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

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.


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-clickconjugation 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 nat Re complexes were synthesized as non-radioactive reference and the procedure was transferred to radiolabeling chemistry using technetium-99m. Scheme 1. Direct and indirect approach of the traceless Staudinger Ligation.

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 phosphanecontaining 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 (Et 4 N) 2 [Re(CO) 3 Br 3 ] as Re-tricarbonyl source [26][27][28] to the desired rhenium complexes fac-[Re(CO) 3 4a]Br and fac-Re(CO) 3 4b]Br, which are intended to be used as non-radioactive references for later radiolabeling with 99m Tc.
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 Figures 1 and 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]. 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 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).

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 (Et 4 N) 2 [Re(CO) 3 Br 3 ] in methanol (last step in Scheme 2) to yield the desired rhenium complex fac-[Re(CO) 3 4a]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 1   In contrast, when reacting 4b with (Et 4 N) 2 [Re(CO) 3 Br 3 ] under the conditions described for fac-[Re(CO) 3 4a]Br, a complex mixture of different complexation products was obtained, which were difficult to separate. The non-radioactive reference complex fac-[Re(CO) 3 4b]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 31 P 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.

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. 31 P 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 (Et 4 N) 2 [Re(CO) 3 Br 3 ] under argon giving a complex mixture, too. One of them was identified as complex 8. A similar complex was described with PPh 3 as ligand [34]. 31 P 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, indi-cating 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, (Et 4 N) 2 [Re(CO) 3 Br 3 ] 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) 3 4a]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) 3 4a]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).

Radiolabeling with Technetium-99m
For the following radiolabeling with technetium-99m, a two-step procedure was applied, first to generate the 99m Tc(CO) 3 species from the 99m Tc-pertechetate solution using a tricarbonyl kit and secondly to perform the final complexation with the DPA-containing ligands [36].  3 ] + (t R = 14.1 min). Two by-products were found, either at t R = 4.2 min (9%) or at t R = 16.4 min (4%), which are identified as backoxidized TcO 4 and an unknown 99m Tc-species, respectively. Both by-products were removed after simple purification using a LiChrolut RP-18 cartridge. The identification of the 99m Tc-complex was confirmed with the respective rhenium complex fac-[Re(CO) 3 4a]Br (t R = 19.8 min). Additionally, the UV signal of ligand 4a was found at t R = 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 t R = 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 99m Tc-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 (Et 4 N) 2 [Re(CO) 3 Br 3 ]. 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.

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) 3 4a]Br with a Bruker-Nonius Apex Kappa-II at T = −150 • C. Graphitemonochromated Mo K α radiation (λ = 0.71073 Å) was used in all cases. The structures were solved by direct methods and refined against F 2 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 O 2 C-C 6 H 4 I moiety of one of the two independent molecules is disordered with two different orientations. Furthermore, crystals of 9 contain co-crystallized CHCl 3 molecules and those of fac-[Re(CO) 3 4a]Br cocrystallized 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) 3 4a]Br.

2-Iodophenyl 4-(chloromethyl)benzoate 3a.
KO t Bu (594 mg, 5.29 mmol) and 2iodophenol (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 NH 4 Cl 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 Na 2 SO 4 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.
[ 99m Tc]TcO 4 (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-[ 99m Tc(CO) 3 (H 2 O) 3 ] + (analytical radio HPLC: t R = 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.

Conclusions
A proof-of-concept study was carried out to use the direct variant of the traceless Staudinger ligation for radiolabeling purposes with a 99m Tc-tricarbonyl core. For this purpose, phosphane 4b with benzoate moiety was prepared and functionalized by introducing a dipicolylamine residue as tridentate ligand. Reactions with (Et 4 N) 2 [Re(CO) 3 Br 3 ] 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-[ 99m Tc(CO) 3 (H 2 O) 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 99m Tc-tricarbonyl core.
Supplementary Materials: The following are available online, NMR spectra of compounds, radiolabeling procedure and radio HPLC chromatograms.