Orthogonal synthesis of a versatile building block for dual functionalization of targeting vectors

: Dual functionalization of targeting vectors, such as peptides and antibodies, is still synthetically challenging despite the increasing demand for such molecules serving multiple purposes (i.e., optical and nuclear imaging). Our strategy was to synthesize a versatile building block via the orthogonal incorporation of chemical entities (e.g., radio-nuclide chelator, ﬂ uorescent dye, cytotoxic drugs, click handle, and albumin binder) in order to prepare various dual functionalized biovectors. The functional groups were introduced on the building block using straightforward chemical reactions. Thus, an azidolysine and a biogenic lysine were installed into the building block to allow the coupling of the second functional group and the regioselective conjugation to the biovector via the strain-promoted azide – alkyne cycloaddition, while the ﬁ rst functional group was inserted during the solid-phase peptide synthesis. To extend the applicability of the building block to large bio-molecules, such as antibodies, a DBCO-maleimide linker was clicked to the azidolysine to present a maleimide group that could react with the exposed sulfhydryl groups of the cysteine residues. To exemplify the possibilities o ﬀ ered by the building block, we synthesized two dual-functionalized compounds containing a 2,2 ′ ,2 ″′ ,2 ‴ -(1,4,7,10-tetraazacyclodode-cane-1,4,7,10-tetrayl) tetraacetic acid chelator and an albumin binder ( 4a ) to extend the blood half-life of radiolabeled bio-vectors or a click handle ( 4b ) to enable the late-stage click reaction; 4a and 4b were conjugated to a model cyclic peptide bearing a short thiolated linker at the N -terminal position, in a single step via the thiol – maleimide Michael addition. Both dual-functionalized peptides, 9a and 9b , were obtained rapidly in high chemical purity ( > 95%) and labeled with [ 111 In]InCl 3 . Both radiopeptides showed good stability in mouse serum and PBS bu ﬀ er.


Introduction
In the past decade, peptides and antibodies have gained a lot of attention for targeted imaging and therapy of different types of cancers [1][2][3].The low molecular weight of peptides confers multiple advantages compared to larger biomolecules, such as antibodies.For instance, peptides are cleared faster from the blood circulation, they present faster pharmacokinetics (PK), and they have better penetration into the malignant tissue [4].However, antibodies are known to exhibit a remarkable binding selectivity and affinity to the targeted antigens.Therefore, peptides and antibodies are getting more and more used for a panoply of applications for which chemical modifications of these biomolecules are required.Several functional groups have been recently considered to modify these biovectors, such as imaging probes (i.e., radiometal chelator and fluorescent dye) for non-invasive molecular imaging and fluorescenceguided surgery, cytotoxic drugs for small molecule-or antibody-drug conjugates, PK modifiers (i.e., albumin binder and cleavable linker) to improve their PK.The trend was initially to develop a mono-functionalized compound for each application.However, due to the rise of combined applications (i.e., multimodality imaging, targeted therapy, and companion diagnostic) and cost-effectiveness, the dual functionalization of biomolecules is getting more and more popular.
The introduction of these functional groups into these biomolecules was usually achieved via the amidation of lysine residues present in the peptide or protein sequence [5,6].However, this approach is either synthetically challenging or lacks regioselectivity.For peptide vectors, it is possible to select appropriate protecting groups during the solid-phase peptide synthesis (SPPS) to thwart these limitations.However, few functional groups, such as fluorescent cyanine dyes or trans-cyclooctene (TCO) click handles, are too sensitive to bear the harsh conditions occurring during the synthesis (e.g., acidic and basic conditions) [7,8].Therefore, alternative synthetic routes are required to functionalize these biovectors under friendly conditions.We recently described the synthesis of a multifunctional single-attachment-point to prepare dual fluorescent/nuclear-labeled biovectors [9,10].Herein, we report the applicability of a building block allowing the dual functionalization of biovectors.The synthetic approach was based on the chemoselective attachment of the functional groups using straightforward chemical reactions, such as the strain-promoted azide-alkyne cycloaddition (SPAAC), the thiol-maleimide Michael addition or the inverse electron-demand Diels Alder (IEDDA) reaction.These reactions are known to be orthogonal, to occur under biologically friendly conditions, and the end-product is stable [11].
As a proof-of-concept, we synthesized two building blocks, 4a and 4b, functionalized with a 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) chelator for complexation of imaging and therapeutic radionuclides (e.g., indium-111, gallium-68, lutetium-177, and actinium-225).Additionally, an azidolysine and a lysine were incorporated into the building block to provide two attachment points dedicated to the coupling of the second functional group and conjugation of the building block to the biovector.The side-chain of the lysine was used to attach an albumin-binding moiety (4a) or a TCO (4b), whereas a dibenzocyclooctyne (DBCO)-maleimide linker was clicked to the azide group.The linker was inserted to increase the distance between the building block and the biovector and to provide a maleimide group that could react selectively with the cysteine residues present in antibodies.To demonstrate the versatility of our dual-functionalized building block, 4a and 4b were conjugated to a model cyclic peptide.The final compounds, 9a and 9b, were radiolabeled with [ 111 In]InCl 3 , and their stability in PBS buffer and mouse serum was investigated.

General information
The chemicals and solvents were obtained from commercial suppliers and used without further purification unless specified.SPPS, based on the Fmoc strategy, was performed manually using manual reaction vessels (Chemglass, Vineland, United States).DOTA-NHS-ester was acquired from Macrocyclics (Plano, TX, USA) and [ 111 In]InCl 3 (370.0MBq/mL in HCl, pH 1.5-1.9)was purchased from Curium (Petten, The Netherlands).High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses were acquired on an Agilent 1260 Infinity II LC-MS system (Middelburg, The Netherlands).Instant thin-layer chromatography (iTLC) analysis was performed on plates of silica gel-impregnated glass fiber sheets.The plates were eluted with sodium citrate (0.1 M, pH 5).They were analyzed using a Brightspec bSCAN radio-chromatography scanner (Antwerp, Belgium) supplied with a sodium iodide detector.The radioactive samples were counted using a Perkin Elmer Wizard 2480 gamma counter (Waltham, MA, USA).Radioactivity determination was performed using the VDC-405 dose calibrator (Comecer, Joure, The Netherlands).Quality control of the compounds was carried out using an analytical column from Agilent (Poroshell 120, EC-C18, 2.7 μm, 3.0 × 100 mm) under a gradient elution of acetonitrile (ACN) from 5 to 100% in H 2 O, containing 0.1% formic acid (FA) using a flow rate of 0.5 mL/min for 8 min.Purification of compounds 3, 4a, and 9a was performed using a preparative Agilent 1290 Infinity II HPLC system and an Agilent 5 Prep C18 preparative column (50 × 21.2 mm, 5 μm) with a gradient elution of ACN (5-95% in H 2 O containing 0.1% FA for 8 min) using a flow rate of 10 mL/ min for 10 min.Purification of compounds 8, 4b, and 9b was carried out using a semi-preparative Alliance HPLC system from Waters (Etten-Leur, The Netherlands).Purification of compound 8 was performed using a semi-preparative column (Phenomenex C-18 Luna ® , 250.0 × 10.0 mm, 5 μm) with an isocratic elution of ACN (35% in H 2 O containing 0.1% trifluoroacetic acid (TFA) using a flow rate of 3 mL/min for 30 min.Purification of compounds 4b and 9b was performed using the C18 Luna ® semi-preparative column with a gradient elution of ACN (10-90% in H 2 O, containing 0.01% TFA) using a flow rate of 3 mL/min for 30 min.Quality control of the radiolabeled compounds, as well as the stability studies, were carried out on a Waters Acquity Arc ultra-high performance liquid chromatography (UHPLC) system equipped with a diode array detector and a radio-detector from Canberra (Zelik, Belgium) using a Phenomenex C-18 Gemini ® analytical column (250.0 × 4.6 mm, 5 μm) with a gradient elution of ACN (5-95% in H 2 O, containing 0.1% TFA) using a flow rate of 1 mL/min for 30 min.Loading of Fmoc-Lys(Boc)-OH (0.5 mmol, 2 equiv.)onto the Rink Amide 4-methylbenzhydrylamine (MBHA) resin (370 mg; average loading capacity, 0.678 mmol/g) was carried out using solutions of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU; 0.25 M, 2 mL, 2 equiv.)and N,N-diisopropylethylamine (DIPEA; 0.50 M, 2 mL, 4 equiv.) in DMF.The beads were shaken for 2 h at room temperature (rt).Capping of the beads was achieved using a mixture of acetic anhydride (1.18 mL, 12.5 mmol, 50 equiv.)and 1 M DIPEA (2.19 mL, 12.5 mmol, 50 equiv.)for 30 min at rt.The Nterminal Fmoc protecting group was removed by treatment of the resin with a solution containing 20% piperidine in DMF (6 mL) for 13 min at rt. Fmoc-Lys(N 3 )-OH (198 mg, 0.5 mmol, 2 equiv.)was coupled following the protocol described earlier.
The resin was treated twice with the cleavage cocktail, and all liquid phases were pooled.TFA was removed by air stream, and the peptide was precipitated using ice-cold diethyl ether and collected by centrifugation.A second treatment of the crude product with TFA/DCM (2 mL, v/v = 1:1) and triethylsilane (51 μL, 0.32 mmol, 2.5 equiv.)for 30 h at rt was carried out to complete the removal of the tBu protecting groups.The solvents were evaporated under reduced pressure.The compound was treated with ice-cold diethyl ether for precipitation and collected by centrifugation.The residue was dissolved in H 2 O/ACN (3 mL, v/v = 1:1) and purified by semi-preparative HPLC.Pure compound 8 was obtained as a white solid (32.6 mg, 9.5%).Analytical retention time: t R = 3.9 min (Figure S4a).S4b).

Stability studies in PBS buffer and mouse serum
The 111 In-labeled peptides (∼0.3 MBq) were incubated in either PBS (250 μL) or mouse serum (125 μL) (Merck, Haarlerbergweg, The Netherlands) at 37°C.The inertness of the radiopeptides was checked at 1 and 3 h post incubation.Precipitation of the proteins in mouse serum was achieved by the addition of an equal volume of ACN to the mixture.The vial was vortexed and centrifuged at 10,000 rpm for 20 min.Stability studies in both media were monitored by radio-HPLC (Figures S8 and S9).

Results and discussion
Our synthetic approach aimed at the design of a building block allowing the incorporation of two different functional groups into the chemical structure of peptide-based biovector.The building block featured three different attachment points.A DOTA chelator was first introduced during SPPS since the chelator is not sensitive to the harsh reaction conditions experienced during the synthesis, such as the strong acidic conditions employed during the cleavage of the peptide from the resin.The DOTA chelator was chosen due to its clinical relevance and its capability to complex a broad variety of radioisotopes.However, other chelators (e.g., NOTA, DFO) could have been coupled to the building block [13].Then, an azidolysine and a natural lysine residue were introduced into the building block to offer two additional attachment points.The presence of the two different attachment points warranted the orthogonal attachment of the building block to the biovector and the incorporation of the second functional group.The SPAAC click reaction was employed with the azidolysine due to its selectivity, favorable PK, and orthogonality allowing the obtention of the desired compounds under friendly conditions [14].The second functional group was introduced on the building block via an amide formation with the remaining lysine.Our synthetic route confirmed that the desired products could be obtained in the liquid phase and allowed the incorporation of sensitive functional groups at the last step of the synthesis.Several investigations have demonstrated that the incorporation of two different functional groups can be performed by coupling onto lysine residues directly inserted into the amino acid sequence of a peptide [5,6,15].However, the success of this approach is mainly depending on the presence of other lysine residues in the original peptide sequence.Moreover, regioselectivity of the introduction of the functional groups on the biovector is sparsely achieved, unless different protecting groups for the side-chain amino groups of the lysine residues are considered during the synthesis.However, this approach significantly complicates the synthetic pathway, and the deprotection conditions are not always compatible with the inserted functional groups (i.e., removal of an ivDde-protecting group with hydrazine and groups containing a carbonyl function).Our chemical approach overcomes these limitations by allowing the orthogonal incorporation of the functional groups via straightforward chemical reactions, known to occur in smooth reaction conditions and with high chemical yields.Furthermore, the regioselective attachment of the building block on the biovector enables to selection of a position at which the chemical modification has less influence on the biochemical properties of the parent peptide.
The orthogonality of the building block was confirmed by performing a SPAAC click reaction with a DBCO-maleimide linker prior to its conjugation to the biovector.We decided to add a linker to avoid the steric hindrance that could be caused by the bulky building block.The linker provides more space between the building block and the peptide, allowing better coupling efficiency and less effect on the binding properties of the peptide.However, the addition of the linker would likely not be required with larger biovectors, such as antibodies and proteins.Furthermore, the derivatization of the building block with a maleimide group enhances its utility, as it enables the conjugation of the building block to antibodies or antibody fragments.Indeed, those large biomolecules are usually functionalized with a chelator, a fluorescent dye, or a drug on a cysteine via the thiol-maleimide Michael addition.Therefore, the maleimide version of our building block could facilitate the dual functionalization of these large and sensitive biovectors [16].
Versatile MSAP agent for multifunctionalized biovectors  5 Compound 3 was a key intermediate in our synthetic approach due to its ability to be derivatized with a broad variety of functional groups, such as fluorescent dyes (IRDye800CW, Cy5 derivatives), pharmacokinetic modifiers, and cytotoxic drugs.A large library of compounds could therefore be obtained by the conjugation of various chemical entities to the free lysine.As a proof-of-concept, we decided to functionalize 3 with either an albumin-binding moiety or a click handle.The 4-(p-iodophenyl)butanoic acid was selected as it has been widely applied to extend the blood circulation of radioligands (e.g., prostate specific membrane antigen, folic acid, and DOTA-TATE) by binding to albumin with a good affinity [17][18][19][20].The TCO group was chosen due to its high sensitivity towards acidic conditions, requiring flexibility in the synthesis of the building block and its incorporation into 3 at the last step of the synthesis [21].TCO was incorporated into the building block to facilitate pretargeting applications [22].Compounds 4a and 4b were obtained by the SPAAC click reaction between the azido intermediate 2 and the DBCO-maleimide linker, followed by the conjugation of the activated succinimidyl AB-NHS and TCO-NHS esters (Scheme 1).This route provided 4a and 4b with 50 and 29% yield, respectively, after HPLC purification.The purity of 4a and 4b was determined by analytical HPLC and was found to be higher than 97 and 98%, respectively.
As mentioned earlier, the thiol group of the cysteine residues is commonly used to conjugate diverse functional groups to antibodies.Therefore, to mimic this situation, we attached a short thiolated linker at the N-terminal position of our model cyclic peptide JR11.We chose to demonstrate the application of our building block on a peptide since, like antibodies, peptides are constituted of amino acids but their chemistry is less challenging.Compound 8 was obtained by Fmoc-based SPPS on a Rink Amide MBHA resin, followed by cyclization of the peptide on resin by treatment with Tl(TFA) 3 , Fmoc deprotection, conjugation of the succinimidyl ester of (tritylthio)acetic acid at the N-terminus, cleavage, and deprotection of the side-chain protective groups [12].Compound 8 was obtained in 9.5% yield after HPLC purification with a purity higher than 95% (determined by analytical HPLC; Scheme 2).
The final dual-functionalized compounds 9a and 9b were prepared by the conjugation of either 4a or 4b with 8 (Scheme 2).The thiol-maleimide Michael reaction was performed in a mixture of H 2 O/ACN, and the conversion of compound 8 into the desired compounds 9a and 9b was observed in only 30 min at 37°C.During the reaction, we added a co-solvent, namely ACN, to improve the solubility of the peptides and the building block, but this reaction can typically be performed under biologically friendly conditions.Compounds 9a and 9b were obtained 76 and 35% yield, respectively, after HPLC purification.The difference in chemical yields might be explained by the difference in the purification methods and the need to use less acidic solvent to purify 9b in order to avoid its isomerization into the cis-isomer.It has been shown that cis-cyclooctene (CCO) is less reactive than TCO toward tetrazines during the IEDDA reaction [21].9a and 9b were characterized by MS, and their purity, determined by analytical HPLC, was higher than 96 and 95%, respectively.
Subsequently, 9a and 9b were radiolabeled with [ 111 In] InCl 3 using gentisic and ascorbic acid.Both substances are quenchers, known to protect the radiolabeled peptides against radiolysis [23].It has been reported that ascorbic acid reacts with the main radicals formed by gentisic acid and reduces them into the original gentisic acid found in the initial dosage form [24]. Kolliphor, a well-known solubilizer and emulsifier, was added to the radiolabeling   In-9b at 3 h).It might be explained by the presence of the carbamate bond between the TCO and the side chain of the lysine residue.Indeed, it has been previously reported that the carbamate linkage is sensitive to radiolysis [25].
The distribution coefficient was determined by the shake-flask method.Both radiopeptides displayed a LogD 7.4 value higher than the parent peptide DOTA-JR11 (−0.59 and −0.66 for [ 111 In]In-9a and [ 111 In]In-9b vs −2.5 for [ 177 Lu]Lu-DOTA-JR11) [26].The introduction of lipophilic chemical groups, such as DBCO, AB, and TCO in the chemical structure of the DOTA-JR11 logically affected the hydrophilicity of the final molecules.
In the previous examples, a chelator, an albuminbinding moiety, and a click handle were introduced into the dual-functionalized building block.However, the presence of the azide and amine groups on the molecule confers full orthogonality to attach selectively various chemical entities.We showed that the insertion of a maleimide into our building block offers the opportunity to functionalize other biovectors, such as antibodies, in a single step through the thiol-maleimide Michael reaction.The reaction proceeded quickly in biologically friendly conditions (aqueous medium and physiological temperature), allowing the radiolabeling of the building block in harsh reaction conditions (i.e., heating in acidic solution) before its conjugation to a fragile biomolecule [27].

Conclusion
In our study, we presented a versatile approach allowing the orthogonal incorporation of different functional groups (DOTA chelator, AB, and TCO) into a building block that can be attached to biovectors in a single step via a SPAAC or thiol-maleimide Michael reaction.Our proof-of-concept was demonstrated by the preparation of two different dual-functionalized building blocks, 4a and 4b, which were coupled to the free sulfhydryl group of a cyclic peptide.Both final compounds, 9a and 9b, were obtained in very high chemical purities, and successfully radiolabeled with [ 111 In]InCl 3 .We confirmed that the chemical remodeling of the peptides did not hamper the complexation of the radionuclide by the chelator, and high stability of the final radiopeptides was demonstrated in PBS and mouse serum.Consequently, this synthetic approach could be very valuable for the functionalization of radiolabeled biovectors.
O, 2 h, rt and (ii) TFA/DCM, 30 h, rt, 9.5%; (e) 4a or 4b, H 2 O/ACN, 37°C, 76% and 35%, respectively.(f) [ 111 In]InCl 3 , 9a or 9b, gentisic/ascorbic acid, sodium acetate, 20 min, 90°C, 96.3, and 96.1% respectively.Versatile MSAP agent for multifunctionalized biovectors  7 mixture in order to improve the solubility of the radiolabeled peptides and reduce their stickiness.Radiolabeling of both compounds with [ 111 In]InCl 3 , using a molar activity of 50 MBq/nmol, was successful and comparable to what has been recently published with other DOTA-JR11 analogs [5].It confirms that the chemical modifications did not hamper the efficiency of the complexation of 111 In 3+ by the DOTA chelator.The RCY and RCP of both radiopeptides are presented in Table 1.[ 111 In]In-9a and [ 111 In]In-9b were obtained in 96.3 and 96.1% RCY and 94.9 and 86.0%RCP, respectively.Next, we determined the stability of both radiopeptides in PBS (pH 7.