compound 24 (1166 mg, 2 mmol) was submitted to reaction by general procedure 2. The
product is an orange solid (554 mg, 45% yield). The eluent for purification by column
chromatography was acetone / petroleum ether 1:4 ‰ 1:2.
1
MP (not corr.) = 90-92°C; - H NMR (300 MHz, CDCl ), δ = 7.52 (d, J = 15.1 Hz, 2H), 6.79
3
(s, 4H), 6.51 (d, J = 15.1 Hz, 2H), 5.75 (s, 2H), 5.25 (s, 3H), 4.10 (m, 12H), 3.57 (m, 8H),
3
.41 (m, 4H), 1.47 (s, 18H), 1.47 (s, 36H); - MS (ESI, CH Cl /MeOH + 10 mmol NH OAc):
2
2
4

ACCEPTED MANUSCRIPT
+
+
+
1
253.6 (100%, MNa ), 1231.6 (49%, MH ), 1131.3 (51%, MH -boc); - HRMS (PI-LSIMS
+
+
FAB, glycerine): calc. for (M+H ) : 1231.6596, found: 1231.6594; - Molecular Weight =
1
231.46 g/mol; - Molecular Formula = C H N O
61 94 6 20
General procedure 3
The whole reaction and purification sequence was conducted under protection from light. The
boc-protected curcumin (0.2 mmol) was dissolved in dichloromethane (10 mL, dried over
CaCl ). Trifluoroacetic acid in dichloromethane (10%, 2.7 mL, 2.4 mmol) cont. TIS (5%) was
2
added dropwise. After stirring for 4 h at room temperature under moisture protection, the
suspension was partitioned between six blue caps and diethylether was added to a total
volume of 15 mL per cap. The product was settled with the aid of a centrifuge (60 mins, 4400
rpm, 0°C) and the supernatant was discarded. The precipitate was re-suspended in
diethylether, settled again and the supernatant was decanted off. This washing step was
repeated once using diethylether. Afterwards the product was dried at reduced pressure to
give yellow powder.
A small column was packed with ion exchanger (Amberlite IRA-958). The resin was rinsed
with acidic sodium chloride solution (10 % aqueous NaCl cont. 0.1 % HCl, 100 mL) washed
salt free with dilute hydrochloric acid (0.1 %) and conditioned with dilute hydrochloric acid
(0.1 %) / methanol / acetonitrile 9:2:1.
The curcumin TFA salt (0.2 mmol) was dissolved in dilute hydrochloric acid (0.1 %) /
methanol / acetonitrile 9:2:1 (2 mL) and was slowly passed through the column (height 10
cm, diameter 1 cm; transferred with 2x 2 mL eluent) of the conditioned anion exchanger
(Amberlite IRA-958) eluting with dilute hydrochloric acid (10 mL, 0.1 %). The aqueous
solution was lyophilized to give the product as yellow-orange solid.

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(1E,6E)-1,7-bis(4-(2-aminoethoxy)phenyl)hepta-1,6-diene-3,5-dione hydrochloride (2)
Deprotection of compound 25 yields a yellow powder (93 mg, 0.2 mmol, quant.).
1
MP (not corr.) > 200°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.60 (m, 6H), 7.04 (d,
J = 6.9 Hz, 4H), 6.67 (d, J = 13.8 Hz, 2H), 4.32 – 4.17 (m, 4H), 3.46 – 3.34 (m, 4H); - MS
+
2+
+
(
ESI, MeCN/H O + 0.06 % TFA): 198.1 (100%, (M+2H ) ), 352.2 (9%, MH -C H N),
2
2
5
+
+ 2+
3
95.2 (2%, MH ); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+2H ) : 198.1019,
found: 198.1028; - Molecular Weight = 467.39 g/mol; - Molecular Formula =
C H N O Cl
2
23
28
2
4
(1E,6E)-1,7-bis(4-(2-aminoethoxy)-3-methylphenyl)hepta-1,6-diene-3,5-dione hydrochloride
(3)
Deprotection of compound 26 yields an orange powder (91 mg, 0.183 mmol, 92 %)
1
MP (not corr.) > 200°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.60 (d, 14.3 Hz, 2H),
7
.55 – 7.37 (m, 4H), 6.99 (d, J = 6.8 Hz, 2H), 6.68 (d, J = 14.3 Hz, 2H), 4.28 (m, 4H), 3.48 –
3
.39 (m, 4H), 2.31 (s, 6H); - MS (ESI, MeCN/H O + 0.06 % TFA): 212.1 (100%,
2
+
2+
+
+
(
M+2H ) ), 380.2 (6%, MH -C H N), 423.2 (2%, MH ); - HRMS (PI-LSIMS FAB,
2 5
+
2+
glycerine): calc. for (M+2H ) : 212.1176, found: 212.1173; - Molecular Weight = 495.44
g/mol; - Molecular Formula = C H N O Cl
2
5
32
2
4
2
(1E,6E)-1,7-bis(4-(2-aminoethoxy)-3-methoxyphenyl)hepta-1,6-diene-3,5-dione
hydrochloride (4)
Deprotection of compound 27 yields a yellow powder (94 mg, 0.178 mmol, 89 %)

ACCEPTED MANUSCRIPT
1
MP (not corr.) > 200°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.61 (d, J = 15.7 Hz,
2
6
0
H), 7.33 (s, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 15.7 Hz, 2H),
.03 (s, 1H), 4.33 – 4.22 (m, 4H), 3.95 (s, 6H), 3.45 – 3.34 (m, 4H); - MS (ESI, MeCN/H O +
2
+
2+
+
+
.06 % TFA): 228.1 (100%, (M+2H ) ), 412.2 (5%, MH -C H N), 455.2 (3%, MH ); -
2
5
+
2+
HRMS (PI-LSIMS FAB, glycerine): calc. for (M+2H ) : 228.1125, found: 228.1133; -
Molecular Weight = 527.44 g/mol; - Molecular Formula = C H N O Cl
2
5
32
2
6
2
(1E,6E)-1,7-bis(4-(2-aminoethoxy)-2-methoxyphenyl)hepta-1,6-diene-3,5-dione
hydrochloride (5)
Deprotection of compound 28 yields a yellow powder (92 mg, 0.174 mmol, 87 %)
1
MP (not corr.) > 200°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.63 (d, J = 15.8 Hz,
2
3
H), 7.52 -7.02 (m, 6H), 6.77 (d, J = 15.7 Hz, 2H), 4.35 – 4.25 (m, 4H), 3.87 (s, 6H), 3.42 –
+
2+
.32 (m, 4H); - MS (ESI, MeCN/H O + 0.06 % TFA): 228.1 (100%, (M+2H ) ), 412.2 (5%,
2
+
+
+ 2+
MH -C H N), 455.2 (3%, MH ); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+2H ) :
2
5
2
28.1128, found: 228.1133; - Molecular Weight = 527.44 g/mol; - Molecular Formula =
C H N O Cl
2
5
32
2
6
2
(1E,6E)-1,7-bis(4-(3-aminopropoxy)-3-(benzyloxy)phenyl)hepta-1,6-diene-3,5-dione
hydrochloride (6)
Deprotection of compound 29 yields an orange powder (106 mg, 0.150 mmol, 75%)
1
MP (not corr.) > 175°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.57 (m, 2H), 7.48 –
7
.33 (m, 12H), 7.28 (m, 2H), 7.08 (m, 2H), 6.68 (m, 2H), 5.13 (s, 4H), 4.23 (m, 4H), 3.16 (t, J
6.4 Hz, 4H), 2.16 (m, 4H); - MS (ESI, MeCN/H O + 0.06 % TFA): 286.1 (100%,
=
2
+
2+
+
+
(
M+2H ) ), 545.3 (4%, MH -C H N), 635.3 (1%, MH ); - HRMS (PI-LSIMS FAB,
2 5

ACCEPTED MANUSCRIPT
+
2+
glycerine): calc. for (M+2H ) : 318.1594, found: 318.1605; - Molecular Weight = 707.7
g/mol; - Molecular Formula = C H N O Cl
3
9
44
2
6
2
(1E,6E)-1,7-bis(4-(2-(2-aminoethoxy)ethoxy)-3-methoxyphenyl)hepta-1,6-diene-3,5-dione
hydrochloride (7)
Deprotection of compound 30 yields a yellow sticky solid (107 mg, 0.174 mmol, 92 %).
1
MP (not corr.) = 118 - 120°C; - H NMR (300 MHz, MeOD), δ = 7.60 (d, J = 15.7 Hz, 2H),
7
.28 (s, 2H), 7.20 (d, J = 8.2 Hz, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.70 (d, J = 15.8 Hz, 2H), 4.22
(dd, J = 5.2, 3.3 Hz, 4H), 3.91 (s, 6H), 3.94 – 3.88 (m, 4H), 3.83 – 3.76 (m, 4H), 3.16 (t, J =
+
2+
3
.2 Hz, 4H); - MS (ESI, MeCN/H O + 0.06 % TFA): 272.1 (100%, (M+2H ) ), 500.2 (3%,
2
+
+
+ 2+
MH -C H N), 543.3 (3%, MH ); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+2H ) :
2
5
2
72.1387, found: 272.1397; - Molecular Weight = 615.55 g/mol; - Molecular Formula =
C H N O Cl
2
9
40
2
8
2
(1E,6E)-1,7-bis(3,4-bis(2-aminoethoxy)phenyl)hepta-1,6-diene-3,5-dione hydrochloride (8)
Deprotection of compound 31 yields an orange powder (118 mg, 0.179 mmol, 90 %)
1
MP (not corr.) > 166°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.61 (d, J = 15.8 Hz,
2
6
0
H), 7.37 (s, 2H), 7.35 – 7.28 (m, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 15.8 Hz, 2H),
.02 (s, 1H), 4.32 (dd, J = 9.8, 4.9 Hz, 8H), 3.43 (t, J = 4.8 Hz, 8H); - MS (ESI, MeCN/H O +
2
+
3+
+ 2+
.06 % TFA): 171.8 (100%, (M+3H ) ), 235.6 (43%, (M+2H ) -C H N), 257.1 (44%,
2
5
+
2+
+
+ 2+
(
M+2H ) ), 513.3 (15%, MH ); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+2H ) :
57.1396, found: 257.1388; - Molecular Weight = 658.44 g/mol; - Molecular Formula =
C H N O Cl
2
27
40
4
6
4

ACCEPTED MANUSCRIPT
(1E,6E)-1,7-bis(3,4,5-tris(2-aminoethoxy)phenyl)hepta-1,6-diene-3,5-dione hydrochloride (9)
Deprotection of compound 32 yields an orange powder (141 mg, 0.166 mmol, 83 %)
1
MP (not corr.) > 160°C (decomp.); - H NMR (300 MHz, MeOD), δ = 7.62 (d, J = 15.8 Hz,
2
H), 7.12 (s, 4H), 6.84 (d, J = 15.9 Hz, 2H), 4.41 – 4.29 (m, 8H), 4.27 – 4.20 (m, 4H), 3.50 –
3
.40 (m, 8H), 3.39 – 3.33 (m, 4H); - MS (ESI, MeCN/H O + 0.06 % TFA): 158.6 (47%,
2
+
4+
+ 3+
+ 2+
(
M+4H ) ), 211.1 (100%, (M+3H ) ), 273.1 (6%, (M+2H ) -C H N), 316.2 (24%,
2 5
+
2+
+
+ 2+
(
M+2H ) ), 631.3 (12%, MH ); - HRMS (PI-LSIMS FAB, glycerine): calc. for (M+2H ) :
16.1761, found: 316.1771; - Molecular Weight = 849.50 g/mol; - Molecular Formula =
C H N O Cl
3
31
52
6
8
6
Absorption and emission spectra. Each spectrum was recorded in the range from 200 nm to
00 nm in 1 nm steps in a quartz cuvette. The spectrum of blank buffer medium or Millipore
6
water was subtracted.
Determination of Polarity. The polarity of the compounds was estimated via their octanol-
water partition coefficient at 25 °C by the shake-flask method. 5 mL of 50 µM photosensitizer
in 10 mM Sörensen buffer pH 6.8 were mixed with 5 mL of n-octanol and agitated vigorously
for 10 min. Both layers were separated using a centrifuge (25°C, 4000 rpm, 5 min). The
absorbance of the aqueous solution before and after mixing with n-octanol was measured in a
quartz cuvette with a thickness of 10 mm. The partition coefficient was calculated from the
concentrations corresponding to these values. In this concentration range the absorbance
shows linear behavior. As the absorption intensity of curcumin is dependent on the polarity of

ACCEPTED MANUSCRIPT
the solvent,[43] only the values for the aqueous solutions were used for calculation of the
coefficient.
Determination of photostability and singlet oxygen quantum yields
Compound 4 was dissolved in pure water at concentration of 2 µM. For the estimation of the
photostability the solution was irradiated with 107 mW in a time span of 20 minutes.
Illumination occurred by a tunable laser system (NT242-SH/SFG, Ekspla, Lithania) with a
wavelength of 405 nm. Absorption spectra were recorded in distinct time intervals.
The oxygen consumption of the solutions was measured by an oxygen microsensor (Presens,
Regensburg, Germany) with a starting concentration of oxygen in water of 270 µM (air
saturated).
Singlet oxygen signals were recorded with a high sensitive NIR-system described elsewhere
[
44-46] using an appropriate laser system . The singlet oxygen signal of 20 seconds was
recorded over 20 minutes to estimate the stability of singlet oxygen generation depending on
absorption of radiation and the energy transfer from the PS to oxygen in the ground state.
1
The O quantum yield Φ of 4 was determined using 5,10,15,20-Tetrakis(1-methyl-4-
2
∆
pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP) with a Φ = 0.77 ± 0.10 as a reference
∆
PS. [47] Therefore the absorbed energy of a TMPyP solution (c = 0.5 µM)and a solution of 4
(2 µM) were compared to their emitted singlet oxygen luminescence at 1270 nm as described
before. [44] Both PS were excited at 405 nm generated by a tunable laser system (NT 242-
SH/SFG, Ekspla, Vilnius, Lithuania) with different excitation powers of 107mW, 162mW,
2
55mW and 283 mW for 50 seconds.
Experimental procedure for PDI. All experiments were performed under sterile conditions
using a laminar flow bench (Biosafe 4-130, Ehret, Emmendingen, Germany). After adding the

ACCEPTED MANUSCRIPT
photosensitizer experimentation was done under subdued light.
Bacterial strains and growth conditions. A volume of 20 mL medium was inoculated with
Escherichia coli (ATCC25922, Manassas,Virginia). The suspension was incubated overnight
at 37 °C under constant agitation (175 rpm) on a shaker (MAXQ4000, Thermo Scientific,
Dubuque, Iowa, USA). For growing of E. coli Todd-Hewittt Bouillon (Carl Roth, Karlsruhe,
Germany) with a supplement of 0.3 % yeast extract (AppliChem, Darmstadt, Germany) was
used as medium.
The volume of overnight culture was diluted to 20 mL bacterial solution with absorption 0.05
and incubated for two hours at 37 °C under constant agitation (175 rpm) on a shaker. The
resulting absorption (600 nm) was between 0.3 and 0.45.
Staphylococcus aureus (ATCC25923; Manassas,Virginia) was grown aerobically at 37°C in
Mueller-Hinton broth (Gibco Life Technologies GmbH, Eggenstein, Germany). Bacteria
originated from culture shaking overnight (5 mL) harvested by centrifugation (3000 rpm, 10
min; Megafuge 1.0, Heraeus Sepatech, Osterode, Germany). The cells were resuspended in
distilled water at an optical density of 0.6, which was measured at 600 nm with a spectrometer
(SPECORD 50 PLUS, Analytik Jena, Jena, Germany) and corresponded to a bacterial count
8
of 10 bacterial cells per mL.
Light sources and illumination parameters. Two different systems were used for
illumination. A LED light source (consisting of an array of 432 LED 435-12-30, Roithner
Lasertechnik, Vienna, Austria) with a wavelength emission of 430 nm – 435 nm was used for
phototoxicity experiments screening efficacy with all curcumin derivatives with E. coli. The
-2
intensity was set to 9.4 mW cm . A non-coherent light source (Partial Body UV Therapy

ACCEPTED MANUSCRIPT
System UV 802 L with BlueV tubes; Waldmann AG, Villingen-Schwenningen, Germany)
emitting light from 380 nm – 480 nm was used for follow-up experiments with S. aureus and
-2
E. coli. The intensity was adjusted to 17.5 mW cm .
The effective radiant exposure of the light sources was calculated as follows:
Photosensitizer solutions. The photosensitizers used for the bacteria were diluted in distilled
water or Dulbecco`s Phosphate Buffered Saline (DPBS) to get the final concentrations
-1
0
/1/5/10/25/50/100/250/500 µML .
Phototoxicity assay against bacteria. Three different controls were performed for each PS.
The “light only” control (LO) was illuminated but not incubated using the medium without
photosensitizer. The “photosensitizer only” control (PS only) was incubated with
photosensitizer, but was not illuminated. This control was performed with the highest used
concentration and longest incubation period and in addition with a concentration and
incubation period most preferable for practice use. The “double negative” control (Co-/-) was
neither incubated with photosensitizer nor illuminated.
After two hours the culture of E. coli was splitted into aliquots of 1800 µL each. Cells were
then harvested by centrifugation at 20 °C, 830 rcf, for 5 min (5417R Centrifuge, Eppendorf,
Hamburg, Germany) and resuspended in DPBS. Different volumes of the respective
photosensitizer stock solution were added to the suspensions, constantly resulting in a final
volume of 1800 µL. The obtained suspensions of different photosensitizer concentrations

ACCEPTED MANUSCRIPT
were again incubated on the shaker immediately (37 °C, 175 rpm; MAXQ4000, Thermo
Scientific, Schwerte, Germany). Doublets of 500 µL were transferred into a 24-well
microplate (Cellstar, Greiner Bio-One, Frickenhausen, Germany). The PS only and co-/-
controls were transferred into a separate microplate wrapped with aluminum foil. Illumination
−2
−2
was done with a LED-Array device from the bottom (9.4 mW cm , 33.8 J cm ), under
constant agitation with a shaking device (MTS4, IKA, Staufen, Germany). After illumination
dilution series (1:10) were done with DPBS. Fifty microliters of each dilution step were plated
on 1.5 % Agar (Agar-Agar, Kobe I, Roth, Karlsruhe, Germany) with 3 % Todd-Hewitt
Bouillon (Carl Roth, Karlsruhe, Germany) and a supplement of 0.3 % yeast extract
(AppliChem, Darmstadt, Germany). The inoculated agar plates were incubated at 37 °C
overnight and bacterial colonies were counted.
8
For the follow-up experiments an aqueous solution of approximately 10 S. aureus or E. coli
cells per mL were placed into a 96-well microtiter plate (25 mL each) and incubated with
different concentrations of photosensitizer solution. Suspensions were immediately irradiated
−
2
with 20 mW cm . Time and dose for the respective experiment are given in the discussion.
Aliquots of a serial dilution were plated on Mueller - Hinton agar and incubated for 24 h at
3
7°C.
The survival of the bacteria was determined by counting the numbers of colony forming units
CFU) 24 h after illumination using the Miles, Misra and Irwin technique. [48] All
(
experiments were replicated at least three times. The relative inactivation was calculated by
dividing the CFU count of the double negative control (Co -/-, no incubation with PS and no
illumination) by the CFU value of the particular sample. The detection limit was defined as 10
CFU/mL.

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Results & Discussion
Spectroscopic Characterization and Photophysics
Compounds 2 to 9 were investigated for their photophysical stability and absorption
characteristics. All measurements were conducted in Sörensen buffer (0.01 M) at pH = 7. The
molar extinction coefficients for compounds 2 to 9 are summarized in table 1.
The di-ketone group in the curcumin structure exhibits keto-enol tautomerism, which can
exist in different types of conformers depending on the environment. [49] In aqueous solution
the keto-enol form is more stabilized than the diketo form. [50] The intense light absorption
maximum (λmax) at 420 nm is attributed to the keto-enol form and can be assigned to π–π*
transitions, the shoulder at approx. 350 nm to n–π* transitions. Detailed information about the
isomeric forms of alkylated curcumin and the impact of the isomerism on the spectra are
described elsewhere.[51, 52]
All substances, except 2 and 5, show comparable absorption characteristics in the range from
4
10 nm to 430 nm (figure 2). Spectrum of 9 deviates downwards and in comparison to the
other curcuminoids, the peak wavelength is about 10 nm blue shifted.

ACCEPTED MANUSCRIPT
Figure 2: absorption spectra of derivatives 2 -9