PH-controlled coordination mode rearrangements of clickable Huisgen-based multidentate ligands with [MI(CO)3] + (M = Re, 99mTc)

Article abstract of DOI:10.1021/ic302330u

The viability of the Huisgen cycloaddition reaction for clickable radiopharmaceutical probes was explored with an alkyne-functionalized 2-[(pyridin-2-ylmethyl)amino]acetic acid (PMAA) ligand system, 3, and fac-[M^I(OH₂)₃(CO)₃]⁺ (M = Re, ^99mTc). Two synthetic strategies, (1) click, then chelate and (2) chelate, then click, were investigated to determine the impact of assembly order on the reactivity of the system. In the click, then chelate approach, fac-[M^I(OH₂)₃(CO)₃]⁺ was reacted with the PMAA ligand clicked to the benzyl azide, 5, to yield two unique coordination species, fac-[M^I(CO) ₃(O,N_amine,N_py-5)], M = Re (8), ^99mTc (8A), and fac-[M^I(CO)₃(N _tri,N_amine,N_py-5)], M = Re (9), ^99mTc (9A), where coordination is through the triazole (N _tri), central amine (N_amine), pyridine (N_py), or carboxylate (O). Depending on the reaction pH, different ratios of complexes 8(A) and 9(A) were observed, but single species were obtained of (O,N _amine,N_py) coordination, 8(A), in basic pHs (>9) and (N_tri,N_amine,N_py) coordination, 9(A), in slightly acidic pHs (<4). In the chelate, then click approach, the (O,N _amine,N_py) coordination of [M^I(CO) ₃]⁺ was preorganized in the alkyne-functionalized fac-[M^I(CO)₃(O,N_amine,N_py-3)], M = Re (6), ^99mTc (6A), followed by standard Cu^I-catalyzed Huisgen click conditions at pH ≈ 7.4, where the (O,N _amine,N_py) coordination mode remained unchanged upon formation of the triazole product in the clicked molecule. Despite the slow substitution kinetics of the low-spin d⁶ metal, the coordination modes (O,N_amine,N_py) and (N_tri,N _amine,N_py) were found to reversibly intraconvert between 8(A) and 9(A) based upon changes in pH that mirrored the (O,N _amine,N_py) coordination in basic pHs and (N _tri,N_amine,N_py) coordination in acidic pHs. Comparison of the Re and ^99mTc analogs also revealed faster intraconversion between the coordination modes for ^99mTc.

Full text of DOI:10.1021/ic302330u

Article
pubs.acs.org/IC
pH-Controlled Coordination Mode Rearrangements of “Clickable”
Huisgen-Based Multidentate Ligands with [MI(CO)3]+ (M = Re, 99mTc)
Shalina C. Bottorff,† Adam L. Moore,† Ariana R. Wemple,† Dejan-Kresimir Bucar,‡
̌
̌
Leonard R. MacGillivray,‡ and Paul D. Benny*,†
†Department of Chemistry, Washington State University, P.O. Box 644630, Pullman, Washington 99164, United States
‡Department of Chemistry, University of Iowa, Iowa City, Iowa 52242-1294, United States
S
* Supporting Information
ABSTRACT: The viability of the Huisgen cycloaddition
reaction for clickable radiopharmaceutical probes was explored
with an alkyne-functionalized 2-[(pyridin-2-ylmethyl)amino]-
acetic acid (PMAA) ligand system, 3, and fac-
[MI(OH2)3(CO)3]+ (M = Re, 99mTc). Two synthetic strategies,
(1) click, then chelate and (2) chelate, then click, were investigated
to determine the impact of assembly order on the reactivity of
the system. In the click, then chelate approach, fac-
[MI(OH2)3(CO)3]+ was reacted with the PMAA ligand
“clicked” to the benzyl azide, 5, to yield two unique coordination
species, fac-[MI(CO)3(O,Namine,Npy-5)], M = Re (8), 99mTc
(8A), and fac-[MI(CO)3(Ntri,Namine,Npy-5)], M = Re (9), 99mTc
(9A), where coordination is through the triazole (Ntri), central amine (Namine), pyridine (Npy), or carboxylate (O). Depending on
the reaction pH, different ratios of complexes 8(A) and 9(A) were observed, but single species were obtained of (O,Namine,Npy)
coordination, 8(A), in basic pHs (>9) and (Ntri,Namine,Npy) coordination, 9(A), in slightly acidic pHs (<4). In the chelate, then
click approach, the (O,Namine,Npy) coordination of [MI(CO)3]+ was preorganized in the alkyne-functionalized fac-
[MI(CO)3(O,Namine,Npy-3)], M = Re (6), 99mTc (6A), followed by standard CuI-catalyzed Huisgen “click” conditions at pH
≈ 7.4, where the (O,Namine,Npy) coordination mode remained unchanged upon formation of the triazole product in the clicked
molecule. Despite the slow substitution kinetics of the low-spin d6 metal, the coordination modes (O,Namine,Npy) and
(Ntri,Namine,Npy) were found to reversibly intraconvert between 8(A) and 9(A) based upon changes in pH that mirrored the
(O,Namine,Npy) coordination in basic pHs and (Ntri,Namine,Npy) coordination in acidic pHs. Comparison of the Re and 99mTc
analogs also revealed faster intraconversion between the coordination modes for 99mTc.
detection, adjacent coordination donors in close proximity to
the metal can impact the species of [MI(CO)3]+ complex
observed and overall stability through intraconversion of the
ligand coordination. A plethora of donor combinations found in
biological systems (e.g., peptides, enzymes) can generate an
endless number of possible coordination modes on the metal
that is dependent on the strength of the donors, charge, and
coordination ring size.26,27 For example, the sequence of six
histidine residues (Histag) has been reported to coordinate fac-
[MI(OH2)3(CO)3]+ through the imidazole donors. However,
the exact coordination of the metal with the Histag sequence
remains unknown.28−33
Recent investigations have focused on understanding the
interactions of fac-[MI(OH2)3(CO)3]+ with more complex
multidentate systems containing four or more coordination
donors to address key factors influencing complex formation:
(1) favored coordination modes (i.e., tripodal or linear), (2)
coordination ring size (5 vs 6), (3) preferential donor selection
INTRODUCTION
■
The second- and third-row congeners of group VII present a
unique combination for both diagnostic imaging (99mTc) and
radiotherapy (186/188Re) from isostructural complexes.1−4 99mTc
has been a primary staple in diagnostic nuclear medicine due to
favorable nuclear properties (t1/2 = 6.02 h, γ = 140 keV (89%)),
availability of the 99Mo/99mTc generator, and versatile metal
coordination chemistry.5−9 The organometallic precursor fac-
[MI(OH2)3(CO)3]+ (M = Re, 99mTc) popularized by Alberto
has gained considerable attention in recent years due to facile
preparation through the IsoLink kit developed by Covidien,
wide pH stability of the precursor, and labile nature of the
coordinated waters for a variety of ligand substitution strategies
(e.g., mono-, bi-, and tridentate, 2 + 1, cyclopentadien-
yl).1−3,10−18
In particular, tridentate ligand systems (e.g., dipyridylamine,
histidine, cysteine) provide a saturated coordination sphere for
fac-[MI(OH2)3(CO)3]+ and generally have increased in vitro
and in vivo stability compared to a multiligand approach (e.g.,
monodentate or 2 + 1).19−25 As these ligand systems are
incorporated into biological targeting vectors for disease
Received: October 24, 2012
Published: March 4, 2013
© 2013 American Chemical Society
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Table 1. Crystallographic Data for Compounds 6−8
6
7
8
formula
Mr
C14H11N2O5Re
473.45
(C22H21N5O5Re)+·(CF3O3S)−
770.71
C21H18N5O5Re
606.60
cryst syst
space group
a, Å
monoclinic
C2/c
monoclinic
P21/c
triclinic
Pi
̅
33.026(4)
6.8209(8)
14.1322(15)
90
14.8352(16)
14.5582(16)
12.6615(14)
90
7.6866(9)
11.9837(13)
12.6489(14)
66.640(5)
86.619(5)
74.588(5)
1029.8(2)
2
b, Å
c, Å
α, deg
β, deg
γ, deg
V, Å3
Z
112.645(5)
90
103.232(5)
90
2938.1(6)
8
2662.0(5)
4
D
calcd, g cm−3
2.141
1.923
1.956
F(000)
μ(Mo Kα), mm−1
1792
1504
588
8.296
4.722
5.946
T, K
125(2)
125(2)
125(2)
cryst size, mm
range of indices
0.28 × 0.23 × 0.04
−38 → 38
−8 → 8
−16 → 16
9485
0.23 × 0.08 × 0.05
−17 → 17
−17 → 17
−15 → 15
17 505
0.23 × 0.22 × 0.18
−9 → 9
−14 → 14
−15 → 15
6822
no. of reflns collected
no. of unique reflns
Rint
2584
4682
3555
0.0414
0.0483
0.0136
no. of reflns with I > 2σ(I)
no. of parameters
R(F), F > 2σ(F)
wR(F2), F > 2σ(F)
R(F), all data
2452
3675
3492
199
371
289
0.0333
0.0276
0.0182
0.0916
0.0627
0.0435
0.0345
0.0447
0.0187
wR(F2), all data
0.0930
0.0672
0.0437
Δr (max, min) eÅ−3
2.388, −1.807
1.859, −1.033
2.170, −0.776
(i.e., soft vs hard), (4) synthetic yields, and (5) complexation
kinetics.34,35 Understanding the ligand interactions with the fac-
[MI(OH2)3(CO)3]+ provides essential insight for designing
new ligand systems for predicting complex formation.
system for [MI(CO)3]+ that yields a neutral complex and can
be functionalized to incorporate an alkyne group similarly to
the DPA system.21,47−50 In the clicked tetradente product,
replacement of a pyridine with a harder carboxylate donor in
PMAA allows comparison of the coordination mode of
[MI(CO)3]+ between the electrostatic charge neutralization of
the carboxylate donor in a neutral complex vs the softer
aromatic nitrogen donor of 1,2,3-triazole in a cationic complex.
Furthermore, Lipowska et al. observed different coordination
species formed by [ReI(CO)3]+ with several polyaminocarbox-
ylate ligands (≥4 donors) based on the initial reaction pH and
adjustment of the pH with acid or base.35 In acidic conditions,
coordination of the carboxylate donors in the polyaminocar-
boxylate ligands was favored over the amine donors. This trend
is reversed to favor amine coordination over carboxylate donors
with the same ligand under basic conditions. Adjustment of
solution pH led to coordination mode intraconversion that
correlated with the speciation observed in the initial reaction
pH studies. Diverging from the inherent stability of a saturated
tridentate complex and seemingly inert nature of the low-spin
d6 center of [MI(CO)3]+, the coordination rearrangement
illustrates potential substitution of ligands by adjacent donors as
a function of pH change.
The Huisgen “click” reaction of a CuI-catalyzed cycloaddition
of an alkyne and an azide to generate a triazole has gained
tremendous acclaim as a facile method for coupling two
molecules.36−40 Schibli and Mindt reengineered the use of the
Huisgen triazole “click” product in an elegant ligand design
strategy, “click to chelate”, to generate a series of tridentate
ligands for the [MI(CO)3]+ core that can be readily
incorporated into biomolecules.41−45 Alternatively, we exam-
ined the effectiveness of the Huisgen reaction to “click” an
alkyne-functionalized known potent tridentate ligand system,
dipyridylamine (DPA), for [MI(CO)3]+ to an azide.46 Two
strategies (chelate, then click and click, then chelate) were
investigated to ascertain the effect of the coordination of
[MI(CO)3]+ on the click reaction and the impact of the
adjacent “click” product on complex formation and stability. In
both strategies, coordination of the triazole to [MI(CO)3]+ was
not observed through either initial complexation or intra-
molecular rearrangement based on X-ray and solution studies.
The preference for pyridine coordination in DPA ligands
over triazole coordination prompted our further investigation
to assess the binding strength of the triazole donor in
competition with other donor systems for complex selectivity
and intramolecular rearrangement in a tetradentate system. The
asymmetric ligand, 2-[(pyridin-2-ylmethyl)amino]acetic acid
(PMAA), represents another potent linear tridentate ligand
In this article, we utilized the alkyne-functionalized PMAA
ligand system to explore the effectiveness of the click reaction
with [MI(CO)3]+ through both chelate, then click and click, then
chelate radiolabeling strategies, utilizing benzyl azide as a model
azide system. Comparison of both strategies revealed that the
synthetic route and reaction conditions can impact the
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Article
observed product formation, where two different coordination
species, fac-[MI(CO)3(O,Namine,Npy-5)], M = Re (8), 99mTc
(8A), and fac-[MI(CO)3(Ntri,Namine,Npy-5)], M = Re (9), 99mTc
(9A), where coordination is through the triazole (Ntri), central
amine (Namine), pyridine (Npy), or carboxylate (O), were
identified in the reaction. Furthermore, examination of the
changes in solution pH revealed the rearrangement of the
coordination geometry of the ligand about the [MI(CO)3]+
center to favor (O,Namine,Npy) coordination in basic conditions
and (Ntri,Namine,Npy) coordination in acidic conditions. This
observation was confirmed in X-ray structural studies and
solution characterization of the Re and 99mTc analogs.
Table 2. Selected Bond Lengths (Angstroms) and Angles
(degrees) for fac-[ReI(CO)3(O, Namine,Npy-3)], 6, and fac-
[ReI(CO)3(O, Namine,Npy-5)], 8
6
8
Re(1)−N(1)
Re(1)−N(2)
Re(1)−O(4)
Re(1)−C(1)
Re(1)−C(2)
Re(1)−C(3)
C(13)−C(14)
C(11)−O(4)
C(11)−O(5)
N(2)−C(10)
N(2)−C(9)
N(3)−N(4)
2.176(3)
2.229(3)
2.120(3)
1.898(4)
1.906(4)
1.932(4)
1.176(8)
1.263(6)
1.229(6)
1.496(6)
1.491(6)
2.177(3)
2.241(3)
2.120(2)
1.918(3)
1.897(3)
1.909(3)
1.366(5)
1.289(4)
1.220(4)
1.495(4)
1.500(4)
1.318(4)
1.346(4)
76.24(10)
82.02(9)
78.68(9)
96.90(12)
170.87(12)
98.08(12)
173.13(12)
97.43(13)
94.26(12)
96.36(12)
94.05(12)
175.53(12)
115.5(3)
110.7(3)
112.5(3)
113.8(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 material fac-[ReI(OH2)3(CO)3]-
(SO3CF3) was prepared by literature methods from Re2(CO)10
purchased from Strem.51 Methyl 2-(pyridine-2-ylmethylamino)acetate
(1) was prepared according to previous reports.47,52 UV−vis spectra
were obtained using a Varian Cary 50 spectrophotometer (1 cm path
N(4)−N(5)
N(1)−Re(1)−N(2)
N(1)−Re(1)−O(4)
N(2)−Re(1)−O(4)
N(2)−Re(1)−C(1)
N(2)−Re(1)−C(2)
N(2)−Re(1)−C(3)
N(1)−Re(1)−C(1)
N(1)−Re(1)−C(2)
N(1)−Re(1)−C(3)
O(4)−Re(1)−C(1)
O(4)−Re(1)−C(2)
O(4)−Re(1)−C(3)
N(2)−C(10)−C(11)
N(2)−C(9)−C(8)
N(5)−C(15)−C(16)
N(2)−C(12)−C(13)
75.61(12)
81.49(12)
78.43(12)
96.67(15)
171.36(14)
99.42(15)
94.32(15)
96.94(16)
174.51(15)
174.19(15)
96.33(16)
95.32(15)
114.8(4)
1
length). H and 13C NMR spectra were recorded on a Varian 300
MHz instrument at 25 °C in CDCl3, CD3OD, or (CD3)2SO as
described. Elemental analyses were performed by Quantitative
Technologies, Inc. Separation and identification of compounds were
conducted on a Perkin-Elmer Series 200 High Pressure Liquid
Chromatograph (HPLC) equipped with a UV−vis Series 200 detector
and a Radiomatic 610TR detector. Utilizing an Agilent Zorbex 5 μm
particle and 30 cm SB-C18 column, the compounds were separated
with a reverse-phase gradient system beginning with an aqueous eluent
(A) gradually shifting to methanol (MeOH) according to the
following method: 0−3.0 min (100% A), 3.0−9.0 min (75% A, 25%
MeOH), 9.0−20.0 min (25−100% MeOH linear gradient), and 20.0−
30.0 min (100% MeOH) at a flow rate of 1.0 or 10.0 mL/min for
separation. Trifluoroacetic acid (0.1%, TFA, pH 1.7), triethylamine
phosphate buffer (TEAP, 10 mM, pH 3), phosphate buffer (PB, 10
mM, pH 7.4), and sodium borate buffer (10 mM, pH 9) were utilized
as aqueous eluents (A) for pH control. 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.
X-ray Crystallography. Crystals of compounds 6−8 were
collected at 125(2) K on a Nonius KappaCCD diffractometer using
MoKα irradiation (λ = 0.71073 Å) in a series of phi and omega scans.
Data collection, cell refinement, and data reduction were performed
using Collect53 and HKL Scalepack/Denzo.54 Crystal structures were
solved by direct methods and refined by the least-squares method on
F2 using SHELXS-97 and SHELXL-97, respectively.55 All non-
hydrogen atoms were refined anisotropically. All hydrogen atoms
bonded to carbon atoms were located in the Fourier-difference
electron-density map, fixed in geometrically constrained riding
positions, and isotropically refined. Crystallographic data of the
investigated compounds are listed in Table 1. Selected bond distances
and angles are listed in Tables 2 and 3.
111.0(3)
Table 3. Selected Bond Lengths (Angstroms) and Angles
(degrees) for fac-[ReI(CO)3(Ntri,Namine,Npy-3)](OTf), 7
Re(1)−N(1)
Re(1)−N(2)
Re(1)−C(1)
Re(1)−C(2)
Re(1)−C(3)
N(3)−Re(1)
C(21)−O(4)
C(21)−O(5)
N(2)−C(10)
N(2)−C(9)
N(2)−C(20)
N(3)−N(4)
N(4)−N(5)
C(11)−C(12)
C(11)−N(3)
2.173(4)
2.269(4)
1.909(5)
1.931(5)
1.928(5)
2.143(4)
1.197(5)
1.344(5)
1.515(5)
1.511(5)
1.494(5)
1.334(5)
1.345(5)
1.361(6)
1.365(6)
N(1)−Re(1)−N(2)
N(1)−Re(1)−N(3)
N(2)−Re(1)−N(3)
N(2)−Re(1)−C(1)
N(2)−Re(1)−C(2)
N(2)−Re(1)−C(3)
N(1)−Re(1)−C(1)
N(1)−Re(1)−C(2)
N(1)−Re(1)−C(3)
N(3)−Re(1)−C(1)
N(3)−Re(1)−C(2)
N(3)−Re(1)−C(3)
N(2)−C(10)−C(11)
N(2)−C(9)−C8)
78.11(13)
77.23(13)
76.54(13)
171.57(15)
97.39(16)
95.95(16)
96.15(17)
95.42(16)
172.94(17)
96.34(16)
171.22(16)
97.77(16)
109.4(6)
112.1(3)
Methyl 2-(Prop-2-ynyl(pyridine-2-ylmethyl)amino)acetate,
2. In a 100 mL round-bottom flask, 1 (0.200 g, 1.11 mmol) was
dissolved in acetonitrile (20 mL) and K2CO3 (0.307 g, 2.22 mmol)
was added. Propargyl bromide (0.119 mL, 1.33 mmol) in acetonitrile
(10 mL) was added slowly (1 mL/min). The reaction was brought to
reflux and stirred overnight. Solvent was removed in vacuo leaving a
dark oil. Oil was redissolved in deionized water (50 mL) and extracted
with dichloromethane (4 × 50 mL). Organic extracts were combined,
dried with anhydrous sodium sulfate, filtered, and dry loaded on silica
gel for column purification (acetone:hexanes gradient 0−30%) (0.133
N(2)−C(20)−C(21)
N(5)−C(13)−C(14)
115.1(4)
109.9(3)
123.42, 122.45, 78.43, 73.97, 59.65, 54.18, 51.78, 43.04.
λmax(CH3OH)/nm (ε/dm3 mol−1 cm−1): 286 (7800), 259 (12 600).
υmax/cm−1: 1639 (CO); 2362 (CC). m/z 218.1 (M+, 100%). Anal.
Calcd for C12H14N2O2: C, 66.04; H, 6.47; N, 12.84. Found: C, 65.70;
H, 5.53; N, 12.62.
2-(Prop-2-yn-1-yl(pyridin-2-ylmethyl)amino)acetic Acid, 3.
Compound 2 (0.244 g, 1.12 mmol) was dissolved in methanol (9
mL) and cooled to 0 °C. After dropwise addition of LiOH (3 mL, 1.0
M), the reaction was stirred overnight at room temperature. Solvent
was removed in vacuo to yield a dark oil, which was redissolved in
1
g, 55%). H NMR [δ (ppm), CDCl3]: 8.56−8.53 (m, 1H), 7.67 (dt,
1H, J = 1.8, 7.8), 7.50−7.31 (m, 1H), 7.15−7.19 (m, 1H), 3.92 (s,
2H), 3.71 (s, 3H), 3.56 (d, 2H, J = 2.4), 3.49 (s, 2H), 2.27 (t, 1H, J =
2.4). 13C NMR [δ (ppm), CDCl3]: 171.28, 158.37, 149.34, 136.79,
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deionized water (15 mL), and the pH was adjusted to 6.5 with 1.0 M
HCl. Solvent was again removed in vacuo, and the oil was taken up in
0.1% TFA for purification by preparative HPLC to yield the product as
dropwise to a 0.1 M fac-[ReI(OH2)3(CO)3](OTf)(aq) solution (4.78
mL, 0.478 mmol) stirring in a scintillation vial. The vial was sealed and
stirred at room temperature for 16 h which produced a light brown
precipitate. Solid was filtered and dried in vacuo. Complex 7 was
recrystallized as an off-white solid by slow addition of diethyl ethyl
1
a yellow oil (0.159g, 70%). R.T. = 11.5 min. H NMR [δ (ppm),
CD3OD]: 8.78−8.75 (m, 1H), 8.48 (dt, 1H, J = 1.8, 7.8), 8.01−7.98
(m, 1H), 7.94−7.89 (m, 1H), 4.35 (s, 2H), 3.73 (s, 2H), 3.68 (d, 2H, J
= 2.7), 2.77 (t, 1H, J = 2.2). 13C NMR [δ (ppm), CD3OD]: 172.47,
154.70, 145.64, 141.94, 126.14, 125.68, 77.02, 75.17, 54.86, 54.59,
1
ether to a saturated solution in dichloromethane (0.126 g, 41%). H
NMR [δ (ppm), (CD3)2SO]: 8.68 (d, 1H, J = 3.3), 8.23 (s, 1H), 8.04
(t, 1H, J = 4.6), 7.69 (d, 1H, J = 4.7), 7.42 (t, 1H, J = 3.9), 7.34−7.32
(m, 3H), 6.93−6.91(m, 2H), 5.65 (ABq, 2H, ΔδAB = 0.01, J = 9.0),
5.13 (ABq, 2H, ΔδAB = 0.07, J = 10.0), 4.89−4.76 (m, 4H), 3.77 (s,
3H). 13C NMR [δ (ppm), (CD3)2SO]: 196.47, 195.97, 194.87, 169.66,
161.05, 152.41, 150.43, 141.26, 135.25, 129.53, 129.18, 127.96, 126.42,
124.69, 124.52, 68.61, 67.72, 58.79, 54.86, 52.86. λmax(CH3OH)/nm
(ε/dm3 mol−1 cm−1): 260 (11 000), 284 (7000). υmax/cm−1: 2359
(Ntri), 2029 and 1908 (CO), 1741 (OCH3). m/z 622.2 (M+, 100%),
620.2 (58%). Anal. Calcd for C22H21N5O5Re·CF3SO3·0.5CH2Cl2: C,
35.26; H, 2.74; N, 8.84. Found: C, 35.05; H, 2.31; N, 8.88.
43.41. λmax(CH3OH)/nm (ε/dm3 mol−1 cm−1): 260 (2800). υmax
/
cm−1: 1727, 1663 (CO); 2361 (CC). m/z 205.0 (M + H+, 100%).
Anal. Calcd for C11H12N2O2·1.2CF3COOH: C, 47.19; H, 3.90; N,
8.21. Found: C, 47.20; H, 4.01; N, 8.31.
Methyl 2-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)(pyridin-2-
ylmethyl)amino)acetate, 4. To a 25 mL round-bottom flask was
added 2 (0.200 g, 9.15 mmol) in methanol (2 mL) followed by benzyl
azide (0.153 g, 9.15 mmol). Sodium ascorbate (0.045 g, 0.183 mmol)
in 1 mL of H2O and copper(II) acetate (0.021 g, 0.915 mmol) in 1 mL
of water was then added to the solution and allowed to stir at room
temperature for 2 h. Sodium sulfide (0.071 g, 0.915 mmol) was added
and allowed to stir for an additional 30 min. The filtrate was dried in
vacuo and purified by preparatory HPLC for isolation as a pale yellow
oil (0.157 g, 49%). 1H NMR [δ (ppm), CD3OD]: 8.41−8.38 (m, 1H),
7.90 (s, 1H), 7.74 (dt, 1H, J = 1.8, 7.7), 7.56 (d, 1H, J = 7.8), 7.38−
7.22 (m, 6H), 5.55 (s, 2H), 3.97 (s, 2H), 3.93 (s, 2H), 3.63 (s, 3H),
3.40 (s, 2H). 13C NMR [δ (ppm), CD3OD]: 172.88, 160.04, 149.39,
146.05, 138.70, 136.81, 129.99, 129.53, 129.07, 125.31, 124.85, 123.85,
60.08, 54.89, 51.92, 49.62. λmax(CH3OH)/nm (ε/dm3 mol−1 cm−1):
286 (7800) 260 (12 600). υmax/cm−1: 2031 and 1910 (CO), 2360
(Nt r i ). m/z 351. 2 (M+ , 100%). Anal. Calcd for
C19H21N5O2·CF3COOH·H2O: C, 52.17; H, 5.00; N, 14.48. Found:
C, 52.38; H, 3.95; N, 14.26.
fac-[ReI(CO)3(O,Namine,Npy-5)], 8. Chelate, then click. A 25 mL
scintillation vial was charged with fac-[ReI(CO)3(O,Namine,Npy-3)], 6
(0.050 g, 0.105 mmol), and benzyl azide (0.014 g, 0.105 mmol)
dissolved in tert-butyl alcohol (4 mL). To this mixture, sodium
ascorbate (0.004 g, 0.021 mmol) in water (2 mL) was added followed
by copper(II) acetate (0.002 g, 0.0105 mmol) in water (2 mL). This
mixture was stirred vigorously and heated at 70 °C for 90 min. The
mixture was neutralized and then extracted with dichloromethane (3 ×
20 mL). Organic extracts were combined and dried over anhydrous
Na2SO4 and then reduced in volume to ca. 5 mL. Product 8 was
precipitated by addition of ether to yield an off white solid that was
collected by vacuum filtration (0.052 mg, 81%).
Click, then chelate. Compound 5 (0.035 g, 0.104 mmol) was
dissolved in methanol (5 mL) and added dropwise to 0.1 M fac-
[ReI(OH2)3(CO)3](OTf)(aq) (1.03 mL, 0.104 mmol) stirring in a 25
Methyl 2-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)(pyridin-2-
ylmethyl)amino)acetic Acid, 5. Compound 4 (0.255 g, 1.12 mmol)
was dissolved in methanol (9 mL) and cooled to 0 °C. After dropwise
addition of LiOH (3 mL, 1.0 M), the reaction was stirred overnight at
room temperature. Solvent was removed in vacuo, the dark oil was
redissolved in deionized water (15 mL), and the pH was adjusted to
6.5 using 1.0 M HCl. Product was evaporated to dryness and purified
by recrystallization from methanol/diethyl ether to produce an off-
mL scintillation vial. pH was adjusted to 8 with 0.1 M NaHCO3(aq)
;
then the vial was sealed and allowed to stir at room temperature for 12
h. A light brown precipitate was observed and collected by vacuum
filtration. Complex 8 was recrystallized as an off-white solid by slow
addition of diethyl ether to a saturated solution in methanol (0.046 g,
74.2%). 1H NMR [δ (ppm), (CD3)2SO]: 8.75 (d, 1H, J = 5.4), 8.43 (s,
1H), 8.13 (t, 1H, J = 7.6), 7.72 (d, 1H, J = 7.6), 7.57 (t, 1H, J = 6.5),
7.39−7.34 (m, 5H), 5.68 (s, 2H), 4.68 (ABq, 2H, ΔδAB = 0.05, J =
1
white solid (0.151 g, 62%). H NMR [δ (ppm), CD3OD]: 8.45−8.43
(m, 1H), 7.94 (s, 1H), 7.75 (dt, 1H, J = 1.8, 7.8), 7.61 (d, 1H, J = 8.1),
7.37−7.24 (m, 6H), 5.57 (s, 2H), 3.85 (s, 2H), 3.81 (s, 2H), 3.10 (s,
2H). 13C NMR [δ (ppm), CD3OD]: 178.97, 160.37, 149.75, 146.16,
138.78, 136.82, 130.02, 129.55, 129.11, 125.05, 124.79, 123.81, 60.19,
59.12, 54.90. λmax(CH3OH)/nm (ε/dm3 mol−1 cm−1): 260 (3700).
υmax/cm−1: 1597 (CO); 2361 (Ntri). m/z 337.1 (M+, 100%). Anal.
Calcd for C18H19N5O2·1.25H2O: C, 60.07; H, 6.02; N, 19.45. Found:
C, 60.23; H, 5.29; N, 19.14.
13.9), 4.62 (ABq, 2H, ΔδAB = 0.11, J = 15.9), 3.60 (ABq, 2H, ΔδAB
=
0.44, J = 16.6). 13C NMR [δ (ppm), (CD3)2SO]: 197.46, 197.29,
197.11, 178.39, 159.08, 152.00, 140.46, 140.25, 135.74, 128.79, 128.22,
128.10, 126.74, 125.88, 124.13, 68.18, 61.88, 60.98, 52.91.
λmax(CH3OH)/nm (ε/dm3 mol−1 cm−1): 245 (10 000), 263 (9000),
288 (6000), 322 (3000). υmax/cm−1: 2342 (Ntri), 2019, and 1905
(CO), 1649 (COO). m/z 606.1 (M+, 100%), 604.1 (58%). Anal.
Calcd for C21H18N5O5Re·0.1CH2Cl2: C, 41.20; H, 2.98; N, 11.38.
Found: C, 41.25; H, 2.62; N, 11.36.
fac-[ReI(CO)3(O,Namine,Npy-3)], 6. Compound 3 (0.033 g, 0.161
mmol) was dissolved in deionized water (5 mL) and stirred. A stock
solution of 0.1 M fac-[ReI(OH2)3(CO)3](OTf)(aq) (1.61 mL, 0.161
mmol) was added, and the pH was adjusted to 6 with sodium
bicarbonate (0.1 M). The reaction was refluxed 3 h and then cooled to
room temperature. The reaction mixture was concentrated in vacuo
(ca. 3 mL) and placed in the refrigerator overnight. The light brown
precipitate was collected by filtration and washed with cold water.
Recrystallization was achieved by slow addition of diethyl ether to a
fac-[ReI(CO)3(Ntri,Namine,Npy-5)](CF3CO2), 9. Compound 5 (0.050
g, 0.148 mmol) was dissolved in methanol (3 mL) and added dropwise
to 0.1 M fac-[Re(OH2)3(CO)3](OTf)(aq) (1.48 mL, 0.148 mmol)
stirring in a 25 mL scintillation vial. The vial was sealed, and the
reaction was stirred at room temperature for 12 h. The reaction
mixture was concentrated in vacuo and extracted with dichloro-
methane (3 × 5 mL) and back extracted (2 × 5 mL) with water.
Solvent was removed in vacuo. The resulting oil was dissolved in 4 mL
of 25% methanol in water, pH adjusted with TFA (0.100 mL, 1.3
mmol), and lyophilized to yield an off-white solid (0.055 g, 52.9%). 1H
NMR [δ (ppm), (CD3)2SO]: 8.66 (d, 1H, J = 5.5), 8.21 (s, 1H), 7.99
(t, 1H, J = 7.9), 7.67 (d, 1H, J = 7.9), 7.38 (t, 1H, J = 6.7), 7.31−7.29
(m, 3H), 6.93−6.90 (m, 2H), 5.62 (ABq, 2H, ΔδAB = 0.01, J = 15.0),
5.14 (ABq, 2H, ΔδAB = 0.08, J = 16.8), 4.85 (ABq, 2H, ΔδAB = 0.03, J
= 16.6), 4.71 (ABq, 2H, ΔδAB = 0.02, J = 16.9). 13C NMR [δ (ppm),
(CD3)2SO]: 196.17, 195.66, 194.54, 170.35, 160.92, 151.91, 150.22,
140.74, 134.88, 129.09, 128.73, 127.57, 125.88, 124.24, 124.11, 68.24,
67.88, 58.36, 54.48. λmax(CH3OH)/nm (ε/dm3 mol−1 cm−1): 260 (11
000), 284 (6900). υmax/cm−1: 2360 (Ntri), 2018 and 1904 (CO). m/z
608.2 (M+ , 100%), 606.2 (58%). Anal. Calcd for
1
concentrated solution of 6 in dichloromethane (0.066 g, 87%). H
NMR [δ (ppm), CD3OD]: 8.75 (d, 1H, J = 3.1), 8.15 (t, 1H, J = 4.6),
7.80 (d, 1H, J = 4.7), 7.58 (t, 1H, J = 3.8), 4.67, (ABq, 2H, ΔδAB
=
0.02, JAB = 9.4), 4.32 (ABq, 2H, ΔδAB = 0.04, JAB = 9.8), 3.77 (s, 1H),
3.59 (ABq, 2H, ΔδAB = 0.24, JAB = 10.2). 13C NMR [δ (ppm),
(CD3)2SO]: 197.25, 197.16, 196.98, 178.15, 158.82, 152.08, 140.53,
125.97, 124.19, 80.01, 77.52, 68.32, 61.10, 56.77. λmax(CH3OH)/nm
(ε/dm3 mol−1 cm−1): 261 (10 200). υmax/cm−1: 2360 (CC), 2018,
1907, and 1863 (CO), 1650 (COO). m/z 475.1 (M + H+, 100%),
473.1 (58%). Anal. Calcd for C14H11N2O5Re·0.25CH2Cl2: C, 34.58;
H, 2.34; N, 5.66. Found: C, 34.38; H, 2.02; N, 5.61.
fac-[ReI(CO)3(Ntri,Namine,Npy-4)](OTf), 7. Compound 4 (0.140 g,
0.398 mmol) was dissolved in deionized water (5 mL) and added
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C21H19N5O5Re·CF3CO2·1.3 CF3CO2H: C, 35.39; H, 2.35; N, 8.06.
Found: C, 35.59; H, 2.34; N, 7.95.
Scheme 1
1H NMR Titration. Approximately 5 mg of pure fac-
[ReI(CO)3(O,Namine,Npy -5)], 8, was dissolved in ∼1 mL of deuterated
DMSO ((CD3)2SO). Neat trifluoroacetic acid was titrated into the
DMSO solution at the following equivalents, 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5,
4, 4.5, 5, 5.5, 6, 8, 10, 15, 25, and 45, and analyzed by subsequent
Fourier transfer scans (16) between 0 and 15 ppm. Addition of TFA to
the solution was continued until the sample resulted in complete
conversion of 8 to fac-[ReI(CO)3(Ntri,Namine,Npy-5)], 9.
General [99mTcI(OH2)3(CO)3]+ Radiolabeling Procedure. A
ligand solution (100 μL, 10−4 M) and buffer solution (800 μL, 0.01
M) were added to a sealable vial (5.0 mL). Depending on the type of
labeling experiment, the buffer system varied according to the desired
pH: sodium borate (pH 9), phosphate (pH 7.4), TEAP (pH 3.0), or
0.1% TFA (pH 1.7). The vial was sealed, and the solution was
degassed by bubbling with a stream of nitrogen for ∼10 min. The
[
99mTcI(OH2)3(CO)3]+ precursor solution (100 μL), prepared
according to the Covidien IsoLink kit specifications, was added to
the degassed solution, and the sample was heated for 30 min at 70 °C.
The reaction mixture was then allowed to cool on an ice bath prior to
injection and analysis by radio-HPLC.
General Procedure for the “chelate, then click” [99mTcI(CO)3]+
Reaction. Formation of the Huisgen triazole “click” product was
a c c o m p l i s h e d b y r e a c t i n g t h e p r e l a b e l e d f a c -
[
99mTcI(CO)3(O,Namine,Npy-3)], 6A, alkyne complex with benzyl
azide under reduced CuI-catalyzed conditions. To a sealable 5 mL
vial, sodium borate (pH = 9, 10 mM, 600 μL), benzyl azide (1 mM,
100 μL (methanol)), and sodium ascorbate (0.2 mM, 100 μL) were
added and degassed with N2 for ∼10 min. Via syringe, 6A (10 μCi, 100
μL) was added to the vial followed by copper(II) acetate (0.1 mM, 100
μL). The solution was stirred at room temperature for 1 h and then
sampled by γ-radio-HPLC to yield the clicked products, fac-
CH2 singlet (5.55 ppm) from addition of benzyl azide that also
correlated with 13C NMR shifts. Deprotection of 2 and 4 was
achieved by base hydrolysis through treatment with a mixture
of LiOH and MeOH. Conversion of 2 to the free carboxylic
acid was achieved with a 1:3 ratio of LiOH:MeOH to produce
3 in near quantitative yields as observed by the loss of the
methyl ester signal in 1H NMR for 2 at 3.71 ppm and loss of a
CH3 mass in the mass spectrometry analysis. The methyl ester
of the triazole “click” product 4 was also deprotected under
analogous LiOH:MeOH conditions to generate the free
carboxylic acid in the “click” product 5 that was isolated in
reasonable yields (62%). Production of 5 was confirmed by loss
of the methyl ester signal of 4 at 3.63 ppm in 1H NMR analysis
and loss of a CH3 mass in mass spectrometry analysis.
Compound 5 could also be directly prepared by conducting the
“click” reaction with 3, although significantly lower yields were
obtained possibly due to coordination of CuI to the PMAA
ligand (data not shown).
Synthesis of the Rhenium Complexes. Two synthetic
routes (chelate, then click or click, then chelate) were investigated
to determine the [ReI(CO)3]+ product speciation observed
based on assembly of the click reaction (Scheme 2). Each
strategy presents a unique route to prepare the same product.
In the chelate, then click approach the coordination mode of the
[MI(CO)3]+ complex is limited by the inherent nature of the
tridentate ligand prior to undergoing the click reaction.
However, the click, then chelate approach is driven by the
kinetics and thermodynamics of complexation, donor selection
by the metal center, and overall charge of the molecule. This
approach initially presents a number of possible tridentate
coordination modes from a tetradentate system.
[
[
9 9 m T c I ( C O ) 3 ( O , N a m i n e , N p y - 5 ) ] , 8 A , o r f a c -
99mTcI(CO)3(Ntri,Namine,Npy-5)], 9A.
Intraconversion between 8A and 9A. Coordination intra-
conversion between the two modes of fac-[99mTcI(CO)3(O,Namine,Npy-
5)], 8A, or fac-[99mTcI(CO)3(Ntri,Namine,Npy-5)], 9A, was achieved by
adjusting the pH of the solution. Depending on the initial reaction pH,
the reaction mixture containing 8A, 9A, or both species was adjusted
to the desired pH (i.e., ∼1.7 or 9) with 1 M NaOH(aq) or HCl(aq)
.
Sample was allowed to stir at room temperature for 15 min prior to
monitoring by γ-radio-HPLC.
RESULTS AND DISCUSSION
■
Ligand Synthesis. General preparation of the asymmetrical
PMAA-functionalized ligands and their “click” products for
[MI(CO)3]+ (M = Re, 99mTc) complexes are illustrated in
Schemes 1−3. The PMAA methyl ester ligand, 1, was
synthesized in an analogous procedure to previous reports.47,52
Incorporation of an alkyne into the ligand system was carried
out by alkylating 1 with propargyl bromide in the presence of
K2CO3 to generate the alkyne-functionalized PMAA ligand, 2,
in moderate yields (55%). Characterization of 2 demonstrated a
single inclusion of an alkyne at the amine position through
1
mass spectrometry analysis, and H NMR showed the CH2
adjacent to the alkyne as a doublet at 3.56 ppm and a triplet of
the alkyne proton at 2.27 ppm. Conversion of the alkyne in 2 to
the Huisgen “click” triazole product was achieved utilizing
standard copper “click” conditions and benzyl azide as a model
system for cyclization. Compound 2 was reacted with 1 equiv of
benzyl azide in the presence of 10 mol % of copper(II) acetate
and 20 mol % of sodium ascorbate at room temperature to
yield the “click” product 4 in reasonable yields (48%). 1H NMR
analysis confirmed cycloaddition by the disappearance of the
alkyne triplet and the appearance of the triazole proton (7.90
ppm), additional aromatic resonances, and the appearance of a
In the chelate, then click approach, the overall design involved
complexation of the alkyne-functionalized PMAA ligand, 3,
with fac-[ReI(OH2)3(CO)3]+ followed by cycloaddition of the
alkyne with benzyl azide. In the first step, the alkyne-
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Scheme 2
Scheme 3
functionalized PMAA ligand, 3, was reacted with fac-
[ReI(OH2)3(CO)3]+ in aqueous conditions to produce the
corresponding fac-[ReI(CO)3(O,Namine,Npy-3)], 6, as a precip-
1
itate in good yields (63%). H NMR characterization of 6
reveled significant shifts and splitting from the free ligand upon
[ReI(CO)3]+ coordination. The methylene protons in the free
ligand, 3, appeared as singlets; upon coordination of the PMAA
ligand to the [ReI(CO)3]+ core to produce 6, the methylene
protons exhibited asymmetric HA/HB coupling that appeared as
AB quartets (4.67, 4.32, and 3.59 ppm). The terminal alkyne
was observed as a triplet (2.77 ppm) in 3, whereas upon
coordination to form 6 there was a downfield shift and
decoupling leading to a singlet (3.77 ppm). IR analysis of 6
exhibited bands indicative of the facial coordination of
[ReI(CO)3]+ with an asymmetric ligand (2018, 1907, 1863
cm−1) and the coordinated carboxylate (1650 cm−1). Single
crystals of 6 were analyzed by X-ray diffraction and determined
to pack in the C2/c spacegroup with one molecule in the
asymmetric unit cell (Figure 1). The complex exists in a
distorted octahedral coordination geometry with facially
coordinated carbonyl ligands and the tridentate ligand 3
oriented about the metal center consistent with other
[ReI(CO)3]+ complexes.21,24,34 The coordinated PMAA ligand
Figure 1. X-ray structure of 6 with hydrogen atoms omitted for clarity.
Ellipsoids are drawn at 50% probability.
in 6 exhibited expected bond angles (N(1)−Re(1)−N(2),
75.61(12)°; N(1)−Re(1)−O(4), 81.49(12)°; N(2)−Re(1)−
O(4), 78.43(12)°) and distances for ligand and the
[MI(CO)3]+ core (Re(1)−N(1), 2.176(3) Å, Re(1)−N(2),
2.229(3) Å, Re(1)−O(4), 2.120(3) Å) (Table 2). These angles
and distances were comparable to analogous [ReI(CO)3]+
PMAA complexes. In particular, the alkyne moiety of 6 was
observed to be directed away from the metal center with a
C(13)−C(14) 1.176(8) Å bond length, typical for carbon−
carbon triple bonds.
The second reaction of the chelate, then click approach, the
Huisgen “click” reaction, was carried out by reacting 6 with
benzyl azide under basic conditions (pH ≈ 7−9) to yield the
expected triazole cycloaddition product fac-
1
[ReI(CO)3(O,Namine,Npy-5)], 8, in moderate yields. H NMR
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and X-ray diffraction of 8 clearly confirmed [ReI(CO)3]+
proton was observed as a singlet at 8.21 ppm, and the
remaining methylene protons were observed at 5.14, 4.85, and
4.71 ppm. The splitting of the benzylic methylene protons is
characteristic of the 1,2,3-triazole-coordinated species and
yielded further support for structural assignment.41
maintained the PMAA coordination mode under these
1
conditions, where triazole coordination was not observed. H
NMR yielded a singlet at 8.43 ppm, a multiplet at 7.34−7.39
ppm, and a singlet at 5.68 ppm corresponding to the 1,2,3-
triazole proton, aromatic benzyl protons, and benzylic
methylene protons, respectively, accompanied by upfield shifts
for the remaining methylene AB quartets to 4.68, 4.62, and 3.60
ppm. The HA/HB splitting of methylene’s of the PMAA ligand
illustrate the [ReI(CO)3]+ coordination to 5 via O,Namine,Npy.
The IR spectrum of 8 exhibited primary bands of the
asymmetric facial carbonyls (2019 and 1905 cm−1) and the
coordinated carboxylate (1649 cm−1).
While repeated attempts to isolate single crystals of 9 were
unsuccessful for structural analysis, an analogous chelate that
maintains the specific coordination (Ntri,Namine,Npy) mode
predicted for 9 would provide a single isomer for comparison
of characterization data. Utilizing the protected methyl ester
“clicked” ligand, 4, complex formation was limited to a single
coordination mode of Ntri,Namine,Npy-4 with [ReI(CO)3]+ by
eliminating the possibility of carboxylate coordination and
engaging the triazole participation in coordination. In a 1:1
ratio, reaction of 4 with fac-[ReI(OH2)3(CO)3]+ at room
temperature for 16 h produced the cationic fac-
[ReI(CO)3(Ntri,Namine,Npy-4)]+, 7, in good to excellent yields.
1H NMR analysis of 7 revealed splitting of the benzyl aromatic
protons into two multiplets (7.34−7.32 and 6.93−6.91 ppm)
and the benzylic methylene protons into an AB quartet (5.65
ppm). The 1,2,3-triazole proton was observed as a singlet at
8.23 ppm upon coordination. The remaining methylene
protons also formed an AB quartet (5.13 ppm) and a multiplet
(4.89−4.76 ppm), while the methyl ester protons were
observed at 3.77 ppm, confirming Ntri,Namine,Npy coordination
without loss of the methyl ester. Comparison of the 1H spectra
of 7 and 9 exhibits similar splitting patterns, coupling constants,
and chemical shifts, indicating a similar coordination environ-
ment. IR of 7 showed bands corresponding to the CO (2029
and 1908 cm−1) and methyl ester (1741 cm−1). Colorless
needles of 7 suitable for X-ray diffraction analysis were obtained
through slow diffusion of diethyl ether into a saturated solution
of 7 in dichloromethane (Figure 3). Crystallizing in the P21/c
space group with one molecule and triflate counterion, the
coordination environment around the rhenium center consisted
of three facially arranged carbonyl ligands and 4. The linear
ligand 4 is oriented on the metal through the pyridine nitrogen
(N(1)), the tertiary nitrogen (N(2)), and the nitrogen of the
1,2,3-triazole moiety (N(3)), completing the pseudooctahedral
Single crystals of 8 were analyzed by X-ray diffraction analysis
and determined to pack in the P-1 space group (Figure 2). The
Figure 2. X-ray structure of 8 with hydrogen atoms omitted for clarity.
Ellipsoids are drawn at 50% probability.
octahedral structure confirmed the facial orientation of the
[ReI(CO)3]+ core and ligand 5 occupying the remaining three
sites. The ligand, 5, is found coordinated to the metal through
the carboxylic acid (Re(1)−O(4), 2.120(2) Å), amine (Re(1)−
N(2), 2.241(3)), and pyridine donors (Re(1)−N(1),
2.177(3)). The 1,2,3-triazole ring and benzyl substituent is
observed pointed away from the rhenium center. Analogous to
the structure of 6, the coordination mode of 8 consists of two
constrained 5-membered coordination rings (76.24(10)° for
N(1)−Re(1)−N(2) and 78.68(9)° for N(2)−Re(1)−O(4)) at
the ReI(CO)3+ core. The angles between N(2)−C(12)−C(13)
and N(2)−C(8)−C(9) of the diastereotopic methylene
carbons are not strained at angles of 110.7(7)° and 113.8(3)°.
In the click, then chelate strategy, the alkyne−PMAA was
“clicked” under the analogous CuI-catalyzed conditions with
benzyl azide to generate the triazole PMAA product for
subsequent complexation with fac-[MI(OH2)3(CO)3]+. Reac-
tion of the ligand 5 with fac-[ReI(OH2)3(CO)3]+ led to
formation of two products depending on the pH of the reaction
mixture. Careful control of the reaction pH allowed for
isolation of each of the products independently. Under slightly
acidic pHs (3−5), the product fac-[ReI(CO)3(Ntri,Namine,Npy-
5)], 9, containing coordination of the triazole, amine, and
1
pyridine donors was observed. H NMR analysis of 9 revealed
splitting of the benzyl aromatic protons into two multiplets
(7.31−7.29 and 6.93−6.90 ppm) and the benzylic methylene
protons into an AB quartet (5.62 ppm). The 1,2,3-triazole
Figure 3. X-ray structure of 7 with hydrogen atoms and triflate
counterion omitted for clarity. Ellipsoids are drawn at 50% probability.
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Figure 4. 1H NMR ((CD3)2SO) of 8 converting to 9 by addition of trifluoroacetic acid: 1, 0 equiv of TFA; 2, 2 equiv of TFA; 3, 4 equiv of TFA; 4,
10 equiv of TFA; 5, 25 equiv of TFA; 6, 45 equiv of TFA.
geometry. The methyl ester substituent bound to N(2) is
directed away from the rhenium center similar to complexes 6
and 8. The Re(1)−N(3) bond length of 2.143(4) Å falls
between the average distances of 2.12−2.15 Å reported
previously for rhenium 1,2,3-triazole coordination trans to a
carbonyl ligand.45,56 Similar to 8, the 1,2,3-triazole bond lengths
of 7 were observed between 1.334(5) and 1.365(6) Å, with the
shortest distance between N(3)−N(4) and N(3)−C(11)
holding the longest. Complex 7 contains two 5-membered
coordination rings generated from the pyridine, amine, and
1,2,3-triazole coordination with the metal center. The limitation
of the 5-membered ring size leads to a slight constriction of
these rings on the metal center with observed angles for N(1)−
Re(1)−N(2) and N(2)−Re(1)−N(3)) at 78.11(13)° and
76.54(13)°, respectively. Similarly, the methylene moieties,
N(2)−C(10)−C(11) and N(2)−C(9)−C(8), exhibited mod-
est to no strain at angles of 109.4(6)° and 112.1(3)°,
respectively.
confirmed the product isolated under this approach directly
correlated with the [ReI(CO)3]+ residing on the (O,Namine,Npy)
donors of the PMAA portion of ligand 5. The observation
confirmed the second coordination isomer produced from the
ligand system that correlated with the chelate, then click
approach. In both cases, the pyridine ring maintained
coordination to the [ReI(CO)3]+ center, and the analogous
(O,Namine,Ntri) complex was not detected.
Intraconversion of 8 and 9. During the isolation and
characterization of products 8 and 9, it was noted that two
peaks could be observed in the HPLC chromatogram of the
reaction mixture using a standard mobile phase of 0.1% TFA/
methanol (pH ≈ 1.7). This eluent system proved to be a
challenge in accurately understanding formation and con-
version of 8 and 9 that will be addressed in greater detail in the
subsequent 99mTc labeling section. Initially, the mixture was
considered to be diastereomeric complexes formed upon
coordination, but independent isolation of these two species
confirmed their identity as coordination isomers rather than
diastereomers. An attempt to drive the kinetic product to the
thermodynamic product, conversion of 8 into 9 or vice versa,
through excessive heating and extended reaction times did not
yield significant changes in the starting compounds.
Significantly different results were obtained when the
reaction of 5 with fac-[ReI(OH2)3(CO)3]+ was carried out
under slightly basic conditions (pH 7−9). The neutral product
fac-[ReI(CO)3(O,Namine,Npy-5)], 8, was isolated from the
reaction mixture as the single product. 1H NMR analysis
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However, pH changes did significantly alter the relative ratio
of complexes 8 and 9 as noted by changes in the intensity of
their respective peaks during HPLC analysis. This pH
sensitivity toward complex speciation was found to be a critical
issue in the reaction, identification, and purification of the
complexes. The relative speciation of 8 and 9 was found to be
impacted by the initial reaction pH and by addition of acid or
base to the purified compounds, suggesting [ReI(CO)3]+ could
undergo a coordination mode rearrangement from one mode
to the other (Scheme 3). In the chelate, then click approach, the
coordination mode of the PMAA ligand was preorganized and
limited to (O,Namine,Npy) in 6. When the cycloaddition of 6
with benzyl azide was conducted at pHs > 7, the complex still
maintained the (O,Namine,Npy) coordination mode. However,
when the click reaction with 6 was carried out at slightly acidic
pHs, both coordination isomers 8 (O,Namine,Npy-5) and 9
(Ntri,Namine,Npy-5) were observed. The appearance of 9 in the
reaction mixture clearly indicated the coordination mode of the
initial complex (O,Namine,Npy) of 6 could undergo an
intramolecular rearrangement to the (Ntri,Namine,Npy) mode.
Lower pHs were examined using this chelate, then click approach
to increase formation of 9; however, the inherent efficiency of
the “click” reaction was greatly diminished at more acidic pHs.
Using the click, then chelate approach, complete formation of 9
was achieved by directly reacting the “clicked” ligand 5 with fac-
[ReI(OH2)3(CO)3]+ under acidic conditions (pH ≈ 3−5).
Direct intraconversion between 8 and 9 or between 9 and 8
could be achieved by adjusting the pH of the solution with acid
or base from the isolated pure complexes. Partial conversion of
8 to 9 was initially observed when analysis of 8 by HPLC (0.1%
TFA/methanol) yielded two peaks in the chromatogram rather
than the expected single peak. Complete conversion from 8
into 9 could be achieved by the molar equivalent addition of
TFA to the reaction that maintained pH < 2. Conversion was
noted to occur readily at room temperature. Conversion of 9
into 8 could be achieved by addition of base (OH−) to the
purified complex 9. Complete conversion under basic
conditions occurred at pH > 8, but it occurred at slightly
slower rates than conversion of 8 to 9.
switch coordination modes in metal complexes solely based on
pH.64−69 To our knowledge, limited studies have been reported
with systems that examine the impact of pH on the speciation
of [99mTcI(CO)3]+ complexes. One example with [ReI(CO)3]+
analogs by Lipowska and co-workers utilized a number of
aminocarboxylate ligands to achieve similar intraconversion
upon rhenium coordination and pH variance.35 In their system,
acidifying the solution initiated carboxylate coordination and
loss of amine coordination as confirmed with NMR solution
studies and X-ray structural analysis. The corresponding
converse reaction involving addition of base yielded coordina-
tion rearrangement to favor amine coordination at basic pH’s.
However, in this work the opposite trend was observed with 8
and 9, where amine coordination was favored at low pH and
carboxylate coordination at high pH. This difference between
the experiments may be attributed to the coordination strength
of an aromatic to alkyl amine and the difference in pKa’s of
tertiary amines versus that of aromatic 1,2,3-triazole amines. An
S-functionalized Huisgen triazole tetradentate cysteine deriva-
tive, S-(1-benzyl-1-H-[1,2,3]triazol-4-ylmethyl)cysteine, yielded
two products upon complexation with [MI(CO)3]+.45 It was
proposed that 1,2,3-triazole coordination participation may
have competed with the amine, sulfur, and carboxylate groups
of cysteine to yield two coordination isomers; however, no
experiments examining pH dependence on the reaction
formation were conducted. In a similar system, the tetradentate
ligand (EtSEtN(CH2COOH)2) was explored with both
[ReI(CO)3]+ and [ReI(CO)2NO]2+, where tridentate coordi-
nation or CO displacement by thioether coordination was
observed.70 Additional studies performed on tridentate
complexes of both synthons indicated that no intraligand
exchange was observed based on changes in pH.
99mTc Solution Studies. The PMAA ligand has been
utilized with fac-[99mTcI(OH2)3(CO)3]+ to yield neutral
[
99mTcI(CO)3(PMAA)] complexes in excellent yields (>95%)
at 10−5 M ligand concentrations.71 However, the majority of
PMAA analogs have been functionalized through the central
tertiary amine utilizing a linker extension that is sufficiently long
enough to eliminate the possibility of coordination isomers. In
this study, the PMAA analogs as noted in the rhenium studies
can participate in the coordination sphere and were investigated
further with [99mTcI(CO)3]+. These studies provided a valuable
comparison of the species formation as substitution kinetics
and differences in micro- vs macroscale synthesis as they can
significantly vary between the group 7 congeners. Following a
similar preparation pathway, the corresponding routes (chelate,
then click or click, then chelate) were investigated with fac-
Conversion of 8 to 9 was studied by titrating acid into a
1
1
solution of 8 and analyzing by H NMR. In H NMR, a series
of scans was collected as concentrated TFA was added in small
increments to a solution of 8 in (CD3)2SO. Analysis of the
spectrum showed partial conversion of 8 to 9 in as little as 0.5
equiv of acid, although complete conversion to 9 was not
achieved until 45 equiv of TFA was added to the sample tube.
The methylene protons adjacent to the COOH and the triazole
proton provided critical fingerprinting for examining the
transition of the species. Most notably, as the coordination
changes from the carboxylate in 8 to the triazole in 9, the
triazole proton (A) shifts upfield to 8.21 ppm (Figure 4). The
benzylic protons (E) have a slight upfield shift and show
splitting to an AB quartet upon formation of 9. The remaining
CH2’s (B, C, and D) maintain AB quartet splitting. The
methylene protons adjacent to the coordinated pyridine (B)
did not show changes in splitting or shifting upon addition of
acid. This observation is consistent with X-ray structure
analysis, where the pyridine donor remains coordinated
throughout the coordination rearrangement.
[
99mTcI(OH2)3(CO)3]+ and the PMAA ligand system for
comparison with the [ReI(CO)3]+ analogs (Scheme 1).
In the chelate, then click approach, reaction of 3 with fac-
[
99mTc(OH2)3(CO)3]+ was carried out at 70 °C for 30 min to
generate
a single peak corresponding to fac-
[99mTcI(CO)3(O,Namine,Npy-3)], 6A, in excellent yields. The
HPLC retention time of 6A correlated with the Re analog 6 in
the TFA/methanol HPLC system. The click reaction of 6A
with benzyl azide was carried out under analogous conditions
and pH as the Re analogs. It was anticipated that both 99mTc
coordination isomers fac-[99mTcI(CO)3(O,Namine,Npy-5)], 8A,
and fac-[99mTcI(CO)3(Ntri,Namine,Npy-5)], 9A, would be ob-
served under their respective pH’s. As noted in the Re
preparation, the pH associated with the mobile phase utilized in
the HPLC gradient system could inadvertently shift the
speciation of the complex, which would be more pronounced
The impact of pH on the potentially labile ligands provides
fascinating insight into complex speciation that has been
observed in other metals and ligand types based on solution
changes.57−63 Only a handful of ligands have been observed to
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in the 99mTc systems. To circumvent this issue, several mobile
phases 0.1% TFA (pH 1.7), TEAP (10 mM, pH 3), PB (10
mM, pH 7.4), and sodium borate buffer (10 mM, pH 9)
specific to the pH of the reaction conditions were used to
maintain the pH in order to not alter the speciation observed at
a particular pH. In each system, the retention times of the
99mTc analogs could be directly related to the isolated Re
analogs as controls.
The click reaction of 6A and benzyl azide was carried out at
pH 7.4 at 70 °C for 30 min to yield a single peak, 8A, matching
the Re analog 8. Performing the “click” reaction at different
pH’s between 5 and 9 yielded only the single product 8A when
analyzed with the corresponding pH-matched eluent. The
“click” reaction was also conducted at pH 9 at 70 °C at several
reaction times (30, 60, and 90 min). Partial cycloaddition was
observed at 30 (60%) and 60 min (85%), and complete
formation occurred after 90 min (100%), Figure 5.
(similar to imidazole) is predicted to be more suited for the TcI
center compared to the hard carboxylate donor.
In the click, then chelate approach, direct complexation of 5
with fac-[99mTcI(OH2)3(CO)3]+ was explored under several pH
conditions. Reacting 5 with fac-[99mTcI(OH2)3(CO)3]+ under
acidic conditions (pH 1.7) produced only 9A. When the
reaction of 5 with fac-[99mTcI(OH2)3(CO)3]+ was conducted at
pH 7.4 both products 8A and 9A were identified in the HPLC
chromatogram. This observation suggests the initial kinetics of
complex formation may drive the speciation, where the
protonation state of the ligand or solution impacts the
thermodynamic product formation. The reaction mixture
produced at pH 7.4 was also found to undergo intraconversion
between the two products based on the pH experiments
mentioned above.
CONCLUSIONS
■
A new class of alkyne-functionalized PMAA complexes has
been explored for complexation with [MI(CO)3]+ (M = Re,
99mTc) that can undergo cycloaddition with an organic azide via
the Huisgen “click” reaction. The two general strategies (click,
then chelate and chelate, then click) revealed similar complexes
were formed through the different synthetic routes, but slight
differences in the reaction conditions affected the overall
product formation. In the click, then chelate approach, the
preorganized coordination mode of (O,Namine,Npy)-5 with
[MI(CO)3]+ was maintained during the “click” reaction
conducted at neutral to slightly basic reaction pHs. In the
chelate, then click approach, two coordination modes of
(O,Namine,Npy)-5 and (Ntri,Namine,Npy)-5 were observed during
incubation of [MI(CO)3]+ at neutral pH with the “clicked”
ligand 5. The observed differences between the two approaches
is most likely related to the kinetics of complex formation
compared to the stability of the coordination bond at neutral
pH. Interestingly, the Re and 99mTc complexes with 5 displayed
a pH-dependent coordination mode speciation, where
[MI(CO)3]+ favored carboxylate coordination in
(O,Namine,Npy)-5 at basic pHs and the triazole donor in
(Ntri,Namine,Npy)-5 at acidic pHs. This observation suggests that
the preference of the [MI(CO)3]+ favors coordination of
aromatic amines over charge neutralization with a deprotonated
carboxylate ligand. An important aspect of this system is that
both complexes readily undergo a pH-reversible intramolecular
coordination rearrangement of the ligand on the [MI(CO)3]+
center based on solution changes by addition of acid or base.
pH reversibility of the coordination species may provide a
unique functionality in future pH-sensing applications.
Figure 5. Radiochromatagram of crude reaction mixture, click, then
chelate (blue, front), run in phosphate buffer (10 mM, pH 7.4);
purified 8A (red, middle) run in borate buffer (10 mM, pH 9.0);
purified 9A (green, back) run in TFA (0.1% aq, pH 1.7).
It was noted that incomplete conversion of complex 8A to
9A was observed on the HPLC by running a TFA/methanol
method that resulted in a mixture of 8A (40%) and 9A (60%).
Independent isolation of 8A by peak collection from the TFA/
methanol method and subsequent reinjection on the HPLC
yielded both peaks in a 20:80 (8A:9A) mixture on the first
reinjection and 100% conversion to 9A on the second iteration.
Complete conversion could also be achieved by direct
acidification of a solution of 8A to a pH of 1.7 at room
temperature to generate only product 9A in less than 15 min.
Conversion of 9A to 8A was accomplished in a similar
manner as the previous rearrangement by adjusting the pH of
the solution to basic to achieve conversion. Injection of a
purified sample of 9A on the HPLC using sodium borate/
methanol pH 9 gradient system produced both 8A and 9A.
Isolated by peak collection, 9A was reinjected on the borate
system to eventually give the single product of 8A after two
iterations. Adjusting the solution to pH 9 with a hydroxide
solution also generated complex 8A, but it appeared to take
longer to convert in this direction. The observed difference may
be related to the changes in coordination strength of the softer
triazole donor to the carboxylate as the complex shifts from a
cationic to a neutral complex. The aromatic triazole donor
ASSOCIATED CONTENT
* Supporting Information
■
S
Complete X-ray structural information for 6, 7, and 8 (CCDC
nos. 912683, 912684, 912685) 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.
AUTHOR INFORMATION
Corresponding Author
*Phone: (509)-335-3858. Fax: (509)-335-8867. E-mail:
bennyp@wsu.edu.
■
Notes
The authors declare no competing financial interest.
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(31) Tavare,
́
R.; Torres Martin De Rosales, R.; Blower, P. J.; Mullen,
ACKNOWLEDGMENTS
■
G. E. D. Bioconjugate Chem. 2009, 20, 2071−2081.
(32) Zahnd, C.; Kawe, M.; Stumpp, M. T.; de Pasquale, C.;
Tamaskovic, R.; Nagy-Davidescu, G.; Dreier, B.; Schibli, R.; Binz, H.
K.; Waibel, R.; Pluckthun, A. Cancer Res. 2010, 70, 1595−1605.
(33) Tolmachev, V.; Hofstrom, C.; Malmberg, J.; Ahlgren, S.;
The authors would like to thank the Office of Science, U.S.
Department of Energy, Radiochemistry and Radiochemistry
Instrumentation Program (DE-FG02-08-ER64672), the Na-
tional Science Foundation Research Experiences for Under-
graduates (REU) program (0851502), and Washington State
University for financial assistance. We greatly appreciate Dr.
Mary Dyszlewski at Covidien for providing the IsoLink kits for
the 99mTc studies.
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Hosseinimehr, S. J.; Sandstrom, M.; Abrahmsen
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