New photodynamic molecular beacons (PMB) as potential cancer-targeted agents in PDT

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New Photodynamic Molecular Beacons (PMB) as

potential cancer-targeted agents in PDT

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a

a,b

c

A. Stallivieri, L. Colombeau, J. Devy, N. Etique,

c

a

b

a

b

,a

C. Chaintreuil, B. Myrzakhmetov, M. Achard, F. Baros, P. Arnoux, R. Vanderesse, and C. Frochot*

LRGP, UMR 7274 and LCPM, UMR 7375, Université de Lorraine, CNRS, Nancy, France. SiRMA, UMR

a

b

c

7

369, Université de Reims Champagne-Ardenne, CNRS, Reims, France.

Bioorganic & Medicinal Chemistry

journal homepage: www.els evier.com

New Photodynamic Molecular Beacons (PMB) as potential cancer-targeted

agents in PDT

Aurélie Stallivieri, ^‡^,^aLudovic Colombeau, ^‡^,^a^,^bJérôme Devy, Nicolas Etique, Carine Chaintreuil,

c

b

a

b

Bauyrzhan Myrzakhmetov, Mathilde Achard, Francis Baros, Philippe Arnoux, Régis Vanderesse, and

,

a

Céline Frochot*

a

b

c

Laboratoire Réactions et Génie des Procédés (LRGP), UMR 7274, Université de Lorraine, CNRS, Nancy, France.

Laboratoire de Chimie Physique Macromoléculaire (LCPM), UMR 7375, Université de Lorraine, CNRS, Nancy, France.

Laboratoire Signalisation des Récepteurs Matriciels (SiRMA), UMR 7369, Université de Reims Champagne-Ardenne, CNRS, Reims, France.

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

Further improvements in Photodynamic therapy (PDT) necessitate that the dye targets more

selectively tumour tissues or neovascularization than healthy cells. Different enzymes such as

matrix metalloproteinases (MMPs) are overexpressed in tumour areas. Among these MMPs,

gelatinases (MMP-2 and MMP-9) and its activator MMP-14 are known to play a key role in

tumour angiogenesis and the growth of many cancers such as glioblastoma multiforme (GBM),

an aggressive malignant tumour of the brain. These last years, the concept of photodynamic

Available online

molecular beacons (PMB) became interesting for controlling the photosensitizer’s ability to

1

generate singlet oxygen ( O

2

) close to target biomolecules as MMPs. We report herein novel

PMBs triggered by MMP-2 and/or MMP-9 and/or MMP-14, comprising a photosensitizer and a

singlet oxygen quencher linked by MMP cleavable peptide linker (H-GRIGFLRTAKGG-OH).

First of all, we focused on the synthesis and the photophysical study of different derivatives

photosensitizer-peptide. This preliminary work concluded on an influence of the nature and the

distance from the peptide, but not of the position of the photosensitizer in these derivatives on

the proteolytic enzymatic action. The nature of the quencher used (a blackberry quencher (BBQ-

Keywords:

Keyword_1 enzymatic-activatable photosensitizer

Keyword_2 matrix metalloproteinase

Keyword_3 photomolecular beacon

Keyword_4 photodynamic therapy

Keyword_5 singlet oxygen quencher

6

50) or a black hole quencher (BHQ3)) does not influence the enzymatic action. We also studied

the influence of an additionnal PEG spacer. Finally, the synthesis, the singlet oxygen quenching

efficiency and the enzymatic activation of these new MMP- cleavable-PMBs were compared.

2

Corresponding AuthorPoly

*

Céline Frochot: phone, (+33)372 743 780; e-mail, celine.frochot@univ-lorraine.fr

‡

These authors contributed equally to this work

1. Introduction

There are various approaches to treat cancer and an alternative or adjuvant treatment to classical cancer therapies such as

radiotherapy, surgery and chemotherapy is Photodynamic therapy (PDT) which is an innovative modality for the treatment of localized

tumours. This modality uses an efficient process which requires a photosensitizer (PS), oxygen and light. When photosensitizers are

exposed to a specific wavelength of light, the formation of the excited triplet state occurs and this triplet PS can transfer energy to

1

1 1

2 2

oxygen to form excited singlet oxygen ( O ). O is highly reactive and can react with various cellular constituents causing cell damage

and eventually cell death. It is noteworthy that even if some photosensitizers used for PDT present certain tumor selectivity, they

2

accumulate also in healthy tissues resulting in uncomfortable adverse effects.

For some years now, research is focused on a controllable singlet oxygen generation, because of the limited lifetime and diffusion

1

3-5

2

distance of O (between 10 and 300 nm, according to different estimates ), with high selectivity and localization to enhance

photodynamic efficiency by directly targeting diseased cells and tissues. Further research in this field brought the concept of a new class

of activatable molecules named photodynamic molecular beacons (PMB) as smart drugs. Different types of activation mechanisms are

6

already known such as the environment-activation, the enzymatic-activation or nucleic acid-activation. Alternatively, the linker may be

7

an antisense oligonucleotide (hairpin) loop, which is opened by hybridization to complementary mRNA. These molecular

1

constructions are capable of controlling O

activation mechanism.

2

production that responds specifically to certain cancer biomarkers according to an

8

,9

Enzymes are catalytic, diverse, and central to all facets of cellular functions and are, therefore, excellent targets. Enzyme

overexpression is correlated with diseases in many cases leading to the development of the enzymatic-activatable photosensitizers in

6

targeted therapies. In that case, the photosensitizer is linked to a quencher moiety (Q) and remains photodynamically inactive until the

1

0

cleavage of the linker by specific enzymes, upon which both the PDT activity and PS photophysical properties are restored. The

development of this type of probes for cancer imaging or cancer treatment using fluorescence resonance energy transfer (FRET) has

1

been well described in the literature.

Nowadays, only few photodynamic molecular beacons for treatment purposes have been designed, as summarized in Table S1 in

supporting information.

1

2

The first enzyme-based activatable PS was described by Krinick et al. in 1994. This polymeric activatable PS used the chlorin-e

self-quenching. This photosensitizer with a methacrylamide backbone was activated by cathepsin B. A similar approach was used to

6

1

3

generate a polylysine-based chlorin-e

6

activatable PS. Enzymatic cleavage at free lysine residues resulted in a loss of self-quenching

generation. Coupling of multiple photosensitizer molecules onto a polymeric backbone via enzyme-cleavable amino

1

and increased O

2

acid sequences was interesting for the PMB concept because of the self-quenching of the PS molecules due to high local concentration

of the photosensitizer. The first PMB, using a quencher different than the PS, was reported by Zheng et al. using a caspase-3 linker

between Pyropheophorbide (a) and a carotenoid or a Black Hole Quencher as quenching molecule. Then, PMB targeting new different

1

4,15

substrates as Fibroblast Activation Protein or Phospholipases were synthetized for the treatment and/or diagnosis of cancers.

Recently, photosensitizer-conjugated gold nanorods via a protease (MMP-2)-cleavable peptide linker demonstrated to be useful for

1

6

protease-mediated fluorescence imaging and subsequent photodynamic therapy for various cancers. Matrix metalloproteinase (MMP)-

9

,17

based beacons were reported in vitro and in vivo, with high selectivity.

Subsequently, Verhille et al. provided a report on the

synthesis, photophysical properties and biological evaluation of a new family of MMP-cleavable PMBs based on the use of a new pair

photosensitizer/quencher, tetraphenylchlorin (TPC)/blackberry quencher (BBQ-650) separated by different lengths of peptides and

1

spacers. Fluorescence intensity and O

cleaved by the targeting enzymes.

2

production of TPC were efficiently quenched in the different PMBs but none of them were

18

Following the latter case, we decided as preliminary work to study the influence of the nature and the position of the photosensitizer

on a peptide substrate of gelatinases (MMP-2 and MMP-9) and MMP-14 (called MT1-MMP as well) on the enzymatic cleavage.

Matrix metalloproteinases (MMPs) play a pivotal role in malignant behavior of cancer cells such as rapid tumor growth, invasion,

1

9

metastasis, and angiogenesis by degrading extracellular matrix (ECM). Among MMPs, there is increasing evidence that the MMP-14

2

0-22

expression level is most closely associated with invasiveness and malignancy of tumors

and that gelatinases (MMP-2 and MMP-9)

23-25

have a key role in tumour angiogenesis and the growth of many cancers.

tumor of the brain, expresses high levels of MMP-14, MMP-2 and MMP-9.

Glioblastoma multiforme (GBM), an aggressive malignant

2

6,27

Because of its ability to activate MMP-2 and MMP-13

to degrade a wide range of extracellular matrix components and to up-regulate vascular endothelial growth factors (VEGF), MMP-14

might be of central importance in the growth of human glioblastoma and could represent an interesting target for anti-cancer

2

8,29

treatments.

Several MMP-14 cleavable peptides are listed in the literature and in Table S2 (supplementary data). Kridel et al.

30

provided a structural basis for selective and non-selective substrate recognition by MMP-14. The peptide SGRIGFLRTA showed the

highest specificity and selectivity for MMP-14. This substrate was reported as highly selective for MMP-14, among the commonly

studied extracellular MMPs associated with tumor generation and development, such as MMP-2 and MMP-9 (with specificity constants

−1

30

m

(kcat/K ) for MMP-14 and MMP-9 of 777 200 and 20 000 M .s , respectively). Zhu et al. synthesized an MMP-14 fluorogenic probe

that consisted of an MMP-14 substrate and near-infrared (NIR) dye (Cyanine 5.5)-quencher (Black Hole Quencher 3) pair, which

exhibited rapid, efficient boosts in fluorescence upon cleavage by MMP-14 in tumor-bearing mice (Figure S1 in supporting

information). Amino acids including Glycine and Lysine were incorporated in the peptide, chosen previously in Kridel publication, as

3

1,32

residues for dye conjugation and as spacers between substrate and dye molecules.

The results on the influence of the nature, the distance from the peptide and the position of the photosensitizer on the MMP-14-

cleavable peptide on the enzymatic activation pushed us to synthesize novel PMBs. Here, we report the detailed chemical synthesis,

characterization, photophysical properties and the enzymatic activation of the different derivatives PS-peptide and the novel PMBs.

2

. Results and discussion

2

.1. Synthesis.

Our aim was to synthesize novel enzyme-based activatable photodynamic molecular beacons using the MMP14 substrate peptide H-

GRIGFLRTAKGG-OH. This study was basically divided in three steps: 1) choice and synthesis of the photosensitizers; 2) coupling to

the peptide; 3) coupling to the quencher. Furthermore, from the beginning of our study, we wondered if the presence on the same

molecule of two hindered groups (ie PS and Quencher) would still allow the PMBs’ cleavage by the MMPs. Thus, we decided to

compare two PMBs differing only by the presence or not of a spacer. However, the spacer arm must not be too long to maintain the

FRET effect. We chosed 2-amino-ethoxy-ethoxyacetic acid (so called PEG in this study) which is ≈ 15-17 Å length and can also

increase the solubility of the PMB in aqueous medium.

2

.1.1. Photosensitizers.

2

Photosensitizers holding a carboxylic acid function were chosen as the starting materials in order to be coupled to the NH -terminal

or to the lysine NH

2

-lateral of specific peptides and so to yield first the derivatives photosensitizer-peptide. Pyropheophorbide (a)

(

Pyro(a)-COOH) was obtained commercially from Frontier Scientific. The 5-(4-carboxypheny)-10,15,20-tetraphenylporphyrin (P1-

3

COOH 1) was synthesized by a commonly used method previously described by Lindsey (Scheme 1). 1 was prepared via acid-

catalyzed condensation reaction from a statistical mixture of distilled pyrrole, benzaldehyde and 4-carboxybenzaldehyde. This mixture

was oxidized with p-chloranil to produce the porphyrin 1 (11 % yield) that was afterwards reacted with N-hydroxysuccinimide to afford

34

the activated ester (P1-NHS 2) which was further coupled to Fmoc-Lys-OH following our general procedure to lead to modified

building-block Fmoc-Lys(P1)-OH (3) with a yield of 82 % (Scheme 1).

HO

O

(i), (ii)

O

N

HN

N

+

N

H

O

HO

NH

4.0 eq

3.0 eq

1.0 eq

P1-COOH 1

(iii)

O

N

HN

N

O

N

O

N

HN

N

(

iv)

HN

NH

O

Fmoc-NH

OH

P1-NHS 2

O

Fmoc-Lys(P1)-OH 3

Scheme 1. Synthesis of P1-COOH (1), P1-NHS (2) and Fmoc-Lys(P1)-OH (3): (i) BF

3

2

.OEt , DCM, 2 h. (ii) p-chloranil, 60°C, 2 h. (iii) NHS, DCC, DCM, 45°C,

4

h. (iv) Fmoc-Lys-OH.HCl in DMF, TEA, DCM, 24 h.

2

.1.2. Photosensitizers-peptide and peptide-quencher.

The peptide H-GRIFGLRTAKGG-OH (4) was synthesized by classical solid-phase peptide synthesis (SPPS) using Fmoc strategy,

double coupling for each amino acids except for the first arginine and glycine (four and six coupling were respectively necessary) and

finally, cleaved from the Wang resin. To obtain the derivatives PS-GRIGFLRTAKGG-OH (5, 6) and H N-PEG-GRIGFLRTAKGG-

2

OH (7), the photosensitizer or the PEG arm had to be coupled to the deprotected N-terminus peptide on solid phase in DMF before the

cleavage from the resin. Compound P1-PEG-GRIGFLRTAKGG-OH (8) was obtained by successive couplings still on solid phase of

the PEG arm and compound

1 on the growing peptide. Compounds H-GRIGFLRTAK(P1)GG-OH (9) and H-

GRIGFLRTAK(BBQ)GG-OH (10) were synthesized by SPPS and cleaved on a Wang resin as described for 4. Fmoc-Lys(P1)-OH (3)

and Fmoc-Lys(BBQ)-OH were coupled as classical amino acids during SPPS procedure, except that the coupling in these cases took

longer time (respectively 5 and 7 days). During the coupling of the PSs and all further steps, light exposure was minimized by sealing

the reaction vessel in alumina foil to limit the occurrence of unwanted side photo-reactions. Compounds H-LRTAK(P1)GG-OH (11),

H-GRIGF-OH (12), H N-PEG-GRIGF-OH (13), PS-GRIGF-OH (14, 15) and P1-PEG-GRIGF-OH (16) were synthesized as references

2

to determine the best HPLC conditions allowing the detection of the enzymatic cleavage. 11 was obtained during the synthesis of 9 by

simply interrupting the synthesis after Leucine’s coupling. 12-16 were prepared starting from a Fmoc-Phe-Wang resin (Scheme 2).

H-GRIGFLRTAK(BBQ)GG-OH 10

Pyro(a)-GRIGFLRTAKGG-OH

P1-GRIGFLRTAKGG-OH

6

5

H-GRIGFLRTAK(P1)GG-OH

H-LRTAK(P1)GG-OH

9

11

Fmoc-Lys(P1)-OH coupling (i), (ii)

or

P1 or Pyro(a) coupling (i), (ii)

Cleavage (iii)

H-GRIGFLRTAKGG-OH

4

Fmoc-Lys(BBQ)-OH coupling (i), (ii)

Cleavage (iii)

Cleavage (i), (iii)

(

Pbf)

(Pbf) (Boc)

O

Coupling cycles

(i), (ii)

O

Fmoc

N

Fmoc-GRIGFLRTAKG

N

H

O

PEG coupling (i), (ii)

Cleavage (iii)

O

OtBu

PEG coupling (i), (ii)

P1 or Pyro(a) coupling (ii)

Cleavage (iii)

H N-PEG-GRIGFLRTAKGG-OH 7

2

P -PEG-GRIGFLRTAKGG-OH

1

8

Coupling cycles

(i), (ii)

H-GRIGF-OH

12

H N-PEG-GRIGF-OH 13

O

2

Fmoc

N

P1-GRIGF-OH

14

H

O

Cleavage (iii)

Pyro(a)-GRIGF-OH 15

P1-PEG-GRIGF-OH 16

Scheme 2. Synthesis of compounds (4-16). (i) Fmoc deprotection: 20% piperidine in DMF. (ii) Coupling: HBTU, NMM, DMF, 16 h. (iii) Cleavage:

TFA/TIS/H O.

2

Scheme 3 shows the synthesis of the peptide-quencher H-GRIGFLRTAK(BHQ3)GG-OH (19). First, the peptide sequence was

synthesized following a classical Fmoc SPPS protocol on a 2-Chlorotrityl resin. The protected peptide was first cleaved from the resin

with a solution of DCM/TFE/acetic acid (4/2/2, v/v/v) and then treated with 3% TFA to remove the Mtt protecting group of the lysine

leading to the intermediate (17). The free εNH

to afford the intermediate (18) whose all the remaining protecting groups were subsequently removed by i) TFA with 5% TIS and ii)

0% piperidine in DMF affording 19.

2

of lysine was reacted with the commercially available activated quencher BHQ3-NHS

2

(

Pbf)

(Pbf) (Mtt)

Cleavage from resin (i)

Mtt deprotection (ii)

(Pbf)

(NH₂)

O

Fmoc-GRIGFLRTAKG

N

H

Fmoc-GRIGFLRTAKGG-OH 17

O

OtBu

BHQ3-NHS coupling (iii)

=

2-Chlorotrityl resin

Side-chains

deprotection (iv)

(Pbf)

(Pbf) (BHQ3)

H-GRIGFLRTAK(BHQ3)GG-OH 19

Fmoc-GRIGFLRTAKGG-OH 18

Fmoc deprotection (v)

OtBu

Scheme 3. Synthesis of H-GRIGFLRTAK(BHQ3)GG-OH (19). (i) Cleavage: DCM/TFE/acetic acid. (ii) Mtt deprotection: 3% TFA, 5% TIS in DCM. (iii)

BHQ3-NHS, DMSO with 2% DIPEA, 16 h, under N . (iv) Side-chains deprotection: 95 % TFA, 5% TIS. (v) Fmoc deprotection: 20% piperidine in DMF.

2

.1.3. Photodynamic molecular beacon (PMB).

As represented in Schemes 4 and 5, PS and quencher are conjugated to the opposite ends of the MMP14-specific cleavable peptide

1

2

linker to keep them in close proximity, enabling FRET and O quenching to make this construct not only optically silent but also

photodynamically inactive. Five PMBs have been synthesized. In this preliminary study we did not try to optimize the synthesis

conditions. According to the photophysical measurements and the cleavage tests, the synthesis of the best candidate would be improved

to increase the yield. For the synthesis of PMBs, two different methods were considered:

a) P1-GRIGFLRTAK(BBQ)GG-OH (20) was totally synthesized on solid support: BBQ was directly added during the SPPS

procedure thanks to the commercially available Fmoc-Lys(BBQ)-OH. Next, the coupling of 1 on the deprotected N-terminus of the

growing peptide was performed on solid phase in DMF before the cleavage from the Wang resin using a mixture TFA/TIS/water

(

Scheme 4).

H

N

O

(Pbf) (Pbf)(BBQ)

Fmoc

(i), (ii)

O

H2N GRIGFLRTAKG N

Coupling cycles

H

O

OtBu

=

Wang resin

P1-COOH coupling (ii)

(Pbf) (Pbf)(BBQ)

Cleavage

O

P1-GRIGFLRTAK(BBQ)GG-OH

PMB 20

1P GRIGFLRTAKG N

(

iii)

H

O

OtBu

Scheme 4. Synthesis of H-GRIGFLRTAK(BBQ)GG-OH (20): (i) Fmoc deprotection: 20% piperidine in DMF. (ii) Coupling: HBTU, NMM, DMF. (iii)

Cleavage: TFA/TIS/H O.

2

b) A second method was used for the synthesis of the PMBs 24-27. First, the peptide sequence was synthesized following the Fmoc

solid phase peptide synthesis (SPPS) protocol on a 2-Chlorotrityl resin. The Fmoc protecting group was then removed by 20%

piperidine in DMF and the PS was conjugated to the N-terminal of peptide. The PS-peptide was cleaved from the resin by a solution of

DCM/TFE/acetic acid (4/2/2, v/v/v) and then treated with 3% TFA to remove the Mtt protecting affording the PS-peptide, the

intermediates without (21, 22) or with PEG (23). After the subsequent coupling of the corresponding quencher, all the remaining

protecting were removed by TFA with 5% TIS. The final product was purified by HPLC and characterized by UV-visible spectroscopy

and ESI mass spectrometry.

Cleavage from resin (iii)

Mtt deprotection (iv)

(Pbf)

(Pbf) (Mtt)

(Pbf)

(Pbf) (NH₂)

O

PS-GRIGFLRTAKGG

N

H

PS-GRIGFLRTAKGG-OH

1, 22

O

2

OtBu

BHQ3 or BBQ coupling (v)

Side-chains

deprotection ((vi)

Fmoc deprotection (i)

PS coupling (ii)

%

24 15

25 17

26 13

Pyro(a)-GRIGFLRTAK(BHQ3)GG-OH

Pyro(a)-GRIGFLRTAK(BBQ)GG-OH

P1-GRIGFLRTAK(BHQ3)GG-OH

(

Pbf)

(Pbf) (Mtt)

O

Fmoc-GRIGFLRTAKGG

N

H

O

OtBu

P1-PEG-GRIGFLRTAK(BHQ3)GG-OH 27 13

BHQ3 coupling (v)

Side-chains

deprotection ((vi)

Fmoc deprotection (i)

PEG and PS coupling (ii)

Cleavage from resin (iii)

Mtt deprotection (iv)

(Pbf)

(Pbf) (Mtt)

(Pbf)

(Pbf) (NH₂)

O

P1-PEG-GRIGFLRTAKGG

N

H

P1-PEG-GRIGFLRTAKGG-OH

23

O

OtBu

Scheme 5. Synthesis of PMBs 24-27. (i) Fmoc deprotection: 20% piperidine in DMF. (ii) Coupling: HBTU, NMM, DMF. (iii) Cleavage from resin:

DCM/TFE/acetic acid. (iv) Mtt deprotection : 3% TFA, 5% TIS in DCM. (v) BBQ-NHS or BHQ3-NHS, DMSO with 2% DIPEA, 16 h, under N . (vi) Side-

2

chains deprotection: 95 % TFA, 5% TIS.

2.2. Photophysical properties.

In order to obtain FRET between the donor and the acceptor, dye excited state transfers energy to quencher ground state generating

an excited quencher. A requirement to have FRET is that the fluorescence emission spectrum of the donor (here, PS) must overlap the

3

5

1

absorption spectrum of the acceptor (here, the quencher). BBQ and BHQ3 are suitable as dual fluorescence and O

2

quencher for both

studied PSs (Figure 1).

Figure 1. Spectral overlap of PS fluorescence emission (red porphyrin, purple Pyropheophorbide (a)) with BBQ650 and BHQ3 absorption (yellow BHQ3, blue

BBQ650) in ethanol. Spectra are normalized.

The fluorescence spectra of BBQ and BHQ3 in ethanol are presented in Figure S2 in supporting information. The absorption spectra

of all the compounds are presented in Figure 2.

-6

-5

Figure 2. Absorption spectra of compounds with P1-COOH (1) (4.05 x 10 mol/L) and with Pyro(a)-COOH (1.06 x 10 mol/L) in ethanol.

As expected, the absorption spectra of the porphyrinic compounds are composed of high Soret band at 414 nm and four Q-bands. For

Pyro(a)-COOH compounds, the Q-bands are more intense especially QI and is shifted to the red part of the spectrum (670 nm). In the

PMBs, a large band centered at 650 nm can be observed due to the presence of the quencher BHQ3 (PMBs 24 and 26-27) or at 570 nm

due to the presence of BBQ (PMBs 20 and 25).

The fluorescence spectra of all the compounds are presented in Figure 3.

Figure 3. Fluorescence spectra of compounds with P1-COOH (1) and with Pyro(a)-COOH in ethanol (A414nm = 0.2).

As expected, the fluorescence spectra are typical of porphyrin with two bands at 650 nm and 720 nm and of Pyro(a)-COOH with an

intense band at 670 nm and a small one at 730 nm. After the grafting of the quencher, all the PMBs present a very weak fluorescence

due to the energy transfer between the photosensitizer and the quencher. With this kind of construction, it is commonly said that FRET

9

occurs. Zheng et al. in 2007 synthesized a Pyro(a)-GPLGLARK(BHQ3). They claim that Pyro(a) and BHQ3 are conjugated to the

1

opposite ends of the MMP-7-specific cleavable peptide linker to keep them in close proximity, enabling FRET and O

2

quenching to

make this construct not only optically silent but also photodynamically inactive. Other publications of the same team describe a FRET

36

mechanism. For example, Stefflova et al. in 2006 synthesized Pyro(a)-GDEVDGSGK-(BHQ3). They claimed that FRET occurs

1

4

between both entities. It is the same for Lo et al. in 2009 with Pyro(a)-TSGPNQEQK(BHQ3). The quenching data of FAP-PP by free

BHQ3 were further analyzed using the Stern-Volmer equation and the Stern-Volmer plots for both solutions. In DMSO, the Stern-

4

-1

3

Volmer plot has a linear relationship giving a high quenching constant (Ksv) 5.3 x 10 .mol .dm ), validating the choice of BHQ3 as an

excellent quencher for Pyro(a)’s fluorescence. However, in the buffer solution, the plot shows an upward curvature suggesting that the

Pyro’s fluorescence is quenched by both dynamic and static quenching mechanisms.

We did Stern-Volmer plots for BHQ3 (Figure S3 in supporting information). As expected the fluorescence intensity decreases with

4

-1

4

-1

the addition of the quencher and we find a high quenching constant of 4.6 x 10 .mol .L for the porphyrin and 2.5 x 10 .mol .L for the

1

4

pyropheophorbide (a) (slightly different than the value obtained by Lo et al. in 2009 ). We also recorded fluorescence lifetime that did

not changed upon addition of the quencher : 11.2 ns for the porphyrin and 7.2 for the pyropheophorbide. We can then conclude that it is

a static quenching.

1

The O

2

emission spectra are presented in Figure 4.

1

Figure 4. O

2

emission of compounds with P1-COOH (1) and with Pyro(a)-COOH in ethanol (A414nm = 0.2).

1

No change can be observed after grafting of the peptide onto the photosensitizers. As expected, a drastic decrease of O

2

is observed

after the coupling of the quencher (Figure 5).

-

6

-1

Figure 5. Fluorescence lifetime of compounds with P1-COOH (1) and with Pyro(a)-COOH in ethanol (λexc = 408 nm, C = 1 x 10 mol.L ).

The decays of P1-COOH and of Pyro(a)-COOH are monoexponential, but a second, shorter lived and dominating component is

observed for 20, 26 and 27, as well as for 24 and 25 (Figure 6). This is consistent with quenching but the fact that the longer-lived

emission can still be seen probably indicates that the constructs exist as a mixture of conformers in which the photosensitizer-quencher

distance is different. This is in agreement with the quenching values of Table 2. We could not observe a total quenching.

1

-6

-1

Figure 6. O

2

lifetime of compounds with P1-COOH (1) and with Pyro(a)-COOH in ethanol (λexc = 408 nm, C = 1 X 10 mol.L ).

1

As expected and seen in Figure 6, no O

2

can be detected in the PMBs.

All the photophysical properties of the PS-peptide derivatives are summed up in Table 1.

Table 1. Summary of PS and PS-peptide derivatives photophysical properties in ethanol.

Product

λ

QI

nm)

ε

QI

φ

f

φ

∆

τ

F

τ

∆

(± 1 µs)

−1

(L.mol .cm )

(

(± 0.02)

0.10

0.11

0.09

0.10

0.09

0.27

0.34

0.33

(± 0.05)

0.64

0.67

0.61

0.62

0.66

0.58

0.63

0.52

0.56

0.52

(± 0.1 ns)

10.0

10.2

10.1

10.2

10.1

9.8

(

1) P1-COOH

645

668

4 276

14

5) P1-GRIGFLRTAKGG-OH

9) H-GRIGFLRTAK(P1)GG-OH

8) P1-PEG-GRIGFLRTAKGG-OH

3 606

16

4 347

18

4 934

14

(

14) P1-GRIGF-OH

4 262

15

11) H-LRTAK(P1)GG-OH

16) P1-PEG-GRIGF-OH

4 197

14

3 618

10.1

6.4

14

-

Pyro(a)-COOH

24 285

30 788

31 709

14

(

6) Pyro(a)-GRIGFLRTAKGG-OH

6.7

13

(

15) Pyro(a)-GRIGF-OH

6.7

12

ε: molar extinction coefficient; Q: Q band; λQI: wavelength of band QI; λexc: excitation wavelength; ϕf: fluorescence quantum yield; ϕ∆: luminescence

quantum yield; τf: fluorescence lifetime; τ∆: singlet oxygen lifetime.

Photophysical properties of PMBs are listed in Table 2. For all PMBs synthesized, efficient quenching of fluorescence intensity and

thus quantum yields were observed with values decreasing by 80-93% for all PMBs as compared to the corresponding photosensitizers.

Generation of singlet oxygen dramatically decreased: PMBs quantum yields were observed from 0-0.25 compared to the corresponding

PS from 0.52 (Pyro(a)-COOH) and 0.64 (P1-COOH 1). If the fluorescence quenching never reaches 100%, the singlet oxygen

quenching is better since we could obtain 100% singlet oxygen quenching for 26, 27 and 24. These three PMBs could be used safely in

their uncleaved state.

Table 2. PMBs’ photophysical properties in ethanol.

1

ϕf

% Fluo

ϕ∆

% O

2

Τf

τ∆

R0

Product

(±0.02) inhibited

(±0.05)

inhibited

(± 0.1 ns)

(± 1 µs)

(Å)

(

-

(

-

(

1)

P1-COOH

0.10

-

0.64

-

10.0

14

-

BBQ

-

8.4 0.7

9.7 1.7

9.2 0.9

9.4 1.2

6.4

-

20)

26)

27)

P1-GRIGFLRTAK(BBQ)GG-OH

P1-GRIGFLRTAK(BHQ3)GG-OH

P1-PEG-GRIGFLRTAK(BHQ3)GG-OH

Pyro(a)-COOH

0.01

0.02

0.27

0.02

0.03

90

80

0.13

0

80

100

0

34.6

41.3

-

0

80

-

0

100

-

0

0.52

0

15

0

24)

25)

Pyro(a)-GRIGFLRTAK(BHQ3)GG-OH

Pyro(a)-GRIGFLRTAK(BBQ)GG-OH

93

100

6.5

49.9

40.3

88

0.12

77

5.8 0.8

0

f ∆ f ∆ 0

λexc: excitation wavelength; ϕ : fluorescence quantum yield; ϕ : luminescence quantum yield; τ : fluorescence lifetime; τ : singlet oxygen lifetime; R : Förster

radius.

2

.3. Enzymatic cleavage.

All enzymatic cleavages were monitered firstly by HPLC to compare the retention times with the expected standards (11-16)

prepared before, and secondly by LC-MS to confirm the mass of the detected degradation products. Different substrate concentrations

-

1

(

2.5, 10, 50 and 100 µmol.L for 4, 5, 6) were tested using MMP-2, MMP-9 and MMP14 active enzymes in order to determine the best

-1

HPLC conditions. We determined that 100 µmol.L was a suitable concentration for both peptide and peptide derivatives, provided that

a small amount of DMSO was used to increase the solubility (Table 3).

Table 3. Enzymatic activity results for the free peptides, PS-peptides and quencher-peptides (C = 100

-

1

µmol.L ).

Compound

MMP (hydrolysis %)

MMP-2

26

MMP-9

MMP-14

Peptides

4

7

5

9

8

6

-

99

20

-

ε

-

100

PS-peptides

-

3

ε

2

5

Peptide-quencher

10

-

31

86

1

9

26

2

.3.1. Peptides.

The free peptides H-GRIGFLRTAKGG-OH (4) and H

MMP-14 and a significant reduction of the peak intensity was observed by HPLC. On the opposite for both peptides, a new peak was

observed which matches with the cleaved peptides H-GRIGF-OH (12) and H N-PEG-GRIGF-OH (13), respectively as assessed by the

2

N-PEG-GRIGFLRTAKGG-OH (7) were efficiently cleaved by MMP-2 and

2

retention times and LC-MS. These results showed that adding a PEG spacer at the N-terminus in 7 did not impact the enzymatic

activity. Finally and at the same concentration, MMP-9 was inactive for both peptides

2

.3.2. Photosensitizer-peptides.

Concerning the 2 possible positions of P1 in compounds 5 and 9, none preferred linkage has been evidenced since no enzymatic

activity occurred with the 3 different MMP. However and more interestingly, the incorporation of a PEG spacer in 8 induced a slight

activity by MMP-2 and 9 if compared to 5. This slight activity resulted in the apparition of a new peak in HPLC and which matched

with the P1-PEG-GRIGF-OH (16) as assessed by LC-MS. Finally, the nature of the PS seems to play a role and the comparison of 5

and 6 in Table 3 clearly indicated that Pyro(a) containing PMB is more cleaved than P1 containing PMB. A new peak was detected

corresponding to the cleaved fragment Pyro(a)-GRIGF-OH (15).

2

.3.3. Peptide-quenchers.

MMP-14 were found to efficiently cleave the peptide-quencher H-GRIGFLRTAK(BBQ)GG-OH 10 with a degree of hydrolysis

equal to 31%. A significant decrease of the peak intensity of 10 was observed by HPLC and a new distinct peak was detected by LC-

MS which matched to the cleaved fragment H-LRTAK(BBQ)GG-OH. The same cleavage was observed for H-

GRIGFLRTAK(BHQ3)GG-OH 19 by MMP-2 (26% hydrolysis) and more particularly by MMP-14 with a 86% hydrolysis level. A

new peak corresponding to H-LRTAK(BHQ3)GG-OH clearly is detected by LC-MS and HPLC.

2

.3.4. Photodynamic molecular beacon.

In a first trial, we used the same conditions than we used for the peptides with a concentration of PMB of about 100 µM (ratio

peptide/MMP = 16 000). In these conditions, none of the PMBs was cleaved (data not shown). In order to increase the degradation rate,

we chosed a ratio peptide/MMP of 250. The enzymatic activities of MMP-2, -9 and -14 with PMBs are presented in Figure 7.

Figure 7. Enzymatic cleavage with MMP-2, 9, 14 with a ratio PMB/enzyme = 250 for all PMBs

Figure 7 raises some comments:

i) Whatever the nature of the PS and the quencher, MMP-9 was the less active enzyme for all the PMB. Contrary to what was

observed in Table 3 for the differents constituents, the best MMP’s activity was attributed to MMP-2 followed by MMP-14 (except 25)

ii) Concerning the nature of the quencher, comparisons between 20 and 26 and between 24 and 25 suggested that BHQ3 induced a

better hydrolysis and this observation is in agreement with the data reported in Table 3 for the peptide-quenchers (two last lines).

iii) Concerning the nature of the PS, comparisons between 20 and 25 and between 24 and 26 showed that Pyro(a) was a better

candidate than P1 accordlingly with the results of 5 and 6 in Table 3.

iv) The addition of the PEG spacer in 27 (compared to 26) increased the hydrolysis of PMB which even exceeded that of compound

2

4. This fact could indicate that P1-PEG could be used in place of the expensive Pyro(a) and that the steric hindrance seems to play a

key-role in the recognition of such molecular constructions.

Substrate cleavage by MMPs depends on the catalytic domain of MMPs. It consists of a 5-stranded β sheet, 3 α helices and

connective loop. To be cleaved, the peptide must coordinate with the catalytic zinc and the pocket “S1” located to the right of the zinc

3

7

atom. The pocket S1 is variably hydrophobic according to the MMPs and gives their substrate specificity. As it was described by Zhu

38

et al., for MMP-13, it could be of great interest to use molecular modelling to study the interaction of our probes with crystal structure

of MMPs.

Nevertheless, to validate the use of PMB in vivo, we performed in vitro cell cleavage assays on PMB 20 which were tested on

conditioned media and cells. For the cells, we decided to use B16F1 cells as they produced MMP-2,-9 and in high proportion MT1-

3

9

MMP.

Figure 8 indicates that MMP-2 and MMP-9 from B16F1 conditioned media are able to cleave PMB 20. Interestingly, results

obtained from cells, where MMP-14 is largely found, indicated that MMP-14 seems to increase the cleavage of the PMB 20.

These data demonstrate the proteolytic activation of PMB 20 by B16F1 cells. As it was described by Liu et al., in the case of breast

cancer metastasis, the possibility to use this type of compound is very promising by limiting the nonspecific accumulation of active

4

0

compound in normal tissues, preserving critical organs and allowing an aggressive approach against tumoral cells.

41

Considering that glioblastoma multiforme (GBM) expressed MMP-2 and/or MMP-9 and/or MMP-14, it seems that our PMB could

be of interest for developing new approaches to fight against this type of cancers.

Figure 8. Enzymatic cleavage of PMB 20 with conditioned media and B16F1 cells

3

. Conclusion

Photodynamic therapy is an innovative treatment modality against cancer. One of the drawbacks is the lack of selectivity of the

photosensitizer. Several researches focus on the development of targeted photosensitizers or the use of nanoparticles to improve the

accumulation of the photosensitizer into the tumor or the neo-vessels. In this article, we prefer focusing on the development of

macrostructures that lead to selective production of singlet oxygen upon activation by MMP-2, -9 and -14 that are over-expressed in the

tumoural environment. 5 PMBs were successfully synthesized and three of them were well cleaved by MMP-2 and MMP-14. As MMP-

2

is activated by MMP-14, PMBs developed here possess the expected selectivity for a future biological application. Nevertheless, in

order to optimize the sensitivity of PMB, it will be necessary to improve the cleavage efficiency. In this way, molecular docking

experiments will be realized. These studies should allow a better understanding of the difference of reactivity with the enzymes of all

the compounds synthesized here and optimize their future developments.

4

. Experimental section

4

.1. Chemicals and materials.

Pyro(a)-COOH) was purchased from Frontier Scientific. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

(

HBTU), all amino acid derivatives and the different resins for peptide synthesis were purchased from Senn Chemicals AG or Iris

Biotech. Fmoc-Lys(BBQ650)-OH and BBQ650-NHS were purchased from Berry & Associates and the black hole quencher 3

carboxylic acid succinimidyl ester (BHQ3-NHS) was obtained from Biosearch Technologies. 2-[2-(Fmoc-amino)ethoxy]ethoxyacetic

acid (called here PEG) and Fmoc-Lys-OH.HCl were purchased from Fluka. Other chemicals were obtained from Sigma-Aldrich.

The MMP-2 Human recombinant active (PF023), MMP-9 Human recombinant active (PF140) and MMP-14 Human recombinant

catalytic domain (475935) were purchased from Merck Millipore.

1

All the compounds were obtained with at least 95% purity as assessed by HPLC, H NMR and mass spectrometry. All the

compounds (and particularly the peptides) are sensitive to temperature. So we did not measure the melting points.

Reverse-phase high-performance liquid chromatography (HPLC) were performed on Prostar HPLC (Varian). Analytical HPLC were

done with a Pursuit 5-C18 column (2.5 µm, 4.6 × 150 mm, Varian) and preparative HPLC with Pursuit 5-C18 column (5 µm, 21.2 ×

1

50 mm, Varian), using a photodiode array detector (UV-Visible detection and fluorescence detector (Varian). Liquid chromatography

coupled to Mass spectrometry (LC-MS) experiments for the cleavage tests were performed on a LCMS-2020 Shimadzu (electrospray

ionization ESI) with a photodiode array detector (UV-Visible detection, SPD-M20A detector). High resolution mass spectrometry

(

HRMS) experiments were performed on a micro-Tof Bruker (electrospray ionization ESI+, 50–1000 in low and 50–2500 in width).

1

NMR spectra ( H, COSY and TOCSY) were recorded on a BRUKER AVANCE spectrometer at 300 MHz.

4

.2. Synthesis of photosensitizers

.2.1. P1-COOH: 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (1)

5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (1). A stream of nitrogen in dichloromethane (700 mL) was let for 30 min. 3.5 mL

of freshly distilled pyrrole (50 mmol), 3.8 mL of benzaldehyde (37.5 mmol) and 4-carboxybenzaldehyde (1.875 g, 12.5 mmol) were

poured to the DCM solution. After 15 min, 0.615 mL of boron trifluoride diethyl etherate (5 mmol) was added and the reaction was

stirred for 2 h. Then, p-chloranil (9.225 g, 37.5 mmol) was introduced. The mixture was stirred at 60°C, overnight, in the dark and

under nitrogen. After cooling to room temperature, the solvent was removed. The crude product was purified by column

chromatography on silica gel (DCM/EtOH, 97/3, v/v), yielding compound 1 (738.5 mg, 11%) as purple crystals. R

f

= 0.35 (DCM/EtOH

1

=

97:3, v/v). H NMR (300 MHz, DMSO-d

H, Ho-phenyl -COOH), 8.84 (s, 8H, Hβ-pyrrole), 13.24 (br, 1H, C-OOH). HRMS (ESI+): m/z calcd. for C45

59.2476. UV/Vis (EtOH): λmax(log ε) = 414 (5.49), 512 (4.13), 546 (3.81), 588 (3.65), 645 nm (3.43).

6

): δ (ppm) = -2.92 (s, 2H, NH), 7.83 (s, 9H, Hm- and p-phenyl), 8.24 (d, 6H, Ho-phenyl), 8.37 (dd,

+

4

6

30 4 2

H N O [M+H] 659.2442; found

4

.2.2. P1-NHS: 5-(4-carboxyphenyl succinimidyl ester)-10,15,20-triphenyl-21, 23-porphyrin (2)

-(4-carboxyphenyl succinimidyl ester)-10,15,20-triphenyl-21,23-porphyrin (2). In the dark and under inert atmosphere, compound

1) (498.5 mg, 0.76 mmol) was dissolved in 50mL of DCM and N-hydroxysuccinimide (177.2 mg, 1.54 mmol) and

5

(

N,N’-hexylcarbodiimide (318.1 mg, 1.54 mmol) were added. The mixture was stirred at 40°C, for 4 h under argon. Purification of the

crude material was performed using a silica gel column with DCM, and compound (2) was obtained as a purple solid with a yield of

1

7

5% (377.3 mg). R

f

= 0.79 (DCM/EtOH = 97:3, v/v). H NMR (300 MHz, DMSO-d

6

): δ (ppm) = -2.91 (s, 2H, NH), 3.00 (s, 4H, CH

2

),

.84 (s, 9H, Hm- and p-phenyl), 8.22 (d, 6H, Ho-phenyl), 8.50 (dd, 4H, Ho-phenyl-CO), 8.85 (s, 8H, Hβ-pyrrole). HRMS (ESI+): m/z calcd. for

+

49 33 5 4

C H N O [M+H] 756.2605; found 756.2588. UV/Vis (EtOH): λmax(log ε) = 415 (5.70), 512 (4.35), 545 (3.99), 589 (3.82), 645 nm

(

3.66).

4

1

.2.3. Fmoc-Lys(P1)-COOH: 2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-[5-(4-carboxyphenyl)-

0,15,20triphenylporphyrin]-hexanoic acid (3).

In the dark under a nitrogen atmosphere, to a solution of Fmoc-Lys-OH.HCl (82.8 mg, 0.20 mmol) in a minimum of DMF were

added N-hydroxysuccinimide activated porphyrin (2) (152.4 mg, 0.20 mmol) and triethylamine (0.21 mmol, 28.3 µL) in 10 mL of

DCM. After been stirred at ambient temperature for 24 h, the solvent was removed and the crude material was purified by column

chromatography on silica gel using EtOH/DCM: 5/95 (v/v) as the eluent. Solvent was removed in vacuo and the residue recrystallized

1

using CHCl

3

/n-hexane. The pure compound was isolated as a purple solid (166.5 mg, 82%). R

f

= 0.45 (DCM/EtOH = 95:5, v/v). H

NMR (300MHz, DMSO-d

6

): δ (ppm) = 12.59 (br, 1H, OH), 10.52 (s, 1H, NH-COOH), 8.82 (d, 8H, Hβ-pyrrole), 8.24 (d + s, 10H, 6H

o

+

4

H

o-m phenyl-CO), 7.82 (s, 9H, Hp-m phenyl-CO), 7.71 et 7.33 (2 x m, 8H, Hphenyl-Fmoc), 5.75 (s, 1H, NH), 4.29 (m, 3H, 2HCH2-Fmoc + 1HCHα-Fmoc),

.02 (m, 1H, HCH-Fmoc), 3.44 (s, 2H, HCH2ε-Lysine), 1.70 (m, 6H, HCH2 α,β,δ), -2.9 (s, 2H, NH-pyrrole). HRMS (ESI+): m/z calcd. for

+

66 52 6 5

C H N O [M + H] , 1009.3999; found 1009.4051. UV/Vis (EtOH): λmax(log ε) = 414 (5.16), 512 (4.14), 546 (3.74), 588 (3.55), 645

nm (3.37).

4

.3. Synthesis of peptides

4

.3.1. H-GRIGFLRTAKGG-OH (4).

The side chains of arginine, threonine and lysine were respectively protected by 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

(

Pbf), 5-tert-butyl ester (OtBu) and N-tert-butyloxycarbonyl (Boc) groups. 4 was synthesized on a multichannel peptide synthetizer

ResPep XL, Intavis AG Bioanalytical Instrument, Germany), according to a classical Fmoc/tBu solid phase methodology, using a

Fmoc-Gly-Wang resin on a 100 µmole scale. Double coupling, except for the first arginine and glycine (four and six coupling were

respectively necessary), were performed using a 3-fold excess of N-Fmoc-amino acid, activation reagents 2-(1H-benzotriazol-1-yl)-

1

,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3 eq.), N-methylpyrrolidone NMP (3 eq.) and N-methylmorpholine (NMM)

(

9 eq.) in dimethylformamide (DMF). Final Fmoc protection was removed with 20 % piperidine in DMF and the peptide on resin was

dried under vacuum. Then, the peptide was cleaved from the resin using TFA/triisopropylsilane (TIS)/water (92.5/5/2.5%) for 2 h. The

resin was filtered and washed with 2 mL of TFA and 20 mL of DCM. The filtrate was dried under vacuum and lyophilisated. The crude

product was further purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25

1

min, followed by isocratic acetonitrile for 15 min. R

300 MHz, DMSO-d ): δ (ppm) = 0.79 (6H, CH (δ+γ), Ile), 0.84 (6H, δCH

βCH , Ala), 1.31 (2H, γCH , Lys), 1.40 (2H, γCH , Arg1), 1.41 (1H, γH, Ile), 1.46 (2H, γCH

Arg1), 1.51 (3H, δCH + βH, Lys), 1.57 (1H, βH, Arg2), 1.59 (2H, βH, Leu), 1.61 (1H, βH, Arg1), 1.68 (1H, βH, Ile), 1.69 (1H, βH,

Lys), 1.72 (1H, βH, Arg2), 2.71 (1H, βH, Phe), 2.71 (2H, εCH , Lys), 3.01 (1H, βH, Phe), 3.07 (2H, δCH , Arg1), 3.10 (2H, δCH

Arg2), 3.50-3.80 (8H, αH, Gly), 4.05 (1H, βH, Thr), 4.18 (1H, αH, Ile), 4.19 (1H, αH, Lys), 4.22 (1H, αH, Thr), 4.28 (1H, αH, Ala),

.29 (1H, αH, Leu),4.30 (1H, αH, Arg2), 4.43 (1H, αH, Arg1), 4.50 (1H, αCH , Phe), 7.19 (5H, Haromatic, Phe),7.68 (1H, εNH,

Arg1),7.78 (1H, NH, Thr), 7.81 (2H, εNH, Lys), 7.94 (1H, NH, Phe), 7.93 (1H, εNH, Arg2), 7.96 (1H, NH, Ala), 7.98 (1H, NH, Ile),

t

= 8.2 min. Pure product was isolated as a white powder (27.8 mg, 23 %). H NMR

(

6

3

, Leu), 1.02 (3H, γCH

3

, Thr), 1.10 (1H, γH, Ile), 1.22 (3H,

3

2

, Arg2), 1.48 (1H, γH, Leu), 1.49 (1H, βH,

2

,

4

2

8

.14 (1H, NH, Lys), 8.17 (1H, NH, Arg2), 8.19 (1H, NH, Leu), 7.85-8.26 (5H, NH, Gly), 8.49 (1H, NH, Arg1). HRMS (ESI+): m/z

+

2

+

93 19

calcd. for C54H N O14 [M+H] 1232.7149; found 1232.7172, [M+2H] 616.8647; found 616.8669.

4

.3.2. H-GRIGF-OH (12).

The same method was used starting from a Fmoc-Phe-Wang resin. R

t

= 8.6 min. Pure product was isolated as a white powder (10.5

(δ+γ), Ile), 1.08 (1H, γH, Ile), 1.42 (1H, γH, Ile), 1.47 (2H, γCH

Arg), 1.53 (1H, βH, Arg), 1.67 (1H, βH, Arg), 1.67 (1H, βH, Ile), 2.81 (1H, βH, Phe), 2.99 (1H, βH, Phe), 3.06 (2H, δCH , Arg),3.58

2H, αH, Gly), 3.74 (2H, αH, Gly), 4.16 (1H, αH, Ile), 4.33 (2H, αCH , Phe), 4.41 (1H, αH, Arg), 7.23 (5H, Haromatic, Phe), 8.01 (1H,

NH, Phe), 8.08 (1H, NH, Ile), 8.20 (1H, εNH, Arg), 8.22 (3H, NH, Gly), 8.61 (1H, NH, Arg). HRMS (ESI+): m/z calcd. for C25

1

mg, 38 %). H NMR (300 MHz, DMSO-d

6

): δ (ppm) =0.76 (6H, CH

3

2

,

2

(

2

40 8 6

H N O

+

[

M+H] 549.3144; found 549.3170.

4

.3.3. H N-PEG-GRIGFLRTAKGG-OH (7).

2

The peptide H-GRIGFLRTAKGG-Wang resin was synthesized as previously described. 2-[2-(Fmoc-amino)ethoxy]ethoxyacetic

acid (PEG, 2 eq., 77 mg) was then added with HBTU (1.5 eq.), NMM (9 eq.) and NMP (3 eq.) in DMF. The reactor was shaken

overnight in the dark. Fmoc protection was removed with 20% piperidine in DMF and the peptide on resin was dried under vacuum,

then cleaved using TFA/TIS/water (92.5/5/2.5%) for 2 h. The acidic resin was filtered and washed with 2 mL of TFA and 20 mL of

DCM. The filtrate was dried under vacuum and lyophilisated. The crude product was further purified by preparative HPLC using

acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25 min, followed by isocratic acetonitrile for 15 min. R

t

= 7.9

(δ+γ), Ile),

, Lys), 1.40 (2H, γCH , Arg1),

+ βH, Lys), 1.57 (1H, βH, Arg2),

.59 (2H, βH, Leu), 1.61 (1H, βH, Arg1), 1.68 (1H, βH, Ile), 1.69 (1H, βH, Lys), 1.72 (1H, βH, Arg2), 2.71 (1H, βH, Phe), 2.71 (2H,

, Lys), 3.01 (1H, βH, Phe), 3.07 (2H, δCH , Arg1), 3.10 (2H, δCH , Arg2), 3.50-3.80 (8H, αH, Gly), 3.61 (2H, CH , PEG), 3.71

2H, CH , PEG), 3.79 (2H, CH , PEG), 3.94 (2H, CH , PEG), 4.05 (1H, βH, Thr), 4.18 (1H, αH, Ile), 4.19 (1H, αH, Lys), 4.22 (1H, αH,

Thr), 4.28 (1H, αH, Ala), 4.29 (1H, αH, Leu), 4.30 (1H, αH, Arg2), 4.43 (1H, αH, Arg1), 4.50 (1H, αCH

Phe), 7.68 (1H, εNH, Arg1), 7.78 (1H, NH, Thr), 7.81 (2H, εNH, Lys), 7.94 (1H, NH, Phe), 7.93 (1H, εNH, Arg2), 7.96 (1H, NH, Ala),

1

min. Pure product was isolated as a white powder (14 mg, 10 %). H NMR (300 MHz, DMSO-d

6

): δ (ppm) = 0.79 (6H, CH

, Ala), 1.31 (2H, γCH

, Arg2), 1.48 (1H, γH, Leu), 1.49 (1H, βH, Arg1), 1.51 (3H, δCH

3

0

1

.84 (6H, δCH

.41 (1H, γH, Ile), 1.46 (2H, γCH

3

, Leu), 1.02 (3H, γCH

3

, Thr), 1.10 (1H, γH, Ile), 1.22 (3H, βCH

3

2

εCH

2

(

2

, Phe), 7.19 (5H, Haromatic,

7

.98 (1H, NH, Ile), 8.14 (1H, NH, Lys), 8.17 (1H, NH, Arg2), 8.19 (1H, NH, Leu), 7.85-8.26 (5H, NH, Gly), 8.49 (1H, NH, Arg1).

2+ 3+ 4+

104 20

HRMS (ESI+): m/z calcd. for C60H N O17 [M+2H] 689.4017; found 689.3970, [M+3H] 459.9369; found 459.9350, [M+4H]

3

45.2045; found 345.2032.

4

.3.4. H N-PEG-GRIGF-OH (13).

2

The same method was used starting from a Fmoc-Phe-Wang resin. . 2-[2-(Fmoc-amino)ethoxy]ethoxyacetic acid (PEG, 2 eq., 77

mg) was then added with HBTU (1.5 eq.), NMM (9 eq.) and NMP (3 eq.) in DMF. The reactor was shaken overnight in the dark. Fmoc

protection was removed with 20% piperidine in DMF and the peptide on resin was dried under vacuum, then cleaved using

TFA/TIS/water (92.5/5/2.5%) for 2 h. The crude product was further purified by preparative HPLC using acetonitrile/water (0.1 %

TFA) [10:90] to 100 % acetonitrile gradient in 25 min, followed by isocratic acetonitrile for 15 min. R

t

= 8.2 min. Pure product was

(δ+γ), Ile), 1.05 (1H, γH, Ile), 1.45

, Arg), 1.53 (1H, βH, Arg), 1.67 (1H, βH, Arg), 1.75 (1H, βH, Ile), 2.90 (1H, βH, Phe), 3.00 (1H, βH,

, Arg), 3.61 (2H, CH , PEG), 3.71 (2H, CH , PEG), 3.79 (2H, CH , PEG), 3.80 (2H, αH, Gly), 3.94 (2H, CH

PEG), 4.15 (1H, αH, Ile), 4.20 (2H, αH, Gly), 4.35 (1H, αH, Arg), 4.40 (2H, αCH , Phe), 7.23 (5H, Haromatic, Phe), 7.53 (1H, εNH, Arg),

.80 (2H, NH, Gly), 7.85 (1H, NH, Ile), 8.15 (1H, NH, Phe), 8.17 (1H, NH, Arg), 12.80 (br, -OH). HRMS (ESI+): m/z calcd. for

1

isolated as a white powder (18 mg, 26 %). H NMR (300 MHz, DMSO-d

1H, γH, Ile), 1.47 (2H, γCH

Phe), 3.06 (2H, δCH

6 3

): δ (ppm) = 0.76 (6H, CH

(

2

,

2

7

C

+

2+

31 51 9 9

H N O [M+H] 694.3883; found 694.3913, [M+2H] 347.6978; found 347.6978.

4

.4. General procedures for the conjugates photosensitizer-peptide syntheses

.4.1. Synthesis of PS-GRIGFLRTAKGG-OH (5) and (6). Procedure A

The peptide H-GRIGFLRTAKGG-Wang resin was synthesized as previously described. The corresponding PS (1.2 eq.) was then

added with HBTU (1.5 eq.) and NMM (2 eq.) in DMF. The reactor was shaken for 3 days in the dark. The resin obtained was dried

under vacuum and then cleaved using TFA/TIS/water (92.5/5/2.5%) for 2 h. The acidic resin was filtered and washed with 2 mL of

TFA and 20 mL of DCM. The filtrate was dried under vacuum and lyophilisated. The crude product was further purified by preparative

HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25 min, followed by isocratic acetonitrile for 15

min.

1

P1-GRIGFLRTAKGG-OH (5). R

DMSO-d ): δ (ppm) = -2.95 (2H, NH, P1-CO), 0.85 (6H, CH

.23 (3H, βCH , Ala), 1.32 (2H, γCH , Lys),1.52 (3H, δCH

.72 (1H, βH, Lys), 1.55, 1.80 (8H, 2 x γCH + 2 x 2 βH, Arg), 1.85 (1H, βH, Ile), 2.73 (2H, εCH

, Arg), 3.95 (1H, βH, Thr), 4.00, 4.15, 4.35, 4.50 (8H, αH, Gly), 4.19 (1H, αH, Ile), 4.20 (1H, αH, Lys),

.20 (1H, αH, Thr), 4.25 (1H, αH, Leu),4.32 (1H, αH, Ala), 4.33 (1H, αH, Arg2), 4.47 (1H, αH, Arg1), 4.52 (1H, αCH , Phe), 7.25 (5H,

aromatic, Phe), 7.57 (1H, εNH, Arg1), 7.65 (1H, εNH, Arg2),7.65 (2H, εNH, Lys), 7.69 (1H, NH, Thr),7.85 (1H, NH, Ile), 7.89 (1H, NH,

t

= 19.7 min. Pure product was isolated as a red powder (41.2 mg, 22 %). H NMR (300 MHz,

6

3

(δ+γ), Ile), 0.85 (6H, δCH

+ βH, Lys), 1.55 (1H, γH, Ile), 1.60 (1H, γH, Leu), 1.70 (2H, βH, Leu),

, Lys), 2.74 (1H, βH, Phe), 2.99 (1H,

3 3

, Leu),1.00 (3H, γCH , Thr),1.10 (1H, γH, Ile),

1

3

2

βH, Phe), 3.10 (4H, 2 x δCH

2

4

2

H

Ala), 7.93 (1H, NH, Phe), 7.95 (9H, Hm- and p-phenyl, P1-CO), 7.95 (1H, NH, Lys), 8.10 (1H, NH, Leu), 8.15 (1H, NH, Arg1), 7.67, 7.97,

8

.22, 9.18 (4H, NH, Gly), 8.25 (1H, NH, Arg2), 8.25 (6H, Ho-phenyl, P1-CO), 8.34 (4H, Ho-phenyl-CO, P1-CO), 8.84 (8H, Hβ-pyrrole, P1-CO).

2

+

3

+

121 23

HRMS (ESI+): m/z calcd. for C99H N O15 [M+2H] 936.9707; found 937.4809, [M+3H] 624.9877; found 624.9914. UV/Vis

(

EtOH): λmax(log ε) = 414 (5.49), 512 (4.13), 546 (3.79), 588 (3.60), 645 nm (3.56).

1

Pyro(a)-GRIGFLRTAKGG-OH (6). R = 18.7 min. Pure product was isolated as a dark green powder (47 mg, 27 %). H NMR (300

t

MHz, DMSO-d ): δ (ppm) = -2.14, 0.68 (2H, NH, Pyro(a)), 0.72 (6H, CH (δ+γ), Ile), 0.95 (6H, δCH , Leu), 0.99 (1H, γH, Ile), 1.02

3H, γCH , Thr), 1.23 (3H, βCH , Ala), 1.32 (2H, γCH , Lys), 1.37 (1H, γH, Ile), 1.45 (8H, γCH , Arg1), 1.48 (1H, γH, Leu), 1.52 (3H,

δCH + βH, Lys), 1.57 (8H, γCH , Arg2), 1.60 (2H, βH, Leu), 1.62 (3H, C : CH , Pyro(a)), 1.67 (1H, βH, Ile), 1.70 (1H, βH, Lys),1.72

2H, βH, Arg1), 1.78 (2H, βH, Arg2), 1.79 (3H, C18 : CH , Pyro(a)), 2.15 (4H, C17, Pyro(a)), 2.72 (1H, βH, Phe), 2.80 (2H, εCH , Lys),

.01 (1H, βH, Phe), 3.05 (2H, δCH , Arg1), 3.12 (2H, δCH , Arg2), 3.21, 3.44, 3.62 (3H x 3, C12-2-7, Pyro(a)), 3.75 (2H, C : CH

Pyro(a)), 4.03 (1H, βH, Thr), 4.05, 4.23 (1H x 2, C17-18, Pyro(a)), 4.07-4.53 (8H, αH, Gly), 4.12 (1H, αH, Ile),4.21 (1H, αH, Lys),4.25

1H, αH, Thr),4.32 (1H, αH, Leu),4.32 (1H, αH, Ala),4.32 (1H, αH, Arg1), 4.40 (1H, αH, Arg2), 4.51 (1H, αCH , Phe), 5.15 (2H, C13

CH , Pyro(a)), 6.20, 6.24 (1H x 2, C : =CH , Pyro(a)), 7.15 (5H, Haromatic, Phe), 7.55 (1H, εNH, Arg1), 7.60 (1H, εNH, Arg2), 7.68 (2H,

εNH, Lys), 7.78 (1H, NH, Thr), 7.80 (1H, NH, Ile), 7.87 (1H, NH, Phe), 7.93 (1H, NH, Ala), 8.00(1H, NH, Arg1), 8.01 (1H, NH, Lys),

.70-8.10 (4H, NH, Gly), 8.09 (1H, NH, Arg2), 8.10 (1H, C : -CH=, Pyro(a)), 8.27 (1H, NH, Leu), 8.90, 9.44, 9.71 (1H x 3, C20-10-5

Pyro(a)). HRMS (ESI+): m/zcalcd. for C87 16 [M+2H] 874.9910; found 874.9900, [M+3H] 583.6631; found 583.6633.

UV/Vis (EtOH): λmax(log ε) = 411 (4.91), 509 (3.98), 539 (3.96), 610 (3.90), 668 nm (4.59).

6

3

(

3

2

8

3

(

3

2

3

2

8

2

,

(

2

:

2

3

2

7

3

,

2

+

3+

125 23

H N O

4

.4.2. P1-PEG-GRIGFLRTAKGG-OH (8). Procedure B

The peptide H-GRIGFLRTAKGG-Wang resin was synthesized as previously described. 2-[2-(Fmoc-amino)-ethoxy]ethoxyacetic

acid (PEG, 2 eq., 77 mg) was then added with HBTU (1.5 eq.), NMM (9 eq.) and NMP (3 eq.) in DMF. The reactor was shaken

overnight in the dark. Fmoc protection was removed with 20% piperidine in DMF and the peptide on resin was dried under vacuum.

P1-COOH (1) (2 eq., 132 mg) was then added with HBTU (3 eq.), NMM (9 eq.) and NMP (3 eq.) in DMF. The reactor was shaken for

3

days in the dark. The resin obtained was dried under vacuum and then cleaved using TFA/TIS/water (92.5/5/2.5%) for 2 h. The acidic

resin was filtered and washed with 2 mL of TFA and 20 mL of DCM. The filtrate was dried under vacuum and lyophilisated. The crude

product was further purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25

1

min, followed by isocratic acetonitrile for 15 min. R

t

= 18.5 min. Pure product was isolated as a red powder (24.6 mg, 12 %). H NMR

(

300 MHz, DMSO-d

1H, γH, Ile), 1.25 (3H, βCH

+ βH, Lys), 1.50 (1H, γH, Leu), 1.62 (2H, βH, Leu), 1.65 (1H, βH, Lys), 1.68 (1H, βH, Ile), 2.76 (1H, βH, Phe), 2.89 (2H, εCH

Lys), 2.99 (1H, βH, Phe), 3.10 (4H, 2 x δCH , Arg),3.60-3.85 (8H, αH, Gly), 3.61-3.85 (10H, CH , PEG), 3.99 (1H, βH, Thr), 4.19 (1H,

αH, Ile), 4.28 (1H, αH, Lys), 4.29 (1H, αH, Arg2), 4.30 (1H, αH, Thr), 4.33 (1H, αH, Leu), 4.35 (1H, αH, Ala), 4.35 (1H, αH, Arg1),

6

): δ (ppm) =-2.95 (2H, NH, P1-CO), 0.80 (6H, CH

3

(δ+γ), Ile), 0.88 (6H, δCH

3

, Leu), 1.03 (3H, γCH

3

, Thr), 1.15

3

, Ala), 1.29 (2H, γCH , Lys), 1.45 (1H, γH, Ile), 1.45- 1.58 (8H, 2 x γCH

2

+ 2 x 2 βH, Arg), 1.47 (3H,

δCH

2

,

2

4

2

.60 (1H, αCH , Phe), 7.20 (5H, Haromatic, Phe), 7.58 (1H, εNH, Arg1), 7.62 (1H, εNH, Arg2), 7.75 (2H, εNH, Lys), 7.75 (1H, NH, Thr),

7

.82 (1H, NH, Ile), 7.84 (9H, Hm- and p-phenyl, P1), 7.97 (1H, NH, Phe), 8.02 (1H, NH, Ala), 8.09 (1H, NH, Lys),8.10 (1H, NH, Arg1), 8.18

(

H

6

1H, NH, Leu), 8.00, 8.10, 8.15,8.92 (4H, NH, Gly), 8.15 (1H, NH, Arg2), 8.22 (6H, Ho-phenyl, P1), 8.31 (4H, Ho-phenyl-CO, P1), 8.84 (8H,

2+

3+

β-pyrrole, P1). HRMS (ESI+): m/z calcd. for C105H N O18 [M+2H] 1009.5148; found 1009.5179, [M+3H] 673.3457; found

132 24

4+

73.3474, [M+4H] 505.2611; found 505.2601. UV/Vis (EtOH): λmax(log ε) = 414 (5.45), 512 (4.14), 546 (3.88), 588 (3.77), 645 nm

(

3.69).

4

.4.3. P1-PEG-GRIGF-OH (16).

Adapted from the previous procedure B. R

1

t

= 20.5 min. Pure product was isolated as a red powder (51.2 mg, 38 %). H NMR (300

(δ+γ), Ile), 1.05 (1H, γH, Ile), 1.39 (1H, γH, Ile), 1.45 (2H, γCH , Arg),

.67 (1H, βH, Ile), 1.67 (2H, βH, Arg), 2.87 (1H, βH, Phe), 3.04 (1H, βH, Phe), 3.06 (2H, δCH , Arg), 3.60 (2H, αH, Gly1), 3.61-3.85

10H, CH , PEG), 3.82 (2H, αH, Gly2), 4.20 (1H, αH, Ile), 4.42 (2H, αCH , Phe), 4.45 (1H, αH, Arg), 7.23 (5H, Haromatic, Phe), 7.43

1H, εNH, Arg), 7.84 (9H, Hm- and p-phenyl, P1-CO), 7.93 (1H, NH, Ile), 7.98 (1H, NH, Gly2), 8.07 (1H, NH, Arg), 8.10 (1H, NH,

MHz, DMSO-d

1

6

): δ (ppm) = -2.87 (2H, NH, P1), 0.78 (6H, CH

3

2

(

2

Phe),8.22 (6H, Ho-phenyl, P1), 8.31 (4H, Ho-phenyl, P1), 8.85 (8H, Hβ-pyrrole, P1), 8.90 (1H, NH, Gly1). HRMS (ESI+): m/z calcd. For

2+

76 79 13

C H N O10 [M+2H] 667.8109; found 667.8057. UV/Vis (EtOH): λmax(log ε) = 414 (5.47), 512 (4.26), 546 (4.01), 588 (3.84), 645

nm (3.56).

4

.4.4. Synthesis of PS-GRIGF-OH (14 and 15). Procedure C

The peptide H-GRIGF-Wang resin was synthesized as previously described. The corresponding PS (1.2 eq.) was then added with

HBTU (1.5 eq.) and NMM (2 eq.) in DMF. The reactor was shaken for 3 days in the dark. The resin obtained was dried under vacuum

and then cleaved using TFA/TIS/water (92.5/5/2.5%) for 2 h. The acidic resin was filtered and washed with 2 mL of TFA and 20 mL of

DCM. The filtrate was dried under vacuum and lyophilisated. The crude product was further purified by preparative HPLC using

acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25 min, followed by isocratic acetonitrile for 15 min.

1

P1-GRIGF-OH (14). R

ppm) = -2.91 (2H, NH, P1), 0.85 (6H, CH

.80 (2H, βH, Arg), 2.88 (1H, βH, Phe), 3.07 (1H, βH, Phe), 3.15 (2H, δCH

1H, αH, Ile), 4.46 (2H, αCH , Phe), 4.50 (1H, αH, Arg), 7.22 (5H, Haromatic, Phe),7.50 (1H, εNH, Arg),7.83 (9H, Hm- and p-phenyl, P1),7.87

1H, NH, Ile),8.15 (1H, NH, Phe), 8.17 (1H, NH, Gly2), 8.22 (6H, Ho-phenyl, P1), 8.25 (1H, NH, Arg), 8.35 (4H, Ho-phenyl, P1), 8.85 (8H,

t

= 22.2 min. Pure product was isolated as a red powder (41.2 mg, 22 %). H NMR (300 MHz, DMSO-d

6

): δ

, Arg), 1.77 (1H, βH, Ile),

, Arg), 3.73 (2H, αH, Gly2), 4.10 (2H, αH, Gly1), 4.22

(

1

(

3 2

(δ+γ), Ile), 1.14 (1H, γH, Ile), 1.57 (1H, γH, Ile), 1.57 (2H, γCH

2

+

2+

H

68 12 7

β-pyrrole, P1), 9.17 (1H, NH, Gly1). HRMS (ESI+): m/z calcd. for C70H N O [M+H] 1189.5407; found 1189.5341, [M+2H]

5

95.2740; found 595.2713. UV/Vis (EtOH): λmax(log ε) = 414 (5.61), 512 (4.24), 546 (3.88), 588 (3.71), 645 nm (3.51).

1

Pyro(a)-GRIGF-OH (15). R

DMSO-d ): δ (ppm) = -1.94, 0.65 (2H, NH, Pyro(a)), 0.69 (6H, CH

Arg), 1.62 (3H, C : CH , Pyro(a)), 1.65 (2H, βH, Arg), 1.67 (1H, βH, Ile), 1.79 (3H, C18 : CH

.87 (1H, βH, Phe), 3.00 (1H, βH, Phe), 3.05 (2H, δCH , Arg), 3.21, 3.44, 3.61 (3H x 3, C12-2-7, Pyro(a)), 3.60 (2H, αH, Gly2), 3.70 (2H,

: CH , Pyro(a)), 3.75 (2H, αH, Gly1), 4.12 (1H, αH, Ile), 4.29 (1H, αH, Arg), 4.35 (2H, αCH , Phe), 4.42, 4.57 (1H x 2, C17-18

Pyro(a)), 5.15 (2H, C13 : CH , Pyro(a)), 6.21, 6.39 (1H x 2, C : =CH , Pyro(a)), 7.10 (5H, Haromatic, Phe), 7.42 (1H, εNH, Arg), 7.75 (1H,

NH, Ile),7.95 (1H, NH, Arg), 8.02 (1H, NH, Phe), 8.05 (1H, NH, Gly2), 8.05 (1H, C : -CH=, Pyro(a)), 8.20 (1H, NH, Gly1), 8.90, 9.44,

t

= 21.7 min. Pure product was isolated as a dark green powder (9.2 mg, 9 %). H NMR (300 MHz,

6

3

(δ+γ), Ile), 0.96 (1H, γH, Ile), 1.35 (1H, γH, Ile), 1.47 (2H, γCH

2

,

8

3

, Pyro(a)), 2.10-2.30 (4H, C17, Pyro(a)),

2

C

8

2

,

2

3

2

3

+

9

1

.71 (1H x 3, C20-10-5, Pyro(a)), 12.20 (1H, -COOH, Phe). HRMS (ESI+): m/z calcd. for C58

065.5646, [M+2H] 533.2871; found 533.2853. UV/Vis (EtOH): λmax(log ε) = 411 (4.92), 509 (3.96), 539 (3.93), 610 (3.88), 668 nm

72

H N

12

O

8

[M+H] 1065.5669; found

2+

(

4.58).

4

.4.5. H-GRIGFLRTAK(P1)GG-OH (9) et H-LRTAK(P1)GG-OH (11). Procedure D

9

and 11 were synthesized on the multichannel peptide synthetizer, according to a classical Fmoc/tBu solid phase methodology,

using Fmoc-Gly-Wang resin. Same method than for the peptide 4 except that the coupling of Fmoc-Lys(P1)-OH 3 (2 eq.) was the only

one to be stirred for 5 days. On a 100 µmol scale, final Fmoc protection was removed with piperidine and the peptide on resin was dried

under vacuum. Then, the peptide is cleaved from the resin using TFA/TIS/water (92.5/5/2.5%) for 2 h. The acidic resin was filtered and

washed with 2 mL of TFA and 20 mL of DCM. The filtrate was dried under vacuum and lyophilisated. The crude product was further

purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25 min, followed by

isocratic acetonitrile for 15 min. For the enzymatic cleavage, the compounds 6 and 11 were compared by analytic HPLC using another

solvent system than for the purification which cannot allow the separation of the two compounds. Acetonitrile/water (0.1 % TFA)

[

t

10:90] to [70:30] % gradient in 45 min, followed by isocratic acetonitrile/water [70:30] % for 5 min was used. R are given according

to this last gradient described.

1

H-GRIGFLRTAK(P1)GG-OH (9). R

t

= 39.9 min. Pure product was isolated as a red powder (32.4 mg, 17 %). H NMR (300 MHz,

(δ+γ), Ile), 0.82 (6H, δCH , Leu), 1.05 (3H, γCH , Thr), 1.10 (1H, γH, Ile),

, Lys), 1.49 (1H, γH, Ile), 1.52 (1H, γH, Leu), 1.68 (2H, βH, Leu), 1.69 (3H, δCH + βH, Lys),

.79 (1H, βH, Ile), 1.82 (1H, βH, Lys), 1.45-1.85 (8H, 2 x γCH + 2 x 2 βH, Arg), 2.73 (1H, βH, Phe), 3.00 (1H, βH, Phe), 3.00 (2H,

, Arg1), 3.15 (2H, δCH , Arg2),3.42 (2H, εCH , Lys), 3.57, 3.65, 3.79, 3.90 (8H, αH, Gly), 4.05 (1H, βH, Thr), 4.19 (1H, αH, Ile),

.30 (1H, αH, Lys), 4.35 (1H, αH, Thr), 4.32 (1H, αH, Ala), 4.38 (1H, αH, Leu), 4.39 (1H, αH, Arg2), 4.42 (1H, αH, Arg1), 4.56 (1H,

αCH , Phe), 7.19 (5H, Haromatic, Phe), 7.55 (1H, εNH, Arg1), 7.65 (1H, εNH, Arg2), 7.79 (1H, NH, Thr), 7.82 (9H, Hm- and p-phenyl, P1),

.97 (1H, NH, Ala), 8.00 (1H, NH, Ile), 8.00 (1H, NH, Phe), 8.09 (6H, Ho-phenyl, P1), 8.15 (2H, εNH, Lys), 8.20 (1H, NH, Leu), 8.20

1H, NH, Arg1), 8.22 (4H, Ho-phenyl, P1), 7.99, 8.13, 8.22, 8.31 (5H, NH, Gly), 8.52 (1H, NH, Arg2), 8.84 (1H, NH, Lys), 8.84 (8H, Hβ-

DMSO-d

6

): δ (ppm) = -2.95 (2H, NH, P1), 0.80 (6H, CH

3

1

.25 (3H, βCH , Ala), 1.48 (2H, γCH

3

2

δCH

2

4

2

7

(

2

+

3+

121 23

pyrrole, P1). HRMS (ESI+): m/z calcd. for C99H N O15 [M+2H] 936.9779; found 936.9771, [M+3H] 624.9877; found

6

24.9873.UV/Vis (EtOH): λmax(log ε) = 414 (5.51), 512 (4.28), 546 (3.70), 588 (3.90), 645 nm (3.64).

1

H-LRTAK(P1)GG-OH (11). R = 39.3 min. Pure product was isolated as a red powder (16.9 mg, 13 %). H NMR (300 MHz, DMSO-

t

d

6

): δ (ppm) = -2.92 (2H, NH, P1),0.87 (6H, δCH , Leu), 1.04 (3H, γCH , Thr), 1.27 (3H, βCH , Ala), 1.45 (2H, γCH , Lys), 1.55 (2H,

, Arg), 1.60 (3H, γH + 2 βH, Leu), 1.62 (1H, βH, Arg), 1.72 (3H, δCH + βH, Lys), 1.76 (1H, βH, Arg), 1.79 (1H, βH, Lys), 3.14

2H, γCH , Arg), 3.42 (2H, εCH , Lys), 3.72-3.88 (4H, αH, Gly), 3.80 (1H, αH, Leu), 4.08 (1H, βH, Thr), 4.30 (1H, αH, Thr), 4.30 (1H,

3

2

γCH

2

(

2

αH, Lys), 4.40 (1H, αH, Ala), 4.51 (1H, αH, Arg), 7.59 (1H, εNH, Arg),7.83 (9H, Hm- and p-phenyl, P1), 7.95 (1H, NH, Ala), 7.97 (1H, NH,

Thr), 8.10 (2H, εNH, Lys), 8.15 (1H, NH, Leu),8.17 (6H, Ho-phenyl, P1), 8.20-8.30 (2H, NH, Gly),8.37 (4H, Ho-phenyl, P), 8.68 (1H, NH,

+

Arg), 8.84 (1H, NH, Lys), 8.84 (8H, Hβ-pyrrole, P1).HRMS (ESI+): m/z calcd. for C74

[

H

83

N

19

O

10[M+H] 1342.6520; found 1342.6419,

2

+

M+2H] 671.8296; found 671.8287. UV/Vis (EtOH): λmax(log ε) = 414 (5.31), 512 (4.14), 546 (4.02), 588 (3.84), 645 nm (3.63).

4

.4.6. . General procedures for the conjugates peptide-quencher syntheses

.4.6. 1. H-GRIGFLRTAK(BBQ)GG-OH (10).

Same procedure than procedure A except that the coupling of Fmoc-Lys(BBQ)-OH (1 eq., 50 mg) was stirred for 7 days. On 50

µmol scale, final Fmoc protection was removed with piperidine and the peptide on resin was dried under vacuum. The resin obtained

was dried under vacuum and then cleaved using TFA/TIS/water (92.5/5/2.5%) for 2h. The acidic resin was filtered and washed with 2

mL of TFA and 20 mL of DCM. The filtrate was dried under vacuum and lyophilisated. The crude product was further purified by

preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25 min, followed by isocratic

acetonitrile for 15 min. Rt = 14.2 min. Pure product was isolated as a dark blue powder (3 mg, 3 %). HRMS (ESI+): m/z calcd. for

C86H127N25O20 [M+2H]2+ 915.9917; found 915.9938, [M+3H]3+ 610.9969, found 611.0002.

4

.4.6. 2. H-GRIGFLRTAK(BHQ3)GG-OH (19).

Fisrt step: synthesis of H-GR(Pbf)IGFLR(Pbf)T(OtBu)AKGG-OH (17). Fmoc-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(Mtt)GG-2-

Chlorotrityl resin was synthesized starting from H-Gly-2-Chlorotrityl resin and using a classical SPPS protocol. 50 µmol of peptide was

cleaved from the resin using DCM/TFE/acetic acid (4/2/2) for 2h. The resin was filtered and washed with 20 mL of DCM. The filtrate

was dried under vacuum and lyophilisated. The protected peptide was treated with 3% TFA (30 µL) and 5% TIS (50 µL) in anhydrous

DCM (920 µL) for 1 h at room temperature to deprotect the methyltrityl (Mtt) group on the εNH of the lysine. The filtrate was

concentrated and precipitated by adding anhydrous diethyl ether. The crude product was further purified by preparative HPLC using

acetonitrile/water (0.1 % TFA) [10:90] to 100% acetonitrile gradient in 45 min, followed by isocratic acetonitrile for 15 min. R

t

= 33.7

min. Pure Fmoc-GR(Pbf)IGFLR(Pbf)T(OtBu)AKGG-OH (17) was obtained as white powder. (MS (ESI+): m/z calcd. for

2

+

C

99

H

143

N

19

O

22

S

2

[M+2H] 1008, found 1009.

Second step: coupling with BHQ3-NHS ester. 17 (63.8 mg, 0.032 mmol) was dissolved in 2 mL of anhydrous DMSO with 20 µL of

N,N-diisopropylethylamine (DIPEA). The mixture was treated with BHQ3 carboxylic acid succinimidyl ester (BHQ3-NHS) (25 mg, 1

eq.) at ambient temperature overnight under argon. The reaction was quenched by precipitation with diethyl ether (20 mL) to give

+

176 25 23 2

Fmoc-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(BHQ3)GG-OH (18). HRMS (ESI+): m/z calcd. for C132H N O S [M] 2543,2807; found

2

+

2

543,2608, [M+H] 1272.1440, found 1272.1405.

Third step: removing protections. 18 was first treated with 95% TFA (950 µL) and 5% TIS (50 µL) to remove the OtBu and Pbf-

protected groups. The N-terminal Fmoc protection was removed with NMM/DMF (1:1), milder than piperidine for highly sensitive

BHQ3 (20.7 mg in 3 mL, 2 days, at room temperature under N atmosphere). The crude product was further purified by preparative

HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100% acetonitrile gradient in 45 min, followed by isocratic acetonitrile for 15

min. R = 19.7 min. Pure product H-GRIGFLRTAK(BHQ3)GG-OH (19) was isolated as a dark blue powder (9.1 mg, 49 %). HRMS

2

t

+

2+

3+

(

126 25

ESI+): m/z calcd. for C87H N O15 [M+2H] 880.9966; found 880.9960, [M+3H] 587.6668, found 587.6656.

4

.4.7. .General procedures for the PMB syntheses

.4.7. 1. P1-GRIGFLRTAK(BBQ)GG-OH (20).

H-GRIGFLRTAK(BBQ)GG-Wang resin following a classical SPPS protocol. After removal of the N-terminal Fmoc, P1-COOH (1)

1.2 eq., 40 mg) was then added with HBTU (1.5 eq.) and NMM (2 eq.) in DMF. The reactor was shaken for 5 days in the dark. The

(

resin obtained was dried under vacuum and then cleaved using TFA/TIS/water (92.5/5/2.5%) for 2 h. The acidic resin was filtered and

washed with 2 mL of TFA and 20 mL of DCM. The filtrate was dried under vacuum and lyophilisated. The crude product was further

purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [50:50] to 100 % acetonitrile gradient in 25 min, followed by

isocratic acetonitrile for 15 min. R = 23.1 min. Pure product was isolated as a dark blue powder (13 mg, 11 %). HRMS (ESI+): m/z

t

3

+

4

+

calcd. for C131

155 29

H N O21 [M+3H] 824.4057; found 824.4064, [M+4H] 618.5561; found 618.5562.

4

.4.7. 2. Pyro(a)-GRIGFLRTAK(BHQ3)GG-OH (24) and Pyro(a)-GRIGFLRTAK(BBQ)GG-OH (25).

Fisrt step: synthesis of H-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(Mtt)GG-2-Chlorotrityl resin. The side chains of arginine, threonine and

lysine were respectively protected by 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), tert-butyl ester (OtBu) and Methyltrityl

Mtt) groups. H-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(Mtt)GG-OH was synthesized on a multichannel peptide synthetizer, according to a

classical Fmoc/tBu solid phase methodology, using a H-Gly-chlorotrityl resin. Double coupling, except for the last arginine and glycine

four and six coupling were respectively necessary), were performed using a 3-fold excess of N-Fmoc-amino acid, activation reagents

(

HBTU (3 eq.), NMP (3 eq.) and NMM (9 eq.) in DMF.

Second step: coupling of Pyro(a)-COOH. After the last Fmoc group had been removed from the peptide-resin with 20% piperidine

in DMF, the resin was washed with DMF. Pyro(a) was then coupled to the N-terminal glycine of the growing peptide (Pyro(a)-

COOH/HBTU/NMM/peptide = 1.2:1:2:1 mole ratio) in DMF. The mixture was shaken for a day at room temperature. The dark-green

resin was washed with DMF and DCM followed by drying in vacuum. Then, the Pyro(a)-protected peptide was cleaved from the resin

using DCM/2,2,2-trifluoroéthanol(TFE)/acetic acid (4/2/2) for 2 h. The resin was filtered and washed with 20 mL of DCM. The filtrate

was dried under vacuum and lyophilisated. The Pyro(a)-protected peptide was treated with 3% TFA (30 µL) and 5% TIS (50 µL) in

anhydrous DCM (920 µL) for1h at room temperature to deprotect the methyltrityl (Mtt) group on the εNH of lysine. The filtrate was

concentrated and precipitated by adding anhydrous diethyl ether to give Pyro(a)-GR(Pbf)IGFLR(Pbf)T(OtBu)AKGG-OH (21) which

2

+

165 23 22 2

was not purified but whose mass was verified by HC-MS; MS (ESI+): m/z calcd. for C117H N O S [M+2H] 1155; found 1156,

3

+

[

M+3H] 770, found 771.

Third step: coupling with BHQ3-NHS ester or BBQ650-NHS ester and removing of last protections. (21) (49.4 mg, 0.022 mmol)

was dissolved in 2 mL of anhydrous DMSO with 20 µl of N,N-diisopropylethylamine (DIPEA). The mixture was treated with BHQ3

carboxylic acid succinimidyl ester (BHQ3-NHS) (17.3 mg, 1eq.) or BBQ650 carboxylic acid succinimidyl ester (BBQ-NHS) (15.7 mg,

1

eq.) at ambient temperature overnight under argon. The reaction was quenched by precipitation with diethyl ether (20 mL) to give

Pyro(a)-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(BHQ3 or BBQ)GG-OH. This last compound was then treated with 95% TFA (950 µL) and

% TIS (50 µL) to remove the OtBu and Pbf-protected groups. The filtrate was concentrated and precipitated by adding anhydrous

diethyl ether. The crude product was further purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to [60:40]

5

gradient in 25 min, followed by isocratic [60:40] for 15 min. Pure product Pyro(a)-GRIGFLRTAK(BHQ3)GG-OH 24 was isolated as a

2

+

dark blue powder (7.5 mg, 15 %). R

[

t

= 19.4 min. HRMS (ESI+): m/z calcd. for C120

158 29

H N O17 [M+2H] 1139.1229; found 1139.1200,

3

+

M+3H] 759.7510; found 759.7531. Pure product Pyro(a)-GRIGFLRTAK(BBQ)GG-OH 25 was isolated as a dark blue powder (25.3

mg, 49 %). R = 20.6 min. HRMS (ESI+): m/z calcd. for C119H N O22 [M+2H] 1174.1180; found 1174.1198, [M+3H] 783.0811;

2+

3+

t

1

6

0

2

9

4+

found 783.0848, [M+4H] 587.5626; found 587.5641.

.4.7. 3. P1-GRIGFLRTAK(BHQ3)GG-OH (26).

4

P1-GR(Pbf)IGFLR(Pbf)T(OtBu)AKGG-OH (22) (32.1 mg, 0.013 mmol) was dissolved in 2 mL of anhydrous DMSO with 20 µL

of DIPEA. The mixture was treated with BHQ3 carboxylic acid succinimidylester (BHQ3-NHS) (10.4 mg, 1 eq.) at ambient

temperature overnight under argon. The reaction was quenched by precipitation with diethyl ether (20 mL) to give the intermediate

peptide P1-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(BHQ3)GG-OH. This last compound was then treated with 95% TFA (950 µL) and 5%

TIS (50 µL) to remove the OtBu- and Pbf-protected groups. The filtrate was concentrated and precipitated by adding anhydrous diethyl

ether. The crude product was further purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile

gradient in 25 min, followed by isocratic acetonitrile for 15 min. Pure product P1-GRIGFLRTAK(BHQ3)GG-OH 26 was isolated as a

+

2+

dark blue powder (4 mg, 13 %). R = 21.8 min. HRMS (ESI+): m/z calcd. for C132H N O16 [M+2H] 1201.1098; found 1201.1008,

t

1

5

4

2

9

3

+

4+

[

M+3H] 801.0756; found 801.0710, [M+4H] 601.0585; found 601.0543.

4

.4.7.4. P1-PEG-GRIGFLRTAK(BHQ3)GG-OH (27).

First step: synthesis of H-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(Mtt)GG-2-Chlorotrityl resin

Second step: coupling of PEG arm and 1. 2-[2-(Fmoc-amino)-ethoxy]ethoxyacetic acid (PEG, 2 eq., 77 mg) was then added with

HBTU (1.5 eq.), NMM (9 eq.) and NMP (3 eq.) in DMF. The reactor was shaken overnight in the dark. Fmoc protection was removed

with 20% piperidine in DMF and the peptide on resin was dried under vacuum. P1-COOH (1) (2 eq., 132 mg) was then added with

HBTU (3 eq.), NMM (9 eq.) and NMP (3 eq.) in DMF. The reactor was shaken for 5 days in the dark at room temperature. The resin

was washed with DMF and DCM followed by drying in vacuum. Then, the P1-PEG-protected peptide was cleaved from the resin using

DCM/TFE/acetic acid (4/2/2) for 2 h. The resin was filtered and washed with 20 mL of DCM. The filtrate was dried under vacuum and

lyophilisated. The P1-PEG-protected peptide was treated with 3% TFA (30 µL) and 5% TIS (50 µL) in anhydrous DCM (920 µL) for

1

h at room temperature to deprotect the methyltrityl (Mtt) group on the εNH of lysine. The filtrate was concentrated and precipitated by

adding anhydrous diethyl ether to give P1-PEG-GR(Pbf)IGFLR(Pbf)T(OtBu)AKGG-OH (23) (MS (ESI+): m/z calcd. for

C

2

+

3+

4+

135

H

172

24

N O

24

S

2

[M+2H] 1290; found 1290, [M+3H] 860; found 861, [M+4H] 645; found 645).

Third step: coupling with BHQ3-NHS ester and removing of last protections. (23) (46.6 mg, 0.018 mmol) was dissolved in 2 mL of

anhydrous DMSO with 20 µL of DIPEA. The mixture was treated with BHQ3 carboxylic acid succinimidyl ester (BHQ3-NHS) (14.2

mg, 1eq.) at ambient temperature overnight under argon. The reaction was quenched by precipitation with diethyl ether (20 mL) to give

P1-PEG-GR(Pbf)IGFLR(Pbf)T(OtBu)AK(BHQ3)GG-OH. This last compound was then treated with 95% TFA (950 µL) and 5% TIS

(

50 µL) to remove the OtBu- and Pbf-protected groups. The filtrate was concentrated and precipitated by adding anhydrous diethyl

ether. The crude product was further purified by preparative HPLC using acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile

gradient in 25 min, followed by isocratic acetonitrile for 15 min. Pure product P1-PEG-GRIGFLRTAK(BHQ3)GG-OH 27) was

+

2+

isolated as a dark blue powder (6 mg, 13 %). R

t

= 21.3 min. HRMS (ESI+): m/z calcd. for C138

H

165

N

30

O

19 [M+2H] 1273.6467; found

3+

4+

1

273.6392, [M+3H] 849.4336; found 849.4306, [M+4H] 637.3270; found 637.3233.

For the enzymatic cleavage, PMBs were compared by analytic HPLC using another solvent system than for the purification which

cannot allow the separation of the two compounds. Acetonitrile/water (0.1 % TFA) [10:90] to 100 % acetonitrile gradient in 25 min,

followed by isocratic acetonitrile for 15 min was used. R for PMBs are given according to this last gradient described.

t

4

.5. Studies with the active enzymes MMP-2, MMP-9 and MMP-14

The buffer used for cleavage contains 50 mM TRIS (tris(hydroxymethyl)aminomethane), 150 mM NaCl, 5mM CaCl

2

, pH 7.5. For

each cleavage study, the activity of MMPs was confirmed by their ability to cleave the peptide 5. The sample solutions of peptides, PS-

peptides, peptide-quenchers or PMBs were prepared as 2 mM stock solution in DMSO.

For the ratio enzyme/peptide = 16 000: The composition of each sample was as follows: (i) 10 µL from a 2 mM sample stock

solution without MMP; (ii) 10 µL from a 2 mM sample stock solution with 1 µL of MMP-2 (1.2 µM, M

a 2 mM sample stock solution with 1 µL of MMP-9 (1.2 µM, M = 83 000 Da); and (iv) 10 µL from a sample stock solution with 1 µL

of MMP-14 (1.2 µM, M = 22 500 Da). 189 µL of buffer was added in each sample to obtain a final volume of 200 µL. The cleavage

w

= 66 000 Da); (iii) 10 µL from

w

solutions were let for incubation during 8 h at 37°C.

For the ratio enzyme/peptide = 250 the composition of each sample was as follows: (i) 10 µL from a 300 µM sample stock solution

without MMP; (ii) 10 µL from a 300 µM sample stock solution with 1 µL of MMP-2 (1.2 µM, M

stock solution with 1 µL of MMP-9 (1.2 µM, M = 83 000 Da); and (iv) 10 µL from 300 µM sample stock solution with 1 µL of MMP-

4 (1.2 µM, M = 22 500 Da). 189 µL of buffer was added in each sample to obtain a final volume of 200 µL. The cleavage solutions

w

= 66 000 Da); (iii) 10 µL of sample

w

1

w

were left for incubation during 8 h at 37°C.

These solutions were analyzed first by HPLC using fluorescence and UV-Visible detectors and references solution of fragments 11-

1

6 for the retention times. LC-MS experiments were conducted to confirm the mass of the hydrolysis products (and therefore their

nature). Digest solutions were desalting and concentrated for MS analysis using OMIX C18 Pipette TIS. Table 4 gathers the detection

parameters used for the enzymatic activity by HPLC.

Table 4. Detection parameters used for enzymatic activity.

Fluorescence

Compounds

UV1 (nm)

UV2 (nm)

Excitation (nm)

Emission (nm)

Peptides 4, 7

214

415

256

415

650

600

256

415

256

415

280

650

280

650

670

PS-peptides 5, 6, 8, 9, 11, 16

Peptide-quencher 10, 19

PMBs 20, 26, 27

2

4, 25

4

.6. Cells experiments.

B16F1 cells, a murine melanoma cell line, were provided by ATCC (CRL6323). Cells were grown and maintained in Roswell Park

Memorial Institute (RPMI) 1640 media supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5%

CO2 in a humidified incubator.

For the preparation of conditioned media, cells were seeded in 24 well plates until 90% of confluency was reached. Then, cells were

washed two times in PBS and 300 µL of serum-free RPMI was added. After 24 h, the culture media were harvested, centrifuged for 10

min at 500 g to eliminate cellular debris and then mixed with PMB for 24 h at 37°C.

PMB cleavage was also tested by incubating PMB on confluent monolayer of B16F1. Before mixing cells and PMB, cells were

seeded in 24 well plates until 90% of confluency was reached. Then, cells were washed two times in PBS and 300 µL of serum-free

RPMI containing 100 µM of PMB was added.

Enzymatic cleavage was detected by HPLC.

4

.7. Photophysical Properties

The compounds were dissolved in DMSO in order to obtain 1 mg/mL solutions. The bulk solutions were then diluted in the solvent

of interest (in EtOH) so as to obtain adequate absorption of the final solutions (A ~ 1 for the Soret band for determination of absorption

coefficients and A ~ 0.2 for the Soret band for the other measurements described). Measurements were done in 10 mm quartz cuvettes.

Absorption spectra were recorded on a Perkin-Elmer (Lambda EZ 210) double beam UV-Visible spectrophotometer. The ratio of

DMSO in the final solution was always less than 1%. The absorption spectrum of the solution of known concentration was measured

between 200 and 800 nm. The operation was repeated 3 times. The epsilon values were calculated and averaged from the Beer Lambert

law:

ꢂ

ꢀ

=ꢁ

(1)

ꢃ.ꢄ

−

1

−1

with A: absorbance; ε: molar extinction coefficient in L.mol .cm ; l = size of the cuvette, 1 cm; C = concentration of the solution.

Fluorescence spectra were recorded on a Horiba Jobin Yvon Fluorolog-3 (FL3-222) spectrofluorimeter equipped with a 450 W

Xenon lamp, a thermostated cell compartment (25°C), a R928 (HAMAMATSU Japan) UV-visible photomultiplier and a liquid nitrogen

cooled InGaAs infrared detector (DSS-16A020L Electro-Optical System Inc, Phoenixville, PA, USA). The excitation spectrometer is a

SPEX double grating monochromator (1200 grating/mm blazed 330 nm). The fluorescence was measured by the UV-Visible detector

1

through a SPEX double grating monochromator (1200 grating/mm blazed 500 nm). The O

2

production was measured by the IR

detector through a SPEX double grating monochromator (600 grating/mm blazed 1 µm). All spectra were recorded using four optical

4

2

faces quartz cells. Fluorescence quantum yields (Φ

f

) were determined using tetraphenylporphyrin (TPP) solution in toluene (Φ = 0.11 )

f

43

or methylene blue solution in methanol (Φ = 0.04 ) as fluorescence standards. Rose Bengal or methylene blue were chosen as

f

1

44

2 ∆ ∆

reference solutions thanks to their high O quantum yields in ethanol (Φ (Rose Bengal) = 0.68 and Φ (methylene blue) = 0.52 );

methylene blue for the phthalocyanine derivatives and rose de Bengal for the others. The absorbance value at the excitation wavelength

of the reference and the sample solutions were set to around 0.2.

The quantum yield of fluorescence is determined thanks to the equation (2):

ꢇ_ꢈꢊꢋ_ꢉ

^ꢇꢈꢉ ꢊꢋ

ꢌ

Φ = Φ ꢁ. ꢁ.

ꢁ. ( )²

(2)

ꢅ

ꢅꢆ

ꢌ

ꢉ

where Φ and Φf0, I and If0, DO and DO , n and n are the fluorescence quantum yields, the fluorescence intensities, the optical

f

0

45,46

densities at the excitation wavelength and the refraction indices of the sample and of the reference respectively.

The quantum yield of singlet oxygen production is determined thanks to the equation (3):

ꢇ

ꢊꢋ_ꢉ

Φ = Φ ꢁ. ꢁ.

(3)

ꢍ

ꢍꢆ

ꢇ_ꢎ_ꢉꢊꢋ

where Φ

∆ ∆ 0

and Φ∆0, I and I∆0, DO and DO are the luminescence quantum yields, the luminescence intensities and the the optical

47

densities at the excitation wavelength of the sample and of the reference respectively.

The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined by the

Förster radius (R ). The magnitude of R is dependent on the spectral properties of the donor and acceptor dyes. The efficiency of FRET

is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable to the dimensions

0

48

of biological macromolecules. Typical effective distances between the donor and acceptor molecules are in the 10 to 100 Å range. The

theoretical calculation of Förster radius can be calculated from the following equation (4):

1

/6

2

−

4

ꢏ = 0.211(ꢐ ꢑ ꢒ ꢔ(ꢕ))

(4)

2

0ꢁ

ꢓ

0

where R is the Förster radius expressed in angstrom (Ǻ), k is the orientation factor taken as 2/3, n is the refraction index of ethanol

taken as 1.3594, Φ

f

is the fluorescence quantum yield of the donor (here the PS). The overlap integral J is described in equation (5):

ꢞ

ꢉ

ꢜ

ꢖ

ꢗꢘ(ꢙ)ꢚꢛ(ꢙ)ꢙ ꢝꢙ

ꢞ

ꢉ

ꢔ(ꢕ) =ꢁ

(5)

ꢖ

ꢗꢘ(ꢙ)ꢝꢙ

-1

where F (λ) is the emission spectra of the donor (PS), ꢀ (ꢕ) is the extinction coefficient of the acceptor (Quencher) in M .cm and

ꢕ is the wavelength in nm.

D

ꢟ

49

Fluorescence lifetimes were measured on a Time-Correlated Single Photon Counting (TCSPC) using for excitation: a pulsed laser

diode LDH-P-C-405 coupled with a driver PDL 800-D (both PicoQuant GmbH, Berlin, Germany) emitting at 408 nm (FWHM < 70 ps,

1

MHz) and an avalanche photodiode SPCM-AQR-15 (EG & G, Vaudreuil Canada) coupled with a 650 nm (± 10 nm) interference

long-wave pass filter as detection system. The acquisition system was a PicoHarp 300 module with a four channels router PHR-800

both PicoQuant GmbH, Berlin, Germany). Fluorescence decays were recorded using the single photon counting method (four optical

(

faces quartz cells for pure products in ethanol). Data was collected up to 1000 counts accumulated in the maximum channel and

analyzed using TCSPC software Fluofit (PicoQuant GmbH, Berlin, Germany) based on iterative reconvolution using a Levensberg-

Marquandt algorithm, allowing to obtain the multi-exponential profiles (one or two exponentials in our cases).

Singlet oxygen lifetime measurements have been performed on a TEMPRO-01 spectrophotometer (Horiba JobinYvon – Palaiseau

France) composed with a pulsed diode excitation source SpectraLED-415 emitting at 415 nm, a cuve compartment, a Seya-Namioka

type emission monochromator (600 – 2000 nm) and a H10330-45 near-infrared photomultiplier tube with thermoelectric cooler

(

HAMAMATSU) as detection system. The system is monitored by a single photon counting controller FluoroHub-B and the software

DataStation and DAS6 (Horiba Jobin Yvon).

Conflict of interest

All authors of this manuscript declare no competing financial interest.

Acknowledgements

We thank Cancéropôle Grand-Est for its financial support for the grant “Emergence en Recherche Translationnelle et Clinique en

oncologie”. We thank Jean-Claude André for his advice.

A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at

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Article Doi

New photodynamic molecular beacons (PMB) as potential cancer-targeted agents in PDT

DOI: 10.1016/j.bmc.2017.12.034

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