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Article

Strategic Evaluation of the Traceless Staudinger Ligation for Radiolabeling with the Tricarbonyl Core

by
Constantin Mamat
1,2,*,
Christian Jentschel
1,
Martin Köckerling
3 and
Jörg Steinbach
1,2
1
Helmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400, D-01328 Dresden, Germany
2
Fakultät Chemie und Lebensmittelchemie, Technische Universität Dresden, D-01062 Dresden, Germany
3
Institut für Chemie, Anorganische Festkörperchemie, Universität Rostock, Albert-Einstein-Straße 3a, D-18059 Rostock, Germany
*
Author to whom correspondence should be addressed.
Molecules 2021, 26(21), 6629; https://doi.org/10.3390/molecules26216629
Submission received: 23 September 2021 / Revised: 22 October 2021 / Accepted: 27 October 2021 / Published: 1 November 2021
(This article belongs to the Special Issue Technetium and Rhenium in Chemistry and Their Advanced Applications)
Browse Figures
Versions Notes

Abstract

:
The traceless Staudinger ligation with its two variants is a powerful biorthogonal conjugation method not only for the connection of biomolecules, but also for the introduction of fluorescence- or radiolabels under mild reaction conditions. Herein, the strategic evaluation of the traceless Staudinger ligation for radiolabeling 99mTc using the fac-[Tc(CO)3]+ core is presented. A convenient and high-yielding three-step synthetic procedure of dipicolylamine-based phosphanols as ligands for the mild radiolabeling was developed. The labeling was accomplished using a tricarbonyl kit and a 99mTc-pertechnetate generator eluate showing 87% radiochemical conversion. The respective rhenium-based, non-radioactive reference compounds were synthesized using (Et4N)2[Re(CO)3Br3] as precursor. All products were analyzed by NMR, MS, and elemental analysis. Additional XRD analyses were performed.

Graphical Abstract

1. Introduction

The traceless Staudinger ligation belongs to the biorthogonal click conjugation reactions and is widely used, e.g., to connect small molecules to (bio)macromolecules like peptides, proteins, or carbohydrates, for cyclization processes to construct large sized lactams, or for the preparation of molecular rods [1,2,3,4,5]. In general, two variants of the traceless Staudinger ligation are described for (radio)labeling purposes [6,7]. Both approaches are shown in Scheme 1. The direct variant is based on a modified phosphane containing the (radio)label, which is reacted with an azide-containing biomolecule. The second, indirect variant, uses an azide-functionalized molecule bearing the (radio)label and a biomolecule, which is connected to the phosphane unit. Due to the smooth reaction conditions, the insertion of any label, such as fluorescent dyes [8] or radionuclides like fluorine-18 [9], iodine-131, or radiometals [7], is also possible without the need for a catalyst in contrast to other Cu-catalyzed click reactions.
In radiopharmacy, the convenient preparation of organometallic precursors bearing the fac-[Tc(CO)3]+ core [10,11] opens a convenient route to design new radiopharmaceuticals for single photon emission computed tomography (SPECT) [12]. Especially bio(macro)molecules like peptides, proteins, or antibodies can be labeled under mild conditions when using Tc- and Re-tricarbonyl precursors in combination with click chemistry methods [13]. This has been applied in the past using the “click-to-chelate” approach for instance [14,15,16], but never with the Staudinger ligation. The drawback of this Cu-click-conjugation method lies in the need of a copper catalyst, which must be removed prior to in vivo applications [16,17,18,19] to minimize toxicity.
To overcome this obstacle, the application of the traceless Staudinger ligation, known as a strong and powerful copper-free ligation method, was evaluated for radiolabeling with technetium-99m. For this purpose, phosphanes are required equipped with a chelator to insert the M(CO)3 core. The 2,2′-dipicolylamine (DPA) moiety [20,21,22], which is known to be an excellent tridentate chelator for the M(CO)3 core [23], was chosen and connected to the phosphanol skeleton. The corresponding natRe complexes were synthesized as non-radioactive reference and the procedure was transferred to radiolabeling chemistry using technetium-99m.

2. Results and Discussion

2.1. Preparation of Phosphanes

Azide-functionalized target molecules and label-containing phosphanes with benzoate moiety are required for the direct variant of the traceless Staudinger ligation (first line in Scheme 1). Two ways were elaborated in the past describing the functionalization of phosphanol 2b or the preparation of functionalized 2-iodophenyl esters from 2a followed by introduction of the phosphane residue [24,25]. Moreover, the dipicolylamine moiety, which is used as a chelating unit for the tricarbonyl core, was mandatory to connect to phosphanol skeleton by a benzoate linker. The synthesis route to the final phosphane-containing ligands with the dipicolylamine-chelating group is shown in Scheme 2.
In the first step, 4-(chloromethyl)benzoyl chloride (1) was reacted with 2-iodophenol (2a), phosphanol 2b, and the borane-protected phosphanol 2c to give the building blocks 3a–c in high yields (97%, 84%, and 82%). Compound 3a was synthesized to use the alternative route for the phosphane preparation, whereas 3c was synthesized to avoid side-reactions like unwanted phosphonium-salt formation or oxidation of the phosphorus. Afterwards, 3a–c were reacted with 2,2′-dipicolylamine in a nucleophilic substitution to yield the final DPA-ligands 4a (86%) and 4b (65%). Borane-containing compound 4c could not be isolated due to decomposition. The last step involved the reaction of (Et4N)2[Re(CO)3Br3] as Re-tricarbonyl source [26,27,28] to the desired rhenium complexes fac-[Re(CO)34a]Br and fac-Re(CO)34b]Br, which are intended to be used as non-radioactive references for later radiolabeling with 99mTc.
The building blocks 3a–3c and ligand 4a were grown as single-crystals to establish their molecular structures from XRD analyses. The molecular structures of the compounds are presented in Figure 1 and Figure 2. All the bond lengths and angles are within their expected ranges. As expected, the average C–P–C angle in 3b is significantly smaller (102.2°) than the ideal tetrahedral angle, whereas in 3c the bonding angles around the phosphorous have an average of 109.4° with larger C–P–B and smaller C–P–C angles [24,25].

2.2. Preparation of the Non-Radioactive Rhenium-Reference Compounds

Due to the lanthanide contraction, the ionic radii of Tc and Re are comparable, which leads to a similar coordination behaviour and complexation chemistry [12]. Rhenium compounds were classically used as non-radioactive reference for technetium-99m-tracers, or as therapeutic pendant when using rhenium-186/-188 [29,30,31] according to the theranostic concept [32,33]. For this purpose, ligand 4a was reacted with (Et4N)2[Re(CO)3Br3] in methanol (last step in Scheme 2) to yield the desired rhenium complex fac-[Re(CO)34a]Br in 95%. Its structure was confirmed by MS, NMR, and XRD analysis (Figure 3). Single crystals were obtained upon slow evaporation of the methanolic reaction mixture. Furthermore, in the 1H NMR spectrum, the facial arrangement of the DPA moiety induces a splitting of the methylene signals of the two picolyl units in fac-[Re(CO)34a]Br into two doublets, which are found downfield-shifted at δ = 4.73 and 5.84 ppm (singlet of 3.86 ppm for ligand 4a) with a coupling constant of 2J = 16.3 Hz.
In contrast, when reacting 4b with (Et4N)2[Re(CO)3Br3] under the conditions described for fac-[Re(CO)34a]Br, a complex mixture of different complexation products was obtained, which were difficult to separate. The non-radioactive reference complex fac-[Re(CO)34b]Br, which is required for later radiolabeling, was not found. For instance, compound 5 as oxidized species with bound fac-Re(CO)3 core could be identified (box in Scheme 3), but no ligation was possible anymore with 5. A downfield-shifted signal of δ = 28.1 ppm was found in the 31P NMR and a peak at m/z = 880 was obtained from an ESI-MS analysis. To avoid, e.g., the oxidation of the phosphorous and the formation of other side products, the reaction was repeated under argon, with different solvents and temperatures, but without more success. In fact, a broad product mixture was obtained, because complexes of the fac-Re(CO)3 core are also able to be formed with the phosphorus of ligand 4b. NMR spectra of all compounds can be found in the Supplementary Materials.

2.3. Attempt to Identify the Side Products from Complexation

In order to prove the complexation behavior of such compounds and to identify components of the above-mentioned product mixture, two different sample phosphanes 2b and 6 [24] were chosen and treated under different reaction conditions with different starting materials as pointed out in Scheme 3. 31P NMR is an ideal tool to analyze such compounds and their behavior in terms of their oxidation state. Sample compound 6 was first oxidized to compound 7 to check the oxidation process during the complexation procedure. Next, phosphane 6 was reacted with (Et4N)2[Re(CO)3Br3] under argon giving a complex mixture, too. One of them was identified as complex 8. A similar complex was described with PPh3 as ligand [34]. 31P NMR spectra of all synthesized compounds were compared (Figure 4). A signal of δ = −14.9 ppm was determined for phosphane 6 whereas a downfield-shifted signal of δ = 28.0 ppm was found for the oxidized species 7. Furthermore, a downfield-shifted signal of δ = 23.1 ppm was found for complex 8, indicating a coordinative bond of the phosphorus to the rhenium atom. An ESI-MS analysis of 8 revealed that phosphane 6 is twice coordinated as monodentate ligand (m/z = 1118). To further confirm the reaction behavior of the used phosphanes and to further elucidate the complexation pattern, (Et4N)2[Re(CO)3Br3] was directly reacted with phosphanol 2b yielding complex 9. Interestingly, ligand 2b is bidentate [35] in Re-complex 9 in contrast to ligand 5. This finding is supported by an XRD analysis of single crystals grown from complex 9 (Figure 5).
In both structurally characterized rhenium complexes, the metal atoms are coordinated in a distorted octahedral geometry. The coordination environment in fac-[Re(CO)34a]Br (Figure 3) consist of three carbonyl-carbon atoms and three nitrogen atoms, of which two are aromatic nitrogen atoms (pyridine, N1 and N2) and one aliphatic (N3). As expected, a carbonyl ligand is bound on the opposite (trans) site of each nitrogen atom. N3 has a significantly longer bond to Re1 (2.233(1) Å) than N1 and N2 (2.169(2) and 2.171(2) Å). The Re–C (carbonyl) bond lengths are found in a narrow range of 1.914(2) to 1.831(2) Å. Because of these differences in bond lengths and due to steric restrictions of ligand 4a, the cis arranged bond angles around Re1 differ from ideally 90° and range from 78.05(5)° to 97.88(7)°. In complex 9 (Figure 5), the Re1 atom is also octahedrally coordinated. The deprotonated bidentate ligand 2b is coordinated via the oxygen and the phosphorus atom to the central Re atom. The remaining coordination sites are occupied with the carbon atoms of three bound carbonyl ligands and additionally one nitrogen atom of an acetonitrile ligand. As in fac-[Re(CO)34a]Br, a trans arrangement of the different ligands is observed. The longest Re–ligand distance is found for P1, the next shortest for N1 and the shortest for O1. The lengths of the trans-Re–C (carbonyl) bonds follow the same order. The longest is found for C19 (1.952(6) Å, trans to P1), the next shorter for C21 (trans to N1, 1.915(6) Å), and the shortest for C20 (1.906(6) Å, trans to O1).

2.4. Radiolabeling with Technetium-99m

For the following radiolabeling with technetium-99m, a two-step procedure was applied, first to generate the 99mTc(CO)3 species from the 99mTc-pertechetate solution using a tricarbonyl kit and secondly to perform the final complexation with the DPA-containing ligands [36]. For this purpose, approximately 500 MBq of [99mTc]TcO4− in 1 mL saline was added to the kit and heated at 100 °C for 30 min. The resulting fac-[[99mTc]Tc(CO)3(H2O)3]+ complex was reacted with ligands 4a,b in a MES buffer/ethanol mixture at pH 6.2 for 30 min at 100 °C. After cooling to rt, purification was done using cartridge separation.
In the case of radiolabeling with ligand 4a, complex fac-[[99mTc]Tc(CO)34a]+ was obtained showing a peak at tR = 20.2 min in the radio-HPLC chromatogram with 100% conversion of reduced [[99mTc]Tc(CO)3(H2O)3]+ (tR = 14.1 min). Two by-products were found, either at tR = 4.2 min (9%) or at tR = 16.4 min (4%), which are identified as back-oxidized TcO4– and an unknown 99mTc-species, respectively. Both by-products were removed after simple purification using a LiChrolut RP-18 cartridge. The identification of the 99mTc-complex was confirmed with the respective rhenium complex fac-[Re(CO)34a]Br (tR = 19.8 min). Additionally, the UV signal of ligand 4a was found at tR = 15.2 min. All chromatograms for this radiolabeling procedure are shown in Figure 6. Details of the radiolabeling procedure can be found in the Supplementary Materials.
In the case of radiolabeling with ligand 4b, minimum five peaks were found in the radio-HPLC chromatogram (Figure 7) independent of the change of reaction parameters like solvent (ACN, THF, DMF, DMSO, EtOH), temperature (25–100 °C) and labeling time. Beside the known by-product peaks at tR = 4.1 and 16.4 min, two main signals at 16.9 min (24%) and at 19.2 min (45%) occurred together with two by-products at 14.0 min (11%, remaining 99mTc-precursor) and at 16.4 min (7%). At this point, it was not possible for us to further identify the single fractions from the radiolabeling mixture, at least due to the missing non-radioactive reference compound (see discussion of the corresponding Re-complex earlier). These findings are in accordance to the complexation trials of ligand 4b with (Et4N)2[Re(CO)3Br3]. In addition to the oxidized ligand 4b, we found complexes where the phosphane moiety as monodentate ligand is bound to the Re.
To overcome the problem of additional complexation by the phosphane moiety during the complexation step with the M(CO)3 core, the functionalities of labeling unit and targeting molecule have to be changed. This enables the alternative way to apply the indirect approach of the traceless Staudinger ligation (Scheme 4) for future investigations, which seems to be more promising, because organic azides are not described as ligands for the tricarbonyl core and methods to connect phosphanes to bio(macro)molecules are also known in the literature [5,37]. Moreover, the phosphanol moiety can be easily connected to pharmacological target molecules of interest with a carboxylic function.

3. Materials and Methods

3.1. General

All reagents were purchased from commercial suppliers and were used without further purification. Analytical TLC was performed on pre-coated Silica Gel 60 F254 plates (Merck, Darmstadt, Germany) and results read under UV-light (λ = 254 nm). NMR spectra were recorded on an Agilent DD2-400 MHz system (OneNMR™ Probe) (Santa Clara, CA, USA) at 400 (1H), 101 (13C), 128 (11B), and 376 MHz (31P) or on an Agilent 600 MHz system (OneNMR™ Probe) at 600 (1H), 151 (13C), 192 (11B) and 243 MHz (31P). Chemical shifts are reported in ppm with tetramethylsilane (1H, 13C), BF3-OEt2 (11B) and H3PO4 (31P) as internal standard, respectively. MS spectra were obtained on a Micromass Quattro-LC spectrometer (Waters, Eschborn, Germany) using electron spray (ESI) as the ionization method. Melting points were recorded on a Galen III apparatus and are uncorrected (Cambridge Instruments/Leica, Wetzlar, Germany). Analytical HPLC was performed on a HPLC system (Perkin Elmer, Rodgau, Germany), equipped with a reverse phase column (Jupiter 4µ Proteo C18 90A (4.6 × 250 mm)), a UV-diode array detector (λ = 254 nm) and a Gabi Star scintillation radiodetector (Raytest, Straubenhardt, Germany) at a flow rate of 1 mL/min (eluent: acetonitrile/water + 0.1% TFA). The radioactive compounds were identified using analytical radio-HPLC by comparison of the retention time of the reference compound.

3.2. X-ray Diffraction Studies

X-ray diffraction data of 3a, 3b, 3c, and 9 single-crystals were collected with a Bruker-Nonius Apex-X8 CCD diffractometer with a crystal temperature of −100 °C and those of 4a and fac-[Re(CO)34a]Br with a Bruker-Nonius Apex Kappa-II at T = −150 °C. Graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) was used in all cases. The structures were solved by direct methods and refined against F2 on all data by full-matrix least-squares using the SHELX suite of programs (version 2014/2) [38,39,40]. All non-hydrogen atoms were refined anisotropically; all hydrogen atoms bound to C atoms were placed on calculated positions and refined using riding models. The asymmetric units of 3c and 4a contain two of the respective title molecules. In crystals of 4a, the O2C-C6H4I moiety of one of the two independent molecules is disordered with two different orientations. Furthermore, crystals of 9 contain co-crystallized CHCl3 molecules and those of fac-[Re(CO)34a]Br co-crystallized methanol molecules. All information about the X-ray diffraction structure determinations is deposited as cif files with the CCDC data center. The data can be obtained free of charge from the Cambridge Crystallographic Data Centre: CCDC 2064011 for 3a; CCDC 2064270 for 3b, CCDC 2067941 for 3c, CCDC 2071545 for 4a, CCDC 2069525 for 9, and CCDC 2080217 for fac-[Re(CO)34a]Br.

3.3. Chemical Syntheses

2-Iodophenyl 4-(chloromethyl)benzoate 3a. KOtBu (594 mg, 5.29 mmol) and 2-iodophenol (2a, 1.1 g, 4.81 mmol) were dissolved in anhydrous THF (10 mL). A solution of 4-(chloromethyl)benzoyl chloride (1, 1.0 g, 5.29 mmol) in 5 mL of anhydrous THF was added dropwise and the resulting mixture was allowed to stir overnight. Afterwards, a saturated NH4Cl solution (10 mL) was added, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4 and the solvent was removed under reduced pressure. Purification was done via column chromatography (silica gel, petroleum ether/ethyl acetate 10/1) to obtain 3a as a colorless solid (1.8 g, 97%); mp 116 °C; Rf 0.47 (petroleum ether/ethyl acetate 5/1); 1H NMR (400 MHz, C6D6): δ 3.92 (s, 2H, CH2), 6.44 (dt, 3J = 7.7 Hz, 4J = 1.6 Hz, 1H, H-4), 6.87 (t, 3J = 8.0 Hz, 1H, H-5), 6.94–6.99 (m, 3H, H-6, Hmeta), 7.53 (dd, 3J = 7.9 Hz, 4J = 1.5 Hz, 1H, H-3), 8.17 (d, 3J = 8.3 Hz, 2H, Hortho); 13C NMR (101 MHz, C6D6): δ 45.1 (CH2), 90.9 (C-2), 123.6 (C-6), 127.7 (C-4), 128.9 (Cmeta), 129.5 (Cipso), 131.0 (Cortho), 139.7 (C-3), 143.0 (Cpara), 152.0 (C-1), 163.7 (C=O); MS (ESI +): m/z = 395 [M+ + Na]; Anal. calcd. for: C14H10ClIO2 (372.59): C 45.13, H 2.71; found C 45.65, H 2.64.
2-(Diphenylphosphano)phenyl 4-(chloromethyl)benzoate 3b. KOtBu (53 mg, 0.47 mmol) and (2-hydroxyphenyl)diphenylphosphane (2b, 100 mg, 0.36 mmol) were dissolved in anhydrous THF (3 mL). A solution of 4-(chloromethyl)benzoyl chloride (1, 88 mg, 0.47 mmol) in 1 mL of anhydrous THF was added dropwise and the resulting mixture was allowed to stir overnight. Afterwards, a saturated NH4Cl solution (10 mL) was added, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4, and the solvent was removed under reduced pressure. Purification was done via column chromatography (silica gel, petroleum ether/ethyl acetate 5/1) to obtain 3b as a colorless solid (130 mg, 84%); mp 154 °C; Rf 0.66 (petroleum ether/ethyl acetate 3/1); 1H NMR (400 MHz, CDCl3): δ 4.60 (s, 2H, CH2), 6.86 (dd, 3JH,P = 4.5 Hz, 3J = 7.7 Hz, 1H, H-3), 7.18 (t, 3J = 7.4 Hz, 1H, H-4), 7.28–7.35 (m, 11H, H-6, Hortho, Hmeta, Hpara), 7.37 (d, 3J = 8.3 Hz, 2H, Hmeta’), 7.43 (t, 3J = 7.7 Hz, 1H, H-5), 7.82 (d, 3J = 8.3 Hz, 2H, Hortho’); 13C NMR (101 MHz, CDCl3): δ 45.5 (CH2), 122.6 (d, 3JC.P = 1.6 Hz, C-6), 126.4 (C-4), 128.5 (Cmeta’), 128.7 (d, 3JC,P = 7.3 Hz, Cmeta), 129.2 (Cpara), 130.0 (Cortho’), 130.1 (d, 1JC,P = 106.7, Cipso), 130.7 (Cortho), 133.7 (d, 2JC,P = 2.0 Hz, C-5), 134.2 (d, 2JC,P = 20.6 Hz, Cortho), 135.5 (d, 4JC,P = 10.2, C-3), 142.8 (Cpara’), 152.8 (d, 2JC,P = 16.3 Hz, C-1), 163.9 (C=O); 31P NMR (162 MHz, CDCl3): δ −14.4 ppm; MS (ESI+): m/z = 431 (28) [M+ + H], 453 (12) [M+ + Na]; Anal. calcd. for: C26H20ClO2P (430.86): C 72.48, H 4.68; found C 72.70, H 4.85.
2-(Diphenylphosphano)phenyl 4-(chloromethyl)benzoate borane adduct 3c. KOtBu (254 mg, 2.26 mmol) and (2-hydroxyphenyl)diphenylphosphane borane (2c, 440 mg, 1.51 mmol) were dissolved in anhydrous THF (5 mL). A solution of 4-(chloromethyl)benzoyl chloride (427 mg, 2.26 mmol) in 2 mL of anhydrous THF was added dropwise and the resulting mixture was allowed to stir overnight. Afterwards, a saturated NH4Cl solution (10 mL) was added, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4, and the solvent was removed under reduced pressure. Purification was done via column chromatography (silica gel, petroleum ether/ethyl acetate 5/1) to obtain 3c as a colorless solid (548 mg, 82%); mp 128 °C; Rf 0.34 (petroleum ether/ethyl acetate 5/1); 1H NMR (400 MHz, C6D6): δ 1.67–2.63 (m, 3H, BH3), 3.88 (s, 2H, CH2), 6.74 (t, 3J = 7.5 Hz, 1H, H-4), 6.81 (d, 3J = 8.3 Hz, 2H, Hmeta’), 6.89–6.93 (m, 6H, Hortho, Hmeta), 7.03 (t, 3J = 7.8 Hz, 1H, H-5), 7.17 (ddd, 4JH,P = 3.9 Hz, H-6), 7.29 (ddd, 4J = 1.6 Hz, 3J = 7.3 Hz, 3JH,P = 11.6 Hz, H-3), 7.69–7.76 (m, 4H, Hpara), 7.80 (d, 3J = 8.3 Hz, 2H, Hortho’); 13C NMR (101 MHz, C6D6): δ 45.1 (CH2), 124.7 (d, J = 4.9 Hz, C-6), 126.0 (d, J = 9.1 Hz, C-4), 128.4 (Cmeta’), 128.9 (d, 3J = 10.2 Hz, Cmeta), 129.4 (C-5), 130.9 (Cortho’), 131.2 (4JC,P = 2.4 Hz, Cpara), 132.6 (d, J = 2.0 Hz, C-3), 133.5 (d, 2J = 9.8 Hz, Cortho), 134.9 (d, 1JC,P = 7.3 Hz, Cipso), 143.0 (Cpara’), 153.2 (d, 1JC,P = 3.2 Hz, C-1), 163.3 (C=O); 31P NMR (162 MHz, C6D6): δ 20.9 ppm; 11B NMR (MHz, C6D6): δ −36.6 ppm; MS (ESI+): m/z = 467 (20) [M+ + Na]; Anal. calcd. for: C26H23BClO2P (444.70): C 70.22, H 5.21; found C 70.41, H 5.31.
2-Iodophenyl 4-((bis(pyridin-2-ylmethyl)amino)methyl)benzoate 4a. Compound 3a (500 mg, 1.34 mmol) was dissolved in acetone (6 mL), NaI (221 mg, 1.47 mmol) was added and the mixture was stirred for 1 h at rt. Afterwards, 2,2′-dipicolylamine (335 mg, 1.68 mmol) was added and the resulting mixture was stirred overnight at rt. Thereafter, the solvent was removed, saturated hydrogen carbonate solution (20 mL) was added, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4 and the solvent was removed under reduced pressure. Purification was performed via column chromatography (silica gel, ethyl acetate → ethyl acetate/EtOH 10/1) to obtain 4a as a colorless solid (620 mg, 86%); mp 90 °C; Rf 0.41 (chloroform/methanol 9/1); 1H NMR (400 MHz, C6D6): δ 3.60 (s, 2H, CH2N), 3.86 (s, 4H, CH2Pyr), 6.44 (dt, 3J = 7.7 Hz, 4J = 1.4 Hz, 1H, H-4), 6.65 (dd, 3J = 4.9 Hz, 3J = 7.4 Hz, 2H, Pyr), 6.87 (dt, 3J = 8.0 Hz, 4J = 1.4 Hz, 1H, H-5), 7.02 (dd, 3J = 8.0 Hz, 4J = 1.4 Hz, 1H, H-6), 7.13 (dd, 3J = 7.6 Hz, 4J = 2.0 Hz, 2H, Pyr), 7.37 (d, 3J = 7.7 Hz, 2H, Pyr), 7.41 (d, 3J = 8.2 Hz, 2H, Hmeta), 7.54 (dd, 3J = 7.8 Hz, 4J = 1.4 Hz, 1H, H-3), 8.31 (d, 3J = 8.2 Hz, 2H, Hortho), 8.48 (d, 3J = 4.9 Hz, 2H, Pyr); 13C NMR (101 MHz, C6D6): δ 58.3, 60.2 (CH2), 91.1 (C-2), 122.0 (C-5-Pyr), 122.9 (C-3-Pyr), 123.7 (C-6), 127.5 (C-4), 128.6 (Cipso), 129.3 (Cmeta), 129.4 (C-5), 130.9 (Cortho), 136.0 (C-4-Pyr), 139.6 (C-3), 146.5 (Cpara), 149.5 (C-6-Pyr), 152.1 (C-1), 160.0 (C-2-Pyr), 164.2 (C=O); MS (ESI+): m/z = 536 (100) [M+ + H]; Anal. calcd. for: C26H22IN3O2 (535.38): C 58.33, H 4.14; found C 58.36, H 4.11.
2-(Diphenylphosphino)phenyl 4-((bis(pyridin-2-ylmethyl)amino)methyl)benzoate 4b. Under argon, compound 3b (250 mg, 0.58 mmol) was dissolved in anhydrous THF (5 mL), NaI in catalytic amounts and 2,2′-dipicolylamine (193 mg, 0.97 mmol) was added and the resulting mixture was stirred at 60 °C for 5 h. Thereafter, the solvent was removed, saturated hydrogen carbonate solution (20 mL) was added, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4, and the solvent was removed under reduced pressure. Purification was done via column chromatography (silica gel, ethyl acetate) to obtain compound 4b as a pale yellow solid (224 mg, 65%); mp 77 °C; Rf 0.45 (chloroform/methanol 9/1); 1H NMR (400 MHz, CDCl3): δ 3.73 (s, 2H, CH2N); 3.81 (s, 4H, CH2Pyr), 6.85 (dd, 4J = 4.2 Hz, 3J = 7.4 Hz, 1H, H-4), 7.13–7.18 (m, 3H, H-1, Pyr), 7.25–7.34 (m, 11H, Hortho, Hmeta, Hpara, H-5), 7.38–7.43 (m, 3H, Hmeta’, H-2), 7.55 (d, 3J = 7.9 Hz, 2H, Pyr), 7.68 (dt, 4J = 1.7 Hz, 3J = 7.7 Hz, 2H, Pyr), 7.80 (d, 3J = 8.3 Hz, 2H, Hortho’), 8.53 (d, 3J = 4.4 Hz, 2H, Pyr); 13C NMR (101 MHz, CDCl3): δ 58.4 (NCH2Ar), 60.2 (NCH2Py), 122.2 (C-Py-5), 122.7 (C-6), 123.0 (C-Py-3), 126.2 (C-5), 128.1 (C-i’), 128.7 (d, 3JC,P = 7.4 Hz, C-m, C-m’), 129.1 (C-p), 123.0 (C-2), 130.3 (C-o’), 130.8 (d, 1JC,P = 15.0 Hz, C-i), 133.6 (C-4), 134.2 (d, 2J = 20.7 Hz, C-o), 135.6 (d, 2JC,P = 10.2 Hz, C-3), 136.6 (C-Py-4), 145.3 (C-p’), 149.2 (C-Py-6), 152.9 (d, 2JC,P = 17.0 Hz, C-1), 159.4 (C-Py-2); 164.2 (C=O); 31P NMR (162 MHz, CDCl3): δ −14.8; MS (ESI+): m/z (%) = 594 (100) [M+ + H], 616 (60) [M+ + Na]; Anal. calcd. for: C38H32N3O2P (593.65): C 76.88, H 5.43; found C 76.91, H 5.51.
fac-[Re(CO)34a]Br. Compound 4a (100 mg, 0.14 mmol) and (NH4)2[Re(CO)3Br3] (143 mg, 0.14 mmol) were dissolved in methanol (5 mL) and the resulting mixture was stirred at 60 °C for 6 h. Afterwards, the solvent was removed and the crude product was dissolved in chloroform (15 mL), the organic phase was washed with water (2 × 10 mL) and dried over Na2SO4. Afterwards, petroleum ether was added to precipitate the complex. Compound 5a was obtained as a colorless solid (157 mg, 95%); mp 215°C; 1H NMR (400 MHz, CDCl3): δ 4.73 (d, 2J = 16.3 Hz, 1H, CH2Pyr), 4.96 (s, 2H, CH2N), 5.84 (d, 2J = 16.3 Hz, 1H, CH2Pyr), 7.02 (dt, 3J = 4.9 Hz, 3J = 7.5 Hz, 1H, H-4), 7.19 (t, 3J = 6.5 Hz, 2H, H-5-Pyr), 7.24–7.28 (m, 1H, H-6), 7.41 (dt, 3J = 7.5 Hz, 4J = 2.0 Hz, 1H, H-5), 7.79–7.88 (m, 3H, H-3, H-4-Pyr), 7.93 (d, 3J = 8.1 Hz, 2H, Hmeta), 8.01 (d, 3J = 8.0 Hz, 2H, H-3-Pyr), 8.37 (d, 3J = 8.1 Hz, 2H, Hortho), 8.63 (d, 3J = 5.6 Hz, 2H, H-6-Pyr); 13C NMR (101 MHz, CDCl3): δ 68.1 (CH2Pyr), 72.0 (CH2N), 90.5 (C-2), 123.3 (C-6), 125.5, 125.6 (C-3Pyr/C-5Pyr), 127.9 (C-4), 129.6 (C-5), 130.9 (Cipso), 131.4 (Cortho), 133.3 (Cmeta), 137.6 (Cpara), 139.6 (C-3), 140.6 (C-4Pyr), 150.8 (C-6Pyr), 151.3 (C-1), 160.4 (C-2Pyr), 163.7 (C=O), 195.2, 195.8 (3 × C≡O). MS (ESI+): m/z = 804 (65) [M+ − Br, 185Re], 806 (100) [M+ − Br, 187Re]; Anal. calcd. for: C29H22BrIN3O5Re (884.93): C 39.33, H 2.84, N 4.75; found C 39.36, H 2.81, N 4.77. Analytical HPLC (254 nm): tR = 19.8 min.
2-(Diphenylphosphoryl)phenylbenzoate 7. Compound 6 [24] (210 mg, 0.44 mmol) was dissolved in DMF (4 mL), NaN3 (113 mg, 1.75 mmol) and water (0.5 mL) were added, and the solution was stirred at 80 °C for 4 h. Afterwards, the solvent was removed and the crude product was dissolved in chloroform (20 mL), the aqueous phase was washed with water (3 × 15 mL), dried over Na2SO4 and the solvent was removed. Purification was performed by column chromatography (petroleum ether/ethyl acetate, 1/1) to yield compound 6 (161 mg, 90%) as a colorless oil. Rf 0.29 (petroleum ether/ethyl acetate, 1/1). 1H NMR (400 MHz, CDCl3): δ 6.78–6.84 (m, 1H, H-5), 6.95 (ddd, 3J = 14.7 Hz, 3J = 7.8 Hz, 4J = 1.6 Hz, 1H, H-3), 7.07 (ddd, 3J = 8.4 Hz, 3J = 5.0, 4J = 0.7 Hz, 1H, H-6), 7.39–7.63 (m, 10H, H-m’, H-m, H-p, H-p’, H-4), 7.81–7.88 (m, 4H, H-o), 8.15-8.19 (m, 2H, H-o’) ppm; 13C NMR (101 MHz, CDCl3): δ = 107.7 (d, JC,P = 110.4 Hz), 119.3 (d, JC,P = 13.3 Hz), 119.6 (d, JC,P = 8.8 Hz), 126.5, 127.4, 128.2 (CHAr), 129.0 (d, JC,P =12.5 Hz, CHAr), 129.3 (d, JC,P = 2.2 Hz, CHAr), 131.6, 132.9 (d, JC,P 2.9 = Hz), 133.3 (d, JC,P = 10.7 Hz, CHAr), 133.4 (d, JC,P = 11.3 Hz), 135.0 (d, JC,P = 2.5 Hz), 137.0 (d, JC,P = 2.6 Hz), 164.2 (d, JC,P = 17.3 Hz, C=O), 176.1 (d, JC,P = 8.6 Hz) ppm; 31P NMR (162 MHz, CDCl3): δ 29.7 ppm; MS (ESI+): m/z (%) = 399 [M+ + H], 421 [M+ + Na]; Anal. calcd. for: C25H19O3P (398.40): C 75.37, H 4.81; found C 75.28, H 4.85.
fac-[Re(CO)3(5)2Br] 8. Compound 6 (102 mg, 0.36 mmol) and (Et4N)2[Re(CO)3Br3] (274 mg, 0.36 mmol) were dissolved in methanol (8 mL) and stirred for 7 h at 60 °C. Afterwards, the solvent was removed and the crude product purified using RP column chromatography (water/acetonitrile 10:1 → 1:1). After lyophilization, compound 8 (23 mg, 19%) was obtained as colorless solid. Rf 0.54 (RP-18, methanol). 1H NMR (400 MHz, C6D6): δ 6.78 (t, 1H, 3J = 7.5 Hz, H-p’), 6.89–7.09 (m, 10H, H-m, H-m’, H-p, H-4, H-6), 7.17–7.22 (m, 1H, H-5), 7.52 (dd, 1H, 3JH,P = 7.4 Hz, 3J = 12.5 Hz, H-3), 7.75–7.81 (m, 4H, H-o), 8.01 (d, 2H, 3J = 7.4 Hz, H-o’) ppm; 13C NMR (101 MHz, C6D6): δ 124.5 (d, 3JC,P = 5.9 Hz, C-6), 125.6 (d, 3JC,P = 11.3 Hz, C-4), 128.4 (c-m), 128.6 (C-p), 129.4 (C-5), 130.8 (C-o’), 131.5 (d, 1JC,P = 2.6 Hz, C-2), 132.2 (d, 1JC,P = 9.8 Hz, c-o), 133.3 (d, 2JC,P = 4.0 Hz, C-3), 134.6 (d, 1JC,P = 8.3 Hz, C-i), 153.7 (C-1), 163.9 (C=O) ppm (missing signals are under the solvent signal); 31P NMR (162 MHz, C6D6): δ 23.1 ppm; IR (ATR): ν = 3536 (OH), 3403 (OH), 2029 (C≡O), 1909 (C≡O) cm−1; MS (ESI+): m/z 1118 [M+ + H, 81Br], 1116 [M+ + H, 79Br].
fac-Acetonitrile-tris-carbonyl-(2-(diphenylposphino)phenolato)-rhenium(I) 9. Under argon, 2-(diphenylphosphano)phenol (2b, 99 mg, 0.36 mmol) and (Et4N)2[Re(CO)3Br3] (274 mg, 0.36 mmol) were dissolved in methanol (8 mL) and stirred for 7 h at 60 °C. Afterwards, the solvent was removed and the crude product purified using RP column chromatography (water/acetonitrile 10:1 → 1:1). After lyophilization, compound 9 (79 mg, 38%) was obtained as a colorless solid. Rf 0.62 (RP-18, methanol); mp 108 °C; 1H NMR (400 MHz, C6D6): δ 6.53–6.60 (m, 1H, H-4), 6.86–7.04 (m, 6H, H-3, H-6, H-m), 7.18–7.23 (m, 1H, H-5), 7.28–7.38 (m, 2H, H-p), 7.59–7-69 (m, 2H, H-o), 7.76 (dd, 3J = 7.8 Hz, 3J = 11.8 Hz, 2H, H-o) ppm; 13C NMR (101 MHz, C6D6): δ 113.0 (d, 1JC,P = 53.6 Hz, C-2), 115.7 (d, 3JC,P = 5.8 Hz, C-6), 122.3 (d, 3JC,P = 7.6 Hz, C-4), 129.0 (d, 3JC,P = 10.7 Hz, C-m), 129.0 (d, 3JC,P = 9.8 Hz, C-m), 459.8 (C-p), 123.6 (C-p), 132.8 (d, 2JC,P = 11.6 Hz, C-o), 133.4 (d, 2JC,P = 10.7 Hz, C-o), 133.7 (C-5), 134.0 (C-3), 180.7 (d, 2JC,P = 25.4 Hz, C-1) ppm; 31P NMR (202 MHz, C6D6): δ = 32.9 ppm; IR (ATR): ν = 3399 (OH), 2020 (C≡O), 1923 (C≡O), 1884 (C≡O) cm−1; MS (ESI+): m/z (%) = 549 (100) [M+-CH3CN]; Anal. calcd. for: C23H17NO4PRe (588.57): C 46.94, H 2.91, N 2.38; found C 46.64, H 2.66, N 2.01.

3.4. Radiolabeling Procedure

The preparation of boranocarbonate and the tricarbonyl kit (content: 17 mg Na-tartrate, 3.5 mg Na2B4O7, 3.2 mg Na2CO3, 8.1 mg K2[CO2BH3]) was described earlier; see ref. [41]. 99mTc was used as Na[99mTc]TcO4 generator eluate obtained by elution from a 99Mo/99mTc generator (Polatom, Otwok, Poland) using a 0.9% NaCl solution. [99mTc]TcO4 (1 mL, 500 MBq) was added to the tricarbonyl kit. Afterwards, the solution was heated for 30 min at 100 °C, cooled down to rt for 15 min, and was ready to use without further purification. Next, the pH of the fac-[99mTc(CO)3(H2O)3]+ (analytical radio HPLC: tR = 14.1 min) complex solution was adjusted to 6.2 by adding MES buffer (500 µL). Ligand 4a or 4b (200 µg) was dissolved in 200 µL of ethanol and added. The resulting reaction mixture was stirred at 100 °C for 30 min. Radio-HPLC showed 78% product formation. After cooling to rt and filtration, the solution was added to a RP-18 cartridge (LiChrolut RP-18 (40–63 µm), 500 mg) for purification. The cartridge was washed with water (3 × 2 mL) and eluted with ethanol (2 mL) to yield the final radiotracer.
The resulting radiotracer was analyzed via HPLC using a Jupiter 4µ Proteo C18 90A (4.6 × 250 mm) column (phenomenex, Aschaffenburg, Germany). Eluents were purified water + 0.1% TFA (solvent A) and acetonitrile + 0.1% TFA (solvent B) with a gradient of 95% (A) 0–3 min, 95% → 5% (A) 3–20 min, 5% (A) 20–25 min, 5 → 95% (A) 25–28 min, 95% (A) 28–45 min (flow rate: 1 ml/min). Analytical radio HPLC for fac-[[99mTc]Tc(CO)34a]+: tR = 20.2 min.

4. Conclusions

A proof-of-concept study was carried out to use the direct variant of the traceless Staudinger ligation for radiolabeling purposes with a 99mTc-tricarbonyl core. For this purpose, phosphane 4b with benzoate moiety was prepared and functionalized by introducing a dipicolylamine residue as tridentate ligand. Reactions with (Et4N)2[Re(CO)3Br3] to prepare the non-radioactive reference led to a mixture of products, including the oxidized 4b and different Re-complexes, which could not be identified completely. The same situation was found for the radiolabeling of 4b with fac-[99mTc(CO)3(H2O)3]+. Due to the high number of by-products during complexation, which were mandatory to separate, and the resultant low yields, it must be stated that this variant of the Staudinger ligation is not sufficient for radiolabeling purposes. Future work will entail concentrating on the change of the functionalities to prepare target molecules containing the phosphanol residue and azide-functionalized DPA-containing radiolabeling building blocks for chelation of the 99mTc-tricarbonyl core.

Supplementary Materials

The following are available online, NMR spectra of compounds, radiolabeling procedure and radio HPLC chromatograms.

Author Contributions

Conceptualization, C.M. and J.S.; methodology, C.M.; validation, C.J., M.K. and C.M.; formal analysis, M.K. and C.J.; investigation, C.J. and M.K.; resources, M.K. and J.S.; data curation, C.J., M.K., and C.M.; writing—original draft preparation, C.M. and M.K.; writing—review and editing, all authors; visualization, C.M. and M.K.; supervision, J.S. and M.K.; project administration, C.M. and J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are included in this paper.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

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Molecules 26 06629 sch001 550
Scheme 1. Direct and indirect approach of the traceless Staudinger Ligation.
Scheme 1. Direct and indirect approach of the traceless Staudinger Ligation.
Molecules 26 06629 sch001
Molecules 26 06629 sch002 550
Scheme 2. Synthesis of the 4-(chloromethyl)benzoate-building blocks 3a–c, the 2,2′-dipicolylamine-containing ligands 4a,b and their rhenium complexes fac-[Re(CO)34a]Br and fac-[Re(CO)34b]Br.
Scheme 2. Synthesis of the 4-(chloromethyl)benzoate-building blocks 3a–c, the 2,2′-dipicolylamine-containing ligands 4a,b and their rhenium complexes fac-[Re(CO)34a]Br and fac-[Re(CO)34b]Br.
Molecules 26 06629 sch002
Molecules 26 06629 g001 550
Figure 1. Molecular structures with atom labelling scheme of the 4-(chloromethyl)benzoate derivatives 3a–c, ORTEP plots with 50% probability level (in the case of 3c only one of the two symmetry independent molecules is shown).
Figure 1. Molecular structures with atom labelling scheme of the 4-(chloromethyl)benzoate derivatives 3a–c, ORTEP plots with 50% probability level (in the case of 3c only one of the two symmetry independent molecules is shown).
Molecules 26 06629 g001
Molecules 26 06629 g002 550
Figure 2. Molecular structure with atom labelling scheme of 4a. ORTEP plot with 50% probability level (only one of the two symmetry independent molecules is shown).
Figure 2. Molecular structure with atom labelling scheme of 4a. ORTEP plot with 50% probability level (only one of the two symmetry independent molecules is shown).
Molecules 26 06629 g002
Molecules 26 06629 g003 550
Figure 3. Structure of the rhenium complex fac-[Re(CO)34a]Br (Ortep-Plot with 50% probability level, any co-crystallized methanol molecules are omitted for clarity).
Figure 3. Structure of the rhenium complex fac-[Re(CO)34a]Br (Ortep-Plot with 50% probability level, any co-crystallized methanol molecules are omitted for clarity).
Molecules 26 06629 g003
Molecules 26 06629 sch003 550
Scheme 3. Synthesis of different phosphorous compounds 6–9 for structure elucidation of products obtained from the reaction of ligand 4b with (Et4N)2[Re(CO)3Br3].
Scheme 3. Synthesis of different phosphorous compounds 6–9 for structure elucidation of products obtained from the reaction of ligand 4b with (Et4N)2[Re(CO)3Br3].
Molecules 26 06629 sch003
Molecules 26 06629 g004 550
Figure 4. Comparison of 31P NMR spectra. (A): phosphane 6 (violet), (B): oxidized species 7 (green), and (C): phosphane complex 8 (red). All spectra were recorded in benzene-d6.
Figure 4. Comparison of 31P NMR spectra. (A): phosphane 6 (violet), (B): oxidized species 7 (green), and (C): phosphane complex 8 (red). All spectra were recorded in benzene-d6.
Molecules 26 06629 g004
Molecules 26 06629 g005 550
Figure 5. Molecular structure of the rhenium complex 9 (Ortep-Plot with 50% probability level, co-crystallized trichloromethane molecule is omitted for clarity).
Figure 5. Molecular structure of the rhenium complex 9 (Ortep-Plot with 50% probability level, co-crystallized trichloromethane molecule is omitted for clarity).
Molecules 26 06629 g005
Molecules 26 06629 g006 550
Figure 6. HPLC chromatograms (UV-/γ-traces) of ligand 4a (blue, tR = 15.2 min), the 99mTc-tricarbonyl precursor (orange, tR = 14.1 min), fac-[[99mTc]Tc(CO)34a]+ (green, tR = 20.2 min), and the non-radioactive Re-reference fac-[Re(CO)34a]Br (violet, tR = 19.8 min).
Figure 6. HPLC chromatograms (UV-/γ-traces) of ligand 4a (blue, tR = 15.2 min), the 99mTc-tricarbonyl precursor (orange, tR = 14.1 min), fac-[[99mTc]Tc(CO)34a]+ (green, tR = 20.2 min), and the non-radioactive Re-reference fac-[Re(CO)34a]Br (violet, tR = 19.8 min).
Molecules 26 06629 g006
Molecules 26 06629 g007 550
Figure 7. (Radio) HPLC chromatograms of ligand 4b (blue, tR = 15.2 min), the 99mTc-tricarbonyl precursor (gray, tR = 14.1 min), and the reaction mixture after radiolabeling (green).
Figure 7. (Radio) HPLC chromatograms of ligand 4b (blue, tR = 15.2 min), the 99mTc-tricarbonyl precursor (gray, tR = 14.1 min), and the reaction mixture after radiolabeling (green).
Molecules 26 06629 g007
Molecules 26 06629 sch004 550
Scheme 4. Future approach to use the indirect variant of the traceless Staudinger Ligation.
Scheme 4. Future approach to use the indirect variant of the traceless Staudinger Ligation.
Molecules 26 06629 sch004
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Mamat, C.; Jentschel, C.; Köckerling, M.; Steinbach, J. Strategic Evaluation of the Traceless Staudinger Ligation for Radiolabeling with the Tricarbonyl Core. Molecules 2021, 26, 6629. https://doi.org/10.3390/molecules26216629

AMA Style

Mamat C, Jentschel C, Köckerling M, Steinbach J. Strategic Evaluation of the Traceless Staudinger Ligation for Radiolabeling with the Tricarbonyl Core. Molecules. 2021; 26(21):6629. https://doi.org/10.3390/molecules26216629

Chicago/Turabian Style

Mamat, Constantin, Christian Jentschel, Martin Köckerling, and Jörg Steinbach. 2021. "Strategic Evaluation of the Traceless Staudinger Ligation for Radiolabeling with the Tricarbonyl Core" Molecules 26, no. 21: 6629. https://doi.org/10.3390/molecules26216629

APA Style

Mamat, C., Jentschel, C., Köckerling, M., & Steinbach, J. (2021). Strategic Evaluation of the Traceless Staudinger Ligation for Radiolabeling with the Tricarbonyl Core. Molecules, 26(21), 6629. https://doi.org/10.3390/molecules26216629

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