On-resin Diels–Alder reaction with inverse electron demand: an efficient ligation method for complex peptides with a varying spacer to optimize cell adhesion

Solid phase peptide synthesis (SPPS) is the method of choice to produce peptides. Several protecting groups enable specific modifications. However, complex peptide conjugates usually require a rather demanding conjugation strategy, which is mostly performed in solution. Herein, an efficient strategy is described using an on-resin Diels-Alder reaction with inverse electron demand (DARinv). This method is compatible with the standard Fmoc/tBu strategy and is easy to monitor. As a proof of concept a titanium binding peptide was modified with a cyclic cell binding peptide (RGD) by DARinv on a solid support applying different tetrazines and alkenes. The generated bulky DARinv linkers were employed to act as the required spacer for RGD mediated cell adhesion on titanium. In vitro studies demonstrated improved cell spreading on DARinv-conjugated peptides and revealed, in combination with molecular dynamics-simulation, new insights into the design of spacers between the RGD peptide and the surface. Performing the DARinv on resin expands the toolbox of SPPS to produce complex peptide conjugates under mild, catalyst free conditions with reduced purification steps. The resulting conjugate can be effectively exploited to promote cell adhesion on biomaterials.


Introduction
Peptide conjugates are perfect tools to selectively address various targets in medical applications and analytical investigations. Solid phase peptide synthesis (SPPS) provides a variety of orthogonal protecting groups, which facilitate the modification of peptides with fluorophores, drugs, carbohydrates, fatty acids or reporter molecules. [1][2][3] Nevertheless, the efficient synthesis of multifunctional peptides with several complex bioactive moieties such as cyclic peptides is still challenging. Chemoselective click reactions offer the possibility to selectively ligate biomolecules without the use of further protecting groups in benign solvents resulting in high yields. 4,5 Therefore click chemistry is a convenient method to upgrade traditional SPPS to obtain multifunctional peptides. [6][7][8] It was shown that copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), oxime ligation and recently the Staudinger-phosphite reaction are applicable for on resin synthesis to obtain branched and functionalized molecules. [9][10][11][12] Click reactions on a solid support reduce purification steps, enable automation or protect sensitive amino acids like L-3,4-dihydroxylphenylalanine (DOPA) from oxidation. 10 Another bioorthogonal cycloaddition is the Diels-Alder reaction with inverse electron demand (DAR inv ) that can be performed in water without catalysts or hazardous sideproducts and reagents. 13 This irreversible reaction between an alkene (dienophile) and a tetrazine (diene) has been successfully used to synthesize peptide conjugates by us and others for various medical applications. [14][15][16][17] However, solubility problems or instability of tetrazines can lead to difficulties. 17,18 An on-resin approach can overcome these limitations and decrease purification and cleavage steps, thus increasing the yield. DAR inv -conjugates mostly consist of bulky linker-units between two conjugated molecules with different distances and flexibilities depending on the applied dienes and dienophiles. This could be beneficial for several in vitro investigations where distances are required to enable binding of ligands to proteins or reporter molecules. [19][20][21] RGD-peptides bind to receptors in the cell-membrane and thus can mediate cell-adhesion on surfaces to improve healing and osseointegration of e.g. titanium implants. 22,23 It could be shown that a minimum distance between the RGD-peptide and the surface is crucial for successful cell adhesion. 24 Hence, DAR inv -conjugated RGD-peptides present an efficient approach to promote cell attachment on surfaces such as titanium.
Herein, it is demonstrated that the DAR inv can be expanded to exploit polymer-bound synthesis compatible with standard Fmoc/tBu-SPPS (Fig. 1). The applicability of two tetrazines and four different dienophiles was investigated by ligating a celladhesive-and a titanium-binding-peptide to the solid support. The obtained products with a varying linker length were then applied to improve cell adhesion on coated titanium. Moreover, molecular dynamics (MD)-simulation was used to assess the DAR inv -linker conformation and thus discuss the cell response in theory.
Monitoring of the Diels-Alder reaction with inverse electron demand (DAR inv ) on resin by photometry Diene modified resin (approximately 1 µmol) was transferred into a 96-well plate and swollen in water. Subsequently, the water was removed and the dissolved dienophile (30 mM; 100 µL) was added. An absorption scan from 430-600 nm of the diene modified resin was carried out with a plate reader (Tecan, Infinite® 200 PRO series). If the absorption maximum at 540 nm was diminished, the reaction was complete (8a: 5 h, 8b: 2 h, 8c: 7 d).  1 General approach of the DAR inv on resin. The hydrophobic tetrazine remains on the solid support to avoid solubility problems. After incubation with the dienophile, N 2 is released and the desired product is formed. The conjugate can be further modified on resin or cleaved off by TFA-treatment. The resulting mixture of the isomers can be oxidized.

Cleavage of peptides from the resin and purification
Final and sample cleavage of the peptides was performed with TFA/scavenger (9 : 1 v/v) by shaking for 2 h at RT. Scavenger mixtures were used as follows: TIS/H 2 O (1 : 1 v/v) for peptide 6 and TBP 2, thioanisole/thiocresol (1 : 1 v/v) and thioanisole/ 1,2-ethanedithiol (7 : 3 v/v) for 3-5, conjugates 8a,b and H 2 O for peptides 1a,b and 8c. Peptides were precipitated and washed with diethyl ether. The dissolved peptide was analyzed and lyophilized. Isolation of the obtained peptides was carried out by RP-HPLC on a Phenomenex Jupiter Proteo column (90 Å/4 µm, 22 mm × 250 mm) using linear gradients of eluent B in eluent A (A: 0.1% TFA in H 2 O, B: 0.08% TFA in ACN). The identity and purity of the isolated products was verified by MALDI-ToF (Bruker Daltonics) and ESI-HCT (high-capacity ion trap, Bruker Daltonics) mass spectrometry and analytical RP-HPLC (Varian VariTide RPC (200 Å, 6 µm), Phenomenex Jupiter Proteo (90 Å/4 µm and 300 Å, 5 µm) and Grace Vydac (300 Å, 5 µm)) with linear gradients of eluents A and B. The detected m/z signals were in agreement with the calculated molecular weights and the peptide products were obtained with a purity of ≥90% (Table 1).
Titanium foil (Sigma Aldrich, thickness: 0.127 mm) was cut into round pieces and etched in H 2 SO 4 (30%)/H 2 O 2 (1 : 1, v/v) for 7 min. The slides were then washed with water and PBS buffer (Dulbecco's phosphate) and sterilized in 70% ethanol/ water (v/v) under ultrasonic irradiation for 15 min. After washing twice with PBS, the Ti-plates were incubated overnight at room temperature with a 1 µM solution of peptides dissolved in PBS as well as PBS (untreated Ti) and fibronectin (25 µg mL −1 in PBS) as controls.
To investigate the initial cell behavior to the synthesized peptide coatings, cells were resuspended in media without FCS (to prevent adsorption of serum proteins) and seeded on coated and washed Ti-plates. After adhesion for 6 h, the plates were washed twice with PBS and subsequently the cells were fixed with 4% paraformaldehyde in PBS for 30 min. The plates were washed with PBS and the cells were permeabilized with 1% triton X-100 in PBS for 1 min. Next, the slides were washed and cells were stained with phalloidin-tetramethylrhodamineisothiocyanate ( phalloidin-TRITC, Sigma Aldrich) and HOECHST 33342 (Sigma Aldrich). Fluorescence microscopy (Axio Observer microscope, Zeiss) was performed with mounted Ti-plates on glass slides. For each plate, three representing pictures were taken at 20 fold magnification. The average cell area was determined by manually outlining each cell on one representing picture of each triplicate (using the Software Axio Vision 4.8, Zeiss). Data were analyzed from at least 3 independent experiments and presented as mean ± SEM.

Molecular dynamics of the linker
Molecular dynamics simulations were carried out using the Gromacs-4.6 package. 25 The simulation system consisted of the respective linker with a C-terminal amidated and N-terminal acetylated lysine on the RGD-peptide side and a C-terminal amidated and N-terminal acetylated lysine/cysteine on the titanium binding site. The linker was parameterized with antechamber 26 and acpype 27 for the GAFF force field. The charges were derived with the antechamber sqm method. The simulations were carried out under periodic boundary conditions in TIP3P water with 150 mM NaCl at 300 K for 50 ns after equilibration with NVT and NPT ensemble for 50 ps each. The trajectories were analyzed with the g_dist tool by measuring the C α -C α distance and plotting the values in a histogram with a bin size of 0.5 Å.

Results and discussion
Peptide modification with tetrazines The titanium binding peptide (TBP, 1) was synthesized on a TentaGel resin to enable the DAR inv on the resin in aqueous solution. The sequence was built up of spacer units such as polyethylene glycol (PEG) and β-Ala. DOPA (L-3,4-dihydroxyphenylalanine), which shows high affinity and stability to titanium and other surfaces, was used to anchor the peptide to the biomaterial. 28 Functional groups were introduced by using Lys, Pra (L-propargylglycine) and Cys to allow the multifunctional modifications of TBP. 29 Two dienes (2a,b) were coupled to TBP after selective side chain deprotection of Lys (Fig. 2). The result of the reaction was studied by RP-HPLC and MALDI-ToF-MS analysis (Fig. 4d). The tetrazine stability in 1a and 1b was tested in solution and on resin over 7 d in an aqueous environment and yielded low fragmentation (see the ESI †). As a side reaction, the undesired DAR inv between the alkyne functionality of Pra and the applied tetrazines was observed, mainly for peptide 1a in solution. On resin, this reaction occurred only to a small extent. Cleavage of 1b from the resin resulted in fragmentation induced by water, which was slightly increased after incubation of the resin in water for 7 d. 30 Nevertheless, possible degradation of tetrazines by the cleavage of the peptide from the resin is circumvented by the DAR inv on resin.

Peptide modification with dienophiles
Different dienophiles were introduced to the cyclic cell binding peptide c[RGDfK]. The peptide was synthesized on an acid labile chlorotrityl resin (Fig. 3). The modification with different dienophiles was either realized by the incorporation of alkene containing amino acids (4,5) or by Lys-side chain modification (3,6). The dienophile functionalized peptides were cleaved under mild acidic conditions to retain side chain protecting groups. Subsequently, the selective head to tail cyclization was performed in solution. Peptides were finally purified by RP-HPLC after complete deprotection by TFA. The Reppe-anhydride, which has already been used in previous studies, was coupled at the Lys-side chain of the c[RGDfK]peptide to yield compound 3. [31][32][33] To facilitate the introduction of dienophiles during SPPS, we used terminal alkenes, which are present in common protecting groups like Alloc and Ally. A 6-maleimidohexanoic acid modified peptide (6) was synthesized to explore the feasibility of this easily accessible dienophile for the DAR inv on resin (Fig. 3).

Ligation of peptides by DAR inv on resin
To perform the DAR inv on the solid phase, the dienophile 3 was dissolved in H 2 O (c = 30 mM) and added to the resin bound peptides 1a and b. A disadvantage of reactions on the solid phase is the often complicated monitoring of the reaction. However, herein we demonstrate a direct photometric measurement of the reaction process on resin. The DAR inv was monitored by measuring the specific absorbance of the tetrazines at 540 nm directly on the resin by photometry (Fig. 4b  and c). The cycloaddition with peptide 1b was complete after 2 h. Tetrazine 2a resulted in a slower reaction with full conversion after 5 h (see the ESI †), which can be explained by the presence of stronger electron withdrawing groups in 2b compared to 2a. Higher reactivity of tetrazines often results in lower stability. 34 During the DAR inv on resin with peptide 3 we could not detect the differences in diene stability. However, for reactions with longer incubation times, as for peptide 6, lower stability of tetrazine 2b compared to 2a was observed (see the ESI †). Furthermore, combining the DAR inv with additional   reactions as the CuAAC lead to stability problems for compound 8b originated from tetrazine 2b as well (see the ESI †). Contrarily, 8a was stable under the CuAAC on resin and was recently used to synthesize a multifunctional cell adhesive titanium coating. 29 This suggests that the choice of the tetrazine can strongly influence the stability of the resulting DAR invproduct as shown also for metabolic degradation. 14 After completion of the reaction, the dienophile containing peptide 3, which was used in excess, was fully recovered by lyophilization. Cleavage from the resin and subsequent analysis showed the generation of the desired dihydropyridazine 8b (Fig. 4e).
Notably, the DAR inv -product was stable under the conditions of the Fmoc-cleavage with piperidine in DMF, which underlines the compatibility with Fmoc/tBu-based-SPPS. Conjugate 8b was partially oxidized to the corresponding pyridazine (Fig. 1). Complete oxidation can be achieved by incubation with isoamyl nitrite and acetic acid, which can be performed on resin and in solution (Fig. 5). 35 Terminal alkenes were successfully applied to react with tetrazines by a DAR inv . 36,37 Incubation of the tetrazine containing peptide 1a and dienophile 4 or 5 respectively could show the formation of the desired DAR inv product, which was however cleaved after cycloaddition (for the hypothesized mechanism see the ESI †). The reaction of these terminal alkene bearing peptides with tetrazine 1b resulted only in low conversion of around 10% as a result of low reactivity and stability of the tetrazine (see the ESI †). Alternative tetrazines could be applied to overcome this problem to establish an easy introduction of dienophiles in SPPS. Moreover, the suggested mechanism could be probably applied as a decaging method as described recently for proteins or drugs, since the active chemotherapeutic RGD-peptide is released upon fragmentation. 38,39 Maleimides are classical dienophiles in normal Diels-Alder reactions. 40 However, the DAR inv between peptide 1a and the  maleimide modified RGD (6) resulted in the stable product 8c.
The reaction completed rather slowly after 7 d (Fig. 6). This reactivity towards tetrazines should be considered when using maleimide modified tetrazines or a combination of DAR inv with a thiol-maleimide addition. [41][42][43] Moreover, this finding gives rise to the potential use of maleimides as easily accessible dienophiles in the DAR inv . However, alternative tetrazines should be tested to increase the reaction rate. The outcome of all the studied reactions between the different tetrazines and dienophiles is summarized in Table 2.
The DAR inv on a solid support displays the following advantages. Tetrazines are often hardly soluble in aqueous solution. Therefore, the reaction has to be carried out in organic solvents or the synthesis strategy has to be changed by the introduction of solubility increasing molecules such as short PEG units. 18 Performing the DAR inv with a tetrazine containing peptide bound to a solid phase overcomes this problem. Besides the advantages of water as a solvent for environmental and biochemical issues it is also favorable for kinetic reasons. 44 It is known that water can increase the proximity of hydrophobic molecules like the used diene and dienophiles. 31 Hence the reaction can be accelerated. 45 Additionally, the water-swellable TentaGel resin facilitates the synthesis of rather long or branched peptides compared to the synthesis carried out on non-PEG-based resins. Furthermore, cleavage and purification steps are reduced since the crude resin bound peptides can be directly used for the ligation. This is especially beneficial for peptides that consist of rather cost intensive amino acids such as DOPA. DAR inv -reagents as the dienophile carrying RGD peptide are easily recovered by lyophilization and can be thus used in excess without loss of yield owing to purification steps.

DAR inv -linkers as spacers to improve cell adhesion
RGD-mediated cell adhesion is strongly dependent on the distance between the integrin ligand and the corresponding surface. Various spacers as polyglycine, polyproline, aminohexanoic acid or PEG have been used so far. 46,47 Using bulky dienes and dienophiles circumvents the introduction of additional spacer moieties. Hence we applied the DAR inv with rather large dienes and dienophiles to efficiently introduce a distinct spacer. To test the influence of the varying lengths of tetrazines 2a and 2b, cell adhesion studies with the peptide conjugates 8a and 8b were performed. Furthermore, peptide 8d, synthesized by maleimide-thiol Michael addition, was tested to compare the tested DAR inv -linker with a shorter spacer (Fig. 7a). Negative controls, containing RAD were synthesized to proof the RGD-mediated cell adhesion (see the ESI †). The increased cell size is an indicator of successful cell attachment and is dependent on the RGD-surface distance. 47 Therefore, cell spreading was determined by measuring the average cell area on coated Ti. Interestingly, conjugate 8b showed the significantly highest average cell size compared to all the tested coatings, although its spacer is shorter than that of peptide 8a (Fig. 7b). By MD-simulation the maximum length (from Cα of Lys in RGD to Cα of Lys in TBP and respectively to Cα of Cys in TBP 2) as well as the most likely present spacer distance were calculated. These studies revealed that the longest spacer is in fact present in 8a with 34 Å. However, this linker is more prone to collapse and interacts intramolecularly, thus it only reaches a distance of around 12 Å for the most stable conformations (Table 3, see also the ESI †). Peptide 8b consists of a spacer with a maximum length of 30 Å. The mostly occurring distance is 20 Å, thus cell spreading is more advanced on 8b than on 8a. The linker of 8d was, as anticipated, the shortest with a maximum length of 21 Å and the most stable conformations showed a distance of 16 Å. Although the most likely distance of 8d is higher than that of 8a, cell spreading is more pronounced on 8a. This can be explained by the higher average length of 19 Å for 8a compared to 16 Å for 8d ( Fig. 7d and Table 3).  Previous studies already have suggested that a longer, more rigid and extended spacer (e.g. polyproline) favors RGDmediated cell adhesion, however until now this has not been assessed by MD-simulation. 46 In addition, the herein described MD-simulations support the assumption that too long spacers impair cell adhesion because of their pronounced flexibility. 46,48 Nevertheless, a long and flexible linker should be favored over a too short spacer as their average length may be higher. These results highlight that estimating the distance of a RGD peptide to its surface only by the extended conformation of a linker molecule is not sufficient. It is rather suggested that the three-dimensional length is evaluated. We suggest that a suitable linker consists of two short flexible ends, connected by a long rigid structure (see the ESI †). The flexible parts in the spacer may be important to freely orientate the RGD-peptide on the surface towards the cell membrane.

Conclusion
The Diels-Alder reaction with inverse electron demand (DAR inv ) could be successfully employed to ligate peptides on a solid support. This method describes an efficient strategy to complex peptide conjugates without using catalysts or additive reagents and it is easily monitored. Low solubility as well as yield loss and potential fragmentation through cleaving tetrazine modified peptides from the resin are thereby circumvented. Furthermore, maleimide was found to be an easily accessible dienophile alternative for DAR inv ligations on resin. Large DAR inv -reagents could be applied to generate a spacer that might be useful for several biochemical applications. MDsimulations gave insights into the theoretical conformations of different DAR inv -linkers, thereby providing crucial information for the choice of a distinct diene-dienophile pair. Thereby, tetrazine 2b in the DAR inv product 8b resulted in the most enhanced cell adhesion because of its less flexible structure. With the here presented DAR inv on resin the toolbox of orthogonal reactions for SPPS is broadened.