Synthesis of a porphyrin with histidine-like chelate: an efficient path towards molecular PDT/SPECT theranostics

Article abstract of DOI:10.1039/d0cc03958f

The goal of “personalised” medicine has seen a growing interest in the development of theranostic agents. Bifunctional, and targeted-trifunctional, theranostic water-soluble porphyrins with a histidine-like chelating group have been synthesisedviacopper-catalysed azide-alkyne cycloaddition (CuAAC) “click” chemistry in high yield and purity. They are capable of photodynamic treatment and [^99mTc(CO)₃]⁺complexation for single-photon emission computed tomography (SPECT) imaging, with a radiochemical yield of >95%. The toxicity and phototoxicity were evaluated on HT-29 cells, DU145, and DU145-PSMA cell lines, with the targeted theranostic showing more potent phototoxicity towards DU145-PSMA expressing cells.

Full text of DOI:10.1039/d0cc03958f

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Commun., 2020, DOI: 10.1039/D0CC03958F.
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S. J. Connon, M. O. Senge et al.
Conformational control of nonplanar free base porphyrins:
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DOI: 10.1039/D0CC03958F
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Synthesis of a porphyrin with histidine-like chelate: an efficient
path towards molecular PDT/SPECT theranostics
a
a
b,c
b,c
a
Steven Y. Yap, Huguette Savoie, Isaline Renard, Benjamin P. Burke, Harry C. Sample, Saul
Received 00th January 20xx,
Accepted 00th January 20xx
a
b,c
a
b,d
Michue-Seijas, Stephen J. Archibald, Ross W. Boyle, * and Graeme J. Stasiuk. *
DOI: 10.1039/x0xx00000x
www.rsc.org/
The goal of “personalised” medicine has seen a growing interest in monitoring of the accumulation of PDT drug within the body,
the development of theranostic agents. Bifunctional, and facilitating the tuning of treatment by light irradiation when
targeted-trifunctional, theranostic water-soluble porphyrins with the accumulation of drug is at a maximum, ultimately
a histidine-like chelating group have been synthesised via copper- improving treatment outcomes.
catalysed azide-alkyne cycloaddition (CuAAC) “click” chemistry in
Single-photon emission computed tomography (SPECT) is
high yield and purity. They are capable of photodynamic a highly sensitive imaging technique that requires the
9
9m
+
treatment and [ Tc(CO)
emission computed tomography (SPECT) imaging, with
radiochemical yield of >95%. The toxicity and phototoxicity were obtained using a gamma camera and computed to provide a
3
]
complexation for single-photon administration of a radiotracer. SPECT requires the radiotracer
a
to biodistribute/accumulate in the body. Images are then
9
9m
evaluated on HT-29 cells, DU145, and DU145-PSMA cell lines, with 3D image of the location of the radiotracer in the body. Tc is
the targeted theranostic showing more potent phototoxicity often used in SPECT imaging, since it has desirable nuclear
towards DU145-PSMA expressing cells.
properties with a half-life, t1/2, of 6 hours and a main gamma
6
emission, E
γ
, at 140 keV (89% emission). Its half-life is long
Theranostics, a term given to the combination of therapy
enough to manipulate the isotope to prepare various
radiopharmaceuticals and its gamma emission allows
(thera-) and diagnostic (-nostic) modalities into a single
construct or molecule, has seen a growing interest in the field
of medicine.1 Conventional treatments often involve a “one-
size-fits-all” technique, and can have significant differences in
the treatment outcomes between patients. Theranostics
naturally lends itself towards the goal of “personalised”
medicine, capable of optimising and improving treatment
outcomes.1
established chemistry. Its availability from a 99Mo/99mTc
generator and relative low cost, have made it the most
,2
6
commonly used radioisotope in diagnostic nuclear medicine.
Schibli has shown that alkyne/azide-modified amino acids
“click”, via copper-catalyse azide alkyne cycloaddition (CuAAC),
with other azide/alkyne-functionalised molecules, to form a
histidine-like structure that can efficiently complex
Porphyrins are excellent candidates for molecular
theranostic agents. There are multiple studies documenting
their use in medicine, especially as a photosensitiser in the
field of photodynamic therapy (PDT). Porphyrins, when
combined with molecular oxygen and visible light (400 – 700
nm), are capable of delivering potent and selective
99m
+ 7
[
3
Tc(CO) ] . The utilisation of “click” chemistry unlocks the
possibility to use a high yielding and high purity “click” reaction
to combine two moieties together, opening the way towards
1
,2
molecular SPECT theranostic medicine.
Despite the potential utility of 99mTc-labelled porphyrins,
there are limited reports on the combination of PDT with
1
,3–5
photodynamic treatment to tumour tissues.
a diagnostic modality with a PDT drug, this allows the careful
By combining
8–13
SPECT,
histidine-like structures as a
our interest in developing porphyrin molecular theranostics,
with only one example on the usage of conjugated
99m
13
Tc chelate. Herein, following
1
4–
1
7
we report the synthesis of two water-soluble porphyrins
a. Department of Chemistry and Biochemistry, Faculty of Science and Engineering,
University of Hull, HU6 7RX. r.w.boyle@hull.ac.uk
conjugated with histidine-like ligands using CuAAC “click”
chemistry in high yields and with minimal impurities, and show
b.
Department of Biomedical Sciences, University of Hull, Cottingham Road, Hull,
9
9m
+
that they are capable of complexing [ Tc(CO)
3
] with good
HU6 7RX, UK.
c.
Positron Emission Tomography Research Centre, University of Hull, Cottingham
radiochemical yields.
Road, Hull, HU6 7RX.
Department of Imaging Chemistry and Biology, School of Biomedical Engineering
d.
and Imaging Sciences, King’s College London, Fourth Floor Lambeth Wing, St
Thomas’ Hospital, London SE1 7EH, UK. E-mail: graeme.stasiuk@kcl.ac.uk
Electronic Supplementary Information (ESI) available: Experimental Section and
spectras. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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Journal Name
N
N
O
O
View Article Online
DOI: 10.1039/D0CC03958F
(iv)(v)
N
N
N
N
O
N
N
N
N
OH
Zn
N
N
Zn
N
N
N
H2N
N
NH2
N
(i)
N
M
CO
N
OC
CO
3Cl
3Cl
N
N
OH
O
OH
O
N
N
N
N
N
Zn
N3
O
OH
O
OH
2
[M(2)]
O
O
NH
NH
NH
NH
O
N
O
N
H
H
N
3Cl
N
HN
HN
N
O
O
HO
O
HO
O
(iv)(v)
N
Zn
N
N
N
N
N
N
O
1
Zn
O
HN
M
N
H
N
N
N
N
N
(ii)(iii)
N
N
N
OR
N
O
OC
CO
3Cl
CO
3Cl
N
N
M = (iv) Re
(v) 99mTc
R = -CH (3), -H(4)
3
[M(4)]
9
9m
Scheme 1 Synthesis of conjugate 2 and conjugate 3 and the subsequent complexation of natural rhenium and Tc. (i) 1:1 t-butanol:water, D-propargylglycine, CuSO
ascorbate, microwave (20 mins, 75 W, 50 °C). (ii) 6, 1:1 t-butanol:water, CuSO , sodium ascorbate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, microwave (3 hours, 75 W, 70
C). (iii) water, LiOH, room temperature 3 hours. (iv) phosphate buffer pH 7, [Re(CO) Br ][NEt (H O)
, 65 °C, 2 hrs. (v) [99mTc(CO) ]+, pH 7.4, 90 °C, 30 mins.
4
, sodium
4
°
3
3
4
]
2
3
2
3
Initially, a water-soluble azide-appended porphyrin 1 was conjugatable linker, 5. This linker allows further conjugation
synthesised via 5 steps as previously reported (detail in with a targeting moiety. We have chosen a well-described
1
8
Supplementary information).
Commercially available
small molecule dipeptide that acts as an inhibitor against
D-propargylglycine
was prostate specific membrane antigen (PSMA), which is
1
9
20
conjugated with porphyrin 1 using CuAAC (Scheme). With overexpressed in metastatic prostate cancer. The targeting
excess D-propargylglycine, quantitative conversion of dipeptide has the structure: lysine-urea-glutamate, with the
porphyrin 1 to conjugate 2 was shown using HPLC analysis, glutamate moiety responsible for targeting and binding to the
2
1,22
with a clear shift in HPLC retention time from 10.8 mins to 7.5 pharmacophore pocket of PSMA.
Peptide conjugation and
mins (Figure S1 A). Subsequently, purification of conjugate 2 subsequent deprotection of tert-butyl groups using
was carried out using a counter-ion exchange technique trifluoroacetic acid (TFA) yielded the clickable 6 capable of
1
4
previously reported by our group. Briefly, the solubility of the targeting PSMA.
porphyrin can be controlled by exchanging the anionic
Using similar CuAAC reaction conditions for the synthesis
counter-ions. Exchange of the iodide anions with of conjugate 2, quantitative conversion of porphyrin 1 to 3 was
hexafluorophosphate rendered the conjugate insoluble in observed, with a clear shift in HPLC retention time from 10.8
water. This allowed filtration and removal by washing of to 8.6 mins (Figure S1 B). Using the same counter-ion exchange
excess starting materials and reagents. Subsequent anionic technique, 3 was purified, isolated, and characterised.
conversion to chloride yielded the water-soluble conjugate 2. Deprotection of methyl ester on 3 was carried out in aqueous
The identity of conjugate 2 was further confirmed using NMR LiOH yielding trifunctional water-soluble conjugate 4 in
and mass spectrometry.
quantitative yield.
We envisaged introduction of an additional targeted
functionality could be facilitated by using lysine, instead of
glycine, which has an amino group side chain. However, there
is no commercially available propargyl lysine, hence,
modification of commercially available N(ε)-Boc-Lys(OMe) was
required. Scheme 2 shows the modification of N(ε)-Boc-
Lys(OMe) to introduce an alkyne-functionality and also a
[
[
99mTc(2)]
Re(2)]
100
1
0000
5
0
0
5
000
0
O
O
O
0
0
5
5
10
15
20
25
25
O
O
NHBoc
HN
N
HN
(i)(ii)(iii)
O
(iv)(v)
HN
Time/ minutes
O
[
99mTc(4)]
250
200
150
100
OH
O
O
O
40000
H2N
N
H
N
H
O
[Re(4)]
O
O
O
O
NH
NH
O
HO
2
0000
0
HO
O
5
0
0
5
6
10
15
20
Scheme 2 Synthesis of trifunctional-propargyl lysine. (i) DMF, K CO , propargyl bromide,
2 3
room temperature, 48 hours. (ii) DCM, TFA, room temperature, 2 hours. (iii) DMF,
disuccinimidyl suberate, triethylamine, room temperature, overnight. (iv) DMF, Figure
Time/ minutes
99mTc(2)] and 99mTc(4)] and UV-vis HPLC
1 Overlays of radio-HPLC of [ [
triethylamine, PSMA-dipeptide inhibitor, room temperature, overnight. (V) DCM, TFA, chromatograms [Re(2)] and [Re(4)]. Reaction conditions: [L] = 150 µM, t = 30 mins, T =
room temperature, 3 hrs.
99 °C, pH 7.4.
2
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1
99m
[
[
Tc(2)]
9m
Tc(4)]
9
literature has shown the stability of histidine and histidine-like
View Article Online
7
,25,28
DOI: 10.1039/D0CC03958F
complexes against serum.
5
0
Toxicity and phototoxicity of 2 and [Re(2)] were
evaluated on human adenocarcinoma (HT-29) cells. Cells were
incubated with 2 and [Re(2)] at varying concentrations (1-18
µM) for 1 hour and irradiation was carried out using a constant
dose of visible light (20 J cm ; 400 – 700 nm). The results
obtained were compared with a non-irradiated control. Under
these conditions, both conjugate 2 and [Re(2)] were capable of
inducing >50% cell death at 4.5 µM with minimal dark toxicity
observed in the non-irradiated controls (Figure 3 and Figure
S6).
0
0
2
4
6
time/ hour
-2
Figure 2 Serum stability of radiolabelled conjugates, [99mTc(2)] and [99mTc(4)].
Chemically and physically similar rhenium was complexed
with conjugate 2 and 4 (Scheme 1).
commonly used surrogate for technetium as there is no stable
technetium isotopes. In addition, the availability of β-particle
emitting rhenium 186/188 isotopes further expand the utility
of 2 and 4 as radiotherapeutic agent.
rhenium was carried out to produce non-radioactive
complexes for use as HPLC standards and in biological
cytotoxicity evaluations. With a slight excess of rhenium at 65
2
3,24
Rhenium is a
2
5
As 4 and [Re(4)] have a targeting molecule which inhibits
PSMA, two different human prostate cancer cell lines were
used to evaluate the toxicity and phototoxicity of 4 and
2
6
Complexation with
[
Re(4)], namely DU145, which does not express PSMA, and
2
9
DU145-PSMA, a PSMA-expressing variant. Initially, the level
of PSMA expression on both cell lines was determined by flow
cytometry analysis (Figure S5). Varying concentration of 4 and
°
C for 2 hours, HPLC analysis of the reaction mixture showed
quantitative conversion of conjugates 2 and 4 to their
corresponding complexes, [Re(2)] and [Re(4)], exhibited by a
clear shift in HPLC with retention times of 10.2 mins and 10.8
mins, respectively (Figure S1). C NMR spectroscopy showed
the addition of three carbonyl carbon peaks between δ 197-
[
3
Re(4)] (1.5 – 24 µM) were incubated with these cell lines for
0 mins prior to visible light irradiation. Results obtained were
also compared with non-irradiated controls (Figure 3 and
Figure S6). Without irradiation, both 4 and [Re(4)] show
minimal toxicity at these concentration with more than 86%
cell survival in both DU145 and DU145-PSMA. Under
irradiation with visible light, both 4 and [Re(4)] were capable
1
3
1
99 for both complexes, [Re(2)] and [Re(4)]. High resolution
mass spectrometry of [Re(2)] and [Re(4)] also gave their
corresponding mass peak, giving evidence for the successful
complexation of rhenium.
[Re(2)] in HT29
[Re(2)] in HT29
Successful synthesis of conjugate 2 and 4 and their
1
00
50
0
corresponding
rhenium
complexes
prompted
(H O)
the
]+ was
radiolabelling with 99mTc. Firstly, [99mTc(CO)
3
2
3
prepared according to the literature method (Figure S2 and
Figure S3).27 After careful optimisation of reaction conditions –
reaction time, pH, reaction temperature, and conjugate
concentration, both conjugate 2 and 4 can be produced in
>
95% radiochemical yield (RCY) at a concentration of 150 µM
at pH 7.4, 99 °C for 30 mins, as determined using radio-HPLC.
Radio-HPLC analysis showed the formation of a single
radiolabelled product which matches well with the retention
time of the rhenium complexes (Figure 1), giving evidence for
the formation of the desired radiolabelled complexes,
1
10
Concentration/ µM
Non-irradiated
Irradiated
[Re(4)] in DU145
Re(4)] in DU145-PSMA
[
Re(4)] in DU145
9
9m
99m
[
Tc(2)] and [ Tc(4)]. With these optimised radiolabelling
[
[Re(4)] in DU145-PSMA
-1
conditions, specific activities of 76 GBq µmol and 96 GBq
-1
µmol were achieved for conjugates 2 and 4, respectively. The
lipophilicity of the tracers were determined using the shake-
flask method and were found to be -0.57±0.08 and -1.31±0.01
100
9
9m
99m
for [ Tc(2)] and [ Tc(4)], respectively. This indicates
9
9m
[
Tc(4)] is more hydrophilic due to the presence of extra
carboxylate groups.
The stability of the radiolabelled complexes, [99mTc(2)]
and [99mTc(4)], were tested against 50% foetal bovine serum
5
0
0
(
FBS) with an activity of 35-40 MBq (at concentration of 460-
1
10
9
9m
99m
5
30 nM for [ Tc(2)] and 360-420 nM for [ Tc(4)]).
Concentration/ µM
9
9mTc(2)] shows 94% of 99mTc still associated and [ Tc(4)] Figure 3 Percentage cell survival of HT-29 (top) and DU145 and DU145-PSMA (botom),
shows remarkable stability against 50% FBS with >99%
99m
[
9
9m
irradiated (red) and non-irradiated (black) cells, as determined using MTT assay. Cells
were incubated with varying concentration of [Re(2)] (top) and [Re(4)] (bottom) and
irradiated cells received 20 J cm-2 white light. MTT assays were carried out in three
quadruplicates.
Tc
still associated after incubation for 6 hours (Figure 2). The high
9
9m
serum stability of these
Tc conjugates was expected as
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of inducing death in both DU145 and DU145-PSMA cells, with
more potent phototoxicity towards DU145-PSMA. At a
concentration of 6 µM, 85% cell survival was observed for 4 in
DU145 cells with light irradiation, while only 17% cell survival
was observed in DU145-PSMA cells under similar conditions.
Similarly, [Re(4)] showed minimal phototoxicity towards
DU145 cells at a concentration of 1.5 µM with >90% cell
survival, but was capable of inducing near 70% cell death in
DU145-PSMA under similar conditions. These results show, 4
and [Re(4)] are capable of inducing greater cell death in
5
6
7
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and [99mTc(4)] show the validity of a click to chelate method to 14 S. Y. Yap, T. W. Price, H. Savoie, R. W. Boyle and G. J. Stasiuk,
bifunctional theranostic and trifunctional targeted theranostic
Chem. Commun., 2018, 54, 7952–7954.
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1
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Conflicts of interest
There are no conflicts to declare.
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ChemComm
View Article Online
DOI: 10.1039/D0CC03958F
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