Structure Derivatization of IgG-Binding Peptides and Analysis of Their Secondary Structure by Circular Dichroism Spectroscopy

Abstract

Mid-sized cyclic peptides are a promising modality for modern drug discovery. Their larger interaction area coupled with an appropriate secondary structure is more suitable than small molecules for binding to the target protein. In this study, we conducted a structure derivatization of an immunoglobulin G (IgG)-binding peptide (15-IgBP), a β-hairpin-like cyclic peptide with a twisted β-strand and assessed the effect of the secondary structure on IgG-binding activity using circular dichroism (CD) spectra analysis. As a result, derivatization at the Ala5 and Gly9 positions affected the secondary structure of 15-IgBP, in particular the appearance of a small positive peak in the 220–240 nm region characteristic of 15-IgBP in the CD spectrum. Maintaining this peak at a moderate level may be important for the expression of IgG binding activity. We found the small methyl group at Ala5 to be crucial for retaining the preferred secondary structure; we also found Gly9 could be replaced by D-amino acids. By integrating these findings with previous results of the structure–activity relationship, we obtained four potent affinity peptides for IgG binding (K_d = 4.24–5.85 nM). Furthermore, we found the Gly9 position can be substituted for D-Lys. This is a new potential site for attaching functional units for conjugation with IgG for the preparation of homogeneous antibody–drug conjugates.

Introduction

Affinity peptides targeting the fragment crystallizable (Fc) region of immunoglobulin G (IgG) are well-utilized in pharmaceutical applications, particularly in the preparation of antibody–drug conjugates (ADCs) for homogeneous modifications.^1–14) So far, a number of peptides with strong IgG Fc-binding properties have already been reported^15–17) (Fig. 1). However, their secondary structures are generally diverse. Among such IgG-binding peptides, the IgG binding domain of Protein G consists of a combination of α-helical and β-strand structures (K_d < 10 nM, PDB: 1FCC).^18–20) In contrast, the IgG binding domain of Protein A, known as the B-domain (K_d = 10–50 nM, PDB: 1FC2),²¹⁾ and its derivative Z-domain (K_d = 14.9 nM)²²⁾ are composed of a three-helix bundle structure. Moreover, a group of shortened peptide derivatives such as Z33 (K_d = 43 nM)²³⁾ or Z34C (K_d = 20.0 nM, PDB: 5UBX),^24,25) developed from the Z-domain, form helix-loop-helix structures. Additionally, several β-hairpin-like cyclic peptides with at least one disulfide bond have been developed using a phage display method. These include Fc-III (K_d = 185 nM, PDB: 1DN2),^26,27) FcBP-III (K_d = 2.2 nM),²⁷⁾ Fc-III-4C (K_d = 2.45 nM),²⁸⁾ and peptide 1/Lys8Arg (Fig. 2a, Ac-GPDC*AYHRGELVWC*TFH-NH₂, *disulfide) (K_d = 29 nM, PDB: 6IQG).^1,29) However, despite having different secondary structures and interaction modes, these peptides bind to the same region spanning the CH2 to CH3 regions of Fc.

Fig. 1. Secondary Structure of IgG Binding Peptides

Fig. 2. a) The Primary Structure of Peptide 1/Lys8Arg and Lys8Leu; b) Crystal Structure of the Region Surrounding the Core Cyclic Structure (from Asp3 to Thr15) of Peptide 1/Lys8Arg (PDB: 6IQG); Side Chains of Ala5, His7, Arg8, Glu10, Val12, and Trp13 Direct to the Antibody Surface; c) The Hydrogen Bonding Network around the β-Bulge Structure; The Dotted Arrows Indicate Hydrogen Bonding from Amide-NH to Carbonyl Oxygen; Residues of the Same Color (White or Gray) Have Their Side Chains Pointing in the Same Direction

Among these various IgG binding peptides, the β-hairpin-like cyclic peptides look promising for the development of drugs or chemical tools because of their relatively smaller molecular size (i.e., shorter sequences with 13–17 amino acid residues), while maintaining a high binding affinity comparable to other longer peptides (33–58 amino acid residues). These β-hairpin-like cyclic peptides partially exhibit a unique secondary structure with a twisted β-strand, called the β-bulge (Figs. 2b, 2c). This structure is sometimes observed in proteins but is less common in peptides.^30–32) The side chains of successive amino acids on a typical β-strand are oriented in alternating directions on the sheet. Conversely, the twisted β-bulge of these peptides results in consecutive side chains facing the same direction, as seen in peptide 1/Lys8Arg, where the side chains of Va12 and Trp13 face the antibody surface (Figs. 2b, 2c). As a result, two-thirds of the amino acids (Ala5, His7, Arg8, Glu10 Val12, and Trp13) in the cyclic structure (from Cys4 to Cys14) are oriented toward the antibody surface, contributing to a strong binding affinity despite the small size of the peptide. Thus, in the structure derivatization, maintenance of the secondary structure plays a crucial role in achieving potent binding affinity.

We previously performed a structure–activity relationship (SAR) study of β-hairpin-like IgG-binding cyclic peptide 1 (Ac-GPDC*AYHKGELVWC*TFH-NH₂, *disulfide, K_d = 225 nM).^33,34) As a result, we found shortened peptides, 15-IgBP (Ac-DC*AYHKGELVWC*TFH-NH₂, *disulfide, K_d = 267 nM) retained affinity, and Lys8Leu (K_d = 8.19 nM) and His17(2-Pya) (2-pyridylalanine) (K_d = 76 nM) to be more potent substitutes.^33,34) During the SAR study, the peptide derivatives with a twisted β-bulge structure showed random coil-like spectra in circular dichroism (CD) analysis with more than 50% random coil content according to the Reed’s reference, in contrast to their β-hairpin-like structure.³⁵⁾ Additionally, the presence of a distinct small positive peak at 230 nm in the CD spectra was observed in derivatives possessing strong binding affinity^33,36,37) (Fig. 3a). Therefore, this small CD peak may be important for maintaining the preferred secondary structure and serve as one of the suitable indicators of derivatization based on the desired secondary structure.

Fig. 3. a) The CD Spectra of 15-IgBP Derivatives with a Derivatization of the Ala5 Position; b) Correlation of Taft’s Steric Factor (Es) and CD Signal of Ala5-Substituted Peptides at the Wavelength of 230 nm (Correlation Coefficient R = −0.9422); c) The Value Used to Calculate the Correlation

The Es value was from Ref. 39).

Therefore, in the present study, we used the CD spectral signal as an indicator and performed further SAR study of 15-IgBP. 15-IgBP is the starting point of our comprehensive SAR study of the IgG-binding peptide, with the goal of obtaining more potent peptides. As a result, important properties at positions Ala5 and Gly9, which influence the peptide secondary structure, were newly identified as evidenced by the CD spectral data. We also discovered a new peptide derivative with an improved affinity, achieving nanomolar K_d values while retaining the unique secondary structure.

Results and Discussion

Synthesis of Peptide Derivatives

In this study, derivatives of 15-IgBP were newly synthesized by a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (SPPS).³⁸⁾ The Fmoc group of SAL resin was deprotected with 20% piperidine in N,N-dimethylformamide (DMF) for 10 min twice. Then, the first Fmoc-protected amino acid was condensed by the reaction with Fmoc-His(Trt)-OH, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole (HOAt), and N,N-diisopropylethylamine (DIPEA) for 30 min. Following this, the deprotection of Fmoc and coupling of the next Fmoc-amino acids was repeated to obtain the desired protected peptide resin. Finally, the N-terminal amino group was acetylated with acetic anhydride and DIPEA for 15 min. Then, after washing and drying the resin, cleavage of the peptide from the resin and its final deprotection were performed by treatment with trifluoroacetic acid (TFA) : triisopropylsilane (TIS) : 1,3-dimethoxybenzene (DMB)³⁴⁾ (40 : 1 : 2) for 3 h at room temperature. An intramolecular disulfide bridge was constructed by treating crude peptides with Npys-OMe^39,40) (2–5 equivalent (equiv.)) in a solution of 30–60% CH₃CN/H₂O. After monitoring the cyclization reaction completion by HPLC, the solution was lyophilized and triturated with Et₂O to remove excess reagent. Then, after drying the residual solid in vacuo, synthetic peptides were purified by reverse phase HPLC. The peptide derivatives, obtained with >95% purity, were used for the evaluation of the binding affinity to IgG and secondary structure analysis by CD spectroscopy.

Derivatization of Ala5

The side chain of Ala at position 5 in the 15-IgBP (Ala5) is understood to be directed toward the IgG surface and fill the space between the peptide chain and IgG surface.¹⁾ Furthermore, based on the crystal structure of a related derivative (PDB: 6IQG),¹⁾ the carbonyl oxygen of Ala5 forms intramolecular hydrogen bonds with the amide-NH of Val12 and Trp13 thereby creating a structural feature of β-bulge³⁰⁾ (Fig. 2c). Thus, the substitution of Ala5 was expected to affect the secondary structure of the peptide.

To investigate the role of Ala5 in detail, five peptide derivatives of 15-IgBP with bulky side chains or without side chains at this amino acid position were synthesized and evaluated for their IgG binding affinity (Table 1). As a result, peptides with bulky aliphatic side chains at position 5, with or without branched structures (Ala5Abu, Ala5Nva, Ala5Val, and Ala5Leu), completely lost their binding affinity (K_d > 2000 nM). Additionally, Ala5Gly with no side chain also lost binding affinity (K_d > 2000 nM). These results suggest that Ala5 with a methyl group as a side chain is essential for IgG binding affinity. Interestingly, all Ala5 substitutes caused changes in the CD signals observed at wavelengths of 220–240 nm (Fig. 3a), which is typically associated with peptide derivatives that have preferable interactions with IgG.²⁸⁾ In addition, the changes at 230 nm showed a strong correlation with the bulkiness parameter (correlation coefficient R = −0.9422), that is, Taft’s steric factor (Es)⁴¹⁾ (Figs. 3b–c). Therefore, the small methyl group of Ala residue contributes to maintaining the preferred conformations for binding and further derivatization of this position of Ala5 would require side chain structures with a bulkiness similar to the methyl group, such as a fluorine (Es = 0.78),⁴²⁾ to maintain the secondary structure. Alternatively, since all substitutes tested lost the binding affinity to IgG due to changes in secondary structure, it is conceivable that these side chain structures may be compatible with the binding site of IgG, if the preferable secondary structure was achieved independently of the side chain structure at the Ala5 position.

Table 1. Binding Affinity of 15-IgBP Derivatives Involving a Substitution at the Ala5 Position

Substitution	Kd (nM)	kon (s−1·µM−1)	koff (s−1)
None (15-IgBPa))	267 ± 4	0.741 ± 0.003	0.198 ± 0.001
Ala5Abu	>2000	—	—
Ala5Nva	>2000	—	—
Ala5Val	>2000	—	—
Ala5Leu	>2000	—	—
Ala5Gly	>2000	—	—

a) The values are from Ref. 33).

Derivatization of Gly9

Next, we focused on Gly9, located in the β-turn region of the hairpin-like cyclic peptide 15-IgBP, as depicted in the crystal structure (PDB: 6IQG) shown in Fig. 2b. During the previous Ala-scanning study of 17-residues peptide 1, the substitution of Gly9 with Ala resulted in a secondary structure change on CD analysis and a decrease in affinity to IgG (peptide 1/Gly9Ala, K_d = 1720 nM).³³⁾ Thus, this Gly9 position is also expected to be influential for the secondary structure of the peptide.

As shown in Table 2, consistent with the results of Ala substitution in the 17-residue peptide 1, the substitution of Gly9 with Ala on the 15-residue peptide decreased affinity compared to 15-IgBP (Gly9Ala, K_d = 1660 nM).³³⁾ However, substitution with D-Ala maintained binding affinity (Gly9D-Ala, K_d = 334 nM). Therefore, D-amino acid would be acceptable in this position. In the CD spectra analysis, Gly9Ala and Gly9D-Ala showed different CD spectra at a wavelength of 230 nm (Fig. 4). In particular, the CD spectra of Gly9D-Ala was similar to that of the original 15-IgBP, although the spectrum is shifted slightly upwards, indicating the secondary structure, including β-bulge, was retained. This is similar to what we observed in the X-ray crystal structure of the related derivative Peptide 1 (Figs. 2b, 2c). Conversely, the difference in the CD spectra of Gly9Ala from 15-IgBP suggests the changed secondary structure.

Table 2. Binding Affinity of 15-IgBP Derivatives Involving a Substitution at the Gly9 Position

Substitution	Kd (nM)	kon (s−1·µM−1)	koff (s−1)
None (15-IgBPa))	267 ± 4	0.741 ± 0.003	0.198 ± 0.001
Gly9Ala	1660 ± 10	0.245 ± 0.001	0.406 ± 0.001
Gly9D-Ala	334 ± 10	0.868 ± 0.009	0.290 ± 0.003

a) The values are from Ref. 33).

Fig. 4. CD Spectra Analysis of IgG Binding Peptides with Gly9 Position Substitution

To understand the difference between L-Ala and D-Ala substitutions at the Gly9 position on the secondary structure, a molecular modeling study was performed, focusing on the dihedral angles of the main chain (φ and ψ) based on X-ray crystallographic data (PDB: 6IQG).¹⁾ The dihedral angles at Gly9 in a type I′ β-turn⁴³⁾ were found to be φ = 90.8° and ψ = −25.2° (Fig. 5). According to the Ramachandran plot, it is difficult for L-amino acids to form these dihedral angles, but D-amino acids which possess an inverse preference can adopt these dihedral angles.⁴⁴⁾ Therefore, the configuration of D-amino acid is likely acceptable at the Gly9 position for preserving the peptide’s secondary structure required for the binding to IgG.

Fig. 5. Dihedral Angle of Gly9 from the Modeling Structure of the 15-IgBP Derivative Based on X-Ray Crystal Structure (PDB: 6IQG)

The antibody is shown as a ribbon.

This finding allowed us to carry out the D-amino acid substitution at the Gly9 position (Table 3). Substitutions with bulky β-branched structures (Gly9D-Ile and Gly9D-Val, K_d = 538 and 875 nM, respectively) or aromatic structures (Gly9D-Phe, Gly9D-His, Gly9D-Trp, and Gly9D-Tyr, K_d = 1230, 676, 920, and 1220 nM, respectively) resulted in reduced binding affinity compared to 15-IgBP (K_d = 267 nM). These substitutions affected the secondary structure as indicated by the disappearance of the small positive peak around 230 nm in the CD spectra (Supplementary Fig. S1a). On the other hand, Gly9D-Lys, Gly9D-Pro, Gly9D-Arg, or Gly9D-Ser, which involve D-amino acids with cationic or preference property to form a turn structure,⁴³⁾ showed stronger affinities (K_d = 105, 187, 152, and 211 nM, respectively) than the original 15-IgBP. Gly9D-Asp and Gly9D-Glu showed weaker affinities (K_d = 703 and 525 nM, respectively), although they are also turn-preferable amino acids. The CD spectra of these seven derivatives showed a similar pattern to that of 15-IgBP except for Gly9D-Pro, which maintained the pattern but showed a spectrum with both the 230 nm maximum and 200 nm minimum values amplified (Supplementary Figs. S1b, c). These results suggest that the Gly9 position prefers a cationic structure rather than an anionic structure, probably because this position is surrounded by anionic residues, such as Glu382, which likely create electrostatic interactions, although the side chain is located on the opposite side of Fc surface. On the other hand, substituted derivatives with other neutral D-amino acids (Gly9D-Leu, Gly9D-Met, Gly9D-Asn, Gly9D-Gln, and Gly9D-Thr) showed similar binding affinity (K_d = 263–352 nM) and CD spectra to the original 15-IgBP. These results suggest that while the introduction of a side chain to the D-amino acid at position Gly9 maintains a favorable secondary structure, the binding affinity is influenced by whether the properties of the side chain are suitable for the surrounding environment at the IgG binding site.

Table 3. Binding Affinity of 15-IgBP Derivatives Involving a Substitution at the Gly9 Position to D-Amino Acids

Substitution	Kd (nM)	kon (s−1·µM−1)	koff (s−1)
None (15-IgBPa))	267 ± 4	0.741 ± 0.003	0.198 ± 0.001
Gly9D-Ile	538 ± 2	0.408 ± 0.001	0.220 ± 0.000
Gly9D-Val	875 ± 4	0.293 ± 0.001	0.256 ± 0.001
Gly9D-Phe	1230 ± 50	0.232 ± 0.009	0.287 ± 0.001
Gly9D-His	676 ± 3	0.364 ± 0.002	0.256 ± 0.001
Gly9D-Trpb)	920 ± 32	—	—
Gly9D-Tyr	1220 ± 10	0.277 ± 0.001	0.338 ± 0.001
Gly9D-Lys	105 ± 1	1.20 ± 0.01	0.125 ± 0.001
Gly9D-Pro	187 ± 1	0.684 ± 0.003	0.128 ± 0.000
Gly9D-Arg	152 ± 1	0.882 ± 0.005	0.134 ± 0.001
Gly9D-Ser	211 ± 3	0.744 ± 0.008	0.157 ± 0.001
Gly9D-Asp	703 ± 6	0.423 ± 0.002	0.298 ± 0.002
Gly9D-Glu	525 ± 2	0.538 ± 0.002	0.282 ± 0.000
Gly9D-Leu	343 ± 1	0.536 ± 0.001	0.184 ± 0.000
Gly9D-Met	291 ± 1	0.619 ± 0.002	0.180 ± 0.000
Gly9D-Asn	352 ± 1	0.656 ± 0.002	0.231 ± 0.000
Gly9D-Gln	263 ± 1	0.679 ± 0.003	0.179 ± 0.000
Gly9D-Thr	338 ± 1	0.648 ± 0.002	0.219 ± 0.000

a) The values are from Ref. 33). b) Steady-state analysis was applied because of the low reliability of a fitting curve (U-value >14).

Regarding the kinetics among the four derivatives with a strong affinity (Gly9D-Lys, Gly9D-Pro, Gly9D-Arg, and Gly9D-Ser), cationic peptides (Gly9D-Arg and Gly9D-Lys) showed slightly faster association rates (k_on = 0.882 and 1.20 s⁻¹, respectively) compared to Gly9D-Pro and Gly9D-Ser (k_on = 0.684 and 0.744 s⁻¹, respectively), while Gly9D-Pro showed a slow binding feature among these peptides.

Combination of SAR Results

In our previous SAR study on 15-IgBP, substitution of Lys8Leu, where the Lys at position 8 in 15-IgBP was substituted to Leu, afforded a strong IgG-binding affinity (K_d = 8.19 nM).³³⁾ Therefore, the superior substitutions found in the present Gly9 derivatization were combined to the Lys8Leu peptide, and four derivatives_—Lys8Leu/Gly9D-Arg, Lys8Leu/Gly9D-Lys, Lys8Leu/Gly9D-Pro, and Lys8Leu/Gly9D-Ser—were newly synthesized. As a result, these four derivatives exhibited IgG-binding affinities (Table 4, K_d = 4.24–5.85 nM) more potent than Lys8Leu (K_d = 8.19 nM). They showed CD spectra similar to 15-IgBP with a small positive peak around 230 nm (Supplementary Fig. S1d), suggesting that these potent Lys8Leu derivatives possess a β-bulge as the secondary structure. Interestingly, a wide range of kinetic values (k_on and k_off) were observed among these derivatives. The k_on values ranged from 1.13 to 2.39 s⁻¹·µM⁻¹ and k_off values from 0.00658 to 0.0135 s⁻¹, while their potent binding affinity remained comparable. The k_off values of combined peptides, including the Lys8Leu substitution, remained consistent with the results in Table 3 for the substitutions on 15-IgBP (k_off = D-Ser > D-Arg > D-Pro > D-Lys at Gly9 position). Therefore, replacing the cationic Lys at position 8 with the hydrophobic Leu has minimal effect on the mode of interaction of the residue at Gly9 position with the antibody surface, despite the residues being adjacent to each other. These results suggest that the amino acid at position 9 can be selected according to the intended use of the IgG binding peptide. In other words, Gly9D-Lys can be used for applications where the slow-binding/slow-release feature is intended. Gly9D-Ser, on the other hand, is suitable for applications where the fast-binding/fast-release is used.

Table 4. Binding Affinity of the 15-IgBP Derivatives

Substitution	Kd (nM)	kon (s−1·µM−1)	koff (s−1)
Lys8Leua)	8.19 ± 2.07	1.47 ± 0.00	0.0116 ± 0.0010
Lys8Leu/Gly9D-Arg	5.25 ± 0.02	1.78 ± 0.01	0.00936 ± 0.00003
Lys8Leu/Gly9D-Lys	5.85 ± 0.01	1.13 ± 0.00	0.00658 ± 0.00001
Lys8Leu/Gly9D-Pro	4.24 ± 0.03	2.09 ± 0.01	0.00885 ± 0.00004
Lys8Leu/Gly9D-Ser	5.66 ± 0.02	2.39 ± 0.01	0.0135 ± 0.0000

a) The values are from Ref. 33).

Notably, the side chain of D-amino acids at Gly9 position of 15-IgBP is important for maintaining the secondary structure rather than interacting with the IgG surface. It is noteworthy that Lys8Leu/Gly9D-Lys, which showed one of the strongest affinities among all peptide derivatives synthesized up to this stage, has a modifiable ε-amino group that is far from the main chain and less likely to affect the secondary structure. Therefore, the ε-amino group of D-Lys could be a new crosslinking site for attaching functional molecules such as fluorophores and drugs to make IgG more functional. Meanwhile, previous studies by other groups have selected Lys8, Glu10, C-terminal, or N-terminal as modification sites. Although the ε-amine group is located far side from the Fc protein and longer crosslinker should be required for the crosslinking reaction, the approximately 50-fold higher binding affinity of Lys8Leu/Gly9D-Lys (K_d = 5.85 nM) compared to 15-IgBP (K_d = 267 nM) is beneficial for the crosslinking reaction.

Conclusion

In this study, we performed structure derivatizations of IgG-binding peptide 15-IgBP at the Ala5 and Gly9 positions. A total of 28 peptides were synthesized and the propensity of the secondary structure was evaluated for its IgG-binding affinity. From CD spectra analysis, we found that both the Ala5 and Gly9 positions have a significant effect on the secondary structure of IgBP. Furthermore, four peptides (Lys8Leu/Gly9D-Arg, Lys8Leu/Gly9D-Lys, Lys8Leu/Gly9D-Pro, and Lys8Leu/Gly9D-Ser), which combine the findings of the present study with previously discovered potent Lys8Leu, exhibited similar sub-nanomolar K_d values (K_d = 4.24–5.85 nM) but different binding kinetics. Importantly, the ε-amino group of D-Lys at the Gly9 position would be a novel site for attaching functional units such as fluorophores and drugs, and for inserting crosslinking structures applicable for ADC preparation. Consequently, IgG-binding peptides obtained in this study, together with their secondary structural information, have the potential for application in drug development.

Experimental

General Information

Reagents and solvents were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan), Sigma-Aldrich (St. Louis, MO, U.S.A.), Tokyo Chemical Industries (Tokyo, Japan), and Nacalai tesque (Kyoto, Japan). Amino acids and resin were purchased from Watanabe Chemical Industries (Hiroshima, Japan). The following building blocks were used for the amino acids whose functional groups required protection at the side chains: Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-D-Glu(OtBu)-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-D-Trp(Boc)-OH. Herceptin was purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Mass spectra were obtained on a Waters MICRO MASS LCT–premier (electrospray ionization (ESI)).

Synthesis of IgG-Binding Peptide Derivatives

IgG-binding peptides were synthesized by a Fmoc-based solid-phase peptide synthesis (SPPS) method using an automatic peptide synthesizer (Prelude). The Fmoc group of Fmoc-SAL resin (40 µmol) was deprotected with 20% piperidine in DMF for 10 min twice. The peptide chains were elongated with the Fmoc-protected amino acid (5 equiv.), HATU (5 equiv.), HOAt, (5 equiv.), and DIPEA (10 equiv.) for 30 min. The deprotection of Fmoc and coupling of corresponding Fmoc-amino acid were repeated to lengthen the desired peptide. The N-terminal amino group was acetylated with acetic anhydride and DIPEA for 15 min. After washing and drying the resin, cleavage from the resin and final deprotection was performed by treatment with TFA:TIS:DMB³⁴⁾ (40 : 1 : 2) for 3 h. An intramolecular disulfide bridge was constructed in solution by treatment of crude peptide with Npys-OMe^39,40) (2–5 equiv.) in 30–60% CH₃CN in H₂O. After the reaction was complete, as monitored by HPLC, the solution was lyophilized and washed with Et₂O to remove the reagent. After drying, the residual solid of synthetic peptides was purified by reverse phase HPLC. The purity of synthesized peptides was analyzed by reverse-phase chromatography (COSMOSIL 5C18 AR-II, 4.6ID × 150 mm) using a binary solvent system with a linear gradient starting from 10% MeCN in 0.1% TFA aq. to 50% MeCN in 0.1% TFA aq. at a flow rate of 0.9 mL/min with detection at UV 230 nm.

Evaluation of IgG Binding Affinity

The binding kinetics were determined based on previously reported conditions using a Biacore T-200 system.³³⁾ Herceptin was dissolved in an acetate buffer (pH 5.5) and immobilized by premixed N-hydroxysuccimimide and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC·HCl) onto a CM5 sensor chip. The analytes (peptide derivatives) were adjusted to the desired concentration by a serial dilution in a running buffer (HBS-EP; 0.01 M N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% tween 20, pH 7.4). The sensorgrams were obtained with an association time of 180 s, a dissociation time of 600 s, and a flow rate of 50 µL/min. To determine the binding kinetics (k_on, k_off and K_d), the obtained sensorgrams were analyzed by the Biacore T200 Evaluation software Ver.1.0, using a 1 : 1 binding model or steady state analysis.

CD Spectra Analysis

The CD spectra of the peptide derivatives were measured based on previously described conditions³³⁾ using a Jasco J-1500CD spectrometer (JASCO, Japan) and a quartz cell with a 0.5 cm path length. Spectra were collected between 190–250 nm with a scan speed of 100 nm/min, a response time of 1 s, and a bandwidth of 1 nm. The peptides were dissolved in a 10 mM phosphate buffer (pH 7.4) with a concentration of 2.5 µM.

Molecular Modeling

The crystal structure of IgG binding peptides [binding domain of protein G (PDB: 1FCC),²⁰⁾ B-domain (PDB: 1FC2),²¹⁾ Z34C (PDB: 5UBX),²⁵⁾ and core cyclic structure (from Asp3 to Thr15) of peptide 1/Lys8Arg (PDB: 6IQG)¹⁾] were obtained from the Protein Data Bank. The structures were depicted by molecular operating environment (MOE) software or UCSF Chimera.⁴⁵⁾ The energy minimization process was performed using the Amber10:EHT force field.

Acknowledgments

The authors acknowledge Mr. H. Fukaya and Ms. M. Okuyama of the Tokyo University of Pharmacy and Life Sciences for the mass spectral analysis and peptide synthesis, respectively. This work was supported by the Japan Society for the Promotion of Science (JSPS), KAKENHI, a Grant-in-Aid for Scientific Research (B) 15H04658, Basic Science and Platform Technology Program for Innovative Biological Medicine (AMED, JP18am0301006).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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