Photo-initiated thiol-ene click reactions as a potential strategy for incorporation of [MI(CO)3]+ (M = Re, 99mTc) complexes

Article abstract of DOI:10.1021/ic302771f

Click reactions offer a rapid technique to covalently assemble two molecules. In radiopharmaceutical construction, these reactions can be utilized to combine a radioactive metal complex with a biological targeting molecule to yield a potent tool for imaging or therapy applications. The photo-initiated radical thiol-ene click reaction between a thiol and an alkene was examined for the incorporation of [M^I(CO)₃]⁺ (M = Re, ^99mTc) systems for conjugating biologically active targeting molecules containing a thiol. In this strategy, a potent chelate system, 2,2′-dipicolylamine (DPA), for [M^I(CO)₃]⁺ was functionalized at the central amine with a terminal alkene linker that was explored with two synthetic approaches, click then chelate and chelate then click, to determine the flexibility and applicability of the thiol-ene click reaction to specifically incorporate ligand systems and metal complexes with a thiol containing molecule. In the click then chelate approach, the thiol-ene click reaction was carried out with the DPA chelate followed by complexation with [M^I(CO)₃]⁺. In the chelate then click approach, the alkene functionalized DPA chelate was first complexed with [M ^I(CO)₃]⁺ followed by the conduction of the thiol-ene click reaction. Initial studies utilized benzyl mercaptan as a model thiol for both strategies to generate the identical product from either route to provide information on reactivity and product formation. DPA ligands functionalized with two unique linker systems (allyl and propyl allyl ether) were prepared to examine the effect of the proximity of the chelate or complex on the thiol-ene click reaction. Both the thiol-ene click and coordination reactions with Re, ^99mTc were performed in moderate to high yields demonstrating the potential of the thiol-ene click reaction for [M ^I(CO)₃]⁺ incorporation into thiol containing biomolecules.

Full text of DOI:10.1021/ic302771f

Article
pubs.acs.org/IC
Photo-initiated Thiol-ene Click Reactions as a Potential Strategy for
Incorporation of [MI(CO)3]+ (M = Re, 99mTc) Complexes
Thomas R. Hayes,† Patrice A. Lyon,† Elsa Silva-Lopez,† Brendan Twamley,‡ and Paul D. Benny*,†
†Department of Chemistry, Washington State University, P.O. Box 644630, Pullman, Washington 99164, United States
‡Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343, United States
S
* Supporting Information
ABSTRACT: Click reactions offer a rapid technique to covalently
assemble two molecules. In radiopharmaceutical construction, these
reactions can be utilized to combine a radioactive metal complex with a
biological targeting molecule to yield a potent tool for imaging or therapy
applications. The photo-initiated radical thiol-ene click reaction between a
thiol and an alkene was examined for the incorporation of [MI(CO)3]+ (M
= Re, 99mTc) systems for conjugating biologically active targeting
molecules containing a thiol. In this strategy, a potent chelate system,
2,2′-dipicolylamine (DPA), for [MI(CO)3]+ was functionalized at the
central amine with a terminal alkene linker that was explored with two
synthetic approaches, click then chelate and chelate then click, to determine
the flexibility and applicability of the thiol-ene click reaction to specifically
incorporate ligand systems and metal complexes with a thiol containing molecule. In the click then chelate approach, the thiol-ene
click reaction was carried out with the DPA chelate followed by complexation with [MI(CO)3]+. In the chelate then click
approach, the alkene functionalized DPA chelate was first complexed with [MI(CO)3]+ followed by the conduction of the thiol-
ene click reaction. Initial studies utilized benzyl mercaptan as a model thiol for both strategies to generate the identical product
from either route to provide information on reactivity and product formation. DPA ligands functionalized with two unique linker
systems (allyl and propyl allyl ether) were prepared to examine the effect of the proximity of the chelate or complex on the thiol-
ene click reaction. Both the thiol-ene click and coordination reactions with Re, 99mTc were performed in moderate to high yields
demonstrating the potential of the thiol-ene click reaction for [MI(CO)3]+ incorporation into thiol containing biomolecules.
chemistry has offered a viable alternative for the formation of
the linker between these molecules without these limita-
tions.7−9 Defined as reactions that are stereospecific, high-
yielding, easy to purify, and occur rapidly to combine two
orthogonal compounds, “click” reactions are well suited to the
facile formation of a covalent linker and have been applied to
many branches of organic chemistry including polymer
chemistry, protein chemistry, combinatorial chemistry, and
drug development.10,11 A number of reactions (e.g., oxirane and
aziridine ring-openings, additions of thiols, and hydrazone
condensations) have been proposed as potential “click”
reactions.12 Due to its facile nature, the CuI-catalyzed Huisgen
1,3 dipolar cycloaddition “click” reaction between an azide and
alkyne remains one of the most utilized in polymer synthesis,
post-translational modifications (e.g., glycosylation), peptide
synthesis, and combinatorial library synthesis.13 Recently, the
Huisgen cycloaddition reaction has been specifically imple-
mented in the synthesis of [MI(CO)3]+ (M = Re, 99mTc)
imaging agents in several strategies from incorporating the
triazole product into chelate design (e.g., click to chelate)14−17
or as a coupling strategy to append chelates or complexes to a
INTRODUCTION
■
Technetium-99m (γ = 140 keV, t1/2 = 6.0 h) is the primary
radionuclide used in clinical diagnostic imaging for single
photon emission computed tomography (SPECT). The
versatile chemistry of the transition metal has led to a number
of proposed labeling strategies, ligands and complexes.
Recently, the low valent organometallic species fac-
[
99mTcI(OH2)3(CO)3]+ has gained significant notoriety because
of its small molecular volume, tridentate coordination,
increased redox stability, and reproducible preparation
compared to midvalent TcV oxo/dioxo complexes.1−4 Selective
delivery of [99mTcI(CO)3]+ to a particular cellular target can be
achieved by attachment of a targeting moiety through a
bifunctional chelator (BFC) that functions to coordinate the
[
99mTcI(CO)3]+ core and to provide a covalent linker between
the two moieties. The design and function of BFCs and linkers
have been of increasing interest to mitigate the effects of the
lipophilic [99mTcI(CO)3] core on the phamokinetics of the
targeting moiety.5,6
Traditionally, attachment of the BFC to the targeting moiety
has been achieved by formation of amides through an activated
ester and an amine to yield the covalent product. However,
these reactions can pose synthetic challenges and often require
additional protection and deprotection steps. Recently, “click”
Received: December 17, 2012
Published: February 27, 2013
© 2013 American Chemical Society
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targeting moiety.18 More specifically in coupling strategies, the
BFC can be complexed with [MI(CO)3]+ prior to the click
reaction to give the desired “click” product (chelate then click)
or the “click” reaction is carried out between the targeting
moiety and the BFC prior to Re/99mTc coordination (click then
chelate). The chelate then click method offers a distinct
advantage for preparing radiolabeled compounds separately
under reaction conditions typical for [99mTcI(CO)3]+ complex-
ation (>90 °C, 30 min) prior to the click reaction. This offers
the advantage of the targeting moiety being subjected to mild
conditions in comparison to the coordination reactions and
simplifies the removal of excess copper because of the
occupation of the chelation site by the [99mTcI(CO)3]+ metal
center.
Each strategy takes advantage of the fast reaction rates and
selectivity of the CuI-catalyzed Huisgen click reaction.
However, the use of the CuI-catalyst presents a challenge in
each application as purification is required to remove cytotoxic
copper salts found in solution or bound to the chelate system
prior to in vitro and in vivo usage. To circumvent the copper
toxicity issues, the “click” chemistry field has shifted toward
copper-free “click” reactions, which utilize molecules with ring
strain or functional groups with increased reactivity to carry out
these reactions without the use of metal catalysts.19 Although
many “click” reactions fall under this copper-free classification
(e.g., hydrazine/hydrazone, Michael addition, Diels−Alder),
the ring strained cyclooctyne Huisgen cycloaddition has gained
considerable interest because of similarities in reactivity and
preparation of azide containing molecules to yield a 1,2,3
triazole ring.20 While this approach offsets the need for a Cu
catalyst, the cyclooctyne or dibenzylcyclooctyne adds tremen-
dous lipophilicity and steric bulk to the click product, which can
negatively impact the solubility and pharmacokinetics of the
compound.21
A method to make BFCs that can be attached through facile
“click” chemistry and has a minimal effect on the
pharmacokinetics of the targeting moiety is of interest in the
assembly of radiopharmaceuticals. Thiols (R-SH) present an
attractive moiety for functionalization by BFCs due their
biological availability in amino acids (e.g., cysteine and
homocysteine), peptides, and antibodies. Several thiol-based
“click” reactions (e.g., S-alkylation22,23 and the Michael addition
of thiols to acrylates or maleimides24,25) have been previously
applied in radiopharmaceutical synthetic strategies for the
incorporation of metal chelators. However, both methods have
potential drawbacks because of limitations in reaction specificity
and product stability. Alkylation of thiols with alkyl bromides
can exhibit cross reactivity with other nucleophilic functional
groups (e.g., amines) to yield non-thiol specific products. In
Michael addition reactions of maleimides, specificity for thiols is
maintained at reasonable pHs (6−8), but the thioether linkange
can undergo retro-Michael reactions under physiological
conditions. This instability can lead to decoupling of the BFC
from the targeting molecule limiting the effectiveness of the
click reaction.26
macrocyclic compounds.32 An advantage of the thiol-ene
reaction is that reactions can be carried out neat or in small
volumes with reaction times amenable to radiopharmaceutical
applications.28,33 The combination of the biological availability
of thiols (e.g., cysteine and homocysteine) and alkene
functionalization strategies similar to the alkyne moieties used
in Huisgen reactions suggest the radical thiol-ene reaction as an
attractive technique for the conjugation of biological targets
with BFCs.
In this work, we examine the feasibility of the radical thiol-
ene “click” reaction for the formation of BFCs for the
[MI(CO)3]+ core. The BFC was generated by functionalizing
the central amine of 2,2′-dipicolylamine (DPA), a potent
chelator for the [MI(CO)3]+ core, with a linker containing a
terminal alkene (allyl or propyl allyl ether). The functionalized
DPA chelate and the corresponding [MI(CO)3]+ complexes
were conjugated to a model thiol, benzyl mercaptan, using a
photo-initiated radical generator, 2,2-dimethoxy-2-phenylaceto-
phenone (DMPA), to determine the potential for thiol-ene
click reactions with these compounds. Two overall strategies
(click then chelate and chelate then click) were explored using the
thiol-ene reaction to determine the most effective route of
assembly of the final product of both reactions. Macroscale
reactions were carried out with [ReI(CO)3]+ analogues for
standard chemical characterization and compared to the
radioactive complexes of [99mTcI(CO)3]+.
EXPERIMENTAL SECTION
■
All reagents and organic solvents of reagent grade or better were used
as purchased from Aldrich, Acros, or Fluka without further
purification. Rhenium starting materials ReI(CO)5Br, and fac-
[ReI(OH2)3(CO)3](SO3CF3), were prepared by literature methods
from Re2(CO)10 purchased from Strem.34,35 99mTc was obtained in the
form of Na[99mTcO4]−, and the [99mTcI(OH2)3(CO)3]+ complex was
prepared using a commercially available Isolink kit from Covidien.
Compound 5 was prepared as previously reported.36 UV−vis spectra
were obtained using a Varian Cary 50 spectrophotometer (1 cm path-
1
length). H and 13C NMR spectra were recorded on a Varian 400
MHz instrument at 25 °C in CD3OD or CDCl3. Elemental analyses
were performed by Intertek Pharmaceutical Services. FT-IR spectra
were obtained on a Thermo Nicolet 6700 FT-IR with an ATR cell and
analyzed with OMNIC 7.1 software. Mass spectra were obtained on a
Thermo-Finnigan LCQ Advantage ESI-MS. Irradiation of samples at
366 nm was performed using a 18W Blak-Ray lamp Model UVL-21.
Separation and identification of compounds were conducted on a
Perkin-Elmer Series 200 High Performance Liquid Chromatography
(HPLC) equipped with a UV/vis Series 200 detector and a
Radiomatic 610TR detector. Utilizing a Varian Pursuit XRs 5 μm
particle and 250 × 4.6 mm C-18 column, the compounds were
separated with a reverse phase gradient system beginning with 0.1%
trifluoroacetic acid (TFA) aqueous eluent gradually shifting to
methanol. HPLC analysis was performed using 0−3.0 min (100%
TFA), 3.0−9.0 min (75% TFA, 25% MeOH), 9.0−20.0 min (25% to
100% MeOH linear gradient), 20.0−25.0 min (100% MeOH) at a flow
rate of 1.0 mL/min.
Purification of compounds was conducted on a Hitachi preparatory
HPLC. Utilizing a Varian Pursuit XRs 5 μm particle and 250 × 21.2
mm C-18 column, the compounds were separated with a reverse phase
gradient system beginning with 0.1% TFA aqueous eluent gradually
shifting to methanol. Two methods were used for HPLC purification
of compounds; preparatory HPLC method 1 was performed using 0−
3.0 min (100% TFA), 3.0−6.0 min (40% TFA, 60% MeOH), 6.0−22.0
min (60% to 100% MeOH linear gradient), 22.0−28.0 min (100%
MeOH) at a flow rate of 10.0 mL/min, and preparatory HPLC
method 2 was performed with 0−3.0 min (100% TFA), 3.0−6.0 min
(60% TFA, 40% MeOH), 6.0−22.0 min (40% to 100% MeOH linear
The radical thiol-ene click reaction yields a product which is
both stable and thiol specfic. In the thiol-ene reaction, an alkene
and a thiol are reacted in the presence of a radical initiator to
produce a stable thioether linker. While the primary use of the
thiol-ene reaction has been in the synthesis of polymers,
dendrimers,27 and other macromolecules,28 this reaction has
recently been used in the functionalization of biomolecules
such as peptides29 and sugars30,31 and in the formation of
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gradient), 22.0−28.0 min (100% MeOH) at a flow rate of 10.0 mL/
min.
8.67 (d, 2H, J = 5.9 Hz); 13C NMR (100 MHz, CDCl3) δ 57.2, 59.8,
117.8, 121.8, 122.7, 135.2, 136.3, 148.9, 159.6 ; IR: 2359, 2342, 1931
cm−1; UV (λmax 266 nm, CH3CN): εmax 10900 M−1 cm−1; MS (m/z):
[M]+ 510.1.
N,N-bis(pyridin-2-ylmethyl)prop-2-en-1-amine, 1. DPA
(0.565 mL, 3.04 mmol) was dissolved in CH3CN (10 mL). Cs2CO3
(1.560 g, 4.79 mmol) and allyl bromide (0.368 mL, 4.09 mmol) were
then added, and the resulting suspension was stirred for 3 h at room
temperature. The reaction mixture was then filtered through Celite
and concentrated to dryness. The resulting oil was dissolved in
chloroform, and the resulting precipitate was removed by filtration.
Concentration in vacuo of the remaining solution gave 1 as a brown oil
(0.699 g, 96%).
3-(Allyloxy)-N,N-bis(pyridin-2-ylmethyl)propan-1-amine, 6.
Compound 5 (0.420 g, 1.63 mmol) was dissolved in dry THF (10
mL) under N2. The solution was cooled to 0 °C and a 60% dispersion
of NaH (0.196 g, 4.90 mmol) was added. After 30 min allyl bromide
(0.424 mL, 4.90 mmol) was added and the reaction was allowed to
warm to room temperature. After 5 h, DI H2O (2 mL) was added and
the solution was stirred for 10 min. The reaction was then
concentrated to dryness and the residue was dissolved in CH2Cl2
(50 mL). This was then washed with 0.1 M NaOH (10 mL) and the
organic layer was extracted with 1 M HCl (2 × 30 mL). The aqueous
layer was washed with hexanes (30 mL) and then basified with NaOH
to a pH > 10. The aqueous layer was extracted with CH2Cl2 (2 × 30
mL) and the combined organic extract was washed with brine (30
mL), dried over anhydrous MgSO4, filtered, and the filtrate was
concentrated in vacuo to give pure product 6 as a brown oil (0.454 g,
94% yield).
Anal. Calcd for C18H23N3O·0.05CH2Cl2: C, 71.46; H, 7.66; N,
13.85. Found: C, 71.40; H, 7.53; N, 13.83; 1H NMR (400 MHz,
CDCl3) δ 1.79 (p, 2H, J = 6.4 Hz), 2.60 (t, 2H, J = 7.1 Hz), 3.39 (t,
2H, J = 6.3 Hz), 3.77 (s, 4H), 3.83 (d, 2H, J = 4.3 Hz), 5.11 (m, 2H),
5.79 (m, 1H), 7.08 (t, 2H, J = 5.1 Hz), 7.48 (d, 2H, J = 7.8 Hz), 7.59
(dt, 2H, J = 7.4, 1.5 Hz), 8.45 (d, 2H, J = 5.1 Hz); 13C NMR (100
MHz, CDCl3) δ 27.4, 51.3, 60.4, 68.4, 71.7, 116.7, 121.8, 122.0, 135.0,
136.4, 148.9, 159.8; IR: 2360, 1590 cm−1; UV (λmax 252 nm, MeOH):
εmax 6143 M−1 cm−1; MS (m/z): [M+Na]+ 320.2.
3-(Benzylthio)-N,N-bis(pyridin-2-ylmethyl)propan-1-amine,
7. Compound 6 (0.100 g, 0.336 mmol) was dissolved in MeOH (50
μL). Benzyl mercaptan (0.099 mL, 0.841 mmol) and DMPA (17 mg,
0.067 mmol) were added and the solution was irradiated with a 366
nm UV lamp for 1 h at room temperature. The reaction was then
diluted with H2O and MeOH and purified by preparatory HPLC using
prep HPLC method 2. The solution was then dried in vacuo and the
resulting oil was dissolved in H2O (30 mL), basified with 1 M NaOH
to a pH > 10 and extracted with CH2Cl2 (3 × 20 mL). The combined
organic layers were dried with anhydrous MgSO4, filtered, and
concentrated in vacuo to give product 7 as a brown oil (0.097 g, 68%).
1H NMR (400 MHz, CDCl3) δ 1.70 (m, 4H), 2.35 (t, 2H, J = 7.5
Hz), 2.55 (t, 2H, J = 7.1 Hz), 3.31 (m, 4H), 3.60 (s, 2H), 3.74 (s, 4H),
7.05 (m, 2H), 7.19 (m, 5H), 7.44 (d, 2H, J = 7.9 Hz), 7.56 (t, 2H, J =
7.4 Hz), 8.44 (d, 2H, J = 4.7 Hz); 13C NMR (400 MHz, CDCl3) δ
27.5, 28.2, 29.4, 36.3, 51.4, 60.6, 69.0, 69.3, 122.0, 122.9, 127.0, 128.6,
128.9, 136.5, 138.6, 149.1, 160.0; IR: 1589, 1473 cm−1; UV (λmax 262
nm, MeOH): εmax 7216 M−1 cm−1; MS (m/z): [M+H]+ 422.3.
fac-[ReI(CO)3(7)](CF3CO2), 8. Method A (Click then chelate):
Compound 7 (0.064 g, 0.152 mmol) was dissolved in MeOH (5 mL).
ReI(CO)5Br (0.0678 g, 0.167 mmol) was added to solution and the
reaction was then heated to reflux for 18 h. The solution was cooled to
room temperature and concentrated to dryness. The resulting oil was
then purified using preparatory HPLC method 2. Fractions from the
prep HPLC were checked for purity by analytical HPLC (R.T. = 22.2
min). The fractions were concentrated to dryness and gave complex 8
as a yellow oil (0.047 g, 74%).
1H NMR (400 MHz, CDCl3) δ 3.11 (d, 2H, J = 6.3 Hz), 3.81 (s,
4H), 5.12 (m, 2H), 5.85 (m, 1H), 7.04 (m, 2H), 7.52 (d, 2H, J = 8.0
Hz), 7.64 (dt, 2H, J = 1.9 Hz, 7.7 Hz), 8.52 (d, 2H, J = 4.6 Hz); 13C
(100 MHz, CDCl3) 57.2, 59.8, 117.8, 121.8, 122.7, 135.2, 136.3, 148.9,
159.6; MS m/z: [M+H]+ 240.2
3-(Benzylthio)-N,N-bis(pyridin-2-ylmethyl)propan-1-amine,
2. Compound 1 (0.100 g, 0.418 mmol) was dissolved in MeOH (50
μL). Benzyl mercaptan (0.124 mL, 1.05 mmol) and DMPA (21 mg,
0.084 mmol) were added and the solution was irradiated with a 366
nm UV lamp for 1 h at room temperature. The reaction was diluted
with H2O and MeOH and purified by preparatory HPLC method 2.
The solution was dried in vacuo and the resulting oil was dissolved in
H2O (30 mL), basified with 1 M NaOH to a pH > 10 and extracted
with CH2Cl2 (3 × 20 mL). The combined organic layers were dried
with anhydrous MgSO4, filtered, and concentrated in vacuo to give
product 2 as a brown oil (0.113 g, 75%).
1H NMR (400 MHz, CDCl3) δ 1.77 (p, 2H, J = 7.4 Hz), 2.39 (t,
2H, J = 7.4 Hz), 2.60 (t, 2H, J = 7.1 Hz), 3.65 (s, 2H), 3.78 (s, 4H),
7.14 (m, 2H), 7.26 (m, 5H), 7.46 (d, 2H, J = 7.8 Hz), 7.63 (dt, 2H, J =
7.8, 2.0 Hz), 8.52 (d, 2H, J = 4.0 Hz); 13C NMR (100 MHz, CDCl3) δ
26.8, 29.0, 36.1, 53.3, 60.4, 121.9, 122.8, 126.9, 128.4, 128.8, 136.4,
138.5, 140.0, 159.7 ; IR: 1588, 1567, 1494 cm−1; UV (λmax 262 nm,
MeOH): εmax 9480 M−1 cm−1; MS m/z: [M+H]+ 364.2.
fac-[ReI(CO)3(2)](CF3CO2), 3. Compound 2 (0.056 g, 0.154 mmol)
was dissolved in MeOH (5 mL). ReI(CO)5Br (0.0688 g, 0.170 mmol)
was added to the solution and the reaction was then heated to reflux
for 18 h. The solution was cooled to room temperature and
concentrated to dryness. The resulting oil was then purified by
preparatory HPLC method 2. Fractions from the prep HPLC were
checked for purity by analytical HPLC (R.T. = 21.7 min). The solution
was concentrated to dryness and to give complex 3 as a brown oil
(0.040 g, 72%).
Anal. Calcd for C27H25N3O5F3SRe·C2F3O2H·1.5H2O: C, 39.23; H,
3.29; N, 4.73. Found: C, 39.15; H, 3.14; N, 4.79; 1H NMR (400 MHz,
CDCl3) δ 2.21 (m, 2H), 2.58 (t, 2H, J = 6.3 Hz), 3.78 (m, 4H), 4.16,
5.64 (ABq, 4H, J = 16.4 Hz), 7.19 (m, 3H), 7.31 (dd, 2H, J = 7.8 Hz),
7.39 (d, 2H, J = 7.0 Hz), 7.78 (m, 4H), 8.64 (d, 2H, J = 5.4 Hz); 13C
NMR (100 MHz, CDCl3) δ 24.1, 28.6, 36.3, 66.9, 69.7, 124.6, 125.5,
128.3, 128.7, 129.1, 138.4, 140.1, 150.9, 160.7, 195.7; IR: 2026, 1900
cm−1; UV (λmax 262 nm, MeOH): εmax 11005 M−1cm−1; MS (m/z):
[M]+ 634.2.
fac-[ReI(CO)3(1)](CF3CO2), 4. Compound 1 (0.050 g, 0.208 mmol)
was dissolved in MeOH (3 mL). A 0.1 M solution of fac-
[ReI(OH2)3(CO)3](SO3CF3) (2.5 mL, 0.250 mmol) was added and
the pH was adjusted to 6 with saturated NaHCO3. Additional MeOH
was added until all solids were dissolved and the solution was heated
to 50 °C for 1 h. The solution was then cooled to room temperature
and concentrated to dryness. The resulting solid was then purified by
preparatory HPLC using preparatory HPLC method 1. Fractions from
the prep HPLC were checked for purity by analytical HPLC (R.T. =
19.7 min). The solution obtained using this method was then
concentrated in vacuo to give complex 4 as a yellow oil (0.096 g, 74%).
Crystals were obtained of complex 4 with the triflate counterion prior
to HPLC purification by slow evaporation from a acetonitrile,
methanol, toluene solution.
Method B (Chelate then click): Complex 9 (33 mg, 0.05 mmol)
was dissolved in MeOH (100 μL). Benzyl mercaptan (15 mL, 0.125
mmol) and DMPA (2.5 mg, 0.001 mmol) was then added and the
solution was irradiated at 366 nm for 1 h at room temperature. The
reaction was then rotovapped to dryness and the resulting oil was
washed with Et2O (3 × 2 mL). The brown oil was then purified by C-
18 sep-pak using H2O/MeOH gradient (0−50%). Fractions were
identified by analytical HPLC (R.T. = 22.2 min) The resulting solution
was then concentrated to dryness to give 8 as a yellow oil (10.8 mg,
27%).
Anal. Calcd for C18H17N3O5F3Re·0.5C2F3O21H: C, 37.05; H, 2.59;
N, 6.17. Found: C, 36.87; H, 2.61; N, 6.15; H NMR (400 MHz,
CDCl3) δ 4.25 (d, 2H, J = 7.1 Hz), 4.48, 5.31 (ABq, 4H, J = 16.5 Hz),
5.70 (m, 2H), 6.34 (m, 1H), 7.23 (t, 2H, J = 7.0 Hz), 7.83 (m, 4H),
Anal. Calcd for C30H31N3O6F3SRe·0.5C2F3O2H: C, 43.20; H, 3.68;
1
N, 4.87. Found: C, 43.23; H, 3.83; N, 4.91; H NMR (400 MHz,
CDCl3) 1.79 (p, 2H, J = 7.1 Hz), 2.20 (m, 2H), 2.48 (t, 2H, J = 7.0
Hz), 3.50 (m, 4H), 3.66 (s, 2H), 3.83 (m, 2H), 4.40, 5.45 (ABq, 4H, J
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= 16.4 Hz), 7.21 (m, 7H), 7.72 (d, 2H, J = 7.5 Hz), 7.79 (t, 2H, J = 7.4
Hz), 8.64 (d, 2H, J = 5.4 Hz); 13C NMR (100 MHz, CDCl3) δ 25.6,
28.3, 29.2, 36.4, 67.4, 69.0, 69.4, 124.8, 125.5, 127.0, 128.6, 128.9,
138.5, 140.6, 150.9, 161.1; IR: 2026, 1900, 1688, 1485 cm−1; UV (λmax
262 nm, MeOH): εmax 9558 M−1 cm−1; MS (m/z): [M]+ 692.1.
fac-[ReI(CO)3(6)](CF3CO2), 9. Compound 6 (0.050 g, 0.190 mmol)
was dissolved in MeOH (3 mL). A 0.1 M solution of fac-
[ReI(OH2)3(CO)3](SO3CF3) (2.0 mL, 0.200 mmol) was added and
the pH was adjusted to 6 with saturated NaHCO3. Additional MeOH
was added until all solids were dissolved and the solution was heated
to 50 °C for 1 h. The solution was then cooled to room temperature
and concentrated to dryness. The resulting oil was then purified using
preparatory HPLC method 1. Fractions from the prep HPLC were
checked for purity by analytical HPLC (R.T. = 20.7 min). The
fractions were then concentrated in vacuo to give complex 9 as a
yellow oil (0.083 g, 73%).
spacer. These linker systems were selected to probe the role of
spatial proximity of the terminal alkene in relation to the metal
center as coordination through η1 (end-on) and η2 (sideways)
can positively or negatively impact the overall thiol-ene reaction
yield and product formation. In addition, through bond
activation of the alkene can be examined by varying the linker
length. Two synthetic routes (click then chelate and chelate then
click) were employed in the preparation of the alkene
functionalized DPA and the thiol-ene click DPA product and
their corresponding complexes to determine the most effective
route of assembly for the final product.
Ligand Synthesis. In either route, functionalization of the
DPA ligand with the corresponding alkene linker was the initial
step. The overall synthesis of the alkene DPA ligand to its
corresponding thiol-ene conjugate is detailed in Scheme 1.
Anal. Calcd for C23H25N3O6F3Re·C2F3O2H: C, 37.79; H, 3.04; N,
5.28. Found: C, 37.47; H, 3.17; N, 5.49; 1H NMR (400 MHz, CDCl3)
δ 2.21(m, 2H), 3.58 (t, 2H, J = 5.4 Hz), 3.88 (m, 2H), 4.00 (m, 2H),
4.49, 5.15 (ABq, 4H, J = 16.4 Hz), 5.25 (m, 2H), 5.90 (m, 1H), 7.25
(t, 2H, J = 7.0 Hz), 7.65 (d, 2H, J = 7.8 Hz), 7.84 (t, 2H, J = 7.4 Hz),
8.68 (d, 2H, J = 4.7 Hz); 13C NMR (100 MHz, CDCl3) δ 25.7, 66.7,
67.5, 68.9, 72.1, 117.4, 124.5, 125.7, 134.4, 140.6, 151.1, 160.7, 195.7;
IR: 2361, 2029, 1907 cm−1; UV (λmax 254 nm, MeOH): εmax 7952 M−1
cm−1; MS (m/z): [M]+ 568.2.
a
Scheme 1. Synthesis of Complexes 3 and 3a
Complexation of Ligands with [99mTcI(CO)3]. A 10−3
M
solution of ligand (1, 2, 6, or 7) dissolved in MeOH (100 μL) was
added to a 10 mM pH 7.4 phosphate buffer (800 μL) in a sealable vial.
The solution was then sealed, and the vial was sparged with N2 for 5
min. A solution of [99mTcI(OH2)3(CO)3]+ (100 μL) was then added
to give a final ligand concentration of 10−4 M, and the solution was
heated to 70 °C for 30 min. The reaction was then cooled to room
temperature and analyzed by radio-HPLC.
Thiol-ene “click” Reactions with [99mTcI(CO)3]+ Complexes.
Solutions of 99mTc complex 4a or 9a (500 μL) prepared as described
above were concentrated to dryness under a stream of dry N2 in a 2
mL vial. Benzyl mercaptan (100 μL) was added neat or as a 1 or 2 M
solution in MeOH to the vial. DMPA (250 μg) was then added, and
the resulting solutions were irradiated with a 366 nm UV lamp for 1 h.
Reaction solutions were then diluted with MeOH (1 mL) and
analyzed by radio-HPLC.
X-ray Crystallography. Crystals of complex 4 were removed from
the flask, a suitable crystal was selected, attached to a glass fiber, and
data were collected at 90(2) K using a Bruker/Siemens SMART APEX
instrument (Mo Kα radiation, λ = 0.71073 Å) equipped with a
Cryocool NeverIce low temperature device. Data were measured using
ω scans 0.3° per frame for 15 s, and a full sphere of data was collected.
A total of 2400 frames were collected with a final resolution of 0.83 Å.
Cell parameters were retrieved using SMART37 software and refined
using SAINTPlus38 on all observed reflections. Data reduction and
correction for Lp and decay were performed using the SAINTPlus
software. Absorption corrections were applied using SADABS.39 The
structure was solved by direct methods and refined by least-squares
method on F2 using the SHELXTL40 program package. The structure
was solved in the space group P2(1)/c by analysis of systematic
absences. All non-hydrogen atoms were refined anisotropically. No
decomposition was observed during data collection. Details of the data
collection and refinement are provided in the Supporting Information.
a
Conditions: (a) Allyl bromide, Cs2CO3, CH3CN, 3 h. (b) Benzyl
mercaptan, DMPA, MeOH, 366 nm, 30 min. (c) Re complex - fac-
[ReI(OH2)3(CO)3](SO3CF3), NaHCO3, MeOH, H2O, 50 °C, 1 h. (d)
99mTc complex - fac-[99mTcI(CO)3]+, MeOH, H2O, 70 °C, 30 min. (e)
Re complex - ReI(CO)5Br, MeOH, reflux, 18 h. (f) 99mTc complex -
fac-[99mTcI(OH2)3(CO)3]+, MeOH, H2O, 70 °C, 30 min. (g) Benzyl
mercaptan, DMPA, 366 nm, 1 h.
Alkylation of DPA was achieved by reacting allyl bromide
yielding ligand 1 in excellent yields (96%) as confirmed by
standard chemical analysis. In particular, H NMR showed the
1
appearance of the alkene protons at 5.12 and 5.84 ppm and a
doublet corresponding to the allylic methylene at 3.11 ppm.
These alkene peaks are particularly relevant as these signals
disappear upon conversion to alkyl carbons after the thiol-ene
reaction. In the click then chelate route, the thiol-ene reaction
was conducted using 2,2-dimethoxy-2-phenylacetophenone
(DMPA), which homolytically cleaves under irradiation with
366 nm UV light. This radical initiator was used in preference
to azobisisobutyronitrile (AIBN) or other heat-initiators
because of potential complications with heat sensitive targeting
RESULTS AND DISCUSSION
■
The chelate system (2,2′-dipicolylamine, DPA) was selected for
these studies because of its high efficiency and stability of
complexes formed with [MI(CO)3]+ (M = Re, 99mTc).5,6,41 To
investigate the feasibility of using the thiol-ene “click” reaction
for the generation of [MI(CO)3]+ radiopharmaceuticals, the
central amine of DPA was functionalized with two unique
linker systems containing a terminal alkene moiety either by
direct allylation of the central amine or through a propyl ether
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a
Scheme 2. Synthesis of Complexes 8 and 8a
a
Conditions: (a) Allyl bromide, NaH, THF, 0 °C to r.t., 5 h. (b) Benzyl mercaptan, DMPA, MeOH, 366 nm, 30 min. (c) Re complex - fac-
[ReI(OH2)3(CO)3](SO3CF3) NaHCO3, MeOH, H2O, 50 °C, 1 h. (d) 99mTc complex - fac-[99mTcI(OH2)3(CO)3]+, MeOH, H2O, 70 °C, 30 min.
(e) Re complex - ReI(CO)5Br, MeOH, reflux, 18 h. (f) 99mTc complex - fac-[99mTcI(OH2)3(CO)3]+, MeOH, H2O, 70 °C, 30 min. (g) Benzyl
mercaptan, DMPA, 366 nm, 1 h.
molecules and to take advantage of potentially mild “click”
reaction conditions. Ligand 1 was irradiated with 366 nm UV
light for 30 min in the presence of DMPA and benzyl
mercaptan to yield the product ligand 2 in 75% yield which was
identified by standard chemical analysis. The 1H NMR
spectrum of 2 confirmed the loss of the allylic peaks previously
observed in 1 and the formation of a triplet at 2.60 ppm
integrating for two protons indicating the attachment of the
benzyl mercaptan at the terminal position of the alkene,
producing the expected thiol-ene product. MS analysis of 2
gave the expected (m/z) for the [M+H]+ ion of 364.2
supporting the addition of the benzyl mercaptan to the alkene
moiety of ligand 1.
To incorporate an ether moiety into the linker, synthesis of
the extended ligand system and its thiol conjugates was
performed in an analogous manner as detailed in Scheme 2.
DPA-propanol, 5, was prepared according to known literature
methods36 and was utilized as the central species for allylation.
Treatment of 5 with allyl bromide and NaH gave the extended
linker containing ligand system 6 in excellent yields (94%).
Identification of 6 by 1H NMR analysis indicated the
attachment of the allyl group by the appearance of alkene
protons at 5.11 and 5.79 ppm. Conversion of ligand 6 to the
thiol-ene product via the click then chelate approach was
achieved by irradiation with 366 nm light in the presence of
DMPA and benzyl mercaptan to give product 7 in good yield
(68%). Characterization of the product confirmed the
formation of the thioether bond. 1H NMR analysis of 7
indicated the disappearance of the alkene protons at 5.11 and
5.79 ppm observed in ligand 6. Conjugation of the sulfur in
benzyl mercaptan to the terminal alkene position was
confirmed by the presence of a triplet at 2.55 ppm
corresponding to the CH2 adjacent to the sulfur (CH2-CH2-
S-), not a doublet corresponding to addition at the internal
carbon of the alkene. MS analysis of 7 gave the expected (m/z)
for the [M+H]+ ion of 422.3 confirming the addition of benzyl
mercaptan to the alkene moiety of 6.
synthesis of complex 3 (Scheme 1) and the extended linker
system 8 (Scheme 2). Using the click then chelate approach, the
“clicked” thiol-ene complex fac-[ReI(CO)3(2)]+, 3, was
achieved by reacting the clicked thioether ligand 2 with
ReI(CO)5Br in refluxing methanol for 18 h. Preparatory HPLC
purification of the reaction mixture gave 3 with the
trifluoroacetate counterion in 72% yield. Complexation of
[ReI(CO)3]+ to the DPA chelate caused significant downfield
shifts and splitting patterns in the 1H NMR spectrum similar to
those observed for other fac-[ReI(CO)3DPA]+ complexes.42 In
3, the methylene protons in the DPA chelator were split into an
AB quartet at 4.16 and 5.64 ppm due to Re complexation
compared to the free ligand observed as a singlet at 3.78 ppm.
Additionally, the triplet corresponding to the methylene
protons of the linker adjacent to the central amine (N-CH2-
CH2) was shifted downfield from 2.60 to 3.78 ppm. The
methylene protons in the pentet (CH2-CH2-CH2) at 1.77 ppm
also experienced significant deshielding resulting in a multiplet
at 2.21 ppm due to [ReI(CO)3]+ binding. While not as
pronounced as CH2 shifts, the protons in the pyridine rings
were also shifted downfield slightly by 0.2−0.3 ppm. IR showed
characteristic bands at 2026 and 1900 cm−1 indicative of a facial
[MI(CO)3]+ complex with a symmetric chelator. While
thioether ligands have been proposed as suitable donors for
[MI(CO)3]+, analysis of the reaction mixture did not indicate
complexes containing the S bound metal center.17,43,44 This is
most likely due to the smaller coordination ring size (5
member) and the strong preferential potency of the DPA
chelator for [ReI(CO)3]+.
To examine the chelate then click methodology, the overall
process involved the complexation of the alkene containing
DPA ligand 1 with [ReI(CO)3]+ followed by the photochemical
reaction of the complex under thiol-ene conditions with benzyl
mercaptan. In the first step, the synthesis of fac-
[ReI(CO)3(1)]+, 4, was accomplished by treating 1 with fac-
[ReI(OH2)3(CO)3](SO3CF3) to give the product in 74% yield
1
after isolation. As mentioned above, the H NMR spectrum of
Synthesis of Rhenium Complexes. Both strategies (click
then chelate and the chelate then click) were examined in the
4 showed significant shifts and splitting from the free ligand
because of coordination of the [ReI(CO)3]+ core. Of particular
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interest, the N-CH2-CH adjacent to the central amine of the
alkene linker experienced a substantial change in chemical shift
moving from 3.11 ppm in the free ligand to 4.25 ppm upon
complexation. The alkene protons of the terminal CH2 and the
CH groups exhibited downfield shifts. While terminal alkenes
are well-known to form end-on η1 or side-on η2 complexes, the
solution data of 4 does not indicate direct involvement of the
alkene in the Re coordination sphere based on the absence of
structural changes in the NMR splitting patterns.
Single crystals of 4 were obtained as the triflate salt by slow
solvent evaporation to yield suitable quality for X-ray diffraction
analysis. Complete experimental parameters and tables of bond
angles and distances can be found in the Supporting
Information (SA 1−5). The crystals were found to pack in a
monoclinic P2(1)/c space group with one molecule in the unit
cell. Consistent with other fac-[ReI(CO)3(DPA)]+ complexes,
4 exists in a distorted octahedral coordination geometry with
the expected facial arrangement of the carbonyl ligands and the
tridentate ligand 1 (Figure 1).18,45 The coordinated DPA ligand
While solubility issues hindered further investigation of 4 via
the chelate then click route, the incorporation of an additional
ether group into the linker backbone provided improved
solubility for both the ligands and the respective complexes. In
the click then chelate approach, ReI(CO)5Br was reacted with
the thiol-ene click product of the extended ligand system 7 in
refluxing methanol for 18 h to form the corresponding DPA
coordinated fac-[ReI(CO)3(7)]+ complex 8 as a single species.
The complex was isolated by preparatory HPLC purification
1
with the trifluoroacetate counterion in 74% yield. H NMR
analysis displayed significant downfield shifts and ligand
splitting patterns upon complexation that were consistent
with the coordination of the [ReI(CO)3]+ core to the DPA
chelate. IR stretches observed at 2026 and 1900 cm−1
confirmed the formation of the [ReI(CO)3]+ core from the
decarbonylation of the ReI(CO)5Br starting material. The M+
parent ion of 8 was observed at 692.1 m/z in the positive mode
in the MS analysis.
The chelate then click approach was further examined with the
extended linker system to understand the thiol-ene product
formation in the presence of the [ReI(CO)3]+ core. In the first
step, the alkene containing complex was prepared by reacting
ligand 6 with fac-[ReI(OH2)3(CO)3](SO3CF3) to yield the
desired complex fac-[ReI(CO)3(6)]+, 9. Purification of 9 as the
trifluoroacetate complex was achieved by preparatory HPLC in
reasonable yields (73%). 1H NMR analysis exhibited
comparable changes in splitting and chemical shifts as observed
in complexation of 1 with [ReI(CO)3]+. Coordination to 6
resulted in only modest shifts in the alkene proton peaks
indicating a decreased through bond or direct interaction
between the metal center and the alkene.
In the next step of chelate then click, the thiol-ene click
reaction was evaluated by observing the conversion of the
alkene in 9 into the thiol-ene product in 8. Using conditions
similar to those described in the ligand synthesis, 9 was
irradiated for 1 h in the presence of DMPA and benzyl
mercaptan at 366 nm. The product was isolated using C-18
chromatography to give a 27% isolated yield of complex 8,
which was confirmed to have identical spectra (1H NMR, MS)
as that obtained by the click then chelate approach. A important
observation of the reaction is that the isolated complex did not
exhibit significant decomposition of the [ReI(CO)3]+ core due
photolytic induced decarbonylation. This suggests the stability
of the rhenium CO ligands under the thiol-ene reaction
conditions as bicarbonyl or monocarbonyl complexes were not
detected. These positive results of successful thiol-ene product
formation and stability of the [ReI(CO)3]+ core under reaction
conditions confirmed that the thiol-ene reaction could be
applied in a chelate then click approach and facilitated further
investigation with 99mTc analogues.
Figure 1. X-ray structure of complex 4, hydrogens and the triflate
anion have been omitted for clarity. Ellipsoids are drawn at 40%
probability. Selected bond lengths: C(1)−C(2) 1.323(6) Å, N(4)−
Re(1) 2.229(4) Å, N(7)−Re(1) 2.178(3) Å, N(14)−Re(1) 2.182(3)
Å. Selected bond angles: C(1)−C(2)−C(3) 122.5°, N(7)−Re(1)−
N(14) 79.70(13)°, N(7)−Re(1)−N(4) 77.92(13)°, N(14)−Re(1)−
N(4) 77.44(13)°.
in 4 exhibited expected bond distances for pyridine and amine
with the [MI(CO)3]+ core of Re(1)−N(4), 2.229(4) Å,
Re(1)−N(7), 2.178(3), Re(1)−N(14), 2.182(3) Å and bond
angles of N(7)−Re(1)−N(14) 79.70(13)°, N(7)−Re(1)−
N(4) 77.92(13)°, N(14)−Re(1)−N(4) 77.44(13)° that are
observed with other fac-[ReI(CO)3(DPA)]+ complexes. In
accordance to the NMR data, the structure of 4 confirms the
lack of bond participation of the alkene linker as it is directed
away from the metal center. The alkene C(1)−C(2) bond
maintains a typical bond length of 1.323(6) Å and bond angle
of 122.5(4)° that is consistent with carbon−carbon double
bonds. In the second step of the chelate then click approach,
several attempts were made to conduct the click reaction using
thiol-ene conditions of complex 4 with various solvent systems
(i.e., methanol and CH2Cl2). The poor solubility of rhenium
complex 4 limited the effective solution concentration and
inhibited the assessment of the efficiency of coupling under the
thiol-ene conditions on a macroscopic level.
[
99mTcI(CO)3]+ Complexation and Thiol-Ene Click
Reactions. Similar to the Re analogues, both strategies (click
then chelate and chelate then click) were investigated with fac-
[
99mTcI(OH2)3(CO)3]+ in the complexation of the thiol-ene
clicked ligands and the thiol-ene click reaction. Generally, the
radiolabeling experiments were conducted by incubating fac-
[
99mTcI(OH2)3(CO)3]+ in a 10−4 M ligand solution for 30 min
at 70 °C (Table 1). The reaction mixture was analyzed using
radio-HPLC to identify and to quantify product formation
(Table 1). The radioactive chromatographic peaks observed in
the 99mTc reactions in the chromatograms were also correlated
with the retention times of the corresponding rhenium
analogues discussed previously.
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a
click reaction was identified by a significant peak shift in the
HPLC chromatogram (Figure 2) that permitted the determi-
nation of relative radiochemical yields from the peak area from
the starting complexes to the corresponding “click” products
from the total observed counts. Photoirradiation of 4a or 9a
without the presence of the thiol did not exhibit any change or
new peak formation which indirectly suggests the
Table 1. Radiolabeling Yields of 1, 2, 6, and 7
reactant
product
radiochemical yield
1
2
6
7
4a
3a
9a
8a
99%
89%
94%
98%
a
Conditions: Complexation at 10−4 M ligand concentration, fac-
[
99mTcI(CO)3]+ core does not undergo photo-induced
[
99mTcI(OH2)3(CO)3]+, pH 7.4 phosphate buffer, 30 min, 70 °C.
decarbonylation under the reaction conditions.
Several concentrations of the thiol model ligand, benzyl
mercaptan, were investigated to determine product formation
and evaluate the concentration limitations of the reaction.
Experiments containing (8.5 (neat), 2.0, or 1.0 M) benzyl
mercaptan were examined with both 4a and 9a to compare
reactivity differences based on the influence of linker variation
and proximity to the 99mTc center (Table 2). In the highest
In the click then chelate approach, the previously prepared
thiol-ene click ligands 2 and 7 were complexed with fac-
[
99mTcI(OH2)3(CO)3]+ to generate the corresponding com-
plexes fac-[99mTcI(CO)3(2)]+, 3a, and fac-[99mTcI(CO)3(7)]+,
8a, in 89% and 98% yield, respectively. In both cases, a single
chromatographic peak was observed that corresponded directly
to the rhenium analogues (Figure 2). While the kinetics of
Table 2. Thiol-ene Reactions with 99mTc-alkene Complexes
a
(4a and 9a) with Benzyl Mercaptan
complex
thiol conc (M)
product
% conversion
4a
9a
9a
9a
neat
neat
2
3a
8a
8a
8a
52%
87%
88%
64%
1
a
Conditions: Benzyl mercaptan (neat or in MeOH), DMPA (250 μg),
b
366 nm, 1 h, r.t. Following irradiation at 366 nm, 1 h, r.t.
thiol concentration (8.5 M), conversion of (4a to 3a) or (9a to
8a) yielded the thiol-ene product formation in reasonable to
good yields of 52% and 87%, respectively. Comparison of the
two ligand systems chromatograms revealed the conversion of
9a to 8a occurred in greater radiochemical yield, but it also
proceeded with significantly less side products formation than
were observed in the conversion of 4a to 3a. While the Re
analogue 8 did not exhibit significant interaction or degradation
during the thiol-ene click reaction condition, the close
proximity of the truncated linker during the radical addition
may have an ancillary effect on the metal center, but the specific
role is unclear at this time. When the thiol-ene click reaction
was conducted at lower benzyl mercaptan concentrations (2.0
and 1.0 M) in methanol, conversion of 4a to 3a was minimally
observed. However, conversion of 9a to 8a at lower benzyl
mercaptan concentration maintained good to excellent product
formation, where no decline in yield was observed at 2 M
concentrations (88%) compared to a reduced yield at 1 M
(64%). On the basis of these initial studies, the role of the
linker has a critical effect on the overall formation of the thiol-
ene click product.
While the coupling in the chelate then click approach can be
achieved, the thiol concentrations examined were significantly
higher than desired for radiopharmaceutical applications with
biomolecules in the 10−3−10−5 M range. While the literature
demonstrates the thiol-ene reaction can be conducted in
macroscopic quantities as low as 10−3 M in equal molar ratio,
the inherent challenge of clicking a radiolabeled probe at
extremely low concentrations (10−9−10−12 M) with another
moiety provides additional restrictions on the reaction. The
disparity in thiol and 99mTc-alkene concentrations may
potentially be improved with increased radicals to facilitate
the reaction driven by the concentration differences or type of
thiol ligand utilized to enhance reactivity. While these findings
demonstrate proof of concept for the potential of the thiol-ene
Figure 2. HPLC chromatograms (bottom) at 220 nm for Re complex
9 (red) and 8 (black). 99mTc radiochromatograms (top) showing the
coordination reaction of 9a (blue), and the thiol-ene reaction of 9a to
form 8a (green) using the chelate then click approach.
complexation of 99mTc is generally faster than the Re analogues,
multiple peaks that can be related to different coordination
species (e.g., NPyNAmineS), cleavage of linker, or degradation of
the chelate were not observed in the γ-chromatograms.46
Overall, the results demonstrate the formation and stability of
this ligand system under the aforementioned labeling
conditions to generate the 99mTc products in excellent yields,
which are comparable to other DPA functionalized systems.
In the chelate then click approach, the first step involved the
formation of the 99mTc complex with an alkene functional
group available for further reactivity using the thiol-ene click
reaction. Ligands 1 and 6 were reacted with fac-
[
99mTcI(OH2)3(CO)3]+ using the general labeling conditions
mentioned above to form the corresponding 99mTc complexes
fac-[99mTcI(CO)3(1)]+, 4a, and fac-[99mTcI(CO)3(6)]+, 9a, in
near quantitative yields of 99% and 94%, respectively. The next
step involved the conversion of the alkene to the thioether by
the introduction of thiol using the photochemical initiated
radical thiol-ene click reaction. The general reaction conditions
involved incubation of the solutions of 4a or 9a, benzyl
mercaptan, and DMPA, followed by 366 nm irradiation for 1 h
at room temperature. The product formation of the thiol-ene
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click reaction with [99mTcI(CO)3]+ complexes, additional
studies are still needed to examine this reaction to achieve
conditions with improved reaction yields prior to implementa-
tion of the chelate then click approach as an effective direct
coupling strategy.
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CONCLUSIONS
■
We describe the first application of the radical initiated thiol-
ene click reaction with organometallic [MI(CO)3]+ complexes
that was explored utilizing click then chelate and chelate then click
design strategies for incorporating into a thiol containing
molecule. In the click then chelate approach, the thiol-ene
reaction was found to be a viable technique to produce
thioether linkages between allylic DPA conjugates that was
conducive to further radiolabeling conditions to yield an
efficient and well-defined Re or 99mTc product. This approach
provides a facile and effective method for functionalizing thiol
containing molecules that can readily be translated without
further modification. In the chelate then click approach, it was
demonstrated that the first step of [MI(CO)3]+ complex
formation with linkers containing an alkene could be efficiently
formed and maintained their reactivity toward benzyl
mercaptan in the photo-initiated thiol-ene radical reaction.
However, subtle differences in the subsequent thiol-ene click
reaction revealed the importance of the linker for both
solubility and reactivity. Overall, the extended propyl ether
linker exhibited higher yields and overall stability compared to
the shorter linker. While the thiol concentrations examined
within are higher than desired for immediate application,
additional studies are needed to address this issue to improve
the relevancy in the chelate then click approach. These initial
results illustrate the promising potential of the thiol-ene click
reaction in the assembly of macroscopic molecules as well as
low concentration reaction conditions exhibited in radio-
chemical samples.
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ASSOCIATED CONTENT
* Supporting Information
Complete X-ray structural information for 4 is available as a
CIF file and additional characterization data of selected
complexes as a PDF. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
(21) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666−
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AUTHOR INFORMATION
Corresponding Author
*E-mail: bennyp@wsu.edu. Phone: (509)-335-3858. Fax:
(509)-335-8867.
■
(24) Banerjee, S. R.; Babich, J. W.; Zubieta, J. Chem. Commun. 2005,
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
2008, 130, 5062−5064.
■
(28) Hoyle, C. E.; Bowman, C. N. Angew. Chem., Int. Ed. 2010, 49,
1540−1573.
The Bruker (Siemens) SMART APEX diffraction facility was
established at the University of Idaho with the assistance of the
NSF-EPSCoR program and the M. J. Murdock Charitable
Trust, Vancouver, WA, U.S.A. This worked was funded in part
by WSU College of Sciences Undergraduate Student Research
Mini-grant and DOE, Radiochemistry and Radiochemistry
Instrumentation Program (#DE-FG02-08-ER64672).
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