Selective covalent capture of collagen triple helices with a minimal protecting group strategy

Collagens and their most characteristic structural unit, the triple helix, play many critical roles in living systems which drive interest in preparing mimics of them. However, application of collagen mimetic helices is limited by poor thermal stability, slow rates of folding and poor equilibrium between monomer and trimer. Covalent capture of the self-assembled triple helix can solve these problems while preserving the native three-dimensional structure critical for biological function. Covalent capture takes advantage of strategically placed lysine and glutamate (or aspartate) residues which form stabilizing charge–pair interactions in the supramolecular helix and can subsequently be converted to isopeptide amide bonds under folded, aqueous conditions. While covalent capture is powerful, charge paired residues are frequently found in natural sequences which must be preserved to maintain biological function. Here we describe a minimal protecting group strategy to allow selective covalent capture of specific charge paired residues which leaves other charged residues unaltered. We investigate a series of side chain protecting groups for lysine and glutamate in model peptides for their ability to be deprotected easily and in high yield while maintaining (1) the solubility of the peptides in water, (2) the self-assembly and stability of the triple helix, and (3) the ability to covalently capture unprotected charge pairs. Optimized conditions are then illustrated in peptides derived from Pulmonary Surfactant protein A (SP-A). These covalently captured SP-A triple helices are found to have dramatically improved rates of folding and thermal stability while maintaining unmodified lysine–glutamate pairs in addition to other unmodified chemical functionality. The approach we illustrate allows for the covalent capture of collagen-like triple helices with virtually any sequence, composition or register. This dramatically broadens the utility of the covalent capture approach to the stabilization of biomimetic triple helices and thus also improves the utility of biomimetic collagens generally.

Peptide N-terminal Acetylation. After coupling all the amino acid residues and the final cycle of deprotection, peptide was acetylated with acetic anhydride. The molar ratio of the acetylation reagents was 1:8:4 (resin/acetic anhydride/DiEA). The acetylation reagents were mixed and added into the DCM-washed resin and reacted for 45 mins. Acetylation was repeated once more before cleavage. After acetylation, the resin was washed 3 times with DCM, 10 mL for each (3*10 mL) and continued with cleavage. Ninhydrin or chloranil test was carried out to confirm complete acetylation.
Peptide Cleavage. The peptide on resin was mixed with the cleavage cocktail (90% TFA/2.5% Anisole/2.5% Milli Q H2O/2.5% TIPS/2.5% EDT by volume) and reacted for 3 hours. The cleaved peptide was then drained into a clean r.t flask and half the amount of cleavage cocktail was added to wash the resin; all the solution was collected into the flask. The TFA was blown off with nitrogen gas. Approximately 50 mL of ice-cold diethyl ether was added into the peptide solution to triturate the peptide. The mixture was then centrifuged to obtain the white pellet of the crude peptide. The white pellet was washed once more with 30 mL of cold ether to further remove the cleavage cocktail. After that, the crude peptide was covered with Kim wipes and left to dry in the fume hood. The acid labile side chain protecting groups were removed during cleavage. The side chains of the internal lysine, aspartic acid and glutamic acid were orthogonally protected and were removed after successful covalent capture.

Peptide Purification and Characterization 2.1 Peptide Purification
The dried crude peptide was dissolved in milli Q H2O to a concentration of approximately 18 mg/mL. The peptide solution was purified with reversed-phase HPLC (Varian PrepStar220) using a preparative C-18 column. The mobile phase is acetonitrile with 0.05% TFA by volume (solvent B) vs milli Q H2O with 0.05% TFA by volume (solvent A). The void time of the system is 2.50 mins. The collected fractions of purified peptides were rotovapored of the acetonitrile and frozen and then lyophilized to remove the moisture. The purified peptides were reinjected into the UPLC to confirm the purity. The UPLC gradient was 0.75 min-6.75 min, 5-35% solvent B.

Mass spectrometry
Bruker AutoFlex Speed MALDI ToF and Bruker MicroToF ESI LC-MS System were used to characterize the mass-to-charge ratio of ions of peptides. For MALDI spectrometry measurement, 1.5 uL of 10 mg/mL matrix CHCA in 50% (v.v.) MQ H2O, 50% (v.v) acetonitrile and 0.1% (v.v) TFA was mixed with 1.5 ul 20 mg/mL peptide solutions for sample co-crystallization. For LC-MS measurement, samples were prepared into a 0.5 mg/mL concentration in MQ H2O. The measured and the expected massto-charge ratio was listed in Table S1. *"cc" represents covalent capture. The covalent capture samples were removed of the respective protecting groups. The spectra comparison of trimers before and after covalent capture are listed below.    Figure S9. a) UPLC trace of purified peptide SP-A2_ K. The gradient of solvent B is 0.75-6.75 min 5-35% B. b) ESI mass spectrum of purified peptide SP-A2_K. Figure S10. Maldi mass spectrum of covalently captured triple helices with a) ivDde protecting groups, b) OAll protecting groups and c) OBzl protecting groups. Triple helices samples are pre-purification and pre-deprotecting.

ESI mass spec of ccSP-A1_K and ccSP-A2_K
Figure S11. ESI mass spectra of covalently captured SP-A triple helices with ivDde protecting groups. a) ccSP-A1 _K b) ccSP-A2 _K. . UPLC traces of purified covalently captured triple helices. The gradient is 5-35% B 0.5-6.5 min. a) ccSP-A1 from the covalent capture of SP-A1_K triple helix followed by the ivDde protecting group removal. b) ccSP-A2 from the covalent capture of SP-A2_K triple helix followed by the ivDde removal.

Covalent Capture
The covalent capture procedures were referred to the literature. 1 In detail, HPLC purified peptide was dissolved in 100 mM MES buffer pH 6.1 to result in a 3 mM peptide concentration. The pure peptide content was approximately 76% according to our previous analysis. 12 mM Hydroxybenzotriazole and 120 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) solutions were prepared in 100 mM MES buffer. After the monomer peptide folded into collagen triple helices and the thermal stability was confirmed with circular dichroism, the peptide solution was mixed with EDC and HOBt solutions in a 1:1:1 volume ratio. The resulted solution has the peptide concentration of 1 mM in 4 mM HOBt and 40 mM EDC. The mixture was kept at 5 °C and was vortexed every hour at the first 4 hours of covalent capture reaction. After 4 days of covalent capture reaction, the reaction was quenched with different conditions (described below) and continued with removal of the side chain protection groups of the internal charged amino acid residues. EDC and HOBt need to be removed with Spin column, HPLC, or quenching reagents before sample characterization with mass spectrometry.

Deprotection of the Side Chain Protecting Groups on Internal Charged Amino Acid Residues
Scheme S1. Reaction Scheme of the Selective Covalent Capture using ivDde, OBzl and OAllyl Protecting Group Scheme S1. Successful deprotection condition of template triple helices. Before the deprotection, the reaction solution was treated with spin column (MWCO 3K) to remove part of EDC and HOBt.

Deprotection of ivDde protecting group (Condition 1 and 8 in the main text)
Condition 1. Removal of ivDde from monomer peptide. The reaction works for either crude peptide or purified peptide. The monomer peptide was dissolved into 100 mM MES buffer till a concentration of approximately 10 mg/mL. Add equal volume of 1M hydroxyamine (NH2OH). Mix and agitate the reaction solution overnight at room temperature. Then quench hydroxyamine with 1M HCl till the solution is acidic.
Condition 8. Removal of ivDde from covalently captured trimer. After covalent capture, we used the Pierce™ Protein Concentrators PES, 3K MWCO, 0.5 mL to remove EDC and HOBt to avoid extra iso-peptide bond formation after the deprotection. The sample solution was centrifuged three times, each at 12 000 rcf for 30 mins. If uses 3K MWCO, 2-6 mL concentrator, the samples were treated at 4700 rcf for 30 mins for 3 time. Between each centrifugation, certain volume of 100 mM MES buffer was added into the sample tube to bring the volume of solution to the starting point. After centrifugation, collect the sample solution and equal volume of 1M NH2OH was added into the solution for ivDde removal. The solution was mixed, reacted, and quenched as described in Condition 1.

Deprotection of allyl ester protecting group (Condition 2, 9, 10)
Condition 2. Removal of allyl easter from monomer peptide. The reaction conditions refer to literature with modification. 2 According to our trials, the condition works only for purified peptide. The peptide was prepared into approximately 10 mg/mL solution in 50 mM phosphate buffer (pH 7). 25 eq. of [Pd(All)Cl]2 catalyst and 25 eq. of L-Glutathione reduced (GSH) were added into the solution. The pH was adjusted to be 8 with 1M NaOH. The solution was mixed well and agitated at 37 °C for 30 mins. To quench the reaction, 40 eq. dithiothreitol (DTT) was added. The catalyst then precipitated, and the sample solution was centrifuged three times to remove the catalyst. The reaction solution was purified with HPLC.
Condition 9. The covalently captured reaction solution was pretreated with Pierce™ Protein Concentrators PES, 3K MWCO as described previously. Similarly, 25 eq. of [Pd(All)Cl]2 catalyst and 25 eq. of L-Glutathione reduced (GSH) were added into the solution. The pH was adjusted to be 8 with 1M NaOH. The solution was mixed well and agitated at 45 °C for > 12 hrs. After quenching the reaction with DTT, peptide was purified and characterized. We observed incomplete deprotection.
Condition 10. The covalently captured reaction solution was pretreated with Pierce™ Protein Concentrators PES, 3K MWCO as described previously. Certain amount of 1M K2CO3 was added into the solution till the concentration of K2CO3 was approximately 80 mM. The pH was confirmed to be approximately 11. The solution was agitated at 45 °C for 2 hrs. The reaction was quenched with 1M HCl till the solution was acidic. (Condition 3, 4, 5, 11, 12) Condition 3. Removal of benzyl ester from peptide monomers with Pd black. Dissolve the peptide in glacier acetic acid till a concentration of 10 mg/mL. Add Pd black into the solution. Purge the reaction solution three times with N2 and vacuum. Add a H2 balloon into the reaction solution. Agitate the reaction solution and follow the reaction progress with UPLC. To quench the reaction, centrifuge the solution three times to get rid of Pd catalyst.

Deprotection of benzyl ester protecting group
Condition 4 and 5. Removal of benzyl ester from the monomer peptide with saponification. These conditions work for either purified or crude peptide solutions. Dissolve the peptide in MQ H2O till a concentration of 10 mg/mL. In condition 4, add 1M NaOH till pH close to 11; in condition 5, add 1M K2CO3 till the pH close to 11. Mix and agitate the reaction solution at 45 °C for 1 hr. Add 1M HCl to quench the reaction till the solution is acidic.
Condition 11 and 12. Removal of benzyl ester from the covalently captured trimer. The samples need to be pretreated with Pierce™ Protein Concentrators PES, 3K MWCO as described above. The deprotection and workup procedures are as described in conditions 4 and 5.

Deprotection of Dmab protecting group (Condition 6,7, 13, 14)
Condition 6 and 7. Removal of Dmab protecting group from monomer peptides. Purified peptides were dissolved into 10 mg/mL concentration in MQ H2O. For condition 6, 2% wt. hydrazine was added. For condition7, equal volume of 1M NH2OH was added into the solution. The reaction solutions were mixed and reacted at r.t. To quench the reaction, 1M HCl was added till the solution was acidic. Our data suggest the hydrazine condition can realize almost quantitative conversion within 30 mins and that was 2 hrs for hydroxyamine condition. No side product was observed.
Condition 13 and 14. Removal of Dmab protecting group from the covalently captured trimer. The covalently captured trimer solution was pretreated with Pierce™ Protein Concentrators PES, 3K MWCO as described above. In condition 13, 2% wt. hydrazine was added into the solution and in condition 14, equal volume of 1M hydroxyamine was added. The reaction was mixed and agitated at r.t. for 2 hrs in condition 13 and 6hrs in condition 14. We observed the cyclized side product as the major species. Figure S13. UPLC traces of OAll removal from covalently captured triple helix ccKGE(Obzl). The deprotection was carried out at in 80 mM aqueous K2CO3 solutions at a) room temperature, b) 35 °C and c) 45 °C. The percentage of expected product was calculated by integrating the product peak area eluted at 2.6 min.  Figure S14. UPLC traces of OBzl removal from covalently captured triple helix ccKGE(Obzl). The deprotection was carried out at in 80 mM aqueous K2CO3 solutions at a) room temperature, b) 35 °C and c) 45 °C. The percentage of expected product was calculated by integrating the product peak area eluted at 2.5 min. Figure S15. UPLC traces of OBzl removal from covalently captured triple helix ccKGE(Obzl). The deprotection was carried out at 45 °C in a) 20 mM b) 40 mM c) 60 mM and d) 80 mM aqueous K2CO3 solutions. The percentage of expected product was calculated by integrating the product peak area eluted at 2.5 min.

Circular Dichroism
The circular dichroism (CD) data were collected on a Jasco J-810 spectropolarimeter equipped with a Peltier temperature controller. Spectrum measurement was collected at 5 °C. The spectra scanning used 1 mm cuvette and a peptide concentration of 0.03 mM. The melting curves were collected from 5 °C to 85/95 °C with a heating rate of 10 °C/hour at the wavelength that gives the maximum MRE value of each sample. The melting data was collected using 1 mm cuvette with a peptide concentration of 0.3 mM to obtain better signal. For the refolding experiment, the sample was kept at 85/95 °C for 30 mins to fully denature the folded triple helices and then the temperature was cooled down to 5 °C with a cooling rate of 10°C/hour. In some cases, the volume of the sample solution decreased from 200 uL to approximately 185 uL by the completion of the CD experiment. The volume change was caused by evaporation of the solvent, despite efforts to prevent this, at high temperature. Therefore, we assume the concentration as well as the MRE values presented in the main text of the refolded sample are approximately 8% higher than reality. The first-order derivatives of the melting curves were calculated with Savitzky-Golay smoothing algorithm, and temperature where the minimum derivative value appears was regarded as the melting temperature. The molar residue ellipticity (MRE) value was calculated with the equation, MRE = (θ × m)/ (c × l × nr × 10) where θ represents the experimental ellipticity in millidegrees, m is the molecular weight of the peptide (g/mol), c is the peptide concentration (milligrams/milliliter), l is the path length of the cuvette (cm), and nr is the number of amino acid residues in the peptide. CD spectrum Figure S16. CD spectra of folded supramolecular triple helix. a) CD spectra of triple helices from unprotected and protected KGE peptides. b) CD spectra of triple helices from SP-A1 peptides with and without protecting groups. c) CD spectra of triple helices from unprotected and protected SP-A2 peptides.