ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 975-983 © The Author(s) 2025. This article is an open access publication.
Published in Russian in Biokhimiya, 2025, Vol. 90, No. 7, pp. 1063-1072.
975
Novel Branched Polyamine-Modified Chlorin
with a Photoinduced Antimicrobial Activity
Nikita V. Suvorov
1,a
*, Galina M. Gamenyuk
1
, Ekaterina A. Safonova
1
,
Margarita A. Shagabaeva
1
, Viktoria V. Shchelkova
2
, Sergey I. Tikhonov
1
,
Dmitriy A. Minakov
1
, Nadezhda V. Konovalova
1
, Yuri L. Vasil’ev
1,3
,
and Mikhail A. Grin
1
1
Institute of Fine Chemical Technologies, MIREA – Russian Technological University, 119571 Moscow, Russia
2
A. N. Kosygin Russian State University, 117997 Moscow, Russia
3
I. M. Sechenov First Moscow State Medical University (Sechenov University), 119048 Moscow, Russia
a
e-mail: suvorov.nv@gmail.com
Received May 5, 2025
Revised June 18, 2025
Accepted June 23, 2025
Abstract— Multiple drug resistance is one of the major threats to global health. One of the approaches to
solving this problem is antimicrobial photodynamic therapy, however, currently used photosensitizers are
not sufficiently effective against pathogens. Polycationic molecular constructs enhance the binding and pen-
etration of photosensitizers into poorly permeable gram-negative bacteria. Such conjugates can be obtained
by introducing polyethyleneimines into a photosensitizer molecule. In this study, we synthesized a branched
tetraamine and introduced it into the pyrrole ringA of the natural chlorin molecule and assessed invitro the
photoinduced toxicity of the new photosensitizer against Staphylococcusaureus, Enterococcus faecalis, Pseu-
domonas aeruginosa, and Escherichia coli bacteria. Compared to its structural precursor, the obtained chlorin
with a branched polyamine residue demonstrated an increased bactericidal effect when irradiated with light.
DOI: 10.1134/S0006297925601431
Keywords: chlorophyll A, chlorin e
6
, photodynamic therapy, antimicrobial therapy, antibiotic resistance
* To whom correspondence should be addressed.
INTRODUCTION
Multiple drug resistance of microorganisms is
one of the serious threats to global health. The devel-
opment of bacterial resistance to existing drugs and
the shortage of structures that can potentially form
the basis for new antibiotics have made the search
for new approaches to combating antibiotic-resistant
pathogens a priority. Among the new innovative meth-
ods is antimicrobial photodynamic therapy (APDT),
which is equally effective against antibiotic-sensi-
tive and antibiotic-resistant pathogens [1]. APDT uses
photosensitizers (PSs) in combination with visible
light of a certain wavelength for their excitation. The
transfer of energy from a PS to molecular oxygen
results in the formation of reactive oxygen species,
including singlet oxygen as the main cytotoxic agent
that kills bacteria by oxidizing their vital intracellu-
lar structures. The principal advantage of APDT is
its ability to oxidatively destroy multiple structures
within the target microbial cells, preventing them
from developing resistance to subsequent cycles of
photodynamic exposure. In addition, since the bacte-
ricidal effect of APDT is local, APDT does not exert a
systemic destructive effect on the body’s obligate mi-
croflora. Therefore, APDT simultaneously solves two
main problems of modern antibiotic therapy: it over-
comes the high drug resistance of pathogenic micro-
organisms and eliminates the possibility of systemic
drug toxicity [2].
Previous studies have shown that gram-positive
and gram-negative bacteria differ in their susceptibil-
ity to APDT. While neutral, anionic, and cationic PS
molecules bind equally well to gram-positive bacte-
ria, only cationic or neutral PSs bind to gram-negative
bacteria. Several studies have demonstrated that the
SUVOROV et al.976
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
most effective photosensitizers for APDT contain pos-
itively charged groups [3].
A traditional method of synthesizing cationic PSs
involves quaternization of nitrogen atoms in aliphatic
amine residues located on the periphery of the tetra-
pyrrole macrocycle. As a rule, such alkylammonium
derivatives can easily enter bacterial cells and, there-
fore, exhibit high photoinduced cytotoxicity. Several
tetrapyrrole compounds (derivatives of porphyrins
and their hydrogenated analogs containing alkylam-
monium substituents) have been described and their
antimicrobial activity has been studied [4-6]. Another
approach to developing cationic PSs for APDT is the
synthesis of pyridyl-containing pigments and their
subsequent quaternization with alkyl halides. This
approach reduces the “dark” toxicity of PSs while
maintaining their high photoinduced activity. Various
tetrapyrrole macroheterocyclic compounds have been
described, including derivatives of natural chlorophyll
A containing one or more positively charged pyridin-
ium groups at the macrocycle periphery [7-11]. Also,
chlorin derivatives containing other heterocyclic frag-
ments have been synthesized [12]. Our research group
has previously produced chlorin and bacteriochlorin
PSs modified with the residues of nicotinic and ison-
icotinic acids which exhibited a high photoinduced
toxicity against gram-negative and gram-positive bac-
teria in biofilms, as well as in infected wound models
invivo [13-15].
Development of polycationic molecular constructs
should facilitate internalization of PSs in gram-nega-
tive bacteria. One method of their production is in-
troduction of various polyethyleneimines into a PS
molecule. Hamblin et al. [16] described the synthesis
of several chlorin e
6
derivatives containing fragments
of both linear and branched polyethyleneimines [16].
The resulting cationic PSs, and particularly a deriv-
ative with a highly branched, high-molecular-weight
amine, demonstrated a high antimicrobial activity
against pathogenic microorganisms. However, despite
their high biological activity, these conjugates remain
difficult to synthesize and purify, which hinders their
further use. The high-molecular-weight nature of the
initial polyamines implies formation of a mixture of
PS molecules with different molecular weights, which
significantly limits the prospects of their clinical ap-
plication. In this study, we introduced a low-molecu-
lar-weight branched polyamine into the chlorin mole-
cule and investigated the photoinduced antimicrobial
toxicity of the resulting derivate.
MATERIALS AND METHODS
Equipment and materials. Chlorin e
6
trimethyl
ester (5) was obtained from pheophorbide A methyl
ester according to a previously described procedure
[17]. Oxidation of chlorin e
6
trimethyl ester vinyl
group to produce carboxyl derivative 6 was per-
formed as described in [18]. Organic solvents were
purified and prepared according to standard proce-
dures. Kieselgel 40/60 silica gel and Kieselgel 60 silica
gel (Merck, Germany) were used for column chroma-
tography and preparative thin-layer chromatography
(TLC), respectively. Analytical TLC was performed on
Kieselgel 60 F
245
plates (Merck). Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker
DPX300 spectrometer in CDCl
3
. Residual signals of
1
H
nuclei were used to calibrate the scale. The experi-
ments were performed according to standard Bruker
procedures. High-resolution mass spectra were record-
ed on an Orbitrap Elite mass spectrometer (Thermo
Scientific, USA). Absorption spectra were recorded
on a Shimadzu UV1800 UV/VIS spectrophotometer in
CH
2
Cl
2
. To measure the quantum yield of singlet oxy-
gen generation, we used 5,6,11,12-tetraphenyltetracene
as a trap and red light-emitting diode (LED) as a light
source (λ  =  660  ±  20nm, Ps  =  5  mW/cm
2
) as described
in [19]. For biological studies, LED-based irradiator
(λ  =  660  ±  10  nm, Ps  =  10  mW/cm
2
) was used as a
source of red light.
Synthesis of polyamine 3. tert-Butyl (2-amino-
ethyl)carbamate (2) (1.246 g; 7.777 mmol) was dis-
solved in tetrahydrofuran (THF) (60 ml) under an
argon atmosphere. Ethyl 3-bromo-2-(bromomethyl)
propanoate (1) (532 mg; 1.942 mmol) in THF (18 ml)
was added dropwise to the resulting solution over
6 min with vigorous stirring. The reaction mixture
was stirred for 12 h at room temperature in an in-
ert atmosphere, while the reaction was monitored by
analytical TLC [Rf  =  0.30; Hex (hexane) /EtOAc (ethyl
acetate) as an eluent, 2/1, v/v]. Next, di-tert-butyl di-
carbonate (1.693 g; 7.768 mmol) was added to the
reaction mixture at 0°C. The mixture was stirred for
another 12  h at room temperature under an inert
atmosphere. The solvent was removed in  vacuo. The
desired product3 was isolated by column chromatog-
raphy (isocratic elution; Hex/EtOAc, 2/1, v/v). As a re-
sult, 128 mg of product3 was obtained with a yield of
10.6%.
1
H NMR (300 MHz, CDCl
3
, δ, ppm): 5.08 (2H, m,
2xNH); 4.14-4.07 (2H, q, OCH
2
CH
3
); 3.35-3.09 (13H, m,
CH, 2xCHCH
2
N, 2xNCH
2
CH
2
NH, 2xNCH
2
CH
2
NH); 1.44-
1.41 (36H, m, 12xCH
3
); 1.25-1.21 (3H, t, OCH
2
CH
3
).
13
C  NMR (75 MHz, CDCl
3
, δ, ppm): 173.27; 155.88-
155.84; 129.39; 80.30; 79.04; 68.36; 60.88; 47.94; 44.79-
44.75; 39.34; 30.79-27.68; 22.10; 14.03. ESI-HRMS m/z
calculated [M+H]
+
: 633.4075; found: 633.4050; calculat-
ed [M+Na]
+
: 655.3894; found: 655.3865.
Synthesis of polyamine 4. Compound 3 (64 mg;
0.104 mmol) was dissolved in a mixture of THF
(0.83ml) and methanol (0.83 ml). Next, potassium hy-
droxide (113.7 mg; 2.026 mmol) dissolved in distilled
NOVEL CHLORIN WITH BRANCHED POLYAMINE 977
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
water (0.83 ml) was added to the resulting solution.
The reaction mixture was stirred at room temperature
for 4h, while the reaction was monitored by analytical
TLC [Rf = 0.15; eluent, EtOAc/Ethanol, 9/1, v/v]. After
complete conversion of the starting reagent, the mix-
ture was diluted with distilled water (5 ml) and citrate
buffer (3  ml, pH  5). The product was isolated from
the reaction mixture by extraction with ethyl acetate
(50  ml). The organic layer was dried over anhydrous
Na
2
SO
4
and evaporated in  vacuo. As a result, 41  mg
of compound4 was obtained (yield, 65.1%). ESI-HRMS
m/z calculated [M+H]
+
: 605.3762; found: 605.3763; cal-
culated [M+Na]
+
: 627.3581; found: 627.3571.
Synthesis of chlorin 7. 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) solution (71.75  mg; 0.189  mmol) and triethyl-
amine (26.3  μl) in N,N-dimethylformamide (DMF) (5  ml)
were added to derivative 6 (113  mg; 0.172 mmol).
The reaction mixture was stirred under argon atmo-
sphere at room temperature for 60 minutes, after
which a solution of Boc-ethylenediamine 2 (30.3 mg;
0.189 mmol) in DMF (1 ml) was added to the mix-
ture. After 15 min, the reaction mixture was evapo-
rated under reduced pressure, and the residue was
redissolved in CH
2
Cl
2
(50 ml) and washed with water
(2×100 ml). The organic layer was dried over Na
2
SO
4
and evaporated invacuo. The product was isolated by
preparative TLC (CH
2
Cl
2
/MeOH (methanol), 20/1, v/v)
resulting in 135 mg of compound 7 with a yield of
98%.
1
H NMR (300 MHz, CDCl
3
, δ, ppm): 9.80 (H, s,
10-H); 9.63 (H, s, 5-H); 8.81 (H, s, 20-H); 7.20 (H, m,
3
2
-NH); 5.36-5.21 (2H, q, J  =  19.6 Hz,15
1
-CH
2
a
, 15
1
-CH
2
b
);
5.21 (H, m, 3
5
-NH); 4.53-4.40 (2H, m, 18-H, 17-H); 4.28
(3H, s, 13
1
-COOCH
3
); 3.87 (2H, m, 8
1
-CH
2
a
, 8
1
-CH
2
b
);
3.78 (3H, s, 15
2
-COOCH
3
); 3.65 (3H, s, 17
3
-COOCH
3
); 3.65-
3.56 (2H, m, 3
3
-CH
2
); 3.65-3.56 (2H, m, 3
4
-CH
2
); 3.56 (3H,
s, 12-CH
3
); 3.22 (3H, s, 7-CH
3
); 2.65-2.51 (2H, m, 17
2
-CH
2
a
,
17
2
-CH
2
b
); 2.28-2.16 (2H, m, 17
1
-CH
2
a
, 17
1
-CH
2
b
); 1.74
(3H, d, J  =  Hz, 18-CH
3
); 1.71 (3H, t, J  =  Hz, 8
2
-CH
3
); 1.37
(9H, s, 3
6
-C(CH
3
)
3
); -1.49 (H, I-NH); -1.80 (H, III-NH).
ESI-HRMS m/z calculated [M+H]
+
: 798.400; found:
798.400. UV/VIS (CH
2
Cl
2
) λ
max
, nm (ε, M
−1
 cm
−1
) 400
(121000); 501 (11220); 531 (4700); 614 (4560); 668 (43900).
Synthesis of chlorin 8. Compound 7 (47 mg;
0.059  mmol) was dissolved in freshly prepared 10%
CF
3
COOH in CH
2
Cl
2
; the reaction mixture was stirred
under argon atmosphere for 30min, then diluted with
CH
2
Cl
2
(50 ml) and washed with 5% Na
2
CO
3
solution
(2×100 ml). The organic layer was dried over Na
2
SO
4
and evaporated in  vacuo. The residue was redissolved
in DMF (1.5 ml), and the resulting solution was add-
ed to a solution of compound 4 (41  mg; 0.068  mmol)
with HBTU (28  mg; 0.074  mmol) and triethylamine
(TEA) (7.6  mg; 0.075  mmol) in DMF (2 ml). The re-
action mixture was stirred under argon atmosphere
for 16 h. After complete conversion of compound 4,
the reaction mixture was evaporated in vacuo; the
residue was redissolved in CH
2
Cl
2
and washed with
distilled water (4×100  ml). The organic layer was
dried over Na
2
SO
4
and evaporated in vacuo. The tar-
get compound 8 was purified by preparative TLC in
CH
2
Cl
2
/MeOH (45/1, v/v). Yield, 13 mg; 17.1%.
1
H  NMR
(300 MHz, CDCl
3
, δ, ppm): 9.40 (H, s, 10-H); 9.36 (H,
s, 5-H); 8.79 (H, s, 20-H); 6.97 (H, m, 3
2
-NH); 6.56 (H,
m, 3
5
-NH); 5.44-5.27 (2H, q, J  =  18.7 Hz, 15
1
-CH
2
a
,
15
1
-CH
2
b
); 4.50-4.43 (2H, m, 18-H, 17-H); 4.29 (3H,
s, 13
1
-COOCH
3
); 3.81 (3H, s,15
2
-COOCH
3
); 3.67 (3H, s,
17
3
-COOCH
3
); 3.45 (3H, s, 12-CH
3
); 3.31 (3H, s, 7-CH
3
);
2.64-2.50 (2H, m, 17
2
-CH
2
a
, 17
2
-CH
2
b
); 2.26-2.15 (2H, m,
17
1
-CH
2
a
, 17
1
-CH
2
b
); 1.79 (3H, d, J  =  7.8Hz, 18-CH
3
); 1.79
(3H, t, J  =  8.6Hz, 8
2
-CH
3
); 1.44 (36H, s, Boc). ESI-HRMS
m/z calculated [M+H]
+
: 1284.7006; found: 1284.7059.
UV/VIS (CH
2
Cl
2
) λ
max
, nm (ε, M
−1
 cm
−1
) 401 (122000);
501 (11240); 530 (4700); 612 (4500); 667 (43840).
Synthesis of conjugate 9. Compound 8 (10 mg;
0.0078 mmol) was dissolved in freshly prepared 50%
CF
3
COOH in CH
2
Cl
2
, and the reaction mixture was
stirred under argon atmosphere for 60 min. The re-
action progress was monitored by analytical TLC in
the CH
2
Cl
2
/MeOH mixture (20  :  1, v/v). The reaction
mixture was diluted with CH
2
Cl
2
(50 ml) and washed
with 10% Na
2
CO
3
solution (2×100ml). The organic lay-
er was dried over Na
2
SO
4
and evaporated in vacuo
to obtain product 9 (5 mg; yield, 72.5%). ESI-HRMS
m/z calculated [M+H]
+
: 884.4909; found: 884.4898.
UV/VIS (CH
2
Cl
2
) λ
max
, nm (ε, M
−1
cm
−1
): 401 (121300);
501 (11250); 534 (4730); 613 (4540); 668 (43900).
Photoinduced toxicity assay. To study the anti-
microbial activity, the following reference strains of
gram-positive and gram-negative bacteria were used:
Staphylococcus aureus ATCC 25923, Enterococcus faeca-
lis ATCC 29212, Pseudomonas aeruginosa ATCC 27853,
and Escherichia coli ATCC 25922. The initial concen-
tration of the microbial suspension in the experiments
was 3×10
3
CFU/ml. Cells in the positive control groups
were kept under identical conditions and exposed to
light but without preincubation with the PS. Cells in
the negative control groups were neither exposed to
light nor incubated with the PS. Microbial suspension
was added to the wells of a microplate (150  μl per
well), and solutions of compounds5 and 9 were added
so that the final PS concentration reached 0.44, 1.32,
and 3.96 μM, respectively . The cells were incubat-
ed with PSs for 30 min and then irradiated with red
light (λ  =  660  nm; P
s
  =  10  mW/cm
2
) until a light dose
of 10J/cm
2
was achieved. After irradiation, the micro-
plates were incubated for 24 h at 37°C. To determine
microbial survival rate, bacterial cells were seeded on
a dense agar medium with a 2-μL bacteriological loop.
After 24-h incubation, the number of grown colonies
was determined. The experiments were carried out
in triplicate.
SUVOROV et al.978
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
RESULTS AND DISCUSSION
As the initial PS molecule, we selected chlorin e
6
trimethyl ether, which is a derivative of natural chlo-
rophyll A. Drugs based on this class of compounds
have proven to be effective as PSs in photodynamic
therapy and are actively used in clinical practice [20-
24]. Using the information on how the position of a
substituent affects the biological activity [25-27], we
introduced the polyamine residue into pyrrole A of
the chlorin macrocycle. To synthesize the chlorin con-
jugate with the polyamine, we acquired a branched
tetraamine and chemically modified the vinyl group
of the chlorin macrocycle.
As a polyamine for conjugation with chlorin, we
selected symmetrical N,N′-bis(2-aminoethyl)-1,3-pro-
panediamine containing a carboxyl group in posi-
tion2. The synthesis of this compound from ethylene-
diamine and 3-bromo-2-(bromomethyl)propanoic acid
was described in earlier papers [28], but we opted
for an alternative method in order to simplify puri-
fication of the target compound, increase its purity,
and reduce formation of transalkylation by-products.
The starting compound for the polyamine syn-
thesis was ethyl 3-bromo-2-(bromomethyl)propa-
noate (1) obtained from diethyl malonate according
to a previously described method [29, 30]. As a result
of 1-[(tert-butoxycarbonyl)amino]-2-aminoethane (2)
alkylation with dibromo derivative 1 and subsequent
treatment of the reaction mixture with di-tert-butyl
dicarbonate, branched polyamine 3 was obtained
(Scheme 1). To attach the latter to chlorin, the es-
ter group was hydrolyzed under alkaline conditions
to obtain derivative 4. The structure of the resulting
tetraamine was confirmed by high-performance liq-
uid chromatography coupled with mass spectrometry
(HPLC/MS). A molecular ion corresponding to this
structure was found in the high-resolution mass spec-
trum of compound 4.
Ethylenediamine was used as a linker to intro-
duce polyamine 4 into chlorin molecule (Scheme 2).
The amidation of chlorin 6 with 1-[(tert-butoxycar-
bonyl)amino]-2-aminoethane 2 was carried out using
HBTU to obtain the target compound 7 with a high
yield. The transformations of substituents in pyrrole
A were accompanied by noticeable changes in the
Scheme  1. Reagents and conditions: (i) TEA, THF, 24 h; (ii) Boc
2
O, 1 h, 5°C then 12 h, 20°C; (iii) KOH, H
2
O/THF/MeOH, 4  h,
20°C; (iv) H
2
O, citrate buffer (pH 5), 20°C.
Scheme  2. Reagents and conditions: (i) OsO
4
, THF, NaIO
4
/H
2
O, Ar, 0.75  h, 20°C; (ii) THF, NH
2
SO
2
OH, dimethyl sulfoxide
(DMSO), NaClO
2
/H
2
O, Ar 0.5  h, 20°C; (iii) HBTU, TEA, DMF, Ar, then 2, 0.3  h, 20°C; (iv) 10% CF
3
COOH/CH
2
Cl
2
, Ar, 0.5  h, 20°C;
(v) DMF, 4, HBTU, TEA, Ar, 12  h, 20°C; (vi) 50% CF
3
COOH/CH
2
Cl
2
, Ar, 0.5 h, 20°C, then Na
2
CO
3
/H
2
O.
NOVEL CHLORIN WITH BRANCHED POLYAMINE 979
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 1. UV/VIS spectra of compounds 5-7 and conjugate 9 in CH
2
Cl
2
.
spectral properties of chlorin e
6
derivatives. Thus, ox-
idation of the chlorin vinyl group to a formyl group
was accompanied by a bathochromic shift by 30 nm
(up to 693 nm) of the maximum in the electronic ab-
sorption spectrum. Subsequent oxidation to carboxylic
acid 6 was accompanied by a hypsochromic shift of
the absorption maximum by 10 nm, and then by an-
other shift by 15 nm during amidation (Scheme 1).
After removal of the protecting Boc-group in com-
pound7, it was coupled with the tetraamine moiety4
using HBTU to produce conjugate 8. The structure of
this compound was confirmed by NMR spectroscopy.
The
1
HNMR spectrum of the conjugate contained sig-
nals from the polyamine moiety (protons of CH, CH
2
-
groups in the region of 3.4-3.1 ppm; Boc – 1.45 ppm)
and amide bonds (6.98 and 6.57 ppm), as well as a
set of signals characteristic of the chlorin macrocycle.
The shift of the signal of the meso-proton of chlorin
in position 5 of the macrocycle to a stronger field also
indicated successful conjugation. A molecular ion cor-
responding to the calculated value was detected in the
high-resolution mass spectrum of compound 8.
To remove the protecting Boc-groups, compound8
was treated with a 50% solution of trifluoroacetic acid
in dichloromethane, followed by washing the reac-
tion mixture with a sodium carbonate solution and
isolation of chlorin 9 as a free amine. A signal corre-
sponding to the calculated value for the divalent ion
was detected in the high-resolution mass spectrum of
compound 9. Therefore, we have successfully intro-
duced a branched tetraamine fragment into pyrroleA
of chlorin e
6
trimethyl ester.
Studying the photophysical properties of the ob-
tained compound showed that the introduction of the
tetraamine residue into pyrrole A led to an insignifi-
cant bathochromic shift of the long-wave absorption
maximum from 664  nm (original chlorin e
6
trimeth-
yl ester) to 669  nm (Fig.  1). The quantum yield of
the singlet oxygen generation was measured using
5,6,11,12-tetraphenyltetracene as a trap according to
a previously described method [19]. Modification of
position 3 led to a slight decrease in this parameter
(Table 1). The lack of obvious differences in the pho-
tophysical parameters of PS molecules 5 and 9 allowed
us to evaluate the effect of the polyamine fragment on
the compound internalization into pathogenic bacteri-
al cells in  vitro.
The photoinduced antimicrobial cytotoxicity of
the obtained chlorin 9 was assessed against a number
of gram-positive and gram-negative bacteria (S.  au-
reus, E.  coli, E.  faecalis, P.  aeruginosa) (Fig.  2) using
original chlorin e
6
trimethyl ester (5) for comparison.
The water- soluble forms of the PSs were obtained as
emulsions using Kolliphor ELP surfactant according
to the method previously described by our research
group [31]. Bacterial cells were incubated for 30 min
with the PS solutions at the concentrations of 0.44, 1.32,
and 3.96  μM. To assess the photocytotoxic effect, the
cells were irradiated with red light (λ  =  660  ±  10  nm,
P
s
  =  10  mW/cm
2
) until a light dose of 10  J/cm
2
was
achieved. Bacterial cells that were irradiated in the
absence of PS were used as a control group.
Table 1. Photophysical parameters of the obtained
conjugate 9 and original chlorin e6 trimethyl ester (5)
Compound
Absorption
maximum, λ, nm
Singlet oxygen
quantum yield
5 664 0.55 ± 0.01
9 669 0.49 ± 0.01
SUVOROV et al.980
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 2. Photoinactivation of gram-positive S.aureus and E.faecalis (a, c) and gram-negative P. aeruginosa and E.coli (b, d)
using photosensitizers 5 and 9.
No suppression of bacterial growth was detected
upon irradiation with red light in the absence of PS,
while exposed to light of cells that had been prein-
cubated with compounds 5 or 9 resulted in the con-
centration-dependent inhibition of bacterial growth.
Tetraamine-modified chlorin 9 exhibited greater pho-
toinduced cytotoxicity than the original compound  5
(Fig.  2), which was evidenced by the fact that the com-
pound 9 at a concentration of 3.96  μM caused stable
photoinactivation of gram-positive bacteria S.  aureus
and E.  faecalis. Virtually no photoinactivation of the
gram-negative bacterium P.  aeruginosa was found for
PS 5 at the concentrations of 0.44  μM and 1.32  μM,
whereas the photosensitizing effect of PS 9 on this
strain was an order of magnitude greater over the
entire range of the studied concentrations. The bac-
teriostatic effect of compounds 5 and 9 toward light-
irradiated E.  coli was similar. It should be noted
that in the studied concentration range, the efficacy
of both compounds against gram-negative bacteria
was lower than against gram-positive ones. This is
consistent with previous studies, in which inactiva-
tion of gram-negative bacteria required higher PS
concentrations and higher light doses [16, 32]. Nev-
ertheless, our findings suggest that the introduction
of low-molecular-weight branched tetraamine into
the chlorin molecule increases its affinity to bacterial
cell wall.
CONCLUSION
In this study, we synthesized a new PS contain-
ing a branched polyamine residue in the pyrrole A
of chlorin macrocycle and examined its photoin-
duced antimicrobial activity. The introduction of a
polyamine structural fragment into the chlorin mol-
ecule facilitated its internalization into pathogenic
cells and, therefore, consequently enhanced the pho-
toinduced cytotoxicity of the resulting compound.
NOVEL CHLORIN WITH BRANCHED POLYAMINE 981
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
The properties of the photosensitizer can presumably
be improved by quaternization of polyamine nitrogen
atoms to produce alkylammonium salts. We plan to
test this hypothesis in future studies.
Abbreviations. APDT, antimicrobial photody-
namic therapy; Boc,tert-butoxycarbonyl; DMF,N,N-di-
methylformamide; ESI, electrospray ionization;
HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate; HRMS, high-resolution
mass spectrometry; NMR,nuclear magnetic resonance;
TLC,thin-layer chromatography.
Acknowledgments. This work was performed
using the equipment of the Shared Science and
Training Center for Collective Use (RTU MIREA)
and supported by the Ministry of Science and High-
er Education of the Russian Federation within the
framework of agreement no. 075-15-2025-548 from
18.06.2025.
Contributions. N.V.S., M.A.G., and Yu.L.V. devel-
oped the concept and supervised the study; G.M.G.,
E.A.S., M.A.S., D.A.M., and V.V.S. performed the experi-
ments; N.V.S., M.A.S., and M.A.G. discussed the results;
N.V.S. wrote the draft of the article; N.V.K. and D.A.M
edited the manuscript.
Funding. The work was supported by the Min-
istry of Science and Higher Education of the Russian
Federation (Government Assignment 075-00727-25-05
from 20.03.2025; FSFZ-2024-0013).
Ethics approval and consent to participate.
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
Open access. This article is licensed under a Cre-
ative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution,
and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s)
and the source, provide a link to the Creative Com-
mons license, and indicate if changes were made. The
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use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license,
visit http://creativecommons.org/licenses/by/4.0/.
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