A porphycene-gentamicin conjugate for enhanced photodynamic inactivation of bacteria

Article abstract of DOI:10.1016/j.bioorg.2020.103661

A novel photoantimicrobial agent, namely 2-aminothiazolo[4,5-c]-2,7,12,17-tetrakis(methoxyethyl)porphycene (ATAZTMPo-gentamicin) conjugate, has been prepared by a click reaction between the red-light absorbing 9-isothiocyanate-2,7,12,17-tetrakis(methoxyethyl)porphycene (9-ITMPo) and the antibiotic gentamicin. The conjugate exhibits submicromolar activity in vitro against both Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli, respectively) upon exposure to red light and is devoid of any cytotoxicity in the dark. The conjugate outperforms the two components delivered separately, which may be used to enhance the therapeutic index of gentamicin, broaden the spectrum of pathogens against which it is effective and reduce its side effects. Additionally, we report a novel straightforward synthesis of 2,7,12,17-tetrakis(methoxyethyl) porphycene (TMPo) that decreases the number of steps from nine to six.

Full text of DOI:10.1016/j.bioorg.2020.103661

Journal Pre-proofs
A porphycene-gentamicin conjugate for enhanced photodynamic inactivation
of bacteria
Ingrid Nieves, Cormac Hally, Cristiano Viappiani, Montserrat Agut, Santi
Nonell
PII:
S0045-2068(19)31428-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103661
YBIOO 103661
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 October 2019
8 February 2020
10 February 2020
Please cite this article as: I. Nieves, C. Hally, C. Viappiani, M. Agut, S. Nonell, A porphycene-gentamicin
conjugate for enhanced photodynamic inactivation of bacteria, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.103661
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A porphycene-gentamicin conjugate for enhanced photodynamic inactivation of bacteria
Ingrid Nieves,a Cormac Hally,a,b Cristiano Viappiani,b Montserrat Aguta and Santi Nonell*,a
a Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017, Barcelona, Spain
b Dipartamento di Scienze Matematiche, Fisiche e Informatiche, Università di Parma, Parco Area delle Scienze 7A, 43124,
Parma, Italy
* Corresponding author: santi.nonell@iqs.url.edu
Abstract
A
novel
photoantimicrobial
agent,
namely
2-aminothiazolo[4,5-c]-2,7,12,17-
tetrakis(methoxyethyl)porphycene (ATAZTMPo-gentamicin), conjugate has been prepared by a click
reaction between the red-light absorbing 9-isothiocyanate-2,7,12,17-tetrakis(methoxyethyl)porphycene
(9-ITMPo) and the antibiotic gentamicin. The conjugate exhibits submicromolar activity in vitro
against both Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli,
respectively) upon exposure to red light and is devoid of any cytotoxicity in the dark. The conjugate
outperforms the two components delivered separately, which may be used to enhance the therapeutic
index of gentamicin, broaden the spectrum of pathogens against which it is effective and reduce its
side
effects.
Additionally,
we
report
a
novel
straightforward
synthesis
of
2,7,12,17-tetrakis(methoxyethyl) porphycene (TMPo) that decreases the number of steps from nine to
six.
Keywords: antimicrobial resistance; photodynamic therapy; porphycene; singlet oxygen;
photosensitizer conjugates; amphiphilicity.

1. Introduction
Antimicrobial resistance (AMR) is an increasingly serious threat to the gains made in health and
development.1 It has been estimated that in the EU in 2015 about 33,000 people died from infectious
diseases caused by multidrug-resistant bacteria.2 Current antibiotics are often insufficiently because of
AMR, so, alternative anti-infective approaches are urgently needed and do not cause resistance
themselves. Antimicrobial photodynamic therapy (aPDT) is a potential alternative treatment that is
currently gaining a lot of attention due to its broad spectrum of action, its multitarget mechanism of
action, its efficiency against antibiotic-resistant strains, and its very low potential for eliciting
resistance by itself.3 aPDT is a local treatment based on the generation of reactive oxygen species
(ROS), particularly singlet oxygen (1O2),4,5 upon exposure of a photoantimicrobial drug, referred to as
the photosensitizer (PS), to light of a suitable wavelength. In the presence of oxygen, 1O2 or other ROS
are generated and cause oxidative damage to a large number of cell components, leading to cell death.
Damage to the host tissue is minimized by exposing the lesion to light very soon after PS delivery,
before it can be uptaken by the host tissue.6 Although aPDT is effective against both Gram-positive
and Gram-negative microorganisms, photoinactivation of the latter requires, in general, the use of
chemical agents to permeabilise their more complex outer wall, e.g., Tris/EDTA or polycationic
agents, as well as cationic PSs that bind to the negatively-charged residues on the outer wall.7,8
Among the PSs being currently scrutinized for aPDT applications, porphycenes stand out as excellent
candidates owing to their broad and strong absorption and fluorescence in the red/near-IR spectral
1
region, where tissue is most permeable to light (600–900 nm), and to a high O2 photoproduction
efficiency.3 Porphycenes have been mostly applied to photodynamic therapy of cancer and only
recently have begun to be explored for aPDT applications.4 In the cancer field, Richert and co-workers
described that 2,7,12,17-tetrakis(methoxyethyl)porphycene (TMPo), showed excellent fluorescence
and photosenzitising properties, adequate solubility in biological media, efficient uptake by cancer
cells, and very promising photodynamic activity in mice assays.10 Nevertheless, antimicrobial
photoinactivation studies performed with TMPo revealed the limitations imposed by a lack of
hydrophilicity and positive charges, thus requiring either solubilization agents such as Pluronic

F-12711 or coupling of the porphycene core with cationic polylysine moieties12,13 for effective delivery
to bacteria.
A current trend to tackle multi-drug resistant bacterial infections is to administer a combination of
antibiotics14 that target different cellular components. From this perspective, the combination of aPDT
with antibiotics seems ideally suited to overcome the majority of resistance mechanisms owing to the
multitarget nature of aPDT, and has the added benefit that those bacteria that might survive the
photodynamic insult will still be exposed to the antibiotic, precluding their regrowth.15 Early studies
combining aPDT with antibiotics have observed promising effects ranging from additive to
synergistic.16–20 Of particular interest is the approach by Cahan et al. who have linked covalently the
PS Rose Bengal to the antibiotics 6-aminopenicillanic acid and kanamycin.16 We pursued a similar
approach linking antimicrobial peptides to a porphycene and a series of porphyrins.21,22
In this work, we have linked the aminoglycoside antibiotic gentamicin to the porphycene TMPo.
Gentamicin is a well-established antibiotic, effective against Gram-negative bacteria but with limited
activity against Gram-positive microorganisms. However, many bacteria have developed resistance to
this antibiotic23 and there is still no selective method for combating this resistance nor the nephrotoxic
and ototoxic side effects.24 Since gentamicin contains residues that will be protonated at physiological
pH, conjugation of gentamicin to TMPo is expected to enhance the pharmacological and
photoantimicrobial activity of TMPo due to a higher aqueous solubility and higher affinity for the
negatively-charged outer cell wall of Gram-negative bacteria..25 On the other hand, conjugation may
also lead to a reduction in the amount of gentamicin required for treating pathogenic bacterial
infections and hence its side effects in adults.24
Herein, we present a new straightforward synthetic route for the TMPo scaffold, the synthesis and
photophysical characterisation of the novel ATAZTMPo-gentamicin conjugate, and we explore its
potential as a photoantimicrobial agent for the photoinactivation of Gram-positive and Gram-negative
bacteria.

2. Results
2.1 Synthesis
Synthesis of TMPo
The synthesis of TMPo was first reported by Vogel and co-workers, requiring nine synthetic steps.10,26
This classical approach consisted in the formation of a -pyrrole by means of Fischer methodology or
Knorr condensation, followed by two coupling reactions, namely an Ullmann coupling, which
afforded the 2,2’-bipyrrole moiety, and a subsequent McMurry coupling. Nevertheless, Vogel’s route
and other recent alternative approaches27,28 for the preparation of porphycenes are associated with
harsh reaction conditions, poor regioselectivity, poor yields, and a large number of steps.
As part of our continuous efforts to simplify the synthesis of porphycenes, this work describes a novel
three-step synthetic strategy for the preparation of gram quantities of alkoxyalkyl-2,2’-bipyrroles
and apply it to the synthesis of TMPo, decreasing from nine to six the number of steps required.
The synthesis of the '-alkoxyalkyl-2,2’-bipyrrole 4 (Figure 1) was an extension of our previously
reported one-pot access to 'aryl 2,2’-bipyrroles,29 by a tandem reaction based on the oxidative
dimerization of a 2-(trimethylstannyl) pyrrole generated in situ by a van Leusen-type methodology,
which is unprecedented in the literature for alkoxyalkyl substituents. For this purpose, the
commercially available 3-methoxypropanol 1 was chosen as starting material. Gratifyingly, oxidation
of 1 in the presence of the mild oxidant 2-iodoxybenzoic acid (IBX)30 led to the corresponding
aldehyde 2 in excellent yield (85%), in contrast to other common oxidants reported in literature, such
as pyridinium chlorochromate (37%).31 The aldehyde 2 was then elongated by Horner-Wadsworth-
Emmons (HWE) olefination to give the α,β-unsaturated ester 3 as a mixture of isomers (E:Z; 98:2).
The stereochemical assignment of the new double bond was made by comparison of the coupling
constants. Then, ethyl 5-methoxypent-2-enoate 3 was subjected to the pyrrole [3+2] ring-forming
process, in the presence of tosylmethyl isocyanide (TosMIC) and trimethyltin chloride under basic
conditions, leading to the intermediate 2-(trimethylstannyl)pyrrole. Subsequent addition of copper (II)
nitrate afforded the desired 2,2’-bipyrrole 4 in a poor yield. The reaction yield could be improved by

fine tuning the reaction stoichiometry. Specifically, optimum yields were obtained when TosMIC was
increased from 1 to 1.5 equiv., and copper (II) nitrate was added stepwise (1 equiv. per 40 min). Under
those conditions, the first 4,4’-alkoxyalkyl functionalised 2,2’-bipyrrole 4 was obtained in 25% yield,
which is in excellent agreement with those described for alkoxyaryl bipyrroles (25% for 4-methoxy-
and 27% for 3,5-dimethoxy bipyrroles).29
Figure 1 Synthesis of 2,2’bipyrrole 4. Reagents and conditions: (a) IBX, EtOAc, refluxed, 12h, 85%; (b) Triethyl
phosphonoacetate, NaH, THF, 0 ºC, 2 h, 75% (E:Z;98:2); (c) i) TosMIC, BuLi, Me3SnCl, THF, -78 ºC to RT, 2 h; ii)
Cu(NO3)2·3H2O; RT, 2 h, 25%.
With 2,2’-bipyrrole 4 in hand, we examined the stability of the methoxyethyl group under a direct
saponification and decarboxylation reaction (Figure 2). Thus, the use of sodium hydroxide in ethylene
glycol32 provided successfully the α-free bipyrrole 5 in just one step, which was subjected to next
reaction without further purification due to its tendency to polymerise. Finally, conventional
Vilsmeier-Haack formylation, followed by McMurry reductive cyclisation afforded the porphycene
TMPo in 25% yield, which reproduces that reported by Richert et al.10
Figure 2 Synthesis of TMPo. Reagents and conditions: (a) NaOH, ethylene glycol, 180 ºC, 1.5 h, 85%; (b) i) POCl3, DMF,
60 ºC, 1 h; ii) NaOAc, H2O, 85 ºC, 1 h, 80%; (c) i) TiCl4, Zn, Cu2Cl2, THF, refluxed, 5 h; ii) K2CO3, H2O, 0 ºC to RT, 1 h,
25%.

Synthesis of ATAZTMPo-gentamicin conjugate
TMPo has been functionalised at 9-position with an isothiocyanate group (9-ITMPo). 9-
Isothiocyanateporphycenes undergo a click reaction with amino-substituted compounds yielding a
thiourea that spontaneously evolves to a near-IR absorbing 2-aminothiazolo[4,5-c]porphycene
(ATAZPo).33,34 We hypothesized that this would be the case also when 9-ITMPo would be allowed to
react with the less-hindered primary amino-group of gentamicin, enabling conjugation through a one-
step reaction.
As depicted in Figure 3, the synthesis of 9-ITMPo was envisaged by using the methodology developed
by Vogel and co-workers, consisting in a mono-nitration of TMPo at the meso position (9-NTMPo)
and subsequent Zinin reduction of the nitro group to the amine (9-ATMPo).35 The resulting 9-ATMPo
was subjected to 1,1′-thiocarbonyldi-2(1H)-pyridone33 in DCM, which afforded the corresponding 9-
ITMPo in 80% yield over three-steps.
Figure 3 Synthesis of 9-ITMPo. Reagents and conditions: (a) AgNO3, acetic acid glacial : DCM (10:7), 55 ºC, 20 min,
100%; (b) NaOH, Na2S2O4, DMC, reflux, 1 h, 95%; (c) 1,1′-thiocarbonyldi-2(1H)-pyridone, DCM, RT, 16 h, 80%.
Gentamicin sulphate is a mixture of several congeners, in which gentamicin C1, C1a, and C2 are the
three major components of the complex (Figure 4, A). Despite all these distinct forms, we aimed to
single couple the less hindered primary C6’-amino group of gentamicin’s purpurosamine moiety with
9-ITMPo, in order to alter as little as possible the pharmacokinetic properties of the antibiotic.36 For
this purpose, a high molar excess of the aminoglycoside was added to the reaction mixture, and basic
pH was adjusted with potassium carbonate powder to ensure the basic state of the primary amino-
groups.37 The reaction conversion could be monitored visually due to the distinct colour progression
that the 18-electron system of 9-ITMPo (dark-green) suffers when it transformed into the

corresponding thiourea-intermediate (dark-blue) and it subsequently evolved to the 22 aromatic
thiazolo-conjugate ATAZTMPo-gentamicin (light green) (Figure 4, B).
Figure 4 [A] Chemical structures of gentamicin C1, C1a, and C2; [B] Click reaction-mediated conjugation of antibiotic
gentamicin. Reagents and conditions: (a) gentamicin, K2CO3, ethanol absolute, RT, 5 days, 80%.
The mono-conjugated ATAZTMPo-gentamicin was then purified from the antibiotic excess, and from
undesired porphycene by-products, by flash column chromatography on C18-reversed phase (RP)
silica-gel (see Experimental Section for details). Subsequent RP-HPLC analytical characterisation of
the pure fraction was challenging since retention of the conjugate in RP-HPLC was largely observed
due to its intrinsic amphiphilic character. Gratifyingly, after carefully tuning the elution conditions, the
desired compound was detected showing a chromatographic profile similar to those reported by
Woiwode et al. for gentamicin-Texas red probe as a mixture of species of the aminoglycoside (Figure
S1 and S2).37 Finally, the m/z mass values of the eluted peaks at retention time around 3.4 min
confirmed the single-conjugate for the most abundant charge state of z = 1 and z = 2.37,38
2.2 Photophysical characterisation
The photophysical properties of 9-ITMPo and ATAZTMPo-gentamicin in methanol are summarised
in Table 1. As depicted in Figure 5, the absorption spectra of 9-ITMPo and ATAZTMPo-gentamicin
show the characteristic Soret and Q bands of asymmetric porphycenes in methanol.39 Additionally, the
cyclised conjugate is endowed with red-shifted absorption and fluorescence spectra in comparison
with its 18- precursor as a result of the expanded 22-electronic delocalisation system. The observed
spectral changes of ATAZTMPo-gentamicin correlate with the results described by Planas and co-

workers for fused tetraphenylporphycenes,33 in which absorbance of the compounds shifted to the
near-IR, where tissues are most transparent to light and present less autofluorescence.40
Figure 5 (A,C) Absorption and (B,D) fluorescence spectra of 9-ITMPo (A,B) and ATAZTMPo-gentamicin (C,D) in
methanol respectively. Insets: (A,C) Excitation spectra of the fluorescence observing at 640 and 715 nm when indicated.
(B,D) Time-resolved fluorescence of either compound exciting at 596 nm. Signal, fit and instrument response function (IRF)
at 640 and 715 nm (exc= 596 nm).
In sharp contrast, the fluorescence spectrum of the conjugate exhibits two emission bands compared to
the single emission of the precursor 9-ITMPo. Further details were obtained from time-resolved
fluorescence analysis. While 9-ITMPo fluorescence decays with monoexponential kinetics (τs = 3.4 ns;
Table 1), ATAZTMPo-gentamicin decays with a biexponential kinetics indicating two different
populations of the excited singlet state of the PS. The fluorescence decay kinetics presented one major
component (73%, 22-electron system) which decays much faster, τ1 = 1.9 ns, in contrast to the minor
excited-state specie with lifetime τ2 = 6.1 ns (27%, 18-electron system). These values were obtained
by deconvolution of the spectrum of time resolved emission spectra (Figure S3). Laser flash photolysis
determined triplet lifetimes of either compounds to be in tens of microseconds range in argon-
saturated methanol (Figure S4).

Table 1 Summary of optical and photophysical properties of 9-ITMPo and ATAZTMPo-gentamicin in MeOH
c
d
e
f
g
Compound
9-ITMPo
λmax a/ nm ε b/ M-1cm-1 λF / nm
ΦF
τs / ns
τT / µs
ΦΔ
640
664
6.8 x 103
659
0.26
0.10
3.4
74
66
0.59
0.29
τ1 = 1.9 (73 %)
τ2 = 6.1 (27%)
ATAZTMPo-gentamicin
1.2 x 104 643, 713
a
b
c
Maximum absorption wavelength at the Q-region. Molar extinction coefficient at λmax
.
Wavelength of fluorescence. d
Fluorescence quantum yield, meso-tetraphenylporphyrin as reference (ΦF (toluene) = 0.11).41 e Singlet state lifetime in air-
f
saturated methanol, (amplitudes). Triplet state lifetime in argon saturated methanol (µs) obtained by transient absorption
spectroscopy. g Singlet oxygen quantum yield, phenalenone as reference (ΦΔ (MeOH) = 0.97).42
A unique feature of thiazoloporphycenes is the existence of an equilibrium between 18 and 22
electronic systems, whereby the 18 form is stabilized in strong hydrogen-bond donating (HBD)
34
solvents, while the 22system is favoured in weaker- or non-HBD solvents. In this regard, the
absorbance and the fluorescence of ATAZTMPo-gentamicin were measured in three solvents of
different HBD capacity, such as phosphate-buffered saline (PBS; pH 7.4), methanol (MeOH), and
dimethyl sulfoxide (DMSO). As shown in Figure 6, the absorption spectra in PBS loses much of its
structure and exhibits lower molar absorption coefficients, which is consistent with the aggregation
that several porphyrin-like compounds tend to experience in aqueous media. The absorbance spectrum
suffers a hypochromic shift in comparison with organic solvents and its bands are less structured and
broader. Moreover, the fluorescence spectra in PBS shows a single emission band at λ = 640 nm,
typical of the 18- electronic system. Consistent with this, the fluorescence excitation spectrum
(Figure S5) indicates that the emission originates from a monomeric species with 18- electronic
system, as observed previously for a related porphycene.8 A gradual conversion to the near-IR
absorbing species is observed when non-aqueous and weaker- (MeOH) or non-HBD (DMSO) solvents
are used. Thus, the ATAZTMPo-gentamicin conjugate presents a major near-infrared emission band at
approximately λ = 720 nm in DMSO, corresponding to the 22-expanded electronic system, while in
MeOH it shows an intermediate behaviour, which is in concordance with the previously reported for
tetraphenyl analogues.34 To further support this interpretation, compound 2-N-butylaminothiazolo-

[4,5-c]-tetrakis-2,7,12,17-(methoxyethyl)porphycene (ATAZTMPo-butylamine, see ESI for details)
was also studied since it is the PS’s analogue without the antibiotic moiety. It also shows a dual
emission in methanol. The ratio of the short-to-long wavelength bands is smaller than for the
gentamicin conjugate but is also strongly solvent dependence: it increases in hexafluoro-2-propanol
(HFIP, a strong HBD solvent) and decreases in DMSO (see Figure S6 in the supplementary
information), following the same trend as that observed for the gentamicin conjugate. This confirms
that the ratio of the two emissions reflects the equilibrium between the 18 and 22 electronic
systems, and suggests that gentamicin stabilizes the zwitterionic 18 system, likely by forming
hydrogen bonds through its free amino groups.
Figure 6 Top: Equilibrium between the 18- and 22- forms of ATAZTMPo-gentamicin. Bottom: Absorbance (left panel)
and normalised fluorescence emission spectra (right panel) of ATAZTMPo-gentamicin (8 µM) in MeOH (full green line),
DMSO (dotted blue line) and PBS (dashed red line).
1
The ability ATAZTMPo-gentamicin to sensitise the production of O2 was studied by direct
observation of its phosphorescence at 1275 nm in methanol, heavy water and dimethyl sulfoxide.
Table 2 collects the values of the singlet oxygen quantum yield () measured (Figure S7).

Table 2 Singlet oxygen quantum yields of ATAZTMPo-gentamicin in air-saturated methanol, heavy water and dimethyl
sulfoxide.
Solvent
D2Oa
0.02
CH3OHb
0.29
(CH3)2SOc
0.36

a PNS was used as reference ( =1.03)43; b PN used as reference (=0.97)42; c Fullerene C60 used as reference (=1)44.
The  of ATAZTMPo-gentamicin presents different efficiencies depending on the polarity and the
H-bond donor properties of the solvent in which the conjugate is dissolved. To begin, the  in heavy
water is very low ( = 0.02) in comparison with the values in organic solvents mainly due to the
aggregation of the compound. The slight differences in  in the two organic solvents can be due to
the stabilization of different proportions of the two electronic systems (18 and 22) caused by the
different polarity and HBD strength of the solvents. If these species have different , the overall
yield of the compound would reflect this equilibrium in its overall (and measurable) quantum yield.
Up to date it has not been possible to elucidate the individual  values.
For comparison, the  value of ATAZTMPo-butylamine in methanol was also measured and found
higher (0.46) than that of the gentamicin conjugate (0.29) (Figure S8). This considerable difference in
quantum yields may be due to photoinduced electron transfer (PET) quenching of the
thiazoloporphycene excited states by gentamicin, a common observation in porphyrinoids
substituted with moieties containing amino groups.45,46 The amino group directly attached to
thiazoloporphycene ring is unlikely to participate in the PET process due to the delocalization of its
electron pair across the macrocycle, hence PET quenching is not expected in the butylamine
derivative.
2.3 Photoinactivation studies
To assess the potential of the ATAZTMPo-gentamicin conjugate as a novel photoantimicrobial agent,
tests against representative members of Gram-positive (Staphylococcus aureus) and Gram-negative
(Escherichia coli) bacteria were performed. Taking into consideration the survival fraction of both
strains when they were dark-incubated with different gentamicin concentrations upon experimental

conditions (see Figure S9), the photoinactivation of ATAZTMPo-gentamicin was first attempted in
E. coli at 8 µM (3.8 µg mL-1) and 45 J·cm-2 light fluence (Figure 7). Additionally, ATAZTMPo-
butylamine was tested under the same conditions either alone or combined with gentamicin, but not
covalently bound. Surprisingly, only the conjugate ATAZTMPo-gentamicin was able to
completely eliminate the strain without showing any dark toxicity, whereas the PS and the
antibiotic alone failed to effectively inactivate the bacteria. The concentration used (8
M, 3.8

g/ml) is close to the reported minimum inhibitory concentration (MIC; 4 
g/ml).47 However,
MIC values are determined at inoculum sizes (CFUs/mL) 100-1000-fold more diluted than those
used in this work and, in addition, the antimicrobial effect is measured after incubating the drug
for 24 h, while only 1-h contact was allowed in our experiments. Therefore, the lack of dark
toxicity is not surprising.
Figure 7 E. coli photoinactivation studies in PBS upon red light irradiation (λ = 638 ± 9 nm) (orange bars). Grey bars
represent dark controls. ATAZTMPo-butylamine was added from a 5 mM stock in N,N-dimethylacetamide. Light doses: 45
J·cm-2. Working concentration: 8 μM. Incubation time: 1 h. Data correspond to the mean ± SD after performing three
triplicates.
In order to further explore the antimicrobial effect of ATAZTMPo-gentamicin, photoinactivations
were performed upon diff erent concentrations and light fluences against E. coli (Figure 8, left

panel) and S. aureus (Figure 8, right panel). Among the whole conditions tested, the best ratio
concentration/light dose with the highest antibacterial activity was found for S. aureus at 80 nM
(45 J·cm−2) or 800 nM (30 J·cm−2), where the strains were completely eliminated. Curiously, in
contrast with the tendency observed for bacterial inactivation with gentamicin alone (see Figure
S7), photoinactivations of E. coli required more amount of conjugate and higher light-fluences to
observe significant photosensitizing activity against the bacterial cells. Thus, complete killing
was observed at 800 nM upon irradiation with 45 J·cm-2 of red light, whereas, a smaller killing
efficiency (5 log10) was detected when exposed to 30 J·cm-2. Noteworthy, the dark toxicity in
both strains was below 1-log10 CFU mL−1, a desired characteristic for aPDT photoantimicrobials.
Figure 8 E. coli (left panel) and S. aureus (right panel) photoinactivation studies with ATAZTMPo-gentamicin upon red
light irradiation (λ = 638 ± 9 nm). Light doses: 30 J·cm−2 (filled triangles), 45 J·cm−2 (filled circles). Working concentrations:
0.8 (only for S. aureus), 8, 80, 800 and 8000 nM. Incubation time: 2 h. Dark controls are represented with grey empty
symbols. Data correspond to the mean ± SD after performing three triplicates.
Experiments on mammalian (HeLa) cells show that this conjugate does not present any dark
toxicity at the highest concentration tested (8 M) even after 24 h incubation, conditions much
harsher than those used in the bacteria photoinactivation studies (1h incubation; data not shown).
3. Discussion and Conclusions
A novel straightforward synthesis of TMPo, that reduces from nine to six the number of steps
necessary to obtain this compound, has been described. Key to this development has been the one-pot
synthesis of the unprecedented ’-alkoxyalkyl-2,2’-bipyrrole from an alkoxyalkyl Michael acceptor

via a 2-(trimethylstannyl)-pyrrole intermediate. Furthermore, a click reaction between 9-ITMPo
derivative and the antibiotic gentamicin has afforded a cyclised photoactive ATAZTMPo-gentamicin
conjugate as a novel antimicrobial agent for aPDT. The in vitro photoinactivations indicate that
ATAZTMPo-gentamicin is a powerful broad-spectrum near-IR photoantimicrobial agent devoid
of dark toxicity and effective at submicromolar concentrations. Stemming from the results
observed, the antibiotic moiety of the conjugate does not seem to exert any bactericidal effect; all
cell death being attributable to the photodynamic activity of the porphycene. Rather, gentamicin
enhances the solubility of the porphycene and endows it with amphiphilic character. We suspect
that this allows the conjugate to cross the bacterial wall, causing damage not only to the outer
wall, as the butyl conjugate would also do, but also to the inside of the bacteria, and therefore
exhibiting a larger biological activity.
These promising results are evidenced when compared to previous antibacterial studies reported
in the literature which used TMPo, or by combining gentamicin with other PSs. For instance,
Masiera and co-workers required 5 µM of TMPo in Pluronic F-127 solution and 54 J·cm-2 light-
dose to effectively inactive E. faecalis,11 or Polo et al. observed about 4 log10 of bacterial lethality
using 1 µM of TMPo linked to oligomeric polylysine residues in Gram-positive (S. aureus) and
Gram-negative (E. coli) bacteria.13 Gentamicin (3 µM) has been previously been combined with
5-aminolevulinic acid (5-ALA) aPDT, against S. aureus biofilms, resulted in 50% of lethality
when irradiated with 250 J·cm-2.48 Recently, Pérez-Laguna and co-workers49 described a
synergistic bactericidal effect against S. aureus by combination of 1.5-15 µM gentamicin with 30-
70 nM Rose-Bengal under irradiation with a 37 J·cm-2 light-dose, reducing the bacteria growth by
6 log10.
Data for PS-antibiotic conjugates are scarce. Some few examples, such as porphyrin derivatives
linked to antimicrobial peptides21,50,51 or Rose-Bengal bound to Kanamycin52 have been reported,
where the concentrations of the corresponding conjugate needed to induce significant
photosensitizing activity (1.5 - 80 µM) were much higher than that of ATAZTMPo-gentamicin
conjugate (in the nanomolar range).

In summary, the photoinactivations performed with the novel ATAZTMPo-gentamicin conjugate
described here, have demonstrated that the concentration of antibiotic required to achieve a
bactericidal effect is significantly lower than that required for gentamicin alone, in both Gram-positive
and Gram-negative bacteria. Finally, the lack of any dark toxicity observed at photochemically active
doses paves the way to develop novel phototherapeutic strategies to eradicate antibiotic resistance,
which has become one of the major threats to human health according to the World Health
Organization (WHO).
4. Experimental Section
Chemicals
General remarks: All chemicals and dry solvents were purchased from commercial sources and used
as received unless otherwise noted. Synthesis grade or HPLC–grade solvents were used for
extractions and purifications. Zinc dust was activated by stirring with dilute HCl during 10-15
minutes, and then washed several times with distilled H2O, EtOH and absolute Et2O before rigorous
drying.53
All reactions were monitored by TLC analysis using POLYGRAM® SIL G/UV254 pre-coated
polyester sheets (0.2 mm-thickness) (Machery-Nagel). UV light was used as the visualizing agent (at
λ = 254 nm or λ = 365 nm), and a 5% (w/v) ethanolic solution of phosphomolybdic acid or vanillin as
developing agents. Flash column chromatography was carried out with the indicated solvents using
flash-grade silica-gel 60 Å (37-70 μm). Yields refer to chromatographically and spectroscopically
pure compounds, unless otherwise stated.
13
NMR spectra were recorded at RT on a Varian Mercury 400 (1H at 400 MHz and C at 100.6 MHz)
spectrometer. The chemical shifts (δ) are reported in parts per million (ppm) relative to the solvent
signal, and coupling constants (J) are reported in Hertz (Hz). The following abbreviations are used to
1
define the multiplicities in H NMR spectra: s = singlet, d = doublet, t = triplet, q = quartet, dd =
doublet of doublets, ddd = doublet of doublet of doublets, m = multiplet, br = broad signal and app =
apparent, or combinations of these descriptive names. Carbon spectra assignments are supported by

HSQC editing. Infrared spectra were recorded in a Nicolet Magna 560 FTIR spectrophotometer.
Values are reported in wave numbers (cm-1). Mass Spectrometry (MS) for reported compounds was
conducted on an Agilent Technologies 5975 mass spectrometer operating in electron ionization (EI)
mode at 70 eV and at 4 kV accelerating potential. New compounds were characterized by an HPLC-
MS Agilent 1260 infinity Series chromatograph equipped with photodiode array detector (200–700
nm) and coupled to an Agilent 6130 quadrupole LC/MS. Separation was achieved using a XBridgeTM
C18 (4.6 x 75 mm, 2.5µm particle size) column. The flow rate was 1 mL/min, column temperature 45
ºC. The DAD detector was set at 380 nm matching the Soret band of porphycenes. API-ES positive
ionization mode at 15 V cone voltage was used. Fragmentations were scanned from 0 to 1800 m/z.
Samples (200 ppm in MeOH) were analysed using a 20 μL volume injection. m/z ratios are reported
in atomic mass units.
Synthetic procedures
3-Methoxy-1-propanal (2) To a solution of 3-methoxy-1-propanol 1 (25 mL, 262.0 mmol) in 700
mL of EtOAc, 2-iodoxybenzoic acid (IBX) (88 g, 315.0 mmol, prepared according to literature)30 was
added in one portion; the mixture was refluxed overnight and then cooled to RT. The white
precipitate was removed by filtration through a Celite® pad and further rinsed with 3 x 100 mL of
EtOAc. The solvent of the organic layers was distilled at 80 ºC (atm) giving 3-methoxy-1-propanal as
unique product (19.6 g, 222.0 mmol, 85%). The physical and spectroscopic data of 3-methoxy-1-
propanal 2 were identical to those reported in the literature.31,54
1
TLC (CH2Cl2:MeOH; 95:5), Rf: 0.46. H NMR (CDCl3): δ 9.79 (td, J = 2.0, 1.0, 1H), 3.72 (tapp, J =
13
6.0, 2H), 3.36 (s, 3H), 2.66 (td, J = 6.0, 2.0, 2H). C NMR (CDCl3): δ 201.2 (C), 66.4 (CH2), 59.1
(CH3), 44.0 (CH2).
(E)-Ethyl 5-methoxypent-2-enoate (3) Triethyl phosphonoacetate (64 mL, 306.0 mmol) was slowly
added to a suspension of NaH (60% in mineral oil, 13 g, 327.0 mmol) in anhydrous THF (400 mL) at
0 °C. After 30 min of stirring at 0 ºC, a solution of 3- methoxy-1-propanal 2 (19 g, 215.0 mmol) in
anhydrous THF (80 mL) was added. The reaction mixture was stirred at 0 °C for 1.5 h before being
quenched by saturated NH4Cl (200 mL). The product was extracted with EtOAc (3 x 150 mL). The

combined extracts were washed with brine, dried and concentrated. The crude was redissolved in
cyclohexane and the mixture was washed with saturated NH4Cl solution (3 x 100 mL), brine (3 x 100
mL) and water (3 x 100 mL). The organic extracts were dried over anhydrous MgSO4 and
concentrated under reduced pressure to afford ethyl 5-methoxypent-2-enoate as a yellowish oil (26 g
(E:Z; 98:2), 162 mmol, 75%) without further purification. The physical and spectroscopic data of
ethyl 5-methoxypent-2-enoate 3 were identical to those reported in the literature.55
1
TLC (Cyclohexane:EtOAc; 8:2), Rf: 0.44 (E); 0.52 (Z). H NMR (CDCl3): (Data for only E) δ 6.95
(dt, J = 15.5, 7.0, 1H), 5.89 (dt, J = 15.5, 1.5 Hz, 1H), 4.18 (q, J = 7.0, 2H), 3.50 (t, J = 6.5, 2H), 3.35
(s, 3H), 2.52 – 2.42 (qd, J = 6.5, 1.5, 2H), 1.28 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3): (Data for only
E) δ 166.6 (C), 145.7 (CH), 123.1 (CH), 70.9 (CH2), 60.4 (CH2), 58.9 (CH2), 32.7 (CH2), 14.4 (CH3).
Diethyl 4,4'-bis(2-methoxyethyl)-1H,1'H-[2,2'-bipyrrole]-3,3'-dicarboxylate (4) n-BuLi (1.6 M in
hexane, 96 mL, 154.0 mmol) was added to a solution of p-toluenesulfonylmethyl isocyanide
(TosMIC) (16.7 g, 85.0 mmol) in anhydrous THF (250 mL) at -78 °C. After 5 min of stirring at -78
°C, 125 mL of Me3SnCl (1 M in THF, 125.0 mmol) was added dropwise. After another 5 min of
stirring at -78 °C, a solution of ethyl 5-methoxypent-2-enoate 3 (9.0 g, 57.0 mmol) in anhydrous THF
(100 mL) was added dropwise. The temperature of the reaction mixture was allowed to rise to RT in
30 min, and stirring was continued for 2 h. To this solution, 14.0 g of Cu(NO3)2·3H2O (57.0 mmol)
was added in a single portion. The mixture was stirred at RT for 40 minutes, and then added an
additional 57.0 mmol of Cu(NO3)2·3H2O. The solvent was removed under reduced pressure and the
residue was dissolved in EtOAc. The organic layer was washed with 10% ammonia (3 x 500 mL),
water (3 x 500 mL) and brine. The solvent was dried over MgSO4 and concentrated under reduced
pressure. The crude was purified by flash chromatography (CH2Cl2:EtOAc; stepwise gradient from 0
to 5% of EtOAc) to yield the corresponding bipyrrole 4 as a yellow solid (2.7 g, 7.0 mmol, 25%).
TLC (Cyclohexane:EtOAc; 8:2), Rf: 0.67. 1H NMR (CDCl3): δ 13.61 (br s, 2H), 6.66 (d, J = 2.5, 2H),
4.36 (q, J = 7.0, 4H), 3.59 (t, J = 7.0, 4H), 3.37 (s, 6H), 3.03 (td, J = 7.0, 1.0, 4H), 1.41 (t, J = 7.0,
13
6H). C NMR (CDCl3): δ 168.8 (C), 130.6 (C), 124.0 (C), 117.4 (CH), 109.6 (C), 73.5 (CH2), 60.9
(CH2), 58.8 (CH3), 28.6 (CH2), 14.4 (CH3). IR (KBr): ʋ, cm-1 3500–3200 (st N–H), 2978 (=C-H),

2930, 2891, 2869 (st C-H), 1652 (st C=O), 1564 (st C=C), 1417, 1181 (st as CO-O), 1119 (st as O-C-
C). ESI-MS m/z C20H28N2O6 [M+Na]+ Found: 415.1795; Calculated: 415.18; [M+H]+ Found:
393.1880; Calculated: 393.20.
4,4'-Bis(2-methoxyethyl)-1H,1'H-2,2'-bipyrrole (5) Diethyl 4,4'-bis(2-methoxyethyl)-1H,1'H-[2,2'-
bipyrrole]-3,3'-dicarboxylate 4 (2.7 g, 6.8 mmol), ethylene glycol (50 mL) and sodium hydroxide (5.4
g, 136 mmol) were combined in a 500 mL two-necked round-bottom flask fitted with a reflux
condenser. The system was degassed for 1 hour with a stream of N2, and the mixture was heated to
180 ºC for 90 minutes under N2 atmosphere. The mixture was allowed to cool to 100 ºC and then 100
mL of water (previously degassed) was added. The resulting slurry was allowed to cool to RT and the
mixture was extracted with CH2Cl2 (3 x 300 mL), water (3 x 300 mL) and brine. The organic extracts
were dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the
corresponding bipyrrole as a dark-green powder without further purification (1.4 g, 5.8 mmol, 85%).
The physical and spectroscopic data of 4,4'-bis(2-methoxyethyl)-1H,1'H-2,2'-bipyrrole 5 were
identical to those reported in the literature.56
1
TLC (Cyclohexane:EtOAc; 8:2), Rf: 0.31. H NMR (CDCl3): δ 8.00 (br s, 2H), 6.59 – 6.55 (m, 2H),
6.07 (dd, J = 2.5, 1.5, 2H), 3.58 (t, J = 7.0, 4H), 3.38 (s, 6H), 2.75 (t, J = 7.0, 4H). 13C NMR (CDCl3):
δ 126.1 (C), 121.8 (C), 115.4 (CH), 104.3 (CH), 73.9 (CH2), 58.7 (CH3), 27.6 (CH2).
IR (KBr): ʋ, cm-1 3600-3200 (st N–H), 2924, 2860 (st C-H), 1109 (st C-O). MS (EI) m/z (rel
intensity) C14H20N2O2 248.2 (100%).
4,4'-Bis(2-methoxyethyl)-1H,1'H-[2,2'-bipyrrole]-5,5'-dicarbaldehyde (6) To a solution of 1.2 g
(5.0 mmol) of 4,4'-bis(2-methoxyethyl)-1H,1'H-2,2'-bipyrrole 5 in dry DMF (25 mL), phosphoryl
chloride (1.7 mL, 19.0 mmol) was added dropwise at 0 ºC, under argon. The solution was heated to
60 ºC for 1 h and then cooled at RT. The resulting mixture was chilled in an ice/water bath while
adding a solution of 28 g of NaOAc in 220 mL of water. This biphasic mixture was then heated at 85
ºC for 1 hour, wherein the dialdehyde precipitated out as yellow flakes. The suspension was cooled to
0 ºC, filtered and the residue was washed with cold water. The precipitate was then purified by flash
column chromatography (EtOAc; 100%) to yield the desired dialdehyde as a yellow powder (1.2 g,

4.0 mmol, 80%). The physical and spectroscopic data of 4,4'-bis(2-methoxyethyl)-1H,1'H-[2,2'-
bipyrrole]-5,5'-dicarbaldehyde 6 were identical to those reported in the literature.56
TLC (EtOAc), Rf: 0.5. 1H NMR (CDCl3): δ 12.26 (br s, 2H), 9.70 (s, 2H), 6.58 (d, J = 2.5, 2H), 3.65
(t, J = 6.5, 4H), 3.39 (s, 6H), 3.07 (t, J = 6.5, 4H). 13C NMR (CDCl3): δ 177.8 (CH), 136.2 (C), 131.7
(C), 130.7 (C), 111.9 (CH), 73.0 (CH2), 58.9 (CH3), 26.2 (CH2). IR (KBr): ʋ, cm-1 3268 (st N–H),
3175 (st =C-H), 2982, 2926, 2876 (st C-H), 1649, 1607 (st C=O), 1116 (st C-O).
MS (EI) m/z (rel intensity) C16H20N2O4 304.2 (100%).
2,7,12,17-Tetrakis(methoxyethyl)porphycene (TMPo) To a slurry of low-valent titanium reagent,
generated by reduction of titanium tetrachloride (3.6 mL, 33.0 mmol) in anhydrous THF (200 mL)
with activated zinc (4.3 g, 65.5 mmol) and CuCl (1.0 g, 10.0 mmol) by refluxing 3 h, a solution of
4,4'-bis(2-methoxyethyl)-1H,1'H-[2,2'-bipyrrole]-5,5'-dicarbaldehyde 6 (400 mg, 1.3 mmol) in
anhydrous THF (200 mL) was added dropwise slowly over 1 h under reflux conditions with vigorous
stirring. The reaction mixture was heated under reflux for an additional 1 h and then hydrolyzed by
slow addition of 10% aqueous potassium carbonate (ca. 175 mL) to the ice cooled reaction mixture.
The reaction mixture was stirred with 150 mL of CH2Cl2 under oxygen atmosphere for 30 min and
then filtered through Celite® pad to remove the metal excess. The residue was extracted with 200 mL
of CH2Cl2, and the combined organic layer were washed with water (3 x 200 mL). Following drying
over anhydrous MgSO4, the solvent was rotoevaporated and the residue was chromatographed on
silica gel (CHCl3; 100%) to afford a dark violet-blue fraction. The red fluorescing compound was
finally purified by trituration with hot hexane giving the TMPo porphycene as long violet needles.
Yield: 25% (90 mg, 150 µmol). The physical and spectroscopic data of TMPo were identical to those
reported in the literature.56
TLC (CHCl3), Rf: 0.1. 1H NMR (CDCl3): δ 9.72 (s, 4H), 9.34 (s, 4H), 4.34 – 4.31 (m, 16H), 3.62 (s,
12H), 3.10 (br s, 2H). 13C NMR (CDCl3): δ 143.7 (C), 141.0 (C), 134.3 (C), 123.8 (CH), 110.9 (CH),
74.2 (CH2), 59.2 (CH3), 29.0 (CH2). IR (KBr): ʋ, cm-1 3600-3300 (st N-H), 2868, 2806 (st C–H),
1561 (st C=C), 1460, 1391 (as C-H), 1117 (st C-O). MS (EI) m/z (rel intensity) C32H38N4O4 542.3
(54%).

9-Nitro-2,7,12,17-tetrakis(methoxyethyl)porphycene (9-NTMPo) AgNO3 (50 mg, 275 µmol) was
added in one portion to a solution of 2,7,12,17-tetrakis(methoxyethyl)porphycene (150 mg, 275
µmol) in AcOH/CH2Cl2 100/70 mL. The mixture was vigorously stirred at 55 ºC for 20 min,
monitoring the reaction conversion by TLC (CH2Cl2:EtOAc; 75:25). The resulting dark green mixture
was cooled down at RT and filtered through a Celite® pad. The combined filtrate was washed with
H2O, the organic layer was neutralised with an aqueous solution of NaHCO3 (10%), dried over
anhydrous MgSO4 and concentrated under reduced pressure. Flash column chromatography
purification with silica-gel (CH2Cl2:EtOAc; stepwise gradient from 0 to 30% of EtOAc) gave
compound 9-NTMPo as a dark green solid (160 mg, 275 µmol, 100%). The physical and
spectroscopic data of 9-NTMPo were identical to those reported in the literature.35
TLC (CH2Cl2:EtOAc; 75:25), Rf: 0.2. 1H NMR (CDCl3): δ 9.52 (s, 1H), 8.93 (s, 1H), 8.81 (dd, JAB
=
12.0, 2H), 8.66 (s, 1H), 8.48 (s, 1H), 8.35 (s, 1H), 4.26 (t, J = 6.5, 2H), 4.16 (t, J = 7.0, 4H), 4.11 (t, J
= 7.0, 2H), 4.02 (t, J = 6.5, 2H), 3.99 – 3.89 (m, 6H), 3.68 (s, 3H), 3.61 (s, 3H), 3.61 (s, 3H), 3.59 (s,
3H), 1.45 (s, 1H), 0.89 (s, 1H). 13C NMR (CDCl3): δ 143.9 (C), 143.4 (C), 141.8 (C), 141.7 (C), 140.8
(C), 138.0 (C), 137.5 (C), 137.3 (C), 135.8 (C), 135.4 (C), 134.7 (C), 132.9 (C), 132.0 (C), 126.5
(CH), 123.8 (CH), 122.9 (CH), 122.8 (CH), 112.0 (CH), 110.6 (CH), 105.3 (CH), 73.5 (3 x CH2),
73.1 (CH2), 59.1 (3 x CH3), 59.0 (CH3), 29.2 (CH2), 28.5 (CH2), 28.4 (CH2). IR (KBr): ʋ, cm-1 2821,
2869 (st C–H), 1560 (st C=C), 1519 (as NO2), 1461 (δ C-H), 1336 (sym NO2), 1190 (st C-N), 1117
(st C-O), 998, 960, 870, 812. MS (EI) m/z (rel intensity) C32H37N5O6 587.3 (60%)
9-Amino-2,7,12,17-tetrakis(methoxyethyl)porphycene (9-ATMPo) An aqueous solution of NaOH
5 M (55 mL) was added to a solution of 9-NTMPo (160 mg, 275 µmol) in CH2Cl2 (100 mL),
followed by the addition of 65 mL of Na2S2O4 (11.4 g, 65 mmol) dissolved in water. The two-phase
mixture was heated under reflux for 45 min, monitoring the reaction conversion by TLC
(CH2Cl2:EtOAc; 75:25). The resulting crude was washed with water and the organic phase was dried
over anhydrous MgSO4 and concentrated under reduced pressure. The resulting dark blue solid (145
mg, 260 µmol, 95%) was used without further purification. The physical and spectroscopic data of
9-NTMPo were identical to those reported in the literature.35

1
TLC (CH2Cl2:EtOAc; 75:25), Rf: 0.3. H NMR (CDCl3): δ 9.41 (d, J = 11.0, 1H), 9.09 (d, J = 11.0,
1H), 8.96 (s, 1H), 8.94 (s, 1H), 8.84 (s, 1H), 8.81 (s, 1H), 8.42 (s, 1H), 6.22 (br s, 2H), 5.46 (s, 1H),
4.90 (s, 1H), 4.30 – 4.16 (m, 8H), 4.13 (dd, J = 8.0, 6.5, 4H), 3.90 (dt, J = 11.5, 6.5, 4H), 3.61 (s, 3H),
3.60 (s, 3H), 3.55 (s, 3H), 3.46 (s, 3H).b 13C NMR (CDCl3): δ 145.8 (C), 145.3 (C), 141.6 (C), 141.5
(C), 140.8 (C), 139.0 (C), 138.3 (C), 136.8 (C), 136.6 (C), 133.6 (C), 132.6 (C), 132.5 (C), 132.4 (C),
124.5 (CH), 123.6 (CH), 122.5 (CH), 121.1 (CH), 114.0 (CH), 106.9 (CH), 101.2 (CH), 75.6 (CH2 x
3), 74.5 (CH2), 74.0 (CH2 x 3), 73.6 (CH2 x 2), 59.4 (CH3), 59.1 (CH3 x 3), 31.1 (CH2), 28.8 (CH2 x
2), 28.75 (CH2 x 2), 28.7 (CH2 x 2). IR (KBr): ʋ, cm-1 3350, 3241 (st N-H), 2921, 2865 (st C-H), 1646
(δ N-H), 1553 (st arom C=C), 1463, 1357 (δ C-H), 1326, 1188 (st C-N), 1113 (st C-O), 993, 891, 812,
625, 470. MS (EI) m/z (rel intensity) C32H39N5O4 557.3 (70%).
9-Isothiocyanate-2,7,12,17-tetrakis(methoxyethyl)porphycene (9-ITMPo) 1,1′-Thiocarbonyldi-
2(1H)-pyridone (125 mg, 540 µmol) was added to a solution of 9-ATPPo (100 mg, 180 µmol) in
CH2Cl2 (25 mL). The mixture was stirred during 16 h at room temperature. The solvent was
evaporated and the residue was chromatographed on silica gel (EtOAc; 100%) to afford a dark blue-
green compound (85 mg, 140 µmol, 80%).
1
TLC (EtOAc), Rf: 0.42. H NMR (CDCl3): δ 9.66 (d, JAB = 11.0, 1H), 9.62 (d, JAB = 11.0 Hz, 1H),
9.35 (s, 1H), 9.22 – 9.19 (m, 2H), 9.16 (s, 1H), 9.14 (s, 1H), 4.37 – 4.21 (m, 14H), 4.15 (tapp, J = 6.5,
13
2H), 3.63 (s, 3H), 3.62 (s, 3H), 3.62 (s, 6H), 3.52 (br s, 1H), 2.79 (br s, 1H). C NMR (CDCl3): δ
143.9 (C), 143.3 (C), 141.0 (C), 140.5 (C), 140.2 (C), 139.3 (C), 138.3 (C), 137.6 (C), 135.1 (C),
133.7 (C), 133.5 (C), 133.1 (C), 132.7 (C), 124.7 (CH), 123.6 (CH), 123.4 (CH), 122.8 (CH), 114.9
(C), 111.4 (CH), 111.1 (CH), 110.4 (CH), 73.9 (CH2), 73.3 (CH2), 72.9 (CH2), 59.2 (2 x CH3), 59.1 (2
x CH3), 30.6 (CH2), 28.8 (2 x CH2), 28.2 (CH2). IR (KBr): ʋ, cm-1 3439 (st N-H), 2921, 2870 (st C–
H), 2134 (st SC≡N), 1116 (st C-O), 966 (st sym N=C=S), 889, 811 (st C-S). ESI-MS m/z
C33H37N5O4S [M+H]+ Found:600.3877; Calculated: 600.26.
2-gentamicin-thiazolo[4,5-c]2,7,12,17-tetrakis(methoxyethyl)porphycene
(ATAZTMPo-
gentamicin) To a mixture of gentamicin sulphate (500 mg, 830 µmol) and K2CO3 (150 mg, 1.1
mmol) in ethanol (10 mL), 9-ITMPo (5.0 mg, 8 µmol) in ethanol (2 mL) was added dropwise. The

resulting solution was stirred at RT for 5 days until no conversion was observed by TLC. The green
mixture was concentrated under reduced pressure, and the resulting crude was purified by flash
column chromatography on C18-RP silica-gel (glacial acetic acid:Methanol; 5:95); stepwise gradient
from 0 to 100% of MeOH) to afford the desired ATAZTMPo-gentamicin conjugate in 75% (8 mg, 6
µmol) yield. The pure fraction isolated was analysed by RP-HPLC-MS in gradient elution mode
applying the following gradient profile: 0 - 0.5 min: 100% A; 0.5 - 2 min: 0 - 100% B; 2 - 7 min:
100% B; 7 - 9 min: 0 - 100% A; 9 - 15 min: 100% A; Mobile phase A: Water; B: MeOH; both with
0.1% formic acid (v/v). ATAZTMPo-gentamicin, as a mixture of congeners due to gentamicin, was
isolated at retention time around 3.4 min (Figure S1). The MS survey scan at retention time of 3.576
min confirmed the single-conjugate for the most abundant charge state of z = 1 and z = 2 of
ATAZTMPo-(C1)gentamicin congener, as well as the pick at 3.375 min the z = 1 and z = 2 of
ATAZTMPo-(C1,1a)gentamicin congener can be observed along with other conjugate fragmentations
(Figure S2).
TLC (CHCl3:MeOH:NH3
30%); 70:30:10), Rf: 0.82. ESI-MS m/z ATAZTMPo-(C1)gentamicin
(liq
C53H76N10O11S [M+H]+ Found: 1061.5555; Calculated: 1061.5494; [M+2H]2+ Found: 531.2758;
Calculated: 531.2780. ATAZTMPo-(C1,1a)gentamicin C52H74N10O11S [M+H]+ Found: 1047.5403;
Calculated: 1047.5338; [M+2H]2+ Found: 524.2782; Calculated: 524.2703.
2-N-butylaminothiazolo[4,5-c]2,7,12,17-tetrakis(methoxyethyl)porphycene
(ATAZTMPo-
butylamine) N-butylamine (0.62 mL, 6.2 mmol) was added dropwise to a solution of 9-
isothiocyanate-2,7,12,17-tetrakis(methoxyethyl)porphycene (9-ITMPo, 15 mg, 0.025 mmol) in
CH2Cl2 (5 mL). The mixture was stirred 24 h at room temperature. Then, the mixture was
concentrated under reduced pressure and the crude was purified by flash column chromatography
(CH2Cl2:MeOH; stepwise gradient from 0 to 2% of MeOH) to afford the green ATAZTMPo-
butylamine in 80% (13 mg, 0.02 mmol) yield.
1
TLC (CH2Cl2:MeOH; 95:5), Rf: 0.7. H NMR (CDCl3): δ 9.43 (d, J = 11.0, 1H), 9.31 (d, J = 11.0,
1H), 9.15 – 9.03 (m, 2H), 8.98 (s, 1H), 8.79 (s, 1H), 5.65 (t, J = 5.5, 1H), 4.77 (s, 1H), 4.37 (tapp, J =
7.0 Hz, 2H), 4.32 – 4.16 (m, 8H), 4.05 (t, Japp = 7.0 Hz, 2H), 3.88 (s, 1H), 3.72 - 3.64 (m, 2H), 3.61 (s,

3H), 3.60 (s, 3H), 3.59 – 3.57 (m, 2H), 3.57 (s, 3H), 3.54 (s, 3H), 1.86 (dt, J = 15.0, 7.5, 2H), 1.61 (td,
J = 15.0, 7.5, 2H), 1.10 (t, J = 7.5, 3H). 13C NMR (CDCl3): δ 164.0 (C), 143.6 (C), 142.9 (C), 142.1
(C), 140.3 (C), 139.4 (C), 139.2 (C), 138.8 (C), 137.2 (C), 136.1 (C), 135.4 (C), 133.2 (C), 132.7 (C),
130.1 (C), 125.3 (CH), 124.0 (CH), 123.2 (CH), 123.0 (CH), 120.5 (C), 111.3 (CH), 108.7 (CH), 74.1
(CH2), 73.9 (CH2), 72.3 (CH2), 59.0 (CH3), 58.9 (CH3), 58.8 (CH3), 45.2 (CH2), 32.4 (CH2), 31.8
(CH2), 31.4 (CH2), 28.7 (CH2), 28.6 (CH2), 20.3 (CH2), 13.9 (CH3). IR (KBr): ʋ, cm-1 3390 (st N-H),
2921, 2870 (st C-H), 1640 (δ N-H), 1550 (st arom C=C), 1465, 1360 (δ C-H), 1326, 1190 (st C-N),
1115 (st C-O), 985, 890, 812. ESI-MS m/z C37H46N6O4S [M+Na]+ Found: 693.3204; Calculated:
693.3199; [M+H]+ Found: 671.3377; Calculated: 671.3379.
Spectroscopic techniques
All spectroscopic measurements were carried out in 1 cm quartz cuvettes (Hellma, Germany) in air-
saturated solutions, unless otherwise stated, at room temperature using spectroscopic grade solvents.
Absorption spectra were recorded using a double beam UV-Vis-NIR Varian Cary 6000i
spectrophotometer (Varian, Palo Alto, CA, USA). Absorption coefficients were derived from the
slopes of Beer-Lambert plots.
Fluorescence emission and excitation spectra were registered using a a Spex Fluoromax-4
spectrofluorometer (Horiba Jobin-Yvon, Edison, NJ, USA). Fluorescent quantum yields were
determined by plotting the integrated fluorescent intensity of a series of solutions of increasing
concentration versus the amount of light absorbed (1-10-Abs), obtaining a linear correlation. By means
of Equation 1, slopes of the desired compound and reference (meso-tetraphenylporphyrin; F =
0.11)41 were compared, and corrected by the refractive index of the solvents used.
2
푆푙표푝푒
·푛
·푛
푆푎푚푝푙푒
푆푎푚푝푙푒
훷퐹 =
(1)
2
푆푙표푝푒
푅푒푓푒푟푒푛푐푒
푅푒푓푒푟푒푛푐푒
Time-resolved fluorescence decays were measured using a Fluotime 200 time-correlated fluorescence
lifetime spectrophotometer (PicoQuant GmbH, Berlin, Germany), equipped with a red sensitive
photomultiplier. Excitation was achieved by means of a 596 nm picosecond diode laser working at 10

MHz repetition rate. The counting frequency was always below 1%. Fluorescence lifetimes were
analysed using PicoQuant FluoFit 4.6.6 data analysis software.
Transient absorption spectra (Figure S4) were monitored by nanosecond laser flash photolysis using a
Q-switched Nd:YAG Laser (Surelite I-10, Continuum) with right-angle geometry and an analysing
beam produced by a Xe lamp (PTI, 75 W) in combination with a dual-grating monochromator (mod.
101, PTI) coupled to a UV-Vis radiation detector (PTI 710). The signal was fed to a Lecroy
WaveSurfer 454 oscilloscope for digitizing and averaging (typically 10 shots) and finally transferred
to a PC for data storage and analysis.
Singlet oxygen generation was studied by time‐ resolved near‐ infrared phosphorescence by means
of a customised setup.57 Briefly, a pulsed Nd:YAG laser (FTSS355‐ Q, Crystal Laser, Berlin,
Germany) working at 1 kHz repetition rate at 355 nm (0.5 μJ per pulse) was used to excite the
sample. A 1064‐ nm rugate notch filter (Edmund Optics) and an uncoated SKG‐ 5 filter (CVI Laser
Corporation) were placed in the laser path to remove any NIR emission. The light emitted by the
sample was filtered with a 1000‐ nm long‐ pass filter (Edmund Optics) and later by a narrow
bandpass filter at 1275 nm (BK‐ 1270‐ 70‐ B, bk Interferenzoptik). A thermoelectric‐ cooled NIR-
sensitive photomultiplier tube assembly (H9170‐ 45, Hamamatsu Photonics, Hamamatsu, Japan) was
used as detector. Photon counting was achieved with a multichannel scaler (NanoHarp 250,
1
PicoQuant). The time dependence of the O2 phosphorescence with the signal intensity S(t) is
described by Equation 2, in which T and Δ are the lifetimes of the photosensitizer triplet state and of
1O2 respectively, and S0 a preexponential parameter proportional to 
푡
푡
−
⁄
휏
휏
∆
푆1275 푡 = 푆1275 0 × 휏 −휏 × (푒푥푝−
− 푒푥푝
)
(2)
⁄
휏
∆
푇
( )
( )
∆
푇
The plotting of S0 versus the absorbed energy at different concentrations yields also a linear
relationship. The ΦΔ values of the different samples were obtained by comparison of the slopes of
such plots with an appropriate reference compound using Equation 3.
푆푙표푝푒
푠푎푚푝푙푒
훷훥,sample = 훷훥,ref
×
(3)
푆푙표푝푒
푟푒푓

Microbial strains, culture conditions and photodynamic inactivation studies
The microorganisms studied were Staphylococcus aureus ATCC 29213 as a Gram-positive model
and Escherichia coli ATCC 25922 as Gram-negative bacteria.
Bacterial cells were grown overnight in an orbital shaker at 37 °C in Tryptic Soy Broth (TSB)
medium. An aliquot was then suspended in fresh TSB and set to grow in exponential phase at 37 °C
to achieve approximately 108 colony forming units (CFU·mL-1). They were later centrifuged (5000
rpm, 10 min) and resuspended in PBS (pH = 7.4). The cells were incubated with the drug, delivered
in PBS, in the dark for 1 hour under the same growth conditions.
Three independent experiments were done for each photoinactivation treatment, which in turn was
carried out in duplicate. The average and SD values were calculated.
Cells were then irradiated from the top in 96-well plates by means of a LED-lamp (Sorisa Photocare;
635 ± 15 nm; 17 mW·cm-2). The irradiance of the lamp was regularly monitored with an Ophir AN/2
Laser Power Meter (Ophir Optronics Solutions Ltd, Har Hotzvim, Israel) to ensure the light dose
delivered.
Since no significant statistical difference was obtained when the photoinactivation was performed
with and without removal of the excess of ATAZTMPo-gentamicin or the PS by centrifugation (data
not shown), the procedure was followed without removal of the excess of the conjugate.
Controlling the cell irradiation without PS was also performed in order to exclude any inactivation
due to light or heating effects, along with the evaluation of the toxicity of ATAZTMPo-gentamicin in
the dark and controls using gentamicin. After illumination, the samples were serially diluted and
streaked on agar plates and incubated in the dark for 18 h at 37 °C.
5. Associated Content
Supporting Information
Bacterial inactivations with gentamicin, characterisations, and further spectroscopic data (PDF).

6. Acknowledgements
This work was supported by the Spanish Ministerio de Economia y Competitividad through grant No.
CTQ2016-78454-C2-1-R.
C. H. thanks the SUR del DEC de la Generalitat de Catalunya and the FSE for his predoctoral
fellowship (grants No. 2017 FI_B_00617, 2018 FI_B1 00174 and 2019 FI_B2 00167).
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