68Ga/DOTA- and 64Cu/NOTA-phthalocyanine conjugates as fluorescent/PET bimodal imaging probes

Article abstract of DOI:10.1021/bc400257u

In this paper, we describe the synthesis and characterization of a series of new bimodal probes combining water-soluble sulfonated zinc phthalocyanine (ZnPc) as a fluorescence imaging unit and either ⁶⁸Ga/1,4,7,10- tetraazocyclododecane-N,N′N″,N′″-tetraacetic acid (DOTA) or ⁶⁴Cu/1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for PET imaging. The two moieties were linked through aliphatic chains of different lengths to modulate amphiphilicity. Labeling of DOTA- or NOTA-ZnPc conjugates with ⁶⁸Ga (t_1/2 = 68 min) and ⁶⁴Cu (t _1/2 = 12.7 h) was performed at 100 C for 15 min with >90% efficiency for all conjugates. In vitro plasma stability assays demonstrated high stability of the ⁶⁴Cu/NOTA-ZnPc conjugate, which remained intact over a 24 h time period, and reasonably high stability of the ⁶⁸Ga/DOTA-ZnPc conjugate, which released up to 7% of free ⁶⁸Ga over a 3 h period. Based on in vitro plasma stability results, we performed biodistribution studies on two ⁶⁴Cu-labeled derivatives, which allowed us to select a single candidate for preliminary in vivo experiments. Fluorescence and PET imaging confirmed the potential of these novel conjugates to act as bimodal probes.

Full text of DOI:10.1021/bc400257u

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Article
pubs.acs.org/bc
64
68Ga/DOTA- and Cu/NOTA-Phthalocyanine Conjugates as
Fluorescent/PET Bimodal Imaging Probes
Elena Ranyuk,† Rejean Lebel,†,∥ Yves Berube-Lauziere,‡,∥ Klaus Klarskov,§ Roger Lecomte,†,∥
́
́
́
̀
Johan E. van Lier,*,†,∥ and Brigitte Guerin#,†,∥
́
∥Centre d’imagerie molec
Faculty of Medicine and Health Sciences, Universite
́
ulaire de Sherbrooke (CIMS), Departments of Nuclear Medicine and Radiobiology and Pharmacology,
†
§
́
de Sherbrooke; 3001 12th Avenue North, Sherbrooke, Queb
́
ec, Canada J1H
5N4
‡Department of Electrical and Computer Engineering, Universite
Canada J1K 2R1
́
de Sherbrooke, 2500 boulevard Universite,
́
Sherbrooke, Quebec,
́
S
* Supporting Information
ABSTRACT: In this paper, we describe the synthesis and
characterization of a series of new bimodal probes combining
water-soluble sulfonated zinc phthalocyanine (ZnPc) as a
fluorescence imaging unit and either 68Ga/1,4,7,10-tetraazocy-
clododecane-N,N′N″,N′″-tetraacetic acid (DOTA) or 64Cu/
1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for PET
imaging. The two moieties were linked through aliphatic
chains of different lengths to modulate amphiphilicity.
Labeling of DOTA− or NOTA−ZnPc conjugates with 68Ga
(t1/2 = 68 min) and 64Cu (t1/2 = 12.7 h) was performed at 100
°C for 15 min with >90% efficiency for all conjugates. In vitro
plasma stability assays demonstrated high stability of the 64Cu/NOTA-ZnPc conjugate, which remained intact over a 24 h time
period, and reasonably high stability of the 68Ga/DOTA-ZnPc conjugate, which released up to 7% of free 68Ga over a 3 h period.
Based on in vitro plasma stability results, we performed biodistribution studies on two 64Cu-labeled derivatives, which allowed us
to select a single candidate for preliminary in vivo experiments. Fluorescence and PET imaging confirmed the potential of these
novel conjugates to act as bimodal probes.
tissues and quantitation. Consequently, bimodal imaging
probes that use optical imaging and positron emission
tomography (PET) or MRI in tandem are attractive, as they
take advantage of strengths of each imaging modality. For
example, after lesion localization and surgery planning using
PET or MRI, optical imaging may guide surgical procedure.
Also, the combination of PET and FI can provide
complementary information in small animal models where
light penetration is less of an issue.
Water-soluble sulfonated metallo-phthalocyanines (MPc) are
NIR fluorophores that possess high extinction coefficients,
favorable quantum yields, and display extraordinarily high
photostabilities.4 These qualities make them particularly
attractive for FI applications along with other important
characteristics such as high water-solubility and reduced
aggregation as compared to nonsubstituted Pc, as well as
tumor-localizing properties and low toxicity. There are several
examples in the literature where phthalocyanine fluorescence
was used to study in vivo fluorescence kinetics and localization
of Pc.5,6 Several sulfonated metallo-phthalocyanine analogues
INTRODUCTION
■
A steadily increasing number of scientific papers and the rising
interest at scientific meetings attest that there has been a surge
in research on multimodal contrast agent development over the
past few years.1 Multimodal contrast agents can be visualized
using different imaging techniques in parallel. No single
molecular imaging modality is perfect and capable of providing
all necessary information to answer a particular question.2 For
example, it is difficult to obtain quantitative information using
fluorescence imaging (FI) alone, since it is limited by depth of
light penetration and scatter of emitted light photons; magnetic
resonance imaging (MRI) has high resolution and exquisite soft
tissue contrast, yet it suffers from very low sensitivity;
radionuclide-based imaging techniques are very sensitive and
highly quantitative, but they have poor spatial resolution.
Therefore, combining different molecular imaging modalities
offers synergistic advantages over any single modality allowing
high-resolution, high-sensitivity investigations of specific bio-
logical activities.3
Among prevailing imaging modalities used in biomedical
research, optical imaging is currently the most widely used
method.3 Strengths of this technique include high sensitivity
and direct detection of signals with simple optical devices, while
the major disadvantage is poor light penetration through soft
Received: May 24, 2013
Revised: July 26, 2013
Published: August 12, 2013
© 2013 American Chemical Society
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have been demonstrated to be preferentially retained in
tumors7−11 with the amphiphilic derivatives showing the
highest tumor-to-nontumor ratios.7,11 Furthermore, sulfonated
Pc are effective photosensitizers (PS) for photodynamic
therapy (PDT) of cancer to kill tumor cells in vitro12−17 and
cause tumor regression in vivo.7,18−24 Several Pc-based PS have
been investigated in preclinical and clinical trials and their
potential therapeutic application in oncology was demonstra-
ted.25,26
abbreviations were used to describe spin multiplicity: s =
singlet, d = doublet, t = triplet, q = quintet, dd = doublet of
doublets, m = multiplet. Mass spectra were acquired in positive
linear ion mode using a MALDI-TOF spectrometer (TofSpec
2E, Micromass/Waters). Samples were diluted with a saturated
solution of α-cyano-4-hydroxycinnamic acid, prepared in 50%
(v/v) aqueous acetonitrile containing 0.1% trifluoroacetic acid
(TFA). Visible absorption spectra were recorded in 1 cm quartz
cells. Analytical reverse phase HPLC was performed on a C18
semipreparative reverse phase column (10 × 250 mm, 5 μm) at
a flow rate of 2 mL/min using a linear gradient of 0% to 100%
MeOH in phosphate buffer (10 mM, pH 5.0) over 40 min.
Purification of the target conjugates was performed on the same
system. The identity of all new compounds was confirmed by
MALDI-TOF and MALDI-TOF/TOF mass-spectrometry for
HRMS, UV−vis spectroscopy, as well as by 1H, 13C NMR, and
HRMS for the N-Boc-oct-7-yne. Due to aggregation and the
presence of various regioisomers, the 1H NMR signals of
compounds 5−13 were not sufficiently resolved even at
relatively low concentrations to obtain any accurate data. For
We recently reported the synthesis of 64Cu-labeled
sulfonated phthalocyanines ([64Cu]CuPc) and their biodis-
tribution using PET imaging.11 The amphiphilic derivatives
(disulfonated Pc and trisulfonated Pc bearing a lipophilic
hexynyl chain) gave the highest tumor-to-background ratios.
Unfortunately, CuPc are not fluorescent in vivo as opposed to
ZnPc, so only a mixture of a fluorescent ZnPc and a
corresponding radioactive [64Cu]CuPc could be used as a
bimodal imaging probe,27 assuming that biodistribution of both
ZnPc and CuPc is the same and does not depend on the central
metal ion or in vivo concentration. Combining multimodal
functionality in a single probe unit is not necessary for all
applications, but there are advantages to this arrangement. A
single multimodal probe helps ensure the same pharmacoki-
netics and localization of signal for each modality and
eliminates additional stress on the body’s blood clearance
mechanisms that can accompany administration of multiple
doses of agents.1
For this reason, we proceeded to prepare conjugates
combining water-soluble fluorescent ZnPc and a multidentate
chelating agent like DOTA (1,4,7,10-tetraazocyclododecane-
N,N′N″,N′″-tetraacetic acid) or NOTA (1,4,7-triazacyclono-
nane-1,4,7-triacetic acid). DOTA and NOTA derivatives are
commercially available and their corresponding metal com-
plexes are widely used as contrast agents for MRI as well as for
PET and SPECT imaging. Besides, DOTA forms a stable
1
these reasons, H and 13C NMR data are not reported for
phthalocyanine derivatives 2−13. Phthalocyanine derivatives
can only be analyzed by MALDI-TOF. In reflector mode, a
mass accuracy below 60 ppm can be readily obtained with the
instrument. Copper-64 (t1/2 = 12.7 h) was produced on a 19
MeV cyclotron by proton bombardment of an enriched Ni-64
plated rhodium disk (22 mm diameter, 1 mm thickness).31 The
target material was dissolved in HCl to give [64Cu]CuCl2 and
converted to [64Cu]Cu(OAc)2 by dissolution in 0.1 M
ammonium acetate buffer (0.1 mL), pH 5.5. The 68Ga(III)
was eluted from the 68Ge/68Ga generator (68Ge t1/2 = 270.8
days) using ∼6−7 mL of a solution of 0.1 N hydrochloric acid
(HCl). Then, radioactive material was concentrated and
purified on a cation exchange resin following a literature
method.30,32 The 68Ga(III) was eluted from the resin with 800
μL of an 97.6% acetone/0.05 N HCl mixture (pH 2.3). Then,
500 μL of HEPES buffer (1 M, pH 4.0) was added and the
resulting solution of 68Ga(III) was used immediately for
radiolabeling of DOTA-ZnPc conjugates. Radio thin-layer
chromatography (TLC) was performed on an Instant Imager
scanner (Bioscan, DC, U.S.A.) using C18 TLC plates and 15%
methanol in sodium citrate buffer (1 M, pH 5.5) as a
developing solvent. Fluorescence spectra were acquired in
methanol in an Infinite M1000 PRO microplate reader.
General Procedure for Boc-Protection of Acetylenic
Amines. To a stirred solution of an acetylenic amine (3 mmol)
in a THF:H2O (1:1, 20 mL) mixture were sequentially added
Na2CO3 (3 equiv, 0.95 g, 9 mmol) and Boc2O (1.5 equiv, 0.98
g, 4.5 mmol). After 3 h, the reaction mixture was extracted with
EtOAc (2×). The combined organic layers were then dried
(MgSO4), filtered, and concentrated under reduced pressure.
The residue was purified on silica gel utilizing 10% EtOAc/
hexane.
complex with gallium(III) (formation constant, log KML
=
21.33)28 and therefore is widely used in preparation of 68Ga-
based PET probes as a chelating agent for 68Ga(III), a
radioisotope with high positron-emitting fraction (89% of its
total decay) and half-life of 68 min. NOTA has been shown to
form highly stable complexes with both Ga3+ and Cu2+ ions.29,30
In the current study, we report the synthesis of a series of
new potential bimodal probes combining a water-soluble
fluorescent sulfonated zinc phthalocyanine and a DOTA/
NOTA moiety providing a stable chelating environment for
68Ga3+ or 64Cu2+ ions for PET-imaging. ZnPc and the
polyazamacrocyclic moieties are linked together through an
aliphatic chain of different lengths to modulate the amphiphilic
character of the conjugates. Characterization, radiolabeling with
68Ga and 64Cu of the DOTA/NOTA-Pc conjugates, and in vitro
stability of the resulting fluorescent/PET probes are presented.
An imaging experiment with the most promising candidate is
also described to confirm its potential as a bimodal probe for
fluorescence and PET imaging.
N-Boc-3-Butynylamine. Prepared according to the general
procedure from 3-butynylamine. The product was obtained as a
colorless oil (0.35 g, 70% yield). The characterization data for
N-Boc-3-butynylamine were in agreement with previously
reported data.33
N-Boc-5-Hexynylamine. Prepared according to the general
procedure from 5-hexynylamine. The product was obtained as a
colorless oil (0.38 g, 65% yield). The characterization data for
N-Boc-5-hexynylamine were in agreement with previously
reported data.34
EXPERIMENTAL SECTION
■
General Methods. Unless otherwise stated, all reactions
were carried out under argon atmosphere. All chemicals and
1
solvents were used as supplied without further purification. H
and 13C NMR spectra were recorded on a 60/300 MHz
spectrometer in CDCl3 with the chemical shifts reported as δ in
ppm and coupling constants expressed in Hz. The following
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+
N-Boc-7-Octynylamine. Prepared according to the general
procedure from 7-octynylamine. The product was obtained as a
colorless oil (0.43 g, 64% yield): 1H NMR (300 MHz, CDCl3)
δ 1.32−1.59 (m, 17H), 1.97 (t, 2H, J = 3.0 Hz), 2.22 (td, 2H, J
= 6.0, 3.0 Hz), 3.14 (m, 2H), 4.54 (br. s, 1H); 13C NMR (75.5
MHz, CDCl3) δ 18.3, 26.3, 28.4, 30.0, 40.7, 68.2, 79.2, 84.6,
to give the desired product ZnPc-Cn-NH3 OTf− (n = 1, 3, 5),
5−7, without the need of further purification.
Zinc (4-Aminobut-1-ynyl)phthalocyanine Trisulfonate,
Ammonium Salt (ZnPc-C4−NH3 OTf, 5). Prepared according
+
to the general procedure from ZnPc-C4−NHBoc, 2. The
product was obtained as a dark-green solid (7.1 mg, quant.):
MALDI-TOF MS (m/z): [(M+H)+ ] calcd for
C36H22N9O9S3Zn 886.2, found 886.3, [(2M+H)+] calcd for
C72H43N18O18S6Zn2 1771.4, found 1769.7; MALDI-TOF
HRMS calcd for C36H22N9O9S3Zn [(M+H)+] 883.9994,
found: 883.9924 (Δm 8 ppm); UV−vis: λmax (DMF): 680,
613 (sh), 348; HPLC (675 nm): four close peaks, tR 19.0−25.2
min.
156.0; MALDI-TOF HRMS calcd for C8H16N [(M+H)+
-
BOC] 126.1283, found: 126.1209 (Δm 59 ppm).
General Procedure for Sonogashira Cross-Coupling of
ZnPc 1 and a Boc-Protected Acetylenic Amine. Pd(OAc)2
(1.1 mg, 50 mol.%), (o-tolyl)3P (4.5 mg, 150 mol.%),
phthalocyanine 1 (10 mg, 0.01 mmol), and a Boc-protected
acetylenic amine (0.1 mmol, 10-fold excess) were assembled in
a round-bottomed flask sealed with a septum. The reaction flask
was evacuated and purged with argon three times. Sub-
sequently, 1 μL of degassed DMF was added under argon,
followed by addition of DIPEA (∼30 μL, ∼15 equiv) degassed
beforehand by gentle bubbling Ar through it for 30 min. The
reaction mixture was stirred at 70 °C for 1−2 h. Thereafter,
solvent was evaporated under reduced pressure to 50−100 μL.
Phthalocyanines were precipitated using ethyl acetate, washed
subsequently with large amounts of ethyl acetate and methanol,
and dried in vacuo to give the desired crude product ZnPc-Cn-
NHBoc (n = 1, 3, 5), 2−4. This product was used for the next
step without further purification. The identity of the products
was confirmed by MALDI-TOF for their unprotected
analogues and UV−vis spectrometry.
Zinc (6-Aminohex-1-ynyl)phthalocyanine Trisulfonate,
Ammonium Salt (ZnPc-C6−NH3 OTf, 6). Prepared according
+
to the general procedure from ZnPc-C6−NHBoc, 3. The
product was obtained as a dark-green solid (7.4 mg, quant.):
MALDI-TOF MS (m/z): [(M+H)+ ] calcd for
C38H26N9O9S3Zn 914.3, found 914.4, [(2M+H)+] calcd for
C76H51N18O18S6Zn2 1827.6, found 1826.0; MALDI-TOF
HRMS calcd for C38H26N9O9S3Zn [(M+H)+] 912.0307,
found: 912.0186 (Δm 13 ppm); UV−vis: λmax (DMF): 680,
613 (sh), 348; HPLC (675 nm): three close peaks, tR 20.9−
24.8 min.
Zinc (8-Aminooct-1-ynyl)phthalocyanine Trisulfonate,
Ammonium Salt (ZnPc-C8−NH3 OTf, 7). Prepared according
+
to the general procedure from ZnPc-C8−NHBoc, 4. The
product was obtained as a dark-green solid (7.5 mg, quant.):
MALDI-TOF MS (m/z): [(M+H)+ ] calcd for
C40H30N9O9S3Zn 942.3, found 942.9, [(2M+H)+] calcd for
C80H59N18O18S6Z2 1885.6, found 1883.5; MALDI-TOF HRMS
calcd for C40H30N9O9S3Zn [(M+H)+] 940.0620, found:
940.0494 (Δm 13 ppm); UV−vis: λmax (DMF): 680, 614
(sh), 349; HPLC (675 nm): four close peaks, tR 24.5−28.4 min.
General Procedure for the Synthesis of DOTA/NOTA-
Zinc (N-Boc-4-aminobut-1-ynyl)phthalocyanine Trisulfo-
nate, Sodium Salt (ZnPc-C4−NHBoc, 2). Prepared according
to the general procedure from 1 and N-Boc-3-butynylamine.
The product was obtained as a dark-blue powder (9.2 mg,
88%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C36H22N9O9S3Zn 886.2, found 886.3, [(2M+H)+] calcd for
C72H43N18O18S6Zn2 1771.4, found 1769.7; UV−vis: λmax
(DMF): 680, 614 (sh), 353; HPLC (675 nm): four close
peaks, tR 30.3−32.3 min.
+
ZnPc Conjugates. To a solution of ZnPc-Cn-NH3 OTf− (n =
Zinc (N-Boc-6-aminohex-1-ynyl)phthalocyanine Trisulfo-
nate, Sodium Salt (ZnPc-C6−NHBoc, 3). Prepared according
to the general procedure from 1 and N-Boc-5-hexynylamine.
The product was obtained as a dark-blue powder (9.7 mg,
90%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C38H26N9O9S3Zn 914.3, found 914.4, [(2M+H)+] calcd for
C76H51N18O18S6Zn2 1827.6, found 1826.0; UV−vis: λmax
(DMF): 680, 614 (sh), 356; HPLC (675 nm): four close
peaks, tR 31.0−33.2 min.
Zinc (N-Boc-8-aminooct-1-ynyl)phthalocyanine Trisulfo-
nate, Sodium Salt (ZnPc-C8−NHBoc, 4). Prepared according
to the general procedure from 1 and N-Boc-7-octynylamine.
The product was obtained as a dark-blue powder (10.0 mg,
90%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C40H30N9O9S3Zn 942.3, found 942.9, [(2M+H)+] calcd for
C80H59N18O18S6Z2 1885.6, found 1883.5; UV−vis: λmax
(DMF): 680, 613 (sh), 355; HPLC (675 nm): four close
peaks, tR 29.8−33.8 min.
General Procedure for Boc-Deprotection of ZnPc-Cn-
NHBoc (n = 1, 3, 5), 2−4. To a suspension of the carbamate
ZnPc-Cn-NHBoc (n = 1, 3, 5), 2−4 (0.007 mmol), in CH2Cl2
(5 mL) at room temperature was added trifluoroacetic acid (2
mL). Immediately the product precipitated as a dark-green
solid, and the reaction was stirred 30 min more to ensure
completion of the reaction. As confirmed by RP-HPLC, the
product was isolated by filtration, washed subsequently with
large amounts of ethyl acetate and methanol and dried in vacuo
1, 3, 5), 5−7 (0.008 mmol), in a 5:1 mixture (1.2 mL) of DMF
and Et3N was added DOTA-NHS ester (3 equiv, 0.024 mmol,
18.2 mg) or NOTA-NHS ester (3 equiv, 0.024 mmol, 9.6 mg).
If after 30 min of stirring, dissolution of Pc was not complete,
deionized water was added in 100 μL portions. The reaction
was stopped after MALDI-TOF analysis revealed complete
disappearance of the starting amino-substituted Pc (12−24 h).
When the reaction was incomplete, 2 equiv more of NHS-ester
of DOTA (12.1 mg, 0.016 mmol) or NOTA (6.4 mg, 0.016
mmol) were added and the stirring continued until completion
(6−12 h). After that the solvent was evaporated under reduced
pressure to a final volume of 50−100 μL, Pc was precipitated
using 1 N HCl, filtrated off, and washed subsequently with large
amounts of water and MeOH to give the desired crude
conjugate DOTA/NOTA-Cn-ZnPc (n = 1, 3, 5), 8−13 in acidic
form. The solid was dissolved in a minimum volume of 1 M
NaOH, diluted with deionized water, and purified on
preconditioned (methanol followed by water) Sep Pack C18
cartridge eluted with water and methanol−water (1:4, v/v).
The blue-green methanol−water fraction was evaporated in
vacuo and the dark-blue residue was redissolved in a minimum
volume of water and freeze-dried to yield the pure product. The
analytical sample was further purified by semipreparative RP-
HPLC using linear gradient of MeOH in phosphate buffer (10
mM, pH 5) over 40 min.
Zinc 4-[(4,7,10-Tri(carboxymethyl)-1,4,7,10-tetraazacyclo-
dec-1-yl)acetamido]but-1-ynyl) Phthalocyanine Trisulfonate,
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Sodium Salt (DOTA-C4−ZnPc, 8). Prepared according to the
67%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C52H49N12O14S3Zn 1227.6, found 1127.8, [(M-CH2COOH
+H)+] calcd for C50H47N12O12S3Zn 1169.6, found 1170.7;
MALDI-TOF HRMS calcd for C52H49N12O14S3Zn [(M+H)+]
1225.1945, found: 1225.2158 (Δm 17 ppm);UV−vis λmax
(DMF): 681, 615 (sh), 352; HPLC (675 nm): three close
broad peaks, tR 28.6−31.6 min.
general procedure from ZnPc-C4−NH3 OTf−, 5, and DOTA-
+
NHS ester. The product was obtained as a blue-green solid (7.2
mg, 65%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C52H48N13O16S3Zn 1272.6, found 1273.2, [(M-CO2+H)+]
calcd for C51H48N13O14S3Zn 1227.6, found 1228.1, [(2M
+H)+] calcd for C104H95N26O32S6Zn2 2544.2, found 2544.0;
MALDI-TOF HRMS calcd for C52H48N13O16S3Zn [(M+H)+]
1270.1796, found: 1270.2090 (Δm 23 ppm); UV−vis λmax
(DMF): 680, 614 (sh), 348; HPLC (675 nm): two close broad
peaks, tR 22.1−23.9 min.
General Procedure for the 68Ga-Radiolabeling. A
solution of DOTA-ZnPc conjugate 8−10 (10 μL, 430 μM)
in deionized water was added to ∼500 μL of HEPES buffer/
acetone containing ∼165 MBq (∼4.5 mCi) of 68Ga(III). The
reaction vial was heated at 95−100 °C for 15 min. Reaction
progress was followed by radio-TLC to confirm completion of
the radiolabeling. The resulting blue solution was passed
through a preconditioned (methanol followed by water)
reversed-phase Sep-Pak C18 cartridge. The cartridge was
washed with water (10 mL). Thereafter, the blue radioactive
product was eluted with 2 mL of MeOH. The 68Ga/DOTA-Cn-
Pc fraction was collected, evaporated, and counted in a
Capintec radioisotope calibrator (Capintec, Inc., NJ, USA) to
determine the specific activity of the product. The solvent was
evaporated to dryness to yield the 68Ga/DOTA-Cn-Pc (14−16)
as a blue solid (radiochemical purity >98%, as checked by
radio-TLC) in ∼60% decay-corrected yield.
Zinc 6-[(4,7,10-Tri(carboxymethyl)-1,4,7,10-tetraazacyclo-
dec-1-yl)acetamido]hex-1-ynyl) Phthalocyanine Trisulfo-
nate, Sodium Salt (DOTA-C6−ZnPc, 9). Prepared according
to the general procedure from ZnPc-C6−NH3 OTf−, 6, and
+
DOTA-NHS ester. The product was obtained as a blue-green
solid (8.0 mg, 70%): [(M+H)+] calcd for C54H52N13O16S3Zn
1300.7, found 1300.9, [(M-CH2COOH+H)+] calcd for
C52H50N13O14S3Zn 1242.6, found 1243.2; UV−vis λmax
(DMF): 681, 614 (sh), 351; HPLC (675 nm): four close
peaks, tR 26.1−28.6 min.
Zinc 8-[(4,7,10-Tri(carboxymethyl)-1,4,7,10-tetraazacyclo-
dec-1-yl)acetamido]oct-1-ynyl) Phthalocyanine Trisulfonate,
Sodium Salt (DOTA-C8−ZnPc, 10). Prepared according to the
general procedure from ZnPc-C8−NH3 OTf−, 7, and DOTA-
+
Non-radiolabeled analogues Ga/DOTA-Cn-ZnPc (14−16)
were prepared in a similar manner using GaCl3.
Compound Ga/DOTA-C4−ZnPc, 14. MALDI-TOF MS (m/
z): [(M+H)+] calcd for C52H46GaN13O16S3Zn 1340.3, found
1340.6; UV−vis λmax (DMF): 680, 614 (sh), 347.
Compound Ga/DOTA-C6−ZnPc, 15. MALDI-TOF MS (m/
z): [(M+H)+] calcd for C54H50GaN13O16S3Zn 1368.4, found
1368.6; UV−vis λmax (DMF): 680, 614 (sh), 350.
Compound Ga/DOTA-C8−ZnPc, 16. MALDI-TOF MS (m/
z): [(M+H)+] calcd for C56H54GaN13O16S3Zn 1396.4, found
1396.3; UV−vis λmax (DMF): 681, 614 (sh), 353.
NHS ester. The product was obtained as a blue-green solid (8.5
mg, 73%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C56H56N13O16S3Zn 1328.7, found 1329.1, [(M-CO2+H)+]
calcd for C51H48N13O14S3Zn 1284.7, found 1284.3, [(2M
+H)+] calcd for C55H56N13O14S3Zn 2656.4, found 2654.9;
MALDI-TOF HRMS calcd for C56H56N13O16S3Zn [(M+H)+]
1326.2422, found: 1326,1923 (Δm 38 ppm); UV−vis λmax
(DMF): 682, 617 (sh), 354; HPLC (675 nm): six close peaks,
tR 28.2−31.2 min.
Zinc 4-[(4,7-Di(carboxymethyl)-1,4,7-triazacyclononan-1-
yl)acetamido]but-1-ynyl) Phthalocyanine Trisulfonate, So-
dium Salt (NOTA-C4−ZnPc, 11). Prepared according to the
General Procedure for the 64Cu-Radiolabeling. A
solution of NOTA-ZnPc conjugates 11−13 (20 μL, 800 μM)
in deionized water was added to a solution of [64Cu]Cu(OAc)2
(∼130 MBq; ∼3.5 mCi) in 150−200 μL of ammonium acetate
buffer (0.1 M, pH 5.5). The reaction mixture was heated at 95−
100 °C for 15 min. Radio-TLC revealed product formation in
decay-uncorrected yields ranging from 90% to 100%. The
resulting blue solution was passed through a reversed-phase
Sep-Pak C18 cartridge and the product was absorbed on the
cartridge. The cartridge was then washed with water (3 × 10
mL); thereafter, the blue radioactive product was eluted with 2
mL of MeOH. The 64Cu/NOTA-Cn-Pc fraction was collected,
evaporated, and counted in a Capintec radioisotope calibrator
(Capintec, Inc., NJ, USA) to determine the specific activity of
the product. The solvent was evaporated to dryness to give
64Cu/NOTA-Cn-Pc (17−19) as a blue solid (radiochemical
purity >95%, as checked by radioactive TLC) in ∼70% decay-
uncorrected yield.
Non-radiolabeled analogues Cu/NOTA-Cn-ZnPc (17−19)
were prepared in a similar manner using Cu(II) acetate.
Compound Cu/NOTA-C4−ZnPc, 17. MALDI-TOF MS (m/
z): [(M+H)+] calcd for C48H39CuN12O14S3Zn 1233.0, found
1233.2; UV−vis λmax (DMF): 681, 614 (sh), 347.
Compound Cu/NOTA-C6−ZnPc, 18. MALDI-TOF MS (m/
z): [(M+H)+] calcd for C50H43CuN12O14S3Zn 1261.1, found
1261.1; UV−vis λmax (DMF): 682, 616 (sh), 349.
general procedure from ZnPc-C4−NH3 OTf−, 5, and NOTA-
+
NHS ester. The product was obtained as a blue-green solid (7.2
mg, 70%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C48H41N12O14S3Zn 1171.5, found 1171.9, [(M-CO2+H)+]
calcd for C47H41N12O12S3Zn 1127.5, found 1127.9; MALDI-
TOF HRMS calcd for C48H41N12O14S3Zn [(M+H)+]
1169.1319, found: 1169, 1992 (Δm 58 ppm);UV−vis λmax
(DMF): 681, 614 (sh), 353; HPLC (675 nm): three broad
close peaks, tR 24.6−27.0 min.
6-[(4,7-Di(carboxymethyl)-1,4,7-triazacyclononan-1-yl)-
acetamido]hex-1-ynyl) Phthalocyanine Trisulfonate, Sodium
Salt (NOTA-C6−ZnPc, 12). Prepared according to the general
+
procedure from ZnPc-C6−NH3 OTf−, 6, and NOTA-NHS
ester. The product was obtained as a blue-green solid (6.6 mg,
64%): MALDI-TOF MS (m/z): [(M+H)+] calcd for
C50H45N12O14S3Zn 1199.6, found 1200.0, [(M-CH2COOH
+H)+] calcd for C48H43N12O12S3Zn 1141.5, found 1142.2;
MALDI-TOF HRMS calcd for C50H45N12O14S3Zn [(M+H)+]
1197.1632, found: 1197.2046 (Δm 35 ppm);UV−vis λmax
(DMF): 682, 615 (sh), 352; HPLC (675 nm): four close
broad peaks, tR 26.5−28.7 min.
8-[(4,7-Di(carboxymethyl)-1,4,7-triazacyclononan-1-yl)-
acetamido]oct-1-ynyl) Phthalocyanine Trisulfonate, Sodium
Salt (NOTA-C8−ZnPc, 13). Prepared according to the general
+
procedure from ZnPc-C8−NH3 OTf−, 7, and NOTA-NHS
ester. The product was obtained as a blue-green solid (7.1 mg,
1627
dx.doi.org/10.1021/bc400257u | Bioconjugate Chem. 2013, 24, 1624−1633