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Whether Native Chemical Ligation

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PEGylation (protein-polymer conjugation) is considered as the gold standard for modulating the functions of different proteins, the self-assembly of hierarchical structures, and the delivery of various therapeutic agents. Such protein conjugates were known to prolong the circulating time of different therapeutic molecules. PEGylation keeps the target protein active by ensuring optimum shielding. It is contended that controlling the topology of such protein-polymer conjugates could have important clinical correlates. For example, different studies have reflected that cyclized proteins could enhance the thermal stability and resist protease digestion of the respective therapeutic molecules (Hou et al., 2016). Although there have been recent developments in designing protein-polymer conjugates, there are various limitations of such preparations. Hou et al. (2016) stated that “generation of heterogeneous populations and poor molecular weight control due to non-selective chemical ligation methods impede the functioning and viability of such conjugates.”

The authors emphasized that the key hurdle in preparing such conjugates is not the lack of appropriate mechanisms for ligating the target macromolecules, but the redundant and costly methods of introducing the orthogonal functionalities in such conjugates. Although PEGylation is considered a gold standard in designing protein-polymer conjugates, repeated injections could enhance their blood clearance which might reduce their viability as a therapeutic system.

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Moreover, administration of PEGylated products could elicit antibody formation which might lead to undesirable antigen-antibody reactions. Hence, alternative methods to PEG are explored in preparing protein-polymer conjugates. In this regard, polypeptides or poly amino acids (PAA) have shown promising results in designing protein-polymer conjugates in comparison to PEG (Polyethylene glycol). This is because such molecules mimic the protein backbone, exhibit versatile functionalities of their side-chains, and ensure tunable degradability. For example, the fusion of unstructured polypeptides such as XTEN has shown to improve the pharmacological properties of different target proteins. However, fusion proteins based on PAA ligation require genetic modifications and are limited to the number of naturally occurring amino acids.

On the contrary, recent focus has shifted towards the synthesis of poly amino acids by the ring-opening polymerization (COP) of alpha-amino acid N-carboxy anhydrides. Such preparations exhibit intriguing stimulus-responsive, hierarchical self-assembly, and secondary structure formation properties. Initially, ROP was achieved with organometallic catalysts and metal-free initiators based on primary-amine derived initiators were used to synthesize PAAs under normal or specialized polymerization environments. Likewise, N-trimethylsilyl amines (N-TMS) such as hexamethyldisilazane (HMDS) are also effective in forming PAAs. Such agents act by making a nucleophilic attack on the carboxyl end of the N-carboxy anhydrides (NCAAs) to generate an amide bond and regulating the chain propagation through the addition of trimethylsilyl carbamate (TMSC) group at their amino terminal. However, such thiol-mediated or N-TMS-based ROP exhibit poor control and slow rate of polymerization of NCAAs (Yuan et al., 2016).

However, transforming the thioester group of PAAs with different functional groups might impose critical challenges within laboratory settings. Such challenges stem from the requirements of moisture-free experimental conditions and introduction of complex functional groups. Hence, only a few reports have confirmed the reproducibility and viability of labeled protein-PAA conjugates. On the other hand, no study has been conducted to explore the topological control of such conjugates and their viability across in-vivo and in-vitro cellular preparations.

The present study explored the effectiveness of natural/native chemical ligation (NCL) for conjugating ionic helical polypeptides (which are PAAs and used as carriers of thioester groups) with a given protein of interest (green fluorescent protein; eGFP). The study further explored whether such preparations could be easily delivered into the cytoplasm of target cells. The findings of the present study might unfold therapeutic areas of interest such as targeted drug delivery and genesis of transgenic cells. The NCL that was used in this study was sortase A. Sortase A is an enzyme that is isolated from the bacterium Staphylococcus aureus.

Materials and Methods

Purification of the conjugate of P (OEG3Glu-5% C6F13-DMA)20 -L- eGFP and P (OEG3Glu-5% C8F17-DMA)20 -L- eGFP by superdex 200 and MONO-SCG-e GFP(6 mg/ml, 500 μL, 1.0 equiv.) and P (OEG3Glu-5% C6F13-DMA)20 -L, (10 mM,128μL, 3.0 equiv.)P (OEG3Glu-5% C8F17-DMA)20 -L, (10 mM, 32μL, 3.0 equiv.) were mixed with TCEP and MPAA as the ratio of protein/MPAA/TCEP =1:9:18. The reactions were purified by Ni-Column chromatography, and the conjugates were purified in superdex 200. The solution was further purified by MONO-S (50 mM CH3CHOONa and 50 mM CH3CHOONa with 1M NaCl pH 6.0) and was analyzed in 12.5% SDS-PAGE.

Circularization of eGFP-Del1 with L-p(OEG3ene-Glu-NMe2)20
P(OEG3-Glu)20-eGFP4-LPETGGLEH6 (3.5 mg/ mL) and sortase A (0.1 equiv.) in the Tris・HCl buffer (50 mM, pH ~7.5) were incubated at room temperature overnight and purified by NiNTA affinity column (elution buffer: 20 mM Tris-HCl, 500 mM NaCl, 20 mMS15imidazole, pH 8.0). The product was confirmed by 12.5% SDS-PAGE.

Purification of OEG3ene

The impurities of OEG3ene was removed by dissolving it in a minimal amount of EtOAc and loaded onto a 2.4 x 10 cm silica column. The buffer was PE: EtOAc=5:1 SML (Sortase mediated ligation) of TEV-INFa-LPETG and TEV-eGFP-LPETG with PEG-GGG CG-eGFP-LPETG(2 mg/ml, 4mg/mL,6mg/mL30μL, 1.0 equiv.) and TEV-INFa-LPETG(4 mg/mL 30 uL ) and PEG-GGG (10 mM, 32μL, 10.0 equiv.) were mixed with sortase A in the ratio of protein/sortase A =1:0.1/1:1 and analyzed under 12.5% SDS-PAGE.

Synthesis of SPh-P(OEG3-Glu)50, SPh-P(OEG3-Glu)20-Gly3

In a glovebox, OEG3ene-Glu NCA (50.0 mg, 0.2898 mmol, 50 equiv) dissolved in anhydrous DMF (500 μL) was added to a PhS-TMS stock solution in DMF (5.8 μL × 0.5 M, 1.0 equiv) and stirred for 36 h at room temperature. Upon complete consumption of the monomer, an aliquot of the reaction mixture was diluted to 10 mg/mL in DMF and injected to GPC for molecular weight (MW) and polydispersity index (PDI) analysis. To obtain purified PAA-SPh, the reaction solution was poured into diethyl ether (50 mL), and the precipitate was separated by centrifugation, washed extensively by diethyl ether (50 mL × 2), and dried under vacuum. Typical yields were weighted at ∼60%.

Purification of P (OEG3-Glu)20-G3-eGFP-LPETG

CG-eGFP (12.4 mg/ mL, 600 uL, 1.0 equiv.) and Phs-P (OEG3-Glu-DMA)20-L-G3,(10 mM,57.4 μL, 5.0 equiv.) were mixed with TCEP and MPAA at the ratio of protein/MPAA/TCEP =1:9:18. The reactions were purified by MONO-S (50 mM CH3CHOONa and 50 mM CH3CHOONa with 1M NaCl pH 6.0) and analyzed by 12.5% Native gel.

Synthesis of OEG3ene-Glu

3.36 mL sulfuric acid was added drop-wise to a solution of L- Glu(5.65 g) suspended in OEG3ene-OH (19.3 g)at 0 oC for 10 min. After stirring at room temperature, the overnight viscous solution was poured slowly into a solution of triethylamine and isopropanol to yield a white precipitate. The precipitate was collected by centrifugation and then dissolved in methanol. After filtration, the filtrate was combined, and the solvent was removed under vacuum to obtain a white solid.

Cyclization of P (OEG3-Glu)20-G3-eGFP-LPETG

P (OEG3-Glu)20-G3-eGFP-LPETG(1.5 mg/ml, 100 μL, 1.0 equiv.) was mixed with sortase A (as a ratio of protein/sortase A =1:0.1) and analyzed under 15% SDS-PAGE and Native PAGE.

Synthesis of SPh-P (OEG3-Glu)50-3%C8F17, SPh-P(OEG3-Glu)100-3%C8F17

In a glovebox, OEG3ene-Glu NCA (100.0 mg, 0.2898 mmol, 20 equiv, 50 equiv and 100 equiv) was dissolved in anhydrous DMF (1000 μL) and added to a PhS-TMS stock solution in DMF (34.8 μL × 0.5 M, 1.0 equiv). The contents were stirred for 12 h at room temperature. Upon complete consumption of the monomer, an aliquot of the reaction mixture was diluted to 10 mg/mL in DMF and injected to GPC for molecular weight (MW) and polydispersity index (PDI) analysis. To obtain purified PAA-SPh; the reaction solution was poured into diethyl ether (50 mL), and the precipitate was separated by centrifugation, washed extensively by diethyl ether (50 mL × 2), and dried under vacuum. Typical yields were weighted ∼60%.

Preparation of EGxGlu NCAs

Anhydrous THF (50 mL) was added to a mixture of OEG3ene-Glu (1 g, 3.13mmol) and triphosgene (0.37 eq) under nitrogen. The solution was then heated to 50-degree centigrade for 5h, after which, the solvent was removed under vacuum. The yellow oil was then dissolved in a minimal amount of EtOAc and loaded onto 2.4 x 10 cm silica column slurry packed with 25 % EtOAc in petroleum ether. The NCA was eluted with 50% EtOAc in petroleum ether.

Cyclization of P (OEG3-Glu) 20-G3-eGFP-LPETG

P (OEG3-Glu) 20-G3-eGFP-LPETG (1.5 mg/ml, 1000μL, 1.0 equiv.) were mixed with sortase A (with a ratio of protein/sortase A =1:0.1) . The reaction was analyzed under 15% SDS-PAGE.

Intracellular Trafficking of P (OEG3ene-DMA)50 -3%C8H2F17-L- eGFP and P(OEG3ene-DMA)100 -3%C8H2F17-L- eGFP in HeLa
The target cells were seeded in a glass-bottomed culture chamber at dimensions of 10 × 104 cell/well. The cells were allowed to attach for 24 h at 37 ºC with 5% CO2. The culture medium was removed, and opti-men (1 mL) containing P (OEG3ene-DMA)100 -3%C8H2F17-L- eGFP (final concentration:2 uM) was added to the wells. The cells were incubated for 5 h and washed with heparin sodium (1.0 mg/mL, 1.0 mL) for 3 times. Finally, the cells were dyed with Lyso tracker and Hoechst life and incubated for 15 min at 37 ºC with 5% CO2. The cells were washed with 1XPBS three times viewed under a confocal microscope.
Intracellular Trafficking of circ (P(OEG3ene-Glu-DMA)20-eGFP) in HeLa with grafting yield of 70%

The process for analyzing intracellular trafficking of P(OEG3ene-DMA)50 -3%C8H2F17-L- eGFP and P(OEG3ene-DMA)100 -3%C8H2F17-L- eGFP in HeLa was repeated with circ(P(OEG3ene-Glu-DMA)20-eGFP) conjugate that exhibited a 70% grafting yield.

Results
Purification of the conjugate of P (OEG3Glu-5% C6F13-DMA)20 -L- eGFP and P (OEG3Glu-5% C8F17-DMA)20 -L- eGFP by superdex 200 and MONO-S

Fig.1: MONOS of conjugates of each peak

Fig.2 12.5% of SDS-PAGE of different peaks

Fig.3: Confocal microscopy of P (OEG3Glu-5% C6F13-DMA)20 -L- eGFP and P (OEG3Glu-5% C8F17-DMA)20 -L- eGFP
Although the fractions were purified, the conjugate of P (OEG3Glu-5% C6F13-DMA)50 –L and P (OEG3Glu-5% C8F17-DMA)50 -L did not escape from the lysosomes.Circularization of eGFP-Del1 with L-p (OEG3ene-Glu-NMe2)20
Fig.4: 12.5% SDS-PAGE of Circular of eGFP-Del1 with L-p (OEG3ene-Glu-NMe2)20
Fig 4 reflected that the circularization of eGFP-Del1 with L-p (OEG3ene-Glu-NMe2)20 was not appropriately achieved. Hence, the OEG3ene product was purified to repeat the circularization.
Purification of OEG3ene

Fig.5: NMR of OEG3ene
The figure reflected that the impurities were successfully removed from OEG3ene.
SML (Sortase mediated ligation) of TEV-INFa-LPETG and TEV-eGFP-LPETG with PEG-GG

Fig.6:12.5% SDS-PAGE of SML of TIL with PEG-GGG at different times

Fig.7:12.5% SDS-PAGE of SML of eGFP with PEG-GGG at different times
Fig.8:12.5% SDS-PAGE of SML of eGFP with PEG-GGG at different time

Fig.9:12.5% SDS-PAGE of SML of eGFP with PEG-GGG at different times

Fig.10:12.5% SDS-PAGE of SML of eGFP with PEG-GGG in 12 h
The SML of eGFP with PEG-GGG was effective with a ratio of 0.1:1 (protein: sortase).
Synthesis of SPh-P(OEG3-Glu)50, SPh-P(OEG3-Glu)20-Gly3

Fig.11: GPC of PhS-P(OEG3ene-Glu-DMA)50
The polymerization was a failure because the PDI of PhS-P(OEG3ene-Glu-DMA)50 was 1.069 and the MW of the conjugate was 23250 g/mol.
Purification of P (OEG3-Glu)20-G3-eGFP-LPETG

Fig. 12: Separation of P(OEG3-Glu)20-G3-eGFP-LPETG by superdex 200

Fig. 13: 12.5% Native PAGE of P (OEG3-Glu)20-G3-eGFP-LPETG by Superdex 200

Fig. 14: Separation of P (OEG3-Glu)20-G3-eGFP-LPETG (Peak 2 of Fig.1) by MONOS

Fig. 15: 12.5% Native PAGE of P (OEG3-Glu)20-G3-eGFP-LPETG by MONO S
Native PAGE of P (OEG3-Glu) 20-G3-eGFP-LPETG by MONOS showed that the conjugates were purified and were ready for cyclization.
Synthesis of OEG3ene-Glu

Fig. 16:1H NMR (400 MHz, CDCl3) of OEG3ene-Glu before recrystallization
Cyclization of P (OEG3-Glu) 20-G3-eGFP-LPETG

Fig. 17:12.5% Native PAGE of cyclization of P(OEG3-Glu)20-G3-eGFP-LPETG at different times.

Fig. 18:15% SDS-PAGE of cyclization of P (OEG3-Glu)20-G3-eGFP-LPETG in different time
The native PAGE electrophoresis suggested that the cyclization of P (OEG3-Glu) 20-G3-eGFP-LPETG could be appropriately achieved. Considering P (OEG3-Glu) 20-G3-eGFP-LPETG as ideal candidates for cyclization PAAs such as SPh-P(OEG3-Glu)50-3%C8F17, and SPh-P(OEG3-Glu)100-3%C8F17 were synthesized.
Synthesis of SPh-P(OEG3-Glu)50-3%C8F17, SPh-P(OEG3-Glu)100-3%C8F17

Fig. 19.GPC of PhS-P(OEG3ene-Glu-DMA)50

Fig. 20:1H NMR (400 MHz, CDCl3) of SPh-P(OEG3-Glu)50-3%C8F17

Fig. 21.GPC of PhS-P(OEG3ene-Glu-DMA)100

Fig. 22:1H NMR (400 MHz, CDCl3) of SPh-P(OEG3-Glu)100-3%C8F17
The PDI of PhS-P (OEG3ene-Glu-DMA) 50 was 1.038 with an MW of 15340 g/mol. The PDI of PhS-P(OEG3ene-Glu-DMA)100 was 1.05 with an MW of 29660 g/mol. The percentage of grafting achieved was 100%.
Extraction of EGxGlu NCAs

Fig. 23:1H NMR (400 MHz, CDCl3) of OEG3ene-Glu NCACyclization of P (OEG3-Glu)20-G3-eGFP-LPETG

Fig. 24:15% SDS-PAGE of cyclization of P (OEG3-Glu)20-G3-eGFP-LPETG in different time
Findings on Intracellular trafficking

Fig.1: Confocal microscopy of P(OEG3ene-DMA)100-L-eGFP with 2 uM
Fig.2: Confocal microscopy of P(OEG3ene-DMA)50-L-eGFP with 2 uM

Fig. 25: Confocal microscopy of circ(P(OEG3ene-Glu-DMA)20-eGFP) with different time
Figure 1 reflects that P (OEG3ene-DMA) 100 -3%C8H2F17-L- eGFP cannot escape from the lysosomes. However, figure 2 reflected that the conjugate P (OEG3ene-DMA) 50 -3%C8H2F17-L- eGFP could escape from the lysosomes when the functional status of the target cell deteriorates. Fig 3 reflected that the conjugate of circ (P (OEG3ene-Glu-DMA) 20-eGFP) could not escape from the lysosomes in either 6h or 18 h under 70% grafting.

Discussion and Conclusion

Yuan et al. (2016) emphasized the use of S-trimethylsilyl amines (S-TMS) in place of N-trimethylsilyl amines for mediating the ROP of NCAAs. The authors highlighted that S-TMS-based ROP of NCAAs would be more effective in ensuring ROP of NCAAs in preference to N-TMS-based methods. This is because the sulfur atom is a more powerful nucleophilic candidate than nitrogen, and the sulfur-silicon bond is more reactive than the nitrogen-silicon bond for prompting ring opening. Hence, S-TMS-based ROP ensures faster chain initiation than the N–TMS based ROP of NCAAs. On the other hand, the S-TMS based ROP would generate a reactive thioester at the C-terminal end of the PAA. Such groups are easily transformable into other functional groups by incorporating peptides or amino acids through native or natural chemical ligation.

In one study, Hou et al. (2016) showed that a two-step polymerization reaction that leads to the formation of hetero-telechelic block PAAs are potential candidates for the synthesis of site-specific topological protein-PAA conjugates. Such two-step polymerizations produce two orthogonal chemical handles. The two orthogonal chemical handles include a phenyl thioester site for NCL and a polyglycine site for sortase-mediated ligation at the carboxyl and amino terminals of the PAAs respectively. Such process eliminates the need of prefunctionalization of the initiator units or post-polymerization modification of the respective end products. The authors concluded that “NCL or sortase-based protein conjugates exhibit well-defined topological properties such as improved thermo stability and resistance to protease-mediated digestion.

The present study explored the effectiveness of natural/native chemical ligation (NCL) for conjugating ionic helical polypeptides (which are PAAs and used as carriers of thioester groups) with a given protein of interest (green fluorescent protein; eGFP). The present study explored two broad aspects; which are the most effective ROP that undergoes effective conjugation with NCA and whether such cyclical conjugates can exhibit intracellular trafficking. The study showed that although the conjugate of P (OEG3Glu-5% C6F13-DMA) 50 –L and P (OEG3Glu-5% C8F17-DMA)50 –L was achieved; it did not escape from the lysosomes. On the other hand, the cyclization of P (OEG3-Glu) 20-G3-eGFP-LPETG could be appropriately achieved. Considering P (OEG3-Glu) 20-G3-eGFP-LPETG as ideal candidates for cyclization PAAs such as SPh-P(OEG3-Glu)50-3%C8F17, and SPh-P(OEG3-Glu)100-3%C8F17 were synthesized. The PDI of PhS-P (OEG3ene-Glu-DMA) 50 and PhS-P(OEG3ene-Glu-DMA)100 was appropriate. These findings suggest that such PAAs could be effective as conjugates because of viable surface topologies. Regarding intracellular trafficking, the P (OEG3ene-DMA) 50 -3%C8H2F17-L- eGFP conjugate escaped from the lysosomes when the functional status of the target cell was compromised. Hence, future studies should be carried out in functionally viable target cells to assess the intracellular trafficking of P (OEG3ene-DMA) 50 -3%C8H2F17-L- eGFP. On the contrary, circularized P (OEG3ene-Glu-DMA) 20-eGFP was not released from the lysosomes.

The present study reflected that certain conjugates were purified and viable, but they were not efficient to exhibit intracellular trafficking. However, such possibilities may be relooked with a higher percentage of grafting and under the functional integrity of the transfected cells.

References

Yuan, J, Sun, Y, Wang, J, and Lu, H (2016). Phenyl Trimethylsilyl Sulfide-Mediated Controlled Ring-Opening Polymerization of αAmino Acid NCarboxyanhydrides, Biomacromolecules 17, 891−896
Hou, Y., Yuan, J, Zhou, Y, Yu, J, and Lu, H (2016). A Concise Approach to Site-Specific Topological Protein−Poly (amino acid) Conjugates Enabled by in Situ-Generated Functionalities, J. Am. Chem. Soc. 138, 10995−11000

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