Enzymatic transhalogenation of dendritic RGD peptide constructs with the fluorinase†

The substrate scope of fluorinase enzyme mediated transhalogenation reactions is extended. Substrate tolerance allows a peptide cargo to be tethered to a 5’-chloro-5’-deoxynucleoside substrate for transhalogenation by the enzyme to a 5’-fluoro-5’-deoxynucleoside. The reaction is successfully extended from that previously reported for a monomeric cyclic peptide (cRGD) to cargoes of dendritic scaffolds carrying two and four cyclic peptide motifs. The RGD peptide sequence is known to bind upregulated αVβ3 integrin motifs on the surface of cancer cells and it is demonstrated that the fluorinated products have a higher affinity to αVβ3 integrin than their monomeric counterparts. Extending the strategy to radiolabelling of the peptide cargoes by tagging the peptides with [F]fluoride was only moderately successful due to the poor water solubility of these higher order peptide scaffolds although the strategy holds promise for peptide constructs with improved solubility.


Introduction
The fluorinase enzyme (E.C. 2.5.1.63)4][5][6] The fluorinase catalyses the combination of fluoride ion and S-adenosyl-L-methionine 1 (SAM) to generate 5′-fluoro-5′-deoxyadenosine 2 (FDA) and L-(S)-methionine 3 (L-Met) (Scheme 1A). 1,7We have recently demonstrated that the enzyme has a specificity tolerance at the 2-position of the adenine base. 5Replacement of hydrogen with an acetylene group at this position, as shown in Scheme 1B, gave the modified substrate ClDEA 4 which was an efficient substrate for transhalogenation in the presence of Se-adenosyl-L-selenomethionine (SeSAM). 7This transformation was utilised to prepare [ 18 F]FDEA [ 18 F]-6 under ambient aqueous conditions.The pendant acetylene moiety was then efficiently reacted with an azido-cRGD (cyclic arginyl-glycinylaspartyl) peptide using the CuAAC (Cu-catalysed alkyne-azide cycloaddition) click reaction, to generate a [ 18 F]-radiolabelled cRGD construct. 6ile two step strategies involving prosthetic groups remain widely used for radiolabelling of peptides and proteins with fluorine-18, 8 it is attractive to consider the development of straightforward, "last step" radiolabelling protocols.Such protocols need to be relatively rapid due to the short half-life of fluorine-18 (t 1/2 = 109.7 min), and be conducted under conditions that preserve the structural and functional integrity of the biomolecule. 9The poor nucleophilicity of aqueous [ 18 F]fluoride ion, the form most readily prepared on a cyclotron with an [ 18 O]H 2 O target, 10 often precludes direct labelling of biomolecules using a "last step" approach.To circumvent the limited reactivity of aqueous [ 18 F]fluoride, fluoride sequestering strategies have been explored.For example boron, 11 aluminium 12 and silicon 13 based methods have been developed in an effort to achieve "last step" radiolabelling of peptides by bioconjugation approaches which secure these elements to the peptide in advance.The strategies have proven successful and these technologies are being further developed, 14,15 but there are challenges as the conditions required to achieve fluoride ion sequestration require various combinations of low pH, organic solvents and heating.The fluorinase has the capacity to catalyse C-F bond formation at ambient temperature and near neutral pH.Within this context, we have previously demonstrated 5 a fluorinase enzyme-based system for "last step" radiolabelling of a small cRGD peptide 7.The peptide was tethered to a 5′-chloro-5′-deoxynucleoside substrate through a PEG linker prior to enzymatic radiolabelling with [ 18 F]fluoride, as shown in Scheme 1B.This proved successful and the radiolabelled construct [ 18 F]-8 was shown to be stable to defluorination in vivo (in a rat), and the cRGD moiety retained high affinity for α V β 3 integrins.In order to explore this approach further, we wished to examine whether the substrate scope at the C-2 position would extend to larger, more complex peptides.
The search for high affinity ligands for biological receptors has led to the development of imaging agents based on multivalent antibody fragments 16 or multivalent small peptides. 17ultimeric interactions play a key role in recognition events between biological ligands and receptors. 18Weak non-covalent interactions between monovalent ligands and individual recep-tors can be enhanced by attaching multiple ligands to a single scaffold. 19,20Incorporating the RGD (arginyl-glycinyl-aspartyl) tripeptide motif into cyclic pentapeptides gives cRGDs, a class of high affinity ligands for α V β 3 integrin, a cell surface protein identified as a biomarker of angiogenesis and strongly associated with malignant tumours. 21,22Multimeric cRGD peptides are validated constructs employed for PET imaging using either fluorine-18 or heavy metal PET isotopes. 19,23,24Usually the cRGD peptide is conjugated to a metal chelating group for co-ordination to radiometals, often at the final step in the synthesis of such a tracer.6][27][28] However, two multimeric cRGDbased radiotracers have been reported recently, synthesised using a "last step" radiofluorination protocol.These are the aluminium chelates, [ 18 F]alfatide I 14,29 and [ 18 F]alfatide II 15,30 which were prepared using a kit-like 31 methodology and they have been evaluated in humans. 14,15][34][35] Herein, we report our initial results on "last step" fluorination of structurally complex multimeric cRGD peptides with the fluorinase enzyme.A primary objective at this stage was to establish if the fluorinase could tolerate higher molecular weight cargoes attached to the C-2 position of the adenine base.Although successful biotransformations could be achieved with fluoride, it became apparent that reduced solubility of higher molecular weight substrates proved challenging for efficient radiolabelled experiments, where the fluoride-18 concentrations are necessarily low.

Synthesis of di-and tetra-alkynes for a CuAAC reaction
A multimeric peptide substrate for the fluorinase requires an enzyme recognition/reaction site attached to a linker to extend the peptide cargo away from the surface of the enzyme.In order to assemble such a substrate, we envisaged a strategy similar to that used for the synthesis of 5 and 7 (Scheme 1B). 5 A Sonogashira coupling was identified for coupling a 2-iodonucleoside to an alkynyl-PEG linker, which had been prefunctionalised with either a protected dialkyne or a protected tetraalkyne.Following deprotection to the free terminal alkynes, a multi-CuAAC reaction with an azide bearing cRGD peptide, similar to that reported by Liskamp et al., 34 should furnish multimeric cRGD substrates.The synthesis of the protected alkynes and halogenated 2-iodo-nucleosides is summarised in Schemes 2 and 3 respectively.
The dendrimeric core was prepared by dialkylation of methyl 3,5-dihydroxybenzoate 9 with propargyl bromide (Scheme 2). 34The acetylenes of the newly installed propargylic ethers were then silylated to furnish ester 10.Ester 10 was then reduced to the corresponding benzylic alcohol and periodinane oxidation 36 generated aldehyde 11 which was used directly in future reductive amination reactions without further purification.The linker was prepared by monoalkylation of tetraethylene glycol 12 with propargyl chloride.The free hydroxyl was converted to the corresponding tosyl ester and then nucleophilic displacement by azide gave the corresponding azido-PEG.Reduction under Staudinger conditions 37 gave amine 14.With both aldehyde 11 and amine 14 in hand, attention turned to coupling the linker to the multimeric core.
The reductive amination after condensation of amine 14 with aldehyde 11 38 generated both the mono-and di-alkylated products 15 and 16.Separation of the two products by column chromatography gave the di-alkyne linker-core assembly 15 in 35% yield (based on aldehyde 11), while the terta-alkyne linker-core assembly 16 was formed in 21% yield (based on aldehyde 11).
With these linker-core assemblies in hand, attention turned to the synthesis of the iodinated coupling partners.2-Iodoadenosine 17, 5 was treated with thionyl chloride in pyridine, followed by ammonia in aqueous methanol to furnish the 5′-chloro-5′-deoxy adenosine 18, as shown in Scheme 3. 39 For the synthesis of the fluorinated nucleoside, 2-iodoadenosine 17 was protected as its 2′,3′-acetonide 19 before being fluorinated by the action of TsF and TBAF in refluxing THF.Deprotection to generate 20 was achieved in good yield with catalytic anhydrous TsOH in refluxing MeOH. 40onogashira couplings [41][42][43] of the halogenated 2-iodonucleosides 18 and 20 with acetylenes 15 and 16 were now

Evaluation of multimeric RGDs as substrates for the fluorinase
With the multimeric cRGD fluorinase substrates in hand, enzyme catalysed transhalogenation reactions were explored.ClDEA-PEG-(RGD) 2 30 (40 µM) was added to a mixture of L-SeMet (75 µM) and potassium fluoride (50 mM) in a 1 mL volume to explore the transformation illustrated in Fig. 1A.The reaction was initiated by addition of the fluorinase (1 mg mL −1 ).
Aliquots of the reaction were removed for HPLC assay over a 4 h period in order to assess conversion.The HPLC traces in Fig. 1B show the time-dependent appearance of the product at t R = 8.2 min, a peak which was identified as FDEA-PEG-(RGD) 2 31 by LC-MS comparison with the synthetic reference previously prepared.The mass spectra of the HPLC peaks corresponding to the multimeric 5′-chloro substrates and 5′-fluoro products are shown in Fig. 1C and D respectively.The peaks for the product contain multiply charged ions of 31 3+ , and [M + 4H] 4+ respectively), confirming its identity as FDEA-PEG-(RGD) 2 31.
A series of control experiments, where the reaction was conducted in the absence of either enzyme or L-SeMet, did not result in any conversion of 30 to 31, confirming an enzyme catalysed process.In the absence of fluoride, the consumption of 30 was observed, however, no fluorinated product was produced.This result is consistent with the generation of a SeSAM intermediate, which cannot be converted to product 31 in the absence of fluoride.
A similar analysis was conducted for the enzymatic reaction with ClDEA-PEG-(RGD) 4 32, as illustrated in Fig. 2A.ClDEA-PEG-(RGD) 4 32 (20 µM) was added to a mixture of L-SeMet (75 µM) and potassium fluoride (50 mM), in a total volume of 1 mL.The reaction was initiated by the addition of the fluorinase (1 mg mL −1 ) and monitored over 4 h.The resultant HPLC profile (Fig. 2B) indicated that 32 was consumed, while a new peak, at t R = 9.2 min, steadily increased throughout the assay.The retention time of this new peak was identical to that of synthetic FDEA-PEG-(RGD) 4 33, and its identity was further confirmed by LC-MS.
Tolerance of the fluorinase to these large multimeric peptides suggests that the C-2 position of a chlorinated nucleoside represents a site for the attachment of a diverse range of peptide cargos for use in enzymatic fluorination.The constructs explored here consist of 10 and 20 amino acids, and they were efficiently fluorinated under neutral aqueous conditions.With the knowledge that the multimeric constructs were suitable substrates for fluorinase catalysed transhalogenation, it was of interest to evaluate the effect the multiple RGD motifs on binding affinity to α V β 3 integrin.

Binding affinities of RGD multimers to immobilised
The binding affinities of the fluorinated multimers 31 and 33 to α V β 3 integrin were determined by their ability to displace a biotin-labelled cRGD peptide from immobilised α V β 3 integrin in an ELISA assay. 45The data are shown in Table 1 including the IC 50 values of some reference peptides. 5,6The ELISA data indicate that for this series the multimers have a progressively higher integrin affinity (lower IC 50 ) as the number of cRGD ligands increases e.g., monomeric 8 (IC 50 = 74 nM), dimeric 31 (IC 50 = 60 nM) and tetrameric 33 (IC 50 = 24 nM).The IC 50 values measured for the multimeric constructs are also lower than the parent azido-peptide 29 (IC 50 = 90 nM) and the multimeric cRGD compounds have significantly lower IC 50 values compared with reference compounds containing linear RGDcontaining peptides.
Going from one to two cRGD motifs resulted in a marginal increase in binding affinity however progression to a tetramer showed a significant decrease in IC 50 and gave a compound with the highest affinity of those tested.The linker between the two or four cRGD units is not so extended and steric constraints will likely prevent simultaneous multivalent binding of cRGDs to more than one surface site.Instead the increased affinity is most likely due to an increased effective molarity at the surface. 46Some multimeric constructs reported in the literature have IC 50 values in the 1-100 nM range, 24 similar to this study although a direct comparison of different assays and dissimilar peptide motifs make accurate comparison difficult.

Enzymatic radiolabelling of multimers with fluorine-18
The ability of the fluorinase to employ these large peptide assemblies of 10 or 20 amino acids as substrates opened up prospects of labelling these peptides with [ 18 F]fluoride for PET.Radiolabelling trials of ClDEA-PEG-(RGD) 2 30 (0.04 mM) was initially investigated using similar reaction conditions to the assays described with [ 19 F]fluoride, but with an aqueous [ 18 F]fluoride solution generated on the cyclotron.The enzyme  33  24 ± 18 0.011 a Affinities for these constructs were previously reported, and were measured using an identical assay.
concentration was increased from 1 mg mL −1 (29 µM) to 10 mg mL −1 (290 µM), as it had previously been shown that high enzyme concentrations improves conversions in radiolabelling experiments. 47Notably [ 18 F]fluoride ion concentrations are necessarily very low in the pico-molar range.Radiochemical incorporations (RCI) were found to be low under these conditions, and only traces of the product, [   By contrast, ClDEA-PEG-(RGD) 2 30 and ClDEA-PEG-(RGD) 4 32 were found to be poorly soluble in water, and concentrations of 0.6 mM were not achievable in buffer.The upper concentration limit for the peptides was found to be ∼0.3 mM.ClDEA-PEG-(RGD) 2 30 and ClDEA-PEG-(RGD) 4 32 were incubated in separate experiments with L-SeMet (0.08 mM) and [ 18 F]fluoride in water (138 µL-160 µL, 15.2-35.4MBq).Some precipitation of the substrate was observed upon addition of aqueous [ 18 F]fluoride.The reaction mixtures were incubated at 37 °C for 30 minutes, and then the enzyme was heat denatured and the mixture centrifuged.A sample of the clarified supernatant from each reaction was analysed by HPLC, and the resultant HPLC radio-chromatograms are shown in Fig. 3.
Radio-HPLC of the two reaction mixtures (Fig. 3A and B) revealed the presence of the respective [ 18 F]-products, along with a broad peak (t R = 2.2 min), identified as [ 18 F]fluoride. 49he peak at t R = 13.7 min in Fig. 3A was identified as [ 18

Conclusions
Enzymatic transhalogenations using the fluorinase was explored with relatively complex di-and tetra-cRGD constructs.This exploited the previously identified substrate tolerance at C-2 of the adenine base in the substrate motif.The multimeric assemblies, ClDEA-PEG-(RGD) 2 30 and ClDEA-PEG-(RGD) 4 32, were assessed as fluorinase substrates and both were found to undergo efficient enzyme-catalysed transhalogenations.The multimers also showed higher affinities to immobilised α V β 3 integrins, which opened up the prospect of using them as cancer imaging agents in [ 18 F]-radiolabelled form.This proved only partially successful due to the relatively poor solubility of the multimeric constructs and low [ 18 F]-fluoride concentrations.It follows that the development of this approach to larger peptides or proteins will depend on using more highly soluble peptide substrates in buffer.The peptides used here are 'click' derived with low solubility, however the approach should prove more useful with natural peptides and antibodies which have evolved high water solubility.
F]-31 by spiking and comparison of the retention time to that of a [ 19 F]-reference sample.Similarly the peak at t R = 10.1 min in Fig. 3B was identified as [ 18 F]-33 relative to a [ 19 F]-reference.[ 18 F]-FDA [ 18 F]-2 was also observed in this experiment, which must arise as a consequence of low levels of L-AdoMet (SAM) bound to the enzyme and residual from the purification process.This outcome only arose in the reactions of low conversion.Reactions were also conducted with the addition of DMSO 1% (v/v) as a co-solvent, however this did not improve the radiochemical conversions and extending the reaction time or increasing the concentration of L-SeMet had no significant effect.Therefore despite the efficient conversions observed in the "cold" experiments at high [ 19 F]fluoride ion concentrations, low [ 18 F]fluoride concentrations require to be compensated for by high substrate concentrations, and this limited the efficiency of the radiolabelled experiments.

Table 1
IC 50 values of selected RGD-containing peptides measured as the ability to compete with c(RGDfK[PEG-PEG-biotin]) for binding to immobilised α V β 3 integrin.Results are the average ± standard error (s.e.) from three independent experiments, each performed in triplicate.Q is the normalised affinity of the peptides, referenced to GRGDSPK 18 F]FDEA-PEG-(RGD) 2[18 F]-31 were observed by radio-HPLC.Optimisation trials were carried out with ClDA 4 to [ 18 F]FDA [ 18 F]-2 as a substrate, and the data suggest that an important factor for improving radiochemical incorporations is substrate concentration.A RCI of greater than 90% was observed when ClDA 4 was incubated at a concentration of 0.6 mM, compared with a 10% RCI at 0.04 mM (see ESI †).