Application of cysteinyl prolyl ester for the synthesis of cyclic peptides containing an RGD sequence and their biological activity measurement

Cysteinyl RGD-peptidyl cysteinyl prolyl esters, which have different configurations at the cysteine and proline residues, were synthesized by the solid-phase method and cyclized by the native chemical ligation reaction. Cyclization efficiently proceeded to give cyclic peptides, regardless of the difference in the configuration. The peptides were further derivatized to the corresponding desulfurized or methylated cyclic peptides at the Cys residues. The inhibition activity to αvβ6 integrin binding was then analyzed by ELISA. The results showed that the activity varied depending on the difference in the configuration and modification of the cysteinyl prolyl ester (CPC) moiety, demonstrating the usefulness of this method in the search for a good inhibitor of the protein–protein interaction.


Introduction
Cyclic peptides are promising leads for drugs regarding their stability to proteinases as well as the rigidity of the cyclic structure.In fact, many cyclic peptides are currently used as therapeutics (Muttenthaler et al., 2021;Ji et al., 2023).Therefore, efficient methods have been developed to obtain cyclic peptides via the amide bond as well as the disulfide bond, oxime bond, and other bond formations (Malesevic et al., 2004;Tulla-Puche and Barany, 2004;White and Yudin, 2011;Hemu et al., 2013;Terrier et al., 2017;Gless and Olsen, 2018;Shimodaira et al., 2018;Arbour et al., 2019;Chow et al., 2019;Reguera and Rivera, 2019;Sarojini et al., 2019;Serra et al., 2020).We also developed efficient cyclization methods based on our cysteinyl prolyl ester (CPE) (Kawakami et al., 2022), which was originally developed for the synthesis of the peptide thioester (Kawakami and Aimoto, 2007).In the CPE method, a peptide with the cysteinyl prolyl ester at the C-terminus is converted to the peptide thioester via the N-to-S acyl shift, followed by diketopiperazine formation.Therefore, if a peptide has the N-terminal Cys and C-terminal CPE, in which the configuration of the cysteinyl proline residue in CPE is the same, the cyclic peptide was obtained by the native chemical ligation reaction (Dawson et al., 1994) as shown in Figure 1A.In this case, the Cys-Pro sequence was liberated from the sequence.The yield of the desired cyclic monomer was moderate due to the formation of a cyclic dimer and trimer.In contrast, when the configuration in the Cys-Pro sequence of CPE is different (LD or DL), the ligation with the N-terminal cysteine efficiently occurs at the Pro, keeping the CPC sequence via thiolactone formation (Figure 1B).Remarkably, this procedure gives the desired cyclic peptide in an excellent isolated yield without the production of cyclic dimers and oligomers, showing the efficiency of this method for cyclic peptide synthesis.In addition, we can design a cyclic peptide library with different absolute configurations at the CPC moiety, leading to different biological activities.Two cysteine residues can be further modified, such as by desulfurization and alkylation, which can further increase the number of libraries.To demonstrate these possibilities, the method was used for the synthesis of cyclic hexapeptides containing the RGD sequence, and the effect of the configuration differences on their biological activities was analyzed by the integrin-binding assay.
Integrins are heterodimeric transmembrane receptors and engage in cell adhesion.Integrins are composed of α and β subunits (Hynes, 2002;Kechagia et al., 2019).So far, 18 kinds of α-subunits and 8 kinds of β-subunits have been identified, and from these subunits, 24 kinds of integrin species are formed.Integrins can interact with many ligands, including extracellular matrix (ECM) proteins.Many of the integrins recognize short peptide sequences within the ligands.Depending on the type of recognition motif, integrins are classified into five types: the RGD-binding integrin, laminin-binding integrin, collagen-binding integrin, leukocytebinding integrin, and EMILIN-binding integrin.As the aberrant integrin-ligand interaction is known to be involved in various diseases, the integrin inhibitor attracts much attention as a therapeutic.
We synthesized cyclic hexapeptides with the Arg-Gly-Asp (RGD) sequence containing CpC, Cpc, cPC, and cPc (lowercase letters denote D-amino acids) (Figure 2).The desulfurized and methylated derivatives of these peptides were also prepared, and their inhibitory activity regarding the integrin-ligand interaction was analyzed.

Synthesis of the linear peptides containing the Arg-Gly-Asp sequence
We first synthesized linear precursors, the peptide prolyl esters with different configurations, using the fluorenylmethoxycarbonyl protecting group (Fmoc) method, as shown in Figure 3. Glycolic acid was introduced to the Fmoc-NH-SAL-PEG resin using the N,N'diisopropylcarbodiimide-1-hydroxybenzotriazole (DIC-HOBt) method.Fmoc-Pro-OH or Fmoc-D-Pro-OH was then introduced by 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU/DIEA).The next two amino acid residues were introduced as a dipeptide to avoid the elimination of the C-terminal dipeptide by diketopiperazine formation (Kawakami and Aimoto, 2007).The remaining amino acids were introduced using the Fmoc method to obtain the protected peptide resins with the desired sequences.The resins were then treated with the trifluoroacetic acid (TFA) cocktail to deprotect the peptides.The obtained crude peptide was purified by reversed-phase (RP)-HPLC to obtain the desired linear peptides, 2a−2d, in high purity (Supplementary Figure SI-1).The yields of the peptides were around 30%.

Cyclization reaction
The linear peptide was then cyclized under the native chemical ligation reaction condition using the CPE method.In brief, the linear precursor was dissolved in a sodium phosphate buffer containing 20 mM TCEP and 20 mM ascorbic acid (pH 7.8) at the concentration of 2 mM, and the reaction mixture was kept at 37 °C.In spite of the relatively high concentration of peptides, intramolecular cyclization proceeded without the formation of a dimer and oligomers within 4 h, as shown in Figure 4, indicating that the peptide cysteinyl prolyl ester tends to assume a cyclizable conformation, at least in the case of the hexapeptide.The cyclized peptides, 3a-3d, were isolated by RP-HPLC, as shown in Supplementary Structure of the cyclic peptides.

Desulfurization a) Use of VA044 as a radical initiator
The desulfurization reaction was first attempted by the prevalent conditions using VA-044 as the initiator (Wan and Danishefsky, 2007).The cyclic peptides were dissolved in degassed sodium phosphate buffer (pH 6.8) containing 10 mM VA-044, 0.10 M TCEP・HCl, and 50 mM MESNa (pH 6.8) at a 1.0 mM concentration and shaken at 37 °C.Excess amounts of VA044, MESNa, and TCEP are required for this method.Unfortunately,  RP-HPLC profile of the desulfurization reaction using VA-044 as a radical initiator.4a' and 4d' denote the peaks of peptides in which one of the cysteine residues in the sequence was desulfurized.
the large peaks derived from these reagents were eluted close to the peptides, which made monitoring the progress of the reaction by RP-HPLC difficult (Figure 5).The TCEP-sulfide (TCEP = S) formed by the reaction of TCEP with MESNa eluted very close to the peptides.Therefore, we examined the use of another desulfurization reaction.b) Use of NaBEt 4 as the radical initiator Sun et al. recently reported the use of NaBEt 4 as the radical initiator for desulfurization (Sun et al., 2022).As TCEP = S is derived only from the reaction of TCEP and cysteine residues, the amount is low, and monitoring the reaction by RP-HPLC would be much easier.The peptide was dissolved in 0.10 M citric acid containing 0.10 M TCEP•HCl (pH 5.2), and NaBEt 4 was added to the mixture to the final concentration of 0.10 M in the solution.As expected, the efficient monitoring of the reaction was achieved, as shown in Figure 6.After 1 min, the reaction was complete.The mixture was purified by RP-HPLC to obtain the desulfurized peptides, 4a-4d (Supplementary Figure SI-3).The isolated yield of the product was around 70%, indicating a clean reaction.

Methylation
In this experiment, the cyclization and methylation were sequentially performed in one pot.The cyclization of linear peptides was carried out in the same manner as described in Section 2.2 Cyclization, and the reaction proceeded similarly.Without isolating the product, CH 3 I in dimethylformamide (DMF) was added, and the reaction mixture was shaken for 30 min at room temperature.The reaction efficiently proceeded, and the doubly methylated peptide was obtained, as shown in Figure 7. Isolation by RP-HPLC gave the methylated peptides 5a-5d in high purity (Supplementary Figure  in yields of around 45%.

Inhibition of integrin-ligand binding by the cyclic peptides
Among the RGD-binding integrins, we selected α v β 6 integrin for measurement of the inhibition activity of the cyclic peptides.The α v β 6 integrin is reported to recognize the sequence beyond the RGD motif, RGDLXXL (Dong et al., 2014).Therefore, the inhibition activity of the cyclic peptides is expected to vary depending on the configuration and the modification at the CPC moiety.In addition, the inhibitor of the α v β 6 integrin is still under development (John et al., 2020), while an efficient inhibitor, cilengitide, exists for the α v β 3 and α v β 5 integrins (Stupp and Ruegg, 2007).The latency-associated peptide (LAP, TGF-β1) was used as a ligand for α v β 6 integrin and coated on the ELISA plate, to which the α v β 6 integrin and cyclic peptides were added to measure the inhibitory activity of the cyclic peptides by ELISA.The inhibitory activity of the commonly used RGD tripeptide was also measured as a control.
As shown in Figure 8, the parental cyclic peptides, 3a to 3d, retained stronger or comparable inhibitory activity than the RGD control peptide, indicating that the RGD sequence in these cyclic peptides is recognized by α v β 6 integrin.Peptides 3a and 3b are stronger than 3c and 3d, showing that the L-configuration is favored for the penultimate residue to Asp.In addition, the residue also requires hydrophobicity as the inhibitory activity of the desulfurized peptides 4a to 4d decreased compared to the parental peptides 3a to 3d.This fact is also supported by the increased inhibitory activity of the methylated peptides 5a to 5d, which are more hydrophobic than the parental peptides.These results agree with the fact that the α v β 6 integrin recognizes the sequence RGDLXXL, where the penultimate amino acid to Asp is Leu (Dong et al., 2014).Therefore, there is a possibility that modification at the cysteine residues by more bulky hydrophobic groups will further increase the inhibitory activity, demonstrating the utility of the CPC method for the efficient peptide design.RP-HPLC profile of the desulfurization reaction.

Conclusion
The cyclic peptide synthesis using the CPE method has been established using the RGD integrin-binding motif as a model.The linear precursor was easily synthesized by the Fmoc solid-phase method and cyclized by the native chemical ligation reaction at a relatively high 2 mM concentration without the formation of dimeric and oligomeric cyclic peptides.In addition, the desulfurization and alkylation of the parental peptides at the Cys residues also efficiently proceeded, which led to an increase in the number of cyclic peptides with different conformations.The inhibition of the α v β 6 integrin-binding activity measurement showed that the activity depends on the configuration of the CPC motif as well as the desulfurization and methylation at this  Inhibitory activity of cyclic peptides for binding of the α v β 6 integrin with LAP (TGF-β1) under the following conditions: 1 µM peptide concentration, 10 nM integrin αvβ6 in the presence of 1.0 mM Mn 2+ , and LAP (TGF-β1) 10 nM.
motif, demonstrating the usefulness of this method for the design of cyclic peptide inhibitors.
4 Experimental procedure
The Fmoc group quantification was performed using a dried resin sample (200-500 μg), which was treated with 20% piperidine/ DMF (50 µL) for 30 min.The mixture was diluted with DMF (5.0 mL), and the absorbance at 301 nm was measured by a V-730BIO (JASCO, Tokyo).The content of the Fmoc group (x mmol) in the 1 g of resin was calculated by the following equation: where a is the weight of the resin in mg, b is the volume of the diluted solvent in mL, c is the absorbance at 301 nm, and ε is the molar extinction coefficient (ε = 7,800 (mol L −1 ) −1 cm −1 ).The cDNA encoding the extracellular region of the human integrin β6 (Met1-Ile708) was amplified using the total RNA extracted from the human colon adenocarcinoma cell line WiDr as a template.The cDNA was digested with BamHI and PmeI and ligated into the corresponding restriction sites of the pEF-BASE-His6 expression vector (REF: PMID11323715).An expression vector for the extracellular region of the human integrin av with an ACID α-helical coiled-coil peptide was kindly gifted from Dr. Junichi Takagi (Institute for Protein Research, Osaka University) (REF: PMID 12230977).A FLAG tag was fused to the C-terminal site of the integrin αv-ACID sequence as previously described (REF: PMID 16324831).An expression vector for the human LAP (TGFβ1) was constructed as previously described (REF: PMID 27033701).Recombinant human αvβ6 and LAP were expressed using the FreeStyle ™ 293 Expression System and purified from the resultant supernatant, as previously shown (REF: PMID 16324831 & 27,033,701).A rabbit pAb against the ACID/BASE coiled-coil region was produced as previously described (REF: PMID 16413178).The pAb was biotinylated using EZ-Link ™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer's instructions.

Integrin inhibition assay with synthetic peptides
The integrin inhibition assay was performed as follows.The 96-well Nunc MaxiSorp ™ flat-bottom plate was coated overnight with 50 µL of a 10 nM LAP solution at 4 °C.After washing with 200 µL of Tris-buffered saline [20 mM Tris-HCl (pH 7.4) containing 137 mM NaCl] (TBS) containing 0.1% (v/v) Tween-20 (Sigma) and 3% (w/v) BSA (Sigma) (3% BSA/TBS-T), the plate was blocked with 200 µL of 3% BSA/TBS-T for 1 h at room temperature.After washing three times with TBS containing 0.1%(v/v) Tween-20 (Sigma), 0.3% (w/v) BSA (Sigma), and 1 mM manganese chloride (Wako) (Mn-Wash), 50 µL of 10 nM αvβ6 integrin preincubated with 1 µM peptide in Mn-Wash for 1 h at room temperature was added to the wells and incubated with gentle agitation for 1 h at room temperature.The bound αvβ6 integrin was detected by the biotinylated anti-Velcro polyclonal antibody and horseradish peroxidaseconjugated streptavidin.In brief, after washing three times with the Mn-Wash, the plate was incubated with 50 µL of 1.5 μg/mL biotinylated anti-Velcro antibody for 0.5 h at room temperature with gentle agitation.The bound antibody was detected by horseradish peroxidase-conjugated streptavidin (0.53 μg/mL), followed by colorimetrically measuring the amount of bound αvβ6 integrin using o-phenylenediamine (0.4 mg/mL; FUJIFILM Wako) and H 2 O 2 (0.006%) in a 25 mM citric acid/50 mM Na 2 HPO 4 buffer.The colorimetric reaction was stopped with 100 μL of 2.5 M H 2 SO 4 , and the absorbance of the chromogenic substrate was measured at 490 nm using a microplate reader (Molecular Devices EMax).A 490 -A 650 was used to calculate the inhibitory activity.The inhibition activities of the synthetic peptides for the αvβ6 integrin were calculated according to the following formula: where A p is the absorbance at 490 nm of the bound αvβ6 integrin with synthetic peptide, A i is the absorbance at 490 nm of the bound αvβ6 integrin without synthetic peptide (control), and A b is the absorbance at 490 nm of 1 mM manganese chloride as a background.
The inhibitory activity was calculated as the mean of three wells per analysis.All the measurements were performed with N = 3, and the final inhibitory activity was calculated as the mean ± standard error.

FIGURE 3
FIGURE 3Synthetic route of the linear precursor and the RP-HPLC profile of crude peptides.

Figure
Figure SI-2, in yields of around 50%, proving the efficient cyclization of the linear peptides by using this method.

FIGURE 4 RP
FIGURE 4RP-HPLC profile of the cyclization reaction progress.

FIGURE 7
FIGURE 7RP-HPLC profile of the cyclization and subsequent methylation reaction.